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The bile canalicular membrane contains an ATP-dependent phosphatidylcholine translocator which is regulated by taurocholate
HEPATOLOGY Vol. 22, N o . 4, P t . 2, 1995
AASLD
753 CLONING OF A LIVER-SPECIFICMRP-HOMOLOGUE-WHICHIS DEFICIENT
ABSTRACTS
754
IN THE TR- RAT. CC Paulusma'. PJ Bosma'. CTM Bakker', M Otter ~. GJR Zaman'. and RPJ O~de Elferink'. "Dept.Gastrointestinal and Liver Diseases, JDept.Biochemistry, Academic Medical Centre, *The Netherlands Cancer Institute, Amsterdam. The Netherlands.
THE BILE CANALICULAR MEMBRANE CONTAINS AN ATPDEPENDENT PHOSPHATIDYLCHOLINETRANSLOCATOR WHICH IS REGULATED BY TAUROCHOLATE AT Nies. Z Gatmaitan. IM Arias. Dept. of Physiology, Tufts University, Boston Mdr2 P-glycoprotein, the dominant Pgp in bile canalicular plasma membrane, is involved in phospholipid secretion. Mice with a disruption at the mdr2-1ocus lack phosphatidylcholine (PC) in bile, and ATP-dependent PC translocation was observed in secretory vesicles of yeast transfected with murine mdr2. We investigated whether a PC translocating protein which is functionally similar to murine Mdr2, exists in rat canalicular membrane vesicles (CMV). Asymmetric PC distribution in inner and outer plasma membrane leaflets was measured fluorometrically (Ruetz & Gros, 1994, Cell, 77: 1-24) in inside-out and right-side-out CMV. CMV were labeled with NBD (nitrobenzoxadiazol)-caproyl-PC or NBD-dodecaoyl-PC and fluorescence was measured at Xem, 540 nm; ~.ex, 470 nm. Sodium hydrosulfite, a water-soluble membrane-impermeable anion, decreased fluorescence, reflecting reduction of NBD-PC to non-fluorescent ABD (aminobenzoxadiazol)-PC in the outer leaflet'. Upon addition of detergent, the inner leaflet became accessible to hydrosulflte, resulting in a further decrease in fluorescence. Results: PC translocation was specific for CMV and phosphatidylcholine, and required ATP. PC translocation was concentration-, time- and temperature-dependent resulting in 200 pmol PC translocated/mg protein/30 min at 37 °C. ATPyS, a non-hydrotyzable ATP analogue, did not support translocatiou. ATP-dependent PC translocation was 13-fold greater in inside-out CMV compared to results in right-side-out CMV, indicating that PC movemem ill vivo is from the inner to the outer leaflet of the canalicular membrane. Taurocholate enhanced ATP-dependent PC translocation hs vitro in a dose-dependent manner (25(I pmol PC translocated/mg protein/30 min at Ill }aM, and 400 pmol PC at 250 ~tM taurocholale). Concluskm: An ATP-dependem mechanism translocates PC from the inner to the outer leaflet of rat bile canalicular plasma membrane, and is functionally similar to murine Mdr2 as expressed in yeast secretory vesicles. The results suggest lfiat taurocholate is required to remove PC from the outer leaflet into die cauaticulus, and that PC facilitates micelle fornmtion lhereby protecting the biliary epithelium from the detergent activity of bile acids.
