Inhibition Of O2 Consumption Research Articles

Restricted O2 consumption in pea roots induced by hexanoic acid is linked to depletion of Krebs cycle substrates.

Plant roots are exposed to hypoxia in waterlogged soils, and they are further challenged by specific phytotoxins produced by microorganisms in such conditions. One such toxin is hexanoic acid (HxA), which, at toxic levels, causes a strong decline in root O2 consumption. However, the mechanism underlying this process is still unknown. We treated pea (Pisum sativum L.) roots with 20 mM HxA at pH 5.0 and 6.0 for a short time (1 h) and measured leakage of key electrolytes such as metal cations, malate, citrate and nonstructural carbohydrates (NSC). After treatment, mitochondria were isolated to assess their functionality evaluated as electrical potential and O2 consumption rate. HxA treatment resulted in root tissue extrusion of K+ , malate, citrate and NSC, but only the leakage of the organic acids and NSC increased at pH 5.0, concomitantly with the inhibition of O2 consumption. The activity of mitochondria isolated from treated roots was almost unaffected, showing just a slight decrease in oxygen consumption after treatment at pH 5.0. Similar results were obtained by treating the pea roots with another organic acid with a short carbon chain, that is, butyric acid. Based on these results, we propose a model in which HxA, in its undissociated form prevalent at acidic pH, stimulates the efflux of citrate, malate and NSC, which would, in turn, cause starvation of mitochondrial respiratory substrates of the Krebs cycle and a consequent decline in O2 consumption. Cation extrusion would be a compensatory mechanism in order to restore plasma membrane potential.

Boosting intracellular sodium selectively kills hepatocarcinoma cells and induces hepatocellular carcinoma tumor shrinkage in mice

Pharmacological treatments for advanced hepatocellular carcinoma (HCC) have a partial efficacy. Augmented Na+ content and water retention are observed in human cancers and offer unexplored targets for anticancer therapies. Na+ levels are evaluated upon treatments with the antibiotic cation ionophore Monensin by fluorimetry, ICP-MS, 23Na-MRI, NMR relaxometry, confocal or time-lapse analysis related to energy production, water fluxes and cell death, employing both murine and human HCC cell lines, primary murine hepatocytes, or HCC allografts in NSG mice. Na+ levels of HCC cells and tissue are 8-10 times higher than that of healthy hepatocytes and livers. Monensin further increases Na+ levels in HCC cells and in HCC allografts but not in primary hepatocytes and in normal hepatic and extrahepatic tissue. The Na+ increase is associated with energy depletion, mitochondrial Na+ load and inhibition of O2 consumption. The Na+ increase causes an enhancement of the intracellular water lifetime and death of HCC cells, and a regression and necrosis of allograft tumors, without affecting the proliferating activity of either HCCs or healthy tissues. These observations indicate that HCC cells are, unlike healthy cells, energetically incapable of compensating and surviving a pharmacologically induced Na+ load, highlighting Na+ homeostasis as druggable target for HCC therapy.

Abstract LB078: Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) inhibits hormone therapy resistant breast cancer cells, in part by Inhibiting CYP3A4 arachidonic acid epoxygenase activity

