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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.

Hepatic bilirubin excretion requires UDP-glucuronosyltransferase-mediated glucuronidation. Patients with type I Crigler-Najjar syndrome and mutant rats (Gunn strain) inherit deficiency of UDP-glucuronyltransferase activity toward bilirubin as an autosomal recessive trait and, as a result, exhibit marked nonhemolytic unconjugated hyperbilirubinemia throughout postnatal life. Heterozygous carriers of the trait have normal serum bilirubin levels. Because of placental excretion of unconjugated bilirubin, type 1 Crigler-Najjar syndrome patients and Gunn rats are not jaundiced in utero, making prenatal diagnosis difficult. Here we report a diagnostic method in Gunn rats based on genomic DNA analysis for prenatal recognition of deficiency of UDP-glucuronyltransferase activity toward bilirubin in Gunn rats and identification of heterozygous carriers. We and others have shown that two distinct messenger RNA species (UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible phenol-UDP-glucuronyltransferase messenger RNA) in Gunn rat liver contain identical deletions of a single guanosine residue in their common 3' regions. Loss of the restriction site for the endonuclease BstNI, which results from this deletion, was used as the basis for a diagnostic test. Female heterozygous Gunn rats were mated with male homozygous Gunn rats. Genomic DNA was extracted from the chorionic aspect of placenta of 17-day fetuses or from leukocytes from normal rats, obligate heterozygotes and homozygous Gunn rats. The DNA was sequentially digested with the restriction enzymes EcoRI and BstNI and subjected to Southern-blot analysis with a double-stranded DNA probe for the common region of UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible UDP-glucuronyltransferase messenger RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

The contraction of hepatic endothelial cell fenestrae after exposure to serotonin is associated with an increase in intracellular Ca2+ which is derived from extracellular Ca2+, is inhibited by pertussis toxin and is not associated with activation of phosphoinositol turnover or cAMP. Using cell-attached and excised patches in primary cultures of rat hepatic endothelial cells, we identified a serotonin-activated channel with conductance of 26.4+/-2.3 pS. The channel was also permeant to Na+, K+ and Ca++ but not to anions. In cell-attached patch recordings, addition of serotonin to the bath significantly increased channel activity with Ca2+ or Na+ as the charge-carrying ions. This channel provides a mechanism whereby serotonin can raise the cytosolic Ca2+ concentration in hepatic endothelial cells.

Dubin-Johnson patients, mutant Corriedale sheep and TR- and EHBR mutant rats have recessively inherited defective bile canalicular secretion of many nonbile acid organic anions. The human and ovine mutants have black livers and lysosomal pigment accumulation. The livers in TR- and EHBR mutant rats are not black, and sparse lysosomal pigment accumulation is seen. Previously, we postulated that the unidentified pigment in the Dubin-Johnson syndrome results from the accumulation of tyrosine, phenylalanine and tryptophan metabolites, such as metanephrine, which are normally secreted in bile as organic anions. We tested this hypothesis in TR- rats. 3H-epinephrine was injected intravenously; control rats secreted 2.80% +/- 0.52% of the injected dose in bile as compared with 0.19% +/- 0.07% in TR- rats. From 82% to 90% of biliary radioactivity was due to polar conjugates in control rats and mutant rats. TR- rats retained more of the injected dose in the liver, particularly in lysosomes, and secreted more in urine than did control rats. After feeding control and TR- rats for 4 mo with a rat chow diet supplemented with 4% tyrosine, tryptophan and phenylalanine, the liver did not become grossly black; however, histological and electron microscopic study revealed dense lysosomal pigment accumulation in TR- rats. Intraportal injection of metanephrine resulted in the appearance of black liver in TR- rats that persisted for at least 2 hr and was not associated with pigment accumulation by light or electron microscopic examination.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of nucleoside loss in bile is physiologically desirable because hepatocytes are the main source of nucleosides for animal cells which lack de novo nucleoside biosynthesis. We have demonstrated a Na+ gradient-energized, concentrative nucleoside transport system in canalicular membrane vesicles (CMV) from rat liver by studying [3H]adenosine uptake using a rapid filtration technique. The Na(+)-dependent nucleoside transporter accepts purine, analogues of purine nucleosides and uridine; exhibits high affinity for adenosine (apparent Km, 14 microM); is not inhibited by nitrobenzylthioinosine or dipyridamole, and is present in CMV but not in rat liver sinusoidal membrane vesicles. Adenosine transport in right side-out CMV was substantially greater than with inside-out CMV. CMV also contain abundant ecto-ATPase and ecto-AMPase (5'-nucleotidase). These ectoenzymes were shown to degrade nucleotides into nucleosides which were conserved by the Na(+)-dependent nucleoside transport system.

