Nitric oxide and cerebral blood flow responses to hyperbaric oxygen.

We have tested the hypothesis that cerebral nitric oxide (NO) production is involved in hyperbaric O(2) (HBO(2)) neurotoxicity. Regional cerebral blood flow (rCBF) and electroencephalogram (EEG) were measured in anesthetized rats during O(2) exposure to 1, 3, 4, and 5 ATA with or without administration of the NO synthase inhibitor (N(omega)-nitro-L-arginine methyl ester), L-arginine, NO donors, or the N-methyl-D-aspartate receptor inhibitor MK-801. After 30 min of O(2) exposure at 3 and 4 ATA, rCBF decreased by 26-39% and by 37-43%, respectively, and was sustained for 75 min. At 5 ATA, rCBF decreased over 30 min in the substantia nigra by one-third but, thereafter, gradually returned to preexposure levels, preceding the onset of EEG spiking activity. Rats pretreated with N(omega)-nitro-L-arginine methyl ester and exposed to HBO(2) at 5 ATA maintained a low rCBF. MK-801 did not alter the cerebrovascular responses to HBO(2) at 5 ATA but prevented the EEG spikes. NO donors increased rCBF in control rats but were ineffective during HBO(2) exposures. The data provide evidence that relative lack of NO activity contributes to decreased rCBF under HBO(2), but, as exposure time is prolonged, NO production increases and augments rCBF in anticipation of neuronal excitation.     

Activation of matrix metalloproteinase-2 by overexpression of manganese superoxide dismutase in human breast cancer MCF-7 cells involves reactive oxygen species

Matrix metalloproteinases (MMPs) participate in cell migration and remodeling processes by affecting the extracellular matrix. MMP-2 is thought to be involved in cancer cell invasiveness. It has been proposed that the activity of MMP-2 can be modulated by intracellular reactive oxygen species (ROS)/reactive nitrogen species. We hypothesized that manganese superoxide dismutase (MnSOD) could mediate MMP-2 activity by changing the intracellular ROS level and that nitric oxide ((.)NO) may be involved in this process. Human breast cancer MCF-7 cells were stably transfected with plasmids containing MnSOD cDNA. A 2-30-fold increase of MnSOD protein and activity was observed in four clones. Our data demonstrated that overexpression of MnSOD stimulated the activation of MMP-2 with a corresponding elevation of ROS. A decrease in ROS by ebselen, a glutathione peroxidase mimetic, or by transduction of adenovirus containing human catalase or glutathione peroxidase cDNA abolished the effect of MnSOD on MMP-2 activation. Treatment of MCF-7 cells with antimycin A or rotenone increased intracellular ROS production and MMP-2 activation simultaneously. Our data also showed a suppression of endothelial nitric-oxide synthase expression that was accompanied by decreased (.)NO production in MnSOD-overexpressing cells. However, the changes in endothelial nitric-oxide synthase and (.)NO did not correlate with the MnSOD activity. Corresponding changes of MMP-2 activity after the addition of a NOS inhibitor (N(G)-amino-l-arginine) or a (.)NO donor ((Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate) to the cells suggested the possibility that (.)NO may be involved in the MnSOD-mediated MMP-2 activation pathway. These results indicate that MnSOD induces MMP-2 activity by regulation of intracellular ROS and imply that signaling pathways involving (.)NO may also be involved in the MnSOD mediation of MMP-2 activity.     

Vascular reactivity and endothelial NOS activity in rat thoracic aorta during and after hyperbaric oxygen exposure

Accumulating evidence suggests that hyperbaric oxygen (HBO) stimulates neuronal nitric oxide (NO) synthase (NOS) activity, but the influence on endothelial NOS (eNOS) activity and vascular NO bioavailability remains unclear. We used a bioassay employing rat aortic rings to evaluate vascular NO bioavailability. HBO exposure to 2.8 atm absolute (ATA) in vitro decreased ACh relaxation. This effect remained unchanged, despite treatment with SOD-polyethylene glycol and catalase-polyethylene glycol, suggesting that the reduction in endothelium-derived NO bioavailability was independent of superoxide production. In vitro HBO induced contraction of resting aortic rings with and without endothelium, and these contractions were reduced by the NOS inhibitor N(omega)-nitro-l-arginine. In addition, in vitro HBO attenuated the vascular contraction produced by norepinephrine, and this effect was reversed by N(omega)-nitro-l-arginine, but not by endothelial denudation. These findings indicate stimulation of extraendothelial NO production during HBO exposure. A radiochemical assay was used to assess NOS activity in rat aortic endothelial cells. Catalytic activity of eNOS in cell homogenates was not decreased by HBO, and in vivo HBO exposure to 2.8 ATA was without effect on eNOS activity and/or vascular NO bioavailability in vitro. We conclude that HBO reduces endothelium-derived NO bioavailability independent of superoxide production, and this effect seems to be unrelated to a decrease in eNOS catalytic activity. In addition, HBO increases the resting tone of rat aortic rings and attenuates the contractile response to norepinephrine by endothelium-independent mechanisms that involve extraendothelial NO production.     

