Kidney Cytosol Research Articles

Alteration of acetohexamide reductase activities in kidney microsomes and cytosol of cadmium-treated rats

We examined the alteration of acetohexamide reductase activities in kidney microsomes and cytosol of cadmium (Cd)-treated rats. Acetohexamide reductase activity in kidney microsomes of male rats was markedly decreased by treatment with Cd at a dose of 1.23 mg/kg body weight. However, the decreased enzyme activity was completely restored by repeated treatment with testosterone propionate. Therefore, it is reasonable to assume that the treatment with Cd indirectly affect the androgen-dependent acetohexamide reductase activity in kidney microsomes of male rats, possibly by depressing androgen production. In the case of female rats, unlike male rats, the microsomal enzyme activity was little detectable, and was unaffected by the treatment with Cd. Furthermore, Cd treatment had no significant effect on acetohexamide reductase activity in kidney cytosol of male or female rats.

Purification to Apparent Homogeneity and Properties of Pig Kidneyl-Fucose Kinase

L-Fucokinase was purified to apparent homogeneity from pig kidney cytosol. The molecular mass of the enzyme on a gel filtration column was 440 kDa, whereas on SDS gels a single protein band of 110 kDa was observed. This 110-kDa protein was labeled in a concentration-dependent manner by azido-[32P]ATP, and labeling was inhibited by cold ATP. The 110-kDa protein was subjected to endo-Lys-C digestion, and several peptides were sequenced. These showed very little similarity to other known protein sequences. The enzyme phosphorylated L-fucose using ATP to form beta-L-fucose-1-P. Of many sugars tested, the only other sugar phosphorylated by the purified enzyme was D-arabinose, at about 10% the rate of L-fucose. Many of the properties of the enzyme were determined and are described in this paper. This enzyme is part of a salvage pathway for reutilization of L-fucose and is also a valuable biochemical tool to prepare activated L-fucose derivatives for fucosylation reactions.

Purification of a heat-stable activator protein for ADP-ribosylation factor-dependent phospholipase D

A heat-stable activator for ADP-ribosylation factor (ARF)-dependent phospholipase D (PLD) was purified to near homogeneity from rat kidney cytosol by a sequential column chromatography. The purified activator has a molecular mass of 23 kDa on SDS-PAGE. Using a partially purified ARF-dependent PLD from rat kidney, the activator synergistically stimulates PLD with ARF in time- and dose-dependent manner. In the absence of ARF, the activator has little or no effect. The purified activator also stimulates PLD under several conditions including permeabilized cell system, suggesting that the activator is a physiologically relevant regulator of PLD.

Identification and characterization of a novel synthetic cannabinoid CP 55,940 binder in rat brain cytosol.

We have detected the presence of a specific [3H] CP 55,940 binder in the cytosol of rat cerebral cortex. Competition studies showed that only cold CP 55,940 and to a lesser extent delta9THC was able to compete with [3H] CP 55,940; little competition was observed with either delta8THC or anandamide. Scatchard analysis of the data indicate the presence of two distinct binding components having affinity constants (Kd) of 0.97 +/- 0.03 nM, 5.83 +/- 0.08 nM, and Bmax of 3.31 +/- 0.06 pmol/mg protein, 22.2 +/- 1.2 pmol/mg protein respectively. The cytosolic CP 55,940 binder was heat stable up to 30 degrees C. Besides the brain cytosol, lesser amounts of binding were also detected in the spleen, and testis. Liver, kidney and muscle cytosol preparations were found to be devoid of this binder. Unlike the previously characterized brain membrane cannabinoid receptor, this binder was found to be salt, sulfhydryl blocking reagents and nucleotide resistant. Interestingly, dithiothreitol (DTT), a protein-disulfide group reducing agent, inhibited the binding of [3H] CP-55,940 to the receptor and approximately 80% binding inhibition was obtained at a 5 mM concentration. Western blot analysis using anti-receptor antibody reveal the presence of a 95-110, 50 and 38 kDa band in the brain, spleen and testis cytosolic preparations. In conclusion, we have identified the presence of a novel CP 55,940 binder in rat cerebral cortex cytosol possessing biochemical properties distinct from those previously observed using rat cerebral cortex membrane cannabinoid receptor.

