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

Abstract

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.

Annexin II Modulates Volume-activated Chloride Currents in Vascular Endothelial Cells

“…the plasma membrane (trace a), the whole cell current shows a prominent inwardly rectifying K ϩ current at negative potentials, which was blocked by substituting external KCl by CsCl (trace b). The residual current in these cells is mainly a Cl Ϫ current and has been extensively described elsewhere (1,3,23,24). This limited run down of I Cl, vol is consistent with our observations in a previous report where we showed that the volume-activated Cl Ϫ current runs down very slowly by about 15% after 60 min (24).…”

“…The residual current in these cells is mainly a Cl Ϫ current and has been extensively described elsewhere (1,3,23,24). This limited run down of I Cl, vol is consistent with our observations in a previous report where we showed that the volume-activated Cl Ϫ current runs down very slowly by about 15% after 60 min (24). The currents activated by a hypotonic challenge (HTS) are slightly outwardly rectifying and reverse around Ϫ10 mV.…”

Hypotonicity activates transcription through ERK-dependent and -independent pathways in renal cells

“…In skate and clam erythrocytes, swelling increased phosphorylation of the anion exchanger band 3 (37) as well as other proteins (41). In endothelial cells, however, inhibitor studies failed to show a relationship between de novo phosphorylation and Cl Ϫ conductance in response to cell swelling (53).…”

Signalling Mechanisms Involved in Volume Regulation of Intestinal Epithelial Cells

“…For Jurkat T lymphocytes however, strong evidence exists that the Srclike p56 lck tyrosine kinase isboth essential and sufficient for the activation of volume-sensitive anion channels [15][16][17]. Activation of VRACs through a mechanism involving protein tyrosine phosphorylation is not universally observed in all cell models studied: both in ROS 17/2.8 osteoblasts and in CPAE cells tyrosine kinase inhibitors were found ineffective [18,19].…”