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The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation.
Szücs, G; Heinke, S; De Greef, C; Raeymaekers, L; Eggermont, J; Droogmans, G; Nilius, B.
Afiliação
  • Szücs G; KU Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Leuven, Belgium.
Pflugers Arch ; 431(4): 540-8, 1996 Feb.
Article em En | MEDLINE | ID: mdl-8596697
We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (ICl,vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation of ICl,vol occurred at a hypotonicity of 27.5+/-1.2%. Run-down of the current upon repetitive activation was less than 15% within 60 min. Stimulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (-)-indolactam did not affect ICl,vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 micromol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19-31 pseudosubstrate peptide, did not influence ICl,vol. Trifluoperazine and tamoxifen fully blocked ICl,vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 micromol/l respectively. This inhibitory effect is probably not mediated by the calmodulin-antagonistic action of these compounds, because it occurs at free intracellular [Ca2+] of 50 nmol/l, which are below the threshold for calmodulin activation. The tyrosine kinase inhibitor herbimycin A (1 micromol/l) and genistein (100 micromol/l) did not affect ICl,vol. Exposing CPAE cells to lysophosphatidic acid (1 micromol/l), an activator of p42 MAPkinase and the focal adhesion kinase p125(FAK) in endothelial cells, neither evoked a Cl- current nor affected ICl,vol. Neither wortmannin (10 micromol/l), an inhibitor of MAP kinases and of PI-3 kinase, nor rapamycin (0.1 mmol/l), which interferes with the p70S6 kinase pathway, affected ICl,vol. Exposure of CPAE cells to heat or Na-arsenite, both activators of a recently discovered stress-activated tyrosine phosphorylation pathway, neither activated a current nor affected the hypotonic solution-induced Cl- current. We conclude that none of the studied phosphorylation pathways is essential for the activation of the Cl- current induced by hypotonicity.
Assuntos
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Endotélio Vascular / Canais de Cloreto Limite: Animals Idioma: En Ano de publicação: 1996 Tipo de documento: Article
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Endotélio Vascular / Canais de Cloreto Limite: Animals Idioma: En Ano de publicação: 1996 Tipo de documento: Article