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 (PGI(2)) 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. PGI(2) 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 PGI(2) and NO alone or synergistically raised cAMP, only NO was able to increase intracellular cGMP levels. 3 Treatment of megakaryocytes with PGI(2) 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 PGI(2) 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, PGI(2) and NO regulate opposite megakaryocyte survival responses through a delicate balance between intracellular cyclic nucleotide levels and caspase-3 activity control.
Pozner
Roberto Gabriel
RG
Department of Thrombosis and Haemostasis, Hematological Research Institute, National Academy of Medicine, National Research Council (CONICET), Pacheco de Melo 3081 (1425), Buenos Aires, Argentina.
Negrotto
Soledad
S
D'Atri
Lina Paola
LP
Kotler
Mónica Lidia
ML
Lazzari
María Angela
MA
Gomez
Ricardo Martin
RM
Schattner
Mirta
M
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D011464:Epoprostenol / Q000494:pharmacology*; D006801:Humans; D008533:Megakaryocytes / Q000187:drug effects* / Q000378:metabolism; D009569:Nitric Oxide / Q000037:antagonists & inhibitors* / Q000494:pharmacology*
D009569:Nitric Oxide; D011464:Epoprostenol