Regulation by membrane potential of phosphatidylinositol hydrolysis ...

Vol. 268, No. 15, Issue of May 25, pp. 11065-11072,1993 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation by Membrane Potential of Phosphatidylinositol Hydrolysis in PancreaticIslets* (Received for publication, October 1, 1992, and in revised form, January 22, 1993)

Trevor J. Biden$, Aidan G . M. Davison, and Monica L. Prugue From the Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney 2010, New South Wales, Australia

In pancreatic islets stimulated with carbamylcholine hydrolysis, initiated by occupation of specific cell-surface (carbachol),hydrolysis of bothphosphatidylinositol receptors, plays an important role in many examples of cel(PtdIns) and phosphatidylinositol bisphosphate (Ptd- lular activation (1,2). The underlying molecular interactions as the inositol mon- are also broadly understood the occupied receptor interacts InsPa) occurs and can be measured ophosphates Ins(l)P1, or Ins(4)P1, respectively (Biden, with a specific G-protein, which in turn activates a particular T. J., Prugue, M. L., and Davison, A. G . M. (1992) Biochem. J. 285, 541-549). Our current aim was to isoform of phospholipase C (3). The resultant hydrolysis of PtdInsP2 liberates bothDAG, which activates protein kinase establish whether these two events were independently regulated. Rat islets werelabeled with either [‘Hlino- C, and Ins(1,4,5)P3, which mobilizes Ca2+ from intracellular sitol or [‘Hlarachidonic acid for measurement of InsPls stores (1, 2). Most of the studies which led to the delineation of this by high performance liquid chromatography or diacyll)P1due sequencefocussed on the events whichoccurred following glycerol by TLC, respectively. The rise in Ins( to carbachol (1min) was inhibited by 50%by concom- application of a singleclass of external stimuli.However, cells itantly raising extracellular KC1 ([K+],) from 6 to 30 in vivo are likely to be subjected to a variety of concurrent mM, thereby depolarizing the islets. Similiar results, stimuli, and it is therefore important to examine how these obtained in theabsence of extracellular Ca2+, exclude might modulate PtdInsP2 hydrolysis. For electrically excitable the involvement of voltage-gated Ca2+channels. Conversely, hyperpolarization, by lowering[K’], to 3 mM, cells, an obvious first step would be an evaluation of the increased by 30%the rise in Ins(l)P1. In fact, over theeffects due to changes in membrane potential.However, this [K+]. range of 3 to 48 mM, stimulated Ins(l)P1 accu- has only been addressed in the human leukemic HL60 cell line which, althoughnot electricallyexcitable,diddisplay mulation was directly proportional to the calculated membrane potential. In contrast, raising[K+], from 6 attenuated PtdInsP2hydrolysis under conditions inwhich the to 4 8 mM exerted no significant effect on carbachol- membrane potentialwas completely collapsed(4).Better docstimulatedIns(4)P1levels,andboth Ins(l)P1and umented is the direct stimulation of PtdInsPz hydrolysis in Ins(4)P1 wereunaffected in the absence of carbachol. pancreatic islets ( 5 ) and adrenal chromaffincells (6) exposed also inhib- to depolarizing K+ concentrations, but this is the The rises in Ins(l)P1(but not Ins(4)P1) were consequence ited by depolarization with thesodium pump inhibitor, of Ca2+ influx through voltage-dependent Ca2+ channels ouabain, or the K+channel blocker, tolbutamide. Stimulated diacylglycerol accumulation and insulin secre- rather than aneffect of depolarization per se. In pancreatic islets, nutrient secretagogues such as glucose tion (20 min) showed a biphasic dependency on [K+],, periodic being less pronounced at 6 mM than at either3 or 30 also depolarize the plasma membrane and initiate mM KCl. This reflectsa selective potentiationof PtdIns bursts in electrical activity which are accompanied by the and PtdInsPz hydrolysis, due, respectively, to hyper- gating of voltage-dependent Ca2+ channels(7). On the other polarization and the gatingof voltage-dependent Ca2+ hand, the neurotransmitteracetylcholine, which acts asa cochannels. The differential regulation of these two hy- stimulus with nutrients during the cephalic phase of insulin drolytic events is probably important for independent @-cellsurface control of the activation of protein kinase C and Ca2+ secretion, binds to muscarinic receptors on the mobilization and might play a role in modulating the and promotes PtdInsP2 hydrolysis (8). This has been demonstrated experimentally as an increased production of both secretory responsein vivo. InsP3 (9, 10)and DAG (11, 12), as well astheenhanced release of Ca2+ from intracellular stores (9) and translocation of protein kinase C (13). Recently we have shown that musIt isnow well established that the stimulation of PtdInsPz‘ carinic agonists also stimulate the hydrolysis of PtdIns, an * This work was supported by research grants from the Diabetes effect which is rapid in onset (5s) and increasingly predominant in comparison to PtdInsP2 breakdown during prolonged Australia Research Trustandthe Juvenile Diabetes Foundation International. The costs of publication of this article were defrayed stimulation (14). In the current study, we demonstrate that in part by the payment of page charges. This article must therefore the hydrolysis of PtdIns, but not that of PtdInsP2, is exbe hereby marked “aduertisernent” in accordance with 18 U.S.C. tremely sensitive to alterations in plasma membrane potenSection 1734 solelyto indicate this fact. tial, and additionally thatonly PtdInsP2 breakdown is stim$ Recipient of a JuvenileDiabetes Foundation International Career ulated by Ca2+ influx. In differentiating between these two Development Award. The abbreviations used are: PtdInsP2, phosphatidylinositol 4,5- sources of DAG, we also demonstrate a potential role for bisphosphate; PtdIns, phosphatidylinositol; KRB, Krebs-Ringer bi- PtdIns hydrolysis in the stimulation of insulin secretion by carbonate; InsP,, inositol monophosphate (with isomeric configura- muscarinic receptor agonists.

tions as indicated); InsPz, inositol bisphosphate; Inspa, inositol trisphosphate; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; G-protein, GTP-binding proteins; DAG, diacylglycerol; [K’],, extracellular K+ concentration; [Ca2+Ii,free cytosolic Ca2+concentration; KATpchannel, ATP-dependent K+ channel.

