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Phospholipid alterations in rat hepatocyte membranes in cholestasis and cytoprotective transporter protein changes
AASLDA933
April 2000
161
163
PHOSPHOLIPID ALTERATIONS IN RAT HEPATOCYTE MEMBRANES IN CHOLESTASIS AND CYTOPROTECTIVE TRANSPORTER PROTEIN CHANGES. Hideyuki Hyogo, Susumu Tazuma, Minoru Sakomoto, Kuniharu Nakai, Yasumasa Asamoto, Kazuhiko Tsuboi, Shigeyuki Yasumiba, Yasushi Sunami, Hidenori Ochi, Michael Muller, Goro Kajiyama, Hiroshima Univ, Hiroshima, Japan; Hiroshima Univ, Hiroshima Unive, Japan; Groningen Univ, Groningen, Netherlands. Background: Biliary components are transported by hepatic adenosine triphosphate-binding cassette (ABC) transporters that are located in canalicular membranes. Physiological transporter function is related to membrane fluidity, which is modulated by the phospholipid composition of the lipid bilayer. Aims:To clarify whether cholestasis alters transporter function by modifying membrane phospholipid species and/or expression of ABC transporters, and if it does, to determine whether it is associated with cytoprotective systems. Methods:Canalicular and sinusoidal membrane vesicles (CMV and SMV, respectively) were isolated from rat liver 6 h or 3 days after bile duct ligation, followe-d by membrane lipid analysis using gas-liquid chromatography, membrane fluidity assessment using fluorescence polarization (OPH, 1,6-diphenyl-I,3,5-hexatriene), and analyzing transporter protein expression by Western blotting and reverse-transcriptase polymerase chain reaction (RT-PCR) of mRNA levels. Results:Canalicular membrane fluidity decreased in a marked time-dependent manner. Sinusoidal membranes showed biphasic changes: increased fluidity at 6 h and a decrease at 3 days. In CMV, the saturated/unsaturated fatty acid (stu) ratio increased for phosphatidylcholine (PC). The reverse occurred in SMV. The Stu ratio for sphingomyelin (Sph) showed the opposite pattern. P-glycoprotein expression was markedly increased at both 6 hand 3 days, while expression of multidrug resistance protein 2 (mrp2) and bile salt export pump (bsep) decreased. Mdrla/lbl2 and mrpl mRNA were increased, whereas mrp2 mRNA was decreased time-dependently. Interestingly, bsep mRNA showed a biphasic pattern: increased at 6h and decreased by 3 days. Conclusions:Unique reciprocal changes were found in membrane fluidity and phospholipid fatty acid composition between CMV vs. SMV or PC vs. Sph. Changes in ABC transporter protein expressions and mRNA levels were found to represent responses against cholestasis. We conclude that the lipid bilayer plays an important role in hepatocellular-protection by modulating the lipid constitution both in sinusoidal and canalicular membrane, along with the changes of ABC transporters. This study was supported, in part, by the Ministry of Education of the Japanese Government, awarded to Or. Tazuma (No. 10670477).
OATP3 IS AN APICAL BILE ACID TRANSPORTER IN RAT SMALL INTESTINE. Holly C. Walters, Ann L. Craddock, Hisae Fusegawa, Mark Willingham, Paul Oawson, Wake Forest Univ Sch of Medicine, Winston-Salem, NC. Transport of bile acids from the intestinal lumen into the portal circulation is mediated by a series of transporters located on the enterocyte apical and basolateral membranes. The Apical SodiumlBile Acid Cotransporter (ASBT) is responsible for ileal bile acid absorption. In contrast, the carrier responsible for bile acid uptake in the proximal small intestine has not been identified. An organic anion transporting polypeptide subtype 3 (oatp3) cONA was cloned from rat small intestine and studied to further define the components of the intestinal transport system. In transfected COS cells, rat oatp3 mediated sodium-independent, OIDS-inhibited uptake of taurocholate (Km "" 30 ILM) and transported both conjugated and unconjugated bile acids. In transiently transfected COS and stably transfected MOCK cells, oatp3-mediated uptake was more rapid for glycine than taurine conjugates and for conjugated dihydroxy than trihydroxy bile acids. In contrast to the bile acids, oatp3 did not transport folate, methotrexate, digoxin, or microcystin, In polarized monolayers of the stably transfected MOCK cells, oatp3 mediated apical, but not basolateral, uptake of taurocholate. Reverse-transcription-coupled polymerase chain reaction analysis revealed that rat oatp3 but not oatpl or 2 mRNA was expressed in small intestine. By RNase protection assay, oatp3 mRNA was readily detected down the length of the small intestine as well as in brain, lung, and retina. Antibodies were raised against a fusion protein encompassing the carboxyl terminal 47 amino acids of rat oatp3. By immunoblotting and immunofluorescence analysis, oatp3 protein was localized to the apical brush border membrane of intestinal epithelial cells. These findings suggest that rat oatp3 mediates the apical sodium-independent absorption of bile acids previously described for the proximal small intestine.
