Protein kinase C activation increases endothelial nitric oxide release in mesenteric arteries from orchidectomized rats

The aim of the present study was to assess the effect of endogenous male sex hormones on endothelial nitric oxide synthase (eNOS) expression, release and function of the endothelial nitric oxide (NO), as well as to assess the regulatory action of protein kinase C (PKC) on acetylcholine (ACh)-induced endothelial NO release. For this purpose, superior mesenteric arteries from control and orchidectomized male Sprague-Dawley rats were used. eNOS expression and basal-and ACh-induced NO release were similar in arteries from both groups of rats. Orchidectomy decreased the vasodilator effect induced by ACh but did not alter that induced by sodium nitroprusside (SNP). The superoxide anion scavenger, superoxide dismutase (SOD), or the membrane-permeable mimetic of SOD, tempol, only enhanced ACh-induced relaxation in arteries from orchidectomized rats. ACh-induced TXA(2) formation was higher in arteries from orchidectomized than from control rats. Neither the PKC activator, phorbol 12,13-dibutyrate (PDBu), nor the non-selective PKC inhibitor, calphostin C, modified basal- or ACh-induced NO release in arteries from control rats. In arteries from orchidectomized rats, basal- and ACh-induced endothelial NO release were increased by PDBu but decreased by calphostin C. Both G?6976, a PKC inhibitor that is partially selective for conventional PKC isoforms, as well as PKCzeta pseudosubstrate inhibitor (PKCzeta-PI) decreased both basal- and ACh-induced NO release in arteries from orchidectomized rats. Neither PDBu nor calphostin C modified the vasodilator response induced by ACh in arteries from control rats. In segments from orchidectomized rats, PDBu enhanced the ACh-induced response, but this response was not modified by calphostin C, G?6976 or PKCzeta-PI. The vasodilator response induced by SNP was not altered by the PKC activators or inhibitors in any artery from either group. These results show that endogenous male sex hormone deprivation does not affect the eNOS expression or the endothelial NO release induced by ACh, but does decrease the vasodilator action of ACh, by increasing NO metabolism and TXA(2) formation. In addition, PKC seems to modulate eNOS activity only in mesenteric arteries from orchidectomized rats, in which conventional and PKCzeta isoforms are involved in the positive regulation of eNOS.     

Aldose reductase inhibitor improves insulin-mediated glucose uptake and prevents migration of human coronary artery smooth muscle cells induced by high glucose

We examined involvement of the polyol pathway in high glucose-induced human coronary artery smooth muscle cell (SMC) migration using Boyden's chamber method. Chronic glucose treatment for 72 hours potentiated, in a concentration-dependent manner (5.6 to 22.2 mol/L), platelet-derived growth factor (PDGF) BB-mediated SMC migration. This potentiation was accompanied by an increase in PDGF BB binding, because of an increased number of PDGF-beta receptors, and this potentiation was blocked by the aldose reductase inhibitor epalrestat. Epalrestat at concentrations of 10 and 100 nmol/L inhibited high glucose-potentiated (22.2 mmol/L), PDGF BB-mediated migration. Epalrestat at 100 nmol/L inhibited a high glucose-induced increase in the reduced/oxidized nicotinamide adenine dinucleotide ratio and membrane-bound protein kinase C (PKC) activity in SMCs. PKC inhibitors calphostin C (100 nmol/L) and chelerythrine (1 micromol/L) each inhibited high glucose-induced, PDGF BB-mediated SMC migration. High glucose-induced suppression of insulin-mediated [(3)H]-deoxyglucose uptake, which was blocked by both calphostin C (100 nmol/L) and chelerythrine (1 micromol/L), was decreased by epalrestat (100 nmol/L). Chronic high glucose treatment for 72 hours increased intracellular oxidative stress, which was directly measured by flow cytometry using carboxydichlorofluorescein diacetate bis-acetoxymethyl ester, and this increase was significantly suppressed by epalrestat (100 nmol/L). Antisense oligonucleotide to PKC-beta isoform inhibited high glucose-mediated changes in SMC migration, insulin-mediated [(3)H]-deoxyglucose uptake, and oxidative stress. These findings suggest that high glucose concentrations potentiate SMC migration in coronary artery and that the aldose reductase inhibitor epalrestat inhibits high glucose-potentiated, PDGF BB-induced SMC migration, possibly through suppression of PKC (PKC-beta), impaired insulin-mediated glucose uptake, and oxidative stress.     

