Nitric oxide-cyclic GMP system potentiates glucose-induced rise in cytosolic Ca2+ concentration in rat pancreatic beta-cells.

Involvement of nitric oxide (NO) in the regulation of insulin secretion from pancreatic beta-cells was investigated by measuring cytosolic Ca2+ concentration ([Ca2+]i) in isolated rat pancreatic beta-cells. At 7.0 mM glucose, L-arginine (0.1 mM) elevated [Ca2+]i in about 50% of the beta-cells examined. The response was partially inhibited by an NO synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMA; 0.1 mM), suggesting that part of the response was mediated by the production of NO from L-arginine. D-Arginine at higher concentrations (3 or 10 mM) also increased [Ca2+]i at 7.0 mM glucose; however, the response was not affected by L-NMA (0.1 mM). Similar [Ca2+]i elevation was produced by NO (10 nM) and sodium nitroprusside (SNP; 10 microM) at 7.0 mM glucose. The SNP-induced increase in [Ca2+]i was abolished by nicardipine (1 microM), suggesting that the [Ca2+]i response is mediated by Ca2+ influx through L-type voltage-operated Ca2+ channels. In the presence of oxyhemoglobin (1 microM), the [Ca2+]i elevation induced by NO (10 nM) was abolished. Neither degradation products of NO, NO2- nor NO3-, caused any changes in [Ca2+]i. 8-Bromo-cyclic GMP (8-Br-cGMP; 3 mM) and atrial natriuretic peptide (0.1 microM) elevated [Ca2+]i at 7.0 mM glucose. We conclude that NO, which is produced from L-arginine in pancreatic islets, facilitates glucose-induced [Ca2+]i increase via the elevation of cGMP in rat pancreatic beta-cells. NO-cGMP system may physiologically regulate insulin secretion from pancreatic beta-cells.     

Selective activation of Ca2+ influx by extracellular ATP in a pancreatic beta-cell line (HIT)

The action of exogenous ATP on cytoplasmic free Ca2+ ([Ca2+]i) was studied in insulin secreting cells using fura-2. Stimulation of clonal pancreatic beta-cells (HIT) with ATP (range 2-20 microM) evoked a sustained elevation in [Ca2+]i. ATP selectively promoted Ca2+ influx and not Ca2+ mobilization since (1) the effect required external Ca1+ and (2) was observed in cells in which internal stores were depleted with ionomycin (3) the rate of Mn2+ influx, measured as the quenching of the fura-2 signal, was accelerated by ATP. The action of ATP was unaffected by the voltage-sensitive Ca2+ channel blockers nifedipine and verapamil as well as by a depolarizing concentration of K+. The effect on [Ca2+]i was highly specific for ATP since AMP, ADP, adenosine 5'-[gamma-thio]triphosphate, adenosine 5'-[beta, gamma-methylene]triphosphate, GTP and adenosine were ineffective. In normal pancreatic islet cells, both exogenous ATP (range 0.2-2 microM) and ADP induced a transient Ca2+ elevation that did not require external Ca2+. The nucleotide specificity of the effect on [Ca2+]i suggests that ATP activates P2 gamma purinergic receptors in normal beta-cells. Thus, ATP evokes a Ca2+ signal in clonal HIT cells and normal islet cells by different transducing systems involving distinct purinoreceptors. A novel mechanism for increasing [Ca2+]i by extracellular ATP is reported in HIT cells, since the nucleotide specificity and the selective activation of Ca2+ influx without mobilization of internal Ca2+ stores cannot be explained by mechanisms already described in other cell systems.     

Enhanced intracellular calcium induced by urocortin is involved in degranulation of rat lung mast cells.

