The lipid composition of plasma membrane subfractions originating from the three major functional domains of the rat hepatocyte cell surface

1. The neutral and phospholipid compositions of three rat liver plasma membrane subfractions originating predominantly from the three major functional domains of the hepatocyte viz the blood sinusoidal, contiguous and bile canalicular fractions, were determined. 2. The sinusoidal and canalicular plasma membrane subfractions, both of which were vesicular, contained a higher lipid to protein weight ratio than the contiguous plasma membrane subfraction that consisted of membrane strips, junctional complexes and some larger vesicles. The three plasma membrane subfractions contained a similar neutral lipid to phospholipid ratio. The highest unesterified cholesterol content was associated with the canalicular plasma membrane subfraction. 3. The phospholipid profiles of the three subfractions were generally similar. However, the canalicular plasma membrane subfraction contained a higher proportion of sphingomyelin than the other subfractions. 4. Correlations between the neutral and phospholipid composition of the subfractions and membrane integrity and function are discussed, especially with respect to a possible role of lipids in governing the resilience of the canalicular plasma membrane to the action of bile salts.     

Ethinylestradiol treatment induces multiple canalicular membrane transport alterations in rat liver

We investigated the effects of 17 alpha-ethinylestradiol treatment of rats on various transport functions in isolated basolateral and canalicular liver plasma membrane vesicles. Both membrane subfractions were purified to a similar degree from control and cholestatic livers. Although moderate membrane lipid alterations were predominantly observed in basolateral vesicles, no change in basolateral Na+/K(+)-ATPase activity was found. Furthermore, while Na(+)-dependent taurocholate uptake was decreased by approximately 40% in basolateral vesicles, the maximal velocity of ATP-dependent taurocholate transport was decreased by 63% in canalicular membranes. In contrast, only minimal changes or no changes at all were observed for electrogenic taurocholate transport in "cholestatic" canalicular membranes and total microsomes, respectively. However, canalicular vesicles from cholestatic livers also exhibited marked reductions in ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione and in Na(+)-dependent uptake of adenosine, while in the same vesicles HCO3-/SO4- exchange and Na+/glycine cotransport activities were markedly stimulated. These data show that in addition to the previously demonstrated sinusoidal transport abnormalities ethinylestradiol-induced cholestasis is also associated with multiple canalicular membrane transport alterations in rat liver. Hence, functional transport alterations at both polar surface domains might ultimately be responsible for the inhibitory effects of estrogens on the organic anion excretory capacity and on bile formation in rat liver.     

Greater emotional distress is associated with accelerated CD4+ cell decline in HIV infection

The hepatic transport of the immunosuppressive Cyclosporin A (CyA) was studied using liposomal phospholipid membranes, freshly isolated rat hepatocytes and bile canalicular plasma membrane vesicles from rat liver. The Na(+)-dependent, saturable uptake of the bile acid 3H-taurocholate into isolated rat liver cells was apparently competitively inhibited by CyA. However, the uptake of CyA into the cells was neither saturable, nor temperature-dependent nor Na(+)-dependent, nor could it be inhibited by bile salts or CyA-derivatives, indicating passive diffusion. In steady state depolarization fluorescence studies, CyA caused a concentration-dependent decrease of anisotropy, indicating a membrane fluidizing effect. Ion flux experiments demonstrated that CyA dramatically increases the permeability of Na+ and Ca2+ across phospholipid membranes in a dose- and time-dependent manner, suggesting a iontophoretic activity that might have a direct impact on cellular ion homeostasis and regulation of bile acid uptake. Photoaffinity labeling with a [3H]-labeled photolabile CyA-derivative resulted in the predominant incorporation of radioactivity into a membrane polypeptide with an apparent molecular weight of 160,000 and a minor labeling of polypeptides with molecular weights of 85,000-90,000. In contrast, use of a photolabile bile acid resulted in the labeling of a membrane polypeptide with an apparent molecular weight of 110,000, representing the bile canalicular bile acid carrier. The photoaffinity labeling as well as CyA transport by canalicular membrane vesicles were inhibited by CyA and the p-glycoprotein substrates daunomycin and PSC-833, but not by taurocholate, indicating that CyA is excreted by p-glycoprotein. CyA uptake by bile canalicular membrane vesicles was ATP-dependent and could not be inhibited by taurocholate. CyA caused a decrease in the maximum amount of bile salt accumulated by the vesicles with time. However, initial rates of [3H]-taurocholate uptake within the first 2.5 min remained unchanged at increasing CyA concentrations. In summary, the data indicate that CyA does not directly interact with the hepatic bile acid transport systems. Its cholestatic action may rather be the result of alterations in membrane fluidity, intracellular effects and an interaction with p-glycoprotein.     

