The thioredoxin/thioredoxin reductase system has been studied as regenerative machinery for proteins inactivated by oxidative stress in vitro and in cultured endothelial cells. Mammalian glyceraldehyde-3-phospliate dehydrogenase was used as the main model enzyme for monitoring the oxidative damage and the regeneration. Thioredoxin and its reductase purified from bovine liver were used as the regenerating system. The physiological concentrations (2 -14 pM) of reduced thioredoxin, with 0.125 pM thioredoxin reductase and 0.25 mM NADPH, regenerated H202-inactivated glyceraldehyde-3-phosphate dehydrogenase and other mammalian enzymes almost completely within 20 min at 37°C. Although the treatment of endothelial cells with 0.2-12 mM H 2 0 2 for 5 min resulted in a marked decrease in the activity of glyceraldehyde-3-phosphate dehydrogenase, it had no effect on the activities of thioredoxin and thioredoxin reductase. Essentially all of the thioredoxin in endothelial cells at control state was in the reduced form and 70-85% remained in the reduced form even after the H 2 0 2 treatment. The inactivated glyceraldehyde-3-phosphate dehydrogenase in a cell lysate prepared from the H,02-treated endothelial cells was regenerated by incubating the lysate with 3 mM NADPH at 37 "C and the antiserum raised against bovine liver thioredoxin inhibited the regeneration. The inhibition of thioredoxin reductase activity by 13-cis-retinoic acid resulted in a decrease in the regeneration of glyceraldehyde-3-phosphate dehydrogenase in the H202-treated endothelial cells. The present findings provide evidence that thioredoxin is involved in the regeneration of proteins inactivated by oxidative stress in endothelial cells.

The mitochondrial protein SP-22 has recently been reported to be a member of the thioredoxin-dependent peroxide reductase family, suggesting that it may be one of the antioxidant systems in mitochondria, which are the major site of reactive oxygen intermediate generation. The aim of this study was to examine whether SP-22 is involved in mitochondrial antioxidant mechanisms and whether its expression is induced by oxidative stresses, particularly those in mitochondria. The expression of SP-22 protein was enhanced by about 1.5-4.6-fold when bovine aortic endothelial cells (BAEC) were exposed to various oxidative stresses, including mitochondrial respiratory inhibitors which increased the superoxide generation in BAEC mitochondria. The expression of SP-22 mRNA increased 2.0-3.5-fold with a peak at 3-6 h after exposure to Fe2+/dithiothreitol or a respiratory inhibitor, antimycin A. BAEC with an increased level of SP-22 protein caused by pretreatment with mild oxidative stress became tolerant to subsequent intense oxidative stress. On the other hand, BAEC that had been depleted of SP-22 with an antisense oligodeoxynucleotide against SP-22 mRNA became more labile to oxidative stress than control BAEC. The induction of SP-22 protein by oxidative stress in vivo was demonstrated in an experimental model of myocardial infarction in rat heart. These findings indicate that SP-22 functions as an antioxidant in mitochondria of the cardiovascular system.

A possible involvement of thioltransferase (also known as glutaredoxin) in the regenerative reaction of proteins inactivated by oxidative stress were examined in vitro using the enzyme purified from bovine liver. Thioltransferase at physiological concentrations, together with glutathione, glutathione reductase and NADPH, regenerated the oxidatively damaged proteins with a comparable potency to that of thioredoxin. Experiments performed with protein substrates with their critical cysteine residues oxidized differently, that is, phosphofruktokinase and glyceraldehyde 3-phosphate dehydrogenase with mixed disulfide bonds and glyceraldehyde 3-phosphate dehydrogenase with sulfenyl or sulfinyl groups, indicated that thioltransferase regenerated the proteins inactivated by mixed disulfide formation more efficiently than thioredoxin, whereas thioredoxin preferentially regenerated the proteins inactivated by monothiol oxidation to sulfenic or sulfinic acid. These findings suggested that thioltransferase exerted regenerative effects on oxidatively damaged proteins like its cognate protein, thioredoxin, but with different substrate specificity, and their relative contribution to the regeneration reaction is dependent on the form of the oxidized thiols of the damaged proteins.

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Objective: To examine whether or not estrogens induced the expression of protein thiol/disulfide oxidoreductases such as protein disulfide isomerase (PDI), thioredoxin (Trx), Trx reductase, and glutaredoxin (Grx) in vascular endothelial cells. Methods:The regenerative effects of the protein thiol/disulfide oxidoreductases, PDI, Trx and Grx, on oxidatively damaged proteins were assayed using H 2 O 2 -inactivated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reporter enzyme. The induction of protein thiol/disulfide oxidoreductases and the accumulation of protein adducts generated by lipid peroxidation were examined by Western blotting in estrogen-treated bovine aortic endothelial cells (BAECs). Results: Reduced PDI, Trx and Grx regenerated the H 2 O 2 -inactivated GAPDH in vitro. The levels of these protein disulfide oxidoreductases in BAECs were increased by pretreatment with 0.01-10 mmol/l 17b-estradiol, the largest increase (about fourfold of the control) being found for PDI. Other sex hormones such as progesterone and testosterone did not affect the contents of these oxidoreductases in BAECs. 4-Hydroxy-2-nonenal (HNE)-protein adducts, which are generated by lipid peroxidation, were accumulated in BAECs exposed to paraquat, whereas the pretreatment of BAECs with 17b-estradiol suppressed their accumulation. Conclusions:The estrogen-mediated induction of the protein thiol/disulfide oxidoreductases such as PDI, Trx, Trx reductase and Grx suggested a possible involvement of these oxidoreductases in the antioxidant protection of estrogen observed in the vascular system.

SummaryBackground: Plasma nitritehitrate (NOx) is a stable end product of the vasodilator nitric oxide (NO). However, there are few reports about plasma NOx levels in humans.Hypotlzrsis: The purpose of this study was to assess the availability of plasma NOx for evaluating basal endogenously-synthesized or endothelium-derived NO, and to examine whether NOx levels are lowered in patients with coronary artery disease (CAD) or its risk factors.Metlzods: Plasma NOx levels were measured using an automated system based on the Griess reaction. NOx levels for a 24-h period reproducibly became lowest at 6 A.M. in restricted healthy volunteers, and became stable in inpatient volunteers at 6 A.M. within 4 days after admission.Remlts: Based on these findings, NOx levels at 6 A.M. in inpatients can be considered as the basal levels. In 40 inpatients suspected of CAD (28 men, 12 women; mean age 60 f 1 1 years), the basal levels of NOx were not related to CAD and its risk factors, except for hypercholesteroleniia. The NOx level of patients with hypercholesterolemia was significantly lower than that ofpatients with normal cholesterol (n = 16,34 16 pmol/l vs. n = 24,49 k 23 pmoVI, p < 0.03). Furthermore, the NOx levels coirelated negatively with the total cholesterol and low-density lipoprotein cholesterol levels (r = -0.40, p < 0.01 ; r = -0.37, p < 0.003, respectively), but not with other lipid fraction levels.

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