Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function.

Subsaturating levels of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS), can lead to endothelial dysfunction as a result of decreased production of nitric oxide. Furthermore, insufficient BH(4) can also result in NOS-uncoupled production of reactive oxygen intermediates, such as superoxide anion and hydrogen peroxide. Nitric oxide and superoxide react rapidly to form peroxynitrite, which may be the reactive species responsible for many of the toxic effects of nitric oxide. Here we show that BH(4) is a primary target for peroxynitrite-catalyzed oxidation because at pH 7.4, physiologically relevant concentrations of BH(4) are oxidized rapidly by low concentrations of peroxynitrite. Peroxynitrite oxidizes BH(4) to quinonoid 5,6-dihydrobiopterin and a large proportion of the quinonoid isomer readily loses its side chain to form 7,8-dihydropterin which is not a cofactor for nitric oxide synthase. Thus, abnormally low levels of BH(4) can promote a cycle of its own destruction mediated by nitric oxide synthase-dependent formation of peroxynitrite. This mechanism might contribute to vascular endothelial dysfunction induced by oxidative stress.     

Increased vascular biosynthesis of tetrahydrobiopterin in apolipoprotein E-deficient mice

Previous studies suggested that loss of tetrahydrobiopterin (BH(4)) may play an important role in the pathogenesis of vascular endothelial dysfunction induced by diabetes and hypertension. In contrast, controversial results have been reported regarding BH(4) metabolism in experimental models of atherosclerosis. Therefore, the present study was designed to characterize the expression and activity of GTP-cyclohydrolase I, a rate-limiting enzyme in biosynthesis of BH(4), during atherogenesis. BH(4) levels were significantly increased in atherosclerotic aortas of apolipoprotein E (apoE)-deficient mice as compared with wild-type mice after 5 mo of Western diet treatment. This increase was further significantly enhanced in apoE-deficient mice fed for 9 and 14 mo. Removal of the endothelium almost eliminated BH(4) in wild-type mice but not in apoE-deficient mice, suggesting that a major component of increased BH(4) synthesis is localized in the vascular media of apoE-deficient mice. Oxidative products of BH(4) were low and did not differ between wild-type and apoE-deficient mice over the course of this study. Increased protein expression and enzymatic activity of GTP-cyclohydrolase I were detected in aortas of apoE-deficient mice (P < 0.05), providing molecular mechanisms responsible for elevation of vascular BH(4). In contrast to aortas, we did not detect any change in levels of BH(4) and in GTP-cyclohydrolase I expression in the brain. Our results demonstrate selective increase of intracellular BH(4) levels via elevation of GTP-cyclohydrolase I activity in vascular tissue of apoE-deficient mice.     

Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxide synthase activity in vascular endothelial cells

Tetrahydrobiopterin (BH4), which is an essential cofactor for nitric oxide synthase (NOS), is generally accepted as an important molecular target for oxidative stress. This study examined whether hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species (ROS), affects the BH4 level in vascular endothelial cells (ECs). Interestingly, the addition of H(2)O(2) to ECs markedly increased the BH4 level, but not its oxidized forms. The H(2)O(2)-induced increase in the BH4 level was blocked by the inhibitor of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of BH4 synthesis. Moreover, H(2)O(2) induced the expression of GTPCH mRNA, and the inhibitors of protein synthesis blocked the H(2)O(2)-induced increase in the BH4 level. The expression of the inducible isoform of NOS (iNOS) was slightly induced by the treatment with H(2)O(2). Additionally, the L-citrulline formation from L-arginine, which is the marker for NO synthesis, was stimulated by the treatment with H(2)O(2), and the H(2)O(2)-induced L-citrulline formation was strongly attenuated by NOS or GTPCH inhibitor. These results suggest that H(2)O(2) induces BH4 synthesis via the induction of GTPCH, and the increased BH4 is coupled with NO production by coinduced iNOS. H(2)O(2) appears to be one of the important signaling molecules to regulate the BH4-NOS system.     