162 OCTN2 IS NOT A CANALICULAR ORGANIC CATION TRANSPORTER. Laura M. Stack, Kris L. Snow, Richard H. Moseley, VA Med Ctr, Ann Arbor, MI; Univ of Michigan Med Ctr, Ann Arbor, MI. Although we have previously described the functional characteristics of canalicular organic cationic drug transport, identification of this pathway for the biliary excretion of drugs at the molecular level has been unsuccessful to date. A Na+-dependent carnitine transporter, hOCTN2, has been recently isolated from human kidney and a human placental trophoblast cell line; mutations in the gene encoding this transporter result in primary systemic carnitine deficiency. hOCTN2 is a homolog of the organic cation transporter hOCTNI which, when expressed in human embryonic kidney (HEK)293 cells, transports the model organic cation, tetraethylammonium (TEA), in a pH-dependent manner. Although expressed in human fetal liver, OCTNI is expressed strongly only in adult kidney, trachea, and bone marrow. These characteristics suggest that OCTNI acts as an organic cation/proton antiporter at the renal apical membrane. On the other hand, hOCTN2, in addition to Na+-dependent camitine transport, exhibits pHdependent TEA transport and is expressed in adult liver. Therefore, we examined L-carnitine transport in rat liver plasma membrane vesicles to determine whether OCTN2 functions as a canalicular organic cation transporter. Canalicular (cLPM) and basolateral (bILPM) plasma membrane vesicles were prepared from rat liver by the method of Meier, et al. Uptake of [14C]L-carnitine was assessed by a rapid vacuum filtration method. Uptake of 50 pM 4CjL-carnitine in the presence of an inwardly directed Na + gradient was not significantly different than uptake in the presence of an inwardly directed K+ gradient in cLPM vesicles. Furthermore, in contrast to recent reports in isolated hepatocytes, no Na+-dependent Lcarnitine transport was demonstrated in blLPM vesicles. CONCLUSION: Although OCTN2 is expressed in liver, it does not mediate organic cation transport at the canalicular domain and other candidate transporters, including a proposed homolog of synaptic vesicle monoamine transporters, need to be identified. At the same time, studies are underway to characterize the intracellular localization of OCTN2 in the liver.
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164 THE MOLECULAR AND IMMUNOHISTOCHEMICAL EXPRESSION OF MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN (MRP) 3 IN THE LIVER AND INTESTINE OF PATIENTS WITH CHOLESTASIS AND ITS BIOLOGICAL SIGNIFICANCE. Junnichi Shoda, Koji Oda, Junichi Kamiya, Yuji Nimura, Hiroshi Suzuki, Yuichi Sugiyama, Hiroshi Miyazaki, Toru Kawamoto, Naomi Tanaka, The Univ of Tsukuba, Ibaraki, Japan; The Univ of Nagoya, Aichi, Japan; The Univ of Tokyo, Tokyo, Japan; Niigata Coli of Pharmacy, Niigata, Japan. Background: In contrast to down-regulation of plasma membrane transporter proteins, MRP3 is induced and functions as a transporter for bilirubin conjugate and bile acid in experimental cholestasis (mC 1999;274: 15181). Although there is an extensive expression of MRP3 mRNA in human liver and intestinal tissues, the immunohistochemical localization as well as biological function of MRP3 protein has not been fully defined in human cholestasis. Aims: To determine the molecular and immunohistochemical expression of MRP3 in the liver and intestine of patients with obstructive cholestasis (OC). Methods: The liver and small intestinal tissues from 14 patients with DC and 18 control subjects (CONs) were analyzed for the mRNA and immunohistochemical expression levels of MRP3. Furthermore, the expression of MRP2, sister of P-glycoprotein (SPGP), and ileal bile acid transporter (iBAT) were analyzed in the specimens. Results: The mRNA level of MRP3 was significantly higher (!OI :t 5, M :t SEM, % of G3POH) in the liver with OC than in that of CONs (64 :t 3; P
April 2000
161
163
