Nmol O2 Research Articles

Mitochondrial affinity for ADP is twofold lower in creatine kinase knock‐out muscles

Adaptations of the kinetic properties of mitochondria in striated muscle lacking cytosolic (M) and/or mitochondrial (Mi) creatine kinase (CK) isoforms in comparison to wild-type (WT) were investigated in vitro. Intact mitochondria were isolated from heart and gastrocnemius muscle of WT and single- and double CK-knock-out mice strains (cytosolic (M-CK-/-), mitochondrial (Mi-CK-/-) and double knock-out (MiM-CK-/-), respectively). Maximal ADP-stimulated oxygen consumption flux (State3 Vmax; nmol O2 x mg mitochondrial protein(-1) x min(-1)) and ADP affinity (K50ADP; microM) were determined by respirometry. State 3 Vmax and of M-CK-/- and MiM-CK-/- gastrocnemius mitochondria were twofold higher than those of WT, but were unchanged for Mi-CK-/-. For mutant cardiac mitochondria, only the of mitochondria isolated from the MiM-CK-/- phenotype was different (i.e. twofold higher) than that of WT. The implications of these adaptations for striated muscle function were explored by constructing force-flow relations of skeletal muscle respiration. It was found that the identified shift in affinity towards higher ADP concentrations in MiM-CK-/- muscle genotypes may contribute to linear mitochondrial control of the reduced cytosolic ATP free energy potentials in these phenotypes.

Oxic microzones and radial oxygen loss from roots of Zostera marina

Oxygen microelectrodes and planar oxygen optodes were used to map the microdistrib- ution of oxygen and the radial oxygen loss (ROL) from roots of Zostera marina kept in natural sedi- ment. Substantial heterogeneity in the oxygen distribution was seen along the roots, with oxygen mainly leaking out from the root tips. Maximum oxygen levels at the root surface reached 19 to 80% of air saturation in the light and the oxygenated zone extended 1 to 2 mm away from the root tip. The oxygen concentration at the root surface decreased to 0-5% of air saturation at positions 3 to 6 mm behind the root apex. The high oxygen levels at the root tip surface were due to an effective barrier to ROL on the older part of the roots and the presence of an effective gas-transport system in the plant, with numerous intercellular spaces extending very close to the apical meristem of the root. Radial dif- fusion of oxygen from the root surface created a dynamic 0 to 1 mm-wide oxic microzone around the ~0.3 mm wide roots of Z. marina that varied with irradiance and distance from the root tip. Root- surface oxygen concentrations and ROL measured 2 mm behind the apex increased with increasing irradiance until ROL saturation was reached at irradiances >400 µmol photons m -2 s -1 . The ROL increased from 16.2 nmol O2 cm -2 h -1 in darkness to 21.6, 28.8 and 36.0 nmol O2 cm -2 h -1 at incident irradiances of 25, 111 and 467 µmol photons m -2 s -1 , respectively. Based on measured steady-state radial oxygen profiles, the total oxygen export from one 6 cm long root of the first actively growing root bundle with a total surface area of 0.56 cm 2 was estimated to be 6.0 to 6.7 nmol O2 h -1 in saturating light. The total subsurface input of plant-mediated oxygen was estimated to be only in the order of 2 to 14% of the total diffusive oxygen uptake (DOU) across the sediment-water interface. The local input of oxygen from the root tip was, however, similar to the DOU at the primary sediment-water interface, and 35 to 43% of the total oxygen loss occurred from the outermost 0 to 3.5 mm of the root tip. The roots of Z. marina grow ~5 mm d -1 , and significant oxygen levels will therefore only be present at a given spot for less than 24 h during root growth. The rhizosphere of Z. marina is thus characterised by a constantly changing mosaic of ephemeral oxic microniches in the reduced sulphidic sediment, leaving behind an anoxic but oxidised zone around the more mature parts of the roots.

