Angiotensin II-induced over-activation of p47phox in fibroblasts friorn hypertensives: which role in the enhanced ERK1/2 responsiveness to angiotensin II?

Background Fibroblasts Eire involved in the remodeling of the heart and of the vasculature associated to arterial hypertension, and an abnormal extracellular signal-regulated kinase 1/2 (ERK1/2) activation by angiotensin II (Ang II) plays a pivotal role in this process. However, the intracellular pathways leading to cell hypertrophy and hyperplasia, as well as to collagen production, are still incompletely known. Objective To investigate the role of superoxide anion (O-2(-)) and of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase in Ang II-stimulated ERK1/2 over-activation in fibroblasts from hypertensive patients. Methods O-2(-) production was measured in skin fibroblasts from hypertensives (HT, n = 11) and from normotensive controls (NT, n = 10) by electron spin resonance technique. ERK1/2 phosphorylation and p47phox NAD(P)H oxidase subunit translocation were measured by western blot. Results Ang II (1 mu mol/l) induced a larger p47phox subunit translocation and increased intracellular O-2(-) production to a larger extent in HT in comparison to NT and this effect was blocked by apocynin, an inhibitor of the NAD(P)H oxidase. Ang II increased ERK1/2 phosphorylation more in HT than in NT. The Ang III-induced ERK1/2 phosphorylation was inhibited by apocynin in a dose-dependent manner in NT, but not in HT. Conclusions The chain of cellular events leading to increased ERK1/2 responsiveness to Ang II in hypertension include an exaggerated response of p47phox, NAD(P)H oxidase and O-2(-), but it is partially resistant to apocynin. Therefore, NAD(P)H-dependent reactive oxygen species (ROS) production is not the only determinant of the exaggerated ERK1/2 responsiveness in fibroblasts of hypertensives (HT)

Green tea attenuates angiotensin II- induced cardiac hypertrophy in rats by modulating reactive oxygen species production and the Src/EGFR/Akt signalling pathway

We previously documented a clear-cut antihypertensive effect of green teat extract (GTE), which was associated with correction of endothelial dysfunction and prevention of left ventricular hypertrophy in an angiotensin II (Ang II)-dependent model of hypertension, but the molecular mechanisms remain to be defined. As several effects of Ang II involve production of reactive oxygen species (ROS) and activation of 2nd messengers, such as mitogen-activated protein kinase (MAPK) and Akt, we investigated the effect of GTE on these signal transduction pathways in Ang II-treated rats. Rats were treated for 2 wk with Ang II infusion (700 mug.kg(-1).d(-1); n = 6, via osmotic minipumps), Ang II plus GTE (6 g/L) dissolved in the drinking water; n = 6), or vehicle (n = 6) to serve as controls. Blood pressure was monitored by telemetry throughout the study. The activation and expression of NAD(P)H oxidase subunits, protein kinase C isoforms, Src, epidermal growth factor receptor (EGFR), Akt, and MAPK were determined in the heart in vitro through immunoprecipitation and western blot analysis with specific antibodies. NAD(P)H oxidase enzymatic activity was measured by cytochrome c reduction assay. GTE blunted Ang II-induced blood pressure increase and cardiac hypertrophy. In Ang II-treated rats, GTE decreased the expression of the NAD(P)H oxidase subunit gp91(phox) and the translocation of Rac-1, as well as NAD(P)H oxidase enzymatic activity. Furthermore, it specifically reduced Ang II-induced Src, EGFR, and Akt phosphorylation. These results show that GTE blunts Ang II-induced cardiac hypertrophy specifically by regulating ROS production and the Src/EGFR/Akt signaling pathway activated by Ang II

Green tea attenuates angiotensin II-induced cardiac hypertrophy in rats by modulating reactive oxygen species production and the Src/EGFR/Akt signalling pathway.

