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Nicholas A. Delamere - One of the best experts on this subject based on the ideXlab platform.
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protein kinase c inhibits na k 2cl cotransporter activity in cultured rabbit nonpigmented ciliary epithelium
American Journal of Physiology-cell Physiology, 1994Co-Authors: J Dong, Nicholas A. DelamereAbstract:We examined the regulation of Na(+)-K(+)-2Cl- transporter activity by protein kinase C (PKC) in a cell line derived from rabbit nonpigmented ciliary epithelium. Na(+)-K(+)-2Cl- cotransporter activity was measured as the rate of bumetanide-sensitive potassium (86Rb) transport. Phorbol 12,13-dibutyrate (PBDu) was used to activate PKC. PBDu inhibited bumetanide-sensitive potassium (86Rb) uptake, with a half-maximal inhibitory concentration of approximately 0.1 microM. The inhibitory effect of PBDu on potassium uptake by the N(+)-K(+)-2Cl- cotransporter was abolished by PCK downregulation and diminished by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a PKC inhibitor. PBDu inhibited Na(+)-K(+)-2Cl- cotransporter-mediated inward potassium (86Rb) transport by approximately 26% in control cells and by 40% in cells pretreated with Ouabain. PKC activation also reduced the rate of bumetanide-sensitive potassium (86Rb) efflux in Ouabain-treated cells but not in control (no oubain) cells. PBDu caused little change of intracellular sodium, potassium, or chloride, suggesting that an alteration of cytoplasmic ion composition is not responsible for the observed PBDu-induced changes in the rate of either inward or outward potassium movement mediated by the Na(+)-K(+)-2Cl- cotransporter.
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Protein kinase C inhibits Na(+)-K(+)-2Cl- cotransporter activity in cultured rabbit nonpigmented ciliary epithelium.
The American journal of physiology, 1994Co-Authors: J Dong, Nicholas A. DelamereAbstract:We examined the regulation of Na(+)-K(+)-2Cl- transporter activity by protein kinase C (PKC) in a cell line derived from rabbit nonpigmented ciliary epithelium. Na(+)-K(+)-2Cl- cotransporter activity was measured as the rate of bumetanide-sensitive potassium (86Rb) transport. Phorbol 12,13-dibutyrate (PBDu) was used to activate PKC. PBDu inhibited bumetanide-sensitive potassium (86Rb) uptake, with a half-maximal inhibitory concentration of approximately 0.1 microM. The inhibitory effect of PBDu on potassium uptake by the N(+)-K(+)-2Cl- cotransporter was abolished by PCK downregulation and diminished by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a PKC inhibitor. PBDu inhibited Na(+)-K(+)-2Cl- cotransporter-mediated inward potassium (86Rb) transport by approximately 26% in control cells and by 40% in cells pretreated with Ouabain. PKC activation also reduced the rate of bumetanide-sensitive potassium (86Rb) efflux in Ouabain-treated cells but not in control (no oubain) cells. PBDu caused little change of intracellular sodium, potassium, or chloride, suggesting that an alteration of cytoplasmic ion composition is not responsible for the observed PBDu-induced changes in the rate of either inward or outward potassium movement mediated by the Na(+)-K(+)-2Cl- cotransporter.
John M Hamlyn - One of the best experts on this subject based on the ideXlab platform.
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upregulation of na and ca2 transporters in arterial smooth muscle from Ouabain induced hypertensive rats
American Journal of Physiology-heart and Circulatory Physiology, 2010Co-Authors: Maria V Pulina, John M Hamlyn, Mordecai P Blaustein, Alessandra Zulian, Roberto Berraromani, Olga Beskina, Amparo Mazzoccospezzia, Sergey G Baryshnikov, Italia Papparella, Vera A GolovinaAbstract:Prolonged Ouabain administration (25 μg·kg−1·day−1 for 5 wk) induces “Ouabain hypertension” (OH) in rats, but the molecular mechanisms by which Ouabain elevates blood pressure are unknown. Here, we...
