Carbamoylcholine regulation of polyphosphoinositide synthesis and hydrolysis in cultured, dispersed, digitonin-permeabilized mouse pancreatic islet cells.

Continuing formation of inositol phosphates during stimulation of pancreatic beta-cells by hormones and neurotransmitters requires the continued synthesis of the polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5 bisphosphate (PIP2) from phosphatidylinositol (PI). In the present study we have investigated how this pathway and the activity of phosphoinositide-specific phospholipase C (PI-PLC) are regulated by carbamoylcholine (CCh), Ca2+, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), GTP gamma S and NaF in 44-h [3H]inositol-labelled, dispersed and digitonin-permeabilized mouse pancreatic islet cells. CCh stimulated not only PI-PLC (G-protein-mediated) but also, by an as yet unknown mechanism, significantly enhanced PI 4-kinase activity, estimated as the PIP:PI ratio, by 100%, and further increased the flux from PI to PIP and PIP2, GTP gamma S and NaF mimicked the effects of CCh on PI-PLC but had no effect on the levels of PIP and PIP2, TPA raised the PIP:PI ratio by 75%. In addition TPA counteracted the CCh stimulation of PI-PLC. There was no effect of 10(-6) mol/l Ca2+ on the levels of PIP and PIP2. Experiments with quinacrine and adenosine confirmed that PI-PLC and PI 4-kinase could be regulated independently of each other. In conclusion, these data point to differential regulation of polyphosphoinositide synthesis and breakdown.

Role of glucose metabolism and phosphoinositide hydrolysis in glucose-induced sensitization/desensitization of insulin secretion from mouse pancreatic islets.

The role of glucose metabolism and phosphoinositide hydrolysis in glucose-induced sensitization/desensitization of insulin secretion was studied. A change in glucose concentration from 5.5 to 16.7 mM during 22-24 h of pre-exposure of mouse islets in TCM 199 culture medium (0.26 mM Ca2+) led to sensitization of glucose-induced insulin secretion. This change in islet responsiveness to glucose was not mediated by increases in glucose utilization ([5-3H]glucose conversion to 3H2O) and glucose oxidation ([U-14C]glucose oxidation to 14CO2). Glucose-induced sensitization of insulin secretion was associated with an increase in glucose-induced phosphoinositide hydrolysis, leading to a significant increase in inositol 1-monophosphate formation, but not in inositol 1,4-bisphosphate or in inositol 1,4,5-trisphosphate plus inositol 1,3,4-trisphosphate formation. Diacylglycerol, which may arise from both phosphoinositide hydrolysis and de novo from glucose metabolism, was, on the other hand, not increased during acute exposure to glucose and not changed after pre-exposure to glucose. At 16.7 mM glucose in TCM 199 medium, a change in Ca2+ concentration from 0.26 to 1.26 mM led to a reduction in glucose-induced insulin secretion. This Ca(2+)-dependent desensitization of insulin secretion in the presence of glucose was associated with a decrease in glucose-induced phosphoinositide hydrolysis, but not with a change in glucose metabolism or diacylglycerol accumulation. In conclusion, it is suggested that glucose-induced sensitization/desensitization of insulin secretion may involve changes in phosphoinositide hydrolysis, but may occur independently of concomitant changes in glucose metabolism or diacylglycerol accumulation.

Exogenous arachidonic acid inactivates protein kinase C in mouse pancreatic islets.

The effect of arachidonic acid on protein kinase C activity and insulin secretion in mouse islets was investigated. Arachidonic acid stimulated protein kinase C activity in islet cytosol and membrane fractions by substituting for phosphatidylserine. Stimulation by arachidonic acid was dependent on either Ca2+ or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, was potentiated by the combined addition of Ca(2+) + 12-O-tetradecanoylphorbol 13-acetate, and did not further increase protein kinase C activity in the presence of saturating concentrations of phosphatidylserine. Arachidonic acid stimulation of protein kinase C was prevented by binding of arachidonic acid to albumin. In the absence of extracellular Ca2+, exogenous arachidonic acid stimulated insulin secretion. Arachidonic acid-induced insulin secretion was not potentiated by 12-O-tetradecanoylphorbol 13-acetate and was not prevented by the protein kinase C inhibitor staurosporine, suggesting that arachidonic acid-induced insulin secretion may occur independently of protein kinase C activation. Arachidonic acid-induced insulin secretion in Ca(2+)-free medium was on the other hand potentiated by addition of extracellular Ca2+. Stimulation of insulin secretion by exogenous arachidonic acid was associated with inactivation of protein kinase C. Inactivation of protein kinase C was also observed in islet homogenate after pre-incubation with arachidonic acid. Arachidonic acid-induced protein kinase C inactivation in islet homogenate was prevented by albumin or MgATP. Inactivation by arachidonic acid in intact islets was, however, not produced during enzyme isolation and was not prevented by inclusion of albumin or MgATP during preparation of protein kinase C extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term fat-feeding-induced insulin resistance in normal NMRI mice: postreceptor changes of liver, muscle and adipose tissue metabolism resembling those of type 2 diabetes.

