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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.)

High-ceiling diuretics (HCD), known potent inhibitors of housekeeping Na(+),K(+),2Cl cotransporter (NKCC1) and renal-specific NKCC2, decrease [Cl(-)](i), hyperpolarize vascular smooth muscle cells (VSMC), and suppress contractions evoked by modest depolarization, phenylephrine, angiotensin II, and UTP. These actions are absent in nkcc1 (/) knock-out mice, indicating that HCD interact with NKCC1 rather than with other potential targets. These findings also suggest that VSMC-specific inhibitors of NKCC1 may be considered potential pharmacological therapeutic tools in treatment of hypertension. It should be underlined that side by side with attenuation of peripheral resistance and systemic blood pressure, HCD blocked myogenic tone (MT) in renal afferent arterioles. Keeping this in mind, attenuation of MT might be a mechanism underlying the prevalence of end-stage renal disease documented in hypertensive African-Americans with decreased NKCC1 activity and in hypertensive patients subjected to chronic HCD treatment. The role of NKCC1-mediated MT in protection of the brain, heart, and other encapsulated organs deserves further investigation.

Contrasting cell volume behaviours (swelling vs. shrinkage) are considered as criteria to distinguish necrosis from apoptosis. In this study, we employed a time-lapse, dual-image surface reconstruction technique to assess the volume of single vascular smooth muscle cells transfected with E1A-adenoviral protein (E1A-VSMC) and undergoing rapid apoptosis in the absence of growth factors or in the presence of staurosporine. After 30- to 60-min lag-phase, serum-deprived E1A-VSMC volume was increased by ~40%, which preceded maximal increments of caspase-3 activity and chromatin cleavage. Swollen cells underwent rapid apoptotic collapse, documented by plasma membrane budding, and terminated in 10-15 min by the formation of numerous apoptotic bodies. Suppression of apoptosis by inhibition of Na(+),K(+)-ATPase and activation of cAMP signalling with ouabain and forskolin, respectively, completely abolished the swelling of serum-deprived E1A-VSMC. In contrast to serum deprivation, apoptotic collapse of staurosporine-treated E1A-VSMC preceded attenuation of their volume by ~30%. Neither transient hyposmotic swelling nor isosmtotic shrinkage triggered apoptosis. Our results show that cell shrinkage can not be considered as ubiquitous hallmark of apoptosis. The involvement of stimulus-specific cell volume perturbations in initiation and progression of apoptosis in vascular smooth muscle cells should be examined further.

In the present work, we compared the outcome of hyperosmotic and isosmotic shrinkage on ion transport and protein phosphorylation in C11-MDCK cells resembling intercalated cells from collecting ducts and in vascular smooth muscle cells (VSMC) from the rat aorta. Hyperosmotic shrinkage was triggered by cell exposure to hypertonic medium, whereas isosmotic shrinkage was evoked by cell transfer from an hypoosmotic to an isosmotic environment. Despite a similar cell volume decrease of 40%-50%, the consequences of hyperosmotic and isosmotic shrinkage on cellular functions were sharply different. In C11-MDCK and VSMC, hyperosmotic shrinkage completely inhibited Na(+),K(+)-ATPase and Na(+),P(i) cotransport. In contrast, in both types of cells isosmotic shrinkage slightly increased rather than suppressed Na(+),K(+)-ATPase and did not change Na(+),P(i) cotransport. In C11-MDCK cells, phosphorylation of JNK1/2 and Erk1/2 mitogen-activated protein kinases was augmented in hyperosmotically shrunken cells by ∼7- and 2-fold, respectively, but was not affected in cells subjected to isosmotic shrinkage. These results demonstrate that the data obtained in cells subjected to hyperosmotic shrinkage cannot be considered as sufficient proof implicating cell volume perturbations in the regulation of cellular functions under isosmotic conditions.

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.

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 NaHCO(3) 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, NaHCO(3) almost completely abolished inhibitory actions of bumetanide on transient depolarization and [Ca(2+)](i) elevation triggered by PE. In bicarbonate-free medium, bumetanide decreased [Cl(-)](i) by approximately 15%; this effect was almost totally abrogated by NaHCO(3). The addition of NaHCO(3) resulted in 2-fold inhibition of NKCC activity and 3-fold attenuation of [Cl(-)](i). These data strongly suggest that extracellular HCO(3)(-) diminishes the NKCC-sensitive component of excitation-contraction coupling via suppression of this carrier., (Copyright 2009 S. Karger AG, Basel.)

Previously, we reported that hyposmotic swelling evoked transient vascular smooth muscle cell (SMC) contraction that was completely abolished by L-type Ca(2+) channel blockers. In contrast, sustained contraction revealed in hyper- and isoosmotically-shrunken SMCs was insensitive to L-type channel blockers and was diminished in Ca(2+)-free medium by only 30-50%. Several research groups reported cell volume-dependent cytoskeleton network rearrangements. This study examines the role of cytoskeleton proteins in cell volume-dependent contraction of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat thoracic aorta. Hyperosmotic shrinkage and hyposmotic swelling were triggered by modulation of medium osmolality; isosmotic shrinkage was induced by VSMR transfer from hypo- to isosmotic medium. The relative content of globular (G) and fibrillar (F) actin was estimated by fluorescence microscopy. Hyperosmotic shrinkage and hyposmotic swelling led to elevation of the F-actin/G-actin ratio by 2.5- and 1.8-fold respectively. Contraction of shrunken and swollen VSMR was insensitive to modulators of microtubules such as vinblastine, colchicine and docetaxel. Microfilament disassembly by cytochalasin B resulted in dramatic attenuation of the maximal amplitude of contraction of hyperosmotically-shrunken and hyposmotically-swollen VSMR, and almost completely abolished the contraction triggered by isosmotic shrinkage. These data suggest that both L-type Ca(2+) channel-mediated contraction of swollen vascular SMC and Ca(2+)(o)-insensitive contractions of shrunken cells are triggered by reorganization of the microfilament network caused by elevation of the F-actin/G-actin ratio.