Signaling mechanisms that mediate nitric oxide production induced by acetylcholine exposure and withdrawal in cat atrial myocytes

Fluorescence microscopy and the NO-sensitive indicator 4,5-diaminofluorescein were used to determine the effects of acetylcholine (ACh) on intracellular NO (NOi) in cat atrial myocytes. Field stimulation (1 Hz) of cells or exposure of quiescent cells to ACh (1 to 10 micromol/L) had no effect on NOi. However, in field-stimulated cells, ACh exposure increased NOi, and ACh withdrawal elicited an additional, prominent increase in NOi production. During ACh exposure, addition of 1 micromol/L atropine increased NOi production similar to ACh withdrawal. ACh-induced increases in NOi were reduced by prior exposure to 1 mmol/L extracellular Ca2+ ([Ca2+]o) and prevented by 0.5 mmol/L [Ca2+]o, 1 micromol/L verapamil, 1 micromol/L atropine, 10 micromol/L L-N5-(1-iminoethyl)ornithine, 10 micromol/L W-7, or incubating cells in pertussis toxin or 10 micromol/L LY294002 (inhibits phosphatidylinositol 3-kinase). Switching to 0.5 mmol/L [Ca2+]o during ACh withdrawal prevented the additional increase in NOi. ACh exposure increased phosphorylation (Ser473) of protein kinase B (Akt), and this effect was blocked by LY294002 and unaffected in low (0.5 mmol/L) [Ca2+]o. Confocal microscopy revealed that ACh exposure increased NOi at local subsarcolemmal sites, and ACh withdrawal additionally increased NOi by recruiting additional subsarcolemmal release sites. Disruption of caveolae by 2 mmol/L methyl-beta-cyclodextrin abolished ACh-induced NOi production. We conclude that in cat atrial myocytes, ACh stimulates NOi release from local subsarcolemmal sites. ACh-induced increases in NOi requires both muscarinic receptor-mediated Gi protein/phosphatidylinositol 3-kinase/Akt signaling and voltage-activated Ca2+ influx for stimulation of calmodulin-dependent endothelial NO synthase activity. Increases in NOi elicited by ACh withdrawal result from the recovery of Ca2+ influx after ACh inhibition. NO signaling elicited by ACh withdrawal stimulates rapid recovery from cholinergic atrial inhibition.     

Low concentrations of sphingosylphosphorylcholine enhance pulmonary artery vasoreactivity: the role of protein kinase C delta and Ca2+ entry

Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC(50) is approximately 100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane-sensitive Ca(2+) entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC delta inhibitor rottlerin, but not by G?6976, which is ineffective against PKC delta. The potentiation could be attributed to enhancement of Ca(2+) entry. SPC also potentiated the responses to prostaglandin F(2 alpha) and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 micromol/L) caused translocation of PKC delta to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca(2+) entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC delta. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.     

Role of soluble epoxide hydrolase in postischemic recovery of heart contractile function

Cytochrome P450 epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which are converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (Ephx2, sEH). To examine the functional role of sEH in the heart, mice with targeted disruption of the Ephx2 gene were studied. Hearts from sEH null mice have undetectable levels of sEH mRNA and protein and cannot convert EETs to DHETs. sEH null mice have normal heart anatomy and basal contractile function, but have higher fatty acid epoxide:diol ratios in plasma and cardiomyocyte cell culture media compared with wild type (WT). sEH null hearts have improved recovery of left ventricular developed pressure (LVDP) and less infarction compared with WT hearts after 20 minutes ischemia. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 to 100 nmol/L) before ischemia abolishes this cardioprotective phenotype. Inhibitor studies demonstrate that perfusion with phosphatidylinositol-3 kinase (PI3K) inhibitors wortmannin (200 nmol/L) or LY294002 (5 micromol/L), the ATP-sensitive K+ channel (K(ATP)) inhibitor glibenclamide (1 micromol/L), the mitochondrial K(ATP) (mitoK(ATP)) inhibitor 5-hydroxydecanoate (100 to 200 micromol/L), or the Ca2+-sensitive K+ channel (K(Ca)) inhibitor paxilline (10 micromol/L) abolishes the cardioprotection in sEH null hearts. Consistent with increased activation of the PI3K cascade, sEH null mice exhibit increased cardiac expression of glycogen synthase kinase-3beta (GSK-3beta) phospho-protein after ischemia. Together, these data suggest that targeted disruption of sEH increases the availability of cardioprotective EETs that work by activating PI3K signaling pathways and K+ channels.     

