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460 The biophysical properties of hepatocyte plasma membrane domains influence water transport: The canalicular membrane is rate-limiting
382A
AASLD ABSTRACTS
4 5 9
EFFECTS OF HUMAN MRP2 MUTATIONS ON ITS TRANSPORT AND ATPASE ACTIVITIES AND ACTIVATION BY URSODIOL. Phillip M Gerk, Yu Yang, University of Kentucky,
Lexington, KY; Richard H Ho, Vanderbilt University Medical School, Nashville, TN; Wei Li, University of Kentucky, Lexington, KY; Richard B Kim, Vanderbilt University Medical School, Nashville, TN; Mary~ Vore, University of Kentucky, Lexington, KY Several mutations in h u m a n MRP2 are clearly associated with Dubin-Johnson syndrome, while the functional and clinical consequences of other MRP2 polymorphisms are unclear. Additionally, it is hypothesized that MRP2 mutations and polymorphisms may result in an altered response to cholestatic challenges or choleretic therapy with drugs like ursodiol. The purpose of this investigation was to determine the functional differences caused by several MRP2 mutations. Methods: The reference sequence (MRP2) was NM_000932, and the DNA mutations A3517T, G4348A, and G1249A encoded for amino acid changes II173F, A1450T, and V417I, in the third m e m b r a n e spanning domain, the second nucleotide binding domain, and the second m e m b r a n e spanning domain, respectively. The full-length coding regions of the reference MRP2 and three mutants sequences were inserted into the baculovirus genome. Sf9 insect cells were infected with the baculovirus stocks, and m e m b r a n e s were isolated from the infected cells expressing MRP2 or the mutants. ATPase activity was measured by determining release of inorganic phosphate by a colorimetric assay. ATP-dependent transport of 3H-E23G (estradiol-/3-(3/3-D-glucuronide)) or 3H-E217G (estradiol-/3-(17/3-D-glucuronide)) was determined by uptake into inside out m e m b r a n e vesicles collected on filters and counted by liquid scintillation. Results: The total expression levels of MRP2 and the three mutants in the infected Sf9 cells were comparable by densitometry of Western blots of three levels of each protein, indicating total expression in Sf9 cells was not influenced by the mutations. MRP2 ATPase activity was significantly stimulated by the uricosuric probenecid l m M , the cholestatic E217G 250/~M, and the mild choleretic E23G lmM. Although the control and stimulated ATPase activities of the V417I mutant were not different from MRP2, the II173F mutant had significantly less baseline and less stimulated ATPase activities, indicating impaired function. A1450T had less probenecid-stimulated ATPase activity, but activity stimulated by E217G and E23G was not different from MRP2. Ursodiol saturably stimulated ATPase activity of MRP2 by 520% with Km -72_+38/~M. Although the three mutants transported 3H-E23G with affinities not significantly different from MRP2 (Km-122_+23 ~zM), the Vmax of II173F was only 5.1% (154_+4 pmol/mg/min) and the Vmax of V417I was 157% (4701_+51 pmol/mg/min) compared to the Vmax of MRP2 (2991_+186 pmol/mg/min); the Vmax of A1450T was not different (2967_+87 pmol/mg/min). Ursodiol activated transport of 3H-E23G (60nM) with a peak effect of 950% at 100~zM; higher ursodiol concentrations (up to lmM) returned transport to near control values. MRP2-mediated ATP-dependent transport of 3H-E217G (44nM) in the presence of ursodiol (100~zM) for the mutants differed from MRP2 (10%, 67%, and 140% for II173F, A1450T, and V417I, respectively). Conclusions: Ursodiol markedly activated MRP2 transport of E23G. Although ursodiol has several anticholestatic effects, these data show its direct effect on transport of MRP2 substrates, and may suggest activation of MRP2 as a target in the treatment of cholestatic