The Dubin-Johnson syndrome is manifested by conjugated hyperbilirubinemia and pigment accumulation in hepatocellular lysosomes. The TR-rat model is a phenotypic model of the Dubin-Johnson syndrome and is characterized by defective ATP-dependent transport of a group of nonbile acid organic anions, including glutathione-S-conjugates and oxidized glutathione, across the bile canaliculus. Similar ATP-dependent transport mechanisms have been described in erythrocytes. Intact erythrocytes and inverted erythrocyte membrane vesicles from Dubin-Johnson patients, TR-rats and appropriate controls were studied with regard to ATP-dependent transport of dinitrophenyl glutathione and oxidized glutathione. No significant differences were observed, indicating that the erythrocyte and canalicular ATP-dependent transporters for these substrates are functionally and potentially genetically distinct.

Gp170 is a transmembrane glycoprotein that is overexpressed in multidrug-resistant tumor cells and is present in the apical plasma membrane domain of small intestinal mucosal cells. The function of Gp170 was studied in the small intestine of the rat. Jejunal and ileal brush border membrane vesicles, but not basolateral membrane vesicles, manifested adenosine triphosphate (ATP)-dependent transport of daunomycin, a substrate for Gp170, and contained a approximately 170-kilodalton protein that reacts with anti-Gp170 monoclonal antibody. Whereas ATP supported daunomycin transport, nonhydrolyzable ATP analogues were ineffective. ATP-dependent daunomycin transport by brush border vesicles was unidirectional (inside to outside) and temperature dependent and was blocked by Gp170 inhibitors but not by taurocholate or bromsulphalein glutathione. Studies using everted small intestine revealed transport of rhodamine 123, a Gp170 substrate, from the serosal surface through the mucosa and inhibition by Gp170 inhibitors. These results suggest that Gp170 in rat small intestinal brush border membrane vesicles is an ATP-dependent efflux pump responsible for the transport of Gp170 substrates into the small intestinal lumen. Gp170 may protect against exogenously derived potentially damaging hydrophobic cations and contribute to the rarity of small intestinal cancer in humans and many animals.

We have previously shown that efflux of cytotoxic drugs from multidrug resistant (MDR) cell lines can be quantitated at the single cell level using a sensitive fluorescence microscopy technique. Based on the structure of compounds which inhibited the efflux of Rhodamine-123 (Rho-123) using this methodology, we hypothesized that the antiemetic, antihistaminic agent benzquinamide (BZQ) would interfere with P-glycoprotein (P-gp) mediated drug transport and potentiate the effects of anticancer agents in MDR cell lines. We show that BZQ interferes with P-gp mediated drug efflux and increases drug accumulation in MDR cells using Rho-123 as a fluorescent probe. BZQ increases the cytotoxicity of chemotherapeutic agents to both human and hamster MDR cell lines in vitro. A slight increase in cytotoxicity to chemotherapeutic agents is also observed in the parental cell lines with BZQ. BZQ increases [3H]daunorubicin accumulation and inhibits the binding of [125I]iodoaryl azidoprazosin to the P-gp in MDR cells. BZQ is a new agent to increase the cytotoxic effects of anticancer agents in MDR cells and may ultimately prove useful as an adjunct in cancer chemotherapy.

