Role of H2O2 and heme-containing O2 sensors in hypoxic regulation of tyrosine hydroxylase gene expression

In the currentstudy, we investigated links betweenO₂-regulatedH₂O₂formation and the hypoxic induction of mRNA for tyrosine hydroxylase(TH), the rate-limiting enzyme in catecholamine synthesis, inO₂-sensitive PC-12 cells. Duringexposure of PC-12 cells to 5% O₂,H₂O₂concentration decreased by 40% as measured with2',7'-dichlorofluorescein (DCF). Treatment withH₂O₂reduced TH mRNA during normoxia and prevented the induction of TH mRNAduring hypoxia. Treatment with catalase orN-(2-mercaptopropionyl)-glycine, areducing antioxidant agent that decreasesH₂O₂concentration, also induced TH mRNA. Deferoxamine (DF), an ironchelator, failed to affectH₂O₂formation but induced TH mRNA in normoxia and hypoxia.CoCl₂ led to a decrease inH₂O₂at 20 h of treatment but induced TH mRNA during normoxia and hypoxiabefore it affectedH₂O₂.In conclusion, TH gene expression correlates inversely withH₂O₂formation. DF and Co²⁺ seem toaffect TH gene expression in themechanism downstream from theH₂O₂formation rather than by interfering with theH₂O₂-generating activity of the O₂ sensor.

     

Photoreduction of 18O2 and H218O2 with Concomitant Evolution of 16O2 in Intact Spinach Chloroplasts: Evidence for Scavenging of Hydrogen Peroxide by Peroxidase

Illuminated intact spinach chloroplasts decomposed one moleculeof H₂¹⁸O₂ which resulted in the evolution of a half moleculeof ¹⁶O₂, but little ¹⁸O₂. The chloroplasts showed the same rateof photoreduction of ¹⁸C₂ as that of the evolution of ¹⁶O₂ withoutaccumulation of H₂¹⁸O₂. These reactions were suppressed by DCMU,and also by several inhibitors of ascorbate peroxidase and dehydroascorbateand monodehydroascorbate reductases in chloroplasts. These observationsindicate that the hydrogen peroxide produced in chloroplastsis reduced to water by a peroxidase using a photoreductant asthe electron donor. The hydrogen peroxide scavenging systemof chloroplasts was inactivated if hydrogen peroxide was addedin the dark, but not if added during the light. (Received May 4, 1984; Accepted July 10, 1984)     

Elevated CO2 reduces O3 flux and O3-induced yield losses in soybeans: possible implications for elevated CO2 studies

Soybeans were grown for three seasons in open-top field chambersto determine (1) whether elevated CO₂ (360 versus 700 µmolmol^–1) alleviates some of the yield loss due to pollutantO₃, (2) whether the partial stomatal closure resulting fromchronic O₃ exposure (charcoal-filtered air versus 1.5 ambientconcentrations) is a cause or result of decreased photosynthesis,and (3) possible implications of CO₂/O₃ interactions to climatechange studies using elevated CO₂. Leaf conductance was reducedby elevated CO₂, regardless of O₃ level, or by exposure to O₃alone. As.a result of these effects on conductance, high CO₂reduced estimated midday O₃ flux into the leaf by an averageof 50% in charcoal-filtered air and 35% in the high O₃ treatment.However, while exposure to O₃ reduced seed yields by 41% atambient CO₂ levels, the yield reduction was completely amelioratedby elevated CO₂. The threshold midday O₃ flux for yield lossappears to be 20–30 nmol m^–2 s^–1 in this study.Although elevated CO₂ increased total biomass production, itdid not increase seed yields. A/C_i curves show a large reductionin the stomatal limitation to photosynthesis due to elevatedCO₂ but no effect of O₃. These data demonstrate that (1) reducedconductance due to O₃ is the result, and not the cause, of reducedphotosynthesis, (2) 700 µmol mol^–1 CO₂ can completelyameliorate yield losses due to O₃ within the limits of theseexperiments, and (3) some reports of increased yields underelevated CO₂ treatments may, at least in part, reflect the ameliorationof unrecognized suppression of yield by O₃ or other stresses. Key words: Stomatal limitation, elevated CO₂, O₃ flux, Glycine max, yield suppression     

