Little is known of the molecular mechanisms of signal transduction in the goblet cells and bicarbonate-secreting principal cells of the excretory duct epithelium of the exocrine pancreas, despite their central role in elaboration of pancreatic juice. The mechanisms through which input from the central nervous system modulates the regulatory effects of gastrointestinal hormones such as secretin are particularly obscure. In the proposed research, the recently developed model of isolated and cultured guinea pig pancreatic duct epithelial cells will be exploited to identify physiological regulators and intracellular messengers that control ductal mucin and bicarbonate secretion and to define the cellular targets of the identified signal transduction cascades. In these studies the effects of potential agonists of ductal bicarbonate secretion on intracellular free Ca2+ and cyclic nucleotide levels will be determined as will potential interactions between cyclic nucleotide- and inositol phospholipid-mediated signaling pathways. The three dimensional kinetics of calcium changes induced by agonists will be examined through the newly developed technology of video rate laser scanning confocal microscopy (LSCM), using fluorescent calcium probes. The cellular targets of identified intracellular signaling molecules will be defined, with particular attention directed to anion and cation exchange mechanisms and anion channels using isotope flux measurements and LSCM. Effects of putative physiological regulators and intracellular messengers on intracellular pH will be examined using LSCM and pH-sensitive fluorochromes. The kinetics of agonist-induced mucin release from goblet cells in isolated pancreatic ducts will be examined through the combined approaches of solid phase radioimmune assay and morphometry and confocal microscopy. These experiments will provide a detailed picture of the intracellular mechanisms through which the parasympathetic nervous system and gastrointestinal hormones regulate bicarbonate and mucin secretion by the epithelium of the pancreatic ducts as well as providing a necessary context for future studies on perturbations of these signaling pathways that occur in pancreatic diseases such as cystic fibrosis and pancrea- titis.