Angiotensin-induced defects in renal oxygenation: role of oxidative stress

We tested the hypothesis that superoxide anion (O(2)(-).) generated in the kidney by prolonged angiotensin II (ANG II) reduces renal cortical Po(2) and the use of O(2) for tubular sodium transport (T(Na):Q(O(2))). Groups (n = 8-11) of rats received angiotensin II (ANG II, 200 ng.kg(-1).min(-1) sc) or vehicle for 2 wk with concurrent infusions of a permeant nitroxide SOD mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol, 200 nmol.kg(-1).min(-1)) or vehicle. Rats were studied under anesthesia with measurements of renal oxygen usage and Po(2) in the cortex and tubules with a glass electrode. Compared with vehicle, ANG II increased mean arterial pressure (107 +/- 4 vs. 146 +/- 6 mmHg; P < 0.001), renal vascular resistance (42 +/- 3 vs. 65 +/- 7 mmHg.ml(-1).min(-1).100 g(-1); P < 0.001), renal cortical NADPH oxidase activity (2.3 +/- 0.2 vs. 3.6 +/- 0.4 nmol O(2)(-)..min(-1).mg(-1) protein; P < 0.05), mRNA and protein expression for p22(phox) (2.1- and 1.8-fold respectively; P < 0.05) and reduced the mRNA for extracellular (EC)-SOD (-1.8 fold; P < 0.05). ANG II reduced the Po(2) in the proximal tubule (39 +/- 1 vs. 34 +/- 2 mmHg; P < 0.05) and throughout the cortex and reduced the T(Na):Q(O(2)) (17 +/- 1 vs. 9 +/- 2 mumol/mumol; P < 0.001). Tempol blunted or prevented all these effects of ANG II. The effects of prolonged ANG II to cause hypertension, renal vasoconstriction, renal cortical hypoxia, and reduced efficiency of O(2) usage for Na(+) transport, activation of NADPH oxidase, increased expression of p22(phox), and reduced expression of EC-SOD can be ascribed to O(2)(-). generation because they are prevented by an SOD mimetic.

A sulphite respiration system in the chemoheterotrophic human pathogen Campylobacter jejuni

The ability to use sulphite as a respiratory electron donor is usually associated with free-living chemolithotrophic sulphur-oxidizing bacteria. However, this paper shows that the chemoheterotrophic human pathogen Campylobacter jejuni has the ability to respire sulphite, with oxygen uptake rates of 23 +/- 8 and 28 +/- 15 nmol O(2) min(-1) (mg cell protein)(-1) after the addition of 0.5 mM sodium sulphite or metabisulphite, respectively, to intact cells. The C. jejuni NCTC 11168 Cj0004c and Cj0005c genes encode a monohaem cytochrome c and molybdopterin oxidoreductase, respectively, homologous to the sulphite : cytochrome c oxidoreductase (SOR) of Starkeya novella. Western blots of C. jejuni periplasm probed with a SorA antibody demonstrated cross-reaction of a 45 kDa band, consistent with the size of Cj0005. The Cj0004c gene was inactivated by insertion of a kanamycin-resistance cassette. The resulting mutant showed wild-type rates of formate-dependent respiration but was unable to respire with sulphite or metabisulphite as electron donors. 2-Heptyl-4-hydroxyquinoline-N-oxide (HQNO), a cytochrome bc(1) complex inhibitor, did not affect sulphite respiration at concentrations up to 25 microM, whereas formate respiration (which occurs partly via a bc(1) dependent route) was inhibited 50%, thus suggesting that electrons from sulphite enter the respiratory chain after the bc(1) complex at the level of cytochrome c. Periplasmic extracts of wild-type C. jejuni 11168 showed a symmetrical absorption peak at 552 nm after the addition of sulphite, demonstrating the reduction of cytochrome c. No cytochrome c reduction was observed after addition of sulphite to periplasmic extracts of the Cj0004c mutant. A fractionation study confirmed that the majority of the SOR activity is located in the periplasm in C. jejuni, and this activity was partially purified by ion-exchange chromatography. The presence of a sulphite respiration system in C. jejuni is another example of the surprising diversity of the electron-transport chain in this small-genome pathogen. Sulphite respiration may be of importance for survival in environmental microaerobic niches and some foods, and may also provide a detoxification mechanism for this normally growth-inhibitory compound.

Lipids That Prime Neutrophils Are Present upon Recognition of the Acute Chest Syndrome.

