Involvement Of Pertussis Toxin-sensitive G-proteins Research Articles

Lysophosphatidylethanolamine stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells: Involvement of pertussis toxin-sensitive G-protein coupled receptor

We investigated whether lysophosphatidylethanolamine (LPE) modulates cellular signaling in different cell types. SK-OV3 ovarian cancer cells and OVCAR-3 ovarian cancer cells were responsive to LPE. LPE-stimulated intracellular calcium concentration ([Ca 2+] i) increase was inhibited by U-73122, suggesting that LPE stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca 2+] i increase by LPE, indicating the involvement of PTX-sensitive G-proteins. Furthermore, we found that LPE stimulated chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells. We examined the role of lysophosphatidic acid receptors on LPE-stimulated cellular responses using HepG2 cells transfected with different LPA receptors, and found that LPE failed to stimulate nuclear factor kappa B-driven luciferase. We suggest that LPE stimulates a membrane bound receptor, different from well known LPA receptors, resulting in chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells.

Effect of lysophosphatidylglycerol on several signaling molecules in OVCAR-3 human ovarian cancer cells: Involvement of pertussis toxin-sensitive G-protein coupled receptor

In this study, we observed that lysophosphatidylglycerol (LPG) stimulated intracellular calcium ([Ca 2+] i ) increase in OVCAR-3 human ovarian cancer cells. LPG-stimulated [Ca 2+] i increase was inhibited by U-73122 but not by U-73343, suggesting that LPG stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca 2+] i increase by LPG, indicating the activation of PTX-sensitive G-proteins. LPG-induced [Ca 2+] i increase was only observed in OVCAR-3 ovarian cancer cells and SK-OV3 ovarian cancer cells among tested several cell types. LPG also induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation in OVCAR-3 ovarian cancer cells. Pertussis toxin did not affect the LPG-induced activation of ERK and Akt phosphorylation. We also found that LPG failed to stimulate NF-κB-driven luciferase activity in exogenously LPA 1, LPA 2, or LPA 3-transfected HepG2 cells. Taken together we suggest that LPG stimulates a membrane bound receptor which is different from well-known LPA receptors (LPA 1, LPA 2, and LPA 3), resulting in at least two different signaling cascades; one involves a pertussis toxin-sensitive and phospholipase C-dependent [Ca 2+] i increase, and the other involves a pertussis toxin-insensitive activation of ERK and Akt in ovarian cancer cells.

Lysophosphatidic acid is a mediator of Trp-Lys-Tyr-Met-Val- d-Met-induced calcium influx

Intracellular calcium (Ca 2+) homeostasis is very strictly regulated, and the activation of G-protein-coupled receptor (GPCR) can cause two different calcium changes, intracellular calcium release, and calcium influx. In this study, we investigated the possible role of lysophosphatidic acid (LPA) on GPCR-induced Ca 2+ signaling. The addition of exogenous LPA induced dramatic Ca 2+ influx but not intracellular Ca 2+ release in U937 cells. LPA-induced Ca 2+ influx was not affected by pertussis toxin and phospholipase C inhibitor (U73122), ruling out the involvement of pertussis toxin-sensitive G-proteins, and phospholipase C. Stimulation of U937 cells with Trp-Lys-Tyr-Met-Val- d-Met (WKYMVm), which binds to formyl peptide receptor like 1, enhanced phospholipase A 2 and phospholipase D activation, indicating LPA formation. The inhibition of LPA synthesis by phospholipase A 2-specific inhibitor (MAFP) or n-butanol significantly inhibited WKYMVm-induced Ca 2+ influx, suggesting a crucial role for LPA in the process. Taken together, we suggest that LPA mediates WKYMVm-induced Ca 2+ influx.

