Rat Cytosol Research Articles

Regulatory effect of regucalcin on nitric oxide synthase activity in rat kidney cortex cytosol: Role of endogenous regucalcin in transgenic rats

The effect of regucalcin, a regulatory protein in Ca2+ signaling, on nitric oxid (NO) synthase activity in the cytosol of kidney cortex of rats was investigated. The presence of calcium chloride (10 micro M) in the enzyme reaction mixture caused a significant increase in NO synthase activity. This increase was significantly prevented by the addition of trifluoperazine (TFP; 20 or 50 micro M), an antagonist of calmodulin, supporting the existence of Ca2+/calmodulin-dependent NO synthase in rat kidney cortex cytosol. NO synthase activity was significantly decreased by the addition of regucalcin (10(-10)-10(-8) M) in the reaction mixture in the absence or presence of calcium chloride (10 micro M). The regucalcin (10(-8) M) effect was not seen in the presence of Nw-nitro-L-argine metylester (NAME; 10(-6) or 10(-5) M), an inhibitor of NO synthase. Regucalcin significantly reduced NO synthase activity in the presence of TFP (50 micro micro M) or EGTA (1 mM) which has a significant inhibitory effect on the enzyme activity. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the reaction mixture caused a significant increase in NO synthase activity. This increase was completely abolished by the addition of regucalcin (10(-7) M). NO synthase activity was not significantly changed in the kidney cortex cytosol of regucalcin transgenic rats overexpressing endogenous regucalcin as compared with that of wild-type rats. However, the effect of calcium chloride (10 micro M) in increasing NO synthase activity in the kidney cortex cytosol of wild-type rats was significantly weakened in regucalcin transgenic rats. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the kidney cortex cytosol of rats.

Suppressive role of endogenous regucalcin in the enhancement of nitric oxide synthase activity in liver cytosol of normal and regucalcin transgenic rats.

The suppressive role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the enhancement of nitric oxide (NO) synthase activity in the liver cytosol of rats was investigated. The enzyme activity was measured in a reaction mixture containing either vehicle or calcium chloride (1-20 microM) in the absence or presence of regucalcin (0.1, 0.25, or 0.5 microM). NO synthase activity was significantly increased by the addition of calcium (5-20 microM). This increase was completely abolished in the presence of trifluoperazine (TFP; 10-50 microM), an antagonist of Ca(2+)/calmodulin. The addition of regucalcin (0.1-0.5 microM) caused a significant fall in the calcium-increased enzyme activity. The effect of regucalcin (0.25 microM) in decreasing NO synthase activity was seen in the presence of ethylene glycol bis-(2-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA, 1 mM) or TFP (20 microM), indicating that regucalcin acts independent on Ca(2+)/calmodulin. NO synthase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture. The effect of the antibody (50 ng/ml) or calcium (10 microM) in elevating NO synthase activity in the liver cytosol of normal rats was not seen in the liver cytosol obtained from regucalcin transgenic rats. Moreover, the increase in NO synthase activity in the liver cytosol of normal rats induced by a single intraperitoneal administration of calcium (5.0 mg/100 g body weight) was significantly enhanced in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. The administration of calcium caused a significant increase in regucalcin level in the liver cytosol of normal rats. The present study demonstrated that endogenous regucalcin plays a suppressive role in the enhancement of NO synthase activity in the liver cytosol of rats.

