Inhibition by extracellular cAMP of phorbol 12-myristate 13-acetate-induced prostaglandin H synthase-2 expression in human pulmonary microvascular endothelial cells - Involvement of an ecto-protein kinase A activity

Exposure of human pulmonary microvascular endothelial cells (HPMECs) to pho rbol 12-myristate 13-acetate (PMA) leads to the increase of prostaglandin H synthase (PGHS)-2 protein levels. Under same conditions and according to i ts constitutive nature, no significant variation of PGHS-1 protein was note d. The elevation of the intracellular cAMP rate is known to enhance PGHS-2 levels through a protein kinase A pathway in various cells. To determine wh ether the extracellular cAMP also regulates the inducible expression of PGH S, cultured HPMECs were exposed to cAMP alone or in combination with PMA. T he PMA-induced PGHS-2 protein was attenuated by the extracellular cAMP, In addition, PGHS-2 activity evaluated through 6-keto-PGF1 alpha generation, w hich was enhanced by PMA was inhibited by extracellular cAMP. Furthermore, in HPMEC medium, PMA-induced PGHS-2 expression was accompanied by the gener ation of a transferable activity (TA) able to abolish platelet aggregation. This resulting TA was dependent from PGHS-2 pathway, because NS-398, a sel ective inhibitor of PGHS-2, suppressed its production. The inhibitory TA re leased by treated HPMECs was also prevented by extracellular cAMP. The spec ific protein kinase A (PKA) inhibitor blocked the extracellular cAMP effect on both PMA-induced 6-keto-PGF1 alpha synthesis and inhibitory TA generati on, suggesting the involvement of PRA signaling at the outer surface of HPM ECs, Accordingly, we established, in phosphorylation experiments, the prese nce of an endothelial ectoprotein kinase activity, able to phosphorylate th e synthetic substrate kemptide in a cAMP-dependent mode. Reverse transcript ion-polymerase chain reaction analysis showed that PIMA-induced PGHS-2 mRNA was markedly reduced by extracellular cAMP. Together, these findings provi de the first experimental evidence that extracellular cAMP is able to reduc e HPMEC PGHS-2 expression in terms of mRNA, protein, and enzyme activity th rough an ecto-PKA pathway. In addition, they outline the potential role of endothelial PGHS-2 in the limitation of platelet activation during inflamma tory processes.