Adrenoceptor antagonists, but not guanethidine, reduce glucopenia-induced glucagon secretion from perfused rat pancreas

This study was designed to investigate (1) whether norepinephrine is released in response to glucopenia in vitro, thereby stimulating glucagon secretion and, (2) the modulating effects of norepinephrine on insulin and glucagon secretion, using isolated perfused rat pancreas preparations. Simultaneous addition of the adrenergic receptor antagonists yohimbine, prazosin and propranolol, each at a concentration of 10⁻⁵ mol/l, significantly potentiated glucose-stimulated insulin secretion (6.23 ± 0.76 vs. 2.11 ± 0.72 (control) nmol/min, P < 0.01), and suppressed glucopenia-induced glucagon secretion (0.59 ± 0.10 vs. 1.34 ± 0.18 (control) ng/min, P < 0.05). Also, 10⁻⁵ mol/l yohimbine alone significantly potentiated glucose-stimulated insulin secretion (4.86 ± 0.50 nmol/min, P < 0.05). The norepinephrine release inhibitor, guanethidine, significantly inhibited tyramine-induced secretion of both norepinephrine (7.86 ± 0.77 vs. 49.7 ± 2.3 nmol/min, P < 0.01) and glucagon (0.31 ± 0.08 vs. 1.21 ± 0.15 ng/min, P < 0.01), but exerted no effects on glucopenia-induced secretion of either norepinephrine or glucagon. We conclude that these results further support the concept that the neurotransmitter norepinephrine is released in response to glucopenia in vitro, and modulates insulin and glucagon secretion. Our data do not, however, provide evidence indicating that glucopenia-induced glucagon secretion is mainly mediated by activation of sympathetic nerve terminals around the -cells in the isolated perfused rat pancreas.     

The interaction of Vasoactive Intestinal Polypeptide (VIP), glucose and arginine on the secretion of insulin,glucagon and somatostatin in the perfused rat pancreas

Summary Vasoactive Intestinal Polypeptide (VIP) increased the release of insulin, glucagon and somatostatin from the perfused rat pancreas. The amount of these hormones released was dependent upon the prevailing glucose concentration. VIP stimulated glucagon release in the absence of glucose, while insulin and somatostatin release were increased by VIP only in the presence of glucose concentrations of 4.4 mmol/l and above. Glucagon secretion stimulated by arginine in the presence of 4.4 mmol/l glucose was potentiated by VIP. In contrast, VIP did not induce any further increase in the secretion of insulin and somatostatin over that stimulated by arginine. At higher concentrations of glucose (6.7, 16.7, and 33.3 mmol/l) VIP continued to stimulate insulin and somatostatin release, this effect being synergistic on early-phase insulin release. The effects of VIP on islet cells thus depend on the levels of modulating nutrients.     

Different effects of hypothermia on insulin and glucagon secretion from the isolated perfused rat pancreas

Summary Two series of experiments with the isolated perfused rat pancreas were performed in parallel. The conditions differed only with respect to temperature, which was 37.5 °C in one series and 28 °C in the other. The lowering of the temperature decreased insulin secretion induced by glucose as well as the insulin response to tolbutamide and acetylcholine. Unlike insulin, glucagon secretion was not significantly modified by hypothermia. Our results suggest that the mechanisms involved in glucagon and insulin secretion are different.Equipe de Recherche Associée au CNRS n 786     

The influence of somatostatin on glucagon and pancreatic polypeptide secretion in the isolated perfused human pancreas

Summary The current study was undertaken to determine whether intraislet somatostatin regulates glucagon or pancreatic polypeptide (PP) secretion in the human pancreas. A high-affinity, high-specificity monoclonal somatostatin antibody (CURE.S6) was used to immunoneutralize somatostatin in the isolated, perfused human pancreas. Single-pass perfusion was performed in pancreata obtained from cadaveric organ donors using a modified Krebs media with either 3.9 or 12.9 mM glucose. Sequential test periods separated by basal periods were performed with infusion of either exogenous somatostatin-14 (SS-14), CURE.S6, or a combined infusion. Infusion of SS-14 did not significantly alter glucagon or PP secretion during low-glucose or high-glucose perfusion. Immunoneutralization of intraislet somatostatin with CURE.S6 resulted in a significant increase of glucagon secretion under low-glucose conditions (ΔX=15±3 pM) (p<0.05), but did not significantly effect glucagon secretion under high-glucose conditions (ΔX=−2±3 pM) (p=NS). PP secretion remained unchanged during CURE.S6 infusion. Combined infusion of SS-14 and CURE.S6 did not significantly alter glucagon or PP secretion. The data suggest that intraislet somatostatin may have an inhibitory role in the regulation of glucagon secretion during low-glucose conditions and that intraislet somatostatin does not regulate PP secretion in the isolated, perfused human pancreas.     

