Unmasking of arachidonate-induced insulin release by removal of extracellular calcium. Arachidonic acid mobilizes cellular calcium in rat islets of Langerhans

Exogenous arachidonic acid does not stimulate insulin release in Ca++-containing medium, but a potent effect was unmasked by extracellular Ca++ depletion. This secretion met several criteria of exocytotic release. It did not require the oxygenation of arachidonate or its esterification into islet membranes, but was potentiated by the presence of 16.7 mM glucose such that 33 microM arachidonate could reverse the inhibitory effects of extracellular Ca++ removal on glucose-induced insulin secretion. Arachidonic acid alone stimulated a rise in intracellular Ca++ concentrations in dispersed islet cells (measured by the fura-2 technique) equal to that induced by 16.7 mM glucose in normal medium. Arachidonic acid may be a critical coupling signal in normal islets.     

Inhibitory effect of clonidine upon adenylate cyclase activity,cyclic AMP production,and insulin release in rat pancreatic islets

Conflicting opinions were recently expressed concerning the possible effect of ₂-adrenergic agonists upon cyclic AMP production in pancreatic islets. In the present: study, clonidine inhibited glucose-induced insulin release from rat pancreatic islets, this inhibitory effect being abolished by idazoxan. Clonidine did not suppress the capacity of forskolin to augment glucose-induced insulin release. In a particulate subcellular fraction derived from the islets, adenylate cyclase was activated by calmodulin (in the presence of Ca²⁺), NaF, GTP,, L-arginine, and forskolin, and slightly inhibited by clonidine. The inhibitory action of clonidine upon basal adenylate cyclase activity was more pronounced in islet crude homogenates. The inhibitory effect of clonidine was antagonized by forskolin whether in the particulate fraction or crude homogenate. At variance with the modest effects of glucagon, D-glucose, L-arginine, or a tumor-promoting phorbol ester upon cyclic AMP production by intact islets, forskolin caused a six-fold increase in cyclic AMP production. Clonidine inhibited cyclic AMP production by intact islets, whether in the absence or presence of forskolin. It is proposed that the inhibitory action of clonidine upon insulin release is attributable , in part at least, to inhibition of adenylate cyclase.     

Rat islet cells have glucose-dependent periodic electrical activity

In order to examine whether rat islet cells have a glucose-dependent plateau/silent phase pattern of electrical activity as seen in mouse islets, intracellular recordings were made in cultured whole rat islets. Rat islet cells responded to glucose stimulation with membrane potential alterations between a polarized silent phase and a depolarized plateau phase associated with spikes. Increasing or decreasing glucose stimulation prolonged or shortened the relative duration of plateau phase, respectively. Removal of glucose from the medium caused membrane hyperpolarization with disappearance of electrical activity while reintroduction of glucose caused membrane depolarization and biphasic onset of electrical activity. These results indicate that rat islet cells have a glucose dependent plateau/silent phase electrical mechanism nearly identical to that seen in mouse islets.     

Inhibitory effect of [Asu1 . 7]-eel calcitonin on glucagon-induced glycogenolysis in perfused rat liver

In an attempt to elucidate the mechanism by which calcitonin modulates glucose metabolism, the effect of elcatonin ([Asu1 . 7]-eel calcitonin), a potent synthetic eel calcitonin analogue, on hepatic glycogenolysis was investigated by use of perfused liver from fed rats. Elcatonin, as infused into the portal vein at concentrations between 10 mU/ml and 200 mU/ml did not affect glucose output into the hepatic venous effluent. At concentrations higher than 100 mU/ml, it inhibited the glycogenolysis stimulated by submaximal concentrations of glucagon which was perfused concurrently. This aspect of elcatonin effect was reproduced by synthetic salmon calcitonin. Though elcatonin showed a marked inhibition of the glycogenolytic activity induced by glucagon at or less than 5.7 X 10(-11) M, the inhibitory effect became undetectable when higher concentrations of glucagon were employed. Elcatonin did not inhibit the glycogenolysis induced by dibutyryl cyclic AMP at near (0.5 microM) or less than half-maximally effective (0.2 microM) concentrations. In addition, it did not inhibit the glycogenolytic activity half-maximally stimulated by alpha-adrenergic agonist (phenylephrine, 0.4 microM) or vasopressin (0.2 mU/ml). Elcatonin inhibited the cyclic AMP production of the tissue induced by glucagon infusion. These data indicate that elcatonin modulates hepatic glycogenolysis by preventing the glucagon effect at a step before cyclic AMP production and with an apparently competitive kinetics. In view of the concept that Ca++ is involved in the glycogenolytic effect of alpha-adrenergic agonist and vasopressin, the fact that elcatonin did not influence the action of these agents suggests that Ca++ fluxes are not involved in the elcatonin effect on liver.     

