Pituitary adenylate cyclase-activating polypeptide and melatonin in the suprachiasmatic nucleus: effects on the calcium signal transduction cascade.

The suprachiasmatic nucleus (SCN) harbors an endogenous oscillator generating circadian rhythms that are synchronized to the external light/dark cycle by photic information transmitted via the retinohypothalamic tract (RHT). The RHT has recently been shown to contain pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitter/neuromodulator. PACAPergic effects on cAMP-mediated signaling events in the SCN are restricted to distinct time windows and sensitive to melatonin. In neurons isolated from the SCN of neonatal rats we investigated by means of the fura-2 technique whether PACAP and melatonin also influence the intracellular calcium concentration ([Ca2+]i). PACAP elicited increases of [Ca2+]i in 27% of the analyzed neurons, many of which were also responsive to the RHT neurotransmitters glutamate and/or substance P. PACAP-induced changes of [Ca2+]i were independent of cAMP, because they were not mimicked by forskolin or 8-bromo-cAMP. PACAP caused G-protein- and phospholipase C-mediated calcium release from inositol-trisphosphate-sensitive stores and subsequent protein kinase C-mediated calcium influx, demonstrated by treatment with GDP-beta-S, neomycin, U-73122, calcium-free saline, thapsigargin, bisindolylmaleimide, and chelerythrine. The calcium influx was insensitive to antagonists of voltage-gated calcium channels of the L-, N-, P-, Q- and T-type (diltiazem, nifedipine, verapamil, omega-conotoxin, omega-agatoxin, amiloride). Immunocytochemical characterization of the analyzed cells revealed that >50% of the PACAP-sensitive neurons were GABA-immunopositive. Our data demonstrate that in the SCN PACAP affects the [Ca2+]i, suggesting that different signaling pathways (calcium as well as cAMP) are involved in PACAPergic neurotransmission or neuromodulation. Melatonin did not interfere with calcium signaling, indicating that in SCN neurons the hormone primarily affects the cAMP signaling pathway.