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R Morgenstern

Publications and source records attributed to R Morgenstern.

At least 109 records · Page 6Linked to original sources
PubMed1992

Mechanism of activation of rat liver microsomal glutathione transferase by noradrenaline and xanthine oxidase.

Activation of glutathione transferase activity in rat liver microsomes under a variety of conditions producing oxidative stress was investigated. Neither hydrogen peroxide (10 mM) (added or produced endogenously by glucose + glucose oxidase) nor duroquinone together with an NADPH-regenerating system (which generates the superoxide anion radical) had any significant effect on the glutathione transferase activity towards 1-chloro-2,4-dinitrobenzene. On the other hand, incubation of microsomes with 1 mM noradrenaline (which autooxidizes and generates superoxide anion radical) gave a 160% activation, as shown earlier (Aniya and Anders, J Biol Chem 264: 1998-2002, 1989). This was taken as an indication that microsomal glutathione transferase could be activated by oxidative stress. Here, we demonstrate that activation by this compound is due to covalent binding (presumably of the quinone formed during autooxidation). The xanthine/xanthine oxidase system, which generates the superoxide anion radical and hydrogen peroxide, increases microsomal glutathione transferase activity, but this activation was not dependent on the presence of xanthine. Western blots of microsomes treated with xanthine oxidase revealed that activation was due to proteolysis (presumably by contaminating proteases in the xanthine oxidase). In conclusion, there is no firm evidence that rat liver microsomal glutathione transferase is activated directly by reduced oxygen species in the microsomal system. The possibility remains that oxidative stress triggers secondary mechanisms such as generation of reactive intermediates and/or activation of proteolysis, which can in turn increase enzyme activity.

Animals↗
PubMed1992

N-acetyl-p-benzoquinone imine-induced protein thiol modification in isolated rat hepatocytes.

Incubation of isolated rat hepatocytes with N-acetyl-p-benzoquinone imine (NAPQI) or 3,5-dimethyl-N-acetyl-p-benzoquinone imine (3,5-Me2-NAPQI) resulted in a concentration-dependent decrease in the protein thiol content of the mitochondrial, cytosolic and microsomal fractions. On a concentration basis, 3,5-Me2-NAPQI induced a more marked depletion of protein thiols than did NAPQI. Sodium dodecyl sulphate-polyacrylamide gel electrophoretic separation of the proteins of each fraction showed that different proteins had different susceptibilities to modification of their cysteine residues by the quinone imines. A few protein bands showed a decreased protein thiol content following incubation with non-toxic concentrations of quinone imines, whereas other proteins were affected by higher concentrations. Concentrations of quinone imines that were highly cytotoxic induced a general loss of protein thiols. NAPQI-induced protein thiol depletion occurred within 5 min and remained essentially unchanged for at least 30 min. In contrast, protein thiol depletion induced by 3,5-Me2-NAPQI increased over the 30-min time course of the experiment. Toxic concentrations of 3,5-Me2-NAPQI caused the formation of high molecular mass aggregates in all three subcellular fractions after 30 min of incubation. The observed crosslinking was not due to protein disulfide formation. However, no aggregate formation was observed after exposure of hepatocytes to NAPQI. One of the major target proteins of quinone imine-induced protein thiol depletion was a 17 kDa microsomal protein that was identified as the microsomal glutathione S-transferase. Exposure of hepatocytes and isolated liver microsomes to the quinone imines resulted in an up to four-fold increase in the specific activity of the microsomal glutathione S-transferase. In conclusion, our results are consistent with the suggestion of a critical role of protein thiol depletion in quinone imine-induced cytotoxicity.

Animals↗
PubMed1992

Studies on the activation of rat liver microsomal glutathione transferase in isolated hepatocytes.