C-Peptide Colocalizes with Macrophages in Early Arteriosclerotic Lesions of Diabetic Subjects and Induces Monocyte Chemotaxis In Vitro

Increased levels of C-peptide, a cleavage product of proinsulin, circulate in patients with insulin resistance and early type 2 diabetes, a high-risk population for the development of a diffuse and extensive pattern of arteriosclerosis. This study tested the hypothesis that C-peptide might participate in atherogenesis in these patients. We demonstrate significantly higher intimal C-peptide deposition in thoracic aorta specimens from young diabetic subjects compared with matched nondiabetic controls as determined by immunohistochemical staining. C-peptide colocalized with monocytes/macrophages in the arterial intima of artery specimen from diabetic subjects. In vitro, C-peptide stimulated monocyte chemotaxis in a concentration-dependent manner with a maximal 2.3+/-0.4-fold increase at 1 nmol/L C-peptide. Pertussis toxin, wortmannin, and LY294002 inhibited C-peptide-induced monocyte chemotaxis, suggesting the involvement of pertussis toxin-sensitive G-proteins as well as a phosphoinositide 3-kinase (PI3K)-dependent mechanism. In addition, C-peptide treatment activated PI3K in human monocytes, as demonstrated by PI3K activity assays. C-peptide accumulated in the vessel wall in early atherogenesis in diabetic subjects and may promote monocyte migration into developing lesions. These data support the hypothesis that C-peptide may play an active role in atherogenesis in diabetic patients and suggest a new mechanism for accelerated arterial disease in diabetes.

Norepinephrine acts via α 2 adrenergic receptors to suppress N-type calcium channels in dissociated rat median preoptic nucleus neurons

The median preoptic (MnPO) nucleus, a key CNS site for hydromineral and cardiovascular homeostasis, receives a dense norepinephrine innervation from brainstem autonomic centers. Since norepinephrine is known to influence neuronal excitability by modulating calcium channel function, we applied whole cell patch clamp techniques to study calcium currents in 116 dissociated MnPO neurons, including 30 cells identified by a retrograde label as projecting to the hypothalamic paraventricular nucleus. Norepinephrine (3–50 μM) suppressed high-voltage-activated calcium currents (HVA I Ca) in 80% of cells, selectively blockable by yohimbine and mimicked by UK14,304 and clonidine. The norepinephrine effect was relieved by strong prior depolarization, indicating a voltage-dependent component. Intracellular GTP-γ-S blocked the effect. Blockade by extracellular NEM suggested involvement of pertussis-toxin sensitive G-proteins. Based on pharmacological properties, these HVA I Cas had the following composition: 40–45% N-type (blockable by ω-conotoxin GVIA); 20–25% L-type (blockable by nimodipine); 15–20% P/Q-type (blockable by ω-agatoxin IVA). Since ∼75% of the norepinephrine effect was blockable with ω-conotoxin GVIA, we conclude that postsynaptic α 2 adrenoceptors preferentially suppress N-type calcium channels, revealing a novel mechanism whereby norepinephrine can modulate excitability in MnPO neurons.

Lack of involvement of pertussis toxin-sensitive G-proteins in norepinephrine-induced vasoconstriction of rat aorta smooth muscle

Several studies have shown that stimulation of pertussis toxin (PTX)-sensitive G-proteins amplified α-adrenoceptor (α-AR) agonist-induced vasoconstriction in small muscular and resistance arteries. The aim of this study was to assess the potential involvement of PTX-sensitive G-proteins in norepinephrine (NE)-induced constriction in a large diameter artery, the rat aorta. PTX (1 μg/mL, 2 hr; 3 μg/mL, 4 hr) did not modify concentration–response curves to NE in endothelium-denuded aortic rings. However, several lines of evidence suggested that aortic smooth muscle cells (SMC) had a PTX-sensitive G-protein pathway. [α- 32P]ADP-ribosylation of G i/o-proteins by PTX (3 μg/mL, 4 hr) was demonstrated in situ in the intact aorta without endothelium. α i/o subunits were identified in vitro by both immunoblotting and ADP-ribosylation experiments in rat aorta SMC membranes. The measurement of G i/o-specific GTPase activity evidenced an effective coupling between NE receptors and G i/o-proteins, as NE induced an increase in basal G i/o-specific GTPase activity (20.7 ± 2.8 vs 7.2 ± 2.2 pmol P i/mg protein at 5 min; P < 0.05 vs basal). Co-immunoprecipitation revealed the in vitro coupling between α 1D-ARs and G i-protein in rat aorta SMC membranes. In conclusion, we identified a PTX-sensitive G i/o-protein pathway in rat endothelium-denuded aorta. We showed an effective coupling between NE receptors and G i-proteins via α 1D-ARs. Since PTX has no effect on NE-induced vasoconstriction, the PTX-sensitive G i-protein pathway does not play a predominant role in NE-induced responses in rat aorta SMC in contrast to small diameter muscular and resistance arteries.

