Effects of docosahexaenoic acid on annular lipid fluidity of the rat bile canalicular plasma membrane.

The role of docosahexaenoic acid (DHA) in the fluidity of the annular lipid regions and their associated membrane-bound proteins is still not as well understood as that in the global (bulk) lipid regions. We therefore studied the effects of dietary DHA on the relationship between annular and global lipid fluidity and membrane-bound enzymes such as 5'-nucleotidase and Mg(2)+-ATPase in the rat bile canalicular membrane. Dietary DHA caused significant increases in 5'-nucleotidase and Mg(2)+-ATPase activity and in global and annular lipid fluidity, a higher increase in fluidity in the annular lipids than the global lipids, and a decrease in the cholesterol-to-phospholipid molar ratio in the canalicular membrane. Plasma total cholesterol and LDL cholesterol decreased, and fecal cholesterol increased in the DHA-fed rats. No changes were observed in oxidative markers, but glutathione peroxidase increased in the liver with DHA feeding. Annular lipid fluidity, but not global lipid fluidity, correlated remarkably well with DHA, synchronously with the activities of 5'-nucleotidase and Mg(2)+-ATPase. The data indicate that the DHA-induced increase in annular lipid fluidity is responsible for the increases observed in the enzyme activity. We therefore concluded that the increased activity of membrane-bound enzymes and transporters induced by DHA and the concomitant increase in annular lipid fluidity comprise one of the mechanisms involved in DHA-induced clearance of plasma cholesterol.     

Effects of ethanol on the incorporation of free fatty acids into cerebral membrane phospholipids

Chronic ethanol exposure is known to affect deacylation-reacylation of membrane phospholipids (PL). In our earlier studies we have demonstrated that chronic exposure to ethanol (EtOH) leads to a progressive increase in membrane phospholipase A2 (PLA2) activity. In the current study, we investigated the effects of chronic EtOH exposure on the incorporation of different free fatty acids (FFAs) into membrane PL. The results suggest that the incorporation of fatty acids into four major PL varied from 9.6 fmol/min/mg protein for docosahexaenoic acid (DHA) into phosphatidylinositol (PI) to 795.8 fmol/min/mg protein for linoleic acid (LA) into phosphatidylcholine (PC). These results also suggest a preferential incorporation of DHA into PC; arachidonic acid (AA) into PI; oleic acid into phosphatidylethanolamine (PE) and PC; LA into PC and stearic acid into PE. Chronic EtOH exposure affected the incorporation of unsaturated fatty acid into PI, phosphatidylserine (PS) and PC. However, EtOH did not affect significantly the incorporation of any of the fatty acids (FA) studied into PE. No significant differences were observed with the stearic acid. It is suggested that acyltransferases may play an important role in the membrane adaptation to the injurious effects of EtOH.     

The effect of CMP on the release of free fatty acids of rat brain in vitro

With CMP, phosphatidylcholine can be converted to diacylglycerols and CDPcholine by reversal of the cholinephosphotransferase that is normally used for synthesis. Incubation of homogenates of rat brains at pH 8 with 20 mM MgCl2 increased the free fatty acid (FFA) levels 30 to 117%. The FFA levels increased 62 to 212% when 4 mM CMP was included. Diacylglycerols were also produced. Hydrolysis of the diacylglycerols to FFA was markedly inhibited by inclusion of 3 mM diisopropylphosphofluoridate in the incubation mixture. The composition of the fatty acids released by CMP resembles that of phosphatidylcholine except for some polyunsaturated fatty acids. These may have been released from the ethanolamine glycerophospholipids. Most of the CMP-stimulated release of FFA was blocked by inclusion of 1 mM CDPcholine in the incubation mixture. Rat brains were labeled by intracerebral injection of [3H]oleic acid. The labeled oleic acid was released primarily from phosphatidylcholine. Thus, measurements of both mass and radioactivity confirm that the reversal of cholinephosphotransferase followed by diacylglycerol lipase can be an important pathway for the liberation of FFA from phosphatidylcholine.     