Postreceptor insulin resistance was studied in liver, muscle and adipose tissue from NMRI mice of both sexes made diabetic by long-term fat-feeding. Intravenous glucose tolerance tests showed a combination of impaired glucose tolerance and increased plasma insulin concentrations consistent with insulin resistance and reduced peripheral and hepatic uptake of glucose. In the morning, the fat-fed mice were normoinsulinaemic and hyperglycaemic. Liver glucokinase activity and glycogen content were reduced whereas lactate dehydrogenase activity was enhanced. Fatty acid synthase activity was decreased but glucose 6-phosphate dehydrogenase and the rate limiting enzyme in fatty acid synthesis, acetyl CoA carboxylase, were both unaffected. In muscle, the proportion of glycogen synthase in the active I-form was decreased. Total glycogen synthase activity was not affected. In isolated adipocytes, basal and insulin-stimulated glucose oxidation, as well as basal and insulin-stimulated lipogenesis from glucose were all severely inhibited, oxidation more so than lipogenesis. It is concluded that insulin resistance and postreceptor metabolic disorders in liver, muscle and adipose tissue from mice made diabetic by long-term fat-feeding are very similar to those demonstrated in human type 2 diabetics and may be studied in more detail and with more ease in this particular animal model.

Fat-induced changes in mouse pancreatic islet insulin secretion, insulin biosynthesis and glucose metabolism.

Insulin secretion, insulin biosynthesis and islet glucose oxidation were studied in pancreatic islets isolated from fat-fed diabetic mice of both sexes. Insulin secretion from isolated islets was studied after consecutive stimulation with alpha-ketoisocaproic acid + glutamine, glucose, forskolin, and 12-O-tetradecanoylphorbol 13-acetate. Glucose-induced insulin secretion was impaired in islets from fat-fed mice. This was associated with a reduction of approximately 50% in islet glucose oxidation. Islet insulin secretion stimulated by the non-carbohydrate secretagogues tended to be higher in the fat-fed mice, but a statistically significant effect was not observed. Pancreatic insulin content was reduced by 50%, whereas the islet insulin and DNA content was unchanged after fat feeding. Proinsulin mRNA was reduced by 35% in islets from fat-fed mice, and was associated with a reduction of approximately 50% in glucose-stimulated (pro)insulin biosynthesis. It is concluded that the insulin secretory response of islets isolated from fat-fed mice is similar to the secretory pattern known from human type 2, non-insulin-dependent diabetics, and that a defect in islet glucose recognition, resulting in decreased glucose oxidation, may be responsible for the observed insulin secretory and biosynthetic defects seen after glucose stimulation.

Ca(2+)- and ATP-dependent reversible inactivation of pancreatic islet phosphoinositide-specific phospholipase C activity.

Phosphoinositide-specific phospholipase C (PI-PLC) activity in whole homogenates of mouse pancreatic islets decreased 60-85% when the homogenates were incubated at 37 degrees C for 1 h in the presence of down to micromolar concentrations of Ca2+. Ca(2+)-induced inactivation was augmented by calmodulin, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in the presence of ATP-Mg, and by Mg2+. Inactivation was inhibited when ATP was removed and completely abolished by trifluoperazine and EGTA. Inactivation was not affected by the non-phosphorylating ATP analogue, AMP-PCP, GMP-PNP, glucose, Zn2+ or a series of protease inhibitors. These observations suggest that PI-PLC in broken cell preparations of pancreatic islets may be inactivated via phosphorylation by Ca(2+)-calmodulin-stimulated protein kinase and/or protein kinase C. Inactivation of PI-PLC was reversible. Reactivation started after approx. 2 h incubation, when the concentration of ATP in the homogenate was below 0.15 x 10(-6) M. PI-PLC activity returned to values approx. 25% higher than the initial values. PI-PLC inactivation via phosphorylation by the mentioned protein kinases may constitute a feedback control on the phosphoinositide response, attenuating subsequent diacylglycerol formation and/or Ca2+ mobilization by inositol trisphosphate.

