- Email: [email protected]
The road to the phosphoinositide-generated second messengers
TIPS - February 1987 [Vol. 81 compliance in reporting clinical events is of the order of 5%. Because of this, usually after one year of monitoring we do an event survey of all the patients in the cohort, asking doctors to check their records retrospectively. In addition, and perhaps more importantly, if scrutiny of any clinical event (performed by at least two experienced clinicians) suggests a clinically important adverse reaction or indeed if there is a case report of such in the literature, we can perform a specific survey related to that event. Much valuable information is
53 gained by this scheme, some of which has been reported in Monitoring for Drug Safety, 2nd edition, (W. H. W. Inman, ed.). Of particular importance is, of Course, our ability to ascribe accurate incidence to events. As with the more recent prescription event monitoring by the Drug Surveillance Research Unit, University of Southampton, the scheme suffers from the drawback of not having properly constructed control groups in the initial event monitoring but these have been incorporated in some of our follow-up surveys. The other drawback is
The road to the phosphoinositidegenerated second messengers is the story ofthe &scovery of the phosphoinositide effect in the early 195Os, a ‘discovery before its time’. Twenty years were to elapse after our initial demonstration of agonist-stimulated phosphatidylinositol (PI) turnover before Bob Michell was to rekindle interest in this effect - an effect which has answered (and posed!) so many questions about signal transduction mechanisms. If only we could have taken a glimpse into the future back then1 wouldnever have allowed an albatross to side-track me from continuing to investigate the true meaning of this fundamentally important phenomenon. This
The early years But to begin at the beginning, this story dates back to 1949, when I arrived at H. A. Krebs’ laboratory in Sheffield to study for a Ph.D in biochemistry. Krebs’ policy was to have a student formulate his own problem, pursue it independently with a minimum of supervision and publish by himself. With some background in gastroenterology which I had received in the laboratory of Warren S. Rehm while attending medical school, I looked into whether I could use pancreatic tissue as a model to study the synthesis and secretion of proteins, using amylase as a measure of these two processes.
Pigeon pancreas s!ices proved to be an excellent system for this purpose. Near the end of my doctoral studies in Krebs’ laboratory, I had become interested in the possible involvement of RNA in protein synthesis. This was based on some cytological studies in the late forties by Caspersson and Brachet showing that RNA levels in a variety of dividing and non-dividing cells were correlated with protein synthetic activity. RNA levels were particularly high in the pancreas. At about that time, Kenneth Burton, who was then a Lecturer in Krebs’ Department, drew my attention to a report by two Russian workers, M. A. Gubemiev and L. I. Il’ina, who showed that on cholinergic stimulation of secretion in pancreas, stomach, and salivary glands in anesthesized dogs (shades of Pavlov) there were marked increases in the incorporation of 32P into ‘nucleoproteins’, isolated by the method of Schmidt and Thannhauser. This method was really designed to isolate phosphorus fractions, including DNA and RNA (separated by alkaline hydrolysis), and not nucleoproteins, but no further details were given. A.pplicationof 32P The use of 32P as a tracer had been adopted by Krebs and his
the relatively small population of New Zealand of approximately 3.3 million. However, the careful scrutiny of events and their followup in individual cases for causality we believe offsets this disadvantage. I.
RALPH
EDWARDS
ZealandMedical Assessor for Medicines Adverse Reactions, University of Otago, Dunedin, New Zealand. New
References
1 Strom,B. L. (1986)TrendsPkatnxacol. Sci. 7, 377-380
associates a year or two earlier. I found that on stimulation of enzyme secretion in pancreas slices, there was about a 100% increase in the incorporation of [32P]orthophosphate into RNA which confirmed the observations of Guberniev and Il’ina on ‘nucleoproteins’, although they observed much greater effects. Near the end of this ._____, I began to suspect that work. perhaps an alkaline hydrolytic product of phospholipids was contaminating the RNA fraction, and this might be responsible for the stimulation of 32P incorporation into RNA. This idea came to me shortly after I had completed my requirements for the Ph.D and was about to set sail with Mabel Hokin for Halifax. 1 did manage to do one experiment, but I did not have time to go through the necessary procedures to rid the lipid fraction of contaminating [32P]orthophosphate and to count the radioactivity. I literally took with me on the boat a rack of about a dozen rather large test tubes containing the ethanol-ether extracts. When we arrived at J. H. Quastel’s laboratory at McGill, we counted the purified total lipid fractions and found an enormous increase in specific radioactivity in the lipids from pancreas slices which had been stimulated with carbachol. With better methods for isolation of nucleic acids from small samples of tissue developed by others in 1952. we were able to show that there was no stimulation of 32P incorporation in RNA. I like the comment of the Russian discoverer of myosin ATPase, V. A. Engelhardt, who knew Gubemiev and Il’ina, when I told him at the XlXth International Physiological
@ 1987. ElsevierSciencePublishersB.V., Amsterdam 0165- 6147/87&QZOQ
TIPS - February 1987 IVol. 81 microsomes (fragments of endoplasmic reticulum and plasma membrane) isolated after incubation of pancreas slices. Using radioautography with [3H]inositol, we showed in the mid-1960s that the effect was predominantly in the endoplasmic reticulum in sympathetic ganglia and pancreas. This has been confirmed more recently with other cell types. It is still not clear how this fits into the overall picture.