Glucose modulates [Ca2+]i oscillations in pancreatic islets via ionic and glycolytic mechanisms

Pancreatic islets of Langerhans display complex intracellular calcium changes in response to glucose that include fast (seconds), slow ( approximately 5 min), and mixed fast/slow oscillations; the slow and mixed oscillations are likely responsible for the pulses of plasma insulin observed in vivo. To better understand the mechanisms underlying these diverse patterns, we systematically analyzed the effects of glucose on period, amplitude, and plateau fraction (the fraction of time spent in the active phase) of the various regimes of calcium oscillations. We found that in both fast and slow islets, increasing glucose had limited effects on amplitude and period, but increased plateau fraction. In some islets, however, glucose caused a major shift in the amplitude and period of oscillations, which we attribute to a conversion between ionic and glycolytic modes (i.e., regime change). Raising glucose increased the plateau fraction equally in fast, slow, and regime-changing islets. A mathematical model of the pancreatic islet consisting of an ionic subsystem interacting with a slower metabolic oscillatory subsystem can account for these complex islet calcium oscillations by modifying the relative contributions of oscillatory metabolism and oscillatory ionic mechanisms to electrical activity, with coupling occurring via K(ATP) channels.     

Ruthenium red-insensitive calcium transport in ascites-sarcoma 180/TG cells.

1. Ruthenium Red-insensitive Ca2+ transport in the mouse ascites sarcoma 180/TG is enriched in a 'heavy' microsomal fraction (microsomes) sedimented at 35 000 g for 20 min. The subcellular distribution of this Ca2+ transport differed from that of Ruthenium Red-sensitive Ca2+ transport and (Na+ + K+)-dependent ATPase activity, but was similar to that of glucose 6-phosphatase. 2. The affinity of this transport system for 'free' Ca2+ is high (Km approx. 6 microM) and that for MgATP somewhat lower (Km approx. 100 microM). Ca2+ transport by the tumour microsomes, by contrast with that by liver microsomes, was greatly stimulated by low concentrations of P1. 3. Although incubation of intact ascites cells with glucagon led to an increase in intracellular cyclic AMP, no stable increase in the initial rate of Ca2+ transport in the subsequently isolated 'heavy' microsomes could be detected as in similar experiments carried out previously with rat liver cells. Reconstitution experiments suggest that a deficiency exists in the tumour microsomal membrane such that an action of glucagon that is normally present in rat liver microsomes is not evoked.     

Evidence of cyclic AMP-independent action of glucagon on calcium mobilization in rat hepatocytes

Glucagon increases the cytoplasmic free calcium concentration as measured by aequorin bioluminescence. It has been proposed by Wakelam et al. (Nature 323 (1986) 68-71) that low concentrations of glucagon mobilize calcium from an intracellular pool by causing polyphosphoinositide breakdown. To identify whether cyclic AMP mediates changes in the cytoplasmic free calcium concentration ([Ca2+]c) induced by glucagon, the effects of forskolin and exogenous cyclic AMP on [Ca2+]c were compared with that of glucagon in aequorin-loaded hepatocytes. Although the magnitudes of the [Ca2+]c responses to 250 microM forskolin and 1 mM 8-bromo cyclic AMP were identical to that of 5 nM glucagon, these two agents induced a more prolonged elevation of [Ca2+]c. Glucagon-induced elevation of [Ca2+]c was accompanied by a smaller increase in cyclic AMP than that induced by forskolin. When the cyclic AMP response to glucagon was potentiated by an inhibitor of phosphodiesterase, 3-isobutyl-1-methylxanthine, the glucagon-induced increase in [Ca2+]c was not affected. Conversely, when the cyclic AMP response to glucagon was reduced by pretreatment of the cells with angiotensin II, glucagon-induced changes in [Ca2+]c were rather enhanced. Furthermore, vasopressin potentiated glucagon-induced changes in [Ca2+]c despite the reduction of the cyclic AMP response to glucagon. In the presence of 1 microM extracellular calcium, angiotensin II did not enhance glucagon-induced changes in [Ca2+]c. These results suggest that at least part of the action of 5 nM glucagon on calcium mobilization is independent of cyclic AMP.     

