Perchlorate stimulates insulin secretion by shifting the gating of L-type Ca2+ currents in mouse pancreatic B-cells towards negative potentials

The effects of the chaotrophic anion perchlorate (ClO4-) on glucose-induced electrical activity, exocytosis and ion channel activity in mouse pancreatic B-cells were investigated by patch-clamp recordings and capacitance measurements. ClO4- stimulated glucose-induced electrical activity and increased the action potential frequency by 70% whilst not affecting the membrane potential when applied in the presence of a subthreshold concentration of the sugar. ClO4- did not influence ATP-dependent K (KATP) channel activity and voltage-gated delayed K+ current. Similarly, ClO4- had no effect on Ca2+-dependent exocytosis. The stimulation of electrical activity and insulin secretion was instead attributable to an enhancement of the whole-cell Ca2+ current. This effect was particularly pronounced at voltages around the threshold for action potential initiation and a doubling of the current amplitude was observed at -30 mV. This was due to a 7-mV shift in the gating of the Ca2+ current towards negative voltages. The action of ClO4- was more pronounced when added in the presence of 0.1 mM BAY K8644, whereas no stimulation was observed when applied at a maximal concentration of the agonist (1 mM). Single-channel recordings revealed that the effect of ClO4- on whole-cell currents was principally due to a 60% increase in the mean duration of the long openings and the number of active channels. We propose that ClO4- stimulates insulin secretion and electrical activity by exerting a BAY K8644-like action on Ca2+ channel gating.     

Polysaccharide isolated from Triticum aestivum stimulates insulin release from pancreatic cells via the ATP-sensitive K+ channel

Traditional natural plants have been used throughout the world for their antidiabetic effects. The aim of the present study was to investigate the stimulating activity of a polysaccharide extract derived from T.?aestivum sprout (TASP) on insulin secretion in?vitro using the RIN-5F pancreatic β-cell line and rat pancreatic islets. In these experiments, TASP (0.1 to 2?mg/ml) augmented glucose-stimulated insulin secretion in a dose-dependent manner in the presence of a stimulatory glucose concentration (16.7?mM), but not of a basal concentration (1.1?mM). Although TASP failed to enhance the high K+-induced insulin secretion, the insulinotropic effect of TASP was significantly inhibited by diazoxide, an opener of ATP-sensitive K+ channel blocking insulin release. TASP potentiated the insulin secretion induced by other secretagogues, such as IBMX and tolbutamide. Moreover, glucose-derived blood insulin levels were significantly elevated by oral administration of TASP to mice, similarly to antidiabetic drugs. We also demonstrated that TASP significantly increased glucose-induced 45Ca2+ uptake and proinsulin mRNA expression in rat islets. Overall, our results suggest that TASP has a stimulating effect on insulin secretion and production in pancreatic β-cells via K+ channel closure and calcium influx. These results suggest that TASP may be useful as a candidate for the therapy of diabetes mellitus.     

Effects of glucose on 45Ca2+ uptake by pancreatic islets as studied with the lanthanum method.

1. Fluxes of 45Ca2+ were studied in pancreatic islets from non-inbred ob/ob-mice. Because La3+ blocked the transmembrane fluxes of 45Ca2+ in islet cells, incubations aimed at measuring glucose-induced changes of the intracellular Ca2+ were ended by washing the islets with 2 mM-La3+ for 60 min. 2. Uptake of 45Ca2+ progressed for 2 hr; the intracellular concentration of exchangable Ca2+ was about 7 m-mole/kg dry wt., as estimated from the isotope distribution at apparent equilibrium in islets exposed to 3 mM D-glucose. Raising the D-glucose concentration to 20 mM enhanced the 45 Ca2+ uptake whether or not the islets had first been equilibrated with the isotope. The stimulatory effect of D-glucose was observed in Tris buffer containing no anions but Cl- as well as in polyanionic bicarbonate buffer. The effect could not be reproduced with equimolar L-glucose. 3. The rate of 45Ca2+ release was the same whether the islets had been pre-loaded in the presence of 3 or 20 mM D-glucose. Thus the 45Ca2+ that had been taken up in response to 20 mM D-glucose appeared to be released much more slowly than the bulk of intracellular 45Ca2+. The release of 45Ca2+ was not significantly influenced by D-glucose during the release period. Incubation for 30 min was require for half of the radioactivity to be released. 4. The rates of insulin secretion were about the same in uni-anionic Tris buffer as in polyanionic bicarbonate buffer. A marked insulin secretory response to 20 mM D-glucose was observed in either buffer. 5. It is concluded that 20 mM D-glucose causes a net uptake of Ca2+ from the extracellular fluid into the interior of the beta-cells. This uptake is probably not regulated at the level of the plasma membrane but more likely reflects an increased affinity of some intracellular phase or compartment for the ion. Because the observed uptake and release of intracellular 45Ca2+ are slow processes in comparison with the rapid effects of extracellular Ca2+ on insulin secretion, insulin secretion may also depend on a more superficial and La3+-displacable Ca2+ pool.     