Inhibitory role of regucalcin in the regulation of nitric oxide synthase activity in rat brain cytosol: involvement of aging

Regucalcin is shown to play a regulatory role in intracellular signaling system in many cells. The protein has been reported to be present in rat brain neurons. The role of regucalcin in the regulation of nitric oxide (NO) synthase activity in the brain cytosol of female young and aged rats was investigated. The presence of regucalcin (10 −9 and 10 −8 M) in the enzyme reaction mixture caused a significant decrease in NO synthase activity in the absence or presence of both calcium chloride (10 μM) and calmodulin (2.5 μg/ml). The effect of regucalcin (10 −8 M) in decreasing brain cytosolic NO synthase activity was not seen in the presence of Nw-nitro- l-argine methyl ester (NAME) (10 −4 M), trifluoperazine (20 μM) or EGTA (1 mM). Regucalcin protein levels were significantly lowered in the brain cytosol of aged (50 weeks old) rats as compared with that of young (5 weeks old) rats. Brain cytosolic NO synthase activity was significantly reduced with increasing age. In aged rat brain cytosol, regucalcin caused a significant decrease in NO synthase activity. The presence of anti-regucalcin monoclonal antibody (10–50 ng/ml) caused a significant increase in NO synthase activity in the brain cytosol of young and aged rats. This elevation was completely blocked by the addition of regucalcin (10 −8 M). The present study demonstrates that endogenous regucalcin has an inhibitory effect on NO synthase activity in the brain cytosol of young and aged rats.

Proteinous amino acids in muscle cytosol of rats' heart after exercise and hypoxia.

Levels of 19 proteinous amino acids and of total free amino acids were assayed by gas-liquid chromatography in cytosols of rat atrial and ventricular heart muscle cardiomyocytes. These amino acids were assayed after the rats had been exposed to either exercise (swimming) or hypoxia (hypobaric pressure of 686 hectoPascals). Out of the total free amino acids levels of arginine, glutamine and cysteine in atrial and ventricular cardiac muscle cytosols of control rats were the highest of all amino acids assayed. The control levels of all other amino acids assayed in atrial or ventricular cardiac muscles ranged from 0.1% to 10.6% of the total free amino acids in the control rats. Physical stress (exercise and hypoxia) significantly reduced the total amount of cytosolic free amino acids in both heart muscles. While hypoxia decreased the levels of arginine in both heart muscles, exercise abolished the level of cysteine in the atrial heart muscle. Decrease in arginine levels, and elimination of cysteine from the heart's atrial muscle after physical stress, may be attributed to its utilization of nitric oxide and to its synthesis of atriopeptin and/or endothelin during stress. No change was recorded in either experimental group in the level of glutamine in heart muscle cytosol. Exercise and hypoxia affect, in different modes, the levels of all other amino acids assayed, except for tryptophan, tyrosine, and histidine, which are precursors of endogenous neurotransmitters. The impact of proteinous amino acids on some bodily functions is discussed.

The influence of dexamethasone on Hsp70 level and association with glucocorticoid receptor in the liver of unstressed and heat-stressed rats

The aim of the present study was to examine the influence of dexamethasone on the levels of heat shock protein Hsp70 and glucocorticoid hormones receptor as well as on the interaction of these two proteins in the liver cytosol and nuclei of unstressed and rats exposed to whole body hyperthermic stress. The results, obtained by quantitative immunoblotting, have shown that dexamethasone provoked a reduction of Hsp70 basal level and an increase in its stress-induced level in the nuclei, supporting the idea that this hormone may be a factor included in the regulation of Hsp70 level both under normal and stress conditions. The cytosolic reduction and nuclear elevation of the glucocorticoid hormones receptor level by dexamethasone were also observed. Co-immunopurification of Hsp70 and glucocorticoid hormones receptor has revealed that the changes of cytosolic and nuclear levels of the two examined proteins resulted in the changes of their interaction within the respective cellular compartments. Thus, 41 ?C heat stress was shown to cause at least two-fold elevation of Hsp70/GR ratio within the glucocorticoid hormones receptor heterocomplexes both in the presence and in the absence of dexamethasone. The results support the view that glucocorticoid hormones signaling pathway and heat shock system are interrelated.