Alteration of Striatal Dopamine Levels Under Various Partial Pressure of Oxygen in Pre-convulsive and Convulsive Phases in Freely-Moving rats

The purpose of this study was to investigate the change in the striatal dopamine (DA) level in freely-moving rat exposed to different partial pressure of oxygen (from 1 to 5 ATA). Some works have suggested that DA release by the substantia nigra pars compacta (SNc) neurons in the striatum could be disturbed by hyperbaric oxygen (HBO) exposure, altering therefore the basal ganglia activity. Such changes could result in a change in glutamatergic and GABAergic control of the dopaminergic neurons into the SNc. Such alterations could provide more information about the oxygen-induced seizures observed at 5 ATA in rat. DA-sensitive electrodes were implanted into the striatum under general anesthesia. After 1 week rest, awaked rats were exposed to oxygen–nitrogen mixture at a partial pressure of oxygen of 1, 2, 3, 4 and 5 ATA. DA level was monitored continuously (every 3 min) by in vivo voltammetry before and during HBO exposure. HBO induced a decrease in DA level in relationship to the increase in partial pressure of oxygen from 1 ATA to 4 ATA (?15 % at 1 ATA, ?30 % at 2 ATA, ?40 % at 3 ATA, ?45 % at 4 ATA), without signs of oxygen toxicity. At 5 ATA, DA level strongly decreases (?75 %) before seizure which occurred after 27 min ± 7 HBO exposure. After the epileptic seizure the decrease in DA level disappeared. These changes and the biphasic effect of HBO were discussed in function of HBO action on neurochemical regulations of the nigro striatal pathway.     

Hyperoxic vasoconstriction in the brain is realized by inactivation of nitric oxide by superoxide anions

We tested a hypothesis that the cerebral blood flow (CBF) is reduced at hyperbaric oxygen due to inactivation of nitric oxide (NO) by superoxide anions (O2). In our experiments, the CBF was measured under hyperbaric oxygenation (HBO) 4ATA after inhibition of NO synthesis and inactivation of O2. The CBF was reduced at HBO exposure. Inhibition of NO--synthase type I and III (NOS) by L-NAME in the air caused the same decreasing of the CBF as at 4 ATA HBO. Hyperbaric vasoconstriction was diminished after NOS inhibition. Intravenous injection of superoxide dismutase (CuZn SOD) increased the CBF in the air and HBO exposure. This effect disappeared at preliminary NOS inhibition. These data suggest that inactivation of NO by O2 is a more effective mechanism of HBO vasoconstriction.     

Nitric oxide-mediated central sympathetic excitation promotes CNS and pulmonary O2 toxicity

In hyperbaric oxygen (HBO(2)) at or above 3 atmospheres absolute (ATA), autonomic pathways link central nervous system (CNS) oxygen toxicity to pulmonary damage, possibly through a paradoxical and poorly characterized relationship between central nitric oxide production and sympathetic outflow. To investigate this possibility, we assessed sympathetic discharges, catecholamine release, cardiopulmonary hemodynamics, and lung damage in rats exposed to oxygen at 5 or 6 ATA. Before HBO(2) exposure, either a selective inhibitor of neuronal nitric oxide synthase (NOS) or a nonselective NOS inhibitor was injected directly into the cerebral ventricles to minimize effects on the lung, heart, and peripheral circulation. Experiments were performed on both anesthetized and conscious rats to differentiate responses to HBO(2) from the effects of anesthesia. EEG spikes, markers of CNS toxicity in anesthetized animals, were approximately four times as likely to develop in control rats than in animals with central NOS inhibition. In inhibitor-treated animals, autonomic discharges, cardiovascular pressures, catecholamine release, and cerebral blood flow all remained below baseline throughout exposure to HBO(2). In control animals, however, initial declines in these parameters were followed by significant increases above their baselines. In awake animals, central NOS inhibition significantly decreased the incidence of clonic-tonic convulsions or delayed their onset, compared with controls. The novel findings of this study are that NO produced by nNOS in the periventricular regions of the brain plays a critical role in the events leading to both CNS toxicity in HBO(2) and to the associated sympathetic hyperactivation involved in pulmonary injury.     

