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January 20, 2005 12:12 Annual Reviews AR237-FM

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6 Jan 2005 22:7 AR AR237-PH67-01.tex AR237-PH67-01.sgm LaTeX2e(2002/01/18) P1: IKH10.1146/annurev.physiol.67.040103.152647

Annu. Rev. Physiol. 2005. 67:1–21doi: 10.1146/annurev.physiol.67.040103.152647

Copyright c© 2005 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on July 16, 2004

UNLOCKING THE SECRETS OF CELL SIGNALING

Michael J. BerridgeThe Babraham Institute, Babraham Research Campus, Babraham, Cambridge CB2 4AT,United Kingdom; email: [email protected]

Key Words calcium, inositol trisphosphate, cyclic AMP, salivary glands

■ Abstract My scientific life has been spent trying to understand how cells com-municate with each other. This interest in cell signaling began with studies on thecontrol of fluid secretion by an insect salivary gland, and the subsequent quest led tothe discovery of inositol trisphosphate (IP3) and its role in calcium signaling, whicheffectively divided my scientific career into two distinct parts. The first part was pri-marily experimental and culminated in the discovery of IP3, which set the agenda forthe second half during which I have enjoyed exploring the many functions of this re-markably versatile signaling system. It has been particularly exciting to find out howthis IP3/Ca2+ signaling pathway has been adapted to control processes as diverse asfertilization, proliferation, cell contraction, secretion, and information processing inneuronal cells.

OUT OF AFRICA

My scientific career was shaped by my early life in Africa. I was born in 1938in a small town called Gatooma (now called Kadoma) in what was then SouthernRhodesia. This country in central Africa has changed its name several times; itbecame Rhodesia and more recently Zimbabwe. My earliest memories are ofEiffel Flats, a small gold-mining town about five miles from Gatooma. It was theAfrican bush with its rich variety of animals and birds that excited me as a youngboy and has continued to fascinate me ever since. This love of nature inspired myinitial interest in academic studies. While at school, I spent some of my spare timewriting notes and drawing pictures of the animals that surrounded me, and thisearly passion evolved into a boyhood fantasy of becoming a game warden in oneof the National Parks.

At school I was fortunate in being taught biology by Pamela Bates who not onlyfostered my scholarly interests but also opened my eyes to the fact that there was anacademic life after school. None of my family had been to university except for adistant aunt who had a degree in child psychology. Because my parents consideredher to be singularly inept at bringing up her own children, they were somewhatdubious about the merits of a university education. However, Miss Bates convincedthem that I had the ability to pursue an academic career and I applied for a place

0066-4278/05/0315-0001$14.00 1

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2 BERRIDGE

at the newly founded University of Rhodesia and Nyasaland in Salisbury to readZoology and Chemistry.

I approached my university courses with great enthusiasm because I had discov-ered that the conservation of African wildlife lay in the emerging field of ecology.I had every intention of going on to do research into big game ecology. Indeed, Ispent one of my vacations working with Dr. Thane Riney, who was one of the firstecologists to work on African wildlife. One of my tasks was to mark giraffes usinga crossbow, which had arrows with paint-filled wax capsules that exploded oncontact with the animal thus spreading colored paint over the hide. We were thenable to determine their home range by plotting out their locations on subsequentsightings. For a young boy fascinated by wild animals, this was exciting work.However, there was something very imprecise about this embryonic field and myenthusiasm for a career in ecology began to wane. A new influence was at work.

The physiology lectures given by Dr. Eina Bursell began to capture my imagina-tion. I can still remember being particularly fascinated by the beauty of the staircasephenomenon in the heart responding to adrenergic stimulation. Little did I knowthat many years later I would understand the molecular signaling mechanisms re-sponsible for this intricate process. Bursell’s lectures brought out the factual detailsand precision that were lacking in my initial foray into ecology. After spendingmy next vacation helping Bursell with his research on water metabolism in tsetseflies, my transformation from a budding big game ecologist to insect physiologistwas complete. I had been bitten by the research bug and was fortunate to gain aplace in the Department of Zoology at Cambridge University to do a Ph.D. withSir Vincent Wigglesworth, the father of insect physiology.

THE FIRST CAMBRIDGE PERIOD

My journey out of Africa was somewhat daunting. Within a short period, I wastransported from the quiet colonial existence of Rhodesia into the hurly burly ofcosmopolitan London and Cambridge. Despite all my reading, nothing could haveprepared me for my new life in this ancient seat of learning. Wigglesworth was afellow of Gonville and Caius College, and he arranged for me to be a member of thiscollege. This was the college of William Harvey, who discovered the circulationof the blood. I felt all the years of academic distinction and tradition crowding inon me, especially as I was given a room in the heart of the college overlookingthe Gate of Honor. A feeling of insecurity was exacerbated by the mischievouspostdoctoral fellows in the laboratory who took great delight in informing me thatSir Vincent took on three students each year and one of them always fell by thewayside. Imagine my apprehension when I found out that the two other studentsin my year were Cambridge graduates who had their projects up and runningwithin weeks, while I was still finding my way around the cavernous spaces of theCambridge Zoology Department. The prediction about the natural wastage of SirVincent’s students turned out to be correct because one of the students droppedout after a few months making me feel marginally more secure.

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IP3 AND Ca2+ SIGNALING 3

Sir Vincent had given me a project to study nitrogen excretion of the Africancotton stainer Dysdercus fasciatus, which enabled me to use my training in bothchemistry and zoology. Once I had settled into my project, I began to enjoy theprocess of doing research especially when I discovered something quite novelabout how these insects excreted nitrogen. The big surprise was that they excretedtheir excess nitrogen as water-soluble allantoin rather than as the insoluble uricacid favored by most insects. It seems that these sap-sucking insects, which haveaccess to a constant supply of water, have no need to waste energy synthesizinguric acid when they can flush out their nitrogen as allantoin in a plentiful flowof urine (1). I also discovered that this copious flow of urine was maximal at thebeginning of each instar when the insects feed avidly and ceases as they prepare forthe next moult. Toward the end of my thesis work I began to be interested in howthis periodic cycle of fluid secretion by the Malpighian tubules was controlled,and this aspect sparked my interest in cell signaling that has continued ever since.Unfortunately, my time in Cambridge ran out, and I was not able to pursue myproject any further because I had to stop doing experiments in order to write upmy thesis. However, some of the preliminary work I had done on the hormonalcontrol of excretion formed the basis of a successful postdoctoral application toProfessor Dietrich Bodenstein, who was chairman of the Department of Biologyat the University of Virginia in Charlottesville.