We previously documented a clear-cut antihypertensive effect of green teat extract (GTE), which was associated with correction of endothelial dysfunction and prevention of left ventricular hypertrophy in an angiotensin II (Ang II)-dependent model of hypertension, but the molecular mechanisms remain to be defined. As several effects of Ang II involve production of reactive oxygen species (ROS) and activation of 2nd messengers, such as mitogen-activated protein kinase (MAPK) and Akt, we investigated the effect of GTE on these signal transduction pathways in Ang II-treated rats. Rats were treated for 2 wk with Ang II infusion (700 mug.kg(-1).d(-1); n = 6, via osmotic minipumps), Ang II plus GTE (6 g/L) dissolved in the drinking water; n = 6), or vehicle (n = 6) to serve as controls. Blood pressure was monitored by telemetry throughout the study. The activation and expression of NAD(P)H oxidase subunits, protein kinase C isoforms, Src, epidermal growth factor receptor (EGFR), Akt, and MAPK were determined in the heart in vitro through immunoprecipitation and western blot analysis with specific antibodies. NAD(P)H oxidase enzymatic activity was measured by cytochrome c reduction assay. GTE blunted Ang II-induced blood pressure increase and cardiac hypertrophy. In Ang II-treated rats, GTE decreased the expression of the NAD(P)H oxidase subunit gp91(phox) and the translocation of Rac-1, as well as NAD(P)H oxidase enzymatic activity. Furthermore, it specifically reduced Ang II-induced Src, EGFR, and Akt phosphorylation. These results show that GTE blunts Ang II-induced cardiac hypertrophy specifically by regulating ROS production and the Src/EGFR/Akt signaling pathway activated by Ang II

Callipeltin A: sodium ionophore effect and tension development in vascular smooth muscle

Callipeltin A is a cyclic depsidecapeptide isolated from the marine sponges Callipelta sp. and Latrunculia sp. that has been previously shown to increase the force of contraction of guinea-pig atria through the inhibition of Na+/Ca2+ exchanger (NCX). We investigated the effect of callipeltin A on guinea-pig aortic rings contracted by procedures that activate NCX in "calcium entry mode". Callipeltin A did not inhibit these contractions. Resting aorta responded to callipeltin A with a remarkable contraction that was concentration-dependent (EC50 0.44microM). This contraction was not inhibited by the calcium channel blocker verapamil and was not mediated by the activation of alpha-adrenergic or endothelin-1 receptors. Pre-incubation of aortic rings with 0.5mM amiloride, an inhibitor of NCX, completely prevented callipeltin A-induced contraction. Furthermore, callipeltin A (EC50 0.51microM) increased Na+ efflux of Na-loaded erythrocytes. 1H and 13C NMR resonances of callipeltin A revealed small but significant changes in the titration with K+ and Na+ salts. It is suggested that the effect of callipeltin A on cardiac and vascular preparations is linked to a Na-ionophore action

First evidence that albumin can directly improve cardiac contractility in cirrhotic rats

A low cardiac output due to cirrhotic cardiomyopathy could be a critical factor in the pathogenesis of hepatorenal syndrome (HRS) in cirrhosis. Albumin together with vasoconstrictor drugs is widely used in the management of HRS however the mechanisms are still unclear. Aim of our study was to verify whether albumin infusion may restore a normal cardiac contractile response to beta\uf020adrenergic agonists and to improve beta\uf020adrenergic signal transduction in an animal model of cirrhosis with ascites. 12 Wistar Kyoto rats were treated for 15 weeks by inhalation with carbon tetrachloride (CCl4 ) to induce cirrhosis and 12 rats were used as control animals. Three days and one day before the sacrifice, albumin was administered as saline solution at a dose of 3g/kg B.W. i.v. to 8 cirrhotic and to 8 control rats. In 4 cirrhotic rats the same amount of saline solution was administered at the same times. Left ventricular contractility was determined in isolated hearts with a beta-agonist, isoproterenol. Cardiac gene expression of beta\uf020adrenergic signalling was performed by Real-Time PCR. Plasma antioxidant capacity was evaluated with Electron Spin Resonance spectroscopy. The maximal response of cardiac contractility induced by isoproterenol (10-8 mol and 10-6 mol) was significantly reduced in the left ventricular tissue of cirrhotic rats in comparison to controls (p<0.01). In albumin-treated cirrhotic rats, isoproterenolinduced cardiac contractility was stronger in comparison to saline-treated cirrhotic rats (p<0.01) and it was not significant different than controls. In hearts from saline-treated cirrhotic rats, gene expression analysis showed a significant overexpression of G protein alpha inhibiting subunit 2 (Galphai2) (p<0.01), of regulator of G-protein signalling (RGS-2) (p<0.01) and of phosphodiesterase-2a (PDE), as compared to control rats. In albumin-treated cirrhotic rats, gene expression of RGS2 and PDE2a was lower in comparison to saline-treated cirrhotic rats alone (p<0.01) and was similar to control animals. No difference was observed for Galphai2 expression between salinetreated and albumin-treated cirrhotic rats. Plasma antioxidant capacity was decreased in cirrhotic rats compared to the control rats (to 57% +-\uf0208%, p<0.01). The administration of albumin almost completely restored the antioxidant level in cirrhotic rats.These results demonstrate for the first time that the administration of albumin improve cardiac contractility and beta adrenergic signal transduction by modulating the expression of RGS2 and PDE2a in cirrhotic rats. All these effects were accompanied by an increase of the level of antioxidant status in these animal

