Bile Canalicular Membrane Research Articles

In vitro effect of free bile acids on the bile canaliular membrane phospholipids in the rat

Liver cell plasma membranes of male rats were isolated and separated into two fractions, one rich in bile canalicular membranes (BCM) and the other comprising the rest of the plasma membrane (PM). Aliquots of BCM, PM, and microsomes were incubated with deoxycholic, chenodeoxycholic, or cholic acid at bile acid - membrane phospholipid mole ratios up to 100, and the phospholipid solubilization from the PM and from microsomes was linear and apparently nonselective, while that from BCM was biphasic and distinctly selective. Phosphatidyl choline and phosphatidyl ethanolamine made up 90% of the phospholipids solubilized from the BCM at a bile acid - membrane phospholipid mole ratio sufficient to solubilize about 50% of the total phospholipids of the BCM. Of particular interest was the observation that the molecular species and fatty acid composition of the phospholipids solubilized from the BCM under these experimental conditions were similar to those of bile obtained from the same animal, and were quite unlike those solubilized at higher bile acid - phospholipids mole ratios. The data are discussed in terms of the mechanism of the biliary secretion of phospholipids.

153: Intrahepatic cholestasis and excessive formation of bile acid intermediates

The patient, a boy and first infant of healthy unrelated parents, was first seen at the age of 2 months because of jaundice. The hyperbilirubinaemia had appeared at the age of 1 month and persisted up to the age of 3 1/2 months when the child died from pulmonary infection. At the age of 2 months serum values were: bilirubine total 16 mg% and direct 12mg%, transaminases GOT 410 IU/l and GPT 170 IU/l and normal alkaline phosphatases. The serum level of bile acids was 67 ug/ml but cholic and chenodeoxycholic acid only made up 15 percent of it. In addition trihydroxycoprostanic acid, dihydroxycoprostanic acid and probably cholestane-tetrol and cholestane-pentol could be detected, At laparotomy, complete opacification of the extrahepatic biliary system was seen. Optical microscopy of the liver showed evidence neither of hepatitis nor of intrahepatic biliary atresia but of cholestasis at the site of the liver cell. Electron microscopy revealed interruptions of the bile canalicular membrane, granular material within the canaliculi and an excessive amount of filaments in the pericanalicular ectoplasm of the hepatocyte. The disturbance of bile acid metabolism could be explained by a block of the 24-hydroxylation of coprostanic acid.

Role of membranes in bile formation. Comparison of the composition of bile and a liver bile-canalicular plasma-membrane subfraction

1. Enzymes, proteins, glycoproteins and lipids of rodent bile were compared with those of a plasma-membrane subfraction originating from the hepatocyte bile-canalicular membrane. 2. Three bile-canalicular glycoprotein enzyme activities were detected in bile. Comparison of the pH optimum and immunoinhibition properties of membrane and bile 5'-nucleotidase activity indicated that they were the same enzyme. Correspondence between membrane and bile alkaline phosphodiesterases also suggested that they were the same enzymes. Activities of Mg2+-stimulated adenosine triphosphatase, a lipid-dependent intrinsic membrane protein, and galactosyltransferase, a Golgi membrane marker, were not detected in bile. 3. Rodent bile contained 15 polypeptide bands that differed radically from those of bile-canalicular membranes. Bands that may correspond in molecular weight to liver plasma-membrane glycoproteins were present at low staining intensities in bile. A major protein of apparent molecular weight 49 500 was present, and albumin was detected by immunodiffusion. 4. The lipid composition of bile and bile-canalicular membrane also differed. Phosphatidylcholine accounted for 82% of rat bile phospholipids, and only trace amounts of phosphatidylinositol, phosphatidylserine and sphingomyelin were present. 5. The results indicate that in healthy animals, the bile-canalicular membrane is refractory to the action of bile acids during the secretory process. The presence of only small amounts of bile-canalicular membrane components, especially glycoprotein enzymes located at the outer face of the membrane, suggests that these are released from the membrane by bile acids after secretion of bile into the canalicular spaces.

