Role of voltage-dependent Na+ channels for rhythmic Ca2+ signalling in glucose-stimulated mouse pancreatic beta-cells.

The putative role of voltage-dependent Na+ channels for glucose induction of rhythmic Ca2+ signalling was studied in mouse pancreatic beta-cells with the use of the Ca2+ indicator fura-2. A rise in glucose from 3 to 11 mM resulted in slow oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i). These oscillations, as well as superimposed transients seen during forskolin-induced elevation of cAMP, remained unaffected in the presence of the Na+ channel blocker tetrodotoxin. During exposure to 1-10 microM veratridine, which facilitates the opening of voltage-dependent Na+ channels, the slow oscillations were replaced by repetitive and pronounced [Ca2+]i transients arising from the basal level. The effects of veratridine were reversed by tetrodotoxin. The veratridine-induced [Ca2+]i transients were critically dependent on the influx of Ca2+ and persisted after thapsigargin inhibition of the endoplasmic reticulum Ca2+-ATPase. Both tolbutamide and ketoisocaproate mimicked the action of glucose in promoting [Ca2+]i transients in the presence of veratridine. It is suggested that activation of voltage-dependent Na+ channels is a useful approach for amplifying Ca2+ signals for insulin release.     

Dual effects of glucose on the cytosolic Ca2+ activity of mouse pancreatic beta-cells

The cytosolic Ca2+ activity of mouse pancreatic beta-cells was studied with the intracellular fluorescent indicator quin2 . When the extracellular Ca2+ concentration was 1.20 mM, the basal cytosolic Ca2+ activity was 162 +/- 9 nM. Stimulation with 20 mM glucose increased this Ca2+ activity by 40%. In the presence of only 0.20 mM Ca2+ or after the addition of the voltage-dependent Ca2+ -channel blocker D-600, glucose had an opposite and more prompt effect in reducing cytosolic Ca2+ by about 15%. It is concluded that an early result of glucose exposure is a lowering of the cytosolic Ca2+ activity and that this effect tends to be masked by a subsequent increase of the Ca2+ activity due to influx of Ca2+ through the voltage-dependent Ca2+ channels.     

Glucose induces temperature-dependent oscillations of cytoplasmic Ca2+ in single pancreatic beta-cells related to their electrical activity

Glucose induces large amplitude oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells. The effects of temperature on these oscillations were examined by monitoring [Ca2+]i continuously in single beta-cells from ob/ob-mice using dual wavelength microfluorometry. The oscillations of [Ca2+]i disappeared when the temperature was increased above 42 degrees C and were reversibly inhibited below 30 degrees C. However, cooling did not prevent a glucose response in terms of the average rise of [Ca2+]i. Since patch clamp studies of single beta-cells have indicated a random occurrence of glucose-induced action potentials at room temperature, it was important to explore how the sugar affected the electrical activity at 37 degrees C. Using the cell-attached configuration of the patch clamp technique for such analyses, the action potentials were found to occur in bursts with durations similar to the large amplitude oscillations of [Ca2+]i.     

Effect of Na/Ca exchange on plateau fraction and [Ca]i in models for bursting in pancreatic beta-cells.

In the presence of an insulinotropic glucose concentration, beta-cells, in intact pancreatic islets, exhibit periodic bursting electrical activity consisting of an alternation of active and silent phases. The fraction of time spent in the active phase over a period is called the plateau fraction and is correlated with the rate of insulin release. However, the mechanisms that regulate the plateau fraction remain unclear. In this paper we investigate the possible role of the plasma membrane Na+/Ca2+ exchange of the beta-cell in controlling the plateau fraction. We have extended different single-cell models to incorporate this Ca2+-activated electrogenic Ca2+ transporter. We find that the Na+/Ca2+ exchange can provide a physiological mechanism to increase the plateau fraction as the glucose concentration is raised. In addition, we show theoretically that the Na+/Ca2+ exchanger is a key regulator of the cytoplasmic calcium concentration in clusters of heterogeneous cells with gap-junctional electrical coupling.     

Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release

In cardiac mitochondria, matrix free Ca²⁺ ([Ca²⁺]_m) is primarily regulated by Ca²⁺ uptake and release via the Ca²⁺ uniporter (CU) and Na⁺/Ca²⁺ exchanger (NCE) as well as by Ca²⁺ buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca²⁺ buffering affects these dynamics under various Ca²⁺ uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca²⁺ buffering on the uptake and release of Ca²⁺, we monitored Ca²⁺ dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca²⁺] ([Ca²⁺]_e) and [Ca²⁺]_m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl₂] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca²⁺]_e and [Ca²⁺]_m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca²⁺-induced changes in mitochondrial bioenergetics. Our [Ca²⁺]_e and [Ca²⁺]_m measurements demonstrate that Ca²⁺ uptake and release do not show reciprocal Ca²⁺ dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca²⁺ buffering system in the matrix compartment. The Na⁺- induced Ca²⁺ release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca²⁺ uptake in the presence of large amounts of CaCl₂, but not by Na⁺- induced Ca²⁺ release.     

