Sulphonylureas do not increase insulin secretion by a mechanism other than a rise in cytoplasmic Ca2+ in pancreatic B-cells

The following sequence of events is thought to underlie the stimulation of insulin release by hypoglycaemic sulphonylureas. Interaction of the drugs with a high-affinity binding site (sulphonylurea receptor) in the B-cell membrane leads to closure of ATP-sensitive K+ channels, depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx and rise in cytoplasmic [Ca2+]i. Recent experiments using permeabilized islet cells or measuring changes in B-cell membrane capacitance have suggested that sulphonylureas can increase insulin release by a mechanism independent of a change in [Ca2+]i. This provocative hypothesis was tested here with intact mouse islets. When B-cells were strongly depolarized by 60 mM K+, [Ca2+]i was increased and insulin secretion stimulated. Under these conditions, tolbutamide did not further increase [Ca2+]i or insulin release, whether it was applied before or after high K+, and whether the concentration of glucose was 3 or 15 mM. This contrasts with the ability of forskolin and phorbol 12-myristate 13-acetate (PMA) to increase release in the presence of high K+. Tolbutamide also failed to increase insulin release from islets depolarized with barium (substituted for extracellular Ca2+) or with arginine in the presence of high glucose. Glibenclamide and its non-sulphonylurea moiety meglitinide were also without effect on insulin release from already depolarized B-cells. In the absence of extracellular Ca2+, acetylcholine induced monophasic peaks of [Ca2+]i and insulin secretion which were both unaffected by tolbutamide. Insulin release from permeabilized islet cells was stimulated by raising free Ca2+ (between 0.1 and 23 microM). This effect was not affected by tolbutamide and inconsistently increased by glibenclamide. In conclusion, the present study does not support the proposal that hypoglycaemic sulphonylureas can increase insulin release even when they do not also raise [Ca2+]i in B-cells.     

Atypical protein kinase C isozyme zeta mediates carbachol-stimulated insulin secretion in RINm5F cells

Carbachol-stimulated insulin release in the RINm5F cell is associated with elevation of the cytosolic Ca2+ concentration ([Ca2+]i) through mobilization of Ca2+ from thapsigargin-sensitive intracellular stores and with the generation of diacylglycerol (DAG). Thus carbachol activates phospholipase C, and this was thought to be the means by which it stimulates insulin secretion. However, when the elevation of [Ca2+]i was blocked by thapsigargin, the effect of carbachol to stimulate insulin release was unchanged. Thus the effect of carbachol to increase [Ca2+]i was dissociated from the stimulation of release. When the role of protein kinase C (PKC) was examined, carbachol-stimulated insulin release was found to be unaffected by phorbol ester-induced downregulation of PKC, using 12-O-tetradecanoylphorbol-13-acetate (TPA), and by the PKC inhibitors staurosporine, bisindolylmaleimide, and 1-O-hexadecyl-2-O-methylglycerol (AMG-C16). These treatments abolished the stimulation of release by TPA. Thus the carbachol activation of PKC appeared also to be dissociated from the stimulation of insulin release. However, when the activation of several different PKC isozymes was studied, an atypical PKC isozyme, zeta, was found to be translocated by carbachol. By Western blotting analysis, carbachol selectively translocated the conventional PKC isozymes alpha and beta (the activation of which is dependent on Ca2+ and DAG) from the cytosol to the membrane. Carbachol also translocated the atypical PKC isozyme zeta, which is insensitive to Ca2+, DAG, and phorbol esters. The PKC inhibitors staurosporine, bisindolylmaleimide, and AMG-C16 blocked the stimulated translocation of PKC-alpha and -beta, but not that of PKC-zeta. Prolonged treatment of the cells with TPA downregulated PKC-alpha and -beta, but not PKC-zeta. Under all these conditions, carbachol-stimulated insulin release was unaffected. However, a pseudosubstrate peptide inhibitor specific for PKC-zeta inhibited the translocation of PKC-zeta and 70% of the carbachol-stimulated insulin secretion. The data indicate that carbachol-stimulated insulin release in RINm5F cells is mediated to a large degree by the activation of the atypical PKC isozyme zeta.     