Bile-canaliculus contraction in rat hepatocyte doublets is postulated to involve activation of an actin-myosin system. We examined this hypothesis by determining the relationship between canalicular contraction and cystolic free Ca2+ ([Ca2+]i) concentration after extracellular addition of ATP or microdialysis of myosin light chain kinase or its Ca(2+)-independent fragment, which retains catalytic activity. After incubation of doublets with 200 mumol/L ATP in the absence of extracellular Ca2+, [Ca2+]i peaked at 40 sec and 71% of canaliculi contracted within 4 min. Decreasing effects were observed with equimolar ADP, AMP and nonhydrolyzable ATP, but no effect was observed with adenosine. The effect of extracellular ATP on [Ca2+]i and canalicular contraction was dose dependent. Addition of extracellular Ca2+ and ATP resulted in a plateau level of [Ca2+]i. Cytochalasin D, which depolymerizes actin filaments, inhibited ATP-induced canalicular contraction, but not the increase in [Ca2+]i. Microdialysis of myosin light chain kinase and its Ca(2+)-independent fragment (but not the heat-denatured fragment, albumin, trypsin plus soybean inhibitor or buffer) into one hepatocyte of a doublet resulted in canalicular contraction in 86% of doublets. Injection of myosin light chain kinase or its Ca(2+)-independent fragment did not increase [Ca2+]i within 5 min. These results indicate that (a) the basolateral plasma membrane of hepatocytes has a P2Y-class purinoceptor, (b) increased [Ca2+]i after incubation with ATP is initially due to mobilization from internal sites and (c) canalicular contraction is directly related to [Ca2+]i and activation of an actin-myosin system. The physiological role of extracellular ATP in canalicular contraction is uncertain.

Gunn rats lack UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. In addition, a UDPGT isoform which is active toward 4-nitrophenol and is induced by 3-methylcholanthrene (3-MC) in normal rats, is produced in a nonfunctional truncated form in Gunn rats due to the deletion of a single guanosine residue in the coding region of its mRNA. The hepatic concentration of bilirubin-UDPGT mRNA was lower in Gunn rats than in congeneic normal rats. However, bilirubin-UDPGT mRNA was of apparently normal length and was induced by clofibrate, a known inducer of bilirubin-UDPGT activity. 3' regions of bilirubin- and 3-MC-inducible UDPGT mRNAs have identical nucleotide sequences; the single base deletion in the 3-MC-inducible UDPGT in Gunn rats occurs within this region. Using oligonucleotide primers corresponding to the identical and unique regions of the two mRNAs, and polymerase chain reaction, we amplified segments of mRNAs for the bilirubin- and 3-MC-inducible UDPGTs from normal and Gunn rat livers. Both amplified DNAs in Gunn rats lacked the restriction site for BstNI. Nucleotide sequence determination revealed that bilirubin- and 3-MC-inducible UDPGT mRNAs in Gunn rats contain an identical frame-shift deletion of a single guanosine residue within the common region of their coding sequences.

The uptake of bilirubin diglucuronide (BDG) into isolated rat hepatocytes was investigated in order to characterize the mechanism by which bile pigments are transported by the liver. The BDG uptake by hepatocytes was saturable. The uptake was inhibited by bilirubin, sulfobromophthalein, and bilirubin monoglucuronide, but not by taurocholate. The uptake was not affected by replacement of sodium with other cations except for choline. Only when sodium was replaced with choline, was significant decrease in uptake observed. When chloride was replaced with nitrate, BDG uptake decreased, but it was not changed by replacement with sulfate. Metabolic inhibitors did not affect BDG uptake significantly. Thus bile pigments share a common sodium-independent and electrogenic potential-dependent transporter in liver cell membranes. A high concentration of albumin interferes with BDG uptake.

To characterize the poorly understood mechanisms of intracellular transport of bile acids, fluorescein isothiocyanate-glycocholate was synthesized and its ring-OH-linked structure established by fast atom bombardment, mass spectroscopy and 13C nuclear magnetic resonance. Biliary secretion of fluorescein isothiocyanate-glycocholate and [14C]-labeled glycocholate in rats was similar, in contrast to the biliary secretion of sodium fluorescein and methylamine-conjugated fluorescein isothiocyanate, which are non-bile acid organic anions. After incubation of cultured hepatocyte doublets with fluorescein isothiocyanate-glycocholate, serial quantitative image analysis of fluorescence was performed in the cellular cytoplasm, perinuclear zone and bile canaliculus. Uptake of fluorescein isothiocyanate-glycocholate into the cytoplasm was inhibited by removal of sodium from the medium and by addition of glycocholate or taurocholate. After preincubation with colchicine, but not lumicolchicine, the proportion of perinuclear to cytoplasmic fluorescein isothiocyanate-glycocholate increased during incubation with fluorescein isothiocyanate-glycocholate. Neither fluorescein isothiocyanate-glycocholate uptake nor canalicular secretion was affected. By confocal microscopy the perinuclear zone containing fluorescein isothiocyanate-glycocholate was identified as the Golgi apparatus by fluorescent colocalization with C6-NBD-ceramide, which specifically identifies the Golgi apparatus. We conclude that colchicine inhibits fluorescein isothiocyanate-glycocholate transfer from the Golgi apparatus to the bile canaliculus. These results suggest that intracellular bile acid transport involves microtubule-dependent vesicular movement from the Golgi apparatus to the bile canaliculus. The role of this process in normal physiology is uncertain.