Differential effects of eNOS uncoupling on conduit and small arteries in GTP-cyclohydrolase I-deficient hph-1 mice

In the present study, we used the hph-1 mouse, which displays GTP-cyclohydrolase I (GTPCH I) deficiency, to test the hypothesis that loss of tetrahydrobiopterin (BH(4)) in conduit and small arteries activates compensatory mechanisms designed to protect vascular wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and BH(4) levels were reduced in the aortas and small mesenteric arteries of hph-1 mice. However, the BH(4)-to-7,8-dihydrobiopterin ratio was significantly reduced only in hph-1 aortas. Furthermore, superoxide anion and 3-nitrotyrosine production were significantly enhanced in aortas but not in small mesenteric arteries of hph-1 mice. In contrast to the aorta, protein expression of copper- and zinc-containing superoxide dismutase (CuZnSOD) was significantly increased in small mesenteric arteries of hph-1 mice. Protein expression of catalase was increased in both aortas and small mesenteric arteries of hph-1 mice. Further analysis of endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) signaling demonstrated that protein expression of phosphorylated Ser(1177)-eNOS as well as basal cGMP levels and hydrogen peroxide was increased in hph-1 aortas. Increased production of hydrogen peroxide in hph-1 mice aortas appears to be the most likely mechanism responsible for phosphorylation of eNOS and elevation of cGMP. In contrast, upregulation of CuZnSOD and catalase in resistance arteries is sufficient to protect vascular tissue from increased production of reactive oxygen species generated by uncoupling of eNOS. The results of our study suggest that anatomical origin determines the ability of vessel wall to cope with oxidative stress induced by uncoupling of eNOS.     

Stimulation of tetrahydrobiopterin synthesis by cyclosporin A in mouse brain microvascular endothelial cells

We examined the effect of the immunosuppressant, cyclosporin A (CsA) on the synthesis of tetrahydrobiopterin (BH4), a cofactor for nitric oxide (NO) synthase and a scavenger of reactive oxygen species (ROS), in mouse brain microvascular endothelial cells. Treatment with CsA increased the BH4 content and the expression of mRNA level of GTP cyclohydrolase I, the rate-limiting enzyme of BH4 synthesis. 2,4-Diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, strongly reduced the CsA-induced increase in BH4 content. Cycloheximide (CHX), a protein synthesis inhibitor, also reduced CsA-induced BH4 synthesis. These findings suggest that CsA stimulates BH4 synthesis via a de novo pathway with the induction of GTP cyclohydrolase I. Moreover, CsA-induced the mRNA level of the inducible type of NO synthase, and stimulated the L-citrulline formation from L-arginine, which is a marker for NO synthesis. The CsA-stimulated L-citrulline formation was attenuated by the co-treatment with GTP cyclohydrolase I inhibitor. The expression of the endothelial type of NO synthase was low under basal condition, and was not affected by the treatment with CsA. These findings suggest that increase in BH4 content induced by CsA is coupled with NO production by inducible type of NO synthase.     

Changes in tetrahydrobiopterin levels in endothelial cells and adult cardiomyocytes induced by LPS and hydrogen peroxide—A role for GFRP?

Alterations in tetrahydrobiopterin (BH4) levels have significant consequences in vascular pathophysiology. However, the mechanisms regulating BH4 remain poorly understood. The activity of GTP cyclohydrolase I (GTPCH-I), the first enzyme in BH4 biosynthesis, is controlled by protein levels, posttranslational modifications and interaction with GTPCH-I feedback regulatory protein (GFRP). This work examined the correlation between GTPCH-I protein levels and activity and changes in BH4 in human endothelial cells (HAECs) and adult rat cardiomyocytes (ARCM). Changes in BH4 were stimulated with LPS in HAECs and ARCM, and with hydrogen peroxide in HAECs only. Biopterin production by HAECs and ARCM were attained with concentrations of LPS >1 microg/ml and responses were nonlinear with respect to LPS concentrations. Western blot analysis demonstrated that induction of biopterin synthesis in HAECs and ARCM by LPS does not entail augmentation of constitutive GTPCH-I protein levels. However, LPS diminished GFRP mRNA, suggesting that disruption of GTPCH-I:GFRP complex enhances de novo biopterin synthesis. Conversely, treatment with hydrogen peroxide increased GTPCH-I and GFRP mRNA levels in HAECs while depleting BH4 and GSH, which was counteracted by catalase. This indicates that GFRP may override increases in GTPCH-I protein inhibiting enzyme activity. This conclusion is further supported by depletion of biopterin in cells transiently transfected with GFRP. Thus, allosteric regulation of GTPCH-I activity in the cardiovascular system maybe an important mechanism regulating BH4 levels through GFRP signaling.     

