Functional partnership of the copper export machinery and glutathione balance in human cells

Cells use the redox properties of copper in numerous physiologic processes, including antioxidant defense, neurotransmitter biosynthesis, and angiogenesis. Copper delivery to the secretory pathway is an essential step in copper utilization and homeostatic maintenance. We demonstrate that the glutathione/glutathione disulfide (GSH/GSSG) pair controls the copper transport pathway by regulating the redox state of a copper chaperone Atox1. GSSG oxidizes copper-coordinating cysteines of Atox1 with the formation of an intramolecular disulfide. GSH alone is sufficient to reduce the disulfide, restoring the ability of Atox1 to bind copper; glutaredoxin 1 facilitates this reaction when GSH is low. In cells, high GSH both reduces Atox1 and is required for cell viability in the absence of Atox1. In turn, Atox1, which has a redox potential similar to that of glutaredoxin, becomes essential for cell survival when GSH levels decrease. Atox1(+/+) cells resist short term glutathione depletion, whereas Atox1(-/-) cells under the same conditions are not viable. We conclude that GSH balance and copper homeostasis are functionally linked and jointly maintain conditions for copper secretion and cell proliferation.     

Correlation between nitric oxide synthase activity and reduced glutathione level in human and murine endothelial cells

Summary The synthesis of nitric oxide (NO), detected as citrulline production, in human (HUVEC) and murine (tEnd.1) endothelial cells correlated with intracellular GSH. tEnd.1, which exhibited an intracellular GSH level 2.5-fold higher than HUVEC, showed a citrulline production (basally and after ionomycin stimulation) 5–8 times higher than human cells. Ionomycinelicited citrulline synthesis in tEnd.1 cells increased 2.4-fold after loading with GSH, and decreased dose-dependently after GSH depletion. Cell loading with N-(2-mercaptopropionyl)-glycine neither significantly increased citrulline production nor relieved the effect of GSH depletion.     

Interplay between glutathione, Atx1 and copper: X-ray absorption spectroscopy determination of Cu(I) environment in an Atx1 dimer

X-ray absorption techniques have been used to characterise the primary coordination sphere of Cu(I) bound to glutathionate (GS⁻), to Atx1 and in Cu₂^I(GS⁻)₂(Atx1)₂, a complex recently proposed as the major form of Atx1 in the cytosol. In each complex, Cu(I) was shown to be triply coordinated. When only glutathione is provided, each Cu(I) is triply coordinated by sulphur atoms in the binuclear complex Cu^I ₂(GS⁻)₅, involving bridging and terminal thiolates. In the presence of Atx1 and excess of glutathione, under conditions where Cu^I ₂(GS⁻)₂(Atx1)₂ is formed, each Cu(I) is triply coordinated by sulphur atoms. Given these constraints, there are two different ways for Cu(I) to bridge the Atx1 dimer: either both Cu(I) ions contribute to bridging the dimer, or only one Cu(I) ion is responsible for bridging, the other one being coordinated to two glutathione molecules. These two models are discussed as regards Cu(I) transfer to Ccc2a.

Influence of a low-selenium diet on erythrocyte glutathione peroxidase and alkane production in exhaled air of rats as a measure of lipid peroxidation in vivo during growth

Erythrocyte glutathione peroxidase activity and alkane production in exhaled air of growing rats were studied as a measure of lipid peroxidation in vivo. When 4-weeks-old, rats were fed a low-selenium (0.05 mg/kg) refined soy concentrate-based diet but adequate in vitamin E and other nutrients. Rats of control groups were fed the same diet supplemented with varying amounts of selenium as sodium selenite. After 10 weeks, erythrocyte glutathione peroxidase activity in the group fed the low selenium diet had decreased to about 40% of the original level. Feeding this diet for a longer period resulted in a slow increase of the glutathione peroxidase level. After about 37 weeks, this level was equal to the initial level. During the same period of rapid growth, ethane and pentane production in the exhaled air of a group of similar animals on the diet containing 0.05 mg Se per kg was slightly although significantly higher compared with the levels of animals on a supplemented (0.4 mg Se per kg) diet. Differences were highest when glutathione peroxidase activity levels in the erythrocytes were lowest and negligible at the start of the experiment and after the period of rapid growth. These results support the view that the seleno-enzyme glutathione peroxidase is active in the defense mechanism of the cell against lipid proxidation.     

