Browsing by Author "ACCATINO, L"

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    ADAPTIVE REGULATION OF HEPATIC BILE-SALT TRANSPORT - EFFECT OF PROLONGED BILE-SALT DEPLETION IN THE RAT
    (1988) ACCATINO, L; HONO, J; MALDONADO, M; ICARTE, MA; PERSICO, R
    Exposure of the liver to increased bile salt flux can increase the bile salt maximum secretory rate (SRm), presumably through the induction of new transport sites. The converse, i.e., the down-regulation of SRm upon bile salt deprivation, has not been demonstrated. We examined the effects of bile salt depletion for 24 h and 48 h on taurocholate SRm and bromsulphalein (BSP) SRm, and on [14C]taurocholate binding to isolated liver surface membranes in unrestrained external biliary fistula rats. Taurocholate SRm was significantly decreased by 35% and 51% in 24-h-depleted and 48-h-depleted rats, respectively, compared with control, sham-operated rats. Maximal taurocholate concentration in bile was also significantly lower in the bile-salt-deprived rats. In contrast, BSP SRm was not significantly different between depleted animals and controls. Bile salt depletion for 24 h and 48 h did not significantly alter liver surface membrane protein recovery and membrance enzyme specific activity including Na+ + K+-ATPase. Specific[14C] taurocholate binding to liver surface membranes was significantly decreased by 25% in 24-h-depleted rats compared with control rats. In contrast to taurocholate SRm, bile salt depletion for 48 h did not result in further reduction of specific taurocholate binding sites. This study demonstrates that taurocholate SRm progressively decreased in 24-h- and 48-h-bile salt-depleted rats, this being consistent with adaptive down-regulation of hepatic bile salt transport. This effect is selective, since BSP SRm was unaltered. The depressed taurocholate SRm can be explained at least in part by decreased bile salt receptor density in liver surface membranes. It appears to be unrelated to either a reduction in membrane surface area (membrane protein recovery and enzyme activity were unchanged in bile salt-depleted rats) or altered Na+ electrochemical gradients (Na+ + K+-ATPase activity was not significantly different between bile salt-depleted and control rats).
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    ADAPTIVE REGULATION OF HEPATIC BILE-SALT TRANSPORT - EFFECTS OF ALLOXAN DIABETES IN THE RAT
    (W B SAUNDERS CO, 1991) ICARTE, MA; PIZARRO, M; ACCATINO, L
    The hepatic transport of bile salts appears to be adaptively regulated by changes in the bile salt pool size and in the flux of bile salt through the liver. The maximum secretory rate of taurocholate increases or decreases when the bile salt pool size is modified by either oral feeding of cholate or taurocholate (up-regulation) or prolonged bile salt depletion through a biliary fistula (down-regulation), respectively. It is not known whether adaptive regulation of hepatic bile salt transport operates under conditions in which the bile salt pool size is modified by endogenous changes in bile acid metabolism. Because experimental diabetes mellitus is associated with alterations in the synthesis of bile acids and total bile salt pool size and composition in the rat, we examined the effects of diabetes mellitus induced by alloxan (5 mg/100 gm body weight, intravenously) and insulin treatment on hepatic bile salt transport and relate the changes to bile salt pool size variations. At 3 days after alloxan injection (DIAB-3 group) both taurocholate maximum secretory rate and pool size were significantly decreased, whereas they were restored to normal values after 6 days of diabetes (DIAB-6 group). Insulinopenic diabetes for 14 days (DIAB-14 group) and for 24 days (DIAB-24 group) resulted in a marked increase of basal bile salt secretory rate (secondary to an increased contribution of cholate conjugates) and an enhanced taurocholate maximum secretory rate compared with control rates (147% and 188% increase, respectively) and with a group (PHARM-control) that received alloxan but did not develop detectable glycosuria (224% and 286% increase, respectively). In contrast, sulfobromophthalein maximum secretory rate was not significantly modified in 14-day diabetic rats compared with control rats. In addition, diabetic rats demonstrated a significant reduction of the bile salt-independent fraction of bile flow. Insulin treatment (3 units/100 gm body wt/day) in diabetic rats from day 0 (alloxan injection) to day 14 (INS-14 group) and from day 14 to day 24 after alloxan administration (INS-24 group) normalized basal bile salt secretion, taurocholate maximum secretory rate and the bile salt-independent fraction of bile flow. Bile salt pool size was significantly greater in DIAB-14 and DIAB-24 groups than in the control group (172% and 216% greater, respectively) and the PHARM-control group (246% and 309% greater, respectively). Insulin treatment prevented, in the INS-14 group, and reversed, in the INS-24 group, the increase of bile salt pool. Cholestyramine administration (5% wt/wt in the diet) to diabetic rats from day 0 (alloxan injection) to day 14 (CHOL-14 group) and from day 14 to day 24 after alloxan administration (CHOL-24 group) prevented and reversed, respectively, bile salt pool and taurocholate maximum secretory rate increase without modifying the hyperglycemia.