liver disease. The MRP2 mutants showed several important differences in activity, including changes in transport Vmax, and transport in the presence of ursodiol, and activation of ATPase activity by ursodiol, probenecid, and the MRP2 substrates E217G and E23G. Further characterization of these MRP2 mutants and others will lead to a better understanding of MRP2 structure and function and may yield new therapeutic approaches. Also, although E217G is an established MRP2 substrate, these data show that its noncholestatic isomer E23G is also an MRP2 substrate, implicating MRP2 in
HEPATOLOGY, October 2003
the disposition of other estrogen metabolites, which can be interconverted to cholestatic compounds like E217G. Disclosures: Phillip M Gerk - No relationships to disclose Richard H Ho - No relationships to disclose Richard B Kim - No relationships to disclose Wei Li - No relationships to disclose Mary E Vore - No relationships to disclose Yu Yang - No relationships to disclose
46O THE BIOPHYSICAL PROPERTIES OF HEPATOCYTE PLASMA MEMBRANE DOMAINS INFLUENCE WATER TRANSPORT: THE CANALICULAR MEMBRANE IS RATELIMITING. Raul A Marinelli, Insh'tuto de Fisiologia~ xperimental,
Consejo Nacional de Invesffgaciones Cienfff-icasy Tecnicas (CONIC~ T), Santa Fe, Argentina; Pamela S Tietz, Ariel J Caride, Bing Q Huang, Mayo Medical School, Clinic and Foundation, Rochester, MN; Sergio A Gradilone, Insh'tuto de Fisiologia~ xperimental, Consejo Nacional de Invesffgaciones Cienfff-icasy Tecnicas (CONIC~ T), Santa Fe, Argenh'na; Nicholas F LaRusso, Mayo Medical School, Clinic and Foundah'on, Rochester, MN Previous work from our laboratory is consistent with an important role for aquaporins (AQPs), a family of water channel proteins, in canalicular bile secretion principally via agonist-induced exocytic insertion of AQPs into the hepatocyte canalicular membrane. To further define the pathways and molecular mechanisms for water movement across hepatocytes, our AIM here was to directlY assess osmotic water permeability (Pf) and activation energy (~ a) in basolateral (BLMV) and canalicular m e m b r a n e (CMV) vesicles using stopped-flow spectrophotometry. Scattered light intensity was used to follow changes in vesicle volume in response to gradients induced by sucrose solutions of different osmolarities. RESULTS: The time course of scattered light for BLMV and CMV fit a single-exponential function. In BLMV, Pf was 108_+4 ~zm.sec-~ (25 °C) with an~ a of 7.7 kcal/mol; in CMV, Pfwas 86_+5/~m.sec -~ (25 °C) with an ~ a of 8.0 kcal/mol. The AQP blocker, dimethylsulfoxide, significantly inhibited the Pf of both basolateral (81_+4 /~m.sec-~; -25%) and canalicular (59_+4 /~m.sec-~; -30 %) vesicles consistent with the presence of AQPs in both vesicle populations. Experimental data for CMV from dibutyryl cAMP-treated hepatocytes fit best to a double-exponential curve implying the presence of two functionally distinct CMV populations; one population had Pf and ~ a values similar to those of CMV from untreated hepatocytes and the other population had a very high Pf -41 (655_135/~m.sec-, 25 o C) and very low~ a (2.8 kcal/mol). Dimethylsulfoxide completely inhibited the high Pf value in this second vesicle population. In contrast, the Pf of BLMV was unaltered by cAMP treatment of hepatocytes. CONCLUSIONS: Our data are consistent with the notion that the canalicular but not the basolateral membrane becomes more permeable to water after a secretory stimulus due to the insertion of AQP molecules. The data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation. Disclosures: Ariel J Caride - No relationships to disclose Sergio A Gradilone - No relationships to disclose Bing Q Huang No relationships to disclose Nicholas F LaRusso No relationships to disclose Raul A Marinelli - No relationships to disclose Pamela S Tietz - No relationships to disclose -
