Physiological characteristics of allo-cholic acid
2003; Elsevier BV; Volume: 44; Issue: 1 Linguagem: Inglês
10.1194/jlr.m200220-jlr200
ISSN1539-7262
AutoresMaria E Mendoza, María J. Monte, María A. Serrano, Marçal Pastor‐Anglada, Bruno Stieger, Peter J. Meier, Manuel Medarde, José J.G. Marı́n,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoThe physiological characterstics of allo-cholic acid (A91), a typically fetal bile acid that reappears during liver regeneration and carcinogenesis were investigated. [14C] Tauro-ACA (TACA) uptake by Chinese hamster ovary cells expressing rat organic anion transporter polypeptide (Oatp)1 or sodium-taurocholate cotransporter polypeptide (Ntcp) was lower than that of [14C]taurocholic acid (TCA). Although TACA inhibited ATP-dependent TCA transport across plasma membrane vesicles from Sf9 cells expressing rat or mouse bile salt export pump (Bsep), no ATP-dependent TACA transport was found. In rats, TACA was secreted into bile with no major biotransformation and it had lower clearance and longer half-life than TCA. In mice, TACA bile output was lower (−50%) than that of TCA, whereas TACA induced 9-fold higher bile flow than TCA. Even though the intracellular levels were lower for TACA, translocation into the hepatocyte nucleus was higher for TACA than for TCA; however, rate of DNA synthesis, expression levels of α-fetoprotein, albumin, Ntcp, and Bsep, cell viability, and apoptosis in rat hepatocytes were similarly affected by both isomers. In conclusion, TACA partly shares hepatocellular uptake system(s) for TCA.Furthermore, in contrast to other "flat" bile acids, TACA is efficiently secreted into bile via transport system(s) other than Bsep and is highly choleretic, hence its appearance during certain situations may prevent accumulation of cholestatic precursors. The physiological characterstics of allo-cholic acid (A91), a typically fetal bile acid that reappears during liver regeneration and carcinogenesis were investigated. [14C] Tauro-ACA (TACA) uptake by Chinese hamster ovary cells expressing rat organic anion transporter polypeptide (Oatp)1 or sodium-taurocholate cotransporter polypeptide (Ntcp) was lower than that of [14C]taurocholic acid (TCA). Although TACA inhibited ATP-dependent TCA transport across plasma membrane vesicles from Sf9 cells expressing rat or mouse bile salt export pump (Bsep), no ATP-dependent TACA transport was found. In rats, TACA was secreted into bile with no major biotransformation and it had lower clearance and longer half-life than TCA. In mice, TACA bile output was lower (−50%) than that of TCA, whereas TACA induced 9-fold higher bile flow than TCA. Even though the intracellular levels were lower for TACA, translocation into the hepatocyte nucleus was higher for TACA than for TCA; however, rate of DNA synthesis, expression levels of α-fetoprotein, albumin, Ntcp, and Bsep, cell viability, and apoptosis in rat hepatocytes were similarly affected by both isomers. In conclusion, TACA partly shares hepatocellular uptake system(s) for TCA. Furthermore, in contrast to other "flat" bile acids, TACA is efficiently secreted into bile via transport system(s) other than Bsep and is highly choleretic, hence its appearance during certain situations may prevent accumulation of cholestatic precursors. Major bile acids (BAs) are mono or polyhydroxylated acidic steroids with a 5β-cholanoyl structure in which the A and B rings are approximately perpendicular; however, minor BAs with unsaturations affecting C5 or with a 5α-cholanoyl configuration (allo-BA) have both rings on the same plane, explaining their designation as "flat" BAs. They are uncommon in healthy adult mammals, but are present in fetuses as well as in lower species. For example, in some migratory species of fishes, such as Petromyzon marinus, allo-BAs are strong specific stimulants of the olfactory epithelium (1Li W.M. Sorensen P.W. Gallaher D.D. The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae.J. Gen. Physiol. 