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In rat vascular smooth muscle cells (RVSMC), 3-h Na + ,K + -ATPase inhibition by ouabain or in K + -free medium resulted in the inversion of the [Na + ] i /[K + ] i ratio and elevation up to 7-fold the content of Egr1, Atf3, Nr4a1 and Ptgs2 mRNAs. Ouabain increased the rate of 45Ca 2+ influx by 2-fold that was abolished by L-type voltage-gated Ca 2+ channel blocker nicardipine, but it was resistant to Na + /Ca 2+ exchanger inhibitor KB-R7943. To study the role of Ca 2+ -mediated signaling in the expression of Na + i /K + i -sensitive genes we used intracellular Ca 2+ chelator BAPTA and incubated RVSMC in Ca 2+ -free medium. The elevation of Nr4a1 and Ptgs2 expression triggered by ouabain was diminished in Ca 2+ -depeleted cells as well as in the presence of nicardipine and calmodulin antagonists A-7 and W-7. Ptgs2 expression was also suppressed by inhibitor of Ca 2+ /calmodulin-dependent protein kinase (CaMKII) KN-93 whereas increment of Nr4a1 content triggered by ouabain was attenuated by inhibitor of Ca 2+ /calmodulin-dependent protein phosphatase (calcineurin, CaN) cyclosporin A. Neither Ca 2+ depletion nor above listed compounds had any impact on the augmented expression of Egr1 and Atf3 in ouabain-treated RVSMC. Our results strongly suggest that dissipation of transmembrane gradient of monovalent cations increases Ptgs2 and Nr4a1 transcription via augment Ca 2+ influx through L-type Ca 2+ channels that, in turn, leads to CaMKII-mediated phosphorylation of CREB and calcineurin-mediated dephosphorylation of NFAT, respectively. Additional experiments should be performed to identify intermediates of Na + i ,K + i -mediated Ca 2+ -independent excitation-transcription coupling involved the regulation of Egr1 and Atf3 expression., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Previously, we reported that Ca(2+) depletion increased permeability of the plasma membrane for Na(+). This study examined the relative impact of [Na(+)]i/[K(+)]i-mediated signaling on transcriptomic changes in cultured vascular smooth muscle cells from rat aorta (VSMC) subjected to Ca(2+)-depletion by extra-(EGTA) and intracellular (BAPTA-AM) Ca(2+) chelators. Na(+),K(+)-ATPase inhibition in K(+)-free medium during 3 h led to elevation of [Na(+)]i and attenuation of [K(+)]i by ∼7- and 10-fold, whereas Ca(2+)-depletion resulted in alteration of these parameters by ∼3- and 2-fold, respectively. Augmented VSMC permeability for Na(+) and elevation of the [Na(+)]i/[K(+)]i ratio was triggered by addition to Ca(2+)-free medium 50 μM EGTA and was not affected by 10 μM BAPTA-AM. Na(+),K(+)-ATPase inhibition and Ca(2+)-depletion changed expression of 3677 and 4610 mRNA transcripts, respectively. We found highly significant (p<10(-12)) positive (R(2)>0.51) correlation between levels of expression of 2071 transcripts whose expression was affected by both stimuli. Among genes whose expression in Ca(2+)-depleted cells was augmented by more than 7-fold we noted cyclic AMP-dependent transcription factor Atf3, early growth response protein Egr1 and nuclear receptor subfamily 4, group A member Nr4a1. Dissipation of transmembrane gradients of monovalent cations in high-K(+), low-Na(+)-medium abolished the increments of the [Na(+)]i/[K(+)]i ratio as well as the augmented expression of these genes triggered by incubation of VSMC in EGTA containing medium. Thus, our results demonstrate, for the first time, that robust transcriptomic changes triggered by Ca(2+)-depletion in the presence of extracellular Ca(2+)-chelators are at least partially mediated by elevation of the [Na(+)]i/[K(+)]i ratio and activation of Ca(2+)i-independent, [Na(+)]i/[K(+)]i-mediated mechanism of excitation-transcription coupling. These results shad a new light on analysis of data obtained in cells subjected to long-term exposure to Ca(2+) chelators., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bumetanide and other high-ceiling diuretics (HCD) attenuate myogenic tone and contractions of vascular smooth muscle cells (VSMC) triggered by diverse stimuli. HCD outcome may be mediated by their interaction with NKCC1, the only isoform of Na(+), K(+), 2Cl(-) cotransporter expressed in VSMC as well as with targets distinct from this carrier. To examine these hypotheses, we compared the effect of bumetanide on contractions of mesenteric arteries from wild-type and NKCC1 knockout mice. In mesenteric arteries from wild-type controls, 100 microM bumetanide evoked a decrease of up to 4-fold in myogenic tone and contractions triggered by modest [K(+)](o)-induced depolarization, phenylephrine and UTP. These actions of bumetanide were preserved after inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester, but were absent in mesenteric arteries from NKCC1(-/-) mice. The data show that bumetanide inhibits VSMC contractile responses via its interaction with NKCC1 and independently of nitric oxide production by endothelial cells.