The effects of spouses' alcohol consumption, gender, and role in a marital dispute on observers' attributions of responsibility and evaluations of the spouses were examined. A major goal of the investigation was to determine whether the effects of alcohol on attributions and evaluations are gender related or role related. Based on the participation of 115 male and 181 female undergraduate students, the results partly supported the role-related effects of alcohol consumption. Respondents rated both husband and wife more negatively when they were depicted as the perpetrators than as victims. Alcohol consumption did not affect evaluations of the spouses as perpetrators. However, drinking by the husband and wife before their victimization was associated with more negative evaluations than when their victimization was not preceded by drinking. It is argued that alcohol consumption of the victim and not the perpetrator may play a more significant legitimizing role for spousal violence.

In order to characterize the mechanism for bilirubin transport in the liver, the uptake of bilirubin diglucuronide (BDG) into purified sinusoidal plasma membrane vesicles was investigated. BDG uptake was saturable, and was inhibited by sulfobromophthalein and unconjugated bilirubin, but was not affected by sodium taurocholate. BDG uptake was sodium-independent and was stimulated by intravesicular bilirubin or BDG (trans-stimulation). BDG transport showed strong potential sensitivity; vesicle inside-negative membrane potential created by different anion gradients inhibited BDG uptake whereas vesicle inside-positive membrane potential generated by potassium gradients and valinomycin markedly stimulated BDG transport. These data suggest that BDG, sulfobromophthalein, and probably unconjugated bilirubin share a common transporter in liver cells which is sodium independent, membrane-potential-dependent and capable of exchange. The direction of transport in vivo may be governed by the intracellular concentration of BDG and of other yet unidentified organic anions sharing this transporter.

Multidrug resistance (MDR) genes encode a family of membrane glycoproteins of approximately 170 kD (P-glycoproteins). In man and mouse, the MDR 1 (mdr 1) genes confer resistance to relatively hydrophobic cationic anti-cancer drugs (i.e., vinblastin, adriamycin). Anti-cancer drug sensitivity is restored by addition of other drugs (i.e., verapamil, reserpine) which are also P-glycoprotein substrates. Transfection of MDR 1 genes produces the resistance phenotype and overexpression of P-glycoprotein. Parenchymal cells in several normal tissues express P-glycoprotein in the secretory domain of the plasma membrane (i.e., bile canaliculus of hepatocytes, brush border of proximal tubular, and small intestinal cells). Studies using plasma membrane vesicles of different sidedness derived from the bile canaliculus and small intestinal brush border permit characterization of P-glycoprotein as a unidirectional, temperature dependent, saturable, ATP-dependent transporter which is competitively inhibited by various anti-cancer drugs and other compounds. Transport studies using single cell fluorescence microscopy with image analysis confirm observations in vesicles. No natural substrate has been identified. Structural studies indicate that the requirements for substrates are molecular weight of 350 to 100, hydrophobicity, two planar rings, and a weak cationic charge. Alternative mechanisms of transport function are considered. The identity of P-glycoproteins in normal rat and human tissues has not been established. Antibody reactions suggest that they may belong to the MDR 2 or 3 class. Studies using everted gut sacs suggest that inhibition of P-glycoprotein may facilitate accumulation of anti-cancer drugs in the tissue.

Hepatic uridinediphosphoglucuronate glucuronosyl transferase (UDPglucuronyltransferase, EC 2.4.1.17) is functionally heterogeneous; 4-nitrophenol and bilirubin are representative substrates for two functional forms of the enzyme. UDPglucuronyltransferase activity for bilirubin and 4-nitrophenol was separated from solubilized rat liver microsomes by DEAE-cellulose chromatography and corresponding enzymes were purified. A radioimmunoassay was developed using a rabbit antiserum against purified rat 4-nitrophenol-specific UDPglucuronyltransferase, which precipitated enzyme activities toward both 4-nitrophenol and bilirubin. After treatment with triiodothyronine (T3) (0.55 mg/kg body weight), hepatic microsomal UDPglucuronyltransferase activity for 4-nitrophenol was increased 400% as compared to controls; the enzyme activity for bilirubin was decreased by 80%; the changes in the substrate-specific enzyme activities were reflected in the enzymatically active fractions separated after DEAE-cellulose chromatography. The changes in enzyme activities paralleled changes in the concentrations of the two corresponding UDP glucuronyltransferase proteins in the chromatographic fractions, as measured by radioimmunoassay. The results indicate that the opposite effects of T3 on the two forms of UDPglucuronyltransferase activity is due to its differential effect on corresponding enzyme proteins.

