Differential effects of protein kinase C on neuronal nitric oxide synthase activity in rat cerebellar slices and in vitro

The effects of neuronal nitric oxide synthase (NOS) of protein kinase C (PKC) activation in rat cerebellar slices and of in vitro phosphorylation by PKC were compared. Incubation of slices with 1-aminocyclopentane-1,3-trans-dicarboxylic acid (trans-ACPD) or phorbol myristate acetate (PMA) in the presence of okadaic acid (OA) shifted the calcium sensitivity of neuronal NOS in the homogenate or in the cytosolic fraction. trans-ACPD promoted translocation of PKC activity to the particulate fraction in the slices. PMA in the presence of OA enhanced phosphorylation of GAP43 protein in the slices. These results ensured that both treatments activated PKC in the slice. However, when neuronal NOS in the slice treated with PMA and OA, in which GAP43 phosphorylation was detected, was immunoprecipitated by a specific antibody, no indication of neuronal NOS phosphorylation was obtained. Nevertheless, PKC phosphorylated partially purified neuronal NOS in vitro. Phosphorylated neuronal NOS showed greater activity than unphosphorylated NOS, but their calcium sensitivity was identical. These data indicated that neuronal NOS is not susceptible to PKC-dependent phosphorylation in cerebellar slices and that the calcium-sensitivity shift of neuronal NOS takes place without direct phosphorylation of neuronal NOS, suggesting the involvement of unknown proteins whose phosphorylation would regulate the calcium sensitivity of neuronal NOS in the cerebellum.