1995; 105: 569-587Google Scholar). These BAs may play an important role as conspecific migratory pheromones produced by larvae to attract adult individuals towards reproductive areas upstream in the river (2Bjerselius R. Li W.M. Teeter J.H. Seelye J.G. Johnsen P.B. Maniak P.J. Grant G.C. Polkinghorne C.N. Sorensen P.W. Direct behavioral evidence that unique bile acids released by larval sea lamprey (Petromyzon marinus) function as a migratory pheromone.Can. J. Fish. Aquat. Sci. 2000; 57: 557-569Google Scholar). Allo-BAs were first described at the beginning of the previous century and little attention has since been paid to the physiological characteristics of these BAs in mammals (3Elliot W.H. Allo bile acids.in: The bile acids. Chemistry, Physiology and Metabolism. Vol. 1. Plenum Press, New York1971: 47-93Google Scholar). Because the cholestatic properties of other BAs that share with allo-BAs their flat structure have been described and the molecular bases of this effect have been studied (4Stieger B. Zhang J. O'Neill B. Sjovall J. Meier P.J. Differential interaction of bile acids from patients with inborn errors of bile acid synthesis with hepatocellular bile acid transporters.Eur. J. Biochem. 1997; 244: 39-44Google Scholar), the first objective of the current study was to investigate the liver handling of allo-cholic acid (ACA) (see structure in the inset of Fig. 1). Flat BAs reappear in both adult humans and in the rat during physiological and pathophysiological liver cell proliferation. They are easily detected in the serum and even more easily in the urine, presumably due to the low ability of the liver to handle them, in patients with hepatocellular carcinoma (5El-Mir M.Y. Badia M.D. Luengo N. Monte M.J. Marin J.J.G. Increased levels of typically fetal bile acid species in patients with hepatocellular carcinoma.Clin. Sci. 2001; 100: 499-508Google Scholar). In rats, flat BAs also reappear during hepatocarcinogenesis (6Monte M.J. Palomero M.F. Sainz G.R. Dominguez M. Diez M. Toraño A. G. Marin J.J. Bile acid secretion during rat liver carcinogenesis.Life Sci. 2000; 66: 1085-1095Google Scholar, 7Mendoza M.E. Monte M.J. El-Mir M.Y. Badia M.D. Marin J.J.G. Changes in the pattern of bile acids in the nuclei of rat liver cells during hepatocarcinogenesis.Clin. Sci. 2002; 102: 143-150Google Scholar) and are transiently elevated during the liver regeneration that follows two-thirds partial hepatectomy (8Monte M.J. El-Mir M.Y. Sainz G.R. Bravo P. Marin J.J.G. Bile acid secretion during synchronized rat liver regeneration.Biochim. Biophys. Acta. 1997; 1362: 56-66Google Scholar, 9Monte M.J. Martinez-Diez M.C. El-Mir M.Y. Mendoza M.E. Bravo P. Bachs O. Marin J.J.G. Changes in the pool of bile acids in hepatocyte nuclei during rat liver regeneration.J. Hepatol. 2002; 36: 534-542Google Scholar). Whether this is a mere epiphenomenon or allo-BAs play a biological role is unknown. The discovery of BAs in the nuclei of rat hepatocytes (10Setchell K.D.R. Rodrigues C.M.P. Clerici C. Solinas A. Morelli A. Gartung C. Boyer J. Bile acid concentrations in human and rat liver tissue and in hepatocyte nuclei.Gastroenterology. 1997; 112: 226-235Google Scholar) together with the observation that the composition of this sub-pool of BAs markedly differs from that of the cytosolic sub-pool (7Mendoza M.E. Monte M.J. El-Mir M.Y. Badia M.D. Marin J.J.G. Changes in the pattern of bile acids in the nuclei of rat liver cells during hepatocarcinogenesis.Clin. Sci. 2002; 102: 143-150Google Scholar, 9Monte M.J. Martinez-Diez M.C. El-Mir M.Y. Mendoza M.E. Bravo P. Bachs O. Marin J.J.G. Changes in the pool of bile acids in hepatocyte nuclei during rat liver regeneration.J. Hepatol. 