Liver from normal Wistar rats was grafted into the liver of homozygous Gunn rats which are deficient in UDP glucuronyl transferase (UDPGT) (bilirubin) activity. After 3 months, UDPGT activity (bilirubin) remained absent in microsomal suspensions of liver from recipient rats and no bilirubin glucuronide was detected in their bile.

Eight-week-old rats had twofold higher hepatic ligandin concentration than 10-day-old animals as determined immunologically and by steroid isomerase and glutathione S-transferase assays. Increased ligandin content was accompanied by parallel increase in subunit synthesis as determined by [3H]leucine incorporation into each subunit relative to incorporation into total cytosolic proteins. The mRNA content for each ligandin subunit was twofold higher in older animals as determined by cell-free in vitro translation followed by immunoprecipitation and dot hybridization using a ligandin cDNA probe. When poly A mRNA from the postmitochondrial fraction of liver from young or old rats was subjected to agarose gel electrophoresis under denaturing conditions and hybridized to ligandin cDNA probe, a single 11 S band was obtained. With RNA from total liver, an additional 13 S band was obtained, suggesting the existence of a precursor form of ligandin mRNA. Since precursor polypeptides were not observed with RNA from total liver in cell-free in vitro translation systems, the precursor form requires processing to the 11 S form before the mRNA becomes functional.

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.

1. The main bilirubin conjugate in bile of spiny dogfish (Squalus Acanthias) and small skate (Raja Erinacea) is bilirubin monoglucuronide. 2. Microsomal preparations from dogfish and small skate liver have similar bilirubin UDPglucuronyltransferase (UDPGT) activity and catalyze the conjugation of bilirubin with glucose from UDPglucose. 3. The activity of bilirubin glucosidation (UDPGT) was 0.5 times UDPG1T activity in dogfish and 0.15 times in skate liver microsomes. 4. Sodium cholate increased UDPGT and UDPG1T activities in dogfish and skate liver microsomal preparations only minimally, but the detergent markedly increased thermolability of UDPGT in skate liver microsomes.

Fatty acids radioactivity was bound to Z protein in liver after administration of['3H]oleate to rats or to a perfused rat liver preparation. Pretreatment withflavaspidic acid (340 mumol/kg), a potent inhibitor of fatty acid binding to hepatic Zprotein in vitri, effectively reduced oleate radioactivity bound to Z by 90.2 plusor minus 4.3% and 85.0 plus or minus 6.2% in the intact rat and perfused liver, respectively. In spite of this effect, pretreatment of rats with flavaspidic acid did notalter plasma clearance, hepatic uptake, and esterification of ['3H]oleate. In contrast, in the perfused liver preparation, infusion of flavaspidic acid (340 mumol/kg)or bromosulphalein (360 mumol/kg) increased uptake of ['3H]oleate at least twofold,and oleate esterification was decreased by 15-30%. These results suggest that the binding of long-chain fatty acids to Z protein is not an obligatory step in their uptakeby the liver and that Z protein may be involved in fatty acid esterification.

Bilirubin and other cholephilic organic anions are bound to albumin in the circulation; their hepatic uptake involves a carrier-mediated process. To investigate the possible role of serum albumin in the transhepatic transport of a cholephilic ligand, plasma clearance of radioactive bilirubin and its biliary excretion as well as its interaction with plasma proteins were compared between normal and mutant analbuminemic rats (NAR). With a tracer amount of 3H-labeled bilirubin, its plasma clearance and biliary excretion were comparable in both animal groups. However, the plasma clearance of a loading dose of the ligand was significantly increased and its biliary recovery was low in NAR as compared with normal animals. In accord with these findings in vivo, gel permeation chromatographic analysis revealed that the bilirubin binding capacity of serum proteins was significantly lower in NAR than in control animals. When bilirubin was administered to NAR as a mixture with equimolar albumin, its plasma disappearance was considerably decreased and its biliary recovery was increased. Similar effects were observed when albumin was replaced by an equimolar amount of glutathione S-transferases (ligandins). These observations indicate that, although ligand-protein interaction in the circulation is important for directing bilirubin to the plasma membranes of the hepatocyte, this mechanism is not specific for albumin.

