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Alterations in glutathione homeostasis in mutant Eisai hyperbilirubinemic rats.
Alterations in Glutathione Homeostasis in Mutant Eisai Hyperbilirubinemic Rats SHELLY C. LU,1 JIAXIN CAI,1 JOHN KUHLENKAMP,2 WEI-MIN SUN,1 HAJIME TAKIKAWA,3 OSAMU TAKENAKA,4 TOHRU HORIE,4 JIAN YI,1 AND NEIL KAPLOWITZ1,2
Eisai hyperbilirubinemic rats (EHBR) are mutant Sprague-Dawley rats that exhibit impaired biliary organic anion and reduced glutathione (GSH) secretion. In addition, liver GSH levels are twice that of age-matched controls. The mechanisms for the defect in biliary GSH secretion and the increase in cell GSH are not fully understood. We previously showed that canalicular membrane-enriched vesicles isolated from EHBR livers exhibited normal GSH transport. In the present study, we examined the steady-state rat canalicular reduced glutathione transporter (RcGshT) messenger RNA (mRNA) and protein levels, as well as the mechanisms for the increase in cell GSH. Both Northern and Western blot analyses of EHBR livers showed nearly identical RcGshT mRNA and polypeptide levels, respectively, as compared with controls. Treatment with phenobarbital, which increased steady-state RcGshT mRNA by five- to sixfold, RcGshT polypeptide, and biliary GSH secretion by onefold in controls, had a smaller effect on steadystate RcGshT-mRNA level in EHBR (by 1.5-fold) and did not increase RcGshT polypeptide or biliary GSH secretion. In examining possible mechanisms for increased liver GSH, both cysteine level and g-glutamylcysteine synthetase (GCS) activity were significantly higher than controls, while the activity of GSH synthetase was unchanged. Northern and Western blot analyses also showed increased steady-state GCS heavy subunit (GCSHS) mRNA and polypeptide levels, respectively. In addition to liver, GSH levels in kidney, duodenal, jejunal, and ileal mucosa of EHBR were 200% to 300% of age-matched control rats. GCS activity was also increased in kidney cytosol of EHBR. Thus, the defect in biliary GSH secretion in EHBR most likely is either at the posttranslational level of RcGshT or in the inhibition exerted by retained endogenous organic anions. In addition, there is a widespread up-regulation of GSH synthesis capacity in the tissues of EHBR. (HEPATOLOGY 1996;24:253-258.)
Abbreviations: EHBR, Eisai hyperbilirubinemic rat; GY, Groningen Yellow; TR0, transport-deficient; GSH, reduced glutathione; RcGshT, rat canalicular reduced glutathione transporter; GCS, g-glutamylcysteine synthetase; mBCl, monochlorobimane; cDNA, complementary DNA; SDS, sodium dodecyl sulfate; GCS-HS, heavy subunit of GCS; ATP, alkaline triphosphate; mRNA, messenger RNA; MRP, multidrug resistance–associated protein; cMOAT, canalicular multispecific organic anion transporter. From the 1Division of Gastrointestinal and Liver Diseases, Department of Medicine, University of Southern California School of Medicine, 2Department of Veteran Affairs Outpatient Clinic, Los Angeles, CA; 3Teikyo University School of Medicine, Tokyo 173, Japan; and 4Eisai Laboratory, Ibaraki 300-26, Japan. Received December 4, 1995; accepted March 27, 1996. Supported by National Institutes of Health grants DK-45334 and DK-30312, V. A. Medical Research Funds, and Professional Staff Association Grant 6-268-0-0, USC School of Medicine. Address reprint request to: Shelly C. Lu, M.D., MUDD Bldg., Rm 410, USC School of Medicine, 1333 San Pablo St., Los Angeles, CA 90033. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2401-0040$3.00/0
Eisai hyperbilirubinemic rats (EHBR) are mutant Sprague-Dawley rats (similar to the Groningen Yellow (GY)/ transport-deficient (TR0) mutant Wistar rats) characterized by an inherited defect in organic anion secretion into bile.1-4 Both mutants resemble the human Dubin-Johnson syndrome and exhibit impaired biliary secretion of organic anions, such as conjugated bilirubin, sulfobromophthalein-reduced glutathione (GSH) conjugate, and sulfated and glucuronidated bile acids.1-4 In addition, biliary GSH excretion is also severely impaired in both mutants when examined in vivo or in perfused livers.1-3 The mechanism for the impairment in biliary GSH secretion has been a subject of controversy. While Oude Elferink et al. suggested the defect is a direct effect of the mutation,1 we demonstrated normal GSH transport in canalicular membrane-enriched vesicles isolated from EHBR livers,2 which supports an indirect or secondary effect of the mutation. Because our laboratory recently cloned the rat canalicular reduced glutathione transporter (RcGshT),5 possible altered expression of RcGshT secondary to the mutation in EHBR can now be examined. Normally, hepatic GSH level is maintained by a balance between synthesis and efflux,6 about half of which is biliary in mature rats.7 GSH synthesis rates are governed by the availability of cysteine and the activity of the rate-limiting enzyme, g-glutamylcysteine synthetase (GCS), which, in turn, is regulated physiologically by feedback-competitive inhibition by GSH (Ki Å 2.3 mmol/L).6,8 In GY/TR0 mutants, liver GSH levels are twice that of age-matched controls.1 One suggested mechanism was retention of GSH because of decreased biliary secretion,1 which, stated another way, predicts that a fall in GSH efflux would be associated with decreased demand for synthesis. To decrease synthesis at the same level of precursors and enzymes would require a rise in cell GSH to slow synthesis by feedback inhibition. Although this is a plausible hypothesis, alternative mechanisms for increased cell GSH have not been excluded (e.g., precursor availability and expression of GCS). Furthermore, failure to secrete GSH in bile might be predicted to cause intestine mucosal GSH to fall, as has been described with bile duct ligation.9 In addition, considering that bile GSH is hydrolyzed in the intestine and amino acid constituents are absorbed, failure to secrete GSH may have systemic consequences (e.g., lowering of kidney GSH levels). In this study, we examined these alternative mechanisms for increased cell GSH and the organ specificity of these changes. MATERIALS AND METHODS Materials. GSH, b-nicotinamide adenine dinucleotide phosphate (reduced form), 5,5*-dithiobis (2-nitrobenzoic acid), sodium ethylenediaminetetraacetic acid, GSH reductase, a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), and HEPES were purchased from Sigma Chemical Co. (St. Louis, MO). 32P-deoxycytidine 5*-triphosphate (3,000 Ci/mmol) was purchased from New England Nuclear (DuPont, Boston, MA). Total RNA isolation kit was obtained from Promega (Madison, WI). Monochlorobimane (mBCl) was purchased from Molecular Probes, Inc. (Eugene, OR). g-(bis) glutamyl-
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cysteine was prepared by enzymatic hydrolysis of oxidized glutathione10 and reduction with dithiothreitol prior to use. The purity of g-(bis) glutamylcystine was confirmed by high-performance liquid chromatography. All other reagents were of analytical grade and were obtained from commercial sources. Animals. Age-matched male Sprague-Dawley rats and EHBRs weighing 265 to 380 g were used for these experiments. EHBRs were maintained by inbreeding male homozygotic and female heterozygotic rats at the Eisai Laboratory (Gifu, Japan) as described.3 All animals received humane care in compliance with the National Research Council’s criteria for human care as outlined in ‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985). In Vivo Measurement of Bile Flow and Biliary GSH Efflux. Rats were fasted overnight for all experiments. Rats were anesthetized with pentobarbital (65 mg/kg) intraperitoneally and subjected to open laparotomy. The body temperature was maintained by placing the animals under a lamp. The bile duct was cannulated with PE10 tubing (Clay Adams, Parsippany, NJ), treated with a retrograde biliary infusion of 20 mmol/kg acivicin, an irreversible inhibitor of gglutamyl transpeptidase, clamped for 1 minute, and allowed to drain for 5 minutes. Following this, bile was collected over 5-minute intervals for a total of 20 to 30 minutes. To prevent in vitro oxidation of reduced GSH, bile was collected into preweighed tubes containing 50 mL of 10% metaphosphoric acid.11 GSH was measured by the recycling method of Tietze,12 and the molecular form was confirmed by high-performance liquid chromatography, according to the method of Fariss and Reed.13 Bile flow rate was measured gravimetrically assuming a specific gravity of 1.0 and is expressed as microliters per minute per gram of liver. At the end of the experiment, the portal vein was flushed and liver was homogenized (33% wt/vol in 0.01 mol/L sodium phosphate, 0.25 mol/L