Differential effects of antioxidants and indomethacin on compound 48/80 induced histamine release and Ca2+ uptake in rat mast cells

The effect of nordihydroguaiaretic acid (NDGA), vitamin E, butylated hydroxytoluene (BHT) and indomethacin on histamine release and Ca2+ uptake in rat mast cells stimulated with compound 48/80 was studied. NDGA inhibited both the release of histamine and Ca2+ uptake in stimulated cells; however, there was no correlation between inhibition of Ca2+ uptake and the amount of histamine release. At a concentration of 5 microM, NDGA completely inhibited Ca2+ uptake, while histamine release was decreased by less than 50%. BHT (50 microM) inhibited both the Ca2+ uptake and histamine release. On the other hand, vitamin E (50 microM) inhibited histamine release by 70% without impairment in Ca2+ uptake. In the absence of the stimulus, vitamin E increased the cell-associated Ca2+; however, it had no effect on spontaneous release of histamine. Indomethacin (3 microM) inhibited Ca2+ uptake in stimulated cells by 50%, but did not affect the release of histamine. The results suggest that a part of Ca2+-influx may not be related to the coupled activation--secretion response and that lipid peroxidation through the lipoxygenase pathway may be involved in secretion of histamine from mast cells.     

Sphingosine-1-phosphate-induced intracellular Ca2+ mobilization in human endothelial cells.

The authors have studied the effect of sphingosine-1-phosphate (S1P) on Ca2+ release from intracellular stores in cultured human umbilical vein endothelial cells (HUVECs). In the presence of extracellular Ca2+, S1P increased intracellular Ca2+ concentration ([Ca2+]i) and this increase was partially inhibited by La3+ (1 microM), indicating that S1P induces Ca2+ influx from extracellular pool and Ca2+ release from intracellular stores. S1P increased [Ca2+]i concentration dependently in Ca2+-free extracellular solution. The Hill coefficient (1.7) and EC50 (420 nM) was obtained from the concentration-response relationship. When caffeine depleted Ca2+ store in the presence of ryanodine, S1P did not induce intracellular Ca2+ release. Furthermore, the Ca2+-induced Ca2+ release inhibitors ruthenium red or dantrolene completely inhibited S1P-induced intracellular Ca2+ release. S1P-induced intracellular Ca2+ release was inhibited by the phospholipase C (PLC) inhibitors neomycin and U73312, or the inositol 1,4,5-triphosphate (IP3)-gated Ca2+ channel blocker aminoethoxybiphenyl borane (2-APB). In contrast, S1P-induced intracellular Ca2+ release was not inhibited by the mitochondrial Ca2+ uptake inhibitor CCCP or the mitochondrial Ca2+ release inhibitor cyclosporin A. These results show that S1P mobilizes Ca2+ from intracellular stores primarily via Ca2+-induced and IP3-induced Ca2+ release and this Ca2+ mobilization is independent of mitochondrial Ca2+ stores.     