Properties of voltage-gated Ca2+ currents measured from mouse pancreatic beta-cells in situ

We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreatic beta-cells within intact mouse islets of Langerhans at 37 degrees C, the typical preparation for studies of glucose-induced "bursting" electrical activity. Cells were impaled with intracellular microelectrodes, and voltage pulses were applied in the presence of tetraethylammonium. Under these conditions, a voltage-dependent Ca2+ current (I(Cav)), containing L-type and non-L-type components, was observed. The current measured in situ was larger than that measured in single cells with whole-cell patch clamping, particularly at membrane potentials corresponding to the action potentials of beta-cell electrical activity. The temperature dependence of I(Cav) was not sufficient to account for the difference in size of the currents recorded with the two methods. During prolonged pulses, the voltage-dependent Ca2+ current measured in situ displayed both rapid and slow components of inactivation. The rapid component was Ca2+-dependent and was inhibited by the membrane-permeable Ca2+ chelator, BAPTA-AM. The effect of BAPTA-AM on beta-cell electrical activity then demonstrated that Ca2+-dependent inactivation of I(Cav) contributes to action potential repolarization and to control of burst frequency. Our results demonstrate the utility of voltage clamping beta-cells in situ for determining the roles of ion channels in electrical activity and insulin secretion.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Glucose inhibits 45Ca efflux from pancreatic beta-cells also in the absence of Na+-Ca2+ countertransport

During perifusion with medium deprived of Ca2+, addition of glucose or omission of Na+ resulted in prompt and quantitatively similar inhibitions of 45Ca efflux from beta-cell rich pancreatic islets microdissected from ob/ob mice. Glucose had no additional inhibitory effect when Na+ was isoosmotically replaced by sucrose or choline+. When K+ was used as a substitute for Na+, the inhibitory effect of Na+ removal on 45Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting 45Ca efflux in the presence or absence of Na+ is difficult to reconcile with the postulate that the Na+-Ca2+ countertransport mechanism is a primary site of action for glucose.     

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.     

Three types of cytoplasmic Ca2+ oscillations in stimulated pancreatic beta-cells

Oscillations of cytoplasmic Ca2+ (Ca2+i) involved in cell regulation have recently attracted considerable attention. In the pancreatic beta-cells an intermediate concentration of glucose (11 mM) induces large oscillations of Ca2+i with periods of 2 to 6 min. Procedures stimulating insulin secretion further, such as raising glucose to 20-30 mM or adding carbachol, ATP, theophylline, glucagon, or forskolin, often changed these oscillations into a steady increase of Ca2+i. In addition, forskolin and glucagon triggered prominent 9- to 14-s Ca2+i spikes during the intervals of increased Cai2+, whereas carbachol and ATP initiated a series of rapid spikes of decreasing magnitude and increasing duration (6-11 s). All types of oscillations depended on the presence of extracellular Ca2+i, but carbachol and ATP also induced single Cai2+ transients in the absence of the cation. The results demonstrate hitherto unknown oscillations of Ca2+i in the pancreatic beta-cell which are dependent in different ways on Ca2+ entry.     

The role of Na+/Ca2+ exchange in the generation of delayed electrical activity of smooth muscle cells

It was shown that electrogenic 3Na+/Ca2+ antiport evoked depolarization of the membranes and Ca2+ ions influx through Ca2+ activated channels which lead to the appearance of stable self-oscillation of the membrane potential (MP), as well as Na+ and Ca2+ ions intracellular concentrations.     

Dependence of Na+-Ca2+ exchange and Ca2+-Ca2+ exchange on monovalent cations

Two mechanisms of passive Ca2+ transport, Na+-Ca2+ exchange and Ca2+-Ca2+ exchange, were studied using highly-purified dog heart sarcolemmal vesicles. About 80% of the Ca2+ accumulated by Na+-Ca2+ exchange or Ca2+-Ca2+ exchange could be released as free Ca2+, while up to 20% was probably bound. Na+-Ca2+ exchange was simultaneous, coupled countertransport of Na+ and Ca2+. The movement of anions during Na+-Ca2+ exchange did not limit the initial rate of Na+-Ca2+ exchange. Na+-Ca2+ exchange was electrogenic, with a reversal potential of about -105 mV. The apparent flux ratio of Na+-Ca2+ exchange was 4 Na+:1 Ca2+. Coupled cation countertransport by the Na+-Ca2+ exchange mechanism required a monovalent cation gradient with the following sequence of ion activation: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+. In contrast to Na+-Ca2+ exchange, Ca2+-Ca2+ exchange did not require a monovalent cation gradient, but required the presence of Ca2+ plus a monovalent cation on both sides of the vesicle membrane. The sequence of ion activation of Ca2+-Ca2+ exchange was: K+ much greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Na+ inhibited Ca2+-Ca2+ exchange when Ca2+-Ca2+ exchange was supported by another monovalent cation. Both Na+-Ca2+ exchange and Ca2+-Ca2+ exchange were inhibited, but with different sensitivities, by external MgCl2, quinidine, or verapamil.     