The acute chest syndrome (ACS) is a form of acute lung injury (ALI) unique to sickle cell disease (SCD), and for children >10 years of age represents the major cause of morbidity and mortality. ALI is thought to be neutrophil (PMN)-mediated; however, the pathogenesis of ACS is not known but is linked to an increase of secretory phospholipase A2-IIA (sPLA2) activity within 24 hours of its development (Styles, Blood 87:2573, 1996). We hypothesize that sPLA2 cleavage of membrane lipids generates biologically active lipophilic compounds that prime PMNs, which provokes ACS in patients with SCD, resulting in increased lipid priming activity during ACS. After obtaining informed consent, heparinized whole blood was drawn from children with SCD when well, at their annual comprehensive visit, and serially when admitted to the hospital, upon admission and every other day until discharge. Plasma was isolated, and the plasma lipids extracted. In selected patients the plasma was treated with buffer or sPLA2 for 1 hour at 37°C prior to lipid extraction. Lipids were assayed for their ability to prime fMLP-activation of the PMN oxidase as measured by the maximal rate of O2− production by the SOD-inhibitable reduction of cytochrome c at 550 nm. The data (mean ± SEM of fold increase over fMLP-treated controls of the lipid priming activity at baseline and over hospitalization) consists of three groups: patients with SCD, patients with SCD treated with daily hydroxyurea (HU), or patients with SCD treated with monthly, red cell exchanges (EX). At baseline, there was slightly more lipid priming activity in untreated SCD patients versus either HU- or EX-treated patients, which was not statistically different (Table). The lipid activity did not increase with vaso-occlusive crisis (VOC) but significantly increased in the one patient with ACS. Moreover, sPLA2 treatment of the plasma obtained 12 hours prior to developing ACS, while this patient was in VOC, demonstrated an increase in lipid priming activity versus the patient's prior samples (0.96- to 1.73-fold control with sPLA2). The lipids from healthy African-American patients, did not exhibit increased activity vs controls (data not shown). We conclude that both HU and EX therapies may diminish the baseline activity of plasma lipids as compared to untreated children with SCD. In addition, sPLA2-treatment of the plasma 12 hours prior to the devlopment of ACS increased the amount of bioactive lipids released. Moreover, the amount of lipid priming activity was significantly increased during ACS as compared to baseline and during VOC. These preliminary results support the concept that the pathogenesis of ACS involves sPLA2 activity, which generates bioactive lipids that may provoke ACS, and need to be extended through the recruitment of more patients and completion of a multi-institutional trial.Lipid priming activity in children with SCDGroup/Clinical StatusBaselineVOCACSSCD1.8±0.81.3±0.12.3±0.2†SCD + HU1.6±0.21.4±0.3no dataSCD + EX1.5±0.21.8±0.2no dataUnits are nmol O2−/min; † = p≤0.05 versus Baseline and VOC.

D 5 dopamine receptor regulation of reactive oxygen species production and blood pressure in mice

OR-45 Key Words: D5 Receptor, Reactive Oxygen Species, Hypertension

Inhibition of Na-K-ATPase in thick ascending limbs by NO depends on O2- and is diminished by a high-salt diet.

A high-salt diet enhances nitric oxide (NO)-induced inhibition of transport in the thick ascending limb (THAL). Long exposures to NO inhibit Na-K-ATPase in cultured cells. We hypothesized that NO inhibits THAL Na-K-ATPase after long exposures and a high-salt diet would augment this effect. Rats drank either tap water or 1% NaCl for 7-10 days. Na-K-ATPase activity was assessed by measuring ouabain-sensitive ATP hydrolysis by THAL suspensions. After 2 h, spermine NONOate (SPM; 5 microM) reduced Na-K-ATPase activity from 0.44 +/- 0.03 to 0.30 +/- 0.04 nmol P(i).microg protein(-1).min(-1) in THALs from rats on a normal diet (P < 0.03). Nitroglycerin also reduced Na-K-ATPase activity (P < 0.04). After 20 min, SPM had no effect (change -0.07 +/- 0.05 nmol P(i).microg protein(-1).min(-1)). When rats were fed high salt, SPM did not inhibit Na-K-ATPase after 120 min. To investigate whether ONOO(-) formed by NO reacting with O(2)(-) was involved, we measured O(2)(-) production. THALs from rats on normal and high salt produced 35.8 +/- 0.3 and 23.7 +/- 0.8 nmol O(2)(-).min(-1).mg protein(-1), respectively (P < 0.01). Because O(2)(-) production differed, we studied the effects of the O(2)(-) scavenger tempol. In the presence of 50 microM tempol, SPM did not inhibit Na-K-ATPase after 120 min (0.50 +/- 0.05 vs. 0.52 +/- 0.07 nmol P(i).microg protein(-1).min(-1)). Propyl gallate, another O(2)(-) scavenger, also prevented SPM-induced inhibition of Na-K-ATPase activity. SPM inhibited pump activity in tubules from rats on high salt when O(2)(-) levels were increased with xanthine oxidase and hypoxanthine. We concluded that NO inhibits Na-K-ATPase after long exposures when rats are on a normal diet and this inhibition depends on O(2)(-). NO donors do not inhibit Na-K-ATPase in THALs from rats on high salt due to decreased O(2)(-) production.

