Plasma stimulation of prostacyclin production by rat smooth muscle cells requires previous induction of phospholipase activity

Human normal platelet poor plasma (PPPn) stimulates prostacyclin (PGI(2)) production in a dose-dependent manner and after 15 to 60 min of incubation time when confluent rat smooth muscle cells (RSMC) were preincubated for 24 hours with fresh culture medium. This PGI(2) production was independent of new protein synthesis, and was not observed in the cells maintained only in exhausted medium. The serum of fresh culture medium also induced a significant and transient increase of prostaglandin endoperoxide synthase (PGHS) activity as a function of preincubation time, which was dependent of protein synthesis. However, neither PGHS activity nor arachidonic acid availability limited the PPPn induced PGI(2), synthesis in RSMC. Moreover, the previous addition of phorbol 13-myristate acetate also allowed the PPPn to induce PGI(2) synthesis, that was significantly inhibited by a specific phospholipase A(2) inhibitor. Furthermore, we found that PPPn induced a significant increase of intracellular calcium, and also stimulated PGI(2) production at short incubation times due to its effect on phospholipases, and not by a direct supply of substrate. We conclude that a previous activation of phospholipase A(2) was necessary to observe a significant and sustained PGI(2) synthesis induced by PPPn in RSMC, and that the increase of intracellular calcium observed with PPPn might stimulate these previously activated phospholipases.