PHOSPHOLIPID ALTERATIONS IN RAT HEPATOCYTE MEMBRANES IN CHOLESTASIS AND CYTOPROTECTIVE TRANSPORTER PROTEIN CHANGES. Hideyuki Hyogo, Susumu Tazuma, Minoru Sakomoto, Kuniharu Nakai, Yasumasa Asamoto, Kazuhiko Tsuboi, Shigeyuki Yasumiba, Yasushi Sunami, Hidenori Ochi, Michael Muller, Goro Kajiyama, Hiroshima Univ, Hiroshima, Japan; Hiroshima Univ, Hiroshima Unive, Japan; Groningen Univ, Groningen, Netherlands. Background: Biliary components are transported by hepatic adenosine triphosphate-binding cassette (ABC) transporters that are located in canalicular membranes. Physiological transporter function is related to membrane fluidity, which is modulated by the phospholipid composition of the lipid bilayer. Aims:To clarify whether cholestasis alters transporter function by modifying membrane phospholipid species and/or expression of ABC transporters, and if it does, to determine whether it is associated with cytoprotective systems. Methods:Canalicular and sinusoidal membrane vesicles (CMV and SMV, respectively) were isolated from rat liver 6 h or 3 days after bile duct ligation, followe-d by membrane lipid analysis using gas-liquid chromatography, membrane fluidity assessment using fluorescence polarization (OPH, 1,6-diphenyl-I,3,5-hexatriene), and analyzing transporter protein expression by Western blotting and reverse-transcriptase polymerase chain reaction (RT-PCR) of mRNA levels. Results:Canalicular membrane fluidity decreased in a marked time-dependent manner. Sinusoidal membranes showed biphasic changes: increased fluidity at 6 h and a decrease at 3 days. In CMV, the saturated/unsaturated fatty acid (stu) ratio increased for phosphatidylcholine (PC). The reverse occurred in SMV. The Stu ratio for sphingomyelin (Sph) showed the opposite pattern. P-glycoprotein expression was markedly increased at both 6 hand 3 days, while expression of multidrug resistance protein 2 (mrp2) and bile salt export pump (bsep) decreased. Mdrla/lbl2 and mrpl mRNA were increased, whereas mrp2 mRNA was decreased time-dependently. Interestingly, bsep mRNA showed a biphasic pattern: increased at 6h and decreased by 3 days. Conclusions:Unique reciprocal changes were found in membrane fluidity and phospholipid fatty acid composition between CMV vs. SMV or PC vs. Sph. Changes in ABC transporter protein expressions and mRNA levels were found to represent responses against cholestasis. We conclude that the lipid bilayer plays an important role in hepatocellular-protection by modulating the lipid constitution both in sinusoidal and canalicular membrane, along with the changes of ABC transporters. This study was supported, in part, by the Ministry of Education of the Japanese Government, awarded to Or. Tazuma (No. 10670477).
OATP3 IS AN APICAL BILE ACID TRANSPORTER IN RAT SMALL INTESTINE. Holly C. Walters, Ann L. Craddock, Hisae Fusegawa, Mark Willingham, Paul Oawson, Wake Forest Univ Sch of Medicine, Winston-Salem, NC. Transport of bile acids from the intestinal lumen into the portal circulation is mediated by a series of transporters located on the enterocyte apical and basolateral membranes. The Apical SodiumlBile Acid Cotransporter (ASBT) is responsible for ileal bile acid absorption. In contrast, the carrier responsible for bile acid uptake in the proximal small intestine has not been identified. An organic anion transporting polypeptide subtype 3 (oatp3) cONA was cloned from rat small intestine and studied to further define the components of the intestinal transport system. In transfected COS cells, rat oatp3 mediated sodium-independent, OIDS-inhibited uptake of taurocholate (Km "" 30 ILM) and transported both conjugated and unconjugated bile acids. In transiently transfected COS and stably transfected MOCK cells, oatp3-mediated uptake was more rapid for glycine than taurine conjugates and for conjugated dihydroxy than trihydroxy bile acids. In contrast to the bile acids, oatp3 did not transport folate, methotrexate, digoxin, or microcystin, In polarized monolayers of the stably transfected MOCK cells, oatp3 mediated apical, but not basolateral, uptake of taurocholate. Reverse-transcription-coupled polymerase chain reaction analysis revealed that rat oatp3 but not oatpl or 2 mRNA was expressed in small intestine. By RNase protection assay, oatp3 mRNA was readily detected down the length of the small intestine as well as in brain, lung, and retina. Antibodies were raised against a fusion protein encompassing the carboxyl terminal 47 amino acids of rat oatp3. By immunoblotting and immunofluorescence analysis, oatp3 protein was localized to the apical brush border membrane of intestinal epithelial cells. These findings suggest that rat oatp3 mediates the apical sodium-independent absorption of bile acids previously described for the proximal small intestine.
