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Hepatocellular uptake of organic cations by the organic anion transporting polypeptides (Oatp1, Oatp2 and OATP-A) and the organic cation transporters (rOCT1 and hOCT1)
Posters
I P/C07105 I
I
CORTICOSTEROIDS MODULATE THE SECRETORY PROCESSES OF INTRAHEPATIC B1LIARY E P I T H E L I U M D. Alvaro, L. Marncci l, A. Gigliozzi, G. Alpini 2, E, Papa, M. Delle Monache, R. Monterubbianesi. L. CaDocaccia, A. Bencdetti I Div. Gastro., Univ. Rome "La Sapienza", Italy. 1Div. Gastro., Univ. Ancona, Italy. 2Texas A&M Univ., Texas, USA.
DOWNREGULATION OF THE HEPATOCELLULAR ORGANIC ANI O N TRANSPORTERS OATP-C AND MRP2 IN PATIENTS W I T H PRIMARY SCLEROSING C H O L A N G I T I S M. Oswald, G.A. KuUak-Ublick, G. Paumgartncr, U. Beucrs Department of Medicine II, Klinikum Grosshadern, University of Munich, Munich, Germany. Background: Formation and secretion of bile depends on the coordinated action of various transporter proteins of the sinusoidal and canalicular membranes of the hepatoeyles. The recently isolated liver-specific organic anion transporter OATP-C (JBC 1999;274:t7159) is a liver-specific membrane transport protein for the basolateral uptake of organic anions from blood into the hepatocyte. The multidmg resistance associated protein 2 MRP2 mediates the canalicular excretion of organic anions into bile. Biliary secretion of organic anions is impaired in chronic cholestatic liver disease. The underlying molecular mechanisms, however, have not yet been eludicated. Therefore, the aim of our study was to investigate the expression of the organic anion transporters OATP-C and MRP2 in patients with primary sclcrosing cholangitis (PSC) (n=4), a chronic cholestatic liver disease as compared to controls (non-cholestatic liver disease, n=4).Methods: The expression of OATP-C, MRP2 and [3-actin (reference transcript) was studied using quantitative RT-PCR: Total RNA was isolated from liver tissue obtained at routine biopsy, reverse transcribed and then subjected to PCR amplification using specific primers. The PCR products were quantified using densitometry. The levels of OATP-C and MRP2 mRNA were normalized for [i-actin mRNA. Results: The level of OATP-CmRNA in liver tissue ofpatients with PSC (OATP-C/J~-actin 1.60 + 0.25) was only 49% of that in liver tissue of controls (OATP-C/[3-actin 3.24 + 0.69; p<0.05). The level of MRP2-mRNA in patients with PSC (MRP2/fl-actin 0.70 + 0.36) was reduced to 27% of controls (MRP2/[3-actin 2.54 + 0.56; p=0.005). In summary, the expression of both, the basolateral and the canalicular organic anion transport proteins, OATP-C and MRP2, was reduced in patients with PSC. Downreguiation of OATP-C might be the consequence of impaired canalicular secretion of organic anions and might, thereby, reduce the organic anion load of cholestatic hepatocytes.
We investigated the expression of glucocorticoid receptors (GcR) and the role of corticosteroids in the regulation of cholangiocyte secretory processes. METHODS: GcR were investigated by immunohistochemistry and RT-PCR. The effects of dexamethasone and budesonide on bicarbonate biliary excretion and I-F/HCO3" transport processes were evaluated in bile fistula rats and isolated bile ducts (IBDU). RESULTS: GcR were expressed in rat cholangiocytes and markedly increased after cholangiocyte proliferation induced by bile duct ligation. A 2-3 day treatment with dexamethasone (1 mg Lid) or budesonide (0.3 mg tid) induced an increased (p<0.05) bicarbonate biliary secretion. IBDU incubated for 24-36 hrs with dexamethasone or budesonide showed an increased (p<0.02) expression (+120 %, western blot) and activity (+60%) of Na+/I-I+ exchanger (NHE1 isoform) and of CI'/HC03" exchanger. CONCLUSIONS: The intrahepatic biliary epithelium express GcR. Corticosteroids enhance the expression and activities of transport processes driving bicarbonate excretion in bile suggesting a potential mechanism for the beneficial effects in the treatment of cholangiopathies. Supported by Grant # 9806210866 from MURST.