EXPERIMENTALPROCEDURES

Islet Isolation and Incubation-Pancreatic islets were isolated from 220-240-g rats by digestion of the exocrine pancreas by ductal infu-

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Islet PtdIns Hydrolysis

sion of collagenase (15). They were purified on a Histopaque 1077 gradient and then hand-picked under a binocular microscope (14). Batches of approximately 600 islets were cultured for 48-72 h inside a modular incubation chamber as previously described (14). Medium 199 (4 ml/batch) was used, supplemented with 10% (v/v) fetal calf serum, 14 mM NaHC03, 11.1mM glucose, 500 IU/ml penicillin, 100 pg/ml streptomycin, and 50 pg/ml gentamycin. For inositol phosphate measurements, and for the determination of specific activity in PtdIns, themedium was supplemented with 30 pCi/ml [2-3H]inositol throughout the culture period; for estimation of [3H]DAG,5 pCi/ml [15-3H]arachidonic acid was included during the final 24h. After culture, the islets were washed and resuspended in a modified KRB mediumwhich contained5 mM NaHC03, 2.8 mM glucose, 1 mM CaC12,0.5% bovine serum albumin, and 10 mM Hepes (pH 7.4). Inositol Phosphate Studies-For inositol phosphate measurements, groups of 50 islets were preincubated for 10 min a t 37 "C in 0.1 ml of KRB. In some studies, ouabain and tolbutamide were added 2 min prior to stimulation while in others, in which neomycin was also included, the preincubation period was extended to 20 min. Experimental additions were made doubly concentrated in another 0.1 ml of KRB. Incubations were terminated with 0.8 mlof 10% (w/v) trichloroacetic acid. After vortexing and extraction at 4 "C for approximately 30 min, the samples were centrifuged to remove nonsoluble material, and the supernatantwas washed three times with a 5foldexcess of diethyl ether. In some studies, these extracts were neutralized, and inositol phosphates were separated exactly as described (14) using 0.6-ml columns of Dowex AGl-X8 (formate) eluted sequentially with 0.1 M formic acid buffers containing increasing strengths of ammonium formate. Radioactivity in the collected fractions was determined by liquid scintillation spectrometry. For analysis by HPLC, extracts were processed prior to injection as already reported (14). A Partisphere PAC (12.5 cm) column was used, prewashed prior to each injection for 10 min at 2.0 ml/min with each of the following: 0.05M H3P01, 2 M ammonium formate (pH 3.7 with H3P04), and water. These columns showed resolution equal to that of Partisphere SAX, but with less variability between individual columns and perhaps better column life. Since the InsPls were the only species measured in the current study, a simple linear gradient of 0 to 0.068 M ammonium formate (pH 3.7) was applied, following an initial 5-min wash with water (all at 1 ml/min). Under these conditions, the resolution of individual InsP, isomers was similar to that already described Ins(l)P1eluting around 2-3 min prior to the putative Ins(2)Pl,which was followed in turn a further 3-5 min later by Ins(4)P1 (14). Although always maintaining this degree of separation, the exact elution times of the InsP,s (in contrast to the InsPzs and InsP3s) tended to vary between runs. Ins(l)P1 was therefore always identified by co-injection of [14C]Ins(3)P1,an enantiomer of Ins(l)P1, andknown to co-elute with it (14). Fractions were collected over 30 s and mixed with 4 ml of scintillant, and radioactivity was determined by liquid scintillation spectrometry. The islet content of PtdIns was also determined in order to asses its specific activity. The trichloroacetic acid-insoluble material from islets incubated as described above was dissolved in 0.5 ml of chloroform/methanol/l2 M HCl (200:100:0.75). Combined extracts from 6 tubes (300 islets total) were then washed three times with 3 ml of chloroform/methanol/0.6 M HC1 (3:48:47),and theorganic phase was dried under Nz (11).After reconstitution, the extract was applied to Silica Gel G plates which weresubsequently developed in chloroform/ methanol/acetic acid/water (65:43:1:3). Spots corresponding to PtdIns, well separated from other phospholipids by this solvent system (16), were cut out and eluted with 0.5 ml of water. The eluates were transferred to glass tubes, dried at 100 "C, and then treated at 190 "C for 4 h with 0.2mlof a solution containing 60% perchloric acid and 5 M NaHZSO, (6:1, v/v). After cooling,0.25ml of 5 mM H2S04was added to thetubes, which were then vortexed and briefly centrifuged to remove any particulate matter. To 0.12mlof the supernatant was added 0.48 ml of a color reagent which consisted of 3 volumes of malachite green (0.045% w/v in water) and 1 volume of ammonium molybdate solution (4.2% w/v in 4 M HCl). Phosphate content was determined by comparison of the Am with those of samples containing 0-6 nmol of Pi which had been processed in parallel with the experimental tubes (17,18). A portion of the sample which had not been used for color development was counted to determine the amount of radioactivity present as [2-3H]inositol. The specific activity of PtdIns was then calculated from these two values. DAG Studies-For measurement of DAG, groups of 50 islets prelabeled with [15-3H]arachidonic acid were distibuted in 0.1 ml of KRB. Alternatively, groups of 100 unlabeled islets were used for

