Gastric Acids

GASTRIC ACID, CALCIUM ABSORPTION, ANDTHEIR IMPACT ON BONE HEALTHSascha Kopic and John P. Geibel

Departments of Surgery and Cellular and Molecular Physiology, Yale School of Medicine,New Haven, Connecticut

LKopic S, Geibel JP. Gastric Acid, Calcium Absorption, and Their Impact on BoneHealth. Physiol Rev 93:189–268, 2013; doi:10.1152/physrev.00015.2012.—Cal-cium balance is essential for a multitude of physiological processes, ranging from cellsignaling to maintenance of bone health. Adequate intestinal absorption of calcium is amajor factor for maintaining systemic calcium homeostasis. Recent observations indi-

cate that a reduction of gastric acidity may impair effective calcium uptake through the intestine.This article reviews the physiology of gastric acid secretion, intestinal calcium absorption, and theirrespective neuroendocrine regulation and explores the physiological basis of a potential link be-tween these individual systems.

I. INTRODUCTION 189II. GASTRIC ACID SECRETION 189III. INTESTINAL CALCIUM ABSORPTION 203IV. REGULATION OF CALCIUM HOMEOSTASIS 212V. THE STOMACH AND CALCIUM 231VI. CONCLUSIONS 238

I. INTRODUCTION

The average adult human body contains �1.6% calcium,which relates to �1,120 g in a 70-kg individual (743). Ninety-nine percent of the calcium is stored in bone and teeth andis therefore inaccessible to most physiological processes(743). Although the amount of the immediately accessible11 g (1%) of calcium may seem miniscule, this fractionrepresents a pivotal constituent of our body. It serves abroad diversity of roles, which range from intracellular sig-naling and maintenance of membrane integrity to musclecontraction and neuronal transmission.

To allow for these calcium-dependent processes to func-tion, our body undertakes extensive measures to keep theintracellular and extracellular calcium concentrations andthe gradient between these two compartments stable. Theextracellular calcium concentration is typically clamped at�1.1 mM, whereas the intracellular environment is kept ata 10,000 times lower concentration. In consequence, rela-tively small disturbances in calcium homeostasis can lead tosevere symptoms, such as cardiac arrhythmias or cognitivedysfunctions. To maintain eucalcemia, our body is there-fore tightly regulating the balance between calcium absorp-tion by the intestine and calcium excretion by the kidney. Inaddition, calcium is deposited in or extracted from bone,which serves as a dynamic calcium reservoir. These threeorgan systems, i.e., the intestine, the kidney, and bone, areprecisely controlled by a complex endocrine network,

which primarily consists of the calcitropic hormones: 1,25-dihydroxyvitamin D [1,25(OH)2-vitamin D], parathyroidhormone (PTH), and calcitonin.

This review mainly focuses on the question as to how calciumenters the body through the intestine and how this mechanismis regulated via the endocrine system. Furthermore, the processof gastric acid secretion as related to calcium homeostasis willbe reviewed in detail. This may seem surprising, as gastric acidsecretion and intestinal calcium absorption are two distinctphysiological processes, which on first examination may notseem to be interdependent. However, recent clinical studiessuggest that there may be a relationship between reduced gas-tric acid secretion and increased risk for sustaining bone frac-tures, which asks the question whether we need gastric acid toabsorb calcium efficiently through the intestine, or whether thestomach exerts endocrine functions that impact bone health.Indeed, it has been put forward several decades ago that gas-tric acid solubilizes calcium that is then complexed with otherdietary constituents, thereby allowing for a more efficient ab-sorption in the intestine (18, 520, 699, 797). Furthermore, it islong known that a partial or complete resection of the stomachresults in decreased bone density, also leading to fractures (58,305, 732, 876). The stomach, the intestine, and bone are there-fore functionally more intertwined than one may initially as-sume. This review will independently analyze the processes ofgastric acid secretion, intestinal calcium absorption, and theirrespective neuroendocrine control and will conclude with acritical attempt at illustrating where these two seemingly inde-pendent organ systems intersect in terms of calcium homeo-stasis and bone health.

II. GASTRIC ACID SECRETION

The stomach is a unique organ that fulfills multiple roles.The main function of the gastric mucosa is to secrete con-

Physiol Rev 93: 189–268, 2013doi:10.1152/physrev.00015.2012

1890031-9333/13 Copyright © 2013 the American Physiological Society

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centrated hydrochloric acid, which provides a chemical bar-rier against ingested pathogens and aids in the digestion offoodstuffs. To achieve these functions, the gastric glandcontains specialized cells that pump protons into the gastriclumen in an effort to acidify the contents of the stomach.These cells are known as parietal cells, or oxyntic cells.Since concentrated acid is a noxious substance, the gastricmucosa has to undertake extensive measures to protect it-self from tissue injury. The protection is accomplished bysecreting mucus from mucus neck cells, but also by tightlyregulating the secretion of acid (see sect. IIB). A variety ofspecialized endocrine cells in the gastric mucosa are in-volved in the regulation of gastric acid secretion. A pertur-bation of either protective mechanism can lead to severetissue damage, resulting in gastric ulcers. This section dis-cusses the process of how gastric acid is secreted by review-ing the molecular mechanism underlying acid secretion inthe parietal cell and its neuroendocrine regulation.

A. Apical Ion Transport in the Parietal Cell

The gastric parietal cell is responsible for acidifying thestomach by secreting concentrated acid. Gastric acid secre-

tion depends on the apical extrusion of three ions. Protonsare pumped into the gastric lumen by a proton pump, thegastric H�-K�-ATPase, to acidify the gastric content to apH of as low as 1. Chloride is secreted via apical chloridechannels to ensure formation of HCl and to provide thecounter-ion conductance to protons. Lastly, potassiumleaves the parietal cell apically in a recycling mechanism,thereby fueling reciprocal proton transport by the H�-K�-ATPase (FIGURE 1). It has been demonstrated in numerousinvestigations that disruption of one of these ion transportmechanism renders the parietal cell incapable of secretinggastric acid (705, 820, 1013, 1029).

1. H�-K�-ATPase

A) STRUCTURE. The gastric H�-K�-ATPase belongs to thefamily of P2-type ATPases, which also includes the ubiqui-tous Na�-K�-ATPase and the sarcoplasmic reticulumCa2�-ATPase (SERCA). As the name implies, it exchangesone intracellular hydrogen ion for one extracellular potas-sium ion at the expense of ATP. ATP is provided to thepump by a large network of mitochondria, which occupyup to 40% of the cell volume, making the parietal cell one ofthe most mitochondria-rich cells in the body (292). In the

K+

PPIsAPAs

Parietal cell

KCNQ1Kir

CFTRCIC-2?SLC26A9

SST

BasolateralApical

Hist

SSTR

K+

H+

Ca2+

cAMP

Cl–

Gast

ACh

CCK2

M3

H2

FIGURE 1. Parietal cell model. The gastric parietal cell is equipped with apical ion transport mechanisms thatallow for the secretion of concentrated hydrochloric acid. Activation of basolateral secretagogue receptorsmainly leads to an increase in either cAMP (histamine) or calcium (acetylcholine, gastrin), causing apicalinsertion and activation of the H�-K�-ATPase. Somatostatin reduces intracellular cAMP levels. ACh, acetyl-choline; APAs, acid pump antagonists; Gast, gastrin; Hist, histamine; PPIs, proton pump inhibitors; SST,somatostatin.

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process of proton extrusion, the H�-K�-ATPase can over-come a massive acid gradient of 6 pH units, which is nec-essary to achieve sufficient gastric acidification. The pumpitself is a heterodimer, consisting of a � subunit and a �subunit, while the individual pumps assemble as (��)4 te-tramers on the parietal cell surface (1). The � subunit con-sists of 10 transmembrane domains and contains the cata-lytic site, which mediates ion exchange. The � subunit sta-bilizes the � subunit and is heavily glycosylated (41, 1105).Mutational analysis of the glycosylated asparagine residuessuggests that these sites are critical for adequate membranedelivery of the entire pump (41, 1105). Furthermore, the �subunit prevents a reversal of ion transport by a “ratchet”-like mechanism, which allows H�-K�-ATPase to pumpagainst the imposed high proton gradient (4, 294). Bothsubunits share a significant degree of homology to Na�-K�-ATPase (697, 1012). This close relationship to other P2-type ATPase has historically been exploited for homologymodeling of H�-K�-ATPase based on the crystal structureof SERCA, which had been acquired in several conforma-tional states (762, 815, 1092, 1093). Recently, however,direct structural information on H�-K�-ATPase has beenobtained by electron crystallography, also in the presence ofthe acid pump antagonist SCH28080 (2–4).

B) TRAFFICKING. In the resting parietal cell, H�-K�-ATPase isstored in tubulovesicles throughout the cell (292). Follow-ing neuronal or hormonal stimulation (see sect. IIB), thesevesicles are postulated to fuse with the apical pole, which ischaracterized by multiple microvilli-lined membrane in-vaginations, the so-called secretory canaliculi (292). Thisdistinct apical morphology of the parietal cells maximizescell surface and thereby allows for insertion of a high num-ber of proton pumps per cell following stimulation. Thechanges in membrane morphology and insertion of H�-K�-ATPase are extremely dynamic to ensure fine regulation ofgastric acid secretion (973). H�-K�-ATPase containingtubulovesicle fusion relies on SNARE complex forma-tion. In particular, the SNARE proteins syntaxin 3/7/12/13, VAMP2/8, and SNAP-25 were implicated to be candi-dates mediating this process (548–550, 624). The func-tional significance of these proteins was, for example,demonstrated in primary rabbit parietal cell cultures ex-pressing a SNAP-25 mutation, which was shown to reducetheir capacity to secrete gastric acid (548).

Apart from SNARE proteins, the small GTPases of the rabfamily (rab2/11a/25/27b) are involved in the regulation ofH�-K�-ATPase vesicle trafficking (147, 293, 386, 387,1049, 1070). Functional data especially substantiate theimportance of rab11a and rab27b. In parallel to SNAP-25defective cells, parietal cells transfected with a rab11a andrab27b mutant secrete acid less effectively (293, 1049).

After stimulation, in the off-phase of gastric acid secretion,H�-K�-ATPase has to be retrieved from the plasma mem-

brane for recycling (336). It is plausible that the initial stepof this process relies on the formation of clathrin-coated pitsand subsequent vesicle budding. Indeed, clathrin was iden-tified fairly early on H�-K�-ATPase containing tubulo-vesicles, although a functional role was not demonstrated(813). One of the multiple clathrin binding proteins is Hun-tingtin interacting protein 1 related (Hip1r) which aids invesicle formation and membrane trafficking (309). It isstrongly expressed in parietal cells, especially in the vicinityof secretory canaliculi (522). Functionally, Hip1r-deficientanimals present with a decreased number of parietal cells,loss of tubulovesicles, and decreased acid output (522,561).

2. Chloride secretion

Apical chloride secretion provides the second componentfor the formation of concentrated HCl and maintains over-all electroneutrality during acid secretion. The importanceof chloride efflux for the process of gastric acid secretionhas been established in the 1980s. Patch-clamp measure-ments demonstrated the presence of chloride conductanceon the apical pole of the parietal cell in Necturus, the humanparietal cell line HGT-1, and rabbit parietal cells (259, 935,940). All reports demonstrated a sensitivity of the chloridecurrent to cAMP or histamine, which is a common secondmessenger promoting acid secretion or a direct acid secre-tagogue, respectively (259, 935, 940). Simple flux measure-ments in isolated parietal cell vesicles had indicated thepresence of a chloride conductance pathway even earlier(232, 895, 1169). In these early experiments, inhibition ofchloride flux with chloride channel blockers also abolishedproton transport which underlines the necessity of intactchloride secretion for acid secretion to take place (232, 895,1169). However, the molecular identity of the chloridepathway remained elusive. Today, at least three candidateshave been put forward as potential mediators of apical chlo-ride secretion in the parietal cell: the cystic fibrosis conduc-tance regulator (CFTR), chloride channel protein 2 (ClC-2),and solute carrier 26 A 9 (SLC26A9) (FIGURE 1).

A) CFTR. CFTR represents a common apical chloride conduc-tance pathway in a broad variety of epithelia, such as theairways, intestine, and pancreas. Its mutation is responsiblefor the most widespread inherited disease, namely, cysticfibrosis (CF), which results in increased mortality due tosecretory defects and concomitant infections. The presenceof CFTR has been confirmed in gastric mucosa by in situhybridization, albeit at low quantities (1044). Nevertheless,functional measurements in isolated gastric glands demon-strated a decreased acid secretory capacity in animals car-rying the most common mutation responsible for CF(�F508) (1013). Furthermore, acid secretion was reducedin wild-type animals when a specific CFTR inhibitor wasapplied (1013). Although these observations may suggest adirect involvement of CFTR in the process of chloride se-cretion, it is plausible that CFTR rather has a regulatory

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effect on H�-K�-ATPase (1013). In other tissues, CFTRcan interact with a variety of ion transport proteins, such asNHE, forming regulatory complexes, making an interac-tion with H�-K�-ATPase plausible (1013).

B) CLC-2. ClC-2 has been proposed as an alternative chloridesecretion pathway to CFTR in other epithelia, such as thelung and intestine (207, 404, 675, 766). ClC-2 has beencloned from rabbit gastric mucosa, which led to the hypoth-esis that the channel may also be involved in acid secretion(706). However, follow-up investigations revealed that therole of ClC-2 is much less clear. The studies revealed con-troversial results regarding the channel’s expression in thegastric mucosa (488, 706, 1001). While the initial observa-tions reported mRNA and cDNA expression in rabbit gas-tric mucosa, no protein could be detected in human and ratgastric glands (488, 706, 1001). The importance of ClC-2 inthe stomach has further been severely challenged by thecreation of a ClC-2 (�/�) animal model. Although ClC-2-deficient animals present with a distinct phenotype charac-terized by testicular and retinal abnormalities, no defect inacid secretion was observed (118).

C) SLC26A9. Lastly, evidence suggests that chloride may leavethe apical pole via SLC26A9, a chloride-bicarbonate anti-porter. Both SLC26A9 and an antiporter from the sameanion exchanger family (SLC26A6) have been detected inthe tubulovesicles of parietal cells (845, 1179, 1180). Con-cerning the functional involvement, the authors speculateabout two potential roles SLC26A9 may play in parietal cellphysiology. Being a chloride-bicarbonate exchanger, its ac-tivation would entail alkalinization of the gastric lumenby bicarbonate efflux and simultaneous chloride uptake(1180). Since this would neutralize H�-K�-ATPase-mediated proton extrusion, it has been suggested thatSLC26A9 activates in the off-phase of acid secretion toneutralize tubulovesicular pH during vesicle retrieval(1180). Alternatively, SLC26A9 may function as a chloridesecretion pathway that contributes to acid secretion. Thishypothesis is based on the observation that SLC26A9 canalso exhibit the behavior of a bona fide chloride channel,rather than an anion antiporter (88, 281). Undoubtedly,further functional investigations are needed to delineate itsexact role in the parietal cell. Its genetic disruption, how-ever, leads to a severely altered parietal cell morphologythat is characterized by dilation of gastric glands, loss oftubulovesicles, and decreased acid output (1180). Althoughthese results do not answer whether SLC26A9 serves as anapical chloride efflux pathway, they indicate that it may benecessary for normal parietal cell function.

3. Potassium recycling

Even before the identification of H�-K�-ATPase, it hasbeen observed that potassium is necessary for acid secretionto take place (335). To prevent the luminal depletion ofpotassium, which would impair proton pumping by H�-

K�-ATPase, potassium has to leak through potassiumchannels or transporters into the gland lumen to ensureadequate supply to H�-K�-ATPase (FIGURE 1). This pro-cess is referred to as potassium recycling. Early flux mea-surements in isolated H�-K�-ATPase containing parietalcell vesicles had already indicated the presence of a largepotassium conductance during H�-K�-ATPase activity(1169). The exact molecular identity of the potassium effluxpathway is, however, under debate. The list of candidatesthat have been put forward to be responsible for potassiumrecycling during acid secretion is long and includes KCNQ1(Kv7.1), KCNJ10 (Kir4.1), KCNJ15 (Kir4.2), KCNJ2(Kir2.1.), and KCC4.

A) KCNQ1. KCNQ1 is a typical “shaker”-like six transmem-brane spanning domain voltage-gated potassium channel(1144). It was initially identified in the heart, where itsmutation can be responsible for cardiac arrhythmias(1144). Yet, studies in KCNQ1 (�/�) animals revealed noelectrocardiographical abnormalities (641). Rather thansuffering from cardiac abnormalities, these animals surpris-ingly exhibited a distinct gastric phenotype with gastric hy-perplasia, dilated gastric glands, vacuolated parietal cells,hypochlorhydria, and hypergastrinemia (641). This obser-vation led to the speculation that KCNQ1 may be the chan-nel responsible for potassium recycling. Subsequently, im-munohistochemical studies confirmed a colocalization ofthe channel with H�-K�-ATPase, and acid secretion wasshown to be inhibited by pharmacological blockade (253,391). Direct measurement of acid secretion in KCNQ1(�/�) mice with modified Ussing chambers (pH stat) laterconfirmed the initially observed hypochlorhydria (1029).Interestingly, luminal substitution of potassium could res-cue the acid secretory deficit, indicating that hypochlorhy-dria ensued from a true lack of apical potassium secretionrather than a general morphological defect of the KCNQ1(�/�) parietal cell (1029).

KCNQ1 is a peculiar channel in that it has a low conduc-tance in acidic environments. In the context of the extremeacidic milieu surrounding the parietal cell, this would im-pede its function as a potassium recycling pathway. Tocircumvent this limitation, KCNQ1 attaches to a regulatorysubunit (KCNE2), which modulates the channel’s gatingproperties and current amplitude (253, 391, 1087). Coas-sembly with KCNE2 activates KCNQ1 at acidic pH valuesand thus facilitates the process of potassium secretion intothe gland lumen (391, 436). The importance of KCNE2 forproper channel function is underlined by the observationthat KNCE2 (�/�) animals display a phenotype similar toKCNQ1 (�/�) mice, i.e., hypochlorhydria, altered parietalcell morphology, and hypergastrinemia (917).

B) KIR CHANNELS. Apart from KCNQ1, several members ofthe inward-rectifier potassium channel (Kir) family havebeen proposed to be involved in gastric acid secretion, albeit



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the amount of functional evidence supporting a role of thesechannels is smaller and the field is divided about the relativecontribution of each channel. Kir 2.1, 4.1, 4.2, and 7.1 wereall confirmed on an mRNA level in gastric mucosa (353,431, 707). On a protein level, immunohistochemistry dem-onstrated colocalization of Kir 2.1, 4.1, and 4.2 with H�-K�-ATPase (353, 431, 556, 707). Cell fractionation exper-iments further indicated trafficking of Kir 4.1 and 4.2 to thecell surface, following parietal cell stimulation (431, 556).A most recent observation monitored acid secretion in Kir

4.1 (�/�) mice (1028). Surprisingly, loss of Kir 4.1 results inaugmented rather than impaired acid secretion, accompa-nied by upregulated H�-K�-ATPase expression (1028).This makes a contribution of Kir 4.1 to potassium recyclinghighly unlikely. Instead, it has been proposed that the chan-nel may balance excessive potassium loss through KCNQ1and may be involved in membrane recycling (1028). In sum-mary, more investigations will be necessary to clarify theroles of the individual Kir channels.

C) KCC4. Apart from being secreted through channels, potas-sium and chloride may exit the parietal cell through trans-porters. This alternative hypothesis is corroborated by arecent observation of Fuji et al. (352). The group reportedthat the K-2Cl cotransporter KCC4 coimmunoprecipitateswith H�-K�-ATPase in apical membrane fractions of pari-etal cells (352). Furthermore, flux measurements in H�-K�-ATPase containing vesicles showed decreased chloride andproton transport under pharmacological blockade ofKCC4, suggesting a functional coupling of KCC4 to H�-K�-ATPase (352). Although the hypothesis that both po-tassium and chloride leave the cell via a transporter is in-triguing, the observation is, as of now, solitary and needsfurther experimental validation.

B. Control of Acid Secretion

Gastric acid secretion is subjected to precise regulation. Thecomplex regulatory machinery that orchestrates the secretionof gastric acid consists of hormonal (gastrin, somatostatin),paracrine (histamine, somatostatin), and neuronal compo-nents (FIGURE 2). The need for this tight regulation is high-lighted by conditions that lead to a hypersecretion of gastricacid, such as Zollinger-Ellisson syndrome (ZES; gastrinoma).Gastric hypersecretion can overcome the measures our bodyundertakes to protect itself from the acid and thereby lead topeptic ulcers. A fine on-demand regulation of acid secretion isthus pivotal to ensure the balance between an adequately lowintragastric pH and tissue protection.

According to the well-established model of acid secretion,the parietal cell is activated by neuronal input from thevagus nerve, endocrine input from gastrin-producing Gcells, and paracrine input from histamine-producing en-terochromaffin-like (ECL) cells (FIGURES 1 AND 2). Thedistinct substances released by these cells, i.e., acetylcho-

line, histamine, and gastrin, directly or indirectly stimulatethe parietal cell by inducing insertion of H�-K�-ATPase atthe apical membrane and are thus commonly referred to asacid secretagogues. The main inhibitor of parietal cell acidsecretion is somatostatin, which is secreted by the D-cellsof the gastric mucosa (FIGURES 1 AND 2). Because of thecomplexity of the network that controls the release ofacid into the stomach, it has been historically challengingto dissect the relative role of each individual regulatorycomponent. Without a doubt, knockout models havegreatly aided us in the last years to gain a more profoundunderstanding of this process, despite their limitations ofchronic compensation. The subsequent chapter aims tosummarize the key players in our canonical model of acidregulation.

1. Cholinergic stimulation/vagus nerve

Since the seminal experiments conducted by Pavlov ondogs, we know that the mere prospect of food ingestion orsham-feeding is sufficient to trigger the secretion of gastricacid (833). This first of three phases of acid secretion iscalled the cephalic phase and is mostly mediated throughthe vagus nerve (595, 725, 910). Hence, before the adventof pharmacological inhibitors, vagotomy has been an effec-tive surgical procedure to control acid-related disorders(301).

The parietal cell receives neuronal input from the vagus nervethat is relayed via cholinergic postganglionic enteric fibers inthe enteric nervous system (ENS) (FIGURES 1 AND 2). In ad-dition, the vagus nerve activates G-cells to release gastrin,resulting in an indirect stimulation of the parietal cell. Di-rect cholinergic activation occurs mostly via muscarinic M3

receptors, which have been identified on the surface of theparietal cell (507, 541, 846). The M3 receptor is a classicseven-transmembrane domain GPCR. Predictably, knock-out of M3 receptors leads to an impairment of gastric acidsecretion and compensatory hypergastrinemia due to nega-tive feedback (9). Following acetylcholine binding, M3 re-ceptor activation mostly causes an increase in intracellularcalcium concentrations (44, 1163). Calcium rises in re-sponse to PLC-mediated IP3 generation and subsequent mo-bilization from intracellular stores (190). The primary ki-nases activated by the M3 receptor are protein kinase C(PKC) and calcium/calmodulin-dependent protein kinase II(CaMKII) (136, 196, 314–316, 773, 774, 1095). Whileactivation of CaMKII has a clear stimulatory effect on acidsecretion, PKC has been reported to have dual effects, al-though reports of an inhibitory role predominate numeri-cally (23, 73, 136, 196, 313, 314, 316, 597, 755, 773,1095). It has been postulated that the expression of differ-ent PKC isoforms may account for this dichotomy (313,314). Current evidence suggests that the PKC-� isoform hasa suppressing effect by trans-inhibiting CaMKII activity,whereas PKC-� increases the baseline levels of intracellularcalcium, thereby sensitizing the parietal cell to subsequent



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stimulation (313, 314). Apart from PKC and CaMKII acti-vation, cholinergic signaling activates parietal cell MAPKs,which is partially a downstream effect of PKC activation(771, 1039, 1062, 1063). MAPK activation seems to have abiphasic effect on acid secretion (acute inhibition andchronic augmentation) and also serves as a mediator oftrophic responses in the parietal cell. For example, pro-

longed MAPK activation (72h) has been shown to serve asa maturation and differentiation signal leading to a trans-formation of parietal cell morphology in vitro (1039). Thechange in morphology is accompanied by a downregulationof H�-K�-ATPase gene expression (1039). As of now, it ischallenging to put these findings into a physiological per-spective.

PPIsAPAs

Parietal cell

D-cell

D-cell

ECL-cell

SST

Somatostatin

Somatostatin

Histamine

Basolateral

Oxyntic mucosa

Lumenal

Hist

SSTR

SSTR

K+

H+

Gast

ACh

CCK2

H,K-ATPase

CCK2

M3

PAC1

H2

SST

Gast

PACAP

D-cell

G-cell

Somatostatin

Gastrin

Circulation

Basolateral

Antrum

Lumenal

LowlumenalpH

CalciumAmino acidsPolyamines

ENS

ENS

ENS

ENS

SSTR

VPAC

SST

PACAPVIP

AChGRP

CaSRCaSR

CCK1 CCK

CaSR

?

FIGURE 2. Neuroendocrine regulation of gastric acid secretion. In addition to direct neuronal regulation, theparietal cell receives paracrine signals from neighboring ECL- and D-cells. Gastrin is produced in the antralmucosa of the stomach and reaches the oxyntic mucosa via the circulation (endocrine regulation). Gastrin-mediated histamine release represents one of the major stimulatory pathways leading to the secretion ofgastric acid (gastrin-histamine axis). The secretion of gastrin is closely tied to intragastric pH (via somatosta-tin), thereby creating a negative-feedback loop. ACh, acetylcholine; APAs, acid pump antagonists; ENS, entericnervous system; Gast, gastrin; Hist, histamine; PPIs, proton pump inhibitors; SST, somatostatin.



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In addition to M3 receptors, M1 receptors have also beenimplicated to play a role in the process of acid secretion.This hypothesis was derived from the observation that theM1 receptor is expressed in gastric mucosa and that itsblocker pirenzepine can inhibit gastric acid secretion (29,466). Most evidence pointed to an expression of M1 onECL-cells, where it was speculated to regulate the release ofhistamine (437, 507). More recent findings somewhat sur-prisingly report that pirenzepine also suppresses acid secre-tion in M1-deficient animals. Furthermore, these animalsshow a normal phenotype in terms of acid output (10).These observations question both the involvement of M1

receptors in acid secretion and the specificity of pirenzepine.

Lastly, knockout studies point towards a contribution of theM5 receptor to the regulation of acid secretion, as its deletioncorrelates with decreased acid output (10). Yet, M5 receptormRNA could only be detected in whole stomach homoge-nates, but not in gastric mucosa per se, making its localizationto the submucosal enteric plexus more likely (10).

2. Gastrin/G-cell

Gastrin has been discovered in 1906 by John S. Edkins, whoinjected gastric extracts of pig and cat stomachs into thejugular vein of cats and observed a subsequent increase inacid secretion (298). Gastrin is a peptide hormone that isproduced in specialized G-cells, located in the antral sectionof the stomach (FIGURE 2) and endocrine cells in the duo-denum, small intestine, colon, pancreas, testis, and pitu-itary. It is the main mediator of the so-called gastric phase ofacid secretion, which initiates when the ingested food entersthe stomach. The gastric phase accounts for the majority ofthe acid secretory response of the stomach.

A) SYNTHESIS. The gastrin cDNA encodes a 101-amino acidpre-pro-hormone that undergoes extensive posttransla-tional processing (113, 519, 551, 552, 1161). In brief, thepre-pro-hormone is first cleaved NH2-terminally to createprogastrin and then truncated to two main core proteins,G17 and G34, which can exist in glycine extended (G17-Gly; G34-Gly) or terminally amidated (G17-NH2, G34-NH2) forms. Furthermore, a fraction of progastrin (�47%in humans) is sulfated at Tyr66 in the course of its passagethrough the Golgi apparatus, thereby giving rise to sulfatedand nonsulfated isoforms of gastrin (22). Sulfation has noinfluence on the acid secretory response, as the affinity tothe gastrin receptor remains unchanged (399, 596). G17-NH2 is the main circulating form that mediates the secre-tory effects of gastrin. Although the glycine-extended formshave a low affinity towards the gastrin receptor (CCK2) andthus play no role in gastric acid secretion (they are four tofive orders of magnitude less potent in inducing acid secre-tion), it is still important to acknowledge their existence(178, 722). First, they serve as substrates for the synthesis ofamidated gastrin and are cosecreted with gastrin by theG-cells (1040, 1051). Second, they potentiate the acid se-

cretory response to amidated gastrin, although they have nointrinsic ability to induce acid secretion (178). Third, pro-gastrin and glycine extended gastrins were shown to act asa proliferative signal, especially in the colon (20, 482, 994,1145). This is also of pathophysiological relevance as bothforms can promote cancer growth by presumably inhibitingapoptosis and inducing angiogenesis (71, 87, 900). For ex-ample, it was shown that overexpression of progastrin inmice is a predisposing factor for the development of colo-rectal or bronchoalveolar cancers (587, 1017).

B) REGULATION OF RELEASE. Gastrin is released by the G-cell inresponse to a variety of stimuli of different origin. Directneuronal stimulation of the G-cell occurs via ACh and gas-trin releasing peptide (GRP), which are released by postgan-glionic neurons of the enteric nervous system. The postgan-glionic fibers themselves receive input from the efferentfraction of the vagus nerve (86, 485). On the other hand,food-related signals, such as calcium, amino acids, andamines, can also directly trigger gastrin secretion (FIGURE 2)

(257). The secretory stimuli culminate in an increase inintracellular calcium concentrations, leading to vesiclefusion and gastrin secretion. The main inhibitory signalfor gastrin secretion is somatostatin, which reaches theG-cells in a paracrine fashion from neighboring D-cells(632, 975).

