Sodium transport inhibition by selective mitochondrial inhibitors in the urinary bladder of the toad

The effects of selective mitochondrial inhibitors on the short-circuit current and oxygen consumption displayed by the isolated urinary bladder of the toad was studied. Three types of compounds were used: (a) electron transfer inhibitors, Amytal, Cyanide and Antimycin A; (b) energy transfer inhibitors Guanidine, Oligomycin and Rutamycin; and (c) uncoupling agents, Carbonyl cyanide m-chlorophenylhydrazone and 2–4 dinitrophenol. The kinetics of inhibition of oxygen consumption indicated that the inhibitors tested were effectively reaching the mitochondria of the bladder cells. Different kinetics of inhibition of short-circuit current were obtained with the various inhibitors tested. Uncouplers and electron transfer inhibitors rapidly blocked the short-circuit current; energy transfer inhibitors only produced a slow and partial inhibition. A site of energy-coupling, tentatively identified with the intermediate formed in the energy transfer reactions closest to the electron transfer chain, is proposed.     

Die Trennung von Zellteilung und Suberinsynthese in dereprimiertem pflanzlichem Speichergewebe durch Tris-(hydroxymethyl-)aminomethan

Summary Differential derepression of the genome of potato tuber cells by slicing of the tuber tissue leads to cell divisions. This mitotic activity is totally suppressed by Tris-(hydroxymethyl)-aminomethane (Tris)-buffer widely used in biochemical research. The blockage is reversible if tissue slices are transferred to water in order to wash out the Tris-ions.The actual reason for the inhibition of mitotic activity by Tris is as yet unknown. As a possible mechanism of action an uncoupling of electron transfer from phosphorylation in the mitochondrial respiratory chain is discussed. However, experiments show no difference in O₂-absorption between Tris-treated tissue and control in water. Moreover the application of several inhibitors of respiration causes exactly the same effects in both tissues. Amytal (blockage of flavoproteids) has no influence on the respiratory rate at any time. Antimycin A (blockage of electron flow from cytochrome b to cytochrome c) as well as HCN (inhibition of cytochrome-oxidase) inhibit respiration of both tissues during the first 24 hours after derepression. Later on the respiration becomes resistant to both inhibitors. So the quality of respiration is assumed to be the same in mitotic active potato slices as in the Tris-treated tissue.Recent results of biochemical analyses of events in carbohydrate breakdown of the tissues in question point to a differential effect of Tris on several enzymes as possible reason for its inhibitory action.     

Coenzyme Q-pool function in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochromec oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochromec reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochromec oxidoreductase is strongly stimulated by added CoQ₃, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of theK _m for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ₉ and CoQ₁₀.     

Hemocytes of the pond snail Lymnaea stagnalis generate reactive forms of oxygen

Macrophagelike hemocytes of the pond snail Lymnaea stagnalis were stimulated in vitro with various particulate agents (latex, Escherichia coli, Staphylococcus saprophyticus, zymosan) and with phorbol myristate acetate in order to determine whether these blood cells show biochemical reactions reminiscent of a respiratory burst. Phagocytic stimulation of the hemocytes resulted in a superoxide dismutase-sensitive reduction of nitroblue tetrazolium, which is indicative of the generation of superoxide anions. Moreover, the hemocytes also produced hydrogen peroxide, and they showed a sodium azide-sensitive diaminobenzidine reaction. The hemocytes displayed a luminol-dependent chemiluminescence that differed for each stimulus used. Zymosan elicited a relatively high dose-dependent response. The chemiluminescence was (partly) inhibited by superoxide dismutase, azide, and cyanide. These data indicate the possible involvement of toxic oxygen intermediates in phagocytic defense reactions of L. stagnalis hemocytes.     

