Calcium channel blockade inhibits platelet activating factor production by human umbilical vein endothelial cells.
An increase in intracellular calcium level is an important signal in the regulation of cellular responses under normal and pathological conditions. Because two key enzymes in the synthetic pathway of platelet activating factor (PAF), phospholipase A2 and acetyltransferase, are calcium dependent, we hypothesized that calcium channel blockade may inhibit agonist-induced PAF synthesis. Primary cultures of human umbilical vein endothelial cells (EC), pre-incubated with [3H]acetate, were exposed to thrombin (5 U/mL) and PAF production was quantitated by incorporation of radiolabel into the EC lipid fraction co-migrating with exogenous PAF in thin-layer chromatography. The effect of pre-incubation with calcium channel blockers (verapamil, diltiazem, 10(-4) M) or buffer was determined. Results (triplicate experiments, * P less than 0.05 vs buffer, P less than 0.05 vs thrombin) demonstrate that pre-incubation with calcium channel blocker markedly inhibits thrombin-induced PAF production (verapamil:buffer 273 +/- 122, thrombin 10,735 +/- 1524*, thrombin + verapamil 178 +/- 91 cpm/plate; diltiazem:buffer 1097 +/- 581, thrombin 15,283 +/- 2661*, thrombin + diltiazem 280 +/- 56 cpm/plate). The effect of diltiazem was dose-dependent (% inhibition: 10(-7) M, 46%; 10(-5) M, 60%; 10(-4) M, 98%). Diltiazem also inhibited bradykinin (10(-8) M) induced PAF synthesis. In calcium-free medium or in the presence of LaCl3 (10(-3) M), the PAF response of EC to thrombin was blunted (buffer 582 +/- 360, thrombin 5394 +/- 1069, thrombin + calcium free medium 1055 +/- 571, thrombin + LaCl3 1271 +/- 58 cpm/plate). We conclude that calcium channel blockers prevent agonist-induced PAF synthesis, possibly by preventing cellular calcium influx and activation of PAF synthetic enzymes. We speculate that this mechanism may underlie, at least in part, the beneficial effect of calcium channel blockade under various pathological conditions.
Tolins
J P
JP
University of Minnesota School of Medicine, Minneapolis.
Melemed
A
A
Sulciner
D
D
Gustafson
K S
KS
Vercellotti
G M
GM
D001920:Bradykinin / Q000494:pharmacology; D002478:Cells, Cultured; D004110:Diltiazem / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D013917:Thrombin / Q000494:pharmacology; D014471:Umbilical Veins; D014700:Verapamil / Q000494:pharmacology*
D010972:Platelet Activating Factor; C028521:lanthanum chloride; D007811:Lanthanum; D014700:Verapamil; D013917:Thrombin; D004110:Diltiazem; D001920:Bradykinin