Svetlana V. Gusakova

Transcriptomic Changes Triggered by Hypoxia: Evidence for HIF-1α -Independent, [Na + ] i /[K + ] i -Mediated, Excitation-Transcription Coupling

This study examines the relative impact of canonical hypoxia-inducible factor-1alpha- (HIF-1α and Na+ i/K+ i-mediated signaling on transcriptomic changes evoked by hypoxia and glucose deprivation. Incubation of RASMC in ischemic conditions resulted in ∼3-fold elevation of [Na+]i and 2-fold reduction of [K+]i. Using global gene expression profiling we found that Na+,K+-ATPase inhibition by ouabain or K+-free medium in rat aortic vascular smooth muscle cells (RASMC) led to the differential expression of dozens of genes whose altered expression was previously detected in cells subjected to hypoxia and ischemia/reperfusion. For further investigations, we selected Cyp1a1, Fos, Atf3, Klf10, Ptgs2, Nr4a1, Per2 and Hes1, i.e. genes possessing the highest increments of expression under sustained Na+,K+-ATPase inhibition and whose implication in the pathogenesis of hypoxia was proved in previous studies. In ouabain-treated RASMC, low-Na+, high-K+ medium abolished amplification of the [Na+]i/[K+]i ratio as well as the increased expression of all tested genes. In cells subjected to hypoxia and glucose deprivation, dissipation of the transmembrane gradient of Na+ and K+ completely eliminated increment of Fos, Atf3, Ptgs2 and Per2 mRNAs and sharply diminished augmentation expression of Klf10, Edn1, Nr4a1 and Hes1. In contrast to low-Na+, high-K+ medium, RASMC transfection with Hif-1a siRNA attenuated increments of Vegfa, Edn1, Klf10 and Nr4a1 mRNAs triggered by hypoxia but did not impact Fos, Atf3, Ptgs2 and Per2 expression. Thus, our investigation demonstrates, for the first time, that Na+ i/K+ i-mediated, Hif-1α- -independent excitation-transcription coupling contributes to transcriptomic changes evoked in RASMC by hypoxia and glucose deprivation.

NKCC1 and NKCC2: The pathogenetic role of cation-chloride cotransporters in hypertension.

This review summarizes the data on the functional significance of ubiquitous (NKCC1) and renal-specific (NKCC2) isoforms of electroneutral sodium, potassium and chloride cotransporters. These carriers contribute to the pathogenesis of hypertension via regulation of intracellular chloride concentration in vascular smooth muscle and neuronal cells and via sensing chloride concentration in the renal tubular fluid, respectively. Both NKCC1 and NKCC2 are inhibited by furosemide and other high-ceiling diuretics widely used for attenuation of extracellular fluid volume. However, the chronic usage of these compounds for the treatment of hypertension and other volume-expanded disorders may have diverse side-effects due to suppression of myogenic response in microcirculatory beds.

Vascular Smooth Muscle Contraction Evoked by Cell Volume Modulation: Role of the Cytoskeleton Network

Previously, we reported that hyposmotic swelling evoked transient vascular smooth muscle cell (SMC) contraction that was completely abolished by L-type Ca2+ channel blockers. In contrast, sustained contraction revealed in hyper- and isoosmotically-shrunken SMCs was insensitive to L-type channel blockers and was diminished in Ca2+-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 Ca2+ channel-mediated contraction of swollen vascular SMC and Ca2+o-insensitive contractions of shrunken cells are triggered by reorganization of the microfilament network caused by elevation of the F-actin/G-actin ratio.

Cation Chloride Cotransporters: Regulation, Physiological Significance, and Role in Pathogenesis of Arterial Hypertension

This review summarizes the data on the functioning of carriers providing electroneutral symport of sodium, potassium, and chloride (Na+,K+,2Cl− cotransport), potassium and chloride (K+,Cl− cotransport), and sodium and chloride (K+,Cl− cotransport) as well as molecular mechanisms of the regulation of these carriers and their physiological significance. We emphasized the involvement of chloride-coupled carriers in the regulation of cell volume and intracellular chloride concentration and novel data on the role of ubiquitous isoform of Na+,K+,2Cl− cotransporter NKCC1 in regulation of vascular smooth muscle contraction and activity of GABAA receptors. Finally, we analyzed the data on activation of NKCC1 in patients with essential hypertension and its role in the long-term maintenance of elevated systemic blood pressure and myogenic response in microcirculatory beds.