Effects of alpha- and beta-adrenoceptor stimulation on 45Ca2+ efflux and insulin secretion from perfused rat islets

Exposure to the beta 2-adrenoceptor agonist terbutaline resulted in a transient stimulation of 45Ca2+ efflux from 45Ca2+ preloaded rat islets perfused in 2 mM Ca2+ and 8.3 mM glucose. Concomitantly, an increase in insulin secretion occurred. Under the same experimental conditions, the alpha-adrenoceptor agonist noradrenaline promptly inhibited insulin release without any apparent influence on 45Ca2+ efflux. In contrast, in a medium containing 2 mM Ca2+ and a low glucose concentration (2.8 mM), terbutaline stimulated insulin secretion without any apparent effects on 45Ca2+ efflux. Noradrenaline had no effect on insulin secretion or 45Ca2+ efflux in this medium. When islets were perfused with 8.3 mM glucose in a Ca2+ deficient medium, with or without addition of the chelating agent EGTA, terbutaline induced a marginal stimulation of insulin secretion and a negligible stimulation of 45Ca2+ efflux. On the contrary, noradrenaline stimulated to an immediate and notable 45Ca2+ efflux in these Ca2+ deficient media. Noradrenaline also clearly inhibited insulin secretion, though less markedly and with a slower onset than in islets perfused in 2 mM Ca2+. When the islets were perfused in a Ca2+ deficient medium with 2.8 mM glucose, terbutaline had a slight insulin releasing effect, but stimulated 45Ca2+ efflux potently. Noradrenaline had no influence on insulin secretion but a weak stimulatory effect on 45Ca2+ efflux. The data suggest that the beta 2-adrenoceptor agonist terbutaline has the ability to stimulate insulin secretion in perfused rat islets, requiring extracellular Ca2+ for the full expression of its effects. These effects may be exerted through a Ca2+-Ca2+ exchange over the cell membrane and/or through cAMP and intracellular Ca2+ perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective alpha 2-adrenoceptor activation by clonidine: effects on 45Ca2+ efflux and insulin secretion from isolated rat islets

A possible role for Ca2+ in the alpha-adrenoceptor-induced inhibition of glucose-stimulated insulin secretion was studied in isolated rat islets by the use of the selective alpha 2-adrenoceptor agonist clonidine. We found that clonidine, in contrast to the alpha 1-adrenoceptor agonist phenylephrine, inhibited glucose-stimulated insulin secretion at dose levels below 10(-6) mol l-1. In islets preloaded with 45Ca2+ and perifused at 2 mmol l-1 Ca2+, clonidine (10(-6) mol l-1) reduced the glucose (13.3 mmol l-1)-stimulated 45Ca2+ efflux during both the first and second phases of insulin secretion. Furthermore, the inhibitory effect of clonidine on glucose (13.3 mmol l-1)-stimulated insulin secretion was partially counteracted by raising the extracellular Ca2+ concentrations. Moreover, the calcium channel agonist Bay K 8644 counteracted the inhibition by clonidine on glucose-stimulated insulin secretion. Our results suggest that selective alpha 2-adrenoceptor-induced inhibition of glucose-stimulated insulin secretion is mediated, at least partially, by restraint of Ca2+-influx. This action might in turn be exerted through interference with the voltage-dependent calcium channels.     

Effects of alpha-adrenoceptor agonists and antagonists on insulin secretion, calcium uptake, and rubidium efflux in mouse pancreatic islets

Epinephrine, norepinephrine or the more selective alpha-2 adrenoceptor agonist, clonidine, inhibited insulin release from isolated pancreatic islets of lean mice or obese mice homozygous for the gene ob. Clonidine was highly effective at 0.1 mumol/l. In contrast, the preferential alpha-1 adrenoceptor agonist, phenylephrine, had no or only a modest effect at 10 mumol/l. The effects of norepinephrine or clonidine were counteracted by yohimbine, a preferential blocker of alpha-2 receptors, but not by prazosine, an alpha-1 receptor blocker. The glucose-stimulated uptake of 45Ca2+ in the islets was only consistently inhibited by epinephrine. This effect was counteracted by yohimbine. Clonidine had no effect on the release of 86Rb+ from preloaded islets. It is concluded that insulin secretion is suppressed by alpha-2 receptor agonism in the pancreatic beta-cells and that this effect is mediated by mechanisms other than the transmembrane fluxes of calcium or potassium ions.     