The mechanism of activation of microsomal glutathione transferase in isolated liver cells by diisapropylidene acetone (phorone) was investigated. Phorone (1 mM) causes a time-dependent increase (up to 2.6-fold) in the glutathione transferase activity of microsomes isolated from treated hepatocytes. Since phorone reacts with sulfhydryl groups, the possibility that this compound activated microsomal glutathione transferase directly was studied. It was found that neither the activity of the purified enzyme nor that in isolated microsomes is affected by phorone. It has been suggested [Masukawa T and Iwata H, Biochem Pharmacol 35: 435-438, 1986] that activation of microsomal glutathione transferase by phorone in vivo is mediated through thiol-disulfide interchange involving oxidized glutathione (GSSG). It is shown here that the glutathione transferase activity of isolated microsomes, which was increased by the addition of 10 mM GSSG, can be decreased to the basal level with 0.1 M dithioerythritol. Dithioerythritol, on the other hand, only marginally decreases the glutathione transferase activity in microsomes isolated from phorone-treated hepatocytes. This finding argues against a role for thiol-disulfide interchange in the activation of the enzyme by phorone. Furthermore, the glutathione depletion caused by phorone does not seem to be responsible for activation per se, since other thiol depletors [e.g. diethylmaleate (DEM)] do not affect the activity of the enzyme. Immunoblot analysis of microsomes isolated from phorone-treated hepatocytes did not reveal any partial proteolysis which might have accounted for the activation. It is suggested that activation of microsomal glutathione transferase by phorone proceeds through a mechanism which might reflect an in vivo regulation of this enzyme. Additional compounds which have been shown to activate the microsomal glutathione transferase in vivo were also tested and significant activation was obtained with 1,2-dibromoethane (1.4-fold) but not with DEM or carbon tetrachloride. Activation was also obtained with 1-chloro-2,4-dinitrobenzene (CDNB) (1.6-fold) and to a small extent with t-butyl hydroperoxide (1.2-fold). The activation by 1,2-dibromoethane and CDNB is probably mediated through covalent binding, considering the known alkylating properties of these compounds. CDNB is the first substrate shown to activate the microsomal glutathione transferase implying that electrophilic compounds which are substrates can increase the rate of their own elimination by reacting with this enzyme. In addition, activation by t-butyl hydroperoxide indicates that oxidative stress can activate microsomal glutathione transferase.

Animals↗
PubMed1992

Studies on glutathione transferases belonging to class pi in cell lines with different capacities for conjugating (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The glutathione transferases (GST) belonging to class pi are primarily responsible for the intracellular detoxification of the highly mutagenic and carcinogenic compound (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). The aim of the present investigation was to study the nature and function of the GST pi gene in relation to the mutagenicity of BPDE in different cell lines. The studies were performed on three cell lines commonly used in toxicological studies, i.e. rat hepatoma cells (H4IIE), human mammary carcinoma cells (MCF-7) and Chinese hamster lung fibroblasts (V79). Western blotting with antisera against GST pi revealed a high level of reaction with cytosol from V79 and H4IIE cells. Furthermore, cytosol from the V79 cells demonstrated low levels of GSTs belonging to the alpha and mu classes, suggesting that a considerable portion of the total capacity of these cells to conjugate chlorodinitrobenzene (CDNB) was provided by GST pi. The level of mRNA for GST pi, as measured by Northern blots, was high in V79 and H4IIE and undetectable in the MCF-7 cell line. Analysis of the DNA fragment patterns using a series of restriction enzymes, revealed that all three cell lines have the pi class gene, although with different band patterns. The findings with H4IIE and MCF-7 cells with respect to their expression of the GST pi gene and their ability to conjugate BPDE were in agreement with the mutagenic effects of BPDE, produced by metabolic activation of (-)-7 beta, 8 alpha-dihydroxybenzo[a]-pyrene in the cells. In contrast, V79 cells although expressing high levels of GST pi, showed no ability to conjugate BPDE or to inhibit the mutagenicity of this compound. Based on these results, we suggest that V79 Chinese hamster lung cells contain a GST pi with a different substrate specificity from those of the human and rat GST pi enzymes.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

Studies on the activity and activation of rat liver microsomal glutathione transferase with a series of glutathione analogues.