Angiotensin II stimulates extracellular signal-regulated kinase activity in intact pressurized rat mesenteric resistance arteries

The activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was assessed in isolated rat mesenteric resistance arteries (200-micrometer diameter) in a pressure myograph and stimulated for 5 minutes by angiotensin II (Ang II, 0.1 micromol/L) with a pressure of 70 mm Hg. ERK1/2 activity was measured by using an in-gel assay, and ERK1/2 phosphorylation was measured by Western blot analysis with use of a phospho-specific ERK1/2 antibody. Ang II (0.1 micromol/L) induced contraction (28% of phenylephrine contraction, 10 micromol/L). ERK kinase inhibitor PD98059 (10 micromol/L) attenuated this contraction by 36% but not that to phenylephrine or K(+) (60 mmol/L). In unpressurized arteries, Ang II increased ERK1/2 activity by 26%, and pressure (70 mm Hg) itself increased ERK1/2 activity by 72%. Ang II and pressure together acted synergistically, increasing ERK1/2 activity by 264%. Thus, in pressurized vessels, Ang II (0.1 micromol/L) increased ERK1/2 activity by 112%, calculated as [(364/172)-1]x100, which was confirmed by a measured 72% increase in ERK1/2 phosphorylation. Ang II type 1 receptor blockade by candesartan (10 micromol/L) abolished the Ang II-induced increase in ERK1/2 activity, but Ang II type 2 receptor blockade (PD123319, 10 micromol/L) did not. The Ang II-induced increase in ERK1/2 activity was inhibited by protein kinase C inhibitors Ro-31-8220 (1 micromol/L) and Go-6976 (300 nmol/L) and tyrosine kinase inhibitors genistein (1 micromol/L, general) and herbimycin A (1 micromol/L, c-Src family). The present findings show for the first time in intact resistance arteries that ERK1/2 activation is rapidly regulated by Ang II, is synergistic with pressure, and is involved in contraction. The ERK1/2 signaling pathway apparently includes upstream protein kinase C and c-Src.     

Endothelin-1 (ET-1)-potentiated insulin secretion: involvement of protein kinase C and the ET(A) receptor subtype

Endothelin-1 (ET-1), a potent vasoconstrictor peptide of endothelial origin, is capable of influencing hormone secretion from endocrine tissues, eg, pancreatic islet cells. We have shown a direct stimulatory effect of ET-1 on insulin secretion from isolated mouse islets of Langerhans. However, it is unknown as to whether the peptide acts through specific receptors on the islet cells and which mechanisms are involved in this insulinotropic action. We have therefore used the specific ET(A) receptor antagonist BQ123, the ET(B) receptor agonist BQ3020, and classic alpha- and beta-adrenergic and cholinergic antagonists. ET-1 (100 nmol/L) stimulated insulin secretion from islets incubated at 8.3, 11.1, 16.7, and 25 mmol/L glucose (P < .05). At 3.3 mmol/L glucose, no alteration in insulin secretion was found. The cholinergic receptor antagonist atropine (5 micromol/L) or the adrenergic receptor antagonists propranolol (5 micromol/L) or phentolamine (5 micromol/L) did not affect ET-1 (100 nmol/L)-stimulated insulin secretion. BQ123 (10 pmol/L to 10 nmol/L) and BQ3020 (1 nmol/L to 1 micromol/L) had no effect on glucose (16.7 mmol/L)-stimulated insulin secretion, but BQ123 counteracted the stimulatory effect of ET-1 (100 nmol/L) at concentrations of 1 nmol/L to 10 micromol/L (P < .01). We also studied the relative role of protein kinase C (PKC) and a Wortmannin-sensitive pathway for ET-1-induced insulin secretion using 12-O-tetradecanoyl phorbol-13-acetate (TPA), Calphostin C, and Wortmannin, respectively. At 5.6 mmol/L glucose, ET-1 (100 nmol/L) had no effect per se, whereas in the presence of 1 micromol/L TPA, which acutely stimulates PKC, the peptide did potentiate insulin secretion (P < .05). Furthermore, the insulinotropic effect of ET-1 at 16.7 mmol/L glucose was counteracted by the PKC inhibitor Calphostin C (P < .05) and by downregulation of PKC by 24 hours of exposure of islets to TPA (0.5 micromol/L, P < .05). Wortmannin (1 micromol/L) did not alter ET-1-potentiated insulin secretion. In conclusion, our results suggest that ET-1 acts through specific ET-1 receptors, most likely the ETA subtype. Furthermore, PKC plays an essential role in the insulinotropic action of ET-1 in mouse islets.     

