Protein kinase A- and C-dependent modulation of murine inducible nitric oxide synthase

Effects of pharmacological modulation of protein kinase A, C and G (PKA, PK C and PKG) were examined on inducible form of nitric oxide synthase (iNOS) expressed in COS cells to elucidate regulatory mechanism of iNOS by protein kinases. Formation of nitric oxide (NO), as an index of NOS activity, was assessed by measurement of nitrite in incubation medium in long term observ ation and by hemoglobin assay method in kinetic study. In long term observa tion (18 hours), activation of PKA by 8-Br-cAMP increased NO formation that was inhibited by N-(2-[p-bromocinnamylamino] ethyl)-5-isoquinolinesulfonam ide (H89). Though activation of PKC by 12-O-tetradecanoyl phorbol-13-acetat e (TPA) decreased NO formation, PKC inhibitor, chelerythrine, failed to inh ibit the decrease. Activation of PKG with 8-Br-cGMP and inhibition with KT5 823 resulted in no change in NO formation. Western blot analysis revealed t hat neither 8-Br-cAMP nor TPA affect iNOS expression. In kinetic study (sho rt term perfusion study), no change in NO formation was observed by 8-Br-cA MP and TPA. These results indicate that,in living cells, PKG does not play a regulatory role in iNOS activity and that PKA and PKC do not directly mod ulate iNOS activity. However, PKA and PKC would possibly modify NOS activit y indirectly via cofactors necessary for NO formation.