COX-2 and cytosolic PLA2 mediate IL-1β-induced cAMP production in human vascular smooth muscle cells

Interleukin (IL)-1 is a potent vasodilator that causes prolonged induction of prostacyclin (PGI(2)) and cAMP synthesis in human vascular smooth muscle cells (HVSMC). The present study investigated IL-1 induction of PG synthetic enzymes in HVSMC and tested their respective roles in PGI(2) and cAMP production. Cyclooxygenase (COX)-1 mRNA was not detectable in either control or IL-l-treated HVSMC, as assessed by RT-PCR. In contrast, COX-2 mRNA was detectable in control HVSMC, increased markedly (16-fold) after 1 h of IL-1 exposure, and increased further (52-fold) after 24 h. COX-2 protein levels, assessed by Western analysis, were increased concomitantly. HVSMC contained mRNA encoding both the secreted and cytosolic forms of phospholipase Az (sPLA(2) and cPLA(2), respectively). IL-I stimulation did not affect sPLA2 mRNA levels, but cPLA(2) mRNA levels increased at 8 h, after the initial induction of PG synthesis. HVSMC constitutively expressed PGI(2) synthase mRNA, and its levels were not affected by IL-1. A selective COX-2 inhibitor, NS-398, reversed IL-l-induced PGI(2) and cAMP production, supporting a role of COX-2 in mediating increased PG synthesis. IL-l-induced cAMP was also reversed by a selective cPLA(2) inhibitor, AACOCF(3), but not by thioetheramide phosphorylcholine, which inhibits sPLA(2) preferentially over cPLA(2), supporting a requirement for cPLA(2)-derived arachidonic acid in IL-1-induced PG synthesis. The delayed induction of cPLA(2) mRNA was also attenuated by NS-398, suggesting that it was secondary to the initial COX-2-induced PG synthesis. Together, the results support the hypothesis that IL-1 induces intracellular PG synthesis in HVSMC via rapid upregulation of COX-2, which utilizes cPLA(2)-derived arachidonic acid to generate PG metabolites that regulate adenylate cyclase.