Effects of Cl- deficiency on the membrane potential in mouse pancreatic beta-cells.

The membrane potential of mouse pancreatic beta-cells was measured with microelectrodes. In the resting cell (3 mM D-glucose), the membrane potential was -63 +/- 3 mV (mean +/- S.E. for four experiments). In the presence of 3 mM D-glucose, total Cl- substitution by isethionate induced a depolarization by 3-4 mV, and readmission of Cl- induced a hyperpolarization by 3-5 mV. At 10 mM glucose, reduction of Cl- to 12 mM by substituting isethionate for Cl- reversibly shifted the repolarization potential by 6-9 mV in the positive direction and stimulated the burst activity during the initial 2-3 min by increasing the fraction of plateau phase. This was followed by a gradual inhibition of electrical activity, including decrease in fraction of plateau phase and slow wave amplitude. Total substitution of Cl- by isethionate or methyl sulphate reversibly shifted the repolarization potential by 3-4 mV in the positive direction and rapidly inhibited the electrical burst pattern without any initial stimulation. Glucose-induced (10 mM) insulin release (15 min) and 45Ca2+ uptake (3 min) were strongly inhibited by reducing the Cl- concentration to 10 mM (isethionate as substitute) and were further inhibited by further reduction of the Cl- concentration. It is suggested that beta-cells are equipped with on electrogenic Cl- flux, which can affect the burst pattern of electrical activity. The inhibitory effects of Cl- substitution may be explained by an influence of Cl- on the voltage-controlled Ca2+ channels.

9-Aminoacridine- and tetraethylammonium-induced reduction of the potassium permeability in pancreatic B-cells. Effects on insulin release and electrical properties.

The effects of 9-aminoacridine and tetraethylammonium on insulin release and rubidium efflux from perifused rat islets were investigated and correlated with their effects on the electrical properties of mouse B cells studied with microelectrode techniques. 9-Aminoacridine (0.05--1 mmol/l) and tetraethylammonium (2--40 mmol/l) produced a dose-dependent, reversible potentiation of glucose-stimulated insulin release. This effect was rapid, affected both phases of secretion and was maximum in the presence of 6 mmol/l glucose, but no longer significant at 20 mmol/l glucose. It was unaltered by atropine or propanolol, and abolished by mannoheptulose or omission of extracellular calcium. 9-Aminoacridine, but not tetraethylammonium, also induced insulin release in the absence of glucose stimulation. Neither drug modified glucose metabolism in islet cells and only 9-aminoacridine increased 45Ca2+ uptake. In the presence of 0, 3 or 6 mmol/l glucose, but no longer at 20 mmol/l glucose, 9-aminoacridine and tetraethylammonium reduced the rate of 86Rb+ efflux from the islets. Both drugs also slightly reduced 86Rb+ uptake by islet cells. In the presence of 11 mmol/l glucose, 9-aminoacridine reduced the amplitude and the duration of the polarization phases between the bursts of electrical activity; concomitantly these periods of spike activity were markedly prolonged. At lower glucose concentrations (3 or 7 mmol/l), 9-aminoacridine progressively depolarized B cells and induced electrical activity in otherwise silent cells. Tetraethylammonium also suppressed the repolarization phases between the bursts of spikes in the presence of a stimulating concentration of glucose. At low glucose, tetraethylammonium produced only a limited and not maintained depolarization. These results show that a reduction of the potassium permeability in pancreatic B cells potentiates the insulin-releasing effect of glucose and may even stimulate secretion. They also suggest that the initial depolarizing effect of glucose is due to a reduction of the potassium permeability, whereas the repolarization at the end of each burst of electrical activity is mediated, at least in part, by an increase in the potassium permeability of B cells.

The effects of cesium chloride on insulin release, ionic fluxes and membrane potential in pancreatic B-cells.