H(2)O(2)-mediated permeability: role of MAPK and occludin

H₂O₂-mediated elevation inendothelial solute permeability is associated with pathological eventssuch as ischemia-reperfusion and inflammation. To understand howH₂O₂ mediates increased permeability, weinvestigated the effects of H₂O₂ administrationon vascular endothelial barrier properties and tight junctionorganization and function. We report that H₂O₂exposure caused an increase in endothelial solute permeability in atime-dependent manner through extracellularly regulated kinase 1 and 2 (ERK1/ERK2) signal pathways. H₂O₂ exposurecaused the tight junctional protein occludin to be rearranged fromendothelial cell-cell junctions. Occludin rearrangement involvedredistribution of occludin on the cell surface and dissociation ofoccludin from ZO-1. Occludin also was heavily phosphorylated onserine residues upon H₂O₂ administration. H₂O₂ mediates changes in ERK1/ERK2phosphorylation, increases endothelial solute permeability, and altersoccludin localization and phosphorylation were all blocked by PD-98059,a specific mitogen-activated protein (MAP) or ERK kinase 1 inhibitor. These data strongly suggest thatH₂O₂-mediated increased endothelial solutepermeability involves the loss of endothelial tight junction integritythrough increased ERK1/ERK2 activation.

     

NADH2- and succinate-dependent O2 uptake in chromatophores from Chromatium vinosum

The O₂ uptake linked to NADH₂ and succinate oxidation was observedin chromatophores from photosynthetically grown Chromatium vinosum. The maximal rate was 60–120 nmoles of O₂ uptake per minper {diaeresis}mole of bacterio-chlorophyll. The rate of O₂uptake linked to NADH₂ oxidation was higher in the neutral-to-acidicpH range than in the alkaline range, whereas that linked tosuccinate oxidation was higher in the alkaline range. The O₂ uptake linked to NADH₂ oxidation was inhibited by rotenone,amytal, antimycin A, KCN and NaN₃, while that linked to succinateoxidation was inhibited by antimycin A, KCN and NaN₃. Malate,citrate, pyruvate, acetate, -ketoglutarate, NADPH₂ and thiosulfatedid not serve as substrates for the O₂ uptake of isolated chromatophores. The rates of the O₂ uptake linked to both NADH₂ and succinateoxidation were not stimulated by adding uncouplers or underphosphorylating conditions. Little or no ATP was synthesizedin the dark, coupled to either NADH₂ or succinate oxidation,in spite of a high activity of photophosphorylation in Chromatiumchromatophores. (Received February 26, 1980; )     

O2 extraction maintains O2 uptake during submaximal exercise with beta -adrenergic blockade at 4,300m

Wholebody O₂ uptake (O₂)during maximal and submaximal exercise has been shown to be preservedin the setting of -adrenergic blockade at high altitude, despitemarked reductions in heart rate during exercise. An increase in strokevolume at high altitude has been suggested as the mechanism thatpreserves systemic O₂ delivery (blood flow × arterialO₂ content) and thereby maintains O₂ at sea-level values. To test thishypothesis, we studied the effects of nonselective -adrenergicblockade on submaximal exercise performance in 11 normal men(26 ± 1 yr) at sea level and on arrival and after 21 days at 4,300 m. Six subjects received propranolol (240 mg/day), and five subjectsreceived placebo. At sea level, during submaximal exercise, cardiacoutput and O₂ delivery were significantly lower inpropranolol- than in placebo-treated subjects. Increases instroke volume and O₂ extraction were responsible for themaintenance of O₂. At 4,300 m,-adrenergic blockade had no significant effect onO₂, ventilation, alveolarPO₂, and arterial blood gases duringsubmaximal exercise. Despite increases in stroke volume, cardiac outputand thereby O₂ delivery were still reduced inpropranolol-treated subjects compared with subjects treated withplacebo. Further reductions in already low levels of mixed venousO₂ saturation were responsible for the maintenance ofO₂ on arrival and after 21 days at4,300 m in propranolol-treated subjects. Despite similarworkloads and O₂,propranolol-treated subjects exercised at greater perceived intensitythan subjects given placebo at 4,300 m. The values for mixed venousO₂ saturation during submaximal exercise inpropranolol-treated subjects at 4,300 m approached thosereported at simulated altitudes >8,000 m. Thus -adrenergicblockade at 4,300 m results in significant reduction in O₂delivery during submaximal exercise due to incomplete compensation bystroke volume for the reduction in exercise heart rate. Total bodyO₂ is maintained at a constant levelby an interaction between mixed venous O₂ saturation, thearterial O₂-carrying capacity, and hemodynamics duringexercise with acute and chronic hypoxia.