Acute effects of alloxan- and streptozotocin-induced insulin deficiency on somatostatin and glucagon secretion by the perfused isolated rat pancreatico-duodenal preparation

Summary The secretion of somatostatin and glucagon by the perfused rat pancreatico-duodenal preparation was examined in situ under control conditions and after the induction of acute insulin deficiency by alloxan or streptozotocin. A 10 min 0.625 mmol/l alloxan perfusion resulted in an immediate and transient increase in basal insulin and glucagon release and a slightly delayed and persistent increase in basal somatostatin secretion. The insulin responses to 16.7 mmol/l glucose, 1 mmol/l theophylline, and 19 mmol/l arginine alone or in combination were virtually eliminated by alloxan treatment, Somatostatin secretion in response to the stimuli was completely inhibited or markedly attenuated. The glucagon-suppressive effect of glucose was unaltered by alloxan and the stimulatory effect of arginine was enhanced. Addition of 1 g/ml porcine insulin to the perfusion medium did not modify the alterations in somatostatin and glucagon responses to arginine. Streptozotocin treatment 90 min prior to the onset of perfusion resulted in changes in somatostatin, glucagon, and insulin responses to glucose and arginine similar to those of alloxan. The present results are consistent with an effect of alloxan and streptozotocin on the D cell similar to that on the B cell, namely, interference with a glucose-mediated effect on hormone secretion.     

Pancreatic polypeptide,glucagon and insulin secretion from the isolated perfused canine pancreas

Summary The release of pancreatic polypeptide (PP) by gut hormones, acetyl choline and adrenaline was investigated in an isolated perfused pancreas preparation. PP was potently released by 1 nmol/l caerulein (186±12%, p<0.001) and gastric inhibitory peptide (GIP) (211±31%, p<0.005) as well as by 1 [mol/l acetyl choline (1097±59%, p<0.001). A significant two-fold release of PP was also evoked by 1 nmol/l vasoactive intestinal peptide (VIP) (129±38%, p<0.02 and gastrin (108±25% p<0.01). Insulin release, induced by high glucose concentration was enhanced by both GIP (210 ±38%, p<(0.01) and VIP (48±5%, p<0.001). In addition GIP enhanced the release of glucagon by 179±18% (p<0.001) at 1.4 mmol/l glucose and by 127±24% (p<0.005) at 8.3 mmol/l glucose. Thus no simple inter-relationship appears to exist between the control of the three circulating islet hormones.     

Time-dependent inhibition of insulin release: glucose-arginine interactions in the perfused rat pancreas

Summary The isolated perfused rat pancreas was stimulated sequentially with arginine or glucose to analyze the time-dependent modulation of insulin release. A 10-min perfusion with arginine (5.0 mmol/l) induced 75% inhibition of the insulin response to repeated arginine stimulation 10 min later. When glucose (8.3 mmol/l) was given as two pulses, inhibition of the second insulin response was less pronounced. The inhibitory effect generated by arginine also suppressed the insulin response to glucose (27.7 mmol/l), and this inhibitory effect persisted for over 80 min. Stimulation for 30 min with glucose (27.7 mmol/l) strongly potentiated the insulin responses to a pair of arginine stimuli given 20 min later. However, despite augmented secretion rates, the insulin response to the second arginine pulse was still inhibited by 75%. When insulin secretion was strongly amplified by two 10 min pulses of the synergistic mixture of arginine (5.0 mmol/l) and glucose (8.3 mmol/l), there was no inhibition of the second insulin response. If glucose (8.3 mmol/l) was present during the first arginine stimulation only, the response to the second arginine pulse was inhibited as in control experiments. However, when glucose was added to the second arginine pulse only, the inhibition generated by the first arginine pulse did not express itself, insulin release remaining similar to control. We conclude that: (1) short stimulations of the pancreas by arginine or glucose generate long-lasting inhibition of the insulin response to subsequent stimulations; (2) synergistic amplification of the insulin response by addition of glucose to arginine obliterates the inhibition; (3) glucose does not suppress the induction of inhibition, it blocks the expression of the inhibitory signal on insulin secretion; (4) these in vitro findings are in keeping with observations in normal and hyperglycaemic man.     