Cytosolic Ca2+ gradients in pancreatic islet-cells stimulated by glucose and carbachol

Digital image analysis was employed to resolve the spatial differences in distribution of cytosolic free Ca2+ concentrations ([Ca2+]i) in mouse pancreatic islet-cells stimulated with glucose and carbachol. Using Indo-1 loaded mouse islet-cells, we have demonstrated that glucose induces steep spatial gradients of [Ca2+]i in isolated mouse islet-cells. Furthermore, the largest [Ca2+]i increase was always spatially restricted to a region just beneath the plasma membrane. Low concentrations of carbachol (0.6 microM) induced steep spatial gradients of [Ca2+]i which originated from the center of the cells. However, 10 microM carbachol increased [Ca2+]i to high levels collapsing the [Ca2+]i gradients in the center of the cells. Different patterns of [Ca2+]i oscillations were observed between dissociated pancreatic islet-cells and mouse pancreatic islets when challenged with 11 mM glucose. Under these conditions we could identify cells within the islet which oscillate with the same pattern as the whole islet. We postulate that "initiators" of insulin release, as glucose, induce greater [Ca2+]i increases at exocytotic sites than those induced by "potentiators", as carbachol.     

The novel insulinotropic mechanism of pimobendan: direct enhancement of the exocytotic process of insulin secretory granules by increased Ca2+ sensitivity in beta-cells

Pimobendan is a new class of inotropic drug that augments Ca2+ sensitivity and inhibits phosphodiesterase (PDE) activity in cardiomyocytes. To examine the insulinotropic effect of pimobendan in pancreatic beta-cells, which have an intracellular signaling mechanism similar to that of cardiomyocytes, we measured insulin release from rat isolated islets of Langerhans. Pimobendan augmented glucose-induced insulin release in a dose-dependent manner, but did not increase cAMP content in pancreatic islets, indicating that the PDE inhibitory effects may not be important in beta-cells. This agent increased the intracellular Ca2+ concentration ([Ca2+]i) in the presence of 30 mM K+, 16.7 mM glucose, and 200 microM diazoxide, but failed to enhance the 30 mM K+-evoked [Ca2+]i rise in the presence of 3.3 mM glucose. Insulin release evoked by 30 mM K+ in 3.3 mM glucose was augmented. Then, the direct effects of pimobendan on the Ca2+-sensitive exocytotic apparatus were examined using electrically permeabilized islets in which [Ca2+]i can be manipulated. Pimobendan (50 microM) significantly augmented insulin release at 0.32 microM Ca2+, and a lower threshold for Ca2+-induced insulin release was apparent in pimobendan-treated islets. Moreover, 1 microM KN93 (Ca2+/calmodulin-dependent protein kinase II inhibitor) significantly suppressed this augmentation. Pimobendan, therefore, enhances insulin release by directly sensitizing the intracellular Ca2+-sensitive exocytotic mechanism distal to the [Ca2+]i rise. In addition, Ca2+/calmodulin-dependent protein kinase II activation may at least in part be involved in this Ca2+ sensitization for exocytosis of insulin secretory granules.     

Temporal and quantitative correlations between insulin secretion and stably elevated or oscillatory cytoplasmic Ca2+ in mouse pancreatic beta-cells

An increase in cytoplasmic Ca2+ in beta-cells is a key step in glucose-induced insulin secretion. However, whether changes in cytoplasmic free Ca2+ ([Ca2+]i) directly regulate secretion remains disputed. This question was addressed by investigating the temporal and quantitative relationships between [Ca2+]i and insulin secretion. Both events were measured simultaneously in single mouse islets loaded with fura-PE3 and perifused with a medium containing diazoxide (to prevent any effect of glucose on the membrane potential) and either 4.8 or 30 mmol/l K+. Continuous depolarization with 30 mmol/l K+ in the presence of 15 mmol/l glucose induced a sustained rise in [Ca2+]i and insulin release. No oscillations of secretion were detected even after mathematical analysis of the data (pulse, spectral and sample distribution analysis). In contrast, alternating between 30 and 4.8 mmol/l K+ (1 min/2 min or 2.5 min/5 min) triggered synchronous [Ca2+]i and insulin oscillations of regular amplitude in each islet. A good correlation was found between [Ca2+]i and insulin secretion, and it was independent of the presence or absence of oscillations. This quantitative correlation between [Ca2+]i and insulin secretion was confirmed by experiments in which extracellular Ca2+ was increased or decreased (0.1-2.5 mmol/l) stepwise in the presence of 30 mmol/l K+. This resulted in parallel stepwise increases or decreases in [Ca2+]i and insulin secretion. However, while the successive [Ca2+]i levels were unaffected by glucose, each plateau of secretion was much higher in 20 than in 3 mmol/l glucose. In conclusion, in our preparation of normal mouse islets, insulin secretion oscillates only when [Ca2+]i oscillates in beta-cells. This close temporal relationship between insulin secretion and [Ca2+]i changes attests of the regulatory role of Ca2+. There also exists a quantitative relationship that is markedly influenced by the concentration of glucose.     