Lowell and Mabel Hokin optimistic
in the early 1950s in Copenhagen.
Congress in Montreal in 1953 that the stimulated 32P turnover was in phospholipids and not in ‘nucleoproteins’. With the characteristic Russian shrug of the shoulders he said, ‘So it’s in the phospholipids,’ which I interpreted as saying that the stimulated 32P incorporation was the important point and why quibble over details; after alI, at this juncture we do not know much about the function of either RNA or phospholipids (the Watson-crick double helix structure of DNA was published that year; nucleic acids had a much brighter immediate future than phospholipids). The 1953 JBC paper arising from this work is often incorrectly quoted as demonstrating the stimulated turnover of phosphatidylinositol (PI). We did not actually show this until 1955 when techniques for measuring radioactivity in individual phospholipids from small samples of tissue became available (Dawson; Marinetti). The discovery of the PI-PA
(phosphatidic) effect launched Mabel Hokin and me on a 1Syear quest for its explanation, as welI as a pursuit of its biochemical mechanism. Mabel Hokin continued this search after I had left the field. I have only recently returned. We made several key observations in the fifties concerning the PI-PA effect, including: (1) the effect occurred with many agonists and in many cell types; (2) the effect for the most part was due to a turnover and not a net synthesis of PI and PA, with the diacylglycerol (DG) moiety being partly or completely conserved; (3) the physiological response, i.e., amylase secretion, was not tightly coupled to PI turnover as shown by different dose-response curves (in the middle sixties I also showed a dissociation of these two processes by omitting Ca*+); (4) PA, in addition to being synthesized by acylation of ar-glycerophosphate (Komberg and Pricer; Kennedy), could be synthesized from ATP and DG; and (5) the PI effect was located in
The PI-PAcycle In the early sixties, with the aid of kinetic studies on the turnover of PI and PA in the avian salt gland on cholinergic stimulation (followed by atropine clamping), we were able to present the first version of the PI-PA cycle (Fig. 1). This cycle also incorporated our knowledge of the enzymes involved in the phosphodiesterase cleavage of PI (Hawthorne) and in PI synthesis (Agranoff; Kennedy). We published these studies in 1964 in a symposium entitled, ‘Metabolism and Physiological Significance of Lipids’, edited by R. M. C. Dawson and D. N. Rhodes (Wiley). I have often regretted that the results and the proposed PI cycle in this paper were not made more generally available by publication in a regular journal. The main points of this cycle were that on cholinergic stimulation of salt gland slices phospholipase C catalysed the breakdown of PI to DG and IP. Diacylglycerol kinase then formed PA. On quenching with atropine, the resting steady-state level of PI was restored at the expense of PA by the sequential actions of PAcytidyl transferase -and PI synthase. Mat -1 Hokin made some key observations in the early 1970s further supporting the PI-PA cycle, including the findings that on stimulation in pancreas, there was a loss in the mass in PI and a rise in the mass of PA. Also, on stimulation, the fatty acid composition of PA approached more closely that of PI. These data gave additional strong support to the view that PA was derived from PI during stimulated turnover of PI and PA. She further found an increase in mass of DG in pancreas on agonist stimulation, although the fatty acid composition of the liberated DG did not agree closely with that of PI. Later studies did
77PS - February 1987 [Vol. 81 show a close similarity in fatty acid compositions between PI and liberated DG in other cell types. At about the same time, Michell showed independently a fall in mass in PI in the parotid gland on stimulation. Mabel Hokin also confirmed the salt gland kinetic data in the pancreas. Her recalculation of some old measurements of PI in the salt gland (expressed as percentages of controls rather as absolute values) showed a significant loss in mass in PI in this tissue as well. The original PI-PA cycle has, of course, been modified to incorporate the polyphosphoinositides. There is still controversy as to wheLher there is dibect phosphodiesteratic cleavage of PI as an additional source of DG. TThepoIyphosphoinositides With regard to early studies on polyphosphoinositides, in the early 1960s many of us studied the turnover of phosphatidylinositol-4phosphate (PIP) and phosphatidyiinositol4,5-bisphosphate (pIP2), in-