AN AMERICAN SOJOURN: POSTDOCTORAL STUDIES ATTHE UNIVERSITY OF VIRGINIA AND AT CASE WESTERNRESERVE UNIVERSITY

During my time in Cambridge I fully intended to return to Africa, but politicsintervened. While I had been studying in England, a vicious civil war had brokenout in Rhodesia, and I would certainly have returned to immediate conscription tofight what I believed to be a completely unnecessary bush war. Instead, I decidedto emigrate to the United States to begin a new life as a postdoctoral fellow at theUniversity of Virginia in Charlottesville, which is a university town very similarto Cambridge. My intention was to use blowfly Malpighian tubules as a modelsystem to study hormone action. While at Cambridge, I learned how to studyMalpighian tubules in vitro from Dr. Arthur Ramsay who had spent his life de-veloping techniques for studying the physiology of these tubular secretory organs(2). The isolated tubule was set up in a drop of saline held under liquid paraffin ina watchmaker’s glass dish. A piece of silk was unraveled under liquid paraffin andindividual fibers were used as fine ligatures to pull the cut end of the tubule out intothe liquid paraffin. The urine that emerged from this cut end was then collectedand its volume determined to provide a measure of the rate of fluid secretion. Ithus had a simple model system to study hormone action because the effects ofadding agents to the bathing medium on the rate of fluid secretion could be easilymeasured. However, there was a problem in that the tubules did not survive for

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4 BERRIDGE

long in vitro. I spent most of my time in Virginia unsuccessfully trying to developa culture medium that would promote longer survival.

I continued with this problem of Malpighian tubule survival when I moved toCase Western Reserve University to work first in Michael Locke’s laboratory inthe Developmental Biology Center and then with Bodil Schmidt-Nielsen in theDepartment of Biology. As I was making little progress with trying to design mediathat would support Malpighian tubules, I began to get increasingly desperate, whenmy luck suddenly changed. One day while dissecting out yet another Malpighiantubule to test out yet another culture medium, I noticed this long clear tube lyingalongside the yellow-white Malpighian tubules. Out of curiosity, I dissected it outof the fly and set it up as for the Malpighian tubules and was astonished by theresult. This tiny little tubule, considerably smaller than the Malpighian tubules,was secreting at rates 50 to 100 times those I had been recording with the tubules.I soon found out that these tubes were the salivary glands that extend down thelength of the fly and secrete a voluminous flow of saliva each time the fly settlesdown to feed. I calculated that at these rates of secretion the fly would pump itselfdry within a short period. This implied that the gland is normally quiescent andis called into action only during feeding. But the glands I had isolated continuedto secrete at high rates for long periods, and this could only mean that there wassomething in my complex medium that was a potent activator of secretion. I soonfound out that the stimulant was 5-hydroxytryptamine (5-HT) (3), a contaminantin fetal bovine albumin fraction V that presumably came from the blood plateletsduring preparation of the albumin fraction. In the absence of 5-HT, the glands weretotally quiescent, but upon addition of this agonist they began to secrete rapidly.My foray into the world of cell signaling had taken a giant step forward; I now hada defined agonist capable of switching the gland on and off. My luck was holdingbecause my research was soon to take another major step forward.

An Introduction to the Second Messenger Concept—CyclicAMP Comes into View

As I made progress developing my model system to study cell signaling, I be-came aware of the work by Earl Sutherland and Ted Rall on cyclic adenosinemonophosphate (cyclic AMP) and their novel second messenger concept (4). Iwas very excited about the idea that cyclic AMP acted as a second messenger tomediate the action of the first messenger that arrived at the cell surface; in the caseof my salivary gland experiments this was 5-HT. Imagine my excitement when Ifound that the stimulatory effect of 5-HT on fluid secretion was exactly duplicatedby the addition of cyclic AMP (3, 5). I had the first steps of an embryonic signalingpathway (Figure 1).

When discussing my latest result with colleagues in the laboratory, I was amazedto find out that Sutherland and Rall had discovered cyclic AMP in the Departmentof Pharmacology just across the road. I plucked up enough courage to go acrossto meet them. Sutherland had left the department but Rall was still there and had

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Figure 1 Model 1 (1968). The first model of the signaling pathway used by5-HT to activate fluid secretion in the insect salivary gland.

become a key player in the field. He was very influential in driving forward theconcept of cyclic AMP functioning as a second messenger in cell signaling. Theold cliche of being in the right place at the right time was never more apt becausemy work was helped enormously by frequent pharmacology tutorials from Rall.He patiently taught me about the action of phosphodieterase inhibitors and theconcept of synergism, which helped me to establish quickly that cyclic AMP wasa key intracellular messenger in the insect salivary gland where it carried outthe stimulatory action of 5-HT. Rall took a great interest in this work because itwas one of the first examples of cyclic AMP functioning in an invertebrate. Mypostdoctoral sojourn in America, which for a long time seemed to be drifting ratheraimlessly, was ending on a high note. However, it was now time to leave my cozypostdoctoral life to find a more permanent position.

I began looking for a job and went for interviews at Rice University in Houstonand various other places. Decision time was fast approaching when a letter arrivedfrom John Treherne in Cambridge offering me a position in a new Unit of Inver-tebrate Chemistry and Physiology he was setting up in the Zoology department.This position seemed just right for me so in 1969 I crossed back over the Atlanticto begin a second period of research in Cambridge.