Abstract Introduction: Small molecule therapeutics of estrogen receptor-positive/HER2-negative breast cancer remains an area of active investigation where novel agents are greatly needed for treatment of hormone therapy resistant metastatic disease. The biguanide hexyl-benzyl-biguanide (HBB) is a potent inhibitor of CYP3A4 arachidonic acid (AA) epoxygenase activity and inhibits breast cancer cell proliferation and MCF-7 breast cancer tumor growth in nude mice. To explore the impact of bioisosteric substitution of the benzyl moiety of HBB with a cubane moiety, we synthesized hexyl-(cuban-1-yl-methyl)-biguanide (HCB) and tested its potency for the inhibition of the cognate CYP3A4 target AA epoxygenase activity as well as breast cancer cell proliferation of hormone therapy sensitive and resistant cell lines. Results: HCB selectively inhibited CYP3A4-mediated biosynthesis of (±)-14,15-EET with an IC50 of 4.7±0.2 uM vs. 64.8±6.5 uM for 8,9-EET and 26.5±1.9 uM for 11,12-EET. At 24 hours, HCB inhibited proliferation of MCF-7 (ER+HER2-), BT474 (ER+HER2+) and MDA-MB-231 (ER-HER2-) cells at IC50 of 8.4±1.2, 11±1.3 and 15±0.9 uM, respectively. At 48 hours, HCB inhibited proliferation of aromatase inhibitor and fulvestrant resistant (LR,FR), and cyclin dependent kinase inhibitor (CDKi) palbociclib resistant (LR,FR,PR) MCF-7 cell lines; LR,FR MCF-7AC1 (IC50 =1.34±0.1 uM) and LR,FR,PR MCF-7AC1 (IC50 =1.64±0.2 uM). Addition of 14,15-EET (1 uM) partially rescues MCF-7 cells from HCB-mediated inhibition of proliferation. OXPHOS is promoted, in part, by EETs. HCB is a potent OXPHOS inhibitor and rapidly inhibits O2 consumption of the MCF-7 and ZR75 (ER+HER2-) cells in a dose-dependent fashion (P<0.05). HCB treatment (10 uM) reduces mitochondrial membrane potential to 57.4±15.3% (P<0.001) of vehicle control in MCF-7 cells. Treatment with HCB at 20 uM for 0.5 hour also causes mitochondrial swelling in MCF-7 cells. HCB (10 uM) activates AMPK within 0.5 hour and increases the level of phosphorylation from 2.4±0.3 to 25.1±6.0 folds in a time dependent fashion in MCF-7 cells from 0.5-24 hours. Conclusion: These results show that HCB inhibits proliferation of ER+HER2- breast cancer cells, in part through inhibition of OXPHOS and suppression of the CYP product 14,15-EET. This inhibition is highly active in hormonal therapy and CDKi resistant ER+HER2- breast cancer cells. These results suggest that HCB is a novel and potent biguanide that has potential to be developed for inhibition of hormone therapy resistant and CDKi resistant breast cancer. Citation Format: Zhijun Guo, Jianxun Lei, Kwon Ho Hong, Beverly Norris, Craig M. Flory, Swaathi Jayaraman, Connor McDermott, Elizabeth Ambrose, Irina Sevrioukova, Tom Poulos, Ilia Denisov, Stephen Sliga, Robert J. Schumacher, Gunda I. Georg, John R. Hawse, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) inhibits hormone therapy resistant breast cancer cells, in part by Inhibiting CYP3A4 arachidonic acid epoxygenase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB078.

Mutational analysis of the Qi-site proton pathway in yeast cytochrome bc1 complex

The respiratory cytochrome bc1 complex functions as a protonmotive ubiquinol:cytochrome c oxidoreductase. Lysine 228 (K228) located within the quinol reduction (Qi) site of the bc1 complex, has been reported as a key residue for proton transfer during the redox chemistry cycle to substrate quinone at Qi. In yeast, while single mutations had no effect, the combination of K228L and F225L resulted in a severe respiratory growth defect and inhibition of O2 consumption in intact cells. The inhibition was overcome by uncoupling the mitochondrial membrane or by suppressor mutations in the region of K228L-F225L. We propose that the K228L mutation introduces energetic (and kinetic) barriers into normal electron- and proton transfer chemistry at Qi, which are relieved by dissipation of the opposing protonmotive force or through the restoration of favourable intraprotein proton transfer networks via suppressor mutation.