Involvement of cAMP-dependent protein kinase and pertussis toxin-sensitive G-proteins in CGRP mediated JNK activation in human neuroblastoma cell line

Calcitonin gene-related peptide (CGRP) is a neuropeptide with potent cardiovascular effects, which include positive inotropic and chronotropic actions, systemic vasodilation, and hypotension in animal and human studies. Human neuroblastoma cells (SK-N-MC) have been used as a model system to study the CGRP receptors and downstream signaling pathways. This investigation was undertaken to study the role of CGRP in the activation of mitogen-activated protein kinases. While exposure of these cells to CGRP had no significant effect on ERK-1 or p38 MAP kinases, JNK activity was stimulated by CGRP in a time- and concentration-dependent fashion. CGRP-mediated JNK-activation was inhibited by CGRP receptor antagonist, CGRP8–37, confirming that this is a receptor-mediated event. In addition, pretreatment of the cells with H-89, protein kinase A inhibitor or pertussis toxin greatly attenuated CGRP-mediated JNK activation suggesting the requirement of cAMP-dependent protein kinase activation and involvement of pertussis toxin-sensitive G-protein in CGRP-mediated JNK activation.

Human Fetal Skin Fibroblast Migration Stimulated by the Autocrine Growth Factor bFGF Is Mediated by Phospholipase A2 via Arachidonic Acid without the Involvement of Pertussis Toxin-Sensitive G-Protein

We reported previously that human fetal skin fibroblast migration into a denuded area was stimulated by an autocrine factor, basic fibroblast growth factor (bFGF). Since the signal transduction pathway of this migration is unknown, we attempted to clarify it by comparing this fibroblast migration with a previously reported bovine endothelial cell migration into a wounded area stimulated by an addition of bFGF, in which the bFGF signal was mediated by phospholipase A2-coupled G-protein and phospholipase A2 (PLA2) via arachidonic acid. Our study demonstrated that pertussis toxin, a specific inhibitor of PLA2-coupled G-protein, did not suppress human fetal skin fibroblast migration, but 2-(p-amylcinnamyl)amino-4-chlorobensoic acid (ONO-RS-082), a PLA2 inhibitor, did. Since ONO-RS-082 is a non-specific PLA2 inhibitor, a cytoplasmic, Ca-dependent PLA2 (cPLA2) inhibitor, AACOCF3, was examined. AACOCF3 suppressed cell migration in certain concentrations. The PLA2 inhibitor-suppressed cell migration was restored by adding arachidonic acid, and cell migration suppressed by anti-bFGF antibodies was restored by adding arachidonic acid. In addition, pertussis toxin did not suppress arachidonic acid release, which shows an action of PLA2, but AACOCF3 did. These results indicate that human fetal skin fibroblast migration stimulated by an autocrine factor, bFGF, was mediated by PLA2 via arachidonic acid without the involvement of PLA2-coupled G-protein.

Leukotoxin and its diol induce neutrophil chemotaxis through signal transduction different from that of fMLP

When injected into animals, leukotoxin (Lx) causes acute lung injury which is associated with neutrophils infiltrating the lung tissues. However, the effect of Lx on neutrophils is still unknown, and recently it has been reported that Lx diol, a hydrolyzed metabolite, should be more potent than Lx in vitro. In this study, the authors examined the effect of Lx and its diol on human neutrophils by assessing their chemotactic response, expression of adhesion molecules, and production of peroxides. Both Lx and its diol induced chemotaxis in human neutrophils via an involvement of pertussis toxin-sensitive G-proteins, but they did not influence the expression of adhesion molecules or the production of peroxides. Furthermore, Lx synergistically affected chemotaxis with N-formyl-methionyl-leucyl-phenylalanine (fMLP), but not with endothelin-1. Neutrophil chemotaxis induced by both Lx and its diol was inhibited by phosphatidylinositol-3-kinase (PI3-K) inhibitors, but not by protein tyrosine kinase (PTK) inhibitors or by protein kinase C (PKC) inhibitors, whereas fMLP-induced chemotaxis was inhibited by PTK inhibitors, but not by PI3-K inhibitors or by PKC inhibitors. These results suggest that neutrophil chemotaxis induced by both Lx and its diol involves pathways different from those induced by fMLP. In conclusion, both leukotoxin and its diol metabolite induce chemotaxis in human neutrophils in a unique way and may act as important bioactive lipids when considering the pathological mechanism of acute lung injury.