Ability of omega-3 fatty acids to restore the impaired glucose tolerance in a mouse model for type-2 diabetes. Different effects in male and female mice.

Mice of both sexes were fed diets with 80 per cent animal or vegetable fat for 3 months. Half of the animals also received SuperEPA, which contains 61% omega-3 fatty acids. At the end of the feeding period the mice receiving animal fat had gained more weight than the controls and the mice receiving vegetable fat, and all fat diet groups, irrespective of sex or kind of diet, had become hyperglycaemic and had impaired intravenous glucose tolerance. The decay in plasma glucose during the tolerance tests was, however, significantly slower in the groups getting animal fat than in the groups getting vegetable fat. Supplementation with omega-3 fatty acids only affected the male mice receiving the animal fat diet. Thus, these mice gained less weight, and both the hyperglycaemia and the impairment of the glucose tolerance were significantly less pronounced in this group than in the male mice fed animal fat without SuperEPA. In the groups eating fat diets, the plasma total cholesterol levels increased 50-100 per cent during the first 2 weeks of the experiment and then plateaued. In both sexes HDL-cholesterol averaged approx. sixty-five per cent of the total cholesterol content at the start of the experiment and was not changed significantly during the feeding period. It is concluded, that omega-3 fatty acids do not seem to be suitable as a general means of ameliorating impaired glucose tolerance. It is further suggested that the fat-fed mouse may be a useful animal model for further studies of the regulation of metabolism in type-2 diabetes.

Characteristics of phosphoinositide-specific phospholipase C activity from mouse pancreatic islets.

In pancreatic islets the bulk of phosphoinositide-specific phospholipase C (PI-PLC) activity was cytosolic. The soluble enzyme was activated by submicromolar concentrations of Ca2+, independent of calmodulin. It was unaffected by glucose and a series of glycolytic intermediates, including glyceraldehyde 3-phosphate. These observations lend support to the hypothesis that glucose-stimulated inositol triphosphate production in islets may be secondary to and provoked by glucose-mediated Ca2+ influx. All four pyridine nucleotides stimulated PI-PLC. Phosphatidylinositol hydrolysis was also stimulated by dioleine and arachidonic acid, and by the polyamines, putrescine and spermine. Phosphatidylinositol hydrolysis was inhibited by chlorpromazine, tetracaine, ATP, 5'-AMP, inorganic pyrophosphate and by phosphatidylinositol 4,5-bisphosphate, phosphatidylcholine and phosphatidylserine--but not affected by phosphatidylethanolamine. The cyclic nucleotides, cAMP and cGMP had no effect on the enzyme, and GTP-gamma-S did not activate the enzyme event at very low Ca2+ concentrations. The diglyceride lipase inhibitor, RHC 80267, and the cyclooxygenase inhibitor, indomethacin, had no effect on PI-PLC activity.

Phorbol-ester-induced down-regulation of protein kinase C in mouse pancreatic islets. Potentiation of phase 1 and inhibition of phase 2 of glucose-induced insulin secretion.