-
AASLD ABSTRACTS
4 5 9
EFFECTS OF HUMAN MRP2 MUTATIONS ON ITS TRANSPORT AND ATPASE ACTIVITIES AND ACTIVATION BY URSODIOL. Phillip M Gerk, Yu Yang, University of Kentucky,
Lexington, KY; Richard H Ho, Vanderbilt University Medical School, Nashville, TN; Wei Li, University of Kentucky, Lexington, KY; Richard B Kim, Vanderbilt University Medical School, Nashville, TN; Mary~ Vore, University of Kentucky, Lexington, KY Several mutations in h u m a n MRP2 are clearly associated with Dubin-Johnson syndrome, while the functional and clinical consequences of other MRP2 polymorphisms are unclear. Additionally, it is hypothesized that MRP2 mutations and polymorphisms may result in an altered response to cholestatic challenges or choleretic therapy with drugs like ursodiol. The purpose of this investigation was to determine the functional differences caused by several MRP2 mutations. Methods: The reference sequence (MRP2) was NM_000932, and the DNA mutations A3517T, G4348A, and G1249A encoded for amino acid changes II173F, A1450T, and V417I, in the third m e m b r a n e spanning domain, the second nucleotide binding domain, and the second m e m b r a n e spanning domain, respectively. The full-length coding regions of the reference MRP2 and three mutants sequences were inserted into the baculovirus genome. Sf9 insect cells were infected with the baculovirus stocks, and m e m b r a n e s were isolated from the infected cells expressing MRP2 or the mutants. ATPase activity was measured by determining release of inorganic phosphate by a colorimetric assay. ATP-dependent transport of 3H-E23G (estradiol-/3-(3/3-D-glucuronide)) or 3H-E217G (estradiol-/3-(17/3-D-glucuronide)) was determined by uptake into inside out m e m b r a n e vesicles collected on filters and counted by liquid scintillation. Results: The total expression levels of MRP2 and the three mutants in the infected Sf9 cells were comparable by densitometry of Western blots of three levels of each protein, indicating total expression in Sf9 cells was not influenced by the mutations. MRP2 ATPase activity was significantly stimulated by the uricosuric probenecid l m M , the cholestatic E217G 250/~M, and the mild choleretic E23G lmM. Although the control and stimulated ATPase activities of the V417I mutant were not different from MRP2, the II173F mutant had significantly less baseline and less stimulated ATPase activities, indicating impaired function. A1450T had less probenecid-stimulated ATPase activity, but activity stimulated by E217G and E23G was not different from MRP2. Ursodiol saturably stimulated ATPase activity of MRP2 by 520% with Km -72_+38/~M. Although the three mutants transported 3H-E23G with affinities not significantly different from MRP2 (Km-122_+23 ~zM), the Vmax of II173F was only 5.1% (154_+4 pmol/mg/min) and the Vmax of V417I was 157% (4701_+51 pmol/mg/min) compared to the Vmax of MRP2 (2991_+186 pmol/mg/min); the Vmax of A1450T was not different (2967_+87 pmol/mg/min). Ursodiol activated transport of 3H-E23G (60nM) with a peak effect of 950% at 100~zM; higher ursodiol concentrations (up to lmM) returned transport to near control values. MRP2-mediated ATP-dependent transport of 3H-E217G (44nM) in the presence of ursodiol (100~zM) for the mutants differed from MRP2 (10%, 67%, and 140% for II173F, A1450T, and V417I, respectively). Conclusions: Ursodiol markedly activated MRP2 transport of E23G. Although ursodiol has several anticholestatic effects, these data show its direct effect on transport of MRP2 substrates, and may suggest activation of MRP2 as a target in the treatment of cholestatic liver disease. The MRP2 mutants showed several important differences in activity, including changes in transport Vmax, and transport in the presence of ursodiol, and activation of ATPase activity by ursodiol, probenecid, and the MRP2 substrates E217G and E23G. Further characterization of these MRP2 mutants and others will lead to a better understanding of MRP2 structure and function and may yield new therapeutic approaches. Also, although E217G is an established MRP2 substrate, these data show that its noncholestatic isomer E23G is also an MRP2 substrate, implicating MRP2 in
