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Biomedical subjects

D J Reed

Publications and source records attributed to D J Reed.

At least 109 records · Page 6Linked to original sources
PubMed1983

The binding mechanism of glutathione and the anti-tumor drug L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125;NSC-163501) to gamma-glutamyltransferase.

The glutathione-protein binding interactions of rat renal gamma-glutamyltransferase (gamma GT) were studied by examining the effect of phenylglyoxal (PGO), a chemical modifying agent for arginyl residues. PGO inactivation of gamma GT followed pseudo-first order kinetics and the rate was dependent upon the concentration of PGO. Glutathione (GSH) protected the enzyme from inactivation by PGO. The anti-tumor drug L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) inactivated purified gamma GT. The inactivation capability of AT-125 was abolished by esterification of the carboxyl moiety and was regained upon incubation of AT-125 methyl ester with a carboxyl esterase. AT-125 and glutathione may bind to gamma GT via the electrostatic interaction of their respective carboxyl group(s) and an arginyl residue at the active site.

Animals↗
PubMed1983

Depletion in vitro of mitochondrial glutathione in rat hepatocytes and enhancement of lipid peroxidation by adriamycin and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU).

Treatment of isolated rat hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and adriamycin (ADR) produced a complete depletion of cellular glutathione accompanied by a significant increase in lactate dehydrogenase (LDH) leakage. Separation of the mitochondrial and cytoplasmic pools of glutathione by digitonin disruption showed that, although BCNU, a specific inhibitor of glutathione, completely depleted the cytoplasmic pool of glutathione, the mitochondrial supply was not entirely expended and LDH leakage was only moderately stimulated. Only after depletion of the mitochondrial supply of glutathione by ADR and BCNU did LDH leakage increase markedly. Measurement of lipid peroxidation, by monitoring malondialdehyde through the thiobarbituric acid procedure, showed that malondialdehyde accumulated more extensively and at a rate mirroring release of LDH from ADR/BCNU treated cells. The time of increase in LDH leakage and malondialdehyde production corresponded to the time of depletion of mitochondrial glutathione to less than 10% of the initial pool size. No such increase in LDH leakage was observed with BCNU or ADU treatment alone or when aminopyrine, an inhibitor of lipid peroxidation, was included. Aminopyrine was found to prevent, in a dose-dependent manner, both LDH leakage and malondialdehyde production stimulated by ADR/BCNU treatment. The protective effect peaked at 5 mM aminopyrine, and higher concentrations produced significant LDH leakage exhibiting LDH release kinetics different than those observed with ADR/BCNU. Although aminopyrine had no effect on the rate or extent of cytoplasmic glutathione depletion by ADR/BCNU treatment, the mitochondrial pool was conserved significantly in those cells protected by aminopyrine. These data suggest that enhanced hepatocyte damage observed after treatment with a combination of ADR and BCNU versus BCNU or ADR alone is due to the extensive depletion of mitochondrial glutathione supported by ADR after glutathione reductase inhibition. Further, enhancement of lipid peroxidation is strongly implicated in the mechanism of adriamycin toxicity.

Aminopyrine↗
PubMed1983

Involvement of arginine residues in glutathione binding to yeast glyoxalase I.

Yeast glyoxalase I was inactivated by arginine-specific reagents. Inactivation by 2,3-butanedione, phenylglyoxal and camphorquinone 10-sulfonic acid followed pseudo first-order kinetics with the rate dependent upon modifier concentration. Extrapolation to complete inactivation showed modification of approx. two of the ten total arginyl residues in the native enzyme, with approx. one residue protected by glutathione (GSH) as determined by [ring-14C]phenylglyoxal incorporation. GSH protected the enzyme from inactivation, whereas methylglyoxal, glutathione disulfide (GSSG) and dithiothreitol afforded partial protection. The hemimercaptal of methylglyoxal and GSH and the catalytic product, S-lactoylglutathione provided substantial protection from inactivation. A methyl ester placed on the glycyl carboxyl moiety of GSH abolished all protective capability which suggests that this functionality is responsible for binding to the enzyme. These results provide the first evidence concerning the molecular binding mode of GSH to an enzyme. Arginyl residues are proposed as anionic recognition sites for glutathione on other GSH-utilizing enzymes.