determination of DAG mass. After a 10-min preincubation at 37 "C, experimental additions were made doubly concentrated in another 0.1 ml of prewarmed KRB. After incubation for 20 min at 37 "C, an extraction mixture of0.6mlof ice-cold chloroform/methanol (1:2) was added, and the tubes were left for 30-60 min on ice. A further 0.6 ml of the extraction mixture was then added, containing 5 pgof 1,2-distearoyl-sn-glycerol(ommitted in mass experiments) and approximately 2,000 cpm of l-oleoyl-2-[l-14C]arachidonyl-sn-glycerol. After extraction overnight at 4 "C, the samples were transferred to glass tubes, to each of which was added 0.4 ml of distilled water and 0.2 ml of chloroform. After vortexing, the tubes were centrifuged, and the aqueous (upper) layer was removed. The samples were washed two more times with 0.2mlof water and 0.2 mlof chloroform, on each occasion removing the aqueous layer (11).The organic layer was dried and stored under N, at -20 "C. The procedure then diverged depending on whether DAG mass or [3H]DAGwas being measured. For the latter, samples were reconstituted in 50 pl of chloroform and spotted on silica gel TLC plates preheated for 30 min at 42 "C. The plates were developed in tanks saturated with a solvent mixture (18) of benzene/chloroform/methanol (8015:5). Under these conditions, the 1,2-DAG (R, = 0.77) is well separated from 1,3-DAG (RF= 0.85) and other neutral lipids (not shown). Spots corresponding to 1,2dioleoyl-sn-glycerolwere visualized by Coomassie Blue staining and removed from the plates, and radioactivity was determined by dual channel liquid scintillation spectrometry. Individual replicates were corrected for recovery (routinely >80%)as calculated using the internal [14C]DAGstandard. For measurement of DAG mass, the DAG kinase method (19) was employed as modified for use with a commercial kit. Briefly, the dried DAG samples were taken up in a detergent solution, sonicated, and then incubated for 30 min at 25 "C with a reagent mix containing DAG kinase, ATP (0.5 mM final), and [y-32P]ATP(1pCi/tube). The incubation was terminat.ed with perchloric acid (1%v/v) and chloroform/methanol(1:2) containing1pg of phosphatidic acid as carrier. After further washing with the perchloric acid, the organic phases were dried under N,, reconstituted inchloroform, and applied to silica gel TLC plates. The latter were developed in chloroform/methanol/ acetic acid (65:15:5), the spots of [32P]phosphatidicacid were identified by autoradiography, and radioactivity was then quantified by liquid scintillation spectroscopy. DAG mass was calculated by comparison of radioactivity in the experimental samples with that of a range of DAG standards carried through the same procedure. Insulin Release-Groups of 10 islets were preincubated for 10 min at 37 "C with 0.5 ml of KRB, after which time experimental additions were made doubly concentrated in another 0.5 ml ofthe same medium. After a further 20 min, the tubes were chilled to 4 "C and 0.8 ml of the medium was removed for estimation of insulin by radioimmunoassay. Data Presentation-Results are generally presented as mean f S.E. Statistical significance was assessed by Students't-test for unpaired data. Curve-fitting and correlation coefficients were determined using Cricketgraph, Cricket Software, Philadelphia, PA. Materials-The sources of all materials were as previously de[15scribed (14) except for ~-oleoyl-2-[l-14C]arachidonyl-sn-glycerol, 3H]arachidonic acid, [y-32P]ATP,3-[1251]iodotyrosyl A14 insulin, and DAG assay kits which were supplied by Amersham Australia Pty Ltd, Sydney, NSW, Australia. Insulin radioimmunoassay reagents were from the following suppliers: guinea pig anti-porcine insulin serum from Linco Research Inc., St. Louis, MO; rat insulin standard from Novo, Copenhagen, Denmark; and sheep anti-guinea pig immunoglobulin from Silenus Laboratories, Melbourne, Victoria, Australia. TLC plates were supplied by Merck AG, Darmstadt, FRG. Ouabain, tolbutamide, 1,2-distearoyl-sn-glycerol, phosphatidic acid, and all other biochemicals and specialized reagents were from Sigma. RESULTS

It haspreviously been established thatdepolarizing agents, such as glucose o r KC1, promote PtdInsPn hydrolysis in pancreatic islets in a manner secondary to the influx of Ca2+ throughvoltage-dependent Ca2+ channels (5). However, a small component of the PtdInsP2 hydrolysis due to glucose appearedto be independent of Ca2+ influx (5, 20). These additionaleffectsmight be explained by the inhibition of Ins(1,4,5)P3 degradation by certain glucose metabolites which has been demonstrated using islet homogenates (21). If this

Islet PtdIns Hydrolysis explanation is valid, the increase in Ins( 1,4,5)P3due to stimulation with a receptor-binding agonist should be potentiated by glucose in a manner independent of Ca2+influx. Testing this postulate was the startingpoint of the current study. Islets were incubated for 1 min at low (2.8 mM) or high (16.7 mM) glucose, either alone or in the presence of 0.5 mM carbachol, in a medium in which the free extracellular Ca2+ concentration was lowered with EGTA. Under these conditions, glucosewas unable to raise InsP3, InsP,, or InsP1, whereas carbachol increased all 3 species (Fig. 1, upperpanel). Both results are consistent with a previous study documenting that carbachol, as opposed to glucose, stimulated PtdInsP, hydrolysis in the absence of extracellular Ca2+ (5). In the combined presence of carbachol and 16.7 mM glucose, InsPB levelswere not increased above those seen with carbachol alone nor was InsP, decreased. These results are therefore inconsistent with the postulatedinhibition of Ins(1,4,5)P3 dephosphorylation by glucose metabolites (21). However, most surprisingly, the carbachol-stimulated increase in InsPl was significantly attenuated by16.7 mM glucose (upper panel). This effect was reproduced when [K’], was raised from 6 to 30 mM (lower panel). This manipulation, as with raising the glucose concentration, had no effect alone on any of the measured inositol phosphate species, nor did it alter the carbachol-stimulated increases in InsP, or InsP3. The attenuation of the rise in InsPl was not secondary to Ca2+ influx, since that should have been abolished in the presence of EGTA and was not simply due to glucose metabolism, since it was reproduced by KCl. It should be noted that InsP, levels are representative of the hydrolysis of both PtdIns and PtdInsP2, whereas only the latter contributes to InsP3 accumulation. Therefore, the simplest interpretation of the current findings is that PtdIns hydrolysis is selectively inhibited by depolarization.