Mastoparan stimulates exocytosis at a Ca(2+)-independent late site in stimulus-secretion coupling. Studies with the RINm5F beta-cell line

Mastoparan, a tetradecapeptide from wasp venom, stimulated exocytosis in a concentration-dependent manner, which was enhanced by pertussis toxin pre-treatment, in the insulin secreting beta-cell line RINm5F. Mastoparan (3-20 microM) also elevated cytosolic free calcium concentration ([Ca2+]i), a rise that was not attenuated by nitrendipine. Divalent cation-free Krebs-Ringer bicarbonate (KRB) medium with 0.1 mM EGTA nullified the mastoparan-induced increase in [Ca2+]i, suggesting that the peptide increased Ca2+ influx but not through the L-type voltage-dependent Ca2+ channel. Depletion of the intracellular Ca2+ pool did not affect the mastoparan-induced elevation of [Ca2+]i. Remarkably, in divalent cation-free KRB medium with 0.1 mM EGTA and 2 microM thapsigargin in which mastoparan reduced [Ca2+]i, the mastoparan-stimulated insulin release was similar to that in normal Ca(2+)-containing KRB medium. Inhibitors of protein kinase C, such as bisindolylmaleimide, staurosporine, and 1-O-hexadecyl-2-O-methyl-rac-glycerol did not suppress the mastoparan-stimulated insulin release. Mastoparan at 10-20 microM did not increase cellular cAMP levels, nor did mastoparan at 5-10 microM affect [3H]arachidonic acid release. In conclusion, although mastoparan increased [Ca2+]i, this increase was not involved in the stimulation of insulin release. Rather, the data suggest that mastoparan directly stimulates exocytosis in a Ca(2+)-independent manner. As GTP-binding proteins (G proteins) are thought to be involved in the process of exocytosis and as mastoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimulatory Ge (exocytosis) protein is likely.     

Dimensionality of depression in acute schizophrenia: a methodological study using the Bech-Rafaelsen Melancholia Scale (BRMES)

Ca2+ plays an essential role in pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated growth hormone (GH) secretion from porcine somatotropes. Here, Indo-1 microfluorimetry was used to investigate the dynamics of free cytosolic Ca2+ concentration ([Ca2+]i) in single porcine somatotropes in response to PACAP38 and PACAP27. We also evaluated the relative contributions of extra- and intracellular Ca2+ sources and of cAMP-dependent protein kinase (PKA) and phospholipase C (PLC). A high proportion of somatotropes responded to PACAP38 (79.4%) and PACAP27 (68.4%) with [Ca2+]i rises that could be followed by a refractory plateau (type 1 response), or by a decrease in [Ca2+]i during which somatotropes were responsive to a subsequent PACAP pulse (type II response). Although Ca2+ profiles in response to both peptides were similar, PACAP38-induced [Ca2+]i rises were higher. Somatotrope response to PACAP38 or PACAP27 was markedly reduced by removing extracellular Ca2+, blocking Ca2+ entry through L-type voltage sensitive Ca2+ channels (VSCC), or inhibiting PKA. Conversely, Ca2+ depletion from intracellular stores or PLC inactivation did not affect the response to PACAP27 but considerably reduced maximal [Ca2+]i induced by PACAP38. We conclude that both peptides stimulate extracellular Ca2+ influx through L-type VSCC by a PKA-dependent mechanism. However, PACAP38 also triggers a PLC-mediated Ca2+ mobilization from intracellular stores, thereby indicating that the two molecular forms of PACAP activate common and distinct second messenger pathways within porcine somatotropes.     