Cs+ decreases K+ permeability in nerve and muscle cells. Its effects on the pancreatic B-cell function were studied with mouse islets. In the presence of 3 mM glucose, Cs+ substitution for K+ steadily inhibited 86Rb+ efflux and hyperpolarized the B-cell membrane. Addition of Cs+ to a K+-medium also inhibited 86Rb+ efflux, but depolarized the B-cell membrane. None of these changes altered insulin release. Substitution of Cs+ for K+ in a medium containing 10 mM glucose caused a Ca2+-dependent stimulation of insulin release and 45Ca2+ efflux, produced an initial fall and a secondary rise in 86Rb+ efflux and augmented the electrical activity in B-cells. Reintroduction of K+ to the medium was followed by a marked and transient inhibition of insulin release, that was blocked by ouabain and accompanied by an inhibition of 45Ca2+ and 86Rb+ efflux and by a hyperpolarization of the B-cell membrane. Addition of Cs+ to a K+ medium containing 10 mM glucose stimulated insulin release, 45Ca2+ efflux and 86Rb+ efflux. It also increased the electrical activity in B-cells. In the absence of Ca2+, however, Cs+ addition decreased the rate of 86Rb+ efflux. The effects of Cs+ on the B-cell function may be explained by its ability to decrease K+ permeability of the plasma membrane, by its inability to activate the sodium pump, and by a third unidentified effect likely brought about by the accumulation of intracellular Cs+.

The ionic, electrical, and secretory effects of endogenous cyclic adenosine monophosphate in mouse pancreatic B cells: studies with forskolin.

Forskolin, an activator of adenylate cyclase, was used to study the effects of endogenous cAMP on the stimulus-secretion coupling in mouse pancreatic B cells. Forskolin produced a rapid, dose-dependent (0.05-50 microM) increase in islet cAMP, which was not influenced by the prevailing concentration of glucose and did not require extracellular Ca2+. At a nonstimulatory concentration of glucose (3 mM), a high concentration of forskolin (20 microM) barely doubled basal insulin release, marginally decreased 86Rb+ efflux from the islets, was without effect on the B cell membrane potential, and did not affect 45Ca2+ uptake (5 min). High concentrations of endogenously formed cAMP thus failed to mimic these early steps of the B cell response to glucose and other stimuli. At a threshold concentration of the sugar (7 mM), 5 microM forskolin slightly depolarized the B cell membrane, induced electrical activity (slow waves and spikes), stimulated 45Ca2+ uptake, and triggered insulin release. At a stimulatory concentration of glucose (10 mM), forskolin potentiated insulin release; half maximal and maximal effects were observed at 1 and 20 microM, respectively. Forskolin also increased the rate of 45Ca2+ and 86Rb+ efflux from islet cells, augmented 45Ca2+ uptake, and potentiated the electrical activity triggered by glucose in B cells. Its effects on insulin release, 45Ca2+ fluxes, and electrical activity were inhibited by omission of extracellular Ca2+ and/or Ca channels blockers. At a high concentration of glucose (25 mM), forskolin augmented the amplitude and the duration of the spikes occurring in the depolarized B cell membrane, slightly increased 45Ca2+ uptake, and potentiated insulin release. At threshold or stimulatory concentrations of glucose, endogenously formed cAMP can thus induce or enhance the Ca2+-dependent events normally triggered by the sugar. It is suggested that, besides its action on intracellular Ca stores, cAMP facilitates Ca2+ influx in B cells, by modulating the gating properties of the Ca channels.

Forskolin, an activator of adenylate cyclase, increases CA2+-dependent electrical activity induced by glucose in mouse pancreatic B cells.

The effects of forskolin, an activator of adenylate cyclase, on the membrane potential of pancreatic B cells have been studied with microelectrodes. Forskolin (5 microM) did not affect the stable resting membrane potential (3 mM glucose). In the presence of 10 mM glucose, forskolin lengthened the slow waves with superimposed spikes and shortened the polarized intervals between them. This caused a marked increase in the frequency of the slow waves and doubled the fraction of time spent at a depolarized level, with spike activity. The frequency of the spikes was not changed. The effects of forskolin were of rapid onset, but were only slowly and partially reversible; they were completely blocked when Ca2+ influx was prevented by cobalt. The results show that forskolin increases electrical events underlain by Ca inward currents and suggest that, besides its action on intracellular Ca stores, cyclic AMP could also modulate the permeability of Ca channels in the plasma membrane of B cells.

Opposite effects of tolbutamide and diazoxide on 86Rb+ fluxes and membrane potential in pancreatic B cells.

