Several observations suggest that in a number of species including man, bile secretion is not fully developed at birth and that, because of this immaturity, the perinatal liver is particularly subject to cholestasis. However, direct evidence for this view is lacking. This proposal aims to achieve an understanding of neonatal biliary physiology, and its postnatal development. Using puppies of 0-56 days of age and adult dogs (rats of different ages will also be used for the conduct of projects # 5-9), we will first examine the time-course of Postnatal Development of Bile Secretory Physiology. This includes: 1) assessment of hepatic bile flow and composition (individual bile acids, electrolytes, phospholipids, and cholesterol) and gallbladder bile composition; 2) determination of the effects of bile acids, secretin, glucagon, and insulin on bile flow and composition both in vivo and in the isolated perfused liver; 3) measurement of the permeability of the biliary tree to polar nonelectrolytes (erythritol, etc.), and that of the extrahepatic bile duct to electrolytes (Na+, HCO3, Cl-, etc.), nonelectrolytes, hydrophilic and hydrophobic bile acids, and unconjugated and conjugated (diglucuronide) bilirubin; 4) studying the structure of hepatic parenchymal and biliary epithelial cells and their surroundings, and that of gallbladder epithelium; 5) measuring the uptake of cholate, taurocholate and glycocholate by hepatocytes isolated from puppies and rats of different ages; and 6) assessing the diffusive permeability of isolated hepatocytes (puppies and rats of different ages) to polar nonelectrolytes. Additionally, since the new born puppy is not responsive to the choleretic effect of secretin, which is thought to be of ductular/ductal origin, we will attempt to Isolate and Characterize a Non-Parenchymal Liver Cell Fraction Enriched in Biliary Epithelial Cells, which is of potential use for studying bile ductular cell function and its postnatal development. Using puppies of 0-28 days of age and adult rats, we will 7) fractionate non-parenchymal liver cells by centrifugal elutriation, 8) characterize the different fractions by biochemical, histochemical and immunofluorescent techniques, and 9) further purify a fraction of biliary epithelial cells using the fluorescence activated cell sorter. These studies will advance knowledge of neonatal biliary physiology and will provide a basis for understanding the pathophysiology of neonatal cholestasis. Furthermore, they will shed light on the mechanisms of hepatic bile formation in the mature biliary system as well.
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