Regulation of calcium fluxes in rat pancreatic islets: Calcium extrusion by sodium-calcium countertransport

Summary The mechanisms by which glucose regulates calcium fluxes in pancreatic endocrine cells were investigated by monitoring the efflux of⁴⁵Ca from prelabeled and perifused rat pancreatic islets. In the absence of both extracellular calcium and glucose, partial or total removal of extracellular sodium decreases the efflux of⁴⁵Ca from prelabeled islets. Glucose also reduces the efflux of⁴⁵Ca from islets perifused in the absence of extracellular calcium. This inhibitory effect of glucose on⁴⁵Ca efflux is decreased by half when the extracellular concentration of sodium is lowered to 24mm. In the absence of extracellular calcium but presence of glucose, partial or even total removal of extracellular sodium fails to decrease the efflux of⁴⁵Ca. At normal extracellular calcium concentration (1mm) partial removal of extracellular sodium dramatically increases⁴⁵Ca efflux from pancreatic islets. This increase in⁴⁵Ca efflux is partially but not totally suppressed by either 16.7mm glucose or cobalt. It is totally suppressed by 4.4mm glucose or by the combination of 16.7mm glucose and cobalt. At normal extracellular calcium concentration, glucose initially reduces and subsequently increases⁴⁵Ca efflux. The initial fall is unaffected by tetrodotoxin but decreased by 50% at low extracellular sodium concentration (24mm). The present results suggest the existence in pancreatic endocrine cells of a glucose-sensitive process of sodium-calcium counter-transport. By inhibiting such a process, glucose may decrease the efflux of calcium from islet cells. The effect of glucose is not mediated by an increase in intracellular sodium concentration. It could contribute to the intracellular accumulation of calcium which is thought to trigger insulin release.This paper is the IVth in a series.     

Calcium antagonists and islet function: VII. Effect of calcium deprivation

Summary The role of extracellular Ca²⁺ in the regulation of islet function is investigated. Decreasing extracellular Ca²⁺ concentrations cause a dose-related inhibition of glucose-induced insulin release. Whereas the efflux of⁴⁵Ca from perifused islets is transiently increased on exposure to Ca²⁺-deprived media, it is unaffected by a partial lowering of the extracellular Ca²⁺ concentration. Under the latter condition, therefore, the observed reduction in the size of the islets' exchangeable calcium pool(s) appears to be due to reduced Ca²⁺ entry. The proper effect of glucose on Ca handling by the islets is apparently not affected by a lowering in the extracellular Ca²⁺ concentration. Nevertheless, in islets exposed to glucose and incubated in Ca²⁺-deprived media, glucose uptake and oxidation and lactate output are decreased, whereas the islet ATP level is increased, as if extracellular Ca²⁺ shortage were to affect not only the cellular pool of Ca regulating insulin release, but also energy-consuming processes possibly located at the cell membrane.     

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/1) 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.     

Activation,but not inhibition,by glucose of Ca2+-dependent K+ permeability in the rat pancreatic B-cell

The effect of glucose on the Ca²⁺-activated K⁺ permeability in pancreatic islet cells was investigated by measuring the rate of ⁸⁶Rb efflux, ⁴⁵Ca efflux and insulin release from perifused rat pancreatic islets exposed to step-wise increased in glucose concentration. When the glucose concentration was raised from intermediate (8.3 or 11.1 mM) to higher values, a rapid and sustained increase in ⁸⁶Rb outflow, ⁴⁵Ca outflow and insulin release was observed. Likewise, in the presence of 8.3 or 16.7 mM glucose, tolbutamide increased ⁸⁶Rb and ⁴⁵Ca efflux, as well as insulin release. In the two series of experiments, a tight correlation was found between the magnitude of the changes in ⁸⁶Rb and ⁴⁵Ca outflow, respectively. It is concluded that, at variance with current ideas, glucose does not inhibit the response to cytosolic Ca²⁺ of the Ca²⁺-sensitive modality of K⁺ extrusion. On the contrary, as a result of its effect upon Ca²⁺ handling, glucose stimulates the Ca²⁺-activated K⁺ permeability.     