Electron microscopic cytochemical characterization of bile canaliculi and bile ductsin vitro

Electron microscopic cytochemical localization of Mg++-activated adenosine triphosphatase (Mg++-ATPase) and 5-nucleotidase (AMPase) was investigated in bile canaliculus-rich and bile duct-containing fractions isolated from rat liver. Comparative cyochemical studies between prefixed and non-prefixed fractions revealed that the activity of both enzymes could be detected in the fractions under appropriate experimental conditions. However, the cytochemical activity of AMPase was much more sensitive to glutaraldehyde than that of Mg++-ATPase. Mg++-ATPase and AMPase reaction products were localized primarily on bile canalicular microvilli, that is, along the outer (luminal) surface of canalicular plasma membranes, but they were never observed on bile ductal microvilli. AMPase was also detectable on lateral hepatic plasma membranes. Mg++-ATPase demonstrated by the cytochemical technique described is a reliable enzyme marker for isolated bile canalicular membranes. At high magnification, Mg++-ATPase reaction product was also observed on the microfilaments surrounding isolated bile canaliculi. The possibility that the reaction product on the pericanalicular microfilaments may result from the hydrolysis of ATP byan actomyosin ATPase-like enzyme associated with these filaments is briefly discussed.

Characterization of rat liver cell plasma membranes.

A method is described by which bile canalicular membranes (BCM) can be prepared, together with canaliculus-free plasma membrane (PM), both essentially free of contamination. The recovery of both fractions together was estimated to be 46%. The concentrations of total lipid, total phospholipid and cholesterol were substantially greater in the BCM, and polyacrylamide gel electrophoresis revealed differences in protein composition. The differences in lipid and protein composition of these two plasma membrane fractions are presumably related to their very different physiological functions.

Liver cell plasma membrane lipids and the origin of biliary phospholipid.

The liver cell plasma membranes of fed male Wistar rats were separated into a fraction rich in bile canaliculi and the remainder of the plasma membrane. Electron-microscopically, the bile canalicular fraction consisted almost exclusively of intact bile canaliculi with thier contiguous membranes. The remaining plasma membrane fraction consisted primarily of vesicles and sheets of membranes essentially free from the bile canaliculi. The bile canalicular membrane fraction contained relatively more total lipid, cholesterol, and phospholipid, and relatively less protein. Although the phospholipid composition of the two fractions was the same, the specific activity of the bile canalicular membrane phosholipids, up to 12 h following in vivo administration of [2-3H]glycerol, was always significantly greater than that of the remaining plasma membranes, and showed a biphasic response not found in the latter. The specific activity of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membranes rose to a peak within 40 min after administration of the label, fell sharply and then rose to a second peak after 120 min. The specific activity of the sphingomyelin and phosphatidylserine plus phosphatidylinositol of the bile canalicular membranes and of all the phospholipids of the remaining plasma membranes diphasic pattern but increased steadily to reach a maximum at 120 min. The specific activity of biliary phosphatidylcholine followed a pattern identical to that of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membrane fraction. These results show that the average rate of turnover of phospholipid in the bile canalicular membranes is considerably greater than that in the remaining plasma membrane and other cell membrane fractions; they indicate that the phospholipid of the bile canalicular membranes exists in two or more pools, turning over a different rates; and they support the concept that biliary phospholipid is derived from the bile canalicular membrane. The results also suggest that bile canalicular phospholipid may be derived from two different sources, in contrast to the remainong plasma membrane.

Isolation and partial characterization of a bile canalicular plasma membrane fraction from normal and regenerating rat liver

A bile canalicular membrane fraction was isolated from 24-hour regenerating rat livers, and its properties were compared to those of homologous fractions prepared from the livers of sham-operated and unoperated controls. These canalicular membrane fractions were found to be closely related in terms of their morphology, their purity, their yield, and their qualitative protein banding profiles on sodium dodecyl sulfate-polyacrylamide gels. However, when a rigorous examination of plasma membrane enzyme marker activities was made, the regenerating liver membranes were shown to possess an increased specific activity of alkaline phosphatase and lower levels of Mg2+ ATPase and 5'-nucleotidase in comparison with control specific activity values.

Mechanism of secretion of biliary lipids. I. Role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lecithin and cholesterol.

