Cyclic AMP Raises Intracellular Ca 2+ in Human Megakaryocytes Independent of Protein Kinase A

Abstract

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.