With regard to the neuronal control of gastrin secretion, it isgenerally thought that vagal stimulation increases the re-lease of gastrin, although some conflicting evidence exists(310, 694). Latest experiments that assessed local gastrinconcentrations utilizing microdialysis, however, clearlyshow an increase in gastrin levels following acute electricalvagal stimulation (310). The vagus nerve then synapses onneurons of the ENS, which are thought to release either theneurotransmitter ACh or GRP on a G-cell, leading to secre-tion of gastrin (295, 635, 694, 960, 978). It should be notedthat a recent investigation failed to observe increased gas-trin levels, following exogenous GRP administration in hu-mans (456). Yet, GRP itself serves as a clear acid secreta-gogue, although potentially not via gastrin (456). Whetherthese conflicting observations are attributable to species dif-ferences (most earlier observations utilized rodent models)remains to be elucidated. The ENS is also thought to medi-ate parietal and G-cell activation in response to mechanicaldistension of the stomach (455, 962, 977). The neurohor-monal response to gastric stretch is an integral part of thegastric phase of acid secretion. Closer examination, how-ever, reveals that the reports are very conflicting in that it isnot clear whether a pure mechanical distension stimulatesor inhibits gastrin release (455, 664, 803, 962, 977). Abiphasic model characterized by initial inhibition of gastrinsecretion under low volumes followed by stimulation underhigh volumes has been suggested, but awaits further confir-mation (977).



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Dietary components, such as amino acids and calcium, candirectly promote the secretion of gastrin and can thus sus-tain acid secretion in the gastric and intestinal phase asdigestion progresses (652, 1074). A rise in serum calciumconcentrations evokes a similar effect. The correlation be-tween calcium and gastrin is discussed in a separate section(see sect. VD2). It has been unclear for a long time as to howthese dietary components activate the G-cell. An involve-ment of the ENS has been proposed as the most likely ex-planation in the past. More recent observations, however,strongly indicate that the calcium-sensing receptor (CaSR)represents the molecular link between luminal dietary con-stituents and G-cell activation (325). The CaSR and its rolein the stomach are discussed separately and shall only besummarized at this point (FIGURE 8) (see sect. IVD). First,the same dietary components, i.e., amino acids, amines, andcalcium, which have all been shown to trigger gastrin re-lease also function as activators of CaSR (652, 1074). Sec-ond, CaSR is expressed on the apical and basolateral side ofthe G-cell, which allows it to act as nutrient sensor both inthe gastric lumen and the circulation (142, 182, 886).Third, direct activation of the CaSR is known to stimulateacid secretion (145, 291, 373). Finally, and most impor-tantly, CaSR (�/�) animals lack the gastrin secretory re-sponse to intraluminal instillation of peptone, calcium, andphenylalanine (325). In light of this evidence, it is highlylikely that CaSR is the long elusive luminal nutrient sensorthat regulates the secretion of gastrin from the G-cell.

The plasma levels of gastrin are closely tied to the intragas-tric pH. Low intraluminal pH is a potent inhibitor of gastrinrelease, which serves as a negative-feedback mechanism toimpede an overproduction of acid. Conversely, a more al-kali intragastric pH induces the secretion of gastrin, whichaccounts for the commonly observed hypergastrinemia instates of acid suppression, such as during proton pumpinhibitor (PPI) therapy. The pH dependency of serum gas-trin levels is mainly relayed via somatostatin, as acid di-rectly stimulates somatostatin release (see sect. IIB4). Soma-tostatin, released by neighboring antral D-cells, in turn actsas the main inhibitor of gastrin secretion (FIGURE 2). Thephysical proximity to G-cells allows for a fine paracrineregulation of gastrin release. Although it is generally ac-cepted that intragastric pH mostly modulates local soma-tostatin levels, the G-cell may also directly sense intragastricpH via CaSR. CaSR is acid sensitive, and it has been shownthat isolated rat G-cells secrete less gastrin when the extra-cellular pH is dropped from 7.4 to 5.5 (569). However,more investigations are needed to substantiate this evi-dence. Furthermore, gastrin release is also inhibited by neu-ronal regulation by the ENS. The neurotransmitter galaninhas been demonstrated to exert a direct inhibitory effect onisolated G-cells (695, 961).

C) CELLULAR EFFECTS. Following secretion, gastrin enters thebloodstream and acts on its target cells in an endocrine

fashion. Its half-life is determined by its rate of eliminationfrom the plasma which mainly occurs by metabolism in thekidney, gut, and brain (419, 420). The importance of renalelimination is corroborated by the observation that patientswith renal failure present with higher plasma gastrin levels(818, 1075).

The two primary target cells of gastrin are the histamine-secreting ECL cell and the parietal cell. Gastrin exerts itsfunctions via binding to the cholecystokinin receptor type 2(CCK2), a seven transmembrane domain G protein-coupledreceptor, which is expressed on mature parietal and ECLcells, but also on gastric stem cells (560, 596, 608, 769, 772,904). On the ECL cell, gastrin binding causes the release ofhistamine, which in turn stimulates the parietal cell in aparacrine fashion (FIGURE 2) (see sect. IIB3) (412). Thisactivation cascade is commonly referred to as the gastrin-histamine axis. Evidence for a direct, i.e., nonhistamine-relayed, activation of H�-K�-ATPase in the parietal cell bygastrin exists, but is far less substantiated (459, 1024,1025). Gastrin may sensitize the parietal cell to subsequentsecretagogue stimulation, rather than acting as a bona fidesecretagogue itself. Canonically it is widely accepted thatgastrin exerts its physiological effects mostly via activationof ECL cells (24, 1131). Knock-out of gastrin leads to asevere impairment of basal and stimulated acid secretion(179, 347). Apart from stimulating acid secretion, gastrinserves as a pivotal proliferative signal for the gastric mucosain general (60, 410, 534, 631, 816). It is commonly ob-served that elevated plasma gastrin levels lead to substantialmucosal proliferation (60, 410, 534, 631, 816). This phe-nomenon has been extensively described in various knock-out animals suffering from hypochlorhydria and concomi-tant hypergastrinemia, but also in patients with ZES (39,515, 584, 1066). The source of mucosal cell proliferation isprogenitor cells located in the isthmus region of the gastricgland (545). Expression of the CCK2 has been confirmed onseveral gastric progenitor cells (560, 769). Furthermore,gastrin has been shown to stimulate cell migration from theprogenitor region along the gastric gland axis (578). Muco-sal hyperplasia thus ensues most likely via a direct activa-tion of precursor cells by gastrin. Although hypergastrine-mia causes a generalized mucosal hyperplasia, ECL cellsseem to be particularly regulated by gastrin, as their relativefraction compared with other mucosal cells increases underprolonged gastrin exposure (60, 410, 631). Conversely, theabsence of the CCK2 almost entirely eliminates matureECL-cells from the gastric mucosa (177, 622). [Somewhatsurprisingly this does not occur when gastrin itself isknocked out (179, 347).] It should be noted that the M3

receptor seems to be necessary as a cofactor mediating thetrophic effects of gastrin, as its absence is associated with anormal mucosal phenotype despite elevated serum gastrinlevels (see above) (9). The mechanism underlying this inter-dependency between gastrin and the M3 receptor is as ofnow elusive. The cholinergic and gastrin systems also seem



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to be intertwined with regard to acid secretion. In the ab-sence of the CCK2 receptor, the parietal cell’s acid secretoryresponse to the secretagogue carbachol (ACh analog) isabolished, while the response to histamine remains intact(543). Again, one can only speculate about the molecularbasis of this interaction.

In conclusion, gastrin is the most important activator ofacid secretion in the stomach. The role of gastrin, and espe-cially its glycine extended forms, has evolved beyond beinga mere acid secretagogue to being an important global reg-ulator of cell growth and differentiation. Furthermore, theregulation of gastrin by the levels of plasma calcium pro-vokes the question as to whether gastrin itself in turn has animpact on global calcium homeostasis. A subsequent sec-tion makes an attempt at addressing this question (see sect.VD2).

3. Histamine/ECL cell

Histamine has been discovered as early as 1910 by Dale,Barger and Laidlow in extracts of ergot fungi (63, 237). In1920, Popielski for the first time described its effect on thesecretion of gastric acid (866). He observed that subcutane-ous administration of histamine resulted in increased acidsecretion (866). Furthermore, he concluded that this effectwas independent of the vagus nerve, as secretion still tookplace after vagotomy and administration of atropine. Thisled Popielski to postulate that histamine exerts its effectsdirectly on the level of the gastric gland (866). The hypoth-esis that histamine acts in a paracrine fashion on parietalcells and that its release is regulated by the levels of gastrinhas been put forward for the first time by Emmelin andKahlson in 1944 (306). At this point, the cellular source ofhistamine was still obscure. It was only in the late 1960sthat histamine had been histochemically localized to theECL cells of the gastric gland (413, 1084).

A) SYNTHESIS AND REGULATION OF RELEASE. Histamine is theeffector of the gastrin-histamine axis and directly stimulatesthe parietal cell to secrete hydrochloric acid (FIGURE 2).Histamine is derived from the amino acid histidine, which isenzymatically converted to histamine by L-histidine decar-boxylase (HDC) (957). The effects of genetic HDC deletionare predictably severe: animals lacking HDC have a lowbasal acid output that does not respond to exogenous ad-ministration of gastrin (1066).

Histamine is stored in secretory granules of the ECL cell andis released into the surrounding milieu in response to stim-ulation by gastrin and neuronal signals. Stimulation by gas-trin occurs via activation of its GPCR CCK2 (772, 904).Gastrin affects the ECL cell in multiple ways. First, gastrinexposure increases the levels of HDC expression by enhanc-ing its transcription and inhibiting its degradation, to allowfor increased synthesis of histamine (268, 331). The molec-ular mechanism underlying increased HDC transcription is

fairly well understood. Following CCK2 activation, in-creased transcription of HDC is mediated via a PKC- andERK-dependent pathway (470, 472). The HDC gene pro-moter is then activated by at least three distinct nuclearfactors which bind to gastrin response elements, resulting ingene transcription (889, 890). Apart from augmenting genetranscription, gastrin regulates the degradation of HDC,which further increases intracellular enzyme levels (331,1214). Second, gastrin enhances the transcription of the vesic-ular monoamine transporter type 2 (VMAT2; SLC18A2),which is responsible for accumulating histamine in the se-cretory vesicles (376). Similarly to HDC, this effect dependson PKC and ERK activation and binding of a nuclear factorto a gastrin response element in the VMAT2 promoter re-gion (164, 1154). It should be mentioned that gastrin reg-ulates the transcription of a plethora of other genes whichserve a diverse array of roles, ranging from growth to me-tabolism (346). Amongst many others these include chro-mogranin A, which is essential for granule packaging and isa precursor of pancreastatin (see sect.VD3) (231). Third,gastrin induces the fusion of secretory granules and therelease of histamine into the gland environment. Secretionfollows a biphasic elevation of intracellular calcium concen-trations after activation of CCK2 (1201). The biphasic in-crease has been proposed to result from initial IP3-mediatedrelease from intracellular stores, which is followed by sub-sequent influx of calcium via L-type calcium channels fromthe extracellular space (1201). The importance of intracel-lular store mobilization has been contested by a differentgroup, which proposed that solely influx trough L-type, andto a lesser extent N-type, calcium channels triggers the se-cretory response (673). Lastly, gastrin has a trophic effecton the ECL cell (see sect. IIB2).

Apart from gastrin, ECL cells are stimulated by pituitaryadenylate cyclase activating polypeptide (PACAP), which isa neuropeptide expressed in the ENS of the gastric mucosa(737, 1054). PACAP has homology to vasoactive intestinalpolypeptide (VIP) and binds to a distinct receptor (PAC-1)on the ECL cell (1207, 1208). Binding of PACAP to PAC-1induces release of histamine (672, 798, 944, 1207). Similarresults have been obtained with VIP, which is attributableto partial agonism at PAC-1 (798, 941). Historically, inves-tigations yielded controversial results with regard to theeffects of exogenously administrated PACAP on acid secre-tion. Both an inhibition and stimulation of acid secretionfollowing PACAP injection are reported (760, 862, 944,1207). This discrepancy is most likely attributable to thefact that PACAP can also act as an agonist of the VIP re-ceptor (VPAC) on the somatostatin-secreting D-cell, lead-ing to a concomitant suppression of acid secretion by soma-tostatin release (1207). Indeed, if an anti-somatostatin an-tibody is injected into rats simultaneously with PACAP,acid secretion is elevated threefold from baseline (comparedwith 1.5-fold in the absence of an anti somatostatin anti-body) (1207). Evidence points to the fact that the PACAP-



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stimulated release of somatostatin is of particular impor-tance in the mouse, as most studies showing a suppressionof acid secretion after PACAP administration were con-ducted in murine models.

PACAP has very similar effects on the ECL cell as gastrin.Similarly to gastrin, PACAP causes histamine release byincreasing intracellular calcium concentrations via calciuminflux through L-type, but also ligand-gated calcium chan-nels (673). In further analogy to gastrin, PACAP upregu-lates the expression of HDC and exerts trophic effects onthe ECL cell (590, 729, 810). Contradictory results withregard to the effects of acetylcholine on histamine releaseexist. It has been reported that acetylcholine can either stim-ulate or has no effect on the secretion of histamine in in vitroexperiments on isolated ECL cells (481, 672, 674, 941,946). In vivo application of muscarinic agonists, followedby measurement of histamine concentrations using micro-dialysis, also yielded no evidence for cholinergic stimulation(798). Conversely, it is well accepted that adrenergic stim-ulation leads to an increase in histamine release; however,the physiological relevance of adrenergic activation of ECLcells is not entirely clear (636, 672, 674, 798, 871, 941).

The ECL cell is inhibited by a variety of substances, themost prominent of which is somatostatin (204, 590, 798,941). Somatostatin is produced in D-cells of the oxynticmucosa and reaches the ECL cell in a paracrine fashionwhere it binds to the somatostatin receptor (SST2 and po-tentially SST5) (FIGURE 2) (570, 873). Receptor bindingleads to inhibition of histamine exocytosis via blockade ofmostly L-type calcium channels (105). This impedes theelevation of intracellular calcium concentrations caused byECL activators, such as gastrin (see above) (873). In addi-tion, somatostatin also inhibits the proliferation of ECLcells (570). Somatostatin can thus be seen as the globalhormonal antagonist to gastrin with regard to ECL cellfunction and proliferation. The neuronal inhibition of ECLcells is mainly carried out by the neuropeptide galanin (105,672, 798, 1209). Galanin is localized to neurons of the ENSand demonstrated an inhibitory effect on histamine secre-tion in in vitro and in vivo models (105, 302, 672, 731, 798,1209). Similarly to somatostatin, the molecular mechanismunderlying its inhibitory effect is an interference with cal-cium signaling via closure of L-type calcium channels (105).Lastly, prostaglandin E and nitric oxide also act as inhibi-tors of histamine release (105, 554, 798, 1002). Althoughneuropeptide YY (PYY) and calcitonin gene-related peptide(CGRP) have also been implicated in playing a role in ECLcell regulation, a detailed discussion is omitted in light ofcontradictory results which range from stimulation to inhi-bition of secretion (672, 674, 798, 1210).

B) CELLULAR EFFECTS. As mentioned earlier, stimulation of theECL cell is translated into an elevation in intracellular cal-cium concentrations, leading to exocytosis of preformed

histamine-containing secretory vesicles. The molecularmechanism of vesicle fusion with the apical membrane re-lies on the formation of the core SNARE complex, consist-ing of syntaxin, synaptobrevin, and SNAP-25. Synaptotag-min presumably acts as a calcium sensor relaying theintracellular calcium signal to the vesicle fusion proteinapparatus. The expression of all SNARE complex pro-teins has been confirmed in the ECL cell (471, 477,1215). In accordance with these findings, introduction ofthe neurotoxins tetanus toxin light chain and botulinumtoxin, which cleave constituents of the SNARE complexapparatus and thereby render it nonfunctional, result ininhibition of histamine secretion (477).

Very small amounts of histamine are sufficient to induceacid secretion. Histamine acts via the H2 receptor on theparietal cell, which has been discovered by Sir J. W. Black in1972 (106). For this seminal discovery, he was laterawarded the Nobel Prize in Physiology and Medicine. TheH2 receptor belongs to the family of seven-transmembranedomain GPCRs. Its activation predominantly leads to in-creases in the intracellular levels of cAMP, but also of cal-cium, which serve as stimulatory signals for H�-K�-ATPasetrafficking (67, 189, 738, 840, 1026, 1143). In analogy,pharmacological agents that elevate cAMP, such as IBMXor forskolin, induce acid secretion (1026, 1191). The in-crease in cAMP is due to activation of adenylate cyclase viaGs. The role of calcium in the process of histamine secretionremains a controversial matter. First, the mechanism lead-ing to histamine-induced increases in intracellular calciumhas been subject of discussion. Evidence exists that hista-mine can, apart from adenylate cyclase, also activate PLC,leading to calcium release from intracellular stores (607,1142, 1143). Conversely, it has been suggested that theobserved increases in intracellular calcium are a byproductof cAMP-mediated PKA activation, which in turn can reg-ulate the opening of calcium channels (144, 189, 840). Sec-ond, it is questionable to what degree the calcium signal isan integral and necessary part of the acid secretory responseto histamine (738, 840). Chelation of the transitory calciumincreases with BAPTA abolishes only the secretory responseof isolated gastric glands to histamine by �40%, while itcompletely eliminates the response to cholinergic stimula-tion (738). Also, live fluorescence imaging in isolated glandsshowed no spatiotemporal correlation between the hista-mine-induced increases in calcium and the onset of acidsecretion, thereby questioning an involvement of calcium inthe secretory response (840).

H2 receptor knockout animals effectively illustrate the sig-nificance of the histamine-gastrin axis in gastric physiology.Lack of the H2 receptor leads to a complete failure of gas-trin or histamine to induce acid secretion (584). The secre-tory response to carbachol, however, remains intact (584).Hypergastrinemia develops as a feedback mechanism withthe aim of reestablishing acid secretion, leading to mucosal



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hypertrophy (584). In light of the central role of the H2

receptor in parietal cell physiology, it has been successfullyused as a pharmacological target with the aim of suppress-ing gastric acid output (see sect. IIC2).

4. Somatostatin/D-cell

Somatostatin was isolated for the first time in 1973 fromovine hypothalamus and characterized as an inhibitor ofgrowth hormone release from the pituitary gland (124). Afew years later somatostatin was identified in endocrinecells of the stomach, which we now know as D-cells (638).

A) SYNTHESIS AND REGULATION OF RELEASE. Somatostatin is apeptide hormone that exists in two primary forms that dif-fer in their respective peptide length. The most abundantform in the gastric mucosa is somatostatin-14 (consisting of14 amino acids), whereas somatostatin-28 only constitutesa minute fraction of the total gastric somatostatin content(198, 1125). The two forms of somatostatin are cleavageproducts of a larger 116-amino acid pre-prohormone (pre-prosomatostatin), which in turn is processed to the 92-amino acid-long prosomatostatin (1000). It should be men-tioned that other cleavage products, such as antrin or so-matostatin-28(1–12) exist and are secreted together withsomatostatin (77, 885). Their physiological significance is,however, less well understood.

Somatostatin is the global antagonist of the acid secreta-gogues. It is produced by intestinal and gastric D-cells, thelatter of which exist in two populations in the stomach (61):an antral population locally inhibits the release of gastrinfrom G-cells, whereas a population localized to the acid-producing oxyntic mucosa directly regulates the parietalcell and inhibits histamine release from the ECL cell (FIG-URE 2) (19). The morphology of the D-cell is characteristicin that it possesses long cytoplasmic processes, which allowit to communicate with and regulate neighboring cells in aparacrine fashion (620, 632). It is worthwhile to distinguishthe two populations of gastric D-cells, as each populationpossesses unique physiological properties (1202).

The antral D-cell is mostly regulated by the local concen-trations of gastrin, cholecystokinin, and intraluminal pH.Gastrin induces somatostatin secretion from D-cells, whichcauses reciprocal inhibition of gastrin release from neigh-boring G-cells, thereby creating a local negative-feedbackloop (976, 1011, 1202). The molecular mechanism under-lying this loop is, however, less clear. CCK2 receptor is, if atall, only expressed at very low levels in the antral mucosa(749, 905, 967). It has been proposed that gastrin stimu-lates somatostatin release in the antrum in a receptor-inde-pendent mechanism (1202). This may be accomplished viadirect cell-cell contacts between the G- and the D-cell,which have been demonstrated with electron microscopy(620). Conversely, evidence for cholecystokinin and itsstimulatory role for somatostatin release via CCK1 is more

substantiated (749, 905, 967, 1202). Cholecystokinin isstructurally closely related to gastrin (both share an identi-cal 5-amino acid COOH terminus) and also exists in vari-ous peptide lengths (767). It is secreted by I-cells of the smallintestine following protein and fat-rich chyme entering theduodenum, and thus represents a classical mediator of theintestinal phase of acid secretion (594). As its name implies,cholecystokinin has originally been described as a stimula-tor of gallbladder contraction; however, its inhibitory influ-ence on gastric acid secretion is now well accepted andextensively described (593, 1203). Cholecystokinin canbind to both the CCK1 and CCK2 receptor with almostequal affinity, whereas the actions of gastrin are almostexclusively mediated by the CCK2 receptor. The dual affin-ity of cholecystokinin would imply a possible stimulatoryeffect on acid secretion via activation of CCK2 on ECL cells;however, in vivo the inhibitory effect mediated by activa-tion of CCK1 and CCK2 on D-cells prevails (593, 966,1202, 1203).

One of the most important stimulators of D-cell secretion isthe intragastric pH. A seminal observation demonstrating acorrelation between gastric acidity and the amount of se-creted somatostatin was made in dogs in the 1970s. It hasbeen shown that the amount of somatostatin directly in-creases in antral venous blood following gastric HCl infu-sion, while somatostatin levels were unaffected in venousblood from the oxyntic mucosa (982). Similar observationswere later made in isolated mouse stomach, however with-out topographic discrimination (975). Two main hypothe-ses as to how somatostatin is regulated by intragastric pHexist. The first states that the D-cell can directly act as a pHsensor, and the second postulates that the pH sensing ismediated by neurons, which in turn act on D-cells. To ac-complish putative direct pH sensing, several antral D-cellsare equipped with a distinct morphological feature. Theypossess apical projections that are in contact with the glan-dular lumen, potentially allowing them to constantly mon-itor the intraluminal milieu (620). These D-cells have beentermed open type. Conversely, the D-cells of the oxynticmucosa are mostly of the closed type, meaning that they areembedded in the mucosa without luminal contact. The mo-lecular identity of the putative apical pH sensor remainselusive. However, the presence of CaSR, which has pHsensing properties, was recently confirmed in preliminarystudies on the D-cell and may represent a possible candidatefor this mechanism (770). Apart from directly acting onD-cells, the effect of pH on somatostatin secretion may bemediated via afferent spinal neurons. Over 80% of the spi-nal afferent neurons contain the neuropeptide CGRP (397,758, 1047, 1116). Perfusion models of antral sleeves haveshown that the acid-induced rise in somatostatin is accom-panied by a concomitant increase in the concentrations ofthe neuropeptide CGRP (708). Furthermore, application ofa CGRP receptor blocker inhibited the release of somatosta-tin following acid exposure (708). As D-cells are known to



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express the CGRP receptor, an involvement of CGRP inacid sensing is plausible (558). Again, this provokes thequestion of how CGRP-containing neurons may sense acid-ity on a molecular basis. The acid-sensitive channels tran-sient receptor potential vanilloid channel (TRPV1) and theacid-sensing ion channel 3 (ASIC3) had been proposed asmolecular acid sensors; however, latest experiments haveshown that the increase in CGRP still occurs in the geneticabsence of the channels (47, 96, 163).

Neuropeptides of the gastric ENS that stimulate the secre-tion of somatostatin include PACAP and VIP, which bothbind to the VPAC receptor expressed on D-cells (199, 657,1207). The presence of VIP and PACAP containing neu-rons, which integrate signals from the vagus nerve, has beendemonstrated in the gastric mucosa (302, 737). Further-more, cholinergic signals can act on the antral D-cell via theM3 receptor to promote secretion of somatostatin (140).This is in sharp contrast to D-cells from the oxyntic mucosathat are inhibited by cholinergic signals (197, 200, 1182).As mentioned earlier, the D-cells in the oxyntic mucosa alsodiffer in their morphology. D-cells in the oxyntic mucosaare of the closed type and have thus not been implicated toparticipate in acid sensing. They exert their acid-suppres-sive effects by the paracrine regulation of ECL and parietalcells. Further functional divergence between antrum and theoxyntic mucosa has been demonstrated in the regulation of thesomatostatin mRNA. For example, suppression of acid secre-tion with omeprazole in fasted animals markedly decreasedsomatostatin mRNA levels in the antrum, whereas the levels inthe oxyntic mucosa were affected to a much lesser extent,which further corroborates the hypothesis that the antral cellsare involved in luminal chemosensation (945).

B) CELLULAR EFFECTS. The effects of somatostatin on its targetcells are mediated by the SST2 receptor. Knockout of the re-ceptor causes a 10-fold increase in basal acid output, whichexemplifies the pivotal role somatostatin plays as a global sup-pressant of acid secretion (715). Somatostatin acts on all maincell types that are involved in the process of acid secretion, i.e.,parietal cells, ECL cells, and G-cells (FIGURE 2). The inhibitionof the G- and ECL cells has been discussed in the respectivesections. In the parietal cell, somatostatin has a clear directinhibitory effect on secretagogue-induced acid secretion (827,1177). This effect is partially attributable to activation of Gi,leading to inhibition of adenylate cyclase and a subsequentdecrease in intracellular cAMP levels (827).

In conclusion, somatostatin acts as the global brake on acidsecretion. By acting on G-cells, ECL cells, and parietal cells,it exerts its inhibitory action on every link in the regulatorychain leading to the secretion of gastric acid.

5. Other substances

A variety of other substances have been shown to haveeither direct or indirect effects on acid secretion. In the

interest of conciseness, their physiological effects will onlybe discussed briefly at this point.

A) SECRETIN. Secretin is a 27-amino acid peptide hormonethat is synthesized in duodenal S-cells and secreted into thecirculation in response to a low duodenal pH or passage ofdigestive products, such as fat (195, 955, 1153). A subpop-ulation of secretin-producing cells is also present in thegastric mucosa, where it may influence acid secretion in aparacrine manner (191–193). Given its secretory stimulus,it is regarded as a classic effector of the intestinal phase ofacid secretion. When it was first discovered in 1902 byBayliss and Starling (interestingly secretin was the first hor-mone ever to be discovered), it was noted that secretin in-duces pancreatic bicarbonate secretion, which leads to abuffering of the gastric acid entering the duodenum (68). Inthe stomach, secretin acts as an inhibitor of gastric motilityand acid secretion (141, 194, 269, 374, 532, 564, 656, 677,1107). The exact mechanism as to how secretin attenuatesthe secretion of acid is not exactly known, and several hy-potheses have been put forward. For example, it has beenshown that secretin induces the secretion of somatostatinfrom isolated D-cells (141). Increases in somatostatin levelswere also observed in isolated perfused stomach models (205,374). Others have proposed that secretin activates vagal pri-mary afferent neurons, which in turn leads to neuronal mod-ulation of acid secretion (656, 659). In opposition to this the-ory, it has also been demonstrated that the inhibitory effects ofsecretin are independent of vagotomy (677).

B) OXYNTOMODULIN. Oxyntomodulin is a peptide hormoneproduced in the mammalian intestine. It is closely related toglucagon and contains its entire amino acid sequence, ex-tended by a COOH-terminal octapeptide (66). In isolatedparietal cells, oxyntomodulin acts as an activator of acidsecretion (959). The integrated response to oxyntomodulinis, however, opposite. Systemic injection decreases gastricacid secretion in rat, cat, and human test subjects (53, 157,285, 524, 525, 965). The inhibitory effect is most likelymediated via somatostatin release (53).

C) SEROTONIN. It was recognized in the early 1950s that se-rotonin was present in the antral mucosa of dog stomachs(323). Serotonin is stored in granules of enterochromaffincells of the antrum (1099). It is released into the circulationand the gastric lumen in response to vagal stimulation (107,649). Intraluminal acidification serves as another stimulusfor serotonin release (1196). Serotonin has an inhibitoryeffect on the secretion of gastric acid (107, 153, 521, 650,720, 903). It is still poorly understood where serotonininterferes with acid secretion.

D) NEUROTENSIN. Neurotensin is a 13-amino acid neuropep-tide that was originally isolated from calf hypothalamus(161). In the periphery, it is also produced and secretedpostprandially by specialized endocrine cells (N-cells) of the



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small intestine (863). Various investigations have demon-strated that neurotensin suppresses the secretion of gastricacid and delays gastric emptying (25, 108, 486, 985). Thishas been shown by direct systemic injection, but also byimmunoneutralization of endogenous neurotensin in a re-verse approach (25, 108, 486, 985). In disagreement withthese findings, other investigators could only inhibit acidsecretion at unphysiologically high serum concentrations of�750 pmol (747). Of note, physiological postprandial neu-rotensin levels were measured to be �15 pmol by the sameinvestigators, questioning the role of neurotensin as a phys-iological endocrine inhibitor of acid secretion (747). Neu-rotensin is also located to nerve fibers of the enteric nervoussystem in the stomach, indicating that it may act as a localneuronal rather than an endocrine regulator. It has beenproposed that neurotensin may induce the secretion of so-matostatin and thereby exert its inhibitory action on acidsecretion (53, 414). Most recently, however, the low-affin-ity neurotenstin type 2 receptor (NTS2) has been identifiedon the parietal cell, suggesting a direct influence.

E) GHRELIN. Ghrelin is a recently discovered 28-amino acidpeptide hormone that is synthesized in P/D1 cells of thefundus (242). Since its discovery, multiple functions havebeen ascribed to it, ranging from being a regulator of appe-tite to being a modulator of bone remodeling. Its effects onbone are described in a separate section of this review (seesect. VD1). Apart from these functions, ghrelin also hasbeen implicated to affect gastric acid secretion, although itremains a matter of discussion in which direction, as periph-erally administered ghrelin has been reported to stimulate,inhibit, or not affect acid secretion (278, 355, 654, 719).The reason for these dichotomic results is largely unclear.The fact that ghrelin circulates in acylated and desacylatedforms adds further complexity to the subject (491). Indeed,acylated ghrelin has been shown to stimulate acid secretionfollowing peripheral injection, whereas desacylated ghrelinremained without effect (938). Further investigations areneeded to clarify the controversy surrounding ghrelin andits influence on gastric acid secretion.