sucrose [pH 7.4]) and processed for GSH measurement by the recycling method of Tietze.12 The effect of phenobarbital on biliary GSH secretion was examined by treating animals with 80 mg/kg of phenobarbital intraperitoneally or saline per day for up to 7 days. Experiments were performed the following morning after the last dose of phenobarbital or saline. Measurement of Kidney and Intestinal GSH Levels. At the end of the in vivo biliary GSH efflux measurement as described above, kidneys and small intestine were removed and placed on ice. The contents of the small intestine were flushed with 5 mL of normal saline, and the small intestine was divided into three equal segments, labeled duodenum, jejunum, and ileum. Each segment was cut open, placed on a piece of ice-cold glass, and the mucosa was gently scraped off by scraping the serosal side of the intestine with a scapula. The mucosa was then placed on dry ice, weighed and homogenized with sucrose-phosphate buffer as described above, and processed for GSH measurement by the recycling method of Tietze.12 Kidneys were also homogenized and processed for GSH. RNA Isolation and Northern Blot Analysis. EHBR and agematched controls were treated with phenobarbital (80 mg/kg in saline intraperitoneally) or saline for 3 days. RNA was isolated from liver biopsies according to the method of Chomczynski and Sacchi.14 RNA (10 mg) was separated on a 1% agarose gel with 18% formaldehyde, blotted onto a nitrocellulose membrane by capillary transfer, and then hybridized to a 1.5-kb complementary DNA (cDNA) derived from a subcloned insert corresponding to nucleotides 1132 to 2623 of the rat canalicular GSH transporter as described previously.5 The cDNA probe was labeled with 32P-deoxycytidine 5*-triphosphate by BCAbest Labeling Kit (Takara, Kyoto, Japan). Hybridization was at 557C overnight in 50% formamide, 61 SSPE (0.18 mol/L NaCl, 10 mmol/L sodium phosphate [pH 7.7], 1 mmol/L ethylenediaminetetraacetic acid), 2% sodium dodecyl sulfate (SDS) containing 100 mg/mL salmon sperm DNA, and 107 dpm labeled insert. Blots were washed twice for 30 minutes in 21 SSC (11 SSC Å 0.15 mol/L NaCl, 15 mmol/L Na citrate), 0.1% SDS at 657C, once for 10 minutes in 0.21 SSC, 0.1% SDS at 657C, and autoradiographed for 4 hours onto an imaging plate. The imaging plate was analyzed with a Bio-Image Analyzer (Bas 2000, Fuji Photo Film Co., Ltd., Tokyo, Japan). Equal loading of RNA samples was ensured by staining total RNAs (18S and 28S) on the same nitrocellulose paper with methylene blue. Northern blot analysis for the heavy subunit of GCS (GCS-HS) was performed according to methods described.15 To ensure equal loading of RNA samples, the same membrane was rehybridized with 32 P-labeled human b-actin cDNA probe (Clontech, Palo Alto, CA). Autoradiography and densitometry (Biomed Instrument model SLR-
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2D Soft Laser Scanning Densitometer, Fullerton, CA) were used to quantitate relative RNA. Results of Northern blot analysis were normalized to b-actin. Western Blot Analysis of RcGshT and GCS-HS. Rabbit polyclonal antibody against a synthetic peptide that corresponds to amino acid residues 158-165 of RcGshT5 was used for Western blotting. The peptide was chosen according to the Hopp and Woods Hydrophilicity Scale.16 Both peptide synthesis and antibody generation were carried out by Bio-Synthesis, Inc. (Lewisville, TX). A cysteine was added at the N-terminus, and the peptide was conjugated to Keyhole Limpet Hemocyanin. Each rabbit received intramuscularly and subcutaneously 1.0 mg of conjugate emulsified in Freund’s complete adjuvant. At 6, 8, and 20 weeks thereafter, each rabbit received booster intramuscular immunization injections of 1.0 mg conjugate in Freund’s incomplete adjuvant. Antibody titers were determined by enzymelinked immunosorbent assay 1 week after each injection. Liver homogenates from EHBR and age-matched controls treated with saline or phenobarbital (80 mg/kg in saline intraperitoneally) for 3 days were used for Western blotting. Protein concentration was determined by the Bio-Rad protein assay and was 80 mg/mL. Liver homogenates containing 100 mg protein were solubilized in equal volumes of sample buffer (285 mmol/L Tris [pH 6.8], 30% glycerol, 6% SDS, 1.5% 2-mercaptoethanol, and 0.01% Bromphenol Blue), subjected to 10% SDS–polyacrylamide gel electrophoresis,17 and electrotransferred to 0.2-mm NitroCellulose membranes using Semidry Transfer Cell (Bio-Rad, Hercules, CA).18 The NitroCellulose membranes were subsequently subjected to the Amplified Alkaline Phosphatase Immun-Blot Assay according to procedures described in the kit (cat. no. 170-6412, Bio-Rad). The first antibody was rabbit antirat RcGshT peptide postimmune serum diluted 1:250 in TBST (10 mmol/L Tris [pH 8.0], 150 mmol/L NaCl, containing 0.05% Tween 20). Preimmune serum showed no reactivity with liver homogenates. The specificity of the anti-RcGshT antiserum has been previously confirmed to identify a 98-kd polypeptide in canalicular-enriched membrane fraction but showed no reactivity with sinusoidal-enriched membrane fraction prepared from a pool of normal rat livers.19 In addition, antibody to a peptide that corresponds to amino acid residues 47-54 of RcGshT5 identified the same protein only in canalicular-enriched membrane fraction (not shown). Western blot analysis for GCS-HS was performed using liver cytosols of EHBR and age-matched controls according to methods described.15 Equivalent loading of protein was assured by Coomasie Blue staining of gels. Measurement of Enzyme Activities of GSH Synthesis Using mBCl. GSH synthesis was measured using liver and kidney cytosol
of EHBR and age-matched controls using mBCl as previously described.20 Liver cytosol was dialyzed overnight using molecularporous membrane tubing (molecular weight cutoff, 12,000-14,000, Spectrum Medical Industries, Inc., Los Angeles, CA) in 1001 volume of 0.01 mol/L sodium phosphate, pH 7.4, to avoid high background with mBCl and to eliminate feedback inhibition exerted by preexisting GSH. After overnight dialysis, cytosolic GSH concentration was decreased by 99% to 99.5%. The GSH synthesis rate was measured by addition of dialyzed iver cytosol (2-3.5 mg protein) to the cuvette containing 100 mmol/L TrisHCl, 150 mmol/L KCl, 20 mmol/L MgCl2 , 2 mmol/L ethylenediaminetetraacetic acid (pH 7.3), glutamate (10 mmol/L), glycine (10 mmol/ L), alkaline phosphate (ATP) (3 mmol/L), cysteine / dithiothreitol (0.1 and 1 mmol/L), plus 100 mmol/L mBCl in a final volume of 2.5 mL at 377C. The difference in initial rate of linear increase in fluorescence over 6 to 8 minutes with or without pretreatment with buthionine sulfoximine (5 mmol/L for 5 minutes at 377C) is equivalent to the rate of GSH synthesis. The change in fluorescence is converted to GSH concentration units by using standard curves. Specifically, formation of fluorescent adducts were monitored by adding mBCl (100 mmol/L) and glutathione-S transferase (0.1 U/mL) to the cuvette containing GSH standards. Standard curves were then generated by applying linear regression to the relationship between fluorescence and GSH concentration. This method assesses the formation of GSH as an end product from two steps—the formation of g-glutamylcysteine from cysteine and glutamate (catalyzed by GCS) and the formation of GSH from g-glutamylcysteine and glycine (catalyzed by GSH synthetase). Because GCS is rate-limiting, this is in essence a measure of GCS activity. To assess only the second step in GSH synthesis, liver cytosol was added to the cuvette containing 100 mmol/L TrisHCl, 150 mmol/L KCl, 2 mmol/L ethylenediaminetetraacetic acid (pH 7.3), substrates g-glutamylcysteine / dithiothreitol (0.2 mmol/L),
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TABLE 1. Effect of Phenobarbital on Biliary GSH Efflux in EHBR Body Weight (g)
Control SDR SDR / 3-D phenobarbital EHBR EHBR / 3-D phenobarbital EHBR / 7-D phenobarbital
264 258 252 247 267
{ { { { {
2 5 4* 4† 3‡
Liver Weight (g)
8.5 9.5 10.2 11.3 11.4
{ { { { {
Biliary GSH Efflux (nmol/g liver/min)
0.3 0.2* 0.3* 0.1‡ 0.1‡
3.40 6.78 0.014 0.008 0.023
{ { { { {
0.26 0.08* 0.001* 0.001† 0.006†
Bile Flow (mL/g liver/min)
1.25 1.95 0.79 0.64 0.81
{ { { { {
0.16 0.07* 0.06* 0.10† 0.05†
NOTE. Results represent mean { SE from 3 to 6 animals for each group. Biliary GSH efflux and bile flow rates were measured after overnight fasting as described in Materials and Methods. Phenobarbital treatment consisted of 80 mg/kg intraperitoneally per day for 3 or 7 days. Abbreviation: SDR, Sprague-Dawley rats. * P õ .05 vs. control SDR; † P õ .05 vs. SDR / phenobarbital; ‡ P õ .05 vs. EHBR by ANOVA, followed by Fisher’s test.