Interactions of peroxynitrite,tetrahydrobiopterin, ascorbic acid,and thiols: implications for uncoupling endothelial nitric-oxide synthase

Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO* production. BH4 has been suggested to be a target for oxidation by peroxynitrite (ONOO-), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO* production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO- with BH4 was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO- reacted with BH4 6-10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO- and BH4 was the trihydrobiopterin radical (BH3.), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO* using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO*-spin trap iron-diethyldithiocarbamate. Bolus ONOO-, the ONOO- donor 3-morpholinosydnonimine, and an inhibitor of BH4 synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO* production. Exogenous BH4 supplementation restored endothelial NO* production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO- prevented uncoupling of eNOS by ONOO-. This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO- and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO- uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles BH3. radical back to BH4.     

Dose dependent effects of reactive oxygen and nitrogen species on the function of neuronal nitric oxide synthase

Reactive nitrogen species (RNS) and oxygen species (ROS) have been reported to modulate the function of nitric oxide synthase (NOS); however, the precise dose-dependent effects of specific RNS and ROS on NOS function are unknown. Questions remain unanswered regarding whether pathophysiological levels of RNS and ROS alter NOS function, and if this alteration is reversible. We measured the effects of peroxynitrite (ONOO-), superoxide (O2.-), hydroxyl radical (.OH), and H2O2 on nNOS activity. The results showed that NO production was inhibited in a dose-dependent manner by all four oxidants, but only O2.- and ONOO- were inhibitory at pathophysiological concentrations (50muM). Subsequent addition of tetrahydrobiopterin (BH4) fully restored activity after O2.- exposure, while BH4 partially rescued the activity decrease induced by the other three oxidants. Furthermore, treatment with either ONOO- or O2.- stimulated nNOS uncoupling with decreased NO and enhanced O2.- generation. Thus, nNOS is reversibly uncoupled by O2.- (50muM), but irreversibly uncoupled and inactivated by ONOO-. Additionally, we observed that the mechanism by which oxidative stress alters nNOS activity involves not only BH4 oxidation, but also nNOS monomerization as well as possible degradation of the heme.     

Three different oxygen-induced radical species in endothelial nitric-oxide synthase oxygenase domain under regulation by L-arginine and tetrahydrobiopterin

Endothelial nitric-oxide synthase (eNOS) plays important roles in vascular physiology and homeostasis. Whether eNOS catalyzes nitric oxide biosynthesis or the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite is dictated by the bioavailability of tetrahydrobiopterin (BH(4)) and L-arginine during eNOS catalysis. The effect of BH(4) and L-arginine on oxygen-induced radical intermediates has been investigated by single turnover rapid-freeze quench and EPR spectroscopy using the isolated eNOS oxygenase domain (eNOS(ox)). Three distinct radical intermediates corresponding to >50% of the heme were observed during the reaction between ferrous eNOS(ox) and oxygen. BH(4)-free eNOS(ox) produced the superoxide radical very efficiently in the absence of L-arginine. L-Arginine decreased the formation rate of superoxide by an order of magnitude but not its final level or EPR line shape. For BH(4)-containing eNOS(ox), only a stoichiometric amount of BH(4) radical was produced in the presence of L-arginine, but in its absence a new radical was obtained. This new radical could be either a peroxyl radical of BH(4) or an amino acid radical was in the vicinity of the heme. Formation of this new radical is very rapid, >150 s(-1), and it was subsequently converted to a BH(4) radical. The trapping of the superoxide radical by cytochrome c in the reaction of BH(4)(-) eNOS(ox) exhibited a limiting rate of approximately 15 s(-1), the time for the superoxide radical to leave the heme pocket and reach the protein surface; this reveals a general problem of the regular spin-trapping method in determining radical formation kinetics. Cytochrome c failed to trap the new radical species. Together with other EPR characteristics, our data strongly support the conclusion that this new radical is not a superoxide radical or a mixture of superoxide and biopterin radicals. Our study points out distinct roles of BH(4) and L-arginine in regulating eNOS radical intermediates. BH(4) prevented superoxide formation by chemical conversions of the Fe(II)O(2) intermediate, and l-arginine delayed superoxide formation by electronic interaction with the heme-bound oxygen.     

