Phosphorylation of endothelial nitric-oxide synthase regulates superoxide generation from the enzyme

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O(2)* generation are unknown. Therefore, we phosphorylated recombinant BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O(2)* generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O(2)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O(2)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.     

In vivo measurement of tumor redox environment using EPR spectroscopy

Solid tumors are characterized by a number of physiological properties such as occurrence of significant hypoxia, large amounts of cellular reducing equivalents, compromised blood-flow and low pH, all of which are distinctly different from normal tissues. Tumor therapeutic regimens such as radiation or chemotherapy attempt to exploit these physiological differences between normal and malignant tissue. Thus, methods that can detect these subtle differences would greatly aid in devising appropriate treatment strategies. Low-frequency in vivo electron paramagnetic resonance (EPR) spectroscopy is capable of providing non-invasive measurements of these parameters in tumors. This requires the use of appropriate exogenously injected free radical reporter molecules (probes), such as nitroxides. In the present study we performed measurements of nitroxide metabolism in RIF-1 murine tumors, in vivo, and demonstrated that the rate of nitroxide decay correlated with the tumor redox environment. The results showed the existence of significantly higher reducing environment in the tumor tissue compared to normal tissue. The dependence of the tumor redox status on the intracellular GSH levels and tissue oxygenation was investigated. The measurement of redox status and its manipulation may have important implications in the understanding of tumor growth and therapy.     

Esterified trityl radicals as intracellular oxygen probes

Triarylmethyl (trityl) radicals exhibit high stability and narrow linewidth under physiological conditions which provide high sensitivity and resolution for the measurement of O₂ concentrations, making them attractive as EPR oximetry probes. However, the application of previously available compounds has been limited by their poor intracellular permeability. We recently reported the synthesis and characterization of esterified trityl radicals as potential intracellular EPR probes and their oxygen sensitivity, redox properties, and enzyme-mediated hydrolysis were investigated. In this paper, we report the cellular permeability and stability of these trityls in the presence of bovine aortic endothelial cells. Results show that the acetoxymethoxycarbonyl-containing trityl AMT-02 exhibits high stability in the presence of cells and can be effectively internalized. The intracellular hydrolysis of AMT-02 to the carboxylate form of the trityl (CT-03) was also observed. In addition, this internalized trityl probe was applied to measure intracellular O₂ concentrations and the effects of menadione and KCN on the rates of O₂ consumption in endothelial cells. This study demonstrates that these esterified trityl radicals can function as effective EPR oximetry probes measuring intracellular O₂ concentration and consumption.     

Activation of Hsp90-eNOS and increased NO generation attenuate respiration of hypoxia-treated endothelial cells

Hypoxia induces various adoptive signaling in cells that can cause several physiological changes. In the present work, we have observed that exposure of bovine aortic endothelial cells (BAECs) to extreme hypoxia (1-5% O(2)) attenuates cellular respiration by a mechanism involving heat shock protein 90 (Hsp90) and endothelial nitric oxide (NO) synthase (eNOS), so that the cells are conditioned to consume less oxygen and survive in prolonged hypoxic conditions. BAECs, exposed to 1% O(2), showed a reduced respiration compared with 21% O(2)-maintained cells. Western blot analysis showed an increase in the association of Hsp90-eNOS and enhanced NO generation on hypoxia exposure, whereas there was no significant accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha). The addition of inhibitors of Hsp90, phosphatidylinositol 3-kinase, and NOS significantly alleviated this hypoxia-induced attenuation of respiration. Thus we conclude that hypoxia-induced excess NO and its derivatives such as ONOO(-) cause inhibition of the electron transport chain and attenuate O(2) demand, leading to cell survival at extreme hypoxia. More importantly, such an attenuation is found to be independent of HIF-1alpha, which is otherwise thought to be the key regulator of respiration in hypoxia-exposed cells, through a nonphosphorylative glycolytic pathway. The present mechanistic insight will be helpful to understand the difference in the magnitude of endothelial dysfunction.     

Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite

Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO(-)) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the "hyperoxic state" of 21% O(2), compared with more physiological O(2) tensions (Po(2)), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O(2)(*-)) generation leading to ONOO(-) formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O(2) consumption rates of bovine aortic ECs sheared (10 dyn/cm(2), 30 min) at 5%, 10%, or 21% O(2) or left static at 5% or 21% O(2). Respiration was inhibited to a greater extent when ECs were sheared at 21% O(2) than at lower Po(2) or left static at different Po(2). Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO(-) scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O(2)(*-) production was higher in ECs sheared at 21% than at 5% O(2), as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O(2) was modestly increased compared with ECs sheared at lower Po(2), suggesting that eNOS activity may be higher at 21% O(2). Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O(2)(*-) production, leads to enhanced ONOO(-) formation intramitochondrially and suppression of respiration.     

