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Pharmacokinetic studies of the drugs administered to subjects with mechanical cholestasis are scarce. The purpose of the present study was to examine the effects of bile duct ligation of 3 days (peak of elevation of serum bile acids and bilirubin) on the systemic and renal PAH clearance and on the expression of cortical renal OAT1 and OAT3 in a rat model. PAH is the prototypical substrate of the renal organic anion transport system. Male Wistar rats underwent a bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BDL rats displayed a significantly lower systemic PAH clearance. Renal studies revealed a reduction in the renal clearance and in the excreted and secreted load of PAH in BDL rats. The OAT1 protein expression in kidney homogenates was not modified, but it decreased in the basolateral membranes from BDL rats. In contrast, OAT3 abundance in both kidney cortex homogenates and in basolateral membranes increased by 3 days after the ligation. Immunocytochemical studies (light microscopic and confocal immunofluorescence microscopic analyses) confirmed the changes in the renal expression of these transport proteins. The present study demonstrates the key role of OAT1 expression in the impaired elimination of PAH after 3 days of obstructive cholestasis.

Isoursodeoxycholic acid (isoUDCA), the 3 beta-epimer of ursodeoxycholic acid (UDCA), may have pharmaceutical potential because of its similar hydrophilicity and in vitro cytoprotection as compared with UDCA. We compared metabolism and effects on cholestasis of UDCA and isoUDCA in experimental cholestasis in rats. Cholestasis was induced by bile duct ligation. For bile flow and biliary bile acid analysis, UDCA or isoUDCA were infused intraduodenally. For the study of chronic effects, chow was supplemented with 2.5 g/kg UDCA or isoUDCA for 3 weeks. Sham-operated animals served as controls. IsoUDCA became completely converted to UDCA in the liver. Choleresis and biliary bile acids were the same after the intraduodenal administration of either compound. Oral administration of UDCA or isoUDCA significantly improved liver biochemistry but not clinical and histological parameters in chronic cholestasis. The decrease of serum cholic acid in control animals was more pronounced after isoUDCA (-93%) than after UDCA (-76%). Only after UDCA, this decrease was compensated by increases of UDCA, beta-muricholic acid (MCA), and Delta(22)-beta-MCA. Our results show that isoUDCA has the same effect on choleresis and liver biochemistry as UDCA. IsoUDCA features pro-drug characteristics of UDCA and causes compared to the latter lower serum bile acid concentrations in non-cholestatic animals.

Stimulated superoxide generation was 2-fold higher in neutrophils from 20 rats with common bile-duct ligation (CBDL) compared to that of 20 sham-operated control rats. In order to study the mechanism of the higher NADPH oxidase activity in CBDL rats, the kinetic parameters of NADPH oxidase were analyzed. The Vmax of the NADPH oxidase in CBDL rat neutrophils was significantly higher than that of control rat neutrophils (10.2 and 5.3 nmol/min, respectively). The membrane and cytosol fractions of the oxidase were studied in a cell-free system. Neutrophil cytosol from CBDL rats added to neutrophil membranes from either CBDL or control rats produced 22.4 +/- 1.6 and 21.0 +/- 1.4 nmol/10(6) cells per 10 min, respectively. When neutrophil cytosol from control rats was mixed with neutrophil membranes from control or CBDL rats the generation of superoxide was 10.6 +/- 1.4 and 10.0 +/- 1.5 nmol/10(6) cells per 10 min, respectively. These results suggest that the cytosol components of the oxidase regulate its activity. By immunoblot analysis it was shown that the amount of the cytosolic factor p47 in neutrophils of CBDL rats is higher than that present in an equal number of neutrophils from control rats.

Data regarding the action of monensin on the concentrations of glycerophospholipids and cholesterol in bile of rats subjected to total biliary diversion for 3 h are reported. After monensin their concentration in bile drops significantly in the first 60 min collections with respect to the control. Differences seem to be produced between the rates of transport to the bile of glycerophospholipids and cholesterol, not sufficiently explained by the inhibition of bile salt uptake determined by monensin at the sinusoidal pole of the hepatocyte.

Acute biliary obstruction in the rat is associated with striking alterations of the plasma level and the physico-chemical properties of plasma lipoproteins. 1. The level of very low density lipproteins (VLDL) in plasma increases from 2 to 3 fold. The chemical composition of VLDL is characterized by a high content of phospholipids and cholesterol and by a diminution of the relative content of triacylglycerols and protein. On cellulose acetate electrophoresis, VLDL show a beta-mobility. 2. The plasma concentration of low density lipoproteins (1.019--1.063 g/ml, LDL2) increases several fold above the control level. Phospholipids and unesterified cholesterol are the major components of this fraction which contains only a minute amount of cholesteryl ester (4%) and triacylglycerols (10%). LDL2 contain a component which migrates to the cathode in 1% agar gel electrophoresis. Separation of LDL2 by gel filtration on 2% agarose column results in the identification of three subfractions. Subfraction I contains a large proportion of cholesterol and triacylgylcerols, subfraction II is rich in unesterified cholesterol and phospholipids whereas subfraction III has a chemical composition fairly similar to that of the control LDL2. 3. The level of high density lipoproteins (HDL) also increases after bile duct ligation. The chemical composition of HDL2 (1.063-1.125 g/ml) is characterized by a high content of unesterified cholesterol and phospholipids and by a remarkable reduction in the content of cholesteryl esters and protein.