The Signaling Pathways of Erythropoietin and Interferon-γ Differ in Preventing the Apoptosis of Mature Erythroid Progenitor Cells

Interferon (IFN)-gamma is a survival factor for mature erythroid progenitor cells. To elucidate related survival mechanisms, we compared the role of phosphatidylinositol 3-kinase (PI3-kinase) in the survival signals of IFN-gamma and erythropoietin (EPO). Human erythroid colony-forming cells (ECFCs) purified from peripheral blood were used, and Ly294002 was used as a PI3-kinase inhibitor. Treating ECFCs with a high concentration of Ly294002 (50 micromol/L) in the presence of EPO and/or IFN-gamma reduced cell viability by inducing apoptosis. However, treating cells with a lower concentration of Ly294002 (10 micromol/L) did not affect the antiapoptotic function of IFN-gamma and abolished the antiapoptotic effect of EPO. Adding IFN-gamma or EPO induced Bcl-x expression in ECFCs, as determined by Western blotting, and expression was suppressed in the presence of Ly294002. We also examined the phosphorylation of the protein kinase Akt, the downstream target of PI3-kinase. EPO stimulation significantly increased the level of Akt phosphorylation, but IFN-gamma did not. These results suggest that IFN-gamma plays a role in preventing the apoptosis of erythroid progenitor cells by affecting Bcl-x expression, thereby reducing the disruption of the mitochondrial transmembrane potential via PI3-kinase pathways that are related to but distinct from the EPO pathway.     

Extracellular signal-regulated kinase, Jun N-terminal kinase, p38, and c-Src are involved in gonadotropin-releasing hormone-stimulated activity of the glycoprotein hormone follicle-stimulating hormone beta-subunit promoter

The role of ERK, Jun N-terminal kinase (JNK), p38, and c-Src in GnRH-stimulated FSHbeta-subunit promoter activity was examined in the LbetaT-2 gonadotroph cell line. Incubation of the cells with a GnRH agonist resulted in activation of ERK, JNK, p38, and c-Src. The peak of ERK activation was observed at 5 min, whereas that of JNK, p38, and c-Src at 30 min, declining thereafter. ERK activation by GnRH is dependent on protein kinase C (PKC), as evident by activation, inhibition, and depletion of 12-O-tetradecanoylphorbol-13-acetate-sensitive PKC subspecies. Ca(2+) influx, but not Ca(2+) mobilization, is required for ERK activation. GnRH signaling to ERK is partially mediated by dynamin and a protein tyrosine kinase, apparently c-Src. ERK activation by GnRH in LbetaT-2 cells does not involve transactivation of epidermal growth factor receptor or mediation via Gbetagamma or beta-arrestin. Once activated by GnRH, ERK translocates to the nucleus. We examined the role of ERK, JNK, p38, and c-Src in GnRH-stimulated ovine FSHbeta promoter, linked to a luciferase reporter gene (-4741oFSHbeta-LUC). The PKC activator 12-O-tetradecanoylphorbol-13-acetate, but not the Ca(2+) ionophore ionomycin, stimulated FSHbeta-luciferase (LUC) activity. Furthermore, down-regulation of PKC, but not removal of Ca(2+), inhibited the GnRH response. Cotransfection of FSHbeta-LUC and the constitutively active forms of Raf-1 and MEK stimulated FSHbeta-LUC activity, whereas the dominant negatives of Ras, Raf-1, and MEK and the selective MEK inhibitor PD98059, abolished GnRH-induced FSHbeta-LUC activity. The dominant negatives of CDC42 and JNK reduced the GnRH response by 36 and 49%, respectively. Incubation of the cells with the p38 or the c-Src inhibitors SB203580 and PP1 also reduced the GnRH response. Surprisingly, two proximal activator protein-1 sites contribute very little to the GnRH response. Thus, PKC, ERK, JNK, p38, and c-Src, but not Ca(2+), are involved in GnRH induction of the ovine FSHbeta gene.     