Cell-specific Induction of Apoptosis by Microinjection of Cytochrome c: Bcl-xL HAS ACTIVITY INDEPENDENT OF CYTOCHROMEc RELEASE

Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-xL activity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.

21-Hydroxy-6,19-oxidoprogesterone: a novel synthetic steroid with specific antiglucocorticoid properties in the rat.

In the rat, the conformationally highly bent steroid 21-hydroxy-6, 19-oxidoprogesterone efficiently displaces [3H]corticosterone from thymus-glucocorticoid receptors and blocks type II receptors in kidney cytosols but competes with neither [3H]aldosterone for kidney-mineralocorticoid receptors nor [3H]progesterone for uterus-progesterone receptors. It evokes Na+ retention only at very high doses (approximately 100 microg/100 g of rat weight) and is unable to induce tyrosine aminotransferase or to increase glycogen deposits in rat liver. When coincubated with corticosterone or dexamethasone, 2.5 microM 21OH-6OP inhibits 80% of tyrosine aminotransferase induction. It may therefore be used experimentally as an antiglucocorticoid virtually lacking mineralocorticoid or glucocorticoid properties as well as affinity for mineralocorticoid or progesterone receptors.

Cysteine conjugate beta-lyase-dependent biotransformation of the cysteine S-conjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A).

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (1, Compound A) is a fluoroalkene formed by the base-catalyzed degradation of sevoflurane that is nephrotoxic in rats. Fluoroalkene 1 is a structural analog of other nephrotoxic haloalkenes that undergo glutathione S-conjugate formation and cysteine S-conjugate beta-lyase-dependent bioactivation to reactive intermediates. The present experiments were designed to study the beta-lyase-dependent biotransformation of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (4) and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine (5) by 19F NMR and UV spectroscopy and GC/MS. Incubation of cysteine S-conjugate 4 with rat kidney cytosol or a pyridoxal model system showed the formation of inorganic fluoride, pyruvate, and 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (9), the expected products of a beta-lyase-catalyzed reaction. The ratio of fluoride to pyruvate ranged from 2.3 to 2.5. The amount of acid 9 formed in the rat kidney cytosol and the pyridoxal model system was, however, less than 5% of the amount of pyruvate formed. Incubation of conjugate 4 with rat kidney cytosol and analysis by 19F NMR spectroscopy showed resonances that were assigned to 3,3,3-trifluorolactic acid (10); the formation of acid 10 was observed in the pyridoxal model only after prolonged incubation (> 18 h). Lactic acid 10 was identified as a degradation product of acid 9. Cysteine S-conjugate 5 was not stable in pH 7.4 buffer and underwent a rapid cyclisation reaction (t1/2 approximately 5 min) to form 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3-thiazol e-4 -carboxylic acid (14). These data show that fluoroalkene 1-derived cysteine S-conjugates are substrates for renal beta-lyase and that acid 9 is formed as a terminal product. Acid 9 is, however, unstable and affords lactic acid 10 as a degradation product.

In vivo and in vitro effects of carbenoxolone on glucocorticoid receptor binding and glucocorticoid activity

Carbenoxolone potentiates the mineralocorticoid activity of endogenous glucocorticoid hormones by inhibiting the enzyme 11β-hydroxysteroid dehydrogenase, which converts cortisol and corticosterone to inactive 11-oxo-derivatives. We addressed the question of whether glucocorticoid activity is also affected by carbenoxolone. Using a rat model involving low dose corticosterone treatment, we found that carbenoxolone neither potentiated nor inhibited the modest increases in blood pressure or reductions in weight gain caused by steroid treatment. Other indices of glucocorticoid activity including white blood cell number, thymus weight, and down regulation of the glucocorticoid receptor were unaffected. In vitro studies with liver and kidney cytosol preparations indicated that carbenoxolone did compete for 3H-dexamethasone binding sites. Carbenoxolone was 5–10 times more effective than glycyrrhetinic acid, 20–30 thousand times less effective than dexamethasone, and is, therefore, approximately 1000 times less effective than corticosterone. Analysis of dexamethasone-binding curves indicated a single class of receptor. We conclude that carbenoxolone at the dose tested does not have intrinsic glucocorticoid activity in vivo, nor does it modulate the activities of corticosterone. Carbenoxolone binds weakly to the glucocorticoid receptor. It is not clear whether this weak affinity accounts for some or any of the direct in vitro effects of high concentrations of carbenoxolone that others have described.

