Synchronization of Ca(2+)-signals within insulin-secreting pseudoislets: effects of gap-junctional uncouplers

The secretory response of the intact islet is greater than the response of individual beta-cells in isolation, and functional coupling between cells is critical in insulin release. The changes in intracellular Ca(2+)([Ca(2+)](i)) which initiate insulin secretory responses are synchronized between groups of cells within the islet, and gap-junctions are thought to play a central role in coordinating signalling events. We have used the MIN6 insulin-secreting cell line, to examine whether uncoupling gap-junctions alters the synchronicity of nutrient- and non-nutrient-evoked Ca(2+)oscillations, or affects insulin secretion. MIN6 cells express mRNA species that can be amplified using PCR primers for connexin 36. A commonly used gap-junctional inhibitor, heptanol, inhibited glucose- and tolbutamide-induced Ca(2+)-oscillations to basal levels in MIN6 cell clusters at concentrations of 0.5 mM and greater, and it had similar effects in pseudoislets when used at 2.5 mM. Lower heptanol concentrations altered the frequency of Ca(2+)transients without affecting their synchronicity, in both monolayers and pseudoislets. Heptanol also had effects on insulin secretion from MIN6 pseudoislets such that 1 mM enhanced secretion while 2.5 mM was inhibitory. These data suggest that heptanol has multiple effects in pancreatic beta-cells, none of which appears to be related to uncoupling of synchronicity of Ca(2+)signalling between cells. A second gap-junction uncoupler, 18 alpha-glycyrrhetinic acid, also failed to uncouple synchronized Ca(2+)-oscillations, and it had no effect on insulin secretion. These data provide evidence that Ca(2+)signalling events occur simultaneously across the bulk mass of the pseudoislet, and suggest that gap-junctions are not required to coordinate the synchronicity of these events, nor is communication via gap junctions essential for integrated insulin secretory responses.     

Alpha-latrotoxin induces exocytosis by inhibition of voltage-dependent K+ channels and by stimulation of L-type Ca2+ channels via latrophilin in beta-cells

The spider venom alpha-latrotoxin (alpha-LTX) induces massive exocytosis after binding to surface receptors, and its mechanism is not fully understood. We have investigated its action using toxin-sensitive MIN6 beta-cells, which express endogenously the alpha-LTX receptor latrophilin (LPH), and toxin-insensitive HIT-T15 beta-cells, which lack endogenous LPH. alpha-LTX evoked insulin exocytosis in HIT-T15 cells only upon expression of full-length LPH but not of LPH truncated after the first transmembrane domain (LPH-TD1). In HIT-T15 cells expressing full-length LPH and in native MIN6 cells, alpha-LTX first induced membrane depolarization by inhibition of repolarizing K(+) channels followed by the appearance of Ca(2+) transients. In a second phase, the toxin induced a large inward current and a prominent increase in intracellular calcium ([Ca(2+)](i)) reflecting pore formation. Upon expression of LPH-TD1 in HIT-T15 cells just this second phase was observed. Moreover, the mutated toxin LTX(N4C), which is devoid of pore formation, only evoked oscillations of membrane potential by reversible inhibition of iberiotoxin-sensitive K(+) channels via phospholipase C, activated L-type Ca(2+) channels independently from its effect on membrane potential, and induced an inositol 1,4,5-trisphosphate receptor-dependent release of intracellular calcium in MIN6 cells. The combined effects evoked transient increases in [Ca(2+)](i) in these cells, which were sensitive to inhibitors of phospholipase C, protein kinase C, or L-type Ca(2+) channels. The latter agents also reduced toxin-induced insulin exocytosis. In conclusion, alpha-LTX induces signaling distinct from pore formation via full-length LPH and phospholipase C to regulate physiologically important K(+) and Ca(2+) channels as novel targets of its secretory activity.     