Bilirubin diglucuronide (BDG) may be formed in vitro by microsomal UDP glucuronosyl transferase (EC 2.4.1.17)-mediated transfer of a second mole of glucuronic acid from UDP-glucuronic acid, or by dismutation of bilirubin monoglucuronide (BMG) to BDG and unconjugated bilirubin, catalyzed by an enzyme (EC 2.4.1.95) that is concentrated in plasma membrane-enriched fractions of rat liver. To evaluate the role of these two enzymatic mechanisms in vivo, [(3)H]bilirubin mono-[(14)C]glucuronide was biosynthesized, purified by thin-layer chromatography, and tracer doses were infused intravenously in homozygous Gunn (UDP glucuronyl transferase-deficient) rats or Wistar rats. Bilirubin conjugates in bile were separated by high-pressure liquid chromatography and (3)H and (14)C were quantitated. In Gunn rats, the (14)C:(3)H ratio in BDG excreted in bile was twice the ratio in injected BMG. In Wistar rats the (14)C:(3)H ratio in biliary BDG was 1.25 +/- 0.06 (mean +/- SEM) times the ratio in injected BMG. When double labeled BMG was injected in Wistar rats after injection of excess unlabeled unconjugated bilirubin (1.7 mumol), the (14)C:(3)H ratio in BDG excreted in bile was identical to the ratio in injected BMG. Analysis of isomeric composition of bilirubin conjugates after alkaline hydrolysis or alkaline methanolysis indicated that the bile pigments retained the IX(alpha) configuration during these experiments. The results indicate that both enzymatic dismutation and UDP glucuronyl transferase function in vivo in BDG formation, and that dismutation is inhibited by a high intrahepatic concentration of unconjugated bilirubin. This hypothesis was supported by infusion of [(3)H]bilirubin-monoglucuronide in isolated perfused homozygous Gunn rat liver after depletion of intrahepatic bilirubin by perfusion with bovine serum albumin (2.5%), and after bilirubin repletion following perfusion with 0.34 mM bilirubin. From 20 to 25% of injected radioactivity was recovered in BDG in bile in the bilirubin-depleted state; only 8-10% of radioactivity was in BDG in bile after bilirubin repletion. After infusion of [(3)H]bilirubin di-[(14)C]glucuronide in homozygous Gunn rats, 5-7% of the injected pigment was excreted in bile as BMG. The (14)C:(3)H ratio in the injected BDG was 10% greater than the (14)C:(3)H ratio in BMG excreted in bile. These results indicate that in vivo, dismutation rather than partial hydrolysis, is responsible for BMG formation. Incubation of [(3)H]bilirubin, BDG and a rat liver plasma membrane preparation resulted in formation of BMG (3.3 nmol/min per mg protein) indicating that dismutation is also reversible in vitro.

The urinary excretion of ligandin, a proximal tubular enzyme and binding protein, was measured by radioimmunoassay in eight normals, six patients receiving radiocontrast media, and six patients in a critical care unit who were considered at high risk for acute renal failure. Ligandinuria was found to occur normally at rates under 5 micrograms/hr. In the patients receiving radiocontrast media, abnormal rates of ligandinuria were found in four patients. In 102 ligandin measurements in the critically ill patients, rates of ligandinuria exceeded normal only once (after contrast media exposure) despite 13 identifiable episodes of potentially nephrotoxic circumstances and two episodes of acute renal failure. Ligandinuria appears more sensitive as a marker for tubular injury from contrast media than from other renal insults.

Forty patients with superficial mycoses were treated with itraconazole, a new triazole derivative, in an open study. In 29 cases the diagnosis was dermatophytosis; in five, candidosis; and in six, pityriasis versicolor. Each patient received 50 mg of itraconazole orally once a day for 30-60 days. Thirty-three patients completed therapy; at the end of therapy, 21.2% were considered clinically cured and 57.6% were considered mycologically cured. Results of direct microscopic examination were negative at 30 days for the patients with pityriasis versicolor; none of the patients with candidosis responded to therapy. Pruritus and other symptoms or signs of infection improved after the 15th day, but even after 60 days of therapy, nine of the 10 patients who finished the trial at that time had residual lesions. The drug was well tolerated. It appears that a dosage of 50 mg of itraconazole daily is not adequate for the treatment of these mycoses.