P/C07/07 I
I P/covoe I
I P/co,/o8 ]
HEPATOCELLULAR UPTAKE OF ORGANIC CATIONS BY THE ORGANIC ANION TRANSPORTING POLYPEPTIDES (Oatpl, Oatp2 AND OATP-A) AND THE ORGANIC CATION TRANSPORTERS (rOCTI and hOCTI) J.E. van Montfoort 1.3, B. Stieger 1, H. Koepsell 2, M. Mi~ller 3, G.M.M. Groothuis 3, D.K.E Metier 3. EJ. Meier ~ 1Die. Clinical Pharmacology and Toxicology, University Hospital, Ziirich, Switzerland. 2Department of Anatomy, University of Wttrzburg, Germany. 2Liver Research Center, Groningen Institute of Drug Studies, The Netherlands. Background: Based on kinetic transport studies organic cations have been divided into small and hydrophilic type 1 and more bulky end amphipathiotype 2 cations. Potentialcandidates for these so far only functionally charactadsed hepatic carders are the OCTs for type 1 cations and Oatps for type 2 cations. Aim: To investigate the discrimination between type 1 and type 2 cations by the OCTs and Oatps in a heterologous expression system and to determine structural features of the substrates necessary for this discrimination, Methods: Uptake studies in water and OCT- or Oatp-cRNAinjected Xenopus laevis oocytes with the type 1 cations tdbutylmethylammonium (TBuMA), azidoprocainamida methoiodide (APM) and azopentyl-quinuclidine (APQ) and the type 2 cations APD-ajmalinium (APDA), rocuronium, N-methyl-quinine (NMQ), N-methyl-quinidine (NMQD) and quinidlne (QD). Results: TBuMA, APM and APQ were taken up by rOCT1 and hOCT1, but not transported by any of the Oatps. Uptake of TBuMA by rOCTI and hOCT1 was saturable and yielded apparent Km values of 34 and 53 pM, respectively. APDA was transported by all investigated carders, while the uptake of rocuronium was restricted to the Oatps. NMQ and NMQD were transported by OATP-A (previously called OATP), rOCT1 and hOCT1, but not by Oatpl and Oatp2. Uptake of NMQ and NMQD by these carders was saturable with apparent Km values of 5 and 26 IJMfor OATP-A, 17 and 7 pM for rOCT1 and 19 and 12 pM for hOCT1, respectively, In contrast to its methyl derivative, QD was not transported by OATP-A, rOCT1 or hOCT1. Conclusions: Type 1 cations are only transported by the OCTs, whereas the type 2 cation rocuronium is only taken up by the Oatps. NMQ, NMQD and APDA belong to an intermediate class of organic cations and can be transported by both OCTs and Oatps. Therefore it is concluded that OCTs are charge specific and transport quaternary ammonium compounds, while Oetps are charge independent and transport anions, neutral compounds and bulky cations with OATP-A having the broadestactivity for the tested cations.
