Significance of Ca2+, Rb+ fluxes, of cAMP and cGMP for the CCK8-modulated insulin release

In rat pancreatic islets the effects of cholecystokinin-8 (CCK8) on glucose-mediated insulin release, 45Ca2+ net uptake, 45Ca2+ efflux, 86Rb+ efflux, cAMP- and cGMP levels were studied. In the presence of a substimulatory glucose concentration (3 mM) CCK8 concentrations of up to 1 microM had no effect on insulin release, but CCK8 at 10 nM potentiated the stimulatory effect of glucose (11.1 mM). 10 nM CCK8 enhanced glucose-stimulated 45Ca2+ net uptake but was ineffective at substimulatory glucose levels. CCK8 had no effect on cAMP and cGMP levels in the presence of 11.1 mM glucose, CCK8 increased 86Rb+ (a measure of K+) in the presence of both 3 and 11.1 mM glucose. This effect was abolished when Ca2+ was omitted from the perifusion medium. CCK8 did not alter glucose (11.1 mM)-stimulated 45Ca2+ efflux rate. These data indicate that (1) CCK8 potentiates glucose-stimulated insulin secretion possibly via an effect on Ca2+ uptake, 2) by affecting Ca2+ uptake, CCK8 enhances K+ efflux, and 3) CCK8 does not mediate its effect via cAMP or cGMP. With respect to 86Rb+ efflux the mechanism of CCK8 action appears to be different from that of glucose. When the mechanism of CCK action on islets is compared with that on exocrine pancreas (data from others) there are similarities (importance of Ca2+ uptake and non-importance of cAMP and cGMP).     

The coupling of metabolic to secretory events in pancreatic islets. The possible role of glutathione reductase

The participation of glutathione reductase in the process of nutrient-stimulated insulin release was investigated in rat pancreatic islets exposed to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU caused a time-and dose-related, irreversible inhibition of glutathione reductase activity. This coincided with a fall in both GSH/GSSG ratio and the thiol content of the islets. Pretreatment of the islets with BCNU inhibited the oxidation of glucose and its stimulant action upon both 45Ca net uptake and insulin release. Although BCNU (up to 0.5 mM) failed to affect the oxidation of L-leucine and L-glutamine, it also caused a dose-related inhibition of insulin release evoked by the combination of these two amino acids. The latter inhibition was apparently not fully accounted for by the modest to negligible effects of BCNU upon 45Ca uptake, 45Ca efflux, 86Rb efflux and cyclic AMP production. Since BCNU failed to inhibit insulin release evoked by the association of Ba2+ and theophylline, these results support the view that glutathione reductase participates in the coupling of metabolic to secretory events in the process of nutrient-stimulated insulin release. However, the precise modality of such a participation, for example the control of intracellular Ca2+ distribution, remains to be elucidated.     

Calcium and pancreatic beta-cell function. The mechanism of insulin secretion studied with the aid of lanthanum.

La3+ was used to study the involvement of Ca2+ in insulin secretion in beta-cell-rich pancreatic islets micro-dissected from non-inbred ob/ob mice. Ultrastructural studies revealed that the localization of La3+ was entirely restricted to the exterior of the cells. Consistent with a membrane action, exposure to La3+ failed to affect glucose oxidation and either the sucrose space or the general ultrastructure of the islets. In contrast, La3+ had marked effects on insulin release and 45Ca fluxes. Exposure to La3+ resulted in pronounced inhibition of insulin release irrespective of the presence or absence of Ca2+, 3-isobutyl-1-methylxanthine or glucose. Perifusion experiments revealed that the inhibitory action was prompt, sustained and readily reversible. Removal of La3+ was associated with a subsequent prolonged stimulatory phase of insulin release even in medium deficient in Ca2+. This action could not be attributed to an increase in cyclic AMP, but was potentiated by 3-isobutyl-1-methylxanthine and abolished by L-adrenaline. La3+ displaced 45Ca from superficially located binding sites and inhibited the uptake and efflux of 45Ca. The stimulatory and inhibitory actions of glucose on 45Ca efflux were also abolished in the presence of 2 mM-La3+ Removal of La3+ was associated with the preferential mobilization of 45Ca incorporated in response to glucose. The results indicate that binding of La3+ to superficial sites in the plasma membrane leads to inhibition of insulin release by suppression of transmembrane Ca2+ fluxes. It is suggested that accumulation of Ca2+ in the cytoplasm accounts for the stimulation of insulin release seen after removal of La3+ from inhibitory binding sites in the beta-cell plasma membrane.     