Formation of bilirubin monoglucuronide from unconjugated bilirubin requires a microsomal enzyme, UDP-glucuronate glucuronyltransferase (EC 2.4.1.17). Conversion of bilirubin monoglucuronide to bilirubin diglucuronide, the major bilirubin conjugate in bile, was studied in subcellular fractions of rat liver. The highest specific activity for bilirubin diglucuronide formation occurred in a fraction highly enriched in plasma membranes. Studies of reaction stoichiometry and utilization of UDP-D-[14C]glucuronic acid revealed that conversion of bilirubin monoglucuronide to bilirubin diglucuronide is not catalyzed by UDP-glucuronyltransferase, and results from transglucuronidation of bilirubin monoglucuronide, with formation of bilirubin diglucuronide and unconjugated bilirubin. When unconjugated bilirubin was infused intravenously into rats at rates exceeding the maximal hepatic excretory capacity, bilirubin monoglucuronide accumulated in serum and bilirubin diglucuronide was found exclusively in bile as the predominant bilirubin metabolite. These results suggest that formation of bilirubin diglucuronide occurs at the surface membrane of the liver cell. Conversion of bilirubin monoglucuronide to bilirubin diglucuronide may play a role in the transport of bilirubin glucuronides from liver to bile.

Canalicular plasma membrane vesicles isolated from rat liver (right side out) were used to study glutathione disulfide (GSSG) transport. GSSG is transported into an osmotically sensitive compartment. Extrapolation to an external osmolarity of infinity indicates 35% binding after 10 min of incubation, e.g. as mixed disulfides between glutathione and SH-groups in membrane proteins. Glutathione disulfide uptake occurred with equal rates in the presence of KCl or NaCl. After equilibration, no significant GSSG concentration gradient across the canalicular membrane was found. The temperature dependence of uptake is consistent with a carrier-mediated transport for GSSG (Q10 X 1.6) and seems to exclude simple diffusion. Concentration dependence of GSSG uptake shows Michaelis-Menten kinetics at concentrations up to 1 mM; a Km of 0.4 mM and a V of 1.1 nmol X min-1 X mg protein-1 is calculated. At higher concentrations a linear dependence is observed without saturation kinetics. It is concluded that transport of glutathione disulfide is mediated by a carrier present in the canalicular membrane of the hepatocyte.

UDPglucuronosyltransferase was solubilized by treating Wistar rat liver microsomes with deoxycholate. Chromatography of this preparation on Bio-Gel P-30 resulted in extraction of 92% of phospholipids and complete loss of enzyme activity. UDPglucuronosyltransferase was reactivated by dialysing this delipidated preparation in the presence of lecithin, a mixture of liver microsomal lipids or microsomal preparations from livers of UDPglucuronosyltransferase-deficient Gunn rats. Virtually complete enzyme reactivation was obtained with regard to glucuronidation and glucosidation of bilirubin; however, the inactivation of UDPglucuronosyltransferase with p-nitrophenol as substrate was irreversible. These findings demonstrate that UDPglucuronosyltransferase with bilirubin as substrate is a lipid-requiring enzyme.

Covalent binding of activated azo dye and methyl cholanthrene carcinogens preferentially occurs to subunit B of ligandin. This subunit contains 7 of ligandin's 9 cysteine residues which, according to Ketterer et al, are the site of covalent carcinogen binding. Subunit B appears to be cytoplasmic and intranuclear. We postulate that binding of activated carcinogens to subunit B may facilitate their transfer from cytoplasm to nucleus. Rat and human primary liver cell cancers often contain ligandin which is released into the circulation. In patients at risk for, or suspected of having primary liver cell cancer, increased serum ligandin concentrations may have diagnostic value.

Ligandinuria is a useful index of acute tubular injury. Ligandin probably enters the urine at the time of initial necrosis and should be looked for soon after the toxic or ischemic event. Periodic examination of perfusates for this substance might yield useful information about techniques for storage of cadaver kidneys.

Australia antigen was found in clams contaminated by drainage of untreated sewage from a coastal hospital. In closed-system aquariums, the antigen was ingested by clams and transmitted to previously uninfected clams. In opensystem aquariums, the titer of Australia antigen decreased with time, suggesting viral concentration rather than replication.