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    ADAPTIVE-CHANGES OF HEPATIC BILE-SALT TRANSPORT IN A MODEL OF REVERSIBLE INTERRUPTION OF THE ENTEROHEPATIC CIRCULATION IN THE RAT
    (1993) ACCATINO, L; HONO, J; KOENIG, C; PIZARRO, M; RODRIGUEZ, L
    The reversibility and time course of the adaptive changes in hepatic bile salt transport related to modifications of the bile salt enterohepatic circulation and bile salt pool size have not been previously studied. For this reason a model of reversible interruption of entero-hepatic circulation was characterized in unrestrained rats, which allowed the study of changes in hepatic bile salt transport following bile salt pool depletion and subsequent restoration by either the de novo synthesis of bile acids or i.v. administration of exogenous taurocholate. Rats subjected to biliary drainage for 24 h through a transduodenal common bile duct cannula, followed by removal of the cannula and restoration of the enterohepatic circulation were studied at 24, 48 and 72 h. Neither light and electron microscopy examination nor plasma biochemical parameters showed evidence of necrosis, fibrosis, cholestasis or inflammatory changes. Maximum taurocholate secretory rate decreased to 50% following 24-h bile salt depletion. After restoration of the enterohepatic circulation maximum taurocholate secretory rate progressively increased to normal values at 72 h, following the normalization of the bile salt pool size, which had a similar composition compared with controls. The same effect was obtained when the native bile salt pool was substituted with exogenous taurocholate. Thus, adaptive down-regulation of hepatic bile salt transport capacity is a reversible process, related to restoration of entero-hepatic circulation and normalization of bile salt pool size.
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    ASSOCIATION OF CANALICULAR MEMBRANE ENZYMES WITH BILE-ACID MICELLES AND LIPID AGGREGATES IN HUMAN AND RAT BILE
    (ELSEVIER SCIENCE BV, 1995) ACCATINO, L; PIZARRO, M; SOLIS, N; KOENIG, CS
    This study was undertaken to gain insights into the characteristics of the polymolecular association between canalicular membrane enzymes, bile acids, cholesterol and phospholipids in bile and into the cellular mechanisms whereby the enzymes are secreted into bile. With this purpose, we studied the distribution of bile acids, cholesterol, phospholipids, proteins and representative canalicular membrane enzymes (alkaline phosphatase, 5'-nucleotidase and gamma-glutamyl transpeptidase), which can be considered specific marker constituents, in bile fractions enriched in phospholipid-cholesterol lamellar structures (multilamellar and unilamellar vesicles) and bile acid-mixed micelles. These fractions were isolated by ultracentrifugation from human hepatic bile, normal rat bile and bile of rats treated with diosgenin, a steroid that induces a marked increase in biliary cholesterol secretion, and were characterized by density, lipid composition and transmission electron microscopy. These studies demonstrate that alkaline phosphatase, T-nucleotidase and gamma-glutamyl transpeptidase are secreted into both human and rat bile where they are preferentially associated with bile acid-mixed micelles, suggesting a role for bile acids in both release of these enzymes and lipids from the canalicular membrane and solubilization in bile. In addition, heterogeneous association of these enzymes with nonmicellar, lamellar structures in human and rat bile is consistent with the hypothesis that processes independent of the detergent effects of bile acids might also result in the release of specific intrinsic membrane proteins into bile.
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    EFFECT OF COMPLETE BILIARY OBSTRUCTION ON BILE-FLOW AND BILE-ACID EXCRETION - POSTCHOLESTATIC CHOLERESIS IN THE RAT
    (1979) ACCATINO, L; CONTRERAS, A; FERNANDEZ, S; QUINTANA, C
    Bile secretory function was studied in rats subjected to a 7-day obstructive cholestasis induced by complete common duct obstruction. Bile flow and bile acid excretion were examined during bile depletion, following the release of the biliary obstruction, and during the infusion of sodium taurocholate at submaximal and saturating rates. A highly significant increase, greater than 100%, in bile flow was evident in cholestatic rats at any bile acid excretory rate when compared to control sham-operated rats. 14C-erythritol clearance measurements performed during bile depletion and during taurocholate infusion suggest that bile flow was mainly of canalicular origin in cholestatic rats. Estimated taurocholate transport maximum (.mu.mol/min per rat) was not statistically different between cholestatic and control rats. Significantly greater taurocholate plasma levels at Tm [maximal excretory capacity] in cholestatic rats suggest a decreased efficiency of the bile acid transport process. The relationship between bile flow and bile acid excretion was nonlinear at low bile acid excretory rates in cholestatic rats. Important changes in bile formation occurred in rats subjected to temporary obstructive cholestatis, which differ from those observed in other models of cholestasis that are associated to a reduction in bile flow and bile acid transport capacity.