Hyperoxic exposure affects the ventilatory response to hypoxia in awake rats

We tested whether hyperbaric O2 (HBO) has an adverse effect on the hypoxic ventilatory drive. Four groups of rats were exposed for 550 min to O2 at 1.67, 1.90, and 2.15 ATA and to air at 1.90 ATA, respectively. Ventilatory parameters (frequency, tidal volume, and minute ventilation) were measured using whole-body plethysmography, before the hyperbaric exposure, immediately after the exposure, and up to 20 days after the exposure. Resting ventilation was not affected after exposure at 1.90 ATA to air or at 1.67 ATA to O2. HBO at 1.90 and 2.15 ATA caused a reduction of frequency and an elevation of tidal volume at different inspired gases: air, 5% CO2 balance O2, 80% O2, and 4.5% O2. However, minute ventilation on the day after the hyperoxic exposure was not different from the control at either air, 5% CO2, or 80% O2 but was markedly attenuated on the first three breaths at 4.5% O2. The hypoxic ventilation decreased to 48 +/- 13 (SD) and 32 + 11% after 1.90 and 2.15 ATA, respectively. The ventilatory parameters recovered in the days after HBO. We conclude that HBO reversibly depresses the hypoxic ventilatory drive, most probably by a direct effect on the carotid O2 chemoreceptors.     

Hydrogen peroxide promotes endothelial dysfunction by stimulating multiple sources of superoxide anion radical production and decreasing nitric oxide bioavailability.

Hydrogen peroxide (H(2)O(2)) is an oxidant implicated in cell signalling and various pathologies, yet relatively little is known about its impact on endothelial cell function. Herein we studied the functional and biochemical changes in aortic vessels and cultured porcine aortic endothelial cells (PAEC) exposed to H(2)O(2). Exposure of aortic rings to 25 or 50 microM, but not 10 microM, H(2)O(2) for 60 min prior to constriction significantly decreased subsequent relaxation in response to acetylcholine (ACh), but not the nitric oxide ((.)NO) donor sodium nitroprusside. Treatment of PAEC with 50 microM H(2)O(2) significantly decreased ACh-induced accumulation of (.)NO, as measured with a (.)NO-selective electrode, yet such treatment increased nitric oxide synthase activity approximately 3-fold, as assessed by conversion of L-arginine to L-citrulline. Decreased (.)NO bioavailability was reflected in decreased cellular cGMP content, associated with increased superoxide anion radical (O(2)(-.)), and overcome by addition of polyethylene glycol superoxide dismutase. Increased cellular O(2)(-.) production was inhibited by allopurinol, diphenyliodonium and rotenone in an additive manner. The results show that exposure of endothelial cells to H(2)O(2) decreases the bioavailability of agonist-induced (.)NO as a result of increased production of O(2)(-.) likely derived from xanthine oxidase, NADPH-oxidase and mitochondria. These processes could contribute to H(2)O(2)-induced vascular dysfunction that may be relevant under conditions of oxidative stress such as inflammation.     

Brain oxygenation and CNS oxygen toxicity after infusion of perfluorocarbon emulsion