F) NITRIC OXIDE. Nitric oxide (NO) is an important signalingmolecule that plays a role in multiple physiological pro-cesses, such as vasodilation or the immune response. In-deed, NO has been shown to mediate the hyperemic re-sponse of the gastric mucosa that occurs during acid secre-tion (553). However, NO also directly influences theproduction of acid. The effect of NO on acid secretion ismost likely inhibitory (81, 82, 555, 1002; opposed by Ref.426). NO is produced by various forms of NO synthases,one of which has been localized at high concentrations incells in the vicinity of parietal cells, allowing for a putativeparacrine regulation (80). NO has been proposed to exertits inhibitory action by either directly inhibiting the parietalcell or by suppressing the release of histamine from ECLcells (81, 82, 555, 1002). Intracellular increases in cGMP

concentrations have been observed in both cell types afterNO exposure, suggesting that guanylate cyclase is an intra-cellular target for NO (82, 1002).

G) INTERLEUKINS. Interleukins (IL) are cytokines that mainlycoordinate immune responses. In particular, IL-1� has beenshown to impact gastric acid secretion. IL-1� is a generalproinflammatory cytokine that plays an important role inthe stomach in the context of Helicobacter pylori infection.H. pylori infection triggers an elevation of IL-1� levels aspart of the host’s immune response (65). Peripheral injec-tion of IL-1� can profoundly suppress gastric acid secretion(912, 947, 1059, 1101, 1132). Multiple explanations forthis observation have been put forward. It has been sug-gested the IL-1� acts in the CNS, as intrathecal injectionalso has an acid-suppressive effect (948, 949). Others havesuggested that IL-1� promotes formation of prostaglandinsor NO, which in turn inhibit acid secretion (312, 947,1101). Yet, a direct effect on parietal cells and ECL cells isthe most likely explanation, as both cell types express theIL-1 receptor and have been shown to be inhibited in theirfunction in isolated cell models (69, 70, 872, 958).

C. The Pharmacological Suppressionof Acid Secretion

Decreasing gastric acidity is indicated in many pathologicalcontexts, including gastric reflux disease or peptic ulcer dis-ease. This target can be achieved by two main pharmaco-logical approaches: 1) the inhibition of gastric acid secre-tion or 2) the intraluminal neutralization of already secretedgastric acid (antacids). Gastric acid secretion can be atten-uated by either directly blocking its final molecular effector,namely, H�-K�-ATPase (PPIs and acid pump antagonists),or by interfering with the neurohormonal signaling path-way leading to its secretion (H2 antagonists). The followingsection attempts to discuss the four most common sub-stance classes employed to increase intragastric pH.

1. Direct pharmacological inhibition of H�-K�-ATPase

The inhibition of H�-K�-ATPase-mediated proton trans-port represents the main contemporary pharmacologicalstrategy for reducing gastric acidity. An increase of gastricpH is the main factor ameliorating acid-related disordersand has been show to directly correlate with healing rates of,for example, GERD (74). Two main substance groups exerttheir acid-reducing effect via inhibiting H�-K�-ATPase func-tion: PPIs and acid pump antagonists (APAs). Both substancesachieve this aim by distinct mechanisms.

Omeprazole was the first clinically available PPI (324). Thefirst patent on omeprazole was filed in 1979 by the Swedishcompany Astra AB (today AstraZeneca). The introductionas a prescription PPI followed in 1989. Today, the omepra-zole enantiomer esomeprazole (S-omeprazole) generates the



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second highest revenue of all pharmaceuticals in the UnitedStates and is only surpassed by the statin atorvastatin (512).Furthermore, in the United States, PPIs are available asover-the-counter formulations, making them accessible forthe broad public. This is partially made possible by the highsafety profile of PPIs with a low incidence of unspecificadverse effects. Recently, however, concerns about thelong-term effects of chronic acid suppression have emergedwith regard to its impact on bone health (see sect. VA).

PPIs are delivered as pro-drugs through the bloodstream tothe parietal cell. They are weak bases (pKa �4), which caneasily pass the cell membrane and accumulate in acidic com-partments, such as the secretory canaliculus of the parietalcell. The pro-drug is then converted to the pharmacologi-cally active cyclic sulphenamide by the acidic pH in thesecretory canaliculus (670, 1007, 1135). Their specific ac-cumulation in acidic milieus and their pH-catalyzed conver-sion to active substances confers specificity and thus a highsafety profile to PPIs. Once activated, PPIs bind covalentlyvia disulfide bonds to H�-K�-ATPase, thereby inhibiting itscapacity to pump protons (89, 90, 1005, 1006, 1008). Thepattern of the cysteine residues, which are involved in PPIbinding, differ among the respective members of the PPIfamily: cysteine-813 reacts with all PPIs. In addition,omeprazole reacts with cysteine-892, lansoprazole withcysteine-321, and pantoprazole and tenatoprazole, respec-tively, with cysteine-822 (89, 90, 1005, 1006, 1008). Sincethe binding is covalent and irreversible, the inhibitory effectof PPIs lasts long beyond their plasma half-life, which usu-ally ranges between 0.5 and 2 h depending on the specificPPI (581, 1036). PPIs are generally metabolized by the he-patic cytochrome P-450 system, in particular CYP2C19and CYP3A4. This is of particular clinical importance, asCYP2C19 polymorphisms are known to exist. These poly-morphisms can impact the pharmacokinetics of PPIs byaffecting the metabolic rate of CYP2C19, which may haveconsequences for the optimal therapeutic regimen (582).Esomeprazole and rabeprazole seem to be less dependent onCYP2C19 metabolism (516, 983). Apart from the patternof cysteine reactivity, half-life and metabolism, PPIs alsovary in oral bioavailability (581).

Suppression of acid secretion can never be complete, asH�-K�-ATPase is subjected to a constant turnover (half-life�50 h) and needs to be stimulated for the conversion of thePPI to take place (936). Nevertheless, PPIs are highly effec-tive in reducing gastric acidity. Depending on the PPI andthe regimen, overall intragastric pH can be elevated by sev-eral pH units, up to a pH of 6 (compared with 1–2 atbaseline) (137, 425, 1036). For an excellent summary of PPIefficacy, please refer to Reference 1036.

APAs represent the second class of H�-K�-ATPase inhibi-tors. Unlike PPIs, they do not undergo irreversible binding,but rather act as potassium competitive antagonists. The

duration of inhibition is thus directly dependent on theplasma concentration of the inhibitor. As predicted by ho-mology modeling, mutational analysis, but also recentstructural data, APAs bind in the luminal cavity of H�-K�-ATPase in the vicinity of the potassium entry site where theyexert their inhibitory action (2, 42, 761, 1104, 1106). Al-though the inhibition of acid secretion has been shown to bevery effective, these substances are generally not in clinical use(577). For example, clinical trials of the APA AZD08650 haveshown no additional therapeutic effect compared with the PPIgold-standard, which resulted in abandonment of the drug in aclinical setting (262, 539).

2. H2 antagonists

The development of H2 blockers is inseparably intertwinedwith Sir Black’s discovery of the H2 receptor on the gastricparietal cell at the Smith Kline and French Laboratories(now GlaxoSmithKline) (106). In his original publication,Sir Black also describes burimamide as a competitive H2

antagonist that can effectively inhibit pentagastrin-stimu-lated gastric acid output in human volunteers (106). Furtherdevelopment of the antagonist led to the synthesis of cime-tidine, which was first commercially introduced in 1976 inthe United Kingdom, followed by the United States in 1977.Other commonly used members of H2-antagonist familynow include ranitidine, famotidine, and nizatidine.

H2 antagonists prevent histamine-mediated stimulation ofthe parietal cell by competitively interfering with its recep-tor. Although this effectively terminates the gastrin-hista-mine axis, the parietal cell is still susceptible to cholinergicstimulation via the M3 receptor. This partial inhibitionmainly accounts for the lower clinical efficacy of H2 antag-onists compared with PPIs, which directly target H�-K�-ATPase as the final target of all parietal cell stimuli (384).For example, a meta-analysis concluded that patientstreated for bleeding peptic ulcers are about twice as likely tosuffer from persistent or recurrent bleeding if treated withH2 antagonists compared with PPIs (384). Another meta-analysis also demonstrated a higher efficacy of PPIs in treat-ing esophagitis (83% healing rate with PPIs compared with52% with H2 antagonists)(567). Today, H2 antagonists arelargely superseded by PPIs due to their higher clinical effi-cacy. Furthermore, with the exception of famotidine, H2

antagonists are extensively metabolized in the liver by theCYP-450 system, leading to substantial drug-drug interac-tion profile (for review, see Ref. 506).

3. Antacids

Antacids directly neutralize gastric acid allowing immediateshort-term control of heartburn. They exist in various saltformulations, the most common of which are carbonatesalts, such as CaCO3, MgCO3, or NaHCO3. The use ofcalcium carbonate as a dietary calcium supplement is dis-



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cussed in a separate section (see sect. VC). Although eachformulation possesses its own spectrum of side effects, anotable condition in the context of this review is the milk-alkali syndrome, which is the result of a concomitant over-ingestion of calcium and alkali, such as CaCO3. Althoughthe syndrome became less prevalent with the introductionof modern ulcer therapies, it still poses a significant risk forpatients that may ingest CaCO3 as a calcium supplementfor the prevention of osteoporosis or as an antacid on aregular basis. Milk-alkali syndrome presents with the triadof metabolic alkalosis (carbonate), hpercalcemia (calcium),and renal insufficiency. The above average ingestion of cal-cium leads to increased plasma calcium levels due to excessabsorption and impaired renal secretion. PTH levels are lowdue to negative feedback (6). Hypercalcemia further causesrenal vasoconstriction, which decreases the glomerular fil-tration rate, and renal fluid loss because of activation of theCaSR, which in turn has loop-diuretic-like effects (see sect.IVD3). The activation of the CaSR is further potentiated bythe metabolic alkalosis, which increases its sensitivity tocalcium. All abnormalities are usually reversible after with-drawal of the offending agent and adequate treatment.

III. INTESTINAL CALCIUM ABSORPTION

The intestine is responsible for the absorption of dietarycalcium into our systemic circulation. Although the kidneyalso plays a pivotal role in calcium homeostasis by retainingand balancing systemic calcium via regulating its excretioninto the urine, renal calcium handling will not be the subjectof this review.

Current recommendations suggest that an average 40-yr-old adult should ingest �1,000 mg of calcium on a dailybasis (218). In the United States, this requirement is mostlymet (57). Up to 72% of the dietary calcium intake is attrib-utable to dairy products (218). Typically, the intestine ab-sorbs between 25 and 35% of the ingested calcium (1193).This occurs via two distinct pathways: 1) a paracellularpathway and 2) a transcellular pathway.

Calcium absorption via the paracellular route is tied to adownhill concentration gradient between the luminal andthe extracellular compartment and occurs throughout theentire intestine (although solvent drag induced paracellularflux may also play a role at low luminal calcium concentra-tions; Refs. 266, 1071, 1098). Conversely, transcellular ab-sorption can also take place against an uphill gradient, butrequires molecular machinery in the form of distinct cal-cium transport proteins which are expressed on the apicaland basolateral membranes of the enterocyte. This processdirectly requires energy in the form of hydrolyzable ATPand is alternatively termed “active” transport (versus “pas-sive” paracellular transport). The proximal small intestine,i.e., the duodenum and the jejunum, is the main site fortranscellular calcium absorption (825).

Since a concentration gradient is not a prerequisite for thisprocess, transcellular transport allows us to absorb calciumeven when the calcium concentration in the chyme is fairlylow. The relative importance of each respective absorptionpathway thus alternates with the amount of ingested cal-cium (46, 824, 1224). The rate of paracellular calcium up-take is canonically thought to remain constant, while tran-scellular transport can be upregulated under conditions ofdietary calcium restriction (46, 824, 1224). This regulationoccurs via the active metabolite of vitamin D [1,25(OH)2-vitamin D], which serves as a stimulator for transcellularcalcium uptake to prevent systemic calcium depletion.

A. Transcellular Calcium Absorption

1. Calcium entry

Evidence for active transport of calcium across the intestinalepithelium was established very early. With the use of a cal-cium radioisotope, various groups demonstrated 1,25(OH)2-vitamin D-dependent calcium transport against an imposedconcentration gradient in the small intestine of the rat (952,954, 1150). It has also been observed that active transportcan be induced by a low-calcium diet, which we now knowto stimulate the production of 1,25(OH)2-vitamin D (1133,1134). The degree of transport was highest in the duode-num and decreased in the more distal segments (952). Theduodenum is still considered the primary site where the bulkof transcellular transport occurs. To conduct active transcel-lular calcium absorption, the enterocyte has to be equippedwith an apical calcium entry pathway, a mechanism for cyto-solic calcium shuttling, and a basolateral calcium exit pathway(FIGURE 3). As the cytosolic concentration of free calciumis kept at a constant low level with typical concentrationsof �100 nM, apical calcium influx follows its electro-chemical gradient into the cell. In contrast, the extrusionof calcium on the basolateral membrane against an uphillgradient either directly requires ATP or energy stored inthe sodium gradient.

A) THE SEARCH FOR THE APICAL CALCIUM ENTRY PATHWAY. The mo-lecular identity of the apical calcium entry pathway was un-clear for a long time. Early experiments in isolated duodenalbrush-border vesicles revealed that calcium uptake was pas-sive, saturable, sensitive to ruthenium red, 1,25(OH)2-vitaminD dependent, and functionally optimal at a pH of 7.5 (740).This black box characterization suggested that a “specificcarrier” was responsible for calcium absorption and in ret-rospect already provided us with accurate key characteris-tics of the transient receptor potential vanilloid channeltype 6 (TRPV6), which was later established as the primaryapical calcium uptake channel (740). In subsequent at-tempts to further unravel the nature of the calcium uptakemechanism, various voltage-gated L-type calcium channelblockers were used (474, 717, 838). Although isolated du-odenal cells accumulated less calcium following application



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of the inhibitors and 1,25(OH)2-vitamin D stimulation(717), in vivo calcium entry proved to be fairly insensitive totheir effects (with the exception of verapamil, which dem-onstrated some degree of inhibition if applied at very highconcentration in the millimolar range) (474, 838). The con-flicting reports may be attributable to the different experi-mental models that were used, as isolated single cells do notallow discrimination between apical and basolateral trans-port mechanisms. Furthermore, it has been argued that L-type calcium channels may play a role in the stimulatorypathway of vitamin D, rather than in calcium uptake per se(717). In conclusion, an involvement of L-type calciumchannels seemed rather inconclusive and the identity of thecalcium entry channel remained elusive.

B) TRPV6. The cloning of the calcium transport protein sub-type 1 (CaT1) in 1999 finally marked a turning point in thesearch for the elusive intestinal calcium entry channels(838). The work was pioneered by Hoenderop and col-leagues who had identified the main calcium entry proteinin the kidney (epithelial calcium channel type 1, ECaC) viaan expression cloning strategy a few months earlier (474).

CaT1 was identified by a similar approach. A rat duodenalcDNA library was functionally screened using a calciumuptake assay in a Xenopus oocyte expression system (838).This screening process yielded the 727-amino acid proteinCaT1, which showed a 75% sequence homology to rabbitECaC. Homology analysis also demonstrated a relationshipto the vanilloid reptor type 1 (VR1), a nonspecific cationchannel that is activated by capsaicin, the pungent ingredi-ent in chili peppers, and mostly mediates pain signalingthrough afferent sensory neurons (838). The nomenclaturechanged over time as new channel proteins were identified,and today we consider CaT1, ECaC, and VR1 to be mem-bers of the same transient potential receptor vanilloid(TRPV) ion channel family. Literature now refers to ECaCas TRPV5, to CaT1 as TRPV6, and to VR1 as TRPV1.

The structure of TRPV6 was predicted to have six trans-membrane domains and four ankyrin repeat domains,which serve as cytoskeletal linking sites (838). Channel con-ductance was not dependent on other ions and was inhib-itable by a low extracellular pH (838). Calcium uptake wasreduced by as much as �70% at a pH of 5.5, which con-

PMCA

NCXTRPV6

Ca2+

Transcription

Enterocyte

Transcription

Ca2+Ca2+

Ca2+

Na+

Ca2+

Calbindin-D 9k

RXR VDR

D

D

D

D

VDR

FIGURE 3. Transcellular and paracellular calcium absorption in the intestine. The transcellular intestinalabsorption of calcium relies on apical calcium entry through TRPV6, intracellular calcium transport by calbin-din-D9k, and basolateral calcium extrusion via either NCX or PMCA. 1,25(OH)2-vitamin D regulates most ofthese ion transport proteins on a transcriptional level. 1,25(OH)2-vitamin D passes the plasma membrane ofthe enterocyte and binds to its receptor (VDR), which then heterodimerizes with RXR to initiate transcription.Evidence also suggests that 1,25(OH)2-vitamin D regulates the permeability of tight junctions, which gate theparacellular absorption of calcium. D, 1,25(OH)2-vitamin D.



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firmed the early black box data observations made in intes-tinal brush-border vesicles (838). This behavior seemedcounterintuitive to the investigators, as TRPV6 expressionwas highest in the duodenum, which is exposed to an acidload from the stomach (838). Duodenal pH has been re-ported to be as low as �6.1–6.6 but is even lower acutelyafter gastric emptying (�5.4), which would entail signifi-cant inhibition of TRPV6-mediated calcium uptake (284,290, 838). Conductance was also modestly sensitive (10–15% inhibition) to L-type calcium channel blockers at highconcentrations, which partially clarified the preceding am-biguous observations made by other groups with these in-hibitors (318, 337, 717, 838).

Subsequently, the human analog of rat TRPV6 was cloned(it had 97% sequence homology and was rather confusinglynamed ECaC2), and its expression was confirmed in humanduodenum (64). The generation of a TRPV6 antibody al-lowed for first localization studies, which confirmed an api-cal localization of TRPV6 and thus reaffirmed its role as theprimary apical calcium entry pathway in the intestine(1220). Tissue expression of TRPV6 varies among species(461). In humans, TRPV6 was identified in the duodenum,jejunum, stomach, esophagus, kidney, placenta, mammarygland, pancreas, prostate, testis, and salivary gland, but wasalso found to be upregulated in a series of malignancies,including prostate, breast, colon, and ovarian cancer (461,792, 836, 839, 1168, 1220). The role of TRPV6 in thesetissues mostly remains to be elucidated.

TRPV6 displays some unique biophysical properties. Asmentioned earlier, TRPV6 consists of six transmembranesegments, like many voltage-gated cation channels. Unlikethese channels, TRPV6 lacks the voltage sensor domain inthe fourth �-helix and is in a constitutively open state atresting membrane potential. Analysis of the quarternystructure shows that it assembles in tetramers (476). Theankyrin domains were implicated to be responsible for te-tramer formation; however, more recent structural data ob-tained by crystallography question this hypothesis (311,848). It is thought that heterotetramers can also be formedwith subunits of closely related TRPV5 (476). In contrast tomost other members of the TRP channel family, TRPV6 is avery selective channel for calcium (475, 1058, 1197). Therelative permeability of TRPV6 for calcium over sodium(PCa/PNa) is �100, whereas TRPV1, for example, has aselectivity of PCa/PNa �10 (although this has recently beenshown to be variable) (206, 475, 1197). This high selectiv-ity is crucial for the maintenance of a constant membranepotential in the enterocyte during calcium absorption.TRPV6 is strongly inward rectifying, which has been attrib-uted to magnesium ions plugging the channel pore for out-ward ion movement during states of depolarization (1126).Magnesium also exerts a voltage-independent inhibitory ef-fect on TRPV6 current; however, the mechanism underly-ing this observation is unclear (475, 1126). Furthermore,

TRPV6 gating is sensitive to intracellular calcium. Increasesin calcium were shown to inhibit the channel, resembling anegative-feedback mechanism (475). Although global cal-cium concentrations in the cell largely remain constant, thechannel microenvironment is exposed to fluctuations in lo-cal calcium. This feedback loop may be important for finelytuning the amount of calcium influx and preventing calciumoverload of the enterocyte. The putative mechanism of thisregulation will be discussed later in this section.

In 2001 it was demonstrated that ATP modulates TRPV6activity by preventing channel rundown (475). Recent workby Al-Ansary et al. (15) suggests that ATP directly binds tothe channel, thereby inhibiting inactivation by locking thechannel in the open conformation. Binding of ATP may beantagonized by channel phosphorylation through PKC(15). In conclusion, TRPV6 is precisely regulated by its ownmicroenvironment. Calcium, magnesium, and protons havean inhibitory effect on the channel, whereas ATP preventschannel inactivation.

It is important to remember that apart from being a nutri-ent, calcium is a crucial intracellular messaging molecule.Therefore, the enterocyte has to tightly control its intracel-lular concentration and adapt uptake to energy status andbasolateral extrusion. To ensure this delicate intracellularhomeostasis, TRPV6 is associated with and regulated by avariety of auxiliary proteins. The first protein that has beenidentified to interact with TRPV6 was the S100A10-an-nexin2 complex (1112). The S100A10-annexin2 complexhas been implicated to play a role in protein trafficking andmembrane anchoring. Its association with TRPV6 is essen-tial for channel trafficking to occur, as a disruption of theinteraction leads to cytosolic scattering of the channel(1112). Formation of the S100A10-annexin2 complex andits association with TRPV6 involves activation of PKA andcalcineurinA (CnA) (117). Another protein that is requiredfor TRPV6 membrane insertion is rab11a (1111). In anal-ogy to S100A10-annexin2, perturbation of rab11a bindingresults in decreased surface expression of TRPV6 and chan-nel retention in the cytosol (1111). Furthermore, the proteinkinases SGK1 and WNK3 are known to promote mem-brane insertion of TRPV6; however, their exact site of ac-tion is still unclear (114, 1031). Once trafficked to the mem-brane, physical channel stability is maintained by a varietyof auxiliary proteins. The COOH-terminal tail of TRPV6contains a PDZ protein binding motif. PDZ proteins serveas membrane anchors and protein scaffolds and mediate theassembly of multiprotein complexes, thereby regulatingchannel activity. The PDZ protein sodium hydrogen ex-changer regulating factor (NHERF4) (aka PDZK2) has re-cently been identified to interact with TRPV6 (574, 1113).It has been speculated that NHERF4 serves as a scaffold forTRPV6 at the apical pole, which is underlined by the obser-vation that knockdown of NHERF4 by RNAi leads to de-creased TRPV6 current in HEK293 cells (574, 1113). Apart



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from trafficking to the apical membrane, channel numbercan be regulated by internalization and degradation. Onepossibility of decreasing functional channels at the cell sur-face is protein ubiquitination. The process of ubiquitinationinvolves enzymatic tagging of target proteins, thus directingthem to the proteasome or lysosome for rapid degradation.The degradation tag is transferred to the target protein byE3 ubiquitin protein ligases, such as Nedd4–2. It is alreadywell understood how Nedd4–2-mediated ubiquitinationcan decrease the number, but also directly modulate theactivity, of the epithelial sodium channel (ENaC) in thekidney (825). A recent study demonstrated that a similarmechanism applies to TRPV6 (1212). Coexpression ofNedd4–2 and TRPV6 in oocytes resulted in decreased cal-cium flux and channel numbers (1212). Nedd4–2-mediatedTRPV6 ubiquitination could thus serve as a mechanism ofrapidly regulating channel retrieval from the plasma mem-brane (1212).

Several other proteins have been identified to associate withTRPV6 that do not affect channel numbers, but rather di-rectly regulate channel activity. The nisnap1 gene was iden-tified in the late 1990s; however, its function remained elu-sive (992). Recently, TRPV6 was shown to interact withnisnap1 (971). It is expressed in the intestine, but not thekidney, which is unusual as TRPV6 and TRPV5 are gener-ally regulated by similar auxiliary proteins. Electrophysio-logical measurements showed that nisnap1 inhibits TRPV6without affecting its surface expression (971). Very similarfunctional properties were attributed to RGS2, a proteinthat is mainly known to alter the GTPase activity of Gproteins. In analogy to nisnap1, RGS2 can inhibit TRPV6at the plasma membrane without affecting its traffickingdynamics (970).

As previously discussed, the activity of TRPV6 is finelyregulated by intracellular calcium concentrations. Eleva-tions in intracellular calcium levels exert an autoinhibi-tory effect on channel opening (475). Shortly after theidentification of TRPV6, calmodulin (CaM) was shownto bind to the channel in a calcium-dependent manner,and it was speculated that it mediates the calcium feed-back response (461, 791). Subsequent investigations dem-onstrated that CaM may indeed represent the molecularcalcium sensor (263, 619). Elegant FRET studies reportedthat CaM dynamically associates with TRPV6 in the pres-ence of calcium and that this association is terminated whenintracellular calcium is depleted (263). The association be-tween TRPV6 and CaM also correlates with decreased cur-rent flux through the channel (263). Although the ankyrinrepeat domains of the channel were thought to possiblyserve as binding sites for CaM, more recent structural in-sights provide rebutting evidence for this hypothesis (848).Instead, the COOH-terminal tail of TRPV6 has been sug-gested as the site where CaM binding occurs (263, 619,791). Hence, CaM can tune ion flux through TRPV6 and

most likely mediates the channel’s sensitivity to intracellu-lar calcium.

Phosphorylation by kinases and dephosphorylation byphosphatases represent a common cellular strategy for rap-idly modulating channel gating properties. The non-recep-tor tyrosine kinase Src has emerged as a candidate for thedirect phosphorylation of TRPV6, thereby increasing chan-nel conductance (1042). Src was previously shown to mod-ulate TRPV4 activity (1178). The effects of Src are antago-nized by the phosphatase PTP1B. Both enzymes act on thetyrosine residues Y161/162, which are located in the NH2-terminal tail of the channel (1041).

An interesting interaction without direct relevance for theintestine has been reported between renal TRPV5 andklotho (170). Klotho is a �-glucuronidase with a transmem-brane anchor that can be cleaved, resulting in shedding ofthe enzymatically active domain of the protein into theurine (170, 612). Klotho can then increase TRPV5-medi-ated calcium uptake by hydrolyzing N-linked oligosaccha-rides on extracellular channel domains, thereby preventingchannel retrieval from the plasma membrane (170). Al-though a recent report indicates that klotho can also affectTRPV6 activity in vitro, it is not expressed in the intestineand thus may only interact with renal TRPV6 (612, 683).Although this finding has no implications for intestinal cal-cium uptake, various bacteria and neutrophils produce�-glucuronidase, which may affect TRPV6 activity in theintestine (359, 1004).

Recently, Stumpf et al. (1046) described an association be-tween cyclophilin B (CyB) and TRPV6 in the placenta. Co-expression of both proteins in oocytes increased calciumuptake (1046). CyB was also detected by Western blot anal-ysis in the small intestine and colon; however, colocaliza-tion and functional studies in intestinal tissue remain to beperformed (1046).

C) THE TRPV6 KNOCKOUT CONUNDRUM. As will be discussed inmore detail later, 1,25(OH)2-vitamin D is one of the keyhormonal regulators of systemic calcium homeostasis (seesect. IVA). Increased levels of 1,25(OH)2-vitamin D lead toenhanced intestinal calcium absorption. Shortly after clon-ing of TRPV6, it was recognized that the channel is posi-tively regulated at the mRNA level by 1,25(OH)2-vitamin D(614, 1030, 1109, 1110). The TRPV6 promoter has mul-tiple binding sites for the 1,25(OH)2-vitamin D receptor(VDR) and is thus directly sensitive to increases in1,25(OH)2-vitamin D levels (736). Consequently, VDR-de-ficient animals display a marked decrease in TRPV6 mRNAlevels, whereas administration of 1,25(OH)2-vitamin D inwild-type animals results in an increase in mRNA transcrip-tion (1030, 1109, 1110). In conjunction with the rapidly ex-panding characterization of the channel itself, these observa-tions supported the dogma that TRPV6 is the essential player



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in transcellular calcium uptake and that this pathway isstrongly regulated by 1,25(OH)2-vitamin D.

With the introduction of novel genetic techniques, a TRPV6(�/�) mouse was created in 2007 by Bianco et al. (94). Theanimals presented with decreased intestinal calcium uptake,as measured by serum concentrations of a calcium radioiso-tope following gavage, decreased femoral bone density(9.3%) and increased 1,25(OH)2-vitamin D levels, as a re-sult of feedback regulation (94). These findings were inaccordance with the postulated role of TRPV6 as the pri-mary 1,25(OH)2-vitamin D-sensitive calcium uptake mech-anism in the intestine. However, the generation of anotherTRPV6 (�/�) animal line by Benn et al. (76) a year laterspawned a controversy in the field. In these animals, base-line calcium uptake was identical between wild-type and(�/�) groups. Surprisingly, calcium uptake could be in-creased in the (�/�) group by a low calcium diet and, moreimportantly, by 1,25(OH)2-vitamin D (an observation thatwas also confirmed by another group; Ref. 615) (76). Thesefindings profoundly questioned the role of TRPV6 in cal-cium absorption and suggested that a different moleculartarget of 1,25(OH)2-vitamin D mediates the increase in cal-cium uptake after exposure. All these observations were insharp contrast to the initial report by Bianco et al. (94).However, Benn et al. (76) also reported that compared withwild-type animals calcium uptake was reduced in TRPV6-deficient animals that were fed a low-calcium diet. Thissuggests that the animals may not be able to adequatelyincrease absorption, when confronted with a low dietaryavailability of calcium, which in turn suggests a role ofTRPV6 during states of dietary calcium insufficiency.