ENDOTOXIN AND T U R P E N T I N E BOTH PRODUCE CHOLESTASIS BUT HAVE DIFFERENT EFFECTS ON mRNA FOR TNF-a, l I . ~ , AND Mdrlb E Ott 1, K. Ban~ert 2, H.C. Bisgaard 3, N. Tygstrup 1 IHepatology, Rigshospitalet, Denmark. 2Clin Biochem, Rigshospitalet, Denmark. 3Life Sci. & Chem., Roskilde Univ, Denmark. In 200 g fed male Wistar rats, we compared the effects of systemic inflammation induced by i.p. lipopolysaccadde (LPB) and local aseptic inflammation induced by s.c. turpentine oil (TURP) on the hepatic expression of mRNA for the transporters Ntcp, Oatpl, Oatp2, Mrp2, Bsep, Mdrlb and Mdr2, the cytokines TNF-ct and IL-6, the acute phase proteins ¢t~-acidglycoprotein (Type I) and ~fibdnogen (Type II) and the negative acute phase protein albumin. The table shows mRNA concentrations (relative to saline treated controls=100) 6h and 12h after 15 mg/kg LPS i.p. or 1 ml TURP s.c. LPS TURP 6h (n=6) 12h (n=6) 6h (n=6) 12h (n=6) TNF-c¢ "797±109 ~216+_32 97:1:13"* 125+25" IL-6 #246+37 243+62 #86+2** #86+1" Albumin #58+4 #63¢4 (7,1.glucOprOt. #747+44 #530~90 B-fibrinogen #194+12 ~250-~_43 Mdrlb #331+50 #545+112 138+18" 106+9" Mdr2 #45:1:3 #42~4 #82.~3"* #49±2 Mrp2 #33+3 #43+17 #62.t:5"* ~6±5 Ntcp #57+5 #44+14 #75:1:5 ~63+5 Oatpl #54+4 #43+9 #g0~2** #77+4* Oatp2 #50¢8 #33+9 #53+10 #34+4 Bsep #72:!:4 #64¢8 86+5 #86+_2* : p<0.05 as compared to controls. * or ** : p<0.05 or p<0.005 for LPS versus TURP TNF-tx and IL-6 mRNA increased after LPS, not after TURP. Both compounds produced an acute phase reaction in which mRNA was decreased for albumin, increased for acute phase proteins, and downregulated for most of the transporters. Only LPS increased Mdrlb. Thus, intrahepatic production of TNF-ct and IL-6 is not needed for the inflammatory cholestatic reaction. Mdrlb upregulation is not part of this response.
118
I P/C07105 I
I
CORTICOSTEROIDS MODULATE THE SECRETORY PROCESSES OF INTRAHEPATIC B1LIARY E P I T H E L I U M D. Alvaro, L. Marncci l, A. Gigliozzi, G. Alpini 2, E, Papa, M. Delle Monache, R. Monterubbianesi. L. CaDocaccia, A. Bencdetti I Div. Gastro., Univ. Rome "La Sapienza", Italy. 1Div. Gastro., Univ. Ancona, Italy. 2Texas A&M Univ., Texas, USA.
DOWNREGULATION OF THE HEPATOCELLULAR ORGANIC ANI O N TRANSPORTERS OATP-C AND MRP2 IN PATIENTS W I T H PRIMARY SCLEROSING C H O L A N G I T I S M. Oswald, G.A. KuUak-Ublick, G. Paumgartncr, U. Beucrs Department of Medicine II, Klinikum Grosshadern, University of Munich, Munich, Germany. Background: Formation and secretion of bile depends on the coordinated action of various transporter proteins of the sinusoidal and canalicular membranes of the hepatoeyles. The recently isolated liver-specific organic anion transporter OATP-C (JBC 1999;274:t7159) is a liver-specific membrane transport protein for the basolateral uptake of organic anions from blood into the hepatocyte. The multidmg resistance associated protein 2 MRP2 mediates the canalicular excretion of organic anions into bile. Biliary secretion of organic anions is impaired in chronic cholestatic liver disease. The underlying molecular mechanisms, however, have not yet been eludicated. Therefore, the aim of our study was to investigate the expression of the organic anion transporters OATP-C and MRP2 in patients with primary sclcrosing cholangitis (PSC) (n=4), a chronic cholestatic liver disease as compared to controls (non-cholestatic liver disease, n=4).Methods: The expression of OATP-C, MRP2 and [3-actin (reference transcript) was studied using quantitative RT-PCR: Total RNA was isolated from liver tissue obtained at routine biopsy, reverse transcribed and then subjected to PCR amplification using specific primers. The PCR products were quantified using densitometry. The levels of OATP-C and MRP2 mRNA were normalized for [i-actin mRNA. Results: The level of OATP-CmRNA in liver tissue ofpatients with PSC (OATP-C/J~-actin 1.60 + 0.25) was only 49% of that in liver tissue of controls (OATP-C/[3-actin 3.24 + 0.69; p<0.05). The level of MRP2-mRNA in patients with PSC (MRP2/fl-actin 0.70 + 0.36) was reduced to 27% of controls (MRP2/[3-actin 2.54 + 0.56; p=0.005). In summary, the expression of both, the basolateral and the canalicular organic anion transport proteins, OATP-C and MRP2, was reduced in patients with PSC. Downreguiation of OATP-C might be the consequence of impaired canalicular secretion of organic anions and might, thereby, reduce the organic anion load of cholestatic hepatocytes.