The substrate specificity of rat liver microsomal glutathione transferase toward glutathione has been examined in a systematic manner. Out of a glycyl-modified and eight gamma-glutamyl-modified glutathione analogues, it was found that four (glutaryl-L-Cys-Gly, alpha-L-Glu-L-Cys-Gly, alpha-D-Glu-L-Cys-Gly, and gamma-L-Glu-L-Cys-beta-Ala) function as substrates. The kinetic parameters for three of these substrates (the alpha-D-Glu-L-Cys-Gly analogue gave very low activity) were compared with those of GSH with both unactivated and the N-ethylmaleimide-activated microsomal glutathione transferase. The alpha-L-Glu-L-Cys-Gly analogue is similar to GSH in that it has a higher kcat (6.9 versus 0.6 s-1) value with the activated enzyme compared with the unactivated enzyme but displays a high Km (6 versus 11 mM) with both forms. Glutaryl-L-Cys-Gly, in contrast, exhibited a similar kcat (8.9 versus 6.7 s-1) with the N-ethylmaleimide-treated enzyme but retains a higher Km value (50 versus 15 mM). Thus, the alpha-amino group of the glutamyl residue in GSH is important for the activity of the activated microsomal glutathione transferase. These observations were quantitated by analyzing the changes in the Gibbs free energy of binding calculated from the changes in kcat/Km values, comparing the analogues to GSH and each other. It is estimated that the binding energy of the alpha-amino group of the glutamyl residue in GSH contributes 9.7 kJ/mol to catalysis by the activated enzyme, whereas the corresponding value for the unactivated enzyme is 3.2 kJ/mol. The importance of the acidic functions in glutathione is also evident as shown by the lack of activity with 4-aminobutyric acid-L-Cys-Gly and the low kcat/Km values with gamma-L-Glu-L-Cys-beta-Ala (0.03 and 0.01 mM-1s-1 for unactivated and activated enzyme, respectively). Utilization of binding energy from a correctly positioned carboxyl group in the glycine residue (10 and 17 kJ/mol for unactivated and activated enzyme, respectively) therefore also appears to be required for optimal activity and activation. A conformational change in the microsomal glutathione transferase upon treatment with N-ethylmaleimide or trypsin, which allows utilization of binding energy from the alpha-amino group of GSH as well as the glycine carboxyl in catalysis, is suggested to account for at least part of the activation of the enzyme.

Amino Acid Sequence↗
PubMed1991

2-Bromolisuride, an ergot derivative, with dopamine antagonistic and serotonin agonistic properties.

The open-field test was used to study the involvement of dopaminergic and serotonergic mechanisms in the effects of 2-bromolisuride on locomotor activity in the rat. 2-Bromolisuride produced a dose-dependent inhibition of spontaneous locomotor activity. This is most likely due to an antagonistic action at postsynaptic dopamine receptors. Low doses of 2-bromolisuride potentiated apomorphine-induced hypermotility. This potentiating effect was not mediated by a blockade of presynaptic dopamine receptors, because it was not prevented by 6-OHDA lesion of the nucleus accumbens. The potentiating effect of 2-bromolisuride was completely blocked by the serotonin antagonists cyproheptadine and ritanserin. It is suggested that 2-bromolisuride possesses dopamine antagonistic and serotonin agonistic properties.

Animals↗
PubMed1991

CCK-8 modulates D2 receptor agonist-induced hypermotility in the nucleus accumbens.

The influence of CCK-8 on locomotor effects associated with independent D2 receptor stimulation was studied. To selectively stimulate mesolimbic D2 receptors LY 171555 was injected into the nucleus accumbens of awake rats. Locomotor activity was measured in the open-field test. LY 171555 induced a biphasic effect: low doses stimulated, whereas higher doses inhibited locomotor activity. CCK-8 injected into the posteromedial part of the nucleus accumbens suppressed hyperlocomotion induced by LY 171555. The CCK-8 effect was prevented by the CCK-antagonist L 364,718. Our results indicate that CCK-8 modulates D2 receptor-mediated effects in the mesolimbic system.

Animals↗
PubMed1991

The liver in typhoid fever: always affected, not just a complication.

The course of liver involvement during the first three weeks of typhoid fever was studied in 20 patients. Previous studies of liver involvement in typhoid fever have not considered the time course of changes. In this study, hepatomegaly was found during the 2nd or 3rd wk more often than in the 1st wk (36% vs. 11%), whereas jaundice was detectable in 9% of patients after the 1st wk, but never before. Alkaline phosphatase, AST, and ALT were raised in 100%, 100%, and 91% of cases, respectively, during the 2nd and 3rd wk but during the 1st wk, only 11%, 89%, and 56% had mild increases. This study shows that, although the clinical picture of hepatitis is unusual, liver involvement is invariably present after the 1st wk, and should not be considered as a complication, but as a feature of the disease.

Adolescent↗
PubMed1990

Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function.

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5'-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.

Animals↗
PubMed1990

The influence of tifluadom on cholecystokinin-induced antinociception.