Methyltert-Butyl Ether Causes α2u-Globulin Nephropathy and Enhanced Renal Cell Proliferation in Male Fischer-344 Rats

Methyltert-butyl ether (MTBE), a fuel additive blended into unleaded gasoline to decrease carbon monoxide emissions, induces renal tumors in male, but not female, rats exposed by inhalation to ≥3000 ppm MTBE. A number of chemicals that induce male rat-specific renal tumors also cause a syndrome unique to male rats referred to as α2u-globulin nephropathy (α2u-N). The objective of the present study was to determine if MTBE induces an α2u-N and renal cell proliferation in male F-344 rats. Male and female F-344 rats were exposed to MTBE vapors of 0, 413, 1516, or 3013 ppm for 6 hr/day for 10 consecutive days. Significant proximal tubule necrosis and protein droplet accumulation were observed in kidneys from male rats exposed to 1516 and 3013 ppm MTBE. Significantly greater labeling indices were observed in all groups of MTBE-exposed male rats. α2u-Globulin immunoreactivity was present in and confined to protein droplets in male rat kidney. A mild dose-related increase in α2u concentration in the kidney, as measured by an enzyme-linked immunosorbent assay, was observed in male rats exposed to MTBE, with a statistically significant increase in α2u concentration in male rats exposed to 3013 ppm MTBE. There was a strong positive correlation (r= 0.994) with exposure concentration between cell proliferation and α2u concentration in male rat kidney. No significant differences were observed in female rats for any of these responses. Further analysis of kidney cytosol failed to demonstrate the accumulation of any protein besides α2u in MTBE-exposed male rat kidney. These findings demonstrate that MTBE causes a mild induction of α2u-N and enhanced renal cell proliferation in male, but not female, F-344 rats, suggesting a role for α2u-N in renal tumorigenesis.

Redox modulation of selenium binding proteins by cadmium exposures in mice.

The mechanism of the antagonistic behaviour of selenium (Se) against cadmium (Cd) toxicity is investigated. This study reports the distribution of Cd at the organ and subcellular level after chronic treatments. The possible role of the selenium binding proteins (SBP) during Cd exposure are also evaluated. Kidney concentrates more Cd than liver following 8 weeks of treatment. Simultaneous administration of Se reduced Cd accumulation in Kidney. This affect did not occur in liver. Among the subcellular fractions, the maximum concentrations of both of the elements were found in the cytosol. The overall uptake of 75Se was enhanced in the cytosol of kidney and liver of the Cd treated animals. These observations support a hypothesis that selenium is complexed with cadmium. The increase in the labeling of SBP as a result of Cd exposures may reflect a change in the conformation of the protein molecule. These proteins (SBP) contain a sequence motif, which may be an active redox centre. Also, Cd significantly reduced the glutathione level, thereby disrupting the thiol/disulfide balance. This in turn may affect the redox status of the proteins leading to a 75Se or 75Se-Cd complex with SBP.

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in rat kidney cytosol.

The effect of regucalcin, a novel Ca2+-binding protein, on Ca2+/calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10[-7] M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl2 and calmodulin. However, the activatory effect of both CaCl2 (10 microM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10[-8] to 10[-6] M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl2 (5-50 microM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 microM), an antagonist of calmodulin, caused a partial inhibition of Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10[-7] M). The inhibitory effect of regucalcin (10[-7] M) was not seen in the presence of 20 microM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl2 (10 microM) and regucalcin (10[-7] M). The present results demonstrates that regucalcin has an inhibitory effect on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.

Specific binding of 11-ketotestosterone in an androgen target organ, the kidney of the male three-spined stickleback,Gasterosteus aculeatus