Fluorometric measurement of pyridine nucleotide reduction in the giant axon of the squid

By monitoring the fluorescence of the isolated giant axon of the squid Loligo pealei, it was possible to follow changes in its oxidation-reduction state as caused by the action of anoxia, cyanide, Amytal, and azide. The response to oxygen depletion was very rapid, the NAD within the axon being 90% reduced within 1–2 min. Cyanide and Amytal gave essentially similar results, although somewhat longer periods of time elapsed during their onset and washout periods. The extent of NAD reduction was essentially the same under conditions of anoxia and treatment with cyanide and Amytal. Azide was less effective in this respect, and at comparatively high levels of concentration (25–50 mM) gave values of 40% or less of the reduction observed with the other inhibitors. The application of ouabain and strophanthidin gave no observable NAD reduction. Variations in the time required to consume given quantities of dissolved oxygen before and after stimulation indicated an increase of 10–20% in oxygen uptake rate associated with activity, although this figure appeared to be a function of the surface-to-volume ratio of the axon. A biochemical analysis of axoplasm for oxidized and reduced pyridine nucleotide was made. Fluorometric examination of centrifuged axoplasm indicated that the NAD-NADH was largely confined to the mitochondria of the axon.     

On the nature of electron and energy transport in mitochondria. I. Multiple inhibition of mitochondrial respiration

1. A series of eight classical respiratory-chain inhibitors was studied. The slopes of State-3 respiratory rate versus dose plots are convex for antimycin, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), rotenone and sulfide, and concave for malonate, Amytal, cyanide and azide.

2. Plots of ADP: O ratio versus dose indicate uncoupling effects at higher concentrations of antimycin, HOQNO, cyanide and azide. On the other hand, sulfide and rotenone have no effect on the phosphorylating efficiency. Malonate increases the ADP: O ratio.

3. Two inhibitors can be combined in such a way that the total inhibition should be equal to the inhibition caused by the single inhibitors if each inhibitor affects respiration independently (additivity of inhibition). In practice, however, antagonism and synergism are also found.

4. Additivity of combined inhibition occurs where both inhibitors act on the same enzyme.

5. Antagonism is observed where the two inhibitors act on different enzymes of the same chain.

6. Synergism is found where the two inhibitors act on enzymes in different branches of a forked chain. This turns into normal additivity when the electron flow through both branches is made equal.

7. The results are compatible with the hypothesis that respiratory enzymes are arranged in chains. The possibility that the chains may be cross-linked or branched is discussed.     

Cyanide-insensitive Respiration in Plant Mitochondria

Pathways of electron transport have been studied in mitochondria isolated from hypocotyls of etiolated mung bean seedlings and skunk cabbage spadices that show cyanide-resistant respiratory activity. The residual flux through cytochrome c oxidase is shown to be small in comparison with the flux through an unidentified alternative oxidase that is known to have a high affinity for oxygen. This alternative oxidase is not a cytochrome. Skunk cabbage and mung bean mitochondria contain cytochromes a and a₃ that have absorption peaks differing slightly from those of animal preparations. A slow oxidation-reduction of cytochrome a₃-CN has been demonstrated. Cytochromes b undergo oxidation and reduction in the presence of cyanide but play no essential role in the cyanide-resistant pathway. Antimycin inhibits to an extent similar to that of cyanide; the respiratory chain bifurcates on the substrate side of the antimycin-sensitive site. Evidence is presented for the selective inhibition by thiocyanate, α, α′-dipyridyl, and 8-hydroxyquinoline of the alternative oxidase pathway, which may therefore contain a non-heme iron protein.     