Cilnidipine is a novel slow-acting blocker of vascular L-type calcium channels that does not target protein kinase C

Cilnidipine is a novel dihydropyridine (DHP) antagonist. However, its pharmacological effects on vascular DHP-sensitive L-type channels and protein kinase C (PKC)-mediated arterial contraction is incompletely understood. To address this issue, we studied the effects of cilnidipine on multi-subunit, C-class L-type Ca2+ channels in rat aortic A7r5 cells, as well as on Ca2+ channel (L-type) alpha1C-b and (T-type) alpha1G subunits in the Xenopus oocyte expression system. Cilnidipine dose- and time-dependently inhibited Ba2+ currents in A7r5 cells, with half-maximal inhibitions (IC50) at 10 nmol/l after 10 min. Unlike classical pharmacological Ca2+ channel blockers, cilnidipine's block of Ca2+ currents did not reach steady-state levels within 10 min, indicating steady-state half-maximal inhibition of native, multi-subunit L-type channels at < 10 nmol/l. In contrast, smooth muscle alpha1Cb currents were blocked by cilnidipine at much higher doses (steady-state IC50, 20 micromol/l) whereas alpha1G currents were not inhibited by cilnidipine (30 micromol/l). Cilnidipine dose-dependently inhibited depolarization- and Ca2+-induced contractions of rat aortic rings, with an IC50 of 10 nmol/l at 10 min. However, the onset of the effects was very slow, with approximately 71% inhibition by 3 nmol/l cilnidipine after 90 min exposure to cilnidipine. In contrast, cilnidipine did not inhibit phorbol 12-myristate-13-acetate (100 nmol/l)-mediated contractions. We conclude that cilnidipine represents an extremely slow-acting DHP that targets multi-subunit L-type channels, but not PKC in arterial smooth muscle. Because cilnidipine is less potent in cells expressing the pore-forming alpha1C-b subunit, the data further suggest that this unique slow-acting mechanism of cilnidipine is mediated by a complex interaction of cilnidipine with alpha1C-b and accessory channel subunits.     

Phosphoinositide 3-kinase mediates enhanced spontaneous and agonist-induced contraction in aorta of deoxycorticosterone acetate-salt hypertensive rats

Arteries from deoxycorticosterone acetate (DOCA)-salt and N(omega)-nitro-L-arginine (L-NNA) hypertensive but not normotensive rats develop spontaneous tone. LY294002 and wortmannin, phosphoinositide 3-kinase (PI3-kinase) inhibitors, eliminate spontaneous tone. We hypothesized that PI3-kinase protein and/or activity was increased in hypertension and contributed to the observed enhanced contractility. PI3-kinase activity assays revealed 2-fold higher activity in thoracic aorta from DOCA-salt [systolic blood pressure (SBP)=184+/-5 mm Hg] compared with sham rats (SBP=111+/-2 mm Hg). Western analyses of aortic homogenates revealed the presence of p85alpha, p110alpha, p110beta, and p110delta but not p110gamma PI3-kinase subunits; p110delta protein was elevated in aorta of hypertensive rats as compared with sham. Aortic homogenates from L-NNA rats also had elevated p110beta protein density, but neither L-NNA nor DOCA-salt had differences in p85alpha and p110alpha. Total Akt density was unaltered, but pAkt was significantly lower in homogenates from DOCA-salt rats. LY294002 (20 micromol/L) and nifedipine (50 nmol/L) abolished Ca2+-induced spontaneous tone in aorta from DOCA-salt rats. However, LY294002 did not alter BayK8644-induced contraction, indicating that LY294002 does not inhibit L-type Ca2+ channels directly. PTEN (phosphatase and tensin homolog) and pPTEN were expressed but not different in aorta from DOCA-salt and sham rats. LY294002 corrected the enhanced contraction to KCl and norepinephrine in aorta from DOCA-salt rats. These data support an increase in PI3-kinase activity and p110delta density in aorta from L-NNA and DOCA-salt rats. Importantly, this increase contributes to the enhanced contractility observed in two models of hypertension.     