Arginine↗
PubMed1982

Status of the mitochondrial pool of glutathione in the isolated hepatocyte.

Using the selective membrane-solubilizing properties of digitonin and a rapid centrifugation method to separate cytoplasmic and mitochondrial components, the metabolic state of mitochondrial glutathione was investigated in isolated rat hepatocytes. Two pools of GSH were released from hepatocytes incubated with increasing concentrations of digitonin. The largest pool (about 85% of cellular total) was released simultaneously with lactate dehydrogenase, the other pool with citrate synthase, indicating cytoplasmic and mitochondrial locations, respectively. The t1/2 of the mitochondrial pool was estimated by linear regression analysis to be 30 +/- 3 h, while the cytoplasmic pool turned over with a t1/2 of about 2 +/- 0.1 h. The rate of incorporation of [35S]methionine or cysteine into the cytoplasmic pool of GSH, when corrected for turnover, was 15 times greater than into the mitochondrial pool. Mitochondrial GSH was not depleted after 60 min with 185 microM diethyl maleate with or without 75 microM bis-1,3-(2-chloroethyl)-1-nitrosourea, a specific inhibitor of glutathione reductase, whereas cytoplasmic levels were reduced to 40% and 10% of control values, respectively. In vivo experiments, using L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid to inactive gamma-glutamyl transpeptidase to limit cysteine formation from plasma GSH, demonstrated that in the absence of label reincorporation, liver glutathione exhibits a biphasic turnover. The rates of decay (half-lives) and percentages of total GSH under these conditions correlate well with the half-lives and pool distribution seen in the mitochondrial and cytoplasmic populations of GSH found in the isolated hepatocytes.

Animals↗
PubMed1982

Glutathione conjugation and DNA-binding of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in isolated rat hepatocytes.

Isolated rat liver hepatocytes, previously depleted of glutathione (GSH) by treatment with diethylmaleate, were allowed to incorporate [3H]glycine into their GSH. Incubation of 3H-labelled cells with 14C-labelled (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene ((+/-)-BP-7,8-dihydrodiol) or (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+/-)-BPDE) revealed the formation of double labelled products. This together with evidence from amino acid analysis indicates formation of GSH-conjugates of the highly carcinogenic BP-derivatives. Incubation of hepatocytes isolated from 3-methylcholanthrene (MC) treated rats with 3H-labelled (+/-)-BP-7,8-dihydrodiol or (+/-)-BPDE resulted in binding of radioactivity to DNA. Reduction of the intracellular level of GSH to approximately 40% of the normal level resulted in an approximate 2-fold increase in the DNA-binding of either substrate. In addition there was a concurrent decrease in the amount of GSH-conjugates formed. These data clearly demonstrate that GSH participates in conjugation reactions with carcinogenic (+/-)-BP-7,8-dihydrodiol and (+/-)-BPDE and that the intracellular level of GSH is important in preventing reactive intermediates from reacting with the DNA in intact cells.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1982

Unique characteristics of rat spleen lymphocyte, L1210 lymphoma and HeLa cells in glutathione biosynthesis from sulfur-containing amino acids.

Suspensions of rat spleen lymphocyte, murine L1210 lymphoma and HeLa cells were partially depleted of glutathione (GSH) with diethyl maleate and allowed to utilize either [35S]methionine, [35S]cystine or [35S]-cysteine for GSH synthesis. Lymphocytes preferentially utilized cysteine, compared to cystine, at a ratio of about 30 to 1, which was not related to differences in the extent of amino acid uptake. Only HeLa cells displayed a slight utilization of methionine via the cystathionine pathway for cysteine and GSH biosynthesis. HeLa and L1210 cells readily utilized either cystine or cysteine for GSH synthesis. The three cell types accumulated detectable levels of intracellular cysteine glutathione mixed disulfide when incubated in a medium containing a high concentration of cystine. Various enzyme activities were measured including gamma-glutamyl transpeptidase, GSH S-transferase and gamma-cystathionase. These results support the concept of a dynamic interorgan relationship of GSH to plasma cyst(e)ine that may have importance for growth of various cell types in vivo.