Glucose-stimulated elevation of cytoplasmic calcium is defective in the diabetic Chinese hamster islet B cell

To characterize insulin release and cytoplasmic free Ca2+ ([Ca2+]i) levels in the diabetic Chinese hamster islet B cell, islets from genetically normal (subline M) and diabetic (subline L) hamsters were collagenase isolated. Insulin release and glucose utilization (conversion of D-[5-(3H)]glucose to 3H2O) were measured in whole islets; [Ca2+]i levels were measured in single islet cells using fura-2. The Ca2+ channel agonist, 12 mmol/l perchlorate, ClO4-, increased the subnormal insulin response during 20 mmol/l glucose perifusion, but did not normalize it. Glucose utilization measured over a 2-h period was normal. Glucose induced an initial decrease and then a rise in [Ca2+]i in 85% of the normal (presumably B) cells. In diabetic cells, the [Ca2+]i response was delayed, subnormal and only observed in 23% of the cells. When perchlorate or another Ca2+ channel agonist, 10 mumol/l CGP 28392, was added with glucose, a larger proportion of the diabetic cells (61-67%) showed increased [Ca2+]i and the mean [Ca2+]i response was not different from normal. However, neither perchlorate nor CGP 28392 could normalize glucose-stimulated insulin release, and K(+)-induced insulin release was decreased in diabetic islets. The K(+)-induced [Ca2+]i rise was essentially normal in all the diabetic islet cells. Therefore, the diabetic hamster islet appears to metabolize glucose normally, but has a diminished insulin response to glucose and K+. The Ca2+ channel agonists markedly improve the subnormal [Ca2+]i response but not the insulin response. Glucose-induced elevation of [Ca2+]i and exocytosis appear defective in the diabetic Chinese hamster B cell.     

Protein kinase A-dependent stimulation of exocytosis in mouse pancreatic beta-cells by glucose-dependent insulinotropic polypeptide

The mechanisms by which glucose-dependent insulinotropic polypeptide (GIP) stimulates insulin secretion were investigated by measurements of whole-cell Ca2+ currents, the cytoplasmic Ca2+ concentration, and cell capacitance as an indicator of exocytosis in individual mouse pancreatic beta-cells maintained in short-term culture. GIP produced a 4.2-fold potentiation of depolarization-induced exocytosis. This stimulation of exocytosis was not associated with a change in the whole-cell Ca2+-current, and there was only a small increase (30%) in the cytoplasmic Ca2+ concentration [intercellular free Ca2+([Ca2+]i)]. The stimulatory effect of GIP on exocytosis was blocked by pretreatment with the specific protein kinase A (PKA) inhibitor Rp-8-Br-cAMPS. Glucagon-like peptide-I(7-36) amide (GLP-I) stimulated exocytosis (90%) in the presence of a maximal GIP concentration (100 nmol/l). Replacement of GLP-I with forskolin produced a similar stimulatory action on exocytosis. These effects of GLP-I and forskolin in the presence of GIP did not involve a change in the whole-cell Ca2+-current or [Ca2+]i. GIP was ineffective in the presence of both forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). Under the same experimental conditions, the protein kinase C (PKC)-activating phorbol ester 4-phorbol 12-myristate 13-acetate (PMA) stimulated exocytosis (60%). Collectively, our data indicate that the insulinotropic hormone GIP stimulates insulin secretion from pancreatic beta-cells, through the cAMP/PKA signaling pathway, by interacting with the secretory machinery at a level distal to an elevation in [Ca2+]i.     

Calcium and a mitochondrial signal interact to stimulate phosphoinositide hydrolysis and insulin secretion in rat islets

