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.     

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.     

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.     

Adrenergic-agonist-induced Ca2+ fluxes in rat parotid cells are not Na+-dependent.

We investigated the hypothesis that extracellular Na+ is required for the rapid mobilization of Ca2+ by rat parotid cells after adrenergic stimulation. When Na+ salts in the media were osmotically replaced with either choline chloride (+atropine) or sucrose, efflux of 45Ca2+ from preloaded cells, caused by 10 microM-(-)-adrenaline, was unchanged. Similarly adrenaline stimulated 45Ca2+ uptake into cells under nonsteady-state conditions in the presence or absence of Na+. Monensin, a Na+ ionophore, was able to elicit a modest increase in 45Ca2+ efflux, compared with controls. Studies of net 45Ca2+ flux, performed under near-steady-state conditions, showed that adrenaline caused net 45Ca2+ accumulation, whereas monensin caused net 45Ca2+ release. The effect of monensin required the presence of Na+ in the incubation medium. Both 1 mM-LaCl3 and 0.1 mM-D-600 prevented adrenaline-stimulated 45Ca2+ uptake into cells, but had no effect on monensin-induced changes. We conclude that (1) the rapid mobilization of Ca2+ by adrenergic agonists seen in rat parotid cells does not require a Na+out greater than Na+in gradient and (2) the nature of the monensin effect is quite different from the adrenergic-agonist-induced response.     

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).     

A role for transglutaminase in glucose-stimulated insulin release from the pancreatic beta-cell.

Preincubation of rat islets of Langerhans with the potent inhibitors of islet transglutaminase activity, monodansylcadaverine (30-100 microM) and N-(5-aminopentyl)-2-naphthalenesulphonamide (100-200 microM), led to significant inhibition of glucose-stimulated insulin release from islets. In contrast, the respective N'-dimethylated derivatives of these two compounds, which did not inhibit islet transglutaminase activity, were much less effective as inhibitors of glucose-stimulated insulin release. None of the compounds inhibited rat spleen protein kinase C activity at concentrations which gave rise to inhibition of glucose-stimulated insulin release. When tested for their effects on calmodulin-stimulated bovine heart phosphodiesterase activity, of the compounds that inhibited insulin release, only monodansylcadaverine did not act as an effective antagonist of calmodulin at concentrations (up to 50 microM) that gave rise to significant inhibition of glucose-stimulated insulin release. Furthermore, at 50 microM, monodansylcadaverine did not inhibit methylation of islet lipids. The inhibition of glucose-stimulated insulin release by monodansylcadaverine is therefore likely to be attributable to its interference with islet transglutaminase activity. The sensitivity of islet transglutaminase to activation by Ca2+ was investigated by using a modified assay incorporating dephosphorylated NN'-dimethylcasein as a substrate protein. The Km for Ca2+ obtained (approx. 3 microM) was an order of magnitude lower than previously reported for the islet enzyme [Bungay, Potter & Griffin (1984) Biochem. J. 219, 819-827]. Mg2+ (2 mM) was found to have little effect on the sensitivity of the enzyme to Ca2+. Investigation of the endogenous substrate proteins of islet transglutaminase by using the Ca2+-dependent incorporation of [14C]methylamine into proteins of islet homogenates demonstrated that most of the incorporated radiolabel was present in cross-linked polymeric aggregates which did not traverse 3% (w/v) acrylamide gels. The radiolabelled polymeric aggregates were present in 71 000 g-sedimented material of homogenates, and their formation was transglutaminase-mediated. These findings provide new evidence for the involvement of islet transglutaminase in the membrane-mediated events necessary for glucose-stimulated insulin release.     

The asymmetric effect of lanthanides on Na+-gradient-dependent Ca2+ transport in synaptic plasma membrane vesicles

