Drinking behavior elicited by central injection of angiotensin II: roles for protein kinase C and Ca2+/calmodulin-dependent protein kinase II

Prior studies utilizing neurons cultured from the hypothalamus and brain stem of newborn rats have demonstrated that ANG II-induced modulation of neuronal firing involves activation of both protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). The present studies were performed to determine whether these signaling molecules are also involved in physiological responses elicited by ANG II in the brain in vivo. Central injection of ANG II (10 ng/2 microl) into the lateral cerebroventricle (icv) of Sprague-Dawley rats increased water intake in a time-dependent manner. This ANG II-mediated dipsogenic response was attenuated by central injection of the PKC inhibitors chelerythrine chloride (0.5-50 microM, 2 microl) and Go-6976 (2.3 nM, 2 microl) and by the CaMKII inhibitor KN-93 (10 microM, 2 microl). Conversely, icv injection of chelerythrine chloride (50 microM, 2 microl) and KN-93 (10 microM, 2 microl) had no effect on the dipsogenic response elicited by central injection of carbachol (200 ng/2 microl). Furthermore, injection of ANG II (10 ng/2 microl) icv increases the activity of both PKC-alpha and CaMKII in rat septum and hypothalamus. These data suggest that signaling molecules involved in ANG II-induced responses in vitro are also relevant in physiological responses elicited by ANG II in the whole animal model.     

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

ANG II constricts descending vasa recta (DVR) through Ca(2+) signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 microM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) elevation, and blocked Mn(2+) influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 microM) or phorbol 12,13-dibutyrate (PDBu; 1 microM) induced both vasoconstriction and pericyte [Ca(2+)](cyt) elevation. Diltiazem (10 microM) blocked the ability of PDBu to increase pericyte [Ca(2+)](cyt) and enhance Mn(2+) influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT(2) receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 microM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca(2+)](cyt), divalent ion conductance into the cytoplasm, and ROS generation.     

Involvement of novel protein kinase C isoforms in carbachol-stimulated insulin secretion from rat pancreatic islets

The roles of protein kinase C (PKC) isoforms in cholinergic potentiation of glucose-induced insulin secretion were investigated in rat pancreatic islets. Western-blot analysis showed the presence of PKC-alpha, betaII, delta, epsilon, eta, and zeta, but not PKC-betaI, gamma, or iota, in the islets. Carbachol (CCh) caused translocations of PKC-alpha, betaII, delta, and epsilon from the cytosol to the plasma membrane. CCh facilitated 7-mM glucose-induced insulin secretion from isolated rat islets. The CCh-stimulated insulin secretion was significantly suppressed by the generic PKC inhibitor chelerythrine. In contrast, Go 6976, an inhibitor of conventional PKC isoforms, had no effect on the insulin secretion stimulated by CCh, although it significantly inhibited that induced by phorbol 12-myristate 13-acetate. These results suggest that the novel PKC isoforms activated by CCh, i.e., PKC-delta and/or epsilon, participate in the stimulatory effect of CCh on insulin secretion.     

Proliferation of human breast cancer cells and anti-cancer action of doxorubicin and vinblastine are independent of PKC-alpha

Protein kinase C (PKC) has been considered for a potential target of anticancer chemotherapy. PKC-alpha has been associated with growth and metastasis of some cancer cells. However, the role of PKC-alpha in human breast cancer cell proliferation and anticancer chemotherapy remains unclear. In this study, we examined whether alterations of PKC-alpha by phorbol esters and PKC inhibitors could affect proliferation of human breast cancer MCF-7 cells and the cytotoxic effect of chemotherapeutic agents. Exposure for 24 h to doxorubicin (DOX) and vinblastine (VIN) caused a concentration-dependent reduction in proliferation of MCF-7 cells. However, these two anticancer drugs altered cellular morphology and growth pattern in distinct manners. Phorbol 12,13-dibutyrate (PDBu, 100 nM), which enhanced activities of PKC-alpha, increased cancer cell proliferation and attenuated VIN (1 microM)-induced cytotoxicity. These effects were not affected in the presence of 10 nM staurosporine. Phorbol myristate acetate (PMA, 100 nM) that completely depleted PKC-alpha also enhanced cancer cell proliferation and attenuated VIN-induced cytotoxicity. Three potent PKC inhibitors, staurosporine (10 nM), chelerythrine (5 microM) and bisindolylmaleimide-I (100 nM), had no significant effect on MCF-7 cell proliferation; staurosporine and chelerythrine, but not bisindolylmaleimide-I, attenuated VIN-induced cytotoxicity. Moreover, neither phorbol esters nor PKC inhibitors had an effect on cytotoxic effects of DOX (1 microM) on MCF-7 cell proliferation. Thus, these data suggest that MCF-7 cell proliferation or the anti-cancer action of DOX and VIN on breast cancer cells is independent of PKC-alpha.     

