Opening Of Voltage-dependent Ca2 Research Articles

Spermatozoa motility in bivalves: Signaling, flagellar beating behavior, and energetics

Though bivalve mollusks are keystone species and major species groups in aquaculture production worldwide, gamete biology is still largely unknown. This review aims to provide a synthesis of current knowledge in the field of sperm biology, including spermatozoa motility, flagellar beating, and energy metabolism; and to illustrate cellular signaling controlling spermatozoa motility initiation in bivalves. Serotonin (5-HT) induces hyper-motility in spermatozoa via a 5-HT receptor, suggesting a serotoninergic system in the male reproductive tract that might regulate sperm physiology. Acidic pH and high concentration of K+ are inhibitory factors of spermatozoa motility in the testis. Motility is initiated at spawning by a Na+-dependent alkalization of intracellular pH mediated by a Na+/H+ exchanger. Increase of 5-HT in the testis and decrease of extracellular K+ when sperm is released in seawater induce hyperpolarization of spermatozoa membrane potential mediated by K+ efflux and associated with an increase in intracellular Ca2+ via opening of voltage-dependent Ca2+ channels under alkaline conditions. These events activate dynein ATPases and Ca2+/calmodulin-dependent proteins resulting in flagellar beating. It may be possible that 5-HT is also involved in intracellular cAMP rise controlling cAMP-dependent protein kinase phosphorylation in the flagellum. Once motility is triggered, flagellum beats in asymmetric wave pattern leading to circular trajectories of spermatozoa. Three different flagellar wave characteristics are reported, including “full”, “twitching”, and “declining” propagation of wave, which are described and illustrated in the present review. Mitochondrial respiration, ATP content, and metabolic pathways producing ATP in bivalve spermatozoa are discussed. Energy metabolism of Pacific oyster spermatozoa differs from previously studied marine species since oxidative phosphorylation synthetizes a stable level of ATP throughout 24-h motility period and the end of movement is not explained by a low intracellular ATP content, revealing different strategy to improve oocyte fertilization success. Finally, our review highlights physiological mechanisms that require further researches and points out some advantages of bivalve spermatozoa to extend knowledge on mechanisms of motility.

GIRK4 Mutations R52H and E246K Impair Channel Gating but not Inward Rectification

G-protein inwardly rectifying potassium channels (GIRK) mediates inhibitory transmission via G-protein coupled receptors in heart, brain and adrenal cortex. Of four GIRK subunits (GIRK1-4), GIRK4 (KCNJ5) subunits are abundant in the heart and in adrenal cortex. Recent studies suggest that KCNJ5 mutations are involved in overproduction of aldosterone in primary aldosteronism (PA) and aldosterone-producing adenomas (APA). As of this point the consensus is that most or all GIRK4 mutations cause loss of GIRK channel selectivity, leading to increased permeability to Na+, depolarization of cell membrane, opening of voltage dependent Ca2+ channels and eventually Ca2+-dependent aldosterone secretion. This mechanism has been extended to mutations in cytosolic N and C terminal domains outside the pore region, GIRK4R52H and GIRK4E246K1. Using confocal microscopy, we found that YFP-tagged GIRK1 coexpressed with GIRK4R52H or GIRK4E246K in Xenopus laevis oocytes yield similar expression levels as YFP-GIRK1/GIRK4WT channels. Nevertheless, GIRK1/GIRK4R52H channels had significantly smaller whole-cell currents than GIRK1/GIRK4WT, suggesting impaired gating. Coexpression of Gβγ greatly increased the currents of both mutants, suggesting that Gβγ may “correct” a gating defect. Additionally, analysis of current-voltage curves revealed that both mutations did not affect the reversal potential or inward rectification of GIRK1/4. Our results suggest that previous interpretations of the effects of mutations GIRK4R52H and GIRK4E246K might have been incorrect, and that these mutations impair channel's gating but not selectivity or rectification. Overall, our results urge to reconsider the existing model of the mechanism responsible for aldosterone overproduction in patients with the out-of-pore mutations of KCNJ5, and prompt for looking for alternative explanations.1. Murthy M et al. Hypertension 63, 783-789 (2014).