Lanthanides (La3+, Pr3+ and Tb3+) inhibit Na+-gradient-dependent Ca2+ influx into synaptic plasma membrane vesicles. 50% inhibition is obtained by 7 microM lanthanide concentration. The inhibition of the Na+-gradient-dependent Ca2+ uptake exhibits competitive kinetic behaviour. The apparent Km of the Ca2+ influx is increased from 50 microM in the absence of lanthanides to 118 microM in the presence of La3+, 170 microM in the presence of Pr3+ and 130 microM in the presence of Tb3+. The maximal reaction velocity is not altered (8.35 nmol Ca2+ transported per mg protein per min in the absence of lanthanides and 8.16 nmol/mg per min in the presence of lanthanides). Lanthanides also inhibited Na+-gradient-dependent Ca2+ efflux from synaptic plasma membrane vesicles that were preloaded with Ca2+ in a Na+-gradient-dependent manner. Introduction of La3+ into the interior of the synaptic plasma membrane vesicles by rapid freezing of the vesicles in liquid N2 and slow thawing had no effect on either Na+-gradient-dependent Ca2+ influx or efflux. Synaptic plasma membrane vesicles can be preloaded with Ca2+ also in an ATP-dependent manner. This form of Ca2+ uptake is also inhibited by La3+ though at higher concentrations than the Na+-gradient-dependent Ca2+ uptake. Na+-gradient-dependent efflux from synaptic plasma membrane vesicles preloaded in an ATP-dependent fashion ('inside-out' vesicles) unlike efflux from synaptic plasma membrane vesicles preloaded in a Na+-gradient-dependent manner was not inhibited by La3+. These findings suggest that the inhibition by La3+ is manifested asymmetrically on both sides of the synaptic plasma membrane. Lanthanides are probably not transported via the Na+-Ca2+ exchanger since Tb3+ entry measured by fluorescence of Tb3+-dipicolinic acid complex formation occurred at high Tb3+ concentrations only (1.5 mM or above) and was not Na+-gradient dependent.     

Studies on the interaction of staphylococcal delta-haemolysin with isolated islets of Langerhans.

delta-Haemolysin, a small surface-active polypeptide purified from the culture media of Staphylococcus aureus, was observed to stimulate the release of insulin from isolated rat islets of Langerhans. This effect was dose-dependent and saturable, with the half-maximal response elicited by a delta-haemolysin concentration of 10 micrograms/ml. Stimulation of insulin release by delta-haemolysin (10 micrograms/ml) was not dependent on the presence of glucose in the incubation medium, but was augmented by increasing concentrations of the sugar. The release of insulin in response to delta-haemolysin could be inhibited by depletion of extracellular Ca2+ or by adrenaline (epinephrine) (10 microM) and was readily reversible when delta-haemolysin was removed from the medium. In addition, the response was potentiated by incubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mM). These observations suggest that delta-haemolysin induced a true activation of the beta-cell secretory mechanism. Stimulation of islets of Langerhans with delta-haemolysin was found to be associated with a modest increase in intracellular cyclic AMP levels, although the adenylate cyclase activity of islet homogenates was not increased by delta-haemolysin. delta-Haemolysin was observed to induce a dose-dependent net accumulation of 45Ca2+ by islet cells and to stimulate the efflux of 45Ca2+ from preloaded islets. The efflux of 45Ca2+ was modest in size and short-lived, but dramatically increased in medium depleted fo 40Ca2+. Incubation in the presence of verapamil augmented delta-haemolysin-induced 45Ca2+ efflux and insulin secretion. delta-Haemolysin was found to be a potent 45Ca2+-translocating ionophore in an artificial system. This response was dose-dependent and could be augmented by verapamil. In addition, phosphatidylcholine (25 micrograms/ml) was found to inhibit both delta-haemolysin induced 45Ca2+ translocation and insulin release in a precisely parallel manner. These studies suggest that the ability of delta-haemolysin to stimulate insulin release may be due, in part, to the facilitation of Ca2+ entry into the beta-cells of islets of Langerhans, mediated directly by an ionophoretic mechanism.     

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.     

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.     

Modulation of Ca2+-mediated K+-gating of erythrocyte ghosts by external Ca-EGTA

Using 86Rb+ as a marker for K+ permeability, we find that extracellular Ca-EGTA influences the rate of 86Rb+ efflux from erythrocyte ghosts preloaded with 86Rb+ and "buffered" Ca2+. At an internal free Ca2+, where the rate of 86Rb+ efflux is minimal and uninfluenced by either external EGTA or external Ca2+, external Ca-EGTA at 0.2-0.5 mM can raise the flux rate to as high as can be attained by raising internal Ca2+, in the presence of an excess externally either of Ca2+ or of EGTA. Higher concentrations of Ca-EGTA (up to 1-2 mM) diminish the flux rate. External Ca-EDTA or Mg-EDTA can substitute for Ca-EGTA in enhancing and suppressing flux rate. The peak rate is insensitive to external free Ca2+ but depends on internal Ca2+; internal Mg-EDTA does not substitute for internal Ca-EGTA. Thus, the erythrocyte membrane is asymmetric with respect to its interaction with Ca2+ and Ca-EGTA. Also, 22Na+ does not substitute for 86Rb+. The peak rate of 86Rb+ flux produced by external Ca-EGTA is diminished by chlorpromazine (0.1 mM) and augmented by 1-propranolol (25 microM), in the same way as the rate produced by increasing internal Ca2+. The results suggest that external Ca-EGTA enhances the affinity of internal Ca2+ for its receptor(s) which operate the K+-gate at the inner surface of the membrane. At external concentrations of Ca-EGTA above 1-2 mM, 86Rb+ flux rate again rises with increase of Ca-EGTA. This phenomenon does not depend upon internal Ca2+, is not affected by chlorpromazine or by 1-propranolol, and is associated with an enhanced permeability to 22Na+, inulin, and haemoglobin.     