The inhibitory effect of CR-1409 on amylase secretion and intracellular Ca2+ mobilization in rat pancreatic acini in vitro

The inhibitory effects of CR-1409, a new glutaramic acid derivative developed as a cholecystokinin (CCK) receptor antagonist, on caerulein-stimulated amylase secretion and on intracellular Ca2+ ([Ca2+]i) mobilization were studied in isolated rat pancreatic acini. Pancreatic acini were prepared by collagenase digestion method and loaded with 1 microM fura-2/AM for measurement of the intracellular free Ca2+ concentration. Amylase release was examined by a perifusion method. Stimulation with 10(-10) M caerulein, 10(-5) M carbachol, or 10(-8) M gastrin-releasing peptide (GRP) led to biphasic amylase release and increase in [Ca2+]i. CR-1409 at 1 and 5 microM inhibited, by 50 and 84%, respectively, the amylase secretion and increase in [Ca2+]i induced by 10(-10) M caerulein, and 25 microM CR-1409 completely inhibited both amylase secretion and increase in [Ca2+]i induced by caerulein. However, 25 microM CR-1409 did not inhibit unstimulated secretion of amylase or the secretions induced by carbachol and GRP, which are also mediated by changes in intracellular Ca2+. We conclude that CR-1409 acts as a specific inhibitor of the CCK receptor in the pancreas, and is useful in studies on the involvement of the release and action of CCK in vitro.     

Cyclic AMP potentiates glucose-induced insulin release from mouse pancreatic islets without increasing cytosolic free Ca2+

The effects of various stimulants of insulin release on cytosolic free Ca2+, [Ca2+]i, in dispersed and cultured pancreatic beta-cells from ob/ob-mice were studied using the indicator quin-2, which in itself has only slight effects on the glucose-induced insulin release and the metabolism of the sugar. The resting [Ca2+]i was 158 +/- 7 nM. After increasing glucose to 20 mM there was a lag-period of 1-2 min before [Ca2+]i gradually rose, reaching a new plateau 60% higher after 5-6 min. Increasing intracellular cyclic AMP by adding forskolin did not further increase [Ca2+]i; on the contrary there was a slight temporary reduction despite a doubling of insulin secretion. The maintenance of the beta-cell function was evident from a marked increase of cytosolic [Ca2+]i after depolarization evoked by high extracellular K+. Also dibutyryl cyclic AMP and theophylline lacked the ability to raise [Ca2+]i beyond that obtained by glucose. The results suggest that cyclic AMP potentiates glucose-induced insulin release by sensitizing the secretory machinery to changes of [Ca2+]i rather than by increasing the cytosolic concentration of the ion.     

Somatostatin inhibition of glucose-induced electrical activity in cultured rat islet cells.

Electrophysiological studies of rat islet cells in monolayer culture were undertaken to determine the role of transmembranous ionic fluxes in the inhibitory action of somatostatin on insulin release. In the presence of somatotropin release inhibiting factor (SRIF) (2.5 nM), hyperpolarization occured with or without glucose (16.6 mM) in the medium. SRIF also inhibited the incidence of glucose-induced spike activity. The inhibitory action of SRIF occurred within 5 min and was readily reversible. An increase in extracellular K+ (5-13 mM) or Ca2+ (2.3-4.6 mM) prevented SRIF inhibition of glucose-induced electrical activity. The secretory response of cultured islets to glucose (16.6 mM) was completely inhibited by SRIF (2.5 nM). The presence of high [Ca2+]o or [k+]o enhanced insulin release in the presence of SRIF and glucose. Although phentolamine (5.0 microgram/ml) did not block the inhibition of glucose-induced electrical responses by SRIF, it prevented the inhibitory action of epinephrine (0.2 microgram/ml). It is concluded that the primary action of SRIF is to alter transmembranous cationic fluxes, as manifested by hyperpolarization and a decrease in the incidence of spike activity, which may prevent glucose from eliciting a normal secretory response.     

K-channel blocking drugs induce histamine release and 45Ca uptake in isolated mast cells.

Histamine secretion and 45Ca uptake processes were studied in mast cells treated with four K+ channel blocking drugs in physiological saline and in media containing different ionic concentrations. Quinine, 4-aminopyridine and sparteine were effective as histamine-releasing agents when mast cells were incubated in physiologic saline solution. The dose-response profile obtained was in the range of 0.1-0.5 mM for quinine, 1-10 for 4-aminopyridine and 0.5-5 mM for sparteine and did not show significant differences between purified and unpurified mast cells. By contrast, tetraethylammonium (1-100 mM) did not induce histamine release. The presence of high K+ or Rb+ concentrations in the medium (Tris-K+ or Tris-Rb+, both at 150 mM) displaced the profile obtained to the right in cells stimulated with 4-aminopyridine or sparteine, but abolished histamine release induced by quinine. Additionally, all three K+ channel blockers increased 45Ca uptake in mast cells. The exact mechanism of the action of K+ channel blockers on mast cells is unknown. However, the fact that the drugs used were effective as histamine-releasing and 45Ca uptake promoters suggests both that mast cells might be endowed with a K+ channel activity and that the blockade of this should open certain calcium channels, leading to elevated intracellular Ca2+ levels which in turn activate mast cell secretion.     