Leukotoxin and its diol induce neutrophil chemotaxis through signal transduction different from that of fMLP

When injected into animals, leukotoxin (Lx) causes acute lung injury which is associated with neutrophils infiltrating the lung tissues. However, the effect of Lx on neutrophils is still unknown, and recently it has been reported that Lx diol, a hydrolyzed metabolite, should be more potent than Lx in vitro. In this study, the authors examined the effect of Lx and its diol on human neutrophils by assessing their chemotactic response, expression of adhesion molecules, and production of peroxides. Both Lx and its diol induced chemotaxis in human neutrophils via an involvement of pertussis toxin-sensitive G-proteins, but they did not influence the expression of adhesion molecules or the production of peroxides. Furthermore, Lx synergistically affected chemotaxis with N-formyl-methionyl-leucyl-phenylalanine (fMLP), but not with endothelin-1. Neutrophil chemotaxis induced by both Lx and its diol was inhibited by phosphatidylinositol-3-kinase (PI3-K) inhibitors, but not by protein tyrosine kinase (PTK) inhibitors or by protein kinase C (PKC) inhibitors, whereas fMLP-induced chemotaxis was inhibited by PTK inhibitors, but not by PI3-K inhibitors or by PKC inhibitors. These results suggest that neutrophil chemotaxis induced by both Lx and its diol involves pathways different from those induced by fMLP. In conclusion, both leukotoxin and its diol metabolite induce chemotaxis in human neutrophils in a unique way and may act as important bioactive lipids when considering the pathological mechanism of acute lung injury.

Rat striatal adenosinergic modulation of ethanol-induced motor impairment: possible role of striatal cyclic AMP

We have previously reported the involvement of the striatum in acute ethanol-induced motor incoordination and the striatal adenosinergic modulation of ethanol-induced motor incoordination through A 1 receptor-mediated mechanism(s). The present study, a continuation of our previous work, was carried out to investigate the possible functional correlation between striatal cyclic AMP and ethanol-induced motor incoordination, and its modulation by striatal adenosine in Sprague–Dawley rats. Forskolin (0.1, 0.5 and 1.0 pmol), a known activator of adenylate cyclase, significantly attenuated ethanol-induced motor incoordination in a dose-dependent manner following its direct intrastriatal microinfusion. Forskolin also antagonized the accentuating effect of intrastriatal N 6-cyclohexyladenosine on ethanol-induced motor incoordination. These results suggested that ethanol-induced motor incoordination might be functionally correlated to a decrease in the striatal cyclic AMP levels and that the striatal adenosine A 1 receptors might modulate ethanol-induced motor incoordination through cyclic AMP signaling mechanism(s). Further support to this hypothesis was obtained by the actual measurement of the striatal cyclic AMP levels in the same experimental conditions as in motor coordination studies using high-performance liquid chromatography with fluoroscence detection. Regardless of the method (focused microwave irradiation, cervical dislocation or decapitation into a dry ice–ethanol mixture) used to kill the animals, a significant decrease in the striatal cyclic AMP levels was observed due to ethanol. Intrastriatal adenosine A 1-selective agonist, N 6-cyclohexyladenosine (24 ng), caused a further significant decrease in the striatal cyclic AMP levels in the ethanol- but not in the vehicle-treated animals. The further enhancement in the ethanol-induced decrease in the striatal cyclic AMP levels by intrastriatal N 6-cyclohexyladenosine, therefore, functionally correlated with the observed potentiating effect of intrastriatal N 6-cyclohexyladenosine on ethanol-induced motor incoordination. The effects of intrastriatal N 6-cyclohexyladenosine+ethanol and of ethanol alone on the striatal cyclic AMP levels were blocked by intrastriatal pertussis toxin (500 ng) pretreatment, indicating the involvement of pertussis toxin-sensitive G-proteins (G i, G o) and possibly of the adenosine A 1 receptor coupled to the G-proteins in the striatum. Furthermore, ethanol alone significantly decreased the basal as well as the cyclic AMP-stimulated catalytic activities of the striatal cyclic AMP protein kinase, which were further reduced by intrastriatal N 6-cyclohexyladenosine. The results of the present study therefore support an involvement of a cyclic AMP signaling pathway in the striatal adenosinergic modulation of ethanol-induced motor incoordination at the post-adenosine A 1 receptor level.