Intravenous perfluorocarbon (PFC) emulsions, administered with supplemental inspired O(2), are being evaluated for their ability to eliminate N(2) from blood and tissue prior to submarine escape, but these agents can increase the incidence of central nervous system (CNS) O(2) toxicity, perhaps by enhancing O(2) delivery to the brain. To assess this, we infused a PFC emulsion (Oxycyte, 6 ml/kg iv) into anesthetized rats and measured cerebral Po(2) and regional cerebral blood flow (rCBF) in cortex, hippocampus, hypothalamus, and striatum with 100% O(2) at 1, 3, or 5 atmospheres absolute (ATA). At 1 ATA, brain Po(2) stabilized at >20 mmHg higher in animals infused with PFC emulsion than in control animals infused with saline, and rCBF fell by ~10%. At 3 ATA, PFC emulsion raised brain Po(2) >70 mmHg above control levels, and rCBF decreased by as much as 25%. At 5 ATA, brain Po(2) was ≥159 mmHg above levels in control animals for the first 40 min but then rose sharply; rCBF showed a similar profile, reflecting vasoconstriction followed by hyperemia. Conscious rats were also pretreated with PFC emulsion at 3 or 6 ml/kg iv and exposed to 100% O(2) at 5 ATA. At the lower dose, 80% of the animals experienced seizures by 33 min compared with 50% of the control animals. At the higher dose, seizures occurred in all rats within 25 min. At these doses, administration of PFC emulsion poses a clear risk of CNS O(2) toxicity in conscious rats exposed to hyperbaric O(2) at 5 ATA.     

Prevention of H2O2 generation by monoamine oxidase protects against CNS O2 toxicity.

Toxicity to the central nervous system (CNS) by hyperbaric oxygen (HBO) presumably relates to increased production of reactive oxygen species. The sites of generation of reactive oxygen species during HBO, however, have not been fully characterized in the brain. We investigated the relationship between regional generation of hydrogen peroxide (H2O2) in the brain in the presence of an irreversible inhibitor of catalase, aminotriazole (ATZ), and protection from CNS O2 toxicity by a monoamine oxidase (MAO) inhibitor, pargyline. At 6 ATA of oxygen, pargyline significantly protected rats from CNS O2 toxicity whereas ATZ enhanced O2 toxicity. In animals pretreated with ATZ, HBO inactivated 21-40% more catalase than air exposure in the six brain regions studied. Because ATZ-mediated inactivation of catalase was H2O2 dependent, the decrease in catalase activity during hyperoxia was proportional to the intracellular production of H2O2. Pargyline, administered 30 min before HBO, inhibited MAO by greater than 90%, prevented ATZ inhibition of catalase activity during HBO, and reversed the augmentation of CNS O2 toxicity by ATZ. These findings indicate that H2O2 generated by MAO during hyperoxia is important to the pathogenesis of CNS O2 toxicity in rats.     

Dual effects of copper-zinc superoxide dismutase

Copper-zinc superoxide dismutase (CuZnSOD) specifically catalyzes the removal of superoxide radicals to protect cellular function against the generation of superoxide-dependent hydroxyl radicals ((.)OH). However, an unexpected observation reveals that denatured CuZnSOD (dCuZnSOD) itself induces (.)OH formation. This dCuZnSOD-dependent (.)OH generation was not inhibited by active CuZnSOD, suggesting that it is a superoxide-independent process. Sodium cyanide, histidine, and N,N'-diethyldithiocarbamate abolished (.)OH generation, implying that Cu may be responsible for dCuZnSOD-induced (.)OH formation. Catalase eliminated ()OH generation, suggesting that hydrogen peroxide may be involved in the mechanism of dCuZnSOD-mediated (.)OH production. Furthermore, nitric oxide ((.)NO) completely inhibited dCuZnSOD-induced (.)OH radical generation, indicating that (.)NO is an important (.)OH radical scavenger. Our results shed new light on the effect of dysfunctional CuZnSOD and suggest that structural disorder of the enzyme may be one of the endogenous pathways of toxic (.)OH formation in biological systems.     

Relationship between protein nitration and oxidation and development of hyperoxic seizures.

Recent studies have implicated nitric oxide (NO*) as a mediator of CNS hyperbaric O2 (HBO2) toxicity. One mechanism by which NO* may contribute to HBO2-induced brain toxicity involves a neurotoxic, pro-oxidative action of NO* via the formation of the potent oxidant peroxynitrite (ONOO-). The present study compares: (a) the formation of protein nitrotyrosine as a marker of ONOO- accumulation and (b) protein oxidation as an indicator of reactive oxygen species production during HBO2 exposure. Rats were exposed to 5 atm 100% O2 to pre-convulsive exposure or until the occurrence of electroencephalographic (EEG) seizures. After exposures, brains were analyzed for protein nitrotyrosine (NT) and protein carbonyl measurement by Western blot and for superoxide dismutase (SOD) activity by NBT assay. The results show a significant increase in protein NT, exceeding control level by several fold. There was only a slow and non-significant increase in the quantity of oxidized proteins during the pre-convulsive phase of HBO2 exposure. Levels of both protein NT and protein carbonyls were significantly (p<0.05) elevated after seizures. Total SOD activity was not changed during preconvulsive exposures, but was significantly (p<0.05) elevated post-seizures. The specific neuronal nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI), significantly reduced the increases in seizure-induced protein NT and protein carbonyl and at the same time very effectively (p<0.05) delayed onset of HBO2 seizures. Pre-seizure increases in protein NT might indicate its role in the mechanism of HBO2-induced brain toxicity. This is supported by the observed capacity of 7-NI to inhibit tyrosine nitration and increase time to seizure.     