55 eluding our demonstration of a fall in steady-state level of 32P-labelled polyphosphoinositides on stimulation of salt gland slices with acetylcholine (no time courses were done). We also showed for the first time the presence of the kinases for PI and PIP (in the erythrocyte membrane), as well as the rapid exchange of 32P into the monoesterified phosphates of the polyphosphoinositides in erythrocyte membranes on incubation with [32P] ATP. In the late 196Os, Durell demonstrated the formation of inositol-l-phosphate (IP) and inositol 1,4-biphosphate (II’*) in cholinergically stimulated synaptosomes and suggested that the primary response might be the phosphodiesteratic cleavage of the polyphosphoinositides, releasing inositol phosphates. Abdel-Latif pursued this line in the late 1970s and showed that cholinergic or adrenergic stimulation of iris smooth muscle caused a rapid breakdown of PIP2 which was accompanied by an increase in
Fig. 1. TheoriginalversionofthePCPA cycle. From Hokin, M. R. andHokin, 1. E. (1964) in Metabolism and PhysiologicalSignificance of Lipids (Oatwon, R. M. C. and Rhodes, D. PI., eds), pp. 423434, John Wiley.
Fig. 2. Chromatogram demons!rating incorporation of *P into the responsive in&tide, PI. Right, using cholinergically siimutated pancreas.Left, unstimulated contrd From Hokin and Hokin, J. Biol. Chem. (1958) 233, 8tX-810. IP, IP2 and inositol 1,4,5-trisphosphate (IPa). Abdel-Latif did not believe that polyphosphoinositide breakdown was antecedent to Ca” gating for two reasons. The breakdown was dependent on Ca2+ (later shown to be at conceittrations lower than basal cytosolic leveis) and was stimulated by Ca2+ ionophores. This turned out to be due to a direct release of norepinephrine by ionophores.
A discovery before its time The invitation to write this article has stimulated me to go back and look at our papers published in the early 1950s and I am struck by how naive some of our interpretations of the phospholipid effect were because so many pieces of the puzzle were missing. Very little was known about membrane structure. The classic experiments from the laboratories of Douglas and Katz pointing to the importance of Ca*+ in stimulus-response coupling were not carried out until the early 1960s. We did not know for certain the structure of the ‘phosphoinositide’ which showed increased turnover in pancreas and brain until around 1958, when we showed it was PI (Fig. 2). Although Jordi Folch had identified in brain a phosphoinositide fraction which showed a phosphorus : inositol ratio of 2 and which he called diphosphoinositide (this was presumably a mixture of all three phosphoinositides), it was not until the early 1960s that the identification and the full shwtural elucidation of PIP and PIP;! were made in the laboratories of Ballou and Dawson. The existence
TIPS - Februa y 1987 [Vol. 81
56 of the intracellular ‘trigger pool’ of Ca’+ which rapidly releases part of its stores of Ca2+ on agonist stimulation was not known until the work of Schulz and others in the late 1970s. The use of lithium to amplify the accumulation of inosit01 phosphates was not adopted until the early 1980s. The technique of cell permeabilization to allow entry of phosphorylated compounds into the cell was not developed until the late 1970s and early 1980s. This, of course, permitted Berridge and his associates to demonstrate the release of nonmitochondrial intracellular stores of Ca2+ by IPs, although it would have been possible to show this effect (but perhaps not to fully appreciate it) earlier with microsomes. Perhaps we were led astray mostly when in 1959, we found a dramatic stimulation of PA turnover and a lesser stimulation of PI turnover in the salt gland of the albatross (shipped to us by the US Navy from Midway Island). Since the exclusive function of this organ is to secrete concentrated salt solutions in response to cholinergic stimulation, we were naturally tempted to conclude that the ?I-PA effect was an important component of the Na+ pump these studies preceded, by many years, the work showing Ca2+ involvement in NaCl secretion. Since the membranes of this tissue were also very rich in DG kinase and PA phosphatase, we proposed that if DG kinase were located on the inner surface of the plasma membrane and PA phosphatase situated near its outer surface, perhaps lipid-soluble sodium phosphatidate and DG could shuttle back and forth across the plasma membrane with PA carrying two sodiums per cycle. The PA cycle would thus function as an ATPase. This scheme naturally aroused considerable interest since the data seemed to suggest that PA might be the long-soughtafter Na+ carrier - in those days many of us thought of ion carriers as being small molecules. It also stimulated considerable controversy, partly centered around the predicted stoicldometry of two sodiums per ATP. After the Na+/K+-transporting ATPase was discovered by Skou in 1957, evidence mounted over the next several years (mainly at that @ 1987, Elsevier Science Publishers B.V., Amsterdam
