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In smooth muscles, inhibition of Na+,K+,2Cl- cotransport (NKCC) by bumetanide decreased intracellular Cl- content ([Cl-]i) and suppressed the contractions triggered by diverse stimuli. This study examines whether or not bicarbonate, a regulator of several Cl- transporters, affects the impact of NKCC in excitation-contraction coupling. Addition of 25 mM NaHCO3 attenuated the inhibitory action of bumetanide on mesenteric artery contractions evoked by 30 mM KCl and phenylephrine (PE) by 5 and 3-fold, respectively. In cultured vascular smooth muscle cells, NaHCO3 almost completely abolished inhibitory actions of bumetanide on transient depolarization and [Ca2+]i elevation triggered by PE. In bicarbonate-free medium, bumetanide decreased [Cl-]i by ∼15%; this effect was almost totally abrogated by NaHCO3. The addition of NaHCO3 resulted in 2-fold inhibition of NKCC activity and 3-fold attenuation of [Cl-]i. These data strongly suggest that extracellular HCO3- diminishes the NKCC-sensitive component of excitation-contraction coupling via suppression of this carrier. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: In contrast to rat and human erythrocytes, nucleated erythrocytes from two fish species (Cyprinus carpio and Salmo trutta) underwent almost complete haemolysis in 20min of EDTA addition. Using Ca2+/Mg2+ EGTA-citrate buffer, we observed that half-maximal haemolysis of fish erythrocytes occurs at [Ca2+]o ∼10μM independently of extracellular Mg2+ concentration. Attenuation of [Ca2+]o with EGTA also decreased stability of the plasma membrane of vascular smooth muscle cells (VSMC) and HeLa cells, indicated by a three- to five-fold elevation of lactate dehydrogenase release and passive permeability of plasma membrane for Na+. In VSMC, EGTA lowered [Ca2+]i by ∼20%. This effect was absent in VSMC-loaded with the intracellular Ca2+ chelator BAPTA. In contrast to EGTA, BAPTA did not affect haemoglobin release from fish erythrocytes and passive permeability for Na+ in VSMC. Viewed collectively, our data show that in nucleated cells, extracellular Ca2+ plays a crucial role in the maintenance of plasma membrane integrity. [Copyright &y& Elsevier]

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca2+ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca2+o. Thirty minutes preincubation with bumetanide, a potent Na+,K+,Cl- cotransport (NKCC) inhibitor, decreased Cl-i content, nifedipine-sensitive45Ca uptake and contractions triggered by modest depolarization ([K+]o=36 mM). Elevation of [K+]o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl-i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further. [ABSTRACT FROM AUTHOR]

The Na/K pump of vascular smooth muscle cells (VSMC) and renal epithelial cells (REC) is viewed as a target of digitalis and endogenous ouabain (EO), leading to the development of hypertension. In this study, we compared the effect of ouabain on Na/K pump activity and the intracellular content of monovalent cations in VSMC and REC obtained from rats, humans and dogs. In VSMC from the rat aorta, ouabain inhibited maximal Na/K pump activity measured as the rate of [sup 86]Rb influx in Na[sup +]-loaded cells, with an ID[sub 50] of ~20-30 µM without any differences between two strains of normotensive rats (WKY and BN.lx) and three substrains of spontaneously hypertensive rats (SHR). Half-maximal inhibition of the Na/K pump in REC from the rat inner medullary collecting duct was observed at ~20 µM of ouabain. In contrast to rat cells, half-maximal inhibition of [sup 86]Rb influx in VSMC from human coronary arteries and in REC from the Madin-Darby canine kidney was seen at ~0.03 and 0.1 µM ouabain, respectively. At concentrations lower than 100 µM, ouabain did not affect the intracellular content of exchangeable Na[sup +] and K[sup +] in rat VSMC, measured as the steady-state distribution of [sup 22]Na and [sup 86]Rb, whereas in human VSMC, it increased the intracellular Na[sup +]/K[sup +] ratio with an ID[sub 50] of ~0.5 µM. Keeping in mind that the circulating level of administered digitalis and EO does not exceed 10[sup -9] M, our results strongly suggest that the involvement of these compounds in the pathogenesis of hypertension in rats is not mediated by inhibition of the α1-isoform of the Na/K pump in VSMC and REC. Alternative mechanisms of the involvement of EO and ouabain-like factors in the development of hypertension are considered. [ABSTRACT FROM AUTHOR]