An Abnormal Gene Expression of the beta-Adrenergic System Contributes to the Pathogenesis of Cardiomyopathy in Cirrhotic Rats

IDecreased cardiac contractility and beta-adrenergic responsiveness have been observed in cirrhotic cardiomyopathy, but their molecular mechanisms remain unclear. To study beta-adrenergic-stimulated contractility and beta-adrenergic gene expression patterns, 20 Wistar Kyoto rats were treated with carbon tetrachloride to induce cirrhosis and 20 rats were used as controls. Left ventricular contractility was recorded in electrically driven isolated hearts perfused at constant flow with isoproterenol (10(-10) to 10(-6) M). A cardiac gene expression profile was obtained using a microarray for the myocyte adrenergic pathway. The cardiac contractility maximal response to isoproterenol was significantly reduced in cirrhotic rats in comparison to control rats, whereas the half-maximal effective concentration was not different. In cirrhotic rats, cardiac gene expression analysis showed a significant overexpression of G protein alpha-inhibiting subunit 2 (Galpha(i2)), cyclic nucleotide phosphodiesterase (PDE2a), regulator of G-protein signaling 2 (RGS2), and down-expression of adenylate cyclase (Adcy3). These results indicate that overexpression of Galpha(i2), PDE2a, and RGS2 down-regulates the beta-adrenergic signaling pathway, thus contributing to the pathogenesis of cirrhotic cardiomyopathy

Activation of c-SRC underlies the differential effects of ouabain and digoxin on Ca2+ signaling in arterial smooth muscle cells

Cardiotonic steroids (CTS) of the strophanthus and digitalis families have opposing effects on long-term blood pressure (BP). This implies hitherto unrecognized divergent signaling pathways for these CTS. Prolonged ouabain treatment upregulates Ca(2+) entry via Na(+)/Ca(2+) exchanger-1 (NCX1) and TRPC6 gene-encoded receptor-operated channels in mesenteric artery smooth muscle cells (ASMCs) in vivo and in vitro. Here, we test the effects of digoxin on Ca(2+) entry and signaling in ASMC. In contrast to ouabain treatment, the in vivo administration of digoxin (30 μg·kg(−1)·day(−1) for 3 wk) did not raise BP and had no effect on resting cytolic free Ca(2+) concentration ([Ca(2+)](cyt)) or phenylephrine-induced Ca(2+) signals in isolated ASMCs. Expression of transporters in the α2 Na(+) pump-NCX1-TRPC6 Ca(2+) signaling pathway was not altered in arteries from digoxin-treated rats. Upregulated α2 Na(+) pumps and a phosphorylated form of the c-SRC protein kinase (pY419-Src, ∼4.5-fold) were observed in ASMCs from rats treated with ouabain but not digoxin. Moreover, in primary cultured ASMCs from normal rats, treatment with digoxin (100 nM, 72 h) did not upregulate NCX1 and TRPC6 but blocked the ouabain-induced upregulation of these transporters. Pretreatment of ASMCs with the c-Src inhibitor PP2 (1 μM; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) but not its inactive analog eliminated the effect of ouabain on NCX1 and TRPC6 expression and ATP-induced Ca(2+) entry. Thus, in contrast to ouabain, the interaction of digoxin with α2 Na(+) pumps is unable to activate c-Src phosphorylation and upregulate the downstream NCX1-TRPC6 Ca(2+) signaling pathway in ASMCs. The inability of digoxin to upregulate c-Src may underlie its inability to raise long-term BP

Different effect of ouabain on endothelin-1-induced extracellular signal-regulated kinase stimulation in rat heart and tail artery

Endogenous ouabain may play a role in the control of cardiovascular system function. In this study, we investigated the effects of a long-term ouabain treatment on basal and endothelin-1 (ET-1)-induced phosphorylation of cardiac and vascular extracellular signal-regulated kinases 1 and 2 (ERK-1 and ERK-2), which are involved in several cardiac and vascular physiologic and pathologic conditions. Our results show that the hearts from ouabain-treated rats have a higher basal level of ERK-1 and ERK-2 phosphorylation compared with untreated rats. Perfusion of the hearts with ET-1 increased ERK-1 and ERK-2 phosphorylation both in ouabain-treated and in control rats, with a larger stimulatory effect in ouabain-treated animals. On the contrary, exposure of endothelium-free tail artery to ET-1 increased ERKs phosphorylation both in treated and untreated rats, but this effect was blunted in ouabain-treated rats. These findings demonstrate that ouabain treatment has opposite effects on basal and ET-1-induced ERKs phosphorylation in the heart and in the tail artery of the rat