Incorporation of [32P]orthophosphate into phosphatidylcholine, lysophosphatidylcholine and molecular species of phosphatidylcholine in vivo was observed in liver, plasma and erythrocytes of bile duct-ligated or sham-operated rats. Both the amount and radioactivity of dienoic species of phosphatidylcholine in all tissues examined increased in bile duct-ligated rats as compared to sham-operated rats. The experiments in vivo and in vitro showed that the ratio of lysophosphatidylcholine to phosphatidylcholine transferred to erythrocytes from plasma in sham-operated rats was much higher than that in bile duct-ligated rats. It is suggested that one of the mechanisms by which abnormal erythrocytes appear might be explained by the facilitated and direct transfer of phosphatidylcholine, which is caused by the interaction of erythrocytes with bile acid in bile duct-ligated rat plasma.

Lipid molecules in lipoprotein surfaces exchange with their counterparts in cell plasma membranes. In human or experimental liver disease, plasma lipoprotein surfaces are enriched in cholesterol and deficient in arachidonate; corresponding alterations occur in membrane lipids of erythrocytes. To determine whether similar changes take place in membranes of nucleated cells, the lipid content of plasma and of erythrocyte, liver and kidney membranes was measured in rats with acute (3-day) galactosamine-induced hepatitis or chronic (3-week) biliary obstruction. In both models of liver injury the cholesterol:phospholipid ratio in plasma and in erythrocytes was significantly increased (P less than 0.001). Although this ratio was also elevated in liver and kidney microsomes, only in liver microsomes of obstructed rats was the increase significant (P less than 0.001). However, the cholesterol:phospholipid ratio of kidney brush-border membranes, was significantly higher in bile-duct-ligated rats; presumably, compensating mechanisms limit cholesterol accumulation in intracellular membranes. Kidney brush-border membranes from obstructed rats were deficient in arachidonate as were plasma and erythrocytes. However, arachidonate levels were unchanged in kidney microsomes; renal delta 6-desaturase, the rate-limiting enzyme in the conversion of linoleic acid to arachidonic acid, was increased by 50% (P less than 0.001) and may have counteracted a reduced supply of exogenous lipoprotein arachidonate. We conclude that in experimental liver disease lipoprotein-induced lipid abnormalities can occur in renal membranes, although compensatory mechanisms may operate; the alterations seen, cholesterol accumulation and arachidonate depletion, would be expected to interfere with sodium transport and prostaglandin production, respectively. Our findings support the hypothesis that lipid abnormalities in kidney membranes contribute to the renal dysfunction which is a frequent complication of human liver disease.

The effects of bile-duct ligation on hepatic and intestinal (jejunum) alkaline phosphatase activities were studied using rats and guinea pigs. In ligated rats, the enzyme activity was increased 4.1-fold in the liver after 24 h and 2.8-fold in the intestine after 12 h. In guinea pigs, the hepatic and intestinal enzyme activities were increased 2.3-fold and 1.5-fold after 100 and 24 h, respectively. The intestinal activity was induced sooner after ligation than hepatic activity. The induction of alkaline phosphatase was inhibited by prior treatment of animals with amanitin, an inhibitor of RNA polymerase activity. This result indicates that the induction is associated with de novo enzyme synthesis. The content of cyclic AMP in liver and intestine increased immediately after ligation. The increase in alkaline phosphatase activities was also inhibited by pretreatment with chlorpromazine, an inhibitor of adenylate cyclase activity. Hence, cellular cyclic AMP may be implicated in playing a role in the induction of alkaline phosphatase by bile-duct ligation.

Plasma membranes were isolated from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5'-nucleotidase and (Na+ + K+)-ATPase were used. The yield of plasma membrane was 0.6-0.9 mg protein per g wet weight of liver. The recovery of 5'-nucleotidase and (Na+ +K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the activity of glucose-6-phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5'-nucleotidase, alkaline phosphatase, (Na+ +K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na2+ +K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphate was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.

A single injection of colchicine (1--3 mg/kg body weight) caused a remarkable induction of hepatic alkaline phosphatase, which increased linearly in the homogenate starting at 5--6 h and reached a maximum level (14-fold of the control activity) at 20--22 h after the drug treatment. In the plasma membrane, however, the increase in specific activity and the recovery of alkaline phosphatase were greatly inhibited up to 12 h after the treatment. Such an inhibitory effect of colchicine was confirmed by a combination experiment of the drug treatment with bile duct ligation; in the plasma membrane elevation of the enzyme induced by bile duct ligation was also greatly retarded by colchicine. The subcellular distribution of the enzyme activity in livers was determined among the four groups of rats with or without bile duct ligation and/or colchicine administration taken at 8 h after each treatment. In the control and the bile duct-ligated livers, the highest specific activity was observed in the plasma membrane fraction, while in the colchicine-treated livers, with or without bile duct ligation, the highest activity was found in the Golgi fractions. These results indicate that the Golgi membranes enriched with the induced enzyme were blocked by the drug to prevent migration toward the plasma membranes enriched with the induced enzyme were blocked by the drug to prevent migration toward the plasma membrane, thus demonstrating involvement of the Golgi complex in the translocation route of newly synthesized alklaine phosphatase to the plasma membrane.