glycine (10 mmol/L), cofactors ATP (3 mmol/L) and Mg2/ (20 mmol/ L), plus 100 mmol/L mBCl in a final volume of 2.5 mL at 377C. The difference in initial rate of linear increase in fluorescence over 8 to 10 minutes in the presence of all precursors and cofactors for the second step of GSH synthesis versus only g-glutamylcysteine / dithiothreitol is equivalent to the rate of GSH synthesis catalyzed by GSH synthetase. Cysteine Assay. To measure cysteine concentrations in livers of EHBR and controls, liver biopsies were homogenized in 5% perchloric acid (25% wt/vol) to precipitate the proteins and centrifuged for 3 minutes in a microfuge; supernatant cysteine levels were determined according to the method of Gaitonde.21 Statistical Analysis. Paired Student’s t test was used for comparison within the same animal. For comparison between groups, unpaired Student’s t test (in the case of only two comparisons) or ANOVA followed by Fisher’s test (for multiple comparisons) was used. Two-tailed t tests were used. Significance was defined by P õ .05. RESULTS Biliary GSH Secretion in EHBR. First, we confirmed previous work that both biliary GSH secretion and bile flow in EHBR are significantly lower than age-matched controls.3 Table 1 shows that biliary GSH secretion is almost undetectable in EHBR despite acivicin treatment. This dose of acivicin maximally inhibits GGT activity, because an additional dose of acivicin did not result in higher biliary GSH secretion rates (not shown). High-performance liquid chromatography confirmed near-complete absence of both reduced and oxidized GSH and GSH mixed disulfides in bile samples of EHBR (not shown). Bile flow in EHBR is Ç40% lower than age-matched controls, similar to what was reported in TR0 mutants.1 After 3 days of phenobarbital treatment, which doubled the biliary GSH efflux in control Sprague-Dawley rats, as we previously reported,22 no change in biliary GSH efflux was observed in EHBR mutants. To see if more prolonged phenobarbital treatment would influence biliary GSH efflux in EHBR, some EHBR mutants received 7 days of phenobarbital at the same dose of 80 mg/kg/d. Still, no increase in biliary GSH efflux was observed. A separate group of older EHBR animals weighing 380 to 400 g also exhibited the same defect in biliary GSH secretion and nonresponsiveness to phenobarbital treatment (data not shown). Next, to see if there are abnormalities in RcGshT expression in EHBR, both Northern and Western blot analyses were performed. Figure 1 shows Northern blot analysis comparing EHBR with age-matched controls of liver RcGshT expression with and without 3 days of phenobarbital treatment. The baseline 4.0-kb RcGshT–messenger RNA (mRNA) level in EHBR livers is nearly identical to that of age-matched controls. After phenobarbital treatment, RcGshT-mRNA level increased by 550% in controls but by only 150% in EHBRs. Thus, the transcript for RcGshT is normally present in EHBR but did not respond with the same magnitude of increase after phenobarbital treatment. Western blot analysis using specific anti-RcGshT polyclonal antibody also showed the presence of the 98-kd RcGshT polypeptide in EHBR livers
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in normal amounts (Fig. 2A). Consistent with the observed functional changes after phenobarbital treatment, hepatic RcGshT polypeptide level doubled after phenobarbital treatment in control animals but remained unchanged in EHBR (Fig. 2B). Up-regulation of Cell GSH in EHBR—Organ Specificity and Mechanism. As in GY/TR0 mutants, EHBR exhibited in-
creased liver GSH (Table 2). However, this phenomenon extends beyond the liver, because GSH levels in all segments of the small intestine and kidneys were also 200% to 300% of age-matched controls. We next investigated the mechanism for the increase in liver GSH in EHBR. Parameters most important in determining steady-state GSH level, cysteine availability, and GCS activity,6,8 were first measured. As shown in Table 3, liver cysteine levels in EHBR are 70% higher than controls. Liver GCS activity as measured by GSH synthesis rate in the presence of all precursors of GSH (glycine, glutamate, and cysteine) was also significantly higher in EHBR, while that of the GSH synthetase activity as measured by GSH synthesis rate in the presence of g-glutamylcysteine and glycine was unchanged from controls. When GCS activity was measured at 1 mmol/L cysteine concentration, it was similarly increased in EHBR as at 0.1 mmol/L cysteine concentration (Table 3) (control GCS activity Å 4.29 { 0.19 and EHBR Å 5.98 { 0.26 nmol/mg/min; results represent mean { SE from 5 animals each, P õ .01 by unpaired t test). Since the Km of GCS for cysteine is Ç0.1 mmol/L,20 this would suggest the capacity (Vmax ) of GCS was affected. GCS activity (nmol/mg protein/min) in kidney cytosol of EHBR was also higher than controls (controls Å 2.79 { 0.32, EHBR Å 5.70 { 0.45; results represents mean { SE from 5 animals each, P õ .05 by unpaired t test). To see if increased GCS can be explained by an increase in GCS gene expression, steady-state GCS-HS mRNA and polypeptide levels were measured. As suspected, Northern blot analysis showed a doubling of the steady-state hepatic
FIG. 1. Effect of phenobarbital on RcGshT-mRNA expression in livers of EHBR and age-matched controls. Liver RNA samples obtained from three EHBR and three control rats that had been treated with phenobarbital (80 mg/kg intraperitoneally per day for 3 days) or saline vehicle were analyzed by Northern blot hybridization with a 32P-labeled RcGshT-cDNA probe as described in Materials and Methods. The position of RcGshT (4.0 kb) is as indicated. Pb, phenobarbital.
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HEPATOLOGY July 1996 TABLE 3. Comparison of Parameters Important in Hepatic GSH Synthesis in EHBR and Controls
Controls EHBR
Cysteine level (mmol/g liver)
GCS activity (nmol/mg protein/min)
GSH Synthetase activity (nmol/mg protein/min)
0.17 { 0.01 0.29 { 0.02*
2.67 { 0.47 4.29 { 0.27*
5.34 { 0.35 5.07 { 0.26
NOTE. Results represent mean { SE from five animals each. GCS activity was measured in liver cytosol in the presence of 10 mmol/L each of glutamate and glycine and 0.1 mmol/L cysteine, while GSH synthetase activity was measured in the presence of 10 mmol/L glycine and 0.2 mmol/L g-glutamylcysteine using mBCl (see Materials and Methods for details). Amount of cytosolic protein (mg) per gram of liver was not different: control Å 89 { 4; EHBR Å 91 { 3. * P õ .05 vs. control by unpaired Student’s t test.
FIG. 2. (A) Western blot analysis of RcGshT polypeptide in EHBR and agematched controls. Liver homogenates (100 mg each lane) obtained from EHBR and age-matched control rats (n Å 3 each) were analyzed by Western blot analysis using polyclonal antibodies against a synthetic peptide of RcGshT as described in Materials and Methods. The position of RcGshT (98 kd) is indicated. Preimmune serum showed no reactivity. (B) Effect of phenobarbital on RcGshT polypeptide expression in livers of EHBR and age-matched controls. Liver homogenates (100 mg each lane) obtained from three animals each of EHBR (bottom panel) and controls (top panel) treated with phenobarbital (80 mg/kg intraperitoneally per day for 3 days) or saline vehicle were analyzed by Western blot analysis using polyclonal antibodies against a synthetic peptide of RcGshT as described in Materials and Methods. The position of RcGshT (98 kd) is indicated. Pb, phenobarbital.
GCS-HS–mRNA level in EHBR rats (196% of age-matched controls after normalizing to b-actin; P õ .01 by unpaired t test) (Fig. 3). Semiquantitative Western blot analysis also confirmed the increase in GCS-HS polypeptide level (Fig. 4). DISCUSSION
The failure to secrete GSH in bile of EHBR and GY/TR0 mutants has been a puzzling and controversial issue. We have recently cloned the canalicular GSH transporter, which permits an examination of its expression in EHBR. In the present study, we have found that changes in the steady-state mRNA and polypeptide level of RcGshT could not account for the defect. Thus, alterations of RcGshT transcription, mRNA stability, translation, and polypeptide stability cannot account for the defect. We previously reported that canalicular membrane-enriched vesicles from EHBR exhibited the same Km and Vmax for GSH transport as controls.2 Thus, our previous hypothesis that the defect is accounted for by either failure to secrete endogenous organic anions, which normally trans-stimulate GSH secretion, or retention of these sub-
stances at sufficiently high concentration to also cis-inhibit RcGshT remains plausible. Alternatively, we cannot exclude the possibility that a labile posttranslational modification is somehow enhanced secondary to the primary genetic defect, such as phosphorylation, which inhibits the function of the transporter and is reversed (dephosphorylation) during the membrane vesicle isolation procedure. Another possibility is that the vesicle preps contain intracellular and canalicular membrane, and that the defect in the mutants leads to a secondary block in the transfer of RcGshT from the former to the latter. Finally, a regulatory role of the defective organic anion transporter is conceivable (see below). Further work is required to examine these possibilities. Phenobarbital treatment has been shown to increase biliary GSH secretion,22 the Vmax for GSH transport in canalicular membrane-enriched vesicles,23 and the abundance of RcGshT mRNA.5 We therefore treated EHBR with phenobarbital in the hope of raising RcGshT levels sufficiently to at least partially overcome the possible inhibition by retained organic anions. Unfortunately, the results are not definitive. Although phenobarbital treatment had no effect on biliary GSH secretion in EHBR, it exerted a much smaller induction of RcGshT mRNA for reasons that are unknown. Even in the case of control animals, a 550% increase in the steady-state RcGshT-mRNA level only led to a doubling of the polypeptide level. Thus, it is perhaps not surprising that a much smaller increase in the RcGshT-mRNA level did not alter the steadystate polypeptide level. However, because the expected extent of induction was not achieved, the goal of the experiment could not be achieved. Impaired phenobarbital responsiveness in EHBR has been found by others as well. Treatment with phenobarbital (80 mg/kg intraperitoneally per day for 3 days) increased cytochrome P450 2B and 2C6 protein levels and cytochrome P450 2B1 steady-state mRNA level in control Sprague-Dawley rats, but not in EHBR mutants (Dr. Takemi Yoshida, Personal communication, February 1996). A recent report raised the possibility that the multidrug resistance–associated protein (MRP) might transport GSH.24 Although expression of MRP is low in liver, it has been recently reported that an MRP-like transporter of similar speci-
TABLE 2. Comparison of GSH Level in Multiple Organs of EHBR and Controls Small Intestinal Mucosal GSH (mmol/g)
Controls EHBR
Liver GSH (mmol/g)
Kidney GSH (mmol/g)
Duodenum
Jejunum
Ileum
4.85 { 0.14 10.11 { 0.57*
0.12 { 0.01 0.35 { 0.02*
1.41 { 0.17 3.52 { 0.15*
1.18 { 0.16 3.35 { 0.17*
1.56 { 0.20 3.14 { 0.14*
NOTE. Results represent mean { SE from five animals each. GSH levels in multiple organs were measured as described in Materials and Methods after overnight fasting. * P õ .05 vs. control by unpaired Student’s t test.