The effects of tifluadom, a benzodiazepine-kappa-opioid-receptor agonist, on cholecystokinin-octapeptide (CCK-8)-induced antinociception were investigated in the mouse writhing test. When given alone, tifluadom produced pronounced, dose-dependent analgesia. The antinociceptive effect of intracerebroventricularly injected CCK-8 was potentiated by high doses of tifluadom. In contrast, when tifluadom was applied at low doses which did not induce antinociception, the antinociceptive effect of CCK-8 was completely antagonized. It is concluded that tifluadom acts both as kappa-opioid receptor agonist and as an antagonist at CCK receptors mediating CCK-induced antinociception.

Analgesics↗
PubMed1990

Atypical neuroleptics suppress dopaminergic behavioral supersensitivity.

Seven days after bilateral 6-OHDA denervation of the nucleus accumbens locomotor activity was recorded in rats. 6-OHDA lesion strongly enhanced hypermotility induced by apomorphine (1.0 mg/kg IP) as a sign of behavioral dopaminergic supersensitivity. The potency of the classical neuroleptic haloperidol (0.03-0.25 mg/kg IP) to antagonize apomorphine-induced hypermotility was reduced in 6-OHDA-pretreated rats. The atypical neuroleptics sulpiride (5.0-20.0 mg/kg IP), thioridazine (1.0-5.25 mg/kg IP) and clozapine (0.5-2.0 mg/kg IP) and the 5-HT antagonists cyproheptadine (0.2 mg/kg IP) and ritanserin (0.01 mg/kg IP) suppressed the augmented apomorphine response in 6-OHDA-lesioned animals to the level of the apomorphine effect in controls. It is concluded that the model of denervation supersensitivity is capable of differentiating typical and atypical neuroleptics. The abolition of the 6-OHDA-induced increase of the apomorphine hypermotility by the atypical neuroleptics cannot be explained solely by postsynaptic dopamine receptor antagonism. Serotonergic mechanism may be involved in this action.

Animals↗
PubMed1990

Inhibition studies on rat liver microsomal glutathione transferase.

A set of inhibitors for rat liver microsomal glutathione transferase have been characterized. These inhibitors (rose bengal, tributyltin acetate, S-hexylglutathione, indomethacin, cibacron blue and bromosulphophtalein) all have I50 values in the 1-100 microM range. Their effects on the unactivated enzyme were compared to those on the N-ethylmaleimide- and trypsin-activated microsomal glutathione transferase. It was found that the I50 values were decreased upon activation of the enzyme (5-20-fold), except for S-hexylglutathione, where a slight increase was noted. Thus, the activated microsomal glutathione transferase is generally more sensitive to the effect of inhibitors than the unactivated enzyme. It was also noted that inhibitor potency can vary dramatically depending on the substrate used. The I50 values for the N-ethylmaleimide- and trypsin-activated enzyme preparations are altered in a similar fashion compared to the unactivated enzyme. This finding indicates that these two alternative mechanisms of activation induce a similar type of change in the microsomal glutathione transferase.

Animals↗
PubMed1990

Activation of microsomal glutathione transferase activity by reactive intermediates formed during the metabolism of phenol.

The activity of microsomal glutathione transferase was increased 1.7-fold in rat liver microsomes which carried out NADPH dependent metabolism of phenol. Known phenol metabolites were therefore tested for their ability to activate the microsomal glutathione transferase. The phenol metabolites benzoquinone and 1,2,4-benzenetriol both activated the glutathione transferase in microsomes 2-fold independently of added NADPH. However, NADPH was required to activate the enzyme in the presence of hydroquinone. Catechol did not activate the enzyme in microsomes. The purified enzyme was activated 6-fold and 8-fold by 5 mM benzenetriol and benzoquinone respectively. Phenol, catechol or hydroquinone had no effect on the purified enzyme. When microsomal proteins that had metabolized [14C]phenol were examined by SDS polyacrylamide gel electrophoresis and fluorography it was found that metabolites had bound covalently to a protein which comigrated with the microsomal glutathione transferase enzyme. We therefore suggest that reactive metabolites of phenol activate the enzyme by covalent modification. It is discussed whether the binding and activation has general implications in the regulation of microsomal glutathione transferase and, since some reactive metabolites might be substrates for the enzyme, their elimination through conjugation.

Animals↗