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    EFFECTS OF INCREASED BILIARY RESISTANCE ON BILE SECRETION IN THE RAT
    (1984) ACCATINO, L; GAVILAN, P; CONTRERAS, A; QUINTANA, C
    The effects of increased biliary resistance of bile secretion and biliary permeability properties were studied in rats. High biliary resistance produced significant reductions in bile flow and the secretion rate of cholesterol, but did not modify bile salt and phospholipid secretory rates. Decreased bile flow appeared to be secondary to a marked reduction in bile salt-independent fractions of bile flow and bile salt-dependent flow was simultaneously increased. 14C-erythritol and 3H-inulin clearance studies suggest that decreased net canalicular secretion of water rather than increased water reabsorption at the biliary ducts is the mechanism involved in decreased bile flow. In addition, a marked increase in canalicular permeability to inulin was evident when rats were secreting against increased biliary resistance as well as during recovery.
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    ENHANCED BILIARY-EXCRETION OF CANALICULAR MEMBRANE ENZYMES IN ESTROGEN-INDUCED AND OBSTRUCTIVE CHOLESTASIS, AND EFFECTS OF DIFFERENT BILE-ACIDS IN THE ISOLATED-PERFUSED RAT-LIVER
    (MUNKSGAARD INT PUBL LTD, 1995) ACCATINO, L; FIGUEROA, C; PIZARRO, M; SOLIS, N
    Backgrounds/Aims: Canalicular membrane enzymes are normally released into bile by partially known processes. This study was undertaken to investigate whether hepatocellular cholestasis induced in rats by ethynylestradiol or obstructive cholestasis produced by complete biliary obstruction for 24 h is associated with an increased release of alkaline phosphatase and gamma-glutamyl transpeptidase into bile, and to clarify hen: this process is affected by different bile acids.
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    ENHANCED BILIARY-EXCRETION OF CANALICULAR MEMBRANE ENZYMES IN ETHYNYLESTRADIOL-INDUCED CHOLESTASIS - EFFECTS OF URSODEOXYCHOLIC ACID ADMINISTRATION
    (PERGAMON-ELSEVIER SCIENCE LTD, 1995) ARRESE, M; PIZARRO, M; SOLIS, N; KOENIG, C; ACCATINO, L
    Cholestasis is associated with a marked increase in the release of canalicular membrane enzymes into bile. This phenomenon has been related to an increased lability of these canalicular membrane integral proteins to the solubilizing effects of secreted bile salts. To further characterize the effects of oral ursodeoxycholic acid (UDCA) administration on ethynylestradiol (EE)-induced cholestasis, the influence of this bile acid on changes in biliary excretion of membrane-bound enzymes was investigated Bile flow, basal bile salt and biliary lipid secretory rates, the maximum secretory rate of taurocholate TC SRm), and the biliary excretion of the canalicular membrane-bound ectoenzymes alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (GGT) were measured in rats after EE and/or UDCA administration. The activities of ALP, GGT and Na+,K+-ATPase in purified isolated canalicular and sinusoidal membrane fractions and the ultrastructure of hepatic acinus, including histochemical studies of ALP distribution, were also examined. EE significantly reduced bile flow, bile salt and biliary lipid secretory rates, and TC SRm, and caused dilatation and loss of microvilli at the canalicular pole of hepatocytes. Biliary excretion of ALP increased 2-fold, whereas biliary excretion of GGT was unchanged. The relationship between biliary excretion of ALP or GGT and bile salt secretion (units of enzyme activity secreted per nanomole of bile salt) was greater in EE-treated rats compared with controls (2.1- and 1.5-fold greater for ALP and GGT, respectively), indicating that in EE-induced cholestasis more enzyme was released into bile per nanomole of bile salt. Na+,K+-ATPase activity in sinusoidal membrane fraction was reduced significantly, whereas ALP activity increased in both membrane fractions in EE-treated rats. The histochemical distribution of ALP in the acinus showed a strong reaction in acinar zone 3 and at both the canalicular and sinusoidal membranes. Oral administration of UDCA prevented EE-induced bile secretory failure by normalizing bile flow, bile salt and biliary phospholipid secretory rates, and TC SRm. UDCA also prevented the EE-induced changes in the biliary excretion of enzymes. On the contrary, UDCA did not modify either the enzyme activity in isolated membrane fractions or the morphological or ALP histochemical changes associated with EE administration. These data indicate that in BE-induced cholestasis changes occur at the canalicular membrane, enabling this portion of the plasma membrane to be more susceptible to the solubilizing effect of bile salt, and that oral administration of UDCA prevents bile secretory failure and changes in the biliary excretion of ALP and GGT in EE-treated rats.
