MECHANISM OF CATECHOLAMINE SYNTHESIS INHIBITION BY NEUROPEPTIDE-Y - ROLE OF CA2-KINASES( CHANNELS AND PROTEIN)

We have previously demonstrated that neuropeptide Y (NPY) inhibits dep olarization-stimulated catecholamine synthesis in rat pheochromocytoma (PC12) cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF). The present study uses multiple selective C a2+ channel and protein kinase agonists and antagonists to elucidate t he mechanisms by which NPY modulates catecholamine synthesis as determ ined by in situ measurement of DOPA production in the presence of the decarboxylase inhibitor m-hydroxybenzylhydrazine (NSD-1015), The L-typ e Ca2+ channel blocker nifedipine inhibited the depolarization-induced stimulation of DOPA production by similar to 90% and attenuated the i nhibitory effect of NPY. In contrast, the N-type Ca2+ channel blocker omega-conotoxin GVIA inhibited neither the stimulation of DOPA product ion nor the effect of NPY. Antagonism of Ca2+/calmodulin-dependent pro tein kinase (CaM kinase) greatly inhibited the stimulation of DOPA pro duction by depolarization and prevented the inhibitory effect of NPY, whereas alterations in the cyclic AMP-dependent protein kinase pathway modulated DOPA production but did not prevent the effect of NPY. Stim ulation of Ca2+/phospholipid-dependent protein kinase (PKC) with phorb ol 12-myristate 13-acetate (PMA) did not affect the basal rate of DOPA production in NGF-differentiated PC12 cells but did produce a concent ration-dependent inhibition of depolarization-stimulated DOPA producti on. In addition, NPY did not produce further inhibition of DOPA produc tion in the presence of PMA, and the inhibition by both PMA and NPY wa s attenuated by the specific PKC inhibitor chelerythrine. These result s indicate that NPY inhibits Ca2+ influx through L-type voltage-gated Ca2+ channels, possibly through a PKC-mediated pathway, resulting in a ttenuation of the activation of CaM kinase and inhibition of depolariz ation-stimulated catecholamine synthesis.