neuropeptide-y and chelerythrine

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.

Topics: Alkaloids; Animals; Barium; Benzophenanthridines; Calcium Channel Blockers; Calcium Channels; Cell Differentiation; Electric Conductivity; Enzyme Activation; Enzyme Inhibitors; Nerve Growth Factors; Neuropeptide Y; PC12 Cells; Pertussis Toxin; Phenanthridines; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella

Mammalian circadian rhythms originate in the hypothalamic suprachiasmatic nuclei (SCN), from which rhythmic neural activity can be recorded in vitro. Application of neurochemicals can reset this rhythm. Here we determine cellular correlates of the phase-shifting properties of neuropeptide Y (NPY) on the hamster circadian clock in vitro. Drug or control treatments were applied to hypothalamic slices containing the SCN on the first day in vitro. The firing rates of individual cells were sampled on the second day in vitro. Control slices exhibited a peak in firing rate in the middle of the day. Microdrop application of NPY to the SCN phase advanced the time of peak firing rate. This phase-shifting effect of NPY was not altered by block of sodium channels with tetrodotoxin or block of calcium channels with cadmium and nickel, consistent with a direct postsynaptic site of action. Pretreatment with the glutamate receptor antagonists (DL-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione disodium) also did not alter phase shifts to NPY. Blocking GABAA receptors with bicuculline (Bic) had effects only at very high (millimolar) doses of Bic, whereas blocking GABAB receptors did not alter effects of NPY. Phase shifts to NPY were blocked by pretreatment with inhibitors of protein kinase C (PKC), suggesting that PKC activation may be necessary for these effects. Bathing the slice in low Ca2+/high Mg2+ can block phase shifts to NPY, possibly via a depolarizing action. A depolarizing high K+ bath can also block NPY phase shifts. The results are consistent with direct action of NPY on pacemaker neurons, mediated through a signal transduction pathway that depends on activation of PKC.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Alkaloids; Animals; Benzophenanthridines; Bicuculline; Calcium Channel Blockers; Cations, Divalent; Cell Communication; Circadian Rhythm; Cricetinae; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; GABA Antagonists; Indoles; Ion Channels; Isoquinolines; Male; Maleimides; Mesocricetus; Naphthalenes; Nerve Tissue Proteins; Neuropeptide Y; Phenanthridines; Phorbol Esters; Protein Kinase C; Receptors, GABA-A; Receptors, GABA-B; Receptors, Glutamate; Signal Transduction; Sulfonamides; Suprachiasmatic Nucleus; Tetrodotoxin