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    PHOSPHOLIPIDS AND BILE-ACIDS AS DIFFUSIONAL CARRIERS OF NA+ ACROSS NONPOLAR MEDIA
    (1988) ACCATINO, L; GAVILAN, P
    Phospholipids and bile acids, by virtue of their amphiphilic properties, can interact in nonpolar media forming "inverted" structures (micelles) which presumably have an hydrophilic core and might act as diffusional carriers (ionophores) of electrolytes across low dielectric constant media or lipid membranes. The Na+ ionophoretic capability of various purified phospholipids and the modulating effects of bile acids and phosphatidylcholine was examined by: (a) measurement of 22Na+ partition into the organic phase (chloroform) of a two-phase system and (b) direct measurement of the translocation of 22Na+ across a bulk chloroform phase separating two aqueous phases in a Pressman cell. All phospholipids tested, except for phosphatidylcholine, showed ionophoretic capability for Na+ at micromolar concentrations. Cardiolipin and phosphatidylserine were the most efficient Na+ carriers, comparable with monensin, an established Na+ ionophore. In contrast, cholic acid as well as other bile acids demonstrated only marginal or no Na+ ionophoretic capability. However, hydroxylated bile acids (particularly cholic acid), sodium dodecyl sulfate and Triton X-100, which can induce and stabilize inverted structures in lipid membranes, were able to increase 5- to 8-fold the phospholipid-mediated Na+ transport. Interaction of cardiolipin with Na+ in the chloroform phase followed a rectangular hyperbolic function with an apparent Kd within the physiological Na+ concentration range (16.9 .+-. 5.1 mM). Addition of cholic acid to the cardiolipin-containing organic phase resulted in a 10-fold increase of maximal Na+ uptake and no change in apparent Kd. The effect of cholic acid on both cardiolipin-mediated Na+ partition and Na+ translocation across the chloroform phase showed a marked dependence on pH, being greater at pH 7.4. On the other hand, phosphatidylcholine, which is reported to stabilize phospholipid bilayers and to inhibit formation of inverted structures, inhibited Na+ cardiolipin interactions. Cholic acid addition completely prevented the inhibitory effect of phosphatidylcholine. This study permits us to establish that the acidic phospholipids cardiolipin and phosphatidylserine can act as efficient Na+ ionophores in this in vitro system, at physiological Na+ concentrations and with kinetics comparable to those of systems involved in Na+ transport (e.g. Na+/H+ antiports) in liver surface membrane and other biomembranes. Modulation by bile acids (stimulation) and phosphatidylcholine (inhibition) of cardiolipin-mediated Na+ transport suggests that the underlying mechanism probably involves inverted-mixed micelle formation in the organic phase. The possibility exists that the Na+ ionophoretic properties of acidic phospholipids are relevant to the bile secretory process.