Individual variation in the rate of oxygen consumption by zebrafish embryos

A sensitive microsensor‐based method was used to measure oxygen consumption of individual zebrafish Danio rerio embryos at 6 h intervals from 24 to 75 h post‐fertilization. An increase in oxygen consumption rates from 4·54 to 8·29 nmol O2 h−1 was found during this period. At the individual level the differences in oxygen consumption rates caused the total oxygen consumption from 24 to 75 h post‐fertilization to vary between 0·261 and 0·462 μmol O2 per individual with a mean of 0·379 μmol O2 per individual. A separate carbon mass balance study corroborated the mean total oxygen consumption obtained by yielding a respiratory quotient of 0·80 for this period. These results suggest that there is significant intraspecific variation in the metabolic rate of developing zebrafish embryos, which may influence other early life‐history traits such as growth and starvation resistance.

Energetic cost of development through metamorphosis for the seastar Mediaster aequalis (Stimpson)

Rates of oxygen consumption were measured for embryos, larvae and juveniles of the seastar Mediaster aequalis for 76 days post-fertilization. The rate increased from 0.65 nmol O2 ind−1 h−1 at 6 h after fertilization to 2.8 nmol O2 ind−1 h−1 at day 35. Larvae became competent to metamorphose around day 35 post-fertilization and began to decrease their metabolic rate after this time. Metamorphosed juveniles consumed 0.74 nmol O2 ind−1 h−1. Eggs contained 138.6 µg lipid ind−1 and 12.1 µg protein ind−1. Lipid levels decreased in concentration throughout development while protein levels increased slightly. The lipid levels decreased by 88.5 µg from eggs to day 76 larvae, accounting for 3.5 J of energy. Total oxygen consumption to this point was 3.74 µmol O2 ind−1, accounting for 1.84 J. The energetic demand up to day 76 was met completely through the use of lipid reserves. Metamorphosed juveniles expended 0.5 J more than larvae at the same age. Tubes of the polychaete Phyllochaetopterus prolifica were able to induce metamorphosis in M. aequalis larvae and a non-polar extract of these tubes also triggered metamorphosis. Larvae that are delayed to metamorphose can sustain their metabolic rate with lipid reserves for a limited, yet undetermined, period.

Gut-derived leukotriene B4 following hemorrhagic shock is a pivotal mediator of neutrophil priming

Introduction: The lipid fraction of mesenteric lymph collected after hemorrhagic shock (HS) and reperfusion primes the neutrophil oxidative burst in vitro. Recently, we have identified the culprit agent to be a neutral lipid consistent with Leukotriene B4 (LTB4). We hypothesized that plasma LTB4 increases after HS and that diversion of mesenteric lymph (DML) abrogates this effect. Furthermore, we posited that administration of an LTB4 antagonist blocks the priming effect of mesenteric lymph on neutrophils (PMNs). Methods: Adult male Sprague-Dawley rats (325–375 g) underwent laparotomy with cannulation of the mesenteric lymphatics for lymph diversion (DML) or laparotomy without mesenteric cannulation (Sham). Additionally, DML rats had lymph collected both pre- and post-shock. All rats underwent cannulation of the femoral artery and vein. HS to a MAP of 30 mmHg for 45 minutes was achieved via venous exsanguination. Animals were resuscitated with shed blood and with 2x the shed blood volume of NS over two hours. After resuscitation, plasma was collected for LTB4 measurement by ELISA. Lymph collected in the resuscitation phase was added to isolated PMNs that were then measured for superoxide release in the presence and absence of the LTB4 receptor antagonist CP-105,696. Results: LTB4 concentration in the lymph collected from the DML group measured 281.25 ± 53.81 ng/ml pre-shock, increasing to 692.13 ± 176.91 ng/ml post-shock (p < 0.05). LTB4 plasma concentrations after resuscitation differed based on lymph diversion. LTB4 concentration for DML animals measured 2745 ± 214.77 pg/ml vs. Sham animals, 4012 ± 628.93 pg/ml (p < 0.05). PMN pre-treatment with the LTB4 receptor antagonist completely abrogated superoxide release in response to lymph exposure compared with the untreated control, 1.39 ± 0.23 vs. 4.22 ± 0.68 nmol O2−/3.75 x 105 cells/mL/min respectively (p < 0.05). Conclusion: These data suggest that splanchnic ischemia secondary to HS increases concentrations of the known priming agent LTB4 in mesenteric lymph and the systemic circulation. Furthermore, selective blockade of LTB4 markedly attenuates the effect of mesenteric lymph on PMN priming, indicating a potential therapeutic location for disruption of the dysfunctional PMN priming/activation sequence.