EXPRESSION AND LOCALIZATION OF THE MRP-ENCOOED CONJUGATE EXPORT PUMP IN HUMAN AND RAT LIVER. R. Mayer. 'J. Kartanbeck; M. BQchier, G. Jedlitschky, I. Leler, and D. Keppler. Divisions of Tumor Biochemistry and "Call Biology, Deutsches Krebsforschungszantrum, Heidelberg, Germany. The molecular identity of the conjugate export pump mediating the ATP-dependent transport of glutathione S-conjugates and structurally related amphiphilic organic anions across the canelicular hepatocyte membrane was unknown up to now. Recent work in our laboratory has elucidated the function of the multidrug resistenco-associeted protein (MRP) in non-hepatic cells and shown that it is a pdmary. active ATP-dependent export pump for conjugates of lipophilic compoundswith glutathione and other anionic residues (Jedlitschky el at. (1994) Cancer Res.54:4833-36; Leier at at. (1994) J. Biol. Chem.269:27807-10). We studied the expression and localization of MRP in human and rat liver by reverse transoripiton-PCR, immunoblot analysis, and immunofluorascence microscopy. Reverse transcriptionPCR resulted in two expected cDNA fragments both from human and rat liver. Sequence analysis of the cDNA fragments confirmed the expression of human MRP and rat mrp in liver. Four antibodies against MRP detected the 190 kD-glycoproteln on immunoblots from canelicular as well as basolaterel membranes from human liver. One of these antibodies detected the rat liver homolug (Mrp). Immunofluorescoflce microscopy localized MRP and Mrp to the canelicuier and lateral membrane domain. Mrp was also expressed in the liver of mutant GY/I'R" rats, which are deficient in ~ canaticular ATP-depandant conjugate transpoRer, alternatively termed cMOAT. Immunoblot analysis as well as immunofluorescenco microscopy revealed that Mq) was present only in the lateral but not in the canelicular membrane of mutant rat liver. Conclusions: Our results indicate that the ATP-dependant conjugate export pump expressed in liver is encoded by the MRP gane. The selective absence of Mrp or an isoform of Mq) from the canaUcular membrane is the basis for the hereditary defect of the hepatobiliary excreUon of anionic conjugates in the transport-deficient liver.
INTRODUCTION. Two distinct, ATP-dependent, transport systems have been described for bile salts and non-bile salt organic anions. The non-bile salt organic anion transporter, or canalicular Multispecific Organic Anion Transporter (cMOAT), has been functionally characterized by a naturally occurring mutant rat strain, the TR" rat, which lacks transport of, predominantly, bivalent organic anions. It was recently shown that the human Mu!tidrug Resistance-associated Protein (hMRP1) is an ATPdependent organic anion transporter and it was hypothesized that MRP is identical to cMOAT. Expression of MRP in liver is, however, extremely low and we therefore hypothesized that cMOAT might be a liver-specific MRP homologue. METHODS. A rat hepatocyte cDNA library was screened with a PCR fragment from rat liver which was obtained with degenerate primers based on the hMRP1 sequence. Expression of the obtained transcript and of rat lung MRP (rMRPll in tissues of both Wistar and TR" rats was assessed on Northern blot. RESULTS. We have isolated a liver-specific cDNA from a rat hepatocyte library representing a new member of the ATP-binding cassette (ABC) transporter family. Sequence analysis demonstrated that the protein is homologous to, but different from, hMRP1 and rMRP1. The homology with hMRP1 ranges from 20% outside the nucleotide binding folds (NBF), to 61 % in the first NBF and 69% in the second NBF. Northern blot analysis revealed expression of a 7kb transcript in liver but not in other tissues. Expression of the transcript was virtually absent in TR" rat liver. Northern blotting with a rMRP1 probe revealed expression in many tissues including lung, but a very low signal in liver. CONCLUSIONS. We have cloned a liver-specific homologue of MRP in the rat. Based on the absence of this transcript in the TR" rat we propose that it encodes the canalicular Multispecific Organic Anion Transporter, cMOAT.
