Role of Ca2+ and cyclic AMP in the regulation of the production of prostacyclin by the vascular endothelium.

Abstract

Incubation of primary monolayer cultures of human umbilical vein endothelial cells with buffer, thrombin (0.5 U/ml), ionophore A23187, calcimycin (calcimycin, 10 .mu.M), arachidonic acid (20 .mu.M), or prostaglandin [PG] H2 (4 .mu.M) resulted in prostacyclin (PGI2) production in nanomolar quantities to the extent of 36 .+-. 2, 276 .+-. 13, 485 .+-. 32, 533 .+-. 22 and 532 .+-. 22, respectively, as measured by radioimmunoassay of 6-keto-PGF1.alpha.. Preincubation of the endothelium with 1 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, an antagonist of cytoplasmic Ca2+, or with 4 mM 1-methyl-3-isobutylxanthine (MIX), an inhibitor of cyclic nucleotide phosphodiesterase activity, blocked PGI2 release induced by thrombin or A23187, decreased arachidonic acid-induced release by .apprx. 50%, but had no effect on PGH2-induced release. Radioimmunoassay of cAMP in the endothelium showed that the basal level (1.85 .+-. 0.14 pmol of cAMP per 4.5 .times. 105 cells) was increased by an average of 3.9-fold with 4 mM MIX. PGI2 (0.4 .mu.M) had no significant effect on cAMP levels in the absence of MIX, but caused a 2-fold increase with 4 mM MIX. The stimulation of PGI2 biosynthesis apparently is mediated by Ca2+, increased cAMP inhibits PGI2 production and cAMP phosphodiesterase activity modulates PGI2-induced increases in the intracellular concentration of cAMP.