These controversial findings allow several conclusions andspeculations. 1) As with any model of targeted gene disrup-tion, a compensatory mechanism may be in place thatmasks and distorts the physiological importance of TRPV6.The animals may upregulate other, yet unidentified, cal-cium transport mechanisms to compensate for the loss ofTRPV6 function. 2) Rather than being a constitutivelyactive pathway, transcellular calcium absorption throughTRPV6 only occurs in states of low dietary calcium intake.This hypothesis has been put forward very early and has beenreaffirmed by a recent study that observed an increase in boneturnover in TRPV6 (�/�) animals on a low-calcium diet(46, 666, 824, 1224). Mobilization of bone calcium may benecessary in these animals to maintain systemic calciumconcentrations, which are double challenged by the lack ofTRPV6 and the insufficient calcium intake (666). Further-more, it has been extensively described that a low-calciumdiet induces gene expression of TRPV6 [presumablythrough an increase in 1,25(OH)2-vitamin D levels], sug-gesting a role of the channel during states of insufficientdietary calcium supply (76, 132, 639). 3) 1,25(OH)2-vita-min D may have many more targets in the intestinal mucosathan previously anticipated and may also regulate calcium

uptake through the paracellular pathway (76, 615). In con-clusion, the generation of TRPV6 (�/�) animals has sus-tainably challenged our model of transcellular calcium ab-sorption by questioning the relative importance of TRPV6and by introducing new potential targets of 1,25(OH)2-vitamin D regulation.

D) GENETIC POLYMORPHISMS OF TRPV6. Single nucleotide poly-morphisms (SNPs) are variations in the genomic sequencethat occur in one single base. If the frequency of a SNP or aspecific set of SNPs (haplotype) increases in a populationover time compared with other SNPs, it can be concludedthat this set of SNPs is associated with an evolutionaryadvantage, meaning that this gene locus was under selec-tion. Recent reports from various groups indicate thatTRPV6 was subjected to strong selection (12, 502, 1023,1050). Traces for selection can be found in any non-Africanpopulation (12, 502, 1023). The selective event in Euro-peans has presumably occurred �7,000 years ago andcoincides with the development of agriculture (502). Thiscorrelation also holds true for other populations (502). Ithas been speculated that a change in diet or resistance toemerging disease led to selection and fixation of the nowconserved haplotype (502). Interestingly, selection tookplace in parallel in many populations, pointing towards astrong selective stress that developed independently ineach region (502).

The electrophysiological properties of ancestral and derivedTRPV6 were investigated by two groups (502, 1050).Hughes et al. (502) observed no statistically significant di-vergence in channel behavior. The derived channel onlydisplayed a tendency towards decreased sensitivity to theautoinhibition by calcium, albeit not significant (P ��0.094) (502). The authors speculated that differences inprotein-protein interactions may have led to selection(502). Conversely, Sudo et al. (1050) reported increasedcalcium influx through the derived channel, which may con-stitute an evolutionary advantage by facilitating dietary cal-cium uptake. Regardless of the controversial functionaldata, these insights indirectly underline the importance ofTRPV6 and provide a counterweight to the conclusionsdrawn from the TRPV6 (�/�) animal model. It is unques-tionable that TRPV6 was under parallel independent selec-tion in many regions across the planet. This provokes thequestion of how strong selection can occur for a derivedchannel whose physiological difference to the ancestralform seems to be very subtle, yet complete disruption of thechannel in the mouse model only causes a very mild pheno-type. This inconsistency adequately exemplifies the caveatthat underlies the conclusions drawn from (�/�) animalsand reminds us of the fact that we are far from understand-ing the exact physiology of transcellular calcium uptake.

E) CAV1.3. TRPV6 may not be the only channel mediatingapical calcium absorption in the intestine. A recent investi-



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gation provides another explanation for the initially ob-served partial sensitivity of transcellular calcium uptake toL-type calcium channel blockers. The voltage-gated cal-cium channel Cav1.3, a member of the L-type calcium chan-nel family, was recently identified in the apical membranesof the distal jejunum and proximal ileum (751). Previousobservations were emulated, as the investigators again dem-onstrated a decrease in calcium absorption following appli-cation of L-type calcium channel inhibitors in the corre-sponding segments (751). The authors argued that uptakethrough Cav1.3 may have previously been misinterpreted asparacellular calcium uptake (751). However, it should benoted that the calcium uptake assay used in this report didnot discriminate between transcellular and paracellular cal-cium movement. Subsequently, it has been observed thatL-type inhibitor-sensitive calcium flux is linked to stimula-tion of glucose uptake through the glucose transporter type2 (GLUT2) (692, 750). Cav1.3 may serve as an alternativecalcium entry pathway that is active in states of luminalcalcium abundance and that coregulates glucose absorption(692, 750). Further investigations will be needed to deter-mine the contribution of Cav1.3 to dietary calcium absorp-tion.

2. Calbindin-D 9k

The following section will address the question of whathappens to dietary calcium after it enters the enterocyte. Itshould be considered that transcellular calcium uptake andcytosolic calcium homeostasis are two contradicting re-quirements for the enterocyte. A prominent transcellularflux of free calcium will inevitably interfere with housekeep-ing functions of the cell, such as intracellular signaling.Furthermore, it has been calculated from in vivo data that ifcalcium were to diffuse freely (simple diffusion) through thecytosol, uptake rates would only be 1/70th of the actuallymeasured values (129). Simple diffusion would constitute abottleneck in the process of transcellular calcium absorp-tion if cytosolic calcium concentrations should be kept low.A partial solution to this problem was found as earlyas 1966, when Wasserman et al. (1151) identified a1,25(OH)2-vitamin D-inducible calcium binding protein inthe chick intestine. The authors observed that calcium ra-dioisotopes traveled faster across a cellophane membrane ifsuspended in intestinal homogenates from rachitic, i.e.,1,25(OH)2-vitamin D deficient, chicks than if suspended inthe homogenates from rachitic animals treated with1,25(OH)2-vitamin D (1151). This indicated that calciumwas bound to a protein in the enterocyte and that expres-sion of this protein was controlled by 1,25(OH)2-vitamin D(1151, 1152). Subsequently, the calcium binding proteinwas further characterized (1073, 1149, 1152). Expressionlevels were shown to be highest in the duodenum and togradually decrease in more distal segments, which corre-lated with the degree of calcium uptake that has been at-tributed to each intestinal segment respectively in priorfunctional investigations (1073). The mammalian isoform,

which had a lower molecular mass of �9 kDa (hence thename calbindin-D 9k), compared with the 28 kDa of theavian isoform, was later identified (138, 280, 283, 358).Concerning the functional role of calbindin-D 9k, it hadalready been speculated very early after its discovery that itmay serve as a calcium shuttling protein (951). Indeed, ini-tial calculations and later very basic experimental data con-firmed that calbindin-D 9k may mediate facilitated diffu-sion of calcium between the two poles of the enterocyte, inanalogy to the transport of oxygen by myoglobin in themuscle (321, 602). In a very fundamental investigation,calcium flux was measured between chambers that wereseparated by dialysis membranes. A 51% increase in trans-chamber calcium flux occurred in the presence of calbin-din-D 9k (321). However, it is hard to relate this number tophysiological values, given the simplification underlying theexperimental model. It has subsequently been calculatedthat calbindin-D 9k may facilitate diffusion of calcium by afactor of up to �60 (129). Rather than envisioning calbin-din-D 9k as a protein that individually shuttles calcium ionsacross the cell, one should imagine calbindin-D 9k as acalcium gradient amplifier (129, 602). As the intracellularconcentration of free calcium is in the nanomolar range, theintracellular gradient of free calcium between the apical andbasolateral pole can also only be in the nanomolar range.Diffusional flux, however, directly correlates with the con-centration gradient and can in consequence only be verysmall. Calbindin-D 9k is expressed in the enterocyte in themicromolar range and dynamically binds and releases cal-cium (714). Since the association between calbindin-D 9kand calcium is dynamic, the local concentration of calbin-din-D 9k-bound calcium will directly correlate with theconcentration of free calcium. It is thus the concentrationgradient of calbindin-D 9k-bound calcium throughout thecell that determines the flux rate of calcium. As this gradientcan be in the micromolar range, calbindin-D 9k serves as acalcium gradient amplifier (129, 602). It should be notedthat the real experimental data on calbindin-D 9k are fairlyscarce and that the bulk of scientific effort has gone intomathematical modeling of facilitated diffusion and calciumbinding kinetics (129, 321, 322, 602, 1021).

Functionally, several correlations between calbindin-D 9kand calcium uptake were identified. The calbindin-D 9kcontent of each intestinal segment correlates linearly withits ability for calcium uptake (129, 824, 1021). Althoughthis relation holds true on the experimental and the math-ematical modeling level, it does not prove causality (129,824, 1021). Furthermore, 1,25(OH)2-vitamin D and a low-calcium diet induce calbindin-D 9k on both the mRNA andprotein level, suggesting that it is involved in the process ofregulated calcium absorption (289, 604). In accordancewith these findings, VDR-deficient animals have a de-creased calbindin-D 9k mRNA content that cannot be res-cued by exogenous 1,25(OH)2-vitamin D administration(663, 1194). Although a 1,25(OH)2-vitamin D responsive



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element (VDRE) had been identified in the 5=-flanking re-gion of the calbindin-D 9k gene, later mutational analysisdemonstrated that this site was not essential for transcrip-tional regulation of calbindin-D 9k by 1,25(OH)2-vitaminD (217, 240). Hence, it is not clear how 1,25(OH)2-vitaminD exactly regulates calbindin-D 9k on a molecular level.Interestingly, intestinal calbindin-D 9k mRNA levels can berescued in VDR (�/�) animals by a high-calcium/phospho-rus/lactose diet, while extraintestinal calbindin-D 9kmRNA levels are unaffected (663). This suggests that, apartfrom endocrine regulation through 1,25(OH)2-vitamin D,intestinal calbindin-D 9k is also regulated by local factors(663). A direct short-term stimulatory effect of oral calciumintake on calbindin-D 9k levels had been observed before ina wild-type background (647). The mechanisms underlyingthe effect of diet on calbindin-D 9k levels are, however, stillobscure.

Despite the correlation between calbindin-D 9k levels and1,25(OH)2-vitamin D and the mathematical and experi-mental models, which support its role as a calcium shuttlingprotein, no clear evidence for the involvement of calbin-din-D 9k in transcellular calcium absorption existed. Thishad sparked controversy among investigators. In fact, avery early study concluded that although 1,25(OH)2-vita-min D induced both calbindin-D 9k and calcium absorp-tion, a discrepancy existed in the time course of both effects(424). Later, synthetic 1,25(OH)2-vitamin D derivatives,designed for a maximized effect on cell differentiation andsuppressed calciotropic activity, failed to increase serumcalcium levels while causing a sevenfold increase in calbin-din-D 9k mRNA [native 1,25(OH)2-vitamin D increasesboth serum calcium and calbindin-D 9k mRNA](604). In2006, a calbindin-D 9k (�/�) mouse was created by Kutu-zova et al. (613). The animals presented with no apparentphenotype abnormalities and had normal serum calciumconcentrations (613). Subsequent analysis of these animalsshowed that they responded equally well to 1,25(OH)2-vitamin D with regard to calcium absorption as wild-typeanimals (13). Of note, calcium absorption was assessed bymeasuring serum calcium following gavage of a calciumradioisotope and did not discriminate between transcellularand paracellular absorption (13). A different group repli-cated the findings made in the calbindin-D 9k (�/�) mouseand also did not find any appreciable variation in pheno-type or serum calcium concentrations (639). However, theinvestigators observed that during preweaning, when bothcalcium demand and absorption are at their peak, geneexpression of TRPV6 and the basolateral calcium extruder,plasma membrane calcium ATPase isoform 1b (PMCA1b),were highly induced in the calbindin-D 9k (�/�) animals(128, 639). This has been interpreted as a compensatoryupregulation of calcium transport proteins to ensure suffi-cient uptake in a state of high metabolic demand for cal-cium (639). Subsequently, a TRPV6 and calbindin-D 9kdouble (�/�) animal was created. The mature animals

show no disturbances in calcium homeostasis, although,similarly to TRPV 6 (�/�) animals, they cannot increasecalcium uptake in response to a dietary calcium challenge tothe same extent as wild-type animals (76).

In light of the insights gained through knockout animals,the role of calbindin-D 9k remains somewhat unclear.Again, it is difficult to dissect the physiological function ofcalbindin-D 9k in these animal models, given the assump-tion that the organism will pursue compensation for the lossof a gene product. In conclusion, it is undisputed that cal-bindin-D 9k is regulated by 1,25(OH)2-vitamin D, that itcan bind calcium and that theoretical modeling and veryfundamental experimental models verify that it can facili-tate diffusion of calcium across the enterocyte. The (�/�)models suggest that animals can compensate for the loss ofcalbindin-D 9k and maintain normal calcium homeostasis(13, 76, 613, 639). Furthermore, TRPV6, calbindin-D 9kand double (�/�) animals can still increase their calciumuptake in response to 1,25(OH)2-vitamin D or a low-cal-cium diet, respectively, albeit not to the same extent aswild-type animals (13, 76, 615). One may conclude thatneither of these proteins is necessary for transcellular up-take; however, it seems more likely that 1,25(OH)2-vitaminD has more targets than previously postulated and thatcompensatory mechanisms are in place.

3. Basolateral calcium extrusion

Once calcium reaches the basolateral membrane, it is ex-truded into the extracellular space, which completes theprocess of intestinal absorption. As extracellular calciumconcentrations are higher than cytosolic concentrations,this process requires energy. Two proteins are responsiblefor this task. 1) The PMCA extrudes calcium at the directexpense of ATP, whereas 2) the sodium-calcium exchanger(NCX) utilizes the energy stored in the sodium gradient totransport calcium out of the cell (FIGURE 3). It is this baso-lateral exit process that requires energy during transcellularcalcium uptake and that allows us to absorb calcium againstan uphill gradient when dietary concentrations are low.

A) THE PLASMA MEMBRANE CALCIUM ATPASE. The plasma mem-brane calcium ATPase (PMCA) is a virtually ubiquitousprotein that is responsible for intracellular calcium homeo-stasis by pumping calcium into the extracellular milieu,thereby keeping intracellular concentrations low. Thepump belongs to the family of P-type primary ion transportATPases, which among others also includes gastric H�-K�-ATPase (see sect. IIA1). Four isoforms of the protein exist;however, variety is increased by splicing (562, 1043). ThePMCA1b splice variant is most predominant in the smallintestine and the duodenum in particular, which suggest aninvolvement of this isoform in the process of transcellularcalcium absorption (341, 492). Given the ubiquitous ex-pression of PMCA, a detailed review of its structure and



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function will be omitted. For a current and detailed review,please refer to Reference 1043.

PMCA was first characterized in the 1960s in the membraneof erythrocytes (956). Very early experiments in basolateralmembranes isolated from enterocytes identified a calcium-dependent enzyme with phosphatase activity, which servedas first evidence for PMCA in the intestine (100, 742). Sub-sequent investigations in basolateral vesicles from rat intes-tine concluded that two distinct calcium transport mecha-nisms existed in their membranes: an ATP-dependent(PMCA) and a sodium-dependent (NCX) mechanism (382,457, 573). Furthermore, it was shown that inhibition ofcalmodulin halved the amount of ATP-dependent calciumtransport (573). Today, we know that PMCA is highly reg-ulated by CaM, which can increase both the affinity of thepump to calcium and its turnover speed by a factor of up to10 (526, 627, 628, 756).

Interestingly, it has also been suggested that calbindin-D 9kcan directly stimulate calcium transport via PMCA (1136,1137). In addition to intracellular proteins, PMCA is alsoquantitatively regulated by endocrine factors. 1,25(OH)2-vitamin D can increase both the PMCA mRNA and proteincontent in enterocytes (35, 36, 62, 146, 382, 489, 614, 823,1109; contested by Ref. 1138). The data on the effects of alow-calcium diet on PMCA transcription are more controver-sial, as one study suggests induction whereas two other studiesobserved reduced mRNA levels (146, 583, 639). Of note,these observations were made in different species (chick vs.mouse). The sensitivity of the tissue to 1,25(OH)2-vitamin D isalso reported to decrease with age, which may representanother confounding factor underlying these observations(35, 36). In conclusion, the transcriptional regulation ofPMCA1b by 1,25(OH)2-vitamin D and its decreasing ex-pression along the length of the small intestine correlatewell with the canonical model of transcellular calcium ab-sorption. It is challenging to investigate the functionalcontribution of PMCA1b in the process of calcium ab-sorption, given its role as a housekeeping protein.PMCA1 is crucial during development, which results inembryonic death if knocked out (814, 1198). Conversely,heterozygote (�/�) animals present with no apparentphenotype, albeit parameters linked to calcium homeo-stasis were not assessed (814).

B) THE NCX. Long before the molecular identities of PMCAand NCX were known, it had been observed that calciumuptake in the intestine was dependent on extracellular so-dium (713). The authors concluded that “a sodium, calcium-exchange diffusion carrier may exist at the basal membraneof the cell,” which proved to be a remarkably accurateprediction (713). As mentioned before, later investigationsdelineated between an ATP and a sodium-dependent trans-port of calcium on the basolateral enterocyte membrane(382, 457, 573).

For a recent review on the structure and function of NCX,please refer to Reference 687. In brief, NCX exists in threeisoforms (NCX1–3), which are expressed in a broad varietyof cell types (687). The function of NCX has mainly beeninvestigated in excitatory tissues, given its role as a high-capacity calcium extrusion mechanism following excita-tion. To transport calcium against its strong electrochemi-cal gradient, NCX has to utilize three sodium ions to ac-complish extrusion of one calcium ion (85, 460). NCX1 isthe predominant isoform in the small intestine, where it hasbeen detected on the mRNA and the protein levels (275,688). However, NCX had been postulated to play a moreimportant role during calcium absorption in the kidneythan in the enterocyte, hence not much data on intestinalNCX are available to us (473). Furthermore, genetic dis-ruption of NCX1 is embryonically lethal, which imposessome limitations on our experimental methodology (600).A more recent study provides some functional evidence forNCX in the intestine. By measuring intracellular calciumconcentrations with a fluorescent indicator dye, Dong et al.(275) demonstrated that calcium uptake in a sodium-freeenvironment (to run NCX in its reversed configuration) wassignificantly decreased when NCX was pharmacologicallyinhibited. Despite our knowledge that NCX exists in en-terocytes and that it can extrude calcium under experimen-tal conditions, it is difficult to assess its relative contributionto transcellular calcium absorption.

In addition, NCX is not subjected to regulation by 1,25(OH)2-vitamin D, which further contributes to the ambiguity con-cerning its importance in the process of calcium absorption.This has been observed very early, when exogenous admin-istration of 1,25(OH)2-vitamin D to vitamin D-deficientanimals did not increase sodium-dependent calcium trans-port, while doubling transport through PMCA (382). Fur-thermore, it was recently shown that a calcium-depleteddiet decreases duodenal NCX1 mRNA levels (583).

Of note, two members of the potassium-dependent sodiumcalcium exchanger (NCKX) family, namely, NCKX 3 and4, were also identified in the small intestine (658, 687, 688).However, further functional investigations will be neededto clarify their role.

B. Paracellular Calcium Absorption

If we plot the amount of duodenal calcium absorption as afunction of the luminal calcium concentration, we can ob-serve that the absorption curve is comprised of two distinctkinetic components: a saturable/exponential componentand a nonsaturable/linear component (824, 825, 1134).The saturable component represents transcellular calciumuptake through the enterocyte, as both the number of cal-cium transport proteins and their turnover rate is limited.The nonsaturable component reflects calcium uptakethrough the paracellular pathway. The saturable compo-



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nent is less pronounced in the jejunum and disappears com-pletely in the ileum, indicating that transcellular calciumabsorption is restricted to the proximal segments of theintestine, as discussed previously (825) [this has been con-tested more recently, when transcellular flux was also notedin the ileum (46)]. Compared with the transcellular path-way, the paracellular route has not received much scientificattention. It has been put forward that the rate of paracel-lular calcium absorption is constant across the length of theintestine and is neither sensitive to 1,25(OH)2-vitamin Dnor a low-calcium diet (824, 825, 1224). However, pro-vided that enough dietary calcium is available to saturatethe transcellular pathway, observations indicate that netcalcium absorption is highest in the ileum, which has beenattributed to the sojourn time, rather than alterations inparacellular permeability (290, 710). In the rat, the chymespends some 74% of its transit time in the ileum, whichallows for a long exchange period between lumen andplasma (290). The diffusion rate itself is fairly low and onlyamounts to 2% of the rate if calcium were to diffuse freelybetween intestinal lumen and plasma (290). This effect is aconsequence of the diffusion barrier that tight junctions,which act as functional gating molecules of the paracellularpathway, impose on calcium flux.

Epithelial tight junctions are adhesion points between twoneighboring cells that seal their intercellular space against alumen, thereby restricting ion and water movement be-tween the two compartments. As water cannot be directlytransported, its movement is tied to ion fluxes, which inturn are determined by their respective concentration gra-dients across the epithelium. Each epithelial cell expressesan annulus of tight junction proteins at the apical end of thelateral membrane. Tight junctions are protein complexesthat consist of a variety of intra- and transcellular proteins.A detailed review of their structure would exceed the scopeof this article (for a recent review, please refer to Ref. 999).Briefly, the composition of the involved proteins determinesthe pore size, ion selectivity, and thereby the relative leaki-ness of each tight junction and the whole epithelium ingeneral. Furthermore, tight junctions create a lateral diffu-sion barrier for membrane proteins and help to maintain thefunctional polarity of the epithelial cell. It is important torecognize that tight junctions are not static complexes, butcan be rather dynamically regulated (999). This allows theepithelium to change its ion and water permeability in re-sponse to various stimuli (999).

As discussed previously, the rate of the nonsaturablecomponent of calcium absorption is documented to befairly constant and insensitive to 1,25(OH)2-vitamin D,which indicated that 1,25(OH)2-vitamin D may have noeffect on paracellular transport (824, 825, 1224). How-ever, in the late 1990s, Chirayath et al. (201) postulatedthat 1,25(OH)2-vitamin D can increase paracellular cal-cium flux in confluent Caco-2 cell cultures, which form

an epithelia-like monolayer with tight junctions. Thisconclusion is based on the observations that 1,25(OH)2-vitamin D decreased the transepithelial electric resis-tance, which is often used as a measure of tight junctionpermeability, and induced bidirectional, i.e., also “serosal”to “mucosal,” calcium flux in the cultured monolayers(201). A more recent investigation further substantiatedthis hypothesis. It has been shown that 1,25(OH)2-vitaminD regulates the mRNA levels of some tight junction proteinsin rat duodenum, which may alter their gating characteris-tics (354, 614). For example, the mRNA levels of claudin-3,a protein that directly determines tight junction permeabil-ity, were decreased 2.2-fold following 1,25(OH)2-vitaminD administration (614). Conversely, claudin-2 and -12mRNA and protein levels were increased (354). Functionalobservations in Caco-2 monolayers further demonstratethat overexpression of claudin-2 and -12 increases electricalconductivity and facilitates paracellular calcium flux (354).All of these events are tied to a 1,25(OH)2-vitamin D-me-diated promotion of transcriptional events. However, it isknown that 1,25(OH)2-vitamin D can also exert short-termnongenomic effects (1122) (see sect. IVA6B). A recent inves-tigation presents evidence for augmented solvent-dragparacellular calcium flux after acute administration of1,25(OH)2-vitamin D (1098). Solvent-drag mediated fluxmay occur at low calcium concentrations in the absence ofa calcium gradient. In this model, calcium is draggedthrough the paracellular space by water flux that is fueledby the hyperosmolar milieu in the paracellular space (266,626). 1,25(OH)2-vitamin D was shown to induce this cal-cium flux in the presence of initially equimolar calciumconcentrations between the mucosal and serosal compart-ments (1098).

Over the last years, evidence for the regulation of the para-cellular pathway by 1,25(OH)2-vitamin D has slowly accu-mulated. It is apparent that the canonical dogma whichpostulates that 1,25(OH)2-vitamin D only targets the tran-scellular pathway has to be revisited. Still, more experimen-tal investigations will be needed to ultimately clarify theeffects of 1,25(OH)2-vitamin D on paracellular calciumtransport. Should tight junctions indeed be subjected to regu-lation by 1,25(OH)2-vitamin D, this mechanism may partiallyexplain the sensitivity of calcium uptake in TRPV6 and cal-bindin-D 9k (�/�) animals to 1,25(OH)2-vitamin D.

C. Alternative Models of TranscellularCalcium Absorption

Two alternative models of transcellular calcium absorptionhave been put forward. One suggests that calcium is trans-ported through the enterocyte by vesicles (vesicular trans-port model) rather than facilitated diffusion, and the otherpostulates that 1,25(OH)2-vitamin D can induce intestinal cal-cium absorption in a rapid, nongenomic fashion via a putative1,25(OH)2-vitamin D surface receptor (transcaltachia).



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The assumption that calcium is transported through the cellin vesicles is corroborated by a handful of observations. Ithas been demonstrated that 1,25(OH)2-vitamin D treat-ment increased the number of supranuclear lysosomes inrachitic chicks, compared with nontreated animals (247). Itwas later shown that the calcium content of lysosomes canmore than double following treatment with 1,25(OH)2-vi-tamin D, suggesting that they may play a role in the processof transcellular calcium movement (780). Compared withthe cumulative evidence that is in accordance with our ca-nonical model of calcium absorption, the role of vesiculartransport of calcium is still fairly obscure.

The model of transcaltachia mainly relies on the observa-tion that 1,25(OH)2-vitamin D can exert effects that are tooacute to be attributable to transcriptional events. For exam-ple, intestinal calcium absorption was increased in chickswithin 14 min of 1,25(OH)2-vitamin D exposure, an onsetwhich is too rapid as to be of a genomic nature (785). Itshould be noted that rapid effects of 1,25(OH)2-vitamin Dhave also been suggested to influence the paracellular path-way (see above). Undoubtedly a careful evaluation of theroute of calcium flux, i.e., transcellular versus paracellular,is necessary. At least, isolated intestinal cells respond withincreased uptake of radiolabeled calcium to acute 1,25(OH)2-vitamin D exposure (568). It has been postulated that apartfrom the VDR, a membrane-bound 1,25(OH)2-vitamin D ex-posure receptor, the 1,25(OH)2-vitamin D-MARRS (mem-brane-associated, rapid response, steroid binding) protein, me-diates the acute effects of 1,25(OH)2-vitamin D (568, 779,785).

IV. REGULATION OFCALCIUM HOMEOSTASIS

Eucalcemia is maintained by the concerted effort of vitaminD, PTH, and, to a lesser extent, calcitonin. All three hor-mones can influence serum calcium concentrations by act-ing on the intestine, the kidney, or bone. 1,25(OH)2-vita-min D, the active vitamin D metabolite, primarily modu-lates the intestinal absorption of calcium and will thereforebe discussed in most detail (FIGURE 4). Apart from hor-monal regulators that influence the absorption, excretion,and deposition of calcium, our body needs a mechanismthat allows it to sense the current levels of plasma calcium.This task is fulfilled by the CaSR. It oversees the preciseregulation of the calcitropic hormones.

A. Vitamin D

As discussed previously, vitamin D is one of the key regu-lators of calcium homeostasis. Our body has two sourcesfor vitamin D, namely, a dietary source (vitamin D2�3) andan endogenous source that relies on ultraviolet (UV) lightcatalyzed synthesis in the skin (vitamin D3) (FIGURE 5).

Nomenclature in this case is fairly misleading, as vitamins areper definition substances that cannot be generated by our bodyand have to be ingested from an external source. The fact thatwe can synthesize vitamin D3 in our skin classifies the sub-stance as a prohormone rather than a vitamin. As we will see,our current nomenclature is a byproduct of the historic eventsleading to the discovery of vitamin D.

1. Historical perspective

From a historical perspective, the identification of vitaminD is closely intertwined with attempts to understand thepathophysiology of rickets. Rickets is characterized bychildhood skeletal deformities resulting from inadequateosteoid mineralization and calcification of cartilage due todecreased serum calcium levels during development. Theadult form equivalent of rickets is termed osteomalacia.With the onset of industrialization, rickets became a prev-alent problem in the 18th, 19th, and the beginning of the20th century. In fact, the disease was so widespread at thebeginning of the 20th century that an investigation con-ducted by the German pathologist Schmorl on 386 children,who had died before the age of 4 years, concluded that 90%of them had had rickets (968). Even in present times nutri-tional rickets still remains a major public health concern indeveloping countries (303, 757, 809). The seminal observa-tions that led to the identification of vitamin D were pro-vided by Mellanby in 1919 (733). He observed that dogpups who were fed a severely restricted diet consisting ofporridge or bread were consistently developing rickets(733). Development of rickets could be averted if their dietwas supplemented with cod liver oil, which we now knowto contain a high concentration of vitamin D (733). Mel-lanby (733) concluded that “rickets is a deficiency diseasewhich develops in consequence of the absence of some ac-cessory food factor or factors.” Vitamin A had been discov-ered shortly before, and it was subsequently speculated thatit may represent the factor promoting bone formation(733). This hypothesis was later rebutted by American bio-chemist McCollum who concluded that “a substance whichis distinct from fat-soluble [vitamin] A” must be responsiblefor preventing rickets (727). Furthermore, he stated that hisexperiments “demonstrate the existence of a fourth Vita-min [vitamin D] whose specific property [. . .] is to regulatethe metabolism of bones” (727). In parallel to the unravel-ing of the dietary component of rickets, scientists were in-dependently discovering the importance of sunlight for dis-ease prevention. The Polish pediatrician Raczynski was mostlikely the first to demonstrate evidence for this hypothesis ex-perimentally (881). He kept one dog pup in the shade while alittermate was kept in the sunlight. Both dogs were breastfedby their mother. After 6 wk, the bones of the dog that was keptin the shade contained 36% less calcium (881). These obser-vations were followed up by the German pediatrician Huld-schinsky, who healed rachitic children after exposing themintermittently for 2 months to the UV rays generated by amercury vapor quartz lamp (504). Hess and Unger (447) rep-



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licated these findings by using sunlight treatment. Hess (446)later summed up the impact of these revolutionary observa-tions, which now seem intuitive to us: “We have known that agrowing plant cannot thrive in the dark, but have failed torealize that the same laws apply to growing animals.”