A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF target gene expression

Abnormal accumulation of oncometabolite fumarate and succinate is associated with inhibition of mitochondrial function and carcinogenesis. By competing with α-ketoglutarate, oncometabolites also activate hypoxia inducible factors (HIFs), which makes oncometabolite mimetics broadly utilised in hypoxia research. We found that dimethyloxalylglycine (DMOG), a synthetic analogue of α-ketoglutarate, commonly used to induce HIF signalling, inhibits O2 consumption in cancer cell lines HCT116 and PC12, well before activation of HIF pathways. A rapid suppression of cellular respiration was accompanied by a decrease in histone H4 lysine 16 acetylation and not abolished by double knockdown of HIF-1α and HIF-2α. In agreement with this, production of NADH and state 3 respiration in isolated mitochondria were down-regulated by the de-esterified DMOG derivative, N-oxalylglycine. Exploring the roles of DMOG as a putative inhibitor of glutamine/α-ketoglutarate metabolic axis, we found that the observed suppression of OxPhos and compensatory activation of glycolytic ATP flux make cancer cells vulnerable to combined treatment with DMOG and inhibitors of glycolysis. On the other hand, DMOG treatment impairs deep cell deoxygenation in 3D tissue culture models, demonstrating a potential to relieve functional stress imposed by deep hypoxia on tumour, ischemic or inflamed tissues. Indeed, using a murine model of colitis, we found that O2 availability in the inflamed colon tissue rapidly increased after application of DMOG, which could contribute to the known therapeutic effect of this compound. Overall, our results provide new insights into the relationship between mitochondrial function, O2 availability, metabolic reprogramming and associated diseases.

Antioxidant defences of Spironucleus vortens: Glutathione is the major non-protein thiol

The aerotolerant hydrogenosome-containing piscine diplomonad, Spironucleus vortens, is able to withstand high fluctuations in O2 tensions during its life cycle. In the current study, we further investigated the O2 scavenging and antioxidant defence mechanisms which facilitate the survival of S. vortens under such oxidizing conditions. Closed O2 electrode measurements revealed that the S. vortens ATCC 50386 strain was more O2 tolerant than a freshly isolated S. vortens intestinal strain (Sv1). In contrast to the related human diplomonad, Giardia intestinalis, RP-HPLC revealed the major non-protein thiols of S. vortens to be glutathione (GSH, 776nmol/107 cells) with cysteine and H2S as minor peaks. Furthermore, antioxidant proteins of S. vortens were assayed enzymatically and revealed that S. vortens possesses superoxide dismutase and NADH oxidase (883 and 37.5nmol/min/mg protein, respectively), but like G. intestinalis, lacks catalase and peroxidase activities. Autofluorescence of NAD(P)H and FAD alongside the fluorescence of the GSH-adduct in monochlorobimane-treated live organisms allowed the monitoring of redox balances before and after treatment with inhibitors, metronidazole and auranofin. H2O2 was emitted into the exterior of S. vortens at a rate of 2.85pmol/min/106 cells. Metronidazole and auranofin led to depletion of S. vortens intracellular NAD(P)H pools and an increase in H2O2 release with concomitant oxidation of GSH, respectively. Garlic-derived compounds completely inhibited O2 consumption by S. vortens (ajoene oil), or significantly depleted the intracellular GSH pool of the organism (allyl alcohol and DADS). Hence, antioxidant defence mechanisms of S. vortens may provide novel targets for parasite chemotherapy.

Pseudomonas aeruginosa quorum‐sensing homoserine lactone inhibits O2 consumption and stimulates H+ secretion by airway epithelia (869.2)

P. aeruginosa biofilms accumulate in the airways of cystic fibrosis patients and produce the quorum‐sensing molecule N‐(3‐oxododecanoyl)‐homoserine lactone (C12) that depolarizes mitochondria. We tested whether C12 and P. aeruginosa biofilms change O2 consumption and H+ secretion into the extracellular fluid of JME (CF nasal epithelia, ΔF508CFTR) and CFTR‐corrected JME cells studied in Seahorse X24‐well dishes. C12 caused dose‐dependent (min = 1 µM, max = 50 µM) inhibition of O2 consumption (to 0.6x control) and increases in H+ secretion (to 1.3x control), similar to effects of mitochondrial poisons antimycin + rotenone. As measured by pH‐stat, C12 increased H+ secretion into the apical solution of human tracheal epithelial (HTE) cells in primary culture by 0.3 µeq.cm‐2.hr‐1, similar to rates measured using Seahorse. H+ secretion by HTE cells was blocked by Zn2+. PAO1wt biofilms inhibited O2 consumption and increased H+ secretion by JME cells, while PAO1lasI biofilm and planktonic PAO1wt did not. JME and CFTR‐corrected JME cells behaved similarly during all treatments. These data indicated: (i) P. aeruginosa biofilms, most likely operating through C12, inhibit O2 consumption and stimulate H+ secretion by airway epithelia. (ii) H+ secretion occurs through a Zn2+‐sensitive H+ channel in the apical membrane. (iii) Although there is no direct role for CFTR in these processes, a downward spiral in CF is predicted: increased acidity in CF ASL (no HCO3 secretion) → reduced antimicrobial factors → P. aeruginosa accumulation in biofilms → inhibit mitochondrial metabolism and increase anaerobic glycolysis in epithelia → increased epithelial secretion of H+ → increased acidity in ASL → reduced antimicrobial factors → increased P. aeruginosa accumulation.Grant Funding Source: NIH, CFRI