Effect of the absence of bicarbonate upon intracellular pH and calcium fluxes in pancreatic islet cells

The removal of extracellular HCO⁻₃ together with a decrease in pCO₂, in order to maintain a normal extracellular pH, caused a sustained increase of intracellular pH in rat pancreatic islets. This increase was more marked in glucose-deprived than in glucose-stimulated islets, and was associated with a facilitation of ⁴⁵Ca efflux from the glucose-deprived islets. Such a facilitation was slightly reduced in the absence of extracellular Ca²⁺ and abolished at low extracellular Na⁺ concentration. It failed to occur in glucose-stimulated islets, whether in the presence or absence of extracellular Ca²⁺. The removal of HCO⁻₃ and decrease in the pCO₂ also reduced the magnitude of both the secondary rise in ⁴⁵Ca efflux and stimulation of insulin release normally evoked by an increase in glucose concentration. These findings suggest that changes in intracellular pH affect both the outflow of Ca²⁺ from islet cells as mediated by Na⁺-Ca²⁺ countertransport and the inflow of Ca²⁺ by gated Ca²⁺ channels. The experimental data are also compatible with the view that islet cells are equipped with an active process of bicarbonate-chloride exchange involved in the regulation of intracellular pH.     

Oxidative stress increases potassium efflux from pancreatic islets by depletion of intracellular calcium stores

Oxidative stress to B-cells is thought to be of relevance in declining B-cell function and in the process of B-cell destruction. In other tissues including heart, brain and liver, oxidative stress has been shown to elevate the intracellular free calcium concentration and to provoke potassium efflux. We studied the effect of oxidative stress on Ca²⁺ and K⁺ (Rb⁺) outflow from pancreatic islets using the thiol oxidants DIP and BuOOH. Both compounds reversibly increased ⁸⁶Rb⁺ efflux in the presence of 3 and 16.7 mmol/l glucose. Stimulation of ⁸⁶Rb⁺ efflux was also evident in the absence of calcium. DIP evoked release of ⁴⁵Ca²⁺ from the pancreatic islets both in the presence or absence of extracellular calcium. Employing inhibitors of the calcium-activated potassium channel (K_Ca) and the high conductance K⁺-channel (BK_Ca), the effect of DIP on ⁸⁶Rb⁺ efflux was slightly diminished. Tolbutamide had no effect on ⁸⁶Rb⁺ efflux in the presence of DIP. On the other hand thapsigargin, a blocker of the Ca²⁺-ATPase of the endoplasmic reticulum, completely suppressed the DIP-mediated ⁸⁶Rb⁺ outflow. The data suggest that thiol oxidant-induced potassium efflux from pancreatic islets is mainly mediated through liberation of intracellular calcium and subsequent stimulation of calcium-activated potassium efflux.     

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.     

Effects of maitotoxin on ionic and secretory events in rat pancreatic islets

Maitotoxin (MTX) provoked a dose-dependent increase in both 45Ca efflux and insulin release from rat pancreatic islets perifused in the presence or absence of glucose, provided that Ca2+ was present in the perifusate. The stimulatory effect of MTX on 45Ca outflow was enhanced by CGP 28392. The toxin did not reduce 86Rb outflow and 86Rb inflow. It is suggested that the secretory response to MTX is mediated by direct activation of voltage-dependent Ca2+ channels.     

Na+−K+ pump activity and the glucose-stimulated Ca2+-sensitive K+ permeability in the pancreatic B-cell

A rise in the extracellular concentration of glucose from an intermediate to a high value changes the burst pattern of electrical activity of the pancreatic B-cell into a continuous firing, and yet activates the B-cell Ca2+-sensitive K+ permeability. The hypothesis that glucose exerts such effects by inhibiting the Na+, K+-ATPase was investigated. Ouabain (1 mM) mimicked the effect of 16.7 mM glucose in stimulating 86Rb, 45Ca outflow and insulin release from perifused rat pancreatic islets first exposed to 8.3 mM glucose. The stimulation by ouabain of 86Rb outflow was reduced in the absence of extracellular Ca2+ and almost completely abolished in the presence of quinine, and inhibitor of the Ca2+-sensitive K+ permeability. In the presence of ouabain, a rise in the glucose concentration from 8.3 to 16.7 mM failed to stimulate 86Rb outflow. However, the rise in the glucose concentration failed to inhibit 86Rb influx in islet cells, while ouabain dramatically reduced 86Rb influx whether in the presence of 8.3 or 16.7 mM glucose. These findings do not suggest that inhibition of the B-cell Na+, K+-ATPase represents the mechanism by which glucose in high concentration stimulates 86Rb outflow and induces continuous electrical activity in the B-cell.     

