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, PKC 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 observation and by hemoglobin assay method in kinetic study. In long term observation (18 hours), activation of PKA by 8-Br-cAMP increased NO formation that was inhibited by N-(2-[p-bromocinnamylamino] ethyl)-5-isoquinolinesulfonamide (H89). Though activation of PKC by 12-O-tetradecanoyl phorbol-13-acetate (TPA) decreased NO formation, PKC inhibitor, chelerythrine, failed to inhibit the decrease. Activation of PKG with 8-Br-cGMP and inhibition with KT5823 resulted in no change in NO formation. Western blot analysis revealed that neither 8-Br-cAMP nor TPA affect iNOS expression. In kinetic study (short term perfusion study), no change in NO formation was observed by 8-Br-cAMP 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 modulate iNOS activity. However, PKA and PKC would possibly modify NOS activity indirectly via cofactors necessary for NO formation.