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ficity (canalicular multispecific organic anion transporter [cMOAT]) is present in liver and defective in GY/TR0 rats.25 There are several lines of evidence that dispute the role of MRP or cMOAT as significant GSH transporters. First, erythrocytes have MRP-like activity but do not transport GSH,26 and HeLa cells lack MRP,27 but transport GSH.19 Second, in diabetic rats, there is either an increase or no change in biliary secretion of bilirubin,28,29 which is a substrate for the hepatic MRP-like protein, but a profound selective decrease in biliary GSH excretion is observed.30 Third, BSP-GSH is a substrate for MRP but does not inhibit RcGshT or canalicular GSH secretion.5 Fourth, GSH does not inhibit MRP-mediated ATP-driven GSH S-conjugate31 or leukotriene C4 transport (Dr. D. Keppler, Personal communication, February 1996). Fifth, MRP is a unidirectional efflux pump,27 whereas GSH transport in oocytes expressing RcGshT is bidirectional.5 Sixth, MRP-mediated transport is ATP-driven, whereas RcGshT-mediated GSH transport is not ATP-dependent.2 Although activation of protein kinase C enhances cMOAT activity32 but inhibits canalicular GSH secretion,33 it is not possible to exclude that this reflects a change in substrate specificity of the carrier. The bulk of evidence supports the conclusion that MRP or cMOAT do not transport GSH. As an alternative, a possible role for MRP in regulating the GSH transporters will need to be considered. Other ATP-binding cassette transporters are known to directly and indirectly influence the activity of ion channels,34 and an analogous effect of MRP on GSH transporters is an open possibility. EHBR exhibited a similar marked increase in liver GSH levels as GY/TR0 mutants.1 We found an increase in GCS activity and cysteine concentration in EHBR livers, two of the most important parameters in determining GSH synthesis capacity. In many conditions (i.e., drug-resistant tumor cell lines, treatment with methyl mercury hydroxide) in which GCS activity is increased, there is also an increase in the GCS-HS–mRNA level.35-37 Recently, we have also shown the increase in GCS activity induced by hormones, such as insulin and hydrocortisone,20 or plating cultured rat hepatocytes under low cell density38 correlated with increased GCS-HS– mRNA level.15 Thus, regulation of the GCS-HS gene expression appears critical for GSH homeostasis. Indeed, in the case of EHBR livers, increased GSH levels also correlated with increased steady-state GCS-HS–mRNA and polypeptide levels. Regarding higher hepatic cysteine concentration in EHBR, several possibilities exist. Enhanced sulfur amino acid uptake (methionine and cysteine) and/or activity of trans-sulfuration and/or decreased cysteine utilization in GSH synthesis or other pathways of degradation may be in-
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FIG. 4. Steady-state GCS-HS polypeptide level in EHBR and age-matched control rats. Liver cytosols (50 mg/lane) obtained from three EHBR and three control rats were subjected to SDS–polyacrylamide gel electrophoresis, followed by immunoblotting with preimmune (not shown) and postimmune sera. Rat kidney homogenates (20 mg/lane) were included for comparison. Molecular weight markers (in kd) are shown on the left. The bands at 73 kd represent GCS-HS. A minor band (Mr 63 kd) is also noted in the kidney homogenate, as previously described.39
volved.6 More work will be required to elucidate the exact mechanism. Although the hypothesis that decreased GSH secretion leads to a decreased demand for synthesis, which, according to the feedback regulation, is accomplished by raising cell GSH, remains plausible, the up-regulation in GSH synthetic capacity that we observed would accentuate the GSH level required to accomplish this. It has been reported that bile GSH secretion is important in maintaining intestinal mucosal GSH levels.9 Bile duct ligation leads to a 50% fall in intestinal GSH.9 To our surprise, intestinal GSH was increased in EHBR, and this effect was widespread, including kidney, which presumably reflects a generalized up-regulation of GSH synthesis. In the case of kidney, GCS activity in cytosol was also increased in EHBR. Whether there is a generalized increase in GCS-HS gene expression in all tissues of EHBR remains to be examined. However, it is of interest that the genetic defect in MRPlike polypeptide in GY/TR0 mutants was reported to be liverspecific, whereas the up-regulation in GSH synthetic capacity is generalized; this strongly suggests that the increased GSH level in liver is predominantly due to the up-regulation rather than ‘‘retention.’’ The present study further defines the secondary effects of a mutation in the ATP-driven organic anion transporter on GSH transport and GSH levels. The widespread and unpredicted effects reflect the complexity of the possible secondary effects of genetic mutations, a phenomenon not infrequently encountered in gene knockout models. Thus, our findings underscore the need for caution in interpreting the relationship between phenotypic abnormalities and a genetic mutation. Acknowledgment: The authors appreciate very much the helpful and critical comments of Dr. Ronald Oude Elferink. REFERENCES
FIG. 3. Steady-state GCS-HS–mRNA level in EHBR and age-matched controls. Liver RNA (50 mg each lane) samples were analyzed by Northern blot hybridization with a 32P-labeled GCS-HS–cDNA probe as described in Materials and Methods. The same membrane was then rehybridized with 32P-labeled human b-actin cDNA probe. The molecular size markers GCS-HS (Ç3.7 kb) and b-actin are as indicated.
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1. Oude Elferink RPJ, Ottenhoff R, Liefting W, de Haan J, Jansen PLM. Hepatobiliary transport of glutathione and glutathione conjugate in rats with hereditary hyperbilirubinemia. J Clin Invest 1989;84:476-483. 2. Ferna´ndez-Checa J, Takikawa H, Horie T, Ookhtens M, Kaplowitz N. Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats. J Biol Chem 1992;267:1667-1673. 3. Takikawa H, Sano N, Narita T, Uchida Y, Yamanaka M, Horie T, Mikami T, et al. Biliary excretion of bile acid conjugates in a hyperbilirubinemic mutant Sprague-Dawley rat. HEPATOLOGY 1991;14:352-360. 4. Adachi Y, Kobayashi H, Shouji M, Kitano M, Okuyama Y, Yamamoto T. Functional integrity of hepatocyte canalicular membrane transport of taurocholate and bilirubin diglucuronide in Eisai hyperbilirubinuria rats. Life Sci 1993;52:777-784. 5. Yi J, Lu S, Ferna´ndez-Checa J, Kaplowitz N. Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics. J Clin Invest 1994;93:1841-1845. 6. Kaplowitz N, Aw TY, Ookhtens M. The regulation of hepatic GSH. Annu Rev Pharmacol Toxicol 1985;25:714-744. 7. Ballatori N, Jacob R, Boyer JL. Intrabiliary glutathione hydrolysis: a source of glutamate in bile. J Biol Chem 1986;261:7860-7865.
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8. Richman PG, Meister A. Regulation of g-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem 1975;250: 1422-1426. 9. Aw TY. Biliary glutathione promotes the mucosal metabolism of luminal peroxidized lipids by rat small intestine in vivo. J Clin Invest 1994;94: 1218-1225. 10. Strumeyer D, Bloch K. g-L-glutamyl-L-cysteine from glutathione. Biochem Prep 1962;9:52-55. 11. Eberlie D, Clarke R, Kaplowitz N. Rapid oxidation in vitro of endogenous and exogenous glutathione in bile in rats. J Biol Chem 1981;256:21152117. 12. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969;27:502-522. 13. Fariss MW, Reed DJ. High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives. Methods Enzymol 1987;143:101109. 14. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159. 15. Cai J, Sun W, Lu SC. Hormonal and cell density regulation of hepatic gglutamylcysteine synthetase gene expression. Mol Pharmacol 1995;48: 212-218. 16. Hopp TP, Woods KR. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A 1981;78:3824-3828. 17. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bactriophage T4. Nature 1970;227:680-685. 18. Towbin H, Staehein T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitro-cellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1989;76:4350-4354. 19. Lu SC, Sun W, Yi J, Ookhtens M, Sze G, Kaplowitz N. Role of two recently cloned rat liver GSH transporters in the ubiquitous transport of GSH in mammalian cell lines. J Clin Invest 1996;97:1488-1496. 20. Lu SC, Ge J, Kuhlenkamp J, Kaplowitz N. Insulin and glucocorticoid dependence of hepatic g-glutamylcysteine synthetase and GSH synthesis in the rat: studies in cultured hepatocytes and in vivo. J Clin Invest 1992; 90:524-532. 21. Gaitonde MK. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem J 1967;104:627-633. 22. Kaplowitz N, Eberlie DE, Petrini J, Touloukian J, Corvasce MC, Kuhlenkamp J. Factors influencing the efflux of hepatic glutathione into bile in rats. J Pharmacol Exp Ther 1983;224:141-147. 23. Ferna´ndez-Checa J, Ookhtens M, Kaplowitz N. Selective induction by phenobarbital of the electrogenic transport of glutathione and organic anions in rat liver canalicular membrane vesicles. J Biol Chem 1993;268:1083610841.
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24. Zaman GR, Lankelma J, van Tellingen O, Beijnen J, Dekker H, Paulusma C, Oude Elferink RPJ, et al. Role of glutathione in the export of compounds from cells by the multidrug-resistance–associated protein. Proc Natl Acad Sci U S A 1995;92:7690-7694. 25. Mayer R, Kartenbeck J, Bu¨chler M, Jedlitschky G, Leier I, Keppler D. Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes. J Cell Biol 1995;131:137-150. 26. Kondo T, Dale GL, Beutler E. Glutathione transport by inside-out vesicles from human erythrocytes. Proc Natl Acad Sci U S A 1980;77:6359-6362. 27. Leier I, Jedlitschky G, Buchholz U, Cole SPC, Deeley RG, Keppler D. The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates. J Biol Chem 1994;269:27807-27810. 28. Tun˜on MJ, Gonzalez P, Garcia-Pardo LA, Gonzalez J. Hepatic transport of bilirubin in rats with streptozotocin-induced diabetes. J Hepatol 1991; 13:71-77. 29. Watkins JB, Sherman SE. Long-term diabetes alters the hepatobiliary clearance of acetaminophen, bilirubin and digoxin. J Pharmacol Exp Ther 1992;260:1337-1343. 30. Lee S, Kaplowitz N, Lu SC. Impaired canalicular GSH transport in diabetic rats. Gastroenterology 1994;106:A991. 31. Kobayashi K, Sogame Y, Hara H, Hayashi K. Mechanism of glutathione S-conjugate transport in canalicular and basolateral rat liver plasma membranes. J Biol Chem 1990;265:7737-7741. 32. Roelofsen H, Ottenhoff R, Oude Elferink RP, Jansen PL. Hepatocanalicular organic-anion transport is regulated by protein kinase C. Biochem J 1991;278:637-641. 33. Raiford DS, Sciuto AM, Mitchell MC. Effects of vasopressor hormones and modulators of protein kinase C on glutathione efflux from perfused rat liver. Am J Physiol 1991;24:G578-G584. 34. Inagaki N, Gonoi T, Clement JP IV, Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, et al. Reconstitution of IKATP : an inward rectifier subunit plus the sulfonylurea receptor. Science 1995;270:1166-1170. 35. Mulcahy RT, Untawale S, Gipp JJ. Transcriptional up-regulation of gglutamylcysteine synthetase gene expression in melphalan-resistant human prostate carcinoma cells. Mol Pharmacol 1994;46:909-914. 36. Godwin AK, Meister A, O’Dwyer PJ, Huang CS, Hamilton TC, Anderson ME. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis. Proc Natl Acad Sci U S A 1992;89:3070-3074. 37. Woods JS, Davis HA, Baer RP. Enhancement of g-glutamylcysteine synthetase mRNA in rat kidney by methyl mercury. Arch Biochem Biophys 1992;296:350-353. 38. Lu SC, Ge J. Loss of suppression of GSH synthesis under low cell density in primary cultures of rat hepatocytes. Am J Physiol 1992;263:C11811189. 39. Yan N, Meister A. Amino acid sequence of rat kidney g-glutamylcysteine synthetase. J Biol Chem 1990;265:1588-1593.