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    PLATELET AUTOANTIBODIES IN PATIENTS WITH CHRONIC LIVER-DISEASE
    (1995) PEREIRA, J; ACCATINO, L; ALFARO, J; BRAHM, J; HIDALGO, P; MEZZANO, D
    The thrombocytopenia in chronic liver disease (CLD) has been attributed mainly to hypersplenism, although other factors such as reduced mean life span with increased platelet turnover have also been demonstrated, Immunological abnormalities have been described in the pathogenesis and progression of CLD. In this sense, many studies have reported elevated levels of platelet associated IgG (PAIgG) in patients with CLD, and it has been suggested that PAIgG could represent true antiplatelet antibody. In this study we used a glycoprotein (GP)-specific immunoassay (MACE) to determine whether PAIgG or circulating antiplatelet antibodies, reacted against the GPIIb/IIIa or GPIb/IX complexes, in patients with CLD. Thirty-six patients with CLD of diverse etiology were studied (20 female, mean age 53 years, range 38-75 years). 23 out of 36 patients (64%) had anti-GP antibodies in MACE, Particularly, 12 had anti-GPIb, 4 anti-GPIIb/IIIa, and 7 had both types of autoantibodies, The existence of these anti-GP antibodies was not related with the blood platelet count or etiology of CLD,
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    POSTCHOLESTATIC ALKALINE-PHOSPHATASE ACTIVITY AFTER RELIEF OF BILE-DUCT OBSTRUCTION IN THE RAT
    (W B SAUNDERS CO, 1993) WIELANDT, AM; PIZARRO, M; SOLIS, N; ARRESE, M; ACCATINO, L
    The effects of obstructive cholestasis on the activity of alkaline phosphatase have been extensively studied in serum and liver tissue. However, very little is known about the activity of this enzyme in the postcholestatic condition after relief of the biliary obstruction. The purpose of this study has been to characterize alkaline phosphatase activity in serum, liver and bile in the postcholestatic period and to relate it to changes in bile acid secretory rate. Serum activity and biliary secretory rates of alkaline phosphatase were markedly increased in rats subjected to a reversible obstructive cholestasis for 24 hr or 48 hr and progressively declined along the postcholestatic period to values not significantly different from those of control rats within 48 hr. A significant direct linear relationship between the biliary secretory rates of enzyme activity and bile salts was apparent both in cholestatic groups and in the control groups. The slope of the regression line (units of alkaline phosphatase secreted per micromole of bile salts) was 1.5-fold to 3-fold higher in cholestatic animals. Remarkably, a positive y-intercept of regression lines suggested that a significant fraction of the enzyme was secreted independently of bile salts; this fraction was 18-fold and 34-fold greater in 24-hr and 48-hr cholestatic rats, respectively, compared with that in controls. Sodium taurocholate administered intravenously, either as a bolus or as an infusion at increasing submaximal rates, resulted in parallel increases of bile salt and alkaline phosphatase secretory rates into bile. The enzyme activity secreted per micromole of taurocholate was significantly greater in cholestatic than in control rats. In the liver tissue, increased homogenate and canalicular membrane alkaline phosphatase activity in 24-hr cholestatic rats progressively decreased to reach control values 48 hr after relief of biliary obstruction. This study demonstrates that a marked increase of alkaline phosphatase secretion into bile occurs in the postcholestatic condition. It presents further evidence for bile acid dependency of this process and demonstrates that more enzyme is secreted per micromole of bile salt in the postobstructive condition, probably related to the increased enzyme content in the liver and to an increased lability of the canalicular membrane enzyme to the solubilizing effect of secreted bile acids in cholestatic rats. In addition, this study suggests that alkaline phosphatase might be normally secreted into bile by another process independent of bile salts, which appears to be quantitatively more important in cholestatic than in control rats.
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    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
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    THE EFFECT OF COMPLETE BILIARY OBSTRUCTION ON BILE SECRETION - STUDIES ON THE MECHANISMS OF POSTCHOLESTATIC CHOLERESIS IN THE RAT
    (1981) ACCATINO, L; CONTRERAS, A; BERDICHEVSKY, E; QUINTANA, C
    Rats subjected to obstructive cholestasis apparently demonstrate in the postcholestatic period, after common duct obstruction release, a marked increase in canalicular bile flow relative to bile acid excretion. Changes in canalicular permeability and in (Na+-K+)-ATPase activity in isolated liver surface membranes were studied to determine whether they are associated with postcholestatic choleresis. The clearances of 14C-erythritol and 3H-inulin were simultaneously measured in rats subjected to a 3 day obstructive cholestasis and in controls, during spontaneous choleresis as well as during the i.v. infusion of sodium taurocholate at submaximal and saturating rates. In additional groups of bile duct-ligated rats and controls, liver surface membrane fractions were isolated and the activity of appropriate marker enzymes and (Na+-K+)-ATPase was determined. In the 2 groups 14C-erythritol clearance closely approximated total bile flow, suggesting that bile flow was of canalicular origin. Cholestatic rats showed a 6-fold increase in 3H-inulin clearance compared to controls. Canalicular permeability to inulin is apparently markedly increased in cholestatic rats. (Na+-K+)-ATPase activity was significantly higher in cholestatic rats than in controls in the homogenate (P < 0.001) and liver surface membranes, (P < 0.001). Enhanced choleretic response to bile acids in the postcholestatic period is associated with an increased permeability of canalicular structure to inulin and with a significant increase in homogenate and surface membrane (Na+-K+)-ATPase activity. Some important differences between bile secretory function of rats subjected to obstructive cholestasis and that described in models of bile secretory failure induced by drugs or monohydroxy-bile acids, are pointed out.