Opposing effects of protein kinase A and C on capacitative calcium entry into HL-60 promyelocytes

Abstract

Treatment of HL-60 cells with thapsigargin, a microsomal Ca2+/ATPase inhibitor, led to depletion of intracellular calcium stores followed by capacitative calcium entry. Stimulation of adenylyl cyclase with forskolin enhanced thapsigargin-induced Ca2+ influx. The forskolin effect was confirmed by enhanced fluorescence quenching induced by Mn2+ entry into fura-2 loaded cells. 1,9-Dideoxy-forskolin, an inactive analog of forskolin, did not affect capacitative calcium entry. On the other hand, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, inhibited thapsigargin-induced Ca2+ entry. Histamine and prostaglandin E-2 (PGE(2)) elevated intracellular adenosine 3':5'-cyclic monophosphate (cAMP) levels and enhanced the thapsigargin-induced capacitative calcium entry. Incubation with N-[2-(p-bromocynnamyl-amino)ethyl]-5-isoquinolinesulfonamide (H89), an inhibitor of protein kinase A (PKA), blocked the forskolin effect, and GF109203X, an inhibitor of protein kinase C (PKC), blocked the phorbol 12-myristate 13-acetate effect. The results suggest that protein kinase A regulates capacitative calcium entry positively, but that protein kinase C regulates Ca2+ influx negatively. Furthermore, after differentiation of HL-60 promyelocytes with dimethylsulfoxide to granulocytes, the inhibitory effect of phorbol 12-myristate 13-acetate became more pronounced, whereas the stimulatory effect of prostaglandin E-2 did not change. This result suggests that the regulation of capacitative calcium entry by protein kinase C and protein kinase A develops differently during differentiation. (C) 1998 Elsevier Science Inc.