Effects of monensin on metabolism of isolated rat islets of Langerhans.

Monensin, a univalent ionophore, is a carboxylic acid produced by Streptomyces cinnamonensis. It will complex various alkali-metal ions, but most readily binds Na+. Because of interest in the possible role of Na+ in the regulation of insulin secretion, we examined its effects on several aspects of the metabolism of isolated rat islets of Langerhans. The ionophore inhibited glucose-stimulated insulin release in a concentration-dependent manner, completely inhibiting secretion evoked by 20 mM-glucose at concentrations as low as 0.1 microM in static incubations. In perifusion experiments, both phases of insulin release were equally affected. Monensin (0.1 microM) had no significant effect on glucose oxidation as measured by the generation of 14CO2 from [14C]glucose. Monensin increased the rate of 22Na+ efflux from preloaded islets and net 22Na+ uptake over 30 min, in the absence of changes in islet volume or extracellular space. The ionophore increased the Rb+/K+ permeability of islet cells, as shown by its inhibition of 86Rb+ retention and stimulation of 86Rb+ efflux. At 0.1 microM, monensin abolished glucose-stimulated 45Ca2+ uptake by islets during 5 min incubations, and stimulated 45Ca2+ efflux from preloaded islets perifused with Ca2+-free medium, even in the complete absence of extracellular Na+. Studies of the uptake of 14C-labelled 5,5-dimethyloxazolidine-2,4-dione showed that 0.1 microM-monensin increased net intracellular pH from 7.05 to 7.13. 7 Monensin has widespread, complex, effects on the secretory responses and ion handling by the B cells, which are difficult to interpret in terms solely of actions as a Na+ ionophore.     

The stimulus-secretion coupling of glucose-induced insulin release: effects of valinomycin upon pancreatic islet function.

In isolated rat pancreatic islets, valinomycin (0.01 to 1.0 μm) caused a dose-related facilitation of ⁸⁶Rb⁺ outflow and a dose-related inhibition of the glucose-induced changes in both outflow and net uptake of ⁸⁶Rb⁺. At high concentrations (0.1–1.0 μm), the ionophore also inhibited the oxidation of glucose and endogenous nutrients, decreased the adenylate charge, and lowered the concentration of reduced pyridine nucleotides in the islet cells. However, as little at 1.0 to 10.0 nm valinomycin caused anomalies in the handling of ⁴⁵Ca²⁺ (suppression of the early inhibitory effect of glucose upon ⁴⁵Ca²⁺ efflux, and reduction in the amount of ⁴⁵Ca²⁺ recovered in the islets after an extensive washing procedure) and inhibition of insulin release. Moreover, when the effect of glucose upon K⁺ conductance was abolished by high concentrations of valinomycin (0.1–1.0 μm), the glucose-induced secondary rise in ⁴⁵Ca²⁺ efflux was still observed. These findings suggest that the effects of glucose upon ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ handling, respectively, although normally concomitant with one another, can be dissociated, in part at least, from one another. It is concluded that the glucose-induced reduction in K⁺ outflow may be unnecessary for the sugar to cause a partial remodeling of Ca²⁺ fluxes in the islet cells.     

Na+-H+ exchange in the process of glucose-induced insulin release from the pancreatic B-cell. Effects of amiloride on 86Rb, 45Ca fluxes and insulin release

The effect of amiloride, an inhibitor of Na+-H+ exchange, on intracellular pH (pHi), 86Rb outflow, 45Ca outflow and insulin release from pancreatic rat islets was examined. In the 0.1-1 mM range, amiloride transiently reduced pHi of glucose-deprived islets and allowed glucose to induce a sustained decrease in pHi of the islet cells. Amiloride reproduced the effect of glucose to decrease 86Rb and 45Ca outflow. In the presence of glucose (5.6 mM or more), amiloride (100 microM) acted synergistically with the sugar to reduce K+ outflow, and to stimulate 40Ca inflow and insulin release from perifused islets. These results add strong support to the view that the generation of protons through the metabolism of glucose represents an important step in the process of glucose-induced release. The stimulation by glucose of Na+-H+ exchange apparently masks and even overcomes the glucose-induced decrease in pHi otherwise expected from the increase in catabolic fluxes.     