Fuel metabolism generates multiple signals that interact to stimulate insulin secretion. These studies explored the mechanism by which fuels activate phosphoinositide (PI) hydrolysis and the role of this signal transduction pathway in fuel-stimulated insulin secretion. High potassium (30 mM), which depolarizes the membrane and increases Ca2+ influx, caused only a transient monophasic release of insulin. In contrast, glucose (20 mM) or monomethylsuccinate (MMSucc; 10 mM) markedly stimulated a sustained insulin secretory response, indicating that fuel metabolism generates a signal(s) in addition to Ca2+ influx that is required for a sustained secretory response. On the other hand, diazoxide, an ATP-sensitive K+ channel activator that prevents membrane depolarization and Ca2+ influx in response to fuel metabolism, reduced the secretory responses to glucose and MMSucc to baseline levels, demonstrating that Ca2+ influx was essential to fuel-stimulated insulin secretion. The further addition of high K+ bypassed the diazoxide block and restored insulin secretory rates. The insulin secretory response to glucose or MMSucc in the presence of diazoxide and K+ was inhibited by the Ca2+ channel antagonist nitrendipine and the protein kinase-C inhibitor staurosporine. Changes in PI hydrolysis paralleled those in insulin secretion. High potassium alone induced only a modest 2.5-fold increase in inositol phosphate accumulation. This response was significantly less than that to glucose or MMSucc, which increased inositol phosphate accumulation by 6.8- or 5.2-fold, respectively. Like its effect on secretion, diazoxide markedly reduced glucose- or MMSucc-stimulated PI hydrolysis, and this inhibition was reversed with high K+. In contrast, diazoxide had no effect on receptor-activated PI hydrolysis stimulated by 100 nM cholecystokinin (CCK), and the effects of CCK were not dependent on added fuel, indicating that fuel and CCK activate PI hydrolysis by distinct pathways. These findings demonstrate that mitochondrial metabolism of glucose or MMSucc generates a signal(s) that interacts with Ca2+ influx to stimulate PI hydrolysis and sustained insulin secretion. This pathway of fuel-activated PI hydrolysis is distinct from that of CCK receptor-activated PI hydrolysis. These studies suggest that fuel-activated PI hydrolysis plays an important role in fuel-stimulated insulin secretion.     

Studies on the microsomal cytochrome P450s in the livers of carcinogen-resistant and sensitive rats during long-term administration of 3''-methyl-4-dimethylaminoazobenzene

The fluorescent indicator Fura-2 was used to characterize the store-operated Ca2+ entry in insulin-releasing pancreatic beta-cells. To avoid interference with voltage-dependent Ca2+ entry, the cells were hyperpolarized with 400 microM diazoxide and the channel blocker methoxyverapamil was also present in some experiments. The cytoplasmic Ca2+ concentration ([Ca2+]j) of hyperpolarized mouse beta-cells was strikingly resistant to changes in external Ca2+. In cells exposed to 20 mM glucose, stimulation with 100 microM carbachol induced an initial [Ca2+]j peak followed by a sustained increase due to store-operated influx of the cation. Store-operated influx was also induced by the intracellular Ca(2+)-ATPase inhibitor thapsigargin. In the presence of store-operated influx, [Ca2+]j became markedly sensitive to variations in external Ca2+, but this sensitivity was blocked by La3+. In beta-cells exposed to both Ca2+ and Mn2+ there was slow Mn2+ quenching of the Fura-2 fluorescence, which was accelerated upon stimulation of store-operated influx. This acceleration was reversed by glucose-stimulated filling of the internal Ca2+ stores. The store-operated Ca2+ entry increased markedly during culture of the beta-cells. Activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13 acetate, inhibition of serine/threonine phosphatase by okadaic acid and inhibition of tyrosine kinase by genistein had little effect on the store-operated influx of Ca2+. In beta-cells equilibrated in 5 mM Sr2+, carbachol exposure resulted in a pronounced cytoplasmic Sr2+ ([Sr2+]j) peak due to intracellular mobilization, but little or no sustained elevation. Moreover, after activating the store-operated pathway by exposure to thapsigargin, variations in extracellular Sr2+ between 0-2 mM had only marginal effects on [Sr2+]j. Although the store-operated influx apparently accounts for a minor fraction of the Ca2+ entry, its depolarizing influence may under certain conditions be up-regulated with resulting distortion of the beta-cell function.     

Cyclic-AMP-dependent Ca2+ influx elicited by prostaglandin D2 in freshly isolated nonchromaffin cells from bovine adrenal medulla