The Respiratory Chain of Plant Mitochondria: VII. Kinetics of Flavoprotein Oxidation in Skunk Cabbage Mitochondria

The oxidation kinetics of the two high potential flavo-proteins, one (Fp_hf) fluorescent and the other (Fp_ha) nonfluorescent, in mitochondria from skunk cabbage (Symplocarpus foetidus) spadices have been measured by combined spectrophotometry and fluorimetry. In the absence of respiratory inhibitors, both flavoproteins are oxidized at nearly the same rate with half-times between 120 and 160 milliseconds at 24 C. When slight differences in rate are observed, it is Fp_ha which consistently has the shorter half-time. The presence of 0.3 millimolar KCN has no perceptible effect on the oxidation rate of either component. Antimycin A (2 nanomoles per milligram of protein) increases the oxidation half-time of Fp_ha about 3-fold, but it has no effect on the oxidation half-time of Fp_hf. In contrast to these two inhibitors, m-chlorobenzhydroxamic acid—an inhibitor specific to the cyanide insensitive, alternate oxidase pathway in these mitochondria—increases the oxidation half-time of Fp_hf 10-fold to about 2 seconds, while increasing that of Fp_ha only some 20%. This result implies that the flavoprotein Fp_hf mediates electron transport to the alternate oxidase from the region of the mitochondrial respiratory chain encompassing Fp_ha, ubiquinone, and the cytochromes b. The oxidation rate of cytochrome b₅₅₇ is unaffected by either m-chlorobenzhydroxamic acid or cyanide but is strongly inhibited by antimycin A. This result implies that cytochrome b₅₅₇ plays no direct role in the respiratory pathway to the alternate oxidase and is different from cytochrome b₇ found in mitochondria from the spadices of Arum maculatum.     

Electrophysiology of respiratory chain complexes and the ADP-ATP exchanger in native mitochondrial membranes

Transport of protons and solutes across mitochondrial membranes is essential for many physiological processes. However, neither the proton-pumping respiratory chain complexes nor the mitochondrial secondary active solute transport proteins have been characterized electrophysiologically in their native environment. In this study, solid-supported membrane (SSM) technology was applied for electrical measurements of respiratory chain complexes CI, CII, CIII, and CIV, the F(O)F(1)-ATPase/synthase (CV), and the adenine nucleotide translocase (ANT) in inner membranes of pig heart mitochondria. Specific substrates and inhibitors were used to validate the different assays, and the corresponding K(0.5) and IC(50) values were in good agreement with previously published results obtained with other methods. In combined measurements of CI-CV, it was possible to detect oxidative phosphorylation (OXPHOS), to measure differential effects of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the respective protein activities, and to determine the corresponding IC(50) values. Moreover, the measurements revealed a tight functional coupling of CI and CIII. Coenzyme Q (CoQ) analogues decylubiquinone (DBQ) and idebenone (Ide) stimulated the CII- and CIII-specific electrical currents but had inverse effects on CI-CIII activity. In summary, the results describe the electrophysiological and pharmacological properties of respiratory chain complexes, OXPHOS, and ANT in native mitochondrial membranes and demonstrate that SSM-based electrophysiology provides new insights into a complex molecular mechanism of the respiratory chain and the associated transport proteins. Besides, the SSM-based approach is suited for highly sensitive and specific testing of diverse respiratory chain modulators such as inhibitors, CoQ analogues, and uncoupling agents.     

The Cytochemical Localization of Oxidative Enzymes : I. Diphosphopyridine Nucleotide Diaphorase and Triphosphopyridine Nucleotide Diaphorase

Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 µ in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.     

Fasciola hepatica: cytochrome c oxidoreductases and effects of oxygen tension and inhibitors

Studies were made on the mechanism of respiration in Fasciola hepatica (Trematoda). Respiration was found to be dependent on the oxygen tension. The respiratory enzyme systems, NADH-cytochrome c oxidoreductase (EC 1.6.2.1), succinate-cytochrome c oxidoreductase (EC 1.3.99.1) NADH oxidase and cytochrome c-oxygen oxidoreductase (EC 1.9.3.1) were detected in a mitochondrial preparation, the NADH oxidase activity being markedly stimulated by addition of mammalian cytochrome c. Amytal and rotenone inhibited NADH oxidase activity. Antimycin A inhibited succinoxidase activity only at relatively high concentrations. Azide was inhibitory at high concentrations. However, cyanide was found to stimulate respiration. Hydrogen peroxide was found to be an end product of respiration in F. hepatica.     