Orexin-B augments voltage-gated L-type Ca(2+) current via protein kinase C-mediated signalling pathway in ovine somatotropes

Orexins, orexigenic neuropeptides, are secreted from lateral hypothalamus and orexin receptors are expressed in the pituitary. Since growth hormone (GH) secreted from pituitary is integrally linked to energy homeostasis and metabolism, we studied the effect of orexin-B on voltage-gated Ca(2+) currents and the related signalling mechanisms in primary cultured ovine somatotropes using whole-cell patch-clamp techniques. With a bath solution containing TEA-Cl (40 mM) and Tetrodotoxin (TTX) (1 microM), three subtypes of Ca(2+) currents, namely the long-lasting (L), transient (T), and N currents, were isolated using different holding potentials (-80 and -30 mV) in combination with specific Ca(2+) channel blockers (nifedipine and omega-conotoxin). About 75% of the total current amplitude was contributed by the L current, whereas the N and T currents accounted for the rest. Orexin-B (1-100 nM) dose-dependently and reversibly increased only the L current up to approximately 125% of the control value within 4-5 min. Neither a specific protein kinase A (PKA) blocker (H89, 1 microM) nor an inhibitory peptide (PKI, 10 microM) had any effect on the increase in L current by orexin-B. The orexin-B-induced increase in the L current was abolished by concurrent treatment with calphostin C (Cal-C, 100 nM), protein kinase C (PKC) inhibitory peptide (PKC(19-36), 1 microM), or by pretreatment with phorbol-12,13-dibutyrate (PDBu) (0.5 microM) for 16 h (a downregulator of PKC). Orexin-B also increased in vitro GH secretion in a dose-dependent manner. We conclude that orexin-B increases the L-type Ca(2+) current and GH secretion through orexin receptors and PKC-mediated signalling pathways in ovine somatotropes.     

Aldosterone suppresses insulin signaling via the downregulation of insulin receptor substrate-1 in vascular smooth muscle cells

Clinical reports indicate that patients with primary aldosteronism commonly have impaired glucose tolerance; however, the relationship between aldosterone and insulin signaling pathway has not been clarified. In this study, we examined the effects of aldosterone treatment on insulin receptor substrate-1 expression and insulin signaling pathway including Akt phosphorylation and glucose uptake in rat vascular smooth muscle cells. Insulin receptor substrate-1 protein expression and Akt phosphorylation were determined by Western blot analysis with anti-insulin receptor substrate-1 and phosphorylated-Akt antibodies, respectively. Glucose metabolism was evaluated using (3)H-labeled 2-deoxy-d-glucose uptake. Aldosterone (1-100 nmol/L) dose-dependently decreased insulin receptor substrate-1 protein expression with a peak at 18 hours (n=4). Aldosterone-induced degradation of insulin receptor substrate-1 was markedly attenuated by treatment with the selective mineralocorticoid receptor antagonist eplerenone (10 micromol/L; n=4). Furthermore, degradation was blocked by the Src inhibitor PP1 (20 micromol/L; n=4). Treatment with antioxidants, N-acetylcysteine (10 mmol/L), or ebselen (40 micromol/L) also attenuated aldosterone-induced insulin receptor substrate-1 degradation (n=4). In addition, proteasome inhibitor MG132 (1 micromol/L) prevented insulin receptor substrate-1 degradation (n=4). Aldosterone treatment abolished insulin-induced Akt phosphorylation (100 nmol/L; 5 minutes; n=4). Furthermore, aldosterone pretreatment decreased insulin-stimulated (100 nmol/L; 60 minutes; n=4) glucose uptake by 50%, which was reversed by eplerenone (10 micromol/L; n=4). These data indicate that aldosterone decreases insulin receptor substrate-1 expression via Src and reactive oxygen species stimulation by proteasome-dependent degradation in vascular smooth muscle cells; thus, aldosterone may be involved in the pathogenesis of vascular insulin resistance via oxidative stress.     