Proteolytic processing of rat liver membrane secretory component. Cleavage activity is localized to bile canalicular membranes

Membrane secretory component (mSC) mediates the transcellular movement of polymeric IgA from the sinusoidal to the bile canalicular surface of rat hepatocytes. Prior to or concomitant with arrival at the bile canalicular membrane, mSC is cleaved, producing a soluble proteolytic fragment (fSC) which is released into the bile. Conversion of mSC to fSC occurs at the cell surface of cultured rat hepatocytes (Musil, L. S., and Baenziger, J. U. (1987) J. Cell Biol. 104, 1725-1733), suggesting that vectorial release of fSC into bile in vivo may reflect localization of a mSC-specific protease to bile canalicular membranes. We have established a reconstituted system to examine the process of specific cleavage of mSC to yield fSC and to characterize the protease activity responsible. A membrane fraction highly enriched for endocytic vesicles was found to contain approximately 90% of the [35S]Cys-mSC from metabolically labeled rat liver slices but only 5% of the cellular protein. No cleavage activity was present in these vesicles. Highly enriched bile canalicular membranes were able to mediate cleavage of metabolically labeled mSC to a fragment indistinguishable from authentic fSC. In the absence of nonionic detergent, cleavage was dependent on the presence of polyethylene glycol, presumably to mediate fusion of mSC-enriched membranes with bile canalicular membranes. Following solubilization with nonionic detergent, cleavage was no longer dependent on the addition of polyethylene glycol. Cleavage of mSC was not observed with either intact or detergent-solubilized sinusoidal, microsomal, or lysosomal membranes. We have thus identified a proteolytic activity associated with bile canalicular membranes which has the properties of a membrane protein and is likely to be responsible for production of fSC in vivo. Its highly restricted localization to the bile canalicular membrane would account for the vectorial release of fSC into the bile.     

In vivo identification of aflatoxin-induced free radicals in rat bile

Aflatoxin B1 (AFB1) is a potent hepatocarcinogen. We have recently detected [via electron spin resonance (ESR) spectroscopy] free radicals in vivo in rat bile following AFB1 metabolism using the spin trapping [alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN)] technique. The aim of the present study was to identify the trapped free radical intermediates from the in vivo hepatic metabolism of AFB1. Rats were treated simultaneously with AFB1 (3 mg/kg i.p.) and the spin trapping agent 4-POBN (1 g/kg i.p.), and bile was collected over a period of 1 h at 20 min intervals. On-line high performance liquid chromatography (HPLC) coupled to ESR was used to identify an arachidonic acid-derived radical adduct of 4-POBN in rat bile, and a methyl adduct of 4-POBN from the reaction of hydroxyl radicals with carbon-13-labeled dimethyl sulfoxide ((13)C-DMSO). The effect of metabolic inhibitors, such as desferoxamine mesylate (DFO), an iron chelator, 2-dimethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF) 525A, a cytochrome P-450 inhibitor, and gadolinium chloride (GdCl(3)), a Kupffer cell inactivator, on in vivo aflatoxin-induced free radical formation were also studied. It was found that there was a significant decrease in radical formation as a result of DFO, SKF525A and GdCl(3) inhibition. Trapped 4-POBN radical adducts were also detected in rat bile following the in vivo metabolism of aflatoxin-M1, one of the hydroxylated metabolites of AFB1.     

Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles

Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity. In contrast to effects induced by taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine. These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity. In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion.     

A domain-specific marker for the hepatocyte plasma membrane. III. Isolation of bile canalicular membrane by immunoadsorption

