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 ultracentrifugation, gel filtration chromatography, and electron microscopy, we isolated, purified, and identified lamellar structures (unilamellar 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 unilamellar vesicles and multilamellar bodies within bile canaliculi of rats with cholesterol supersaturated bile. By SDS-PAGE, a characteristic and constant protein profile was found associated to the purified lamellar carriers. One of these proteins, a 130-kDa protein, was isolated from human biliary lamellae and used for preparation of a rabbit polyclonal antibody, which cross-reacted with the homologous rat protein. By Western blotting, it was established that the purified low density fraction of bile-Metrizamide gradients, containing lamellae, was enriched with the 130-kDa protein. The 130-kDa protein was characteristically detected at the canalicular membrane by Western blotting of hepatic subcellular fractions and by immunohistochemistry of rat and human liver biopsies. Amino acid sequencing of the amino terminus of the 130-kDa protein demonstrated a complete identity with aminopeptidase N, a canalicular 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 biliary lamellae are similar to lamellar bodies and surfactant-like material frequently found in other epithelia, suggesting common biogenetic, structural, and functional properties. The identification of aminopeptidase N associated with biliary lamellae is consistent with the involvement of the canalicular membrane in the secretory mechanism of biliary lipids.
Rigotti
A
A
Departamento de Gastroenterología, Pontificia Universidad Católica, Santiago, Chile.
Núñez
L
L
Amigo
L
L
Puglielli
L
L
Garrido
J
J
Santos
M
M
González
S
S
Mingrone
G
G
Greco
A
A
Nervi
F
F
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D001646:Bile / Q000737:chemistry* / Q000378:metabolism; D018826:CD13 Antigens; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism*; D004144:Diosgenin / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008099:Liver / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008148:Lovastatin / Q000031:analogs & derivatives / Q000494:pharmacology; D008297:Male; D008793:Metrizamide; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D019821:Simvastatin
D002352:Carrier Proteins; D011506:Proteins; D002784:Cholesterol; D008148:Lovastatin; D019821:Simvastatin; D000626:Aminopeptidases; D018826:CD13 Antigens; D004144:Diosgenin; D008793:Metrizamide