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.     

Effects of trifluoperazine and pimozide on stimulus–secretion coupling in pancreatic B-cells. Suggestion for a role of calmodulin?

The possible involvement of calmodulin in insulin release was evaluated by studying the effects on intact islets of trifluoperazine and pimozide, two antipsychotic agents known to bind strongly to calmodulin in cell-free systems. Trifluoperazine (10-100mum) produced a dose- and time-dependent inhibition of the two phases of glucose-stimulated insulin release. The effect was not reversible by simple washing of the drug, but could be prevented by cytochalasin B or theophylline. Trifluoperazine also inhibited the release induced by glyceraldehyde, oxoisocaproate, tolbutamide or barium, but not that stimulated by 10mm-theophylline or 1mm-3-isobutyl-1-methylxanthine. Pimozide (0.5-10mum) also produced a dose-dependent inhibition of insulin release triggered by glucose, leucine or barium, but did not affect the release induced by methylxanthines. Glucose utilization by islet cells was not modified by trifluoperazine (25mum), which slightly increased cyclic AMP concentration in islets incubated without glucose. The drug did not prevent the increase in cyclic AMP concentration observed after 10min of glucose stimulation, but suppressed it after 60min. Basal or glucose-stimulated Ca(2+) influx (5min) was unaffected by 25mum-trifluoperazine, whereas Ca(2+)net uptake (60min) was inhibited by 20%. Glucose-stimulated Ca(2+) uptake was almost unaffected by pimozide. In a Ca(2+)-free medium, trifluoperazine decreased Ca(2+) efflux from the islets and did not prevent the further decrease by glucose; in the presence of Ca(2+), the drug again decreased Ca(2+) efflux and inhibited the stimulation normally produced by glucose. In the absence of glucose, trifluoperazine lowered the rate of Rb(+) efflux from the islets, decreased Rb(+) influx (10min), but did not affect Rb(+) net uptake (60min). It did not interfere with the ability of glucose to decrease Rb(+) efflux rate further and to increase Rb(+) net uptake. The results show thus that trifluoperazine does not alter the initial key events of the stimulus-secretion coupling. Its inhibition of insulin release suggests a role of calmodulin at late stages of the secretory process.     

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.     

Effects of chloride deficiency on the pancreatic B-cell response to acetylcholine

Muscarinic stimulation of pancreatic B-cells markedly amplifies insulin secretion through complex mechanisms which involve changes in membrane potential and ionic fluxes. In this study, normal mouse islets were used to evaluate the role of Cl- ions in these effects of acetylcholine (ACh). Whatever the concentration of glucose, the rate of 36Cl- efflux from islet cells was unaffected by ACh. Replacement of Cl- by impermeant isethionate in a medium containing 15 mM glucose did not affect, or only slightly decreased, the ability of ACh to depolarize the B-cell membrane and increase electrical activity, to accelerate 45Ca2+ and 86Rb+ efflux from islet cells, and to amplify insulin release. In the absence of extracellular Ca2+, a high concentration of ACh (100 microM) mobilized intracellular Ca2+ and caused a transient release of insulin and a sustained acceleration of 86Rb+ efflux. None of these effects was affected by Cl- omission or by addition of furosemide, a blocker of the Na+, K+, 2Cl- cotransport. Isethionate substitution for Cl- in a medium containing a nonstimulatory concentration of glucose (3 mM) barely reduced the depolarization of B-cells by ACh, but inhibited the concomitant increase in 86Rb+ efflux. We have no explanation for the latter effect that was not mimicked by furosemide. In conclusion, ACh stimulation of pancreatic B-cells, unlike that of exocrine acinar cells, is largely independent of Cl- and is insensitive to furosemide. The acceleration of ionic fluxes produced by ACh does not involve the Na+, K+, 2Cl- cotransport system.     

Mechanisms of the stimulation of insulin release by arginine-vasopressin in normal mouse islets

The mechanisms by which arginine-vasopressin (AVP) affects pancreatic B-cell function were studied in normal mouse islets. AVP produced a dose-dependent (0.1-1000 nM; EC50 approximately 1-2 nM) amplification of glucose-induced insulin release. This amplification was of slow onset and reversibility. AVP was ineffective when the concentration of glucose was less than 7 mM, but was still very effective in 30 mM glucose. The increase in insulin release produced by AVP was accompanied by small accelerations of 86Rb and 45Ca efflux from islet cells. Omission of extracellular Ca2+ accentuated the effect of AVP on 86Rb efflux, attenuated that on 45Ca efflux, and abolished that on release. Under no condition did AVP inhibit 86Rb efflux. AVP did not significantly affect cAMP levels, but increased inositol phosphate levels in islet cells, even in the absence of extracellular Ca2+. AVP did not affect the membrane potential in unstimulated B-cells and augmented glucose-induced electrical activity only slightly. This was not due to a direct action on ATP-sensitive K+ channels as revealed by patch-clamp recordings (whole cell and outside-out patches). In conclusion, AVP is not an initiator of insulin release, but it potently amplifies glucose-induced insulin release in normal mouse B-cells. This effect involves a stimulation of phosphoinositide metabolism, and presumably an activation of protein kinase C, rather than a change in cAMP levels or a direct control of the membrane potential.     