Potassium ion concentration alters glucagon secretion independently of insulin secretion in the isolated rat pancreas

Summary In the arginine-stimulated perfused rat pancreas, elevated concentrations of potassium ion inhibited glucagon secretion while stimulating the secretion of insulin. Decreased potassium ion produced the reverse effect. The observed inverse correlation between changes in insulin and glucagon secretion (r = -0.64; p<0.001) was suggestive of local interactions between islet hormones, and prompted us to determine whether potassium-induced changes in glucagon secretion were dependent upon concurrent changes in insulin release. We found that when insulin secretion was greatly suppressed, either through acute induction of diabetes with streptozotocin or by utilization of a glucose-free perfusate, no qualitative differences in glucagon responsiveness to altered potassium ion were evident, although the amplitude of these glucagon changes was enhanced. Similarly, when exogenous insulin (20,000 mU/l) was added to the perfusate in order to render potassium-induced changes in endogenous insulin secretion insignificant, glucagon responsiveness to altered potassium ion was maintained. Exogenous insulin alone had no effect on arginine-stimulated glucagon secretion. We conclude that any indirect effects of potassium ion on arginine-stimulated glucagon secretion are not mediated by insulin, but could be related to changes in somatostatin secretion.     

Neurotransmitters partially restore glucose sensitivity of insulin and glucagon secretion from perfused streptozotocin-induced diabetic rat pancreas

Summary To elucidate the mechanisms of insensitivity of hormone secretion to glucose in streptozotocin-induced diabetic rat islets, we investigated the effects of acetylcholine (ACh) and norepinephrine on insulin and glucagon secretion in response to changes in glucose concentration, using perfused pancreas preparations. Basal insulin secretion at a blood glucose level of 5.6 mmol/l was significantly higher and basal glucagon secretion significantly lower in streptozotocin-induced diabetic rats than in controls, and neither high (16.7 mmol/l) nor low (1.4 mmol/l) blood glucose concentrations influenced insulin or glucagon secretion. Addition of 10^–6 mol/l ACh to the perfusate increased glucose-stimulated insulin secretion. Also, 10^–6 mol/l ACh, 10^–7 mol/l norepinephrine, as well as a combination of both, induced marked glucagon secretion, this was suppressed by high blood glucose level. Although simultaneous addition of 10^–6 mol/l ACh and 10^–7 mol/l norepinephrine induced only a slight increase in glucagon secretion in response to glucopenia, there was a significant increase in glucagon secretion in conjunction with an ambient decrease in insulin. Histopathological examination revealed a marked decline in acetylcholinesterase and monoamine-oxidase activities in the islets of streptozotocin-induced diabetic rats. We speculate that reduction of the potentiating effects of ACh and norepinephrine lessens glucose sensitivity of islet beta and alpha cells in this rat model of diabetes.Abbreviations STZ Streptozotocin - STZD streptozotocin-induce diabetic - ACh acetylcholine - AChE acetylcholinesterase - NE norepinephrine - MAO monoamine-oxidase     

Previous exposure to glucose enhances somatostatin secretion from the isolated perfused rat pancreas

Summary Previous exposure to glucose enhances insulin and depresses glucagon secretion by the pancreas. We have investigated whether secretion of somatostatin is also influenced by a glucose priming effect. In perfused rat pancreas from 36 h fasted rats a 5 min pulse of arginine (8 mmol/l) rapidly elicited a peak of somatostatin release. A similar somatostatin response was evoked by a second, identical, pulse of arginine after perfusion with basal glucose (3.9 mmol/l) for 45 min. On the other hand when 27.7 mmol/l D-glucose, was administered for 20 min between arginine pulses, there was significant stimulation of somatostatin secretion. When arginine was re-introduced 15 min after the cessation of the pulse of elevated glucose the magnitude of the arginine-induced peak (min 0–2 of stimulation) was increased from 16.2±4.1 to 33.1±4.7 pg/2 min, p<0.01, relative to the first stimulation with arginine. None of these effects of glucose could be reproduced by Dgalactose. The somatostatin response to arginine was higher in pancreata from fed than from 36 h fasted animals as was also basal release (22.8±5.0 vs 9.0±2.0 pg/min). In the fed state the response to the second pulse of arginine was however reduced by 50% after perfusion with basal glucose. This decrease in responsiveness was counteracted by perfusion with 27.7 mmol/l glucose for 20 min between the arginine pulses. It is concluded that previous exposure to an elevated concentration of glucose enhances D-cell responsiveness to arginine in the fasted as well as the fed state.     