2002; 36: 534-542Google Scholar) and the fact that important changes occur in the nuclear sub-pool during liver carcinogenesis (7Mendoza M.E. Monte M.J. El-Mir M.Y. Badia M.D. Marin J.J.G. Changes in the pattern of bile acids in the nuclei of rat liver cells during hepatocarcinogenesis.Clin. Sci. 2002; 102: 143-150Google Scholar) and regeneration (9Monte M.J. Martinez-Diez M.C. El-Mir M.Y. Mendoza M.E. Bravo P. Bachs O. Marin J.J.G. Changes in the pool of bile acids in hepatocyte nuclei during rat liver regeneration.J. Hepatol. 2002; 36: 534-542Google Scholar) suggest that allo-BAs may be involved in signaling mechanisms related to proliferation, differentiation, viability, or apoptosis of parenchymal liver cells. We therefore extended these studies in an attempt to decipher this question by investigating the ability of ACA to reach the hepatocyte nucleus and to affect the processes mentioned above. Bile acids or sodium salts and methyl cholate (more than 95% pure by TLC), taurine, 3α-hydroxysteroid dehydrogenase, sulfatase, β-glucuronidase, cholylglycine hydrolase, Hoechst-33258, neutral red, culture media, supplements, and antibiotics for hepatocyte and cell line cultures were from Sigma-Aldrich (Madrid, Spain). G418-sulfate (Geneticin) and fetal calf serum were from Boehringer Manheim (Barcelona, Spain). [14C]Taurocholic acid (TCA, specific activity 49 mCi/mmol), [3H]TCA (specific activity 3 Ci/mmol), [14C]taurine (specific activity 108.5 mCi/mmol) and [methyl-14C]thymidine (specific activity 57 mCi/mmol) were from PerkinElmer Life Science (Pacisa & Giralt, Madrid, Spain). 3α,7α,12α-trihydroxy-5α-cholanoic acid (ACA) was synthesized from methyl ester (9% yield) by the method described by Iida et al. (11Iida T. Nishida S. Chang F.C. Niwa T. Goto J. Nabara T. Potential bile acid metabolites. XXI. A new synthesis of allo-chenodeoxycholic and allo-cholic acids.Chem. Pharm. Bull. 1993; 41: 767-768Google Scholar). The trimethylsilyl ether derivative of methyl-ACA was prepared and analyzed by gas-chromatography mass-spectrometry tandem (GC-MS) (5El-Mir M.Y. Badia M.D. Luengo N. Monte M.J. Marin J.J.G. Increased levels of typically fetal bile acid species in patients with hepatocellular carcinoma.Clin. Sci. 2001; 100: 499-508Google Scholar) using commercial ACA (Toronto Research Chemicals, Ontario, Canada) as standard. Both the retention-times in GC (data not shown) and mass spectra (Fig. 2)were consistent with the assigned identity of the product. The purity was approximately 99%. The radiolabeled derivative [14C]tauro-allo-cholic acid (TACA) (259 μCi/mmol specific activity) was obtained by conjugating ACA with [14C]taurine (12Tserng K. Hachey D. Klein P. An improved procedure for the synthesis of glycine and taurine conjugates of bile acids.J. Lipid Res. 1977; 18: 404-407Google Scholar). [14C]TACA was purified to more than 98% by liquid-solid extraction in octadecylsilane cartridges followed by preparative TLC. Intermediate compounds in the reactions and the final products were dissolved in CDCl3 and characterized by proton nuclear magnetic resonance (1H-NMR) at 200 MHz using tetramethylsilane as internal standard. Figure 1 depicts the 1H-NMR spectrum for the methyl-ACA, indicating the most characteristics signals. The characteristic shifts (δ) for methyl-ACA and TACA: the hydrogens in β disposition geminal to the α-hydroxyl groups on three, seven, and 12 and the methyl groups (18, 19, and 21), as well as the