Insulin release from pancreatic islets of fetal rats mediated by leucine b-BCH, tolbutamide, glibenclamide, arginine, potassium chloride, and theophylline does not require stimulation of Ca2+ net uptake

In pancreatic islets of fetal rats the effect of glucose (3 and 16.7 mM), glyceraldehyde (10 mM), leucine (20 mM), b-BCH (20 mM), tolbutamide (100 micrograms/ml), glibenclamide (0.5 and 5.0 micrograms/ml) arginine (20 mM), KCl (20 mM) and theophylline (2.5 mM) on 45Ca2+ net uptake and secretion of insulin was studied. All compounds tested failed to stimulate 45Ca2+ net uptake. However, in contrast to glucose and glyceraldehyde, leucine, b-BCH, tolbutamide, glibenclamide, arginine, KCl and theophylline significantly stimulated release of insulin. This effect could not be inhibited by the calcium antagonist verapamil (20 microM). Elevation of the glucose concentration from 3 to 5.6 mM did not alter 86Rb+ efflux of fetal rat islets but inhibited 86Rb+ efflux of adult rat islets. Stimulation of 86Rb+ efflux with tolbutamide (100 micrograms/ml), leucine (20 mM) or b-BCH (20 mM) in the presence of 3 mM glucose was also ineffective in fetal rat islets. Our data suggest that stimulation of calcium uptake via the voltage dependent calcium channel is not possible in the fetal state. They also provide evidence that stimulators of insulin release which are thought not to act through their metabolism, initiate insulin secretion from fetal islets by a mechanism which is different from stimulation of calcium influx.     

Calcium and pancreatic beta-cell function. 4. Evidence that glucose and phosphate stimulate calcium-45 incorporation into different intracellular pools.

beta-Cell-rich pancreatic islets were microdissected from ob/ob-mice and used for studies of 45Ca uptake and washout. Irrespective of whether the experiments were performed at 21 or 37 degrees C both glucose and phosphate stimulated the net uptake of lanthanum-nondisplaceable 45Ca. The stimulatory effect of phosphate was additive to that produced by glucose. 45Ca incorporated in response to phosphate differed from that taken up in the presence of 20 mM glucose in being easily washed out although it was not affected by the glucose concentration of the washing medium. The efflux of 45Ca was reduced after introducing phosphate into a medium used to perifuse islets which had accumulated 45Ca in response to 20 mM glucose. This suggests that the outward calcium transport can be influenced also by intracellular trapping of the cation. The glucose-stimulated insulin release was inhibited by phosphate; an effect reversed by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. It is concluded that a common effect of glucose and phosphate is to trap calcium in the pancreatic beta-cells but that there are fundamental differences between their effects on intracellular distribution of calcium and on insulin release.     

Glucose-, calcium- and concentration-dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets.

