PACAP-stimulated Catecholamine Synthesis and Secretion in NS-1 Pheochromocytoma Cells: A Model for PACAP Action at the Splanchnicoadrenal Synapse

Abstract ID 129492 Poster Board 166 Historically, acetylcholine (ACh) has been considered as the major, if not the sole, secretagogue released by the splanchnic nerve, controlling catecholamine secretion from chromaffin cells of the adrenal medulla both basally and under stress (flight-or-flight) response. However, pituitary adenylate cyclase-activating peptide (PACAP) is found along with ACh at splanchnic nerve terminals and is required for stress-induced catecholamine secretion in vivo (Hamelink et al., Proc Natl Acad Sci USA. 2002;99(1):461–6) and for high-frequency/stress levels of splanchnic nerve firing from the adrenal medulla ex vivo (Smith and Eiden, J Mol Neurosci. 2012;48(2):403-12) (Stroth et al., Endocrinology. 2013;154(1):330–9). The realization that PACAP is the major secretagogue for stress-induced catecholamine secretion from chromaffin cells initiated intense investigation of the mechanism of action of this neurotransmitter in stimulating both catecholamine secretion and compensatory neobiosynthesis in response to stress. Several groups have identified changes in calcium mobilization through which PACAP causes enhanced secretion, but the primary signaling pathway for stimulus-secretion-synthesis whereby PACAP regulates both catecholamine secretion and maintenance of catecholamine stores in adrenal gland remains unaddressed. Here, we use cultured Neuroscreen-1 (NS-1) cells, a substrate-adherent PC12 subclone (Emery et al., Sci. Signal. 2013;6:ra51) to examine the signaling pathways leading to PACAP induction of catecholamine (dopamine) biosynthesis and secretion. PACAP at concentrations as low as 1 nM causes prompt increase in levels of L-Dopa and dopamine (DA), indicative of enhanced catecholamine biosynthesis, within one hour in NS-1 cells. DA release is increased approximately 8-fold during this time. The increase in L-Dopa production, but not the secretion of DA, is mimicked by forskolin and the cell-permeable cAMP analog 8-CPT-cAMP, indicating that biosynthesis, but not secretion, is a wholly cAMP-dependent process, consistent with Gs-coupled adenylate cyclase activation by PACAP. The elevation of L-Dopa production was not blocked by either the MEK inhibitor U0126 or the Epac inhibitor ESI-09, indicating that neither of these pathways is involved in signaling for DOPA biosynthesis, and suggesting the involvement of protein kinase A in activation of its classic substrate, tyrosine hydrolase, a rate-limiting enzyme for catecholamine biosynthesis. PACAP-induced secretion is blocked by the calcium channel blocker, nifedipine, but only partially, suggesting that additional calcium channels may be involved in DA exocytotic secretion initiated by PACAP in this system. NS-1 cells, amenable to genetic manipulation, may be an appropriate model for unraveling the signaling mechanisms of PACAP's actions on catecholamine biosynthesis and release, required for paraphysiological regulation of the adrenal medulla in vivo.