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This was further investigated by performing HPLC analysis of the InsPl isomers accumulating during 1-min stimulation with carbachol (Fig. 2). We have previously shown that, under these defined conditions, the rise in Ins(l)P1reflects enhanced hydrolysis of PtdIns (14). That work also demonstrated that Ins(4)P1 was an extremely sensitive measure of carbacholstimulated PtdInsP, breakdown, giving a fold increase over basal which was an order of magnitude greater than the corresponding increase in Ins(1,4,5)P3 (14). In the current investigation, the increase in Ins(4)Pl due to carbachol was unaffected by raising [K+], to 30 mM, whereas the rise in Ins(l)P1was attenuated by approximately 60%. This strongly suggests that PtdInshydrolysis is indeed selectively inhibited by depolarization. Moreover, since these experiments were performed in the presence of extracellular Ca2+,the inability of 30 mM KC1 alone to increase Ins(l)P1 suggests that the hydrolysis of PtdIns is not directly initiated by an increase in [Ca2+],.On the other hand, PtdInsP, hydrolysis is stimulated under these conditions ( 5 ) , and, accordingly, it might be considered surprising that high KC1 did not increase Ins(4)P1. However, we have shown previously that although 30 mM KC1 initiates a prompt (2 s) increase in Ins(1,4,5)P3, there was a delay of more than 1 min before a corresponding rise was recorded in InsPl (5).Carbachol, on the other hand,being a stronger stimulus, generated sufficient Ins(1,4,5)P3for the dephosphorylation of the latterto InsPl tobe apparent within 10 s (5). Two further depolarizing agents were next employed in order to demonstrate that theinhibition of PtdIns hydrolysis was truly due to depolarization and not simply a function of raised [K’], which was fortuitously reproduced byglucose. These compounds do not act simply by altering the equilibrium potential across the plasma membrane for K’. Instead, tolbutamide selectively closes KAT^ channels present in the insulin-secreting p cells of the pancreatic islet (7), andouabain inhibits the sodium pump, thereby producing a depolarization which is associated with raised intracellular Na’ (22). Yet as shown in Fig. 3, both of these agents attenuatedthe ability of carbachol to trigger PtdIns hydrolysis without significantly altering ( p > 0.1) the accumulation of Ins(4)P1. A more detailed investigation of the dependence of carbachol-stimulated InsPl accumulation on membrane potential was next carried out. In this instance, extracellular Ca2+was depleted with EGTA, and [K’], was varied from 3 to 48 mM. As shown in Fig. 4 (upper panel) stimulated Ins(4)P1 levels declined in a linear fashion over this range ( R = 0.95; p < A

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FIG. 1. Effects of 16.7 m M glucose (upperpanel) or 24 m M KC1 (lowerpanel)on inositol phosphate levels in isolated islets incubated in either the presence or absence of 0.5 m M carbachol. Isolated islets were labeled to isotopic equilibrium with [3H] inositol and, following a 10-min preincubation, stimulated for 1 min in KRB medium containing 0.5 mM CaC12, 2 mM EGTA, and other additions as described. Separations of inositol phosphates were by Dowex anion exchange chromatography. For further details, see “Experimental Procedures.” All results represent 9-11 individual observations. Values significantly different ( p < 0.05) from the glucose 2.8 mM control are indicated by an asterisk.

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I n sI(n4 s) (P1l ) P l FIG. 2. Effect of 24 m M KC1 on the levels of Ins(4)P1 and Ins( l)P1in islets incubated in either the presence or absence of 0.5 m M carbachol. Incubations were performed as described in the legend to Fig. 1 except that the CaC12 concentration was 1 mM and EGTA was omitted. InsP, isomers were quantified by HPLC using an ammonium formate gradient and a PartispherePAC column. Each bur represents the average of 8 individual determinations. Unless otherwise indicated, values marked by an asterisk ( p < 0.05) are significantly different from those of the glucose 2.8 mM control.

Islet PtdIns Hydrolysis

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Carbachol Carbachol & ouabain Carbachol & tolbutamide

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I n sI (n4s)(P1l) P l FIG. 3. Effects of 0.1 mM oubain and 50 pg/ml tolbutamide on the levels of Ins(4)P1and Ins( I)P1in islets stimulated with 0.5 mM carbachol. Experiments were performed as described in the legend to Fig. 2. Each column represents the average of 9 individual determinations. Results are expressed as a percentage of the incremental stimulation due to carbachol alone. Columns marked with an asterisk are significantly different ( p < 0.01) from the carbachol response.

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3 mM significantly potentiated the ability of carbachol to stimulate PtdIns hydrolysis by almost 30% ( p < 0.05 uersus 6 mM [K'],). The logarithmic relationship demonstrated here is reminiscentof that between [K'], and membrane potential. Assuming that the equilibrium potential for K' across the plasma membrane is the sole determinant of membrane potential, then the latter, according to the Nernst equation, shouldchange by 61.5 mV forevery10-fold alterationin [K'],. However, in the pancreatic @ cell, K' is not the sole determinant of membrane potential, which accordingly has been shown to vary by 47 mV for each change in [K'],of 1 logarithmic unit (23). Assuming an intracellular K' concentration of 120 mM (23), the membrane potential in islet cells can be calculated from a given [K'], as 47 log[K'],/log[K+]i. Accordingly, the data shown in Fig. 4 (upper panel) were uersus membrane potential replotted as Ins(l)P1, or Ins(4)P1, (lozoerpanel). The Ins(l)P, datacould be fitted highly significantly to a straight line ( R = 0.99; p < 0.01) which demonstrated that PtdIns hydrolysis was directly proportional to the prevailing membrane potential. This means that changes of as little as plusor minus 6 mV, from the resting potential of approximately 60 mV, would respectively inhibit or potentiate PtdIns hydrolysis by approximately 10%. In contrast, the Ins(4)P1 data were best fitted to a secondorder polynomial function ( R = 0.93; p < 0.05). However, it is apparent that, even with total collapse of the membrane potential, PtdInsPp hydrolysis would be maximally inhibited by only 50%. In order to obtain an estimate of what these changeswould actually represent in termsof DAG mass, thespecific activity of PtdIns was determined under these labeling conditions. This should also represent the specific activity in the other phosphoinositides, and in the inositol phosphates, since the isletsare labeled to isotopicequilibrium ( 5 ) . As shown in TableI,thenetincreasesinbothIns(l)P1andIns(4)P1 amounted to approximately 0.5 pmol/islet following a 1-min stimulation with carbachol. As discussed above, the former can be used as a quantitative estimate of PtdIns hydrolysis under the conditions of these experiments. Similarly, Ins(4)P1 was previously found to accountfor approximately half of the totalincreasein radioactivitydue tostimulatedPtdInsP? hydrolysis in pancreatic islets (14). Thiswould imply that in the present study PtdIns and PtdInsPBwould have contributed around 0.5 and 1.0 pmol of DAG/islet, respectively, t o the total increase inDAG following stimulation. When DAG mass was measured directly, using a n enzymatic assay, the TABLE I

0

40 60 80 Calculated Membrane Potential (-mV) FIG.4. Dose dependences of 0.5 mM carbachol-stimulated Ins(l)P1 and Ins(4)P1 accumulation uersus [K+]. (upper panel) or calculated membrane potential (lower panel). Experiments were performed as described in the legend to Fig. 1 except that InsPl isomers were separated by HPLC. Results are expressed as the stimulated increment in InsPl normalized to the 6 mM [K'], response and are means of 5-7 individual determinations. Membrane potential was calculated from [K+]. as described in the text.