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chronic Ouabain treatment induces vasa recta endothelial dysfunction in the rat
American Journal of Physiology-renal Physiology, 2009Co-Authors: Kristie Payne, Whaseon Leekwon, Zhong Zhang, John M Hamlyn, Mordecai P Blaustein, Moo H Kwon, Thomas L PalloneAbstract:Descending vasa recta (DVR) are 15-μm vessels that perfuse the renal medulla. Ouabain has been shown to augment DVR endothelial cytoplasmic Ca2+ ([Ca2+]CYT) signaling. In this study, we examined the expression of the Ouabain-sensitive Na-K-ATPase α2 subunit in the rat renal vasculature and tested effects of acute Ouabain exposure and chronic Ouabain treatment on DVR. Immunostaining with antibodies directed against the α2 subunit verified its expression in both DVR pericytes and endothelium. Acute application of Ouabain (100 or 500 nM) augmented the DVR nitric oxide generation stimulated by acetylcholine (ACh; 10 μM). At a concentration of 1 mM, Ouabain constricted microperfused DVR, whereas at 100 nM, it was without effect. Acute Ouabain (100 nM) did not augment constriction by angiotensin II (0.5 or 10 nM), whereas l-nitroarginine methyl ester-induced contraction of DVR was slightly enhanced. Ouabain-hypertensive (OH) rats were generated by chronic Ouabain treatment (30 μg·kg−1·day−1, 5 wk). The acute endothelial [Ca2+]CYT elevation by Ouabain (100 nM) was absent in DVR endothelia of OH rats. The [Ca2+]CYT response to 10 nM ACh was also eliminated, whereas the response to 10 μM ACh was not. The endothelial [Ca2+]CYT response to bradykinin (100 nM) was significantly attenuated. We conclude that endothelial responses may offset the ability of acute Ouabain exposure to enhance DVR vasoconstriction. Chronic exposure to Ouabain, in vivo, leads to hypertension and DVR endothelial dysfunction, manifested as reduced [Ca2+]CYT responses to both Ouabain- and endothelium-dependent vasodilators.
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Ouabain is secreted by bovine adrenocortical cells.
Endocrinology, 1994Co-Authors: James Laredo, Bruce P. Hamilton, John M HamlynAbstract:Ouabain is a specific inhibitor of the sodium pump. This steroid has been found in the mammalian circulation in significant amounts and may be of adrenal origin. Secretion of Ouabain from adrenal cells has been little studied and the purpose of the present work was to determine the adrenal distribution of Ouabain, aldosterone and cortisol, and to characterize the effects of ACTH and angiotensin II on the secretion of these steroids in primary cultures of bovine adrenocortical cells. In fresh bovine adrenals, the cortical to medullary ratios for aldosterone, cortisol and Ouabain were 14, 4.25 and 2.5, respectively. All three steroids were detected in elevated amounts in the conditioned medium of primary cultures of adrenocortical cells. Reverse phase HPLC of the secreted Ouabain immunoreactivity showed it was isopolar with commercial Ouabain. In the presence of 10 nM ACTH or angiotensin II, the secretion of all three steroids increased significantly with similar time courses. The stimulated secretion of Ouabain exceeded the intracellular content of this steroid in either control or activated cells by 3-5 fold. The amount of angiotensin II stimulated Ouabain secretion was greater from cells incubated in larger volumes. These results show that Ouabain is enriched in the bovine adrenal cortex, and is secreted by primary cultures of these cells. The secretion of Ouabain is increased by ACTH and angiotensin II, is due to either de novo synthesis or transformation of an intracellular precursor that is not overtly immunoreactive, and is feedback regulated by either Ouabain itself or a cosecreted factor. These cells may be useful to study stimulus-secretion coupling and the biosynthetic pathway of Ouabain.
Joseph I. Shapiro - One of the best experts on this subject based on the ideXlab platform.