The kidney of male three-spined stickleback,Gasterosteus aculeatus, hypertrophies during the breeding season and produces a "glue" which is used in the building of the nest. This hypertrophy is androgen dependent, with 11-ketotestosterone (11KT) being more effective than other tested steroids in stimulating this secondary sexual character. In the present study kidneys were excised from stickleback males that had been castrated two days earlier. The purpose of this gonadectomy was to reduce the endogenous levels of androgens without allowing time for the kidney to regress. Tissue fragments were incubated with tritiated 11KT with and without unlabelled steroids at increasing concentrations. Displaceable specific 11KT binding was found in kidney tissue fragments whereas only non-specific binding was observed when liver and muscle were investigated in a similar way. Unlabelled 11KT displaced specifically bound, tritiated 11KT with an ED50-value (50% of displaceable binding) of 28 nM. Similar ED50 values were found for 17\-hydroxy-5α-androstane-3,11-dione (29 nM) and 5α-dihydrotestosterone (20 nM), whereas higher ED50 concentrations were estimated for testosterone (T; 203 nM) and progesterone (69 nM). No displacement of tritiated 11KT was found for the other investigated substances tested; estradiol, 17α,20β-dihydroxy-4-pregnen-3-one, flutamide or cyproterone acetate. No specific binding to kidney tissue fragments could be detected when labelled T was used instead of labelled 11KT. Specific binding of 11KT or T was not found either in the kidney cytosol or nuclear extracts. However, using the kidney membrane fraction a displacement of tritiated 11KT with unlabelled 11KT (10(-6)M) was observed. In conclusion there is a specific binding of 11KT in the stickleback kidney. The absence of binding in liver and muscle, the ED50 value observed and the displacement with some, but not all steroids are consistent with a receptor function. The presence of binding in membrane fractions, but not in cytosol or nuclear extracts suggests that the binding is not related to classic steroid receptors.

The cytosolic 47-kilodalton protein that binds to the 3' untranslated region of epidermal growth factor transcripts responds to orchiectomy in a tissue-specific fashion.

The submaxillary gland (SMG) of the male mouse is known to contain more epidermal growth factor (EGF) messenger RNA (mRNA) than that of the female. In contrast, the SMG of female mice contains more of a cytosolic 47-kDa RNA binding protein that binds to a unique 23-b sequence at the end of the 3' untranslated region (3' UTR) of EGF, which includes the canonical polyadenylation signal in an AU-rich region containing a potential mRNA destabilizing sequence. Testosterone treatment can change the activity of this 47-kDa protein within 1 day, which precedes the change produced in the level and in the polyadenylation of EGF mRNA in female mice. In the kidney, however, neither the 47-kDa protein nor the polyadenylation pattern changed after testosterone. We now report that 2 weeks following orchiectomy of Balb/c mice, mature EGF peptide immunoreactivity levels fall by 96% (P < 0.0002), and mRNA levels fall by 76% (P < 0.0001). Orchiectomy also enhances the RNA binding activities of the 47-kDa protein in SMG cytosol but not in the kidney: UV cross-linking to 3' UTR RNA is increased by 67% (P < 0.01) and shifting of the gel mobility of the RNA is increased by 47% (P < 0.05). The polyadenylation pattern of EGF transcripts also changes in SMG but not in kidney cytosol. After orchiectomy, the population of EGF transcripts in the SMG with short, heterogenous poly-A tails (<50 A's) decreases by 31% (P < 0.03), whereas transcripts with long poly-A tails of approximately 50, 70, 100, and 200 A's all increase and become more distinct, resembling the pattern found in the normal female. Thus, 2 weeks after orchiectomy, the responses in EGF expression, EGF mRNA polyadenylation, and the activities of the 47-kDa 3' UTR binding protein are the reciprocal of the responses produced by injecting female mice with testosterone for 5 days, providing further evidence that circulating androgens regulate EGF expression posttranscriptionally in a sexually dimorphic, tissue-specific fashion.

High affinity thyroid hormone-binding protein in human kidney: kinetic characterization and identification by photoaffinity labeling.