Mouse islets were used to define the glucose-dependence and extracellular Ca2+ requirement of muscarinic stimulation of pancreatic beta-cells. In the presence of a stimulatory concentration of glucose (10 mM) and of Ca2+, acetylcholine (0.1-100 microM) accelerated 3H efflux from islets preloaded with myo-[3H]inositol. It also stimulated 45Ca2+ influx and efflux, 86Rb+ efflux and insulin release. In the absence of Ca2+, only 10-100 microM-acetylcholine mobilized enough intracellular Ca2+ to trigger an early but brief peak of insulin release. At a non-stimulatory concentration of glucose (3 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ and 86Rb+ efflux in the presence and absence of extracellular Ca2+. However, only 100 microM-acetylcholine marginally increased 45Ca2+ influx and caused a small, delayed, stimulation of insulin release, which was abolished by omission of Ca2+. At a maximally effective concentration of glucose (30 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ influx and efflux only slightly, but markedly amplified insulin release. Again, only 100 microM-acetylcholine mobilized enough Ca2+ to trigger a peak of insulin release in the absence of Ca2+. The results thus show that only high concentrations of acetylcholine (greater than or equal to 10 microM) can induce release at low glucose or in a Ca2+-free medium. beta-Cells exhibit their highest sensitivity to acetylcholine in the presence of Ca2+ and stimulatory glucose. Under these physiological conditions, the large amplification of insulin release appears to be the result of combined effects of the neurotransmitter on Ca2+ influx, on intracellular Ca2+ stores and on the efficiency with which Ca2+ activates the releasing machinery.     

Effect of somatostatin on glucose-induced 45Ca uptake in the pancreatic islets.

This study was designed in an attempt to elucidate a mechanism of somatostatin inhibition of glucose-induced Ca+ uptake by rat pancreatic islets. Rat pancreatic islets were perifused with Krebs-Ringer bicarbonate (KRB) buffer containing 16.7 mM of glucose with somatostatin (2 micrograms/ml) or/and diltiazem HCl (2 x 10(-5) M). Somatostatin inhibited preferentially the early phase of glucose-induced insulin release, whereas diltiazem HCl inhibited the late one. And the concomitant presence of the submaximal concentration of somatostatin (2 micrograms/ml) and diltiazem HCl (2 x 10(-5 M) provided the completely additive inhibition of glucose-induced insulin release. Rat pancreatic islets were incubated with KRB buffer supplemented with 16.7 mM of glucose and 45CaCl2 (10 muCi/ml) for 5--60 min and the biphasic 45Ca uptake by pancreatic islets was obtained. Somatostatin (500 ng/ml-4 micrograms/ml) gave the suppressive effect on the early phase of glucose-induced 45Ca uptake, but the higher concentration (2 micrograms/ml) of somatostatin did not impair the late phase of 45Ca uptake by pancreatic islets. On the other hand, diltiazem HCl did suppress the late phase of glucose-induced 45Ca uptake dose-dependently, but did not suppress the early phase (2 x 10(-5) M). These data indicate that somatostatin suppresses the early phase of glucose-induced Ca2+ uptake preferentially to the late one and has a different action mechanism from Ca antagonist on glucose-induced insulin release.     

The stimulus-secretion coupling of glucose-induced insulin release. Cationic and secretory effects of menadione in the endocrine pancreas.

1. Menadione (2-methyl-1,4-naphthoquinone) inhibits insulin release evoked in the rat endocrine pancreas by glucose or glyceraldehyde, but fails to affect the secretory response to Ca2+, Ba2+, theophylline or gliclazide. The inhibitory effect of menadione upon glucose-induced insulin release is a dose-related, rapid and reversible phenomenon, menadione and glucose acting apparently as competitive antagonists. Menadione affects both the early and late phase of the secretory response to glucose. Menadione also antagonizes in a dose-related fashion the ability of glucose to reduce 86Rb efflux, to provoke 86Rb accumulation, to cause biphasic changes in 45 Ca efflux and to stimulate 45 Ca net uptake in pancreatic islets. 2. It is concluded that menadione impairs the insulinotropic action of glucose and other nutrients by impeding the remodelling of cationic fluxes normally provoked by these secretagogues in islet cells. Menadione, however, does not affect the capacity of divalent cations to activate the effector system which controls the release of secretory granules. Menadione may therefore represent a valuable tool to elucidate the mechanism by which glucose normally modifies the movement of cations in the pancreatic B-cell.     