The functional significance of sodium channels in pancreatic beta-cell membranes

1. The existence and functional significance of Na channels in pancreatic beta-cell membranes were investigated by studying the effects of the plant alkaloid veratridine on the temporal release of insulin from perfused isolated rat islets of Langerhans.2. 100 muM veratridine evoked a sustained threefold increase in insulin release which was almost completely inhibited by 3 muM tetrodotoxin (TTX). This action of TTX was rapidly reversible.3. The simultaneous presence of 100 muM propranolol, 100 muM phenoxy-benzamine and 10 muM atropine did not alter the magnitude of the response to 100 muM veratridine, indicating that the action of veratridine on the beta-cells was direct and was not mediated via the release of neurotrans-mitters from nerve endings within the islets.4. (45)Ca uptake by isolated islets in static incubation was increased almost threefold by 100 muM veratridine. This increase was completely inhibited by the simultaneous presence of 3 muM TTX.5. Replacement of Na(o) by choline caused a transient fourfold increase in insulin release which was associated with an increase in the uptake of (45)Ca from the extracellular space of similar magnitude. Subsequent exposure of islets to 100 muM veratridine still evoked some insulin release but this only achieved 32% of that secreted by islets exposed to veratridine in medium of normal [Na](o).6. The addition of 2.5 mM CoCl(2) to the medium caused a 62.5% inhibition of veratridine-mediated insulin release.7. In Ca-free medium supplemented with 1 mM EGTA, 100 muM veratridine evoked insulin release of equal magnitude and of similar temporal relationship to that obtained in the presence of normal [Ca](o).8. A twofold increase in insulin release that occurred in the 15 min period immediately following exposure to 1 mM ouabain was completely independent of [Ca](o). Subsequent ouabain-evoked release became increasingly dependent on [Ca](o).9. Tetrodotoxin (3 muM) inhibited the first phase of insulin release evoked by 16.7 mMd-glucose by 37% and the second phase by 20%.10. Both Na and Ca appear capable of entering through Na channels opened in the beta-cell membrane by veratridine. The increase in [Na](i), resulting from the veratridine mediated increase in P(Na+), causes depolarization of the beta-cell membrane with a consequent opening of voltage-sensitive, Co(2+)-blockable channels for additional Ca entry. An increase in [Na](i) also increases [Ca](i) by altering the equilibria of intracellular Ca-sequestering mechanisms. The small but significant reduction of glucose-mediated insulin release by TTX indicates that glucose has a rather weak action on the Na channel and a more pronounced effect on the voltage-dependent Co(2+)-blockable Ca channel.     

The interaction between cAMP-dependent and cAMP-independent mechanisms in mediating the somatostatin inhibition of insulin secretion in isolated rat pancreatic islets.

To characterize the intracellular mechanisms by which somatostatin modulates the insulin secretion, studies were performed with isolated rat pancreatic islets at 12 mmol l-1 glucose. Somatostatin (0.1-1000 nmol l-1) inhibited the glucose-induced insulin secretion concentration-dependently. Increasing intracellular cAMP concentration either with dibutyryl-cAMP (1 mmol l-1) or by the adenylate cyclase activator forskolin (20 mumol l-1) partly reversed the inhibition by somatostatin (100 nmol l-1). Neither somatostatin (100 nmol l-1) nor dibutyryl-cAMP (1 mmol l-1 were able to affect the low insulin secretion observed in the absence of extracellular Ca2+. To study cAMP-independent mechanisms of somatostatin, the experiments were performed with and without dibutyryl-cAMP (1 mmol l-1) present. Both somatostatin (100 nmol l-1) and the Ca(2+)-channel blocker verapamil (25 mumol l-1) inhibited the insulin secretion both with and without dibutyryl-cAMP present. An additional inhibition of the insulin secretion was observed when somatostatin was combined with verapamil in the absence, but not in the presence of dibutyryl-cAMP. We conclude that somatostatin inhibits the glucose-induced insulin secretion both by cAMP-dependent mechanism which requires extracellular Ca2+, and by cAMP-independent/verapamil-sensitive Ca(2+)-channel-dependent mechanism.     

