Prostacyclin prevents nitric oxide‐induced megakaryocyte apoptosis

1 We have previously demonstrated that nitric oxide (NO) triggers CD34+‐derived megakaryocyte apoptosis. We here show that prostacyclin (PGI2) inhibits PAPA/NO‐induced megakaryocyte death detected by fluorescent microscopy and flow cytometry. 2 The cAMP‐specific phosphodiesterase inhibitor, Ro 20‐1724, and the permeable analog dibutyryl‐cAMP also delayed apoptosis. PGI2 effect was fully prevented when adenylyl cyclase activity was suppressed by SQ 22536, and partially reversed by the permeable protein kinase A inhibitor PKI 14‐22 amide. ELISA showed that while both PGI2 and NO alone or synergistically raised cAMP, only NO was able to increase intracellular cGMP levels. 3 Treatment of megakaryocytes with PGI2 abolished both basal and NO‐raised cGMP levels. Addition of 8‐pCPT‐cGMP or activation of soluble guanylyl cyclase by BAY 41‐2272 induced cell death in a concentration‐dependent manner, and ODQ, an inhibitor of guanylyl cyclase, prevented both PAPA/NO‐ or BAY 41‐2272‐induced apoptosis. Specific cGMP phosphodiesterase inhibition by Zaprinast or suppression of adenylyl cyclase by SQ 22536 enhanced the PAPA/NO proapoptotic effect. 4 PGI2 completely inhibited NO‐mediated generation and the increased activity of the cleaved form of caspase‐3. 5 In conclusion, our results demonstrate that contrary to their well‐known direct and synergistic inhibitory effects on platelets, PGI2 and NO regulate opposite megakaryocyte survival responses through a delicate balance between intracellular cyclic nucleotide levels and caspase‐3 activity control. British Journal of Pharmacology (2005) 145, 283–292. doi:10.1038/sj.bjp.0706200