The clinical significance of type 1 fiber predominance

Having recently demonstrated that taurine supplementation prevents total parenteral nutrition (TPN)-induced cholestasis, we chose to use this model to examine plasma membrane composition in relation to bile formation. Male guinea pigs received daily a mixture of glucose and of the amino acid solution Travasol with or without added taurine (1.2 mM). After 3 days, bile was collected and liver plasma membrane fractions enriched in sinusoidal lateral membrane and bile canalicular membrane domains were isolated. In animals receiving TPN alone, bile flow and biliary secretory rate of bile acid and bicarbonate decreased significantly compared with controls. Although membrane ATPases (Na+K+ and Mg+) were unchanged, TPN induced an increase in the lipid to protein ratio and a decrease of polyunsaturated fatty acids, in conjunction with a higher content of diene conjugates in sinusoidal lateral membrane fractions. Taurine corrected these changes and, in addition, reduced significantly the cholesterol to phospholipid ratio in both membrane fractions. The data show that changes in liver cell membranes occur in TPN-induced cholestasis and suggest that free radical injury may play a role. As taurine prevented cholestasis as well as membrane changes, it is suggested that taurine should be added to amino acid solutions used for parenteral nutrition.     

Patterns of optic disk damage in patients with early focal visual field loss

The role of cysteinylglycine S-conjugate dipeptidases in the intrahepatic mercapturic acid pathway was investigated in rat liver. Subcellular compartmentation studies and liver perfusions were performed using monochlorobimane and bimane S-conjugates as model compounds. The major part (over 95%) of total hepatic cysteinylglycine S-conjugate dipeptidase activity was located in the cytosol. Lower specific activity appeared in the canalicular plasma membrane fraction. Similar hepatic localization of dipeptidase activity was seen in the guinea pig. In intact rat liver perfused with monochlorobimane, the major products were the glutathione S-conjugate (mBSG) and the cysteinylglycine S-conjugate (mBCG) in bile. Minor amounts of the cysteine S-conjugate (mBCys) and the mercapturic acid (mBNAc) were formed, indicating a limitation in further metabolism of the dipeptide S-conjugate in the biliary space. However, when the dipeptide S-conjugate was offered to the sinusoidal space in liver perfusions, substantial uptake and conversion to mBNAc was observed, and only trace amounts of the infused dipeptide appeared in bile. The data suggest that cytosolic cysteinylglycine S-conjugate dipeptidase as identified here is involved in hepatic mercapturic acid formation from sinusoidal cysteinylglycine S-conjugates. This is especially of significance for species such as guinea pig and human, in which dipeptide S-conjugates are generated in the sinusoidal domain of the liver due to the presence of high gamma-glutamyltranspeptidase activity.     

