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Characterization of the human Na+-dependent bile acid transporter (NTCP) using a recombinant vaccinia system
HEPATOLOGY V o k 22, No. 4, P t . 2, 1995
AASLD ABSTRACTS
849 EXPRESSION OF THE HUMAN Na+-DEPENDENT BILE ACID TRANSPORTER (NTCP) USING A RECOMBINANT VACCINIA SYSTEM. RB Kim. LM Affati~ato. MM Roden. and GR Wilkinson. Department of Pharmacology, Vanderbilt University, Nashville, TN. Transport of bile acids from the portal circulation into the hepatocytes is an important step in their overall entero-hepatic recircniation. Recently, cDNA encoding a human Na+/bile acid cotransporter (NTCP) has been cloned and expressed in Xenopus laevis oocytes. However, expression of the protein in a mammalian cell line, that more closely reflects its human origin and function, has yet to be reported. Therefore, we utilized the recombinant vaccinia virus (VTF-7), and Hela ceils as a host for the expression of NTCP cDNA. This virus expresses T7-RNA polymerase and cotransfection of cDNA with a sense insert downstream from the T7-promotor results in high expression levels. After cloning the full length NTCP cDNA from RNA derived from a human liver, this was ligated into a pTM1 vector in the proper orientation. The pTM1 vector was chosen because it has strong start and stop sequences for the T-7 R N A polymerase, and produces efficient cap-independent translation of T7 transcripts. The NTCP/pTM1 vector was transfected into Hela cells and [3H]-taurocholate transport studies were carried out 16 hours later. A control pTM1 vector without the cDNA insert failed to show any transport activity. However, the uptake of taurocholate mediated by NTCP/pTM1 was found to be rapid and reached saturation by 15 minutes. Kinetic analysis reveal an apparent Km of 7.9 ttM and a Vmax of 49 pmo1/mg protein/min. The observed Km is similar to that reported using the Xenopus laevis system, but is much lower than the rodent protein(Ntcp): Furthermore, the results show that recombinant vaccinia-based systems are well suited for the expression of individual bile acid transporters and have the sensitivity to allow for detailed functional studies without concerns about the presence of other transporters with overlapping substrate specificities, as is the case when using isolated hepatocyte or membrane vesicles.
8 5 1 CHARACTERIZATION O F THE HUMAN Na+-DEPENDENT BILE ACID TRANSPORTER (NTCP) USING A RECOMBINANT VACCINIA SYSTEM. RB Kim. LM Affati~ato. JM Nadeau. and GR Wilkinson. Dept. of Pharmacology, Vanderbilt University, Nashville, TN. The human Na+/bile acid cotransporter (NTCP) is thought to be important in the uptake of bile acids from the portal circulation. Although the transport of the prototypic substrate taurocholate is inhibited by related bile acids, little else is known regarding the protein's physiological function or non-bile acid substrates. Accordingly, we have carried out studies using the recombinant vaccinia virus (VTF-7) to express the human NTCP cDNA and to investigate the functional effects of related bile acids and several drugs. The constructs NTCP/pTMI or pTM1 vector alone as a control were cotransfected with the vaccinia virus and expressed in Hela cells. A variety of bile acids (100#M) such as chenodeoxycholate, deoxycholate, cholate, and glycocholate, as well as the anionic dye, bromosulphothalein (100#M) were found to inhibit [3H]-taurocholate transport. Additionally, quinidine (100#M) enhanced NTCP-mediated transport by up to 70% (p <0.05), while procainamide (100#M) was without effect. Similarly a 40% (p<0.01) enhancement of transport was also seen with 1% ethanol. Other drugs such as tricyclic antidepressants, and those with calcium entry blocking properties, such as nifedipine, had no effect. However, the B-blocker, propranolol (100tzM), produced nearly 50% reduction (p<0101) in taurocholate transport. Lovastatin, a drug previously suggested to be a substrate for bile acid transporter(s~, failed to inhibit NTCP-mediated taurocholate transport. A number of di and tripeptides were also tested and found to be without effect, although a modest (15%) reduction was seen with the renin-inhibitor oligopeptide EMD51921 (10#M). This was not seen with another renin inhibitor U71038 (100#M). We conclude that functioning of the human Na+/bile acid cotransporter may be modulated positively or negatively by a variety of drugs and possess a broader substrate specificity than just bile acids. Accordingly, it may have a role in the hepatic disposition of some xenobiotics.
