PAC1hop receptor activation facilitates catecholamine secretion selectively through 2-APB-sensitive Ca2+ channels in PC12 cells

PACAP is a critical regulator of long-term catecholamine secretion from the adrenal medulla in vivo, however the receptor or pathways for Ca2+ entry triggering acute and sustained secretion have not been adequately characterized. We have previously cloned the bovine adrenal chromaffin cell PAC1 receptor that contains the molecular determinants required for PACAP-induced Ca2+ elevation and is responsible for imparting extracellular Ca2+ influx-dependent secretory competence in PC12 cells. Here, we use this cell model to gain mechanistic insights into PAC1hop-dependent Ca2+ pathways responsible for catecholamine secretion. PACAP-modulated extracellular Ca2+ entry in PC12 cells could be partially blocked with nimodipine, an inhibitor of L-type VGCCs and partially blocked by 2-APB, an inhibitor and modulator of various transient receptor potential (TRP) channels. Despite the co-existence of these two modes of Ca2+ entry, sustained catecholamine secretion in PC12 cells was exclusively modulated by 2-APB-sensitive Ca2+ channels. While IP3 generation occurred after PACAP exposure, most PACAP-induced Ca2+ mobilization involved release from ryanodine-gated cytosolic stores. 2-APB-sensitive Ca2+ influx, and subsequent catecholamine secretion was however not functionally related to intracellular Ca2+ mobilization and store depletion. The reconstituted PAC1hop-expessing PC12 cell model therefore recapitulates both PACAP-induced Ca2+ release from ER stores and extracellular Ca2+ entry that restores PACAP-induced secretory competence in neuroendocrine cells. We demonstrate here that although bPAC1hop receptor occupancy induces Ca2+ entry through two independent sources, VGCCs and 2-APB-sensitive channels, only the latter contributes importantly to sustained vesicular catecholamine release that is a fundamental characteristic of this neuropeptide system. These results emphasize the importance of establishing functional linkages between Ca2+ signaling pathways initiated by pleotrophic signaling molecules such as PACAP, and physiologically important downstream events, such as secretion, triggered by them.