Neuropeptide Y receptors involved in calcium channel regulation in PC12 cells.

Our laboratory has previously used NGF-differentiated PC12 cells as a sympathetic neuronal model to investigate the effects of NPY on catecholamine synthesis and release. We have additionally used these cells to demonstrate the NPY-induced inhibition of Ca2+ channels which was suggested by those studies. In the present work, multiple NPY, PYY, and PP analogs are utilized to further define the receptor subtypes involved in this Ca2+ channel modulation. We find that in PC12 cells NPY and PP modulate Ca2+ channels through Y1, Y2, Y3, and Y4 receptors. In addition, we show that these receptors are differentially coupled to N, L, and non-N, non-L Ca2+ channel subtypes. The results of the present study in combination with our previous investigations demonstrate an intriguing and complex role for NPY and PP in the modulation of sympathetic neurotransmission.

Neuropeptide Y inhibition of calcium channels in PC-12 pheochromocytoma cells.

We previously demonstrated, using rat PC-12 pheochromocytoma cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF), that neuropeptide Y (NPY) inhibits catecholamine synthesis as well as release. Inquiry into the mechanisms of these inhibitions implicated distinct pathways involving reduction of Ca2+ influx through voltage-activated Ca2+ channels. In the present investigation the effects of NPY on whole cell Ba2+ currents were examined to obtain direct evidence supporting the mechanisms suggested by those studies. NPY was found to inhibit the voltage-activated Ba2+ current in NGF-differentiated PC-12 cells in a reversible fashion with an EC50 of 13 nM. This inhibition was pertussis toxin sensitive and resulted from NPY modulation of L- and N-type Ca2+ channels. The inhibition of L-type channels was not seen with < 1 nM free intracellular Ca2+ or when protein kinase C (PKC) was inhibited by chelerythrine or PKC-(19-31). Furthermore, the effect of NPY on L-type channels was mimicked by the PKC activator phorbol 12-myristate 13-acetate. These studies demonstrate that, in addition to inhibition of N-type Ca2+ channels, in NGF-differentiated PC-12 cells NPY inhibits L-type Ca2+ channels via an intracellular Ca(2+)- and PKC-dependent pathway.

Mechanism of catecholamine synthesis inhibition by neuropeptide Y: role of Ca2+ channels and protein kinases.

We have previously demonstrated that neuropeptide Y (NPY) inhibits depolarization-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 Ca2+ channel and protein kinase agonists and antagonists to elucidate the mechanisms by which NPY modulates catecholamine synthesis as determined by in situ measurement of DOPA production in the presence of the decarboxylase inhibitor m-hydroxybenzylhydrazine (NSD-1015). The L-type Ca2+ channel blocker nifedipine inhibited the depolarization-induced stimulation of DOPA production by approximately 90% and attenuated the inhibitory effect of NPY. In contrast, the N-type Ca2+ channel blocker omega-conotoxin GVIA inhibited neither the stimulation of DOPA production nor the effect of NPY. Antagonism of Ca2+/calmodulin-dependent protein kinase (CaM kinase) greatly inhibited the stimulation of DOPA production 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. Stimulation of Ca2+/phospholipid-dependent protein kinase (PKC) with phorbol 12-myristate 13-acetate (PMA) did not affect the basal rate of DOPA production in NGF-differentiated PC12 cells but did produce a concentration-dependent inhibition of depolarization-stimulated DOPA production. In addition, NPY did not produce further inhibition of DOPA production in the presence of PMA, and the inhibition by both PMA and NPY was attenuated by the specific PKC inhibitor chelerythrine. These results indicate that NPY inhibits Ca2+ influx through L-type voltage-gated Ca2+ channels, possibly through a PKC-mediated pathway, resulting in attenuation of the activation of CaM kinase and inhibition of depolarization-stimulated catecholamine synthesis.

Neuropeptide Y inhibits depolarization-stimulated catecholamine synthesis in rat pheochromocytoma cells.

In PC12 rat pheochromocytoma cells differentiated with nerve growth factor (NGF), neuropeptide Y inhibited depolarization-stimulated catecholamine synthesis as determined by in situ measurement of 3,4-dihydroxyphenylalanine (DOPA) production in the presence of the decarboxylase inhibitor m-hydroxybenzylhydrazine (NSD-1015). The inhibition by neuropeptide Y was concentration-dependent and was prevented by pretreatment with pertussis toxin, suggesting the involvement of a GTP-binding protein of the Gi or Go subtype. The neuropeptide Y analog [Leu31,Pro34]neuropeptide Y also caused inhibition of DOPA production, but was less potent than neuropeptide Y itself, while peptide YY and neuropeptide Y-(13-36) had no significant effect. This pattern is most consistent with the involvement of the neuropeptide Y Y3 receptor subtype. In PC12 cells differentiated with dexamethasone, neuropeptide Y also caused a concentration-dependent inhibition of DOPA production, while peptide YY was again without effect. Neuropeptide Y had no effect on DOPA production in undifferentiated PC12 cells. These results indicate that neuropeptide Y can modulate catecholamine synthesis in addition to its modulatory effects on catecholamine release.