time from the laboratories of Post, Glynn, and Albers) that this enzyme was the molecular machine for effecting Na+ and K+ transport across the plasma membrane. We were thus able to put the PA cycle hypothesis to the test. We compared the turnover of PA in salt gland homogenates with the catal;rtic activity of the Na+/K+-ATPase; the latter was orders of magnitude higher. Thus, PA could not be an intermediate in the Na+/K+-ATPase. The net effect of our studies on Na+ transport in the salt gland was to shift me by the mid-1960s into the Na+/K+-ATPase field. Onreflection I think that many who followed closely the PA cycle hypothesis lost sight of the interesting facts about PI and PA after the demise of the PA cycle. However, some felt differently. Bob Parks commented to me shortly after the second messenger functions of IP3 and DG were discovered, ‘When I first heard you present that data [in the early years], I thought, boy, that has to be important.’ In a somewhat different vein, another colleague at that time put it this way, ‘You have the treatment for a disease without knowing what the disease is.’ It is to the great credit of Bob Michell that he stimulated a revival of interest in the PI effect with his 1975 review and with his dogged determination - against much opposition - to see through to the end his Ca’+-gating hypothesis. Apparently, there was some derision of the PI effect during those years. At a recent meeting, John Fain said that when he arrived at Berridge’s laboratory in the late 197Os,the common attitude towards the PI response (presumably not that of Berridge) was
Conunonlink to K+ channels Evidence that a single GTP-binding protein (e.g. GJ regulates current flow through K+ channels coupled to multiple receptors has recently been reported in Nature (Vol. 325, pp. 259-262). Kazuhiko Sasaki and Makoto Sato have demonstrated in sea slug Aplysia that islet activating protein blocks irreversibly all K+-depen-
0165 - 6147/87&02.Nl
that it was ‘Hokin’s hokum and Michell’s folly’. Naturally, I am elated and I know I speak for Mabel Hokin as well, when I think that what we started over 30 years ago has led to the discovery of two phosphoinositide-derived second messengers - IP3 (Berridge) and DG (Nishizuka) and all that this implies. Additional inositol phosphate compounds formed on stimulation are rapidly being found. I suspect that there may be more phosphoinositide-derived second messengers to come. For example, current studies from Majerus’ laboratory and our own are beginning to suggest that inositol 1: 2(cyclic)-4,5+isphosphate may be another second messenger for Ca2+ release. Provocative studies on the involvement of the phosphoinositide system in fertilization, growth, and oncogene action are emerging. Functions for phosphoinositides other than as secondmessenger generators are beginning to surface. There should be plenty for all of us to do for some time to come. LOWELL E. HOKIN Depnrtment of Pharmacology, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706, USA.
Further reading 1 Benidee. M. I. and Irvine, R. F. (1984) Naturev3i2,3G-321 2 Hokin, L. E. (1985) Annu. Reo. Biochem. 54,205-235 3 Downes, C. P. and Michell, R. H. (1985) in Molecular Mechanisms of Transmembrane SignaJJing(Cohen, P. and Houslay, M. D., eds), pp. 3-56, Elsevier 4 Nishizuka, Y. (1986) Science 233,305-310 5 Abdel-Latif, A. A. (1986) Pknrmacol. Rev. 38,227-272 6 Kennedy, E. P. (1986) in Lipids in Membranes: Past, Present, and Future (Op den Kamp, J. A. F., Roelofsen, B. and Wirtz, K. W. A., eds), pp. 171-206, Elsevier
dent responses to dopamine, histamine and acetylcholine, and that in the presence of GTPyS these agonists opened all K+channels coupled with each type of receptor. These results strongly suggest that all of the agonistinduced increases in K+-conductance were produced by a GTPdependent activation of a common mediator such as Gi or another IAP substrate. (Similar responses have also been seen with opiate and ar2 receptors in the cat locus coeruleus neurone.)