taurine methylenes 25 and 26, are shown in Table 1. Moreover, as expected, the signal corresponding to the hydrogen geminal to the 3α-hydroxyl changed from a broad multiplet (axial disposition) in cholic acid (CA) (data not shown) to a narrow multiplet (equatorial disposition) in ACA (Fig. 1).TABLE 11H-NMR assignments3α,7α,12α-Trihydroxy-5α- Cholanoic Acid Methyl EsterGroupaNumbering of groups refers to those of positions for C in the usual bile acid structure of a cholanoic acid. Solvent was CDCl3.Number of HShift (δ) Multiplicity3β-H1 4.05 m7β-H1 3.83 m12β-H1 3.95 m18-Me3 0.68 s19-Me3 0.77 s21-Me3 0.98 dJ = 5.4 HzCOOMe3 3.66 s3α,7α,12α-Trihydroxy-5α-Cholanoyl-Taurine3β-H1 3.85 m7β-H1 3.67 m12β-H1 3.81 m18-Me3 0.59 s19-Me3 0.69 s21-Me3 0.90 dJ = 5.4 Hz25-H2 2.87 t26-H2 3.47 tδ is given in ppm. Tetramethylsilane was used as internal reference.a Numbering of groups refers to those of positions for C in the usual bile acid structure of a cholanoic acid. Solvent was CDCl3. Open table in a new tab δ is given in ppm. Tetramethylsilane was used as internal reference. Chinese hamster ovary (CHO) cells that had previously been stably transfected with the cDNA for rat sodium-taurocholate cotransporter polypeptide (Ntcp) (13Schroeder A. Eckhardt U. Stieger B. Tynes R. Schteingart C.D. Hofmann A.F. Meier P.J. Hagenbuch B. Substrate specificity of the rat liver Na+-bile salt cotransporter in Xenopus laevis oocytes and in CHO cells.Am. J. Physiol. 1998; 274: G370-G375Google Scholar) or organic anion transporter polypeptide (Oatp1) (14Eckhardt U. Schroeder A. Stieger B. Hochli M. Landmann L. Tynes R. Meier P.J. Hagenbuch B. Polyspecificity substrate uptake by the hepatic organic anion transporter Oatp1 in stably transfected CHO cells.Am. J. Physiol. 1999; 276: G1037-G1042Google Scholar) and wild-type CHO cells were used in uptake studies. Cells were grown in DMEM supplemented with 10% fetal calf serum, 2 mM l-glutamine, 50 μg/ml l-proline, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.5 μg/ml amphotericin B at 37°C in an atmosphere of 5% CO2-95% air. Selective media contained additional 400 μg/ml geneticin. The expression of above-mentioned carriers was induced by incubating the transfected cells for 24 h with culture medium supplemented with 5 mM butyrate (15Palermo D.P. De Graaf M.E. Marotti D.R. Rehberg E. Post L.E. Production of analytical quantities of recombinant proteins in Chinese hamster ovarian cells using sodium butyrate to elevate gene expression.J. Biotechnol. 1991; 19: 35-48Google Scholar). Once grown to confluence on 60 mm diameter dishes, the cells were rinsed three times with pre-warmed (37°C) sodium- or choline-containing Earle's balanced saline solution supplemented with 5.5 mM d-glucose. Cells were then incubated at 37°C in the presence of 20 μM [14C]TACA or [14C]TCA for 15 min. Transport was stopped with 2 ml of ice-cold sodium- or choline-containing Earle's solution. After two additional washing steps with stop solution, the cells were digested in 1 ml Lowry solution (100 mM NaOH, 189 mM Na2CO3) for 2 h. Cells were harvested by scraping to measure radioactivity and total protein concentrations. Plasma membrane vesicles from insect Sf 9 cells expressing rat or mouse bile salt export pump (Bsep) were obtained as previously described (16Gerloff T. Stieger B. Hagenbuch B. Madon J. Landmann L. Roth J. Hofmann A.F. Meier P.J. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver.J. Biol. Chem. 1998; 273: 10046-10050Google Scholar, 17Noe J. Hagenbuch B. Meier P.J. St-Pierre M.V. Characterization of the mouse bile salt export pump overexpressed in the baculovirus system.Hepatology. 