Glucose dependent stimulation by prostaglandin D2 of glucagon and insulin in perfused rat pancreas

Effects of prostaglandin D2 on pancreatic islet function in perfused rat pancreas were examined in comparison with those of prostaglandin E2, which has hitherto been suggested to be a modifier of pancreatic hormone release. In the presence of 2.8 mM glucose, only glucagon release was strongly stimulated by 14 microM of prostaglandin D2, while release of both glucagon and insulin was augmented by 14 microM of prostaglandin E2. When the glucose concentration was elevated to 11.2 mM, insulin release was accelerated by 14 microM of prostaglandin D2 but there was no effect upon glucagon release. Again, release of both glucagon and insulin was augmented by 14 microM of prostaglandin E2 in the presence of 11.2 mM of glucose. The regulation of glucagon and insulin release through prostaglandin D2 is apparently adapted to glycemic changes, and may be a physiological modulator of pancreatic islet function.     

Effect of Na/Ca exchange on plateau fraction and [Ca]i in models for bursting in pancreatic beta-cells.

In the presence of an insulinotropic glucose concentration, beta-cells, in intact pancreatic islets, exhibit periodic bursting electrical activity consisting of an alternation of active and silent phases. The fraction of time spent in the active phase over a period is called the plateau fraction and is correlated with the rate of insulin release. However, the mechanisms that regulate the plateau fraction remain unclear. In this paper we investigate the possible role of the plasma membrane Na+/Ca2+ exchange of the beta-cell in controlling the plateau fraction. We have extended different single-cell models to incorporate this Ca2+-activated electrogenic Ca2+ transporter. We find that the Na+/Ca2+ exchange can provide a physiological mechanism to increase the plateau fraction as the glucose concentration is raised. In addition, we show theoretically that the Na+/Ca2+ exchanger is a key regulator of the cytoplasmic calcium concentration in clusters of heterogeneous cells with gap-junctional electrical coupling.     

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Stimulation of adenylate cyclase in the heart of Aplysia californica by biogenic amines

The effects of serotonin (5-HT), dopamine (DA), several peptides including FMRFamide and arginine vasotocin, the diterpene forskolin and Ca2+ were examined on adenylate cyclase in a particulate fraction from hearts of Aplysia californica. Enzyme activity was stimulated 6-7-fold by 5-HT (EC50, 1 microM) in the presence of GTP. Several 5-HT analogs particularly 5-methoxytryptamine and 5-methoxy-N-N-dimethyltryptamine were also active. The stimulatory action of 5-HT was antagonized by the 5-HT receptor blockers methergoline and metitepine and by the DA receptor blocker chlorpromazine. Dopamine had weak stimulatory action (EC50, 10 microM) and an efficacy relative to that of 5-HT of 0.3. The action of DA was antagonized by chloropromazine and metitepine. Several peptides including FMRFamide and arginine vasotocin had no effect on adenylate cyclase when tested over the concentration range 0.1-100 microM. The enzyme was stimulated 6-fold by the diterpene forskolin (EC50, 2 microM). 5-HT-stimulated activity was strongly inhibited by Ca2+. Calmodulin had no action on the enzyme in the presence of Ca2+.     