Effects of Selected Inhibitors of Protein Kinases and Phosphatases on Cellular Respiration: An In Vitro Study

Inhibitors of protein kinases/phosphatases are known to alter cellular metabolism. Effects of these rapidly identified small molecules on cellular respiration (mitochondrial O2 consumption) have not been adequately investigated, especially in healthy organs. This in vitro study measured cellular respiration in tissues from C57BL/6 mice with and without GSK2126458 (PI3K/mTOR inhibitor), BEZ235 (PI3K/mTOR inhibitor), GDC0980 (PI3K/mTOR inhibitor), GSK1120212 (trametinib, MEK inhibitor), sorafenib, regorafenib (multikinase inhibitors), and cyclosporine (calcineurin inhibitor). Cellular respiration was measured by the phosphorescence oxygen analyzer, aided by the O2 probe Pd(II)-meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. Cyanide inhibited O2 consumption, confirming the oxidation occurred in the respiratory chain. Renal cellular respiration decreased 26-34% in the presence of 10 μM GSK2126458 (p<0.001), 10 μM BEZ235 (p<0.001), or 1.0 μM GDC0980 (p<0.001). Liver cellular respiration decreased 20-32% with 10 μM GSK2126458 (p=0.048), 0.1 μM BEZ235 (p=0.028), or 0.1 μM GDC0980 (p=0.016). Heart cellular respiration decreased 19-27% with 10 μM GSK2126458 (p=0.078), 10 μM BEZ235 (p=0.040), or 10 μM GDC0980 (p=0.036). GSK1120212, sorafenib, regorafenib, and cyclosporine had no effects on cellular respiration. Thus, cellular bioenergetics (the biochemical processes involved in energy conversion) is interconnected with PI3K/PTEN/Akt/mTOR; and inhibitors of this cascade impair cellular respiration. This biomarker (cellular respiration) senses the activity/toxicity of this class of molecularly targeted agents.

Inhibition of murine cardiomyocyte respiration by amine local anesthetics

The hydrophobic amino acyl amide-linked local anesthetics (e.g., lidocaine and bupivacaine) impose potent cardiac toxicity and direct mitochondrial dysfunction. To investigate these adverse events, an in vitro system was employed to measure their effects on O2 consumption (cellular respiration) by murine myocardium. Specimens were collected from the ventricular myocardium and immediately immersed in ice-cold Krebs-Henseleit buffer saturated with 95% O2:5% CO2. O2 concentration was determined as a function of time from the phosphorescence decay rates of Pd(II)-meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. Myocardial O2 consumption was linear with time (zero-order kinetics); its rate (k, in μMO2min(-1)), thus, was the negative of the slope of [O2] vs. time. Cyanide inhibited O2 consumption, confirming the oxidation occurred in the respiratory chain. Lidocaine and bupivacaine produced immediate and sustained inhibition of cellular respiration at plasma concentrations of the drugs (low micromolar range). Bupivacaine was twice as potent as lidocaine. The inhibition was dose-dependent, saturating at concentrations ≥30μM. At saturating doses, lidocaine produced ~20% inhibition and bupivacaine ~40% inhibition. Cellular ATP was also decreased in the presence of 30μM lidocaine or bupivacaine. The studied amines inhibited myocardial cellular respiration. This effect is consistent with their known adverse events on mitochondrial function. The described approach allows accurate assessments and comparisons of the toxic effects of local anesthetics on heart tissue bioenergetics.