Tryptophan metabolism through the kynurenine pathway in rat brain and liver slices

We hypothesized that hyperbaric oxygen (HBO) enhances tryptophan (TRP) flux through the kynurenine (KYN) pathway because oxygen is a substrate for four pathway enzymes. Our objective was to compare the biosynthesis of KYN pathway intermediates by rat brain and liver slices with air or HBO as the gas phase. One-millimeter thick liver and brain slices were obtained from male Sprague-Dawley rats and incubated individually in chambers containing Hanks'-HEPES- buffer with (3)H-TRP (30 Ci/mmol) for 2 h (37 degrees C) in either room air or oxygen (1.2 or 5.2 atmospheres absolute [ATA] oxygen). After incubation, tissue was snap-frozen and analyzed for protein content while medium was extracted for high-performance liquid chromatography analysis. Radiolabeled nicotinamide adenine dinucleotide (NAD) was produced by brain and liver; liver (with air as the gas phase) also produced quinolinic acid (QA). HBO at 1.2 and 5.2 ATA caused increased QA and NAD from liver slices. HBO did not affect KYN metabolism in brain slices, although there was decreased production of NAD during high oxygen. We conclude that rat brain and liver contain the complete KYN pathway and that HBO enhances KYN flux in liver tissue.     

Nitric oxide and carbon dioxide in neurotoxicity induced by oxygen under pressure

Hyperbaric oxygen (HBO2) causes CO2 retention in the brain that leads to the increase in cerebral blood flow (CBF) by poorly understood mechanisms. We have tested the hypothesis that NO is implicated in CBF-responses to hypercapnia under hyperoxic conditions. Alert rats were exposed to HBO2 at 5 ata and blood flow in the striatum measured by H2 clearance every 10 min. Acetazolamide, the inhibitor of carbonic anhydrase, was used to increase brain PCO2. CBF responses to acetazolamide administration (30 mg/kg, i.p.) were assessed in rats breathing air at 1 ata or oxygen at 5 ata with and without NOS inhibition (L-NAME, 30 mg/kg, i.p.). In rats breathing air, acetazolamide increased CBF by 34 +/- 7.4% over 30 min and by 28 +/- 12% over 3 hours while NOS inhibition with L-NAME attenuated acetazolamide-induced cerebral vasodilatation. HBO2 at 5 ata reduced CBF during the first 30 min hyperoxia, after that CBF increased by 55 +/- 19% above pre-exposure levels. In acetazolamide-treated animals, no HBO, induced vasoconstricton was observed and striatal blood flow increased by 53 +/- 18% within 10 min of hyperbaric exposure. After NOS inhibition, cerebral vasodilatation in response to acetazolamide during HBO2 exposure was significantly attenuated. The study demonstrates that NO is implicated in acetazolamide (CO2)-induced cerebral hyperemia under hyperbaric oxygen exposure.     

Hyperbaric Hyperoxia Accelerates Fracture Healing in Mice

Increased oxygen tension influences bone metabolism. This study comprised two main experiments: one aimed to determine the bone mineral apposition and bone formation rates in vivo under hyperbaric hyperoxia (HBO), and the other aimed to evaluate the effects of exposure to HBO on fracture healing. In experiment 1, male mice were exposed to HBO [90 min/day at 90% O₂ at 2 atmospheres absolute (ATA) for 5 days]. In experiment 2, an open femur fracture model was created in mice, followed by exposure to HBO 5 times/week (90 min/day at 90% O₂ at 2 ATA) for 6 weeks after surgery. In experiment 1, HBO treatment significantly increased the mineral apposition and bone formation rates in the lumbar vertebra and femur and type 1 collagen alpha 1 and alkaline phosphatase mRNA expression in the lumbar vertebra. In experiment 2, at 2 weeks after fracture, the fracture callus was significantly larger in the HBO group than in the non-HBO group. Furthermore, at 4 and 6 weeks after fracture, radiographic findings showed accelerated fracture healing in the HBO group. At 6 weeks after fracture, femur stiffness and maximum load were significantly higher in the HBO group than in the non-HBO group. Urinary 8-hydroxy-2′-deoxyguanosine and plasma calcium concentrations were not significantly different between groups. These results suggest that exposure to HBO enhances bone anabolism and accelerates fracture healing without causing oxidative DNA damage or disruption of plasma calcium homeostasis.     

