Bile acids play a crucial role in the promotion of bile flow and the luminal absorption of fatty acids via micellar formation. In addition, bile acid represent the major route for the excretion of cholesterol. We have previously identified that the interactions of bile acids with the cytosolic 3 alpha hydroxysteroid dehydrogenases (HSD) is an important determinant in their ultimate uptake and excretion by hepatocytes. In addition to bile acid transport, 3 alpha HSD is required for bile acid synthesis from cholesterol. My FIRST Award proposal will focus on further characterization of this important enzyme by identifying its gene and examining its regulation. A full length complementary DNA (cDNA) clone for 3 alpha HSD will be isolated and sequenced from a rat liver specific cDNA library. This full length cDNA probe will be crucial for subsequent studies to identify its chromosome location, tissue distribution and molecular regulation. Specific goals include: 1) Isolation and nucleic acid sequence analysis of the entire cDNA clone for hepatic 3 alpha HSD. 2) Chromosomal mapping of the 3 alpha HSD in man and rat. 3) Identification and amino acid sequence analysis of the bile acid binding site of the 3 alpha HSD utilizing a photoactivated bile acid probe 4) Quantization of organ distribution and message size in male and female rats by northern blot analysis. 5) Ontogeny of 3 alpha HSD expression in the liver and intestine of male and female rats. 6) Diurnal variation of 3 alpha HSD expression in male and female rats. 7) Regulation of hepatic 3 alpha HSD by steroid hormones and bile acids in tissue culture and 8) Influence of bile acid load and synthesis on mRNA levels in the intact rat. These studies will significantly advance the characterization of the 3 alpha HSD and its physiological regulation. The work proposed will lay the foundation for more advanced studies on the regulation of this protein in future studies.