We previously reported that prostaglandin D2 (PGD2) specifically elevates intracellular cyclic AMP in nonchromaffin cells isolated from bovine adrenal medulla (Biochim. Biophys. Acta (1989) 1011, 75-80). Here we again found that PGD2 increased intracellular Ca2+ concentration ([Ca2+]i) in freshly isolated nonchromaffin cells and investigated the cellular mechanisms of PGD2-induced [Ca2+]i increase using the Ca2+ indicator fura-2 and a fluorescence microscopic imaging system. Treatment of the cells with PGD2 receptor agonists BW245C and ZK110841 resulted in both marked stimulation of cyclic AMP formation and an increase in [Ca2+]i. The [Ca2+]i response was also induced by bypassing of the receptor with forskolin, a direct activator of adenylate cyclase, but not by PGE2 or PGF2 alpha both of which are devoid of the ability to generate cyclic AMP in the cells. These cyclic AMP and [Ca2+]i responses induced by PGD2 were completely blocked by the PGD2 receptor antagonist BWA868C. The time-course of cyclic AMP production stimulated by PGD2 coincided with that of the [Ca2+]i increase. While the Ca(2+)-mobilizing hormone bradykinin stimulated a rapid inositol phosphate accumulation in nonchromaffin cells, PGD2 did not stimulate it significantly. Removal of extracellular Ca2+ markedly reduced the Ca2+ response to PGD2 in magnitude and duration, but did not alter the peak [Ca2+]i response to bradykinin. These results demonstrate that PGD2 receptor activation induces the increase in [Ca2+]i via cyclic AMP mainly by increasing the Ca2+ influx from the outside, unlike inositol trisphosphate which causes release of Ca2+ from internal stores.     

Evidence for contribution by increased cytoplasmic Na+ to the insulinotropic action of PACAP38 in HIT-T15 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is localized to pancreatic nerve terminals and stimulates insulin secretion. The insulinotropic effect of PACAP38 in insulin-producing HIT-T15 cells is accompanied by increases in cellular cAMP and cytoplasmic Ca2+ ([Ca2+]cyt). As also intracellular Na+ is important for insulin secretion after glucose and other cAMP forming peptides, we examined the Na+ dependence of the insulinotropic effect of PACAP38 in HIT-T15 cells. We found that PACAP38 (100 nM)-induced insulin secretion was diminished by approximately 50% by removal of extracellular Na+ (replaced by equimolar N-methyl-D-glucamine). In contrast, removal of Na+ did not diminish the formation of cellular cAMP (measured by radioimmunoassay) or the increase in [Ca2+]cyt (measured in FURA-2AM-loaded cell suspensions) induced by PACAP38. Furthermore, PACAP-38 increased the cytoplasmic Na+ ([Na+]cyt) in single HIT-T15 cells as measured by the fluorophore sodium-binding benzofran isophthalate. This increase was reduced by removal of extracellular Na+ and by inhibition of protein kinase A by H-89. We conclude that the insulinotropic action of PACAP38 is Na+-dependent. We propose that PACAP38 opens plasma membrane Na+ channels by an action partially mediated by cAMP and protein kinase A, and the subsequent raise in [Na+]cyt elicits insulin secretion by an as yet unsolved mechanism.     

Cytoplasmic Ca2+ oscillations in pancreatic beta-cells

In the last 15 years it has been a growing interest in the cyclic variations of circulating insulin [46]. After the suggestion that this phenomenon may be due to oscillations of the beta-cell membrane potential [8,39], it was demonstrated that [Ca2+]i oscillates in the glucose-stimulated beta-cell with a similar frequency to that of pulsatile insulin release. The present review describes four types of [Ca2+]i oscillations in the pancreatic beta-cell. The slow sinusoidal oscillations, referred to as type-a, are those which most closely correspond to pulsatile insulin release. Although not affecting the properties of the type-a oscillations in individual beta-cells, the concentration of glucose is a determinant for their generation and further transformation into a sustained increase. Accordingly, cytoplasmic Ca2+ is regulated by sudden transitions between oscillatory and steady-state levels at threshold concentrations of glucose, which are characteristic for the individual beta-cell. This behaviour explains the observation of a gradual recruitment of previously non-secreting cells with increase of the extracellular glucose concentration [44]. However, it still remains to be elucidated how the sudden transitions between these three states translate into the co-ordinated slow oscillations of [Ca2+]i in the intact islet. Cyclic variations of circulating insulin require a synchronization of the [Ca2+]i cycles also among the islets in the pancreas. It is still an open question by which means the millions of islets communicate mutually to establish a pattern of pulsatile insulin release from the whole pancreas. The discovery that the beta-cell is not only the functional unit for insulin synthesis but also generates the [Ca2+]i oscillations required for pulsatile insulin release has both physiological and clinical implications. The fact that minor damage to the beta-cells prevents the type-a oscillations with maintenance of a glucose response in terms of raised [Ca2+]i reinforces previous arguments [54] that loss of insulin oscillations is an early indicator of type-2 diabetes. Further analyses of the [Ca2+]i oscillations in the beta-cells should include not only the mechanisms for their generation and subsequent propagation within or among the islets but also how modulation of their frequency affects the insulin sensitivity of various target cells. The latter approach may be important in the attempts to maintain normoglycemia under conditions minimizing the vascular effects of insulin supposed to precipitate hypertonia and atherosclerosis [70,71,77].     