850
319A
EXPRF~SION OF THE RAT LIVER Na+/TAUROCHOLATE COTRANSPORTER (ntcp) IS REGULATED IN VIVO BY RETENTION OF BILIARY CONSTITUENTS, BUT NOT THEIR DEPLETION. C. Gartun~. S. Schuele: S. Schlosser. and J. L. Bover. Liver Center, Yale University School of Medicine, New Haven, CT. Expression and function of the hepatic mcp are down-regulated in several rat models of cholestaals. To test whether retandon and/or depletion of biliary constituents are involved in ntcp regulation, protein mass and steadystate mRNA levels of ntcp were quantified by Western and Northern analysis in rats with either choledochocaval fistulas (CCF) (retention model) or chronic bile fistulas (depletion model). In addition, ntcp mRNA levels were assessed in rats with selective bile duct ligation (SBDL), a cholestatic model, in which both obstructed and non-obstructed lobes can be compared in the same liver. Results: One and 3 days after CCF, ntcp mRNA expression specifically declined by 76 + 4% (p<0.005) and 31 + 9% (p<0.05), respectively returning to control levels by 7 days. In contrast, mRNA levels of cholesterol 7a-hydroxylase (Ch7cx-OH), an enzyme known to be regulated by bile acids, remained unchanged at 1 day but gradthlly increased 1.9 and 9.3-fold, respectively after 3 and 7 days of CCF. mRNA levels of Na, K-ATPase and GAPDH did not decline during this period. Protein expression of both ntcp and Na, K-ATPase was assessed by Western blots and remained unchanged for up to 7 days of CCF. In rats with bile fistulas for 0.5, 1, 2, 4 and 7 days, both ntcp protein and mRNA expression remained unaltered, while Ch7c~-OH mRNA increased 3-4-fold over controls. In SBDL rats, ntcp mRNA levels were down-regulated by 40 + 10% (p < 0.05) only after 12 and 24 hours in !igated lobes, and mRNA levels returned to control values in these lobes after 2 and 4 days. Ntcp mRNA expression remained unchanged in the non-obstructed lobes at any time. When data from CCF & SBDL rats were combined, serum bile acids correlated linearly with ntcp mRNA (r= .62, p < .0005) over a 0-110 #mol/l range. Conclusions: Ntcp is constitutively expressed and remains uneffected by either depletion or increased flux of biliary constituents. However, retention of biliary constituents results in rapid down-regulation of ntcp mRNA consistent with the concept that hepatocytes may be protected from bile acid toxicity during cholestasis by this mechanism.
852 INCREASEDNA÷/TAUROCHOLATE('TO) COTRANSPORTIN BASOLATERAL LIVER PLASMA MEMBRANE VESICLES (BLPM) POST PARTUM (PP) IS DUE TO OVEREXPRESSIONOF NA+/TC COTRANSPORTPOLYPEPTIDE (NTCP). Y Liu. B Stieger. P Meier. and M Vore. Dept Pharmacology, U Kentucky, Lexington, KY; Division Clinical Pharmacology & Toxicology, U Hospital, Zurich, Switzerland. Na*/-FC cotransport in bLPM and Ntcp mRNA are increased PP (Biochem J. 303:33) and following treatement of ovariectomized rats (OVX) with ovine prolactin (oPRL) (Am. J. Phsiol. 268:G11). The aim of the present studies was to determine 1) Km and Vm=( for Na*/TC cotransport in bLPM in PP and pregnant rats (P) relative to normal non-pregnant rats (NP), 2) if expression of Ntcp is correlated with changes in steady state Ntcp mRNA and Na÷/TC cotransport in NP, P, and PP, and 3) if PRL treatment increases Ntcp in bLPM relative to solvent treatment (SOL). Methods: Na+/TC cotransport was determined in bLPM from NP, P and PP (n=3-6). Ntcp in bLPM was determined using Western blot analysis (n=3-4). PRL (n=6) or solvent (SOL, n=4) was administered to ovariectomized rats (OVX) for seven day via osmotic minipump. Contour plots of the joint 95% confidence limits were generated to detect statistical differences in Km and Vm~. *, p<.05. ReSults: Vmax (pmol/mg/5 S) of Na+/TC cotransport was increased 1.7 fold (*) in PP (426) relative to NP (250) and P (240). Kms (/~M) were not different among NP (15), P (18), and PP (15). Ntcp expression in PP was increased 2 fold (*) relative to P and NP, and Ntcp expression was increased 1.4 (*) fold in PRL relative to SOL. Concluslons: The increase in Ntcp expression represents a major mechanism for the increased Na÷/TC cotransport activity in PP and following PRL treatment. However, there is no difference in Ntcp expression or in Na+/TC cotransport activity in bLPM in P vs NP rats.