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involvement of reactive oxygen species in a feed forward mechanism of na k atpase mediated signaling transduction
Journal of Biological Chemistry, 2013Co-Authors: Anna P Shapiro, Steven T Haller, Vinai Katragadda, Jiang Tian, Venkatesha Basrur, Deepak Malhotra, Nader G. Abraham, Joseph I. ShapiroAbstract:Abstract Cardiotonic steroids (CTS, such as Ouabain), signaling through Na/K-ATPase, regulate sodium reabsorption in the renal proximal tubule (RPT) and impairment of this mechanism is implicated in Dahl salt-sensitive hypertension. We report here that reactive oxygen species (ROS) are required to initiate Ouabain-stimulated Na/K-ATPase/c-Src signaling. Pre-treatment with the antioxidant N-acetyl-L-cysteine (NAC) prevented Ouabain-stimulated Na/K-ATPase/c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3 (NHE3), and inhibition of active transepithelial 22Na+ transport. Disruption of the Na/K-ATPase/c-Src signaling complex attenuated Ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of Ouabain and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, Ouabain stimulated direct carbonylation of two amino acid residues in the actuator (A) domain of the Na/K-ATPase α1 subunit. Taken together, the data indicates that carbonylation modification of the Na/K-ATPase α1 subunit is involved in a feed-forward mechanism of regulation of Ouabain-mediated RPT Na/K-ATPase signal transduction and subsequent sodium transport.
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Ouabain induces endocytosis of plasmalemmal na k atpase in llc pk1 cells by a clathrin dependent mechanism
Kidney International, 2004Co-Authors: Jiang Liu, Deepak Malhotra, Riad Kesiry, Sankaridrug M Periyasamy, Zijian Xie, Joseph I. ShapiroAbstract:Ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells by a clathrin-dependent mechanism. Background We have demonstrated that Ouabain causes dose- and time-dependent decreases in 86 Rb uptake in porcine proximal tubular (LLC-PK1) cells. The present study addresses the molecular mechanisms involved in this process. Methods Studies were performed with cultured LLC-PK1 and Src family kinase deficient (SYF) cells. Results We found that 50 nmol/L Ouabain applied to the basal, but not apical, aspect for 12 hours caused decreases in the plasmalemmal Na/K-ATPase. This loss of plasmalemmal Na/K-ATPase reverses completely within 12 to 24 hours after removal of Ouabain. Ouabain also increased the Na/K-ATPase content in both early and late endosomes, activated phosphatidylinositol 3-kinase (PI(3)K), and also caused a translocation of some Na/K-ATPase to the nucleus. Immunofluorescence demonstrated that the Na/K-ATPase colocalized with clathrin both before and after exposure to Ouabain, and immunoprecipitation experiments confirmed that Ouabain stimulated interactions among the Na/K-ATPase, adaptor protein-2 (AP-2), and clathrin. Potassium (K) depletion, chlorpromazine, or PI(3)K inhibition all significantly attenuated this Ouabain-induced endocytosis. Inhibition of the Ouabain-activated signaling process through Src by 4-Amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4- d ]pyrimidine (PP2) significantly attenuated Ouabain-induced endocytosis. Moreover, experiments performed in SYF cells demonstrated that Ouabain induced increases in the endocytosis of the Na/K-ATPase when Src was reconstituted (SYF+), but not in the Src-deficient (SYF-) cells. Conclusion These data demonstrate that Ouabain stimulates a clathrin-dependent endocytosis pathway that translocates the Na/K-ATPase to intracellular compartments, thus suggesting a potential role of endocytosis in Ouabain-induced signal transduction as well as proximal tubule sodium handling.
J Dong - One of the best experts on this subject based on the ideXlab platform.
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protein kinase c inhibits na k 2cl cotransporter activity in cultured rabbit nonpigmented ciliary epithelium
American Journal of Physiology-cell Physiology, 1994Co-Authors: J Dong, Nicholas A. DelamereAbstract:We examined the regulation of Na(+)-K(+)-2Cl- transporter activity by protein kinase C (PKC) in a cell line derived from rabbit nonpigmented ciliary epithelium. Na(+)-K(+)-2Cl- cotransporter activity was measured as the rate of bumetanide-sensitive potassium (86Rb) transport. Phorbol 12,13-dibutyrate (PBDu) was used to activate PKC. PBDu inhibited bumetanide-sensitive potassium (86Rb) uptake, with a half-maximal inhibitory concentration of approximately 0.1 microM. The inhibitory effect of PBDu on potassium uptake by the N(+)-K(+)-2Cl- cotransporter was abolished by PCK downregulation and diminished by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a PKC inhibitor. PBDu inhibited Na(+)-K(+)-2Cl- cotransporter-mediated inward potassium (86Rb) transport by approximately 26% in control cells and by 40% in cells pretreated with Ouabain. PKC activation also reduced the rate of bumetanide-sensitive potassium (86Rb) efflux in Ouabain-treated cells but not in control (no oubain) cells. PBDu caused little change of intracellular sodium, potassium, or chloride, suggesting that an alteration of cytoplasmic ion composition is not responsible for the observed PBDu-induced changes in the rate of either inward or outward potassium movement mediated by the Na(+)-K(+)-2Cl- cotransporter.