Pituitary adenylate cyclase-activating polypeptide causes rapid Ca2+ release from intracellular stores and long lasting Ca2+ influx mediated by Na+ influx-dependent membrane depolarization in bovine adrenal chromaffin cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.     

Inhibitory effect of forskolin on myosin phosphorylation-dependent and independent contractions in bovine tracheal smooth muscle

In bovine tracheal smooth muscle, carbachol (CCh, 1 microM) and high K+ (72.7 mM) induced sustained increases in cytosolic Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction. Forskolin (FK, 1-10 microM) inhibited the CCh-induced increase in [Ca2+]i, MLC phosphorylation and force in parallel. In contrast, FK inhibited the high K(+)-induced contraction and MLC phosphorylation without changing [Ca2+]i. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), CCh (10 microM) and caffeine (20 mM) induced transient increase in [Ca2+]i and contractile force by releasing Ca2+ from cellular store. FK strongly inhibited the CCh-induced Ca2+ transient, but failed to inhibit the caffeine-induced Ca2+ transient. In the absence of external Ca2+, 12-deoxyphorbol 13-isobutylate (DPB, 1 microM) induced sustained contraction without increase in [Ca2+]i and MLC phosphorylation. FK inhibited this contraction without changing [Ca2+]i. In permeabilized muscle, Ca2+ induced contraction in a concentration-dependent manner. FK (10 microM) and cAMP (1-100 microM) shifted the Ca(2+)-force curve to the higher Ca2+ levels. CCh with GTP, GTP gamma S or DPB enhanced contraction in the presence of constant level of Ca2+. Forskolin and cAMP also inhibited the enhanced contractions in the permeabilized muscle. In the permeabilized, thiophosphorylated muscle, ATP induced contraction in the absence of Ca2+. cAMP (300 microM) had no effect on this contraction. These results suggest that forskolin inhibits agonist-induced contraction in tracheal smooth muscle by multiple mechanisms of action; 1) inhibition of MLC phosphorylation by reducing Ca2+ influx and Ca2+ release, 2) inhibition of MLC phosphorylation by changing the MLC kinase/phosphatase balance, and 3) inhibition of regulatory mechanism which is not dependent on MLC phosphorylation.     

The delta-opioid receptor regulates activity of ryanodine receptors in the human neuroblastoma cell line SK-N-BE

Recent studies have demonstrated that opioid agonists affect the cytosolic Ca2+ concentration ([Ca2+]i) either by regulating plasma membrane Ca(2+)-channel activity or by mobilizing intracellular Ca2+ stores. The present report documents the [Ca2+]i increase induced by opioid agonists in a human neuroblastoma cell line, SK-N-BE, expressing delta-opioid receptors. In the presence, as well as in the absence, of extracellular Ca2+, opioid agonists enhanced significantly [Ca2+]i, whereas carbachol, known to mobilize specifically inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores, acted only in the presence of extracellular Ca2+. The opioid-induced increase in [Ca2+]i was not affected by treatments modifying the trimeric Gl, Go, and Gs protein transduction mechanisms or the activity of adenylyl cyclase. The Ca(2+)-ATPase pump-inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid-induced [Ca2+]i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK-N-BE cells, delta-opioid receptors mobilize Ca2+ from intracellular ryanodine-sensitive stores and the mechanism involved is independent of Gl/Go Gs proteins and protein kinase A activation.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Applying task analysis in design specification: current problems and a solution