Integrating Genetics and Genomics in Primary Aldosteronism

Primary aldosteronism (PA) is the most common form of secondary hypertension, with an estimated prevalence of ≈10% in referred patients and 4% in primary care1,2 but as high as 20% in patients with resistant hypertension.3,4 It is characterized by hypertension with low plasma renin and elevated aldosterone that is often seen with hypokalemia. PA occurs as a result of a dysregulation of the normal mechanisms controlling adrenal aldosterone production. The 2 major causes are aldosterone-producing adenomas (APAs) and bilateral adrenal hyperplasia (BAH), also called idiopathic hyperaldosteronism. The early detection of PA has an enormous impact on clinical outcome and survival, given the major cardiovascular adverse effects of aldosterone excess, which are independent of blood pressure,5,6 and predict outcome after surgical treatment.7,8 Patients with PA have a significantly higher risk of nonfatal myocardial infarction, atrial fibrillation, and stroke compared with age-, sex-, and blood pressure–matched essential hypertensives.5 Furthermore, changes in cardiac structure and function9 and partially reversible renal dysfunction10 have been described, whereas the increased occurrence of metabolic abnormalities in patients with PA is still a matter of debate.11,12 Although the 2008 guidelines for the management of PA have been pivotal for homogenizing screening procedures and treatment among specialized centers,13 there remain a few critical issues related to diagnosis, subtype differentiation, and treatment of nonsurgically correctable forms. Reliable diagnostic and prognostic biomarkers are lacking for more sensitive and specific screening, as well as new therapeutic avenues, because medical and/or surgical treatment of PA leads to normotension in only a minority of patients. This may come from a better understanding of the pathogenic mechanisms of the disease, in particular, identification of the genetic and molecular determinants leading to the development of APA and …

Effect of Mn2+ on the development of tension induced in guinea-pig taenia coli by Bay K 8644.

We have studied the effects of Mn2+ on the contractile response induced by Bay K 8644, a dihydropyridine Ca2+ agonist, on guinea-pig taenia coli. Mn2+ (5 mM) completely inhibited Bay K 8644 (10(-6) M)-induced rhythmic contraction and contracture to baseline values in normal Ca2+ medium; thereafter, the contraction progressively increased to about 90% of the K+ (60 mM)-induced tonic response. In Ca2+-free medium in the presence of Bay K 8644 Mn2+ also evoked contraction and a concomitant increase in Mn2+ influx into the cytoplasm. These results suggest that during the opening of voltage-dependent Ca2+ channels activated by Bay K 8644, Mn2+ can enter cytoplasm through the channels and induce contraction in taenia coli.

Monitoring the [ATP]/[ADP] Ratio in Beta-Cells During Glucose Stimulated Insulin Secretion Using the Genetically Encoded Fluorescent Reporter Perceval

Pancreatic beta-cells secrete insulin in response to elevated blood glucose levels. Glucose stimulated insulin secretion depends on glucose metabolism that produces ATP. The resulting increase in [ATP]/[ADP] ratio closes ATP-sensitive potassium (KATP) channels, which leads to membrane depolarization and opening of voltage-dependent Ca2+ channels. This causes an elevation of intracellular free Ca2+ and insulin exocytosis. Insulin is secreted in a pulsatile manner, which is thought to be regulated in part by oscillations in glucose metabolism. Such metabolic oscillations would also lead to oscillations in the [ATP]/[ADP] ratio and hence regulate KATP channel activity.Oscillations in [ATP]/[ADP] ratio have been demonstrated using biochemical and luciferase assays, but neither approach allows measurements of such oscillations in single cells. Perceval is a recently developed fluorescent protein biosensor for [ATP]/[ADP] ratio, and it permits direct measurement of [ATP]/[ADP] ratios inside living cells. We use Perceval in combination with quantitative confocal and two-photon excitation microscopy for direct measurement of the [ATP]/[ADP] ratio in beta-cells during glucose stimulated insulin secretion. For this purpose we have developed an adenoviral vector to express Perceval specifically in the beta-cells of intact mouse islets. Dynamic changes in [ATP]/[ADP] ratio can be correlated with glucose metabolism (by simultaneous imaging of Perceval fluorescence and NAD(P)H autofluorescence) and with intracellular free Ca2+ levels (by simultaneous imaging of Perceval fluorescence and the calcium sensor, FuraRed). This data allows us to test hypotheses regarding the role of localized subcellular signaling complexes and putative microdomains of glucose metabolism, [ATP]/[ADP] ratio, and Ca2+ dynamics in the regulation of glucose stimulated insulin secretion.