Several lines of evidence indicate that ligandin consists of two different subunits. The protein dissociates into two components that are detected by electrophoresis in a discontinuous sodium dodecyl sulfate system, or in acid-urea gels, and by isoelectric focusing in the presence of urea. The apparent molecular weights of the two polypeptides are 25,000 and 22,000. Alkylated or succinylated ligandins also exhibit subunit heterogeneity and resolved into two bands in these electrophoretic systems. Cross-linked ligandin showed only one band in sodium dodecyl sulfate-gel electrophoresis indicating that the two subunits are part of a heterodimeric protein rather than monomers of two different proteins. No dansylated terminal amino acids were detected suggesting that the NH2-terminal residues of both chains are blocked. One mole of arginine or phenylalanine was released per mole of ligandin after digestion with carboxypeptidase B or A, respectively. Tryptic maps of succinylated ligandin were consistent with identical disposition of arginine residues in both chains, but several additional tryptic peptides were obtained with native ligandin as compared to the predicted number if both subunits were identical. These observations are consistent with the possibility that both subunits contain common sequences and that a small peptide of about 25 to 30 amino acid residues is cleaved from the COOH-terminal of the larger subunit to produce the smaller subunit.

Bilirubin diglucuronide, the major pigment in human bile is formed in two steps. Bilirubin is converted to bilirubin monoglucuronide by transfer of the glucuronosyl moiety of uridine diphosphoglucuronic acid catalyzed by the microsomal enzyme, uridine diphosphoglucuronate glucuraonosyl transferase (UDP glucuronyl transferase, EC 2.4.1.17). Bilirubin monoglucuaronide is converted to bilirubin diglucuronide in vitro by two enzymatic mechanisms: (a) UDP glucuronyl transferase-mediated transfer of a second mole of glucuronic acid form UDP-glucuronic acid to bilirubin monoglucuronide; (b) dismutation of 2 moles of bilirubin monoglucuronide to 1 mole of bilirubin diglucuronide and 1 mole of unconjugated bilirubin, catalyzed by bilirubin monoglucuronide dismutase (bilirubin glucuronoside glucuronosyl transferase EC 2.4.1.95). Assay methods for the three enzymatic mechanisms in human liver homogenate by high-pressure liquid chromatography analysis of underivatized bilirubin tetrapyrroles have been developed. UDP glucuronyl transferase was activated in five human liver homogenates with digitonin, Triton X-100, or UDP-N-acetylglucosamine. Greatest activation was observed with Triton X-100. The pH optimum for conversion of bilirubin to bilirubin monoglucuronide was 7.4, and UDP glucuronyl transferase activity was 625 +/- 51 nmoles per 20 min per gm liver. At high initial bilirubin concentrations (342 microM), the product of UDP glucuronyl transferase assay with bilirubin as substrate was predominantly bilirubin monoglucuronide. At lower initial bilirubin concentrations (6.5 to 34 microM), up to 15% bilirubin diglucuronide was formed. Glucuronyl transferase-mediated UDP glucuronic acid-dependent conversion of bilirubin monoglucuronide to diglucuronide was assayed using UDP-14-C-glucuronic acid. The pH optimum was 7.4, and the rate was 21 +/- 7 nmoles per gm liver per 20 min. The rate of bilirubin diglucuronide formation by enzymatic dismutation of bilirubin monoglucuronide was 470 +/- 112 nmoles per gm liver per min. The pH optimum was 6.6. The products of enzymatic dismutation were of the IX alpha configuration.

Physical methods and chemical modifications were used to discriminate between the bilirubin-binding capacity and glutathione-S-transferase activity of ligandin which was purified from rat liver. Binding of bilirubin occurs at a primary high affinity site (KA = 5 X 10(7) M-1) and at a secondary, lesser affinity site (KA = 3 X 10(5) M-1). Circular dichroism and fluorescence-quenching methods were used to distinguish between these sites. Cross-linked as well as reduced and alkylated ligandin lost high affinity bilirubin-binding capacity, but retained glutathione-S-transferase activity, bilirubin binding at a secondary site, and immunological reactivity. Succinylation of ligandin abolished catalytic activity and bilirubin binding at high and low affinity sites, but not immunological reactivity. Catalytic activity was unaffected by concentrations of bilirubin which saturated the primary binding site. These results suggest that the high affinity site at which bilirubin is bound to ligandin is independent from the site at which catalytically reactive substrates bind. The latter substrates probably interact at the secondary bilirubin binding site where bilirubin competitively inhibits glutathione-S-transferase activity.