Oxygen-induced radical intermediates in the nNOS oxygenase domain regulated by L-arginine, tetrahydrobiopterin, and thiol

Fully coupled nitric oxide synthase (NOS) catalyzes formation of nitric oxide (NO), l-citrulline, NADP+, and water from l-arginine, NADPH, and oxygen. Uncoupled or partially coupled NOS catalyzes the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite, depending on the availability of cofactor tetrahydrobiopterin (BH4) and l-arginine during catalysis. We identified three distinct oxygen-induced radical intermediates in the ferrous endothelial NOS oxygenase domain (eNOSox) with or without BH4 and/or l-arginine [Berka, V., Wu, G., Yeh, H. C., Palmer, G., and Tsai, A.-L. (2004) J. Biol. Chem. 279, 32243-32251]. The effects of BH4 and l-arginine on the oxygen-induced radical intermediates in the isolated neuronal NOS oxygenase domain (nNOSox) have been similarly investigated by single-turnover stopped-flow and rapid-freeze quench EPR kinetic measurements in the presence or absence of dithiothreitol (DTT). Like for eNOSox, we found different radical intermediates in the reaction of ferrous nNOSox with oxygen. (1) nNOSox (without BH4 or l-Arg) produces superoxide in the presence or absence of DTT. (2) nNOSox (with BH4 and l-Arg) yields a typical BH4 radical in a manner independent of DTT. (3) nNOSox (with BH4 and without l-Arg) yields a new radical. Without DTT, EPR showed a mixture of superoxide and biopterin radicals. With DTT, a new approximately 75 G wide radical EPR was observed, different from the radical formed by eNOSox. (4) The presence of only l-arginine in nNOSox (without BH4 but with l-Arg) caused conversion of approximately 70% of superoxide radical to a novel radical, explaining how l-arginine decreases the level of superoxide production in nNOSox (without BH4 but with l-Arg). The regulatory role of l-arginine in nNOS is thus very different from that in eNOS where substrate was only to decrease the rate of formation of superoxide but not the total amount of radical. The role of DTT is also different. DTT prevents oxidation of BH4 in both isoforms, but in nNOS, DTT also inhibits oxidation of two key cysteines in nNOSox to prevent the loss of substrate binding. This new role of thiol found only for nNOS may be significant in neurodegenerative diseases.     

Reactivity of tetrahydrobiopterin bound to nitric-oxide synthase.