RETURN TO THE ZOOLOGY DEPARTMENTIN CAMBRIDGE

This period in Cambridge started off very differently from the first. As I was fa-miliar with the quaint ways of the Zoology department, I soon had my researchprogram in full swing. The work I had done in Cleveland on cyclic AMP provideda robust working hypothesis to guide my new research program. The next obviousstep was to determine how cyclic AMP carried out its second messenger role instimulating fluid secretion. I approached this problem by characterizing the elec-trophysiological properties of the secretory response in the hope that it might tellme something about how cyclic AMP acted on the ionic mechanisms responsible

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6 BERRIDGE

for fluid secretion. My research student William Prince, who was a talented elec-trophysiologist, helped to get this project started. We designed a small Perspexperfusion chamber that enabled us to monitor the trans-epithelial potential whileadding and removing components of the putative signaling pathway (Figure 1)such as 5-HT and cyclic AMP or pharmacological agents such as the methyl xan-thines (theophylline and caffeine). When the glands were at rest, the lumen had aslightly positive potential relative to the bathing medium and depolarized rapidlyin response to 5-HT. Fully expecting to observe the same response following theaddition of cyclic AMP, we were greatly surprised to find exactly the opposite.Instead of depolarizing, the potential was found to hyperpolarize (6). Subsequentexperiments revealed that cyclic AMP activated an electrogenic potassium pump,which was the prime mover for fluid secretion. Chloride followed passively, andthis transport of KCl created the osmotic gradient to drive the flow of water. Sub-sequent resistance measurements showed that the passive flux of chloride wasfacilitated by a 5-HT-dependent increase in chloride conductance that occurredindependently of cyclic AMP (7). It seemed that 5-HT was having two actions:one mediated by cyclic AMP to drive potassium transport and a second mechanismfacilitating the movement of chloride.

UNCOVERING A ROLE FOR CALCIUM

It soon became apparent that Ca2+ might regulate the passive flux of chloride. Thenotion that Ca2+ might function as a second messenger was already well estab-lished in muscle cells and had begun to be investigated in secretory cells by BillDouglas prior to the discovery of cyclic AMP (8). However, the notion that Ca2+

might be a more universal intracellular messenger was swept aside by the excite-ment surrounding cyclic AMP, which was rapidly promoted as a universal secondmessenger capable of regulating almost every cellular process imaginable. It grad-ually became apparent, however, that cyclic AMP was not the only messengeroperating in cells and Howard Rasmussen at Yale was very much at the forefrontof reintroducing the concept that Ca2+ could also function as a second messenger(9), an idea that gathered pace in the 1970s. It so happened that in 1971, Rasmussenwas on a sabbatical in the Department of Pharmacology in Cambridge. Once again,I was very fortunate to benefit from the advice of an expert in this emerging fieldof Ca2+ signaling. Indeed, we set up a collaboration to study Ca2+ signaling inthe insect gland, which clearly showed that 5-HT was having a profound effect onCa2+ dynamics. In particular, it suggested that Ca2+ was being released from aninternal store (10). We subsequently used both electrophysiological and pharma-cological techniques to demonstrate that the blowfly salivary gland had two 5-HTreceptors operating through separate second messengers (Figure 2) (11–13). Thesignaling system was becoming a lot more complicated than originally envisagedin that the salivary gland was using two independent systems to regulate secretion(Figure 2). One receptor used cyclic AMP to drive potassium transport, whereasthe other employed Ca2+ to open chloride channels.

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Figure 2 Model 2 (1973). 5-Hydroxytryptamine (5-HT) operates through two sig-naling pathways. One pathway uses cyclic AMP to activate a potassium pump, whereasthe other releases Ca2+ from an internal store to activate chloride channels. The bigquestion at the time was how the receptor on the cell surface gained access to theinternal store.

Searching for a Ca2+-Mobilizing Second Messenger

The most puzzling aspect of the putative Ca2+ messenger system in the blowflywas the source of Ca2+, much of which was derived from an internal store (11).Similar observations were also being made on various mammalian cells so the huntwas on to find the messenger that connected cell surface receptors to the internalstore. The question was easy to define but its solution seemed totally intractable.The aspect that frustrated me most was not having any idea of how to approachthe problem. Finally, a way forward began to emerge from a somewhat unlikelydirection when I became aware of the work by Hokin & Hokin on inositol lipids(14). In 1953, the Hokins discovered that an external agonist stimulated the turnoverof phosphatidylinositol (PI) (14). This came to be known as the PI response. It wasshown subsequently that the agonist was stimulating the hydrolysis of PI. Althoughthis PI response had been measured in many different cells in response to manydifferent stimulants, its function remained somewhat mysterious. This began tochange in 1975 when Bob Michell wrote an extraordinarily insightful and scholarlyreview where he argued that lipid hydrolysis was responsible for Ca2+ signaling(15). A major part of his argument centered on pharmacological observationsindicating that certain receptor types used cyclic AMP as a messenger, whereasothers seemed to work through Ca2+, and it was the latter group of receptors thatalso gave a PI response. Because I had shown that the salivary gland was under dualregulation of 5-HT receptors coupled to either cyclic AMP or Ca2+ (Figure 2), I wasintrigued by Michell’s idea that the PI response might be linked to a Ca2+-signalingpathway. My research suddenly lurched from physiology to biochemistry.

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8 BERRIDGE

Not being a biochemist, I was somewhat daunted by the prospect of starting towork on inositol lipid metabolism, but the transition was greatly helped along itsway by John Fain who joined my laboratory as a sabbatical visitor in 1978. Mostprevious work in this field had used the incorporation of 32P into PI as a way ofmeasuring the PI response. This method necessitated the extraction and separationof the labeled lipids. The small size of the insect gland made this technique wellnigh impossible so we turned our attention to using 3H-inositol. We found thatthis label was rapidly incorporated into PI and, more significantly, the label couldbe released from the lipid during agonist stimulation of intact glands and couldbe collected in the bathing medium. This meant that we could begin to monitorthe PI response at regular time intervals in intact glands simply by measuring theefflux of 3H-inositol (16). The efflux was very small when the glands were at rest,but upon addition of 5-HT there was a dose-dependent increase in the efflux of3H-inositol.