20

0.05). However, the slope of this line was very shallow, and Ins(4)Pl was not significantly reduced even at 48 mM[K'], as compared to its level at the normal physiological concentration of 6 mM. In marked contrast, the decline in Ins(l)Pl was best fitted to a logarithmic curve ( R = 0.99; p < 0.01) and,consistent with theexperimentsconductedin 1 mM extracellular Ca", was significantly decreased at 30 mM uersus 6 mM KC1 ( p < 0.05). Moreover, decreasing the [K'], to

Comparison of mass increases in Ins(l)P1and Ins(4)Pl or DAG in pancreatic islets Stimulatedfor 1 min with 0.5 mM carbachol Groups of 50 (A) or 100 islets (B) were preincubated for 10 min and then stimulated for 1 min in KRB medium containing 0.5 mM CaC12, 2 mM EGTA, and other additions as described. Inositol phosphates (A) were separated and quantified by anion exchange HPLC. Inositol phospholipids were separated by TLC, and thePi content of PtdIns was determined by a colorimetric assay. This was used to determine the specific activity of PtdIns (31 pCi/pmol) from which the mass of Ins(l)P1and Ins(4)P1 were calculated. Results are the mean of three individual determinations. DAG mass (B) was determined by assay with DAG kinase. For further details see "Experimental Procedures." Results are the means of 6 individual determinations. Mass Metabolite

Stimulated

Control pmol/islet

1.81 f 0.12" 1.28f 0.06 A. Ins(l)P1 0.61 f 0.07" 0.10 f 0.01 Ins(4)PI 1.99 0.05" 1.38 f 0.17 B. DAG a Significantly different ( p < 0.02) from the unstimulated control.

Islet Ptdlns Hydrolysis net increase of 0.6 pmol/islet was less than half that predicted from the InsPl data. However, the calculated basal levels of DAG accumulation agreed very well using the two methods. The results described in Figs. 2-4 createa compelling argument that thecarbachol-stimulated hydrolysis of PtdIns would be attenuated during depolarization. However, under those conditions, PtdInsP:! hydrolysis would also be stimulatedasa result of the gating of voltage-dependent Ca2+ channels ( 5 ) . Therefore,net phosphoinositide breakdown should be a function of the two competing effects. The results of experiments shown in Fig. 5 suggest that this is indeed the case. In these studies, the islets were incubated for 20 min at a [K'],of 3, 6, or 30 mM, in the presence or absence of carbachol. In keeping with an earlierstudy ( 5 ) , PtdInsPz hydrolysis (as measured by InsP3 accumulation) was stimulated by approximately 50% by raising [K'Ie from 6 to 30 mM (upper panel).Carbachol alone was more potent than raised [K+Ie, and the combined effects of both stimuli on InsPs were additive (upper panel). Consistent with earlier results (Fig. 4), lowering [K'Ie from 6 to 3 mM did not affect basal or stimulatedInsP3 (upperpanel). InsPl was also measured under these conditions (lower panel). This is comprised of Ins(l)P1 and Ins(4)P1 andtherefore reflects the overall contribution of the hydrolysis of both PtdIns and PtdInsPz. In the absence of carbachol, the net effect on InsPl of raising [K'Ie from 3 to 6 to 30 mM (lower panel) was very similar to that on InsPB(upper panel)and presumably reflects the Ca2+stimulated PtdInsPz hydrolysis. Conversely, lowering [K'], from 6 to 3 mM, while not affecting basal InsP1, significantly increased the response due to carbachol, as would be expected as a resultof the potentiation of PtdIns hydrolysis. However, raising [K'Ie from 6 to 30 mM significantly increased carbachol-stimulated InsP, levels, suggesting that theinhibition of PtdIns hydrolysis, which would be expected to occur over this range, was more than compensated for by theCa2+stimulated hydrolysis of PtdInsP2. 0.8

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0.4

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The ability of alterations in [K'], to modulate either basal or carbachol-stimulated DAG accumulation over 20 min was next examined. Although this time pointwas chosen to allow comparison with the InsPl data, DAG levels would be expected to have declined after 20 min from a peak occurring earlier in the time course (11, 12). Therefore, in order to measure what were likely to be small changes, we have monitored [3H]arachidonic acid-labeled DAG rather than total DAG mass. It has previously been shown that carbachol promotes the preferential accumulation ofDAG enriched in arachidonic acid in pancreaticislets (11,12). As shown in Fig. 6, basal DAG was significantly elevated at 30 uersus 6 mM KC1. Although at 6 mMKC1, carbachol induced only a 12% rise in islet DAG,which failed to reach significance, the responses at both 3 and 30 mM [K'], were significantly increased by approximately 20%. As described above, there are probably two components underlying this biphasic response: PtdIns hydrolysis at 3 mM KC1 and PtdInsPz hydrolysis at 30 mM KC1. However, there is one discrepency between the measurements of DAG and InsP1: for DAG, the combined effect of carbachol and 30 mM KC1 was not significantly different from that of 30 mM KC1 alone, whereas for InsPl the responses were additive. Another series of experiments was undertaken to determine whether the modulation of carbachol-stimulated PtdIns hydrolysis correlated with changes in insulin secretion. Islets were incubated once again for 20 min at 3, 6, or 30 mM KC1 in the presence or absence of carbachol. The results presented in Fig. 7 are broadly consistent with those already obtained on the accumulation of DAG and InsP]. Although there was a tendency toward a higher secretory rate at 3 uersus 6 mM KCI, this was small ( 4 0 % ) and not statistically significant ( p > 0.10). In contrast, raising [K'Ip to 30 mM resulted in a highly significant (30%)increase in insulin release. Carbachol significantly stimulated secretion at 6 mM [K'],by nearly 40%, and this response was further augmented at both 3 and 30 mM [K'], (both p C 0.05 uersus carbachol at 6 mM KC1). Insulin secretion in the combined presence of 30 mM KC1 and carbachol was significantly greater ( p < 0.05) than that due to high KC1 alone, but the responses were less than additive. The failure of 1-min stimulationwith 30 mM KC1 to elevate Ins(l)P1(Fig. 2) was earlier cited as anindication that PtdIns hydrolysis was not triggered by a rise in [Caz+li.A final series of experiments were undertaken to corroborate this conclu130 0