The effect of cholera toxin (CTX), an activator of the adenylate cyclase-coupled G protein α[sub S] subunit, was studied on cultured vascular smooth muscle cell (VSMC) proliferation. Continuous exposure (48 h) to CTX as well as 2-min pretreatment of VSMC with CTX led to the same level of cAMP production, inhibition of DNA synthesis, and arrest in the G[sub 1] phase without induction of necrosis or apoptosis in VSMC. Protein kinase A (PKA) activity in CTX-pretreated cells was transiently elevated by 3-fold after 3 h of incubation, whereas after 48 h it was reduced by 2-fold compared with baseline values without modulation of the expression of its catalytic α subunit. The PKA inhibitors H89 and KT 5720 did not protect VSMC from the antiproliferative effect of CTX. Two-dimensional electrophoresis was used to analyze the influence of CTX on protein phosphorylation. After 3 h of incubation of CTX-pretreated cells, we observed both newly-phosphorylated and dephosphorylated proteins (77 and 50 protein species, respectively). After 24 h of incubation, the number of phosphorylated proteins in CTX-treated cells was decreased to 39, whereas the number of dephosphorylated proteins was increased to 106. In conclusion, brief exposure to CTX leads to full-scale activation of cAMP signaling and evokes VSMC arrest in the G[sub 1] phase.Key words: vascular smooth muscle, proliferation, cholera toxin, cAMP, protein kinase A.L'effet de la toxine de choléra (TXC), qui active la sous-unité α[sub S] de la protéine G couplée à l'adénylate cyclase, a été étudié sur la prolifération des cellules vasculaires musculaires lisses en culture. Une exposition continue (48 h) à la TXC ainsi qu'un traitement des cellules pendant 2 min a produit une accumulation d'AMPc similaire, une inhibition de la synthèse d'ADN et un arrêt des cellules en phase G[sub 1] sans induction de nécrose ou d'apoptose. L'activité de la protéine kinase A (PKA) a augmenté transitoirement de 3 fois après 3 h d'incubation en présence de TXC; après 48 h, l'activité de la PKA était 2 fois inférieure bien que l'expression de la sous-unité catalytique α de la PKA n'était pas affectée. Les inhibiteurs de la PKA H89 et KT 5720 n'ont pas empêché l'effet antiproliférateur de la TXC. L'électrophorèse bi-dimensionnelle a été utilisée pour analyser l'influence de la TXC sur la phosphorylation protéique: après 3 h d'incubation, la TXC a induit la phosphorylation de 77 protéines et la déphosphorylation de 50 autres. Après 24 h, le nombre de protéines phosphorylées par la TXC était diminué à 39 et le nombre de protéines déphosphorylées était augmenté à 106. En conclusion, une exposition des cellules vasculaires musculaires lisses à la TXC mène à une activation complète de la voie de signalisation de l'AMPc et conduit à un arrêt de croissance cellulaire.Mots clés : vasculaires musculaires lisses, prolifération, toxine de choléra, cAMP, protéine kinase A. [ABSTRACT FROM AUTHOR]

This study examined the contribution of cAMP signaling to the modulation of vascular smooth muscle cell (VSMC) proliferation by adenosine. At a concentration of 1 mM, adenosine inhibited [[sup 3] H]-thymidine uptake, measured as the initial rate of isotope influx, by 10-fold. Diminution of [[sup 3] H]-thymidine uptake by adenosine was independent of the presence of A[sub 1] - and A[sub 2] -receptor antagonists, indicating that adenosine competes with thymidine for plasma membrane transporter-binding sites. Considering these results, in order to estimate [[sup 3] H]-thymidine DNA labeling, VSMCs were preincubated with adenosine for 48 h, and adenosine was then omitted during the subsequent 2 h of incubation in [[sup 3] H]-thymidine-containing medium. In serum-depleted VSMCs, preincubation with 100 μM or 1,000 μM adenosine augmented DNA synthesis by approximately 6- and 3-fold, respectively, whereas the increment of DNA synthesis triggered by serum was decreased in the presence of adenosine by 20–30%. Both cAMP production and inhibition of DNA synthesis by adenosine in serum-supplied cells were independent of the presence of the A[sub 1] -antagonist 1,2-dipropyl-8-cyclopentylxanthine (DPCPX), but were abolished by the A[sub 2] -antagonist 1,3-dimethyl-7-propylxanthine (DMPX). In contrast, the activation of DNA synthesis in serum-depleted cells by adenosine was decreased in the presence of DPCPX and DMPX by approximately 30 and 40%, respectively. Both in serum-supplied and -depleted VSMCs, dose-dependent elevation of cAMP production with an adenylate cyclase activator, forskolin, reduced DNA synthesis by up to 40–60%. Thus, our results show that in addition to suppressing thymidine uptake, adenosine depresses the DNA synthesis triggered by serum-derived growth factors and stimulates DNA synthesis in serum-depleted cells. These data also suggest that the inhibition of DNA synthesis is mediated by cAMP production where the activation of DNA synthesis is independent of cAMP signaling.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In this article, the author reflects on the study by H. A. Lee and colleagues on the increases methylation of Na+,K+,2Cl- contransporter (Nkcc1) promoter in rats. He states that the data of the study suggest that maintaining hypomethylation in Nkcc1 promoter lies the development of hypertension. Furthermore, he notes that adenosine monophosphate-raising vasodilators inhibit the vascular smooth muscle cells (VSMC) NKCC1.