Calcium ionophore (A23187)- and arachidonic acid-stimulated prostaglandin release from microvascular endothelial cells: effects of calcium antagonists and calmodulin inhibitors.
Calcium ionophore (A23187)-stimulated prostaglandin (PG) E2 and I2 (measured as 6-keto PGF1 alpha) release from cultured rabbit coronary microvessel endothelial (RCME) cells in a time- and dose-dependent manner. A23187-stimulated PG release was reduced by the calcium channel blockers nifedipine, verapamil and diltiazem and by the intracellular calcium blocker, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate. A23187-stimulated PG release was significantly reduced by lowering the calcium concentration in the buffer to concentrations of 0.2 mM or less. A23187-stimulated 45Ca uptake was not inhibited by nifedipine (0.5 microM), diltiazem (10 micron) or verapamil (50 microM) although these same concentrations of calcium channel blockers significantly inhibited A23187-stimulated PG release. However, these concentrations of calcium blockers did inhibit K+ (10 mM)-valinomycin (5 microM)-stimulated 45Ca uptake, indicating that, although RCME cells probably have voltage-dependent calcium channels and although calcium influx via these channels is blocked by the calcium channel blockers, voltage-dependent calcium influx plays little or no role in A23187-stimulated 45Ca influx and PG release. KCl-valinomycin significantly stimulated PG release, but this increase was not significantly affected by either nifedipine (0.5 microM) or diltiazem (10 microM) despite complete inhibition of KCl-valinomycin-stimulated 45Ca influx. Verapamil (50 microM) exhibited a small but significant suppression of KCl-valinomycin-stimulated PG release. These observations most likely indicate that calcium influx by voltage-dependent calcium channels plays little or no role in the events leading to either A23187- or KCl-valinomycin-stimulated PG release. The calmodulin antagonists, trifluoperazine and calmidazolium, also reduced A23187-stimulated PG release. In vitro studies of porcine pancreatic phospholipase (PL) A2 activity suggested that the inhibitory actions of trifluoperazine, but not the calcium antagonists, may be mediated by direct inhibitory actions on PLA2. Studies with [3H]arachidonic acid (AA)- and [14C]stearic acid-prelabeled RCME cells suggested that A23187 stimulated both PLA2 and PLC activity, leading to the release of AA. Studies with exogenous AA indicated that reducing calcium availability by reducing buffer calcium concentrations resulted in an enhanced conversion of exogenous AA to PGs. RCME cells incubated in nominally calcium-free buffer exhibited a decreased rate of AA incorporation. The observed increase in AA conversion to PGs in low calcium buffer suggests that calcium may stimulate AA uptake and acylation as well as AA release.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000378:metabolism; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D008833:Microcirculation; D011453:Prostaglandins / Q000378:metabolism*; D011817:Rabbits; D014268:Trifluoperazine / Q000494:pharmacology*; D014634:Valinomycin / Q000494:pharmacology
D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D002147:Calmodulin; D007093:Imidazoles; D011453:Prostaglandins; D014634:Valinomycin; D014268:Trifluoperazine; D016718:Arachidonic Acid; D000001:Calcimycin; C031938:calmidazolium; D002118:Calcium