Characterisation of somatostatin release from the pancreas

Summary The effect of calcium on somatostatin secretion was investigated in the isolated, perfused canine pancreas preparation and compared with those of acetylcholine, glucose, isoproterenol and arginine. Calcium (5 mmol/l) stimulated somatostatin release in a typical biphasic response pattern being about 5 times as potent as acetylcholine (1 mol/l), arginine (5 mmol/l), and isoproterenol (2 ng/ml) while the release of insulin and glucagon in response to calcium and the other secretagogues were of the same magnitude. Somatostatin release increased progressively when perfusate calcium was increased step-wise from 0 through 1.25 and 2.5 to 5.0 mmol/l. Calcium stimulated the secretion of somatostatin in the absence of glucose. The stimulatory effect of calcium was, however, modulated by the glucose concentration being about twice as large at 200 mg/100 ml as at 25 mg/100 ml glucose in the perfusion medium.     

In situ binding of islet hormones in the isolated perfused rat pancreas: evidence for local high concentrations of islet hormones via the islet-acinar axis

Summary Insulin and somatostatin reportedly affect pancreatic acinar cell function via specific receptor binding. Theoretically peri-insular levels depend on the islet-acinar portal system, but the actual hormone levels have never been demonstrated. Rat pancreata were perfused anterogradely or retrogradely with ¹²⁵I-insulin, -somatostatin, or -glucagon (each, 10^–11 mol/l). Tracer binding was determined from differences between influx and efflux radioactivity. Saturable binding was observed for insulin and somatostatin, but not for glucagon. Binding in the absence of unlabelled peptides was significantly higher during retrograde perfusion than during anterograde perfusion for insulin (25.9±2.6 vs 16.0±2.1%, mean±SD; each, n=4; p<0.001) and somatostatin (18.4±2.0 vs 13.6±1.2%; each, n=3; p<0.05). Non-specific binding was similar in both directions. These findings are attributable to endogenous hormones acting as unlabelled ligands competing with the tracers during anterograde perfusion. This conclusion was supported by the demonstration that endogenous insulin stimulation by d-glucose, but not by l-glucose, caused a decrease in labelled insulin binding only during anterograde perfusion. Displacement curves obtained during retrograde perfusion showed that interstitial concentrations of insulin and somatostatin were 7.5×10^–9 and 1.1×10^–9 mol/l, respectively. Thus, the exocrine pancreas is indeed exposed to locally high concentrations of islet hormones.Abbreviations SS Somatostatin - TCA trichloroacetic acid     

Glucagon-like peptide-1, but not glucose-dependent insulinotropic peptide,inhibits glucagon secretion via somatostatin (receptor subtype 2) in the perfused rat pancreas

Aims/hypothesis  The glucose-lowering effect of glucagon-like peptide-1 (GLP-1) is based not only upon its potent insulinotropic actions but also on its ability to restrain glucagon secretion. Surprisingly, the closely related glucose-dependent insulinotropic peptide (GIP) stimulates glucagon release. We examined whether the islet hormone somatostatin, which strongly inhibits glucagon secretion, is involved in this divergent behaviour. Methods  At 1.5 mmol/l glucose and therefore minimal insulin secretion, the glucagon, insulin and somatostatin responses to 20 mmol/l glucose, GLP-1, GIP and somatostatin were studied in the presence of a high-affinity monoclonal somatostatin antibody and of a highly specific somatostatin receptor subtype 2 (SSTR2) antagonist (PRL-2903) in the isolated perfused rat pancreas. Results  In control experiments, GLP-1 at 1 and 10 nmol/l reduced glucagon secretion significantly to 59.0 ± 6.3% (p < 0.004; n = 5; SSTR2 series; each vs pre-infusion level) and to 48.0 ± 2.6% (p < 0.001; n = 6; somatostatin antibody series) respectively. During somatostatin antibody administration, GLP-1 still inhibited glucagon secretion significantly, but the effect was less pronounced than in control experiments (p < 0.018). Co-infusion of the SSTR2 antagonist completely abolished the GLP-1-induced suppression of glucagon secretion. In contrast, neither the GIP-induced stimulation of glucagon release nor its inhibition by 20 mmol/l glucose was altered by somatostatin antibody or SSTR2 antagonist administration. Conclusions/interpretation  We conclude that GLP-1 is capable of inhibiting glucagon secretion even in the absence of secretory products from the beta cell. It is highly likely that this is mediated via somatostatin interacting with SSTR2 on rat alpha cells. In contrast, GIP and glucose seem to influence the alpha cell independently of somatostatin secretion.     