Characterization of human recombinant somatostatin sst5 receptors mediating activation of phosphoinositide metabolism.

1. We have functionally characterized the human recombinant somatostatin (SRIF) sst5 receptor in Chinese hamster ovary-K1 (CHOsst5) cells by measuring total [3H]-inositol phosphate ([3H]-InsPx) accumulation, in the presence of 10 mM LiCl, in cells labelled with [3H]-myo-inositol. 2. In CHOsst5 cells, SRIF, SRIF-28 and the cyclic hexapeptide, L-362,855, produced time- and concentration-related increases in [3H]-InsPx accumulation, with similar potency (pEC50 values of 6.5, 6.8 and 7.2, respectively). L-362,855 behaved as a partial agonist, producing approximately 30% of the SRIF maximum response. The other peptide analogues of SRIF, BIM-23027 and BIM-23056, were inactive as agonists. 3. Increasing concentrations of L-362,855 increased [3H]-InsPx accumulation and simultaneously produced rightward shifts of SRIF concentration-effect curves, with an estimated pKp value of 7.6, confirming that it was acting as a partial agonist. 4. BIM-23056, but not BIM-23027, potently antagonized SRIF-induced [3H]-InsPx accumulation, with an estimated pKB value of 7.4. BIM-23056 did not antagonize [3H]-InsPx accumulation induced by uridine 5'-triphosphate (UTP). 5. SRIF- but not UTP-induced [3H]-InsPx accumulation was inhibited by increasing concentrations of pertussis toxin (0.01-100 ng ml-1), indicating the involvement of pertussis toxin-sensitive G-proteins. 6. These findings show that the human recombinant sst5 receptor, when stably expressed in CHO-K1 cells, is able to mediate activation of phosphoinositide metabolism in a pertussis toxin-sensitive manner. In this system L-362,855 behaved as a partial agonist while BIM-23056 was a specific antagonist. These agents should provide useful tools for functionally characterizing endogenous SRIF receptors.

Chemotactic agonists induce cytokine generation in eosinophils.

Recent studies have shown that eosinophils are capable of generating and releasing cytokines, providing a novel biologic aspect of eosinophils for regulating allergic inflammation by an autocrine or paracrine mechanism. Eosinophils synthesize various cytokines; however, the physiologic stimuli that trigger eosinophils to generate cytokines have not been fully elucidated. We examined the effect of chemotactic agonists on eosinophil cytokine generation by employing the determination of IL-8 as the main parameter. Both C5a and FMLP stimulated eosinophils to release IL-8, whereas platelet-activating factor and C-C chemokines did not exert any significant effects. On a molar basis, C5a was two orders of magnitude more potent than FMLP. The generation of IL-8 by chemoattractants was absolutely dependent on the presence of cytochalasin B. Pertussis toxin completely attenuated C5a- and FMLP-induced IL-8 production, indicating the involvement of pertussis toxin-sensitive G-proteins in the signal-transduction process leading to these responses. Experiments of in situ hybridization and PCR amplification revealed that both C5a and FMLP promoted eosinophil IL-8 production through transcriptional gene activation. Pyrrolidine dithiocarbamate completely abrogated chemoattractant-induced IL-8 production, indicating the involvement of NF-kappa B in the cytoplasmic/nuclear signal-transduction process. Furthermore, chemoattractant-induced cytokine production was not limited to IL-8; C5a and FMLP but not platelet-activating factor induced significant secretion of granulocyte-macrophage-CSF from eosinophils. These results indicate that C5a and FMLP stimulate eosinophils to elaborate cytokines, which could be an important mechanism in the regulation of allergic inflammation.

Inhibitory action of somatostatin on cAMP dependent pepsinogen secretion from rat gastric chief cells: involvement of pertussis toxin-sensitive G-protein.

