The studies described in this proposal examine the mechanisms responsible for secretion of fluid and electrolytes by intrahepatic bile duct epithelial (BDE) cells. Ductular secretion accounts for 13-40% of bile volume and modification of bile composition, but little is known of the cellular mechanisms responsible. Recent observations using patch clamp recording techniques suggest that C1-channels in the apical membrane may play a key role in regulation of secretion. Using patch clamp and other techniques, the long term objective of the first working hypothesis is to identify and characterize the role of intracellular signalling pathways in regulation of BDE cell membrane C1- (and other) channels.
The Specific Aims are: i) to evaluate the distinctive properties of Ca2+- and cAMP-dependent C1- currents and determine the role of regulatory phosphorylation by Ca2+- and cAMP-dependent protein kinases in current activation; ii) to identify the ion channels which contribute to these currents, with emphasis on an ~8 pS C1-channel activated by cAMP in the intact cell; iii) to determine whether cAMP-dependent currents are related to endogenous expression of cystic fibrosis transmembrane conductance regulator (CFTR), a putative C1- channel protein; and iv) to evaluate the role of GTP-binding (G) proteins in inhibition of high conductance anion channels, and in activation of whole cell C1- currents. The long term objective of the second working hypothesis is to identify and physiologic factors which contribute to independent regulation of duct cell secretion.
The Specific Aims are i) to develop model systems for investigation of receptor-mediated regulation of transepithelial transport; ii) to define the cAMP-dependent and -independent mechanisms responsible for activation of C1- channels by secretin; and iii) to evaluate the role in regulation of secretion of extracellular adenosine and ATP which, through purinergic receptors, increase adenylyl cyclase activity and intracellular Ca2+, respectively. Investigation of membrane C1-and other channels provides a focus for physiologic and pharmacologic regulation of bile duct cell secretion which contributes directly to the volume and composition of bile, and abnormal regulation may also account for the cholestatic manifestations of cystic fibrosis and other primary disorders affecting biliary epithelia.
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