53 gained by this scheme, some of which has been reported in Monitoring for Drug Safety, 2nd edition, (W. H. W. Inman, ed.). Of particular importance is, of Course, our ability to ascribe accurate incidence to events. As with the more recent prescription event monitoring by the Drug Surveillance Research Unit, University of Southampton, the scheme suffers from the drawback of not having properly constructed control groups in the initial event monitoring but these have been incorporated in some of our follow-up surveys. The other drawback is
The road to the phosphoinositidegenerated second messengers is the story ofthe &scovery of the phosphoinositide effect in the early 195Os, a ‘discovery before its time’. Twenty years were to elapse after our initial demonstration of agonist-stimulated phosphatidylinositol (PI) turnover before Bob Michell was to rekindle interest in this effect - an effect which has answered (and posed!) so many questions about signal transduction mechanisms. If only we could have taken a glimpse into the future back then1 wouldnever have allowed an albatross to side-track me from continuing to investigate the true meaning of this fundamentally important phenomenon. This
The early years But to begin at the beginning, this story dates back to 1949, when I arrived at H. A. Krebs’ laboratory in Sheffield to study for a Ph.D in biochemistry. Krebs’ policy was to have a student formulate his own problem, pursue it independently with a minimum of supervision and publish by himself. With some background in gastroenterology which I had received in the laboratory of Warren S. Rehm while attending medical school, I looked into whether I could use pancreatic tissue as a model to study the synthesis and secretion of proteins, using amylase as a measure of these two processes.
Pigeon pancreas s!ices proved to be an excellent system for this purpose. Near the end of my doctoral studies in Krebs’ laboratory, I had become interested in the possible involvement of RNA in protein synthesis. This was based on some cytological studies in the late forties by Caspersson and Brachet showing that RNA levels in a variety of dividing and non-dividing cells were correlated with protein synthetic activity. RNA levels were particularly high in the pancreas. At about that time, Kenneth Burton, who was then a Lecturer in Krebs’ Department, drew my attention to a report by two Russian workers, M. A. Gubemiev and L. I. Il’ina, who showed that on cholinergic stimulation of secretion in pancreas, stomach, and salivary glands in anesthesized dogs (shades of Pavlov) there were marked increases in the incorporation of 32P into ‘nucleoproteins’, isolated by the method of Schmidt and Thannhauser. This method was really designed to isolate phosphorus fractions, including DNA and RNA (separated by alkaline hydrolysis), and not nucleoproteins, but no further details were given. A.pplicationof 32P The use of 32P as a tracer had been adopted by Krebs and his
the relatively small population of New Zealand of approximately 3.3 million. However, the careful scrutiny of events and their followup in individual cases for causality we believe offsets this disadvantage. I.
RALPH
EDWARDS
ZealandMedical Assessor for Medicines Adverse Reactions, University of Otago, Dunedin, New Zealand. New
References
1 Strom,B. L. (1986)TrendsPkatnxacol. Sci. 7, 377-380
associates a year or two earlier. I found that on stimulation of enzyme secretion in pancreas slices, there was about a 100% increase in the incorporation of [32P]orthophosphate into RNA which confirmed the observations of Guberniev and Il’ina on ‘nucleoproteins’, although they observed much greater effects. Near the end of this ._____, I began to suspect that work. perhaps an alkaline hydrolytic product of phospholipids was contaminating the RNA fraction, and this might be responsible for the stimulation of 32P incorporation into RNA. This idea came to me shortly after I had completed my requirements for the Ph.D and was about to set sail with Mabel Hokin for Halifax. 1 did manage to do one experiment, but I did not have time to go through the necessary procedures to rid the lipid fraction of contaminating [32P]orthophosphate and to count the radioactivity. I literally took with me on the boat a rack of about a dozen rather large test tubes containing the ethanol-ether extracts. When we arrived at J. H. Quastel’s laboratory at McGill, we counted the purified total lipid fractions and found an enormous increase in specific radioactivity in the lipids from pancreas slices which had been stimulated with carbachol. With better methods for isolation of nucleic acids from small samples of tissue developed by others in 1952. we were able to show that there was no stimulation of 32P incorporation in RNA. I like the comment of the Russian discoverer of myosin ATPase, V. A. Engelhardt, who knew Gubemiev and Il’ina, when I told him at the XlXth International Physiological
@ 1987. ElsevierSciencePublishersB.V., Amsterdam 0165- 6147/87&QZOQ
TIPS - February 1987 IVol. 81 microsomes (fragments of endoplasmic reticulum and plasma membrane) isolated after incubation of pancreas slices. Using radioautography with [3H]inositol, we showed in the mid-1960s that the effect was predominantly in the endoplasmic reticulum in sympathetic ganglia and pancreas. This has been confirmed more recently with other cell types. It is still not clear how this fits into the overall picture.