In order to relate this hydrolysis of PI to Ca2+ signaling, it was necessary to havesome way of measuring the latter. At that time, however, there were no establishedmethods for measuring intracellular Ca2+. I began to dabble with Ca2+-sensitivemicroelectrodes, but these proved to be difficult to prepare and were decidedly unre-liable (17). I thus searched for an alternative, and in a separate series of experimentscarried out with Herb Lipke, another visitor to the laboratory, I devised a method ofmeasuring Ca2+ entry into the insect gland by monitoring the transepithelial flux of45Ca (18), which proved invaluable as a way to study Ca2+ signaling. As I now hadmethods for monitoring both Ca2+ signaling and inositol lipid hydrolysis in intactcells, I could begin to see whether these two processes were related. The first thingwe did was to compare their sensitivities to 5-HT. The dose response curve forinositol efflux lay to the left of that for Ca2+ flux, which in turn lay to the left of thesecretory response. This sensitivity sequence was entirely consistent with Michell’snotion (15) that the PI response generated the Ca2+ signal responsible for cell stim-ulation (Figure 3).

Because there had been many false leads regarding the function of the PI re-sponse, I felt we needed more direct evidence that it was playing such a centralrole in the signaling sequence. The fact that inositol was leaking out of the glands(Figure 3), particularly when they were being stimulated, suggested a way of test-ing the hypothesis more directly. At this stage the hypothesis was based on theassumption that 5-HT was stimulating the hydrolysis of PI to produce diacyl-glycerol (DAG) and inositol-1-phosphate (IP1), (Figure 3). The latter was thenhydrolyzed to inositol (Figure 3), which was then recycled back into PI in orderto maintain the membrane level of this precursor lipid. Because our experimentsrevealed that a proportion of this inositol was being lost from the cell, we reasonedthat the cell would begin to run out of it, thus compromising Ca2+ signaling ow-ing to a decline in the level of PI. This is exactly what happened. When glandswere stimulated for 2 h with a high dose of 5-HT and washed repeatedly to re-move the inositol that escaped into the medium (thus reducing the resynthesisof PI), there was a dramatic desensitization of Ca2+ signaling (19). What was

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Figure 3 Model 3 (1979). 5-HT has two actions. One is to act through Receptor 1 (R1)to produce cyclic AMP. The other is to stimulate Receptor 2 (R2) to hydrolyze phos-phatidylinositol (PI) to produce inositol-1-phosphate (IP1) and diacylglycerol (DAG).Lithium inhibits the enzyme that hydrolyzes IP1 to inositol, which is either incorpo-rated back into PI or lost from the cell. This PI response was proposed to play a rolein releasing Ca2+ from the internal store through an unknown mechanism.

particularly pleasing was the fact that Ca2+ signaling could be rescued by incu-bating the glands in inositol thus enabling them to reconstitute the level of the PIrequired for signaling (20). It was this experiment that convinced me that the PIresponse was directly linked to Ca2+ signaling, exactly as proposed by Michellin 1975 (15). But what was the mechanism? The answer finally came through asomewhat circuitous route that began with some studies on lithium (Li+).

LITHIUM PROVIDES AN ENTREE TO THEINOSITOL PHOSPHATES

In attempting to find an inroad into the relationship between the PI response andCa2+ signaling, I stopped doing experiments and began an exhaustive search of thelarge and at times somewhat tedious PI literature in the hope of finding a new wayforward. During the course of this reading, I came across some interesting workon Li+ and inositol phosphate metabolism that had been largely ignored (21, 22).Allison and his colleagues found that Li+ was a potent inhibitor of the inositolmonophosphatase that hydrolyzed IP1 to inositol (Figure 3). I was immediatelystruck by the potential significance of this observation because it was this Li+-sensitive step that released the free inositol that we were measuring to monitor PIhydrolysis. This inhibitory action of Li+ has some very unusual properties, which

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10 BERRIDGE

may explain its therapeutic action in controlling manic-depressive illness and mayalso have a bearing on its teratogenic action of distorting axis formation in earlydevelopment (23) that I discuss further below. The fact that Li+ was able to inhibita putative signaling pathway immediately struck a chord because it reminded meof my earlier studies on cyclic AMP, where the methyl xanthine inhibitors of itsmetabolism had proved so useful in determining its second messenger function.Therefore, I began to examine whether Li+ might prove equally valuable for un-derstanding more about the PI response. The inositol efflux method we had used tomonitor the PI response offered a simple way of testing the inhibitory effect of Li+.Because this inositol was derived from the IP1 produced by hydrolyzing PI, Li+

should reduce the efflux by inhibiting the hydrolysis of IP1 to inositol (Figure 3).That is exactly what happened. Li+ induced a rapid reduction in the efflux of 3H-inositol from prelabeled glands (24). What came as a surprise, however, was thevery large overshoot of inositol efflux that occurred when Li+ was withdrawn. Thislarge surge in free inositol that flooded into the bathing medium resulted from theinositol monophosphatase suddenly hydrolyzing the IP1 that had built up behindthe Li+ block. The fact that Li+ resulted in a large accumulation of inositol phos-phates provided an entree into the next phase of my research on inositol phosphatemetabolism.

Inositol Trisphosphate Comes into View

As a physiologist, I had always tried to avoid biochemical techniques that in-volved destroying the cell. The inositol efflux method described earlier had provedvery effective in enabling the PI response to be monitored continuously in theintact cell. However, in order to study the inositol phosphates that were lockedup within the cell, there was no escape from breaking the cell open to measurethese PI metabolites after separating them out on anion-exchange columns (25).As expected, there was a large accumulation of IP1 during Li+ inhibition, but un-expectedly there were two additional peaks running after the IP1. On the basis ofprevious observations, these two peaks were likely to be inositol 1,4-bisphosphate(IP2) and inositol 1,4,5-trisphosphate (IP3). The whole story was about to get a lotmore complex and difficult, especially because there were no commercially avail-able inositol phosphates to use as standards to verify the preliminary identificationof the unknown peaks.