     

Sulfhydryls associated with H2O2-induced channel activation are on luminal side of ryanodine receptors

The mechanism underlying H₂O₂-inducedactivation of frog skeletal muscle ryanodine receptors was studiedusing skinned fibers and by measuring single Ca²⁺-releasechannel current. Exposure of skinned fibers to 3-10 mM H₂O₂ elicited spontaneous contractures.H₂O₂ at 1 mM potentiated caffeine contracture.When the Ca²⁺-release channels were incorporated into lipidbilayers, open probability (P_o) and open timeconstants were increased on intraluminal addition ofH₂O₂ in the presence of cis catalase,but unitary conductance and reversal potential were not affected.Exposure to cis H₂O₂ at 1.5 mM failedto activate the channel in the presence of trans catalase.Application of 1.5 mM H₂O₂ to the transside of a channel that had been oxidized by cisp-chloromercuriphenylsulfonic acid (pCMPS; 50 µM) still led to anincrease in P_o, comparable to that elicited bytrans 1.5 mM H₂O₂ without pCMPS.Addition of cis pCMPS to channels that had been treated with orwithout trans H₂O₂ rapidly resulted inhigh P_o followed by closure of the channel. Theseresults suggest that oxidation of luminal sulfhydryls in theCa²⁺-release channel may contribute toH₂O₂-induced channel activation and musclecontracture.

     

H(2)O(2) and ethanol act synergistically to gate ryanodine receptor/calcium-release channel

We examined theeffect of low concentrations of H₂O₂ on theCa²⁺-release channel/ryanodine receptor (RyR) to determineif H₂O₂ plays a physiological role in skeletalmuscle function. Sarcoplasmic reticulum vesicles from frog skeletalmuscle and type 1 RyRs (RyR1) purified from rabbit skeletal muscle wereincorporated into lipid bilayers. Channel activity of the frog RyR wasnot affected by application of 4.4 mM (0.02%) ethanol. Openprobability (P_o) of such ethanol-treated RyRchannels was markedly increased on subsequent addition of 10 µMH₂O₂. Increase of H₂O₂to 100 µM caused a further increase in channel activity. Applicationof 4.4 mM ethanol to 10 µM H₂O₂-treated RyRsactivated channel activity. Exposure to 10 or 100 µMH₂O₂ alone, however, failed to increaseP_o. Synergistic action of ethanol andH₂O₂ was also observed on the purified RyR1 channel, which was free from FK506 binding protein (FKBP12).H₂O₂ at 100-500 µM had no effect onpurified channel activity. Application of FKBP12 to the purified RyR1drastically decreased channel activity but did not alter the effects ofethanol and H₂O₂. These results suggest thatH₂O₂ may play a pathophysiological, butprobably not a physiological, role by directly acting on skeletalmuscle RyRs in the presence of ethanol.

     

H2O2-mediated permeability II: importance of tyrosine phosphatase and kinase activity

We previously reportedthat exposure of endothelial cells to H₂O₂results in a loss of cell-cell apposition and increased endothelialsolute permeability. The purpose of this study was to determine howtyrosine phosphorylation and tyrosine phosphatases contribute tooxidant-mediated disorganization of endothelial cell junctions. Wefound that H₂O₂ caused a rapid decrease in total cellular phosphatase activity that facilitates a compensatory increase in cellular phosphotyrosine residues.H₂O₂ exposure also results in increasedendothelial monolayer permeability, which was attenuated by pp60, aninhibitor of src kinase. Inhibition of protein tyrosinephosphatase activity by phenylarsine oxide (PAO) demonstrated a similarpermeability profile compared with H₂O₂,suggesting that tyrosine phosphatase activity is important inmaintaining a normal endothelial solute barrier. Immunofluorescence shows that H₂O₂ exposure caused a loss ofpan-reactive cadherin and -catenin from cell junctions that was notblocked by the src kinase inhibitor PP1.H₂O₂ also caused -catenin to dissociate fromthe endothelial cytoskeleton, which was not prevented by PP1. Finally,we determined that PP1 did not prevent cadherin internalization. Thesedata suggest that oxidants like H₂O₂ produce biological effects through protein phosphotyrosine modifications bydecreasing total cellular phosphatase activity combined with increasedsrc kinase activity, resulting in increased endothelial solute permeability.