It was later recognized that it was sufficient to irradiate thefood administered to animals rather than the whole animalto prevent development of or heal rickets. Thus initially“inert” dietary substances with no antirachitic propertiescould be activated by UV light (385, 448–450, 505, 1037).It was concluded that irradiation caused the conversion of abiological precursor to an active form and that the samemechanism was physiologically taking place in the skin.Initially, it was speculated that cholesterol may serve as this

pro-vitamin. It was Windaus and Hess (in collaborationwith Rosenheim) who were the first to uncover its exactmolecular identity. They stated: “We conclude from ourexperiments with complete certainty that ergosterol [. . .]represents the anti-rachitic provitamin (1166). In 1928,Windaus received the Nobel Prize in Chemistry for “theservices rendered through his research into the constitutionof the sterols and their connection with the vitamins.” Theirradiation product of ergosterol was later purified andnamed vitamin D2 (ergocalciferol) (43, 896, 1164, 1167).Although these findings solved the question as to how UVirradiation generates Vitamin D2 from ergosterol, which hasantirachitic properties if ingested, the molecular mechanismsunderlying the antirachitic effects of cutaneous sunlight expo-sure still remained obscure. Since ergosterol is an exclusive

Parathyroid glands

Bone

Intestine

Kidney

Stimulates PTH secretion

Increases renal calcium absorption

Stimulates 1,25(OH)2-D synthesis

Increases renalcalcium absorption

Increases bone resorption

Kidney

Stimulates 1,2

eases renal

Hypocalcemia

PTH

Increases intestinalcalcium absorption

1,25(OH)2-D

FIGURE 4. Endocrine regulation of serum calcium levels. Calcium homeostasis is mainly regulated by PTH and1,25(OH)2-vitamin D. Both hormones act at their respective target organs to increase serum calcium levels.



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component of yeast and fungal membranes, a different precur-sor substance had to exist in animal skin. Again, it wasWindaus and colleagues who identified 7-dehydrocholesterolas the provitamin in porcine skin, which is converted to vita-min D3 (cholecalciferol) under irradiation (1165). After thesediscoveries, industrially produced vitamin D has rapidly beenused in medical applications and as a food fortification. To-

day, the main portion of dietary vitamin D ingestion in theUnited States stems from fortified dairy products (150).

2. Intestinal vitamin D absorption

To exert its antirachitic effects, dietary vitamin D has to beabsorbed into our circulation. Early everted gut sack exper-

7-dehydrocholesterol

Previtamin D

Isomerization

ER:CYP2R1

Mitochondria: CYP27A1

Mitochondria: CYP27B1

Calcium

PTH

Intestinal uptake

Glomerularfiltration

Vitamin D

Vitamin D

Vitamin D

25(OH)-vitamin D

25(OH)-vitamin D

1,25(OH)2-vitamin D

DBP

UV-B (290-315nm)

1,25(OH)2-vitamin D

Megalin

Keratinocyte

Hepatocyte

25(OH)-vitamin D DBP

25(OH)-vitamin D DBP

Vitamin D

Dietary vitamin D

DBP

Target organs

Proximal tubule

FIGURE 5. Vitamin D metabolism. Vitamin D can either be synthesized in the skin or absorbed from our diet.It is then transported to the liver where it undergoes 25-hydroxylation by one of two hepatic enzymes (CYP27A1or CYP2R1). During transport through the circulation, vitamin D is bound to a carrier protein (DBP). The25(OH)-vitamin-D-DBP complex passes the glomerular filter and is scavenged from the primary urine by theapical megalin receptor of the proximal tubule. Here, 25(OH)-vitamin D is converted to the active vitamin Dmetabolite 1,25(OH)2-vitamin D. DBP, vitamin D binding protein.



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iments demonstrated that this process has linear, nonsatu-rable, and passive kinetics, suggesting that no specific car-rier mechanism for vitamin D is in place (484). These ob-servations were later replicated in in vivo models (483).Absorption is highest in the proximal and mid small intes-tine (484). Since vitamin D is fat soluble, its absorptionmechanism is similar to that of dietary lipids. In an aqueousmedia, vitamin D aggregates in micelle-like structures(735). Its absorption is aided by the secretion of bile acids,which is underscored by the observation that patients suf-fering from cholestasis can present with vitamin D defi-ciency and develop bone disease, such as osteomalacia orosteoporosis (245, 445, 563, 721, 894, 953, 1018, 1080).Apart from bile salts, formation of mixed micelles contain-ing monoglycerides and free fatty acids represents anotherfactor that aids in vitamin D absorption (884, 1082; con-tested by Ref. 483). These substances increase micelle size,which promotes the solubilization of vitamin D, therebyincreasing uptake (884). Clinically, pancreatic insuffi-ciency, causing an impairment of triglyceride breakdownthrough insufficient lipase secretion, leads to decreased vi-tamin D absorption (1080, 1127). This is a particular prob-lem in cystic fibrosis patients, who often develop pancreaticand concomitant vitamin D insufficiencies (33, 230, 625,678, 924).

Following uptake into the enterocyte, vitamin D is packedinto chylomicrons and secreted into the lymph (288, 953). Ithas been demonstrated that after intestinal administrationof radioactive labeled vitamin D3, up to 90% of the recov-ered radioactive tracer was associated with the chylomicronfraction of the collected intestinal lymph (288). Further-more, patients suffering from the autosomal recessive chy-lomicron retention disease (Anderson disease; OMIM246700), which causes impairment of chylomicron process-ing and secretion in the enterocyte, can present with insuf-ficient levels of fat-soluble vitamins, such as vitamins D andE (535). The chylomicron remnants, which include vitaminD, are then scavenged by the liver after the lymph has en-tered the circulation through the thoracic duct (287, 407).However, it has been demonstrated in vitro and in vivo inhepatectomized and normal rats that vitamin D can directlytransfer from chylomicrons to a vitamin D binding protein(DBP; see sect. IVA5) in the blood plasma (286, 288). It hastherefore been suggested that at least a fraction of hepaticvitamin D uptake is mediated through DBP rather thanchylomicrons (286; contested by Ref. 407). In the liver,vitamin D is then hydroxylated at its 25 position to25(OH)-vitamin D. The metabolism of vitamin D will bethe subject of a later section.

The intestinal absorption of 25(OH)-vitamin D has beeninvestigated by many groups, with the aim of optimizingvitamin D administration in a therapeutic context by by-passing the first metabolic step in the liver (219, 245, 287,445, 645, 721, 1018, 1019, 1127). In general, enteric up-

take of 25(OH)-vitamin D is more effective than that ofvitamin D, which is partially attributable to a comparablylower dependency on bile acid secretion (219, 245, 287,645, 1018, 1019). The observation that patients withcholestasis still absorb 25(OH)-vitamin D effectively,whereas vitamin D absorption is impaired, corroboratesthis hypothesis (1018). There is some controversy with re-gard to the transport of 25(OH)-vitamin D following itsabsorption (287, 701, 1019). It has been argued that it maybe transported predominantly in the protein fraction of thelymph (287), i.e., not in chylomicrons, or that it is directlyabsorbed into the portal blood (701, 1019).

3. Cutaneous vitamin D synthesis

Cutaneous synthesis is our second source of vitamin D.Cutaneous production depends on exposure to UVB (290–315 nm) light (FIGURE 5). The UVB photons convert 7-de-hydrocholesterol, which is located in the plasma membraneof keratinocytes, to previtamin D3 (1117–1120). This de-pendency on sunlight causes a seasonal variation in vitaminD3 production, with synthesis being low during the wintermonths when the radiation angle of the sun flattens (187,478, 538, 1035, 1156). In consequence, changes in latituderesult in similar variability in production. Further factorsthat decrease production include pigmentation of the skinand application of sunscreen (209, 723), whereas an in-crease in altitude promotes production (478). Followingconversion from 7-dehydrocholesterol, previtamin D3

isomerizes to vitamin D3 (479). The isomerization processis temperature dependent and fairly slow (479, 1117). It hasbeen calculated that the half-life for the formation of vita-min D3 is �2.5 h (1085). Interestingly, in vitro experimentsconducted in isotropic medium demonstrated that theisomerization rate was 10 times slower than in in vivo ex-periments (1085). This was later attributed to the fact thatamphiphatic interactions with phospholipids of the cellmembrane stabilize the previtamin D3 conformer whichthen isomerizes to vitamin D3 (1086). The cellular microen-vironment of the reaction thus greatly optimizes the isomer-ization to vitamin D3. After its synthesis, vitamin D3 isbound to DBP and carried though the bloodstream to itstarget organs. Observations made in patients indicate thatthe vitamin D3 plasma levels peak �2 days after sunlightexposure, which is due to the slow isomerization rate in theskin (7).

4. Vitamin D metabolism and its regulation

Irrespective of the source (endogenous or exogenous), vita-min D is metabolized in the liver to 25(OH)-vitamin D(FIGURE 5). Evidence for the existence of biologically activevitamin D metabolites emerged in the 1960s (686, 799).25(OH)-vitamin D was identified by means of injecting ratswith radiolabeled vitamin D and subsequent silicic acid col-umn chromatography of lipid extracts from serum and var-



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ious tissues (686). Chromatography revealed the presenceof a vitamin D metabolite in serum, liver, bone, and feces inrats and in human serum (256, 686). Furthermore, it wasshown that the vitamin D metabolite was biologically activeby reverting rickets in diseased animals and by increasingintestinal calcium transport and bone metabolism (686,752). The metabolite was characterized as 25(OH)-vitaminD and subsequently successfully synthesized (111, 112). Itwas soon recognized that hepatectomized rats were not ef-fectively converting vitamin D to 25(OH)-vitamin D, sug-gesting that the liver was the main organ responsible for25-hydroxylation (864, 865). The ability of the liver tometabolize vitamin D was also confirmed in perfused liversand tissue homogenates (490). Historically, a long contro-versy existed with regard to the subcellular localization ofthe enzyme that hydroxylates vitamin D (25-hydroxylase)in the liver, as enzyme activity was observed in both mito-chondrial and microsomal fractions of liver homogenates(93, 102, 103, 258, 696, 1053). Extensive investigationsindicated that both the microsomal and mitochondrial 25-hydroxylase are members of the cytochrome P-450 family(102–104, 696, 835). Both fractions demonstrated distinctenzymatic kinetics. The mitochondrial enzyme was charac-terized as a low-affinity high-capacity enzyme, whereas themicrosomal fraction displayed high-affinity low-capacitycharacteristics (103, 104, 356, 696).

The mitochondrial 25-hydroxylase (CYP27A1) was firstpurified to homogeneity from rabbit liver mitochondria(235). It was demonstrated that this cytochrome P-450 wasnot specific to vitamin D and could also hydroxylate othersubstrates, most notably cholesterol (27-hydroxylation),which represents an important step in the formation of bileacids (235). In retrospect, CYP27A1 had been purified 4years earlier; however, only the 27-hydroxylation of cho-lesterol had been investigated and its effects on vitamin Dhad remained obscure (1162). Subsequently, the enzyme’scDNA was cloned from rabbit, rat, and human, and its dualrole in vitamin D and steroid conversion was confirmed (26,149, 1048, 1103). The full gene structure was identified afew years later (646). Although no crystal structure of theenzyme is available to us, a homology model based on otherCYP family members has been proposed (874). In the liver,CYP27A1 is expressed on the mRNA level in hepatocytes,endothelial, stellate, and Kupffer cells (1078). Furthermore,CYP27A1 expression was confirmed in a variety of othertissues, including duodenum, adrenal glands, kidney, lung,vascular endothelium, brain, retina, skin, muscle, and os-teoblasts; however, their potential contribution to vitaminD metabolism remains unclear (26, 51, 135, 184, 370, 383,508, 640, 898, 979). Interestingly, an extrahepatic conver-sion of vitamin D had been suggested previously to occur inthe kidney and the intestine (1097). Marked differences inCYP27A1 activity can be observed between males and fe-males. For example, CYP27A1 enzyme activity and mRNAexpression were demonstrated to be increased in female rats

(933, 1078). Higher expression in females was also con-firmed in biopsy samples from human subjects (370). Aregulation via sex hormones may underlie this phenome-non, as injection of estradiol was shown to induceCYP27A1 activity (933). Interestingly, seasonal variationsin expression were also observed, which may represent aconfounding factor for decreased 25(OH)-vitamin D levelsduring the winter months (370).

It should be noted that CYP27A1 can also hydroxylatevitamin D3 at other positions (402, 950). These includepositions 27 and 26; however, the ratio for 25-:27-:26-hydroxylation has been estimated to be only 100:15:3,which demonstrates that 25-hydroxylation of vitamin D3 isthe most essential reaction catalyzed by the enzyme (950).More importantly, CYP27A1 can also use its own product25(OH)-vitamin D as a substrate to further act as a 1�-hydroxylase and produce the hormonally active form ofvitamin D, namely, 1,25(OH)2-vitamin D (50, 51, 950). Aswill be discussed later, this reaction is normally catalyzed inthe kidney by another CYP family member (CYP27B1). Atthe moment, it is not clear what the physiological signifi-cance of 1�-hydroxylation by CYP27A1 is.

Mutations in CYP27A1 cause the autosomal recessive dis-order cerebrotendinous xanthomatosis (CTX; OMIM213700). The disease was first described in 1937 and ischaracterized by cholestanol deposits that are most promi-nent in tendons, especially the Achilles tendon, the brain,and the lung. Patients present with progressive neurologicdefects, atherosclerosis, and cataracts and commonly sufferfrom diarrhea. The inadequate bile acid synthesis was firstnoted in 1974 by Setoguchi et al. (993). Shortly after thecDNA of CYP27A1 was cloned, it was demonstrated thatmutations of this enzyme were responsible for CTX (148).In agreement with the dual role of CYP27A1, CTX patientsalso suffer from osteoporosis, low 25(OH)-vitamin D lev-els, and impaired intestinal calcium absorption (83, 320).Three CYP27A1 mutations that are known to cause CTXand still lead to protein expression were recreated in vitro,and enzymatic activity was assayed. Depending on the ex-pression system, these mutants showed lower or higher 25-hydroxylation activity than the wt enzyme, which led theauthors to questions the enzyme’s role in vitamin D metab-olism (403). It should be considered that 1) many more(�38) mutations underlying CTX exist, 2) by far not allpatients exhibit disturbances in bone or vitamin D homeo-stasis, and 3) the investigated mutant enzymes may onlycause disturbances in cholesterol, rather than vitamin Dmetabolism (83, 320, 403). In the light of these limitations,it should be questioned whether CTX represents an aptmodel system to evaluate CYP27A1 in the context of vita-min D metabolism.

With the introduction of novel genetic tools, a cyp27a1(�/�) mouse was created in 1998 (918). However, the phe-



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notype of CTX could not be reproduced, albeit fecal bileacid content was markedly decreased (918). Rather surpris-ingly, serum 25(OH)-vitamin D levels were increased in(�/�) animals, which either pointed to compensatory up-regulation of other, maybe microsomal, enzymes or a non-involvement of cyp27a1 in vitamin D metabolism in themouse (918). The (�/�) mouse model was later character-ized in more detail, but no new conclusions with regard tovitamin D were drawn (902).

Another caveat that needs to be considered when assessingthe physiological importance of CYP27A1 for vitamin Dmetabolism is that the enzyme cannot 25-hydroxylate di-etary vitamin D2 (402). This observation underlines thenecessity for another 25-hydroxylase which physiologicallymetabolizes vitamin D2. The microsomal 25-hydroxylasewas a clear candidate for this process; however, it has onlyrecently received more extensive scientific attention.

The molecular identity of the microsomal 25-hydroxylasewas unclear, until it was unmasked by Cheng et al. (184) in2003 as CYP2R1. CYP2R1 is expressed on the mRNA levelin a plethora of tissues, but most prominently in the liverand testes (184). As illustrated by a recent investigation, thetestes may play a role in calcium homeostasis. Patients withtesticulopathy (and concomitant lower CYP2R1 expres-sion) were shown to have decreased 25(OH)-vitamin D lev-els and osteoporosis (333). The protein’s crystal structurehas recently been resolved in complex with vitamin D3

(1045). Unlike CYP27A1, CYP2R1 has been shown to 25-hydroxylate both vitamin D2 and vitamin D3, which may bea solution to the enigma of vitamin D2 metabolism (184).The physiological relevance of CYP2R1 was underscoredby the characterization of a patient who presented with low25(OH)-vitamin D levels and was shown to have a transi-tion mutation in the CYP2R1 gene (183). Furthermore, aremarkable large-scale study has recently tried to establish acorrelation between 25(OH)-vitamin D status and the ge-notype of 33,996 individuals. It was found that lower25(OH)-vitamin D levels correlated with variants in theCYP2R1 gene (1146) (a smaller study came to similar con-clusions, Ref. 139). This represents an outstanding finding,as the influence of CYP27A1 mutations on 25(OH)-vitaminD production is far less conclusive. Given the dispropor-tionate quantity of scientific data, it is at this moment dif-ficult to evaluate the relative contribution of each 25-hy-droxylase to vitamin D metabolism, but more and moreevidence for the importance of CYP2R1 is accumulating.

The regulation of 25-hydroxylation has been the subject ofcontroversy in the past, which is partially due to the factthat multiple enzymes may metabolize vitamin D, and thatit was challenging to experimentally discriminate betweenthese enzyme entities. Thus evidence which supports (91,92, 741, 1078) and questions (258, 1097) the existence of25-hydroxylase regulation can be found. A detailed analysis

of these observations is beyond the scope of this review, butthe most recent investigation should be considered in moredetail, given the advances in our experimental repertoire: ithas been demonstrated in the rat that 1,25(OH)2-vitamin Dcan downregulate hepatic CYP27A1 transcription with aconcomitant reduction in enzyme activity (1078). Theseobservations strongly corroborate the hypothesis that the25-hydroxylation step is subjected to negative-feedbackregulation. The exact mechanism of this regulatory mecha-nism remains elusive, especially since the CYP27A1 gene isnot under control of a VDRE (369, 988, 1078).

Following its synthesis, 25(OH)-vitamin D binds to DBPand is transported to the kidney, where it undergoes furtherconversion to 1,25(OH)2-vitamin D, the hormonally activeform of vitamin D (FIGURE 5). Historically, 1,25(OH)2-vitamin D was first identified in the nuclei of intestinal cellsas an uncharacterized vitamin D metabolite (429, 634). Thebiological activity of the metabolite was determined to bemuch higher than that of vitamin D, and it was eventuallyisolated and identified as 1,25(OH)2-vitamin D (480, 586,633, 800). 1,25(OH)2-vitamin D is up to 10 times morepotent than vitamin D. The importance of the kidney for thesynthesis of 1,25(OH)2-vitamin D was soon discovered, asnephrectomized rats were neither able to convert 25(OH)-vitamin D, nor absorb calcium effectively (339, 458).Briefly thereafter, patients with chronic renal insufficiencywere shown to lack the capability to metabolize 25(OH)-vitamin D, which further established the role of the kidneyas the major conversion site (724). The impairment of1,25(OH)2-vitamin D synthesis in the course of chronicrenal deficiency is a cofactor in the development of renalosteodystrophy, a bone mineralization deficiency due toderanged mineral balance. Of note, the kidney is not theexclusive site of CYP27B1 expression. 1�-Hydroxylase hasbeen detected in a variety of other tissues, including theplacenta, decidua, skin, brain, vascular endothelium, pan-creas, colon, but also in monocytes and dendritic cells (451–453, 603, 1204–1206). The role of extrarenal 1,25(OH)2-vitamin D synthesis is not entirely clear. Beyond modulatingcalcium homeostasis, vitamin D has been shown to have immu-nomodulatory and antiproliferative effects (see sect. IVA6).Given these observations, it has been speculated that extrare-nal local 1,25(OH)2-vitamin D production and paracrine se-cretion may represent important factors for the maintenanceof the “barrier function” in these tissues (451, 453).

In the kidney, 1�-hydroxylation of 25(OH)-vitamin Dtakes place in the inner mitochondrial membrane of theepithelial cells of the proximal tubule (393).

The cellular uptake mechanism of 25(OH)-vitamin D by thetubule cells will be subject of a later section. The 1�-hy-droxylase responsible for the conversion is also a member ofthe cytochrome P-450 family (CYP27B1) and was firstcloned in 1997 by St-Arnaud et al. from rat cDNA (1034).



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The human homolog was cloned shortly thereafter (349,748). Interestingly, mapping of the CYP27B1 gene revealedthat the locus was identical to the gene locus of the auto-somal recessive disorder pseudo vitamin D deficiency rick-ets, type 1A (PDDR1A, OMIM 264700) (1034). PDDR ischaracterized by low serum calcium, secondary hyperpara-thyroidism, and low 1,25(OH)2-vitamin D levels (869) (ofnote, the original article wrongly suggests an autosomaldominant inheritance pattern). Patients can exhibit ricketsor osteomalacia due to increased mobilization of calcium.The gene locus of PDDR1A had been mapped by linkageanalysis; however, before the cloning of CYP27B1, it wasnot clear whether a defect in the enzyme itself or distur-bances in its regulation were responsible for the disease(617, 618, 1223). The clear-cut phenotype of PPDR1A ef-fectively illustrates the pivotal role of CYP27B1 and thelack of redundancy at this essential step of vitamin D me-tabolism. The characteristic features of PDDR1A can essen-tially be emulated if cyp27b1 is knocked out in a mousemodel (238, 822). The serum calcium levels and the second-ary hyperparathyroidism can be normalized if these animalsare fed a high-calcium, phosphorus, lactose diet, albeit bonegrowth remains impaired (239).

The activity of CYP27B1 is subjected to tight hormonalregulation. The key regulator of 1,25(OH)2-vitamin D syn-thesis is PTH, which is secreted by the parathyroid glands inresponse to low serum calcium concentrations in an effortto increase calcium uptake and release bone calcium intothe circulation. The regulation of calcium homeostasis bythe parathyroid glands and the CaSR will be covered insubsequent sections (see sect. IV, B and D). Apart from thisstimulatory input, CYP27B1 is under negative control by itsown product. 1,25(OH)2-vitamin D represses CYP27B1 ona transcriptional level (116, 125, 443, 591, 764). Althoughstudies in (�/�) animals suggest that the VDR is essentialfor autoinhibition to take place, the promoter region ofCYP27B1 does not include a canonical VDRE (125, 591,764). It is thus most likely that the transcriptional regu-lation through 1,25(OH)2-vitamin D is indirect (125,591). Alternatively, so-called E-box-type elements wererecently proposed to act as negative VDREs (575). More-over, CYP27B1 can be directly regulated by the localcalcium concentrations. High extracellular calcium inhib-its 1,25(OH)2-vitamin D synthesis, whereas low calciumconcentrations induce its production (109). It has been pro-posed that changes in calcium modulate VDR expressionand thereby the sensitivity of cell to the local negative feed-back by 1,25(OH)2-vitamin D (702). Some evidence sug-gests that the CaSR mediates the regulatory effects of cal-cium on CYP27B1 activity (FIGURE 8) (702). Other factorsthat regulate CYP27B1 activity include fibroblast growthfactor 23 (FGF23), calcitonin, prolactin, sex steroids (atleast in avian species), and phosphate (716, 913, 1067,1068, 1217).

Apart from regulating the synthesis of the vitamin Dmetabolites, our body also tightly controls their degrada-tion. The first step of vitamin D catabolism is 24-hy-droxylation, which is carried out by the mitochondrialenzyme CYP24A1. The primary site for vitamin D catab-olism is the kidney, but CYP24A1 is also strongly expressedin other extrarenal tissues, such as the intestine, osteoblasts,keratinocytes, prostate, placenta, brain, and heart (11, 181,796). CYP24A1 can hydroxylate both 25(OH)-vitamin Dand 1,25(OH)2-vitamin D, thereby creating 24,25(OH)2-vitamin D and 1,24,25(OH)3-vitamin D, respectively (14).24-Hydroxylation is followed by a series of oxidation/re-duction reactions which finally yield the excretable productcalcitroic acid (703, 893). At least in the proximal tubule ofthe kidney, the regulation of CYP24A1 is reciprocal to thatof CYP27B1. 1,25(OH)2-vitamin D upregulates CYP24A1,thereby stimulating its own breakdown, whereas PTH inhibitsCYP24A1 (37, 180, 812, 1069, 1222). While 1,25(OH)2-vi-tamin D increases the transcription of CYP24A1 (the gene hastwo upstream VDREs), PTH exerts its inhibitory effects bydecreasing CYP24A1 mRNA stability (1222). In osteoblasticand distal convoluted tubule cell lines, PTH has synergisticeffects with 1,25(OH)2-vitamin D in inducing CYP24A1 (37,1189). In analogy to CYP27B1, CYP24A1 is further regulatedby FGF23, calcitonin, and phosphate (361, 513, 1175).

5. Vitamin D transport and cellular uptake

The hypothesis that vitamin D may be bound to a carriersubstance in serum was first expressed in the late 1950s,when it was demonstrated that the alpha fraction of humanserum had high anti-rachitic properties (1079). It was latershown that DBP is identical to the group specific component(Gc) protein, which had been characterized independentlyby Hirschfeld and colleagues around the same time (236,464, 465). DBP (�58 kDa; 458 amino acids) is synthesizedin the liver and is closely related to albumin and �-fetopro-tein, which are all derived from the same ancestral gene(221, 875, 1158, 1187, 1188). The crystal structure of DBPhas been resolved at a resolution of 2.3 Å in complex with25(OH)-vitamin D (1121). DBP can bind vitamin D and allof its metabolites (408). There is, however, a difference inthe relative affinity for the vitamin D steroids, with theaffinity for 25(OH)-vitamin D being highest (Kd �10�8 M),followed by 1,25(OH)2-vitamin D and vitamin D (Kd

�10�7 M) (408). In humans, vitamin D2 and D3 metabo-lites are bound with equal affinity to DBP (406). Given itslong plasma half-life, 25(OH)-vitamin D is also measuredas the primary clinical parameter to assess the vitamin Dstatus of patients. Although the plasma concentration ofDBP is �4–8 �M, only �5% of the binding sites are occu-pied by vitamin D sterols (408, 409). DBP has a very fastturnover rate. It has been estimated that up to 28% of DBPare replaced every day (557). This turnover entails a highdemand for synthesis output by the liver. In consequence,patients with liver disease demonstrate lower DBP and totalvitamin D levels than healthy subjects (97, 409). This rela-



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tionship is reversed during pregnancy, when the levels ofDBP and vitamin D sterols are increased (97, 409, 609, 685,892). Of note, DBP is not the exclusive carrier substance forvitamin D sterols. Albumin and lipoproteins were shown totransport a fraction (�15%) of vitamin D (1015).

Given its multitude of functions, DBP has been regarded as anessential protein. Indeed, an analysis of over 80,000 humanserum samples showed that DBP was present in all of them,which led to the hypothesis that deleterious mutations ofDBP were lethal (213). Rather surprisingly, DBP (�/�)mice thrive well with growth curves identical to theirlittermates, although their 25(OH)-vitamin D and1,25(OH)2-vitamin D levels (total) are severely decreased(937). If challenged with a low vitamin D diet, the DBP(�/�) animals develop secondary hyperparathyroidism,leading to defects in bone mineralization (937). It is un-clear, however, to what extent albumin and lipoproteinscompensate for the loss of the primary vitamin D carriersubstance.

Not much is known about the cellular uptake of vitamin D.As all other steroids, free vitamin D can passively diffusethrough the plasma membrane because of its lipohilic na-ture. However, due to the high concentration of DBP and itsaffinity towards vitamin D ligands, only a fraction of vita-min D circulates in the free form. For example, 0.003% of25(OH)-vitamin D is transported as an unbound sterol inserum, which raises the question whether passive diffusionrepresents a sufficient uptake pathway (98). At least25(OH)-vitamin D has been proposed to be delivered tothe proximal kidney tubule for further conversion to1,25(OH)2-vitamin D by a different and remarkable mech-anism. Rather than diffusing passively, it has been proposedthat the 25(OH)-vitamin D-DBP complex passes the glo-merular filter and is endocytosed by the epithelial cell of theproximal tubule (FIGURE 5) (805). The endocytotic processis mediated by megalin (aka gp330), which is a multifunc-tional clearance receptor on the luminal membrane. Micelacking functional megalin were shown to lose DBP andvitamin D in the urine and develop vitamin D deficiency(805). A kidney specific megalin (�/�) animal was recentlycreated, and the observations made in the global (�/�)animal, which had a very low perinatal survival rate of 2%,could essentially be replicated (643). Furthermore, defectsin cubulin, a membrane protein that colocalizes with mega-lin, cause a similar phenotype (806). The experimental dataare further supported by clinical observations made in pa-tients suffering from Fanconi syndrome. Fanconi syn-drome is a global reabsorption deficiency of the proximaltubule, which can develop as a result of heavy metalpoisoning and/or drugs or may have inherited causes.These patients were shown to lose DBP in their urine,which may reflect the inability of the tubule cell to endo-cytose DBP (805, 1076, 1100). A similar endocytoticuptake mechanism has been proposed for mammary

cells, which can also convert 25(OH)-vitamin D to1.25(OH)2-vitamin D(926).

6. Cellular effects of vitamin D

The cellular effects of 1,25(OH)2-vitamin D can be catego-rized into two major pathways, which are defined by theirrespective speed of onset: 1) slow genomic responses and2) rapid nongenomic responses. Both pathways requirebinding of 1,25(OH)2-vitamin D to its intracellular recep-tor, the VDR (503, 787). The VDR (NR1I1, nuclear recep-tor subfamily 1, group I, member 1) belongs to the super-family of nuclear receptors, which amongst others alsoincludes the estrogen, testosterone, or glucocorticoid recep-tors. VDR was first identified in the late 1960s in the chro-matin fraction of chick intestinal mucosa, where1,25(OH)2-vitamin D increases the rate of intestinal cal-cium uptake (430). The 427-amino acid protein (molecularmass 48.3 kDa) was cloned in 1988 by Baker et al. (59). Thecrystal structure of the VDR is available to us with1,25(OH)2-vitamin D bound to the receptor’s ligand bind-ing domain (914).

A) GENOMIC EFFECTS. Following ligand binding, nuclear recep-tors typically act as transcription factors and induce or re-press the transcription of certain target genes. In the case ofVDR, 1,25(OH)2-vitamin D binds to the receptor, whichsubsequently heterodimerizes with the retinoid X receptor(RXR) (FIGURE 3) (1027, 1083). The VDR-RXR complexthen interacts with a VDRE in the 5= promoter region of theregulated gene resulting in transactivation. Alternatively, ithas been proposed that VDR can bind to the VDRE beforeligand binding occurs (920).