ラットの糖質及び脂質代謝に及ぼすガーリック抽出物腹腔内投与の効果

The effects of garlic components extracted with organic solvents on the carbohydrate and fat metabolism of rats were investigated. Rats fed on a diet prepared according to the AIN-76 formula were given either the garlic extract or saline for 5 weeks via an intra-peritoneal injection. The liver weight of the garlic group decreased, but their thyroid glands increased significantly in wet weight compared to the control group. The weight of the carcass was suppressed in the garlic group, and crude fat content in the carcass significantly decreased. The glycogen synthetase activity in the liver cytosol of the garlic-supplemented rats was also significantly decreased, as was the plasma insulin concentration, while the plasma glucagon concentration of the garlic group was elevated. It is suggested from these data that dietary supplementation with the garlic extract might have promoted the catabolism of glucose and/or fatty acid (combustion) in the rats.

Suppressive role of endogenous regucalcin in the regulation of nitric oxide synthase activity in heart muscle cytosol of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein of Ca2+ signaling, in the regulation of nitric oxide (NO) synthase activity in the cytosol of rat heart muscle was investigated. The addition of calcium chloride (5-20 microM) into the enzyme reaction mixture containing the heart cytosolic protein caused a significant increase in NO synthase activity. The Ca2+ effect was significantly inhibited by trifluoperazine (TFP; 20 or 50 microM), an antagonist of calmodulin, indicating the existence of Ca2+/calmodulin-dependent NO synthase activity in rat heart muscle cytosol. NO synthase activity was significantly decreased by the addition of regucalcin (10(-9) or 10(-8) M). This effect was also seen in the presence of calcium chloride (10 microM), TFP (50 microM) or EGTA (1 mM), a chelator of Ca2+. Meanwhile, the effect of regucalcin (10(-8) M) in decreasing NO synthase activity was not seen in the presence of Nw-nitro-L-arginine methylester (NAME; 10(-6) or 10(-5) M), an inhibitor of the enzyme. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the enzyme reaction mixture caused a significant increase in NO synthase activity. This effect was completely abolished by the addition of regucalcin (10(-7) M). NO synthase activity was not significantly changed in the heart muscle cytosol of transgenic rats overexpressing endogenous regucalcin as compared with that of wild-type rats. However, the effect of calcium (10 micro M) addition in increasing NO synthase activity was significantly weakened in the heart muscle cytosol of regucalcin transgenic rats. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the heart muscle cytosol of rats.

Proteomic analysis of the rat liver

Rat is a useful, widely used animal model for biological and toxicity studies. We analyzed total and cytosolic rat liver proteins by applying proteomics technologies. The proteins were separated by two-dimensional electrophoresis employing broad and narrow range immobilized pH gradient strips, followed by MALDI-MS analysis of the tryptic digests. Two hundred and seventy-three different gene products were identified, of which approximately 60% were enzymes with a broad spectrum of catalytic activities. Most of the identified proteins were detected in other rat protein samples as well, which were analyzed in our laboratory. Fifteen gene products were detected for the first time. These were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. In average, approximately five to 10 spots corresponded to one gene product. The database includes a large number of proteins known to be involved in toxicology-relevant pathways and may be useful in toxicity prediction studies.

Stereochemical features of the hydrolysis of 9,10-epoxystearic acid catalysed by plant and mammalian epoxide hydrolases.