Corticotropin-releasing factor (CRF), which activates the hypothalamic-pituitary- adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. The purpose of this study was to investigate the effect of urocortin (UCN), a 40-amino-acid CRF family peptide, on degranulation and intracellular calcium of rat lung mast cells. The activation and degranulation of mast cells were observed by Toluidine blue staining and transmission electron microscope. The intracellular calcium was investigated using confocal laser scanning microscopy and flow cytometry. The results indicated that all the three different concentrations of UCN (0.1, 1 and 10 microM) significantly induced the activation and degranulation of rat lung mast cells in vitro. This effect was markedly blocked by selective CRF receptor 1 (CRF-R1) antagonist antalarmin, but not by specific CRF receptor 2 (CRF-R2) antagonist antisauvagine-30 (anti-Svg-30). The results also showed that UCN caused a rapid peak increase in [Ca(2+)](i) at point of 300s after UCN treatment, followed by a decrease to a sustained plateau phase. The peak increase in [Ca(2+)](i) induced by UCN was significantly inhibited by antalarmin, but not by anti-Svg-30. This effect of UCN on [Ca(2+)](i) in rat lung mast cells was also found by flow cytometry. Regression analysis revealed a positive correlation between mast cells degranulation extent and the maximum value of [Ca(2+)](i) (P < 0.01). Taken together, our present study suggested that UCN induced the increase of [Ca(2+)](i) and degranulation of rat lung mast cells through CRF-R1. These findings may have implications for the pathophysiology of allergic and inflammatory lung disorders such as asthma, which is closely associated with mast cell activation and degranulation.     

Urocortin II inhibits the apoptosis of mesenteric arterial smooth muscle cells via L-type calcium channels in spontaneously hypertensive rats.

Urocortin (UCN) II, a newly isolated corticotropinreleasing- factor (CRF) related peptide, has been found to have potent cardiovascular protective effects. To investigate the mechanisms of its vascular protective effects, we exposed mesenteric arterial smooth muscle cells (MASMC) from spontaneously hypertensive rats (SHR) to UCN II to observe the change in cell apoptosis using TUNEL assay and measured intracellular calcium concentration ([Ca2+]i) using confocal laser scanning microscope. In addition, effects of UCN II on L-type calcium currents (ICa,L) were also measured using whole-cell patch clamp. Our results showed that UCN II concentration-dependently, but time-independently inhibited cell apoptosis. Astressin 2B, a special CRF 2 receptor antagonist, had no influence on this inhibition. Hypoxia or Bay K8644, the L-type calcium channel activator, induced the apoptosis of MASMC from SHR. Pretreatment of the cells with UCN II diminished the effects of hypoxia or Bay K8644. UCN II was also observed to reduce [Ca2+]i increase induced by KCl or Bay K8644. UCN II concentration-dependently inhibited ICa,L, which was not affected by astressin 2B. It did not affect the activation of ICa,L, but markedly shifted the inactivation curve to the left. In conclusion, UCN II inhibits the apoptosis of MASMC from SHR via inhibiting L-type calcium channels.     

Expression of iNOS gene in macrophages stimulated with 17beta-estradiol is regulated by free intracellular Ca2+.

17Beta-estradiol has potent Ca2+ ionophore capability and its signaling in macrophages is mediated through binding to surface and genomic receptors, resulting in transient nitric oxide (NO) elaboration. We decided to examine if the transient release of NO is due to Ca2+ influx pattern or the quenching effect of superoxide (*O2-) through peroxynitrite formation. Differential chelation of intracellular Ca2+ ([Ca2+]i) showed that NO generation was favored by [Ca2+]i concentration of 237 nM. Application of an estrogen receptor antagonist ICI 182 780 resulted in attenuation of estradiol mediated NO release. Studies directed at identifying the possible role of *O2-; in the attenuation of NO showed no supportive evidence. Inhibition of extracellular Ca2+ channel or extracellular and intracellular Ca2+ channels showed data consistent with a case for optimum Ca2+ influx signal favoring iNOS gene expression, accompanied by an elevation in iNOS protein. These data show that Ca2+ influx pattern determines macrophage NO elaboration.     

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.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Separate locations of urocortin and its receptors in mouse testis: function in male reproduction and the relevant mechanisms.