A Simple Cranial Window Technique for Optical Monitoring of Cerebrocortical Microcirculation and NAD/NADH Redox State. Effect of Mitochondrial Electron Transport Inhibitors and Anoxic Anoxia

Abstract: Fluorescence of NADH and vascular volume of the brain cortex of chloralose-anesthetized cats were measured by surface fluororeflectometry. A cranial window and superfusion technique was elaborated for the topical inhibition of mitochondrial electron transport in the brain cortex by amytal (inhibits at site I) and cyanide (inhibits at site III). The changes in NAD/NADH redox state and CVV evoked by these electron transport inhibitors were compared with those elicited by anoxic anoxia. Amytal (10^-3-10^-1 M ) and cyanide (10^-5-10^-2 M ) resulted in a concentration-dependent and reversible increase in cortical NAD reduction and vascular volume, but the cerebrocortical vessels were almost completely dilatated long before maximum NAD reduction was reached. Cyanide at 10^-2 M increased cortical NAD reduction and vascular volume as much as anoxic anoxia. Amytal at 10^-1 M induced approximately half of the NAD reduction evoked by 10^-2 M cyanide or anoxic anoxia, but resulted in only slightly less vasodilatation than that following cyanide and anoxic anoxia. Since amytal inhibits mitochondrial electron transport at site I—and cyanide and anoxia at site III—but induces a comparable degree of vasodilatation, it is concluded that cytochrome oxidase cannot be the single molecular oxygen sensor in the brain cortex.     

Effect of coenzyme Q10 and vitamin E on brain energy metabolism in the animal model of Huntington's disease

The neuropathological and clinical symptoms of Huntington's disease (HD) can be simulated in animal model with systemic administration of 3-nitropropionic acid (3-NP). Energy defects in HD could be ameliorated by administration of coenzyme Q(10) (CoQ(10)), creatine, or nicotinamid. We studied the activity of creatine kinase (CK) and the function of mitochondrial respiratory chain in the brain of aged rats administered with 3-NP with and without previous application of antioxidants CoQ(10)+vitamin E. We used dynamic and steady-state methods of in vivo phosphorus magnetic resonance spectroscopy ((31)P MRS) for determination of the pseudo-first order rate constant (k(for)) of the forward CK reaction, the phosphocreatine (PCr) to adenosinetriphosphate (ATP) ratio, intracellular pH(i) and Mg(i)(2+) content in the brain. The respiratory chain function of isolated mitochondria was assessed polarographically; the concentration of CoQ(10) and alpha-tocopherol by HPLC. We found significant elevation of k(for) in brains of 3-NP rats, reflecting increased rate of CK reaction in cytosol. The function of respiratory chain in the presence of succinate was severely diminished. The activity of cytochromeoxidase and mitochondrial concentration of CoQ(10) was unaltered; tissue content of CoQ(10) was decreased in 3-NP rats. Antioxidants CoQ(10)+vitamin E prevented increase of k(for) and the decrease of CoQ(10) content in brain tissue, but were ineffective to prevent the decline of respiratory chain function. We suppose that increased activity of CK system could be compensatory to decreased mitochondrial ATP production, and CoQ(10)+vitamin E could prevent the increase of k(for) after 3-NP treatment likely by activity of CoQ(10) outside the mitochondria. Results of our experiments contributed to elucidation of mechanism of beneficial effect of CoQ(10) administration in HD and showed that the rate constant of CK is a sensitive indicator of brain energy disorder reflecting therapeutic effect of drugs that could be used as a new in vivo biomarker of neurodegenerative diseases.     