Synthesis, characterization, and direct aqueous superoxide anion scavenging of a highly water-dispersible astaxanthin-amino acid conjugate

The aqueous solubility and/or dispersibility of synthetic carotenoid analogs can be improved by varying the chemical structure(s) of the esterified moieties. In the current study, a highly water-dispersible astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) derivative was synthesized by esterification to the amino acid L-lysine, and subsequently converted to the tetrahydrochloride salt. Deep violet, evenly colored aqueous suspensions were obtained with addition of the novel derivative to USP purified water up to a maximum of 181.6 mg/mL. These aqueous suspensions were obtained without the addition of heat, detergents, co-solvents, or other additives. At higher concentrations (above 181.6 mg/mL), the dispersion became turbid and viscous. There was no saturation point up to 181.6 mg/mL. The direct superoxide scavenging ability of the tetrahydrochloride dilysine astaxanthin salt was also evaluated by electron paramagnetic resonance (EPR) spectroscopy in a well-characterized in vitro isolated human neutrophil assay. The novel derivative was an extremely potent (micromolar concentration) aqueous-phase scavenger, with near-complete suppression of the superoxide anion signal (as detected by spin-trap adducts of DEPMPO) achieved at 100 microM. To the authors' knowledge, this novel carotenoid derivative exhibits the greatest aqueous dispersibility yet described for a natural and/or synthetic C40 carotenoid, and as such, will find utility in those applications for which aqueous-phase singlet oxygen quenching and direct radical scavenging are required.     

Vascular NAD(P)H oxidase is distinct from the phagocytic enzyme and modulates vascular reactivity control

An NAD(P)H oxidase has been hypothesized to be the main source of reactive oxygen species (ROS) in vessels; however, questions remain about its function and similarity with the neutrophil oxidase. Therefore, vascular superoxide generation was measured by electron paramagnetic resonance spectroscopy using the spin-trap 5,5'-dimethly-pyrroline-N-oxide in aortas from wild-type (WT) and gp91(phox)-deficient mice (gp91(phox)-/-), which do not have a functioning neutrophil NADPH oxidase. There was no significant difference between radical adduct formation by WT or gp91(phox)-/- mouse aortas either at baseline or after stimulation with NADPH or NADH. Also, spin-adduct formation was identical in the 100,000-g pellets obtained from WT and gp91(phox)-/- mouse aortas. SOD mimetics and the flavoenzyme inhibitor diphenyleneiodonium blocked spin-adduct formation from both intact vessels and particulate fractions. Other pharmacological inhibitors of metabolic pathways involved in ROS generation had no effect on this phenomenon. To examine the role of this enzyme in vascular tone control, aortic rings were suspended in organ chambers and preconstricted with phenylephrine to reach half-maximal contraction. Exposure to NADPH elicited a 20% increase in vascular tone, which was decreased by SOD mimetics in a concentration-dependent manner, suggesting that superoxide was responsible for this phenomenon. NADH had no effect on vascular tone. Thus superoxide is generated in the vessel wall by an NAD(P)H-dependent oxidase, which modulates vascular contractile tone. This enzyme is structurally and genetically distinct from the neutrophil NADPH oxidase.     

A real-time electrochemical technique for measurement of cellular hydrogen peroxide generation and consumption: evaluation in human polymorphonuclear leukocytes

There has been a long-standing need for sensitive and specific techniques for hydrogen peroxide (H(2)O(2)) measurement. We describe the development and application of a highly sensitive electrochemical sensor, utilizing a membrane-coated platinum microelectrode, suitable for real-time measurement of hydrogen peroxide generation and consumption in biochemical or cellular systems. This sensor provides high sensitivity enabling measurement of hydrogen peroxide down to 5-10 nM concentrations. We demonstrate that it can be used to measure the magnitude and time course of H(2)O(2) generation from the NADPH oxidase in leukocytes as well as the rate of H(2)O(2) degradation. After human polymorphonuclear leukocytes (PMNs) were activated by phorbol 12-myristate acetate, H(2)O(2) concentration increased with time and reached a peak concentration, from 5 to 15 microM in PMNs prepared from different individuals, within 3 to 8 min, then decreased slowly. The H(2)O(2) concentration in the solution is less than the total H(2)O(2) generation from the activated PMNs because a part of H(2)O(2) generated is decomposed. H(2)O(2) in solution, generated from the PMNs, was rapidly consumed after the activated PMNs were treated with 10 microM diphenylene iodonium (DPI). The rate of H(2)O(2) consumption was measured following the addition of exogenous H(2)O(2). The total production of H(2)O(2) from the activated PMNs was calculated from the measured H(2)O(2) concentration and the rate of H(2)O(2) consumption. This technique enables sensitive and continuous real-time measurement of H(2)O(2) concentration and total H(2)O(2) generation in cellular or enzyme systems without addition of any detection reagents.     