We investigated the expression of glucocorticoid receptors (GcR) and the role of corticosteroids in the regulation of cholangiocyte secretory processes. METHODS: GcR were investigated by immunohistochemistry and RT-PCR. The effects of dexamethasone and budesonide on bicarbonate biliary excretion and I-F/HCO3" transport processes were evaluated in bile fistula rats and isolated bile ducts (IBDU). RESULTS: GcR were expressed in rat cholangiocytes and markedly increased after cholangiocyte proliferation induced by bile duct ligation. A 2-3 day treatment with dexamethasone (1 mg Lid) or budesonide (0.3 mg tid) induced an increased (p<0.05) bicarbonate biliary secretion. IBDU incubated for 24-36 hrs with dexamethasone or budesonide showed an increased (p<0.02) expression (+120 %, western blot) and activity (+60%) of Na+/I-I+ exchanger (NHE1 isoform) and of CI'/HC03" exchanger. CONCLUSIONS: The intrahepatic biliary epithelium express GcR. Corticosteroids enhance the expression and activities of transport processes driving bicarbonate excretion in bile suggesting a potential mechanism for the beneficial effects in the treatment of cholangiopathies. Supported by Grant # 9806210866 from MURST.
P/C07/07 I
I P/covoe I
I P/co,/o8 ]
HEPATOCELLULAR UPTAKE OF ORGANIC CATIONS BY THE ORGANIC ANION TRANSPORTING POLYPEPTIDES (Oatpl, Oatp2 AND OATP-A) AND THE ORGANIC CATION TRANSPORTERS (rOCTI and hOCTI) J.E. van Montfoort 1.3, B. Stieger 1, H. Koepsell 2, M. Mi~ller 3, G.M.M. Groothuis 3, D.K.E Metier 3. EJ. Meier ~ 1Die. Clinical Pharmacology and Toxicology, University Hospital, Ziirich, Switzerland. 2Department of Anatomy, University of Wttrzburg, Germany. 2Liver Research Center, Groningen Institute of Drug Studies, The Netherlands. Background: Based on kinetic transport studies organic cations have been divided into small and hydrophilic type 1 and more bulky end amphipathiotype 2 cations. Potentialcandidates for these so far only functionally charactadsed hepatic carders are the OCTs for type 1 cations and Oatps for type 2 cations. Aim: To investigate the discrimination between type 1 and type 2 cations by the OCTs and Oatps in a heterologous expression system and to determine structural features of the substrates necessary for this discrimination, Methods: Uptake studies in water and OCT- or Oatp-cRNAinjected Xenopus laevis oocytes with the type 1 cations tdbutylmethylammonium (TBuMA), azidoprocainamida methoiodide (APM) and azopentyl-quinuclidine (APQ) and the type 2 cations APD-ajmalinium (APDA), rocuronium, N-methyl-quinine (NMQ), N-methyl-quinidine (NMQD) and quinidlne (QD). Results: TBuMA, APM and APQ were taken up by rOCT1 and hOCT1, but not transported by any of the Oatps. Uptake of TBuMA by rOCTI and hOCT1 was saturable and yielded apparent Km values of 34 and 53 