Mitochondrial Oxygen Consumption by the Foreskin and its Fibroblast Rich Culture = إستهلاك الأوكسجين في المايتوكوندريا من قبل القلفة و الخلايا الليفية

This study investigated the feasibility of using a phosphorescence oxygen analyser to measure cellular respiration (mitochondrial O2 consumption) in foreskin samples and their fibroblast-rich cultures. Foreskin specimens from normal infants were collected immediately after circumcision and processed for measuring cellular respiration and for culture. Cellular mitochondrial O2 consumption was determined as a function of time from the phosphorescence decay of the Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. In sealed vials containing a foreskin specimen and glucose, O2 concentration decreased linearly with time, confirming the zero-order kinetics of O2 consumption by cytochrome oxidase. Cyanide inhibited O2 consumption, confirming that the oxidation occurred mainly in the mitochondrial respiratory chain. The rate of foreskin respiration (mean ± SD) was 0.074 ± 0.02 μM O2 min(-1) mg(-1) (n = 23). The corresponding rate for fibroblast-rich cultures was 9.84 ± 2.43 μM O2 min(-1) per 10(7) cells (n = 15). Fibroblast respiration was significantly lower in a male infant with dihydrolipoamide dehydrogenase gene mutations, but normalised with the addition of thiamine or carnitine. The foreskin and its fibroblast-rich culture are suitable for assessment of cellular respiration. However, the clinical utility of foreskin specimens to detect disorders of impaired cellular bioenergetics requires further investigation.

725 Lymphocyte Respiration in Children with Trisomy 21

AimsThis study aimed to measure lymphocyte mitochondrial O2 consumption (cellular respiration) in children with trisomy 21.MethodsPeripheral blood mononuclear cells were isolated from whole blood of trisomy 21 and control children...

Electron Paramagnetic Resonance Highlights That the Oxygen Effect Contributes to the Radiosensitizing Effect of Paclitaxel

BackgroundPaclitaxel (PTX) is a potent anti-cancer chemotherapeutic agent and is widely used in the treatments of solid tumors, particularly of the breast and ovaries. An effective and safe micellar formulation of PTX was used to administer higher doses of PTX than Taxol® (the current commercialized drug). We hypothesize that PTX-loaded micelles (M-PTX) may enhance tumor radiosensitivity by increasing the tumor oxygenation (pO2). Our goals were (i) to evaluate the contribution of the “oxygen effect” to the radiosensitizing effect of PTX; (ii) to demonstrate the therapeutic relevance of the combination of M-PTX and irradiation and (iii) to investigate the underlying mechanisms of the observed oxygen effect.Methodology and Principal FindingsWe used (PEG-p-(CL-co-TMC)) polymeric micelles to solubilize PTX. pO2 was measured on TLT tumor-bearing mice treated with M-PTX (80 mg/kg) using electron paramagnetic resonance (EPR) oximetry. The regrowth delay following 10 Gy irradiation 24 h after M-PTX treatment was measured. The tumor perfusion was assessed by the patent blue staining. The oxygen consumption rate and the apoptosis were evaluated by EPR oximetry and the TUNEL assay, respectively. EPR oximetry experiments showed that M-PTX dramatically increases the pO2 24 h post treatment. Regrowth delay assays demonstrated a synergy between M-PTX and irradiation. M-PTX increased the tumor blood flow while cells treated with M-PTX consumed less oxygen and presented more apoptosis.ConclusionsM-PTX improved the tumor oxygenation which leads to synergy between this treatment and irradiation. This increased pO2 can be explained both by an increased blood flow and an inhibition of O2 consumption.