The role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lipids was investigated by using the isolated perfused rat liver model. Labeled lecithin precursors ((3H)-palmitic acid, (14C)linoleic acid, (3H)choline, and 32PO4) and a cholesterol precursor ((3H)mevalonic acid) were administered with and without sodium taurocholate. The incorporation pattern of these labeled precursors into linoleyl and arachidonyl lecithins and cholesterol fractions of microsomes, bile canaliculi, and bile were examined at 30-min intervals up to 90 min. Marker enzymes and electron microscopy indicated that isolated subfractions of plasma membranes were enriched with bile canaliculi (less than 10 percent microsomal contamination). Taurocholate significantly stimulated the incorporation of 32PO4, (3H)choline, (3H)palmitic acid, and (14C)linoleic acid into linoleyl and arachidonyl lecithin with parallel incorporation curves for microsomal and bile canalicular membranes throughout the 90-min study period. During the 30-60-min period, however, these same lecithin fractions in bile significantly exceeded the specific activity of the membrane lecithins. The enzyme CDP-choline diglyceride transferase was virtually absent from canaliculi relative to microsomes, indicating that canaliculi lack the capacity for de novo lecithin synthesis. Incorporation of (3H)mevalonic acid into membranous and biliary cholesterol followed a pattern similar to that for lecithin. These data provide evidence that (a) biliary lecithin and cholesterol are derived from a microsomal subpool regulated by the flux of enterohepatic bile acids, (b) the role of the bile canalicular membranes with respect to biliary lipids is primarily transport rather than synthesis, and (c) lecithin and cholesterol are transported together from microsomes to bile. The findings are consistent with the existence of a cytoplasmic lipid complex within the hepatocyte which is actively involved in the intermembrane transport of biliary lipid.

α-Naphthyl-isothiocyanate-induced cholestasis in the rat: studies of liver plasma membrane enzymes

Oral administration of a single dose of alpha-naphthyl-isothiocyanate (ANIT) to rats produced a conjugated hyperbilirubinaemia, maximal at 2 days and which subsided by 7. The activities of 3 liver plasma membrane enzymes, Mg-2+-ATPase, (Na-+-K-+)-ATPase and 5-nucleotidase, and serum bilirubin levels were studied for up to 7 days after treatment. Activities of the 3 enzymes were significantly decreased at 2 days after treatment and returned to normal by 7, thus varying inversely with the degree of hyperbilirubinaemia. Enzyme histochemistry used to demonstrate canalicular localization of Mg-2+-ATPase in sections of whole liver and of isolated plasma membrane pellets showed that the reduction in activity was not a uniform partial loss, but represented a range of reductions in most canaliculi with a few retaining normal staining intensity. The results suggest that after ANIT intoxication there is a membrane lesion which may be responsible for the observed hyperbilirubinaemia due to the failure of secretion of biliary constituents into the canaliculus. However, more direct studies are necessary to determine whether any one of these enzymes is directly involved in the transport of biliary constituents across the bile canalicular membrane.

Function and Control of Liver Alkaline Phosphatase

Abstract Partial hepatectomy causes a large rise in the activity of liver alkaline phosphatase that takes place exclusively in the plasma membrane. The activity of the membrane phosphatase can be raised in the livers of unoperated rats by infusing the animals with glycine, heparin, phosphorylcholine, phosphatidylcholine, or cortisol. The effects of glycine and heparin are additive. l-Serine is as effective as glycine but none of the other amino acids tested has activity. Any of several compounds that are known to increase the supply of fatty acids to the liver can substitute for heparin. The increase in phosphatase activity is accompanied by rises in the rates of excretion of choline and phosphatidylcholine into the bile. Folate blocks the increases in the phosphatase activity and in the excretion of choline that are ordinarily produced by a mixture of glycine and heparin or by cortisol. Neither phosphorylcholine nor alkaline phosphatase activity is detectable in samples of freshly collected rat bile. The membrane phosphatase is able to hydrolyze phosphorylcholine. Taken together, these observations have suggested that at least one of the functions of the alkaline phosphatase of the liver membrane is to hydrolyze phosphorylcholine so that choline can be transported across the bile canalicular membrane. The level of the enzyme activity appears to be controlled by the quantity of phosphorylcholine that is available for excretion into the bile. Partial hepatectomy leads to alterations in 1-carbon and phospholipid metabolism that result in increases in liver phosphorylcholine and in the activity of the membrane phosphatase. The rise in alkaline phosphatase activity is not essential for the entry of the liver cell into the period of DNA replication.

Canalicular bile formation in the isolated perfused rat liver.

Canalicular bile formation in the isolated perfused rat liver.

Canalicular bile production in dogs.

Canalicular bile production in dogs.