Mechanism of peroxynitrite interaction with cytochrome c

Kinetics of the reaction of peroxynitrite with ferric cytochrome c in the absence and presence of bicarbonate was studied. It was found that the heme iron in ferric cytochrome c does not react directly with peroxynitrite. The rates of the absorbance changes in the Soret region of cytochrome c spectrum caused by peroxynitrite or peroxynitrite/bicarbonate were the same as the rate of spontaneous isomerization of peroxynitrite or as the rate of the reaction of peroxynitrite with bicarbonate, respectively. This means that intermediate products of peroxynitrite decomposition, (.)OH/(.)NO(2) or, in the presence of bicarbonate, CO(3)(-)(.)/(.)NO(2), are the species responsible for the absorbance changes in the Soret band of cytochrome c. Modifications of the heme center of cytochrome c by radiolytically produced radicals, (.)OH, (.)NO(2) or CO(3)(-)(.), were also studied. The absorbance changes in the Soret band caused by radiolytically produced (.)OH or CO(3)(-)(.) were much more significant that those observed after peroxynitrite treatment, compared under similar concentrations of radicals. (.)NO(2) produced radiolytically did not interact with the heme center of cytochrome c. Cytochrome c exhibited an increased peroxidase-like activity after reaction with peroxynitrite as well as with radiolytically produced (.)OH, (.)NO(2) or CO(3)(-)(.) radicals. This means that modification of protein structure: oxidation of amino acids and/or tyrosine nitration, facilitates reaction of H(2)O(2) with the heme iron of cytochrome c, followed by reaction with the second substrate.     

NO and cGMP mediate angiotensin AT2 receptor-induced renal renin inhibition in young rats

We hypothesized that angiotensin subtype-2 receptor (AT(2)R) inhibits renal renin biosynthesis in young rats via nitric oxide (NO). We monitored changes in renal NO, cGMP, renal renin content (RRC), and ANG II in 4-wk-old rats in response to low sodium (LNa(+)) intake alone and combined with 8-h direct renal cortical administration of AT(1) receptor blocker valsartan (VAL), AT(2)R blocker PD123319 (PD), NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME), NO donor S-nitroso-N-acetyl penicillamine (SNAP), or guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,2-alpha] quinoxaline-1-one (ODQ). In addition, we monitored renal endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in response to VAL or PD. LNa(+), VAL, PD, l-NAME, and ODQ increased RRC, ANG II, and renin mRNA. PD and l-NAME decreased NO and cGMP, while SNAP reduced RRC, ANG II, renin mRNA, and reversed the effects of PD. PD also reduced eNOS and nNOS protein and mRNA. Combined treatment with PD, l-NAME, or ODQ and VAL reversed the effects of VAL and caused further increase in RRC, ANG II, renin mRNA, and protein. ODQ reversed the effects of SNAP. These data demonstrate that the renal AT(2) receptor decreases renal renin biosynthesis and ANG II production in young rats. Reversal of the PD effects by SNAP and SNAP effects by ODQ confirms that NO and cGMP mediate the AT(2) receptor inhibition of renal renin production.     