Normalization of intracellular Ca(2+) induces a glucose-responsive state in glucose-unresponsive beta-cells

Although intracellular Ca(2+) in pancreatic beta-cells is the principal signal for insulin secretion, the effect of chronic elevation of the intracellular Ca(2+) concentration ([Ca(2+)](i)) on insulin secretion is poorly understood. We recently established two pancreatic beta-cell MIN6 cell lines that are glucose-responsive (MIN6-m9) and glucose-unresponsive (MIN6-m14). In the present study we have determined the cause of the glucose unresponsiveness in MIN6-m14. Initially, elevated [Ca(2+)](i) was observed in MIN6-m14, but normalization of the [Ca(2+)](i) by nifedipine, a Ca(2+) channel blocker, markedly improved the intracellular Ca(2+) response to glucose and the glucose-induced insulin secretion. The expression of subunits of ATP-sensitive K(+) channels and voltage-dependent Ca(2+) channels were increased at both mRNA and protein levels in MIN6-m14 treated with nifedipine. As a consequence, the functional expression of these channels at the cell surface, both of which are decreased in MIN6-m14 without nifedipine treatment, were increased significantly. Contrariwise, Bay K8644, a Ca(2+) channel agonist, caused severe impairment of glucose-induced insulin secretion in glucose-responsive MIN6-m9 due to decreased expression of the channel subunits. Chronically elevated [Ca(2+)](i), therefore, is responsible for the glucose unresponsiveness of MIN6-m14. The present study also suggests normalization of [Ca(2+)](i) in pancreatic beta-cells as a therapeutic strategy in treatment of impaired insulin secretion.     

Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells

The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.     

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.     

Depolarizing stimuli reduce Ca(2+)/calmodulin-dependent protein kinase II activity in islets of Langerhans

Elevations in intracellular Ca(2+) ([Ca(2+)](i)) initiate insulin secretion from pancreatic beta-cells, but the secretory responses become rapidly desensitised to maintained elevations in [Ca(2+)](i). We have investigated the mechanisms underlying the Ca(2+) desensitization of insulin secretion using electrically permeabilized rat islets of Langerhans. Measurements of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) enzyme activity and immunoreactivity in permeabilized islets demonstrated Ca(2+)-induced reductions in enzyme activity which could not be attributed to reductions in CaMK II immunoreactive protein. Measurements in intact islets demonstrated that the Ca(2+)-induced reduction of CaMK II activity was also operative in intact cells, suggesting that this mechanism may have pathophysiological implications for beta-cell function.     

Glucose-mediated Ca(2+) signalling in single clonal insulin-secreting cells: evidence for a mixed model of cellular activation

Using clonal insulin-secreting BRIN-BD11 cells, we have assessed whether the graded response of the whole cell population to glucose can be accounted for by a dose-dependent recruitment of individual cells, an amplification of the response of the recruited cells or both. Cytosolic free Ca(2+) concentration ([Ca(2+)](i)) is an established index of beta-cell function. We used fura-2 microfluorescence techniques to assess the [Ca(2+)](i) responsiveness of single BRIN-BD11 cells to glucose and other secretagogues. Glucose (1-16.7 mM) evoked oscillatory [Ca(2+)](i) rises in these cells resembling those found in parental rat pancreatic beta-cells. The percentage of glucose-responsive cells was 11% at 1 mM and increased to 40-70% at 3-16.7 mM glucose, as assessed by a single-stimulation protocol. This profile was unrelated to possible differences in the cell cycle, as inferred from experiments where the cultured cells were synchronized by a double thymidine block protocol. Individual cells exhibited variable sensitivities to glucose (threshold range: 1-5 mM) and a variable dose-dependent amplification of the [Ca(2+)](i) responses (EC(50) range: 2-10 mM), as assessed by a multiple-stimulation protocol. Glyceraldehyde and alpha-ketoisocaproic acid had glucose-like effects on [Ca(2+)](i). The data support a mixed model for the activation of insulin-secreting cells. Specifically, the graded secretory response of the whole cell population is likely to reflect both a recruitment of individual cells with different sensitivities to glucose and a dose-dependent amplification of the response of the recruited cells.     