Xylem sap flow as a major pathway for oxygen supply to the sapwood of birch (Betula pubescens Ehr.)

ABSTRACTThe role of xylem sap flow as an aqueous pathway for oxygen supply to the wood parenchyma of Betula pubescens saplings was investigated. Using micro‐optode sensors the oxygen status of the sapwood was quantified in relation to mass flow of xylem sap. Sap flow was gradually reduced by an increasing oxygen depletion in the root space. The effect of sap flow on radial O2 transport between stem and atmosphere was assessed by a stoichiometrical approach between respiratory CO2 production and O2 consumption. Restriction of sap flow set in 36.5 h after the onset of O2 depletion, and was complete after 71 h. Interruption of sap flow drastically increased the O2 deficit in the sapwood to 70%. Sap flow contributed about 60% to the total oxygen supply to the sapwood. Diurnal O2 flow rates varied between 3 and 6.3 nmol O2 m−2 leaf area (LA) s−1 during night‐ and daytime, respectively. Maximum O2 flow rates of 20 nmol O2 m−2 LA s−1 were reached at highest sap flow rates of 5.7 mmol H2O m−2 LA s−1. Sap flow not only affected the oxygen status of the sapwood but also had an effect on radial O2 transport between stem and atmosphere.

Diffusive boundary layers and photosynthesis of the epilithic algal community of coral reefs

The effects of mass transfer resistance due to the presence of a diffusive boundary layer on the photosynthesis of the epilithic algal community (EAC) of a coral reef were studied. Photosynthesis and respiration of the EAC of dead coral surfaces were investigated for samples from two locations: the Gulf of Aqaba, Eilat (Israel), and One Tree Reef on the Great Barrier Reef (Australia). Microsensors were used to measure O2 and pH at the EAC surface and above. Oxygen profiles in the light and dark indicated a diffusive boundary layer (DBL) thickness of 180–590 μm under moderate flow (~0.08 m s−1) and >2,000 μm under quasi-stagnant conditions. Under light saturation the oxygen concentration at the EAC surface rose within a few minutes to 200–550% air saturation levels under moderate flow and to 600–700% under quasi-stagnant conditions. High maximal rates of net photosynthesis of 8–25 mmol O2 m−2 h−1 were calculated from measured O2 concentration gradients, and dark respiration was 1.3–3.3 mmol O2 m−2 h−1. From light–dark shifts, the maximal rates of gross photosynthesis at the EAC surface were calculated to be 16.5 nmol O2 cm−3 s−1. Irradiance at the onset of saturation of photosynthesis, E k, was <100 µmol photons m−2 s−1, indicating that the EAC is a shade-adapted community. The pH increased from 8.2 in the bulk seawater to 8.9 at the EAC surface, suggesting that very little carbon in the form of CO2 occurs at the EAC surface. Thus the major source of dissolved inorganic carbon (DIC) must be in the form of HCO3 −. Estimates of DIC fluxes across the DBL indicate that, throughout most of the daytime under in situ conditions, DIC is likely to be a major limiting factor for photosynthesis and therefore also for primary production and growth of the EAC.

Electron spin resonance characterization of vascular xanthine and NAD(P)H oxidase activity in patients with coronary artery disease: relation to endothelium-dependent vasodilation.