     

Response of Wheat Seedlings to Low O2 Concentrations in Nutrient Solution: I. GROWTH, O2 UPTAKE AND SYNTHESIS OF FERMENTATIVE END-PRODUCTS BY ROOT SEGMENTS

Root growth of 7-d-old wheat (Triticum aestivum cv. Gamenya)seedlings was impaired at dissolved O₂ concentrations of 0.01and 0.055 mol m^–3 O₂, while growth at 0.115 mol m^–3O₂ was the same as that in continuously aerated controls (0.26mol m^–3 O2). Oxygen uptake by apical (0–2 mm), expanding (2–4mm) and expanded (10–12 mm) tissues of the roots decreasedbelow 0.16, 0.09 and 0.05 mol m^–3 O₂, respectively. Thishierarchy is consistent with the metabolic rates of these tissues.There was a small (c. 9%) inhibition of O₂ uptake and some netsynthesis of ethanol and alanine in root apices at 0.115 molm^–3 O₂. Significant amounts of anaerobic end-productsaccumulated at 0.055 mol m^–3 O₂ and even more so at 0.01mol m^–3 O₂, indicating that oxidative phosphorylationwas strongly inhibited. Net alanine synthesis increased in fully expanded (10–16mm) tissues exposed to <0.003–0.01 mol m^–3 O₂,and this increase was accompanied either by a proportionallysmaller increase in the concentration of other free amino acidsor by a net decrease in free amino acid levels excluding alanine.This suggests that alanine was synthesized as an end-productof anaerobic catabolism and did not accumulate simply becauseof decreased net protein synthesis. Comparing the carbon flow to CO₂, ethanol, lactate and alaninein roots at 0.01 mol m^–3 O₂ with carbon loss as CO₂ inaerated roots suggests that carbon flow to products of metabolismwas not greatly enhanced due to O₂ deficiency. This infers,but does not prove that, in wheat, generation of energy duringperiods of O₂ deficiency is not enhanced due to a Pasteur effect. Key words: Anaerobic, fermentation, oxygen, wheat     

Dormancy and impotency of cocklebur seeds I. CO2, C2H4 O2 and high temperature

High O₂ tensions, CO₄, C₂H₄ and high temperatures were effectivenot only in breaking the dormancy of cocklebur (Xanthium pennsylvanicumWallr.) seeds but also in increasing the germination potentialof the nondormant but small seeds. There were few qualitativedifferences in response to these factors between the dormantand impotent seeds. Unlike CO₂, however, enriched O₂ and C₂H₄were stimulative even at the low temperature of 13°C. Germination induced by CO₂, C₂H₄ and high temperature treatmentswas lowered when endogenously evolved C₂H₄ or CO₂ was removed,whereas the effect of O₂ enrichment was not affected by theirremoval. CO₂ and high temperatures remarkably stimulated C₂H₄production, whereas O₂ enrichment had no such effect. C₂H₄ productivity was lower in the dormant than non-dormantseeds, suggesting that the after-ripening is characterized byincreasing C₂H₄ production. (Received August 20, 1974; )     

Preparation of Radioactive Starch, Glucose and Fructose from C14O2

Radioactive starch, glucose and fructose have been preparedfrom tobacco leaves after assimilation of C¹⁴O₂. The apparatusused for photosynthesis consisted of a shallow Perspex leafchamber connected to a closed gas system, in which C¹⁴O₂ wasgenerated from BaC¹⁴O₂. Six leaves, area 14 to 18 sq. dm. whenexposed to bright sunlight with an initial CO₂ concentrationof 8 to 10 per cent., assimilated 3.35 g. of C¹⁴O₂ in 8 to 10hours. At least 80 per cent. of the C¹⁴O₂ supplied appearedin the leaves as starch and sugar and over 80 per cent. of theradioactivity was accounted for in these carbohydrates. Thespecific activity per m. atom of carbon of the isolated productswas 85 to 90 per cent. of that of the C¹⁴O₂. Small amounts ofradioactive carbon were also incorporated in the leaf proteinand in the celluose, hemicellulose and polyuronides.     