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Eisai hyperbilirubinemic rats (EHBR) are mutant Sprague-Dawley rats that exhibit impaired biliary organic anion and reduced glutathione (GSH) secretion. In addition, liver GSH levels are twice that of age-matched controls. The mechanisms for the defect in biliary GSH secretion and the increase in cell GSH are not fully understood. We previously showed that canalicular membrane-enriched vesicles isolated from EHBR livers exhibited normal GSH transport. In the present study, we examined the steady-state rat canalicular reduced glutathione transporter (RcGshT) messenger RNA (mRNA) and protein levels, as well as the mechanisms for the increase in cell GSH. Both Northern and Western blot analyses of EHBR livers showed nearly identical RcGshT mRNA and polypeptide levels, respectively, as compared with controls. Treatment with phenobarbital, which increased steady-state RcGshT mRNA by five- to sixfold, RcGshT polypeptide, and biliary GSH secretion by onefold in controls, had a smaller effect on steadystate RcGshT-mRNA level in EHBR (by 1.5-fold) and did not increase RcGshT polypeptide or biliary GSH secretion. In examining possible mechanisms for increased liver GSH, both cysteine level and g-glutamylcysteine synthetase (GCS) activity were significantly higher than controls, while the activity of GSH synthetase was unchanged. Northern and Western blot analyses also showed increased steady-state GCS heavy subunit (GCSHS) mRNA and polypeptide levels, respectively. In addition to liver, GSH levels in kidney, duodenal, jejunal, and ileal mucosa of EHBR were 200% to 300% of age-matched control rats. GCS activity was also increased in kidney cytosol of EHBR. Thus, the defect in biliary GSH secretion in EHBR most likely is either at the posttranslational level of RcGshT or in the inhibition exerted by retained endogenous organic anions. In addition, there is a widespread up-regulation of GSH synthesis capacity in the tissues of EHBR. (HEPATOLOGY 1996;24:253-258.)
Abbreviations: EHBR, Eisai hyperbilirubinemic rat; GY, Groningen Yellow; TR0, transport-deficient; GSH, reduced glutathione; RcGshT, rat canalicular reduced glutathione transporter; GCS, g-glutamylcysteine synthetase; mBCl, monochlorobimane; cDNA, complementary DNA; SDS, sodium dodecyl sulfate; GCS-HS, heavy subunit of GCS; ATP, alkaline triphosphate; mRNA, messenger RNA; MRP, multidrug resistance–associated protein; cMOAT, canalicular multispecific organic anion transporter. From the 1Division of Gastrointestinal and Liver Diseases, Department of Medicine, University of Southern California School of Medicine, 2Department of Veteran Affairs Outpatient Clinic, Los Angeles, CA; 3Teikyo University School of Medicine, Tokyo 173, Japan; and 4Eisai Laboratory, Ibaraki 300-26, Japan. Received December 4, 1995; accepted March 27, 1996. Supported by National Institutes of Health grants DK-45334 and DK-30312, V. A. Medical Research Funds, and Professional Staff Association Grant 6-268-0-0, USC School of Medicine. Address reprint request to: Shelly C. Lu, M.D., MUDD Bldg., Rm 410, USC School of Medicine, 1333 San Pablo St., Los Angeles, CA 90033. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2401-0040$3.00/0
Eisai hyperbilirubinemic rats (EHBR) are mutant Sprague-Dawley rats (similar to the Groningen Yellow (GY)/ transport-deficient (TR0) mutant Wistar rats) characterized by an inherited defect in organic anion secretion into bile.1-4 Both mutants resemble the human Dubin-Johnson syndrome and exhibit impaired biliary secretion of organic anions, such as conjugated bilirubin, sulfobromophthalein-reduced glutathione (GSH) conjugate, and sulfated and glucuronidated bile acids.1-4 In addition, biliary GSH excretion is also severely impaired in both mutants when examined in vivo or in perfused livers.1-3 The mechanism for the impairment in biliary GSH secretion has been a subject of controversy. While Oude Elferink et al. suggested the defect is a direct effect of the mutation,1 we demonstrated normal GSH transport in canalicular membrane-enriched vesicles isolated from EHBR livers,2 which supports an indirect or secondary effect of the mutation. Because our laboratory recently cloned the rat canalicular reduced glutathione transporter (RcGshT),5 possible altered expression of RcGshT secondary to the mutation in EHBR can now be examined. Normally, hepatic GSH level is maintained by a balance between synthesis and efflux,6 about half of which is biliary in mature rats.7 GSH synthesis rates are governed by the availability of cysteine and the activity of the rate-limiting enzyme, g-glutamylcysteine synthetase (GCS), which, in turn, is regulated physiologically by feedback-competitive inhibition by GSH (Ki Å 2.3 mmol/L).6,8 In GY/TR0 mutants, liver GSH levels are twice that of age-matched controls.1 One suggested mechanism was retention of GSH because of decreased biliary secretion,1 which, stated another way, predicts that a fall in GSH efflux would be associated with decreased demand for synthesis. To decrease synthesis at the same level of precursors and enzymes would require a rise in cell GSH to slow synthesis by feedback inhibition. Although this is a plausible hypothesis, alternative mechanisms for increased cell GSH have not been excluded (e.g., precursor availability and expression of GCS). Furthermore, failure to secrete GSH in bile might be predicted to cause intestine mucosal GSH to fall, as has been described with bile duct ligation.9 In addition, considering that bile GSH is hydrolyzed in the intestine and amino acid constituents are absorbed, failure to secrete GSH may have systemic consequences (e.g., lowering of kidney GSH levels). In this study, we examined these alternative mechanisms for increased cell GSH and the organ specificity of these changes. MATERIALS AND METHODS Materials. GSH, b-nicotinamide adenine dinucleotide phosphate (reduced form), 5,5*-dithiobis (2-nitrobenzoic acid), sodium ethylenediaminetetraacetic acid, GSH reductase, a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), and HEPES were purchased from Sigma Chemical Co. (St. Louis, MO). 32P-deoxycytidine 5*-triphosphate (3,000 Ci/mmol) was purchased from New England Nuclear (DuPont, Boston, MA). Total RNA isolation kit was obtained from Promega (Madison, WI). Monochlorobimane (mBCl) was purchased from Molecular Probes, Inc. (Eugene, OR). g-(bis) glutamyl-
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cysteine was prepared by enzymatic hydrolysis of oxidized glutathione10 and reduction with dithiothreitol prior to use. The purity of g-(bis) glutamylcystine was confirmed by high-performance liquid chromatography. All other reagents were of analytical grade and were obtained from commercial sources. Animals. Age-matched male Sprague-Dawley rats and EHBRs weighing 265 to 380 g were used for these experiments. EHBRs were maintained by inbreeding male homozygotic and female heterozygotic rats at the Eisai Laboratory (Gifu, Japan) as described.3 All animals received humane care in compliance with the National Research Council’s criteria for human care as outlined in ‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985). In Vivo Measurement of Bile Flow and Biliary GSH Efflux. Rats were fasted overnight for all experiments. Rats were anesthetized with pentobarbital (65 mg/kg) intraperitoneally and subjected to open laparotomy. The body temperature was maintained by placing the animals under a lamp. The bile duct was cannulated with PE10 tubing (Clay Adams, Parsippany, NJ), treated with a retrograde biliary infusion of 20 mmol/kg acivicin, an irreversible inhibitor of gglutamyl transpeptidase, clamped for 1 minute, and allowed to drain for 5 minutes. Following this, bile was collected over 5-minute intervals for a total of 20 to 30 minutes. To prevent in vitro oxidation of reduced GSH, bile was collected into preweighed tubes containing 50 mL of 10% metaphosphoric acid.11 GSH was measured by the recycling method of Tietze,12 and the molecular form was confirmed by high-performance liquid chromatography, according to the method of Fariss and Reed.13 Bile flow rate was measured gravimetrically assuming a specific gravity of 1.0 and is expressed as microliters per minute per gram of liver. At the end of the experiment, the portal vein was flushed and liver was homogenized (33% wt/vol in 0.01 mol/L sodium phosphate, 0.25 mol/L sucrose [pH 7.4]) and processed for GSH measurement by the recycling method of Tietze.12 The effect of phenobarbital on biliary GSH secretion was examined by treating animals with 80 mg/kg of phenobarbital intraperitoneally or saline per day for up to 7 days. Experiments were performed the following morning after the last dose of phenobarbital or saline. Measurement of Kidney and Intestinal GSH Levels. At the end of the in vivo biliary GSH efflux measurement as described above, kidneys and small intestine were removed and placed on ice. The contents of the small intestine were flushed with 5 mL of normal saline, and the small intestine was divided into three equal segments, labeled duodenum, jejunum, and ileum. Each segment was cut open, placed on a piece of ice-cold glass, and the mucosa was gently scraped off by scraping the serosal side of the intestine with a scapula. The mucosa was then placed on dry ice, weighed and homogenized with sucrose-phosphate buffer as described above, and processed for GSH measurement by the recycling method of Tietze.12 Kidneys were also homogenized and processed for GSH. RNA Isolation and Northern Blot Analysis. EHBR and agematched controls were treated with phenobarbital (80 mg/kg in saline intraperitoneally) or saline for 3 days. RNA was isolated from liver biopsies according to the method of Chomczynski and Sacchi.14 RNA (10 mg) was separated on a 1% agarose gel with 18% formaldehyde, blotted onto a nitrocellulose membrane by capillary transfer, and then hybridized to a 1.5-kb complementary DNA (cDNA) derived from a subcloned insert corresponding to nucleotides 1132 to 2623 of the rat canalicular GSH transporter as described previously.5 The cDNA probe was labeled with 32P-deoxycytidine 5*-triphosphate by BCAbest Labeling Kit (Takara, Kyoto, Japan). Hybridization was at 557C overnight in 50% formamide, 61 SSPE (0.18 mol/L NaCl, 10 mmol/L sodium phosphate [pH 7.7], 1 mmol/L ethylenediaminetetraacetic acid), 2% sodium dodecyl sulfate (SDS) containing 100 mg/mL salmon sperm DNA, and 107 dpm labeled insert. Blots were washed twice for 30 minutes in 21 SSC (11 SSC Å 0.15 mol/L NaCl, 15 mmol/L Na citrate), 0.1% SDS at 657C, once for 10 minutes in 0.21 SSC, 0.1% SDS at 657C, and autoradiographed for 4 hours onto an imaging plate. The imaging plate was analyzed with a Bio-Image Analyzer (Bas 2000, Fuji Photo Film Co., Ltd., Tokyo, Japan). Equal loading of RNA samples was ensured by staining total RNAs (18S and 28S) on the same nitrocellulose paper with methylene blue. Northern blot analysis for the heavy subunit of GCS (GCS-HS) was performed according to methods described.15 To ensure equal loading of RNA samples, the same membrane was rehybridized with 32 P-labeled human b-actin cDNA probe (Clontech, Palo Alto, CA). Autoradiography and densitometry (Biomed Instrument model SLR-