The influence of down-regulation of protein kinase C on glucose-induced insulin secretion was studied. A 22-24 h exposure of mouse pancreatic islets to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 0.16 microM) in RPMI 1640 culture medium (8.3 mM-glucose, 0.43 mM-Ca2+) abolished TPA (0.16 microM)-induced insulin secretion and led to a potentiation of phase 1 and a decrease in phase 2 of glucose-induced insulin secretion. Thus, although the total insulin release during 40 min of perfusion with glucose (16.7 mM) (45-85 min) was unaffected, the percentage released during phase 1 (45-55 min) was increased from 12.9 +/- 1.5 (4)% in controls to 35.8 +/- 3.9 (4)% in TPA-treated islets (P less than 0.01), and the percentage released during phase 2 (65-85 min) was decreased from 63.2 +/- 3.9 (4)% to 35.3 +/- 1.4 (4)% (P less than 0.005). In contrast, TPA exposure in TCM 199 medium (5.5 mM-glucose, 1.26 mM-Ca2+) caused a total abolition of both phases 1 and 2 of glucose-induced secretion. However, inclusion of the alpha 2-adrenergic agonists adrenaline (10 microM) or clonidine (10 microM), or lowering of the Ca2+ concentration in TCM 199 during down-regulation, preserved and potentiated phase 1 of glucose-induced secretion. Furthermore, perifusion of islets in the presence of staurosporine (1 microM), an inhibitor of protein kinase C, potentiated phase 1 and inhibited phase 2 of glucose-induced secretion. In addition, down-regulation of protein kinase C potentiated phase 1 and inhibited phase 2 of carbamoylcholine (100 microM)-induced insulin secretion at 3.3 mM-glucose, and abolished the potentiating effect of carbamoylcholine (100 microM) at 16.7 mM-glucose. These results substantiate a role for protein kinase C in insulin secretion, and suggest that protein kinase C inhibits phase 1 and stimulates phase 2 of both glucose-induced and carbamoylcholine-induced insulin secretion.

Effect of diacylglycerol lipase inhibitor RHC 80267 on pancreatic mouse islet metabolism and insulin secretion.

The effect of interference with diacylglycerol metabolism was investigated in pancreatic mouse islets. In the presence of the diacylglycerol lipase inhibitor RHC 80,267, glucose-induced insulin secretion was reduced 50-60%; whereas carbacholin-induced insulin secretion was unaffected. Addition of the diacylglycerol kinase inhibitor R 59,022 did not change glucose-stimulated insulin secretion but abolished the inhibition seen in the presence of RHC 80,267. RHC 80,267 increased islet glucose utilisation, measured as formation of tritiated water from 5-[3H]-glucose, 3-fold but did not affect glucose oxidation to CO2, lactate production or islet ATP levels. Glucose utilisation in leucocytes and hepatocytes was not increased by addition of RHC 80,267. Islet lipid production from glucose was augmented 4-fold in the presence of RHC 80,267 but only accounted for about 5% of the increase in glucose utilisation. The activity of adenylate cyclase and phosphoinositide-specific phospholipase C was unaffected by RHC 80,267. Concentrations of RHC 80,267 below 35 mumol/l did not alter the activity of phospholipase A2; whereas higher concentrations of the drug inhibited phospholipase A2 activity approx 25%. The data support the hypothesis that production of arachidonic acid from diacylglycerol may be involved in regulation of insulin secretion.

Stimulation by glucose of cyclic AMP accumulation in mouse pancreatic islets is mediated by protein kinase C.

The mechanism of glucose-stimulated cyclic AMP accumulation in mouse pancreatic islets was studied. In the presence of 3-isobutyl-1-methylxanthine, both glucose and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, enhanced cyclic AMP formation 2.5-fold during 60 min of incubation. Both TPA-stimulated and glucose-stimulated cyclic AMP accumulations were abolished by the omission of extracellular Ca2+. The Ca2+ ionophore A23187 did not affect cyclic AMP accumulation itself, but affected the time course of TPA-induced cyclic AMP accumulation, the effect of A23187 + TPA mimicking the time course for glucose-induced cyclic AMP accumulation. A 24 h exposure to TPA, which depletes islets of protein kinase C, abolished the effects of both TPA and glucose on cyclic AMP production. Both TPA-induced and glucose-induced cyclic AMP productions were inhibited by anti-glucagon antibody, and after pretreatment with this antibody glucose stimulation was dependent on addition of glucagon. Pretreatment of islets with TPA for 10 min potentiated glucagon stimulation and impaired somatostatin inhibition of adenylate cyclase activity in a particulate fraction of islets. Carbamoylcholine, which is supposed to activate protein kinase C in islets, likewise stimulated cyclic AMP accumulation in islets. These observations suggest that glucose stimulates islet adenylate cyclase by activation of protein kinase C, and thereby potentiates the effect of endogenous glucagon on adenylate cyclase.

Insulin release by glucagon and secretin: studies with secretin-glucagon hybrids.