Gender dependency of circadian blood pressure and heart rate profiles in spontaneously hypertensive rats: effects of beta-blockers

This study investigated (i) blood pressure (BP), heart rate (HR), and their relation to urinary NOx and eNOS protein expression in male and female spontaneously hypertensive rats (SHR), as well as (ii) gender-dependent cardiovascular effects of nebivolol (NEB) in comparison to metoprolol (MET) in SHR. BP and HR were measured telemetrically after a single intraperitoneal application of NEB or MET at 07.00 and 19.00 h in male rats and at 19.00 h in proestrus female rats. The two β-blockers varied in time of decreasing BP and HR and also in duration. In males, MET decreased BP and HR for few hours exclusively when applied at the onset of the activity phase (i.e., at 19.00 h), while after its application at 07.00 h, BP and HR were unchanged. In females, MET also caused a short-lasting BP and HR reduction, with the effect being more pronounced than in males. In males, NEB at either dosing time decreased HR and BP to a greater extent than did MET. This effect was evident both during the activity and rest periods and persisted for at least five days. In females, NEB provoked a similar, but more pronounced, effect on BP and HR in comparison to males. These findings demonstrate that significant gender-dependent differences in the circadian profile of BP and HR exist. BP and urinary NOx as well as eNOS expression are inversely correlated, and the cardiovascular effects of NEB and MET vary, depending on the time of application as well as gender.     

Effects of testosterone metabolites on copulation, medial preoptic dopamine, and NOS-immunoreactivity in castrated male rats

The medial preoptic area (MPOA) is an important integrative site for male sexual behavior. Dopamine (DA) is released in the MPOA of male rats shortly before and during copulation. In a previous study, we identified 17beta-estradiol (E(2)) as the metabolite of testosterone (T) that maintains MPOA basal extracellular DA levels. However, the presence of dihydrotestosterone (DHT), an androgenic metabolite of T, is required for the female-induced increase in MPOA DA observed during copulation. Recently, we reported that assays of MPOA tissue DA content showed that castrates actually had more stored DA than did gonadally intact males. Therefore, the reduction in extracellular levels in castrates was not due to decreased availability of DA; most likely it was due to decreased release. Furthermore, T upregulates neuronal nitric oxide synthase (nNOS) in the MPOA. NO has been implicated in the regulation of DA release in the MPOA. It is not known, however, which metabolite(s) of T regulate(s) tissue stores of DA and/or nNOS in the MPOA of male rats. The present experiments were designed to test the following: (1) whether E(2), DHT, or the combination of the two influences MPOA DA tissue levels, an indication of stored DA, in male rat castrates; and (2) whether E(2), DHT, or the combination of the two influences NOS-ir in the MPOA of castrated male rats. The results indicate that E(2) up-regulates nNOS-ir in the MPOA and maintains tissue content of DA at levels similar to those in T-treated rats. DHT did not influence nNOS-ir, while attenuating the effect of castration on tissue DA content.     

Non-steroidal anti-inflammatory agents, tolmetin and sulindac attenuate quinolinic acid (QA)-induced oxidative stress in primary hippocampal neurons and reduce QA-induced spatial reference memory deficits in male Wistar rats

Accumulating evidence suggests that anti-inflammatory agents and antioxidants have neuroprotective properties and may be beneficial in the treatment of neurodegenerative disorders. In the present study, the possible neuroprotective properties of tolmetin and sulindac were investigated using quinolinic acid (QA)-induced neurotoxicity as well as behavioral studies. QA, a metabolite of the tryptophan-kynurenine pathway, significantly induces lipid peroxidation, superoxide anion generation and decreases cell viability in primary hippocampal neurons established from one day old rat pups. However, co-incubation of the neurons with tolmetin or sulindac markedly reduces oxidative stress and enhances cell viability. Animals were trained in a Morris water maze for four consecutive days and thereafter received 0.6 micromol of QA intrahippocampally. The animals were divided into groups and were treated with either tolmetin or sulindac (5 mg/kg twice a day for five days). During test trials, the time taken for each rat to find the submerged platform was recorded over a period of two weeks. Animals were thereafter sacrificed and the hippocampi analyzed for protein carbonyl and glutathione content. The results show that both sulindac and tolmetin reduce the QA-induced spatial memory deficit and sulindac treated animals respond better in the water maze compared to the tolmetin treated animals. Both agents also reduce protein oxidation in rat hippocampus and attenuate the decrease in hippocampal glutathione content induced by QA. This study indicates that the antioxidant properties of tolmetin and sulindac may be beneficial in the treatment of neurodegenerative disorders such as Alzheimer's disease.     