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2D Soft Laser Scanning Densitometer, Fullerton, CA) were used to quantitate relative RNA. Results of Northern blot analysis were normalized to b-actin. Western Blot Analysis of RcGshT and GCS-HS. Rabbit polyclonal antibody against a synthetic peptide that corresponds to amino acid residues 158-165 of RcGshT5 was used for Western blotting. The peptide was chosen according to the Hopp and Woods Hydrophilicity Scale.16 Both peptide synthesis and antibody generation were carried out by Bio-Synthesis, Inc. (Lewisville, TX). A cysteine was added at the N-terminus, and the peptide was conjugated to Keyhole Limpet Hemocyanin. Each rabbit received intramuscularly and subcutaneously 1.0 mg of conjugate emulsified in Freund’s complete adjuvant. At 6, 8, and 20 weeks thereafter, each rabbit received booster intramuscular immunization injections of 1.0 mg conjugate in Freund’s incomplete adjuvant. Antibody titers were determined by enzymelinked immunosorbent assay 1 week after each injection. Liver homogenates from EHBR and age-matched controls treated with saline or phenobarbital (80 mg/kg in saline intraperitoneally) for 3 days were used for Western blotting. Protein concentration was determined by the Bio-Rad protein assay and was 80 mg/mL. Liver homogenates containing 100 mg protein were solubilized in equal volumes of sample buffer (285 mmol/L Tris [pH 6.8], 30% glycerol, 6% SDS, 1.5% 2-mercaptoethanol, and 0.01% Bromphenol Blue), subjected to 10% SDS–polyacrylamide gel electrophoresis,17 and electrotransferred to 0.2-mm NitroCellulose membranes using Semidry Transfer Cell (Bio-Rad, Hercules, CA).18 The NitroCellulose membranes were subsequently subjected to the Amplified Alkaline Phosphatase Immun-Blot Assay according to procedures described in the kit (cat. no. 170-6412, Bio-Rad). The first antibody was rabbit antirat RcGshT peptide postimmune serum diluted 1:250 in TBST (10 mmol/L Tris [pH 8.0], 150 mmol/L NaCl, containing 0.05% Tween 20). Preimmune serum showed no reactivity with liver homogenates. The specificity of the anti-RcGshT antiserum has been previously confirmed to identify a 98-kd polypeptide in canalicular-enriched membrane fraction but showed no reactivity with sinusoidal-enriched membrane fraction prepared from a pool of normal rat livers.19 In addition, antibody to a peptide that corresponds to amino acid residues 47-54 of RcGshT5 identified the same protein only in canalicular-enriched membrane fraction (not shown). Western blot analysis for GCS-HS was performed using liver cytosols of EHBR and age-matched controls according to methods described.15 Equivalent loading of protein was assured by Coomasie Blue staining of gels. Measurement of Enzyme Activities of GSH Synthesis Using mBCl. GSH synthesis was measured using liver and kidney cytosol
of EHBR and age-matched controls using mBCl as previously described.20 Liver cytosol was dialyzed overnight using molecularporous membrane tubing (molecular weight cutoff, 12,000-14,000, Spectrum Medical Industries, Inc., Los Angeles, CA) in 1001 volume of 0.01 mol/L sodium phosphate, pH 7.4, to avoid high background with mBCl and to eliminate feedback inhibition exerted by preexisting GSH. After overnight dialysis, cytosolic GSH concentration was decreased by 99% to 99.5%. The GSH synthesis rate was measured by addition of dialyzed iver cytosol (2-3.5 mg protein) to the cuvette containing 100 mmol/L TrisHCl, 150 mmol/L KCl, 20 mmol/L MgCl2 , 2 mmol/L ethylenediaminetetraacetic acid (pH 7.3), glutamate (10 mmol/L), glycine (10 mmol/ L), alkaline phosphate (ATP) (3 mmol/L), cysteine / dithiothreitol (0.1 and 1 mmol/L), plus 100 mmol/L mBCl in a final volume of 2.5 mL at 377C. The difference in initial rate of linear increase in fluorescence over 6 to 8 minutes with or without pretreatment with buthionine sulfoximine (5 mmol/L for 5 minutes at 377C) is equivalent to the rate of GSH synthesis. The change in fluorescence is converted to GSH concentration units by using standard curves. Specifically, formation of fluorescent adducts were monitored by adding mBCl (100 mmol/L) and glutathione-S transferase (0.1 U/mL) to the cuvette containing GSH standards. Standard curves were then generated by applying linear regression to the relationship between fluorescence and GSH concentration. This method assesses the formation of GSH as an end product from two steps—the formation of g-glutamylcysteine from cysteine and glutamate (catalyzed by GCS) and the formation of GSH from g-glutamylcysteine and glycine (catalyzed by GSH synthetase). Because GCS is rate-limiting, this is in essence a measure of GCS activity. To assess only the second step in GSH synthesis, liver cytosol was added to the cuvette containing 100 mmol/L TrisHCl, 150 mmol/L KCl, 2 mmol/L ethylenediaminetetraacetic acid (pH 7.3), substrates g-glutamylcysteine / dithiothreitol (0.2 mmol/L),
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TABLE 1. Effect of Phenobarbital on Biliary GSH Efflux in EHBR Body Weight (g)
Control SDR SDR / 3-D phenobarbital EHBR EHBR / 3-D phenobarbital EHBR / 7-D phenobarbital
264 258 252 247 267
{ { { { {
2 5 4* 4† 3‡
Liver Weight (g)
8.5 9.5 10.2 11.3 11.4
{ { { { {
Biliary GSH Efflux (nmol/g liver/min)
0.3 0.2* 0.3* 0.1‡ 0.1‡
3.40 6.78 0.014 0.008 0.023
{ { { { {
0.26 0.08* 0.001* 0.001† 0.006†
Bile Flow (mL/g liver/min)
1.25 1.95 0.79 0.64 0.81
{ { { { {
0.16 0.07* 0.06* 0.10† 0.05†
NOTE. Results represent mean { SE from 3 to 6 animals for each group. Biliary GSH efflux and bile flow rates were measured after overnight fasting as described in Materials and Methods. Phenobarbital treatment consisted of 80 mg/kg intraperitoneally per day for 3 or 7 days. Abbreviation: SDR, Sprague-Dawley rats. * P õ .05 vs. control SDR; † P õ .05 vs. SDR / phenobarbital; ‡ P õ .05 vs. EHBR by ANOVA, followed by Fisher’s test.
glycine (10 mmol/L), cofactors ATP (3 mmol/L) and Mg2/ (20 mmol/ L), plus 100 mmol/L mBCl in a final volume of 2.5 mL at 377C. The difference in initial rate of linear increase in fluorescence over 8 to 10 minutes in the presence of all precursors and cofactors for the second step of GSH synthesis versus only g-glutamylcysteine / dithiothreitol is equivalent to the rate of GSH synthesis catalyzed by GSH synthetase. Cysteine Assay. To measure cysteine concentrations in livers of EHBR and controls, liver biopsies were homogenized in 5% perchloric acid (25% wt/vol) to precipitate the proteins and centrifuged for 3 minutes in a microfuge; supernatant cysteine levels were determined according to the method of Gaitonde.21 Statistical Analysis. Paired Student’s t test was used for comparison within the same animal. For comparison between groups, unpaired Student’s t test (in the case of only two comparisons) or ANOVA followed by Fisher’s test (for multiple comparisons) was used. Two-tailed t tests were used. Significance was defined by P õ .05. RESULTS Biliary GSH Secretion in EHBR. First, we confirmed previous work that both biliary GSH secretion and bile flow in EHBR are significantly lower than age-matched controls.3 Table 1 shows that biliary GSH secretion is almost undetectable in EHBR despite acivicin treatment. This dose of acivicin maximally inhibits GGT activity, because an additional dose of acivicin did not result in higher biliary GSH secretion rates (not shown). High-performance liquid chromatography confirmed near-complete absence of both reduced and oxidized GSH and GSH mixed disulfides in bile samples of EHBR (not shown). Bile flow in EHBR is Ç40% lower than age-matched controls, similar to what was reported in TR0 mutants.1 After 3 days of phenobarbital treatment, which doubled the biliary GSH efflux in control Sprague-Dawley rats, as we previously reported,22 no change in biliary GSH efflux was observed in EHBR mutants. To see if more prolonged phenobarbital treatment would influence biliary GSH efflux in EHBR, some EHBR mutants received 7 days of phenobarbital at the same dose of 80 mg/kg/d. Still, no increase in biliary GSH efflux was observed. A separate group of older EHBR animals weighing 380 to 400 g also exhibited the same defect in biliary GSH secretion and nonresponsiveness to phenobarbital treatment (data not shown). Next, to see if there are abnormalities in RcGshT expression in EHBR, both Northern and Western blot analyses were performed. Figure 1 shows Northern blot analysis comparing EHBR with age-matched controls of liver RcGshT expression with and without 3 days of phenobarbital treatment. The baseline 4.0-kb RcGshT–messenger RNA (mRNA) level in EHBR livers is nearly identical to that of age-matched controls. After phenobarbital treatment, RcGshT-mRNA level increased by 550% in controls but by only 150% in EHBRs. Thus, the transcript for RcGshT is normally present in EHBR but did not respond with the same magnitude of increase after phenobarbital treatment. Western blot analysis using specific anti-RcGshT polyclonal antibody also showed the presence of the 98-kd RcGshT polypeptide in EHBR livers
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in normal amounts (Fig. 2A). Consistent with the observed functional changes after phenobarbital treatment, hepatic RcGshT polypeptide level doubled after phenobarbital treatment in control animals but remained unchanged in EHBR (Fig. 2B). Up-regulation of Cell GSH in EHBR—Organ Specificity and Mechanism. As in GY/TR0 mutants, EHBR exhibited in-
creased liver GSH (Table 2). However, this phenomenon extends beyond the liver, because GSH levels in all segments of the small intestine and kidneys were also 200% to 300% of age-matched controls. We next investigated the mechanism for the increase in liver GSH in EHBR. Parameters most important in determining steady-state GSH level, cysteine availability, and GCS activity,6,8 were first measured. As shown in Table 3, liver cysteine levels in EHBR are 70% higher than controls. Liver GCS activity as measured by GSH synthesis rate in the presence of all precursors of GSH (glycine, glutamate, and cysteine) was also significantly higher in EHBR, while that of the GSH synthetase activity as measured by GSH synthesis rate in the presence of g-glutamylcysteine and glycine was unchanged from controls. When GCS activity was measured at 1 mmol/L cysteine concentration, it was similarly increased in EHBR as at 0.1 mmol/L cysteine concentration (Table 3) (control GCS activity Å 4.29 { 0.19 and EHBR Å 5.98 { 0.26 nmol/mg/min; results represent mean { SE from 5 animals each, P õ .01 by unpaired t test). Since the Km of GCS for cysteine is Ç0.1 mmol/L,20 this would suggest the capacity (Vmax ) of GCS was affected. GCS activity (nmol/mg protein/min) in kidney cytosol of EHBR was also higher than controls (controls Å 2.79 { 0.32, EHBR Å 5.70 { 0.45; results represents mean { SE from 5 animals each, P õ .05 by unpaired t test). To see if increased GCS can be explained by an increase in GCS gene expression, steady-state GCS-HS mRNA and polypeptide levels were measured. As suspected, Northern blot analysis showed a doubling of the steady-state hepatic
FIG. 1. Effect of phenobarbital on RcGshT-mRNA expression in livers of EHBR and age-matched controls. Liver RNA samples obtained from three EHBR and three control rats that had been treated with phenobarbital (80 mg/kg intraperitoneally per day for 3 days) or saline vehicle were analyzed by Northern blot hybridization with a 32P-labeled RcGshT-cDNA probe as described in Materials and Methods. The position of RcGshT (4.0 kb) is as indicated. Pb, phenobarbital.
