Svetlana V. Koltsova

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 hypertension: Role in the regulation of vascular smooth muscle contractions and myogenic tone

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.

Excitation-contraction coupling in resistance mesenteric arteries: evidence for NKCC1-mediated pathway.

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.

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.

Myogenic tone in mouse mesenteric arteries: evidence for P2Y receptor-mediated, Na(+), K (+), 2Cl (-) cotransport-dependent signaling

This study examines the action of agonists and antagonists of P2 receptors on mouse mesenteric artery contractions and the possible involvement of these signaling pathways in myogenic tone (MT) evoked by elevated intraluminal pressure. Both ATP and its non-hydrolyzed analog α,β-ATP triggered transient contractions that were sharply decreased in the presence of NF023, a potent antagonist of P2X1 receptors. In contrast, UTP and UDP elicited sustained contractions which were suppressed by MRS2567, a selective antagonist of P2Y6 receptors. Inhibition of Na+, K+, 2Cl− cotransport (NKCC) with bumetanide led to attenuation of contractions in UTP- but not ATP-treated arteries. Both UTP-induced contractions and MT were suppressed by MRS2567 and bumetanide but were insensitive to NF023. These data implicate a P2Y6-mediated, NKCC-dependent mechanism in MT of mesenteric arteries. The action of heightened intraluminal pressure on UTP release from mesenteric arteries and its role in the triggering of P2Y6-mediated signaling should be examined further.

Antifibrotic effects of noscapine through activation of prostaglandin E2 receptors and protein kinase A.

Background: Noscapine is a safe orally available anticough medicine also known to bind microtubules and induce cancer cell death. Results: Noscapine inhibits myofibroblast differentiation and pulmonary fibrosis through prostaglandin receptors and activation of PKA. Conclusion: Noscapine is an antifibrotic drug acting through PKA activation via EP2 prostaglandin receptors. Significance: This study describes a novel antifibrotic function and novel mechanism of action of noscapine. Myofibroblast differentiation is a key process in the pathogenesis of fibrotic disease. We have shown previously that differentiation of myofibroblasts is regulated by microtubule polymerization state. In this work, we examined the potential antifibrotic effects of the antitussive drug, noscapine, recently found to bind microtubules and affect microtubule dynamics. Noscapine inhibited TGF-β-induced differentiation of cultured human lung fibroblasts (HLFs). Therapeutic noscapine treatment resulted in a significant attenuation of pulmonary fibrosis in the bleomycin model of the disease. Noscapine did not affect gross microtubule content in HLFs, but inhibited TGF-β-induced stress fiber formation and activation of serum response factor without affecting Smad signaling. Furthermore, noscapine stimulated a rapid and profound activation of protein kinase A (PKA), which mediated the antifibrotic effect of noscapine in HLFs, as assessed with the PKA inhibitor, PKI. In contrast, noscapine did not activate PKA in human bronchial or alveolar epithelial cells. Finally, activation of PKA and the antifibrotic effect of noscapine in HLFs were blocked by the EP2 prostaglandin E2 receptor antagonist, PF-04418948, but not by the antagonists of EP4, prostaglandin D2, or prostacyclin receptors. Together, we demonstrate for the first time the antifibrotic effect of noscapine in vitro and in vivo, and we describe a novel mechanism of noscapine action through EP2 prostaglandin E2 receptor-mediated activation of PKA in pulmonary fibroblasts.

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.

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 NaHCO₃ 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, NaHCO₃ almost completely abolished inhibitory actions of bumetanide on transient depolarization and [Ca²⁺]_i elevation triggered by PE. In bicarbonate-free medium, bumetanide decreased [Cl^-]_i by ∼15%; this effect was almost totally abrogated by NaHCO₃. The addition of NaHCO₃ resulted in 2-fold inhibition of NKCC activity and 3-fold attenuation of [Cl^-]_i. These data strongly suggest that extracellular HCO₃^- diminishes the NKCC-sensitive component of excitation-contraction coupling via suppression of this carrier.

Transcriptomic changes in Ca2+-depleted cells: Role of elevated intracellular [Na+]/[K+] ratio

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.

Activation of P2Y receptors causes strong and persistent shrinkage of C11-MDCK renal epithelial cells

Purinergic receptors activate diverse signaling cascades and regulate the activity of cell volume-sensitive ion transporters. However, the effects of ATP and other agonists of P2 receptors on cell volume dynamics are only scarcely studied. In the present work, we used the recently developed dual-image surface reconstruction technique to explore the influence of purinergic agonists on cell volume in the C11-Madin-Darby canine kidney cell line resembling intercalated cells from kidney collecting ducts. Unexpectedly, we found that ATP and UTP triggered very robust (55-60%) cell shrinkage that lasted up to 2 h after agonist washout. Purinergic regulation of cell volume required increases in intracellular Ca(2+) and could be partially mimicked by the Ca(2+)-ionophore ionomycin or activation of protein kinase C by 4β-phorbol 12-myristate 13-acetate. Cell shrinkage was accompanied by strong reductions in intracellular K(+) and Cl(-) content measured using steady-state (86)Rb(+) and (36)Cl(-) distribution. Both shrinkage and ion efflux in ATP-treated cells were prevented by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and by the BK(Ca) channel inhibitors charybdotoxin, iberiotoxin, and paxilline. To evaluate the significance of cell-volume changes in purinergic signaling, we measured the impact of ATP on the expression of the immediate-early gene c-Fos. Thirty-minute treatment with ATP increased c-Fos immunoreactivity by approximately fivefold, an effect that was strongly inhibited by charybdotoxin and completely prevented by NPPB. Overall, our findings suggest that ATP-induced cell-volume changes are partially responsible for the physiological actions of purinergic agonists.