Involvement of cyclooxygenase-dependent pathway in contraction of isolated ileum by urotensin II

We previously reported that urotensin II induced biphasic (brief- and long-lasting) contractions and the brief contraction was mediated by acetylcholine release from ganglionic cholinergic neurons in a segment of guinea-pig ileum. In the present work, we studied the mechanism contributing to long-lasting contractions induced by urotensin II. Treatment with 0.1 microM tetrodotoxin, 300 nM omega-conotoxin GVIA (an inhibitor of N-type Ca2+ channels) and 10 microM indomethacin (an inhibitor of cyclooxygenases) markedly inhibited 100 nM urotensin II-induced long-lasting contractions. The addition of 1 microM prostaglandin F2alpha (PGF2alpha) caused a limited brief contraction following long-lasting contraction, while 1 microM PGE2 induced marked biphasic contractions. Treatment with neurotoxins inhibited the long-lasting contractions induced by PGF2alpha and PGE2 without changing the PGE2-induced brief contractions. Treatment with 1 microM atropine markedly inhibited the urotensin II- and PGF2alpha-induced long-lasting contractions, but was less effective on the PGE2 responses. Treatment with a phospholipase A2 inhibitor decreased the urotensin II-induced contractions. These findings suggest that urotensin II induces, at least partially, long-lasting contractions via PG-sensitive cholinergic neurons and muscarinic acetylcholine receptors in the ileum.     

ANG II-mediated inhibition of neuronal delayed rectifier K+ current: role of protein kinase C-alpha

It was previously determined that ANG II and phorbol esters inhibit Kv current in neurons cultured from newborn rat hypothalamus and brain stem in a protein kinase C (PKC)- and Ca2+-dependent manner. Here, we have further defined this signaling pathway by investigating the roles of "physiological" activators of PKC and different PKC isozymes. The cell-permeable PKC activators, diacylglycerol (DAG) analogs 1,2-dioctanoyl-sn-glycerol (1 micromol/l, n = 7) and 1-oleoyl-2-acetyl-sn-glycerol (1 micromol/l, n = 6), mimicked the effect of ANG II and inhibited Kv current. These effects were abolished by the PKC inhibitor chelerythrine (1 micromol/l, n = 5) or by chelation of internal Ca2+ (n = 8). PKC antisense (AS) oligodeoxynucleotides (2 micromol/l) against Ca2+-dependent PKC isoforms were applied to the neurons to manipulate the endogenous levels of PKC. PKC-alpha-AS (n = 4) treatment abolished the inhibitory effects of ANG II and 1-oleoyl-2-acetyl-sn-glycerol on Kv current, whereas PKC-beta-AS (n = 4) and PKC-gamma-AS (n = 4) did not. These results suggest that the angiotensin type 1 receptor-mediated effects of ANG II on neuronal Kv current involve activation of PKC-alpha.     