Once again I was about to benefit from being in the right place. As far as I candetermine, at the time we were doing these studies there were only two sourcesof inositol phosphates that could be used as standards. One source was in ClintBallou’s laboratory in Berkeley, California and the other was a few miles downthe road from me in Rex Dawson’s laboratory at the Babraham Institute. Dawsonand his colleague Robin Irvine were busily working on various aspects of thePI response and in particular identifying the enzymes that hydrolyzed inositollipids. Just as Rall and Rasmussen had proved invaluable in guiding me along thecyclic AMP and Ca2+ paths respectively, Dawson and Irvine were very patient

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Figure 4 Model 4 (1983). The role of inositol 1,4,5-trisphosphate (IP3) as a Ca2+-mobilizing second messenger. 5-HT acts on either Receptor 1 (R1) to produce cyclicAMP or on Receptor 2 (R2) to hydrolze phosphatidylinositol 4,5-bisphosphate (PIP2)to diacylglycerol (DAG) and IP3. The latter diffuses into the cell to release Ca2+ fromthe endoplasmic reticulum.

in helping me come to grips with the rather arcane world of inositol phosphatebiochemistry. More importantly, tucked away in their freezer was a wide rangeof inositol phosphates that they kindly supplied as standards to verify that theunknown peaks coming off my exchange columns were indeed IP2 and IP3 (25).On the basis of what was known previously, the simplest hypothesis was that IP3

was formed first and was sequentially hydrolyzed to IP2, IP1, and then inositol(Figure 4).

For the sequence shown in Figure 4 to be correct, the substrate used by R2 hadto be phosphatidylinositol 4,5-bisphosphate (PIP2) and not the parent lipid PI asoriginally assumed in the earlier model (Figure 3). Indeed, work being done in otherlaboratories had already shown that agonists were able to hydrolyze PIP2 (26, 27).In order to obtain further evidence for the IP3 → IP2 → IP1 → inositol sequence,I developed a rapid perfusion system (28) that enabled me to establish the rate atwhich these inositol phosphates were produced following 5-HT stimulation. Thesestudies on the kinetics of 5-HT-induced inositol phosphate formation revealed thatIP3 and IP2 increased first with no apparent latency, whereas IP1 followed by inositolappeared much later. I was excited by this result because it not only confirmed thatPIP2 was being hydrolyzed but also revealed that IP3 was being generated quickly.In fact, it was the fastest response to 5-HT I had yet recorded. I already knew thatthere was a 1–2 s latency before the onset of the electrical change caused by theCa2+-dependent activation of the chloride channels. I now found the level of IP3

was already elevated by 1 s and, within the limits of my recording system, there

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appeared to be no latency, which suggested that IP3 was being generated as soonas 5-HT bound R2. It was this observation that led me to propose that IP3 mightbe the diffusible messenger that coupled receptor activation to the mobilization ofinternal Ca2+ (29) (Figure 4).

IP3-Induced Ca2+ Mobilization

It is one thing to suggest a mechanism but quite another to prove it. I alreadyknew from my electrophysiological experiments that Ca2+ was coming from aninternal store; the problem was to find a way of testing whether IP3 could releaseCa2+ from some as yet unknown internal store. The experimental techniques tostudy the action of intracellular messengers are now relatively easy, but 20 yearsago such study was much more difficult. Not only was the supply of IP3 verylimited, but also cellular injection techniques were poorly developed. While grap-pling with this problem, I attended a workshop in Amsterdam in December 1982on Biophysical and Biochemical Aspects of Transcellular Transport in AnimalTissues. The title of my lecture was “Phosphatidylinositol Hydrolysis: A GeneralTransducing Mechanism for Calcium-Mobilizing Receptors,” and it was the firsttime that I had publicly put forward the idea that IP3 might be the long sought afterCa2+-mobilizing second messenger. In the same session, Irene Schulz describeda permeabilized pancreatic cell preparation in which she was able to gain accessto the internal Ca2+ stores. This was exactly what I was looking for to test out theproposed function of IP3, and she was more than happy to set up a collaborativestudy. On returning to Cambridge, I contacted Irvine who agreed to prepare thelarge amounts of IP3 that were required for these permeabilized cell experiments.A few weeks after Schulz had received the samples, I had an excited phone callto say that IP3 had mobilized Ca2+. We carried out numerous controls to establishthe specificity of IP3-induced Ca2+ mobilization, and we also identified the en-doplasmic reticulum as its site of action. The paper summarizing our results waspublished in Nature toward the end of 1983 (30).

The initial evidence that IP3 was a Ca2+-mobilizing messenger emerged fromour work on the permeabilized pancreatic acinar cells, and we were anxiousto find out whether it had a similar action in other cell types. Once our paper(30) appeared in Nature, we were contacted by several groups anxious to tryout this new messenger on their cells. We set up a number of collaborations,and within a short period of time we were able to confirm that IP3 releasedCa2+ in many cell types including liver cells (31), Swiss 3T3 cells (32), insulin-secreting cells (33, 34), Limulus photoreceptors (35, 36), and leukocytes (37). Itseemed clear, therefore, that IP3 was the long sought after Ca2+-mobilizing secondmessenger.

It had taken 30 years since the discovery of the PI response by Hokin & Hokinin 1953 (14) to finally find out that one of its functions is to stimulate the releaseof internal Ca2+. However, that is not its only function. Indeed, a few years ear-lier Yasutomi Nishizuka had already shown that DAG, the other product of the

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PI response (Figure 4), also had a signaling role, i.e., it was a potent activator ofa novel protein kinase (38, 39). For those of us involved during that period, oneof the really exciting developments was the realization that the PI response wasspawning two separate signaling cascades: one activated through DAG to stim-ulate protein kinase C and the other using IP3 to stimulate the mobilization ofCa2+ (39–41):

IP3 → Ca2+↗

agonist → receptor → PLC → PIP2 hydrolysis↘

DAG → PKC

THE VERSATILITY AND UNIVERSALITY OFCa2+ SIGNALING

The discovery that IP3 was a universal second messenger controlling Ca2+ sig-naling heralded a new phase in my research career as it provided fresh insightsinto how a variety of cellular processes were regulated. Having spent so muchtime doing biochemistry, I was anxious to return to physiology, which I did byturning my attention to the spatial and temporal aspects of Ca2+ signaling. Oneof the questions I am asked most frequently is how can this messenger regulateso many different processes, in some instances within the same cell. For exam-ple, in smooth muscle cells, Ca2+ can regulate both contraction and relaxation.Likewise, in neurons, Ca2+ can induce both long-term potentiation (LTP) andlong-term depression (LTD) in the same synaptic connection. How is it that Ca2+

can separate out these discrete and sometimes opposing signaling functions? Wenow know that much of this remarkable versatility depends on the way this signalis organized in both time and space (42–45). My interest in these spatiotemporalaspects began with studies on Ca2+ oscillations, and when single cell imaging tech-niques began to appear, attention was also focused on the spatial aspects of Ca2+

signaling.

