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Biochemical and Biophysical Research Communications 268, 636–641 (2000)

doi:10.1006/bbrc.2000.2159, available online at http://www.idealibrary.com on

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oncentrations in Human Parathyroid Cells

adu Mihai, Teresa Lai, George Schofield,* and John R. Farndonepartment of Surgery, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom; and *Department of Biochemistry,chool of Medical Sciences, University of Bristol, Bristol, United Kingdom

eceived December 29, 1999

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Effects of extracellular calcium ([Ca21]ext) on para-hyroid cells are mainly due to the activation of alasma membrane calcium receptor (CaR) coupledith release of intracellular calcium. In addition,igh [Ca21]ext activates the sphingomyelin pathway

n bovine parathyroid cells, generating ceramidesnd sphingosine. This study explored the direct ef-ects of synthetic ceramides on [Ca21]i in humanarathyroid cells. Cells from five parathyroid adeno-as removed from patients with primary hyperpara-

hyroidism were dispersed and maintained in pri-ary culture. Intracellular calcium concentration

[Ca21]i) [Ca21]i was monitored using standard quan-itative fluorescence microscopy in Fura-2/AM-oaded cells. Laser scanning microscopy was used to

onitor the intracellular distribution of a fluores-ent ceramide analogue (BODIPY-C5). After addi-ion of 10 mM C2-ceramide (N-acetyl-D-erythro-phingosine), [Ca21]i increased rapidly (30–60 s) to aeak three times above basal levels in 70% of cells37/55 cells in four experiments). This effect ap-eared to be due to release of Ca21 from intracellulartores rather than Ca21 entry from the extracellularedium. C2-responsive cells had a smaller [Ca21]i

esponse to subsequent stimulation with the CaRgonist—neomycin (1 mM). These responses werepecific to C2 since C6-ceramide (N-hexanoyl-D-rythro-sphingosine) did not affect basal [Ca21]i norhe responses to an increase in [Ca21]ext and to neo-ycin. C5-BODIPY generated intense perinuclearuorescence, suggesting targeting of the ceramideso the Golgi apparatus. These data demonstrate thatndogenous generation of ceramides has the poten-ial to modulate changes in [Ca21]i and secretion inesponse to [Ca21]ext in human parathyroid cells.2000 Academic Press

Key Words: human parathyroid cells; hyperparathy-oidism; C2-, C6-ceramide; sphingosine; Fura-2/AM;5-BODIPY; fluorescence microscopy; confocalicroscopy.

636006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

ain factor controlling secretion from parathyroidells. Changes in [Ca21]ext are sensed by a plasma mem-rane calcium receptor (CaR) (1). High [Ca21]ext andther agents (e.g. neomycin and trivalent cations La31

nd Gd31) stimulate the CaR, which is coupled to the-protein-phospholipase C pathway that leads to gen-ration of inositol 1,4,5-trisphosphate (IP3) and releasef Ca21 from intracellular stores (1). In this way, highCa21]ext is translated into high [Ca21]i and this inhibitsecretion through an as yet unknown mechanism.In bovine parathyroid cells high [Ca21]ext increases the

oncentration of sphingosine (2), one of the intermediatesn the sphingomyelin pathway. In many cell types, thephingomyelin pathway is involved in the regulation ofell proliferation, cell-cell interaction, differentiation andncogenesis (3). Its activation begins with hydrolysis ofembrane sphingomyelin by a neutral sphingomyeli-ase, leading to increased ceramide levels in target cells.he hydrolysis of ceramide by a ceramidase produces freephingosine, which can be further converted tophingosine-1-phosphate (by a sphingosine-kinase) or tophingosine-phosphorylcholine. Although very little isnown about the effects of sphingolipids in the parathy-oid cells, the increase in sphingosine concentration inesponse to [Ca21]ext (2) raises the possibility that thephingomyelin pathway is functionally important inarathyroid cell.The aim of this study was to explore the possibility

hat compounds generated through the sphingomyelinathway modulate the response of human parathyroidells to changes in [Ca21]ext. The effects of syntheticeramides on [Ca21]i were investigated in human para-hyroid cells from patients with primary hyperparathy-oidism. [Ca21]i was measured in individual cells usinghe widely-accepted method of quantitative fluores-ence microscopy in cells loaded with the calcium-arker Fura-2/AM. Confocal microscopy was used toonitor the intracellular distribution of a fluorescent

eramide analogue (C5-BODIPY).