The binding of thyroid hormones and its regulation of NADPH and NADP+ were studied in human kidney cytosol, and a 38-kDa polypeptide (p38) was identified by photoaffinity labeling of cytosol with underivatized [125I]T3, SDS-PAGE, and autoradiography. The cytosolic thyroid hormone binding and p38 photolabeling were strongly activated by NADPH (maximum at 10(-7) M), whereas other nucleotides were less effective or ineffective. NADP+ did not activate T3 binding and p38 photolabeling, provided it was protected from conversion to NADPH by the addition of an exogenous oxidizing enzymatic system (oxidized glutathione plus glutathione reductase). Furthermore, NADP+ inhibited NADPH activation (half-maximum inhibitory effect at approximately 2 x 10(-5)M), and oxidation of NADPH to NADP+ induced dissociation of bound T3. The equilibrium dissociation constant (Kd) of the NADPH-activated cytosolic T3-binding sites was 0.3 nM, similar to the Kd of the nuclear T3 receptors. The kidney contained 200 times more cytosolic NADPH-activated thyroid hormone-binding sites than nuclear T3 receptors. Nonradioactive iodothyronines competed with [125I]T3 for both NADPH-activated binding and p38 photolabeling, with the following order of decreasing affinity: D-isomer of T3 > T3 > T4 > triiodothyroacetic acid > 3'-isopropyl-3,5-diiodothyronine > rT3. NADPH-activated T3 binding and photolabeled p38 were also detected in human heart and liver cytosols, but not in pancreas, cultured fibroblast and erythrocyte cytosols, or plasma. Rat kidney cytosol contained a 35-kDa photolabeled polypeptide homolog to human p38. The native molecular mass of the human photolabeled protein was 50 kDa, whereas that of the rat protein was 60 kDa, as determined by nondenaturing polyacrylamide gel electrophoresis. Two-dimensional PAGE of photolabeled p38 indicated an isoelectric point of 5.3. These findings describe the molecular properties of a NADPH/NADP+-regulated thyroid hormone-binding protein not previously identified in human and rat kidney cytosol.

Cloning and Expression of a Rat Kidney Cytosolic Glutamine Transaminase K That Has Strong Sequence Homology to Kynurenine Pyruvate Aminotransferase

An α-keto acid-dependent cysteineS-conjugate β-lyase of rat kidney was identified as cytosolic glutamine transaminase K by Stevenset al.(J. Biol. Chem.261, 15529–15537, 1986). Subsequently, a rat kidney protein with both glutamine transaminase K- and cysteineS-conjugate β-lyase activities was cloned and sequenced by Perryet al.(Mol. Pharmacol.43, 660–665, 1993). This protein was later shown to be identical with kynurenine pyruvate aminotransferase (Moscaet al., FEBS Lett.353, 21–24, 1994). Thus, kynurenine pyruvate aminotransferase possesses both glutamine transaminase K- and cysteineS-conjugate β-lyase-type activities. We have also cloned a cytosolic glutamine transaminase K from a rat kidney cDNA library and expressed the full-length clone in COS1 cells. The transfected cells exhibit marked increases in activities of both glutamine transaminase K and cysteineS-conjugate β-lyase. The enzyme cloned in the present work is a homodimer. Each subunit has a molecular mass of 45.8 kDa and contains 426 amino acid residues. The sequence of cytosolic glutamine transaminase K obtained in the present work has strong similarities to other aminotransferases, including >90% identity with kynurenine pyruvate aminotransferase. Our preparation of purified rat kidney cytosolic glutamine transaminase K possesses some kynurenine pyruvate aminotransferase activity. Thus, rat kidney cytosol possesses at least two distinct enzymes that catalyze glutamine transaminase K/cysteineS-conjugate β-lyase/kynurenine py- ruvate aminotransferase reactions. The present work underscores the difficulties associated with characterizing aminotransferases with overlapping specificities.

Identification of the GalNAc Kinase Amino Acid Sequence

A new kinase that forms GalNAc-1-P was purified from pig kidney cytosol and identified on gels by labeling with N3-[32P]ATP (Pastuszak, I., Drake, R., and Elbein, A. D. (1996) J. Biol. Chem. 271, in press). A 50-kDa labeled protein was eluted, digested with trypsin, and the sequences of four peptides representing 49 amino acids showed 90% identity to sequence of human galactokinase reported to be on chromosome 15. To resolve this dilemma, activities and substrate specificities of galactokinase and GalNAc kinase from human and pig kidney, as well as of galactokinase from the yeast clone transfected with the cDNA from presumptive human galactokinase, were compared. The purified galactokinases phosphorylated galactose, but not GalNAc, whereas GalNAc kinase also phosphorylated galactose when this sugar was present at millimolar concentrations. Extracts of gal 1(-) yeast clone, transfected with presumptive human galactokinase cDNA, had very low galactokinase activity even when yeast were grown on galactose, but good activity with GalNAc. On the other hand, the wild type yeast phosphorylated galactose, but not GalNAc. These data indicate that the sequence reported for galactokinase on chromosome 15 is that of GalNAc kinase, which can phosphorylate galactose when this sugar is present at millimolar concentrations. This transfection thus allows the yeast mutant to grow slowly on galactose-containing media.