Ca2+ Oscillations

Early on in our studies on the electrical responses of the insect salivary gland to5-HT, we discovered that the transepithelial potential oscillated (6). An interestingfeature of these oscillations is that their frequency varies with agonist concentration(6, 46). Because these changes in frequency occurred over the same range of 5-HTconcentrations that caused changes in fluid secretion, we proposed that the sig-naling system operated through frequency modulation (FM) rather than amplitudemodulation (AM) (47). These potential oscillations depended on changes of resis-tance, which were controlled by Ca2+, thus we speculated that they resulted froman underlying oscillation of Ca2+ (48). When aequorin was introduced to monitorintracellular Ca2+, particularly striking examples of oscillations were revealed inboth liver cells (49) and mammalian oocytes (50–52). Frequency modulation was

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particularly apparent for the liver cell oscillator. What fascinated me most aboutsuch oscillatory activity was its spontaneous nature. In some cases, the level ofCa2+ was found to remain constant for up to 60 s and was then interrupted by aspontaneous Ca2+ spike that returned back to the resting level for another 60 sbefore the next spike occurred and so on. In the case of mammalian oocytes un-dergoing fertilization, these interspike intervals can last for 3–4 min (50–52). Thequestion that intrigued me and still does today is what happens during this inter-spike interval that results in the appearance of the spontaneous spikes? I began tosearch for a mechanism.

When we first became aware of such Ca2+ oscillations, we had little infor-mation about how intracellular Ca2+ was regulated. This, however, did not deterPaul Rapp and me from putting forward a model that attempted to explain theseoscillations on the basis of feedback loops operating between Ca2+ and cyclicAMP (48). However, such models were not particularly plausible because theyfailed to explain adequately how agonists functioned to increase the intracellularlevel of Ca2+. This deficiency was resolved once IP3 had been discovered, andwe were then able to develop more refined models to account for both Ca2+ os-cillations and how frequency might be decoded through protein phosphorylation(53, 54).

An important aspect of these new models was the process of Ca2+-inducedCa2+ release (CICR), which provided the positive feedback to account for therapid regenerative component of each Ca2+ transient. The ryanodine receptors ofmuscle cells were already known to display this process of CICR as first describedin 1970 by Makoto Endo (55), and it was soon shown that IP3 receptors were equallysensitive to Ca2+ (56–58). The process of CICR, therefore, applies just as well toIP3 receptors as it does to ryanodine receptors. Although the IP3-dependent processof CICR nicely explained the regenerative process responsible for the upstroke ofthe Ca2+ spike, it did not address the critical question of what triggered this spikein the first place. Again, an important clue came from work on muscle cells wherea spontaneous release of Ca2+ often occurred when the internal store became over-loaded with Ca2+. Because ryanodine receptors are sensitive to the level of Ca2+

within the lumen of the endoplasmic reticulum, I wondered whether IP3 receptorsmight display a similar sensitivity to store loading. Ludwig Missiaen spent hispostdoctoral period in Cambridge patiently devising methods of loading up suchstores and found that a spontaneous release occurred when the stores were loadedabove a critical point (59). It seems that the IP3 receptors, like their ryanodinereceptor counterparts, are sensitive to the level of Ca2+ within the lumen of theendoplasmic reticulum. Although this remains a controversial area, I believe thatthe loading of the internal store plays a critical role in the timing mechanismof Ca2+ oscillations because it sets the sensitivity of the IP3 receptors and thusdetermines when the next Ca2+ spike will initiate (42, 60). The frequency of Ca2+

oscillations will thus depend upon how quickly the store can be loaded and thisin turn will depend upon the rate at which it enters across the plasma membrane.Such dependence on external Ca2+ may explain why oscillations are so sensitive to

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variations in the external concentration of Ca2+. It may also explain how frequencycan be regulated if one assumes that the rate of Ca2+ entry is controlled by agonistconcentration. The importance of Ca2+ entry processes has been established butthe nature of these processes remains as one of the major unsolved problems inCa2+ signaling.

Spatial Aspects of Ca2+ Signaling

In 1986 I visited Roger Tsien at Berkeley and was shown his new purpose-built,single cell imaging system; immediately I realized the significance of this tech-nology. I obtained the funds to set up such a system in Cambridge and mustacknowledge the help that Tsien provided in guiding us into this new technol-ogy. Within a short time we had our own system running and began to see howCa2+ signals developed in cells in response to external signals. Some of the firstexperiments were performed by Tim Cheek who showed that when chromaffincells were depolarized, the calcium appeared first as a ring confined to the cyto-plasm immediately below the plasma membrane (61). Like many other groups,we were also recording regenerative calcium waves that usually initiated in a dis-crete region of the cell and then propagated through the cytoplasm. A particularlydramatic example of such waves is found in mammalian oocytes following fertil-ization (51, 52). These waves reflect the spatial organization of the calcium spikesdescribed above. We already knew that a process of CICR caused such spikes, andthe next big step was to “see” the elementary events responsible for these calciumwaves.