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Protein kinase C inhibits Na(+)-K(+)-2Cl- cotransporter activity in cultured rabbit nonpigmented ciliary epithelium.
The American journal of physiology, 1994Co-Authors: J Dong, Nicholas A. DelamereAbstract:We examined the regulation of Na(+)-K(+)-2Cl- transporter activity by protein kinase C (PKC) in a cell line derived from rabbit nonpigmented ciliary epithelium. Na(+)-K(+)-2Cl- cotransporter activity was measured as the rate of bumetanide-sensitive potassium (86Rb) transport. Phorbol 12,13-dibutyrate (PBDu) was used to activate PKC. PBDu inhibited bumetanide-sensitive potassium (86Rb) uptake, with a half-maximal inhibitory concentration of approximately 0.1 microM. The inhibitory effect of PBDu on potassium uptake by the N(+)-K(+)-2Cl- cotransporter was abolished by PCK downregulation and diminished by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a PKC inhibitor. PBDu inhibited Na(+)-K(+)-2Cl- cotransporter-mediated inward potassium (86Rb) transport by approximately 26% in control cells and by 40% in cells pretreated with Ouabain. PKC activation also reduced the rate of bumetanide-sensitive potassium (86Rb) efflux in Ouabain-treated cells but not in control (no oubain) cells. PBDu caused little change of intracellular sodium, potassium, or chloride, suggesting that an alteration of cytoplasmic ion composition is not responsible for the observed PBDu-induced changes in the rate of either inward or outward potassium movement mediated by the Na(+)-K(+)-2Cl- cotransporter.
Esteban J. Morcillo - One of the best experts on this subject based on the ideXlab platform.
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Effects of Ouabain on human bronchial muscle in vitro.
Naunyn-Schmiedeberg's archives of pharmacology, 2003Co-Authors: Julio Cortijo, Benjamín Sarriá, Manuel Mata, Emmanuel Naline, Charles Advenier, Esteban J. MorcilloAbstract:The effects of Ouabain, an inhibitor of the plasmalemmal Na+/K+-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with −logEC50=7.16±0.11 and maximal effect of 67±4% of the response to acetylcholine (1 mM). Ouabain (10 μM)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H1-receptors and α-adrenoceptors, or neuronal Na+ channel blockade. The inhibition of Ouabain contraction in tissues bathed in K+-free medium, and the inhibition by Ouabain of the K+-induced relaxation confirm that the contractile action of Ouabain is mediated by inhibition of Na+/K+-ATPase. Furthermore, depolarization (16.4±0.9 mV) was observed in human isolated bronchus by intracellular microelectrode recording. Ouabain (10 μM)-induced contractions were abolished by a Ca2+-free solution but not by blockers of L-type Ca2+ channels. In human cultured bronchial smooth muscle cells, Ouabain (10 μM) produced a sustained increase in [Ca2+]i (116±26 nM) abolished in Ca2+-free medium. Incubation with a Na+-free medium or amiloride (0.1 mM) markedly inhibited the spasmogenic effect of Ouabain thus suggesting the role of Na+/Ca2+ exchange in Ouabain contraction while selective inhibitors of Na+/H+-antiport, Na+/K+/Cl−-antiport, or protein kinase C had no effect. Ouabain (10 μM) failed to increase inositol phosphate accumulation in human bronchus. Ouabain (10 μM) did not alter bronchial responsiveness to acetylcholine or histamine but inhibited the relaxant effects of isoprenaline, forskolin, levcromakalim, or sodium nitroprusside. These results indicate that Ouabain acts directly to produce contraction of human airway smooth muscle that depends on extracellular Ca2+ entry unrelated to L-type channels and involving the Na+/Ca2+-antiporter.