Urocortin (UCN), a newly identified corticotrophin-releasing-factor (CRF) related peptide, has been demonstrated to play important roles in female reproductive system. However, few studies were reported about its effects on male reproduction. This study aimed to investigate the expression profile of UCN and CRF receptors (CRFR) in mouse testis and functions of UCN in male reproduction. Expression of UCN and CRFR mRNA was detected by RT-PCR. Localization of UCN peptide was determined by immunohistochemistry and double-immunostaining. We found that both UCN mRNA and peptide were obviously expressed in mature spermatozoa, whereas CRFR1 and CRFR2 were expressed respectively in spermatocytes and spermatogonia. Double-immunostaining results showed that UCN expression decreased with acrosome reaction (AR) proceeding. UCN significantly inhibited AR initiated by progesterone with chlortetracycline staining and decreased spermatozoa motility concentration-dependently. Pre-incubation of spermatozoa with astressin, a CRFR antagonist, did not affect these inhibitions. In addition, flow cytometry showed that UCN concentration-dependently decreased intracellular Ca(2+) [Ca(2+)](i) in spermatozoa. In summary, UCN located in mouse spermatozoa and exerted inhibitory effects on male reproductive functions including motility and AR. UCN's inhibition on [Ca(2+)](i) via T-type calcium channels might be responsible for these effects.     

Increased internal calcium mobilization in platelets of patients with chronic renal failure.

Platelet free calcium concentrations ([Ca2+]i) were measured with Fura-2 to elucidate the intracellular calcium kinetics in patients with renal disease. There were no significant differences of the resting [Ca2+]i among the control subjects (C) (n = 12), patients with chronic glomerulonephritis (CGN) (n = 8), and patients with chronic renal failure (CRF) (n = 12). In all groups, platelets [Ca2+]i were significantly increased by agonists (thrombin, adenosine diphosphate) compared with their respective basal level. Thrombin-induced [Ca2+]i rise was significantly higher in CRF (840 +/- 265 nM) than in C (600 +/- 163) and CGN (562 +/- 137). Also adenosine diphosphate elicited similar responses. In the presence of calcium chelator in the incubation buffer, the elevation of [Ca2+]i after thrombin stimulation was statistically higher in CRF (469 +/- 85 nM) than in C (275 +/- 60) and CGN (301 +/- 41). These findings suggest that platelets of CRF were capable of increasing [Ca2+]i in response to agonists, through further mobilization of calcium from the intracellular pool rather than the elevation of transmembrane calcium influx.     

Is the osteoclast calcium "receptor" a receptor-operated calcium channel?

Elevated extracellular calcium levels ([Ca2+]e) inhibit osteoclast function by elevating cytosolic free calcium levels ([Ca2+]i), presumably via the activation of a surface Ca2+ "receptor". It is unclear whether or not Ca(2+)-induced [Ca2+]i elevation involves the direct gating, by the putative "receptor", of a divalent cation channel. The results show that [Ca2+]i elevation in response to elevated [Ca2+]e comprises a distinct component of Ca2+ influx, the magnitude of which can be decreased and increased, respectively, by depolarising (100 mM-[K+]) and hyperpolarising (1 microM-[valinomycin]) the osteoclast membrane. In addition, activation of the putative Ca2+ "receptor" by elevated [Ca2+]e causes influx of the related divalent cation, magnesium (Mg2+). We suggest that Ca2+ influx induced by Ca2+ "receptor" activation is a major component of the observed [Ca2+]i response.     