Endothelin-1 promotes Ca2+ antagonist-insensitive coronary smooth muscle contraction via activation of epsilon-protein kinase C

Certain forms of coronary artery disease do not respond to treatment with Ca2+ channel blockers, and a role for endothelin-1 (ET-1) in Ca2+ antagonist-insensitive forms of coronary vasospasm has been suggested; however, the signaling mechanisms involved are unclear. We tested the hypothesis that a component of ET-1-induced coronary smooth muscle contraction is Ca2+ antagonist-insensitive and involves activation of protein kinase C (PKC). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca2+]i was measured in fura-2 loaded cells, and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific PKC antibodies using Western blot analysis. In Hank's solution (1 mmol/L Ca2+), ET-1 (10(-7) mol/L) caused a transient increase in [Ca2+]i (236+/-14 nmol/L) followed by a maintained increase in [Ca2+]i (184+/-8 nmol/L) and 35% cell contraction. The Ca2+ channel blockers verapamil and diltiazem (10(-6) mol/L) abolished the maintained ET-1-induced [Ca2+]i, but only partially inhibited ET-1-induced cell contraction to 18%. The verapamil-insensitive component of ET-1 contraction was inhibited by the PKC inhibitors calphostin C and epsilon-PKCV1-2. ET-1 caused translocation of Ca2+-dependent alpha-PKC and Ca2+-independent epsilon-PKC from the cytosolic to the particulate fraction that was inhibited by calphostin C. Verapamil abolished ET-1-induced translocation of alpha-PKC, but not that of epsilon-PKC. Phorbol 12-myristate 13-acetate (10(-6) mol/L), a direct activator of PKC, caused 22% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that was inhibited by calphostin C, but not by verapamil. KCl (51 mmol/L), which stimulates Ca2+ influx, caused 35% cell contraction and increase in [Ca2+]i (291+/-11 nmol/L) that were inhibited by verapamil, but not by calphostin C, and did not cause translocation of alpha- or epsilon-PKC. In Ca2+-free (2 mmol/L EGTA) Hank's solution, ET-1 caused 15% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that were inhibited by epsilon-PKC V1-2 inhibitory peptide. Thus, a significant component of ET-1-induced contraction of coronary smooth muscle is Ca2+ antagonist-insensitive and involves activation and translocation of Ca2+-independent epsilon-PKC, and may represent a signaling mechanism of Ca2+ antagonist-resistant forms of coronary vasospasm.     

Proteasome inhibitor enhances growth hormone-binding protein release

We used murine Ba/F3 cells transfected with human growth hormone receptor (hGHR) cDNA to investigate the regulatory mechanisms of human growth hormone-binding protein (hGH-BP) release. The extracellular domain of hGHRs were cleaved and released as hGH-BPs (a soluble form of hGHR). The hGH-BP release was enhanced by phorbol 12,13-dibutyrate (PDBu), and suggested to be mediated by activation of PKC, the same as in human IM-9 cells. Thus, Ba/F3 cells have hGH-BP-releasing pathways similar to those of human cells. The proteasome inhibitors MG-132 and clasto-lactacystin beta-lactone also increased hGH-BP release from Ba/F3-hGHR cells, and MG-132 and PDBu synergistically increased hGH-BP release. The results obtained by using three PKC inhibitors G? 6976, GF 109203X and G? 6983 suggest that the enhancement of hGH-BP release by MG-132 and PDBu is mediated by different mechanisms probably involving different PKC isozymes.     

蛋白激酶C及转化生长因子β1信号通路对肝星状细胞激活的影响

目的探讨蛋白激酶C（PKC）活性改变对HSC表达TGF β1的影响及在HSC激活中的作用。方法将肝星状细胞系rHSC-99分为3组：对照组（A组），PKC激动剂佛波酯0．5μmol／L组（B组），PKC抑制剂Calphostin C 100nmol／L组（C组）。加药后0、3、6、12h和24h分别检测各组细胞PKC活性的变化；作用24h后，采用Western blot和RT—PCR方法检测各组细胞TGF β1，Smad 4，Ⅰ、Ⅲ型胶原和α-平滑肌肌动蛋白的表达；采用MTT法检测细胞的增殖情况。结果 佛波酯作用后PKC的活性显著增强，而Calphostin C则抑制PKC的活性。PKC活性增强后，与对照组相比TGF β1及其下游信号分子Smad 4的表达分别升高了4．8倍和13．1倍（P〈0．01）；HSC的Ⅰ、Ⅲ型胶原和α-平滑肌肌动蛋白的表达分别升高了2．4倍、1．8倍和1．3倍（P〈0．01），并促进HSC的增殖；PKC活性被抑制后则能抑制以上作用。结论PKC活性的改变能调控HSC中TGF β1的表达，在HSC的激活中发挥调节作用。     