MATERIAL AND METHODS

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Preparation of dispersed human parathyroid cells. A single para-hyroid adenoma was removed from each of five patients with pri-ary hyperparathyroidism. There were four women and one man,

ge 24–75 years (median 59 years). All patients had hypercalcaemiamean serum calcium 2.96 6 0.31, range 2.61–3.77 mmol/L, refer-nce range 2.20–2.60 mmol/L) and high intact PTH concentrations18.3 6 7.6 pmol/L, range 8.2–46, reference range 2.7–7.0 pmol/L).ntact PTH (1–84) was measured using a chemiluminescent assayImmulite, DPC, Gwynedd, UK) (coefficient of variance 4.7% and.5% for intrassay and interassay precision, respectively).The adenomas were collected from the operating theatre and

tored in ice-cold culture medium (RPMI-1640, supplemented with0% new born calf serum, 1% glutamine, penicillin 100 U/mL, strep-omycin 0.1 mg/mL and amphotericin B 0.25 mg/mL). The outerapsule was removed, the tissue minced with scissors into 1 mmragments and digested for 45 min at 37°C in RPMI 1640 with 2g/mL collagenase (type 2) and 0.2 mg/mL DNase (Sigma). The

igest was thereafter mechanically dispersed by aspiration througheedles of decreasing gauge (No. 19–22). The cell suspension wasltered through a 100 mm mesh stainless steel gauze and centrifugedt 200G for 5 min. Cell cultures were incubated at 37°C, underO2:air mixture (5%:95%) for 24–72 h. All culture surfaces (glass

overslips and petri dishes) were coated with Pronectin F.

Quantitative fluorescence microscopy. Cells grown on glass cov-rslips were immersed in medium containing: NaCl 129 mM, KCl 4.7M, HEPES 11 mM, NaHCO3 4.7 mM, NaH2PO4 2.7 mM, CaCl2 1.8M, MgCl2 1.2 mM and glucose 5.5 mM, at pH 7.4. Cells were

ncubated with 2 mM Fura-2/AM at room temperature for 45–60 minnd then washed with medium and kept until experiments wereerformed (not more than 4 h).The coverslips mounted into a perfusion chamber were transferred

nto the stage of a Nikon DIAPHOT inverted fluorescence micro-cope with a Nikon CF-series UV-Fluor 340 oil immersion objectivenumerical aperture 1.3). Cells were alternatively excited at twoave lengths using interference filters (340 nm and 380 nm)ounted on a wheel turned by a computer-controlled stepper motor.Nikon 455 nm dichroic mirror reflected the 340 and 380 nm light

ut allowed the fluorescent signal of Fura-2 (510 nm) to pass. Cellsere incubated in darkness and exposed alternatively to the appro-riate excitation wavelengths for only 0.3 s to reduce photobleaching.he intervals between stimulations (1–10 s) were computer-ontrolled. During each capture, 10 images were combined, averagednd stored. [Ca21]i was determined using a calibration-by-ratioethod and reported as ratio of fluorescence at 340 and 380 nm

R340/380). All experiments were performed at ambient room tempera-ure (22–25°C).