cis-9,10-epoxystearic acid was used as a tool to probe the active sites of epoxide hydrolases (EHs) of mammalian and plant origin. We have compared the stereochemical features of the hydrolysis of this substrate catalysed by soluble and membrane-bound rat liver EHs, by soluble EH (purified to apparent homogeneity) obtained from maize seedlings or celeriac roots, and by recombinant soybean EH expressed in yeast. Plant EHs were found to differ in their enantioselectivity, i.e. their ability to discriminate between the two enantiomers of 9,10-epoxystearic acid. For example, while the maize enzyme hydrated both enantiomers at the same rate, the EH from soybean exhibited very high enantioselectivity in favour of 9R,10S-epoxystearic acid. This latter enzyme also exhibited a strict stereoselectivity, i.e. it hydrolysed the racemic substrate with a very high enantioconvergence, yielding a single chiral diol product, threo-9R,10R-dihydroxystearic acid. Soybean EH shared these distinctive stereochemical features with the membrane-bound rat liver EH. The stereochemical outcome of these enzymes probably results from a stereoselective attack by the nucleophilic residue on the oxirane ring carbon having the (S)-configuration, leading to the presumed (in plant EH) covalent acyl-enzyme intermediate. In sharp contrast, the reactions catalysed by cytosolic rat liver EH exhibited a complete absence of enantioselectivity and enantioconvergence; this latter effect might be ascribed to a regioselective formation of the acyl-enzyme intermediate involving C-10 of 9,10-epoxystearic acid, independent of its configuration. Thus, compared with soybean EH, the active site of rat liver soluble EH displays a very distinct means of anchoring the oxirane ring of the fatty acid epoxides, and therefore appears to be a poor model for mapping the catalytic domain of plant EHs.

Sulfation of indoxyl by human and rat aryl (phenol) sulfotransferases to form indoxyl sulfate.

The aim of this study was to identify sulfotransferase (SULT) isoform(s) responsible for the formation of indoxyl sulfate from indoxyl (3-hydroxyindole). Indoxyl was incubated together with the co-substrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and either human or rat liver cytosol or recombinant sulfotransferase enzymes. Formation of indoxyl sulfate from indoxyl was measured by HPLC and used for determination of sulfonation rates. Both cytosols sulfonated indoxyl with apparent Km values of 6.8 +/- 0.9 microM for human and 3.2 +/- 0.6 microM for rat cytosol. To help identify the isoform(s) of SULT responsible for indoxyl sulfate formation, indoxyl was incubated with human and rat liver cytosols and PAPS in the presence of isoform-specific SULT inhibitors. No inhibition was observed by DHEA, a specific hydroxysteroid sulfotransferase inhibitor, nor by oestrone, an inhibitor of oestrogen sulfotransferase. However, an aryl (phenol) sulfotransferase inhibitor, 2,6-dichloro-4-nitrophenol (DCNP), inhibited the formation of indoxyl sulfate with a IC50 values of 3.2 microM for human and 1.0 microM for rat cytosol indicating that human and rat aryl (phenol) sulfotransferases are responsible for the formation of indoxyl sulfate. When indoxyl was incubated with SULT1A1*2, a human recombinant aryl SULT, an apparent Km value of 5.6 +/- 1.8 microM was obtained. Kinetic studies with human and rat cytosols and human recombinant SULT1A1*2 gave similar kinetic values indicating that human and rat aryl sulfotransferases efficiently catalyze the formation of indoxyl sulfate, an important uremic toxin metabolite.

Non-Proteinous Amino Acids in Muscle Cytosol of Rats' Heart, After Exercise, Hypoxia or Challenge with Six Selected Cardioactive Drugs

Non-Proteinous Amino Acids in Muscle Cytosol of Rats' Heart, After Exercise, Hypoxia or Challenge with Six Selected Cardioactive Drugs

Downregulation of glucocorticoid receptors of liver cytosols and the role of the inflammatory cytokines in pathological stress in scalded rats