Kinetics of 8-2 fluorotelomer alcohol and its metabolites, and liver glutathione status following daily oral dosing for 45 days in male and female rats

Fluorotelomer alcohols (FTOHs) are raw materials used in the manufacture of polymeric and surfactant products. Based on previous findings from single oral dosing in rats with radiolabeled 8-2 FTOH, glutathione (GSH) depletion and/or the presence of perfluorinated/polyfluorinated acids and aldehyde metabolites was hypothesized to account for the hepatocellular lesions observed in male rats from a 90-day subchronic oral dosing study. Further, the reported nephropathy in female rats from the subchronic experiment was hypothesized to have been initiated by a thiol metabolite produced by degradation of GSH conjugates. In the current investigation, the kinetics of 8-2 FTOH and its metabolites along with liver GSH status were evaluated in the rat following daily oral dosing with 8-2 FTOH for 45 days at 5 and 125 mg/kg/day. Liver GSH stores 1-2 h after dosing were unaffected, suggesting that GSH depletion is not likely a relevant mode of action in the liver. The tissue metabolite data indicate that the liver toxicity mode of action is likely associated with elevated levels of perfluoroalkyl acids found in males, since other polyfluorinated metabolites and 8-2 FTOH were present in livers from female rats at comparable or higher levels. Detection of the N-acetyl cysteine conjugate of the unsaturated parent telomer alcohol in urine from female rats and not male rats provides some evidence to support the mechanistic basis for the observed kidney effects. Further, the increasing levels of perfluorooctanoic acid (PFOA) in plasma from female rats over the 45-day dosing phase, while unexpected, may reflect an increased net absorption of 8-2 FTOH, slow elimination of intermediates in the metabolic pathway between 8-2 FTOH and PFOA, or altered kidney clearance. The results of this study have enhanced our understanding of 8-2 FTOH kinetics and metabolism and potential modes of action in the rat, which will guide the design of future studies for FTOHs and our need to define the mechanistic basis for the observed effects.     

Purification of human erythrocyte glucose 6-phosphate dehydrogenase and glutathione reductase enzymes using 2',5'-ADP Sepharose 4B affinity column material in single chromatographic step

The enzymes of glucose 6-phosphate dehydrogenase and glutathione reductase were purified from human erythrocytes in one chromatographic step consisting of the use of the commercially available resin 2',5'-ADP Sepharose 4B by using different washing buffers. Ammonium sulfate (30-70%) precipitation was performed on the hemolysate before applying to the affinity column. Using this procedure, G6PG, having the specific activity of 22.9 EU/mg proteins, was purified with a yield of 43% and 9150-fold; GR, having the specific activity of 20.7 EU/mg proteins, was purified with a yield of 26% and 8600-fold. The purity of the enzymes was checked on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and each purified enzyme showed a single band on the gel. This procedure has advantages of preventing of enzyme denaturation, short experimental duration, and use of less chemical materials for purification of the enzymes.     

延髓腹外侧头端区注射肾上腺髓质素对大鼠血压、心率和肾交感神经活动的影响

本研究在 3 4只麻醉Sprague Dawley大鼠观察了延髓腹外侧头端区内微量注射肾上腺髓质素 ( 10μmol/L ,2 0 0nl)对平均动脉压 (MAP)、心率 (HR)和肾交感神经放电 (RSNA)的影响。实验结果如下 :( 1)延髓腹外侧头端区内微量注射肾上腺髓质素可引起MAP、HR、和RSNA明显增加 ,分别由 99 0 9± 3 3 2mmHg ,3 70 78± 7 84bpm和 10 0± 0 %增至 113 5 7± 3 64mmHg (P <0 0 0 1) ,3 83 2 8± 7 3 8bpm (P <0 0 0 1)和 12 3 72±2 74% (P <0 0 0 1) ;( 2 )降钙素基因相关肽受体阻断剂CGRP8 3 7( 10 0 μmol/L ,2 0 0nl)不能阻断肾上腺髓质素的上述效应 ;( 3 )静脉注射NO前体L 精氨酸 ( 10 0mg/kg ,0 2ml)可消除肾上腺髓质素的上述效应。以上结果提示 ,肾上腺髓质素作用于延髓腹外侧头端区可产生显著的心血管作用 ,此作用不是由降钙素基因相关肽受体介导 ,但可被NO所阻断