Cyclic AMP raises intracellular Ca(2+) in human megakaryocytes independent of protein kinase A.

The immature megakaryoblastic cell line MEG-01 responds to iloprost with an increase in cytosolic Ca(2+) and cAMP. The Ca(2+) response is almost absent in CHRF-288-11 cells, but cAMP formation is preserved in this more mature megakaryoblastic cell line. Also, in human hematopoietic stem cells, iloprost induces a Ca(2+) response and cAMP formation. The Ca(2+) response is downregulated during megakaryocytopoiesis, but cAMP formation remains unchanged. The Ca(2+) increase may be caused by cAMP-mediated inhibition of Ca(2+) sequestration, because it is (1) independent of Ca(2+) entry; (2) mimicked by forskolin, an activator of adenylyl cyclase, and isobutylmethylxanthine, an inhibitor of phosphodiesterases; and (3) preserved in the presence of inhibitors of protein kinase A and inositol-1,4,5-triphosphate receptors. The small GTPase Rap1 has been implicated in the control of Ca(2+) sequestration. Indeed, Rap1 activation parallels the iloprost- and forskolin-induced Ca(2+) increase and is unaffected by the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid-AM. These findings reveal a novel mechanism for elevating cytosolic Ca(2+) by cAMP, possibly via GTP-Rap1.