Lowell and Mabel Hokin optimistic
in the early 1950s in Copenhagen.
Congress in Montreal in 1953 that the stimulated 32P turnover was in phospholipids and not in ‘nucleoproteins’. With the characteristic Russian shrug of the shoulders he said, ‘So it’s in the phospholipids,’ which I interpreted as saying that the stimulated 32P incorporation was the important point and why quibble over details; after alI, at this juncture we do not know much about the function of either RNA or phospholipids (the Watson-crick double helix structure of DNA was published that year; nucleic acids had a much brighter immediate future than phospholipids). The 1953 JBC paper arising from this work is often incorrectly quoted as demonstrating the stimulated turnover of phosphatidylinositol (PI). We did not actually show this until 1955 when techniques for measuring radioactivity in individual phospholipids from small samples of tissue became available (Dawson; Marinetti). The discovery of the PI-PA
(phosphatidic) effect launched Mabel Hokin and me on a 1Syear quest for its explanation, as welI as a pursuit of its biochemical mechanism. Mabel Hokin continued this search after I had left the field. I have only recently returned. We made several key observations in the fifties concerning the PI-PA effect, including: (1) the effect occurred with many agonists and in many cell types; (2) the effect for the most part was due to a turnover and not a net synthesis of PI and PA, with the diacylglycerol (DG) moiety being partly or completely conserved; (3) the physiological response, i.e., amylase secretion, was not tightly coupled to PI turnover as shown by different dose-response curves (in the middle sixties I also showed a dissociation of these two processes by omitting Ca*+); (4) PA, in addition to being synthesized by acylation of ar-glycerophosphate (Komberg and Pricer; Kennedy), could be synthesized from ATP and DG; and (5) the PI effect was located in
The PI-PAcycle In the early sixties, with the aid of kinetic studies on the turnover of PI and PA in the avian salt gland on cholinergic stimulation (followed by atropine clamping), we were able to present the first version of the PI-PA cycle (Fig. 1). This cycle also incorporated our knowledge of the enzymes involved in the phosphodiesterase cleavage of PI (Hawthorne) and in PI synthesis (Agranoff; Kennedy). We published these studies in 1964 in a symposium entitled, ‘Metabolism and Physiological Significance of Lipids’, edited by R. M. C. Dawson and D. N. Rhodes (Wiley). I have often regretted that the results and the proposed PI cycle in this paper were not made more generally available by publication in a regular journal. The main points of this cycle were that on cholinergic stimulation of salt gland slices phospholipase C catalysed the breakdown of PI to DG and IP. Diacylglycerol kinase then formed PA. On quenching with atropine, the resting steady-state level of PI was restored at the expense of PA by the sequential actions of PAcytidyl transferase -and PI synthase. Mat -1 Hokin made some key observations in the early 1970s further supporting the PI-PA cycle, including the findings that on stimulation in pancreas, there was a loss in the mass in PI and a rise in the mass of PA. Also, on stimulation, the fatty acid composition of PA approached more closely that of PI. These data gave additional strong support to the view that PA was derived from PI during stimulated turnover of PI and PA. She further found an increase in mass of DG in pancreas on agonist stimulation, although the fatty acid composition of the liberated DG did not agree closely with that of PI. Later studies did
77PS - February 1987 [Vol. 81 show a close similarity in fatty acid compositions between PI and liberated DG in other cell types. At about the same time, Michell showed independently a fall in mass in PI in the parotid gland on stimulation. Mabel Hokin also confirmed the salt gland kinetic data in the pancreas. Her recalculation of some old measurements of PI in the salt gland (expressed as percentages of controls rather as absolute values) showed a significant loss in mass in PI in this tissue as well. The original PI-PA cycle has, of course, been modified to incorporate the polyphosphoinositides. There is still controversy as to wheLher there is dibect phosphodiesteratic cleavage of PI as an additional source of DG. TThepoIyphosphoinositides With regard to early studies on polyphosphoinositides, in the early 1960s many of us studied the turnover of phosphatidylinositol-4phosphate (PIP) and phosphatidyiinositol4,5-bisphosphate (pIP2), in-
