Browsing by Author "RIGOTTI, A"

Now showing 1 - 6 of 6
  • No Thumbnail Available
    Item
    BILIARY LIPID SECRETION - IMMUNOLOCALIZATION AND IDENTIFICATION OF A PROTEIN ASSOCIATED WITH LAMELLAR CHOLESTEROL CARRIERS IN SUPERSATURATED RAT AND HUMAN BILE
    (1993) RIGOTTI, A; NUNEZ, L; AMIGO, L; PUGLIELLI, L; GARRIDO, J; SANTOS, M; GONZALEZ, S; MINGRONE, G; GRECO, A; NERVI, F
    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.
  • Thumbnail Image
    Item
    CHOLESTEROL CRYSTALLIZATION-PROMOTING ACTIVITY OF AMINOPEPTIDASE-N ISOLATED FROM THE VESICULAR CARRIER OF BILIARY LIPIDS
    (WILEY, 1993) NUNEZ, L; AMIGO, L; RIGOTTI, A; PUGLIELLI, L; MINGRONE, G; GRECO, AV; NERVI, F
    Different hydrophobic glycoproteins are associated to native biliary vesicles, which are the major carrier of biliary cholesterol. Some of these proteins promote cholesterol crystallization, a key step in cholesterol gallstone formation. This study was specifically conducted to identify the 130 kDa biliary vesicle-associated glycoprotein and to determine its in vitro effect on the cholesterol crystal formation time. The 130 kDa vesicular glycoprotein was identified as aminopeptidase-N by amino acid sequencing and specific enzymatic assay. Polyclonal antibodies raised against aminopeptidase-N allowed us to determine its concentration in human hepatic bile, which varied from 17.3 to 57.6 mug/ml. Aminopeptidase-N showed a concentration-dependent cholesterol crystallization activity when it was added to supersaturated model bile at a concentration range usually found in native bile. Because of this promoting effect on in vitro cholesterol crystal formation, we suggest that biliary aminopeptidase-N may play a critical role in the pathogenesis of cholesterol gallstone disease.
  • No Thumbnail Available
    Item
    EFFECT OF BEAN INTAKE ON BILIARY LIPID SECRETION AND ON HEPATIC CHOLESTEROL-METABOLISM IN THE RAT
    (1989) RIGOTTI, A; MARZOLO, MP; ULLOA, N; GONZALEZ, O; NERVI, F
    We studied the effect of a bean diet on biliary lipid secretion, serum cholesterol concentration, and hepatic cholesterol metabolism in the rat. Rats fed a bean diet for 10-12 days had increased biliary cholesterol output and molar percentage by 300% and 200%, respectively, compared to rats fed an isocaloric and isoproteinic casein diet. Biliary phospholipid output increased 180%. Bile flow and biliary salt output remained in the normal range. Total serum and VLDL cholesterol concentration significantly decreased 27% and 50%, respectively, in the rats fed the bean diet. Hepatic cholesterogenesis was increased 170% in the bean-fed animals. The relative contribution of newly synthesized hepatic cholesterol to total biliary cholesterol increased 200%, and that of endogenous origin only 50%. These results suggested that newly synthetized hepatic cholesterol was preferentially channelled to the biliary cholesterol pathway in bean-fed rats. Although hepatic cholesteryl ester concentration increased 240%, the incorporation of [14C]oleate into hepatic cholesteryl esters was significantly decreased by 30% in isolated hepatocytes of bean-fed animals. These results were consistent with the possibility that the availability of hepatic free cholesterol for biliary secretion was increased in the bean-fed animals. This study demonstrates that bean intake has a profound effect on the metabolic channelling and compartmentalization of hepatic cholesterol, resulting in a significant decrease in total serum and very low density lipoprotein cholesterol concentrations and a high biliary cholesterol output.
  • No Thumbnail Available
    Item
    ISOLATION AND PURIFICATION OF HUMAN BILIARY VESICLES WITH POTENT CHOLESTEROL-NUCLEATION-PROMOTING ACTIVITY
    (1992) MIQUEL, JF; RIGOTTI, A; ROJAS, E; BRANDAN, E; NERVI, F
    1. Cholesterol nucleation is a critical step in the formation of cholesterol gallstones. This nucleation takes place after aggregation and fusion of cholesterol-rich biliary vesicles, a process probably modulated by biliary proteins. The present study was conducted to identify specific proteins associated with native cholesterol-rich biliary vesicles and to explore their effect on the cholesterol-nucleation time of supersaturated artificial bile.
  • Thumbnail Image
    Item
    PROTECTIVE ROLE OF BILIARY CHOLESTEROL AND PHOSPHOLIPID LAMELLAE AGAINST BILE ACID-INDUCED CELL-DAMAGE
    (W B SAUNDERS CO-ELSEVIER INC, 1994) PUGLIELLI, L; AMIGO, L; ARRESE, M; NUNEZ, L; RIGOTTI, A; GARRIDO, J; GONZALEZ, S; MINGRONE, G; GRECO, AV; ACCATINO, L; NERVI, F
  • No Thumbnail Available
    Item
    REGULATION OF BILIARY CHOLESTEROL SECRETION - FUNCTIONAL-RELATIONSHIP BETWEEN THE CANALICULAR AND SINUSOIDAL CHOLESTEROL SECRETORY PATHWAYS IN THE RAT
    (1988) NERVI, F; MARINOVIC, I; RIGOTTI, A; ULLOA, N
    The functional interrelationship between biliary cholesterol secretion, sinusoidal lipoprotein cholesterol secretion and bile salt synthesis was studied in the rat. Diosgenin, fructose, and colestipol in the diet were used to, respectively, influence biliary cholesterol output, VLDL production and bile salt synthesis. In the acute bile fistula rat, biliary cholesterol output was 700% increased by diosgenin and 50% decreased by fructose. In the rats fed both diosgenin and fructose, biliary cholesterol secretion was increased only by .apprx. 200%, whereas biliary bile salts and phospholipid outputs were unchanged. In the isolated perfused liver, VLDL-cholesterol output was 50% reduced by diosgenin alone, but was unchanged following feeding of diosgenin plus fructose. However, the livers of rats fed diosgenin plus fructose exhibited a 700% increase in VLDL-triglyceride production and a 200% increase in VLDL-cholesterol output. A significant reciprocal relationship between VLDL-cholesterol secretion and the coupling ratio of cholesterol to bile salts in bile was observed. Colestipol added to the diet maintained both sinusoidal and biliary cholesterol outputs within the normal range. In the chronic bile fistula rat, colestipol increased bile salt synthesis by 100% while diosgenin and fructose diets had no effect. Similarly, the addition of fructose to the colestipol diet did not decrease bile salt synthesis. These data suggest a reciprocal relationship between biliary cholesterol secretion and hepatic secretion of cholesterol as VLDL particles. The free cholesterol pool used for bile salt synthesis seems functionally unrelated to the pool from which VLDL-cholesterol and biliary cholesterol originate. These findings support the idea that metabolic compartmentalization of hepatic cholesterol is a major determinant of the quantity of cholesterol available for recruitment by the bile salt-dependent biliary cholesterol secretory mechanism.