Decreased insulin secretory response of pancreatic islets during culture in the presence of low glucose is associated with diminished 45Ca2+ net uptake, NADPH/NADP+ and GSH/GSSG ratios

In isolated rat pancreatic islets maintained at a physiologic glucose concentration (5.6 mM) the effect of glucose on parameters which are known to be involved in the insulin secretion coupling such as NADPH, reduced glutathione (GSH), 86Rb+ efflux, and 45Ca++ net uptake were investigated. The insulinotropic effect of 16.7 mM glucose was decreased with the period of culturing during the first 14 days being significant after 2 days though in control experiments both protein content and ATP levels per islet were not affected and insulin content was only slightly decreased. Both NADPH and GSH decreased with time of culture. 86Rb+ efflux which is decreased by enhancing the glucose concentration from 3 to 5.6 mM in freshly isolated islets was not affected by culturing whatsoever, even not after 14 days of culture when there was no longer any insulin responsiveness to glucose. The 45Ca++ net uptake was decreased during culturing. The data indicate (1) that the diminished glucose-stimulated release of insulin during culturing is not due to cell loss or simple energy disturbances, (2) that more likely it is the result of a diminished 45Ca++ net uptake as a consequence of the inability of islet cells to maintain proper NADPH and GSH levels, and (3) that potassium (86Rb+) efflux may not be related to changes of NADPH and GSH.     

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.     

Effects of lactate on pancreatic islets. Lactate efflux as a possible determinant of islet-cell depolarization by glucose.

The secretion of insulin from perifused rat pancreatic islets was stimulated by raising the glucose concentration from 5.6 to 20 mM or by exposure to tolbutamide. The addition of sodium lactate (40 mM) to islets perifused in the presence of glucose (5.6 mM) resulted in a small, transient, rise in the rate of secretion. The subsequent removal of lactate, but not glucose or tolbutamide, from the perifusate produced a dramatic potentiation of insulin release. The rate of efflux of 45Ca2+ was also increased when islets were exposed to a high concentration of glucose or lactate or to tolbutamide, and again subsequently upon withdrawal of lactate. Efflux of 86Rb+ was modestly inhibited upon addition of lactate and markedly enhanced by the subsequent withdrawal of lactate from islets. The output of [14C]lactate from islets incubated in the presence of [U-14C]glucose increased linearly with increasing concentrations of glucose (1-25 mM). It is proposed that the activation of islets by the addition or withdrawal of lactate is not due to increased oxidative flux, but occurs as a result of the electrogenic passage of lactate ions across the plasma membrane, resulting in islet-cell depolarization, Ca2+ entry and insulin secretion. The production of lactate via the glycolytic pathway, and the subsequent efflux of lactate from the islet cells with concomitant exchange of H+ for Na+, could be a major determinant of depolarization and hence insulin secretion, in response to glucose.     

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.     

Activation of insulin-secreting cells by pyruvate and halogenated derivatives.