755
295A
756
IN VITRO EVIDENCE THAT STEROL CARRIER PROTEIN 2 (SCP2) MAY PROVIDE A SCAVENGER PATHWAY FOR PHOSPHATIDYLCHOLINE (PC) SECRETION INTO BILE. AN Leonard and DE Cohen. Harvard Medical School and Brigham & Women's Hospital, Boston, MA. We have shown that submicellar bile salt concentrations markedly stimulate PC-transfer protein (PC-TP) activity in vitro, suggesting an essential role for PC-TP in bile formation (Biochemistry 33:9975, 1994). SCP2, which is also abundant in liver cytosol and may promote delivery of biliary cholesterol to canalicular plasma membrane (CPM), also catalyzes intermembrane transfer of several phospholipid classes (Annu Rev Biochem 60:73, 1991). To investigate the potential contribution of SCP2 to biliary PC secretion, we measured transfer rates of naturally fluorescent, self-quenched parinaroyl (PnA)-PC from PC-rich model endoplssmic reticulum (ER) vesicles (PnA-PC:egg yolk PC 75:25) to model CPM vesicles containing no PC (phosphatidylethanolamine: sphingomyelin: phosphatidylserine: phosphatidylinositol: cholesterol 22:22:10:8:38) following addition of purified bovine SCP2 and/or taurochenodeoxycholate (TCDC). Addition of SCP2 (360nM) resulted in first order time dependent increases in fluorescence (rate constant at 24°C ~ 3 x 10.5 sec% indicating PC transfer from ER to CPM. Submicellar [TCDC] (160 to 470p.M) stimulated SCP2 activity 80-140fold. Transfer rates increased as functions of temperature (13-41 °C), decreased with increasing [ER] (0.9 - 3.7p.M PC), but were not influenced appreciably by [CPM] (17 to 260t1M phospholipid). Arrhenius plots demonstrated that the free energy change (21.5kcal/mol) for activation of SCP2 by TCDC was composed of a relatively small enthalpy term (8.2kcal/mol), reflecting facile departure of PC molecules from membranes, and a larger entropy term (13.3kcal/mol) implying movement of hydrophobic PC monomers in aqueous solution. Compared with bile salt stimulation of bovine PCTP under identical conditions (Biochemistry 33:9975, 1994): 1) PC transfer activity with SCP2 is 30-fold lower than with PC-TP; 2) whereas PC-TP binds PCs tightly during intermembrane transfer, SCP2 promotes monomeric movement of PCs; and 3) as with PC-TP, [ER] modulates SCP2 activity. These results imply that under physiological conditions, hepatocellular selection and transport of biliary PCs is likely mediated by PC-TP. We speculate that redundancies exist in the mechanisms for lipid secretion into bile and that SCP2 may provide an important recruitabte pathway for cytosolic trafficking ot biliary PCs.
AASLD
753 CLONING OF A LIVER-SPECIFICMRP-HOMOLOGUE-WHICHIS DEFICIENT
ABSTRACTS
754
IN THE TR- RAT. CC Paulusma'. PJ Bosma'. CTM Bakker', M Otter ~. GJR Zaman'. and RPJ O~de Elferink'. "Dept.Gastrointestinal and Liver Diseases, JDept.Biochemistry, Academic Medical Centre, *The Netherlands Cancer Institute, Amsterdam. The Netherlands.
THE BILE CANALICULAR MEMBRANE CONTAINS AN ATPDEPENDENT PHOSPHATIDYLCHOLINETRANSLOCATOR WHICH IS REGULATED BY TAUROCHOLATE AT Nies. Z Gatmaitan. IM Arias. Dept. of Physiology, Tufts University, Boston Mdr2 P-glycoprotein, the dominant Pgp in bile canalicular plasma membrane, is involved in phospholipid secretion. Mice with a disruption at the mdr2-1ocus lack phosphatidylcholine (PC) in bile, and ATP-dependent PC translocation was observed in secretory vesicles of yeast transfected with murine mdr2. We investigated whether a PC translocating protein which is functionally similar to murine Mdr2, exists in rat canalicular membrane vesicles (CMV). Asymmetric PC distribution in inner and outer plasma membrane leaflets was measured fluorometrically (Ruetz & Gros, 1994, Cell, 77: 1-24) in inside-out and right-side-out CMV. CMV were labeled with NBD (nitrobenzoxadiazol)-caproyl-PC or NBD-dodecaoyl-PC and fluorescence was measured at Xem, 540 nm; ~.ex, 470 nm. Sodium hydrosulfite, a water-soluble membrane-impermeable anion, decreased fluorescence, reflecting reduction of NBD-PC to non-fluorescent ABD (aminobenzoxadiazol)-PC in the outer leaflet'. Upon addition of detergent, the inner leaflet became accessible to hydrosulflte, resulting in a further decrease in fluorescence. Results: PC translocation was specific for CMV and phosphatidylcholine, and required ATP. PC translocation was concentration-, time- and temperature-dependent resulting in 200 pmol PC translocated/mg protein/30 min at 37 °C. ATPyS, a non-hydrotyzable ATP analogue, did not support translocatiou. ATP-dependent PC translocation was 13-fold greater in inside-out CMV compared to results in right-side-out CMV, indicating that PC movemem ill vivo is from the inner to the outer leaflet of the canalicular membrane. Taurocholate enhanced ATP-dependent PC translocation hs vitro in a dose-dependent manner (25(I pmol PC translocated/mg protein/30 min at Ill }aM, and 400 pmol PC at 250 ~tM taurocholale). Concluskm: An ATP-dependem mechanism translocates PC from the inner to the outer leaflet of rat bile canalicular plasma membrane, and is functionally similar to murine Mdr2 as expressed in yeast secretory vesicles. The results suggest lfiat taurocholate is required to remove PC from the outer leaflet into die cauaticulus, and that PC facilitates micelle fornmtion lhereby protecting the biliary epithelium from the detergent activity of bile acids.
EXPRESSION AND LOCALIZATION OF THE MRP-ENCOOED CONJUGATE EXPORT PUMP IN HUMAN AND RAT LIVER. R. Mayer. 'J. Kartanbeck; M. BQchier, G. Jedlitschky, I. Leler, and D. Keppler. Divisions of Tumor Biochemistry and "Call Biology, Deutsches Krebsforschungszantrum, Heidelberg, Germany. The molecular identity of the conjugate export pump mediating the ATP-dependent transport of glutathione S-conjugates and structurally related amphiphilic organic anions across the canelicular hepatocyte membrane was unknown up to now. Recent work in our laboratory has elucidated the function of the multidrug resistenco-associeted protein (MRP) in non-hepatic cells and shown that it is a pdmary. active ATP-dependent export pump for conjugates of lipophilic compoundswith glutathione and other anionic residues (Jedlitschky el at. (1994) Cancer Res.54:4833-36; Leier at at. (1994) J. Biol. Chem.269:27807-10). We studied the expression and localization of MRP in human and rat liver by reverse transoripiton-PCR, immunoblot analysis, and immunofluorascence microscopy. Reverse transcriptionPCR resulted in two expected cDNA fragments both from human and rat liver. Sequence analysis of the cDNA fragments confirmed the expression of human MRP and rat mrp in liver. Four antibodies against MRP detected the 190 kD-glycoproteln on immunoblots from canelicular as well as basolaterel membranes from human liver. One of these antibodies detected the rat liver homolug (Mrp). Immunofluorescoflce microscopy localized MRP and Mrp to the canelicuier and lateral membrane domain. Mrp was also expressed in the liver of mutant GY/I'R" rats, which are deficient in ~ canaticular ATP-depandant conjugate transpoRer, alternatively termed cMOAT. Immunoblot analysis as well as immunofluorescenco microscopy revealed that Mq) was present only in the lateral but not in the canelicular membrane of mutant rat liver. Conclusions: Our results indicate that the ATP-dependant conjugate export pump expressed in liver is encoded by the MRP gane. The selective absence of Mrp or an isoform of Mq) from the canaUcular membrane is the basis for the hereditary defect of the hepatobiliary excreUon of anionic conjugates in the transport-deficient liver.