Preliminary experiments indicated that target cells were resistant to glucocorticoid (GC) after pathological stress. This study was designed to investigate the alterations in plasma corticosterone level and GC receptor (GR) of liver cytosols, to assess the relative inflammatory cytokines contribution to GC resistant, and to observe the action of α-melanocyte-stimulating hormone (α-MSH) on the potential implications of glucocorticord regulatory effects in burned rats. Male Wistar rats (weight range, 180–200 g) received a 35% total body surface area immersion scald and were randomly divided to receive either tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), polyclonal antibody (pAb), α-MSH, Ac-D-Lys-L-Pro-D-Val (KPV peptide), or saline (control).The binding capacity (Rt) of the steroid-binding sites was measured by radioligand binding assay, using [ 3 H ]dexamethasone as the ligand. We examined plasma levels of IL-1β, TNFα, IL-10, and corticosterone following scald challenge in rats. The Rt of GR (208.45±30.78 fmol/mg of protein) in hepatic cytosol in rats, 12 h later the scald was significantly lower than that (306.71±27.96 fmol/mg of protein) of the control group ( P<0.01). The injections of anti-rat TNFα (257.80±12.82 fmol/mg of protein), IL-1β antibody (254.46±21.21 fmol/mg of protein), α-melanocyte-stimulating hormone (278.32±7.76 fmol/mg of protein) and KPV peptide (263.46±17.46 fmol/mg of protein) might prevent the Rt of GR from decreasing in hepatic cytosols of rats with scald, respectively (all of P<0.05) in vivo. Scald-induced robust increases in plasma IL-1β (214.08±27.25 pg/ml), TNFα (111.18±23.97 pg/ml), IL-10 (177.50±15.79 pg/ml) and corticosterone (2680±443.23 ng/ml) levels after 12 h. The administration of TNFα, IL-1β pAb, α-MSH and KPV might attenuate these increases. These studies suggest that pro-inflammatory cytokines are involved in downregulation of GRs and thus α-MSH and KPV might increase the level of GR in rats with immersion scald.

Hyperthermic stress stimulates the association of both constitutive and inducible isoforms of 70 kDa heat shock protein with rat liver glucocorticoid receptor

Glucocorticoid hormone receptor exists in the cytoplasm of target cells in the form of dynamic multiprotein heterocomplexes with heat shock proteins Hsp90 and Hsp70, and additional components of the molecular chaperone machinery. Whole body hyperthermic stress was previously shown to induce alterations in protein composition of these complexes increasing the share of Hsp70, but participation of individual Hsp70 family members was not investigated. In the present study the association of glucocorticoid receptor with constitutive and inducible forms of Hsp70 in the liver cytosol of rats exposed to 41 °C whole body hyperthermic stress was examined. Immunoprecipitation of glucocorticoid receptor heterocomplexes by monoclonal anti-receptor antibody (BuGR2) followed by quantitative immunoblotting revealed the presence of both nucleocytoplasmic Hsp70 family members, constitutive—Hsc70 and inducible—Hsp72, within the complexes. Immediately after the stress only Hsc70 was found in association with glucocorticoid receptor. However, after the induction of Hsp72 by stress, its appearance within the glucocorticoid receptor heterocomplexes was also recorded and the presence of both Hsp70 forms within the heterocomplexes was evident by the end of examined 24 h period after the stress. This study confirms that heat stress affects protein composition of rat liver glucocorticoid receptor heterocomplexes increasing the share of Hsp70 and shows that this increase could be equally ascribed to constitutive and inducible forms of Hsp70.

Endogenous regucalcin suppresses the enhancement of protein phosphatase activity in the cytosol and nucleus of kidney cortex in calcium-administered rats.

The suppressive role of endogenous regucalcin (RC), which is a regulatory protein of calcium signaling, in the enhancement of protein phosphatase activity (PPA) in the cytosol and nucleus of kidney cortex in calcium-administered rats was investigated. Calcium content in the kidney cortex was significantly increased at 0.5-5 h after a single intraperitoneal administration of calcium chloride solution (10 mg Ca/100 g body weight) to rats. The analysis with Western blotting of RC protein showed that RC levels in the cytosol and nucleus were significantly increased 0.5-5 h after the administration of calcium (10 mg/100 g). PPA toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol and nucleus of kidney cortex. PPA toward three phosphoamino acids in the cytosol and nucleus was significantly increased by the administration of calcium (10 mg/100 g). The presence of anti-RC monoclonal antibody (25 ng/ml) in the enzyme reaction caused a significant increase in PPA toward phosphotyrosine, phosphoserine, and phosphothreonine in the cytosol and nucleus of kidney cortex in normal rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA toward three phosphoamino acids in the cytosol and nucleus was significantly enhanced in calcium-administered rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA in the cytosol and nucleus of normal rats and calcium-administered rats was completely abolished by the addition of RC (10(- 6) M) in the enzyme reaction mixture. The present study suggests that endogenous RC suppresses the enhancement of PPA in the cytosol and nucleus of kidney cortex in calcium-administered rats.

