Augmented gene expression triggered by Na,K-ATPase inhibition: Role of Ca-mediated and -independent excitation-transcription coupling.

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]/[K] ratio and elevation up to 7-fold the content of Egr1, Atf3, Nr4a1 and Ptgs2 mRNAs. Ouabain increased the rate of 45Ca influx by 2-fold that was abolished by L-type voltage-gated Ca channel blocker nicardipine, but it was resistant to Na/Ca exchanger inhibitor KB-R7943. To study the role of Ca-mediated signaling in the expression of Na/K-sensitive genes we used intracellular Ca chelator BAPTA and incubated RVSMC in Ca-free medium. The elevation of Nr4a1 and Ptgs2 expression triggered by ouabain was diminished in Ca-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/calmodulin-dependent protein kinase (CaMKII) KN-93 whereas increment of Nr4a1 content triggered by ouabain was attenuated by inhibitor of Ca/calmodulin-dependent protein phosphatase (calcineurin, CaN) cyclosporin A. Neither Ca 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 influx through L-type Ca 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,K-mediated Ca-independent excitation-transcription coupling involved the regulation of Egr1 and Atf3 expression.