Previous immunolabeling studies (Roman, L.M., and A.L. Hubbard, 1983, J. Cell Biol., 96:1548-1558; Roman, L.M., and A.L. Hubbard, 1984, J. Cell Biol., 98:1488-1496, companion paper) established leucine aminopeptidase (LAP) as a specific marker for the bile canalicular (BC) domain of the rat hepatocyte plasma membrane (PM). In this study, we have isolated membrane from a sonicated PM vesicle fraction using anti-LAP-coated Staphylococcus aureus cells as a solid-phase immunoadsorbent. The extent and specificity of the immunoadsorption were assessed by following the behavior of LAP (the BC marker) and 32P-labeled membrane phospholipids (a uniform membrane marker). The BC fraction obtained was significantly enriched in LAP (yield: greater than 70% of PM-LAP). Alkaline phosphatase, 5'-nucleotidase, and a 110,000-dalton glycoprotein, HA-4, were enriched in the BC fraction to the same extent as LAP (enzyme or antigen/LAP = 1.0). However, alkaline phosphodiesterase I was not enriched to the same degree (enzyme/LAP = 0.5). Contamination of this BC fraction by membrane derived from the sinusoidal domain and endoplasmic reticulum, as determined from the distribution of the asialoglycoprotein receptor and NADH cytochrome c reductase, respectively, was small (less than 13%).     

The effect of phospholipids on the in vitro polymerization of rat brain tubulin

The association of brain tubulin, as measured by the temperature-dependent development of turbidity at 350 nm, is greatly stimulated by the detergent Nonidet P-40 in crude extracts of rat brain tissue. Stimulation of turbidity development is also obtained with partially purified rat brain tubulin treated with Nonidet or other detergents, or preincubated with phospholipase C or D; treatment with bovine pancreatic phospholipase A₂ produces an inhibition. Exogenous phospholipids, diglycerides, other related derivatives, and lipophilic extracts of tubulin and brain supernatants can also alter the turbidity development. In addition, microtubules arising from tubulin obtained in the presence of Tween-20 or Nonidet P-40 exhibit a 50 and 100% increased specific viscosity, respectively, over that of tubulin prepared in the absence of detergent or in the presence of Kyro or Triton N-101. The effectiveness of these detergents in removing phospholipids from tubulin preparations follows a similar pattern: Nonidet P-40 removes 80%, Tween-20 removes 50%, and Kyro or Triton N-101 removes none. The total mass of microtubule formed, as determined by sedimentation, is the same regardless of the effect of the detergents on the viscosity. The microtubules obtained in the presence of Nonidet P-40 have a normal appearance when examined by electron microscopy, and their composition on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is indistinguishable from that of standard tubulin, especially with regard to the minor protein bands always present in the tubulin preparations. The results obtained suggest that the phospholipids associated to brain tubulin preparations might have a role in determining the association of tubulin and/or the final dimensions of the assembled microtubules.     

Influence of dietary bile acids on formation of bile acids in rat

Various taurine-conjugated bile acids were fed to rats at the 1%-level in the diet for 3 or 7 days and the effect on several hydroxylations involved in the biosynthesis and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The 7α-hydroxylation of cholesterol was inhibited by feeding taurocholic acid, taurocheno-deoxycholic acid and taurodeoxycholic acid for 3 as well as 7 days. No marked inhibition was obtained with taurohyodeoxycholic acid or taurolithocholic acid. The 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was inhibited after 3 as well as 7 days by all bile acids except taurohyodeoxycholic acid. With this acid a marked stimulation of 12α-hydroxylation was observed. The effects of the different bile acids on the 7α-hydroxylation of taurodeoxycholic acid were not very marked. The 6β-hydroxylation of lithocholie acid and taurochenodeoxycholic acid was stimulated by taurocholic acid and taurodeoxycholic acid. The reaction was inhibited by taurochenodeoxycholic acid, at least after 7 days. Taurohyodeoxycholic acid inhibited the 6β-hydroxylation slightly and taurolithocholic acid had no effect. The results were discussed in the light of present knowledge concerning mechanisms of regulation of formation and metabolism of bile acids and it was suggested that the mechanisms may be more complex than previously thought.     

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.     