Increased inactivation of nitric oxide by superoxide (O2*-) contributes to endothelial dysfunction in patients with coronary disease (CAD). We therefore characterized the vascular activities of xanthine oxidase and NAD(P)H oxidase, 2 major O2*--producing enzyme systems, and their relationship with flow-dependent, endothelium-mediated vasodilation (FDD) in patients with CAD. Xanthine- and NAD(P)H-mediated O*.- formation was determined in coronary arteries from 10 patients with CAD and 10 controls by using electron spin resonance spectroscopy. Furthermore, activity of endothelium-bound xanthine oxidase in vivo and FDD of the radial artery were determined in 21 patients with CAD and 10 controls. FDD was measured before and after infusion of the antioxidant vitamin C (25 mg/min i.a.) to determine the portion of FDD inhibited by radicals. In coronary arteries from patients with CAD, xanthine- and NAD(P)H-mediated O2*- formation was increased compared with controls (xanthine: 12+/-2 versus 7+/-1 nmol O2*-/ microg protein; NADH: 11+/-1 versus 7+/-1 nmol O2*-/ microg protein; and NADPH: 12+/-2 versus 9+/-1 nmol O2*-/ microg protein; each P<0.05). Endothelium-bound xanthine oxidase activity was increased by >200% in patients with CAD (25+/-4 versus 9+/-1 nmol O2*-/ microL plasma per min; P<0.05) and correlated inversely with FDD (r=-0.55; P<0.05) and positively with the effect of vitamin C on FDD (r=0.54; P<0.05). The present study represents the first electron spin resonance measurements of xanthine and NAD(P)H oxidase activity in human coronary arteries and supports the concept that increased activities of both enzymes contribute to increased vascular oxidant stress in patients with CAD. Furthermore, the present study suggests that increased xanthine oxidase activity contributes to endothelial dysfunction in patients with CAD and may thereby promote the atherosclerotic process.

A novel water-soluble and cell-permeable calpain inhibitor protects myocardial and mitochondrial function in postischemic reperfusion.

The effects of the novel calpain inhibitor A-705239 were studied in isolated perfused rabbit hearts subjected to 45 min of global ischemia, followed by 60 min of reperfusion. During 15 min of perfusion the inhibitor accumulated in myocardial tissue up to 16 times the concentration in the perfusate. Almost complete recovery and survival of heart function (90%) was seen with an inhibitor concentration of 10(-8) M in the perfusion fluid when the compound was administered prior to ischemia. Left ventricular pressure amplitude and coronary flow showed significantly higher values during reperfusion in the presence of the inhibitor. A-705239 significantly reduced the release of creatine kinase, from 166+/-49 U/l in untreated hearts to 44+/-10 U/l, and diminished the release of lactate dehydrogenase from 118+/-20 U/l in untreated hearts to 63+/-4 U/l. Mitochondrial dysfunction following ischemia and reperfusion was markedly attenuated by the inhibitor. Thus, the state 3 respiration rate only decreased to 4.2 in contrast to 2.6 nmol O2/(min x mg s.w.) in untreated hearts, reflecting a reduced damage of oxidative phosphorylation. Furthermore, in the presence of the inhibitor the inner mitochondrial membranes became less permeable as indicated by a smaller leak respiration. The excellent properties of A-705239 should make this compound a valuable tool for further pharmacological studies.

Extracellular signal-regulated kinase inhibition by statins inhibits neutrophil activation by ANCA

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) may modulate cellular inflammatory functions independent of serum cholesterol. We tested the hypothesis that statins decrease respiratory burst activity of human polymorphonuclear neutrophils (PMN) in response to anti-neutrophil cytoplasmic antibodies (ANCA). Neutrophils were isolated from healthy human volunteers, human immunoglobulins were isolated from patients with proteinase-3 (PR3)- and myeloperoxidase (MPO)-ANCA. Superoxide generation was measured by the ferricytochrome C assay and the nitro blue tetrazolium (NBT) test. ANCA antigen expression was measured by flow cytometry and phosphorylation of mitogen-activated protein kinase (MAPK) was assessed by Western blotting. Cerivastatin and simvastatin inhibited respiratory burst activity to ANCA dose-dependently (1 to 25 micromol/L). Tumor necrosis factor-alpha (TNF-alpha)-primed neutrophils released 26.7 +/- 2.8 nmol O2-/0.75 x 106 PMN/45 min and 10 micromol/L simvastatin reduced this amount to 18.0 +/- 2.1 nmol. The inhibitory effect was confirmed by the NBT test. The respiratory burst decrease could not be reversed by 500 micromol/L mevalonic acid (MVA). In this assay, both statins also inhibited the response to human ANCA. PR3-ANCA resulted in 19.4 +/- 2.0 nmol O2- nmol. This amount was decreased to 6.0 +/- 1.2 nmol by preincubation with 10 micromol/L simvastatin (P < 0.01). For MPO-ANCA, the values were 22.6 +/- 2.8 nmol for controls versus 16.7 +/- 3.1 nmol with statin (P < 0.01). By FACS, simvastatin decreased TNF-alpha-mediated ANCA antigen translocation (from 219 +/- 33 to 180 +/- 35 MFI for PR3 and 24.0 +/- 2.4 to 18.3 +/- 1.1 for MPO). Finally, since p38 MAPK and ERK control TNF-alpha priming, we studied the effects of both statins on MAPK. Western blotting showed that statins inhibited TNF-alpha-induced ERK phosphorylation in a dose dependent fashion, but had no effect on p38. These findings demonstrate that HMG-CoA reductase inhibitors decrease respiratory burst activity of human PMN in response to ANCA. This effect was independent of mevalonate, but involved inhibition of ERK activation during TNF-alpha priming. Our data suggest that HMG-CoA reductase inhibitors may help limit inflammatory responses.