pM, respectively. APDA was transported by all investigated carders, while the uptake of rocuronium was restricted to the Oatps. NMQ and NMQD were transported by OATP-A (previously called OATP), rOCT1 and hOCT1, but not by Oatpl and Oatp2. Uptake of NMQ and NMQD by these carders was saturable with apparent Km values of 5 and 26 IJMfor OATP-A, 17 and 7 pM for rOCT1 and 19 and 12 pM for hOCT1, respectively, In contrast to its methyl derivative, QD was not transported by OATP-A, rOCT1 or hOCT1. Conclusions: Type 1 cations are only transported by the OCTs, whereas the type 2 cation rocuronium is only taken up by the Oatps. NMQ, NMQD and APDA belong to an intermediate class of organic cations and can be transported by both OCTs and Oatps. Therefore it is concluded that OCTs are charge specific and transport quaternary ammonium compounds, while Oetps are charge independent and transport anions, neutral compounds and bulky cations with OATP-A having the broadestactivity for the tested cations.
ENDOTOXIN AND T U R P E N T I N E BOTH PRODUCE CHOLESTASIS BUT HAVE DIFFERENT EFFECTS ON mRNA FOR TNF-a, l I . ~ , AND Mdrlb E Ott 1, K. Ban~ert 2, H.C. Bisgaard 3, N. Tygstrup 1 IHepatology, Rigshospitalet, Denmark. 2Clin Biochem, Rigshospitalet, Denmark. 3Life Sci. & Chem., Roskilde Univ, Denmark. In 200 g fed male Wistar rats, we compared the effects of systemic inflammation induced by i.p. lipopolysaccadde (LPB) and local aseptic inflammation induced by s.c. turpentine oil (TURP) on the hepatic expression of mRNA for the transporters Ntcp, Oatpl, Oatp2, Mrp2, Bsep, Mdrlb and Mdr2, the cytokines TNF-ct and IL-6, the acute phase proteins ¢t~-acidglycoprotein (Type I) and ~fibdnogen (Type II) and the negative acute phase protein albumin. The table shows mRNA concentrations (relative to saline treated controls=100) 6h and 12h after 15 mg/kg LPS i.p. or 1 ml TURP s.c. LPS TURP 6h (n=6) 12h (n=6) 6h (n=6) 12h (n=6) TNF-c¢ "797±109 ~216+_32 97:1:13"* 125+25" IL-6 #246+37 243+62 #86+2** #86+1" Albumin #58+4 #63¢4 (7,1.glucOprOt. #747+44 #530~90 B-fibrinogen #194+12 ~250-~_43 Mdrlb #331+50 #545+112 138+18" 106+9" Mdr2 #45:1:3 #42~4 #82.~3"* #49±2 Mrp2 #33+3 #43+17 #62.t:5"* ~6±5 Ntcp #57+5 #44+14 #75:1:5 ~63+5 Oatpl #54+4 #43+9 #g0~2** #77+4* Oatp2 #50¢8 #33+9 #53+10 #34+4 Bsep #72:!:4 #64¢8 86+5 #86+_2* : p<0.05 as compared to controls. * or ** : p<0.05 or p<0.005 for LPS versus TURP TNF-tx and IL-6 mRNA increased after LPS, not after TURP. Both compounds produced an acute phase reaction in which mRNA was decreased for albumin, increased for acute phase proteins, and downregulated for most of the transporters. Only LPS increased Mdrlb. Thus, intrahepatic production of TNF-ct and IL-6 is not needed for the inflammatory cholestatic reaction. Mdrlb upregulation is not part of this response.
118