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0.2

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120 110

-

100

-

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(D

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+

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0 3 6

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"

"

"

Control Carbachol

30

KC1 (mM) KC1 (mM) FIG. 5. Effect of 20-min stimulation with 0.5 mM carbachol on InsPs (upperpanel)and InsPl (lowerpanel)accumulation. Experiments were performed as described in the legend to Fig. 1 except for the 20-min incubation time. Each point represents the mean of 20 individual determinations and is marked by an asterisk where significantly different ( p < 0.05) from the 6 mMKC1 control.

FIG. 6. Effect of a 20-min stimulation with 0.5 mM carbachol on DAG accumulation. Islets were prelabeled with [3H] arachidonic acid but incubated as described in the legend to Fig. 1. DAG was measured by TLC following extraction of neutral lipids as described in detail under "Experimental Procedures." Results represent themeans of 22-26 individual determinations and are expressed as percent of the 6 mM KC1 control which averaged 854 f 81 cpm ( n = 23). Values significantly different ( p < 0.05) from the 6 mMKC1 control are indicated by an asterisk.

11070

Islet PtdIns Hydrolysis c

attenuated by approximately 67% comparedtoisletspretreated in the absence of the drug (lower panel).The rise in InsPl due to 30 mM KC1 was completely abolished by neomycin. These results suggest that, although carbachol stimulates a neomycin-insensitive PtdIns hydrolysis, Ca2+ influx through voltage-dependent Ca2+ channelsdoes not.

2 E 160

8

3

140

4

Control Carbachol

DISCUSSION

In a recent study using rat pancreatic islets, we have dem* onstrated that carbachol stimulates the hydrolysis of both PtdInsP, and PtdIns (14). This conclusion was based upon: E a 0 3 6 30 an analysisof the pathwaysof inositol phosphatemetabolism KC1 (mM) in islet homogenates and the time courses of their accumulaFIG. 7. Effect of 20-min stimulation with0.5 mM carbachol tion in stimulated islets; kinetic analysis of the turnover of on insulin secretion. Incubations were performed using batchesof of 10 islets, preincubated in KRB for 10 min, and then stimulated for a individual inositol phosphates by measuring their rates further 20 minwith additions as described. Results represent the decline from a stimulated steady statefollowing displacement means of 41-43 individual determinations and are expressed as per- of carbachol from its receptor; and the demonstration that cent of the 6 mM KC1 control, which averaged 0.86 ? 0.08 ng/islet/ under conditions inwhich carbachol-stimulated PtdInsPz hy20 min ( n = 43). Values significantly different ( p < 0.001) from the drolysis was attenuated by neomycin pretreatment, the stim6 mM KC1 control are indicated with an asterisk. ulated rise in Ins( l)Pl was unaffected. The latter experiment also establishedthat,atleastduringthefirstminute of stimulation with carbachol, increases in l)P1 Ins( and Ins(4)P1 were uniquely generated asa result of the hydrolysis of PtdIns 800 and PtdInsP2,respectively (14). lnsP3 * These findings have been extended in the current study, firstly by measuring the massof DAG formed (Table I). The 600 basal level of around 1.4 pmol/islet compares very well with v) previous studies using slightly younger rats in which values 0, 400 of 1 pmol/islet were obtained with either the DAG kinase v) method (24) or mass spectroscopy (12). The 50% increase in DAG, measured after 1 min stimulation with carbachol, is O 200 9 also consistent with previous isotopic or mass data (11, 12) E obtained using islets, as is the much smaller increase seen Q 0 0 v after 20 min (Fig. 6). In contrast, total stimulated phosphoinositide hydrolysis, as calculated from the mass increases in nv) InsPls, was predicted to be at least double that of the measS ured rise in DAG (Table I). Since a much better correlation 3000 was foundwithbasal levels, this discrepancy is probably I m explained by an increase in DAG breakdownsecondary to 2000 activation of DAG kinase by both Ca2+ and protein kinase C (25). Thus, the measured accumulation of DAG may underestimate the degree of phosphoinositide hydrolysis. Such a 1000 conclusion might also explain why DAG was notfurther increased by 30 mM KC1 in the presenceof carbachol, whereas n " InsPl levels were raised in an additive manner (Figs. 5 and Control Neomycin FIG. 8. Effect of neomycin pretreatment on the accumula- 6 ) . Building further on this characterization we have demontion of InsPs (upper panel) or InsPl (lower panel) in the presence or absence of 0.5 mM carbachol and 30 mM KCl. strated that the carbachol-stimulatedhydrolyses of PtdInsP2 Batches of 50 islets, labeled with [3H]inositolwere preincubated for and PtdIns are independently regulated by Ca2+influx and 20 min in KRB containing 5 mM neomycin and then stimulated for changes in membrane potential. Two findings suggest that a further 20 min with the additions as described. Inositolphosphates PtdIns hydrolysis was not directly activated by a rise in were measured as described in the legend to Fig. 1. Each point [Ca2+Iisecondarytothegating of votage-dependent Ca2+ represents the mean of 13-14 individualdeterminationsand is marked by an asterisk where it differs significantly ( p < 0.05) from channels. First, a 1-min incubation with 30 mM KC1 did not increase Ins(l)P1levels (Fig. 2). On the other hand, we have the 6 mM KC1 control. previously shown that Ins(1,4,5)P3 was doubled within 2 s of addition of this depolarizing concentration of KC1 (5).Second, sion. Accordingly, islets were first incubated for 20 min in either the presence or absence of 5 mM neomycin and then the rise in InsPl due to a 20-min stimulation at 30 mM [K+] stimulated for a further 20 min, with either carbachol or 30 was completely abolished in the presence of neomycin, sugmM KC1. This concentrationof neomycin has previously been gesting that it was entirely due to PtdInsPz hydrolysis (Fig. shown to cause a 70% inhibition of PtdInsP2 hydrolysis in 8). In contrast, carbachol hasbeen shown to produce a rapid islets stimulated for 1 min with carbachol, but no significant (5-s) rise in Ins(l)P1(14) and a significant increase in InsP1, alteration of PtdIns hydrolysis (14). As shown in Fig. 8, even after neomycin pretreatment, which abolishes the stimneomycin completely abolishedthe abilityof either carbachol ulated accumulation of InsP3 (Fig. 8). Thus, in pancreatic or 30 mM KC1 toincreaseInsPs levels (upper panel). In islets, although the hydrolysis of PtdInsPz is triggered by a rise in [Ca2+Ii, thatof PtdIns is not. This is the converse of contrast, carbachol significantly elevated InsPl in the presence of neomycin ( p < 0.05), although the net increase was the situation in platelets, asdeduced from pulse-chase analy-