A new microcosm to investigate oxygen dynamics at the sea ice water interface

A laboratory sea ice microcosm was developed to enable the cultivation of the ice diatom Fragilariopsis cylindrus in the skeletal layer and bottom 10 cm of sea ice. Growth of diatoms was ensured by continuous flow of new medium beneath the ice. Light was provided from above by a metal halide lamp to simulate a typical natural daylight irradiance spectrum. Oxygen micro-optodes were deployed in the microcosm to measure micro-profiles through the ice water interface and between the ice lamellae of the skeletal layer. Net oxygen production at the ice water interface, at an irradiance of 40 µmol photons m-2 s-1 and -1.9°C, ranged between 0.0064 and 0.0225 nmol O2 cm-2 s-1. Algal biomass increased from 0.03 µg chlorophyll a (chl a) l-1 in the column interior to 42 µg chl a l-1 within 5 mm of the ice water interface. Oxygen micro-profiles revealed diffusive boundary layers (DBLs) which varied between ca. 460 and 1000 µm. DBLs were detected between ice lamellae, the periphery of the ice water interface and extending from the water below the ice through the ice water interface into the spaces between ice lamellae. An additional small-scale horizontal variability of DBLs was also reflected in the net photosynthetic activity. The small-scale patchiness of algae and the differences in DBL thickness were caused by physico-chemical processes (e.g. turbulence, water flow velocity), which in turn were influenced by ice lamellar structure at the ice water interface. These factors were the grounds for the observed variability in net-photosynthesis.

Impact of aging on substrate metabolism by the human heart

BackgroundResults of studies in experimental animals have shown that, with age, myocardial fatty acid metabolism decreases, and glucose metabolism increases. Whether similar changes occur in humans is unknown. MethodsSeventeen healthy younger normal volunteers (six males, 26 ± 5 years) and 19 healthy older volunteers (nine males, 67 ± 5 years) underwent positron emission tomography (PET) under resting conditions in the fasted state. Myocardial blood flow (MBF), myocardial oxygen consumption (MVO2), myocardial fatty acid utilization (MFAU) and oxidation (MFAO), and myocardial glucose utilization (MGU) were quantified by PET with 15O-water, 11C-acetate, 11C-palmitate, and11C-glucose, respectively. ResultsAlthough MBF was similar between the groups, MVO2was higher in the older subjects (5.6 ± 1.6 μmol/g/min) compared with younger subjects (4.6 ± 1.0 μmol/g/min, p < 0.04). Rates of MFAU and MFAO (corrected for MVO2) were significantly lower in older subjects than in younger subjects (MFAU/MVO2: 35 ± 10 vs. 51 ± 20 nmol free fatty acids (FFA)/nmol O2× 10−3, p < 0.005, and MFAO/MVO2: 33 ± 10 vs. 48 ± 18 nmol FFA/nmol O2× 10−3, p < 0.004). In contrast, the rates of MGU corrected for MVO2did not differ between the groups. ConclusionsWith aging, humans exhibit a decline in MFAU and MFAO. Although absolute rates of MGU do not increase, by virtue of the decline in MFAU there is likely an increase in relative contribution of MGU to substrate metabolism. The clinical significance of this metabolic switch awaits further study.