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HEPATOLOGY July 1996 TABLE 3. Comparison of Parameters Important in Hepatic GSH Synthesis in EHBR and Controls
Controls EHBR
Cysteine level (mmol/g liver)
GCS activity (nmol/mg protein/min)
GSH Synthetase activity (nmol/mg protein/min)
0.17 { 0.01 0.29 { 0.02*
2.67 { 0.47 4.29 { 0.27*
5.34 { 0.35 5.07 { 0.26
NOTE. Results represent mean { SE from five animals each. GCS activity was measured in liver cytosol in the presence of 10 mmol/L each of glutamate and glycine and 0.1 mmol/L cysteine, while GSH synthetase activity was measured in the presence of 10 mmol/L glycine and 0.2 mmol/L g-glutamylcysteine using mBCl (see Materials and Methods for details). Amount of cytosolic protein (mg) per gram of liver was not different: control Å 89 { 4; EHBR Å 91 { 3. * P õ .05 vs. control by unpaired Student’s t test.
FIG. 2. (A) Western blot analysis of RcGshT polypeptide in EHBR and agematched controls. Liver homogenates (100 mg each lane) obtained from EHBR and age-matched control rats (n Å 3 each) were analyzed by Western blot analysis using polyclonal antibodies against a synthetic peptide of RcGshT as described in Materials and Methods. The position of RcGshT (98 kd) is indicated. Preimmune serum showed no reactivity. (B) Effect of phenobarbital on RcGshT polypeptide expression in livers of EHBR and age-matched controls. Liver homogenates (100 mg each lane) obtained from three animals each of EHBR (bottom panel) and controls (top panel) treated with phenobarbital (80 mg/kg intraperitoneally per day for 3 days) or saline vehicle were analyzed by Western blot analysis using polyclonal antibodies against a synthetic peptide of RcGshT as described in Materials and Methods. The position of RcGshT (98 kd) is indicated. Pb, phenobarbital.
GCS-HS–mRNA level in EHBR rats (196% of age-matched controls after normalizing to b-actin; P õ .01 by unpaired t test) (Fig. 3). Semiquantitative Western blot analysis also confirmed the increase in GCS-HS polypeptide level (Fig. 4). DISCUSSION
The failure to secrete GSH in bile of EHBR and GY/TR0 mutants has been a puzzling and controversial issue. We have recently cloned the canalicular GSH transporter, which permits an examination of its expression in EHBR. In the present study, we have found that changes in the steady-state mRNA and polypeptide level of RcGshT could not account for the defect. Thus, alterations of RcGshT transcription, mRNA stability, translation, and polypeptide stability cannot account for the defect. We previously reported that canalicular membrane-enriched vesicles from EHBR exhibited the same Km and Vmax for GSH transport as controls.2 Thus, our previous hypothesis that the defect is accounted for by either failure to secrete endogenous organic anions, which normally trans-stimulate GSH secretion, or retention of these sub-
stances at sufficiently high concentration to also cis-inhibit RcGshT remains plausible. Alternatively, we cannot exclude the possibility that a labile posttranslational modification is somehow enhanced secondary to the primary genetic defect, such as phosphorylation, which inhibits the function of the transporter and is reversed (dephosphorylation) during the membrane vesicle isolation procedure. Another possibility is that the vesicle preps contain intracellular and canalicular membrane, and that the defect in the mutants leads to a secondary block in the transfer of RcGshT from the former to the latter. Finally, a regulatory role of the defective organic anion transporter is conceivable (see below). Further work is required to examine these possibilities. Phenobarbital treatment has been shown to increase biliary GSH secretion,22 the Vmax for GSH transport in canalicular membrane-enriched vesicles,23 and the abundance of RcGshT mRNA.5 We therefore treated EHBR with phenobarbital in the hope of raising RcGshT levels sufficiently to at least partially overcome the possible inhibition by retained organic anions. Unfortunately, the results are not definitive. Although phenobarbital treatment had no effect on biliary GSH secretion in EHBR, it exerted a much smaller induction of RcGshT mRNA for reasons that are unknown. Even in the case of control animals, a 550% increase in the steady-state RcGshT-mRNA level only led to a doubling of the polypeptide level. Thus, it is perhaps not surprising that a much smaller increase in the RcGshT-mRNA level did not alter the steadystate polypeptide level. However, because the expected extent of induction was not achieved, the goal of the experiment could not be achieved. Impaired phenobarbital responsiveness in EHBR has been found by others as well. Treatment with phenobarbital (80 mg/kg intraperitoneally per day for 3 days) increased cytochrome P450 2B and 2C6 protein levels and cytochrome P450 2B1 steady-state mRNA level in control Sprague-Dawley rats, but not in EHBR mutants (Dr. Takemi Yoshida, Personal communication, February 1996). A recent report raised the possibility that the multidrug resistance–associated protein (MRP) might transport GSH.24 Although expression of MRP is low in liver, it has been recently reported that an MRP-like transporter of similar speci-
TABLE 2. Comparison of GSH Level in Multiple Organs of EHBR and Controls Small Intestinal Mucosal GSH (mmol/g)
Controls EHBR
Liver GSH (mmol/g)
Kidney GSH (mmol/g)
Duodenum
Jejunum
Ileum
4.85 { 0.14 10.11 { 0.57*
0.12 { 0.01 0.35 { 0.02*
1.41 { 0.17 3.52 { 0.15*
1.18 { 0.16 3.35 { 0.17*
1.56 { 0.20 3.14 { 0.14*
NOTE. Results represent mean { SE from five animals each. GSH levels in multiple organs were measured as described in Materials and Methods after overnight fasting. * P õ .05 vs. control by unpaired Student’s t test.