-

h CI

.-

-

Islet PtdIns Hydrolysis

11071

sis of phosphoinositide pools, and the Ca2+ dependence of tors, might play a role during the hyperpolarization phase purified phospholipaseC isoforms forPtdIns uersus PtdInsPz (29). It is tempting to speculate that sequential modulation in vitro (26). However, such tissue-specific differences should of PtdIns hydrolysis, due to periodic changes in membrane aconsequence of, and indeed further not be surprising given the multipleisoforms of phospholipase potential,mightbe C now known to be expressed differentially in mammalian contribute to, the ongoing development of this electrical activity. cells (27). In many cell types, Ins( 1,4,5)P3-induced Ca2+ mobilization Although not sensitive to Ca", the stimulated hydrolysis K+ channels and thereby commonly stimulates Ca2+-activated of PtdIns was markedly influenced by changes in islet cell membrane potential. There are two reasons to suggest that causes hyperpolarization (30, 31). If applicable to other tissues, the resultsof the present studywould suggest that this this was notanindirect effect butduetoalterationsin membrane potential per se. First, a number of depolarizing hyperpolarization might result in a secondary enhancement agents including glucose, tolbutamide, ouabain, and raised of PtdIns hydrolysis. However, the physiological relevance of necessarily be confined to [K'], all caused an inhibition of carbachol-stimulated PtdIns thecurrentfindingsmightnot hydrolysis (Fig. 3). These various agents act in different ways, potentiation of stimulated DAG formation during hyperpomaking it unlikely that alterations in the concentration gra- larization.Since a depolarization of at least 10-15 mV is K+ or Na+)were obligatorily required for the gating of voltage-dependent Ca2+ channels, dient of a particular ion (such as involved. In this context, it should be stressed that changes the real significance of these results might pertain to small in Ca2+ were clearly not implicated, since the inhibition of ((10 mV) depolarizations which would inhibit PtdIns hyPtdIns hydrolysis wasindependent of the presence or absence drolysis. On the other hand,slightly stronger depolarizations of extracellular Ca2+(Figs. 2 and 4). Second,stimulated would openvoltage-dependent Ca2+ channelsandactivate Ins( l)P1levels were directly proportional to membrane potenPtdInsPz hydrolysis. Such a finely graded reponse would be tial over the calculated rangeof -75 to -20 mV (Fig. 4).Most difficult to demonstrate experimentally but would be consistimportantly,thisindicatesthatnot onlywas stimulated ent with thevariationsinsynapticstrength displayed in PtdIns hydrolysis inhibited by depolarization, but that itwas certain models of neuronal plasticity. Post-synaptic memory also potentiated by hyperpolarization. is known to be inhibited if a phosphoinositide-hydrolyzing The hydrolysis of PtdInsPz, on the other hand, was much stimulus is applied concurrently with a weak depolarization, less sensitivetochangesinmembranepotential over the but potentiated if added during a strong depolarization ( 3 2 ) . physiological range of -20 to -75 mV, and our results predict However, caution should beexercised in applying the current a maximal inhibition of approximately 50%at zero potential. results to othercell types, since the extent towhich nonpanThe latter finding isbroadly consistent with an earlier study creatic tissuesundergo PtdIns hydrolysis is controversial (33, using HL60cells, in which it was shown that the accumulation 34). of Ins(1,4,5)P3, in response to maximal agonist stimulation, The mechanism by which PtdIns hydrolysis is modulated was attenuated by 70% when the membrane potential was by changes in membranepotentialisunknown.Itmight completelycollapsed with gramicidin, or by 50% following represent a site of interaction at any step in the coupling of prolonged treatment with ouabain (4). However, it was not the receptor to the G-protein or G-protein to phospholipase apparent whether changes over a range of 20-40 mV would C. However, perhaps the most likely explanation would be have exerted any significant effect. Moreover, the attentua- that the access of phospholipaseC to its substrate in the tion of Ca2+mobilization by gramicidin or ouabain was less plasma membrane would be acutely dependent on the potenpronounced than the effects on 1,4,5)P3 Ins( and only apparent tial difference across that membrane. PtdInsP2, being more a t submaximal agonist concentrations. Thus, although charged than PtdIns, might be moreexposed at the cytochanges in membrane potential probably exert influence some plasmic face of the lipidbilayer and so less dependent on on PtdInsPzhydrolysis, the resultsof the present study would alterations in plasma membrane potential for regulating the suggest that the regulationof PtdIns hydrolysis is more likely access of phospholipase C. to be physiologically relevant since, in principle, the latter The exact contribution of PtdIns hydrolysis to the regulacould bealtered by 10% following changesinmembrane tion of insulin secretion is difficult to determine. At present, potential of as little as6 mV. the only effectiveway of selectively inhibiting this component The resting membrane potential of the pancreatic p cell is is by depolarization which would exert counteracting stimuapproximately -65 mV (7, 28). Addition of a nutrient stimu- latory effects on secretion, both as a result of enhanced Ca2+ lus, such as glucose, results in the closure of KaTpchannels, influx per se and via the potentiationof PtdInsP2 hydrolysis. of around 10-15 mV which This problem would not be circumvented by removal of extraandaninitialdepolarization developsbetween 1 and 2 min following stimulation(28). cellular Ca2+, since the latter is absolutely required for the However,once thethresholdisreached for thegating of stimulation of insulin secretion by carbachol or acetylcholine voltage-dependent Ca2+ channels, a series of slow waves, or (35,36). Thisis true even forcultured isletswhich, in contrast periodic oscillations in membranepotential,are triggered. to when freshly isolated, secrete insulin in response to musT h e plateau potentialof these waves is in the range of -40 to carinicagonists a t low glucose concentrations(35).Under -30 mV, and the repolarizaton potential is between -60 and hyperpolarizing conditions, the effects of carbachol on PtdIns -50 mV. Superimposed on the elevated plateaus of these slow hydrolysis, DAG accumulation, and insulin secretion were all waves are spikes of burst activity which are associated with increased. This suggests, at the very least, that PtdIns hyc a 2 +influx and may momentarily reach as high as -10 mV drolysisplaysa potential role in the acute stimulation of (7,281. The mechanisms underlying these periodic oscillations insulin release by muscarinic receptoragonists. Confirmation are complex and not completely understood. However, it is of this role awaits the development of more selective means noteworthy that glucose alone may be insufficient for the of differentiating between the consequences of hydrolyzing development of these waves, and a requirement for neuroPtdIns asopposed to PtdInsP2. transmitters and local mediators has been postulated (29). In particular, there evidence is that the generation of Ins(1,4,5)P3 Acknowledgments-We thank Gilbert Meunier and Vickie Theos from PtdInsPz, stimulated by occupation of muscarinic recep- for skilled technical assistance.