I) Intestine. The intestine is one of the primary target sites of1,25(OH)2-vitamin D. 1,25(OH)2-vitamin D upregulatesthe expression of intestinal TRPV6, calbindin-D 9k, andPMCA, which are canonically regarded to mediate the pro-cess of transcellular calcium absorption (FIGURE 3) (see sect.IIIA). Furthermore, 1,25(OH)2-vitamin D may modulatecalcium uptake through the paracellular route (see sect.IIIB). By increasing the amount of absorbed calcium,1,25(OH)2-vitamin D directly elevates serum calcium lev-els. This represents one of the final links in the regulatorychain of calcium homeostasis which starts with sensing oflow calcium levels by the parathyroid gland and ends withincreased synthesis of 1,25(OH)2-vitamin D by the kidney.The significance of 1,25(OH)2-vitamin D for intestinal cal-cium absorption is illustrated by 1,25(OH)2-vitamin D-de-ficient patients, which absorb up to 80% less calcium fromtheir meal compared with healthy individuals (996). Al-though similar results were obtained in animal models,probably one of the more illustrative observations has beenmade in VDR-deficient animals (825, 1110). Deletion ofVDR correlates with a massively impaired capacity to ab-sorb intestinal calcium resulting in low plasma calcium lev-els and hyperparathyroidism (1110). This phenotype is re-



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versible by intestine-specific expression of VDR on a VDR(�/�) background (712, 1181). Thus animals that lack theVDR, except in the intestine, present with normal serumcalcium and PTH levels (1181). In conclusion, it is unques-tionable that 1,25(OH)2-vitamin D and its receptor are thekey regulators of intestinal calcium absorption.

II) Bone. Some evidence exists that 1,25(OH)2-vitamin Dmay have direct influences on the formation of bone (FIG-URE 4). The VDR is expressed in osteoblasts, osteoclasts,and chondrocytes (115, 533, 623, 730). Most of the directeffects of 1,25(OH)2-vitamin D are thought to be mediatedby osteoblasts. 1,25(OH)2-vitamin D has been shown topromote osteoblast differentiation from mesenchymal stemcells and to regulate the synthesis of various osteoblast pro-teins, such as osteocalcin, alkaline phosphatase, collagen I,osteopontin, and RANKL (45, 78, 79, 665, 709, 804, 870,925). In general, however, the effects of 1,25(OH)2-vitaminD on osteoblast maturation and protein synthesis are verypleiotropic and depended on the duration of the exposureand the differentiation stage at which the osteoblast is ex-posed (817). Effects of 1,25(OH)2-vitamin D on the miner-alization of bone have also been reported. For example, anincrease in the mineralization of extracellular matrix wasobserved following concomitant 1,25(OH)2-vitamin D andvitamin K exposure (599, 745). Yet, the overall direct influ-ence of 1,25(OH)2-vitamin D on bone metabolism is some-what obscure. This is illustrated by VDR-deficient animals.Naturally, these animals develop rickets and osteomalaciadue to impaired intestinal calcium absorption. If the ani-mals are, however, maintained normocalcemic, the skeletalphenotype is completely rescued (21, 661). Although, theseobservations question the importance of 1,25(OH)2-vita-min D as a direct regulator of bone metabolism, a subse-quent investigation by Panda et al. (821) came to a differentconclusion. The authors reported that osteoblast numbers,mineral apposition rates, and overall bone volume werereduced in normocalcemic (rescue diet) cyp27b1/VDR(double �/�) animals, suggesting that the 1,25(OH)2-vita-min D system is necessary for intact bone formation (821).The reason for these discrepant findings is generally un-clear; however, differences in the length of exposure to therescue diet have been put forward as a possible cause (821).For a more detailed review of the effects of vitamin D onbone, please refer to References 1033, 1114.

III) Kidney. The kidney is not only the major site of1,25(OH)2-vitamin D synthesis (see above), but also repre-sents a vitamin D target organ. The kidney acts as keyregulator of calcium homeostasis by changing the amountof calcium that is reabsorbed from the primary urine. Themajority of the calcium that is filtered through the glomer-ulus is reabsorbed in the proximal tubule through the para-cellular space, with the amount of absorbed calcium grad-ually decreasing along the nephron. Fine regulation of cal-cium absorption occurs in the distal tubule and collecting

duct. The mechanism by which the distal tubule conductstranscellular calcium absorption is highly analogous to theproximal intestine. The epithelial cells of the distal tubuleexpress TRPV5 (the “sister” channel of TRPV6) as the api-cal calcium entry channel, calbindin-D 28k and NCX1 andPMCA1b as basolateral calcium extruders (203, 474, 610,837, 922). In analogy to the intestine, 1,25(OH)2-vitamin Dupregulates the majority of these proteins in an effort toincrease renal calcium reabsorption (203, 474, 610, 837,922).

B) NONGENOMIC EFFECTS. In contrast to the genomic effects of1,25(OH)2-vitamin D, which have been known for decades,the rapid cellular responses have only recently receivedmore scientific attention. While the transcriptional events of1,25(OH)2-vitamin D take place on a time scale of a fewhours to days, the rapid nongenomic responses occur withinminutes of exposure. One of the first evidences, which inretrospect can be attributed to a nongenomic response, wasthe observation made in 1941 by Selye that intraperitonealinjection of steroids had an anesthetic effect (990). Interest-ingly, the rapid responses to 1,25(OH)2-vitamin D also re-quire the presence of the VDR, as these responses cannot beelicited in VDR deficient animals (503, 787). It should benoted that other proteins such as 1,25(OH)2-vitamin-D-MARRS have also been suggested as candidates for a mem-brane associated 1,25(OH)2-vitamin D receptor (782). At-tempts at identifying the subcellular localization of theVDR in the rapid response context have yielded that VDR isalso present in plasma membrane invaginations, the so-called caveolae (503, 802). These VDR-containing mem-brane microdomains have been identified in multiple tis-sues, including the intestine, kidney, and lung, and are iden-tified by coexpression of caveolin-1, which is used as amarker protein for caveolae (503, 802). Functionally, notmany rapid response effects of 1,25(OH)2-vitamin D havebeen characterized. For example, it has been demonstratedthat 1,25(OH)2-vitamin D can influence ion channel gatingin osteoblasts, modulate the contraction of cardiomyocytes,lead to insulin secretion in pancreatic �-cells via elevatingintracellular calcium, and cause photoprotection in keratin-ocytes (273, 540, 1088, 1200, 1216). In the intestine, thephenomenon of transcaltachia (see sect. IIIC) has been at-tributed to rapid actions of 1,25(OH)2-vitamin D (801).

In addition to 1,25(OH)2-vitamin D, the VDR also bindsthe secondary bile acid lithocholic acid (704). Secondarybile acids are bile acids that have been metabolized by theintestinal gut flora. Lithocholic acid is toxic and has beenimplicated to play a role in intestinal carcinogenesis (601).It has been suggested that the VDR may serve as a second-ary bile acid sensor and induce lithocholic acid breakdownthrough CYP3A activation (537). The noncanonical VDRstimulation by lithocholic acid may thus serve as an auto-protective mechanism (704). Apart from inducing CYP3A,lithocholic acid has been demonstrated to increase expres-



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sion of TRPV6 in intestinal cell lines, which corroborates itsrole as a physiological VDR agonist (517).

We are far from understanding the full spectrum of theeffects of 1,25(OH)2-vitamin D, and unfortunately, thescope of this review does not allow a detailed analysis ofthese processes. In general, our knowledge concerning thephysiological role of 1,25(OH)2-vitamin D is massively ex-panding beyond the horizon of calcium homeostasis. Evi-dence is accumulating that 1,25(OH)2-vitamin D can influ-ence the renin-angiotensin-aldosterone system and maythereby act as a regulator of blood pressure (660, 662,1219). Furthermore, low vitamin D status is associated withan increased incidence of colorectal, ovarian, and breastcancer(363, 364, 642, 984). Vitamin D also acts as a potentmodulator of the immune response and cell proliferation.

B. PTH

Experiments dating back to the beginning of the 1900s havedemonstrated that surgical removal of the parathyroidgland results in tetany. It was recognized very early thatadministration of calcium could ameliorate or prevent themanifestation of tetany, and it was subsequently concludedthat the parathyroid glands play an important role in cal-cium metabolism (690, 826, 939). In 1925, extracts fromthe parathyroids were for the first time shown to controltetany in dogs (216). This finding marked the discovery ofPTH, which is one of the three prime hormones regulatingcalcium homeostasis.

1. Production and secretion

PTH is a 84-amino acid peptide hormone produced in theparathyroid glands. Its amino acid sequence was first estab-lished in 1970 in bovine (126, 788). Cloning of the humancDNA followed a decade later (440). The PTH gene en-codes a 115-amino acid precursor hormone (preproPTH),which is enzymatically cleaved in a two step process to itsmature 84-amino acid secreted form (565). The NH2-ter-minal prepro-signaling sequence is necessary for correcthormone processing and trafficking (340, 546). Thisknowledge is mainly founded on truncation studies andobservations made in patients with mutations in the PTHgene, which can result in familial isolated hypothyroidism,a disorder characterized by hypocalcemia and low PTHlevels (38, 340, 828, 1055). For example, a well-character-ized T-to-C point mutation in the prepro signaling sequencethat causes FIH leads to accumulation of the precursor hor-mone in the ER (243, 546). The impaired processing trig-gers ER stress, ultimately apoptosis and PTH insufficiency(243).

Following its cleavage to mature PTH, the hormone isstored in secretory vesicles and released into the circulationin response to low plasma calcium. The regulation of PTH

secretion is extremely tight, given the body’s need to main-tain the calcium concentration within a narrow window(1.1–1.3 mM). Small alterations in calcium homeostasis canhave deleterious effects, for example, on the excitability ofneurons and muscles. The low plasma half-life of PTH of �5min allows for a precise regulation of this balance (95). Toachieve controlled and rapid on-demand secretion of PTH, theparathyroid is equipped with an ultrasensitive extracellularCaSR, which constantly monitors the plasma calcium levelsand triggers intracellular signaling events and PTH releaseupon imminent drops in calcium levels (FIGURE 6) (see sect.IVD). PTH is further regulated on a transcriptional level by1,25(OH)2-vitamin D, creating a negative-feedback loop(154, 930, 931).

2. PTH1R

PTH exerts its physiological effects via activation of a mem-brane-bound GPCR, the parathyroid hormone receptortype 1 (PTH1R) (PTH2R is mostly expressed in the CNSand tissues that are not involved in calcium handling andwill thus not be reviewed). Of note, PTH1R is not exclu-sively located at the plasma membrane but can also localizeto the cell nucleus. The physiological significance of nuclearPTH1R is currently unclear but may represent a novel sig-naling paradigm for the actions of PTH (830, 856, 857,1155).

Full-length PTH is not required to activate PTH1R. TheNH2-terminal domain of PTH mediates most of the physi-ological effects of the hormone and is responsible for bind-ing in the �-�-�� binding fold of PTH1R (861). This is whyclinically PTH(1–34) is used as a PTH analog with identicalbiological activity (753, 867, 919, 1094). Conversely, NH2-terminal truncation of PTH(1–34) to PTH(2–34) changesthe characteristics to a partial receptor agonist, whereasfurther truncation to PTH(3–34) results in loss of biologicalactivity (1094).

PTH1R was first cloned from opossum in 1991, which wasfollowed by identification of the highly homologous humancDNA shortly thereafter (536, 964). In the nonactivatedstate the receptor is expressed as a homodimer at the cellsurface, which dissociates upon PTH binding (860).PTH1R is a member of the class B (class 2, secretin family)GPCRs. As many other GPCRs, it undergoes N-linked gly-cosylation at four asparagine residues (1218). Mutationalanalysis revealed that site-specific mutation of all four sitesdecreases cell surface expression, whereas impairment offewer glycosylation sites does not seem to have significanteffects on trafficking or ligand binding (1218). Further-more, the extracellular domain of the receptor includes acharacteristic disulfide bond pattern involving six cysteineresidues (392). These residues are thought to be essential forstabilizing the hydrophobic �-�-�� binding pocket forPTH, which is conserved among all members of class B



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GPCRs and has recently been crystallized in the presence ofPTH (861).

Binding of PTH causes activation of at least two distinct Gproteins. G�q/11 mediates intracellular calcium release viaphospholipase C (PLC) activation and increases in inositoltrisphosphate (IP3), whereas G�s activates adenylyl cyclaseleading to rises in cAMP (5, 188, 808, 859).

PTH1R can be regulated on a variety of levels, ranging fromtrafficking and internalization to direct protein interactionsat the cell surface. Desensitization of PTH1R is mediated byGRK2 binding/phosphorylation and �-arrestin binding,which uncouples the receptor from its associated G proteinsand triggers its internalization (267, 326, 330, 1123). For

example, knockout of �-arrestin causes increased and sus-tained levels of the second messenger cAMP in primaryosteoblast cultures upon PTH stimulation (327). PTH1Ralso associates with the scaffolding protein NHERF1,which stabilizes the receptor at the cell membrane and pre-vents its endocytosis and desensitization (1022, 1139). Thiseffect of NHERF1 is partially attributable to a prevention ofan interaction between �-arrestin and PTH1R (1140). Co-localization of NHERF1, �-arrestin, and PTH1R has beendemonstrated and suggests that NHERF1 is constitutivelybound, whereas �-arrestin association is more dependenton receptor activation (580). Interestingly, it has recentlybecome apparent that �-arrestin not only plays a role inreceptor desensitization, but also mediates activation ofdownstream signaling cascades, such as MAPKs, in a G

PLA2

Kinase phosphorylation

Increases VDR expression

PTH

Parathyroid gland

Inhibits PTHsecretion

Reduces PTHsynthesis

Nucleus

Inhibition ofcell growth

RXR VDRD

D

D

VDR

1,25(OH)2-D

Calcium

Calcium

Ca

CaCa

Ca

PLC

IP3

AA

DAG PIP2Gq

ER

FIGURE 6. CaSR signaling in the parathyroid gland. Increased serum calcium levels lead to an inhibition ofPTH secretion. Serum calcium levels are measured by the CaSR receptor. Activation of CaSR causesgeneration of arachidonic acid (AA) metabolites, which inhibit the release of PTH and increase the expressionof VDR, thereby increasing the cell’s sensitivity to the negative feedback exerted by 1,25(OH)2-vitamin D.1,25(OH)2-vitamin D suppresses the synthesis of PTH. Furthermore, CaSR activation inhibits parathyroid glandgrowth.



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protein-independent manner (380). Current scientific effortthus focuses on the development of so-called “biased”PTH1R agonists, which can selectively trigger G protein- or�-arrestin-dependent signaling events, allowing for a moreselective therapeutic repertoire (381, 1160). Furthermore,NHERF may also modulate the cell’s response to PTH bind-ing. In the presence of NHERF2, the cell’s calcium responsevia PLC is augmented, whereas the cAMP response is damp-ened, presumably via recruitment of G�i (698). BothNHERF and �-arrestin provide effective examples of howreceptor-associated proteins can modulate canonical signal-ing events or even, as is the case with �-arrestin, initiatesignaling events in their own right.

PTH1R can also be regulated by its external environment.The extracellular receptor domain can be cleaved by metal-loproteases, resulting in receptor degradation (579). PTHbinding prevents this proteolytic cleavage, thus stabilizingPTH1R at the cell surface. The physiological importance ofthis mechanism is not yet fully understood. However,MMPs are involved in bone remodeling and may in conse-quence locally regulate the sensitivity of osteoblasts to PTH(579).

3. Cellular effects

PTH exerts its effects in two primary target tissues: boneand the kidney.

In the kidney, PTH causes phostphaturia, increases calciumabsorption, and induces the synthesis of 1,25(OH)2-vita-min D. In-detail analysis of renal phosphate handling isbeyond the scope of this review and has been summarizedpreviously (334, 765). In brief, phosphaturia mainly resultsfrom downregulation of the Na-Pi transporter type IIa(NaPi-IIa) at the apical membrane of the proximal tubule,thereby reducing the amount of reabsorbed phosphate fromthe primary urine. Rather than directly modulating thetransporter’s activity, PTH exposure mainly affects thenumber of active cotransporters on the plasma membrane.Activation of basolateral PTH1R causes retrieval of NaPi-IIa and targets it for lysosomal degradation, resulting indiminished Pi reuptake (566, 682, 847). Apart from NaPi-IIa, at least two other apical phosphate transporters arepresent in the proximal tubule: NaPi-IIc and PiT-2 (986,1124). Currently, there is some evidence that PTH can alsoregulate NaPi-IIc and PiT-2, but additional studies remainto be conducted (855, 987). Although a contribution ofthese transporters to renal phosphate reabsorption is highlylikely, knockout studies suggest that NaPi-IIa is responsiblefor �80% of total phosphate transport, thus representingthe major uptake mechanism (72, 468).

It has been recognized for over 30 years that PTH canstimulate the synthesis of the active vitamin D metabolite1,25(OH)2-vitamin D in the kidney (116, 338) (see sect.IVA4). 1,25(OH)2-vitamin D in consequence enhances the

intestinal and renal uptake of calcium in an effort to coun-teract hypocalcemia, which initially led to secretion of PTH.The PTH-stimulated increase in 1,25(OH)2-vitamin D lev-els is achieved on a transcriptional level. PTH upregulatesthe transcription of CYP27B1, the mitochondrial enzymewhich is responsible for the conversion from 25(OH)-vita-min D to 1,25(OH)2-vitamin D. Transcriptional upregula-tion occurs via PTH binding to PTH1R, leading to increasesin the second messenger cAMP and activation of PKA (125,442, 763, 764, 921).

Apart from inducing the synthesis of 1,25(OH)2-vitamin D,PTH can directly upregulate the renal reabsorption of cal-cium. The regulation of calcium absorption by PTH occursin distal segments of the nephron, mostly in the distal con-voluted tubule and the connecting tubule (99, 379, 1003,1172). In close analogy to the duodenum, these segmentsexpress calcium transport proteins, which are responsiblefor mediating the process of active transcellular calciumabsorption in the kidney, namely, TRPV5, calbindin-D28k, and NCX1. PTH can regulate all of these protein levelson a transcriptional level (1108). Interestingly, this seems tobe accomplished independently of 1,25(OH)2-vitamin D,which also positively regulates most of these transporters(see sect. IVA6) (1108). It is therefore difficult to dissect therelative contribution of each of the two hormones in thephysiological regulation of the calcium transport proteins.In addition to transcriptional activation, PTH was shown tocause direct phosphorylation of TRPV5, thereby increasingits opening probability (249). The channel is phosphory-lated at threonine-709 in a PKA-dependent fashion (249).Elevation of intracellular cAMP levels and concomitantPKA activation are classical downstream effects of PTH1Ractivation in the kidney (248, 1172).

In bone, PTH exerts a dichotomous effect depending on thepattern of exposure. It is well documented that pulsatilePTH exposure has anabolic effects on bone mass, whereascontinuous release increases plasma calcium by bone catab-olism (FIGURE 7) (27, 348, 401, 469, 1065). The observa-tion that intermittent PTH administration increases bonemass has led to the use of PTH as a treatment strategy forosteoporosis (621, 671, 897). The enhanced bone forma-tion mainly results from an increase in osteoblast numbers.This phenomenon has been partially attributed to a PTH-mediated induction of osteoblast differentiation and an in-hibition of their apoptosis (75, 274, 518, 530, 531, 684,969, 1032). Multiple mechanisms underlying the anti-apo-ptotic effects of intermittent PTH on osteoblasts have beensuggested. Among others, these include runt-related tran-scription factor 2 (Runx2)-mediated transcription of sur-vival genes and increased DNA repair (75, 969). It hasfurther been shown that fibroblast growth factor 2 (FGF2)is partially needed as an endogenous cofactor for the ana-bolic effects of PTH to take place (934).



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Continuous PTH exposure, on the other hand, mainly af-fects osteoclast numbers and activation, thereby increasingbone turnover. Since osteoclasts are canonically thought tonot express PTH1R (although this view has recently beenchallenged, Ref. 260), the catabolic effects of PTH are re-layed through osteoblast signaling. The PTH-inducedcrosstalk between osteoblasts and osteoclast is mainly me-diated by receptor activator of nuclear factor �B (RANK),osteoprotegrin (OPG), and RANK ligand (RANKL). BothRANKL and OPG are expressed by osteoblasts and exertopposing actions on osteoclasts. RANKL promotes oste-oclastogenesis by binding to RANK on osteoclasts. Con-versely, OPG serves as a soluble decoy receptor for RANKLand thus inhibits its interaction with RANK, thereby sup-pressing osteoclastogenesis. In accordance with this model,RANKL-deficient animals develop osteopetrosis because ofinsufficient osteoclasts activation (592). Sustained PTH ex-posure affects RANK-RANKL signaling by downregulat-ing antiresorptive OPG, while simultaneously stimulat-ing production of RANKL by osteoblasts (FIGURE 7)(350, 497, 679, 689). The enhanced RANK-RANKL sig-naling induces formation of osteoclasts, which in turnleads to enhanced bone resorption and elevates serumcalcium levels.

In the intestine, several observations suggest that PTH mayhave a direct, i.e., non-1,25(OH)2-vitamin-D mediated, ef-fect on intestinal calcium absorption. Both isolated entero-cytes and intestinal loops demonstrated an increase in cal-cium uptake following acute PTH exposure (781, 783,784). However, more investigations are needed to clearlyestablish a direct regulatory role of PTH in the context ofintestinal calcium uptake.

4. PTH fragments

It should be noted that full-length PTH(1–84) is not theonly circulating form of the hormone in the body. VariousPTH fragments can be found in the circulation, which par-tially originate directly from the parathyroid gland and par-tially represent products of peripheral cleavage. The para-thyroid itself releases COOH- and NH2-terminal hormonefragments, which are generated by cysteine proteases (ca-thepsin B and H) in distinct secretory vesicles of the gland(418, 427, 693). Interestingly, the fraction of secreted hor-mone fragments changes with extracellular calcium condi-tions. It has been reported that more fragments are releasedunder conditions of hypercalcemia, when secretion of full-length PTH is suppressed (417, 418, 605, 726). Peripheralproteolysis represents the second source of PTH fragments.This process occurs predominantly in liver and the kidney(127, 234, 989). The group of fragments that have receivedthe most amount of scientific attention is the large NH2-terminally truncated non-PTH(1–84) fragments. PTH(7–84) is the quantitatively major member of this group, whichis secreted by the parathyroids (233). The group of non-PTH(1–84) fragments can represent up to 20% of circulat-ing PTH, but can increase in patients with renal failuredramatically to up to 50% because of impaired renal clear-ance (130, 131, 444, 648). This is of particular interest, as ithas become recently apparent that the non-PTH(1–84)fragments exert biological activity. In general, non-PTH(1–84) fragments antagonize the effects of PTH in its primarytarget tissues, bone and the kidney but also the parathyroidgland directly. It has been shown that PTH(7–84) can in-hibit PTH release from the parathyroid, presumably in anautocrine fashion, despite low serum calcium concentra-

PTH

Intermittent Chronic

PTH1R

Osteoblastproliferation

Osteoblast Osteoclast

Osteoclastdifferentiation

H+

RANKL RANK

OPG

V-ATPase

FIGURE 7. The effects of PTH on bone. PTH has a dual effect on bone. Intermittent PTH exposure causesosteoblast proliferation, leading to an increase in bone mass. Continuous PTH exposure results in RANKLupregulation and concomitant OPG suppression (OPG serves as a decoy receptor for RANKL and prevents itsinteraction with osteoclast RANK). The stimulated RANKL-RANK interaction leads to osteoclast proliferationand increased bone turnover.



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tions (493). In bone, PTH(7–84) blocks the effects of PTHon calcium mobilization in thyroparathyroidectomized rats(786). More detailed investigations revealed that PTH(7–84) reduces calcium release from bone in vivo and inhibitsthe formation of osteoclast-like cells in murine primarymarrow cultures (272). In the kidney, PTH(7–84) can in-hibit the formation of 1,25(OH)2-vitamin D, presumablyvia a posttranscriptional mechanism (768, 1102).

Since activation of PTH1R requires an intact NH2-terminaldomain of PTH, it has been speculated that non-PTH(1–84) fragments exert their function through a pathway dis-tinct of PTH1R. The existence of a COOH-terminal PTHreceptor has thus been postulated (270–272, 786). The mo-lecular identity of this receptor is, however, not yet re-solved. Another mechanism through which non-PTH(1–84) fragments may exert their biological activity is down-regulation of PTH1R. It has been demonstrated thatPTH(7–84) causes internalization of PTH1R, thus offset-ting the effects of PTH by decreasing the number of avail-able receptors at the cell surface (1022).

In conclusion, non-PTH(1–84) fragments act as PTH an-tagonists and are secreted by the parathyroid in response tohypercalcemia.

C. Calcitonin

Calcitonin is a peptide hormone that has been discovered byCopp et al. in 1962 as a factor that reduces serum calciumconcentrations (160, 227). Calcitonin production was ini-tially falsely ascribed to the parathyroid glands, and it wasonly later that the thyroid gland had been established as thesource of calcitonin (463). The primary sites of calcitoninproduction are the parafollicular cells (C-cells) of the thy-roid gland. Calcitonin exerts its hypocalcemic effects pri-marily by inhibition of osteoclast activity. It should benoted that the importance of calcitonin in day-to-day cal-cium homeostasis in humans is rather negligible (see sect.IVC4). For this reason, it will only be reviewed concisely.

1. Production and secretion

Calcitonin is encoded by the CALCA gene and is initiallysynthesized as a 141-amino acid precursor (preprocalci-tonin), which is later processed to the mature 32-amino acidhormone (637). The same gene also encodes the neuropep-tide CGRP. Production of either peptide is dependent ontissue-specific RNA splicing. Although encoded by a differ-ent gene, amylin also belongs to the calcitonin peptide fam-ily. All three peptides, i.e., calcitonin, CGRP, and amylin,share some overlapping functions with regard to osteoclastsuppression (16, 229, 1199). Calcitonin is released from theC-cells in response to rising concentrations of plasma cal-cium. The CaSR is responsible for the molecular process ofcalcium sensing on the parafollicular cells (351, 367, 728).

2. The calcitonin receptor

Calcitonin exerts its physiological functions via activationof the calcitonin receptor. The calcitonin receptor is a seven-transmembrane domain GPCR. In particular, it is a memberof the family B subfamily of GPCRs (669). It shares signif-icant homology with other receptors of the family, whichinclude the PTH, GHRH, PACAP, VIP, secretin, glucagon,and glucagon-like peptide receptors.

The calcitonin receptor is expressed in the two most exten-sively described calcitonin target tissues, i.e., osteoclastsand the kidney, but also in other adult tissues, such as theprostate, CNS, skeletal muscle, and placenta (17, 329, 428,790, 878, 1174). Multiple isoforms of the calcitonin recep-tor occur in the body, which results from different RNAsplicing directed by tissue-specific promoters (17, 30, 389,606). Activation of the calcitonin receptor mostly translatesinto a rise of intracellular cAMP levels via Gs-dependentactivation of adenylate cyclase (167, 332, 435, 669). Al-though the cAMP/PKA pathway appears to be dominant,activation of both PLC and PLD have also been reported(167, 332, 776).

3. Cellular effects

A) OSTEOCLASTS. Shortly after the discovery of calcitonin andits hypocalcemic effects, investigators set out to identify itsphysiological site of action. First evidence for an effect ofcalcitonin on bone metabolism came from experiments onrat embryonic bone in tissue culture. It was observed thatcalcitonin caused a decreased basal release of calcium fromthese preparations (343). These observations served as thefirst evidence of how calcitonin lowers serum calcium levels.Furthermore, calcitonin blocked the resorptive actions ofPTH on bone, albeit only temporarily (342, 343). After 4–6days of combined treatment (calcitonin � PTH), calciumrelease rose again (342). This desensitization to the effectsof calcitonin has been coined as the calcitonin “escape”phenomenon (342). Today we know that the transient ef-fect of calcitonin on osteoclasts is attributable to a down-regulation of surface calcitonin receptors and their synthe-sis (882, 1061, 1129, 1130).

As alluded to before, calcitonin directly inhibits the actionof osteoclasts, causing the balance between bone absorp-tion and formation to shift towards anabolism. Calcitoninexerts its inhibitory effects on osteoclasts via activation ofits receptor, which is expressed in abundance on their sur-face (428, 790, 878). Exposure to calcitonin triggers dis-tinct morphological changes in the osteoclast. Osteoclastsare highly motile cells that resorb bone via formation ofso-called resorptive pits, which are membrane invagina-tions that are luminally acidified by active proton secretion.Calcitonin has been shown to inhibit the formation of theseresorptive bays in vitro (487, 1057). Furthermore, oste-oclast motility is markedly decreased, causing the cell to



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enter a state of functional quiescence (169). Apart fromdirectly altering osteoclast function, calcitonin also affectsosteoclast differentiation. Calcitonin inhibits the formationof multinucleated mature osteoclasts by arresting their dif-ferentiation in more immature stages (1060).

B) KIDNEY. Renal calcium handling is the second organfunction that is influenced by calcitonin. However, it isnot entirely clear whether calcitonin causes calciuria orenhances calcium reabsorption from the urine. The con-flicting results may be to some extent attributable to spe-cies differences. In humans, calcitonin likely increases theexcretion calcium through the urine and thereby acts inconcert with its inhibitory action on osteoclasts to lowerserum calcium levels (31, 32, 143, 215, 819, 1016). Con-versely, most studies demonstrating an increase in therenal reabsorption of calcium and magnesium were con-ducted in rats, rabbits, or mice (84, 158, 159, 265, 304,877, 883, 1225). A single investigation established a cal-cium-conserving effect of calcitonin in human (160). Theprimary site of action for calcitonin in the rat is the thickascending limb (TAL) of the loop of Henle, where calci-tonin has been demonstrated to bind its receptor, in-crease local adenylate cyclase activity, and promote cal-cium reabsorption (166, 168). Apart from enhancing thereabsorption of calcium, calcitonin also promotes thevectorial transport of NaCl in the rat TAL, thereby am-plifying the corticomedullary concentration gradient,which is a prerequisite for the subsequent concentrationof urine in the collecting duct (265, 304). In the rabbit,the calcium-conserving effects of calcitonin seem to bemediated by the distal tubule (1225).