A microsensor for direct measurement of O2 partial pressure within plant tissues3

Widespread use of O₂ microsensors to measure O₂ partial pressure(pO₂) in plant tissues has been limited in part because of difficultyof construction and other technical obstacles. By modifyingpublished techniques, an O₂ microsensor was constructed thatcombined the advantages of Clark-type microsensors with lesscomplicated construction techniques. The specifications andsome performance characteristics of the microsensor are: tipdiameter 1–5 µm; sensitivity 7.5–25 pA kPa^–1;negligible stir-induced current; response time 540 ms. The microsensorcan be used in air or solution, and each sensor can be usedfor several experiments. The sensitivity of the microsensorwas unchanged during measurements over the physiological rangeof pO₂ in intact, growing maize (Zea mays L.) primary roots,and was thus unaffected by cellular fluids and turgor pressure.Use of the microsensor to compare pO₂ profiles in vermiculite-and solution-grown roots is described. The O₂ microsensor couldfind application in studies in which information on tissue pO₂is needed, but for which conventional O₂ probes are too large. Key words: Oxygen microsensor, Zea mays L., roots, oxygen partial pressure     

Physiological and hypoxic O2 tensions rapidly regulate NO production by stimulated macrophages

Nitric oxide (NO) production by inducible NO synthase (iNOS) is dependent on O₂ availability. The duration and degree of hypoxia that limit NO production are poorly defined in cultured cells. To investigate short-term O₂-mediated regulation of NO production, we used a novel forced convection cell culture system to rapidly (response time of 1.6 s) and accurately (±1 Torr) deliver specific O₂ tensions (from <1 to 157 Torr) directly to a monolayer of LPS- and IFN-stimulated RAW 264.7 cells while simultaneously measuring NO production via an electrochemical probe. Decreased O₂ availability rapidly (30 s) and reversibly decreased NO production with an apparent K_mO₂ of 22 (SD 6) Torr (31 µM) and a V_max of 4.9 (SD 0.4) nmol·min^–1·10^–6 cells. To explore potential mechanisms of decreased NO production during hypoxia, we investigated O₂-dependent changes in iNOS protein concentration, iNOS dimerization, and cellular NO consumption. iNOS protein concentration was not affected (P = 0.895). iNOS dimerization appeared to be biphasic [6 Torr (P = 0.008) and 157 Torr (P = 0.258) >36 Torr], but it did not predict NO production. NO consumption was minimal at high O₂ and NO tensions and negligible at low O₂ and NO tensions. These results are consistent with O₂ substrate limitation as a regulatory mechanism during brief hypoxic exposure. The rapid and reversible effects of physiological and pathophysiological O₂ tensions suggest that O₂ tension has the potential to regulate NO production in vivo. inducible nitric oxide synthase; substrate limitation; nitric oxide consumption     

Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

Raising P50 increases tissue PO2 in canine skeletal muscle but does not affect critical O2 extraction ratio