Confocal microscopy. A Leica DM-IRBE inverted epifluorescenceicroscope with phase-contrast (340 Leica objective) was connected

o a laser scanning confocal microscope (Leica TCS-NT) equippedith Krypton/Argon mixed gas laser, generating two wavelengths

568 and 488 nm). The scan head filters were selected to optimise theavelength for FM1-43 and the rhodamine filter set was always used

bandpass filter 514 nm, dichroic 510 nm, emission above 515 nm).he glass coverslips with cells attached were immersed in the exper-

mental medium before being transferred on the microscope stage.Images were collected at equal intervals (30–60 s) using the con-

ocal software. The intensity of fluorescence was represented by aomputer-generated pseudo-colour scale running from black (lowntensity) via blue, green and red to white (high intensity). Alluorescence measurements were expressed in arbitrary units gen-rated from the pixel grey scale. Each image represented the averagef four different frames, collected and averaged at a rate of one frameer second. The imaging parameters were constant for data pre-ented in the same figure but not between figures.

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Chemicals and reagents. RPMI-1640 culture medium, glutamine,enicillin, streptomycin, amphotericin B were from GibcoBRL (Pais-ey, UK). Pronectin F was purchased from TCS Biologicals (Botolphlaydon, Buckingham, UK). Experimental medium was preparedsing chemical of standard purity from Sigma (Poole, Dorset, UK)nd BDH (Poole, Dorset, UK).Fura-2/AM, C2-ceramide (N-acetyl-D-erythro-sphingosine), C6-

eramide (N-hexanoyl-D-erythro-sphingosine), sphingosine (D-erythro-phingosine) and sphingosine-1-phosphate (D-erythro-sphingosine-1-hosphate) were from Calbiochem (Beeston, Nottingham, UK). C5-ODIPY was purchased from Molecular Probes (Eugene, OR).

Statistical analysis. Differences in the responses of individualells at different time-points of the experimental protocol were com-ared using paired Student’s t test.

ESULTS

ffects on [Ca21]i Homeostasis

During incubation in Ca21-free medium C2-ceramide10 mM) induced a transient increase in [Ca21]i in the

ajority of cells (37/55 cells dispersed from two para-hyroid adenomas and analysed in four experiments).Ca21]i increased rapidly (within 30–60 s) to a peakhree times above basal levels (R340/380 1.52 6 0.22 vs..56 6 0.12) (section 1 in Fig. 1). When subsequentlyhallenged with a CaR agonist (neomycin 1 mM), themplitude of the [Ca21]i spike was smaller in C2-esponsive cells compared with C2-nonresponsive cellssection 2, Fig. 1): R340/380 was 1.45 6 0.30 and 1.14 6.22, respectively (p , 0.05). After the Ca21 stores hadeen replenished in the presence of extracellular Ca21

section 3, Fig. 1) the responses to a second adminis-ration of neomycin were similar in both groups of cells

FIG. 1. Relationship between patterns of [Ca21]i responses to2-ceramide and neomycin in human parathyroid cells. Cells wereivided in two groups based on whether [Ca21]i increased or not inesponse to C2-ceramide (filled and empty squares, n 5 21 and n 58 cells, respectively). Horizontal bars mark changes in [Ca21]ext

rom 0 mM (lower bars) to 0.5 mM and 3 mM (upper bars). Filledorizontal bars mark the presence of C2-ceramide (10 mM) in the

ncubation medium. Addition of neomycin (1 mM) is marked byorizontal lines.

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i.e., C2-nonresponsive and C2-responsive). These datauggest that C2 and neomycin access the same intra-ellular Ca21 pool, which was not completely emptiedy the initial incubation with C2 10 mM.The amplitude and dynamic of [Ca21]i responses to2 were similar in Ca21 free medium and at 3 mM

Ca21]ext (sections 5 and 1, Fig. 1), suggesting that Ca21

ntry was not part of C2 response.C6-ceramide (10 mM) had no effect on basal [Ca21]i in

6 cells tested at 0, 0.5 and 3 mM [Ca21]ext. Further-ore, at 0.5 mM [Ca21]ext C6-ceramide did not influ-

nce neomycin-induced responses (Fig. 2): the peak ofCa21]i responses to neomycin in the presence of C6 10M had similar shape and amplitude as the responseefore addition of C6 (section 5 vs. 1 and 3, Fig. 2).A brief (5 min) incubation with C6 10 mM appeared

o improve the responsiveness to high [Ca21]ext (Fig. 3):fter an initial exposure to 3 mM [Ca21]ext, cells had amall [Ca21]i response to a step increase in [Ca21]ext

roviding incubation at low [Ca21]ext was brief (i.e. lesshan 5–10 min) (sections 2 vs. 1, Fig. 4). If C6 ceramide10 mM) was present during incubation at 0.5 mMCa21]ext for 5 min, the [Ca21]i response to stepping to 3M [Ca21]ext was of significantly higher amplitude