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ficity (canalicular multispecific organic anion transporter [cMOAT]) is present in liver and defective in GY/TR0 rats.25 There are several lines of evidence that dispute the role of MRP or cMOAT as significant GSH transporters. First, erythrocytes have MRP-like activity but do not transport GSH,26 and HeLa cells lack MRP,27 but transport GSH.19 Second, in diabetic rats, there is either an increase or no change in biliary secretion of bilirubin,28,29 which is a substrate for the hepatic MRP-like protein, but a profound selective decrease in biliary GSH excretion is observed.30 Third, BSP-GSH is a substrate for MRP but does not inhibit RcGshT or canalicular GSH secretion.5 Fourth, GSH does not inhibit MRP-mediated ATP-driven GSH S-conjugate31 or leukotriene C4 transport (Dr. D. Keppler, Personal communication, February 1996). Fifth, MRP is a unidirectional efflux pump,27 whereas GSH transport in oocytes expressing RcGshT is bidirectional.5 Sixth, MRP-mediated transport is ATP-driven, whereas RcGshT-mediated GSH transport is not ATP-dependent.2 Although activation of protein kinase C enhances cMOAT activity32 but inhibits canalicular GSH secretion,33 it is not possible to exclude that this reflects a change in substrate specificity of the carrier. The bulk of evidence supports the conclusion that MRP or cMOAT do not transport GSH. As an alternative, a possible role for MRP in regulating the GSH transporters will need to be considered. Other ATP-binding cassette transporters are known to directly and indirectly influence the activity of ion channels,34 and an analogous effect of MRP on GSH transporters is an open possibility. EHBR exhibited a similar marked increase in liver GSH levels as GY/TR0 mutants.1 We found an increase in GCS activity and cysteine concentration in EHBR livers, two of the most important parameters in determining GSH synthesis capacity. In many conditions (i.e., drug-resistant tumor cell lines, treatment with methyl mercury hydroxide) in which GCS activity is increased, there is also an increase in the GCS-HS–mRNA level.35-37 Recently, we have also shown the increase in GCS activity induced by hormones, such as insulin and hydrocortisone,20 or plating cultured rat hepatocytes under low cell density38 correlated with increased GCS-HS– mRNA level.15 Thus, regulation of the GCS-HS gene expression appears critical for GSH homeostasis. Indeed, in the case of EHBR livers, increased GSH levels also correlated with increased steady-state GCS-HS–mRNA and polypeptide levels. Regarding higher hepatic cysteine concentration in EHBR, several possibilities exist. Enhanced sulfur amino acid uptake (methionine and cysteine) and/or activity of trans-sulfuration and/or decreased cysteine utilization in GSH synthesis or other pathways of degradation may be in-
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FIG. 4. Steady-state GCS-HS polypeptide level in EHBR and age-matched control rats. Liver cytosols (50 mg/lane) obtained from three EHBR and three control rats were subjected to SDS–polyacrylamide gel electrophoresis, followed by immunoblotting with preimmune (not shown) and postimmune sera. Rat kidney homogenates (20 mg/lane) were included for comparison. Molecular weight markers (in kd) are shown on the left. The bands at 73 kd represent GCS-HS. A minor band (Mr 63 kd) is also noted in the kidney homogenate, as previously described.39
volved.6 More work will be required to elucidate the exact mechanism. Although the hypothesis that decreased GSH secretion leads to a decreased demand for synthesis, which, according to the feedback regulation, is accomplished by raising cell GSH, remains plausible, the up-regulation in GSH synthetic capacity that we observed would accentuate the GSH level required to accomplish this. It has been reported that bile GSH secretion is important in maintaining intestinal mucosal GSH levels.9 Bile duct ligation leads to a 50% fall in intestinal GSH.9 To our surprise, intestinal GSH was increased in EHBR, and this effect was widespread, including kidney, which presumably reflects a generalized up-regulation of GSH synthesis. In the case of kidney, GCS activity in cytosol was also increased in EHBR. Whether there is a generalized increase in GCS-HS gene expression in all tissues of EHBR remains to be examined. However, it is of interest that the genetic defect in MRPlike polypeptide in GY/TR0 mutants was reported to be liverspecific, whereas the up-regulation in GSH synthetic capacity is generalized; this strongly suggests that the increased GSH level in liver is predominantly due to the up-regulation rather than ‘‘retention.’’ The present study further defines the secondary effects of a mutation in the ATP-driven organic anion transporter on GSH transport and GSH levels. The widespread and unpredicted effects reflect the complexity of the possible secondary effects of genetic mutations, a phenomenon not infrequently encountered in gene knockout models. Thus, our findings underscore the need for caution in interpreting the relationship between phenotypic abnormalities and a genetic mutation. Acknowledgment: The authors appreciate very much the helpful and critical comments of Dr. Ronald Oude Elferink. REFERENCES
FIG. 3. Steady-state GCS-HS–mRNA level in EHBR and age-matched controls. Liver RNA (50 mg each lane) samples were analyzed by Northern blot hybridization with a 32P-labeled GCS-HS–cDNA probe as described in Materials and Methods. The same membrane was then rehybridized with 32P-labeled human b-actin cDNA probe. The molecular size markers GCS-HS (Ç3.7 kb) and b-actin are as indicated.
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1. Oude Elferink RPJ, Ottenhoff R, Liefting W, de Haan J, Jansen PLM. Hepatobiliary transport of glutathione and glutathione conjugate in rats with hereditary hyperbilirubinemia. J Clin Invest 1989;84:476-483. 2. Ferna´ndez-Checa J, Takikawa H, Horie T, Ookhtens M, Kaplowitz N. Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats. J Biol Chem 1992;267:1667-1673. 3. Takikawa H, Sano N, Narita T, Uchida Y, Yamanaka M, Horie T, Mikami T, et al. Biliary excretion of bile acid conjugates in a hyperbilirubinemic mutant Sprague-Dawley rat. HEPATOLOGY 1991;14:352-360. 4. Adachi Y, Kobayashi H, Shouji M, Kitano M, Okuyama Y, Yamamoto T. Functional integrity of hepatocyte canalicular membrane transport of taurocholate and bilirubin diglucuronide in Eisai hyperbilirubinuria rats. Life Sci 1993;52:777-784. 5. Yi J, Lu S, Ferna´ndez-Checa J, Kaplowitz N. Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics. J Clin Invest 1994;93:1841-1845. 6. Kaplowitz N, Aw TY, Ookhtens M. The regulation of hepatic GSH. Annu Rev Pharmacol Toxicol 1985;25:714-744. 7. Ballatori N, Jacob R, Boyer JL. Intrabiliary glutathione hydrolysis: a source of glutamate in bile. J Biol Chem 1986;261:7860-7865.
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8. Richman PG, Meister A. Regulation of g-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem 1975;250: 1422-1426. 9. Aw TY. Biliary glutathione promotes the mucosal metabolism of luminal peroxidized lipids by rat small intestine in vivo. J Clin Invest 1994;94: 1218-1225. 10. Strumeyer D, Bloch K. g-L-glutamyl-L-cysteine from glutathione. Biochem Prep 1962;9:52-55. 11. Eberlie D, Clarke R, Kaplowitz N. Rapid oxidation in vitro of endogenous and exogenous glutathione in bile in rats. J Biol Chem 1981;256:21152117. 12. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969;27:502-522. 13. Fariss MW, Reed DJ. High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives. Methods Enzymol 1987;143:101109. 14. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159. 15. Cai J, Sun W, Lu SC. Hormonal and cell density regulation of hepatic gglutamylcysteine synthetase gene expression. Mol Pharmacol 1995;48: 212-218. 16. Hopp TP, Woods KR. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A 1981;78:3824-3828. 17. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bactriophage T4. Nature 1970;227:680-685. 18. Towbin H, Staehein T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitro-cellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1989;76:4350-4354. 19. Lu SC, Sun W, Yi J, Ookhtens M, Sze G, Kaplowitz N. Role of two recently cloned rat liver GSH transporters in the ubiquitous transport of GSH in mammalian cell lines. J Clin Invest 1996;97:1488-1496. 20. Lu SC, Ge J, Kuhlenkamp J, Kaplowitz N. Insulin and glucocorticoid dependence of hepatic g-glutamylcysteine synthetase and GSH synthesis in the rat: studies in cultured hepatocytes and in vivo. J Clin Invest 1992; 90:524-532. 21. Gaitonde MK. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem J 1967;104:627-633. 22. Kaplowitz N, Eberlie DE, Petrini J, Touloukian J, Corvasce MC, Kuhlenkamp J. Factors influencing the efflux of hepatic glutathione into bile in rats. J Pharmacol Exp Ther 1983;224:141-147. 23. Ferna´ndez-Checa J, Ookhtens M, Kaplowitz N. Selective induction by phenobarbital of the electrogenic transport of glutathione and organic anions in rat liver canalicular membrane vesicles. J Biol Chem 1993;268:1083610841.
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24. Zaman GR, Lankelma J, van Tellingen O, Beijnen J, Dekker H, Paulusma C, Oude Elferink RPJ, et al. Role of glutathione in the export of compounds from cells by the multidrug-resistance–associated protein. Proc Natl Acad Sci U S A 1995;92:7690-7694. 25. Mayer R, Kartenbeck J, Bu¨chler M, Jedlitschky G, Leier I, Keppler D. Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes. J Cell Biol 1995;131:137-150. 26. Kondo T, Dale GL, Beutler E. Glutathione transport by inside-out vesicles from human erythrocytes. Proc Natl Acad Sci U S A 1980;77:6359-6362. 27. Leier I, Jedlitschky G, Buchholz U, Cole SPC, Deeley RG, Keppler D. The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates. J Biol Chem 1994;269:27807-27810. 28. Tun˜on MJ, Gonzalez P, Garcia-Pardo LA, Gonzalez J. Hepatic transport of bilirubin in rats with streptozotocin-induced diabetes. J Hepatol 1991; 13:71-77. 29. Watkins JB, Sherman SE. Long-term diabetes alters the hepatobiliary clearance of acetaminophen, bilirubin and digoxin. J Pharmacol Exp Ther 1992;260:1337-1343. 30. Lee S, Kaplowitz N, Lu SC. Impaired canalicular GSH transport in diabetic rats. Gastroenterology 1994;106:A991. 31. Kobayashi K, Sogame Y, Hara H, Hayashi K. Mechanism of glutathione S-conjugate transport in canalicular and basolateral rat liver plasma membranes. J Biol Chem 1990;265:7737-7741. 32. Roelofsen H, Ottenhoff R, Oude Elferink RP, Jansen PL. Hepatocanalicular organic-anion transport is regulated by protein kinase C. Biochem J 1991;278:637-641. 33. Raiford DS, Sciuto AM, Mitchell MC. Effects of vasopressor hormones and modulators of protein kinase C on glutathione efflux from perfused rat liver. Am J Physiol 1991;24:G578-G584. 34. Inagaki N, Gonoi T, Clement JP IV, Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, et al. Reconstitution of IKATP : an inward rectifier subunit plus the sulfonylurea receptor. Science 1995;270:1166-1170. 35. Mulcahy RT, Untawale S, Gipp JJ. Transcriptional up-regulation of gglutamylcysteine synthetase gene expression in melphalan-resistant human prostate carcinoma cells. Mol Pharmacol 1994;46:909-914. 36. Godwin AK, Meister A, O’Dwyer PJ, Huang CS, Hamilton TC, Anderson ME. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis. Proc Natl Acad Sci U S A 1992;89:3070-3074. 37. Woods JS, Davis HA, Baer RP. Enhancement of g-glutamylcysteine synthetase mRNA in rat kidney by methyl mercury. Arch Biochem Biophys 1992;296:350-353. 38. Lu SC, Ge J. Loss of suppression of GSH synthesis under low cell density in primary cultures of rat hepatocytes. Am J Physiol 1992;263:C11811189. 39. Yan N, Meister A. Amino acid sequence of rat kidney g-glutamylcysteine synthetase. J Biol Chem 1990;265:1588-1593.
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