Epinephrine modifications of insulin release and of 86Rb+ or 45Ca2+ fluxes in rat islets

The effects of epinephrine on insulin release, 86Rb+ fluxes, and 45Ca2+ fluxes were measured in rat islets. In the presence of 10 mM glucose, epinephrine did not affect 86Rb+ influx and slightly increased net uptake. It caused a monophasic inhibition of release and a biphasic decrease in 86Rb+ efflux. A maximum effect was observed with 1 microM epinephrine, but release was more markedly inhibited by lower concentrations of the catecholamine than was the efflux. Epinephrine inhibition of release and efflux was reversed by phentolamine and yohimbine but not by prazosin or propranolol. It was mimicked by norepinephrine and clonidine. The inhibition of 86Rb+ efflux persisted when insulin release was prevented by omission of extracellular calcium. Ouabain or high K+ markedly increased 86Rb+ efflux in the presence of glucose and epinephrine; theophylline and quinine had a similar but smaller effect. None of these agents restored insulin release. Epinephrine abolished the insulinotropic effect of arginine without altering the rise in 86Rb+ efflux triggered by the amino acid. Epinephrine abolished insulin release but inhibited 45Ca2+ efflux only partially during stimulation by glucose or by barium plus theophylline. The results show that epinephrine does not inhibit insulin release by activating the Na pump or by increasing K permeability of the B cell membrane. On the contrary, the inhibition of release is accompanied by a decrease in 86Rb+ efflux. Both result from activation of alpha 2-receptors but are not causally related; they could be due to remodeling of Ca2+ fluxes and/or changes in cAMP levels.     

Muscarinic stimulation of guinea pig adrenal chromaffin cells stimulates catecholamine secretion without significant increase in Ca2+ uptake

In isolated guinea pig adrenal chromaffin cells, not only nicotine, but also muscarine stimulated catecholamine (CA) secretion, the stimulation by muscarine being the greater. The secretions of CA by muscarine and nicotine were both dependent on the presence of Ca2+ in the medium, but only the latter was associated with a rapid increase in 45Ca2+ uptake. Experiments with the fluorescent Ca2+ indicator quin 2, showed that muscarine caused an increase in cytoplasmic free Ca2+ concentration [( Ca2+]i). Moreover, the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibited both CA secretion and increase in [Ca2+]i induced by muscarine. These results indicate that in isolated guinea pig adrenal chromaffin cells, nicotine stimulated CA secretion by increasing Ca2+ uptake by the cells, whereas muscarine stimulated CA secretion by mobilizing Ca2+ from the intracellular pool.     

The role of dihydropyridine-sensitive Ca2+ channels in stimulus-evoked catecholamine release from chemoreceptor cells of the carotid body.

The present study utilized an in vitro preparation of the rabbit carotid body, with tissue catecholamine stores labeled by incubation with 3H-tyrosine. The goal was to characterize pharmacologically the voltage-dependent Ca2+ channels present in the type I (glomus) cells of this arterial chemoreceptor organ, and to elucidate their role as pathways for Ca2+ entry. We found that release of 3H-dopamine induced by high external potassium was over 95% dependent on external calcium concentration and that this release was 90-100% inhibited by the dihydropyridine antagonists, nisoldipine and nitrendipine, and was potentiated by the dihydropyridine agonist, BayK 8644. Therefore, any stimulus-induced, calcium-dependent release of 3H-dopamine that was inhibited by nisoldipine and potentiated by BayK 8644, was considered to be supported by Ca2+ entry into the cells via voltage-dependent Ca2+ channels. Significant differences were observed in the release of 3H-dopamine induced by 75 vs 25 mM K+. On prolonged stimulation, release induced by 75 mM K+ was large and transient, whilst that induced by 25 mM K+, although more moderate, was sustained. The release elicited by 75 mM K+ was inhibited approximately 90% by 1.5 mM Co2+ or 625 nM nisoldipine, while release by 25 mM K+ was completely blocked by 0.6 mM Co2+ or 125 nM nisoldipine. Low PO2-induced release of 3H-dopamine was 95% dependent on Ca2+, and was inhibited by nisoldipine (625 nM) in a manner inversely proportional to the intensity of hypoxic stimulation, i.e. 79% inhibition at a PO2 of 49 Torr, and 20% inhibition at PO2 of 0 Torr. BayK 8644 potentiated the release induced by moderate hypoxic stimuli. Release elicited by high PCO2/low pH, or by Na(+)-propionate or dinitrophenol-containing solutions, was approximately 80% Ca(2+)-dependent, and the dihydropyridines failed to modify this release. It is concluded that type I cells possess voltage-dependent Ca2+ channels sensitive to the dihydropyridines, which in agreement with previous electrophysiological data should be defined as L-type Ca2+ channels. Calcium entry which supports the release of 3H-dopamine elicited by moderate hypoxia should occur mainly through these channels while the release induced by strong hypoxic stimuli will be served by Ca2+ entry which occurs in part via voltage-dependent Ca2+ channels, and in part through an additional pathway, probably a Na+/Ca2+ exchanger.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vitamin D3 stimulates calcium-45 uptake by isolated mouse islets in vitro