The phosphorescence oxygen analyzer as a screening tool for disorders with impaired lymphocyte bioenergetics

This study aimed to show the feasibility of using the phosphorescence oxygen analyzer to screen for clinical disorders with impaired cellular bioenergetics. [O2] was determined as function of time from the phosphorescence decay of Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. In sealed vials, O2 consumption by peripheral blood mononuclear cells was linear with time, confirming its zero-order kinetics. Cyanide inhibited O2 consumption, confirming the oxidation occurred in the mitochondrial respiratory chain. The rate of respiration (mean±SD, in μM O2 per min per 107 cells, set as the negative of the slope of [O2] vs. t) for adults was 2.1±0.8 (n=18), for children 2.0±0.9 (n=20), and for newborns (umbilical cord samples) 0.8±0.4 (n=18), p<0.0001. For an 8-year-old patient with reduced NADH dehydrogenase and pyruvate dehydrogenase activities in the muscle, the rate was 0.7±0.2 (n=3) μM O2 per min per 107 cells. For a 3-month-old patient with hepatocerebral mitochondrial DNA depletion syndrome (MDS) with confirmed mutations in the MPV17 gene, the rate was 0.6μM O2 per min per 107 cells. For an18 month-old patient with MDS and confirmed mutations in the POLG gene, the rate was 0.5μM O2 per min per 107 cells. For a 6-year-old patient with MDS and confirmed mutations in the POLG gene, the rate was 0.6μM O2 per min per 107 cells. For 1-week-old patient with congenital lactic acidemia and hypotonia (confirmed mutations in DLD gene), the rate was 1.5μM O2 per min per 107 cells. For three siblings (9-year-old male, 8-year-old male and 2-month-old female) with congenital progressive myopathy, the rates were 0.9, 0.6 and 1.2μM O2 per min per 107 cells, respectively. Four patients with congenital lactic acidemia (with inadequate work-up) were also studied; their rates were 0.2, 1.5, 0.3 and 1.7μM O2 per min per 107 cells. This novel approach permits non-invasive, preliminary assessment of cellular bioenergetics. Potential applications and limitations of this technique are discussed.

Cigarette smoking impairs sperm bioenergetics

The growing consensus on the negative impact of cigarette smoking on fertility prompted us to compare the rate of sperm respiration in smokers and non-smokers. Semen samples from 20 smokers and 58 non-smokers consulting at the andrology laboratory for fertility evaluation were used. Smoking was defined as consumption of at least a half a pack per day. A phosphorescence analyzer that measures O(2) concentration in sperm suspensions as function of time was used to determine the rate of respiration. In a sealed vial, the rate of sperm respiration (k) was defined as -d[O(2)]/dt; where [O(2)] was obtained from the phosphorescence decay rate of a palladium phosphor. [O(2)] in solutions containing sperm and glucose declined linearly with time, showing the kinetics of O(2) consumption was zero-order. Inhibition of O(2) consumption by cyanide confirmed the oxidations that occurred in the sperm mitochondrial respiratory chain. There were no differences (p > 0.28) between smokers and non-smokers for ejaculate volume, motility, concentration, normal morphology, viability and hypo-osmotic swelling test. The rate (mean + or - SD, in microM O(2)/min/10(8) sperm) of sperm mitochondrial O(2) consumption in the smokers was 0.96 + or - 0.58 and in the non-smokers 1.39 + or - 0.67 (p = 0.004). The rate of sperm respiration was significantly lower in smokers. This negative impact of cigarette smoking on sperm aerobic metabolism may, in part, explain the lower rate of fertility in smokers.

Caffeine Impairs HL-60 Cellular Respiration

We investigated the effect of caffeine on mitochondrial O2 consumption in human promyelocytic leukemia (HL-60) cells. A phosphorescence analyzer that measures O2 concentrations in cell suspensions as function of time was used for this purpose. O2 concentrations were determined from the phosphorescence of Pd phosphor, calculated by fitting the phosphorescence decays to exponentials. In sealed vials, O2 concentrations in the cell suspensions containing glucose declined linearly with time, showing zero-order kinetics for O2 consumption. NaCN inhibited O2 consumption, confirming the oxidation occurred in the mitochondrial respiratory chain. A rapid decline in the rate of respiration was observed when 50 μM to 4.0 mM caffeine was added to HL-60 cells in cell growth media (containing 1.41 mM Ca2+) or phosphate-buffered-salts (containing 0.91 mM Ca2+). This reversible inhibition was blocked by verapamil and was concentration-dependent, reaching a plateau (43 + 7% inhibition) at 50 μM caffeine. The inhibition was not observed when cellular Ca2+ stores were depleted. T-cell lymphoma (Jurkat) cells and isolated mitochondria were less sensitive to caffeine. Thus, caffeine is a potent inhibitor of HL-60 respiration. This effect is possibly mediated by Ca2+-flooding into the cytosol and neighboring mitochondria.