Differential vasoconstrictions induced by angiotensin II: role of AT1 and AT2 receptors in isolated C57BL/6J mouse blood vessels

Genetically altered mice are increasingly used as experimental models. However, ANG II responses in mouse blood vessels have not been well defined. Therefore, the aim of this study was to determine the role of ANG II in regulating major blood vessels in C57/BL6J mice with isometric force measurements. Our results showed that in mouse abdominal aorta ANG II induced a concentration-dependent contraction (EC50 4.6 nM) with a maximum contraction of 75.1 +/- 4.9% at 100 nM compared with that of 60 mM K+. Similarly, femoral artery also exhibited a contractile response of 76.0 +/- 3.4% to the maximum concentration of ANG II (100 nM). In contrast, ANG II (100 nM)-induced contraction was significantly less in carotid artery (24.5 +/- 6.6%) and only minimal (3.5 +/- 0.31%) in thoracic aorta. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester and the AT2 antagonist PD-123319 failed to enhance ANG II-induced contractions. However, an AT1 antagonist, losartan (10 microM), completely inhibited ANG II (100 nM) response in abdominal aorta and carotid artery. An AT1 agonist, [Sar1]-ANG II (100 nM), behaved similarly to ANG II (100 nM) in abdominal aorta and carotid artery. RT-PCR analyses showed that mouse thoracic aorta has a significantly lower AT1 mRNA level than abdominal aorta. These results demonstrate that major mouse vessels exhibit differential contractions to ANG II, possibly because of varied AT1 receptor levels.     

Angiotensin II stimulates cardiac L-type Ca(2+) current by a Ca(2+)- and protein kinase C-dependent mechanism

Angiotensin II (ANG II) evokes positive inotropic responses in various species. However, the effects of this peptide on L-type Ca(2+) currents (I(Ca)) are still controversial. We report in this study that the effects of ANG II on I(Ca) differ depending on the mode of patch-clamp technique used, standard whole cell (WC) or perforated patch (PP). No significant effects of ANG II (0.5 microM) were observed when WC in cells dialyzed with high EGTA was used. However, when the intracellular milieu was preserved using PP, ANG II induced a significant 77 +/- 6% increase in I(Ca) (-2.2 +/- 0.3 in control and -3.9 +/- 0.6 pA/pF in ANG II, n = 8, P < 0.05). When WC was used in cells dialyzed with low Ca(2+) buffer capacity (EGTA 0.1 mM), ANG II was able to induce an increase in I(Ca) (-3.5 +/- 0.3 in control vs. -4.8 +/- 0.4 pA/pF in ANG II, n = 13, P < 0.05). This increase was prevented when the cells were also dialyzed with the protein kinase C (PKC) inhibitor chelerythrine (50 microM) or calphostin C (1 microM). The above results allow us to conclude that strong intracellular Ca(2+) buffering prevents the physiological actions of ANG II on cardiac I(Ca), which are also dependent on activation of PKC.     

Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.     

Effect of protein kinase C inhibitors on porcine oocyte activation

The effect of protein kinase C (PKC) inhibitors on porcine oocyte activation by calcium ionophore A23187 was studied. Calcium ionophore applied in a 50 microM concentration for 10 min induced activation in 74% of oocytes matured in vitro. When the ionophore-treated oocytes were exposed to the effect of bisindolylmaleimide I, which inhibits calcium-dependent PKC isotypes (PKC-alpha, -beta(I), -beta(II), -gamma,) and calcium-independent PKC isotypes (PKC-delta, -epsilon), the portion of activated oocytes decreased (at a concentration of 100 nM, 2% of the oocytes were activated). Go6976, the inhibitor of calcium-dependent PKC isotypes PKC-alpha, -beta(I) did not prevent the action of the oocytes treated with calcium ionophore in concentrations from 1 to 100 microM. The inhibitor of PKC-beta(I) and beta(II) isotypes, hispidin, in a concentration of 2 microM-2 mM, was not effective either. The inhibitor of PKC-delta isotype, rottlerin, suppressed activation of the oocytes by calcium ionophore (no oocyte was activated at 10 microM concentration). The PKC-delta isotype in matured porcine oocytes, studied by Western blot analysis, appeared as non-truncated PKC-delta of 77.5 kDa molecular weight, on the one hand, and as truncated PKC-delta, which was present in the form of a doublet of approximately 62.5 and 68 kDa molecular weight, on the other hand. On the basis of these results, it can be supposed that PKC participates in the regulation of processes associated with oocyte activation. Calcium-dependent PKC-alpha, -beta isotypes do not seem to play any significant role in calcium activation. The activation seems to depend on the activity of the calcium-independent PKC-delta isoform.     