Ca2+ Sparks Generate Depolarizing STICs Causing Contraction And Asthmatic Hyperresponsiveness In Airway Smooth Muscle Cells

Ca2+ sparks are well known to be essential for controlling the relaxation of cerebral artery SMCs; however, the functional importance of this local Ca2+ signaling in other types of SMCs remains to be determined. Thus, the aim of this study was to investigate the role of Ca2+ sparks in airway SMCs. Our data reveal that spontaneous Ca2+ sparks could activate spontaneous transient inward currents (STICs) at the resting membrane potential and spontaneous transient outward currents (STOCs) at more positive membrane potentials in mouse airway SMCs. Application of ryanodine to block ryanodine receptors (RyRs) abolished spontaneous Ca2+ sparks without altering the whole-cell cytosolic Ca2+ levels in single airway myocytes and decreased the resting muscle tension in isolated airway rings, whereas activation of RyRs with a low concentration of caffeine had opposite effects. Iberiotoxin, a selective blocker of big-conductance Ca2+-activated K+ channels, eliminated STOCs, but did not affect either spontaneous Ca2+ spark activity or resting muscle tension. In contrast, NPPB, an inhibitor of Cl− channels, reduced resting muscle tension. The effect of NPPB was prevented in the presence of the selective voltage-dependent Ca2+ channel blocker nifedipine. We have also found that the activity of Ca2+ sparks in single asthmatic mouse airway SMCs and in-vivo airway resistance in asthmatic mice were significantly increased. Interestingly, ryanodine caused a stronger relaxation in asthmatic airway smooth muscle. Taken together, these findings suggest that spontaneous Ca2+ sparks can activate Ca2+-activated Cl− channels and then generate STICs, causing membrane depolarization, opening of voltage-dependent Ca2+ channels, extracellular Ca2+ influx and contraction in airway SMCs. Moreover, Ca2+ sparks and attendant STICs are both increased in asthmatic airway SMCs, which may contribute to asthmatic airway hyperresponsiveness.

Regulation of Rho/ROCK signaling in airway smooth muscle by membrane potential and [Ca2+]i

Recently, we have shown that Rho and Rho-activated kinase (ROCK) may become activated by high-millimolar KCl, which had previously been widely assumed to act solely through opening of voltage-dependent Ca(2+) channels. In this study, we explored in more detail the relationship between membrane depolarization, Ca(2+) currents, and activation of Rho/ROCK in bovine tracheal smooth muscle. Ca(2+) currents began to activate at membrane voltages more positive than -40 mV and were maximally activated above 0 mV; at the same time, these underwent time- and voltage-dependent inactivation. Depolarizing intact tissues by KCl challenge evoked contractions that were blocked equally, and in a nonadditive fashion, by nifedipine or by the ROCK inhibitor Y-27632. Other agents that elevate intracellular calcium concentration ([Ca(2+)](i)) by pathways independent of G protein-coupled receptors, namely the SERCA-pump inhibitor cyclopiazonic acid and the Ca(2+) ionophore A-23187, evoked contractions that were also largely reduced by Y-27632. KCl directly increased Rho and ROCK activities in a concentration-dependent fashion that paralleled closely the effect of KCl on tone and [Ca(2+)](i), as well as the voltage-dependent Ca(2+) currents that were measured over the voltage ranges that are evoked by 0-120 mM KCl. Through the use of various pharmacological inhibitors, we ruled out roles for Ca(2+)/calmodulin-dependent CaM kinase II, protein kinase C, and protein kinase A in mediating the KCl-stimulated changes in tone and Rho/ROCK activities. In conclusion, Rho is activated by elevation of [Ca(2+)](i) (although the signal transduction pathway underlying this Ca(2+) dependence is still unclear) and possibly also by membrane depolarization per se.