Pretreatment of Highly Polluted Pharmaceutical Waste Broth by Wet Air Oxidation

The preoxidation of a highly polluted waste pharmaceutical fermentation broth using wet air oxidation (WAO) has been studied as a possible method for the effective removal of organics. The applied (pre)treatment method should enhance the biotreatability of the pharmaceutical fermentation broth in terms of reduced initial toxicity and higher biodegradability. Preliminary experiments in the pilot biological treatment plant were successful only at low organic loads, whereas the system collapses at higher ones. The characterization of the fermentation broth was started by common physicochemical analysis, whereas several bioassays were used to determine its impact on biological treatment plants and the environment. Toxicity prior to and after WAO was determined using the acute Vibrio fischeri test, measurement of inhibition of O2 consumption, and the Daphnia magna acute test. Ready biodegradability of the treated and untreated broth has also been assessed. WAO experiments were accomplished in the 2 L batch rea...

Endothelial nitric oxide synthase plays an essential role in regulation of renal oxygen consumption by NO.

Nitric oxide (NO) regulates renal O2 consumption, but the source of NO mediating this effect is unclear. We explored the effects of renal NO production on O2 consumption using renal cortex from mice deficient (-/-) in endothelial (e) nitric oxide synthase (NOS). O2 consumption was determined polarographically in slices of cortex from control and eNOS-/- mice. NO production was stimulated by bradykinin (BK) or ramiprilat (Ram) in the presence or absence of an NOS inhibitor. Basal O2 consumption was higher in eNOS-/- mice than in heterozygous controls (919 +/- 46 vs. 1,211 +/- 133 nmol O(2). min(-1). g(-1); P < 0.05). BK and Ram decreased O2 consumption significantly less in eNOS-/- mice [eNOS-/-: BK -19.0 +/- 2.8%, Ram -20.5 +/- 3.3% at 10(-4) M; control: BK -29.5 +/- 2.5%, Ram -34 +/- 1.6% at 10(-4) M]. The NO synthesis inhibitor nitro-L-arginine methyl ester (L-NAME) attenuated this decrease in control but not eNOS-/- mice. An NO donor inhibited O2 consumption similarly in both groups independent of the presence of L-NAME. These results demonstrate that NO production by eNOS is responsible for regulation of renal O2 consumption in mouse kidney.

Amino acids counteract the inhibitory effect of fentanyl on hepatocyte oxidative metabolism

Background/Purpose: Hypothermia is common after major surgery in newborns and can be triggered by intraoperative fentanyl analgesia. Recent studies have found that fentanyl inhibits hepatocyte mitochondrial oxidative metabolism, which is proportional to thermogenesis. In adults it has been shown that amino acids have a thermogenic effect, although the biochemical basis of this phenomenon is not known. The aim of this study was to test the hypothesis that amino acids counteract the inhibition of neonatal hepatocyte oxygen consumption by fentanyl. Methods: Hepatocytes were isolated from suckling rats, and O2 consumption was measured polarographically. In experiment A hepatocytes were incubated with (1) palmitate alone (control), (2) palmitate plus fentanyl, (3) palmitate plus fentanyl plus amino acids, and (4) palmitate plus amino acids. In experiment B the effects of essential and nonessential amino acids were tested separately. In experiment C, to investigate whether the effect of amino acids is intramitochondrial, hepatocytes were incubated with amino acids plus inhibitors of mitochondrial respiration. Results: In experiment A, fentanyl significantly inhibited O2 consumption (P =.006). This inhibition was reversed by amino acids (P <.001). In experiment B, both essential and nonessential amino acids reversed the effect of fentanyl (P <.001). In experiment C, there was no difference in O2 consumption in the presence of myxothiazol among the groups indicating that amino acids affect intramitochondrial O2 consumption. Conclusions: (1) Amino acids abolish the inhibitory effect of fentanyl on hepatocyte oxidative metabolism. (2) Amino acids affect intramitochondrial O2 consumption and therefore thermogenesis. (3) Perioperative administration of amino acids in neonates may help to prevent hypothermia and its deleterious effects. J Pediatr Surg 35:736-739. Copyright © 2000 by W.B. Saunders Company.