Intravenous ursodeoxycholic acid reduces cholestasis in parenterally fed newborn piglets

BACKGROUND & AIMS: Cholestasis complicates total parenteral nutrition (TPN) in preterm infants. Ursodeoxycholic acid (UDCA) is used for several cholestatic problems. The hypothesis of this study was that intravenous UDCA prevents TPN-induced cholestasis by (1) maintaining normal basal and stimulated bile flow, (2) altering bile composition, and (3) changing hepatocyte membrane composition and Na+,K(+)-adenosine triphosphatase (ATPase) activity. METHODS: Three groups of piglets were studied: group 1 received sow's milk, groups 2 and 3 received TPN, and group 3 also received 100 mumol.kg-1.day-1 UDCA intravenously. After 3 weeks, basal and stimulated bile flow were measured. Cholesterol, bile acids, phospholipids, and phospholipid fatty acids were analyzed in bile, and fluidity, phospholipid fatty acid composition, and Na+,K(+)-ATPase were analyzed in hepatocyte membranes. RESULTS: Bile acid secretion and basal and stimulated bile flow were similar in control and UDCA-treated animals but reduced to < 50% in the TPN group. Bile acid-dependent and -independent bile flow were lower in the TPN group. UDCA did not normalize abnormalities in TPN-induced bile composition. Sinusoidal but not canalicular membrane fluidity was different in TPN than in control and UDCA-treated animals. UDCA also increased Na+,K(+)-ATPase activity. Bile and membrane phospholipid fatty acids reflected dietary fatty acids. CONCLUSIONS: Intravenous UDCA improves bile flow and reduces bilirubin levels in the serum and liver in piglets with TPN-induced cholestasis.     

Time-related changes of cold-stored rat liver in University of Wisconsin solution. Effect of ursodeoxycholate

Livers of Wistar rats were stored between 0 and 36 hrs. in the University of Wisconsin preservation liquid in order to determine time-related biochemical and morphological hepatic changes. Ursodeoxycholate (100 microM) was also added in the medium to test the hepatoprotective properties of the bile salt. Biochemical assays were performed on hepatic microsomes, plasma and biliary canalicular membranes. Protein and lipid composition of the microsomal and baso-lateral plasma membranes remained stable. Protein and cholesterol content of the biliary canalicular membranes decreased, phospholipid/cholesterol ratio increased between 0 and 36 hrs.; it resulted in a leak of 5'-nucleotidase and leucine amino peptidase activity of these biliary canalicular membranes, especially up to 12 hrs. Between 0 and 36 hrs., the lipid and protein content remained stable in the plasma membranes, as well as both tested enzymatic activities. Observations under electron microscopy showed alterations and underlined fragility of the bile canaliculi, particularly after 24 hrs. preservation. Ultrastructure of sinusoidal membranes showed damaged microvilli. Endoplasmic reticulum remained unchanged, in relation to the stability of the microsomal lipidic, proteic content and hydroxymethylglutaryl-coenzyme A reductase activity, except the decreased protein content after preservation for 36 hrs without ursodeoxycholate. Ursodeoxycholate by itself did not protect against the described disturbances.     

Hepatic metabolism and transport of bilirubin and other organic anions

Most of bilirubin, bile acids and other organic anions are preferentially taken up by the liver and excreted into bile. Recently many transporters on the sinusoidal and canalicular membranes of the hepatocytes have been reported for each ligand. complementary DNA was cloned for human Na+/taurocholate cotransporting polypeptide (NTCP) which mediates sodium dependent secondary active hepatic uptake of bile acids. For the hepatic uptake of non-bile acid-organic anions such as bilirubin, at least 4 transporters are postulated, i.e., bilirubin/BSP binding protein (BBBP), organic anion binding protein (OABP), bilitranslocase, and organic anion transporting polypeptide (OATP). In the hepatocytes, bilirubin is glucuronidated in the endoplasmic reticulum. The gene for UDP-glucuronosyltransferase (UGT) 1 family has been elucidated and differential splicing from several exons 1 (A to J) results in forming isozymes of UGT 1 including bilirubin UGT. At the canalicular membranes, two main ATP-dependent organic anion transporters have been reported, i.e., canalicular bile salt transporter (cBST) for bile acids and canalicular multispecific organic anion transporter (cMOAT) for non-bile acid organic anions. Recently multidrug resistance protein (MRP) is reported closely related to or identical to cMOAT. These canalicular ATP-dependent transporters are called ABC (ATP-binding cassette) transporters.     