The hop cassette of the PAC1 receptor confers coupling to Ca2+ elevation required for pituitary adenylate cyclase-activating polypeptide-evoked neurosecretion

We have identified the single PAC1 receptor variant responsible for Ca2+ mobilization from intracellular stores and influx through voltage-gated Ca2+ channels in bovine chromaffin cells and the domain of this receptor variant that confers coupling to [Ca2+]i elevation. This receptor (bPAC1hop) contains a 28-amino acid "hop" insertion in the third intracellular loop, with a full-length 171-amino acid N terminus. Expression of the bPAC1hop receptor in NG108-15 cells, which lack endogenous PAC1 receptors, reconstituted high affinity PACAP binding and PACAP-dependent elevation of both cAMP and intracellular Ca2+ concentrations ([Ca2+]i). Removal of the hop domain and expression of this receptor (bPAC1null) in NG108-15 cells reconstituted high affinity PACAP binding and PACAP-dependent cAMP generation but without a corresponding [Ca2+]i elevation. PC12-G cells express sufficient levels of PAC1 receptors to provide PACAP-saturable coupling to adenylate cyclase and to drive PACAP-dependent differentiation but do not express PAC1 receptors at levels found in postmitotic neuronal and endocrine cells and do not support PACAP-mediated neurosecretion. Expression of bPAC1hop, but not bPAC1(null), at levels comparable with those of bPAC1hop in bovine chromaffin cells resulted in acquisition by PC12-G cells of PACAP-dependent [Ca2+]i increase and extracellular Ca2+ influx. In addition, PC12-G cells expressing bPAC1hop acquired the ability to release [3H]norepinephrine in a Ca2+ influx-dependent manner in response to PACAP. Expression of PACAP receptors in neuroendocrine rather than nonneuroendocrine cells reveals key differences between PAC1hop and PAC1null coupling, indicating an important and previously unrecognized role of the hop cassette in PAC1-mediated Ca2+ signaling in neuroendocrine cells.     

Ca2+-agonistic effect of a T-type Ca-channel blocker mibefradil (Ro 40-5967)

Here we report that a Ca2+ antagonist mibefradil (Ro 40-5967) which has been shown to be a selective inhibitor of T-type calcium channels increases free calcium concentration ([Ca2+]i) in the cytoplasm of cultured smooth muscle cells isolated from porcine coronary artery. Smooth muscle cells were loaded with Fura 2 and a videoimage system was used to follow the [Ca2+]i responses. It was shown that at a concentration of 1 nM mibefradil induced a transient [Ca2+]i elevation in individual cells and at a concentration of 100 nM this compound stimulated almost all the cells in monolayer. The [Ca2+]i response did not change with the further increase of the mibefradil concentration up to 10 microM. The half-maximal effect was observed at 10 nM. The increase in [Ca2+]i strongly depended on the presence of Ca in the extracellular medium. Calcium antagonists belonging to three different classes--verapamil (phenylalkylamines), diltiazem (benzothiazepines) and amlodipin (dihydropyridines) neither suppressed the mibefradil effect nor mimicked it. These data indicate that mibefradil increased [Ca2+]i acting via a distinct receptor site. We suggest that these receptors are coupled to calcium channels of plasma membrane.     

Neural regulation of parvalbumin expression in mammalian skeletal muscle.

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.     

Two functionally distinct cholecystokinin receptors show different modes of action on Ca2+ mobilization and phospholipid hydrolysis in isolated rat pancreatic acini. Studies using a new cholecystokinin analog, JMV-180.