Nitric oxide mediates protein kinase C isoform translocation in rat heart during postischemic reperfusion

It is controversial whether nitric oxide (NO) is protective or deleterious against ischemia-reperfusion injury. We examined the effect of NO on PKC isoform translocation and protection against ischemia-reperfusion injury in perfused heart. An NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester, 3.0 microM), administered only during reperfusion but not during ischemia, inhibited the translocation of PKC-alpha, -delta and -epsilon isoforms to the nucleus-myofibril fraction and the translocation of PKC-alpha to the membrane fraction after ischemia (20 min) and reperfusion (10 min) in the perfused rat heart. NO donors, 3-morpholinosydnonimine (SIN-1) or S-nitroso-N-acetylpenicillamine (SNAP) activated purified PKC in vitro. SIN-1 also induced PKC isoform translocation in perfused heart. On the other hand, PKC selective inhibitor, calphostin C (0.2 microM) or chelerythrine (1.0 microM), aggravated the contractile dysfunction of ischemic heart during reperfusion, when they were perfused during reperfusion. These data suggest that NO generated during reperfusion following ischemia activates PKC isoforms and may protect the heart against contractile dysfunction in the perfused rat heart.     

Human urotensin II increases coronary perfusion pressure in the isolated rat heart: potentiation by nitric oxide synthase and cyclooxygenase inhibition.

Urotensin II (U-II) is a cyclic peptide, recently cloned in man and present in cardiac tissue and arteries. The effects of human U-II (hU-II) on coronary perfusion pressure (CPP) were investigated in isolated rat hearts perfused retrogradely via the aorta at constant flow. hU-II produced a concentration-dependent increase in CPP (pEC50 8.6 +/- 0.3, n = 8), the maximum increase in CPP (12 +/- 4 mmHg) was obtained at 10(-7) M hU-II. At higher concentrations of hU-II CPP fell back towards baseline. Endothelin-1 produced a maximum increase in CPP of 63 +/- 11 mmHg within the concentration-range studied. Addition of the NO synthase inhibitor L N(G)nitro-arginine methyl ester (10(-4) M) and the cyclooxygenase inhibitor, indomethacin (10(-5) M) to the perfusion solution had no effect on the pEC50 value for hU-II, but significantly increased the maximum constriction (to 34 +/- 7 mmHg, n = 8, p < 0.05) and inhibited the later dilator response to hU-II. These results suggest that receptors for hU-II are present in the coronary vasculature and that smooth muscle contraction is modulated by the release of dilator factors, including NO and prostacyclin. Endothelial function is therefore likely to be of primary importance in modulating the coronary vasoconstrictor effects of hU-II in vivo.     

Age-dependent activation of PKC isoforms by angiotensin II in the proximal nephron

ANG II increases fluid absorption in proximal tubules from young rats more than those from adult rats. ANG II increases fluid absorption in the proximal nephron, in part, via activation of protein kinase C (PKC). However, it is unclear how age-related changes in ANG II-induced stimulation of the PKC cascade differ as an animal matures. We hypothesized that the response of the proximal nephron to ANG II decreases as rats mature due to a reduction in the amount and activation of PKC rather than a decrease in the number or affinity of ANG II receptors. Because PKC translocates from the cytosol to the membrane when activated, we first measured PKC activity in the soluble and particulate fractions of proximal tubule homogenates exposed to vehicle or 10(-10) M ANG II from young (26 +/- 1 days old) and adult rats (54 +/- 1 days old). ANG II increased PKC activity to the same extent in homogenates from young rats (from 0.119 +/- 0.017 to 0.146 +/- 0.015 U/mg protein) (P < 0.01) and adult rats (from 0.123 +/- 0.020 to 0.156 +/- 0.023 U/mg protein) (P < 0.01). Total PKC activity did not differ between groups (0.166 +/- 0.018 vs. 0.181 +/- 0.023). We next investigated whether activation of the alpha-, beta-, and gamma-PKC isoforms differed by Western blot. In homogenates from young rats, ANG II significantly increased activated PKC-alpha from 40.2 +/- 6.5 to 60.2 +/- 9.5 arbitrary units (AU) (P < 0.01) but had no effect in adult rats (46.1 +/- 5.1 vs. 48.5 +/- 8.2 AU). Similarly, ANG II increased activated PKC-gamma in proximal tubules from young rats from 47.9 +/- 13.2 to 65.6 +/- 16.7 AU (P < 0.01) but caused no change in adult rats. Activated PKC-beta, however, increased significantly in homogenates from both age groups. Specifically, activated PKC-beta increased from 8.6 +/- 1.4 to 12.2 +/- 2.1 AU (P < 0.01) in homogenates from nine young rats and from 19.0 +/- 5.5 to 25.1 +/- 7.1 AU (P < 0.01) in homogenates from 12 adult rats. ANG II did not alter the amount of soluble PKC-alpha, -beta, and -gamma significantly. The total amount of PKC-alpha and -gamma did not differ between homogenates from young and adult rats, whereas the total amount of PKC-beta was 59.7 +/- 10.7 and 144.9 +/- 41.8 AU taken from young and adult rats, respectively (P < 0.05). Maximum specific binding and affinity of ANG II receptors were not significantly different between young and adult rats. We concluded that the primary PKC isoform activated by ANG II changes during maturation.     