Glucose 6-phosphate regulates Ca2+ steady state in endoplasmic reticulum of islets. A possible link in glucose-induced insulin secretion

Glucose stimulation of islets is coupled with the rapid intracellular release of myo-inositol 1,4,5-trisphosphate (IP3) and arachidonic acid which in turn mobilize Ca2+ stored in the endoplasmic reticulum (ER). The metabolism of glucose is required for insulin secretion although the link between glucose metabolism and the cellular events resulting in insulin release is unknown. In digitonin-permeabilized islets, glucose 6-phosphate (0.5-4 mM) increased significantly the ATP-dependent Ca2+ content of the ER at a free Ca2+ concentration of 1 microM. At 0.2 microM free Ca2+, glucose 6-phosphate (2-10 mM) had a smaller effect. Glucose, phosphate, mannose 6-phosphate, and fructose 1,6-diphosphate had no effect on the ATP-dependent Ca2+ content of the ER. Glucose 1-phosphate and fructose 6-phosphate also increased ATP-dependent Ca2+ content of the ER, presumably due to conversion to glucose 6-phosphate by islet phosphoglucomutase and phosphoglucoisomerase, respectively. The glucose 6-phosphate increase in the ATP-dependent Ca2+ content of the ER was shown to be mediated by glucose 6-phosphatase localized to the ER. Both arachidonic acid (10 microM) and the Ca2+ ionophore A23187 (2 microM) mobilized Ca2+ stored in the ER by glucose 6-phosphate. However, IP3-induced (10 microM) Ca2+ release from the ER was abolished in the presence of glucose 6-phosphate (0.5-10 mM). We propose that glucose 6-phosphate could provide a regulatory link between glucose metabolism and intracellular Ca2+ regulation by augmenting Ca2+ sequestered in the ER as well as attenuating IP3-induced Ca2+ release. Thus, glucose 6-phosphate would serve as an "off" signal leading to a decrease in intracellular Ca2+ when both the free Ca2+ and glucose 6-phosphate concentrations have increased following glucose stimulus.     

Glucagon decreases cytokine induction of nitric oxide synthase and action on insulin secretion in RIN5F cells and rat and human islets of Langerhans.

Nitric oxide synthase, induced by cytokines in insulin-containing cells, produces nitric oxide which inhibits function and may promote cell killing. Since glucagon was shown to prevent inducible nitric oxide synthase (iNOS) expression in rat hepatocytes it was of interest to examine the action of glucagon (and cyclic AMP) on iNOS induction in insulin-producing cells. Cultured RIN5F cells and primary rat and human islets of Langerhans were treated with interleukin 1beta (IL-1beta) or a combination of cytokines, and were co-treated or pre-treated with glucagon. In RIN5F cells, the activity of iNOS induced by IL-1beta (10 pM, 24 h), was significantly reduced by glucagon (1000 nM), which raises cyclic AMP, and by forskolin (1-10 microM), a non specific activator of adenylate cyclase. Glucagon and forskolin also decreased iNOS expression in RIN5F cells, and rat and human islets, as shown by Western blotting. The inhibitory action of IL-1beta (100 pM, 24 h) on rat islet insulin secretion was partially reversed by 1-h pre-treatment with glucagon (10-1000 nM), while the contrasting stimulatory effect of 48-h treatment with cytokines on insulin secretion from human islets was similarly prevented by glucagon (1000 nM) pre-treatment. These results suggest that glucagon inhibits iNOS expression in insulin-containing cells and imply that glucagon could modulate the inhibitory effects of cytokines.     

Role of voltage-dependent ionic currents in coupling glucose stimulation to insulin secretion in canine pancreatic islet B-Cells