Stimulation of potato tuber respiration by cold stress is associated with an increased capacity of both plant uncoupling mitochondrial protein (PUMP) and alternative oxidase.

The CO2 evolution of intact potato tubers (Solanum tuberosum, L., var. "Bintje") was analyzed during a 10-day period of their warm (25 +/- 2 degrees C) or cold (5 +/- 1 degrees C) storage, to evaluate cold-stress effects on expression and activities of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX). CO2 evolution rates were analyzed at 20 degrees C, to reflect their possible capacities. The 20 degrees C CO2 production declined from 13 to 8 mg kg(-1) h(-1) after 2 days of warm storage and then (after 3 to 7 days) decreased from 8 to 6.5 mg kg(-1) h(-1). In contrast, 20 degrees C CO2 evolution did not change after the first day of cold storage, increased up to 14.5 mg kg(-1) h(-1) after 2 days, and decreased to about 12 mg kg(-1) h(-1) after 3 to 7 days of cold storage. Cold storage increased PUMP expression as detected by Western blots and led to elevated capacities of both PUMP (44%) and CN-resistant AOX (10 times), but not the cytochrome pathway. Since we found that cold storage led to about the same mitochondrial respiration of 40 nmol O2 min(-1) mg(-1) attributable to each of the respective proteins, we conclude that both AOX and PUMP equally contribute to adaptation of potato tubers to cold.

Respiration of steelhead trout sperm: sensitivity to pH and carbon dioxide

Steelhead trout Oncorhynchus mykiss sperm held in seminal plasma or sperm‐immobilizing buffer (pH 8·6) at 10° C consumed O2 at the rate of c. 2 nmol O2 min−1 10−9 sperm; the rate of O2 consumption was not different in sperm held for 4 or 24 h. Decreasing the extracellular pH from 8·5 to 7·5 either by diluting semen with buffer titrated with HCl or by increasing the partial pressure of CO2 in the incubation atmosphere resulted in c. a 40% decrease in the rate of sperm respiration. The data did not, however, support the hypothesis that the precipitous reduction in the capacity for sperm motility that occurs as external pH is reduced is a result of a decrease in cellular metabolism. The rate of O2 consumption of freshly collected semen from different males was not correlated to cellular ATP content or to the proportion of sperm that were motile upon activation; the initial ATP content and sperm motility were positively correlated. The rate of O2 consumption was not significantly increased following sperm activation or by the addition of an uncoupler of oxidative phosphorylation, carbonyl cyanide p‐trifluoromethoxyphenylhydrazone, suggesting that these sperm have little, if any, capacity for increased oxidative metabolism.

Vascular Oxidative Stress and Endothelial Dysfunction in Patients With Chronic Heart Failure

Impaired flow-dependent, endothelium-mediated vasodilation (FDD) in patients with chronic heart failure (CHF) results, at least in part, from accelerated degradation of nitric oxide by oxygen radicals. The mechanisms leading to increased vascular radical formation, however, remain unclear. Therefore, we determined endothelium-bound activities of extracellular superoxide dismutase (ecSOD), a major vascular antioxidant enzyme, and xanthine-oxidase, a potent radical producing enzyme, and their relation to FDD in patients with CHF. ecSOD and xanthine-oxidase activities, released from endothelium into plasma by heparin bolus injection, were determined in 14 patients with CHF and 10 control subjects. FDD of the radial artery was measured using high-resolution ultrasound and was assessed before and after administration of the antioxidant vitamin C (25 mg/min; IA). In patients with CHF, endothelium-bound ecSOD activity was substantially reduced (5.0+/-0.7 versus 14.4+/-2.6 U x mL(-1) x min(-1); P<0.01) and closely related to FDD (r=0.61). Endothelium-bound xanthine-oxidase activity was increased by >200% (38+/-10 versus 12+/-4 nmol O2*- x microL(-1); P<0.05) and inversely related to FDD (r=-0.35) in patients with CHF. In patients with low ecSOD and high xanthine-oxidase activity, a greater benefit of vitamin C on FDD was observed, ie, the portion of FDD inhibited by radicals correlated negatively with ecSOD (r=-0.71) but positively with xanthine-oxidase (r=0.75). These results demonstrate that both increased xanthine-oxidase and reduced ecSOD activity are closely associated with increased vascular oxidative stress in patients with CHF. This loss of vascular oxidative balance likely represents a novel mechanism contributing to endothelial dysfunction in CHF.