In vivo detection of aflatoxin-induced lipid free radicals in rat bile

Human kallikrein hK3 (prostate-specific antigen) is a chymotrypsin-like serine protease which is widely used in the diagnosis of prostate cancer. Assays of the enzymatic activity of hK3 in extracellular fluids have been limited by a lack of sensitive synthetic substrates. This report describes the design of a series of internally quenched fluorescent peptides containing an amino acid sequence based on preferential hK3 cleavage sites in semenogelins. Those were identified by 2-D gel electrophoresis analysis and N-terminal sequencing of semenogelin fragments generated by ex vivo proteolysis in freshly ejaculated semen. These peptides were cleaved by hK3 at the C-terminal of certain tyrosyl or glutaminyl residues with k(cat)/K(m) values of 15000-60000 M(-1) s(-1). The substrate Abz-SSIYSQTEEQ-EDDnp was cleaved at the Tyr-Ser bond with a specificity constant k(cat)/K(m) of 60000 M(-1) s(-1), making it the best substrate for hK3 described to date.     

The exchange of erythrocyte membrane phospholipids with rat liver extracts in vitro

Intact rat or human erythrocytes and their isolated (ghost) membranes were incubated with the high speed supernatant fraction of homogenates derived from ³²P-labeled rat livers. Phospholipid molecules were transferred between the red cell membranes and the liver extracts, as reflected by the convergence of their specific radioactivities with time. Whereas ghosts usually approached isotopic equilibrium with the liver supernatant fraction during a few hours of incubation at 37° C, the exchange of phospholipids by intact cells was no more than one-half, even after 18 hr. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin were all exchanged in both intact cells and ghosts, albeit to different extents. (A control experiment, incubating ³²P-labeled rat erythrocytes or ghosts with unlabeled rat liver extracts, also demonstrated the exchange of all four major phospholipids.) These data may signify that the phospholipids on the cytoplasmic side of the membrane of intact erythrocytes do not exchange with the phospholipids in exogenous liver extracts. If so, all four major phospholipid classes would appear to be present to some extent at both membrane surfaces. The first inference is in agreement with several other studies on this membrane, while the second inference is not.     

Effects of secondary bile acids on the in vitro development of early somite rat embryos

Effects of secondary bile acids--lithocholic (LCA) and deoxycholic (DCA)--on the in vitro development of early somite (10.5 days old) rat embryos were studied. It was shown that an addition to the culture medium of 0.1 mM LCA (final concentration) resulted in 9% growth-retarded and 12% malformed embryos when the duration of exposure was 24 h. When treatment with LCA was prolonged to 48 h, the rate of growth retardation increased to 18% and that of malformations to 40% versus 0.5% for both parameters observed in controls. This could be interpreted as a reversible or time-dependent effect of LCA on the in vitro development of the mammalian embryo. Culture of embryos in medium with 0.5 mM DCA resulted in 22% of growth retardation and 50% of malformations. DCA in 0.1 mM final concentration had only slight and statistically nonsignificant effects. Retardation of growth development could be demonstrated by a decrease in crown-rump length and the number of somites. Among malformed embryos, abnormalities in the development of the neural tube and exencephaly were the most common types of malformations. Abnormalities as well as growth retardation were accompanied by significant pathological changes in structure and perhaps in function of the endodermal visceral yolk sac cells. It could be suggested that secondary bile acids when present in pathophysiological concentrations can affect the embryonic development by direct inhibitory effects and that these effects may be time and dose dependent.     

mdr2 encodes P-glycoprotein expressed in the bile canalicular membrane as determined by isoform-specific antibodies.

We have produced antibodies specific for the three P-glycoprotein (P-gp) isoforms encoded by the mouse mdr1, mdr2, and mdr3 genes. The anti-Mdr2 and anti-Mdr3 antibodies were generated against synthetic peptides derived from the "linker" region, whereas the anti-Mdr1 antibody was raised against a fusion protein containing the amino terminus of Mdr1. Western blot analysis showed that the three antibodies could discriminate between the three isoforms in membrane fractions from Hamster cells transfected with the corresponding full-length or chimeric mdr cDNAs. Immunocytochemistry studies of mdr-transfected cells showed that the three antibodies specifically recognized each P-gp isoform expressed in whole cells. Immunoblotting of normal mouse tissues revealed that the Mdr2 isoform was expressed at very high levels in liver canalicular membrane vesicles (CMV) but not in membrane vesicles prepared from the basolateral (sinusoidal) domain (SMV). Mdr3 was detected in intestinal brush border membrane vesicles and also in CMV, although at levels much lower than Mdr2. Mdr1 was not detected in CMV or SMV but was detected in endometrial tissue from the gravid uterus. Photolabeling experiments with [125I]iodoarylazidoprazosin followed by immunoprecipitation with isoform-specific antibodies indicated that, in CMV, Mdr3 but not Mdr2 could bind the drug analogue.     