The oscillatory behavior of pancreatic islets from mice with mitochondrial glycerol-3-phosphate dehydrogenase knockout

Glucose stimulation of pancreatic beta cells induces oscillations of the membrane potential, cytosolic Ca(2+) ([Ca(2+)](i)), and insulin secretion. Each of these events depends on glucose metabolism. Both intrinsic oscillations of metabolism and repetitive activation of mitochondrial dehydrogenases by Ca(2+) have been suggested to be decisive for this oscillatory behavior. Among these dehydrogenases, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate NADH shuttle, is activated by cytosolic [Ca(2+)](i). In the present study, we compared different types of oscillations in beta cells from wild-type and mGPDH(-/-) mice. In clusters of 5-30 islet cells and in intact islets, 15 mM glucose induced an initial drop of [Ca(2+)](i), followed by an increase in three phases: a marked initial rise, a partial decrease with rapid oscillations and eventually large and slow oscillations. These changes, in particular the frequency of the oscillations and the magnitude of the [Ca(2+)] rise, were similar in wild-type and mGPDH(-/-) mice. Glucose-induced electrical activity (oscillations of the membrane potential with bursts of action potentials) was not altered in mGPDH(-/-) beta cells. In single islets from either type of mouse, insulin secretion strictly followed the changes in [Ca(2+)](i) during imposed oscillations induced by pulses of high K(+) or glucose and during the biphasic elevation induced by sustained stimulation with glucose. An imposed and controlled rise of [Ca(2+)](i) in beta cells similarly increased NAD(P)H fluorescence in control and mGDPH(-/-) islets. Inhibition of the malate-aspartate NADH shuttle with aminooxyacetate only had minor effects in control islets but abolished the electrical, [Ca(2+)](i) and secretory responses in mGPDH(-/-) islets. The results show that the two distinct NADH shuttles play an important but at least partially redundant role in glucose-induced insulin secretion. The oscillatory behavior of beta cells does not depend on the functioning of mGPDH and on metabolic oscillations that would be generated by cyclic activation of this enzyme by Ca(2+).     

Relative contribution of the Na(+)/Ca(2+) exchanger, mitochondria and endoplasmic reticulum in the regulation of cytosolic Ca(2+) and catecholamine secretion of bovine adrenal chromaffin cells

The relative importance of mitochondria, the Na(+)/Ca(2+) exchanger (NCX) and the endoplasmic reticulum (ER) in the regulation of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) were examined in bovine chromaffin cells using fura-2 for average [Ca(2+)](i) and amperometry for secretory activity, which reflects the local Ca(2+) concentration near the exocytotic sites. Chromaffin cells were stimulated by a high concentration of K(+) when the three Ca(2+) removal mechanisms were individually or simultaneously inhibited. When the mitochondrial Ca(2+) uptake was inhibited, the [Ca(2+)](i) decayed at a significantly slower rate and the secretory activity was higher than the control cells. The NCX appears to function only in the initial phase of [Ca(2+)](i) decay and when the ER Ca(2+) pump is blocked. Similarly, the ER had a significant effect on the [Ca(2+)](i) decay and on the secretion only when the NCX was blocked. Inhibition of all three mechanisms leads to a substantial delay in [Ca(2+)](i) recovery and an increase in the secretion. The results suggest that the three mechanisms work together in the regulation of the Ca(2+) near the Ca(2+) channels and exocytotic sites and therefore modulate the secretory activity. When Ca(2+) diffuses away from the exocytotic sites, the mitochondrial Ca(2+) uptake becomes the dominant mechanism.     

Membrane potential dependent modulations of calcium oscillations in insulin-secreting INS-1 cells

This study was undertaken to examine the role of K(+) channels on cytosolic Ca(2+) ([Ca(2+)](i)) in insulin secreting cells. [Ca(2+)](i) was measured in single glucose-responsive INS-1 cells using the fluorescent Ca(2+) indicator Fura-2. Glucose, tolbutamide and forskolin elevated [Ca(2+)](i) and induced [Ca(2+)] oscillations. Whereas the glucose effect was delayed and observed in 60% and 93% of the cells, in a poorly and a highly glucose-responsive INS-1 cell clone, respectively, tolbutamide and forskolin increased [Ca(2+)](i) in all cells tested. In the latter clone, glucose induced [Ca(2+)](i) oscillations in 77% of the cells. In 16% of the cells a sustained rise of [Ca(2+)](i) was observed. The increase in [Ca(2+)](i) was reversed by verapamil, an L-type Ca(2+) channel inhibitor. Adrenaline decreased [Ca(2+)](i) in oscillating cells in the presence of low glucose and in cells stimulated by glucose alone or in combination with tolbutamide and forskolin. Adrenaline did not lower [Ca(2+)](i) in the presence of 30mM extracellular K(+), indicating that adrenaline does not exert a direct effect on Ca(2+) channels but increases K(+) channel activity. As for primary b-cells, [Ca(2+)](i) oscillations persisted in the presence of closed K(ATP) channels; these also persisted in the presence of thapsigargin, which blocks Ca(2+) uptake into Ca(2+) stores. In contrast, in voltage-clamped cells and in the presence of diazoxide (50mM), which hyperpolarizes the cells by opening K(ATP) channels, [Ca(2+)](i) oscillations were abolished. These results support the hypothesis that [Ca(2+)](i) oscillations depend on functional voltage-dependent Ca(2+) and K(+) channels and are interrupted by a hyperpolarization in insulin-secreting cells.     