Cadmium exposure decreases androgen-dependent metabolism of acetohexamide in liver microsomes of male rats through its testicular toxicity.

Administration of cadmium (Cd) at a dose of 1.23 mg/kg (2.0 mg/kg as CdCl(2)) markedly decreased the activity of an enzyme (acetohexamide reductase) catalysing the ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of male rats. However, the decreased enzyme activity was increased by repeated treatment with testosterone propionate (TP). When male rats were castrated and TP was given to the castrated ones, a similar decrease and increase, as described above, were observed in the microsomal enzyme activity. Cd exposure to male rats induced haemorrhage and atrophy of the testes and significantly diminished serum testosterone levels. There was no possibility that Cd accumulated in liver microsomes of male rats causing direct inhibition of the microsomal enzyme activity. We conclude that Cd exposure decreases androgen-dependent metabolism of acetohexamide in liver microsomes of male rats through its testicular toxicity. Cd exposure had no effect on acetohexamide reductase activity in liver cytosol of male rats.

Induction of P-glycoprotein, glutathione-S-transferase and cytochrome P450 in rat liver by atrazine

We studied the effects of intraperitoneally administered atrazine on two hepatic neoplastic markers, P-glycoprotein (P-gp), and glutathione-S-transferase (GST), and several phase I drug-metabolizing enzyme cytochrome P450 (CYP) subfamilies in hepatic microsomes and cytosol of Fischer rats. The P-gp content was increased after 24 h of atrazine administration at 50 mg/kg, and maximum P-gp induction was observed at 300 mg/kg for 3 days. GST-P was induced at a lower dose than P-gp, from 10 mg/kg, but no other form of GST, such as GST1A1, was induced by the same dose. Among the CYP families, CYP1A2 was highly and CYP2B was slightly induced by atrazine while the CYP3A content remained unchanged. The liver plasma membrane marker alkaline phosphatase (AP) was not induced by the same doses. The inductions of P-gp, GST-P and CYP1A2 observed may explain some of the reported tumor-promoting properties and toxicity of atrazine in vivo.

Isolation of DT-Diaphorase [NAD(P)H Dehydrogenase (Quinone)] from Rat Liver Cytosol: Identification of New Enzyme Substrates, Carcinogenic Aristolochic Acids

Cytosolic fractions isolated from liver and kidney of rats treated with β-naphthoflavone, Sudan I, ellipticine, phenobarbital, ethanol, acetone and natural carcinogenic and nephrotoxic nitroaromatics, aristolochic acids, were tested for the activity of DT-diaphorase [NAD(P)H dehydrogenase (quinone), EC 1.6.99.2]. While the most efficient inducers of DT-diaphorase in liver were Sudan I, ellipticine and aristolochic acids, the highest increase in the DT-diaphorase activity in kidney was produced by aristolochic acids. No increase in the enzyme activity was determined after treatment of rats with acetone. DT-Diaphorase was isolated from liver cytosol of Sudan I-treated rats by the procedure consisting of fractionation with ammonium sulfate, gel permeation chromatography on a Sephadex G-150 column, affinity chromatography on an Affi-Gel Blue (Cibracron Blue Agarose) column and re-chromatography on Sephadex G-150. Rat DT-diaphorase catalyzed NAD(P)H-dependent reduction of menadione (vitamin K3), vitamin K1and 4-nitrosophenol as substrates. Moreover, we newly identified two carcinogenic nitroaromatic compounds, aristolochic acids, as other substrates of DT-diaphorase. A selective inhibitor of the human DT-diaphorase, dicoumarol, inhibited the catalytic activity of the rat purified enzyme.