Effect of temperature upon potassium-stimulated insulin release and calcium entry in mouse and rat islets

The effect of cooling to 27 degrees C was studied in islets of Langerhans exposed to 5 and 50 mM potassium in the absence of glucose. Membrane potential and insulin release were measured simultaneously from microdissected mouse islets while 45Ca outflow and insulin release were measured from collagenase-isolated rat islets. Cooling inhibited potassium-induced insulin release in both preparations. However, calcium entry estimated from electrical records and from 45Ca outflow experiments was only slightly affected by decreasing the temperature to 27 degrees C. It is concluded that the inhibition of insulin release caused by cooling to 27 degrees C can, within limits, be dissociated from calcium influx.     

Alterations in pancreatic islet phosphate content during secretory stimulation with glucose

Isolated rat pancreatic islets were perifused and analyzed for phosphate content immediately following the transient increase in the efflux of orthophosphate which occurs when insulin secretion is stimulated by glucose. In some instances, islets were perifused directly following isolation to minimize preparative delay; in others, islets were prelabeled during incubation with [³²P]orthophosphate for 90 min prior to perifusion. In both experimental situations, total islet phosphate content declined 40–50% following exposure to stimulating concentrations of glucose and initiation of enhanced insulin release. In the experiments with prelabeled islets, tissue content of [³²P]orthophosphate fell to a similar extent so that the specific radioactivity of islet orthophosphate was unaffected. Inhibition of heightened insulin release with Ni²⁺ did not modify the decrements in total or radioactive tissue orthophosphate, thus indicating that these responses to islet stimulation reflect events which are proximal to activated exocytosis. Simultaneous analyses for tissue ATP and ADP demonstrated that the efflux in orthophosphate and reduction in tissue orthophosphate content were not mediated via net changes in islet adenine nucleotides. The observations represent the first documentation that a net reduction of tissue inorganic phosphate is one of the early components of stimulus-secretion coupling in isolated pancreatic islets.     

Dissociation between intracellular calcium mobilization and insulin secretion in isolated rat islets of Langerhans

The neuropeptide bombesin provoked a dose-dependent stimulation of 45Ca2+ efflux from pre-loaded islets of Langerhans. This response occurred rapidly, was not sustained and did not depend on the presence of extracellular calcium, suggesting that it resulted from the mobilization of intracellular calcium stores. Under conditions when large increases in 45Ca2+ efflux were observed, bombesin completely failed to stimulate the rate of insulin secretion. Similar results were also obtained with the muscarinic cholinergic agonist, carbachol. The data suggest that the release of calcium from intracellular pools is not sufficient to induce an increase in insulin secretion in normal islet cells.     

32P, 86Rb+ and 45Ca2+ handling by tumoral insulin-secreting cells (RINm5F line)

In perifused tumoral islet cells (RINm5F line), which were prelabelled with either [32P]orthophosphate, 86Rb+ or 45Ca2+, the administration of D-glucose (1.4, 2.8 or 16.7 mM) increased the efflux of 32P, decreased the outflow of 86Rb, increased slightly the efflux of 45Ca from cells perifused in the presence of Ca2+, and decreased modestly the outflow of 45Ca from cells perifused in the absence of Ca2+. D-glucose also stimulated the net uptake of 45Ca2+. When Ba2+ (2 mM) was used, in the absence of Ca2+, instead of D-glucose as an insulin secretagogue, the efflux of 32P was little affected, but the outflow of 45Ca was dramatically increased. These changes are qualitatively similar to those occurring in normal islet cells. Nevertheless, the ionic response to D-glucose appeared, as a rule, less marked in tumoral than normal islet cells. Moreover, the concentration-response relationship was shifted to a lower range of hexose concentrations in the RINm5F cells.     