3T3-L1脂肪细胞对MIN6胰岛素细胞中Kir6.2表达的抑制作用

为明确脂肪细胞对胰岛素细胞中KATP通道表达的直接影响,MIN6胰岛素细胞被分为两组：一组为对照组,一组与分化的3T3-L1脂肪细胞共培养1周。运用半定量RT-PCR方法测定MIN6细胞中KATP通道蛋白Kir6．2的表达变化,Fura-2荧光方法测定MIN6细胞内钙浓度的变化,放射免疫测定方法明确MIN6细胞的胰岛素分泌功能。结果显示,与3T3-L1脂肪细胞共培养1周后,MIN6细胞中Kir6．2的表达明显减少,其表达水平降低为对照组的65．3％。对照组MIN6细胞在0．1mmoi／L甲苯磺丁脲(KATP通道关闭剂)的刺激下,表现为细胞内钙水平显著性升高和胰岛素分泌显著性增加,而共培养组MIN6细胞则失去了甲苯磺丁脲刺激所引起的细胞内钙升高及胰岛素分泌反应。以上实验结果表明,3T3-L1脂肪细胞可以通过分泌一些活性因子直接降低MIN6细胞中KATP通道蛋白的表达和合成,损害MIN6细胞的胰岛素分泌功能。实验结果提示脂肪细胞直接参与2型糖尿病中胰岛β细胞功能障碍的发生。     

NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells

Important questions remain concerning how elevated blood glucose levels are coupled to insulin secretion from pancreatic beta cells and how this process is impaired in type 2 diabetes. Glucose uptake and metabolism in beta cells cause the intracellular Ca(2+) concentration ([Ca(2+)](i)) to increase to a degree necessary and sufficient for triggering insulin release. Although both Ca(2+) influx and Ca(2+) release from internal stores are critical, the roles of inositol 1,4,5-trisphosphate (IP(3)) and cyclic adenosine dinucleotide phosphate ribose (cADPR) in regulating the latter have proven equivocal. Here we show that glucose also increases [Ca(2+)](i) via the novel Ca(2+)-mobilizing agent nicotinic acid adenine dinucleotide phosphate (NAADP) in the insulin-secreting beta-cell line MIN6. NAADP binds to specific, high-affinity membrane binding sites and at low concentrations elicits robust Ca(2+) responses in intact cells. Higher concentrations of NAADP inactivate NAADP receptors and attenuate the glucose-induced Ca(2+) increases. Importantly, glucose stimulation increases endogenous NAADP levels, providing strong evidence for recruitment of this pathway. In conclusion, our results support a model in which NAADP mediates glucose-induced Ca(2+) signaling in pancreatic beta cells and are the first demonstration in mammalian cells of the presence of endogenous NAADP levels that can be regulated by a physiological stimulus.     