2001; 33: 1223-1231Google Scholar). Vesicles were incubated with [3H]TCA, [14C]TACA, or both in the absence or in the presence of 5 mM ATP plus an ATP regenerating system (3 mM phosphocreatine plus 100 μg/ml creatine phosphokinase) for 20 min, and uptake was stopped using a rapid filtration technique, as previously described (17Noe J. Hagenbuch B. Meier P.J. St-Pierre M.V. Characterization of the mouse bile salt export pump overexpressed in the baculovirus system.Hepatology. 2001; 33: 1223-1231Google Scholar, 18Marin J.J.G. Bravo P. El-Mir M.Y. Serrano M.A. ATP-dependent bile acid transport across microvillous membrane of human term trophoblast.Am. J. Physiol. 1995; 268: G685-G694Google Scholar). The study was carried out at a high substrate concentration (100 μM) in order to increase the radioactivity retained by the vesicles on the filters up to an accurate level. Additionally, to accomplish this it was necessary to count three filters together. Non-fasting male and 21-day pregnant Wistar CF rats and male Swiss Albino mice were from the Animal House at the University of Salamanca, Spain. They were fed on commercial pelleted rat or mouse food (Panlab, Madrid, Spain) and water ad libitum. Temperature (20°C) and the light/dark cycle (12 h:12 h) in the room were controlled. All animals received humane care as outlined in the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health Publication No. 8023, revised 1985). Surgery and bile collections were carried out under sodium pentobarbital (Nembutal N.R., Abbot, Madrid, Spain) anesthesia (50 μg/g bwt, ip). Flexible catheters were inserted into the left carotid artery to collect blood samples and into the left jugular vein to administer BAs. The common bile duct was also cannulated to collect bile samples. In mice, the gallbladder was ligated. After administration of a single iv bolus of [14C]TACA or [14C]TCA, blood (in rats) and bile (in rats and mice) samples were collected at the indicated time-points. In some experiments, a single dose of 10 nmol/g bwt [14C]TACA was injected through the penis vein to rats under ether anesthesia. The animals were allowed to recover from anesthesia in a warmed cabinet and were then housed in individual metabolic cages where they had free access to food and water. Six hours after TACA administration, a blood sample was obtained from the tail vein. Serum was ultrafiltered using 10 kD cut-off filters (Amicon Micricon-10, Lexington, MA). Urine samples were collected for 14 days. After this time, animals were anesthetized with sodium pentobarbital and surgically prepared as described above to collect bile samples over 8 h (at 1 h intervals) in order to obtain most of the bile acid pool. Translocation of [14C]TACA or [3H]TCA into the nucleus of adult rat hepatocytes that were isolated by two-steps collagenase method (19Berry M.N. Edward S.A.M. Barrit G.J. Isolated Hepatocytes: Preparation, Properties and Applications. Elsevier, Amsterdam1991Google Scholar) was measured as previously described (9Monte M.J. Martinez-Diez M.C. El-Mir M.Y. Mendoza M.E. Bravo P. Bachs O. Marin J.J.G. Changes in the pool of bile acids in hepatocyte nuclei during rat liver regeneration.J. Hepatol. 2002; 36: 534-542Google Scholar). The degree of differentiation was studied in 21 days fetal liver cells freshly after isolation (20Husson A. Bouazza M. Buquet C. Vaillant R. Hormonal regulation of two urea-cycle enzymes in cultured foetal hepatocytes.Biochem. J. 1983; 216: 281-285Google Scholar) or after 2 or 4 days in primary culture in the presence of none, 10 μM, or 50 μM ACA, CA, or ursodeoxycholic acid (UDCA). Purification of total RNA was performed using the RNAeasy mini kit from Quiagen (Izasa, Barcelona, Spain), following the instructions supplied by the vendor. RNA quality was