BILIARY LIPID SECRETION - IMMUNOLOCALIZATION AND IDENTIFICATION OF A PROTEIN ASSOCIATED WITH LAMELLAR CHOLESTEROL CARRIERS IN SUPERSATURATED RAT AND HUMAN BILE

Feeding a 0.5% diosgenin plus 0.02% simvastatin diet to rats increases biliary cholesterol concentration and saturation to levels generally found in human native supersaturated bile. By using preparative ultrac entrifugation, gel filtration chromatography, and electron microscopy, we isolated, purified, and identified lamellar structures (unilamella r vesicles and multilamellae) as a major biliary cholesterol transport in supersaturated human and rat bile. It was estimated that more than 60% of biliary cholesterol is transported in these lamellar carriers, which were identified by transmission electron microscopy as unilamel lar vesicles and multilamellar bodies within bile canaliculi of rats w ith cholesterol supersaturated bile. By SDS-PAGE, a characteristic and constant protein profile was found associated to the purified lamella r carriers. One of these proteins, a 130-kDa protein, was isolated fro m human biliary lamellae and used for preparation of a rabbit polyclon al antibody, which cross-reacted with the homologous rat protein. By W estern blotting, it was established that the purified low density frac tion of bile-Metrizamide gradients, containing lamellae, was enriched with the 130-kDa protein. The 130-kDa protein was characteristically d etected at the canalicular membrane by Western blotting of hepatic sub cellular fractions and by immunohistochemistry of rat and human liver biopsies. Amino acid sequencing of the amino terminus of the 130-kDa p rotein demonstrated a complete identity with aminopeptidase N, a canal icular transmembrane hydrophobic glycoprotein. These studies show that biliary lipids may acquire an ordered multilamellar structure that is present in the canaliculi of rats with supersaturated bile. These bil iary lamellae are similar to lamellar bodies and surfactant-like mater ial frequently found in other epithelia, suggesting common biogenetic, structural, and functional properties. The identification of aminopep tidase N associated with biliary lamellae is consistent with the invol vement of the canalicular membrane in the secretory mechanism of bilia ry lipids.