55 eluding our demonstration of a fall in steady-state level of 32P-labelled polyphosphoinositides on stimulation of salt gland slices with acetylcholine (no time courses were done). We also showed for the first time the presence of the kinases for PI and PIP (in the erythrocyte membrane), as well as the rapid exchange of 32P into the monoesterified phosphates of the polyphosphoinositides in erythrocyte membranes on incubation with [32P] ATP. In the late 196Os, Durell demonstrated the formation of inositol-l-phosphate (IP) and inositol 1,4-biphosphate (II’*) in cholinergically stimulated synaptosomes and suggested that the primary response might be the phosphodiesteratic cleavage of the polyphosphoinositides, releasing inositol phosphates. Abdel-Latif pursued this line in the late 1970s and showed that cholinergic or adrenergic stimulation of iris smooth muscle caused a rapid breakdown of PIP2 which was accompanied by an increase in
Fig. 1. TheoriginalversionofthePCPA cycle. From Hokin, M. R. andHokin, 1. E. (1964) in Metabolism and PhysiologicalSignificance of Lipids (Oatwon, R. M. C. and Rhodes, D. PI., eds), pp. 423434, John Wiley.
Fig. 2. Chromatogram demons!rating incorporation of *P into the responsive in&tide, PI. Right, using cholinergically siimutated pancreas.Left, unstimulated contrd From Hokin and Hokin, J. Biol. Chem. (1958) 233, 8tX-810. IP, IP2 and inositol 1,4,5-trisphosphate (IPa). Abdel-Latif did not believe that polyphosphoinositide breakdown was antecedent to Ca” gating for two reasons. The breakdown was dependent on Ca2+ (later shown to be at conceittrations lower than basal cytosolic leveis) and was stimulated by Ca2+ ionophores. This turned out to be due to a direct release of norepinephrine by ionophores.
A discovery before its time The invitation to write this article has stimulated me to go back and look at our papers published in the early 1950s and I am struck by how naive some of our interpretations of the phospholipid effect were because so many pieces of the puzzle were missing. Very little was known about membrane structure. The classic experiments from the laboratories of Douglas and Katz pointing to the importance of Ca*+ in stimulus-response coupling were not carried out until the early 1960s. We did not know for certain the structure of the ‘phosphoinositide’ which showed increased turnover in pancreas and brain until around 1958, when we showed it was PI (Fig. 2). Although Jordi Folch had identified in brain a phosphoinositide fraction which showed a phosphorus : inositol ratio of 2 and which he called diphosphoinositide (this was presumably a mixture of all three phosphoinositides), it was not until the early 1960s that the identification and the full shwtural elucidation of PIP and PIP;! were made in the laboratories of Ballou and Dawson. The existence
TIPS - Februa y 1987 [Vol. 81
56 of the intracellular ‘trigger pool’ of Ca’+ which rapidly releases part of its stores of Ca2+ on agonist stimulation was not known until the work of Schulz and others in the late 1970s. The use of lithium to amplify the accumulation of inosit01 phosphates was not adopted until the early 1980s. The technique of cell permeabilization to allow entry of phosphorylated compounds into the cell was not developed until the late 1970s and early 1980s. This, of course, permitted Berridge and his associates to demonstrate the release of nonmitochondrial intracellular stores of Ca2+ by IPs, although it would have been possible to show this effect (but perhaps not to fully appreciate it) earlier with microsomes. Perhaps we were led astray mostly when in 1959, we found a dramatic stimulation of PA turnover and a lesser stimulation of PI turnover in the salt gland of the albatross (shipped to us by the US Navy from Midway Island). Since the exclusive function of this organ is to secrete concentrated salt solutions in response to cholinergic stimulation, we were naturally tempted to conclude that the ?I-PA effect was an important component of the Na+ pump these studies preceded, by many years, the work showing Ca2+ involvement in NaCl secretion. Since the membranes of this tissue were also very rich in DG kinase and PA phosphatase, we proposed that if DG kinase were located on the inner surface of the plasma membrane and PA phosphatase situated near its outer surface, perhaps lipid-soluble sodium phosphatidate and DG could shuttle back and forth across the plasma membrane with PA carrying two sodiums per cycle. The PA cycle would thus function as an ATPase. This scheme naturally aroused considerable interest since the data seemed to suggest that PA might be the long-soughtafter Na+ carrier - in those days many of us thought of ion carriers as being small molecules. It also stimulated considerable controversy, partly centered around the predicted stoicldometry of two sodiums per ATP. After the Na+/K+-transporting ATPase was discovered by Skou in 1957, evidence mounted over the next several years (mainly at that @ 1987, Elsevier Science Publishers B.V., Amsterdam
