Mechanism of phospholipase C-induced release of EDRF from pulmonary artery endothelium.
The mechanism of phospholipase (PL) C-induced release of endothelium-derived relaxing factor (EDRF) was investigated. Bovine pulmonary artery endothelial cells (BPAEC) were treated with phosphatidylinositol (PI)-selective PLCs, nonselective PLCs, or a nonselective PLD. PI-PLCs elicited PI-glycan anchor hydrolysis but did not alter either intracellular Ca2+ ([Ca2+]i) in fura-2-loaded BPAEC or EDRF production in BPAEC-vascular smooth muscle cocultures. In contrast, non-selective PLCs increased [Ca2+]i, an effect prevented by prior exposure to the PLCs, and EDRF production in a time- and concentration-dependent manner. Antibodies raised against PI-glycan anchors did not alter, while heat denaturation abolished, the PLC-dependent effects. Removal of extracellular Ca2+ with [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid both prevented and reversed PLC-stimulated increases in [Ca2+]i and inhibited EDRF production. Although Mn2+ quenched PLC-induced increases in fura-2 fluorescence, high PLC concentrations elicited significant dye loss from fura-2-loaded BPAEC. We conclude that the effects of exogenous PLC on EDRF production are not dependent on release of a membrane PI-glycan-linked moiety. Rather, the PLC actions are mediated by a graded increase in cell membrane permeability, probably related to pore formation by the hemolytic activity of the enzyme, followed by an influx of extracellular Ca2+.
Tracey
W R
WR
Department of Pharmacology, University of Virginia, Health Sciences Center, Charlottesville 22908.
Johns
R A
RA
Romero
G
G
Peach
M J
MJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D016257:Fura-2; D006017:Glycolipids / Q000378:metabolism; D017261:Glycosylphosphatidylinositols; D006868:Hydrolysis; D008345:Manganese / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009994:Osmolar Concentration; D010716:Phosphatidylinositols / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D010738:Type C Phospholipases / Q000494:pharmacology*
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D009569:Nitric Oxide; D008345:Manganese; D010738:Type C Phospholipases; D006152:Cyclic GMP; D002118:Calcium; D016257:Fura-2