AASLD ABSTRACTS
849 EXPRESSION OF THE HUMAN Na+-DEPENDENT BILE ACID TRANSPORTER (NTCP) USING A RECOMBINANT VACCINIA SYSTEM. RB Kim. LM Affati~ato. MM Roden. and GR Wilkinson. Department of Pharmacology, Vanderbilt University, Nashville, TN. Transport of bile acids from the portal circulation into the hepatocytes is an important step in their overall entero-hepatic recircniation. Recently, cDNA encoding a human Na+/bile acid cotransporter (NTCP) has been cloned and expressed in Xenopus laevis oocytes. However, expression of the protein in a mammalian cell line, that more closely reflects its human origin and function, has yet to be reported. Therefore, we utilized the recombinant vaccinia virus (VTF-7), and Hela ceils as a host for the expression of NTCP cDNA. This virus expresses T7-RNA polymerase and cotransfection of cDNA with a sense insert downstream from the T7-promotor results in high expression levels. After cloning the full length NTCP cDNA from RNA derived from a human liver, this was ligated into a pTM1 vector in the proper orientation. The pTM1 vector was chosen because it has strong start and stop sequences for the T-7 R N A polymerase, and produces efficient cap-independent translation of T7 transcripts. The NTCP/pTM1 vector was transfected into Hela cells and [3H]-taurocholate transport studies were carried out 16 hours later. A control pTM1 vector without the cDNA insert failed to show any transport activity. However, the uptake of taurocholate mediated by NTCP/pTM1 was found to be rapid and reached saturation by 15 minutes. Kinetic analysis reveal an apparent Km of 7.9 ttM and a Vmax of 49 pmo1/mg protein/min. The observed Km is similar to that reported using the Xenopus laevis system, but is much lower than the rodent protein(Ntcp): Furthermore, the results show that recombinant vaccinia-based systems are well suited for the expression of individual bile acid transporters and have the sensitivity to allow for detailed functional studies without concerns about the presence of other transporters with overlapping substrate specificities, as is the case when using isolated hepatocyte or membrane vesicles.
8 5 1 CHARACTERIZATION O F THE HUMAN Na+-DEPENDENT BILE ACID TRANSPORTER (NTCP) USING A RECOMBINANT VACCINIA SYSTEM. RB Kim. LM Affati~ato. JM Nadeau. and GR Wilkinson. Dept. of Pharmacology, Vanderbilt University, Nashville, TN. The human Na+/bile acid cotransporter (NTCP) is thought to be important in the uptake of bile acids from the portal circulation. Although the transport of the prototypic substrate taurocholate is inhibited by related bile acids, little else is known regarding the protein's physiological function or non-bile acid substrates. Accordingly, we have carried out studies using the recombinant vaccinia virus (VTF-7) to express the human NTCP cDNA and to investigate the functional effects of related bile acids and several drugs. The constructs NTCP/pTMI or pTM1 vector alone as a control were cotransfected with the vaccinia virus and expressed in Hela cells. A variety of bile acids (100#M) such as chenodeoxycholate, deoxycholate, cholate, and glycocholate, as well as the anionic dye, bromosulphothalein (100#M) were found to inhibit [3H]-taurocholate transport. Additionally, quinidine (100#M) enhanced NTCP-mediated transport by up to 70% (p <0.05), while procainamide (100#M) was without effect. Similarly a 40% (p<0.01) enhancement of transport was also seen with 1% ethanol. Other drugs such as tricyclic antidepressants, and those with calcium entry blocking properties, such as nifedipine, had no effect. However, the B-blocker, propranolol (100tzM), produced nearly 50% reduction (p<0101) in taurocholate transport. Lovastatin, a drug previously suggested to be a substrate for bile acid transporter(s~, failed to inhibit NTCP-mediated taurocholate transport. A number of di and tripeptides were also tested and found to be without effect, although a modest (15%) reduction was seen with the renin-inhibitor oligopeptide EMD51921 (10#M). This was not seen with another renin inhibitor U71038 (100#M). We conclude that functioning of the human Na+/bile acid cotransporter may be modulated positively or negatively by a variety of drugs and possess a broader substrate specificity than just bile acids. Accordingly, it may have a role in the hepatic disposition of some xenobiotics.