A new hepatapeptide cholecystokinin (CCK) analog, JMV-180 (Boc-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester), acts as an agonist at high affinity CCK receptors on rat pancreatic acini to stimulate amylase release but unlike cholecystokinin octapeptide (CCK8) does not act on low affinity CCK receptors to inhibit amylase release (Galas, M. D., Lignon, M. F., Rodriguez, M., Mendre, C., Fulcrand, P., Laur, J., and Martinez, J. (1988) Am. J. Physiol. 254, G176-G188). To investigate the biochemical mechanisms initiated by CCK acting on each class of CCK receptor, the effects of JMV-180 and CCK8 on amylase release, Ca2+ mobilization, and phospholipid hydrolysis were studied in isolated rat pancreatic acini. When acini were loaded with the intracellular Ca2+ chelator BAPTA, amylase release stimulated by both JMV-180 and CCK8 was reduced. Measurement of 45Ca2+ efflux and cytosolic free calcium concentration ([Ca2+]i) by the fluorescence of fura-2-loaded acini in a stirred cuvette showed that JMV-180 induced a concentration-dependent increase but with a maximal response only two-thirds that induced by CCK8. When [Ca2+]i of individual fura-2-loaded acinar cells was measured by microspectrofluorometry, all concentrations of JMV-180 (1 nM-10 microM) induced repetitive transient [Ca2+]i spikes (Ca2+ oscillations). By contrast, stimulation with a high concentration of CCK8 (1 nM) caused a large increase in [CA2+]i followed by a small sustained elevation of [Ca2+]i. The measurement of inositol trisphosphate (IP3) production by both [3H]inositol labeling and 1,4,5-IP3 radioreceptor assay showed that JMV-180 had only minimal effects at 10 microM in contrast to the large increase induced by high concentrations of CCK8 (more than 1 nM). JMV-180 blocked the effect of a high concentration of CCK8 on both [Ca2+]i and 1,4,5-IP3 productions but did not affect the response to carbamylcholine. JMV-180 caused a delayed monophasic stimulation of 1,2-diacylglycerol (DAG) sustained to 60 min without the early increase in DAG observed in response to CCK8. Furthermore, JMV-180 stimulated the release of [3H]choline metabolites, primarily phosphorylated choline, from [3H]choline-labeled acini at low concentrations and to the same extent as CCK8. Since JMV-180 interacts not only with high affinity CCK receptors as an agonist but also with low affinity CCK receptors as a functional antagonist, the present results indicate that the occupancy of high affinity state receptors by CCK induces Ca2+ oscillations, DAG formation from phosphatidylcholine hydrolysis, and amylase release with minimal phosphatidylinositol 4,5-bisphosphate hydrolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytosolic Ca2+ responses to sub-picomolar and nanomolar PACAP in pancreatic beta-cells are mediated by VPAC2 and PAC1 receptors

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates glucose-induced insulin release and increases cytosolic Ca2+ concentration ([Ca2+]i) in islet beta-cells in a concentration-dependent manner with two peaks at 10(-13) and 10(-9) M. PAC1 receptor (PAC1-R) and VPAC2 receptor (VPAC2-R) are expressed in pancreatic beta-cells and thought to be involved in insulin release. We aimed to determine the receptor types involved in the [Ca2+]i responses to 10(-13) and 10(-9) M PACAP. We measured [Ca2+]i in beta-cells and examined comparative effects of PAC1-R-selective agonist maxadilan, its antagonist M65, VPAC2-R-selective agonist Ro25-1553, and native ligands of PACAP and VIP. In the presence of 8.3 mM glucose, maxadilan, Ro25-1553, PACAP, and VIP at 10(-13) and 10(-9) M all increased [Ca2+]i. PACAP and maxadilan elicited greater effects at 10(-9) M than at 10(-13) M both in the incidence and amplitude of [Ca2+]i responses. For VIP and Ro25-1553, in contrast, the effects at 10(-9) and 10(-13) M were comparable. Furthermore, the amplitude of [Ca2+]i responses to 10(-9) M PACAP, but not 10(-13) M PACAP, was suppressed by M65. The results suggest that VPAC2-R and PAC1-R contribute equally to [Ca2+]i responses to sub-picomolar concentrations of PACAP, while PAC1-R has greater contribution to [Ca2+]i responses to nanomolar concentrations of this peptide.     

Single pancreatic beta-cells from normal rats exhibit an initial decrease and subsequent increase in cytosolic free Ca2+ in response to glucose.

