CGMP INHIBITS IP3-INDUCED CA2-DEPENDENT AND CAMP-DEPENDENT PROTEIN-KINASES( RELEASE IN INTACT RAT MEGAKARYOCYTES VIA CGMP)

1. Inhibition of inositol 1,4,5-trisphosphate (IP3) receptor-mediated Ca2+ release by cGMP was examined in intact rat megakaryocytes, by usi ng a combination of single cell fluorescence microscopy to monitor int racellular free calcium ([Ca2+](i)) and flash photolysis of caged seco nd messengers. 2. Sodium nitroprusside (SNP), a nitric oxide (NO) dono r, and the hydrolysis-resistant cGMP analogue 8-(4-chlorophenylthio)gu anosine 3',5'-cyclic monophosphate (pCPT-cGMP) inhibited Ca2+ release induced by photolysis of caged IP3. Neither of them affected the rate of Ca2+ removal from the cytoplasm following photolysis of caged Ca2+. 3. Photolysis of the caged NO donor 3-morpholinosydnonimine (SIN-1) d uring agonist-induced [Ca2+](i) oscillations inhibited Ca2+ release wi thout affecting the rate of Ca2+ uptake and/or extrusion. 4. We conclu de that the inhibition of IP3-induced Ca2+ release is the principal me chanism of NO-caMP-dependent inhibition of [Ca2+](i) mobilization. 5. IPG, a specific peptide inhibitor of cGMP-dependent protein kinase (cG MP-PK), blocked the inhibitory effect of pCPT-cGMP, indicating that th e inhibition of IP3,-induced Ca2+ release by pCPT-cGMP is mediated by cGMP-PK. However, the simultaneous application of both IPG and IP20, a specific peptide inhibitor of cAMP-dependent protein kinase (cAMP-PK) , was required to block the inhibitory effect of SNP. These data stron gly suggest that NO-cGMP-dependent inhibition of [Ca2+](i) mobilizatio n is mediated via the activation of both cQMP-PK and cAMP-PK.