Addition of pyruvate to rat islets perifused in the presence of 5 mM-glucose elicited an immediate pronounced biphasic stimulation of insulin secretion. At lower concentrations of glucose (2.5 mM), only the initial, transient, phase of secretion was observed. Pyruvate inhibited 45Ca2+ efflux from islets at 2.5 mM-glucose and stimulated efflux at 5 mM-glucose. Pyruvate also decreased the rate of efflux of 86Rb+ from perifused islets. A marked stimulation of insulin secretion and 45Ca2+ efflux rate was observed in response to 3-fluoropyruvate and 3-bromopyruvate, compounds which inhibited oxidative metabolism of [14C]glucose and [14C]pyruvate in islets. The stimulatory effects of 3-fluoro- and 3-bromo-pyruvate were associated with enhanced 86Rb+ efflux. Withdrawal of pyruvate or halogenated analogues from the perfusate resulted in a secondary stimulation of insulin release, 45Ca2+ efflux and, to some extent, 86Rb+ efflux rates. Pyruvate, 3-fluoropyruvate and 3-bromopyruvate were all effective in promoting intracellular acidification and a rise in cytosolic Ca2+ concentration, as judged from fluorescence measurements in HIT-T15 cells loaded with 2',7'-biscarboxyethyl-5'(6')-carboxyfluorescein and Quin 2 respectively. It is proposed that oxidative metabolism of pyruvate is not a prerequisite for its stimulatory actions on pancreatic beta-cells. An alternative mechanism of activation by pyruvate and its halogenated derivatives is proposed, based on the possible electrogenic flux of these anions across the cell membrane.     

Distinct effects of various amino acids on 45Ca2+ fluxes in rat pancreatic islets.

The effects of three types of amino acids on 45Ca2+ fluxes in rat pancreatic islets have been compared. Alanine, a non-insulinotropic neutral amino acid, transported with Na+, increased 45Ca2+ efflux in the presence or in the absence of extracellular Ca2+, but not in the absence of Na+. Its effects in Na+-solutions were practically abolished by 7 mM-glucose. Alanine slightly stimulated 45Ca2+ influx (5 min uptake) only when Na+ was present. Two insulinotropic cationic amino acids (arginine and lysine) triggered similar changes in 45Ca2+ efflux. They accelerated the efflux in the presence of Ca2+ and inhibited the efflux in a Ca2+-free medium, whether glucose was present or not. In an Na+-free Ca2+-medium, arginine and lysine markedly accelerated 45Ca2+ efflux, but this effect was suppressed by 7 mM-glucose. Arginine stimulated 45Ca2+ influx irrespective of the presence or absence of glucose and Na+. Leucine, a neutral insulinotropic amino acid well metabolized by islet cells, inhibited 45Ca2+ efflux from the islets in a Ca2+-free medium; this effect was potentiated by glutamine. In the presence of Ca2+ and Na+, leucine was ineffective alone, but triggered a marked increase in 45Ca2+ efflux when combined with glutamine. In an Na+-free Ca2+-medium, leucine accelerated 45Ca2+ efflux to the same extent with or without glutamine. Leucine also stimulated 45Ca2+ influx in the presence or in the absence of Na+, but its effects were potentiated by glutamine only in the presence of Na+. The results show that amino acids of various types cause distinct changes in 45Ca2+ fluxes in pancreatic islets. Certain of these changes involve an Na+-mediated mobilization of cellular Ca2+ from sequestering sites where glucose appears to exert an opposite effect.     

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.     

Effect of thioloxidant diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP) on 45Ca2+ net uptake into rat pancreatic islets

The effect of DIP (an oxidant of glutathione) on 45Ca2+ net uptake induced by a variety of stimulators of insulin secretion was studied in rat pancreatic islets. In addition the effect of exogenous glutathione (GSH) on 45Ca2+ net uptake in response to glucose was tested. DIP (0.1 mM) inhibited the increase of 45Ca2+ net uptake in the presence of glucose (16.7 mM) and glyceraldehyde (10 mM). A similar inhibitory effect could be demonstrated, when 45Ca2+ net uptake was enhanced by tolbutamide (100 micrograms/ml), glibenclamide (0.5 micrograms/ml), b-BCH (20 mM), 2-ketoisocaproate (20 mM), arginine (20 mM) in the presence of 3 mM glucose or by high extracellular potassium (20 mM). The increase of 45Ca2+ net uptake stimulated by leucine (20 mM) plus glucose (3 mM) was further augmented by DIP. Exogenous GSH did not affect 45Ca2+ net uptake in the presence of (5.6-16.7 mM) glucose. It is suggested that 45Ca2+ net uptake of pancreatic islets depends on the redox state of islet thiols regardless of whether uptake is promoted via inhibition of potassium efflux (nutrients, sulfonylureas) or by high potassium and arginine. The voltage sensitive calcium-channel is the site of action of critical thiols. It is possible that these thiols are localized at the inner side of the plasma membrane.     