Current concepts of hepatic uptake, intracellular transport and biliary secretion of bile acids: physiological basis and pathophysiological changes in cholestatic liver dysfunction

Hepatic sinusoidal uptake of bile acids is mediated by defined carrier proteins against unfavourable concentration and electrical gradients. Putative carrier proteins have been identified using bile acid photoaffinity labels and more recently using immunological probes, such as monoclonal antibodies. At the sinusoidal domain, proteins with molecular weights of 49 and 54 kDa have been shown to be carriers for bile acid transport. The 49 kDa protein has been associated with the Na(+)-dependent uptake of conjugated bile acids, while the 54 kDa carrier has been involved in the Na(+)-independent bile acid uptake process. Within the hepatocyte, cytosolic proteins, such as the glutathione S-transferase (also designated the Y protein), the Y binders and the fatty acid binding proteins, are able to bind bile acids and possibly facilitate their movement to the canalicular domain. At the canalicular domain a 100 kDa carrier protein has been isolated and it has been shown by several laboratories that this particular protein is concerned with canalicular bile acid transport. The system is ATP-dependent and follows Michaelis-Menten kinetics. Interference with bile acid transport has been demonstrated by several chemicals. The mechanisms by which these chemicals inhibit bile acid transport may explain the apparent cholestatic properties observed in patients and experimental animals treated with these agents. Several studies have shown that Na+/K(+)-ATPase activity is markedly decreased in cholestasis induced by ethinyloestradiol, taurolithocholate and chlorpromazine. However, other types of interference have been described and the cholestatic effects may be the result of several mechanisms. Cholestasis is associated with several adaptive changes that may be responsible for the accumulation of bile acids and other cholephilic compounds in the blood of these patients. It may be speculated that the nature of these changes is to protect liver parenchymal cells from an accumulation of bile acids to toxic levels. However, more detailed quantitative experiments are necessary to answer questions with regard to the significance of these changes and the effect of various hepatobiliary disorders in modifying these mechanisms. It is expected that the mechanisms by which bile acid transport is regulated and efforts to understand the molecular basis for these processes will be among the areas of future research.     

Assembly of hepatic gap junctions. Topography and distribution of connexin 32 in intracellular and plasma membranes determined using sequence-specific antibodies

The subcellular distribution in rat liver and the topography in intracellular and plasma membranes of connexin 32, a major protein component of gap junctions, was studied using sequence-specific anti-peptide antibodies generated to extracellular and intracellular domains of the protein. The distribution of connexin 32 in liver analyzed using SDS-polyacrylamide gel electrophoresis and Western blotting showed the relative protein levels in the subcellular fractions to be: lateral plasma membranes > Golgi membranes > sinusoidal plasma membranes > lysosomes. Low amounts of connexin 32 were detected in microsomes, endosomes, and bile canalicular plasma membranes. Six highly conserved cysteine residues are located in the amino acid sequences comprising the two extracellular loops of all connexins thus far isolated, and these loops are positioned to extend the channel in the lipid bilayers across the intercellular region of the gap junction. In the present work, the intramolecular disulfide bonds linking the extracellular loops in gap junctions were shown to be present in connexins located in plasma membranes, Golgi, and a microsomal fraction, and it was concluded that the disulfide linkages were formed in the endoplasmic reticulum. In addition, immature configurations of connexin 32, probably occurring during membrane insertion, were detected in liver microsomal fractions. The results contribute to charting of the biogenetic routes followed by connexins in hepatocytes and the general mechanisms of gap junction assembly.     