Amino Acids, Sulfur↗
PubMed1982

Effect of acetazolamide and furosemide on the production and composition of cerebrospinal fluid from the cat choroid plexus.

The effect of acetazolamide and furosemide on choroid plexus (CP) production and electrolyte composition of cerebrospinal fluid (CSF) from the cat CP in situ was investigated. Both drugs decreased CSF production by the CP by 50--90% from a control rate of 0.53 microL . min-1 . mg-1 CP within 1.5--2.5 h after the start of drug treatment. The results were similar whether the drug was administered intravenously or applied directly to the CSF side of the CP. A number of experiments in which the effect of administering drugs via the chamber were studied were continued with the drugs removed by washing the preparation with drug-free artificial CSF and the responses measured. The results demonstrated that the effects of acetazolamide and furosemide were reversed during the 1st h following the washout. Both drugs decreased K concentration of nascent CSF when administered intravenously and furosemide also did so when administered on the CSF side of the CP. It is concluded from these and previous data that acetazolamide and furosemide markedly inhibit the transport mechanism(s) in the cat CP that are responsible for CP secretion which represents about 40--60% of the total CSF production and that K transport is also affected.

Acetazolamide↗
PubMed1981

Turnover of cellular glutathione in isolated rat-kidney cells. Role of cystine and methionine.

Turnover of cellular glutathione in isolated rat kidney cells was studied using cystine or methionine as sulfur donor. In the absence of any sulfur donor a continuous decrease of intracellular reduced glutathione (GSH) during incubation of the cells was observed. This decrease was abolished in the presence of cystine, and, as indicated by incorporation of 35S, there was also a rapid synthesis of GSH. In the presence of gamma-glutamyltransferase inhibitor, the synthesis of intracellular GSH was accompanied by an accumulation of extracellular cysteine-glutathione mixed disulfide whereas only minor amounts of GSH and glutathione disulfide could be detected. The intracellular levels of both the cysteine-glutathione and glutathione disulfides were at all times points very low. Even though the uptake of cystine was rapid and not rate-limiting for GSH synthesis, almost no cystine could be detected intracellularly. An increasing intracellular cysteine concentration was however observed, indicating a rapid reduction of cystine. In contrast to cystine, methionine did not protect from the loss of intracellular GSH and only a low rate of incorporation of 35S into GSH was observed. Methionine was rapidly taken up into the cells but was apparently converted to cysteine only to a very limited extent. This is most likely due to a low activity of the enzyme cystathionase since neither homocysteine nor cystathionine was very effective in supporting GSH synthesis.

Animals↗
PubMed1981

Regulation of lithium in cerebrospinal fluid of the cat by the choroid plexus isolated in situ.

The role of the choroid plexus in regulating the lithium concentration in c.s.f. was studied by the use of the cat choroid plexus isolated in situ in a chamber. LiCl solution (154 mM) was infused i.v. to produce plasma lithium concentrations between 0 and 7.5 m-equiv/l. Over this range of plasma values the c.s.f./plasma ratio remained at 0.58. The plasma and c.s.f. concentrations were constant after about 90 min. Lithium (1.8 or 4.5 m-equiv/l.) was added to the artificial c.s.f. placed in the chamber at the start of a 30-min collection period. The lithium concentration in the chamber fluid was measured at the end of the collection period with the plasma concentration either 0 or greater than that in the chamber. When the lithium gradient was from chamber fluid to blood there was a net loss of lithium from the chamber and the amount was proportional to the gradient. However, when the gradient was in the opposite direction lithium was added to the chamber but the amount added was related to the plasma lithium concentration rather than to the blood-c.s.f. gradient. Altering the potassium concentration in the chamber fluid changed the rate of potassium transport without a comparable effect on lithium movement out of the chamber. Lithium transport by the potassium transport system does not appear to be a major factor in the loss of lithium from the chamber. The data suggest that lithium is transported actively across the choroid plexus into the c.s.f. by the system that is responsible for c.s.f. secretion. Movement from c.s.f. to blood appears to be accomplished primarily by passive diffusion when the c.s.f. concentration significantly exceeds that in the plasma.

Animals↗