Inhibition of inositol 1,4,5-trisphosphate-induced Ca(2+) release by cAMP-dependent protein kinase in a living cell

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