Stem cell mobilization by hyperbaric oxygen

We hypothesized that exposure to hyperbaric oxygen (HBO(2)) would mobilize stem/progenitor cells from the bone marrow by a nitric oxide (*NO) -dependent mechanism. The population of CD34(+) cells in the peripheral circulation of humans doubled in response to a single exposure to 2.0 atmospheres absolute (ATA) O(2) for 2 h. Over a course of 20 treatments, circulating CD34(+) cells increased eightfold, although the overall circulating white cell count was not significantly increased. The number of colony-forming cells (CFCs) increased from 16 +/- 2 to 26 +/- 3 CFCs/100,000 monocytes plated. Elevations in CFCs were entirely due to the CD34(+) subpopulation, but increased cell growth only occurred in samples obtained immediately posttreatment. A high proportion of progeny cells express receptors for vascular endothelial growth factor-2 and for stromal-derived growth factor. In mice, HBO(2) increased circulating stem cell factor by 50%, increased the number of circulating cells expressing stem cell antigen-1 and CD34 by 3.4-fold, and doubled the number of CFCs. Bone marrow *NO concentration increased by 1,008 +/- 255 nM in association with HBO(2). Stem cell mobilization did not occur in knockout mice lacking genes for endothelial *NO synthase. Moreover, pretreatment of wild-type mice with a *NO synthase inhibitor prevented the HBO(2)-induced elevation in stem cell factor and circulating stem cells. We conclude that HBO(2) mobilizes stem/progenitor cells by stimulating *NO synthesis.     

NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II

Recently, it has been shown that the exogenous addition of hydrogen peroxide (H(2)O(2)) increases endothelial nitric oxide (NO(.)) production. The current study is designed to determine whether endogenous levels of H(2)O(2) are ever sufficient to stimulate NO(.) production in intact endothelial cells. NO(.) production was detected by a NO(.)-specific microelectrode or by an electron spin resonance spectroscopy using Fe(2+)-(DETC)(2) as a NO(.)-specific spin trap. The addition of H(2)O(2) to bovine aortic endothelial cells caused a potent and dose-dependent increase in NO(.) release. Incubation with angiotensin II (10(-7) mol) elevated intracellular H(2)O(2) levels, which were attenuated with PEG-catalase. Angiotensin II increased NO(.) production by 2-fold, and this was prevented by Losartan and by PEG-catalase, suggesting a critical role of AT1 receptor and H(2)O(2) in this response(.) In contrast, NO(.) production evoked by either bradykinin or calcium ionophore was unaffected by PEG-catalase. As in bovine aortic endothelial cells, angiotensin II doubled NO(.) production in aortic endothelial cells from C57BL/6 mice but had no effect on NO(.) production in endothelial cells from p47(phox-/-) mice. In contrast, stimulated NO(.) production to a similar extent in endothelial cells from wild-type and p47(phox-/-) mice. In summary, the present study provides direct evidence that endogenous H(2)O(2), derived from the NAD(P)H oxidase, mediates endothelial NO(.) production in response to angiotensin II. Under disease conditions associated with elevated levels of angiotensin II, this response may represent a compensatory mechanism. Because angiotensin II also stimulates O(2)() production from the NAD(P)H oxidase, the H(2)O(2) stimulation of NO(.) may facilitate peroxynitrite formation in response to this octapeptide.     

Effects of pre-exposure to hyperbaric hyperoxia on high-intensity exercise performance

This study comprised 2 main experiments: one was to determine the oxidative DNA damage under hyperbaric hyperoxia (HBO), and the other was to evaluate the effects of pre-exposure to HBO on high-intensity exercise performance. Healthy subjects (n = 8) inspired 100% O2 in an experimental chamber at a pressure of 1.3 atmospheres absolute (ATA) for 50 minutes once per week for 2 weeks. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) was measured as a marker of DNA oxidative damage on day 0 and on days 1, 3, and 5 after each HBO exposure. To investigate the effects of pre-exposure to HBO on high-intensity exercise performance, subjects (n = 6) performed maximal isometric knee extensor exercise (30 repetitions x 2 sets) with and without HBO pre-exposure (100% O2 at 1.3 ATA for 50 minutes). Urinary 8-OHdG did not show any significant change after HBO exposure. Isometric knee extensor torque was significantly lower during the first half of the first set of exercises after HBO pre-exposure compared with the normobaric normoxia (NBO) trial. The decreased torque was associated with the lower integrated electromyography with respect to time. Changes in the degree of ischemia-reperfusion in the vastus lateralis muscle during exercise were larger in the HBO pre-exposure trial than in the NBO trial. Muscle fatigue index, serum lactate concentration, heart rate, and systolic blood pressure showed no differences between the 2 trials. These results indicated that HBO exposure was harmless to DNA, and HBO pre-exposure did not enhance high-intensity exercise performance. As a practical application, athletes who require maximal muscle strength should not inspire high-concentration of O2 just before their competitions because it might, as the case may be, impair their performance.