Respiration of bloodstream forms of the parasite Trypanosoma brucei brucei is dependent on a plant-like alternative oxidase

CoQ links the sn-glycerol-3-phosphate dehydrogenase and oxidase components of the cyanide-insensitive, non-cytochrome-mediated respiratory system of bloodstream African trypanosomes. In this and other characteristics, their respiratory system is similar to the alternative oxidase of plants. The parasites contain 206 ng of CoQ9 mg protein-1 which co-sediments with respiratory activity. The redox state of this CoQ responds in a manner consistent with respiratory function: 60% being in the reduced form when substrate is available and the oxidase is blocked; 13% being in the reduced form when the oxidase is functioning and there is no substrate. The addition of CoQ to aceton-extracted cells stimulates salicylhydroxamic acid-sensitive respiration by 56%. After inhibition of respiration by digitonin-mediated dispersal of the electron transport components, liposomes restore 40% of respiratory activity while liposomes containing CoQ restore 66% of this activity. A less hydrophobic analogue, reduced decyl CoQ, serves as a direct substrate for the trypanosome oxidase supporting full salicylhydroxamic acid-sensitive respiration. After digitonin disruption of electron transport, the nonreduced form of this synthetic substrate can reestablish the chain by accepting electrons from dispersed sn-glycerol-3-phosphate dehydrogenase and transferring them to the dispersed oxidase. Similarities between the alternative oxidase of plants and the oxidase of the trypanosome respiratory system include: mitochondrial location, lack of oxidative phosphorylation, linkage of a dehydrogenase and an oxidase by CoQ, lack of sensitivity to a range of mitochondrial inhibitors, and sensitivity to a spectrum of inhibitors which selectively block transfer of electrons from reduced CoQ to the terminal oxidase but do not block electron transfer to the cytochrome bc1 complex of the mammalian cytochrome chain.     

Ubiquinone synthesis in mitochondrial and microsomal subcellular fractions of Pneumocystis spp.: differential sensitivities to atovaquone

The lung pathogen Pneumocystis spp. is the causative agent of a type of pneumonia that can be fatal in people with defective immune systems, such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), inhibits mitochondrial electron transport and is effective in clearing mild to moderate cases of the infection. Purified rat-derived intact Pneumocystis carinii cells synthesize de novo four CoQ homologs, CoQ7, CoQ8, CoQ9, and CoQ10, as demonstrated by the incorporation of radiolabeled precursors of both the benzoquinone ring and the polyprenyl chain. A central step in CoQ biosynthesis is the condensation of p-hydroxybenzoic acid (PHBA) with a long-chain polyprenyl diphosphate molecule. In the present study, CoQ biosynthesis was evaluated by the incorporation of PHBA into completed CoQ molecules using P. carinii cell-free preparations. CoQ synthesis in whole-cell homogenates was not affected by the respiratory inhibitors antimycin A and dicyclohexylcarbodiimide but was diminished by atovaquone. Thus, atovaquone has inhibitory activity on both electron transport and CoQ synthesis in this pathogen. Furthermore, both the mitochondrial and microsomal fractions were shown to synthesize de novo all four P. carinii CoQ homologs. Interestingly, atovaquone inhibited microsomal CoQ synthesis, whereas it had no effect on mitochondrial CoQ synthesis. This is the first pathogenic eukaryotic microorganism in which biosynthesis of CoQ molecules from the initial PHBA:polyprenyl transferase reaction has been unambiguously shown to occur in two distinct compartments of the same cell.     

The cytochemical localization of oxidative enzymes. I. Diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase

Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 micro in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.     

Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain

Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca²⁺-regulated potassium channel (mitoBK_Ca channel) was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma) U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca²⁺ at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BK_Ca channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BK_Ca channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBK_Ca with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBK_Ca channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.     

Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method

The ultrastructural localization of NADH oxidase, a possible enzyme in the increased oxidative activity of polymorphonuclear leukocytes (PMN) during phagocytosis, was studied. A new cytochemical technique for the localization of H2O2, a product of NADH oxidase activity, was developed. Cerous ions, in the presence of peroxide, form an electron-dense precipitate. Resting and phagocytically stimulated PMN were exposed to cerous ions at pH 7.5 to demonstrate sites of NADH-dependent, cyanide-insensitive H2O2 production. Resting PMN exhibites slight activity on the plasma membrane; phagocytizing PMN had extensive deposits of reaction product localized within the phagosome and on the plasma membrane. Peroxide involvement was demonstrated by the inhibitory effect of catalase on cerium precipitation; the surface localization of the enzyme responsible was confirmed by using nonpenetrating inhibitors of enzymatic activity. A correlative study was performed with an NADH-dependent, tetrazolium-reduction system. As with cerium, formazan deposition on the surface of the cell was NADH dependent, cyanide insensitive, and stimulated by phagocytosis. Superoxide dismutase did not inhibit tetrazolium reduction, as observed cytochemically, indicating direct enzymatic dye reduction without superoxide interposition. These findings, combined with oxygen consumption studies on resting and stimulated PMN in the presence or absence of NADH, indicate that NADH oxidase is a surface enzyme in human PMN. It is internalized during phagocytosis and retains its peroxide-generating capacity within the phagocytic vacuole.     

Severe impairment of nucleotide synthesis through inhibition of mitochondrial respiration

Since de-novo synthesis of pyrimidine nucleotides is coupled to the mitochondrial respiratory chain (RC) via dehydroorotic acid dehydrogenase (DHODH), respiratory chain dysfunction should impair pyrimidine synthesis. To investigate this, we used specific RC inhibitors, Antimycin A and Rotenone, to treat primary human keratinocytes and 143B cells, a human osteosarcoma cell line, in culture. This resulted in severe impairment of de novo pyrimidine nucleotide synthesis. The effects of RC inhibition were not restricted to pyrimidine synthesis, but concerned purine nucleotides, too. While the total amount of purine nucleotides was not diminished, they were significantly broken down from triphosphates to monophosphates, reflecting impaired mitochondrial ATP regeneration. The effect of Rotenone was similar to that of Antimycin A. This was surprising since Rotenone inhibits complex I of the respiratory chain, which is upstream of ubiquinone where DHODH interacts with the RC. In order to avoid unspecific effects of Rotenone, we examined the consequences of a mitochondrial DNA mutation that causes a specific complex I defect. The effect was much less pronounced than with Rotenone, suggesting that complex I inhibiton cannot fully explain the marked effect of Rotenone on pyrimidine nucleotide synthesis.     

Severe Impairment of Nucleotide Synthesis Through Inhibition of Mitochondrial Respiration

Since de‐novo synthesis of pyrimidine nucleotides is coupled to the mitochondrial respiratory chain (RC) via dehydroorotic acid dehydrogenase (DHODH), respiratory chain dysfunction should impair pyrimidine synthesis. To investigate this, we used specific RC inhibitors, Antimycin A and Rotenone, to treat primary human keratinocytes and 143B cells, a human osteosarcoma cell line, in culture. This resulted in severe impairment of de novo pyrimidine nucleotide synthesis. The effects of RC inhibition were not restricted to pyrimidine synthesis, but concerned purine nucleotides, too. While the total amount of purine nucleotides was not diminished, they were significantly broken down from triphosphates to monophosphates, reflecting impaired mitochondrial ATP regeneration. The effect of Rotenone was similar to that of Antimycin A. This was surprising since Rotenone inhibits complex I of the respiratory chain, which is upstream of ubiquinone where DHODH interacts with the RC. In order to avoid unspecific effects of Rotenone, we examined the consequences of a mitochondrial DNA mutation that causes a specific complex I defect. The effect was much less pronounced than with Rotenone, suggesting that complex I inhibiton cannot fully explain the marked effect of Rotenone on pyrimidine nucleotide synthesis.