G Protein-independent Activation of an Inward Na+Current by Muscarinic Receptors in Mouse Pancreatic β-Cells

Depolarization of pancreatic beta-cells is critical for stimulation of insulin secretion by acetylcholine but remains unexplained. Using voltage-clamped beta-cells, we identified a small inward current produced by acetylcholine, which was suppressed by atropine or external Na(+) omission, but was not mimicked by nicotine, and was insensitive to nicotinic antagonists, tetrodotoxin, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DiDS), thapsigargin pretreatment, and external Ca(2+) and K(+) removal. This suggests that muscarinic receptor stimulation activates voltage-insensitive Na(+) channels distinct from store-operated channels. No outward Na(+) current was produced by acetylcholine when the electrochemical Na(+) gradient was reversed, indicating that the channels are inward rectifiers. No outward K(+) current occurred either, and the reversal potential of the current activated by acetylcholine in the presence of Na(+) and K(+) was close to that expected for a Na(+)-selective membrane, suggesting that the channels opened by acetylcholine are specific for Na(+). Overnight pretreatment with pertussis toxin or the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) or guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S) instead of GTP to the pipette solution did not alter this current, excluding involvement of G proteins. Injection of a current of a similar amplitude to that induced by acetylcholine elicited electrical activity in beta-cells perifused with a subthreshold glucose concentration. These results demonstrate that muscarinic receptor activation in pancreatic beta-cells triggers, by a G protein-independent mechanism, a selective Na(+) current that explains the plasma membrane depolarization.

Ca2+ signaling in mouse pancreatic polypeptide cells.

Ca2+ signaling was studied in pancreatic polypeptide (PP)-secreting cells isolated from mouse islets of Langerhans. After measuring the cytoplasmic Ca2+ concentration ([Ca2+]i), the cells were identified by immunocytochemistry. Most PP-cells reacted to carbachol and epinephrine with prompt and reversible elevation of [Ca2+]i, often manifested as slow oscillations. The carbachol effect was muscarinic, because it was inhibited by atropine. Beta-adrenergic elevation of cAMP explains the epinephrine stimulation, which was mimicked by an activator of adenylate cyclase and blocked by an inhibitor of protein kinase A. The responses to carbachol and epinephrine apparently involve depolarization with opening of voltage-dependent Ca2+ channels, because the effects were prevented by the Ca2+ channel antagonist methoxyverapamil and by diazoxide, which activates ATP-dependent K+ (K(ATP)) channels. Being equipped with K(ATP) channels, the PP-cells often responded to tolbutamide or high concentrations of glucose with elevation of [Ca2+]i. Somatostatin reversed the [Ca2+]i elevation obtained by carbachol, epinephrine, tolbutamide, and glucose. These preliminary studies support the idea that glucose has a direct stimulatory effect on the PP-cells, which can be masked by locally released somatostatin. Expressing both K(ATP) channels and voltage-dependent Ca2+ channels, the PP-cells share fundamental regulatory mechanisms with other types of islet cells.

Sulfonylureas enhance exocytosis from pancreatic beta-cells by a mechanism that does not involve direct activation of protein kinase C.