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.     

Rat myometrial Na/K ATPase is increased by serum but not by isoproterenol and relaxin

1. Na/K ATPase activity in rat myometrial cells in culture exhibited a Kapp of 0.93 mM for Rb+ and a Ki of 31 microM for ouabain with respect to Rb+. 2. 86Rb+ uptake was stimulated by serum and monensin but was not affected by the uterine relaxants isoproterenol and relaxin in 0.5-7.5 mM Rb+. Nonetheless, these relaxants elicited significant increases in 45Ca2+ efflux under similar conditions. 3. These data suggest that increased Na/Ca exchange resulting from a stimulation of Na/K ATPase is not involved in the mechanism of action of relaxin and isoproterenol in the uterus.     

Sodium-potassium exchange in sea urchin egg. II. Ionic events stimulating the Na+-K+ pump activity at fertilization

The preceding paper (Ciapa et al., 1984) provided biochemical and kinetic characterization of the Na+-K+ exchange in Paracentrotus lividus eggs. The present work is a study of the ionic events involved in the stimulation of the Na+-K+ transporter after fertilization. Fertilization in low Na+-external medium containing amiloride (0.1 mM) suppresses the stimulation of the net efflux of H+ and 86Rb uptake. Activation of eggs with the ionophore A23187 leads to stimulation of both Na+-H+ exchange and ouabain-sensitive 86Rb influx. When eggs were activated with A23187 in artificial seawater, 86Rb uptake and 24Na influx showed similar saturable kinetics with respect to the external Na+. A23187 treatment of eggs in Na+-free artificial seawater did not stimulate the Na+-K+ exchange until 10 mEq Na+ was added. Activation of eggs by NH4Cl (5 mM) stimulated 86Rb influx and Na+ exit; both fluxes were ouabain sensitive. Monensin increased cell Na+ of unfertilized eggs without any significant increase in intracellular pH: a condition in which 86Rb influx was not markedly stimulated. Addition of 10 mEq Na+ to unfertilized eggs in Na+-free artificial seawater stimulated 86Rb uptake but to a lower extent that did 10 mEq Na+ plus sperm. It is concluded that (1) the stimulation of the Na+-K+ pump at fertilization has an absolute requirement for the Na+-H+ exchange; (2) the alkalinization of eggs resulting from the acid efflux is a prerequisite for the enhancement of the Na+-K+ pump; (3) the amount of Na+ entering eggs at fertilization determines the intensity of the Na+-K+ exchange; (4) early events of fertilization such as exocytosis and calcium release which may be involved in the stimulation of the Na+-K+ pump must necessarily be coupled to cell alkalinization.     

Cadmium-induced insulin release does not involve changes in intracellular handling of calcium

A possible interaction between Cd2+ and Ca2+ as a component in Cd2+-induced insulin release was investigated in beta cells isolated from obese hyperglycemic mice. The glucose stimulated Cd2+ uptake was dependent on the concentration of sugar. This uptake was sigmoidal with a Km for glucose of about 5 mM and was suppressed by both 50 microM of the voltage-activated Ca2+ channel blocker D-600 and 12 mM Mg2+. In the presence of 8 mM glucose 5 microM Cd2+ evoked a prompt and sustained stimulatory response, corresponding to about 3-fold of the insulin release obtained in the absence of the ion. Whereas 5 microM Cd2+ was without effect on the glucose-stimulated 45Ca efflux in the presence of extracellular Ca2+, 40 microM inhibited it. At a concentration of 5 microM, Cd2+ had no effect on the resting membrane potential or the depolarization evoked by either glucose or K+. In the absence of extracellular Ca2+ there was only a modest stimulation of 45Ca efflux by 5 microM Cd2+. Studies of the ambient free Ca2+ concentration maintained by permeabilized cells also indicate that 5 microM Cd2+ do not mobilize intracellularly bound Ca2+ to any great extent. On the contrary, at this concentration, Cd2+ even suppressed inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. The present study suggests that Cd2+ stimulates insulin release by a direct mechanism which does not involve an increase in cytoplasmic free Ca2+ concentration.