Possible involvement of the cholinergic system in hormonal secretion by the perfused pancreas from ventromedial-hypothalamic lesioned rats

Summary Total arginine-induced secretion of insulin, glucagon and somatostatin was studied during a 20 min period in isolated perfused pancreases from control and non-hyperphagic ventromedial hypothalamic (VMH) lesioned rats. Compared to controls pancreases from VMH-lesioned rats secreted more insulin (82±13ng vs 36±9ng) and more glucagon (130±23ng vs 73±14ng) but less somatostatin (0.58±0.18ng vs 1.12±0.14ng). These abnormalities were restored to normal by perfusion with atropine (25 mol/l). Pancreases of both groups were perfused with the cholinergic agonist methacholine (100 mol/l). Again pancreases from VMH-lesioned rats secreted more insulin (157±19ng vs 33±6ng) and more glucagon (95±13 ng vs 57±9 ng) but less somatostatin (0.80±0.15 ng vs 1.30±0.18 ng). These results support the concept that, in pancreases isolated from VMH-lesioned rats increased cholinergic activity may prevail via increased release of endogenous acetylcholine from islet-postsynaptic ganglion cells together with increased numbers of muscarinic receptors on postsynaptic ganglion cells as well as on endocrine cells.     

Effect of a long acting glucagon selective somatostatin analogue on plasma glucose,insulin and glucagon levels in the anaesthetized rat during arginine infusion

Summary The effects of somatostatin and a long acting, glucagon selective somatostatin analog (des-Ala¹Gly²[His^4,5-D-Trp⁸]-somatostatin), were studied during arginine tolerance tests in normal anaesthetized rats. Arginine infusion in control animals resulted in a rapid increase in plasma insulin and glucagon, and an increase of 15±5 mg/dl in plasma glucose. Somatostatin infusion (1 mg/kg/h) resulted in suppression of basal insulin secretion and a decrease in arginine-induced insulin and glucagon release. Glucose levels increased rapidly during the combined arginine-somatostatin infusion reaching a peak of 72±10 mg/dl above basal levels. Similar results were obtained when somatostatin was injected SC (1 mg/kg) at times 0, 15, 30, and 45 minutes (arginine infused from 30–60 minutes). A single injection (1 mg/kg) of the long-acting somatostatin analogue resulted in significant inhibition of basal insulin and glucagon release; during arginine infusion glucagon levels rose only slightly, the insulin response was, however, nearly normal, and only a small arginine-induced increase in glucose levels was observed. Carbohydrate absorption was not influenced by either somatostatin or the analogue.     

Effect of oleic acid on arginine-induced glucagon secretion by the isolated perfused rat pancreas

Summary The isolated perfused rat pancreas was used to investigate the effect of oleic acid on glucagon secretion in response to 10 mmol/l arginine. In the absence of oleic acid and at 2.5 mmol/l calcium, arginine induced a biphasic glucagon secretion. At lower extracellular calcium concentration (1.0 mmol/l), the second phase of glucagon release was reduced, the first phase being unchanged. In the presence of 1,500 μmol/l oleic acid, the glucagon response to arginine was also biphasic, but second phase release was markedly inhibited, the first phase glucagon release being unchanged. Such an effect was not obtained when oleic acid concentration in the medium was 750 μmol/l. These results demonstrate that high concentrations of oleic acid inhibit glucagon secretion in response to arginine from the isolated perfused rat pancreas and support the concept that circulating free fatty acid levels are involved in the control of glucagon secretion. Presented in part at the 13th Annual Meeting of the European Association for the Study of Diabetes, Geneva, 28–30 September, 1977, and published in abstract form in Diabetologia13, 386, 1977.     