The first indication that one might be able to visualize the activity of the indi-vidual building blocks of such calcium signals emerged from work on cardiac cells(62, 63), which revealed the existence of elementary events that were called sparks.These sparks are small bursts of calcium released from a localized group of ryan-odine receptors. They occur at the junctional zones and produce the calcium thatdiffuses onto the myofibrils to activate contraction. Cardiac studies on the micro-scopic activity of ryanodine receptors established the idea that the global calciumsignals we had been studying previously could be broken down into elementaryevents, and this finding greatly increased our understanding of how calcium sig-nals are constructed (43, 44). By pushing our aging imaging system to its limits,Martin Bootman began to get the first indication that the IP3/Ca2+ signaling systemmight be similarly organized into elementary events (64). The elementary eventsproduced by IP3 receptors have been called puffs (65). Bootman then teamed upwith Peter Lipp to characterize these puffs and to show how they are recruited toproduce global calcium signals in HeLa cells (66, 67). For someone who has beenstudying covert Ca2+ signaling for so long, it was and still is a quasi-religiousexperience to visualize the generation of Ca2+ signals in cells. The spiral wavesthat pulsate through the cytoplasm of Xenopus oocytes are objects of great beauty(68). We have come a long way since our initial studies on the insect salivarygland, which provided the first indication that Ca2+ might be oscillating, to the

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detailed molecular and spatial description we have now. We are still in the midst ofexploring the enormous panoply of elementary calcium signaling events that canbe manipulated in many different ways to create the versatility that characterizesCa2+ signaling.

THE ROLE OF IP3/Ca2+ SIGNALING IN CELLULARCONTROL MECHANISMS

Much of my scientific attention at present is focused on trying to understand howthe IP3/Ca2+ signaling pathway functions in cellular control mechanisms. Oneof my first attempts at this was to see whether the new insights we had into themode of action of Li+ (24) might help explain its therapeutic action in controllingmanic-depressive illness or its teratogenic action during early development. Byinhibiting the conversion of inositol phosphate to free inositol with Li+ (Figure 3),we caused the level of inositol to decline, which led to the inositol depletionhypothesis to explain Li+’s therapeutic action in manic-depressive illness (23).Some experimental support for this inositol depletion hypothesis has begun toappear (69). The basic idea is that this illness might be caused by overactive inositollipid signaling, which could be brought back to normal by Li+ acting to slowdown the recycling of inositol. This idea was made all the more plausible becauseLi+ exerts its inhibitory action through an unusual uncompetitive mechanism.The Li+-binding site on the inositol monophosphatase appears only when theenzyme has bound its inositol phosphate substrate. This means that Li+ functionsas a homeostatic drug, i.e., it has little effect if the signaling system is operatingnormally but begins to exert an ever-increasing inhibitory action as inositol lipidsignaling becomes more and more abnormal. I went on to apply this inositoldepletion hypothesis to the teratogenic action of Li+, particularly with regard toits ability to dorsalize Xenopus embryos (23). By extending the same idea thatLi+ is most effective when inositol lipid turnover is high, I proposed that dorso-ventral specification might be set up by a gradient of IP3/Ca2+ signaling, with thehighest level in the ventral region. In the presence of Li+ this high level of IP3/Ca2+

signaling in the ventral region would be suppressed down to that seen in the dorsalregion, which would account for the dorsalization of the developing embryo (23).There now is growing evidence that IP3/Ca2+ signaling does indeed play such arole in dorso-ventral specification (70, 71).

The fact that Li+ might be acting to curb inositol lipid signaling raised the realpossibility that the IP3/Ca2+ signaling pathway might play a prominent role in thebrain. It had long been known that the receptors and enzymes responsible for inosi-tol lipid signaling were strongly expressed in neurons, but their exact function wasunknown. With the discovery that IP3 was a calcium-mobilizing second messenger(30, 40, 41) and that its receptors were located on the endoplasmic reticulum withinthe spines of Purkinje neurons (72), it became apparent that this signaling pathwayhad a role in information processing within the brain (73). In order to discern what

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that function might be, I began to take an interest in the neurobiology of Purkinjeneurons and was immediately struck by the fact that the metabotropic receptorsresponsible for generating IP3 were of central importance in the process of LTD,which is responsible for the motor learning skills that enable us to learn how to ridea bicycle or play the piano. Like many learning mechanisms, LTD occurs when thePurkinje neuron detects the near simultaneous arrival of two synaptic inputs, onecoming from the climbing fibers that innervate the base of the dendritic tree andthe other arriving through the parallel fibers that innervate the spines. The problemwas to identify the molecular coincident detector that enabled the Purkinje cell toassociate the information arriving from these two separate inputs. The more I readabout the system, the more I realized that the IP3 receptor might be that coincidentdetector (74). As noted above, opening of the IP3 receptor depends upon the si-multaneous presence of both IP3 and Ca2+ (56–58), and these are exactly the twomessengers being supplied by the two inputs to the Purkinje neurons. The climbingfiber results in depolarization of the dendrites, which then generates a Ca2+ sig-nal, whereas the parallel fibers activate the metabotropic receptors to generate IP3.When these two messengers are produced at approximately the same time, thereis a large release of Ca2+ within the spine, which induces LTD. Although there arecontenders for coincident detectors in other neurons, there has been considerableexperimental evidence to support the notion that the IP3/Ca2+ signaling pathwaymay be responsible for LTD in Purkinje neurons (75, 76).

What is particularly intriguing about these molecular events is that their tempo-ral characteristics closely match what is known about the timing rule for variousforms of motor learning. For example, the most effective learning occurs when thestimulation of the parallel fibers precedes that of the climbing fibers. Likewise, themost effective release of Ca2+ by the IP3 receptors occurs when the parallel fibersthat generate IP3 are activated 50–200 ms before the climbing fibers that deliver thepulse of Ca2+ (75). It is these subtleties that fascinate me as we continue to probethe many functions of the IP3/Ca2+ signaling pathway in information processingin the brain.

