Critical role of the α1-Na(+), K(+)-ATPase subunit in insensitivity of rodent cells to cytotoxic action of ouabain.

In rodents, ubiquitous α1-Na(+), K(+)-ATPase is inhibited by ouabain and other cardiotonic steroids (CTS) at ~10(3)-fold higher concentrations than those effective in other mammals. To examine the specific roles of the CTS-sensitive α1S- and CTS-resistant α1R-Na(+), K(+)-ATPase isoforms, we compared the effects of ouabain on intracellular Na(+) and K(+) content, cell survival, and mitogen-activated protein kinases (MAPK) in human and rat vascular smooth muscle cells (HASMC and RASMC), human and rat endothelial cells (HUVEC and RAEC), and human and rat brain astrocytes. 6-h exposure of HASMC and HUVEC to 3 μM ouabain dramatically increased the intracellular [Na(+)]/[K(+)] ratio to the same extend as in RASMC and RAEC treated with 3000 μM ouabain.

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.

Ubiquitous [Na+]i/[K+]i-sensitive transcriptome in mammalian cells: evidence for Ca(2+)i-independent excitation-transcription coupling.

Stimulus-dependent elevation of intracellular Ca(2+) ([Ca(2+)](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 Ca(2+) (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).

Hyperosmotic and isosmotic shrinkage differentially affect protein phosphorylation and ion transport.

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.

Low efficiency of functional translation of ouabain-resistant α2-Na(+),K(+)-ATPase mRNA in C7-MDCK epithelial cells.

Na(+),K(+)-ATPase is a heterodimer consisting of catalytic α1-α4 and regulatory β1-β3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na(+),K(+)-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K(+) ((86)Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na(+),K(+)-ATPase.

Molecular origin of Na(+)/Li(+) exchanger: Evidence against the involvement of major cloned erythrocyte transporters.

Numerous studies have demonstrated heightened Na(+)/Li(+) countertransport (NLCT) activity in erythrocytes of patients with essential hypertension or diabetic nephropathy. The same carrier also contributes to the therapeutic action of lithium salt, widely used in the treatment of psychiatric disorders. However, the molecular origin of NLCT remains unknown.

Decreased NKCC1 activity in erythrocytes from African Americans with hypertension and dyslipidemia.

Background: Recent studies demonstrated a key role of ubiquitous isoform of Na+,K+,2Cl- co-transport (NKCC1) in regulation of myogenic tone and peripheral resistance. We examined the impact of race, gender, and plasma lipid on NKCC1 activity in French Canadians and African Americans with hypertension and dyslipidemia.

Methods: NKCC and passive erythrocyte membrane permeability to K+, measured as ouabain-resistant, bumetanide-sensitive, and (ouabain+bumetanide)-resistant 86Rb influx, respectively, were compared in 111 French-Canadian men, 107 French-Canadian women, 26 African-American men, and 45 African-American women with essential hypertension and dyslipidemia.

Cardiotonic steroid-resistant alpha1-Na+,K+-ATPase rescues renal epithelial cells from the cytotoxic action of ouabain: evidence for a Nai+,Ki+ -independent mechanism.

Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood. This study examines the role of Na(+),K(+)-ATPase alpha subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing a variant of the alpha1 isoform, CTS-sensitive alpha1S, were stably transfected with a cDNA encoding CTS-resistant alpha1R-Na(+),K(+)-ATPase, whose expression was confirmed by RT-PCR.

Death of ouabain-treated renal epithelial cells: evidence for p38 MAPK-mediated Na (i) (+) /K (i) (+) -independent signaling.

Recent studies demonstrate that cytotoxic actions of ouabain and other cardiotonic steroids (CTS) on renal epithelial cells (REC) are triggered by their interaction with the Na(+),K(+)-ATPase alpha-subunit but not the result of inhibition of Na(+),K(+)-ATPase-mediated ion fluxes and inversion of the [Na(+)](i)/[K(+)](i) ratio. This study examined the role of mitogen-activated protein kinases (MAPK) in the death of ouabain-treated REC. Exposure of C7-MDCK cells that resembled principal cells from canine kidney to 3 microM ouabain led to phosphorylation of p38 without significant impact on phosphorylation of ERK and JNK MAPK.

Altered phosphorylation of RRXS*/T* motif in ouabain-treated renal epithelial cells is not mediated by inversion of the [Na](i)/[K+](i) ratio.

Recently, we reported that the death of ouabain-treated C7-MDCK cells resembling principal cells from collecting ducts of the Madin-Darby canine kidney (MDCK) is caused by ouabain interaction with Na+,K+-ATPase but is not mediated by inversion of the [Na+](i)/[K+](i) ratio. The mechanism of this intriguing phenomenon remains unknown. We therefore examined the action of ouabain on serine/threonine phosphoproteins as possible intermediates of cell death signaling.

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence.

The mechanisms of cell death signaling triggered by cardiotonic steroids are poorly understood. Based on massive detachment of ouabain-treated Madin-Darby canine kidney (MDCK) cells, it may be proposed that the cytotoxic action of these compounds is mediated by anoikis, i.e.

