Structure and function of P-glycoprotein in normal liver and small intestine.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Biomedical subjects
I M Arias
Publications and source records attributed to I M Arias.
Is the multidrug resistance an ATP channel?
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
The biology of the bile canaliculus, 1993.
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Adenosine Triphosphate↗
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.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Serotonin stimulates a Ca2+ permeant nonspecific cation channel in hepatic endothelial cells.
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.
Animals↗
A nucleoside transporter is functionally linked to ectonucleotidases in rat liver canalicular membrane.
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.
Adenosine↗
Erythrocyte membrane transport of glutathione conjugates and oxidized glutathione in the Dubin-Johnson syndrome and in rats with hereditary hyperbilirubinemia.
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.
Adenosine Triphosphate↗
Defective biliary excretion of epinephrine metabolites in mutant (TR-) rats: relation to the pathogenesis of black liver in the Dubin-Johnson syndrome and Corriedale sheep with an analogous excretory defect.
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)
Animals↗
Prenatal diagnosis of bilirubin-UDP-glucuronosyltransferase deficiency in rats by genomic DNA analysis.
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)
Animals↗
The function of Gp170, the multidrug-resistance gene product, in the brush border of rat intestinal mucosa.
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.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
ATP-dependent organic anion transport system in normal and TR- rat liver canalicular membranes.
Many non-bile acid organic anions enter hepatocytes, where they are converted into polar conjugates and preferentially secreted into bile. ATP-dependent and membrane potential-dependent transport of non-bile acid organic anions across the plasma membrane of rat hepatocytes was studied in canalicular (CMV) or sinusoidal membrane vesicles (SMV) using sulfobromophthalein (BSP), a representative non-bile acid organic anion. Temperature-dependent, osmotically sensitive, saturable, ATP-dependent, and unidirectional (inside to outside) BSP transport was observed in CMV but not in SMV. Apparent Michaelis constants for ATP and BSP were 0.26 mM and 31 microM, respectively. ATP-dependent BSP transport was inhibited by oxidized glutathione, dinitrophenyl-glutathione (GSDNP), BSP glutathione, and bilirubin diglucuronide but not by daunomycin, taurocholate, and reduced glutathione. Inhibition by GSDNP and bilirubin diglucuronide was competitive, with apparent inhibitor constants of 41 and 4.2 microM, respectively. CMV from normal rats demonstrated ATP-dependent and membrane potential-dependent BSP transport that was additive. CMV from TR- rats lacked ATP-dependent BSP transport but retained membrane potential-dependent transport of non-bile acid organic anions as well as ATP-dependent transport mechanisms for organic cations (i.e., daunomycin) and bile acids (i.e., taurocholate).
Adenosine Triphosphate↗
Two distinct mechanisms for bilirubin glucuronide transport by rat bile canalicular membrane vesicles. Demonstration of defective ATP-dependent transport in rats (TR-) with inherited conjugated hyperbilirubinemia.
Bilirubin is conjugated with glucuronic acid in hepatocytes and subsequently secreted in bile. The major conjugate is bilirubin diglucuronide. Using sealed vesicles which are primarily derived from the canalicular (CMV) and sinusoidal (SMV) membrane vesicle domains of the plasma membrane of hepatocytes, we demonstrated that bilirubin glucuronides are transported by CMV by both ATP- and membrane potential-dependent transport systems. In CMV from normal rats, these processes are additive. In CMV from TR- rats, which have an autosomal recessively inherited defect in biliary secretion of nonbile acid organic anions, ATP-dependent transport of bilirubin diglucuronide was absent whereas the membrane potential driven system was retained. Other canalicular ATP-dependent transport systems, which were previously described for organic cations and bile acids, are functionally retained in TR- rats. Our study indicates that bilirubin glucuronides are primarily secreted into the bile canaliculus by an ATP-dependent mechanism which is defective in an animal model of the human Dubin-Johnson syndrome.
Adenosine Triphosphate↗
Benzquinamide inhibits P-glycoprotein mediated drug efflux and potentiates anticancer agent cytotoxicity in multidrug resistant cells.
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.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Molecular basis for the lack of bilirubin-specific and 3-methylcholanthrene-inducible UDP-glucuronosyltransferase activities in Gunn rats. The two isoforms are encoded by distinct mRNA species that share an identical single base deletion.
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.
Animals↗
Rat liver canalicular membrane vesicles contain an ATP-dependent bile acid transport system.
The secretion of bile by the liver is primarily determined by the ability of the hepatocyte to transport bile acids into the bile canaliculus. A carrier-mediated process for the transport of taurocholate, the major bile acid in humans and rats, was previously demonstrated in canalicular membrane vesicles from rat liver. This process is driven by an outside-positive membrane potential that is, however, insufficient to explain the large bile acid concentration gradient between the hepatocyte and bile. In this study, we describe an ATP-dependent transport system for taurocholate in inside-out canalicular membrane vesicles from rat liver. The transport system is saturable, temperature-dependent, osmotically sensitive, specifically requires ATP, and does not function in sinusoidal membrane vesicles and right side-out canalicular membrane vesicles. Transport was inhibited by other bile acids but not by substrates for the previously demonstrated ATP-dependent canalicular transport systems for organic cations or nonbile acid organic anions. Defects in ATP-dependent canalicular transport of bile acids may contribute to reduced bile secretion (cholestasis) in various developmental, inheritable, and acquired disorders.
Adenosine Triphosphate↗
Uptake of bilirubin glucuronides by isolated rat hepatocytes.
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.
Animals↗
Extracellular ATP, intracellular calcium and canalicular contraction in rat hepatocyte doublets.
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.
Adenosine Triphosphate↗
Mechanism of the lack of induction of UDP-glucuronosyltransferase activity in Gunn rats by 3-methylcholanthrene. Identification of a truncated enzyme.
Gunn rats lack UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. 4-Nitrophenol glucuronidation is mediated by several UDPGT isoforms that are distinct from bilirubin-UDPGT, one of which is induced after 3-methylcholanthrene (3-MC) administration in normal, but not in Gunn rats. In normal rats, 3-MC-inducible UDPGT mRNA concentration increased 15-fold in the liver and 3-fold in kidney after 3-MC (140 mg/kg) administration. Concentration of this mRNA is much lower in Gunn rat liver and kidney compared to normal. However, this mRNA was normally induced after 3-MC administration. By RNA blot hybridization, the mRNA in Gunn rat liver and kidney appeared to be of normal size. Nuclear run-on studies showed that the transcription rate for 3-MC-inducible UDPGT was 3-fold higher in Gunn rat liver and kidney than in normal and increased 3- to 5-fold after 3-MC administration. Immunotransblot studies revealed an Mr = 56,000 3-MC-inducible UDPGT in liver and kidney of normal, but not in Gunn rats. However, a new immunoreactive UDPGT band (Mr = 43,000) was present in Gunn, but not in normal rats. Cell-free translation of kidney mRNA from 3-MC-treated Gunn rats showed that the Mr = 43,000 UDPGT is synthesized as an Mr = 45,000 protein. Prior hybridization of the mRNA with an isoform-specific oligonucleotide spanning the initiation codon abolishes synthesis of this protein. These results suggest that a sequence abnormality in the 3-MC-inducible UDPGT mRNA in Gunn rats results in reduced mRNA concentration and synthesis of a truncated, enzymically inactive UDPGT.
Animals↗