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ABSTRACT: 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. In mock-transfected and alpha1R-cells, maximal inhibition of (86)Rb influx was observed at 10 and 1000 muM ouabain, respectively, thus confirming high abundance of alpha1R-Na(+),K(+)-ATPase in these cells. Six-hour treatment of alpha1R-cells with 1000 muM ouabain led to the same elevation of the [Na(+)](i)/[K(+)](i) ratio that was detected in mock-transfected cells treated with 3 muM ouabain. However, in contrast to the massive death of mock-transfected cells exposed to 3 muM ouabain, alpha1R-cells survived after 24-h incubation with 1000 muM ouabain. Inversion of the [Na(+)](i)/[K(+)](i) ratio evoked by Na(+),K(+)-ATPase inhibition in K(+)-free medium did not affect survival of alpha1R-cells but increased their sensitivity to ouabain. Our results show that the alpha1R subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na(+),K(+)-ATPase-mediated Na(+) and K(+) fluxes and inversion of the [Na(+)](i)/[K(+)](i) ratio.
Apoptosis 12/2009; 15(1):55-62. · 4.07 Impact Factor
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ABSTRACT: 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. Maximal increment of p38 phosphorylation was observed at 4 h followed by cell death at 12 h of ouabain addition. In contrast to ouabain, neither cell death nor p38 MAPK phosphorylation were affected by elevation of the [Na(+)](i)/[K(+)](i) ratio triggered by Na(+),K(+)-ATPase inhibition in K(+)-free medium. p38 phosphorylation was noted in all other cell types exhibiting death in the presence of ouabain, such as intercalated cells from canine kidney and human colon rectal carcinoma cells. We did not observe any action of ouabain on p38 phosphorylation in ouabain-resistant smooth muscle cells from rat aorta and endothelial cells from human umbilical vein. Both p38 phosphorylation and death of ouabain-treated C7-MDCK cells were suppressed by p38 inhibitor SB 202190 but were resistant to its inactive analogue SB 202474. Our results demonstrate that death of CTS-treated REC is triggered by Na (i) (+) ,K (i) (+) -independent activation of p38 MAPK.
Apoptosis 09/2009; 14(11):1266-73. · 4.07 Impact Factor
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ABSTRACT: 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 alpha,beta-ATP triggered transient contractions that were sharply decreased in the presence of NF023, a potent antagonist of P2X(1) receptors. In contrast, UTP and UDP elicited sustained contractions which were suppressed by MRS2567, a selective antagonist of P2Y(6) 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 P2Y(6)-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 P2Y(6)-mediated signaling should be examined further.
Purinergic Signalling 05/2009; 5(3):343-9. · 3.16 Impact Factor
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ABSTRACT: 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. Activation of these kinases also occurred in anisomycin-treated cells. Surprisingly, we observed that compounds SP600125 and SB202190, known as potent inhibitors of JNK and p38 in cell-free systems, activated rather than inhibited phosphorylation of the kinases in C11 cells. Importantly, similarly to ATP, all the above-listed activators of JNK and p38 phosphorylation inhibited NKCC. Thus, our results suggest that activation of JNK and/or p38 contributes to NKCC suppression detected in intercalated-like cells from distal tubules after their exposure to P2Y₁ agonists.
Purinergic Signalling 07/2008; 4(2):183-91. · 3.16 Impact Factor