The immune and endocrine mediators that are released during sepsis (e.g., tumor necrosis factor [TNF] alpha, interleukin [IL]-1, IL-6, transforming growth factor [TGF] beta, prostaglandin [PG] E2, catecholamines, vasopressin, glucagon, insulin, and glucocorticoids) can produce inappropriate detrimental cellular responses contributing to exacerbation of septic injury. Examples of such sepsis-related inappropriate responses are: exaggerated hepatic acute-phase protein (APP) expression and release skeletal muscle insulin resistance, and suppressed T-lymphocyte proliferation. The studies discussed in this article present evidence that the generation of the sepsis-related hepatic, skeletal muscle, and T-lymphocyte responses emanate from alterations in intracellular Ca2+ (Ca2+i) homeostasis. In hepatocytes, there is indication of a sepsis-mediated increase in Ca2+ influx from the extracellular milieu leading to a sustained increase in the apparent resting cell Ca2+i concentration ([Ca2+]i) and its depressed elevation on stimulation with Ca2+-mobilizing hormones such as catecholamines and vasopressin. These Ca(2+)- related changes can affect not only the signaling pathways in which Ca2+i itself serves as a signaling component, but also the signaling systems turned on by other sepsis-induced agonists which may not be dependent on Ca2+ signaling. TGF-beta, IL-1, TNF alpha, and IL-6 activate a primarily protein kinase C (PKC)-dependent intracellular signal system for the elicitation of a normal hepatic APP response (APPR). The increased apparent basal [Ca2+]i in sepsis can hypersensitize PKC activation and thus lead to an exaggerated APPR. In the skeletal muscle, an evident increase in Ca2+ membrane flux during sepsis pointed to an increase in the basal [Ca2+]i resulting from a plausible cytokine-mediated overactivation of the voltage-sensitive Ca2+ channels. The increased basal [Ca2+]i can negatively modulate the insulin-mediated stimulation of GLUT4-dependent glucose transport despite the possibility that Ca2+i might not participate as a component in the insulin-receptor-regulated signaling pathway. Increased [Ca2+]i in skeletal myocytes can either directly promote the phosphorylation of GLUT4 or prevent its dephosphorylation, both of which effectively block insulin stimulation of glucose uptake, thereby contributing to insulin resistance. In T lymphocytes, septic injury appears to induce an attenuation in the mitogen and, thus, presumably a T-cell antigen receptor (TCR)-mediated elevation in [Ca2+]i without affecting the basal [Ca2+]i. This decrease in TCR-related Ca2+i mobilization evidently contributes to the suppression of T lymphocyte proliferation during sepsis, probably via an in vivo action of prostaglandin (PG) E2 on the T cells during sepsis. The blockade of PGE2 production after indomethacin administration to septic animals prevents alterations in both T-cell Ca2+i mobilization and proliferation. PGE2 probably acts through its second messenger, cyclic adenosine 3'5'-monophosphate, which can antagonize Ca2+i signaling in T cells.     

Insulin exocytosis and glucose-mediated increase in cytoplasmic free Ca2+ concentration in the pancreatic beta-cell are independent of cyclic ADP-ribose

Stimulation of pancreatic beta-cells by glucose gives rise to an increase in the cytoplasmic free calcium concentration ([Ca2+]i) and exocytosis of insulin. Cyclic adenosine 5'-diphosphate ribose (cADPR), a metabolite of beta-NAD+, has been reported to increase [Ca2+]i in pancreatic beta-cells by releasing Ca2+ from inositol 1,4,5-trisphosphate-insensitive intracellular stores. In the present study, we have examined the role of cADPR in glucose-mediated increases in [Ca2+]i and insulin exocytosis. Dispersed ob/ob mouse beta-cell aggregates were either pressure microinjected with fura-2 salt or loaded with fura-2 acetoxymethyl ester, and [Ca2+]i was monitored by microfluorimetry. Microinjection of beta-NAD+ into fura-2-loaded beta-cells did not increase [Ca2+]i nor did it alter the cells' subsequent [Ca2+]i response to glucose. Cells microinjected with the cADPR antagonist 8NH2-cADPR increased [Ca2+]i in response to glucose equally well as those injected with cADPR. Finally, the ability of cADPR to promote exocytosis of insulin in electropermeabilized beta-cells was investigated. cADPR on its own did not increase insulin secretion nor did it potentiate Ca2+-induced insulin secretion. We conclude that cADPR neither plays a significant role in glucose-mediated increases in [Ca2+]i nor interacts directly with the molecular mechanisms regulating exocytosis of insulin in normal pancreatic beta-cells.