INTRODUCTION. Two distinct, ATP-dependent, transport systems have been described for bile salts and non-bile salt organic anions. The non-bile salt organic anion transporter, or canalicular Multispecific Organic Anion Transporter (cMOAT), has been functionally characterized by a naturally occurring mutant rat strain, the TR" rat, which lacks transport of, predominantly, bivalent organic anions. It was recently shown that the human Mu!tidrug Resistance-associated Protein (hMRP1) is an ATPdependent organic anion transporter and it was hypothesized that MRP is identical to cMOAT. Expression of MRP in liver is, however, extremely low and we therefore hypothesized that cMOAT might be a liver-specific MRP homologue. METHODS. A rat hepatocyte cDNA library was screened with a PCR fragment from rat liver which was obtained with degenerate primers based on the hMRP1 sequence. Expression of the obtained transcript and of rat lung MRP (rMRPll in tissues of both Wistar and TR" rats was assessed on Northern blot. RESULTS. We have isolated a liver-specific cDNA from a rat hepatocyte library representing a new member of the ATP-binding cassette (ABC) transporter family. Sequence analysis demonstrated that the protein is homologous to, but different from, hMRP1 and rMRP1. The homology with hMRP1 ranges from 20% outside the nucleotide binding folds (NBF), to 61 % in the first NBF and 69% in the second NBF. Northern blot analysis revealed expression of a 7kb transcript in liver but not in other tissues. Expression of the transcript was virtually absent in TR" rat liver. Northern blotting with a rMRP1 probe revealed expression in many tissues including lung, but a very low signal in liver. CONCLUSIONS. We have cloned a liver-specific homologue of MRP in the rat. Based on the absence of this transcript in the TR" rat we propose that it encodes the canalicular Multispecific Organic Anion Transporter, cMOAT.
755
295A
756
IN VITRO EVIDENCE THAT STEROL CARRIER PROTEIN 2 (SCP2) MAY PROVIDE A SCAVENGER PATHWAY FOR PHOSPHATIDYLCHOLINE (PC) SECRETION INTO BILE. AN Leonard and DE Cohen. Harvard Medical School and Brigham & Women's Hospital, Boston, MA. We have shown that submicellar bile salt concentrations markedly stimulate PC-transfer protein (PC-TP) activity in vitro, suggesting an essential role for PC-TP in bile formation (Biochemistry 33:9975, 1994). SCP2, which is also abundant in liver cytosol and may promote delivery of biliary cholesterol to canalicular plasma membrane (CPM), also catalyzes intermembrane transfer of several phospholipid classes (Annu Rev Biochem 60:73, 1991). To investigate the potential contribution of SCP2 to biliary PC secretion, we measured transfer rates of naturally fluorescent, self-quenched parinaroyl (PnA)-PC from PC-rich model endoplssmic reticulum (ER) vesicles (PnA-PC:egg yolk PC 75:25) to model CPM vesicles containing no PC (phosphatidylethanolamine: sphingomyelin: phosphatidylserine: phosphatidylinositol: cholesterol 22:22:10:8:38) following addition of purified bovine SCP2 and/or taurochenodeoxycholate (TCDC). Addition of SCP2 (360nM) resulted in first order time dependent increases in fluorescence (rate constant at 24°C ~ 3 x 10.5 sec% indicating PC transfer from ER to CPM. Submicellar [TCDC] (160 to 470p.M) stimulated SCP2 activity 80-140fold. Transfer rates increased as functions of temperature (13-41 °C), decreased with increasing [ER] (0.9 - 3.7p.M PC), but were not influenced appreciably by [CPM] (17 to 260t1M phospholipid). Arrhenius plots demonstrated that the free energy change (21.5kcal/mol) for activation of SCP2 by TCDC was composed of a relatively small enthalpy term (8.2kcal/mol), reflecting facile departure of PC molecules from membranes, and a larger entropy term (13.3kcal/mol) implying movement of hydrophobic PC monomers in aqueous solution. Compared with bile salt stimulation of bovine PCTP under identical conditions (Biochemistry 33:9975, 1994): 1) PC transfer activity with SCP2 is 30-fold lower than with PC-TP; 2) whereas PC-TP binds PCs tightly during intermembrane transfer, SCP2 promotes monomeric movement of PCs; and 3) as with PC-TP, [ER] modulates SCP2 activity. These results imply that under physiological conditions, hepatocellular selection and transport of biliary PCs is likely mediated by PC-TP. We speculate that redundancies exist in the mechanisms for lipid secretion into bile and that SCP2 may provide an important recruitabte pathway for cytosolic trafficking ot biliary PCs.