determined by denaturing formaldehyde agarose gel electrophoresis and the amount was measured spectrophotometrically at 260 nm. Northern blot was performed using 20 μg of denatured RNA, which was size-fractionated by agarose gel electrophoresis. The fractionated RNA was transferred onto a nylon membrane (Biodyne B; Pall Gelman Sciences, Barcelona, Spain) and successively probed with rat Bsep, Ntcp, albumin, and α-fetoprotein (αFP) cDNAs under high stringency conditions using ExpressHyb hybridization solution from Clontech (BD, Madrid, Spain). β-actin cDNA was used as an internal calibrator for comparative purposes among the different experimental conditions. The effect on proliferation, cell viability and apoptosis was determined in adult rat hepatocytes in primary culture. Cells in Williams' medium E, pH 7.4, were seeded at ∼104 cells/cm2 and cultured for 24 h as previously reported (21Martinez-Diez M.C. Serrano M.A. Monte M.J. Marin J.J.G. Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture.Biochim. Biophys. Acta. 2000; 1500: 153-160Google Scholar). The medium was then replaced by fresh one that contained 10 μM or 50 μM ACA, CA, or UDCA. Cells plated in the presence 0.1% DMSO were used as controls. At 69 h after seeding, [14C]thymidine (50,000 dpm/plate) was added. Three hours later, plates were washed twice with PBS and cells were detached with Lowry solution in order to measure total DNA, protein, and radioactivity. Toxicity was determined at the end of the exposure period by the neutral red retention test, as previously described (21Martinez-Diez M.C. Serrano M.A. Monte M.J. Marin J.J.G. Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture.Biochim. Biophys. Acta. 2000; 1500: 153-160Google Scholar). The pro-apoptotic effect of ACA was evaluated using four different approaches. In a first attempt, apoptosis measurements were carried out by the detection of histone-associated DNA fragments using a Cell Death Detection ELISA kit (Roche, Barcelona). Because even the positive control used in these experiments, i.e., glycochenodeoxycholic acid (GCDCA), did not induce a strong pro-apoptotic effect, additional experimental design and evaluation methods were used. Freshly isolated hepatocytes were plated, and after a 1.5 h attachment period they were exposed to 100 μM ACA, CA, or GCDCA for 4 h before carrying out morphological evaluation of apoptosis by: i) staining of nuclei with Hoechst-33258 (5 μg/ml, 20 min in the dark) in 4% paraformaldehyde-fixed cells, followed by fluorescence microscopy to visualize condensed chromatin as well as nuclear fragmentation (22Silva R.F.M. Rodrigues C.M.P. Brites D. Bilirubin-induced apoptosis in cultured rat neural cells is aggravated by chenodeoxycholic acid but prevented by ursodeoxycholic acid.J. Hepatol. 2001; 34: 402-408Google Scholar); ii) DNA extraction and electrophoresis in 1.5% agarose gels to assess DNA ladder formation (23Benz C. Angermuller S. Tox U. Kloters-Plachky P. Riedel H.D. Sauer P. Stremmel W. Stiehl A. Effect of tauroursodeoxycholic acid on bile-acid-induced apoptosis and cytolysis in rat hepatocytes.J. Hepatol. 1998; 28: 99-106Google Scholar, 24Eunok I.M. Choi Y.H. Paik K.J. Suh H. Jin Y. Kim K.W. Yoo Y.H. Kim N.D. Novel bile acid derivatives induce apoptosis via a p53-independent pathway in human breast carcinoma cells.Cancer Lett. 2001; 163: 83-93Google Scholar); and iii) single cell electrophoresis (comet) assays (25De Boeck M. Touil N. De Visscher G. Vande P.A. Kirsch-Volders M. Validation and implementation of an internal standard in comet assay analysis.Mutat. Res. 2000; 469: 181-197Google Scholar). Bile flow was determined gravimetrically. Radioactivity in bile, total serum, ultrafiltered