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 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.
Szücs
G
G
KU Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Leuven, Belgium.
Heinke
S
S
De Greef
C
C
Raeymaekers
L
L
Eggermont
J
J
Droogmans
G
G
Nilius
B
B
D000818:Animals; D016227:Benzoquinones; D002417:Cattle; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D018118:Chloride Channels / Q000378:metabolism / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011493:Protein Kinase C / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives
D016227:Benzoquinones; D018118:Chloride Channels; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D011809:Quinones; D017828:Rifabutin; C020754:herbimycin; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C