The rat liver ecto-ATPase is also a canalicular bile acid transport protein

A approximately 110-kDa glycoprotein purified from canalicular vesicles by bile acid affinity chromatography has been identified as the canalicular bile acid transport protein. Internal amino acid sequence and chemical and immunochemical characteristics of this protein were found to be identical to a rat liver canalicular ecto-ATPase. In order to definitively determine whether these were two activities of a single polypeptide, we examined the possibility that transfection of cDNA for the ecto-ATPase would confer bile acid transport characteristics, as well as ecto-ATPase activity, on heterologous cells. The results show that transfection of the ecto-ATPase cDNA conferred on COS cells de novo synthesis of a approximately 110-kDa polypeptide, as immunoprecipitated by antibody to the purified canalicular bile acid transport protein and conferred on COS cells the capacity to pump out [3H]taurocholate with efflux characteristics comparable with those previously determined in canalicular membrane vesicles (Km = 100 microM; Vmax = 200 pmol/mg of protein/20 s). A truncated ecto-ATPase cDNA, missing the cytoplasmic tail, was targeted correctly to the cell surface but did not confer bile acid transport activity on COS cells. The results of this study also show that the canalicular ecto-ATPase/bile acid transport protein is phosphorylated on its cytoplasmic tail and that its phosphorylation is stimulated by activation of protein kinase C and inhibited by inhibitors of protein kinase C activation. Moreover, inhibition of protein kinase C activation by staurosporine completely abrogates bile acid transport but does not affect ATPase activity. This study, therefore, demonstrates that the rat liver canalicular ecto-ATPase is also a bile acid transport protein, that the capacity to pump out bile acid can be conferred on a heterologous cell by DNA-mediated gene transfer, and that phosphorylation within the cytoplasmic tail of the transporter is essential for bile acid efflux activity but not for ATPase activity.     

Endothelin A-receptor blockade worsens endotoxin-induced hepatic microcirculatory changes and necrosis

BACKGROUND & AIMS: Endothelin 1 is considered to be an important regulator of sinusoidal blood flow and increases during endotoxemia. The purpose of this study was to investigate the role of endothelin 1 in hepatic microcirculation, oxygen transport, and liver injury during endotoxemia. METHODS: Male Sprague-Dawley rats were continuously infused with 2.5 mL/h of saline, 0.8 mg . kg-1 . h-1 of lipopolysaccharide (LPS), 3 mg . kg-1 . h-1 of BQ-485, an endothelin A-receptor antagonist, or LPS plus BQ-485 for 7 hours. RESULTS: BQ-485 infusion had no significant effect on hepatic microcirculation and liver injury. LPS increased the plasma levels of aspartate aminotransferase (AST) and total bilirubin and decreased the hepatic adenosine triphosphate (ATP) level and bile flow rate. LPS + BQ-485 infusion further increased the plasma levels of AST and total bilirubin and decreased the bile flow rate and the hepatic ATP level. Dual-spot microspectroscopy revealed mild decreases in sinusoidal erythrocyte velocity and oxygen transport in the LPS group and profound decreases in these parameters in the LPS + BQ-485 group. Histological examinations revealed massive necrotic changes in the pericentral regions of the LPS + BQ-485 group. CONCLUSIONS: These results suggest that blockade of endothelin A receptors disturbs hepatic microcirculation and oxygen transport and aggravates the necrotic injury induced by endotoxin.     

The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver

Canalicular secretion of bile salts is a vital function of the vertebrate liver, yet the molecular identity of the involved ATP-dependent carrier protein has not been elucidated. We cloned the full-length cDNA of the sister of P-glycoprotein (spgp; Mr approximately 160,000) of rat liver and demonstrated that it functions as an ATP-dependent bile salt transporter in cRNA injected Xenopus laevis oocytes and in vesicles isolated from transfected Sf9 cells. The latter demonstrated a 5-fold stimulation of ATP-dependent taurocholate transport as compared with controls. This spgp-mediated taurocholate transport was stimulated solely by ATP, was inhibited by vanadate, and exhibited saturability with increasing concentrations of taurocholate (Km approximately 5 microM). Furthermore, spgp-mediated transport rates of various bile salts followed the same order of magnitude as ATP-dependent transport in canalicular rat liver plasma membrane vesicles, i.e. taurochenodeoxycholate > tauroursodeoxycholate = taurocholate > glycocholate = cholate. Tissue distribution assessed by Northern blotting revealed predominant, if not exclusive, expression of spgp in the liver, where it was further localized to the canalicular microvilli and to subcanalicular vesicles of the hepatocytes by in situ immunofluorescence and immunogold labeling studies. These results indicate that the sister of P-glycoprotein is the major canalicular bile salt export pump of mammalian liver.     