11072

Islet PtdIns Hydrolysis

REFERENCES 1. Berridge, M. J., and Irvine, R. F. (1989)Nature 341, 197-205 2. Nishizuka, Y. (1986)Science 233,305-312 3. Taylor, S. J., Chae, H. Z., Rhee, S. G., and Exton,J. H. (1991)Nature 360, 516518 4. Pittet, D., Di Virgilio, F., Pozzan, T., Monod, A,, and Lew, D. P. (1990)J. Biol. Chem. 266,14256-14263 5. Biden, T. J., Peter-Riesch, B., Schlegel, W., and Wollheim, C. B. (1987)J. Biol. Chem. 262,3567-3571 6. Eberhard, D. A., and Holz, R. W. (1987)J. Neurochem. 49, 1634-1643 7. Ashcroft, F. M., and Rorsman, P. (1989)Frog. Eiophys. Mol. Biol. 64, 87143 8. Wollheim, C. B., and Biden, T.J. (1986)Ann. N. Y. Acad. Sci. 488, 317222

9. Wiikeim, C. B., and Biden, T. J. (1986)J. Eiol. Chem. 261,8314-8319 10. Morgan, N. G., Rumford, G. M., and Montage, W. (1985)Biochem. J. 228.713-718 11. Peter-Riesch, B., Fathi, M., Schlegel, W., and Wollheim, C.B. (1988)J. Clin. Invest. 81,1154-1161 12. Wolf, B. A,, Easom, R. A., Hughes, J. H., McDaniel, M. L., and Turk, J. (1989)Eiochemistrv 28.4291-4301 13. Easom, R . A., Landt, M., Colca, J. R . , Hughes, J. H., Turk, J., and McDaniel, M. (1990)J.Eiol. Chem. 266,1493S14946 14. Biden, T.J., Prugue, M. L., and Davison, A. G. M. (1992)Eiochem. J. 286, 541-549 15. Sutton, R.,Peters, M., and McShane, P. (1986)Transplantation 42,689691 16. C&&, D. H., Scott, D. L., Gordon, E. A,, and La Belle, E. F. (1985)Cell 42,479-488 17. Van Dongen, C. J., Zwiers, H., and Gispen, W. H. (1985)Anal. Eiochem. 144, 104-109

18. Tokuwa, Y., Takuwa, N., and Rasmussen, H. (1986)J. Eiol. Chem. 261, 14670-14675 19. Preiss, J. -Loomis, C. R., Bishop W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986)J. Biol. Chem. 261,8597-8600 20. Wolf, B. A. Florholmen J., Turk, J., and Mc Daniel, M. L. (1988)J. Eiol. Chem. 283,3565-35y5 21. Rana, R. S., Sekar, M.C., Hokin, H. E., and MacDonald, M. J. (1986)J. Blol. Chem. 261,5237-5240 22. Henquin, J. C., and Meissner, H. P. (1982)J. Physiol. 332,529-552 23. Meissner, H. P., Henquin, J. C., and Preissler, M. (1978)FEES Lett. 94, 87-89 24. Wolf, B. A., Easom, R. A., McDaniel, M. L., and Turk, J. (1990)J. Clin. Invest. 86,482-490 25. Kanoh, H., Yamada, K., and Sakane, F. (1990)Trends Biochem. Sei. 16, 47-50 26. Majerus, P. W Connoll , T M , Deckmyn, H., Ross, T. S., Bross, T. E. Ishii, H., Bahal, V. and Wilson, D.B. (1986)Science 234, 1519: 1526 27. Rhee, S. G., and Choi, K. D. (1992)J. Biol. Chem. 267, 12393-12396 28. Henquin, J. C., and Meissner, H. P. (1984)Experientio 40,1043-1052 29. Ammala, C., Larsson, O., Berggren, P.-O., Bokvist, K., Juntti-Berggren,J., Kindmark, H., and Rorsman, P. (1991)Nature 363,849-852 30. Higashida, H., and Brown, D.A. (1986)Nature 323,333-335 31. Ca iod, T., Field, A. C., Ogden, D. C., and Sandford, C.A. (1987)FEES fett. 217,247-252 32. Artola, A., Brocher S., and Singer W. (1990)Nature 347,69-72 33. Hughes, A.R., and Putney, J. W:, Jr. (1989)J. Biol. Chem. 264, 94009407 34. Fisher, S. K., Heacock, A. M., Seguin, E. B., and Agranoff, B.W. (1990) MOL Pharmacol. 38,54-63 35. Wollheim, C. B., Siegel, E. G., and Sharp, G. W. G. (1980)Endocrinology 107,924-929 36. Hermans, M. P., and Henquin, J. C. (1989)Diabetes 38,198-204

8,