Role of protein kinase C-angiotensin II pathway for extracellular matrix production in cultured human mesangial cells exposed to high glucose levels

The intrarenal renin-angiotensin system has been implicated in the pathogenesis of diabetic nephropathy. This study investigates the mechanisms for glucose-induced increase in angiotensin II (AII) production by human mesangial cells (MCs) in relation to protein kinase C (PKC). We also examine whether locally produced AII mediates extracellular matrix protein production in high-glucose conditions. Human MCs were cultured in 5 or 33 mmol/l glucose for 8 days, and were incubated with or without 5 mmol/l GFX, a PKC inhibitor, 0.1 micromol/l candesartan cilexetil (CC), a specific type 1 AII receptor antagonist, for another 24 h. In addition, MCs grown in 5 mmol/l glucose were incubated with 0.1 micromol/l phorbol-12,13-dibutyrate (PDBu) for 24 h. AII, TGF-beta1, fibronectin and type IV collagen in the culture media were measured by ELISA. The amount of AII secreted from MCs exposed to high-glucose levels was significantly greater (P<0.01) than that in normal glucose levels. The increase in AII production was completely prevented by GFX. The addition of PDBu mimicked the effect of glucose on AII production. The glucose-induced increases in the production of TGF-beta1, fibronectin and type IV collagen were partially, but significantly restored (P<0.01) by CC, while GFX totally abolished these effects of glucose. These results suggest that elevated glucose levels stimulate AII production via mechanisms dependent on glucose-induced PKC activation in human MCs, and that locally produced AII partly mediates the increase in mesangial matrix synthesis in high-glucose conditions.     

Mechanisms of arginine-vasopressin-induced Ca2+ oscillations in beta-cells (HIT-T15): a role for oscillating protein kinase C

We examined the role of protein kinase C (PKC) for the generation of arginine-vasopressin (AVP)-linked Ca2+ oscillations in beta-cells (HIT-T15). Activation of PKC by phorbol-12,13-dibutyrate (PDBu) reduced the frequency and finally abolished AVP-induced Ca2+ oscillations. The PKC inhibitors G? 6976, Ro-32-0432, or chelerythrine converted Ca2+ oscillations to a plateau-like rise in cytosolic free Ca2+, and PKC down-regulation reduced the percentage of cells exhibiting AVP-induced Ca2+ oscillations. Several mechanisms were identified by which PKC could exert feedback on the AVP-linked Ca2+ oscillator. PDBu, but not the PKC inhibitors, inhibited AVP-stimulated inositol 1,4,5-trisphosphate production and mobilization of internal Ca2+. Ca2+ influx through voltage-sensitive Ca2+ channels was attenuated by PDBu and PKC inhibitors, indicating complex PKC-dependent regulation of voltage-sensitive Ca2+ channels involving stimulatory as well as inhibitory components. Furthermore, AVP caused oscillatory translocation of yellow fluorescent protein (YFP)-tagged PKCalpha and PKCbetaIota to the plasma membrane, which paralleled the Ca2+ oscillations in single cells. Repetitive translocation of YFP-PKCalpha and -PKCbetaIota could also be elicited by repetitive release of caged Ca2+. By contrast, AVP-stimulated translocation of YFP-PKCepsilon was monophasic, not synchronized with Ca2+ oscillations, and could not be mimicked by release of caged Ca2+. In conclusion, undisturbed activation of PKCs is a necessary intermediate to generate or maintain AVP-induced Ca2+ oscillations in pancreatic beta-cells. The data further suggest that classical PKCs, predominantly by inhibition of inositol 1,4,5-trisphosphate production, provide the negative feedback required for AVP-induced Ca2+ oscillations to occur that is mediated by their repetitive activation by oscillating Ca2+ concentrations.