HCO3 dependent impact of Na+,K+, 2Cl cotransport in vascular smooth muscle excitation-contraction coupling

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

Ubiquitous [Na+]i/[K+]i-Sensitive Transcriptome in Mammalian Cells: Evidence for Ca2+i-Independent Excitation-Transcription Coupling

Stimulus-dependent elevation of intracellular Ca2+ ([Ca2+]i) affects the expression of numerous genes – a phenomenon known as excitation-transcription coupling. Recently, we found that increases in [Na+]i trigger c-Fos expression via a novel Ca2+i-independent pathway. In the present study, we identified ubiquitous and tissue-specific [Na+]i/[K+]i-sensitive transcriptomes by comparative analysis of differentially expressed genes in vascular smooth muscle cells from rat aorta (RVSMC), the human adenocarcinoma cell line HeLa, and human umbilical vein endothelial cells (HUVEC). To augment [Na+]i and reduce [K+]i, cells were treated for 3 hrs with the Na+,K+-ATPase inhibitor ouabain or placed for the same time in the K+-free medium. Employing Affymetrix-based technology, we detected changes in expression levels of 684, 737 and 1839 transcripts in HeLa, HUVEC and RVSMC, respectively, that were highly correlated between two treatments (p<0.0001; R2>0.62). Among these Na+i/K+i-sensitive genes, 80 transcripts were common for all three types of cells. To establish if changes in gene expression are dependent on increases in [Ca2+]i, we performed identical experiments in Ca2+-free media supplemented with extracellular and intracellular Ca2+ chelators. Surprisingly, this procedure elevated rather than decreased the number of ubiquitous and cell-type specific Na+i/K+i-sensitive genes. Among the ubiquitous Na+i/K+i-sensitive genes whose expression was regulated independently of the presence of Ca2+ chelators by more than 3-fold, we discovered several transcription factors (Fos, Jun, Hes1, Nfkbia), interleukin-6, protein phosphatase 1 regulatory subunit, dual specificity phosphatase (Dusp8), prostaglandin-endoperoxide synthase 2, cyclin L1, whereas expression of metallopeptidase Adamts1, adrenomedulin, Dups1, Dusp10 and Dusp16 was detected exclusively in Ca2+-depleted cells. Overall, our findings indicate that Ca2+i-independent mechanisms of excitation-transcription coupling are involved in transcriptomic alterations triggered by elevation of the [Na+]i/[K+]i ratio. There results likely have profound implications for normal and pathological regulation of mammalian cells, including sustained excitation of neuronal cells, intensive exercise and ischemia-triggered disorders

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

Time- and dose dependent actions of cardiotonic steroids on transcriptome and intracellular content of Na+ and K+: a comparative analysis

Recent studies demonstrated that in addition to Na+,K+-ATPase inhibition cardiotonic steroids (CTSs) affect diverse intracellular signaling pathways. This study examines the relative impact of [Na+]i/[K+]i-mediated and -independent signaling in transcriptomic changes triggered by the endogenous CTSs ouabain and marinobufagenin (MBG) in human umbilical vein endothelial cells (HUVEC). We noted that prolongation of incubation increased the apparent affinity for ouabain estimated by the loss of [K+]i and gain of [Na+]i. Six hour exposure of HUVEC to 100 and 3,000 nM ouabain resulted in elevation of the [Na+]i/[K+]i ratio by ~15 and 80-fold and differential expression of 258 and 2185 transcripts, respectively. Neither [Na+]i/[K+]i ratio nor transcriptome were affected by 6-h incubation with 30 nM ouabain. The 96-h incubation with 3 nM ouabain or 30 nM MBG elevated the [Na+]i/[K+]i ratio by ~14 and 3-fold and led to differential expression of 880 and 484 transcripts, respectively. These parameters were not changed after 96-h incubation with 1 nM ouabain or 10 nM MBG. Thus, our results demonstrate that elevation of the [Na+]i/[K+]i ratio is an obligatory step for transcriptomic changes evoked by CTS in HUVEC. The molecular origin of upstream [Na+]i/[K+]i sensors involved in transcription regulation should be identified in forthcoming studies

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

The Study of the Association of Polymorphisms in <i>LSP1</i>, <i>GPNMB</i>, <i>PDPN</i>, <i>TAGLN</i>, <i>TSPO</i>, and <i>TUBB6</i> Genes with the Risk and Outcome of Ischemic Stroke in the Russian Population

To date, there has been great progress in understanding the genetic basis of ischemic stroke (IS); however, several aspects of the condition remain underexplored, including the influence of genetic factors on post-stroke outcomes and the identification of causative loci. We proposed that an analysis of the results obtained from animal models of brain ischemia could be helpful. To this end, we developed a bioinformatic approach for exploring single-nucleotide polymorphisms (SNPs) in human orthologs of rat genes expressed differentially after induced brain ischemia. Using this approach, we identified and analyzed 11 SNPs from 6 genes in 553 Russian individuals (331 patients with IS and 222 controls). We assessed the association of SNPs with the risk of IS and IS outcomes. We found that the SNPs rs858239 (GPNMB), rs907611 (LSP1), and rs494356 (TAGLN) were associated with different parameters of IS functional outcomes. In addition, the SNP rs1261025 (PDPN) was associated significantly with IS itself (p = 0.0188, recessive model). All these associations were demonstrated for the first time. Analysis of the literature suggests that they should be characterized as being inflammation related. This supports the pivotal role of inflammation in both the incidence of stroke and post-stroke outcomes. We believe the findings reported here will help with stroke prognosis in the future

Effect of high-K⁺, low-Na⁺ medium and <i>Hif-1α</i> siRNA on gene expression triggered by hypoxia and ouabain.

<p>Control non-transfected RASMC or RASMC transfected with scrambled or <i>Hif-1α</i> siRNA were exposed to hypoxia/glucose deprivation or 3 mM ouabain in control or low-Na⁺, high-K⁺ medium for 24 hr and RNAs content of genes listed in the left column was quantified by real-time quantitative RT-PCR as described in Methods. Means ± S.E. obtained in 3 independent experiments performed in triplicate are shown. Mean values for cells incubated in control medium under normoxic conditions were taken as 1.00.</p><p>Effect of high-K⁺, low-Na⁺ medium and <i>Hif-1α</i> siRNA on gene expression triggered by hypoxia and ouabain.</p

Primer sequences for RT-PCR.

<p>Primer sequences for RT-PCR.</p