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.     

The interaction between manganese and calcium fluxes in pancreatic beta-cells.

Electrothermal atomic-absorption spectroscopy was employed for measuring manganese in beta-cell-rich pancreatic islets isolated from ob/ob mice. The efflux from preloaded islets was estimated from the amounts remaining after 30 min of subsequent test incubations in the absence of Mn2+. An increase in the extracellular Mg2+ concentration promoted the Mn2+ efflux and removal of Na+ from a Ca2+-deficient medium had the opposite effect. Addition of 25 mM-K+ failed to affect Mn2+ outflow as did 3-isobutyl-1-methylxanthine and dibutyryl cyclic AMP. Whereas tolbutamide caused retention of manganese, the ionophore Br-X537A promoted an efflux. D-Glucose was equally potent in retaining the islet manganese when the external Ca2+ concentration ranged from 15 microM to 6.30 mM. Subcellular-fractionation experiments indicated a glucose-stimulated incorporation of manganese into all fractions except the microsomes. The effect was most pronounced in the mitochondrial fraction, being as high as 164%. The glucose-induced uptake of intracellular 45Ca was abolished in the presence of 0.25 mM-Mn2+. When added to medium containing 2.5 mM-Mn2+, glucose even tended to decrease 45Ca2+ uptake. The inhibitory effect of Mn2+ was apparent also from a diminished uptake of 45Ca into all subcellular fractions. The efflux of 45Ca2+ was markedly influenced by Mn2+ as manifested in a prominent stimulation followed by inhibition. In addition to demonstrating marked interactions between fluxes of Mn2+ and Ca2+, the present studies support the view that the glucose inhibition of the efflux of bivalent cations from pancreatic beta-cells is accounted for by their accumulation in the mitochondria.     

On the origin of mitochondria: a reexamination of the molecular structure and kinetic properties of pyruvate dehydrogenase complex from brewer''s yeast

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.     

Effects of acute sodium omission on insulin release, ionic flux and membrane potential in mouse pancreatic B-cells

The effects of acute omission of extracellular Na+ on pancreatic B-cell function were studied in mouse islets, using choline and lithium salts as impermeant and permeant substitutes, respectively. In the absence of glucose, choline substitution for Na+ hyperpolarized the B-cell membrane, inhibited 86Rb+ and 45Ca2+ efflux, but did not affect insulin release. In contrast, Li+ substitution for Na+ depolarized the B-cell membrane and caused a Ca2+-independent, transient acceleration of 45Ca2+ efflux and insulin release. Na+ replacement by choline in the presence of 10 mM glucose and 2.5 mM Ca2+ again rapidly hyperpolarized the B-cell membrane. This hyperpolarization was then followed by a phase of depolarization with continuous spike activity, before long slow waves of the membrane potential resumed. Under these conditions, 86Rb+ efflux first decreased before accelerating, concomitantly with marked and parallel increases in 45Ca2+ efflux and insulin release. In the absence of Ca2+, 45Ca2+ and 86Rb+ efflux were inhibited and insulin release was unaffected by choline substitution for Na+. Na+ replacement by Li+ in the presence of 10 mM glucose rapidly depolarized the B-cell membrane, caused an intense continuous spike activity, and accelerated 45Ca2+ efflux, 86Rb+ efflux and insulin release. In the absence of extracellular Ca2+, Li+ still caused a rapid but transient increase in 45Ca2+ and 86Rb+ efflux and in insulin release. Although not indispensable for insulin release, Na+ plays an important regulatory role in stimulus-secretion coupling by modulating, among others, membrane potential and ionic fluxes in B-cells.