R340/380 1.38 6 0.32 vs. 0.92 6 0.26, p , 0.002 ontudents’ paired t-test) (section 3 vs. 2, Fig. 3).Sphingosine (1–50 mM) had no effect on [Ca21]i dur-

ng incubation in Ca21-free medium. At a very highoncentration of sphingosine (100 mM), however, aransient raise in [Ca21]i was observed after 5 min,ith return to basal levels after a further 10–15 min.ollowing incubation with 100 mM sphingosine for 45in the response to the CaR agonist (neomycin 1 mM)

FIG. 2. Incubation with C6 ceramide does not alter the respon-iveness to the CaR agonists neomycin. Symbols represent averageesponse (mean 1 SD) for n 5 66 cells dispersed from a primaryarathyroid adenoma. [Ca21]ext was 0.5 mM during the entire exper-ment. Horizontal lines mark addition of neomycin (1 mM) and theorizontal bar marks the presence of C6 (10 mM) in the incubationedium.

638

as abolished while the responsiveness to 3 mMCa21]ext was maintained.

Sphingosine 10 mM had no significant influence onhe amplitude of [Ca21]i responses to a step from 0.5 tomM [Ca21]ext (Fig. 4).A protocol designed to study the effects of sphin-

osine on capacitative Ca21-entry following neomycin-nduced Ca21 release is illustrated in Fig. 5. The initialequence of the protocol demonstrated the neomycin-nduced [Ca21]i peak during incubation in Ca21-free

edium (section 1, corresponding to release of Ca21

FIG. 3. Incubation with C6-ceramide favours the responsivenesso high [Ca21]ext. Symbols represent average response (mean 1 SD)or n 5 43 cells from a primary parathyroid adenoma. Horizontalars mark changes in [Ca21]ext from 0.5 mM (lower bars) to 3 mMupper bars). The filled horizontal bar marks the presence of C6 (10M) in the incubation medium.

FIG. 4. Sphingosine does not affect the responsiveness of humanarathyroid cells to stepping [Ca21]ext. Symbols represent averageCa21]i changes (mean 1SD) for n 5 31 cells in response to a steprom 3 mM [Ca21]ext to Ca21-free medium. Horizontal bars markhanges in [Ca21]ext 0 mM (lower bars) to 3 mM (upper bars). Theorizontal bar mark addition of sphingosine (10 mM).

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rom intracellular pools). This was followed by an in-rease in [Ca21]i when extracellular Ca21 became avail-ble (section 2, corresponding to Ca21 entry). These twoypes of [Ca21]i changes were further reproduced withimilar amplitude in the absence and in the presence ofphingosine 10 mM (sections 3 and 4 vs. 5 and 6, re-pectively). No effects of sphingosine on the amplitudend dynamic of these responses were observed.Sphingosine-1-phosphate had no effect on [Ca21]i in

uman parathyroid cells.

ffects of Ceramides on Secretion-Coupled MembraneTurnover

Laser scanning microscopy was used to monitor thentracellular distribution of a fluorescent ceramide an-logue (BODIPY-C5). An intense perinuclear fluores-ence was observed within 1–2 min incubation with

mM C5-BODIPY, suggesting targeting of the Golgipparatus.

ISCUSSION

The aim of this study was to investigate the possi-ility that intermediates generated by the sphingomy-lin pathway modulate the responses of parathyroidells to stimulation of the CaR. Such modulation couldrovide a functional linkage between the effects ofCa21]ext on the sphingomyelin pathway (2) and on se-retory responses in parathyroid cells.