Direct superoxide anion scavenging by a disodium disuccinate astaxanthin derivative: Relative efficacy of individual stereoisomers versus the statistical mixture of stereoisomers by electron paramagnetic resonance imaging

Carotenoids are a related group of greater than 600 natural compounds, irrespective of geometric- and stereoisomers, with demonstrated antioxidant efficacy. The carotenoids are broadly divided into "carotenes," or non-oxygen substituted hydrocarbon carotenoids, and "xanthophylls," oxygen-substituted carotenoids. The natural compounds are excellent singlet oxygen quenchers as well as lipid peroxidation chain-breakers; this dual antioxidant capacity is generally attributed to the activity of the polyene chain, and increases with the number of conjugated double bonds along the polyene chain length. However, the poor aqueous solubility of most carotenes and the vast majority of xanthophylls limits their use as aqueous-phase singlet oxygen quenchers and direct radical scavengers. A variety of introduction vehicles (e.g., organic solvents, cyclodextrins) have been used to introduce the insoluble carotenoids into aqueous test systems. Hawaii Biotech, Inc. (HBI) successfully synthesized a novel carotenoid derivative, the disodium disuccinate derivative of astaxanthin (3,3(')-dihydroxy-beta,beta-carotene-4,4(')-dione) in all-trans (all-E) form. The novel derivative is a water-dispersible symmetric chiral molecule with two chiral centers, yielding four stereoisomeric forms: 3R,3(')R and 3S,3(')S (enantiomers), and the diastereomeric meso forms (3R,3(')S and 3(')R,3S). The individual stereoisomers were synthesized at high purity (>90% by HPLC) and compared directly for efficacy with the statistical mixture of stereoisomers obtained from the synthesis from the commercial source of astaxanthin (1:2:1 ratio of 3S,3(')S, meso, and 3R,3(')R, respectively). Direct scavenging of superoxide anion was evaluated in a standard in vitro isolated human neutrophil assay by electron paramagnetic resonance (EPR) imaging, employing the spin-trap DEPMPO. Each novel derivative was tested in pure aqueous formulation and in ethanolic formulation shown to completely disaggregate the compounds in solution. In each case, the ethanolic formulation was a more potent scavenging vehicle. No significant differences in scavenging efficiency were noted among the individual stereoisomers and the statistical mixture of stereoisomers, suggesting that the polyene chain alone was responsible for superoxide scavenging. Dose-ranging revealed that the statistical mixture of stereoisomers of the novel derivative, at millimolar (mM) concentrations, could nearly completely eliminate the superoxide anion signal generated in the activated human neutrophil assay. All ethanolic formulations of the novel derivatives exhibited increased scavenging efficiency over equimolar concentrations of non-esterified astaxanthin delivered in a dimethyl sulfoxide (DMSO) vehicle. These novel compounds will likely find utility in applications requiring aqueous delivery of a highly potent direct radical scavenger.     

In vivo measurement of tumor redox environment using EPR spectroscopy

Solid tumors are characterized by a number of physiological properties such as occurrence of significant hypoxia, large amounts of cellular reducing equivalents, compromised blood-flow and low pH, all of which are distinctly different from normal tissues. Tumor therapeutic regimens such as radiation or chemotherapy attempt to exploit these physiological differences between normal and malignant tissue. Thus, methods that can detect these subtle differences would greatly aid in devising appropriate treatment strategies. Low-frequency in vivo electron paramagnetic resonance (EPR) spectroscopy is capable of providing non-invasive measurements of these parameters in tumors. This requires the use of appropriate exogenously injected free radical reporter molecules (probes), such as nitroxides. In the present study we performed measurements of nitroxide metabolism in RIF-1 murine tumors, in vivo, and demonstrated that the rate of nitroxide decay correlated with the tumor redox environment. The results showed the existence of significantly higher reducing environment in the tumor tissue compared to normal tissue. The dependence of the tumor redox status on the intracellular GSH levels and tissue oxygenation was investigated. The measurement of redox status and its manipulation may have important implications in the understanding of tumor growth and therapy.