Secretin and glucagon potentiate glucose-induced insulin release. We have compared the effects of secretin and glucagon with that of four hybrid molecules of the two hormones on insulin release and formation of cyclic AMP (cAMP) in isolated mouse pancreatic islets. All six peptides potentiated the release of insulin at 10 mM D-glucose, and their effects were indistinguishable with respect to the dynamics of release, dose-response relationship, and glucose dependency. However, measurements of cAMP accumulation in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (10(-4) M) showed that the fold increase compared with glucose alone had the following ranking order: secretin = [Tyr10, Tyr13]-secretin 1.6 less than [Tyr10, Tyr13, Trp25]secretin 1.8 less than glucagon 1.9 less than [Asp3, Glu9, Arg12]glucagon 2.3 = [Asp3, Glu9]glucagon. These results suggest that despite similar potentiating effects of secretin and glucagon on glucose-induced insulin release, their modes of action may be different.

Cytosolic ratios of free [NADPH]/[NADP+] and [NADH]/[NAD+] in mouse pancreatic islets, and nutrient-induced insulin secretion.

When the extracellular concentration of glucose was raised from 3 mM to 7 mM (the concentration interval in which beta-cell depolarization and the major decrease in K+ permeability occur), the cytosolic free [NADPH]/[NADP+] ratio in mouse pancreatic islets increased by 29.5%. When glucose was increased to 20 mM, a 117% increase was observed. Glucose had no effect on the cytosolic free [NADH]/[NAD+] ratio. Neither the cytosolic free [NADPH]/[NADP+] ratio nor the corresponding [NADH]/[NAD+] ratio was affected when the islets were incubated with 20 mM-fructose or with 3 mM-glucose + 20 mM-fructose, although the last-mentioned condition stimulated insulin release. The insulin secretagogue leucine (10 mM) stimulated insulin secretion, but lowered the cytosolic free [NADPH]/[NADP+] ratio; 10 mM-leucine + 10 mM-glutamine stimulated insulin release and significantly enhanced both the [NADPH]/[NADP+] ratio and the [NADH]/[NAD+] ratio. It is concluded that the cytosolic free [NADPH]/[NADP+] ratio may be involved in coupling beta-cell glucose metabolism to beta-cell depolarization and ensuing insulin secretion, but it may not be the sole or major coupling factor in nutrient-induced stimulation of insulin secretion.

Polyamine-enhanced casein kinase II in mouse pancreatic islets.

The occurrence of polyamine-stimulated protein kinase (casein kinase II) in cytosol of mouse pancreatic islets was investigated. Islet protein phosphorylation was enhanced by spermidine, spermine, lysine-rich histone and polylysine; the major endogenous substrates in the cytosol were three proteins of Mr 50,000, 55,000 and 100,000. Cadaverine and putrescine were without effects. A Mr 100,000 protein is a major substrate for Ca2+-calmodulin-dependent protein kinase, and Mr 50,000 and 55,000 proteins are substrates for cyclic adenosine 3',5'-cyclic monophosphate (AMP) dependent protein kinase in mouse islets. However, neither cyclic-AMP-dependent protein kinase inhibitor nor trifluoperazine inhibited polyamine-enhanced protein phosphorylation. Both basal and polyamine-enhanced protein phosphorylation patterns were identical when either [gamma-32P] adenosine 5'-triphosphate (ATP) or [gamma-32P] guanosine 5'-triphosphate (GTP) was used as phosphate donors, indicative of the presence of a polyamine-stimulated casein kinase II in pancreatic islets. It is suggested that polyamines and polyamine-enhanced casein kinase II activity may have an important role in regulation of protein phosphorylation in pancreatic islets.

Potentiation of insulin release in response to amino acid methyl esters correlates to activation of islet glutamate dehydrogenase activity.

Column perifusion of mouse pancreatic islets was used to study the ability of amino acids and their methyl esters to influence insulin release and activate islet glutamate dehydrogenase activity. In the absence of L-glutamine, L-serine and the methyl ester of L-phenylalanine, but neither L-phenylalanine nor L-serine methyl ester, stimulate insulin secretion. In the presence of L-glutamine, however, the effect of L-serine was additive, while the methyl esters of L-serine and L-phenylalanine as well as native L-phenylalanine, potentiated the glucose-stimulated release of insulin. Measurements of islet glutamate dehydrogenase activity showed that only the two methyl esters of L-phenylalanine and L-serine activated the enzyme. It is concluded that the mechanism by which methyl esters of amino acids potentiate insulin release is most likely to be mediated by the activation of pancreatic beta-cell glutamate dehydrogenase activity.