850
319A
EXPRF~SION OF THE RAT LIVER Na+/TAUROCHOLATE COTRANSPORTER (ntcp) IS REGULATED IN VIVO BY RETENTION OF BILIARY CONSTITUENTS, BUT NOT THEIR DEPLETION. C. Gartun~. S. Schuele: S. Schlosser. and J. L. Bover. Liver Center, Yale University School of Medicine, New Haven, CT. Expression and function of the hepatic mcp are down-regulated in several rat models of cholestaals. To test whether retandon and/or depletion of biliary constituents are involved in ntcp regulation, protein mass and steadystate mRNA levels of ntcp were quantified by Western and Northern analysis in rats with either choledochocaval fistulas (CCF) (retention model) or chronic bile fistulas (depletion model). In addition, ntcp mRNA levels were assessed in rats with selective bile duct ligation (SBDL), a cholestatic model, in which both obstructed and non-obstructed lobes can be compared in the same liver. Results: One and 3 days after CCF, ntcp mRNA expression specifically declined by 76 + 4% (p<0.005) and 31 + 9% (p<0.05), respectively returning to control levels by 7 days. In contrast, mRNA levels of cholesterol 7a-hydroxylase (Ch7cx-OH), an enzyme known to be regulated by bile acids, remained unchanged at 1 day but gradthlly increased 1.9 and 9.3-fold, respectively after 3 and 7 days of CCF. mRNA levels of Na, K-ATPase and GAPDH did not decline during this period. Protein expression of both ntcp and Na, K-ATPase was assessed by Western blots and remained unchanged for up to 7 days of CCF. In rats with bile fistulas for 0.5, 1, 2, 4 and 7 days, both ntcp protein and mRNA expression remained unaltered, while Ch7c~-OH mRNA increased 3-4-fold over controls. In SBDL rats, ntcp mRNA levels were down-regulated by 40 + 10% (p < 0.05) only after 12 and 24 hours in !igated lobes, and mRNA levels returned to control values in these lobes after 2 and 4 days. Ntcp mRNA expression remained unchanged in the non-obstructed lobes at any time. When data from CCF & SBDL rats were combined, serum bile acids correlated linearly with ntcp mRNA (r= .62, p < .0005) over a 0-110 #mol/l range. Conclusions: Ntcp is constitutively expressed and remains uneffected by either depletion or increased flux of biliary constituents. However, retention of biliary constituents results in rapid down-regulation of ntcp mRNA consistent with the concept that hepatocytes may be protected from bile acid toxicity during cholestasis by this mechanism.
852 INCREASEDNA÷/TAUROCHOLATE('TO) COTRANSPORTIN BASOLATERAL LIVER PLASMA MEMBRANE VESICLES (BLPM) POST PARTUM (PP) IS DUE TO OVEREXPRESSIONOF NA+/TC COTRANSPORTPOLYPEPTIDE (NTCP). Y Liu. B Stieger. P Meier. and M Vore. Dept Pharmacology, U Kentucky, Lexington, KY; Division Clinical Pharmacology & Toxicology, U Hospital, Zurich, Switzerland. Na*/-FC cotransport in bLPM and Ntcp mRNA are increased PP (Biochem J. 303:33) and following treatement of ovariectomized rats (OVX) with ovine prolactin (oPRL) (Am. J. Phsiol. 268:G11). The aim of the present studies was to determine 1) Km and Vm=( for Na*/TC cotransport in bLPM in PP and pregnant rats (P) relative to normal non-pregnant rats (NP), 2) if expression of Ntcp is correlated with changes in steady state Ntcp mRNA and Na÷/TC cotransport in NP, P, and PP, and 3) if PRL treatment increases Ntcp in bLPM relative to solvent treatment (SOL). Methods: Na+/TC cotransport was determined in bLPM from NP, P and PP (n=3-6). Ntcp in bLPM was determined using Western blot analysis (n=3-4). PRL (n=6) or solvent (SOL, n=4) was administered to ovariectomized rats (OVX) for seven day via osmotic minipump. Contour plots of the joint 95% confidence limits were generated to detect statistical differences in Km and Vm~. *, p<.05. ReSults: Vmax (pmol/mg/5 S) of Na+/TC cotransport was increased 1.7 fold (*) in PP (426) relative to NP (250) and P (240). Kms (/~M) were not different among NP (15), P (18), and PP (15). Ntcp expression in PP was increased 2 fold (*) relative to P and NP, and Ntcp expression was increased 1.4 (*) fold in PRL relative to SOL. Concluslons: The increase in Ntcp expression represents a major mechanism for the increased Na÷/TC cotransport activity in PP and following PRL treatment. However, there is no difference in Ntcp expression or in Na+/TC cotransport activity in bLPM in P vs NP rats.