serum, urine, cell extracts, and isolated nuclei was measured on a Beckman LS-6500 liquid scintillation counter (Beckman Instruments, Madrid, Spain) using Universol Scintillation Cocktail from ICN (Biolink, Barcelona, Spain). The concentrations of 3α-hydroxyl-BAs in bile were measured enzymatically using 3α-hydroxysteroid dehydrogenase (26Talalay P. Enzymatic analysis of steroid hormones.Meth. Biochem. Anal. 1960; 8: 119-143Google Scholar). Biotransformation of [14C]TACA was investigated by TLC of BAs extracted from bile and urine samples (5El-Mir M.Y. Badia M.D. Luengo N. Monte M.J. Marin J.J.G. Increased levels of typically fetal bile acid species in patients with hepatocellular carcinoma.Clin. Sci. 2001; 100: 499-508Google Scholar) using n-butanol-acetone-acetic acid-water (35:35:10:20) as eluent. The presence of total ACA in bile and urine samples was investigated by GC-MS (5El-Mir M.Y. Badia M.D. Luengo N. Monte M.J. Marin J.J.G. Increased levels of typically fetal bile acid species in patients with hepatocellular carcinoma.Clin. Sci. 2001; 100: 499-508Google Scholar) after liquid-solid extraction, enzymatic desulfation, deglucuronation, and deamidation (27Malavolti M. Fromm H. Nsien E. Setchell K.D.R. Albert M.B. Cohen B. Ceryak S. Formation, absorption, and biotransformation of Δ6-lithocholenic acid in humans.Am. J. Physiol. 1993; 264: G163-G171Google Scholar, 28Shoda J. Osuga T. Matsuura K. Mahara R. Tohma M. Tanaka N. Matsuzaki Y. Miyazaki H. Concurrent occurrence of 3β,12α-dihydroxy-5-cholenoic acid associated with 3β-hydroxy-5-cholenoic acid and their preferential urinary excretion in liver diseases.J. Lipid Res. 1989; 30: 1233-1242Google Scholar), and derivatization (29Alme B. Bremmelgaard A. Sjovall J. Thomassen P. Analysis of metabolic profiles of bile acids in urine using a lipophilic anion exchanger and computerized gas-liquid chromatography-mass spectrometry.J. Lipid Res. 1977; 18: 339-362Google Scholar) of BAs. Proteins were measured using a modification of the Lowry method (30Markwell M. Hass S.M. Beiber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Google Scholar) with serum bovine albumin as standard. DNA was measured fluorometrically using Hoechst-33258 (31Labarca C. Paigen K. A simple, rapid, and sensitive DNA assay procedure.Anal. Biochem. 1980; 102: 344-352Google Scholar). The rate of DNA synthesis was determined by [14C]thymidine incorporation into DNA (32Koji T. Terayama H. Arginase as one of the inhibitory principles in the density dependent as well as plasma mediated inhibition of liver cell growth "in vitro".Exp. Cell Res. 1984; 155: 359-370Google Scholar). In order to compare the pharmacokinetics of [14C]TACA and [14C]TCA, model-independent methods based on the theory of statistical moments were used to analyze data on serum concentrations after iv administration of both of these BAs to anesthetized rats. AUCs (areas under the curve) and MRTs (mean residence times) were calculated by numerical integration using the trapezoidal rule, as described by Yamaoka et al. (33Yamaoka K. Nakagawa T. Uno T. Statistical moments in pharmacokinetics.J. Pharmacokinet. Biopharm. 1978; 6: 547-557Google Scholar). Because this was observed over a limited period of time (120 min), extrapolation to infinity (∝ ) was carried out using a monoexponential equation. Clearance (Cl) was calculated as the absolute dose divided by the AUC and the half-life (T1/2) as Ln2/Ke, where Ke (elimination constant) is 1/MRT. Results are expressed as mean ± SE. To calculate the statistical significance of the differences, paired or unpaired Student's t-tests and the Bonferroni method of multiple-range testing were used, as appropriate. Although wild-type CHO cells exhibited low