Intestinal microflora and bile acids. Effect of bile acids on the distribution of microflora and bile acid in the digestive tract of the rat

The population levels of intestinal microflora and bile acid composition in the digestive tract were examined in rats fed bile acids to determine the relationships between gastrointestinal microflora and the host. The population level of Bacteroides was increased in the ceca of rats fed cholic acid or deoxycholic acid. In the ileum, the concentration of conjugated bile acid in rats fed cholesterol, cholic acid, hyodeoxycholic acid or lithocholic acid was higher than that in control rats, and was very low in ceca and feces of all the rats. The concentration of total free bile acid was much higher in the ceca than in the ilea of rats fed hyodeoxycholic acid or lithocholic acid. Cholic acid and deoxycholic acid were found in the ilea, ceca and feces of the cholic acid-fed rats. In the deoxycholic acid-fed rats, cholic acid was localized in the ileum. 7-Ketodeoxycholic acid was also found in the ceca of the cholic acid-fed rats. 12-Ketolithocholic acid was found in the feces of rats fed cholic acid or deoxycholic acid. 3-Ketocholanic acid was found in some samples from the lithocholic acid-fed rats. Therefore, some kinds of bile acids influence the population levels of gastrointestinal microflora and bile acid composition in the intestine.     

Oncostatin M regulates membrane traffic and stimulates bile canalicular membrane biogenesis in HepG2 cells

Hepatocytes are the major epithelial cells of the liver and they display membrane polarity: the sinusoidal membrane representing the basolateral surface, while the bile canalicular membrane is typical of the apical membrane. In polarized HepG2 cells an endosomal organelle, SAC, fulfills a prominent role in the biogenesis of the canalicular membrane, reflected by its ability to sort and redistribute apical and basolateral sphingolipids. Here we show that SAC appears to be a crucial target for a cytokine-induced signal transduction pathway, which stimulates membrane transport exiting from this compartment promoting apical membrane biogenesis. Thus, oncostatin M, an IL-6-type cytokine, stimulates membrane polarity development in HepG2 cells via the gp130 receptor unit, which activates a protein kinase A-dependent and sphingomyelin-marked membrane transport pathway from SAC to the apical membrane. To exert its signal transducing function, gp130 is recruited into detergent-resistant membrane microdomains at the basolateral membrane. These data provide a clue for a molecular mechanism that couples the biogenesis of an apical plasma membrane domain to the regulation of intracellular transport in response to an extracellular, basolaterally localized stimulus.     

Partitioning of bile acids into subcellular organelles and the in vivo distribution of bile acids in rat liver.

1. The subcellular distribution of conjugates of cholic acid and chenodeoxycholic acid between cytosol, nuclei, mitochondria and microsomes in rat liver has been determined. 2. The partition coefficients for the distribution of these bile acids between subcellular fractions and buffer have been measured and used to construct a compartmental model of the amounts of conjugated bile acids present in the different subcellular organelles in vivo. 3. This model indicates that a large percentage of the bile acid in the rat liver is found in the nuclear fraction; 42% of the cholic acid conjugates and 27% of the chenodeoxycholic acid conjugates. Substantial amounts of bile acid are also present in microsomes and mitochondria suggesting that published estimates of the amounts of bile acids in these fractions are underestimates. 4. The model also allows the amount of bile acid which is in free solution in cytosol to be determined; 10.9% of the cholic acid conjugates and 4.1% of the chenodeoxycholic acid conjugates in rat liver were present in this fraction. Knowlege of the amount of free bile acid allows possible roles of the cytosolic bile binding proteins to be assessed.