Hypoglycemic sulfonylureas stimulate insulin release by binding to a regulatory subunit of plasma membrane ATP-sensitive K+ (K(ATP)) channels. The consequent closure of K(ATP) channels leads to depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx, and a rise in intracellular [Ca2+]. Recently, however, it has been suggested that sulfonylureas may have an additional action on secretion, independent of changes in intracellular [Ca2+] but dependent on the activity of protein kinase C (PKC). We have investigated the mechanisms involved in the PKC-dependent effect of sulfonylureas on the secretion machinery in beta-cells. In MIN6 beta-cells permeabilized by streptolysin O, insulin release was stimulated by elevation of [Ca2+] from 10(-8) to 10(-5) mol/l. At a [Ca2+] of 10(-8) mol/l, insulin release from permeabilized beta-cells was stimulated by addition of GTP-gamma-S, or by addition of a phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA, but not GTP-gamma-S, also increased insulin release when [Ca2+] was 10(-5) mol/l. Insulin release from permeabilized beta-cells was stimulated by tolbutamide (0.1-1 mmol/l) at 10(-8) but not at 10(-5) mol/l Ca2+. The effect of tolbutamide was blocked either by inhibition of PKC or when phorbol ester-sensitive PKC isoforms were maximally stimulated by TPA. Meglitinide and glibenclamide also stimulated insulin release from permeabilized beta-cells. To assess the possibility that direct activation of PKC mediates the exocytotic response to sulfonylureas, we studied the effect of tolbutamide and glibenclamide on PKC activity. Purified brain PKC was not activated by tolbutamide or glibenclamide, whether tested in the absence or presence of phosphatidylserine or TPA, or at low or high [Ca2+]; nor was the total PKC activity in extracts of MIN6 beta-cells affected by tolbutamide. Neither tolbutamide nor glibenclamide elicited translocation of any isoform of PKC in intact or permeabilized beta-cells under conditions in which TPA evoked a marked redistribution of PKC alpha- and epsilon-isoforms. We conclude that although the plasma membrane K(ATP) channel-independent stimulation of exocytosis by sulfonylureas may require functional PKC, the mechanism does not involve a direct activation of the enzyme.

A Ca2+-induced Ca2+ release mechanism involved in asynchronous exocytosis at frog motor nerve terminals.

The extent to which Ca2+-induced Ca2+ release (CICR) affects transmitter release is unknown. Continuous nerve stimulation (20-50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequency (MEPP-hump) and intracellular free Ca2+ ([Ca2+]i) in presynaptic terminals (Ca2+-hump) in frog skeletal muscles over a period of minutes in a low Ca2+, high Mg2+ solution. Mn2+ quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by opening of voltage-dependent Ca2+ channels. MEPP-hump depended on extracellular Ca2+ (0.05-0.2 mM) and stimulation frequency. Both the Ca2+- and MEPP-humps were blocked by 8-(N, N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enhanced by CN-. Thus, Ca2+-hump is generated by the activation of CICR via ryanodine receptors by Ca2+ entry, producing MEPP-hump. A short interruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP frequency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous or higher level. Thus, the steady/equilibrium condition balancing CICR and Ca2+ clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP-hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca2+-hump affected little MEPP frequency or [Ca2+]i, but caused a quick increase (faster than MEPP- or Ca2+-hump) after the pause, whose magnitude increased with an increase in pause duration (<1 min), suggesting that Ca2+ entry-dependent inactivation, but not depriming process, explains the decay of the humps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endowed with Ca2+ entry-dependent slow priming and fast inactivation mechanisms, as well as Ca2+ entry-dependent activation, and involved in asynchronous exocytosis. Physiological significance of CICR in presynaptic terminals was discussed.

Synergism between toxin-gamma from Brazilian scorpion Tityus serrulatus and veratridine in chromaffin cells.

Toxin-gamma (Tgamma) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tgamma was approximately 200-fold more potent than veratridine judged from EC50 values, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tgamma (1.2 vs. 12 microg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca2+ concentration ([Ca2+]o), when 30 microM veratridine plus 0.45 microM Tgamma were used. Tgamma (0.45 microM) doubled the basal uptake of 45Ca2+, whereas veratridine (100 microM) tripled it. Again, a drastic synergism in enhancing Ca2+ entry was seen when Tgamma and veratridine were combined; this was particularly pronounced at 5 mM [Ca2+]o. Veratridine induced oscillations of cytosolic Ca2+ concentration ([Ca2+]i) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tgamma elevated basal [Ca2+]i levels, causing only small oscillations. When added together, Tgamma and veratridine elevated the basal levels of [Ca2+]i without causing large oscillations. Tgamma shifted the current-voltage (I-V) curve for Na+ channel current to the left. The combination of Tgamma with veratridine increased the shift of the I-V curve to the left, resulting in a greater recruitment of Na+ channels at more hyperpolarizing potentials. This led to enhanced and more rapid accumulation of Na+ in the cell, causing cell depolarization, the opening of voltage-dependent Ca2+ channels, and Ca2+ entry and secretion.