It has also been speculated that calcitonin may act di-rectly on the collecting duct in a similar fashion to anti-diuretic hormone (ADH or vasopressin), i.e., to concen-trate the urine by increasing the reabsorption of waterfrom the primary urine (251). Indeed, calcitonin wasshown to increase the apical expression of aquaporin 2(AQP2) in principal cells of the collecting duct (119).Apical insertion of AQP2 and subsequent transepithelialwater movement is the primary mechanism by whichADH causes concentration of the urine to lower plasmaosmolarity.

In conclusion, the direct impact of calcitonin on renalcalcium handling is quite vague and may be of minorimportance. However, calcitonin also has another, indi-rect effect on calcium homeostasis. Calcitonin wasshown to be an important regulator of the expression ofCYP27B1, the renal enzyme responsible for the conver-sion of 25(OH)-vitamin D to 1,25(OH)2-vitamin D(1009, 1217). In normocalcemic rats, CYP27B1 mRNAlevels were inducible by calcitonin administration, lead-ing to an increase in the production of 1,25(OH)2-vita-min D (1009, 1217). Furthermore, CYP24A1 was in-

duced in HEK-293 cells following calcitonin exposure,suggesting a regulatory role of calcitonin in vitamin Dcatabolism (361). In addition to its putative direct effecton calcium reabsorption, calcitonin may thus affect nor-mal calcium metabolism indirectly by modulating thelevels of circulating 1,25(OH)2-vitamin D.

4. The relevance of calcitonin forcalcium homeostasis

The relevance of calcitonin for day-to-day calcium balanceis highly debatable. This is corroborated by fundamentalobservations during conditions of decreased or increasedcalcitonin levels, neither of which result in an appreciablephenotype in terms of calcium balance. For example, pa-tients with medullary carcinomas of the thyroid (MTC), atumor of the thyroid C-cells resulting in the hypersecretionof calcitonin, were shown to have normal bone mineraldensities (1176). Animal studies further substantiate thisconundrum. A reduction in serum calcitonin levels by thy-roidectomy in rats did not impact serum calcium levels(223, 1064). In light of this evidence, the question arises asto what the physiological role of calcitonin is.

It has been suggested that calcitonin is an evolutionary rem-nant (462). This is substantiated by the fact that calcitoninfrom other species is more potent than human calcitonin.Teleost calcitonin has the highest biological activity in hu-mans, which may be a result of their higher dependence onthe hormone. For example, salmon calcitonin has an ap-proximately sixfold higher affinity to calcitonin receptorthan human calcitonin (28, 328). Furthermore, it is lesseffectively eliminated by the kidney, resulting in a longerplasma half-life (405). The differences in the biological ac-tivity between calcitonin forms led to the introduction ofsalmon calcitonin as a treatment for skeletal disorders, suchas osteoporosis or Paget’s disease (1077).

Given its ambiguous role in regular calcium homeostasis, ithas been postulated that calcitonin may be of importanceduring states of high calcium demand, such as during lac-tation (1170). It has been demonstrated that calcitonin andCGRP (�/�) mice show greater loss of skeletal mass duringlactation than wt animals (1170). Since calcitonin andCGRP are encoded by the same gene, animals were con-trolled by CGRP substitution, which was without effect(1170). Another mechanism by which calcitonin may beosteoprotective during lactation or pregnancy is by induc-ing the renal synthesis of 1,25(OH)2-vitamin D (1217).

D. The CaSR

The CaSR is a G protein-coupled membrane-bound recep-tor that is the primary sensor for calcium and is the first linkin the regulatory chain of calcium homeostasis. By regulat-ing the release of PTH from the parathyroid to modulate



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current serum calcium levels, it presides over the subsequenthierarchical cascade of vitamin D synthesis and calciumhandling in the vitamin D target organs, such as the intes-tine, kidney, and bone. More recent investigations havedemonstrated that the CaSR is not exclusively expressed inthe parathyroid gland, but is also present locally in thevitamin D target organs. This diverse expression suggeststhat CaSR can modulate organ function locally and outsideof the strict PTH-vitamin D-organ axis. Thus a short andlocal feedback loop is created, which allows the organs torespond rapidly to the local calcium environment. A de-tailed description of the receptor’s role in each tissue isbeyond the scope of this article, but an attempt will be madeto identify the key features of CaSR in these local sites in asubsequent section (FIGURE 8). For excellent in-depth re-views, please refer to Refs. 372, 711, 906.

The importance of CaSR as a regulator of calcium balanceis underlined by clinical pathologies that are caused by itsmutation. Loss-of-function mutations cause familial hy-pocalciuric hypercalcaemia (FHH; OMIM 145980) andneonatal severe hyperparathyroidism (NSHPT; OMIM239200), whereas activating mutations cause autosomaldominant hypocalcemia (OMIM 601198). These muta-tions mostly change the threshold of receptor activation ineither direction. Currently, �300 different mutations arereported, two-thirds of which represent inactivating muta-tions (241). Given its broad expression pattern, the de-ranged sensing of blood calcium levels in these disorders notonly affects the secretion of PTH from the parathyroidglands, but also causes local dysfunction in other organs,such as the kidney where calcium absorption is perturbed.

1. Structure and signaling

The CaSR was first cloned by Brown et al. (134) in 1993from bovine parathyroid using a Xenopus oocyte expres-sion cloning system. Cloning of the human CaSR followed2 years later (366). The CaSR is a 1,028-amino acid proteinthat belongs to the superfamily of classic 7-transmembranedomain G protein-coupled receptors (134, 366). It is mainlyexpressed as a homodimer on the cell surface (55). Thedimerization process has been shown to take place in the ERand is mediated by the formation of disulfide bonds be-tween cysteine residues (C129, C131) and noncovalent in-teractions of leucine residues (L112, L156) in the extracel-lular domain of the receptor (54, 529, 858, 888, 1213).Following assembly in the ER, the CaSR undergoesN-linked glycosylation in the Golgi apparatus, some ofwhich is pivotal for cell surface expression (887). The traf-ficking between ER and Golgi apparatus is regulated by thesmall GTP-binding protein Rab1 (1221). Knockdown andmutations of Rab1 in HEK cells results in decreased num-bers of CaSR at the cell surface (1221). Conversely, theinternalization of CaSR is thought to be mediated by ubiq-uitination by the E3 ligase dorfin (498).

On the cell surface, the CaSR resides in caveolin-1-richplasma membrane domains, which also contain associatedsignaling proteins (571). These signaling complexes areformed with the help of scaffolding proteins. The COOH-terminal tail of CaSR binds to filamin A, an actin bindingprotein (48, 467). Silencing of filamin A with siRNAs re-sults in the attenuation of MAPK signaling by the receptor(496) (see below).

In lack of a crystal structure of CaSR, the exact binding sitesof calcium on the extracellular domain remain subject ofspeculation. So far, applicable structural data has only beenobtained from the metabotropic glutamate receptor type I(mGluR1), which belongs to the same family of type CGPCRs. In this model, glutamate binds to key residueswhich are located in a cavity, embedded in between twolobular domains (LB1 and LB2) of the extracellular tail(611). This structural hallmark has been aptly coined thereceptor’s Venus fly trap module. This motif is conservedamong other GPCRs of the same family. Multiple attemptsto identify the calcium binding sites have been undertakenusing computational homology modeling (499, 500, 1014).These models have postulated between one and five calciumbinding sites (499, 500, 1014). With the employment ofmutational analysis, it has been possible to validate func-tionality of some of these putative binding sites (500). Mon-itoring of the intracellular calcium response to increasingextracellular calcium levels in CaSR transfected HEK cellshad indicated previously that the Hill coefficient for thisresponse was �3.1, suggesting that multiple calcium bind-ing sites may exist (834).

It should be noted that the CaSR can also be stimulated byother polyvalent cations (Mg2�, Pb2�, Cd2�, Fe2�, Ba2�,Ni2�, Co2�, or Gd3

�) and larger polycationic molecules,such as spermine, spermidine, putricine, protamine, andneomycin (134, 416, 880). Furthermore, several substancescan allosterically modify the receptor and potentiate its sen-sitivity to its direct agonists. These include pharmacologicalsmall molecule substances (calcimimetics) that are in clini-cal use for the treatment of conditions, such as secondaryhyperparathyroidism, and L-type amino acids, which en-able the CaSR to act as a nutrient sensor (220). Truncationstudies have demonstrated that the Venus fly trap motif isnecessary for allosteric modification by L-type amino acids(759). The affinity of calcium to the CaSR can also be mod-ulated by changes in extracellular pH (279, 879). An acidicextracellular milieu has been shown to decrease the sensi-tivity of CaSR to its agonists, whereas an increased extra-cellular pH has converse effects (879).

The intracellular domain of CaSR contains five PKC phos-phorylation sites (366). Mutational analysis demonstratedthat PKC-mediated phosphorylation of the CaSR at Thr-888 blunts its response to extracellular calcium, as evi-denced by inhibited calcium release from intracellular



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Differentiationand function

Osteoblast Osteoclast

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? ?

H+

RANKL RANK

V-ATPase

PPIsAPAs

Parietal cell

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H+H,K-ATPase

Circulation

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25(OH)-vitamin D

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H2O

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Na+

NKCC2

K+

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CFTR

CaSR

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Enterocyte

Calcium-sensing receptor

Intestine

BoneStomach

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FIGURE 8. CaSR in the gastrointestinal tract, kidney, and bone. Kidney: the effects of CaSR activation on iontransport in various nephron segments are shown. In the proximal tubule, CaSR stimulates phosphateabsorption and 1,25(OH)2-vitamin D synthesis. In the thick ascending limb of the loop of Henle, CaSR inhibitsapical potassium channels (ROMK), thereby inhibiting NKCC2 (potassium recycling). The resulting changes inthe lumen-positive potential inhibit paracellular calcium uptake. In the distal convoluted tubule, CaSR presum-ably stimulates apical calcium entry through TRPV5. In the collecting duct, CaSR stimulates proton extrusionthrough the V-type ATPase and inhibits urine concentration through AQP2. Stomach: in the parietal cell, CaSRinduces acid secretion by activating H�-K�-ATPase. In the G-cell, CaSR activation results in gastrin secretion.Of note, CaSR serves as a luminal nutrient and calcium sensor on the G-cell. Bone: CaSR on osteoblastspresumably regulates their differentiation and RANKL expression. Intestine: in the intestine, CaSR activationreduces water secretion by inhibiting chloride secretion through CFTR.



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stores (56, 246). The phosphorylation occurs in response toreceptor activation and thus represents an autoinhibitoryfeedback mechanism (246). �-Arrestins are most likely in-volved in the process of PKC-associated desensitization(681, 853). Conversely, dephosphorylation of the Thr-888residue is carried out by a calyculin-sensitive phosphatase,thereby restoring the receptor’s initial sensitivity (246). An-other mechanism of desensitization is mediated by a G pro-tein receptor kinase (GRK), most likely by interfering withG�q regulated pathways (see below) (681, 853).

Once stimulated, the CaSR activates a variety of intracellu-lar signaling cascades. Being a GPCR, most of these pro-cesses are mediated by G proteins. Specifically, G�q/11, G�i,and G�12/13 have been shown to be coupled to the CaSR(40, 175, 495, 849). The expression of all subunits wasconfirmed in bovine parathyroid (1115). G�i mediates thesuppression of cAMP levels by inhibiting adenylyl cyclaseand activates the ERK/MAPK pathway (175, 250, 375,572). Activation of G�q/11 results in increased intracellularcalcium concentrations via activation of PLC and IP3 trig-gered calcium release (133, 1010). As demonstrated in HEKcells, this cascade can also activate further downstreamphospholipase A2 leading to production of arachidonic acidmetabolites (415). G�12/13 is thought to regulate phospho-lipase D and phosphatidylinositol 4-kinase (PI 4-K); how-ever, this interaction has only been demonstrated in heter-ologous cell culture system (494, 495).

2. CaSR in the parathyroid

CaSR regulates parathyroid function at three levels: 1) therelease of PTH from secretory granules, 2) de novo synthe-sis of PTH, and 3) parathyroid cell growth.

Activation of CaSR by increasing plasma calcium results inan inhibition of PTH release, thereby lowering calcium lev-els. It is thought that this response is mediated by the gen-eration of arachidonic acid metabolites via G�q and PLA2

activation (FIGURE 6) (121, 152). Cultured porcine parathy-roid cells demonstrated an increase in arachidonic acid pro-duction after CaSR stimulation while PTH release was in-hibited (121). Furthermore, exogenous administration ofarachidonic acid suppressed PTH release from the parathy-roid cells (121). Similar effects were demonstrated for thearachidonic acid metabolites 12- and 15-hydroxyeicosatet-ranoic acid, suggesting that they represent the downstreameffectors of arachidonic acid production (120).

Apart from directly controlling PTH release, CaSR alsomodulates PTH synthesis. PTH gene transcription is mainlyregulated by 1,25(OH)2-vitamin D. Binding of 1,25(OH)2-vitamin D to the VDR causes a decrease in pre-pro-PTHmRNA levels creating a negative-feedback loop (154, 930,931). However, it was recognized before the identificationof the CaSR that serum calcium can modulate the actions of1,25(OH)2-vitamin D on PTH gene transcription (930). It

was shown that increases in calcium can potentiate the in-hibitory effects of 1,25(OH)2-vitamin D (930). This effect ismost likely mediated by CaSR, whose activation can decreasePTH transcription by augmenting the inhibitory effects of1,25(OH)2-vitamin D. Molecularly this is achieved by upregu-lating the expression of the VDR (151, 162, 362, 653, 916).The current working model states that activation of CaSRcauses an increase of arachidonic acid metabolites and activa-tion of the MAPK pathway, which in turn results in increasedVDR mRNA levels (FIGURE 6) (151). This allows the parathy-roid to adjust its 1,25(OH)2-vitamin D sensitivity to the cur-rent plasma calcium levels.

The molecular mechanisms underlying the trophic effects ofCaSR activation are less clear. Earlier observations had al-ready suggested that hypocalcemia is associated with para-thyroid cell proliferation (778). Currently, the CaSR spe-cific calcimimetics provide the most useful insight into theregulation of parathyroid growth by CaSR. Calcimimeticsadministered in the context of both animal models andclinical studies of hyperparathyroidism demonstrate thatactivation of CaSR leads to a reduction in gland size(510, 589, 746, 1128). Conversely, inactivating muta-tions of CaSR result in parathyroid enlargement. Para-thyroid-selective genetic disruption of G�q was further-more shown to cause moderate hyperparathyroidismwith increased plasma PTH levels and gland hyperplasia,suggesting a role of G�q in the regulation of parathyroidcell growth (849). Similar findings were reported inG�q/11 double KO animals (1159).

3. CaSR in the kidney

The CaSR acts as an important regulator of ion and waterhomeostasis in the kidney. It should be noted that it canexert its effects on calcium transport independently of otherhormonal regulators, such as PTH and 1,25(OH)2-vitaminD. The CaSR is expressed along most of the nephron, albeitin varying subcellular localizations (FIGURE 8) (907, 908).In the proximal tubule, CaSR is localized apically at thebase of the brush border, where it has been implicated toplay a role in phosphate transport (52, 907, 909). The pri-mary regulator of phosphate transport in the proximal tu-bule of the kidney is PTH. In brief, increased PTH levelsinhibit phosphate reabsorption from the lumen. Activationof the apical CaSR can partially reverse the effects of PTHand restore phosphate absorption (52). Conversely, PTHand high phosphate levels reduce CaSR expression (909).Furthermore, it is likely that the CaSR mediates the inhibi-tory effects of calcium on 1,25(OH)2-vitamin D synthesis inthe proximal tubule (109, 702).

In the thick ascending limb of the loop of Henle, CaSR islocated on the basolateral membrane (907). In this nephronsegment, the receptor acts as a major modulator of mon-ovalent and polyvalent ion absorption. Activation of CaSRleads to an inhibition of the apical renal outer medullary



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potassium (ROMK; Kir1.1) channel, mainly through ara-chidonic acid metabolites created by PLA2 (1147, 1148).Apical ROMK releases potassium ions into the lumen,which in turn are needed to fuel apical ion uptake throughthe Na�-K�-2Cl� (NKCC2) cotransporter. By decreasingapical potassium efflux, CaSR inhibits sodium and chlorideuptake through NKCC2 (906). This correlation is reflectedin much earlier observations, which report that calciuminfusions can decrease tubular sodium clearance (300, 718,1052). In addition, impairment of NKCC2 has also impli-cations for calcium absorption. Reduced NKCC2 activitydecreases the lumen-positive potential and negatively af-fects countercurrent multiplication, and in consequence thenephron’s ability to concentrate urine (434). Both mecha-nisms will lead to impaired calcium absorption (434). Cal-cium absorption in the medullary portion of the thick as-cending limb is thought to occur predominantly as passiveuptake through the paracellular route (995). Similar obser-vations have been made when blocking NKCC2 pharmaco-logically with the loop diuretic furosemide (299). It has thusbeen proposed that activation of basolateral CaSR has“loop diuretic-like” effects, reducing NaCl but also calciumabsorption in the kidney (434).

In contrast to the medullary section, the cortical portion ofthe thick ascending limb has been proposed to have pre-dominantly active calcium uptake properties, which are un-der the hormonal regulation of PTH and calcitonin (344,509). PTH increases calcium absorption in this segment,and it has been shown that, similarly to phosphate absorp-tion in the proximal tubule, activation of CaSR can sup-press the effects of PTH (264, 754). The absorption of NaCldoes not seem to be affected by CaSR (264, 754).

The distal convoluted tubule and the connecting tubule areresponsible for the fine-tuning of calcium reabsorption inthe kidney. To achieve this goal, they are equipped withmolecular machinery, similar to that in the duodenum(TRPV5, calbindin-D 28k, NCX1, and PMCA1b) to ab-sorb calcium against its electrochemical gradient throughthe transcellular pathway (680). In analogy to the proximalsmall intestine, these transporters are predominantly regu-lated through 1,25(OH)2-vitamin D, but also PTH. CaSRcolocalizes with TRPV5 in this segment (1090). Its activa-tion causes increase calcium influx through TRPV5 andmay thereby locally and rapidly adapt active absorption tothe urine calcium concentration (FIGURE 8) (1090).

Apart from regulating calcium and phosphate absorption inthe kidney, CaSR modulates proton and water movement inthe collecting duct. In the intercalated cells of the collectingduct, apical V-ATPase acidifies the urine in an effort tomaintain systemic acid-base homeostasis. It has been shownthat luminal calcium and neomycin can induce V-ATPaseactivity via activation of CaSR, thereby causing proton se-cretion into the urine (FIGURE 8) (901). Since the formation

of calcium kidney stones is dependent on luminal pH, it hasbeen speculated that this may represent an autoprotectivemechanism that prevents nephrolithiasis (901). Further-more, stimulation of apical CaSR in the principal cells of thecollecting duct leads to decreased ADH (vasopressin)-stim-ulated water reabsorption through AQP2 (FIGURE 8) (942,943). Taken together, activation of CaSR has diuretic ef-fects via inhibiting NKCC2 in the thick ascending limb ofthe loop of Henle and by inhibiting AQP2-mediated waterreabsorption the collecting duct.

4. CaSR in the gastrointestinal tract

The CaSR is distributed along most of the gastrointestinaltract, ranging from the stomach to the large intestine (186,360, 744, 932, 998). We are now only slowly beginning tounravel its function in this diverse array of tissues. In thestomach, CaSR localizes to the basolateral membrane ofthe acid-secreting parietal cells and to all membranes of thegastrin-secreting G-cells (FIGURE 8) (142, 182, 886). Pri-mary cultures of G-cells were shown to release gastrin afterstimulation of the CaSR with calcium (142, 886). The re-lease is mediated via calcium influx into the cytosol throughnonselective cation channels opening after CaSR stimula-tion (142). These findings provide the molecular basis forthe observation that rises in serum calcium can increaseserum gastrin levels (see sect. IIB2). The apical expression ofCaSR in G-cells theoretically enables it to act as a luminalnutrient sensor modulating gastric acid secretion and otherparameters. In recent studies there is direct evidence show-ing that gastrin levels increase in mice after calcium andL-type amino acid ingestion (325). This effect was abol-ished in CaSR (�/�) animals (325). In healthy human testsubjects, pharmacological stimulation of CaSR leads to aconcomitant increase in gastrin levels and gastric acid out-put (165). CaSR on G-cells was thus postulated to play animportant role in the gastric phase of acid secretion bymaintaining acid output by maintaining gastrin secretion(325).

Apart from being expressed on G-cells, CaSR is also local-ized on the basolateral membrane of the acid-secreting pa-rietal cell, where it exerts effects that are independent ofgastrin and other secretagogues. Activation of parietal cellCaSR has been reported to increase H�-K�-ATPase-medi-ated proton secretion, thereby acidifying the gastric lumen(FIGURE 8) (145, 291, 373). This stimulatory effect wasdemonstrated for direct activators, such as calcium orGd3�, but also allosteric modifiers, such as L-type aminoacids (145, 291, 373). In parallel to other tissues, the intra-cellular activation signal for H�-K�-ATPase is mediated byrises in intracellular calcium, PLC, MAPK, and PKC (899).In conclusion, both rises in luminal and plasma calciumconcentrations can induce gastric acid secretion either indi-rectly through gastrin release or directly through parietalcell activation. The physiological significance of this obser-vation remains the subject of speculation, but may be linked



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to facilitating calcium uptake by increasing acid output. Asdescribed in a subsequent section, it has been speculatedthat gastric acid increases the bioavailability of ingestedcalcium (see sect. V). Alternatively, CaSR may primarilyfunction as a nutrient sensor in the stomach (amino acidsensing), which maintains constant acid output in the gas-tric (apical G-cell sensing) and postprandial (basolateralparietal cell) phase of digestion, when circulating levels ofamino acids are high (325).

In the intestine, functional investigations on CaSR havemainly been conducted in colonic epithelia, where CaSRlocalizes to both the basolateral and apical membranes ofthe colonic crypt (173, 186, 360). Expression patterns varyslightly in the small intestine, with general basolateral ex-pression and additional weak apical expression in the villus(173, 360). Furthermore, CaSR is expressed in both theMeissner’s and Auberach’s plexuses. Early experiments onsingle perfused colonic crypts demonstrated that intracellu-lar calcium concentrations could be increased when expos-ing the crypts to classic CaSR agonists and that forskolin-stimulated fluid secretion could be inhibited (186). This andsubsequent investigations indicate that CaSR plays an im-portant role as a modulator of colonic fluid secretion (185,186). Subsequently, attempts have been made to take ad-vantage of the “constipatory” effects of CaSR activation inpathophysiological settings. Activations of CaSR in thecourse of diarrheagenic enterotoxin exposure was shown todecrease fluid secretion via increased breakdown of cyclicnucleotides (371). Although the potential clinical applica-tions of ameliorating the symptoms of secretory diarrheaare promising, more efforts will have to be made to fullyunravel the physiological role of CaSR in intestinal ion andfluid transport. So far it is not clear whether intestinal CaSRcan modulate calcium absorption, as is the case in the kid-ney.

5. CaSR in bone

It is well established that CaSR is expressed in osteoblasts,osteoclasts, and their respective precursors (172, 174, 542,1183–1185, 1192). The functional role of CaSR in thesecells is, however, less clear. Undoubtedly, both cell lines areexposed to local fluctuations in calcium concentrationsmaking an adaptive response to the calcium environmentplausible. Indeed, changes in extracellular calcium concen-tration have been shown to regulate various cell functions,mostly in in vitro models. Extracellular calcium can stimu-late the proliferation, migration, and differentiation of os-teoblasts (174, 297, 1183, 1184, 1186). Similarly, calciumwas proposed as a differentiation signal for osteoclasts(542, 544, 734). Significant doubt about the in vivo impor-tance of CaSR in bone has emerged with the generation ofthe CaSR (�/�) mice. Although CaSR knockout results inrickets, these animals suffer from severe hyperparathyroid-ism, which did not allow a discrimination between the ef-fects of high PTH and CaSR on bone turnover (365). Con-

comitant genetic ablation of the parathyroid gland or PTHsecretion, however, revealed that the skeletal phenotype ofCaSR single mutation (�/�) could mostly be rescued, sug-gesting that the skeletal abnormalities were due to highcirculating PTH levels rather than CaSR inactivation (598,1096). Furthermore, CaSR does not seem to be the exclu-sive calcium-sensing mechanism in osteoblasts, as changesin extracellular calcium can still elicit functional responsesin CaSR (�/�) osteoblasts (852). This observation has beenattributed to another GPCR with calcium-sensing capabil-ities, namely, GPRC6A (850, 851). Although GPRC6A hasa higher activation threshold for calcium, it also responds tothe CaSR allosteric activator R568 (851). GPRC6A activa-tion may thus represent a confounding factor in most invitro studies on osteoblasts and their modulation by CaSR.Also, GPRC6A knockout leads to osteopenia, further un-derlining the possibility of an alternate calcium-sensingpathway in bone (850). Osteoblasts extracted from theseGPRC6A-deficient animals show decreased sensitivity toextracellular calcium and in vitro mineralization defects(854).

Although these observations have profoundly questionedthe physiological significance of CaSR in bone, closer ex-amination still favors a role of CaSR in bone turnover. Withthe recent advances in genetic methods, an osteoblast-spe-cific CaSR (�/�) model has been created (171). These ani-mals have severely stunted growth and skeletal develop-ment, clearly suggesting an involvement of CaSR in normalosteoblast function (171). The previous conflicting evidencegained from global CaSR (�/�) models with survival rescueby elimination of PTH synthesis have been attributed to thepossible expression of alternate CaSR splice variants, whichmay compensate for the deletion of full-length CaSR inthese animals (171, 915). In an attempt to further elucidatethe function of CaSR in osteoblasts, the reverse approachhas been executed by specifically upregulating CaSR in os-teoblasts with use of a constitutively active receptor mutant(296). Upregulation of CaSR results in bone loss, as evidencedby a decrease in bone volume and density, specifically of tra-becular bone (296). These findings are accompanied by anincreased number of osteoclasts, whereas osteoblast parame-ters were essentially unchanged (296). Activation of CaSR hasbeen speculated to promote RANKL production by osteo-blasts, which serves as an osteoclastogenic signal (296). Osteo-blasts may thus recruit osteoclasts and induce their maturationvia CaSR signaling and increased RANKL expression, whichwould explain the observed increase in bone turnover andosteoclast numbers in the setting of constitutive CaSR activa-tion (241).

V. THE STOMACH AND CALCIUM

Preceding parts of this review have independently summa-rized the physiology of acid secretion, intestinal calciumabsorption, and their respective regulation. The following



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section will attempt to illustrate the functional intersectionsbetween these seemingly unrelated fields. In particular, thequestion of whether acid is needed to absorb calcium effec-tively from the gut or whether the stomach contributes tothe regulation of calcium homeostasis by secretion of anendocrine substance will be investigated.

A. Proton Pump Inhibitors and the Riskof Fracture

In May 2010, the Food and Drug Administration (FDA)released the following safety announcement: “Healthcareprofessionals and users of proton pump inhibitors shouldbe aware of the possible increased risk of fractures of thehip, wrist, and spine with the use of proton pump inhibi-tors, and weigh the known benefits against the potentialrisks when deciding to use them” (319).

Proton pump inhibitors (PPIs, see sect. IIC1) are in wide-spread use for the treatment of acid-related disorders, suchas gastroesophageal reflux disease (GERD) or gastric ulcerdisease. They exert their curative effects by inhibiting theacid output of the stomach. Over the recent years, mostlyepidemiological evidence has accumulated which links theintake of PPIs to an increased risk of sustaining fractures,especially in the elderly population. Yang et al. (1190) pub-lished one of the earliest and largest studies investigatingthis potential correlation in 2006. Examining a populationof over 13,000 hip fracture cases and over 135,000 controlsover the age of 50, the authors concluded that long-term(over 1 year) PPI use was associated with an increase in hipfractures (AOR � 1.44) (1190). Although the likelihood ofsustaining a fracture following PPI intake may seem fairlylow, the implications for public health are substantial. Thishas multiple reasons: PPIs represent the third most com-monly prescribed medication in the United States and arealso available as over-the-counter formulations. Further-more, there is an ongoing debate whether PPIs are overpre-scribed, putting certain populations at unnecessary risk ofside effects. In combination with the high incidence of os-teoporotic fractures, the mean incidence of hip fracturesalone between 1986 and 2005 was 957 per 100,000 womenover the age of 65 per year, a small increase in risk suddenlyhas implications for a very large population (123).

The roots of this controversy may potentially be traced backto the 1940s and 1950s. Before the advent of PPIs, total andpartial gastrectomies or vagotomies were performed to con-trol acid-related disorders. It was soon apparent that pa-tients who underwent these radical surgical procedures de-veloped osteoporosis/-malacia (58, 305, 732, 876). A studythat assessed the prevalence of osteomalacia in gastrecto-mized patients concluded that up to 12% of patients (19%of females) had histologically overt osteomalacia, althoughgeneral disturbances in calcium metabolism were estimatedto occur in up to 28% of patients (208, 368). Other inves-

tigations came to lower prevalence results of �5–10%(1091). The osteomalacia was also shown to translate intoan increased incidence of fractures in these patients (795).Naturally, gastrectomy represents a radical intervention,and the reasons for this correlation may be multifactorial,but reduced acid output may be of significance.

Back in the field of PPIs, the seminal epidemiologic investi-gation by Yang et al. was subsequently followed up by anumber of studies, which also focused on other types offractures, other populations, and other drugs reducing gas-tric acid output, such as H2 receptor antagonists (202, 228,252, 394, 400, 559, 868, 923). Although their conclusionswere somewhat controversial, a recent meta-analysis sup-ports the initial hypothesis that a correlation between PPIintake and fracture risk (hip, spine, and any-site fractures)exists (1195). The meta-analysis considered 11 studies andidentified an overall odds ratio of 1.30 for all fracture typescombined (1195). There was no association between H2

blocker intake and an increase in fracture risk, althoughsome single studies supported a link (228, 1195). Anothermeta-analysis came to a comparable conclusion with regardto an increased fracture risk under PPI exposure (616).