Presence of ATP-pyrophosphohydrolase in mouse pancreatic islets.

The presence of an enzyme that hydrolyzes ATP to AMP and PPi was demonstrated in a 27,000 X g particulate and supernatant fraction of mouse pancreatic islets. The enzyme was stimulated by addition of Ca2+, Zn2+, and Co2+. Addition of calmodulin or trifluoperazine had no effect. In the presence of Ca2+ and Zn2+, the Michaelis constant (Km) for ATP was approximately 0.1 mM and the maximum velocity (Vmax) was close to 90 nmol X min-1 X mg protein-1. After preincubation of the islets for 30 min with 16.7 mM glucose or 5 mM glucose with 1 mM 3-isobutyl-1-methylxanthine (IBMX), a three- to fourfold increase in enzyme activity was seen. Direct addition of IBMX or cAMP to the enzyme assay also had a small stimulatory effect. Preincubation with the insulin secretagogues leucine and alpha-ketoisocaproic acid did not affect the enzyme activity. The possible function of the enzyme in pancreatic islets is discussed in relation to hypotheses given for the function of similar enzyme(s) in other tissues.

An inhibitory role for polyamines in protein kinase C activation and insulin secretion in mouse pancreatic islets.

The occurrence and function of polyamines in protein kinase C activation and insulin secretion in mouse pancreatic islets were studied. Determination of polyamines in mouse islets revealed 0.9 +/- 0.3 (mean +/- S.E.M., n = 6) pmol of putrescine, 11.7 +/- 3.2 (8) pmol of spermidine and 3.7 +/- 0.6 (8) pmol of spermine per islet, corresponding to intracellular concentrations of 0.3-0.5 mM-putrescine, 3.9-5.9 mM-spermidine and 1.2-1.9 mM-spermine in mouse islets. Stimulation of insulin secretion by glucose, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) or the sulphonylurea glibenclamide did not affect these polyamine contents. In accordance with a role for protein kinase C in insulin secretion, TPA stimulated both protein kinase C activity and insulin secretion. Stimulation of insulin secretion by TPA was dependent on a non-stimulatory concentration of glucose and was further potentiated by stimulatory concentrations of glucose, glibenclamide or 3-isobutyl-1-methylxanthine, suggesting that protein kinase C activation, Ca2+ mobilization and cyclic AMP accumulation are all needed for full secretory response of mouse islets. Spermidine (5 mM) and spermine (1.5 mM) at concentrations found in islets inhibited protein kinase C stimulated by TPA + phosphatidylserine by 55% and 45% respectively. Putrescine (0.5 mM) was without effect, but inhibited the enzyme at higher concentrations (2-10 mM). Inhibition of protein kinase C by polyamines showed competition with Ca2+, and Ca2+ influx in response to glucose or glibenclamide prevented inhibition of insulin secretion by exogenous polyamines at concentrations where they did not affect glucose oxidation. It is suggested that inhibition of protein kinase C by polyamines may be of significance for regulation of insulin secretion in vivo and that Ca2+ influx may function by displacing inhibitory polyamines bound to phosphatidylserine in membranes.

Cyclic AMP phosphodiesterase activity in mouse pancreatic islets. Effects of calmodulin and phospholipids.

The activity of cyclic AMP phosphodiesterase in mouse pancreatic islets was investigated. 85% of the total activity was found in a 27000 g supernatant fraction. The phosphodiesterase activity in the supernatant fraction, but not in the particulate fraction, was stimulated approximately 20% by Ca2+ (10(-5)M) and calmodulin (1 microM). The Km (cyclic AMP) of the unstimulated enzyme in the supernatant fraction was 20 microM, and the Vmax was 2 nmol/min X mg protein-1. The possible influence of a range of phospholipids was investigated. PI* and PS (150 micrograms/ml) inhibited the enzyme 20-30% both in the absence and presence of Ca2+/calmodulin, whereas PE, PC and PA did not affect the enzyme activity. ATP (1 mM) did not affect the particulate or supernatant fraction phosphodiesterase either in the absence or presence of Ca2+/calmodulin or Ca2+/phospholipid. It is concluded that, contrary to islet adenylate cyclase, islet cyclic AMP phosphodiesterase may be regulated by Ca2+/calmodulin.