uptake of TCA, this was not negligible for TACA (Fig. 3). Owing to the fact that TACA has a structural similarity with steroid hormones and because CHO cells originiate from a steroidogenic tissue, they may contain intrinsic transport systems for steroids able to carry out the TACA uptake that was observed in wild-type CHO cells. Nevertheless, the expression of Oatp1 or Ntcp by transfected CHO cells was accompanied by a significant enhancement in TACA uptake. The expression of these carriers induced Na+-independent and Na+-dependent uptake of TCA, respectively (Fig. 3). TACA uptake was similar to that found for TCA in Oatp1-expressing CHO cells; however, in Ntcp-expressing CHO cells, although TACA uptake was significantly higher than that seen for wild-type CHO cells, this was not reduced when Na+ was replaced by choline in the incubation medium. Experiments carried out on rat or mouse Bsep-containing plasma membrane vesicles of Sf 9 insect cells revealed that ATP-dependent transport of TCA was inhibited by TACA; however, TACA was not transported by ATP-dependent systems present in these vesicles (Fig. 4).Fig. 4Uptake of [3H]TCA and [14C]TACA separately or together by plasma membrane vesicles obtained from insect Sf 9 cells expressing rat or mouse bile salt export pump (Bsep). Membrane vesicles were incubated with 100 μM of one or both of the radiolabeled bile acids in the absence or in the presence of 5 mM ATP plus an ATP regenerating system (3 mM phosphocreatine plus 100 μg/ml creatine phosphokinase) for 20 min at 37°C. Values are means ± SE from three experiments (carried out in triplicate each). * P < 0.05, as compared to uptake in absence of inhibitor in similar conditions by paired Student's t-test.View Large Image Figure ViewerDownload (PPT) The results obtained in in vitro experiments were consistent with those found in experiments carried out on in vivo models. After administration (iv, 4 nmol/g bwt) of TCA or TACA to anaesthetized rats, the disappearance of TACA from plasma was slower than that of TCA (Fig. 5). Pharmacokinetic analysis indicated that TACA clearance was lower and that it had a longer half-life than TCA (Table 2). Ultrafiltration of serum samples collected 6 h after iv administration of TACA in separate experiments revealed that 80% of this BA was present in the non-ultrafilterable fraction, i.e., presumably bound to serum proteins; however, TACA was rapidly secreted into bile. The total bile output of TACA (78% of dose administered in 2 h) did not differ significantly from that of TCA (Table 2). At the moment of maximum biliary output of 14C, most (94.9 ± 1.2% by T.L.C, n = 4 animals) of the radioactivity secreted into bile was found to be non-biotransformed TACA. Free [14C]taurine accounted for less than 0.5% of this radioactivity. Neither TCA nor TACA induced modifications in bile flow at the dose given to rats (data not shown). To investigate the effect of TACA on bile flow, a higher dose of 25 nmol/g bwt was used. Moreover, to reduce the amount of TACA required to reach this goal, these experiments were carried out on mice (Fig. 6). The amount of TACA secreted into bile in the ensuing 3 h (270 ± 53 nmol/g liver, i.e., 58 ± 5% of the dose) was approximately half (P < 0.01) that found for CA (492 ± 67 nmol/g liver, i.e., 94 ± 3% of the dose; P < 0.01). Conversely, the BA-induced increase in bile flow was much higher for TACA (99.3 ± 2.7 μl/μmol) than for TCA (11.3 ± 1.7 μl/μmol; P < 0.001).TABLE 2Elimination into bile and pharmacokinetic parameters from serum concentrations curves obtained in anesthetized ratsSerumBileAUCClMRTT1/2OutputOutputμmol/l/minml/minminminnmol/g liver% of do
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