time from the laboratories of Post, Glynn, and Albers) that this enzyme was the molecular machine for effecting Na+ and K+ transport across the plasma membrane. We were thus able to put the PA cycle hypothesis to the test. We compared the turnover of PA in salt gland homogenates with the catal;rtic activity of the Na+/K+-ATPase; the latter was orders of magnitude higher. Thus, PA could not be an intermediate in the Na+/K+-ATPase. The net effect of our studies on Na+ transport in the salt gland was to shift me by the mid-1960s into the Na+/K+-ATPase field. Onreflection I think that many who followed closely the PA cycle hypothesis lost sight of the interesting facts about PI and PA after the demise of the PA cycle. However, some felt differently. Bob Parks commented to me shortly after the second messenger functions of IP3 and DG were discovered, ‘When I first heard you present that data [in the early years], I thought, boy, that has to be important.’ In a somewhat different vein, another colleague at that time put it this way, ‘You have the treatment for a disease without knowing what the disease is.’ It is to the great credit of Bob Michell that he stimulated a revival of interest in the PI effect with his 1975 review and with his dogged determination - against much opposition - to see through to the end his Ca’+-gating hypothesis. Apparently, there was some derision of the PI effect during those years. At a recent meeting, John Fain said that when he arrived at Berridge’s laboratory in the late 197Os,the common attitude towards the PI response (presumably not that of Berridge) was
Conunonlink to K+ channels Evidence that a single GTP-binding protein (e.g. GJ regulates current flow through K+ channels coupled to multiple receptors has recently been reported in Nature (Vol. 325, pp. 259-262). Kazuhiko Sasaki and Makoto Sato have demonstrated in sea slug Aplysia that islet activating protein blocks irreversibly all K+-depen-
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that it was ‘Hokin’s hokum and Michell’s folly’. Naturally, I am elated and I know I speak for Mabel Hokin as well, when I think that what we started over 30 years ago has led to the discovery of two phosphoinositide-derived second messengers - IP3 (Berridge) and DG (Nishizuka) and all that this implies. Additional inositol phosphate compounds formed on stimulation are rapidly being found. I suspect that there may be more phosphoinositide-derived second messengers to come. For example, current studies from Majerus’ laboratory and our own are beginning to suggest that inositol 1: 2(cyclic)-4,5+isphosphate may be another second messenger for Ca2+ release. Provocative studies on the involvement of the phosphoinositide system in fertilization, growth, and oncogene action are emerging. Functions for phosphoinositides other than as secondmessenger generators are beginning to surface. There should be plenty for all of us to do for some time to come. LOWELL E. HOKIN Depnrtment of Pharmacology, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706, USA.
Further reading 1 Benidee. M. I. and Irvine, R. F. (1984) Naturev3i2,3G-321 2 Hokin, L. E. (1985) Annu. Reo. Biochem. 54,205-235 3 Downes, C. P. and Michell, R. H. (1985) in Molecular Mechanisms of Transmembrane SignaJJing(Cohen, P. and Houslay, M. D., eds), pp. 3-56, Elsevier 4 Nishizuka, Y. (1986) Science 233,305-310 5 Abdel-Latif, A. A. (1986) Pknrmacol. Rev. 38,227-272 6 Kennedy, E. P. (1986) in Lipids in Membranes: Past, Present, and Future (Op den Kamp, J. A. F., Roelofsen, B. and Wirtz, K. W. A., eds), pp. 171-206, Elsevier
dent responses to dopamine, histamine and acetylcholine, and that in the presence of GTPyS these agonists opened all K+channels coupled with each type of receptor. These results strongly suggest that all of the agonistinduced increases in K+-conductance were produced by a GTPdependent activation of a common mediator such as Gi or another IAP substrate. (Similar responses have also been seen with opiate and ar2 receptors in the cat locus coeruleus neurone.)