INHIBITION OF INOSITOL 1,4,5-TRISPHOSPHATE-INDUCED CA2-DEPENDENT PROTEIN-KINASE IN A LIVING CELL( RELEASE BY CAMP)

Interaction of intracellular free calcium ([Ca2+](i)) and cAMP signali ng mechanisms was examined in intact single megakaryocytes by using a combination of single-cell fluorescence microscopy to measure [Ca2+](i ) and flash photolysis of caged Ca2+, inositol 1,4,5-trisphosphate (IP 3), or cAMP to elevate rapidly the concentration of these compounds in side the cell. Photolysis of caged IP3 stimulated Ca2+ release from an IP3-sensitive store. The cAMP-elevating agent carbacyclin inhibited t his IP3-induced rise in [Ca2+](i) but did not affect the rate of Ca2removal from the cytoplasm after photolysis of caged Ca2+. Photolysis of caged cAMP during ADP-induced [Ca2+](i) oscillations caused the [Ca 2+](i) oscillation to transiently cease without affecting the rate of Ca2+ uptake and/or extrusion. We conclude that the principal mechanism of cAMP-dependent inhibition of Ca2+ mobilization in megakaryocytes a ppears to be by inhibition of IP3-induced Ca2+ release and not by stim ulation of Ca2+ removal from the cytoplasm. Two inhibitors of cAMP-dep endent protein kinase, a specific peptide inhibitor of the catalytic s ubunit of cAMP protein kinase and KT5720, blocked the inhibitory effec t of carbacyclin, indicating that the inhibition of IP3-induced Ca2+-r elease by carbacyclin is mediated by cAMP-dependent protein kinase.