We examined the inhibitory effect of somatostatin on pepsinogen secretion using isolated rat gastric chief cells. Secretin and forskolin significantly increased not only pepsinogen secretion from chief cells but also cellular cAMP accumulation in a dose-dependent fashion. Somatostatin significantly inhibited secretin- and forskolin-induced pepsinogen secretion and secretin-induced cellular cAMP accumulation. However, forskolin-induced cellular cAMP accumulation was not inhibited by somatostatin. The inhibitory effect of somatostatin on secretin-induced pepsinogen secretion was abolished by pretreatment with pertussis toxin, but inhibition of forskolin-, carbachol- and cholecystokinin octapeptide-induced pepsinogen secretion was not. These results suggest that somatostatin inhibits pepsinogen secretion in two ways, one is closely related to the pertussis toxin-sensitive G-protein and the other is not determined.

Involvement of pertussis toxin-sensitive G-proteins on thermic responses to morphine and neurotensin

Involvement of pertussis toxin-sensitive G-proteins on thermic responses to morphine and neurotensin

Inhibitory effect of somatostatin on cAMP accumulation and calcitonin secretion in C-cells: Involvement of pertussis toxin-sensitive G-proteins

The effect of somatostatin on cAMP accumulation and calcitonin secretion in C-cells of the rat medullary thyroid carcinoma cell line rMTC 6–23 was investigated. Intracellular cAMP accumulation as well as calcitonin secretion could be dose-dependently stimulated by rat growth hormone releasing factor (rGRF). The long-acting somatostatin analogue octreotide inhibited rGRF-stimulated cAMP accumulation and calcitonin secretion dose dependently but failed to block 8-bromo-cAMP-stimulated calcitonin secretion. The inhibitory effect of octreotide on rGRF-induced calcitonin secretion was partially abolished by pretreating the cells with pertussis toxin. The octreotide effect was not due to changes in the degradation of cAMP, as it was similarily seen in the presence of isobutylmethylxanthine. Thus we conclude that pertussis toxin-sensitive G-proteins are involved in the cAMP-mediated regulation of calcitonin secretion in C-cells.

Involvement of pertussis toxin-sensitive G-proteins in the hormonal inhibition of dihydropyridine-sensitive Ca2+ currents in an insulin-secreting cell line (RINm5F).

Adrenaline inhibits insulin secretion via pertussis toxin-sensitive mechanisms. Since voltage-dependent Ca2+ currents play a key role in insulin secretion, we examined whether adrenaline modulates voltage-dependent Ca2+ currents of the rat insulinoma cell line, RINm5F. In the whole-cell configuration of the patch-clamp technique, dihydropyridine- but not omega-conotoxin-sensitive Ca2+ currents were identified. Adrenaline via alpha 2-adrenoceptors inhibited the Ca2+ currents by about 50%. Somatostatin which also inhibits insulin secretion was less efficient (inhibition by 20%). The hormonal inhibition of Ca2+ currents was not affected by intracellularly applied cAMP but blocked by the intracellularly applied GDP analog guanosine 5'-O-(2-thiodiphosphate) and by pretreatment of cells with pertussis toxin. In contrast to adrenaline and somatostatin, galanin, another inhibitor of insulin secretion, reduced Ca2+ currents by about 40% in a pertussis toxin-insensitive manner. Immunoblot experiments performed with antibodies generated against synthetic peptides revealed that membranes of RINm5F cells possess four pertussis toxin-sensitive G-proteins including Gi1, Gi2, Go2, and another Go subtype, most likely representing Go1. In membranes of control but not of pertussis toxin-treated cells, adrenaline via alpha 2-adrenoceptors stimulated incorporation of the photo-reactive GTP analog [alpha-32P]GTP azidoanilide into pertussis toxin substrates comigrating with the alpha-subunits of Gi2, Go2, and the not further identified Go subtype. The present findings indicate that activated alpha 2-adrenoceptors of RINm5F cells interact with multiple G-proteins, i.e. two forms of Go and with Gi2. These G-proteins are likely to be involved in the adrenaline-induced inhibition of dihydropyridine-sensitive Ca2+ currents and in other signal transduction pathways contributing to the adrenaline-induced inhibition of insulin secretion.