Effects of glucose on 45Ca2+ outflow, cytosolic Ca2+ concentration and insulin release from freshly isolated and cultured adult rat islets

The present study aimed at comparing the effects of glucose on ionic and secretory events in freshly isolated and 5-7 day cultured rat pancreatic islets. The capacity of glucose to provoke insulin release was severely reduced in islets maintained in culture. Whether in freshly isolated or cultured islets, glucose provoked a marked and sustained decrease in 45Ca2+ outflow from islets deprived of extracellular Ca2+. In the presence of extracellular Ca2+ throughout, the magnitude of the glucose-induced secondary rise in 45Ca2+ outflow was reduced in cultured islets. Glucose provoked a weaker increase in [Ca2+]i in islet cells obtained from cultured islets than in islet cells dissociated from freshly isolated pancreatic islets. On the other hand, the stimulatory effect of carbamylcholine on 45Ca2+ outflow was unaffected by tissue culture. Lastly, in islet cells obtained from cultured islets, the increase in [Ca2+]i evoked by K+ depolarization averaged half of that observed in control experiments. These results indicate that the reduced secretory potential of glucose in cultured pancreatic islets can be ascribed to the inability of the nutrient secretagogue to provoke a suitable increase in Ca2+ influx.     

The coupling of metabolic to secretory events in pancreatic islets: does hexose transport affect cationic fluxes?

Poorly metabolized hexoses, such as 3-O-methyl-D-glucose, 2-deoxy-D-glucose and D-galactose failed to reproduce the inhibition of 86Rb outflow, the early inhibition and secondary rise in 45Ca efflux and the stimulation of insulin release evoked by D-glucose in perifused rat islets. Insulin release induced by either D-glucose or 2-ketoisocaproate was also unaffected by 3-O-methyl-D-glucose. It is concluded that hexose transport in islet cells does not represent in itself a significant determinant of the cationic and secretory response to D-glucose.     

Distinct effects of acetylcholine and glucose on 45calcium and 86rubidium efflux from mouse pancreatic islets

The similarities between the effects of acetylcholine and glucose on phospholipid metabolism in pancreatic islet cells prompted the comparison of their effects on ionic fluxes. Acetylcholine (1 μM) consistently increased ⁴⁵Ca²⁺ efflux from mouse islets, whereas glucose increased it in the presence, but decreased it in the absence of extracellular Ca²⁺. Acetylcholine consistently accelerated ⁸⁶Rb⁺ efflux, and this effect was augmented by Ca²⁺ omission. On the other hand, glucose markedly inhibited ⁸⁶Rb⁺ efflux, except when its concentration was raised from 10 to 15 mM in the presence of Ca²⁺. Unlike their effects on phospholipid metabolism, the ionic effects of the two insulin-secretagogues are thus very different.     

Lactation-induced changes in calcium handling by rat pancreatic islets

Glucose-stimulated insulin release occurred at a lower rate in pancreatic islets removed from lactating than non-lactating rats. This defect was corrected in the presence of either gliclazide or a calcium-agonist. With both agents present, insulin release from islets of lactating rats was greater. When islets were prelabelled with 45calcium, gliclazide stimulated to the same extent 45Ca outflow in islets from lactating and non-lactating rats, respectively. However, when the islets were prelabelled with 45Ca in the presence of gliclazide, the administration of Ba2+ increased effluent radioactivity more markedly in islets from non-lactating than lactating rats. This suggests that lactation favours, in gliclazide-stimulated islets, the sequestration of 45Ca in non-labile subcellular pools. When D-glucose was used instead of Ba2+, the greater lability of 45Ca in islets from non-lactating animals was apparently masked by a lesser efficiency in the metabolism and cationic effects of D-glucose in the non-lactating rats. The calcium-ionophoretic effect of islet extracts was higher in lactating than non-lactating rats. These results support the view that a depletion of endogenous calcium stores accounts, in part at least, for the decreased insulin secretory responsiveness to D-glucose in lactation, since the latter apparently favours the function of those systems involved in either the entry of calcium into or its sequestration within the islet cells.