The study used human parathyroid cells from ade-omas removed during surgical treatment for primaryyperparathyroidism. In this disease, the parathyroidells are less sensitive to changes in [Ca21]ext so that

FIG. 5. Lack of effects of sphingosine on neomycin-induced [Ca21

ddition of neomycin (1 mM) is marked by horizontal lines and sphingo 0.5 mM are marked by the empty horizontal bars.

639

igher concentrations than normal (i.e. hypercalcae-ia) are required to produce the typical inhibition ofTH secretion by [Ca21]ext (4). Normal parathyroid cellsere not available because of ethical issues related toypoparathyroidism following excision of normallands. Because the experimental technique (i.e. fluo-escence microscopy) allowed analysis of responses inndividual cells, each cell acted as its own internalontrol and paired Student’s t-test was used to com-are [Ca21]i in the same cells at different time-points ofhe protocols.

In this study, C2-ceramide (10 mM) increased theCa21]i in over 60% of human parathyroid cells. Theesponse was rapid, occurring in seconds and tran-ient. Furthermore, exposure to C2-ceramide de-reased the subsequent responses to neomycin (Figs.–4). Neomycin is an agonist of the CaR (1) and there-ore mimics activation of CaR by an increase inCa21]ext, activating phospholipase C and initiating IP3-

ediated Ca21 mobilisation (1). It appears, therefore,hat C2-induced rise in [Ca21]i is due to release of Ca21

rom intracellular pools similar/identical with thoseesponsive to generation of inositol 1,4,5-trisphosphateIP3).

Interestingly, C6-ceramide had no effect on [Ca21]i inuman parathyroid cells. Since there are only minimalifferences between the two molecules (i.e. ethanol-nd hexanoil-D-erythro-sphingosine), this observationuggests that the effects of C2-ceramide involve a verypecific recognition system. The possibility that C6 isot taken up by cells cannot be ruled out. Interestingly,he situation is reversed in thrombin activated plate-ets, in which C6 potentiates Ca21 mobilisation andnflux while C2 has no significant effects (5).

ymbols represent average responses (mean 1 SD) for n 5 61 cells.ne by the horizontal bar. Changes in [Ca21]ext from Ca21-free medium

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as unexpected since in other cell types sphingosinenduces IP3 accumulation and Ca21 mobilisation (6, 7)r Ca21 entry (8). Because in some cell types theseffects are temperature-dependent (9), experimentsere carried out during incubation at 37°C and con-rmed the lack of effects on parathyroid cells (data nothown). In parallel experiments it has been shown thatoth ceramides and sphingosine increases [Ca21]i in abroblast cell line (10), demonstrating the biologicalctivity of the synthetic compounds used in these ex-eriments.Sphingosine did not mobilise intracellular Ca21 and

id not influence responses to neomycin (Fig. 5), sug-esting that it does not affect the IP3-dependent Ca21

obilisation. Furthermore, sphingosine did not en-ance [Ca21]i responses to a step from 0.5 mM to 3 mMCa21]ext (Fig. 4), suggesting that it does not influencea21 entry in parathyroid cells. These data appear in

ontrast with those reported in the human Jurkat Tell line, in which sphingosine inhibits Ca21 influx gen-rated by thapsigargin and ionomycin, suggesting thatt activates the Ca21 extrusion process in some cells11).

The lack of effects of sphingosine on [Ca21]ext-inducedhanges in [Ca21]i was unexpected because it is alsonown that sphingosine modulates protein phosphor-lation by inhibiting several kinases, such as proteininase C (12) (which mediates many of its biologicalffects (13)), calmodulin-dependent kinases (14) andnsulin-receptor-tyrosine-kinase (15). It is interestingherefore that sphingosine does not alter responses toeomycin since protein kinase C is known to modulateCa21]ext-responsiveness (16).