Role of calcium in pancreatic islet cell death by IFN-gamma/TNF-alpha

We studied the intracellular events associated with pancreatic beta cell apoptosis by IFN-gamma/TNF-alpha synergism. IFN-gamma/TNF-alpha treatment of MIN6N8 insulinoma cells increased the amplitude of high voltage-activated Ca(2+) currents, while treatment with IFN-gamma or TNF-alpha alone did not. Cytosolic Ca(2+) concentration ([Ca(2+)](c)) was also increased by IFN-gamma/TNF-alpha treatment. Blockade of L-type Ca(2+) channel by nifedipine abrogated death of insulinoma cells by IFN-gamma/TNF-alpha. Diazoxide that attenuates voltage-activated Ca(2+) currents inhibited MIN6N8 cell death by IFN-gamma/TNF-alpha, while glibenclamide that accentuates voltage-activated Ca(2+) currents augmented insulinoma cell death. A protein kinase C inhibitor attenuated MIN6N8 cell death and the increase in [Ca(2+)](c) by IFN-gamma/TNF-alpha. Following the increase in [Ca(2+)](c), calpain was activated, and calpain inhibitors decreased insulinoma cell death by IFN-gamma/TNF-alpha. As a downstream of calpain, calcineurin was activated and the inhibition of calcineurin activation by FK506 diminished insulinoma cell death by IFN-gamma/TNF-alpha. BAD phosphorylation was decreased by IFN-gamma/TNF-alpha because of the increased calcineurin activity, which was reversed by FK506. IFN-gamma/TNF-alpha induced cytochrome c translocation from mitochondria to cytoplasm and activation of caspase-9. Effector caspases such as caspase-3 or -7 were also activated by IFN-gamma/TNF-alpha treatment. These results indicate that IFN-gamma/TNF-alpha synergism induces pancreatic beta cell apoptosis by Ca(2+) channel activation followed by downstream intracellular events such as mitochondrial events and caspase activation and also suggest the therapeutic potential of Ca(2+) modulation in type 1 diabetes.     

Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6)

We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001) J. Cell Biol. 155, 41-51) that ryanodine receptors (RyR) are present on insulin-containing secretory vesicles. Here we show that pancreatic islets and derived beta-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core secretory vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca(2+) concentrations in the secretory vesicle compartment from 40.1 +/- 6.7 to 90.4 +/- 14.8 microm (n = 4, p < 0.01), while having no effect on ER Ca(2+) concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca(2+)-mobilizing agent, decreased dense core secretory vesicle but not ER free Ca(2+) concentrations in permeabilized MIN6 beta-cells, and flash photolysis of caged NAADP released Ca(2+) from a thapsigargin-insensitive Ca(2+) store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 +/- 0.51-fold stimulation to no significant glucose effect; n = 3, p < 0.01), we conclude that RyR I-mediated Ca(2+)-induced Ca(2+) release from secretory vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.     

Calcium dependencies of regulated exocytosis in different endocrine cells

Exocytotic machinery in neuronal and endocrine tissues is sensitive to changes in intracellular Ca(2+) concentration. Endocrine cell models, that are most frequently used to study the mechanisms of regulated exocytosis, are pancreatic beta cells, adrenal chromaffin cells and pituitary cells. To reliably study the Ca(2+) sensitivity in endocrine cells, accurate and fast determination of Ca(2+) dependence in each tested cell is required. With slow photo-release it is possible to induce ramp-like increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that leads to a robust exocytotic activity. Slow increases in the [Ca(2+)](i) revealed exocytotic phases with different Ca(2+) sensitivities that have been largely masked in step-like flash photo-release experiments. Strikingly, in the cells of the three described model endocrine tissues (beta, chromaffin and melanotroph cells), distinct Ca(2+) sensitivity 'classes' of secretory vesicles have been observed: a highly Ca(2+)-sensitive, a medium Ca(2+)-sensitive and a low Ca(2+)-sensitive kinetic phase of secretory vesicle exocytosis. We discuss that a physiological modulation of a cellular activity, e.g. by activating cAMP/PKA transduction pathway, can switch the secretory vesicles between Ca(2+) sensitivity classes. This significantly alters late steps in the secretory release of hormones even without utilization of an additional Ca(2+) sensor protein.     