Na, K-ATPase--its structure, function and intracellular transport

Na, K-ATPase is an integral plasma membrane protein and plays essential roles such as maintaining sodium and potassium ion gradients across the plasma membrane. The enzyme consists of the alpha and the beta subunits with the stoichiometry of one to one. Three alpha subunit and two beta subunit isoforms have been detected in animal cells with the tissue-specific expression of both subunits. Recent advances in molecular biological studies on the Na, K-ATPase enable us to understand the structure-function relationships and mechanisms of intracellular transport of the enzyme. In this article we review the findings deduced from these studies, especially on the assembly and transport to the plasma membrane of the alpha and beta subunits.     

ATP1AL1, a member of the non-gastric H,K-ATPase family, functions as a sodium pump

The human ATP1AL1-encoded protein (an alpha subunit of the human non-gastric H,K-ATPase) has previously been shown to assemble with the gastric H,K-ATPase beta subunit (gH,Kbeta) to form a functionally active ionic pump in HEK 293 cells. This pump has been found to be sensitive to both SCH 28080 and ouabain. However, the 86Rb+-influx mediated by the ATP1AL1-gH,Kbeta heterodimer in HEK 293 cells is at least 1 order of magnitude larger than the maximum ouabain-sensitive proton efflux detected in the same cells. In this study we find that the intracellular Na+ content in cells expressing ATP1AL1 and gH,Kbeta is two times lower than that in control HEK 293 cells in response to incubation for 3 h in the presence of 1 microM ouabain. Moreover, analysis of net Na+ efflux in HEK 293 expressing the ATP1AL1-gH,Kbeta heterodimer reveals the presence of Na+ extrusion activity that is not sensitive to 1 microM ouabain but can be inhibited by 1 mM of this drug. In contrast, ouabain-inhibitable Na+ efflux in control HEK 293 cells is similarly sensitive to either 1 microM or 1 mM ouabain. Finally, 86Rb+ influx through the ATP1AL1-gH,Kbeta complex is comparable to the 1 mM ouabain-sensitive Na+ efflux in the same cells. The data presented here suggest that the enzyme formed by ATP1AL1 and the gastric H,K-ATPase beta subunit in HEK 293 cells mediates primarily Na+,K+ rather than H+,K+ exchange.     

Importance of chronopharmacokinetics in design and evaluation of transdermal drug delivery systems

BACKGROUND: Sodium-potassium-adenosinetriphosphatase (Na,K-ATPase) is the primary membrane enzyme responsible for the reabsorption of sodium ions in the kidney. It is known that in the nephron the major subunit isoforms of Na,K-ATPase are alpha 1 and beta 1. Previous reports on the presence of alpha 2 and alpha 3 isoforms in the kidney were mixed and controversial. METHODS: Techniques of ultrathin cryosectioning and immunoelectron microscopy were used to study the distribution of alpha subunit isoforms (alpha 1, alpha 2, alpha 3) and beta subunit (beta 1 isoform) of Na,K-ATPase in renal tubular cells. Western blot analysis was used to show the presence of the alpha 3 isoform in the extract of kidney mitochondria. RESULTS: We were able to confirm the previous finding that the alpha 1 isoform and the beta 1 isoform were the preponderant isoforms of the alpha and beta subunits of Na,K-ATPase in the basolateral membrane. In addition, we unexpectedly found the presence of the alpha 3 isoform in the mitochondria of rat renal tubular cells. The alpha 2 and alpha 3 isoforms were not observed in either the apical or basolateral membrane. CONCLUSIONS: Both immunoelectron microscopy and Western blot analysis of the rat kidney mitochondria confirm the presence of the alpha 3 isoform of Na,K-ATPase in the rat kidney mitochondria. The function of this enzyme in the mitochondria is not clear at this time.