Purinergic inhibition of Na⁺,K⁺,Cl⁻ cotransport in C11-MDCK cells: Role of stress-activated protein kinases.

Previously, we observed that sustained activation of P2Y₁ leads to inhibition of Na⁺,K⁺,Cl⁻ cotransport (NKCC) in C11 cells resembling intercalated cells from collecting ducts of the Madin-Darby canine kidney. This study examined the role of stress-activated protein kinases (SAPK) in NKCC inhibition triggered by purinergic receptors. Treatment of C11 cells with ATP led to sustained phosphorylation of SAPK such as JNK and p38.

[Na+]i/[K+]i -independent death of ouabain-treated renal epithelial cells is not mediated by Na+,K+ -ATPase internalization and de novo gene expression.

The cytotoxic effect of long-term exposure of renal epithelial cells to ouabain and other cardiotonic steroids (CTS) is mediated by the interaction of these compounds with Na(+),K(+)-ATPase but is independent of the inhibition of Na(+),K(+)-ATPase-mediated ion fluxes. Sustained application of CTS also leads to Na(+),K(+)-ATPase endocytosis and its translocation into the nuclei that might trigger the cell death machinery via the regulation of gene expression. This study examines the role of Na(+),K(+)-ATPase internalization and de novo gene expression in the death of ouabain-treated C7-Madin-Darby canine kidney (MDCK) cells derived from distal tubules of the MDCK.

[Na]i -induced c-Fos expression is not mediated by activation of the 5' -promoter containing known transcriptional elements.

In vascular smooth muscle cells and several other cell types, inhibition of Na(+)/K(+)-ATPase leads to the expression of early response genes, including c-Fos. We designed this study to examine whether or not a putative Na(+) (i)/K(+) (i)-sensitive element is located within the c-Fos 5'-UTR from - 650 to + 103 containing all known response elements activated by 'classic' stimuli, such as growth factors and Ca(2+) (i)-raising compounds. In HeLa cells, the highest increment of c-Fos mRNA content was noted after 6 h of Na(+)/K(+)-ATPase inhibition with ouabain that was abolished by actinomycin D, an inhibitor of RNA synthesis.

Transient activation and delayed inhibition of Na+,K+,Cl- cotransport in ATP-treated C11-MDCK cells involve distinct P2Y receptor subtypes and signaling mechanisms.

In C11-MDCK cells, which resemble intercalated cells from collecting ducts of the canine kidney, P2Y agonists promote transient activation of the Na+,K+,Cl- cotransporter (NKCC), followed by its sustained inhibition. We designed this study to identify P2Y receptor subtypes involved in dual regulation of this carrier. Real time polymerase chain reaction analysis demonstrated that C11-MDCK cells express abundant P2Y1 and P2Y2 mRNA compared with that of other P2Y receptor subtypes.

Modest intracellular acidification suppresses death signaling in ouabain-treated cells.

The signaling cascade resulting in the death of several types of cells treated with ouabain or other cardiotonic steroids (CTS) remains poorly understood. Recently, we observed that ouabain kills epithelial and endothelial cells via its interaction with Na(+), K(+) -ATPase, but independently of inhibition of Na(+), K(+) -ATPase-mediated ion fluxes and inversion of the [Na(+)](i)/[K(+)](i) ratio. Here, we report that the death of ouabain-treated epithelial cells from the Madin-Darby canine kidney (C7-MDCK) and endothelial cells from porcine aortae is suppressed by acidification of medium from pH 7.

Search for intermediates of Na+,K+-ATPase-mediated [Na+]i/[K+]i-independent death signaling triggered by cardiotonic steroids.

Previously, we reported that ouabain and other cardiotonic steroids (CTS) kill renal epithelial and vascular endothelial cells via their interaction with the Na+,K+-ATPase alpha-subunit, but independently of elevation of the [Na+]i/[K+]i ratio. In distinct cell types, side-by-side with inhibition of Na+,K+-ATPase-mediated ion fluxes, CTS trigger [Ca2+]i oscillation, activation of Ras, mitogen-activated protein kinases (MAPK), phosphoinositide-3 kinase (PI3K), and protein kinase C as well as the production of reactive oxygen species and cytoskeleton reorganization. This study examined the potential involvement of the above-listed intermediates in death signaling triggered by ouabain in C7-Madin-Darby canine kidney cells.

Cardiotonic steroids differentially affect intracellular Na+ and [Na+]i/[K+]i-independent signaling in C7-MDCK cells.

Recently, we reported that ouabain kills renal epithelial and vascular endothelial cells independently of elevation of the [Na(+)](i)/[K(+)](i) ratio. These observations raised the possibility of finding cardiotonic steroids (CTS) that inhibit the Na(+),K(+) pump without attenuating cell survival and vice versa. To test this hypothesis, we compared CTS action on Na(+),K(+) pump, [Na(+)](i) content, and survival of Madin-Darby canine kidney cells.