Cellular localization of the inhibitory action of abruquinone A against respiratory burst in rat neutrophils.

1. The possible mechanisms of action of the inhibitory effect of abruquinone A on the respiratory burst in rat neutrophils in vitro was investigated. 2. Abruquinone A caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2.-) generation with IC50 values of 0.33 +/- 0.05 microgram ml-1 and 0.49 +/- 0.04 microgram ml-1, respectively. 3. Abruquinone A also inhibited O2 consumption in neutrophils in response to fMLP/CB and PMA. However, abruquinone A did not scavenge the generated O2.- in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation. 4. Abruquinone A inhibited both the transient elevation of [Ca2+]i in the absence of [Ca2+]o (IC50 7.8 +/- 0.2 micrograms ml-1) and the generation of inositol trisphosphate (IP3) (IC50 10.6 +/- 2.0 micrograms ml-1) in response to fMLP. 5. Abruquinone A did not affect the enzyme activaties of neutrophil cytosolic protein kinase C (PKC) and porcine heart protein kinase A (PKA). 6. Abruquinone A had no effect on intracellular guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels but decreased the adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 7. The cellular formation of phosphatidic acid (PA) and phosphatidylethanol (PEt) induced by fMLP/ CB was inhibited by abruquinone A with IC50 values of 2.2 +/- 0.6 micrograms ml-1 and 2.5 +/- 0.3 micrograms ml-1, respectively. Abruquinone A did not inhibit the fMLP/CB-induced protein tyrosine phosphorylation but induced additional phosphotyrosine accumulation on proteins of 73-78 kDa in activated neutrophils. 8. Abruquinone A inhibited both the O2.- generation in PMA-activated neutrophil particulate NADPH oxidase (IC50 0.6 +/- 0.1 microgram ml-1) and the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free system (IC50 1.5 +/- 0.2 micrograms ml-1) 9. Collectively, these results indicate that the inhibition of respiratory burst in rat neutrophils by abruquinone A is mediated partly by the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways, and by suppressing the function of NADPH oxidase through the interruption of electron transport.

Azide‐stimulated oxygen consumption by the green alga Selenastrum minutum

Dark O2 consumption by the green alga Selenastrum minutum was sensitive to inhibition by the cytochrome pathway respiration inhibitor cyanide in the absence of an alternative oxidase inhibitor, consistent with previous work that suggested that this alga lacks alternative oxidase capacity. In contrast, addition of low concentrations of the cytochrome pathway inhibitor azide (50–750 μM) resulted in a stimulation of dark O2 consumption, while higher concentrations of azide (1–2 mM) partially inhibited O2 consumption. Measurements of changes in cellular levels of pyruvate, malate and pyridine nucleotides upon cyanide addition were consistent with the absence of alternative oxidase capacity, and suggested that cyanide inhibition of O2 consumption was not due to nonspecific effects of cyanide. Addition of salicylhydroxamic acid (SHAM) also resulted in an increase in the rate of O2 consumption. Both azide‐ and SHAM‐stimulated O2 consumption were sensitive to inhibition by 50 mM ascorbate or by cyanide. However, the ubiquinone analogs chloroquine and quinacrine specifically inhibited azide‐stimulated O2 consumption, with only minor effects on SHAM‐stimulated O2 consumption. These results suggest that azide‐stimulated O2 consumption was not mediated by the previously characterized SHAM‐stimulated oxidase, and are consistent with the possibility that azide‐stimulated O2 consumption is mediated by a plasma membrane redox system.