Curtis, Scott E., Thomas A. Walker, W. E. Bradley, andStephen M. Cain. Raising P₅₀increases tissue PO₂ in canineskeletal muscle but does not affect criticalO₂ extraction ratio.J. Appl. Physiol. 83(5):1681-1689, 1997.Affinity of hemoglobin (Hb) forO₂ determines in part the rate ofO₂ diffusion from capillaries tomyocytes by altering capillary PO₂.We hypothesized that a decrease in HbO₂ affinity (increasedP₅₀) would increase capillary and tissue PO₂(Pti_O2) andimprove O₂ consumption duringischemia. To test this hypothesis, blood flow to the pump-perfused lefthindlimb of 18 anesthetized and paralyzed dogs was progressively decreased over 90 min while hindlimb O₂ consumption andO₂ delivery (O₂)and Pti_O2 weremeasured at the muscle surface. Arterial PO₂ was maintained at 150 ± 10 Torr in all dogs. We increased P₅₀by 12.3 ± 0.9 (SE) Torr in nine dogs with RSR-13, an allosteric modifier of Hb. This decreased arterialO₂ saturation to 90-92% butincreased meanPti_O2 from 35.5 ± 11.6 to 44.1 ± 15.2 (SD) Torr(P < 0.05) with no change incontrols (n = 9).O₂ extraction ratio at criticalO₂was 74 ± 2% in controls and 79 ± 1% in RSR-13-treated dogs(P = not significant).Pti_O2 was30-40% higher in the RSR-13-treated group at anyO₂above critical but did not differ between groups below criticalO₂.Perfusion heterogeneity and convergence of the dissociation curvesnear criticalO₂ may have mitigated any effect of increasedP₅₀ onO₂ diffusion. Still, increasingP₅₀ by 12 Torr with RSR-13significantly increased Pti_O2 atO₂values above critical.

     

Pollutant particles enhanced H2O2 production from NAD(P)H oxidase and mitochondria in human pulmonary artery endothelial cells

Particulate matter (PM) induces oxidative stress and cardiovascular adverse health effects, but the mechanistic link between the two is unclear. We hypothesized that PM enhanced oxidative stress in vascular endothelial cells and investigated the enzymatic sources of reactive oxygen species and their effects on mitogen-activated protein kinase (MAPK) activation and vasoconstriction. We measured the production of extracellular H₂O₂, activation of extracellular signal-regulated kinases1/2 (ERK1/2) and p38 MAPKs in human pulmonary artery endothelial cells (HPAEC) treated with urban particles (UP; SRM1648), and assessed the effects of H₂O₂ on vasoconstriction in pulmonary artery ring and isolated perfused lung. Within minutes after UP treatment, HPAEC increased H₂O₂ production that could be inhibited by diphenyleneiodonium (DPI), apocynin (APO), and sodium azide (NaN₃). The water-soluble fraction of UP as well as its two transition metal components, Cu and V, also stimulated H₂O₂ production. NaN₃ inhibited H₂O₂ production stimulated by Cu and V, whereas DPI and APO inhibited only Cu-stimulated H₂O₂ production. Inhibitors of other H₂O₂-producing enzymes, including N-methyl-L-argnine, indomethacin, allopurinol, cimetidine, rotenone, and antimycin, had no effects. DPI but not NaN₃ attenuated UP-induced pulmonary vasoconstriction and phosphorylation of ERK1/2 and p38 MAPKs. Knockdown of p47phox gene expression by small interfering RNA attenuated UP-induced H₂O₂ production and phosphorylation of ERK1/2 and p38 MAPKs. Intravascular administration of H₂O₂ generated by glucose oxidase increased pulmonary artery pressure. We conclude that UP induce oxidative stress in vascular endothelial cells by activating NAD(P)H oxidase and the mitochondria. The endothelial oxidative stress may be an important mechanism for PM-induced acute cardiovascular health effects. mitogen-activated protein kinase; extracellular signal-regulated kinase; p38; vasoconstriction     

Synthesis of Isozymes of Superoxide Dismutase in Maize Leaves in Response to O3, SO2 and Elevated O2

Matters, G. L. and Scandalios, J. G. 1987. Synthesis of isozymesof superoxide dismutase in maize leaves in response to O₃ SO₂and elevated O₂.—J. exp. Bot 38: 842–852. The activities of the enzymes superoxide dismutase (SOD) andcatalase were determined in maize leaves treated with O₃or SO₂for8 h, or with elevated levels of oxygen for up to 96 h. NeitherO₃nor SO₂significantly increased the levels of superoxide dismutaseor catalase activity. However, after 72 h in an atmosphere containing90% oxygen, superoxide dismutase activity was increased, butnot the activities of catalase, ascorbate pcroxidase, and malatedehydrogenase. Immunological analysis showed that amounts ofthe cytosolic superoxide dismutase isozymes, SOD-2 and SOD-4,were increased by the elevated oxygen but not the chroloplast(SOD-1) or mitochondrial (SOD-3) isozymes. Immunoprecipitationof translation products of leaf polysomes indicated that thehigher levels of SOD-2 and SOD-4 were due to increased amountsof polysome-bound mRNA coding for these proteins. The specificresponse of SOD-2 and SOD-4 to 90% oxygen treatments contrastswith the increase in all SOD isozymes in maize leaves treatedwith the herbicide paraquat. Key words: Air pollutants, maize, oxidative stress, oxygen, superoxide dismutase     