Summary Glucose-induced electrical activity in canine pancreatic islet B cells is distinct from that in rodent islets, though both display Ca²⁺-dependent insulin secretion. Rodent islet B cells undergo regular bursts of Ca²⁺-dependent action potentials, while canine islet B cells generate isolated Na⁺-dependent action potentials which often give way to a plateau depolarization. Here we present evidence to reconcile the species difference in electrical activity with the similarity of Ca²⁺ dependence of secretion. (i) In canine B cells increasing glucose concentrations produce membrane depolarization and increasing frequency of Na_o-dependent action potentials until a background membrane potential (-40mV) is reached where Na⁺ currents are inactivated. (ii) Voltage-dependent Ca²⁺ currents are present which are activated over the voltage excursion of the action potential (–50 to +20 mV) and inactivate slowly, (over seconds) in the range of the plateau depolarization (–40 to –25 mV). Hence, they are available to contribute to both phases of depolarization. (iii) Tetrodotoxin (TTX) reduces by half an early transient phase of glucosestimulated insulin secretion but not a subsequent prolonged plateau phase. The transient phase of secretion often corresponds well in time to the period of initial high frequency action potential activity. These latter results suggest that in canine B cells voltagedependent Na⁺ and Ca²⁺ currents mediate biphasic glucose-induced insulin secretion. The early train of Na⁺-dependent action potentials, by transiently activating Ca²⁺ channels and allowing pulsatile Ca²⁺ entry, may promote an early transient phase of insulin secretion. The subsequent sustained plateau depolarization, by allowing sustained Ca²⁺ entry, may permit steady insulin release.     

High external Ca2+ levels trigger membrane potential oscillations in mouse pancreatic beta-cells during blockade of K(ATP) channels.

Glucose depolarizes the pancreatic beta-cell and induces membrane potential oscillations, but the nature of the underlying oscillatory conductance remains unknown. We have now investigated the effects of the Ca2+ ionophore ionomycin and high external Ca2+ concentration ([Ca2+]o) on glucose-induced electrical activity and whole islet intracellular free Ca2+ concentration ([Ca2+]i), under conditions where the K(ATP) channel was blocked (100 microM tolbutamide or 4 microM glibenclamide). Raising [Ca2+]o to 10.2 or 12.8 mM, but not to 5.1 or 7.7 mM, turned continuous electrical activity into bursting activity. High [Ca2+]o (12.8 mM) regenerated a pattern of fast [Ca2+]i oscillations overshooting the levels recorded in tolbutamide. Ionomycin (10 microM) raised the [Ca2+]i and synergized with 5.1 mM Ca2+ to hyperpolarize the beta-cell membrane. The data indicate that a [Ca2+]i-sensitive and sulphonylurea-insensitive oscillatory conductance underlies the beta-cell bursting activity.     

Effects of synthetic cholecystokinin analog on hormone secretion in fetal human pancreatic tissue culture]

We have investigated the effects of Pro-Met-Asp-Phe-NH2 (PMAP) on insulin and glucagon release from human fetal pancreatic microfragments in vitro. Four batches of precultured microfragments were incubated for 24 hrs in medium containing 5.5 mM glucose, 17 mM glucose, 1 microM PMAP or 1 microM PMAP plus 17 mM glucose. PMAP significantly enhanced both basal and glucose-stimulated insulin release (2.2- and 4.1-fold, respectively). Glucagon secretion was markedly inhibited by glucose (17 mM). PMAP neither affected the basal glucagon release nor potentiated the inhibitory action of glucose on glucagon release. Hence, PMAR selectively regulates insulin production in human fetal islet tissue without affecting glucagon production. Our results suggest that the substances similar or related to PMAP may prove to be of clinical value in drug correction of diabetes mellitus.     

The Ca2+ dynamics of isolated mouse beta-cells and islets: implications for mathematical models

[Ca(2+)](i) and electrical activity were compared in isolated beta-cells and islets using standard techniques. In islets, raising glucose caused a decrease in [Ca(2+)](i) followed by a plateau and then fast (2-3 min(-1)), slow (0.2-0.8 min(-1)), or a mixture of fast and slow [Ca(2+)](i) oscillations. In beta-cells, glucose transiently decreased and then increased [Ca(2+)](i), but no islet-like oscillations occurred. Simultaneous recordings of [Ca(2+)](i) and electrical activity suggested that differences in [Ca(2+)](i) signaling are due to differences in islet versus beta-cell electrical activity. Whereas islets exhibited bursts of spikes on medium/slow plateaus, isolated beta-cells were depolarized and exhibited spiking, fast-bursting, or spikeless plateaus. These electrical patterns in turn produced distinct [Ca(2+)](i) patterns. Thus, although isolated beta-cells display several key features of islets, their oscillations were faster and more irregular. beta-cells could display islet-like [Ca(2+)](i) oscillations if their electrical activity was converted to a slower islet-like pattern using dynamic clamp. Islet and beta-cell [Ca(2+)](i) changes followed membrane potential, suggesting that electrical activity is mainly responsible for the [Ca(2+)] dynamics of beta-cells and islets. A recent model consisting of two slow feedback processes and passive endoplasmic reticulum Ca(2+) release was able to account for islet [Ca(2+)](i) responses to glucose, islet oscillations, and conversion of single cell to islet-like [Ca(2+)](i) oscillations. With minimal parameter variation, the model could also account for the diverse behaviors of isolated beta-cells, suggesting that these behaviors reflect natural cell heterogeneity. These results support our recent model and point to the important role of beta-cell electrical events in controlling [Ca(2+)](i) over diverse time scales in islets.     