162 OCTN2 IS NOT A CANALICULAR ORGANIC CATION TRANSPORTER. Laura M. Stack, Kris L. Snow, Richard H. Moseley, VA Med Ctr, Ann Arbor, MI; Univ of Michigan Med Ctr, Ann Arbor, MI. Although we have previously described the functional characteristics of canalicular organic cationic drug transport, identification of this pathway for the biliary excretion of drugs at the molecular level has been unsuccessful to date. A Na+-dependent carnitine transporter, hOCTN2, has been recently isolated from human kidney and a human placental trophoblast cell line; mutations in the gene encoding this transporter result in primary systemic carnitine deficiency. hOCTN2 is a homolog of the organic cation transporter hOCTNI which, when expressed in human embryonic kidney (HEK)293 cells, transports the model organic cation, tetraethylammonium (TEA), in a pH-dependent manner. Although expressed in human fetal liver, OCTNI is expressed strongly only in adult kidney, trachea, and bone marrow. These characteristics suggest that OCTNI acts as an organic cation/proton antiporter at the renal apical membrane. On the other hand, hOCTN2, in addition to Na+-dependent camitine transport, exhibits pHdependent TEA transport and is expressed in adult liver. Therefore, we examined L-carnitine transport in rat liver plasma membrane vesicles to determine whether OCTN2 functions as a canalicular organic cation transporter. Canalicular (cLPM) and basolateral (bILPM) plasma membrane vesicles were prepared from rat liver by the method of Meier, et al. Uptake of [14C]L-carnitine was assessed by a rapid vacuum filtration method. Uptake of 50 pM 4CjL-carnitine in the presence of an inwardly directed Na + gradient was not significantly different than uptake in the presence of an inwardly directed K+ gradient in cLPM vesicles. Furthermore, in contrast to recent reports in isolated hepatocytes, no Na+-dependent Lcarnitine transport was demonstrated in blLPM vesicles. CONCLUSION: Although OCTN2 is expressed in liver, it does not mediate organic cation transport at the canalicular domain and other candidate transporters, including a proposed homolog of synaptic vesicle monoamine transporters, need to be identified. At the same time, studies are underway to characterize the intracellular localization of OCTN2 in the liver.
e
164 THE MOLECULAR AND IMMUNOHISTOCHEMICAL EXPRESSION OF MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN (MRP) 3 IN THE LIVER AND INTESTINE OF PATIENTS WITH CHOLESTASIS AND ITS BIOLOGICAL SIGNIFICANCE. Junnichi Shoda, Koji Oda, Junichi Kamiya, Yuji Nimura, Hiroshi Suzuki, Yuichi Sugiyama, Hiroshi Miyazaki, Toru Kawamoto, Naomi Tanaka, The Univ of Tsukuba, Ibaraki, Japan; The Univ of Nagoya, Aichi, Japan; The Univ of Tokyo, Tokyo, Japan; Niigata Coli of Pharmacy, Niigata, Japan. Background: In contrast to down-regulation of plasma membrane transporter proteins, MRP3 is induced and functions as a transporter for bilirubin conjugate and bile acid in experimental cholestasis (mC 1999;274: 15181). Although there is an extensive expression of MRP3 mRNA in human liver and intestinal tissues, the immunohistochemical localization as well as biological function of MRP3 protein has not been fully defined in human cholestasis. Aims: To determine the molecular and immunohistochemical expression of MRP3 in the liver and intestine of patients with obstructive cholestasis (OC). Methods: The liver and small intestinal tissues from 14 patients with DC and 18 control subjects (CONs) were analyzed for the mRNA and immunohistochemical expression levels of MRP3. Furthermore, the expression of MRP2, sister of P-glycoprotein (SPGP), and ileal bile acid transporter (iBAT) were analyzed in the specimens. Results: The mRNA level of MRP3 was significantly higher (!OI :t 5, M :t SEM, % of G3POH) in the liver with OC than in that of CONs (64 :t 3; P