Effects of atrial natriuretic peptide, angiotensin II and III on norepinephrine uptake in the rat adrenal medulla.

The effects of atrial natriuretic peptide (ANP), angiotensin II (ANG II) and angiotensin III (ANG III) on norepinephrine (NE) uptake were studied in the adrenal medulla of the rat. One microM ANG II and 10 microM ANG III decreased NE uptake while 10 nM and 100 nM ANP increased it. Subthreshold concentrations of ANP (1 nM) blunted the inhibitory effect of 1 microM ANG II but did not modify the inhibitory effect of 10 microM ANG III. The increasing effects of 100 nM ANP on NE uptake were partially reversed by subthreshold concentrations of ANG II (1 nM) and blunted by 1 nM ANG III. The interaction between ANP and the renin-angiotensin system could contribute to modulate the sympathetic function in the adrenal medulla.     

Urotensin II-induced hypotensive responses in Wistar-Kyoto (Wky) and spontaneously hypertensive (Shr) rats

Human urotensin II (hU-II) is a potent vasoactive peptide which modulates some of the functions of the cardiovascular and other systems. The in vivo mechanism of action by which hU-II may influence blood pressure in developmental and pathological conditions, is poorly understood. Herein, the blood pressure effects of hU-II (0.1-10 nmol/kg) injected intravenously (i.v.) were studied on ketamine/xylazine anesthetized male WKY and SHR rats aged 4 and 8 weeks. hU-II elicited dose-dependent decreases in mean arterial pressure in both strains of animals. The hypotensive responses to hU-II were, however, significantly higher in SHR rats, independently of age. Four-week-old SHR rats (which are normotensive) were, however, less responsive than their hypertensive 8-week-old counterparts. A series of pharmacological inhibitors were used to identify putative endogenous (endothelial) factors that might account for the hU-II-mediated hypotension in 8-week-old SHR. These include the non-selective nitric oxide synthase inhibitor L-NAME (5 micromol/kg), the non-selective cyclooxygenase inhibitor meclofenamate (16 micromol/kg), the voltage-sensitive and ATP-sensitive K+-channel inhibitors, 4-aminopyridine (5 micromol/kg) and glybenclamide (10 micromol/kg), the cytochrome P450 CYP2C9 inhibitor sulfaphenazole (15 micromol/kg), the cytoskeletal fixation agent phalloidin (15 micromol/kg), the endothelin ETB receptor antagonist BQ-788(35 micromol/kg), the bradykinin B2 receptor antagonist HOE 140 (0.5 micromol/kg), the angiotensin AT2 antagonist PD 123319(10 micromol/kg) and the UT receptor antagonist urantide (10 micromol/kg). These agents were administered i.v. either at 2.5, 10 or 40 min prior hU-II injection (10 nmol/kg). Among these inhibitors, sulfaphenazole and phalloidin were able to reduce hU-II-induced hypotension. This suggests that the vasodepressor effect of hU-II is mediated by UT receptors and relies in part on the release of epoxide related products; increased microvascular permeability may also contribute to the blood pressure lowering effect of hU-II. Since urantide blocks the constrictor effects of hU-II on isolated aorta, but is inactive against the hypotensive action of hU-II in vivo, the results presented in this paper provide, for the first time, evidence for the existence of two different functional sites for hU-II.     

Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery

The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E-) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor N(omega)-nitro-L-arginine (100 microM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC(50)=9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC(50)=8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC(50)=1-2 microM) and chelerythrine (IC(50)=4-5 microM) and the classical PKC inhibitor G?-6976 (IC(50)=0.3-0.4 microM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential (E(m)) and a shift to the left of the E(m)-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 microM) or SNP (30 nM). PDBu (100-300 nM) increased peak L-type calcium channel (Ca(v)) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 microM) or G?-6976 (200 nM). SNP (500 nM) reduced Ca(v) currents only in the presence of the PKA blocker 8-bromo-2'-O-monobutyryl-cAMPS, Rp isomer (10 microM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Ca(v) activity and classical PKC activity. Both E(m)-dependent and -independent processes contribute to contraction. Our results suggest that one E(m)-independent process is direct enhancement of Ca(v) current by PKC.     

Vascular contractile effect of urotensin II in young and aged rats: influence of aging and contribution of endothelial nitric oxide

To elucidate whether aging influences the vascular contractile effect of urotensin II in rat thoracic aorta, and to evaluate the contribution of endothelial vasodilating substances in mediating the effect of urotensin II, the effect of urotensin II was examined in the vessels of young (2-3-month-old) and aged rat. Isolated rat aortic rings incubated in Krebs-Henseleit solution gassed with 95% O2/5% CO2 were stimulated with urotensin II, and the developed tension was measured. Urotensin II increased the developed tension, which was decreased by aging. In 2-3-months-old young aorta without endothelium, urotensin II (10(-10) to 10(-7)) elicited a concentration-dependent aortic contraction to the maximal response almost equivalent to high KCl-induced contraction (79.4+/-11.3% of KCl(max)). In the presence of endothelium, the urotensin II-induced vasoconstriction in young aorta was significantly attenuated to 33.3+/-4.6% of KCl(max). However, the contractile response was greater in the pretreatment with N(G)-nitro-L-arginine (L-NNA) (100 microM) (50.3+/-8.4% of KCl(max) in endothelial denuded aorta), suggesting the vasorelaxing role of endothelial nitric oxide. In 25-27-months-old aged rat aorta, the urotensin II-mediated contraction was remarkably decreased, both in the presence (6.3+/-2.0% of KCl(max)) and absence (11.7+/-3.0% of KCl(max)) of endothelium. A cyclooxygenase inhibitor, diclofenac (10 microM), did not have any effect on the urotensin II-induced contraction. These results suggest that urotensin II can induce vascular smooth muscle contraction in rat aorta, and there was an aging-related decline in the urotensin II-induced contraction. Endothelial production of nitric oxide in response to urotensin II but not cyclooxygenase metabolites such as prostacyclin may play a role in reducing the vascular constriction especially in young aorta.     

Synergistic effect of high glucose and ANG II on proliferation of mouse embryonic stem cells: involvement of PKC and MAPKs as well as AT1 receptor

This study examined the synergistic effect of high glucose levels and ANG II on proliferation and its related signal pathways using mouse embryonic stem (ES) cells. The combined use of a high glucose concentration (25 mM) and ANG II increased the level of [3H]thymidine/BrdU incorporation, and the number of cells compared with either treatment alone. Each treatment with high glucose or ANG II increased the cell population in the S phase compared with control, and the combined treatment of a high glucose concentration and ANG II significantly increased the number of cells in the S phase according to FACS analysis. Moreover, the high glucose-induced increase in [3H]thymidine incorporation was blocked by inhibiting the ANG II type 1 (AT1) receptor. The combined high glucose and ANG II significantly increased the STAT3 phosphorylation compared with high glucose or ANG II alone. ANG II stimulated the influx of Ca2+ in 25 mM glucose compared with 5 mM glucose. High glucose levels increase the level of PKC alpha, epsilon, and zeta translocation from the cytosol to the membrane fraction. In an examination of other signal pathways, the combined treatment significantly increased the level of p44/42, p38 MAPKs phosphorylation compared with either treatment alone. Indeed, the combined treatment increased the mRNA expression level of the protooncogenes and cell cycle regulatory proteins. In conclusion, the combined treatment of a high glucose concentration and ANG II had a synergistic effect in stimulating mouse ES cell proliferation through the Ca2+/PKC, MAPKs, and the AT1 receptor.     

Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney

We examined the effect of angiotensin II (ANG II) on epithelial Na(+) channel (ENaC) in the rat cortical collecting duct (CCD) with single-channel and the perforated whole cell patch-clamp recording. Application of 50 nM ANG II increased ENaC activity, defined by NP(o) (a product of channel numbers and open probability), and the amiloride-sensitive whole cell Na currents by twofold. The stimulatory effect of ANG II on ENaC was absent in the presence of losartan, suggesting that the effect of ANG II on ENaC was mediated by ANG II type 1 receptor. Moreover, depletion of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM failed to abolish the stimulatory effect of ANG II on ENaC but inhibiting protein kinase C (PKC) abolished the effect of ANG II, suggesting that the effect of ANG II was the result of stimulating Ca(2+)-independent PKC. This notion was also suggested by the experiments in which stimulation of PKC with phorbol ester derivative mimicked the effect of ANG II and increased amiloride-sensitive Na currents in the principal cell, an effect that was not abolished by treatment of the CCD with BAPTA-AM. Also, inhibition of NADPH oxidase (NOX) with diphenyleneiodonium chloride abolished the stimulatory effect of ANG II on ENaC and application of superoxide donors, pyrogallol or xanthine and xanthine oxidase, significantly increased ENaC activity. Moreover, addition of ANG II or H(2)O(2) diminished the arachidonic acid (AA)-induced inhibition of ENaC in the CCD. We conclude that ANG II stimulates ENaC in the CCD through a Ca(2+)-independent PKC pathway that activates NOX thereby increasing superoxide generation. The stimulatory effect of ANG II on ENaC may be partially the result of blocking AA-induced inhibition of ENaC.     

Influence of ANG II on cytoplasmic sodium in cultured rabbit nonpigmented ciliary epithelium

Angiotensin (ANG) II receptors have been reported in the nonpigmented ciliary epithelium (NPE) of the eye. In cultured NPE, we found ANG II caused a dose-dependent rise of cytoplasmic sodium. The sodium increase was inhibited by the AT(1)-AT(2) receptor antagonist saralasin (IC(50) = 3.7 nM) and the AT(1) antagonist losartan (IC(50) = 0.6 nM) but not by the AT(2) antagonist PD-123319. ANG II also caused a dose-dependent increase in the rate of ouabain-sensitive (86)Rb uptake. The ANG II-induced cell sodium increase and (86)Rb uptake increase were reduced by dimethylamiloride (DMA; 10 microM). On the basis of this finding, we propose that Na(+)/H(+) exchange is stimulated by ANG II. Simultaneously, ANG II appears to inhibit H(+)-ATPase-mediated proton export. Thus Ang II (10 nM) did not alter the baseline cytoplasmic pH (pH(i)) but reduced pH(i) in cells that were also exposed to 10 microM DMA. Consistent with the notion of H(+)-ATPase inhibition in ANG II-treated NPE, bafilomycin A(1) (100 nM) (BAF) and ANG II were both observed to suppress the pH(i) increase that occurs upon exposure to a mixture of epinephrine (1 microM) and acetylcholine (10 microM) and the pH(i) increase elicited by depolarization. In ATP hydrolysis measurements, H(+)-ATPase activity (bafilomycin A(1)-sensitive ATP hydrolysis) was reduced significantly in cells that had been pretreated 10 min with 10 nM ANG II. In summary, these studies suggest that ANG II causes H(+)-ATPase inhibition and an increase of cell sodium due to activation of Na(+)/H(+) exchange.