B. Gastric Acid and IntestinalCalcium Uptake

A variety of reasons could theoretically account for theobservation that PPIs increase the likelihood of fractures.The most prominent hypothesis assumes that the reducedacidity in the stomach impairs the intestinal absorption ofdietary calcium. This assumption is based on both patientobservations and experimental animal data. Alas, the num-ber of animal studies, which in contrast to investigations inhumans per default allow more radical experimental de-signs and genetic manipulation, is very small.

1. Effects of gastrectomy, vagotomy, and PPIs onmineral metabolism in humans

Before discussing the reports that try to correlate PPI usewith calcium uptake, it is worthwhile to examine olderliterature on patients that had undergone partial or totalgastrectomy. As discussed previously, these procedures areknown to be linked to bone disease. In contrast to PPIs,which eliminate the singular factor of acid secretion, gas-trectomies also influence gastric emptying, the emulsifica-tion of food stuffs, and food habits. It is thus more difficultto draw conclusions on the influence of acid secretion oncalcium absorption from gastrectomized patients than fromindividuals on PPI therapy. A further caveat lies in the typeof gastrectomy, as different surgical procedures are andwere in use. Some surgeries bypass the duodenum (BillrothII, Roux-en-Y, total gastrectomy), whereas some leave theduodenal passage intact (Billroth I).



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A common finding among gastrectomized patients is theirlow 25(OH)-vitamin D levels, while levels of 1,25(OH)2-vitamin D seem to be increased (101, 244, 378, 511, 794,972, 1081). The pathophysiological reason for this is notentirely clear. It has been argued that bone disease and low25(OH)-vitamin D are a result of impaired vitamin D ab-sorption following surgery; however, the consensus seemsto be that uptake rates of vitamin D is not impaired in thesepatients (245, 378; contested by Ref. 1081). The vitamin Dinsufficiency may also be a byproduct of improper nutrition(378). As fat and milk intolerance can develop, especially insurgeries which exclude the duodenum (Billroth II), achange of dietary habits with insufficient intake of the fat-soluble vitamin D may be an underlying cause. Indeed,long-term longitudinal studies suggest that maintainingbody weight reduces the risk of developing bone diseaseafter gastrectomy, which emphasizes the role of adequatenutrition (667, 668). On the other hand, Billroth I and IIpatients show the same loss in bone density, although Bill-roth II surgery (bypassing of the duodenum) is associatedwith a much higher degree of fat malabsorption (110, 651).A different investigation even concluded that Billroth I pa-tients have a higher loss in bone density than Billroth IIpatients (794). A more recent report suggests that the prob-lem underlying bone disease after gastrectomy may be im-paired calcium absorption, rather than dietary vitamin Ddeficiency (244). It has been shown that high 1,25(OH)2-vitamin D levels accelerate the breakdown of 25(OH)-vita-min D (210–212, 244). The observed low 25(OH)-vitaminD levels in gastrectomized patients may thus be a byproductof increased catabolism, and not insufficient intake,whereas the high 1,25(OH)2-vitamin D levels may representcompensatory upregulation due to insufficient calcium ab-sorption or intake (244). Calcium absorption in gastrecto-mized patients (Billroth I � II) has been reported to be in thelow-normal range, while 1,25(OH)2-vitamin D levels areincreased (794). In line with these findings, secondary hy-perparathyroidism, an indicator of compensatory upregu-lation due to low serum calcium levels, is a known findingafter gastrectomy (101, 244, 1171). Other investigations onintestinal calcium absorption in gastrectomized patientscame to very contradictory results, ranging from increasedto impaired absorption (8, 34, 255, 398, 585, 794). Manyof these reports failed to assess 1,25(OH)2-vitamin D andPTH levels, which means that adaptive mechanisms maymask the insufficient baseline uptake of calcium in the in-testine (8, 34, 255, 398, 585, 794). In conclusion, the exactpathogenesis of postgastrectomy osteopenia remains some-what unclear. The disorder may be attributable to vitaminD insufficiency, impaired calcium absorption, inappropri-ate diet, or a combination of all factors.

The intrusiveness of gastric surgery makes it difficult todissect the influence of gastric acid on these parameters.This is why vagotomized patients are a somewhat more aptpatient population to study the effects of gastric acid on

calcium uptake, albeit the number of studies on this cohortis very limited. Vagotomy abolishes the parasympatheticinput to the stomach and thereby decreases the amount ofsecreted acid. Although the gross anatomy of the stomachremains intact, other parameters, such as gastrin levels, arealso deranged given the important role of the vagus nerve inthe regulation of gastric acid secretion (see sect. IIB). Whilebone disease is generally not reported in these patients, low25(OH)-vitamin D levels are common (514, 793). Similarlyto gastrectomy, the 1,25(OH)2-vitamin D levels are con-comitantly elevated, suggesting adaptive upregulation po-tentially to compensate for decreased calcium absorption(793). As discussed previously, the decreased 25(OH)-vita-min D could be indicative of augmented catabolism of thevitamin (244). Serum calcium is commonly decreased or inthe lower normal range, while intestinal calcium absorptionis increased, presumably in response to elevated 1,25(OH)2-vitamin D (110, 974). Secondary hyperparathyroidism doesnot manifest (793, 974).

In general, the disturbance in mineral metabolism is morepronounced in gastrectomized patients than in vagoto-mized patients, as evidenced by the higher incidence of bonedisease and secondary hyperparathyroidism. It is challeng-ing to draw clear conclusions on the influence of gastric acidon calcium absorption in either patient group. Yet, it isapparent that compensatory mechanisms are in place inthese patients, as evidenced by the increased 1,25(OH)2-vitamin D and PTH levels. Less efficient calcium uptake dueto decreased acid output may be one explanation for this,but without further analysis this conclusion remains specu-lative.

With the advent of PPIs and H2 blockers, the number ofsurgical interventions to control acid-related disorders de-creased massively. Given their high specificity, PPIs selec-tively eliminate gastric acid output. Several investigationsthat try to tie PPI intake to a disturbance in mineral metab-olism exist. Graziani et al. (396) observed in eight healthyvolunteers that postprandial calcium concentrations didnot increase in subjects on a PPI regime (omeprazole 20 mg3� daily), whereas control subjects demonstrated a clearspike in serum calcium levels. Urine calcium excretion wasalso reduced compared with the control group (396). Asimilar effect of PPIs was later observed by two independentgroups in patients undergoing hemodialysis (395, 423). Itshould be noted that neither of these studies directly as-sessed intestinal calcium absorption, but rather measuredserum calcium as an indirectly related parameter. Morerecently, intestinal calcium absorption was measured byO’Connell et al. (807) using a radiolabeled calcium isotope.The investigators reported that 7 days of PPI (omeprazole20 mg 1� daily) intake significantly reduced calcium ab-sorption in elderly women under fasting conditions com-pared with the placebo group. Although these studies sup-port a role of PPIs in reducing calcium uptake, conflicting



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evidence exists. Several investigations that assessed calciumabsorption using radioactive tracers and a whole gut lavagetechnique found no evidence for a decrease in absorptionunder short-term PPI treatment (421, 991, 1173). It is notclear why these discrepancies in the outcomes of the trialsexist. There is, however, variability in the experimentaltechnique to measure calcium uptake, the cohorts investi-gated (young vs. postemenopausal women vs. dialysis pa-tients) and the form of calcium administration (calciumsalts vs. whole meals), which may partially account for thedivergent results. Indeed, different calcium salts are ab-sorbed with different effectiveness in acid suppressed indi-viduals, which will be subject of later discussion (see sect.VC). Furthermore, different populations may have differentcapacities for endocrine compensation.

In summary, it is experimentally difficult to unmask thepotential correlation between a reduction in gastric acidityand calcium absorption, given our body’s high capacity forcompensation. In addition, slight alterations in mineral ho-meostasis may take years to manifest themselves clinically,for example, in osteopenia or fractures. Without followingup on test subjects on a long-term basis, snapshot measure-ments which may still lie within clinically normal range canbe misleading.

2. Effects of gastrectomy, vagotomy, and PPIs onmineral metabolism in the animal model

To further elucidate the problem of osteopenia followinggastrectomy or PPI use, several animal studies tried to rep-licate and expand the observations made in human testsubjects.

For example, Axelson et al. (49) measured serum calciumconcentrations in rats who had undergone parathyroidec-tomy and various surgical procedures to reduce gastric acidoutput (vagotomy, antrectomy, gastrectomy). While para-thyroidectomy alone predictably reduced serum calciumlevels, the gastric operations (with intact parathyroidglands) had little to no effect on calcium concentrations(49). Interestingly, intestinal calcium absorption was evenincreased in the latter group. The authors attributed thisobservation to a compensatory upregulation of PTH secre-tion and concomitant 1,25(OH)2-vitamin D production. Toeliminate this factor, gastrectomy or fundectomy was con-ducted after parathyroidectomy, thereby depriving the ani-mals of their compensatory machinery. This interventionresulted in massive hypocalcemia and death after a fewdays, which led the authors to conclude that acid secretionis important for the maintenance of calcium homeostasis(49). Another investigation in rats that had undergoneantrectomy (Billroth I) observed a significantly decreasedabsorption of calcium (345). Fundectomy did not affectcalcium absorption (927). However, both studies employedthe balance method to calculate calcium absorption, whichis considered less accurate than using radiotracers. In pigs,

total gastrectomy causes massively reduced calcium uptakeand secondary hyperparathyroidism (700). In this study,the duodenum was surgically bypassed by esophagojeju-nostomy, thereby eliminating the site of maximal activecalcium absorption and limiting the conclusion that can bedrawn (700).

In addition, several reports of vagotomy in a rat modelare available to us (49, 307, 308, 928). It has been dem-onstrated that vagotomy alone has no effect on the rate ofintestinal absorption (307, 928). Secondary hyperpara-thyroidism was observed by one group, while PTH levelswere reported to be unaffected by the other group (307,928). 1,25(OH)2-vitamin D was not measured, whichwould have provided further evidence of compensationdue to decreased calcium bioavailability. However, if va-gotomy and parathyroidectomy are performed together,intestinal calcium absorption is significantly impairedcompared with vagotomy or parathyroidectomy alone(307). This is in accordance with the low serum calciumconcentrations found in gastrectomized and parathyroid-ectomized rats (49).

PPIs were also used to relate acid secretion to bone dis-ease in rats. Bone weight did not change in rats that weretreated for 4 wk with omeprazole (841). Alas, calciumabsorption was not measured in these animals, and adecrease in bone weight represents a very terminal andlong-term outcome. A more recent investigation bySchinke et al. (963) demonstrates that mice which havebeen genetically manipulated to be achlorhydric (CCK2

�/�) have decreased serum calcium levels as well as de-velop osteoporosis and secondary hyperparathyroidismin an effort to maintain calcium balance (963). This studyis especially noteworthy, as acid secretion is knocked outselectively in this mouse model while the stomach re-mains intact (stomach morphology). Furthermore, a ge-netic mutation that has been associated with osteopetro-sis (a disease characterized by increased bone density)due to osteoclast malfunction was also shown to causedecreased gastric acid secretion. These patients presentwith lower serum calcium values. Rather than being aproduct of impaired bone resorption (osteoclast defect),the hypocalcemia may thus be related to impaired intes-tinal calcium absorption (gastric acid secretory defect)(963).

C. Calcium Salts

Many investigators have employed calcium salts to de-termine the efficacy of intestinal calcium absorption. Fur-thermore, calcium salts are in wide clinical use as a di-etary supplement. As will be discussed in this section,calcium salts differ in their bioavailability, which notonly represents a potential source of error in experimen-tal designs, but more importantly, has extensive clinicalimplications.



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Calcium salts represent the most common supplementationform of calcium for individuals who do not meet their ad-equate daily intake. The indications for supplementationcan be diverse, but mostly include conditions such as osteo-porosis/-penia or preventative intake after menopause, dur-ing glucocorticoid intake or if lactose intolerant. In the year2000, the National Health Interview Survey concluded that11% of Americans ingest calcium supplements on a dailybasis (739). Females account for 80% of this population,mostly to ensure supply after menopause (739). Calciumsalts exist in multiple formulations. The most commonlyused salts are calcium carbonate, calcium citrate, calciumlactate, and calcium gluconate. Calcium carbonate is themost widely used formulation, because it contains the high-est percentage of elemental calcium per weight (40%),which equates to small tablet size and easier ingestion (997).In comparison, calcium citrate contains 21% elemental cal-cium, calcium lactate 14%, and calcium gluconate 9%(997). However, the calcium salts do not only differ in theircalcium fraction, but are massively divergent with regard totheir solubility in water. Calcium carbonate is the least wa-ter-soluble salt at a neutral pH. For example, calcium citratedissolves 17 times more readily in water than calcium car-bonate (997). Very fundamental in vitro solubility experi-ments have shown that after 1 h in 500 ml of water only 1%of the initial 500 mg of calcium carbonate are dissolved at37°C (997). The solubility of calcium carbonate can begreatly improved by an acidic environment (390, 997). Ad-justing the pH to 5.5 in the same experiment dissolves 86%of the calcium carbonate; further lowering it to 2.5, a valuethat can be expected in the stomach, increases the dissolvedfraction to 100%. Given these differences in solubility, aplethora of studies have investigated the bioavailability ofthe various calcium salts, mostly focusing on the differencebetween calcium carbonate and calcium citrate. Again, theconclusions are heterogenic. Several studies suggest thatcalcium carbonate is absorbed less effectively than the moresoluble calcium citrate (422, 438, 439, 789), while othersconclude that there is no difference in bioavailability (432,433, 520, 547, 831, 832, 891, 997, 1038). A detailed anal-ysis of the individual trials is beyond the scope of this re-view. It suffices to say that there is strong variability in theexperimental methods (direct absorption measurements vs.measurement of postprandial serum calcium vs. urine ex-cretion) and design of the studies (populations; administra-tion in the fasting state vs. with a meal). A confoundingfactor to the results of these studies may be the gastric pH atthe time of the measurement. As discussed earlier, calciumcarbonate is not very soluble at more alkali pH values,which may have implications for patients using these sup-plements while on PPI therapy (997). Furthermore, mealsdramatically affect gastric pH and may change the bioavail-ability of the supplements. Indeed, there seems to be a cor-relation between gastric pH and the absorbability of cal-cium carbonate. The first observation indicative of this as-sociation was made by Ivanovich et al. in the late 1960s

(520). The group reported that absorption of calcium car-bonate was severely impaired in four male patients sufferingfrom achlorhydria (520). Compared with five control sub-jects, who absorbed between 9 and 18% of the ingestedcalcium carbonate, these patients only absorbed 0–2%. In-terestingly, when gastric acid secretion of one of these pa-tients was stimulated by administration of betazol hydro-chloride (a histamine analog), calcium carbonate absorp-tion rose from 2 to 10% (520). This investigation somewhatspawned the entire controversy of whether gastric acid isnecessary to absorb calcium effectively from the intestine. Asimilar investigation was conducted later by Recker(891) in a larger sample of achlorhydric patients. Theinvestigator concluded that 1) control subjects absorbcalcium carbonate and calcium citrate equally well,2) but that achlorhydric patients lose their capability toabsorb calcium carbonate, while calcium citrate absorp-tion is increased (presumably through compensatory up-regulation of the absorption via vitamin D) (891). It isimportant to mention that these absorption assays wereconducted in the fasting state. When the achlorhydricpatients ingested the calcium carbonate salt together witha meal, their calcium uptake normalized. It cannot beconclusively answered which factor of the meal was re-sponsible for the increase, as several food components,such as fiber and protein, are known to affect calciumuptake. However, the authors speculated that the pH(5.8) of the meal was sufficiently low to dissolve theingested calcium carbonate (891). Furthermore, a previ-ously cited study that demonstrated decreased calciumabsorption under PPI therapy employed calcium carbon-ate as source of calcium for the conducted measurements(807). Patients with gastric bypass surgery also absorbcalcium carbonate less effectively than calcium citrate(1089). The importance of acid for the absorption ofcalcium carbonate was also demonstrated in the previ-ously discussed achlorhydric CCK2 (�/�) mouse model(963). The osteoporotic phenotype and secondary hyper-parathyroidism in these achlorhydric mice could only befully rescued by a high-calcium gluconate (2%) diet, butnot by a high-calcium carbonate (2%) diet (963). In thelight of these reports, it is evident that although the bio-availability of calcium salts in the healthy individual maybe equal, an impairment of acid secretion has a negativeeffect on the bioavailability of calcium carbonate, pre-sumably because of decreased solubility. Given the factthat calcium carbonate is the most commonly used for-mulation for calcium substitution therapy, these findingsmay partially account for the statistical correlation be-tween PPI use and the increased risk of sustaining frac-tures.

Another factor that needs to be taken into considerationwhen assessing solubility of calcium salts is the PCO2

(390). In the local milieu of the duodenum, the PCO2 canreach values of up to 300 mmHg, resulting from the



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pancreatic secretion of bicarbonate (929). Solubility ex-periments of calcium carbonate have shown that a highPCO2 negatively affects its solubility, as the carbonateenters equilibrium with CO2 (390). Compared with othercalcium salts, the particularly low bioavailability of cal-cium carbonate in acid suppressed patients may thus be acompounded effect of reduced acid secretion and a highduodenal PCO2.

D. The Endocrine Stomach andCalcium Homeostasis

Apart from being a mere acid secretory organ, the stomachalso plays an important role as an endocrine organ. Itshould be noted that all of the aforementioned surgical orpharmacological interventions, i.e., gastrectomies, vagoto-mies, or pharmacological acid suppression therapy, will in-evitably impact the endocrine functions of the stomach. It istherefore plausible that not only the changes in intragastricpH affect the absorption of calcium, but that the dysregu-lation of the endocrine stomach is responsible for changes incalcium homeostasis. The following section will addresshow hormones that are secreted by the stomach may impactcalcium and bone homeostasis.

1. Ghrelin

Ghrelin has been discovered fairly recently (1999) by Ko-jima and colleagues and is mainly implicated in regulatingfood intake in the hypothalamus (588, 777). Ghrelin levelsare inversely correlated with body mass and elevated inconditions of fasting, such as anorexia nervosa (377). Ghre-lin is mainly synthesized and secreted in a pulsatile mannerby special neuroendocrine cells (P/D1 cells) in the fundicregion of the stomach (242, 777). The influence of ghrelinon gastric acid secretion is discussed in a separate section(see section IIB5E). A few years after its discovery, it wasshown that ghrelin can also directly affect osteoblasts (254,357, 576, 691). Ghrelin induces osteoblast proliferationand differentiation and inhibits their apoptosis (357, 576,691, 1141). It is not entirely clear whether this effect ismediated via the ghrelin surface receptor, the growth hor-mone secretagogue receptor 1a (GHS-R1a), or not. Whilethe receptor is expressed in rat and murine osteoblasts andits pharmacological inhibition abolishes the effects of ghre-lin on differentiation and proliferation, no GHS-R1amRNA could be detected in a human osteoblast cell line(254, 357, 691). It should be noted that this effect is inde-pendent of growth hormone (GH). Ghrelin serves as a po-tent stimulator of GH secretion from the pituitary gland,which in turn acts as an activator of osteoblasts through theGH/IGF-I axis. However, the observations that 1) pharma-cological inhibition of GHS-R1a attenuates the effects ofghrelin and that 2) GH-deficient rats are still sensitive toghrelin, suggest a direct effect on osteoblasts (357). In vivo,the activation of osteoblasts translates to an increase in

bone mineral density (BMD) in rat and murine models (261,357). Ghrelin also promotes the formation of new bonefollowing injury (261). For example, mice that received astandardized bone injury demonstrated 1.6 times more newbone surface if treated with ghrelin compared with controlanimals (261).

Several studies aimed to identify a link between serum ghre-lin levels and BMD in human populations. The most recent,and one of the largest (n � 707 subjects), investigationassessed BMD with peripheral quantitative computed to-mography (pQCT). This technique allows for separateanalysis of trabecular and cortical bone. The results showeda positive correlation between ghrelin and trabecular BMDin elderly men and women (775). A different large-scalestudy, investigating a similar cohort (n � 977) found noassociation using dual-energy X-ray and single-photon ab-sorptiometry (1157). These techniques, however, do notpermit a discrimination between cortical and trabecularbone. Other small-scale studies also came to contradictoryconclusions (388, 811). The reason for these discrepanciesis elusive. Since the formation of trabecular bone representsa more dynamic process, its direct measurement may bemore sensitive to subtle changes than overall bone densitymeasurement (775). Baseline plasma ghrelin levels werealso shown to be inversely correlated to type 1 collagen �C-telopeptide (�CTX), a marker for bone resorption (501).

The source of ghrelin represents another potential caveat. Invitro studies suggest that osteoblasts can also synthesizeghrelin (254, 357). Ghrelin was identified on the mRNAand protein level by two investigations (254, 357). A differ-ent group did not find evidence for ghrelin in osteoblasts(214). This has important implications, as ghrelin may besecreted in an auto-/paracrine fashion, which would makeplasma ghrelin levels less significant for osteoblast activa-tion. On the other hand, (partial) gastrectomy significantlydecreases plasma ghrelin concentrations, which could con-tribute to postgastrectomy osteopenia, although these mayjust be two independent factors. Total gastrectomy causes adrop in plasma ghrelin levels by as much as 70% (528).Partial gastrectomy also severely decreases plasma ghrelin;however, levels normalize depending on the type of resec-tion to 48–88% of the preoperative levels due to compen-satory production in the remaining gastric mucosa (528).This recovery already occurs after 7days. It thus remains tobe elucidated if the slightly decreased ghrelin levels aftersmall gastric resections can account for the long-term phe-nomenon of postgastrectomy osteopenia. Furthermore, inmice, the reduction of bone mass after gastrectomy cannotbe rescued by exogenous administration of ghrelin (277).Ghrelin administration did also not affect markers of boneresorption in gastrectomized patients, although these pa-rameters were only measured very acutely 4 h after ghrelininfusion (501). So far, no data on the effect of chronicghrelin treatment on BMD are available.



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Although decreased ghrelin levels could in theory be a con-tributing factor to the reduction of bone mass followinggastrectomy, it is unknown if PPIs can directly affect ghrelinlevels. A potential link between PPI-related fractures andghrelin levels remains to be investigated. An indirect asso-ciation may be present in patients with Helicobacter pyloriinfections. These infections represent a common indicationfor PPI intake and were suggested to coincide with reducedghrelin in plasma and the gastric mucosa (527). Given therecent discovery of ghrelin’s impact on osteoblast function,many questions still remain to be answered. It is, however,clear that our view of the stomach as a mere acid secretorypouch needs to be expanded to a new level.

2. Gastrin

Gastrin represents one of the main acid secretagogues (seesect. IIB2). It is secreted by specialized G-cells in the antrumof the stomach and the duodenum. The released gastrinenters the circulation and induces acid secretion in gastricparietal cells via the CCK2 receptor. It has been hypothe-sized fairly early that plasma gastrin may have an impact onbone metabolism. Injection of gastrin and its synthetic an-alog pentagastrin was shown to decrease plasma calciumlevels in pigs and rats in the 1970s (222, 980). This effectwas attributed to gastrin-stimulated release of calcitoninfrom the thyroid gland. Indeed, pentagastrin was shown tobe a potent stimulator of calcitonin secretion in variousspecies and is still in clinical use to evaluate thyroid C-cellhyperplasia and medullary carcinomas (155, 156, 226, 441,829). Although a clinical correlation between plasma gas-trin levels and plasma calcitonin has been demonstrated byone study in patients with Zollinger-Ellison syndrome (hy-pergastrinemic patients) and in pigs, it is unclear if nativegastrin, i.e., not pentagastrin, acts as an important secreta-gogue for calcitonin in humans (224, 1020). In fact, otherinvestigations found no association between gastrin andcalcitonin levels in other cohorts (122, 454).

At least in the rat, the hypothesis of the gastrin-calcitoninaxis has been severely challenged. Although gastrin de-creases plasma calcium levels in rats, the same effect occursin (para)thyroidectomized animals, suggesting that calci-tonin is not involved in this process (980). Furthermore,cultured rat thyroid cells could not be stimulated to releasecalcitonin if incubated with gastrin (225). Fundectomy- andomeprazole-induced hypergastrinemia also did not affectcalcitonin levels in rats (843, 927). Interestingly, hypocal-cemia after gastrin injection could not be induced in ratsthat had been (para)thyroidectomized and gastrectomized(844, 981). This observation led to the conclusion that gas-trin may stimulate the release of an unknown substancefrom the rat stomach, which in turn exerts calcitropic ac-tivity. In accordance with this hypothesis, mucosal extractsfrom rat stomachs were shown to have the same hypocal-cemic effects as gastrin and to stimulate uptake of radiola-beled calcium into the bone (844). These findings were also

replicated in chicken (842). The unknown hormone wastentatively named ”gastrocalcin“ (844). When ghrelin wasdiscovered, it was speculated that it might represent a can-didate hormone for gastrocalcin. However, unlike gastro-calcin, ghrelin is not under gastrin control, making thisproposition unlikely (276). Subsequent investigations sug-gested that the origin of gastrocalcin were the gastric ECLcells. ECL extracts can indeed trigger a calcium second mes-senger response in osteoblast (629, 630). Yet, functionalevidence for gastrocalcin-mediated osteoblast activation isstill lacking. A recent report postulates that parathyroidhormone-like hormone (PTHLH) may in fact be gastrocal-cin (676). PTHLH exerts similar physiological effects asPTH by sharing a common receptor and is commonly ele-vated in paraneoplastic syndromes (1056). PTHLH hasbeen identified in ECL cells, and its transcription has beenshown to be inducible by gastrin in parietal cells (523, 676).Of note, PTH causes effects opposite to those assigned togastrin and gastrocalcin, namely, hypercalcemia. Furtherstudies will thus be needed to corroborate this hypothesis.

Whatever the exact effector hormone of gastrin may be,changes in gastrin levels cannot entirely explain the clinicalphenomenon of postgastrectomy osteopenia and PPI-re-lated fractures. Although vagotomy and PPIs undoubtedlyincrease serum gastrin levels through a negative-feedbackmechanism, most partial and all total gastrectomies resultin hypogastrinemia. Yet both hyper- and hypogastrinemicconditions have similar outcomes, i.e., osteopenia and in-creased risks of fractures. It is, of course, plausible thatdifferent factors contribute to this outcome in each individ-ual group. Gastrin may be involved in certain pathologies,but given that its true impact on bone metabolism is some-what elusive, this assumption remains speculative.

Acid suppression

?

Gastrin

Pancreastatin Calcitonin

Calcium solubility

Intestinal absorption

PTH

FIGURE 9. Model summarizing the potential impact of acid sup-pression on calcium homeostasis.



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3. Pancreastatin

Pancreastatin is a cleavage product of chromogranin A thatwas initially isolated from porcine pancreas (1072). GastricECL cells are also known to harbor significant amounts ofchromogranin A and pancreastatin. Pancreastatin is se-creted together with histamine from ECL cells in responseto their neuroendocrine stimulation (see sect. IIB3) (176). Inrat, it has been shown that the serum pancreastatin levelscorrelate with the secretory status of ECL cells. States thatenhance ECL cell secretion, such as gastrin infusion, re-sulted in elevated serum pancreastatin levels (411). In ac-cordance with this hypothesis, and of special relevance forthe topic of this review, PPI therapy also resulted in in-creased serum pancreastatin levels (the ECL is stimulated bygastrin, which in turn is released in response to high gastricpH) (411). These observations led the investigators to con-clude that ECL cells are a major contributor to the serumlevels of pancreastatin in the rat and that these levels changein parallel with ECL cell secretion (411). This is also cor-roborated by the observation that gastrectomy reduces pan-creastatin levels in rats (644).

Pancreastatin exerts a variety of metabolic effects. Apartfrom influencing energy metabolism, pancreasstatin wasshown to affect the secretion of PTH from the parathyroidgland. In isolated bovine and porcine parathyroid cells,pancreastatin has a clear inhibitory effect on PTH secretion(282, 317, 911). The suppression of PTH functions on atranscriptional level (1211). Reduced PTH secretion in turnwould have a potential impact on calcium and bone metab-olism. Whether the same observations are valid for humansis less clear, as pancreastatin failed to inhibit PTH secretionfrom isolated human parathyroid cells (911). Regardless,the volume of data on pancreastatin and its influence on theparathyroid gland is very small, and further investigationswould be necessary to establish this intriguing link. Apartfrom potential modulation of parathyroid secretion, pan-creastatin has also been shown to have an inhibitory effecton gastric acid secretion (655).

In summary, it should be noted that the stomach secretesnot only acid, but also hormones that have been shown todirectly alter calcium and/or bone homeostasis. The secre-tion of these hormones depends on the neuroendocrine ma-chinery that also regulates acid secretion. It is thereforeplausible that the correlation between states of impairedacid secretion and impaired bone mineralization is multi-factorial by depending on intragastric pH and serum levelsof gastric hormones (FIGURE 9).

VI. CONCLUSIONS

We set out in this review to demonstrate that gastric andintestinal physiology are intertwined to regulate calciumabsorption and secretion to maintain bone health. In this

review we have focused on the important role calcium playsas a first and second messenger in the maintenance of bonehealth. By relying on a complex series of receptors, chan-nels, and transport proteins, calcium is tightly controlled atthe cellular and tissue level to ensure its bioavailability tobone. Modulations to any of these pathways by disease,mutation, or pharmaceutical perturbation can lead to clin-ical changes in bone health.

ACKNOWLEDGMENTS

S. Kopic is a Howard Hughes Medical Institute Interna-tional Student Research Fellow. Special thanks to SashkaDimitrievska for her untiring support and critical editing ofthe manuscript.

Address for reprint requests and other correspondence: J. P.Geibel, Yale School of Medicine, 310 Cedar St., BML 238,New Haven, CT 06510 (e-mail: [email protected]).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declaredby the authors.

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