Resveratrol-induced inhibition of insulin secretion from rat pancreatic islets: evidence for pivotal role of metabolic disturbances

Resveratrol is a stilbene present in different plant species and exerting numerous beneficial effects, including prevention of diabetes and attenuation of some diabetic complications. Its inhibitory effect on insulin secretion was recently documented, but the exact mechanism underlying this action remains unknown. Experiments employing diazoxide and a high concentration of K(+) revealed that, in depolarized pancreatic islets incubated for 90 min with resveratrol (1, 10, and 100 microM), insulin secretion stimulated by glucose and leucine was impaired. The attenuation of the insulin secretory response to 6.7 mM glucose was not abrogated by blockade of intracellular estrogen receptors and was found to be accompanied by diminished islet glucose oxidation, enhanced lactate production, and reduced ATP levels. Glucose-induced hyperpolarization of the mitochondrial membrane was also reduced in the presence of resveratrol. Moreover, in depolarized islets incubated with 2.8 mM glucose, activation of protein kinase C or protein kinase A potentiated insulin release; however, under these conditions, resveratrol was ineffective. Further studies also revealed that, under conditions of blocked voltage-dependent calcium channels, the stilbene reduced insulin secretion induced by a combination of glucose with forskolin. These data demonstrate that resveratrol 1) inhibits the amplifying pathway of insulin secretion, 2) exerts an insulin-suppressive effect independently of its estrogenic/anti-estrogenic activity, 3) shifts islet glucose metabolism from mitochondrial oxidation to anaerobic,4) fails to abrogate insulin release promoted without metabolic events, and 5) does not suppress hormone secretion as a result of the direct inhibition of Ca(2+) influx through voltage-dependent calcium channels.     

Effects of calcium manipulation and glucose stimulation on a histochemically detectable mobile calcium fraction in isolated rat pancreatic islets

Pancreatic B-cell calcium as histochemically detectable with glyoxal bis (2-hydroxyanil) = GBHA was studied in isolated islets of fed rats. GBHA has previously been shown by us to detect an ionized or readily ionizable Ca-fraction (GBHA-Ca). In the presence of Ca++ (2.5 mM), high glucose (15 mM) induced a rapid decrease (30%) of islet GBHA-Ca followed by a rise between 30 and 60 min to levels above the initial value. At low glucose (0 or 2.5 mM) GBHA-Ca showed a slight and gradual decline under these conditions. Omission of Ca++ at low glucose rapidly decreased (30%) islet GBHA-Ca. This decrease was markedly inhibited by high glucose, although glucose did not induce insulin secretion under these conditions. Preincubation in the absence of Ca++ (15 min) depleted islet GBHA-Ca, but partial restoration occurred during subsequent incubation with Ca++ at low glucose. By contrast, high glucose completely restored GBHA-Ca within 5 min, followed by a decline and a subsequent rise. Reintroduction of Ca++ also rapidly restored the glucose-induced insulin secretion. These results indicate that islet GBHA-Ca represents a mobile Ca-fraction which is dependent on extracellular Ca++ and which responds very rapidly to glucose stimulation. It is suggested that changes of GBHA-Ca in the B-cells may reflect changes in the Ca pool involved in the insulin secretory mechanism.