Since it was reported that glucose stimulation initially lowers as well as subsequently raises the cytosolic free calcium concentration [( Ca2+]i) in pancreatic islet cells from hyperglycemic ob/ob mice, it has been argued whether the lowering of [Ca2+]i is physiological or artifactual. In the present study, [Ca2+]i in single pancreatic beta-cells from normal rats was measured by Fura-2 microfluorometry. Following elevation of the glucose concentration from 2.8 mM (basal) to 16.7 mM, a bimodal change in [Ca2+]i, an initial decrease and subsequent increase, was demonstrated. When the basal glucose concentration was raised to 5.6 mM, the stimulation with 16.7 mM glucose also induced the decrease in [Ca2+]i in the majority of the cells, though the amplitude of the decrease was reduced. An elevation of the glucose concentration from 2.8 to 5.6 mM induced the decrease in [Ca2+]i but not usually the increase in [Ca2+]i. Removal of extracellular Ca2+ eliminated the increase in [Ca2+]i without affecting the decrease in [Ca2+]i. Thus, the decrease and increase in [Ca2+]i were clearly dissociated under certain conditions. In contrast, mannoheptulose (an inhibitor of glucose metabolism) inhibited both the decrease and increase in [Ca2+]i. These results demonstrate that the glucose-induced bimodal change in [Ca2+]i is a physiological response of islet beta-cells, and that the decrease and increase in [Ca2+]i are generated by mutually-independent mechanisms which are operated through glucose metabolism by islet beta-cells.     

Urocortin 3 elevates cytosolic calcium in nucleus ambiguus neurons

J. Neurochem. (2012) 122, 1129-1136. ABSTRACT: Urocortin 3 (also known as stresscopin) is an endogenous ligand for the corticotropin-releasing factor receptor 2 (CRF(2) ). Despite predominant G(s) coupling of CRF(2) , promiscuous coupling with other G proteins has been also associated with the activation of this receptor. As urocortin 3 has been involved in central cardiovascular regulation at hypothalamic and medullary sites, we examined its cellular effects on cardiac vagal neurons of nucleus ambiguus, a key area for the autonomic control of heart rate. Urocortin 3 (1?nM-1000?nM) induced a concentration-dependent increase in cytosolic Ca(2+) concentration that was blocked by the CRF(2) antagonist K41498. In the case of two consecutive treatments with urocortin 3, the second urocortin 3-induced Ca(2+) response was reduced, indicating receptor desensitization. The effect of urocortin 3 was abolished by pre-treatment with pertussis toxin and by inhibition of phospolipase C with U-73122. Urocortin 3 activated Ca(2+) influx via voltage-gated P/Q-type channels as well as Ca(2+) release from endoplasmic reticulum. Urocortin 3 promoted Ca(2+) release via inositol 1,4,5 trisphosphate receptors, but not ryanodine receptors. Our results indicate a novel Ca(2+) -mobilizing effect of urocortin 3 in vagal pre-ganglionic neurons of nucleus ambiguus, providing a cellular mechanism for a previously reported role for this peptide in parasympathetic cardiac regulation.     

Effect of thrombin on survival of hippocampal neurons

The effect of thrombin on the rat hippocampal neurons death in model of neurotoxicity induced by hemoglobin or glutamate, was studied. Thrombin (10 nM) was shown to inhibit 100-mkM glutamate--or 10-mkM hemoglobin-induced apoptosis of the rat hippocampal neurons. With the aid of PAR1 (protease-activated receptor1) agonist peptide and PAR1 antagonist, the PAR1 was found to be necessary for protective action of thrombin in hippocampal neurons in models of neurotoxicity induced by hemoglobin or glutamate. Because the prolonged elevation [Ca2+] ib neurons is a critical part of neurodestructive processes in CNS, the effect of thrombin on Ca2+-homeostatis of neurons after its injury by the inducer of neuronal apoptosis: a synthetic agonist of the NMDA receptors N-methyl-D-aspartate (NMDA), was studied. We hypothesized that thrombin via receptors PAR may prove to be neuroprotective for the hippocampus. Thrombin was shown to stimulate via PAR1 a transient increase in [Ca2+] in neurons in a concentration-dependent manner. Thrombin (1 nM) decreased the [Ca2+] signal induced by activation of the NMDA-subtype of glutamate receptors. This thrombin effect may be one of the reasons of the protective action of thrombin in hippocampal neurons.