The effects of tolbutamide and diazoxide on 86Rb+ fluxes, 45Ca2+ uptake, insulin release and B cell membrane potential have been studied in rat or mouse islets. In the presence of 3 mM glucose, tolbutamide rapidly and reversibly decreased Rb+ efflux from perifused islets and depolarised B cells. The effect on Rb+ efflux was paradoxically more marked with 20 than 100 micrograms/ml tolbutamide, at least in the presence of extracellular calcium. Addition of tolbutamide to a medium containing 6 mM glucose and calcium increased Rb+ efflux transiently with 20 micrograms/ml and permanently with 100 micrograms/ml. The drug also inhibited Rb+ influx in islet cells, but had little effect on Rb+ net uptake. Diazoxide rapidly, steadily and reversibly increased Rb+ efflux in a dose-dependent manner (20-100 micrograms/ml). When 20 micrograms/ml tolbutamide and diazoxide were combined in the presence of 3 mM glucose, only a slight decrease in Rb+ efflux was observed. The depolarisation of B cells normally produced by tolbutamide was markedly reduced and the electrical activity completely suppressed by diazoxide. In the presence of 10mM glucose, diazoxide increased Rb+ efflux from the islets and hyperpolarised B cells. Tolbutamide, tetraethylammonium and quinine reversed the increase in Rb+ efflux the inhibition of Ca2+ uptake and the suppression of insulin release produced by diazoxide. Tolbutamide rapidly reversed the hyperpolarisation and restored electrical activity. It is suggested that the stimulation and inhibition of insulin release by tolbutamide and diazoxide are due to their respective ability to decrease and to increase the K permeability of the B cell membrane. This change in K permeability leads either to depolarisation and stimulation of Ca2+ influx or to hyperpolarisation and inhibition of Ca2+ influx.

Sparteine increases insulin release by decreasing the K+ permeability of the B-cell membrane.

The effects of sparteine on the pancreatic B-cell function have been studied with mouse islets. In the presence of a non-stimulatory concentration of glucose (3 mM), sparteine (0.2-1 mM) decreased the rate of 86Rb+ efflux from islet cells, depolarized the B-cell membrane, induced a glucose-like electrical activity and stimulated insulin release. This increase in release was observed over a large range of glucose concentrations (3-20 mM), and was most marked in the presence of 10 mM glucose. At this concentration of glucose, the effect of sparteine was already detected with 0.02 mM and was maximal with 0.5 mM. Higher concentrations of sparteine only had a transient effect on insulin release. In the presence of 10 mM glucose, 0.2 mM sparteine decreased 86Rb+ efflux and increased 45Ca2+ efflux from islet cells. The effect on 86Rb+ efflux was only transient in the presence of extracellular calcium, whereas the effect on 45Ca2+ efflux required the presence of extracellular calcium. The electrical activity induced by glucose in B-cells was augmented by sparteine which, at a concentration of 0.5 mM, produced a persistent depolarization with continuous spike activity. The potentiation of insulin release by sparteine was not reversible, but was inhibited by adrenaline and completely blocked by omission of extracellular calcium. Sparteine reversed the increase in 86Rb+ efflux and the decrease in insulin release caused by diazoxide. These results show that sparteine increases insulin release by reducing the K+-permeability of the B-cell membrane.

The non-sulfonylurea moiety of gliquidone mimics the effects of the parent molecule on pancreatic B-cells.

Compound UL-DF 9 corresponds to the non-sulfonylurea moiety of gliquidone, a hypoglycaemic sulfonylurea of the second generation. Its effects on the B-cell function were studied in vitro with mouse islets. In the presence of a non-stimulatory concentration of glucose (3 mM), UL-DF 9 decreased 86Rb+ efflux and accelerated 45Ca2+ efflux from islet cells, depolarized the B-cell membrane and induced an electrical activity similar to that triggered by stimulatory concentrations of glucose, and increased insulin release. The changes in 45Ca2+ efflux and insulin release, but not the inhibition of 86Rb+ efflux, were abolished in the absence of Ca2+. In the presence of 10 mM glucose, UL-DF 9 increased 86Rb+ and 45Ca2+ efflux from the islets, augmented the electrical activity in B-cells, and potentiated insulin release. These changes were suppressed by omission of extracellular Ca2+. Qualitatively similar effects were produced by lower concentrations of gliquidone itself. The data suggest that UL-DF 9 and gliquidone decrease the K+ permeability of the B-cell membrane, thereby causing a depolarization which leads to activation of voltage-dependent Ca channels and Ca2+ influx, and thus eventually increase insulin release. Hypoglycaemic sulfonylureas of the second generation therefore seem to contain a second chemical group that interacts with K channels of B-cells as does the sulfonylurea group itself.

