The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation

Géza Szücs, Stephan Heinke, Christine De Greef, Luc Raeymaekers, Jan Eggermont, Guy Droogmans, Bernd Nilius

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We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (I(Cl,vol)) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation of I(Cl,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 I(Cl,vol). Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 μmol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19-31 pseudosubstrate peptide, did not influence I(Cl,vol). Trifluoperazine and tamoxifen fully blocked I(Cl,vol) with concentrations required for half-maximal inhibition of 3.0 and 2.4 μmol/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 μmol/l) and genistein (100 μmol/l) did not affect I(Cl,vol). Exposing CPAE cells to lysophosphatidic acid (1 μmol/l), an activator of p42 MAPkinase and the focal adhesion kinase p125(FAK) in endothelial cells, neither evoked a Cl- current nor affected I(Cl,vol). Neither wortmannin (10 μmol/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 I(Cl,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.

Original languageEnglish
Pages (from-to)540-548
Number of pages9
JournalPflugers Archiv European Journal of Physiology
Issue number4
Publication statusPublished - Jan 1 1996


  • Endothelium
  • Patch clamp
  • Phosphorylation
  • Volume-activated Cl currents

ASJC Scopus subject areas

  • Physiology
  • Clinical Biochemistry
  • Physiology (medical)

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