Sphingosine metabolites affect Ca21 homeostasis inarious cells. In different cell types sphingosine-1-hosphate releases Ca21 from endoplasmic reticulum17) and sphingosyl-phosphorylcholine activates Ca21

elease from endoplasmic reticulum (18) by acting as aa21-selective channel (19). However, no effects ofphingosine-1-phosphate on [Ca21]i were observed inuman parathyroid cells.In this study, the effects of synthetic ceramides and

phingosine were compared because such experimentsould provide information on whether a specific part ofhe sphingomyelin signalling cascade is predominant.his hypothesis was based on the fact that conversionf ceramides into sphingosine can be evaluated by com-aring the effects of the synthetic compounds: if cellso not possess or do not activate the enzymes necessaryor conversion of ceramides into sphingosine, the ef-ects of the corresponding synthetic compounds williffer while in cells were such a conversion occurs thewo compounds have similar effects. Data presenteduggest therefore that ceramides rather than sphin-osine are the main active components in human para-

640

yelin pathway.How would the present data enhance our under-

tanding of secretory mechanisms in parathyroid cells?n dispersed bovine parathyroid cells high [Ca21]ext

timulates the sphingomyelin pathway (2) and the in-ibition of PTH secretion and PKC activity by en-anced cellular diacylglycerol may result from the ac-ivation of an inhibitory second messenger pathwaynvolving the sphingoid lipids (20). Furthermore, inifferent cell models ceramides modulate both exocyto-is (21) and endocytosis (22). To further explore this,he fluorescent marker FM1-43 was used to monitorhe membrane turnover in response to changes inCa21]ext known to stimulate secretion from parathy-oid cells. Stepping [Ca21]ext from 3 mM to 0.5 mMnduced a rapid uptake of FM1-43 on the plasma mem-ranes followed by internalisation of the dye (corre-ponding to exocytosis and endocytosis, respectively).n preliminary experiments we have found that cellsreincubated with C5-BODIPY showed no subsequentM1-43 uptake in response to a decrease in [Ca21]ext

23). These data suggest therefore that ceramides areotential modulators of exocytosis and/or endocytosisn human parathyroid cells.

The direct effects of ceramides on [Ca21]i and possiblyecretion could also be the mediator for the effects ofa25-dihydroxyvitamin D3, which is a modulator of para-hyroid cell proliferation and secretion. In other cellypes, active vitamin D3 induces rapid sphingomyelinydrolysis (24). Furthermore, differentiation of someells with 1a,25-dihydroxyvitamin D3 is accompanied byphingomyelin turnover (25, 26). A novel cytosolic neu-ral sphingomyelinase has been purified from these cellsnd it has been shown to be activated two hours afterreatment with 1a,25-dihydroxyvitamin D3 (27).

Another hypothesis is suggested by the correlationetween high PTH levels and high circulating levels ofumour necrosis factor alpha (TNF-a) and interleukin(IL-6) in patients with primary hyperparathyroidism

28). In other cell types, TNF-a causes early and re-ersible sphingomyelin hydrolysis, accompanied by theoncomitant generation of ceramide (29) and reduceshe sphingomyelin content in a cell-free system derivedrom HL-60 cells (30). It is possible therefore that PTHtimulates bone cells and monocytes/macrophages toenerate various cytokines (including TNF-a) andhese cytokines in turn modulate PTH secretion viactivation of the sphingomyelin pathway.In conclusion, generation of intermediates by the

phingomyelin pathway may modulate changes inCa21]i and secretion caused by activation of CaR inarathyroid cells from patients with primary hyper-arathyroidism. Together with the previous observa-ion that this is also activated by increased [Ca21]ext (2),hese data suggest that sphingomyelin pathway maylay an important role in the regulation of parathyroid

cell secretion. Whether this modulatory mechanism issr

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ignificant for both normal and adenomatous parathy-oid cells has not been determined.

CKNOWLEDGMENTS

The Overseas Research Student Scheme Award sponsored R.M. asvisiting Ph.D. student from “Carol Davila” University Bucharest,omania. This work was supported by the Joan Greenfield Trust.onfocal microscopy was supported by a Medical Research Council

nfrastructure Award (G45006789) for the Cell Imaging Centre athe School of Medical Sciences, University of Bristol.

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