Over the last few years, we have successfully used a model of cultured non-transformed dog pancreatic duct epithelial cells (PDEC) to study the determinants of pancreatic ductal secretion and characterize different cellular receptors on these cells. In this revised submission, we propose to characterize further the regulation of pancreatic ductal cell secretion by ATP. Hypothesis 1: ATP regulates PDEC secretion through cAMP (in addition to the Ca2+ pathway). We previously reported that ATP and UTP interact with P2Y2 receptors on PDEC to mobilize Ca2+ and stimulate secretion. We recently obtained preliminary data suggesting that ATP interacts with an additional purinergic receptor (from now referred to as P2Y[unk]) to increase intracellular cAMP and stimulate PDEC secretion. Thus, we propose to: a) further define the stimulation of cAMP increase produced by ATP and other nucleotides, b) elucidate the effect of ATP on Cl- channels mediated by the cAMP pathway, c) investigate ATP effects on HCO3- secretion, and d) examine the cAMP-mediated effect of ATP on mucin secretion and exocytosis. Hypothesis 2: ATP has a paracrine and autocrine action on PDEC secretion, through the P2Y2 and P2Y[unk] receptors. P2Y2 receptors, acting via increased [Ca2+]i, are present on both apical and basolateral surfaces of PDEC; preliminary evidence suggest that the P2Y[unk] receptor, acting via cAMP, are confined to the basolateral surface of these cells. These distinct receptors, with separate localization and coupled to different signal transduction pathways, may mediate paracrine and autocrine actions of ATP. Thus we propose to: a) characterize the subtype and confirm the localization of the P2Y[unk] receptor. b) characterize the vectorial release of ATP release from PDEC (autocrine action) c) study the effect of mast cell degranulation, resulting in ATP release, on PDEC secretion (paracrine action).