Vasoconstriction triggered by hydrogen sulfide: Evidence for Na + ,K + ,2Cl - cotransport and L-type Ca 2+ channel-mediated pathway

Objectives This study examined the dose-dependent actions of hydrogen sulfide donor sodium hydrosulphide (NaHS) on isometric contractions and ion transport in rat aorta smooth muscle cells (SMC). Methods Isometric contraction was measured in ring aortas segments from male Wistar rats. Activity of Na+/K+-pump and Na+,K+,2Cl-cotransport was measured in cultured endothelial and smooth muscle cells from the rat aorta as ouabain-sensitive and ouabain-resistant, bumetanide-sensitive components of the 86Rb influx, respectively. Results NaHS exhibited the bimodal action on contractions triggered by modest depolarization ([K+]o=30 mM). At 10−4 M, NaHS augmented contractions of intact and endothelium-denuded strips by ~ 15% and 25%, respectively, whereas at concentration of 10−3 M it decreased contractile responses by more than two-fold. Contractions evoked by 10−4 M NaHS were completely abolished by bumetanide, a potent inhibitor of Na+,K+,2Cl-cotransport, whereas the inhibition seen at 10−3 M NaHS was suppressed in the presence of K+ channel blocker TEA. In cultured SMC, 5×10−5 M NaHS increased Na+,K+,2Cl- - cotransport without any effect on the activity of this carrier in endothelial cells. In depolarized SMC, 45Ca influx was enhanced in the presence of 10−4 M NaHS and suppressed under elevation of [NaHS] up to 10−3 M. 45Ca influx triggered by 10−4 M NaHS was abolished by bumetanide and L-type Ca2+ channel blocker nicardipine. Conclusions Our results strongly suggest that contractions of rat aortic rings triggered by low doses of NaHS are mediated by activation of Na+,K+,2Cl-cotransport and Ca2+ influx via L-type channels.

REGULATION OF CONTRACTILE ACTIVITY OF SMOOTH MUSCLE CELLS BY REACTIVE OXYGEN SPECIES: EVIDENCE FOR CYTOSKELETON-MEDIATED SIGNALING: PP.21.344

Objective: Recent studies showed that the cytoskeleton is the primary target of reactive oxygen species (ROS). This study examines the role of cytoskeleton network in regulation of vascular smooth muscle cell contractions by ROS. Design and Methods: Contractile responses of rat aorta strips triggered by depolarization and activation of α1-adrenergic receptors with high K+-medium and phenylephrine, respectively, were measured as increments of isometric tension. Cytoskeleton elements were modified with colchicines (10 μM), cytochalasin D (0,5 μM) and nocodazole (10 μM). Production of ROS was evoked by hydrogen peroxide (500 μM). Results: Hydrogen peroxide increased contractions triggered by high-K+ -medium by 25,2 ± 2,9% (p < 0,05) and reduced the amplitude of phenylephrine-induced contractile responses by 51,7 ± 2,9% (p < 0,05). Colchicine and cytochalasin D, but not nocodazole increased the relaxing effect of hydrogen peroxide in the reduction of phenylephrine-induced contractions by 83,5 ± 4,1% (p < 0,05) and 89,4 ± 2,9% (p < 0,05), respectively, but did not affect modulation by ROS of contractions triggered by depolarization. Conclusion: Our data demonstrate the involvement of cytoskeleton-mediated signaling in regulation by ROS of vascular smooth muscle cell contractions caused by activation of - α1-adrenergic receptors.

EFFECT OF REACTIVE OXYGEN ON MECHANISMS COUPLING EXCITATION OF SMOOTH MUSCLE CELLS GUINEA PIG URETER: PP.21.345

Objective: Reactive oxygen species claim to the role of signaling molecules, especially towards the realization of oxidative stress. However, the methods and extent of their interaction with key intracellular signaling and effector systems of regulation of excitation-coupling in smooth muscle cells continue to actively explored. This study examines the effect of reactive oxygen species in the mechanisms of regulation by cyclic nucleotides and calcium signaling system of electrical and contractile activity of smooth muscle cells. Design and Methods: The method of double sucrose bridge to study the effect of hydrogen peroxide and nitric oxide on electrical and contractile properties of smooth muscle cells of guinea pig ureter, caused by electric stimulus. To investigate the role of C-kinase branch of the calcium and nitric oxide in the development of the action potential and contractile responses of smooth muscle using phenylephrine and sodium nitroprusside, respectively. Results: Found opposite effect of hydrogen peroxide and cyclic nucleotides on C-kinase branch of the regulation of excitation-coupling in smooth muscle cells. With additional activation protenkinase with phenylephrine changing effects of hydrogen peroxide and sodium nitroprusside: electrical and contractile activity are inhibited. Similar effects have evolved against the background of inhibitors of enzymes break down hydrogen peroxide and cyclic nucleotides. Thus, the C-kinase branch of the calcium signaling system is one of the major intracellular targets, the effect on reactive oxygen species that leads to the inhibition of electrical and contractile activity of smooth muscle cells of guinea pig ureter.