THE VOLUME-ACTIVATED CHLORIDE CURRENT IN ENDOTHELIAL-CELLS FROM BOVINE PULMONARY-ARTERY IS NOT MODULATED BY PHOSPHORYLATION

We employed the patch-clamp technique to investigate the effects of va rious phosphorylation pathways on activation and modulation of volume- activated Cl- currents (I-Cl,I-vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation of I- Cl,I-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. St imulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (-)-indolactam did not affect I-Cl,I-vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 mu mol/ l PMA, or its inhibition by loading the cells with the specific inhibi tory 19-31 pseudosubstrate peptide, did not influence I-Cl,I-vol. Trif luoperazine and tamoxifen fully blocked I-Cl,I-vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 mu mol/l respecti vely. This inhibitory effect is probably not mediated by the calmoduli n-antagonistic action of these compounds, because it occurs at free in tracellular [Ca2+] of 50 nmol/l, which are below the threshold for cal modulin activation. The tyrosine kinase inhibitor herbimycin A (1 mu m ol/l) and genistein (100 mu mol/l) did not affect I-Cl,I-vol. Exposing CPAE cells to lysophosphatidic acid (1 mu mol/l), an activator of p42 MAPkinase and the focal adhesion kinase p125(FAK) in endothelial cell s, neither evoked a Cl- current nor affected I-Cl,I-vol. Neither wortm annin (10 mu mol/l), an inhibitor of MAP kinases and of PI-3 kinase, n or rapamycin (0.1 mmol/l), which interferes with the p70S6 kinase path way, affected I-Cl,I-vol. Exposure of CPAE cells to heat or Na-arsenit e, 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 s tudied phosphorylation pathways is essential for the activation of the Cl- current induced by hypotonicity.