Levels of tetrahydrobiopterin (BH(4)) bound to nitric-oxide synthase (NOS) were examined during multiple turnovers of the enzyme in the presence of an NADPH-regenerating system. Our findings show that NOS-bound BH(4) does not remain in a static state but undergoes redox reactions. Under these experimental conditions, the redox state of BH(4) was determined by the balance between calcium/calmodulin (Ca(2+)/CaM)-dependent oxidation of BH(4) mediated by the uncoupled formation of superoxide/hydrogen peroxide on the one hand and by reductive regeneration of BH(4) on the other hand. BH(4) oxidation was appreciably increased in the presence of arginine. Levels of NOS-bound BH(4) were also examined under single turnover conditions in the absence of an NADPH-regenerating system and in the presence of added superoxide dismutase and catalase to suppress the accumulation of superoxide and hydrogen peroxide. BH(4) oxidation was again dependent on Ca(2+)/CaM. The insensitivity to superoxide dismutase and catalase suggested that the single turnover oxidation of BH(4) did not proceed through superoxide/peroxide, although the involvement of these oxidants could not be definitively excluded. The amount of BH(4) oxidized was highest in the presence of arginine, and this oxidation significantly exceeded that in the presence of N(G)-hydroxy-L-arginine. The findings that single turnover oxidation of BH(4) is stimulated by arginine in the presence of Ca(2+)/CaM and that BH(4) is regenerated are consistent with a role for the pterin as an electron donor in product formation; this role remains to be defined.     

Regulation of Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin Levels and the Role of the Cofactor in Tyrosine Hydroxylation in Primary Cultures of Adrenomedullary Chromaffin Cells

Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N-acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N-acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three-day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl- and 8-bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP-cyclohydrolase, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8-bromo cyclic AMP. The increase in GTP-cyclohydrolase and tetrahydrobiopterin induced by 8-bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP-cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP-cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP-dependent and cyclic AMP-independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation.     

Effects of free radicals and oxidants on myocardial cellular injury

Harmful effects of some biochemically generated free radicals and oxidants, including superoxide anions (O2-), hydroxyl radical (OH.), hydrogen peroxide, hypochlorous acid, and hypohalite radical, on isolated cardiac myocytes were compared in an attempt to identify the exact nature of the free radicals/oxidants responsible for myocardial ischemic-reperfusion injury. All of these free radicals/oxidants, with the exception of O2-, caused significant injury to the myocytes as evidenced by the enhanced lactate dehydrogenase and creatine kinase release as well as by morphologic examinations, simultaneously causing lipid peroxidation and oxidized glutathione release from the cells, OH. being the most detrimental of all.     

Acceleration of adhesion of cancer cells and neutrophils to endothelial cells in the absence of de novo protein synthesis: possible implication for involvement of hydroxyl radicals

The adhesion of colon cancer cells (colo201) and neutrophils to endothelial cells which had been briefly exposed to either hypoxanthine/xanthine oxidase, or hydrogen peroxide, or peroxynitrite was analyzed in the absence of de novo protein synthesis. Such treatments accelerated the adhesions of both colo201 cells and neutrophils to endothelial cells. These effects were blocked by SOD/catalase or EDTA. The results provided evidence that hydroxyl radicals affect the cell surface of endothelial cells and accelerate cell adhesion.     

The cytotoxic effects of diesel exhaust particles on human pulmonary artery endothelial cells in vitro: role of active oxygen species

Diesel exhaust particles (DEP) have been proved to induce serious pulmonary injury, among which lethal pulmonary edema has been assumed to be mediated by vascular endothelial cell damage. In the present study, we investigated the cytotoxic mechanism of DEP on human pulmonary artery endothelial cells focusing on the role of active oxygen species. Endothelial cell viability was assessed by WST-8, a novel tetrazolium salt. Nitric oxide (NO) production was measured by using a new fluorescence indicator, diaminofluorescein-2 (DAF-2). Organic compounds in DEP were extracted by dichloromethane and methanol. DEP-extracts damaged endothelial cells under both subconfluent and confluent conditions. The DEP-extract-induced cytotoxicity was markedly reduced by treatment with SOD, catalase, N-(2-mercaptopropionyl)-glycine (MPG), or ebselen (a selenium-containing compound with glutathione peroxidase-like activity). Thus superoxide, hydrogen peroxide, and other oxygen-derived free radicals are likely to be implicated in DEP-extract-induced endothelial cell damage. Moreover, L-NAME and L-NMA, inhibitors of NO synthase, also attenuated DEP-extract-induced cytotoxicity, while sepiapterin, the precursor of tetrahydrobiopterin (BH(4), a NO synthase cofactor) interestingly enhanced DEP-extract-induced cell damage. These findings suggest that NO is also involved in DEP-extract-mediated cytotoxicity, which was confirmed by direct measurement of NO production. These active oxygen species, including peroxynitrite, may explain the mechanism of endothelial cell damage upon DEP exposure during the early stage.     