Cytosolic [Ca2+] transients in dictyostelium discoideum depend on the filling state of internal stores and on an active sarco/endoplasmic reticulum calcium ATPase (SERCA) Ca2+ pump

Stimulation of Dictyostelium discoideum with cAMP evokes a change of the cytosolic free Ca(2+) concentration ([Ca(2+)](i)). We analyzed the role of the filling state of Ca(2+) stores for the [Ca(2+)] transient. Parameters tested were the height of the [Ca(2+)](i) elevation and the percentage of responding amoebae. After loading stores with Ca(2+), cAMP induced a [Ca(2+)](i) transient in many cells. Without prior loading, cAMP evoked a [Ca(2+)](i) change in a few cells only. This indicates that the [Ca(2+)](i) elevation is not mediated exclusively by Ca(2+) influx but also by Ca(2+) release from stores. Reducing the Ca(2+) content of the stores by EGTA preincubation led to a cAMP-activated [Ca(2+)](i) increase even at low extracellular [Ca(2+)]. Moreover, the addition of Ca(2+) itself elicited a capacitative [Ca(2+)](i) elevation. This effect was not observed when stores were emptied by the standard technique of inhibiting internal Ca(2+) pumps with 2,5-di-(t-butyl)-1,4-hydroquinone. Therefore, in Dictyostelium, an active internal Ca(2+)-ATPase is absolutely required to allow for Ca(2+) entry. No influence of the filling state of stores on Ca(2+) influx characteristics was found by the Mn(2+)-quenching technique, which monitors the rate of Ca(2+) entry. Both basal and cAMP-activated Mn(2+) influx rates were similar in control cells and cells with empty stores. By contrast, determination of extracellular free Ca(2+) concentration ([Ca(2+)](e)) changes, which represent the sum of Ca(2+) influx and efflux, revealed a higher rate of [Ca(2+)](e) decrease in EGTA-treated than in control amoebae. We conclude that emptying of Ca(2+) stores does not change the rate of Ca(2+) entry but results in inhibition of the plasma membrane Ca(2+)-ATPase. Furthermore, the activities of the Ca(2+) transport ATPases of the stores are of crucial importance for the regulation of [Ca(2+)](i) changes.     

Protein kinase A- and C-induced insulin release from Ca2+ -insensitive pools

Insulin secretion is known to depend on an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). However, recent studies have suggested that insulin secretion can also be evoked in a Ca(2+)-independent manner. In the present study we show that treatment of intact mouse islets and RINm5F cells with protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or protein kinase A (PKA) activator forskolin promoted insulin secretion with no changes of [Ca(2+)](i). Moreover, insulin secretion mediated by PMA or forskolin was maintained even when extracellular or cytosolic Ca(2+) was deprived by treatment of cells with ethylene glycol bis(beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxy methyl ester) (BAPTA/AM) in RINm5F cells. The secretagogue actions of PMA and forskolin were blocked by GF109203X and H89, selective inhibitors for PKC and PKA, respectively. PMA treatment caused translocation of PKC-alpha and PKC- epsilon from cytosol to membrane, implying that selectively PKC-alpha and PKC- epsilon isoforms might be important for insulin secretion. Co-treatment with high K(+) and PMA showed a comparable level of insulin secretion to that of PMA alone. In addition, PMA and forskolin evoked insulin secretion in cells where Ca(2+)-dependent insulin secretion was completed. Our data suggest that PKC and PKA can elicit insulin secretion not only in a Ca(2+)-sensitive manner but also in a Ca(2+)-independent manner from separate releasable pools.     

Expression and function of the extracellular calcium-sensing receptor in pancreatic beta-cells

The extracellular calcium-sensing receptor (CaR) was first identified in tissues involved in systemic Ca2+ homeostasis, where it acts to sense changes in circulating Ca2+. It has since been reported that the CaR is expressed in many tissues that are not associated with Ca2+ homeostasis, including the endocrine cells in pancreatic islets of Langerhans. In the present study we have used an insulin-secreting pancreatic beta-cell line (MIN6) to investigate the expression and function of CaR, using the calcimimetic A568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca2+ ([Ca2+]o). Immunocytochemistry, Western blotting and RT-PCR confirmed the expression of CaR in MIN6 cells. CaR activation was associated with rapid and transient increases in [Ca2+]o, which were accompanied by the initiation of a marked but transient insulin secretory response. Stimulation of beta-cell secretory activity had no detectable effect on CaR mRNA levels, but CaR mRNA was markedly reduced by configuring MIN6 cells into islet- like structures. Our data are consistent with an important function for the beta-cell CaR in cell - cell communication within islets to co-ordinate insulin secretory responses.     

Effects of chronic hypoxia on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells.

Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.     

Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.