Islets isolated from ob/ob mice which had been fed a vitamin D-deficient diet released significantly less insulin in response to glucose than did vitamin D-replete islets but showed normal net 45Ca2+ uptake. To determine whether vitamin D3 has a direct effect on the pancreatic B cell, islets from ob/ob mice on a normal diet were exposed to vitamin D3 in vitro for 1 week or only 3 h, and then glucose-stimulated 45Ca2+ uptake and insulin release were measured. Exposure to 1 nM or 1 microM vitamin D3 for 1 week stimulated 45Ca2+ uptake in the presence of 3 mM, but not 20 mM glucose, and did not affect insulin release. Exposure to vitamin D3 for 3 h did not significantly increase net 45Ca2+ uptake although there was a tendency to such an effect (P = 0.10). In conclusion, vitamin D-deficiency in vivo suppressed subsequent glucose-stimulated insulin release in vitro and this effect may be due to a direct effect of the sterol (or one of its metabolites) on calcium handling by the B cell.     

The effect of calcium-blockers nicardipine, darodipine, PN-200-110 and nifedipine on insulin release from isolated rat pancreatic islets

The effect of the calcium-blockers nicardipine, darodipine, PN-200-110 on insulin release from pancreatic islets was studied using nifedipine as a reference compound. All drugs at a concentration of 10(-6)M significantly inhibited insulin release in response to both low (5.5 mM) and high (22 mM) glucose. The present observations support previous reports that calcium blockers of the dihydropyridine series are effective inhibitors of glucose-induced insulin release.     

Participation of Ca2+ and calmodulin in rat pancreatic enzyme secretion induced by secretin, forskolin, and dibutyryl cyclic AMP

The role of Ca2+ and calmodulin in stimulation of the rat pancreatic acini induced by secretin, forskolin, and dibutyryl cyclic AMP (dbcAMP) was studied using W-7, a calmodulin antagonist, and a low Ca2+ medium. The time course of amylase secretion was studied in a perfusion system using dispersed rat pancreatic acini. The amylase release patterns of each secretagogue were as follows: a biphasic amylase release pattern under the stimulation of secretin, a one peak pattern during the stimulation of forskolin and a rapid response after cessation of the stimulation, and a gradual increased pattern during the stimulation of dbcAMP followed by a rapid response. The amylase release under the stimulation by forskolin and dbcAMP was slightly weaker as compared with that of secretin stimulation. The amylase secretion stimulated by secretin (5 X 10(-7) M), forskolin (50 microM), and dbcAMP (2 mM) was inhibited by W-7 (50 microM). In a low Ca2+ medium (4.7-5.1 X 10(-6) M), the secretory rate did not increase during the stimulation by secretin, forskolin, and dbcAMP, and a rapid amylase response remained after cessation of the stimulation of forskolin and dbcAMP. The pretreatment with EDTA (1 mM) suppressed both the gradual amylase release and the rapid response induced by dbcAMP in a low Ca2+ medium. These results suggested that each secretagogue, via cyclic AMP (cAMP), induced a different amylase secretory pattern dependent on an intracellular Ca2+ content, and was mediated by the Ca2+-calmodulin complex.