HEPATOLOGY, October 2003
the disposition of other estrogen metabolites, which can be interconverted to cholestatic compounds like E217G. Disclosures: Phillip M Gerk - No relationships to disclose Richard H Ho - No relationships to disclose Richard B Kim - No relationships to disclose Wei Li - No relationships to disclose Mary E Vore - No relationships to disclose Yu Yang - No relationships to disclose
46O THE BIOPHYSICAL PROPERTIES OF HEPATOCYTE PLASMA MEMBRANE DOMAINS INFLUENCE WATER TRANSPORT: THE CANALICULAR MEMBRANE IS RATELIMITING. Raul A Marinelli, Insh'tuto de Fisiologia~ xperimental,
Consejo Nacional de Invesffgaciones Cienfff-icasy Tecnicas (CONIC~ T), Santa Fe, Argentina; Pamela S Tietz, Ariel J Caride, Bing Q Huang, Mayo Medical School, Clinic and Foundation, Rochester, MN; Sergio A Gradilone, Insh'tuto de Fisiologia~ xperimental, Consejo Nacional de Invesffgaciones Cienfff-icasy Tecnicas (CONIC~ T), Santa Fe, Argenh'na; Nicholas F LaRusso, Mayo Medical School, Clinic and Foundah'on, Rochester, MN Previous work from our laboratory is consistent with an important role for aquaporins (AQPs), a family of water channel proteins, in canalicular bile secretion principally via agonist-induced exocytic insertion of AQPs into the hepatocyte canalicular membrane. To further define the pathways and molecular mechanisms for water movement across hepatocytes, our AIM here was to directlY assess osmotic water permeability (Pf) and activation energy (~ a) in basolateral (BLMV) and canalicular m e m b r a n e (CMV) vesicles using stopped-flow spectrophotometry. Scattered light intensity was used to follow changes in vesicle volume in response to gradients induced by sucrose solutions of different osmolarities. RESULTS: The time course of scattered light for BLMV and CMV fit a single-exponential function. In BLMV, Pf was 108_+4 ~zm.sec-~ (25 °C) with an~ a of 7.7 kcal/mol; in CMV, Pfwas 86_+5/~m.sec -~ (25 °C) with an ~ a of 8.0 kcal/mol. The AQP blocker, dimethylsulfoxide, significantly inhibited the Pf of both basolateral (81_+4 /~m.sec-~; -25%) and canalicular (59_+4 /~m.sec-~; -30 %) vesicles consistent with the presence of AQPs in both vesicle populations. Experimental data for CMV from dibutyryl cAMP-treated hepatocytes fit best to a double-exponential curve implying the presence of two functionally distinct CMV populations; one population had Pf and ~ a values similar to those of CMV from untreated hepatocytes and the other population had a very high Pf -41 (655_135/~m.sec-, 25 o C) and very low~ a (2.8 kcal/mol). Dimethylsulfoxide completely inhibited the high Pf value in this second vesicle population. In contrast, the Pf of BLMV was unaltered by cAMP treatment of hepatocytes. CONCLUSIONS: Our data are consistent with the notion that the canalicular but not the basolateral membrane becomes more permeable to water after a secretory stimulus due to the insertion of AQP molecules. The data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation. Disclosures: Ariel J Caride - No relationships to disclose Sergio A Gradilone - No relationships to disclose Bing Q Huang No relationships to disclose Nicholas F LaRusso No relationships to disclose Raul A Marinelli - No relationships to disclose Pamela S Tietz - No relationships to disclose -
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