CONCLUSION

I have been privileged to live through a period when many of the major signalingpathways used for cell communication were being discovered. I was caught up inthe excitement of the discovery of cyclic AMP, which introduced the concept ofsecond messengers, and this prepared me for my work on the role of IP3 as a secondmessenger linking inositol lipid hydrolysis to Ca2+ signaling. There has been muchexcitement along the way. In addition to the real buzz of making new discoveries inthe laboratory, I have also enjoyed immensely the intellectual challenge of trying touse all this new information to understand how specific cell functions as divergentas fertilization, cell proliferation, muscle contraction, and synaptic plasticity arecontrolled.

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The Annual Review of Physiology is online at http://physiol.annualreviews.org

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Annual Review of PhysiologyVolume 67, 2005

CONTENTS

Frontispiece—Michael J. Berridge xiv

PERSPECTIVES, Joseph F. Hoffman, Editor

Unlocking the Secrets of Cell Signaling, Michael J. Berridge 1

Peter Hochachka: Adventures in Biochemical Adaptation,George N. Somero and Raul K. Suarez 25

CARDIOVASCULAR PHYSIOLOGY, Jeffrey Robbins, Section Editor

Calcium, Thin Filaments, and Integrative Biology of Cardiac Contractility,Tomoyoshi Kobayashi and R. John Solaro 39

Intracellular Calcium Release and Cardiac Disease, Xander H.T. Wehrens,Stephan E. Lehnart and Andrew R. Marks 69

CELL PHYSIOLOGY, David L. Garbers, Section Editor

Chemical Physiology of Blood Flow Regulation by Red Blood Cells:The Role of Nitric Oxide and S-Nitrosohemoglobin, David J. Singeland Jonathan S. Stamler 99

RNAi as an Experimental and Therapeutic Tool to Study and RegulatePhysiological and Disease Processes, Christopher P. Dillon,Peter Sandy, Alessio Nencioni, Stephan Kissler, Douglas A. Rubinson,and Luk Van Parijs 147

ECOLOGICAL, EVOLUTIONARY, AND COMPARATIVE PHYSIOLOGY,Martin E. Feder, Section Editor

Introduction, Martin E. Feder 175

Biophysics, Physiological Ecology, and Climate Change: Does MechanismMatter? Brian Helmuth, Joel G. Kingsolver, and Emily Carrington 177

Comparative Developmental Physiology: An InterdisciplinaryConvergence, Warren Burggren and Stephen Warburton 203

Molecular and Evolutionary Basis of the Cellular Stress Response,Dietmar Kultz 225

ENDOCRINOLOGY, Bert O’Malley, Section Editor

Endocrinology of the Stress Response, Evangelia Charmandari,Constantine Tsigos, and George Chrousos 259

vii

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viii CONTENTS

Lessons in Estrogen Biology from Knockout and Transgenic Animals,Sylvia C. Hewitt, Joshua C. Harrell, and Kenneth S. Korach 285

Ligand Control of Coregulator Recruitment to Nuclear Receptors,Kendall W. Nettles and Geoffrey L. Greene 309

Regulation of Signal Transduction Pathways by Estrogenand Progesterone, Dean P. Edwards 335

GASTROINTESTINAL PHYSIOLOGY, John Williams, Section Editor

Mechanisms of Bicarbonate Secretion in the Pancreatic Duct,Martin C. Steward, Hiroshi Ishiguro, and R. Maynard Case 377

Molecular Physiology of Intestinal Na+/H+ Exchange,Nicholas C. Zachos, Ming Tse, and Mark Donowitz 411

Regulation of Fluid and Electrolyte Secretion in Salivary GlandAcinar Cells, James E. Melvin, David Yule, Trevor Shuttleworth,and Ted Begenisich 445

Secretion and Absorption by Colonic Crypts, John P. Geibel 471

NEUROPHYSIOLOGY, Richard Aldrich, Section Editor

Retinal Processing Near Absolute Threshold: From Behaviorto Mechanism, Greg D. Field, Alapakkam P. Sampath, and Fred Rieke 491

RENAL AND ELECTROLYTE PHYSIOLOGY, Gerhard H. Giebisch, Section Editor

A Physiological View of the Primary Cilium, Helle A. Praetoriusand Kenneth R. Spring 515

Cell Survival in the Hostile Environment of the Renal Medulla,Wolfgang Neuhofer and Franz-X. Beck 531

Novel Renal Amino Acid Transporters, Francois Verrey, Zorica Ristic,Elisa Romeo, Tamara Ramadam, Victoria Makrides, Mital H. Dave,Carsten A. Wagner, and Simone M.R. Camargo 557

Renal Tubule Albumin Transport, Michael Gekle 573

RESPIRATORY PHYSIOLOGY, Carole R. Mendelson, Section Editor

Exocytosis of Lung Surfactant: From the Secretory Vesicle to theAir-Liquid Interface, Paul Dietl and Thomas Haller 595

Lung Vascular Development: Implications for the Pathogenesis ofBronchopulmonary Dysplasia, Kurt R. Stenmark and Steven H. Abman 623

Surfactant Protein C Biosynthesis and Its Emerging Role inConformational Lung Disease, Michael F. Beers and Surafel Mulugeta 663

SPECIAL TOPIC, CHLORIDE CHANNELS, Michael Pusch, Special Topic Editor

Cl− Channels: A Journey for Ca2+ Sensors to ATPases and SecondaryActive Ion Transporters, Michael Pusch 697

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CONTENTS ix

Assembly of Functional CFTR Chloride Channels, John R. Riordan 701

Calcium-Activated Chloride Channels, Criss Hartzell, Ilva Putzier,and Jorge Arreola 719

Function of Chloride Channels in the Kidney, Shinichi Uchidaand Sei Sasaki 759

Physiological Functions of CLC Cl− Channels Gleaned from HumanGenetic Disease and Mouse Models, Thomas J. Jentsch,Mallorie Poet, Jens C. Fuhrmann, and Anselm A. Zdebik 779

Structure and Function of CLC Channels, Tsung-Yu Chen 809

INDEXES

Subject Index 841Cumulative Index of Contributing Authors, Volumes 63–67 881Cumulative Index of Chapter Titles, Volumes 63–67 884

ERRATA

An online log of corrections to Annual Review of Physiology chaptersmay be found at http://physiol.annualreviews.org/errata.shtml

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