Suppressive role of endogenous regucalcin in the regulation of protein phosphatase activity in rat renal cortex cytosol.

The role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the cytosol of rat renal cortex was investigated. Protein phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol of kidney cortex. The addition of regucalcin (50-250 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity toward three phosphoamino acids. The effect of calcium (25 microM) and calmodulin (2.5 microg/ml) in increasing protein phosphatase activity toward three phosphoamino acids was significantly decreased by the addition of regucalcin (100 nM). Protein phosphatase activity toward three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The effect of antibody (25 ng/ml) in increasing the enzyme activity was significantly inhibited by cyclosporin A (10(-5) M) or vanadate (10(-5) M). Regucalcin in the kidney cortex cytosol was clearly decreased by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity toward three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin antibody (25 ng/ml) in increasing protein phosphatase activity toward three phosphoamino acids was not seen in the renal cortex cytocol of saline-administered rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the cytoplasm of rat kidney cortex.

A novel 4 S [3H]β-naphthoflavone-binding protein in liver cytosol of female Sprague‒Dawley rats treated with aryl hydrocarbon receptor agonists

β-Naphthoflavone (β-NF) is a widely used inducer of phase-I and phase-II enzymes controlled by aryl hydrocarbon receptor (AhR). Studies of competitive binding with 3H-labelled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) and benzo[a]pyrene (B[a]P) have shown that β-NF is a high-affinity ligand for AhR and also for polycyclic aromatic hydrocarbon (PAH)-binding protein, both soluble proteins of rat liver in 8 S and 4 S fractions, respectively, of sucrose gradients. This study examined binding of [3H]β-NF to liver cytosolic proteins of female Sprague-Dawley rats. Treatment of rats with β-NF, 3-MC, TCDD or α-naphthoflavone (α-NF) increased the specific [3H]β-NF binding to liver cytosol up to 125-fold that of vehicle (corn oil)-treated rats (< 100 fmol/mg of protein). Sucrose gradients revealed a large 4 S and a small 8 S peak of radioactivity from [3H]β-NF binding to cytosols of β-NF-, 3-MC-, TCDD- or α-NF-treated rats. Whereas co-incubation with the unlabelled β-NF eliminated both peaks, co-incubation with 2,3,7,8-tetrachlorodibenzofuran (TCDF) eliminated only the 8 S peak. The sucrose density gradient from [3H]TCDD binding to cytosol of β-NF- or TCDD-treated rats yielded a small 4 S and a larger 8 S peak; only the latter was abolished by co-incubation with TCDF. Thus, the patterns of sedimentation, distribution and elimination of radioactivity from the 8 S fraction of the liver cytosols from β-NF-, 3-MC-, TCDD- or α-NF-treated rats were characteristic for the AhR, whereas those from the 4 S fraction appeared specific for [3H]β-NF binding. The data indicate that potent AhR agonists, TCDD, 3-MC and β-NF, and to a lesser extent α-NF, a weak AhR agonist, induce a 4 S [3H]β-NF-binding protein in liver cytosol of female rats. α-NF, β-NF and 3-MC were effective competitors (80-85% inhibition) of the [3H]β-NF-specific binding to the β-NF-, 3 MC- or TCDD-induced 4 S protein, whereas several PAHs including B[a]P and benzo[e]pyrene were only weak competitors. The increased [3H]β-NF binding was not associated with glycine N-methyltransferase activity. Hence, the 4 S [3H]β-NF-binding protein described herein differs from the constitutive 4 S PAH-binding protein of rat liver cytosols in the inducibility by β-NF and 3-MC, ligand-binding characteristics, and lack of glycine N-methyltransferase activity. Gel filtration on Sephacryl of liver cytosols from β-NF-treated rats indicated a molecular mass of ≈ 42 kDa for [3H]β-NF-bound protein and suggested that it was derived from a large mass component that before the radioligand binding was eluted with the void volume of the gel and sedimented in a 7 S fraction of the sucrose gradient. The [3H]β-NF binding activity was not eluted with glutathione S-transferase Ya, aldehyde-3-dehydrogenase or DT-diaphorase [NAD(P)H: quinone oxidoreductase] activities, which are AhR-controlled and β-NF-inducible. Further studies are needed to determine the identity and function of this novel protein which may be involved either directly or indirectly (as a carrier protein) in xenobiotic metabolism in vivo.