Partition of the organochlorine insecticide lindane into the human sperm surface induces membrane depolarization and Ca2+ influx.

The effects of the insecticide lindane (the gamma-isomer of 1,2,3,4,5,6-hexachlorocyclohexane) on membrane potential, cytosolic free Ca2+ concentration ([Ca2+]i) and surface biophysical properties were studied in human spermatozoa. The insecticide induces rapid, transient and reproducible membrane depolarization and opening of voltage-dependent Ca2+ channels leading to an increase in [Ca2+]i. In contrast with the effect in somatic cells, lindane did not affect gamma-aminobutyric acid receptor-linked Cl- currents. Ca2+ and K+ currents were found to drive lindane-induced membrane depolarization and repolarization respectively, whereas Na+ and Cl- fluxes appear not to have a role in the phenomenon. The insecticide was still able to produce membrane depolarization both in the combined absence of extracellular Ca2+ and Na+ and in high-K+ buffer, suggesting that lindane alters the membrane dipole potential. In agreement with this, Laurodan and Prodan fluorescence spectroscopy revealed that lindane partition into the sperm plasma membrane lowers water molecular dynamics in the uppermost region of the membrane external leaflet, probably as the result of reordering of water dipoles. We propose that the first effect of lindane partitioning into the sperm plasma membrane is a change in the membrane dipole potential, which results in the activation of membrane-located Ca2+-influx pathways.

Molecular cloning of the cDNA encoding beta-cell calcium/calmodulin-dependent protein kinase II.

Current hypotheses postulate that the metabolism of glucose within islets results in a transient increase in the ATP:ADP ratio, leading to depolarization and opening of voltage-dependent Ca2+ channels, promoting influx of Ca2+ into the β-cell. The increase in intracellular calcium may activate Ca2+-dependent protein kinases, which in turn phosphorylate key components in the secretory machinery. The nature and identity of these components are at present unclear. In addition, the β-cell contains a high (15–50 μM) concentration of calmodulin (CaM) and it has been shown that inhibitors of CaM block insulin secretion. Possible candidates for Ca2+/calmodulin-dependent protein kinases acting as regulatory enzymes in the exocytotic release process are phosphorylase kinase, myosin light chain kinase and CaM kinases.KeywordsNorthern AnalysisMyosin Light Chain KinasePhosphorylase KinaseAutographa Californica Nuclear Polyhedrosis VirusEncode Protein KinaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Effect of low temperatures on glucose-induced insulin secretion and ionic fluxes in rat pancreatic islets.

The direct effect of cold on the inhibition of B cell secretion is well known in hibernating and experimentally hypothermic mammals. This temperature dependency may result from the inhibition of ion transport across the membranes. In order to verify this hypothesis, ionic effluxes and insulin secretion from rat islets loaded with 86Rb+ and 45Ca+ were measured during perifusion. At 37 degrees C, the rise in glucose concentration from zero to 16.7 mmol/l provoked a rapid decrease in 86Rb+ efflux, an early fall and subsequent rise in 45Ca2+ efflux and a typical biphasic pattern of insulin secretion. At 27 degrees C, glucose induced only a very slight increase in insulin secretion, while the fluxes of radioactive ions were not significantly modified in amplitude but were clearly delayed. At 17 degrees C, no insulin response to glucose was observed and the decrease in K+ conductance indicated by 86Rb+ flux decrease was less temperature-dependent than the movement of Ca2+. After supplementary stimulation with a high extracellular concentration of Ca2+, insulin secretion was enhanced at 27 degrees C and reached levels induced by glucose alone at 37 degrees C. An increase in hormone secretion occurred even at 17 degrees C, but only during a first phase of secretion. Regular increases in temperature potentiated insulin secretion and provoked changes in ionic fluxes which suggest that B cell depolarization (86Rb+ flux decrease) induced by glucose can occur at 15 degrees C but cannot induce the opening of voltage-dependent Ca2+ channels (increase in 45Ca2+ efflux) until temperatures higher than 27 degrees C are reached.(ABSTRACT TRUNCATED AT 250 WORDS)