Electrical characteristics of the beta-cells in pancreatic islets.

1. The electrical activity of beta-cells is characterized by a regular burst pattern. 2. The bursts occur in a potential range which is not dependent on external glucose concentration. However, there is a clear dose-response relationship between spike frequency or plateau duration and the glucose concentration. 3. There is a striking biphasic pattern of the electrical activity in response to a sudden and sustained stimulation with glucose. 4. These findings agree well with corresponding investigations on insulin release and strongly support the view that insulin release and electrical activity are directly correlated. 5. Concerning the mechanism of the electrical activity, there is evidence that an electrogenic pump plays an important role in the regulation of the burst pattern and presumably in the regulation of insulin release.

Ionic mechanisms of the glucose-induced membrane potential changes in B-cells.

The ionic basis of the glucose-induced membrane potential changes in pancreatic B-cells was investigated. The results suggest that the initial depolarization of the membrane in response to a stimulation with glucose is due to a decrease of the K permeability. This depolarization seems to open a voltage-dependent Ca-channel and thereby an additional depolarization, the depolarization phase of the slow waves, is initiated. Insulin release is then triggered by the entering Ca ions. The fast spike activity may be a consequence of the exocytotic process. The polarization phase of the slow waves seems to be caused by the activity of an electrogenic Na-K-pump and a calcium-dependent increase of the K permeability. The activity of the Na-pump is considered to be regulated by the intracellular Na concentration.

Cyclic adenosine monophosphate differently affects the response of mouse pancreatic beta-cells to various amino acids.

1. The membrane potential of mouse beta-cells was measured in parallel with 86Rb+ efflux and insulin release from mouse islets during stimulation by three types of amino acids and modulation of their effects by glucose and cyclic adenosine monophosphate (cyclic AMP) (forskolin being used to activate the adenylate cyclase). 2. In the absence of glucose, alanine and arginine accelerated 86Rb+ efflux, whereas leucine decreased it. They all depolarized the beta-cell membrane and slightly increased insulin release. Forskolin had little effect on 86Rb+ efflux, consistently potentiated insulin release but induced electrical activity only in the presence of leucine. 3. The effects of the three amino acids on 86Rb+ efflux and beta-cell membrane potential were not qualitatively altered by a non-stimulatory concentration of glucose (3 mM). However, the release of insulin induced by leucine alone or with forskolin was markedly amplified, in contrast to that of alanine or arginine, which was inhibited. 4. In the presence of a threshold concentration of glucose (7 mM), the three amino acids accelerated 86Rb+ efflux and depolarized the beta-cell membrane. With alanine and arginine, spike activity was transiently observed and coincided with a short-lived increase in insulin release. With leucine, slow waves with superimposed bursts of spikes occurred and were accompanied by a sustained release of insulin. Forskolin alone also triggered slow waves and bursts of spikes, and increased insulin release. Both effects were larger in the presence of arginine, but not in the presence of alanine. Forskolin considerably increased the electrical and secretory effects of leucine. 5. A higher concentration of glucose (10 mM) induced slow waves with bursts of spikes in all cells and stimulated insulin release. Alanine, arginine and leucine increased 86Rb+ efflux, electrical activity and insulin release. However, the changes produced by the three amino acids displayed different time course, amplitude and characteristics. Forskolin potentiated insulin release and electrical activity induced by glucose alone. These effects were not augmented by alanine, but markedly amplified by arginine or leucine. 6. Several conclusions can be drawn from this study. The three types of amino acids depolarize the beta-cell membrane by different mechanisms and produce distinct patterns of electrical activity. Slow waves with bursts of spikes occur only if a decrease in K+ permeability contributes to the depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

The kinetics of electrical activity of beta cells in response to a "square wave" stimulation with glucose or glibenclamide.