Oxidation of tetrahydrobiopterin by peroxynitrite or oxoferryl species occurs by a radical pathway.

The molecular mechanisms of tetrahydrobiopterin (BH4) oxidation by peroxynitrite (ONOO-) was studied using ultra-weak chemiluminescence, electron paramagnetic resonance (EPR) and UV-visible diode-array spectrophotometry, and compared to BH4 oxidation by oxoferryl species produced by the myoglobin/hydrogen peroxide (Mb/H2O2) system. The oxidation of BH4 by ONOO- produced a weak chemiluminescence, which was altered by addition of 50 mM of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN). EPR spin trapping demonstrated that the reaction occurred at least in part by a radical pathway. A mixture of two spectra composed by an intense six-line spectrum and a fleeting weak nine-line one was observed when using ONOO-. Mb/H2O2 produced a short-living light emission that was suppressed by the addition of BH4. Simultaneous addition of POBN, BH4 and Mb/H2O2 produced the same six-line EPR spectrum, with a signal intensity depending on BH4 concentration. Spectrophotometric studies confirmed the rapid disappearance of the characteristic peak of ONOO- (302 nm) as well as substantial modifications of the initial BH4 spectrum with both oxidant systems. These data demonstrated that BH4 oxidation, either by ONOO- or by Mb/H2O2, occurred with the production of activated species and by radical pathways.     

Reactive oxygen species as cardiovascular mediators: Lessons from endothelial-specific protein overexpression mouse models

The term reactive oxygen species (ROS) summarizes several small chemical compounds such as superoxide, peroxynitrite, hydrogen peroxide and nitric oxide. The stoichiometry of the chemical reactions underlying generation and metabolism is subject of tight enzymatic regulation resulting in well balanced steady-state concentrations throughout the healthy body. ROS are short-lived and usually active at the site of production only, e.g. in vascular endothelial cells. Although an increase of vascular ROS-production is considered an important pathogenic factor in cardiovascular diseases, there is evidence for physiological or even beneficial effects as well. We have generated several transgenic mice using the Tie-2 promotor which expresses an enzyme of interest specifically in vascular endothelial cells. Here, we review some results obtained with mice carrying a Tie-2-driven overexpression of catalase or endothelial nitric oxide synthase (eNOS). Tie-2-catalase mice have a strongly reduced steady-state concentration of vascular hydrogen peroxide and show profound hypotension that is not dependent on the bioavailability of endothelial nitric oxide but is completely reversible by treatment with the catalase inhibitor aminotriazole. A similar hypotension was observed in transgenic mice with an endothelial-specific overexpression of eNOS but this hypotension is entirely dependent on vascular eNOS activity. These observations suggest a tonic effect of hydrogen peroxide on vascular smooth muscle. Further studies suggested that hydrogen peroxide promotes the exercise-induced increase of vascular eNOS expression and inhibits the release of endothelial progenitor cells induced by exercise training. In summary, our data support the concept of a dual role of ROS in the vascular system.     

Actions of melatonin in the reduction of oxidative stress

Melatonin was discovered to be a direct free radical scavenger less than 10 years ago. Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, it stimulates several antioxidative enzymes which increase its efficiency as an antioxidant. In terms of direct free radical scavenging, melatonin interacts with the highly toxic hydroxyl radical with a rate constant equivalent to that of other highly efficient hydroxyl radical scavengers. Additionally, melatonin reportedly neutralizes hydrogen peroxide, singlet oxygen, peroxynitrite anion, nitric oxide and hypochlorous acid. The following antioxidative enzymes are also stimulated by melatonin: superoxide dismutase, glutathione peroxidase and glutathione reductase. Melatonin has been widely used as a protective agent against a wide variety of processes and agents that damage tissues via free radical mechanisms.