Alterations in glutathione homeostasis in mutant Eisai hyperbilirubinemic rats.

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 steady-state 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 gamma-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 (GCS-HS) 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 Jul;24(1):253-8)

Stability study on renal type I mineralocorticoid receptor

The purpose of this work is to review stability and activation properties of type I receptor, in order to explain the reasons for its extreme in vitro instability. We demonstrate that the treatment of rat kidney cytosol with H 2O 2 prevents aldosterone binding, DNA/steroid-receptor complex interactions, and prevents the receptor thermal inactivation. In contrast, exogenous sulfhydryl reducing reagents are necessary to insure maximum binding of mineralocorticoid receptor and DNA/steroid-receptor interaction. However, the presence of β-mercaptoethanol in thermal induced incubations reverts the H 2O 2 protection. We also demonstrate that contaminations with free or sequestered iron are harmful for both, receptor binding capacity (in a reversible form) and for hormone-receptor /DNA binding properties (in a partially reversible form). We propose a sulfhydryl oxidative mechanism for type I mineralocorticoid receptor inactivation in which iron contaminants might accelerate this process by oxidative catalysis. We also demonstrate that when thiol groups are blocked by specific reagents such as N-ethyl-maleimide or dithionitrobenzoic acid, type I sites loose binding capacity, but the protein is protected from oxidation as well as inactivation.

Influences of gold on zinc, copper and metallothionein kinetics in liver and kidney of the rat

1. Single, very low dose of gold (500 micrograms/kg body weight) was given intramuscularly to male Sprague-Dawley rats (mean body weight: 200 g) as aurothiokeratinate (Auro-Detoxin(R) dissolved in 0.9% NaCl. 2. Blood, liver and kidney were samples at autopsy 0.5, 1, 2, 3, 6 and 12 h after the gold injection (six animals per time). 12 rats were treated with 0.9% saline only as controls. The zinc (Zn), copper (Cu), gold and metallothionein (MT) concentrations were determined in homogenates and cytosols of liver and kidney using atomic absorption spectrometry and gel chromatography, respectively. 3. The main changes in metal and MT concentrations occurred in the kidney, where an increase of gold was found 0.5 h after the injection, followed by an increase in Cu and MT concentrations after 6 h. Zn homeostasis did not change. 4. The results suggest that gold by itself induces an increase of MT-like peptides in the kidney cytosol, which was accompanied by an increase in the concentration of Cu mainly bound to these MT-like peptides.

Dissociation of Cyclic Inositol Phosphohydrolase Activity from Annexin III

Cyclic inositol phosphohydrolase is a phosphodiesterase that cleaves the cyclic bond of cyclic inositol monophosphate. In 1990, Ross et al. (Ross, T. S., Tait, J. F., and Majerus, P. W. (1990) Science 248, 605-607) purified this enzyme from human placenta and reported that cyclic inositol phosphohydrolase is identical to annexin III. Independent confirmation of this finding has not been provided. The relative distribution of annexin III and cyclic inositol phosphohydrolase activity in rat kidney and spleen indicated that annexin III can be dissociated from cyclic inositol phosphohydrolase activity. Rat spleen contains large quantities of annexin III, but has very little cyclic inositol phosphohydrolase activity. In contrast, rat kidney, one of the richest sources of cyclic inositol phosphohydrolase activity, possesses very little (immunohistochemistry) or no (Western blot) annexin III. Similar to cytosol of human placenta, cytosol of guinea pig kidney contains both annexin III and cyclic inositol phosphohydrolase. On SDS-gel electrophoresis, guinea pig kidney annexin III has a slightly different mobility than the human placental annexin III. Human placental annexin III co-migrates with cyclic inositol phosphohydrolase on ion exchange chromatography, while guinea pig kidney annexin III is clearly dissociated from cyclic inositol phosphohydrolase on ion exchange chromatography. Both guinea pig kidney annexin III and human placental annexin III pellet with the addition of calcium and centrifugation, while cyclic inositol phosphohydrolase activity in both of these tissues remains in the supernatant. Our studies clearly show that cyclic inositol phosphohydrolase and annexin III are two different proteins.