The effect of a "square wave" stimulation with glucose or glibenclamide on the electrical activity of beta-cells has been studied with microelectrode techniques in isolated perifused mouse islets. While glucose evoked a burst activity with periodic oscillations of the membrane potential between two levels, glibenclamide produced a constant depolarization with a continuous spike activity. In both cases the time course of activity was biphasic and resembled closely the corresponding patterns of insulin release. The effect of glibenclamide on the membrane was irreversible. The results provide further evidence for a direct correlation between insulin release and electrical activity. Furthermore, it is suggested that the membrane potential is, at least in part, involved in the mechanism of biphasic insulin release.

Effects of amino acids on membrane potential and 86Rb+ fluxes in pancreatic beta-cells.

The membrane potential of beta-cells was studied with microelectrodes in mouse islets and their potassium permeability was evaluated by measuring 86Rb+ fluxes in rat islets. In the absence of glucose, L-leucine, its metabolite ketoisocaproate, and its nonmetabolized analogue 2-aminonorbornane-2-carboxylic acid (BCH) depolarized beta-cells and triggered bursts of electrical activity like glucose. The effect of leucine was weak, but was potentiated by a low concentration of glucose or by theophylline; the effect of ketoisocaproate was stronger and faster than that of an equimolar concentration of glucose. Arginine alone produced only a fast depolarization of beta-cells, insufficient to trigger electrical activity. Leucine and arginine potentiated the activity induced by glucose. In a glucose-free medium, alanine only slightly depolarized beta-cells, whereas isoleucine and phenylalanine had no effect. Leucine, ketoisocaproate, and BCH reversibly decreased 86Rb+ efflux from islets perifused in the absence of glucose and increased 86Rb+ uptake. By contrast, both in the absence or presence of glucose, arginine increased 86Rb+ efflux and decreased 86Rb+ uptake. It is proposed that leucine, ketoisocaproate, and BCH, as glucose, depolarize beta-cells by decreasing their potassium permeability, whereas arginine acts differently. The appearance of bursts of electrical activity with secretagogues unrelated to glucose suggests that they reflect an intrinsic property of the beta-cell membrane.

The electrogenic sodium-potassium pump of mouse pancreatic B-cells.

1. The contribution of the sodium pump to the membrane potential of mouse pancreatic B-cells was studied with micro-electrodes.2. In 0 or 3 mM-glucose, ouabain rapidly (within 2 min) depolarized the B-cell membrane by an average of 7 mV, whereas K omission hyperpolarized it markedly.3. In 6 or 7 mM-glucose, ouabain still produced depolarization, but K omission had no consistent effect. Both induced electrical activity in certain cells.4. In 10 mM-glucose, withdrawal of ouabain or K re-introduction caused a transient hyperpolarization with suppression of electrical activity. Duration and amplitude of the hyperpolarization increased with the time of pump blockade and with the concentration of ouabain.5. The hyperpolarization following K re-admission was abolished by ouabain and that following ouabain withdrawal was prevented by K omission. Re-admission of various K concentrations showed that the hyperpolarization was not due to depletion of K just outside of the membrane.6. In 10 mM-glucose, the membrane potential of B-cells exhibited repetitive slow waves with bursts of spikes on the plateau. These electrical events were modified by ouabain in a dose-dependent manner. The frequency of the slow waves augmented markedly because of an increase in the slope of the pre-potential and a shortening of the intervals; the slope of their repolarization phase decreased, but their duration was not changed.7. Omission of K increased the slope of the pre-potential and the frequency of the slow waves. It also accelerated their repolarization phase and reduced their duration, likely because of the increase in driving force for K efflux. Increasing K concentration to 8 mM slowed the repolarization phase and lengthened the slow waves without changing their frequency.8. Even when K permeability of the B-cell membrane was increased by high extracellular Ca, ouabain and K omission augmented the frequency of the slow waves.9. In 0 or 10 mM-glucose, ouabain increased (86)Rb(+) efflux from perifused islets, whereas K omission decreased it. In 10 mM-glucose, a marked decrease in (86)Rb(+) efflux accompanied ouabain withdrawal and K re-introduction. The hyperpolarization is thus not due to an increase in K permeability.10. It is concluded that, in pancreatic B-cells, the sodium pump is truly electrogenic, contributes to the resting potential and modulates the slow waves of membrane potential induced by glucose. Rapid changes in insulin release occurring upon inhibition or activation of the sodium pump may thus be due to the changes in B-cell membrane potential.

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells.

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.