Inhibition of insulin and glucagon release from the perfused rat pancreas by cyproheptadine (Periactinol®, Nuran®)

Summary The tricyclic compound cyproheptadine (Periactinol®, Nuran®) inhibited glucose-induced insulin release from the perfused rat pancreas. Tolbutamide-stimulated insulin release was significantly reduced in the presence and completely suppressed in the absence of a substimulatory glucose concentration (5 mM). Arginine produced a slow rise of insulin release, which was completely abolished by cyproheptadine. Furthermore the biphasic glucagon release due to the stimulus was inhibited. Oxidation of ¹⁴C-glucose in isolated islets was unaltered in the presence of cyproheptadine, and pyruvate added to the perfusion medium failed to reverse the inhibitory effect on glucose induced insulin release, indicating that impaired glucose metabolism is not responsible for the inhibition. In addition, the inhibition remained unchanged when phentolamine was present, suggesting that the effect is not mediated by inhibitory adrenergic alpha receptors. Theophylline, in contrast, partly overcame the inhibition. When the calcium concentration of the medium was enhanced, the inhibitory effect of cyproheptadine was still visible, although the relative inhibition had become smaller. The results suggest that cyproheptadine blocks insulin release by affecting a fundamental step of the stimulus-secretion coupling common to peptide hormones. A participation of a calcium-antagonizing effect in the inhibition is discussed.The results were presented in part at the 11th Annual Meeting of the European Association for the Study of Diabetes, Munich, September 1975.     

Divergent effect of glucagon antibodies on arginine and glucose-stimulated insulin secretion in the rat

Summary The effects of glucose and arginine on insulin secretion in the presence of glucagon antibodies were investigated in rats in vivo. In contrast to controls, animals given glucagon antibodies showed an inhibition of arginine-stimulated (p < 0.001), but not glucose-stimulated, insulin secretion. That these effects were not due to incomplete neutralisation of endogenous glucagon is evidenced by the presence of large antibody excess throughout the duration of the experiments. Both the glucagonotropic effect of arginine (319 ± 60ng/l, p < 0.01) and the insulinotropic effect of exogenous glucagon (8.3 ± 0.8 g/l, p < 0.001) were demonstrable under our experimental conditions in the absence of exogenous glucagon antibodies. These observations suggest that different mechanisms are involved in the stimulation of insulin release by arginine and by glucose, and that glucagon may play an important physiological role in the mediation and regulation of insulin secretion by secretogogues, such as arginine.     

High concentration of somatostatin immunoreactivity in chicken pancreas

Summary Extracts of discrete lobes of chicken pancreas were assayed for somatostatin (SRIF), insulin, and glucagon immunoreactivity. The distribution of hormone concentration was correlated appropriately with the known distribution of A, B, and D-cells. Concentrations of all three hormones were highest in the splenic lobe. The glucagon content of the ventral and dorsal lobes was low. Evidence is presented that the SRIF-like material in the pancreatic extracts is very similar to synthetic cyclic SRIF; parallel immunoassay displacement curves and similar chromatographic elution profiles were obtained. The SRIF concentration in chicken pancreas is 21 times higher than that found in the rat. Chicken pancreas may provide a useful model for studies of somatostatin physiology.     

Difference in calcium dependency of insulin,glucagon and somatostatin secretion in response to glibenclamide in perfused rat pancreas

Summary The extracellular calcium requirements for insulin, glucagon and somatostatin release induced by 1 g/ml of glibenclamide have been compared in the perfused, isolated rat pancreas. In the absence of glucose, the drug evoked insulin release equally well at physiological (2.6 mmol/l) and low (0.25 mmol/l) levels of total calcium. In contrast, glibenclamide evoked somatostatin release at 2.6 but not at 0.25 mmol/l of calcium. At 2.6 mmol/l of calcium, glibenclamide evoked bimodal effects (stimulation followed by inhibition) on glucagon secretion. At 0.25 mmol/l of calcium, basal secretory rates of glucagon were elevated and a small stimulatory effect of glibenclamide was seen. Addition of 0.5 mmol/l of EGTA to media with low calcium concentrations uniformly abolished the A, B and D cell secretory responses to glibenclamide. The possible modulation of calcium dependency by a non-stimulatory concentration of glucose was tested by its addition at 3.3 mmol/l to the perfusion media. Glucose enhanced glibenclamide-induced insulin secretion, both at 0.25 and 2.6 mmol/l of calcium. However, at 0.25 mmol/l of calcium, the enhancing effect of glucose was more pronounced than at 2.6 mmol/l. At 2.6 mmol/l of calcium, glucose diminished the somatostatin and abolished the glucagon response to glibenclamide. At 0.25 mmol/l of calcium, glucose did not influence somatostatin release while the presence of the sugar diminished basal and glibenclamide-induced glucagon secretion. The present data confirm the requirement of extracellular calcium for A, B and D cell secretion, demonstrating different calcium dependencies for the cell types and indicate that this dependency can, in part, be modulated by glucose.