A novel 4 S [3H]β-naphthoflavone-binding protein in liver cytosol of female Sprague–Dawley rats treated with aryl hydrocarbon receptor agonists

beta-Naphthoflavone (beta-NF) is a widely used inducer of phase-I and phase-II enzymes controlled by aryl hydrocarbon receptor (AhR). Studies of competitive binding with (3)H-labelled 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) and benzo[a]pyrene (B[a]P) have shown that beta-NF is a high-affinity ligand for AhR and also for polycyclic aromatic hydrocarbon (PAH)-binding protein, both soluble proteins of rat liver in 8 S and 4 S fractions, respectively, of sucrose gradients. This study examined binding of [(3)H]beta-NF to liver cytosolic proteins of female Sprague-Dawley rats. Treatment of rats with beta-NF, 3-MC, TCDD or alpha-naphthoflavone (alpha-NF) increased the specific [(3)H]beta-NF binding to liver cytosol up to 125-fold that of vehicle (corn oil)-treated rats (<100 fmol/mg of protein). Sucrose gradients revealed a large 4 S and a small 8 S peak of radioactivity from [(3)H]beta-NF binding to cytosols of beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats. Whereas co-incubation with the unlabelled beta-NF eliminated both peaks, co-incubation with 2,3, 7,8-tetrachlorodibenzofuran (TCDF) eliminated only the 8 S peak. The sucrose density gradient from [(3)H]TCDD binding to cytosol of beta-NF- or TCDD-treated rats yielded a small 4 S and a larger 8 S peak; only the latter was abolished by co-incubation with TCDF. Thus, the patterns of sedimentation, distribution and elimination of radioactivity from the 8 S fraction of the liver cytosols from beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats were characteristic for the AhR, whereas those from the 4 S fraction appeared specific for [(3)H]beta-NF binding. The data indicate that potent AhR agonists, TCDD, 3-MC and beta-NF, and to a lesser extent alpha-NF, a weak AhR agonist, induce a 4 S [(3)H]beta-NF-binding protein in liver cytosol of female rats. alpha-NF, beta-NF and 3-MC were effective competitors (80-85% inhibition) of the [(3)H]beta-NF-specific binding to the beta-NF-, 3 MC- or TCDD-induced 4 S protein, whereas several PAHs including B[a]P and benzo[e]pyrene were only weak competitors. The increased [(3)H]beta-NF binding was not associated with glycine N-methyltransferase activity. Hence, the 4 S [(3)H]beta-NF-binding protein described herein differs from the constitutive 4 S PAH-binding protein of rat liver cytosols in the inducibility by beta-NF and 3-MC, ligand-binding characteristics, and lack of glycine N-methyltransferase activity. Gel filtration on Sephacryl of liver cytosols from beta-NF-treated rats indicated a molecular mass of approximately 42 kDa for [(3)H]beta-NF-bound protein and suggested that it was derived from a large mass component that before the radioligand binding was eluted with the void volume of the gel and sedimented in a 7 S fraction of the sucrose gradient. The [(3)H]beta-NF binding activity was not eluted with glutathione S-transferase Ya, aldehyde-3-dehydrogenase or DT-diaphorase [NAD(P)H: quinone oxidoreductase] activities, which are AhR-controlled and beta-NF-inducible. Further studies are needed to determine the identity and function of this novel protein which may be involved either directly or indirectly (as a carrier protein) in xenobiotic metabolism in vivo.