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.     

Spatial organization of Ca(2+) entry and exocytosis in mouse pancreatic beta-cells

Secretion from single pancreatic beta-cells was imaged using a novel technique in which Zn(2+), costored in secretory granules with insulin, was detected by confocal fluorescence microscopy as it was released from the cells. Using this technique, it was observed that secretion from beta-cells was limited to an active region that comprised approximately 50% of the cell perimeter. Using ratiometric imaging with indo-1, localized increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by membrane depolarization were also observed. Using sequential measurements of secretion and [Ca(2+)](i) at single cells, colocalization of exocytotic release sites and Ca(2+) entry was observed when cells were stimulated by glucose or K(+). Treatment of cells with the Ca(2+) ionophore 4-Br-A23187 induced large Ca(2+) influx around the entire cell circumference. Despite the nonlocalized increase in [Ca(2+)](i), secretion evoked by 4-Br-A23187 was still localized to the same region as that evoked by secretagogues such as glucose. It is concluded that Ca(2+) channels activated by depolarization are localized to specific membrane domains where exocytotic release also occurs; however, localized secretion is not exclusively regulated by localized increases in [Ca(2+)](i), but instead involves spatial localization of other components of the exocytotic machinery.     

Direct measurements of increased free cytoplasmic Ca2+ in mouse pancreatic beta-cells following stimulation by hypoglycemic sulfonylureas

The effects of the hypoglycemic sulfonylureas tolbutamide and glibenclamide on free cytoplasmic Ca2+, [Ca2+]i, were compared with that of a depolarizing concentration of K+ in dispersed and cultured pancreatic beta-cells from ob/ob mice. [Ca2+]i was measured with the fluorescent Ca2+-indicator quin2. The basal level corresponded to 150 nM and increased to 600 nM after exposure to 30.9 mM K+. The corresponding levels after stimulation with 1 microM glibenclamide and 100 microM tolbutamide were 390 and 270 nM respectively. K+ depolarization increased [Ca2+]i more rapidly than either of the sulfonylureas. It is suggested that the increased [Ca2+]i obtained after stimulation by sulfonylureas is due to depolarization of the beta-cells with subsequent entry of Ca2+ through voltage-dependent channels.     

Dual effect of glucose on cytoplasmic Ca2+ in single pancreatic beta-cells

The cytoplasmic Ca2+ concentration (Ca2+i) was measured in single pancreatic beta-cells from ob/ob-mice using the fluorescent indicator fura-2. Raising the glucose concentration from 3 to 20 mM resulted in 25% initial lowering of Ca2+i, followed by 250% rise above the basal level of 49 +/- 3 nM. Tolbutamide (100 microM) was as effective as glucose in increasing Ca2+i, although its action was more rapid and not preceded by any reduction. The results support the concept that stimulated removal of Ca2+ from the cytoplasm is an essential part of the physiological glucose effect on the pancreatic beta-cells.     

Inhibition of electrical activity in mouse pancreatic beta-cells by the ATP/ADP translocase inhibitor, bongkrekic acid

Bongkrekic acid causes fatal food poisoning which is associated with hyperglycaemia. Here we demonstrate that bongkrekic acid, a potent inhibitor of the mitochondrial ATP/ADP translocase, inhibits glucose-induced electrical activity in the pancreatic beta-cell through the stimulation of ATP-sensitive potassium channel (K-ATP-channel) activity. By comparison of its effects with those of oligomycin, we suggest that bongkrekic acid acts by the inhibition of glucose metabolism and may induce hyperglycaemia by impairing beta-cell function.     

Na+/Ca2+ exchange in coated microvesicles.

Coated microvesicles isolated from bovine neurohypophyses could be loaded with Ca2+ in two different ways, either by incubation in the presence of ATP or by imposition of an outwardly directed Na+ gradient. Na+, but not K+, was able to release Ca2+ accumulated by the coated microvesicles. These results suggest the existence of an ATP-dependent Ca2+-transport system as well as of a Na+/Ca2+ carrier in the membrane of coated microvesicles similar to that present in the membranes of secretory vesicles from the neurohypophysis. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ of the ATP-dependent uptake was 0.8 microM. The average Vmax. was 2 nmol of Ca2+/5 min per mg of protein. The total capacity of microvesicles for Ca2+ uptake was 3.7 nmol/mg of protein. Both nifedipine (10 microM) and NH4Cl (50 mM) inhibited Ca2+ uptake. The ATPase activity in purified coated-microvesicles fractions from brain and neurohypophysis was characterized. Micromolar concentrations of Ca2+ in the presence of millimolar concentrations of Mg2+ did not change enzyme activity. Ionophores increasing the proton permeability across membranes activated the ATPase activity in preparations of coated microvesicles from brain as well as from the neurohypophysis. Thus the enzyme exhibits properties of a proton-transporting ATPase. This enzyme seems to be linked to the ion accumulation by coated microvesicles, although the precise coupling of the proton transport to Ca2+ and Na+ fluxes remains to be determined.