Effect of O2 on the Kinetics of the Flash-Induced 518 nm Absorbance Change in Vivo

The kinetics of the flash induced 518 nm absorbance change (A₅₁₈)in lettuce leaves were found to be dependent on O₂ concentration.(1) Either a lower O₂ partial pressure or the addition of weakred background illumination accelerated the decay of (A₅₁₈)while far-red background light induced a transient acceleration.(2) In the presence of background red light the accelerateddecay could be restored to the original dark level by the additionof O₂. A linear relationship was found between the intensityof red background light and the O₂ pressure required for thisrestoration. (3) The O₂ dependence of (A₅₁₈) decay halftimewas biphasic, the sensitive phase saturating at 0.3 atmospheresO₂ independent of input light energy while the O₂ concentrationneeded to saturate the second phase increased with increasinginput light energy (increasing flash frequency). (4) Treatmentwith N, N'-dicyclohexylcarbodiimide (DCCD) or KCN eliminatedall O₂ and background light effects and DCMU treatment inhibitedall but the sensitive phase of the O₂ dependence on (A₅₁₈) decayhalftime. (5) The extent of the lag phase in the dark recoveryof (A₅₁₈) normally present after preillumination induced accelerationof decay was decreased with added O₂ or KCN. (6) It was concludedthat O₂ competes directly with background red light inducedelectron transport to PS I acceptors to influence the (A₅₁₈)decay. A possible mechanism involving the O₂ sensitive ferredoxin-thioredoxin-reductaseactivation of chloroplast coupling factor 1 ATP-hydrolase activitywas discussed. (Received December 17, 1982; Accepted April 28, 1983)     

Peripheral O2 diffusion does not affect VO2 on-kinetics in isolated in situ canine muscle

To test thehypothesis that muscle O₂ uptake(O₂) on-kinetics islimited, at least in part, by peripheralO₂ diffusion, we determined theO₂ on-kinetics in1) normoxia (Control);2) hyperoxic gas breathing(Hyperoxia); and 3) hyperoxia andthe administration of a drug (RSR-13, Allos Therapeutics), whichright-shifts the Hb-O₂dissociation curve (Hyperoxia+RSR-13). The study was conducted inisolated canine gastrocnemius muscles(n = 5) during transitions from restto 3 min of electrically stimulated isometric tetanic contractions(200-ms trains, 50 Hz; 1 contraction/2 s; 60-70% peakO₂). In all conditions,before and during contractions, muscle was pump perfused withconstantly elevated blood flow (), at a levelmeasured at steady state during contractions in preliminary trials withspontaneous . Adenosine was infusedintra-arterially to prevent inordinate pressure increases with theelevated . was measuredcontinuously, arterial and popliteal venousO₂ concentrations were determinedat rest and at 5- to 7-s intervals during contractions, andO₂ was calculated as · arteriovenous O₂ content difference.PO₂ at 50%HbO₂saturation (P₅₀) was calculated.Mean capillary PO₂(c_O2)was estimated by numerical integration.P₅₀ was higher in Hyperoxia+RSR-13[40 ± 1 (SE) Torr] than in Control and in Hyperoxia (31 ± 1 Torr). After 15 s of contractions,c_O2was higher in Hyperoxia (97 ± 9 Torr) vs. Control (53 ± 3 Torr) and in Hyperoxia+RSR-13 (197 ± 39 Torr) vs. Hyperoxia. Thetime to reach 63% of the difference between baseline and steady-stateO₂ during contractions was 24.7 ± 2.7 s in Control, 26.3 ± 0.8 s in Hyperoxia, and 24.7 ± 1.1 s in Hyperoxia+RSR-13 (not significant). Enhancement ofperipheral O₂ diffusion (obtainedby increasedc_O2at constant O₂ delivery) duringthe rest-to-contraction (60-70% of peakO₂) transition did notaffect muscle O₂on-kinetics.