Protein kinase C modulates calcium sensitivity of nitric oxide synthase in cerebellar slices

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated. Incubation of rat cerebellar slices with the specific metabotropic glutamate receptor agonist, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylate (trans-ACPD) increased cyclic GMP concentration two-fold. The increase was dose-dependently blocked by the protein kinase inhibitors staurosporine and calphostin C. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased cyclic GMP concentration without glutamate receptor activation. The cyclic GMP increases induced by PMA and trans-ACPD were independent of extracellular calcium blocked by N omega-nitro-L-arginine, a specific NOS inhibitor, and were not additive. Measurement of citrulline formation in cerebellar slices confirmed that NOS was activated by trans-ACPD and the activation was blocked by calphostin C. These results suggest that metabotropic glutamate receptor activates NOS through PKC. The calcium dependency of NOS activation was assessed in slices incubated with PMA and okadaic acid. NOS in both PMA-treated and untreated slices had similar activities at 100 nM free calcium, whereas at 25-70 nM free calcium, NOS in PMA-treated slices was more active than that in untreated slices. These results suggest that PKC regulates NO release in resting neurons by modulating the sensitivity of NOS at low calcium concentrations.