Effect of nitric oxide and plant antioxidants on microsomal content of lipid radicals

The antioxidant ability of nitric oxide (NO) generated by a chemical donor and of commercially available antioxidant preparations was assayed. SNAP (S-Nitroso-N-acetylpenicilamine) was used as the NO donor, and Ginkgo biloba, wheat and alfalfa preparations were tested. Lipid peroxidation was assayed by EPR employing a reaction system consisting of rat liver microsomes, ADP, FeCl3, NADPH and POBN in phosphate buffer, pH=7.4. In vitro NO exposure decreased microsomal lipid peroxidation in a dose-dependent manner. The dose responsible for inhibiting the microsomal content of lipid radical adducts by 50% (LD50) for SNAP was 550 microM (NO generation rate 0.1 microM/min). The addition of 50 microM hemoglobin to the incubation media prevented NO effect on lipid peroxidation. The addition of an amount of the antioxidant preparations equivalent to the LD50 doses inhibited lipid peroxidation by 21, 15, and 33% for wheat, alfalfa, ginkgo biloba preparations respectively in the presence of 550 microM SNAP. We detected a decrease in the content of lipid radical adducts after simultaneous supplementation, although it was less than 50%, even when LD50 doses of the products were added. This suggests that NO and the natural antioxidants inhibit lipid peroxidation by a mechanism that has both common and non-shared features.     

The effects of polychlorinated biphenyls on plasma steroid levels and hepatic microsomal enzymes in fish

Aroclor 1254 was administered intraperitoneally (25 mg kg body wt⁻¹ in 1 ml of arachis oil) at weekly intervals for 4 weeks to trout and carp; arachis oil was used as the control. Activities of the following hepatic microsomal enzymes, aminopyrine demethylase, p-nitroreductase, UDP-glucuronyl-transferase and 1-leucyl-β-naphthylamide splitting enzyme were measured in both species; cytochrome P₄₅₀ and microsomal protein contents were also determined.
The changes in the levels of androgens, oestrogens and corticoid hormones were measured in the circulating blood of control and treated groups at weekly intervals. The blood was obtained by cardiac puncture.
Results indicated (a) a significant increase in the activities of all the enzymes measured except 1-leucyl-β-naphthylamide splitting enzyme, (b) cytochrome P₄₅₀ and microsomal protein contents were increased in trout, but not in carp, (c) a significant reduction in the plasma levels of androgens, oestrogens and corticoids in the treated groups, particularly at the end of the fourth week and (d) there was a correlation between increased enzyme activities and a decrease in plasma hormone levels.     

Reduction of Nomega-hydroxy-L-arginine to L-arginine by pig liver microsomes, mitochondria, and human liver microsomes

Nomega-Hydroxy-L-arginine, the intermediate in nitric oxide formation from L-arginine catalyzed by NO synthase, can be released into the extracellular space. It has been suggested that it can circulate and exert paracrine effects. Since it cannot only be used as substrate by NO synthases, but can also be oxidized by cytochrome P450 and other hemoproteins in a superoxide-dependent manner, it has been proposed that it can serve as NO donor. In the present study, the in vitro reduction of Nomega-hydroxy-L-arginine was examined. Pig and human liver microsomes as well as pig liver mitochondria were capable of reducing Nomega-hydroxy-L-arginine to L-arginine in an oxygen-insensitive enzymatic reaction. These results demonstrate that this metabolic pathway has to be considered when suggesting Nomega-hydroxy-L-arginine as NO-precursor. The reconstituted liver microsomal system of a pig liver CYP2D enzyme, the benzamidoxime reductase, was unable to replace microsomes to produce L-arginine from Nomega-hydroxy-L-arginine.     

Oxidation of the ketoxime acetoxime to nitric oxide by oxygen radical-generating systems.

Ketoximes undergo a cytochrome P450-catalyzed oxidation to nitric oxide and ketones in liver microsomes. In addition, nitric oxide synthase (NOS) can catalyze the oxidative denitration of the >C=N-OH group of amidoximes. The objective of this work was to characterize the oxidation of a ketoxime (acetoxime) and to assess the ability of NOS to catalyze the generation of nitric oxide/nitrogen monoxide (*NO) from acetoxime. Acetoxime was oxidized to NO2- (and NO3-) by microsomes enriched with several P450 isoforms, including CYP2E1, CYP1A1, and CYP2B1. Nitric oxide was identified as an intermediate in the overall reaction. Superoxide dismutase and catalase significantly inhibited the reaction. Exogenous iron increased the microsomal generation of NO2- from acetoxime, while metal chelators (desferrioxamine, EDTA, DTPA) inhibited it. A Fenton-like system (Fe2+ plus H2O2, pH 7.4) consumed acetoxime with production of NO2- and NO3-, whereas oxidation by superoxide or by H2O2 was inefficient. The results presented suggest a role for hydroxyl radical-like oxidants in the oxidation of acetoxime to nitric oxide. O-Acetylacetoxime and O-tert-butylacetoxime were not oxidized by a Fenton system or by liver microsomes to any significant extent. Formation of the 5,5'-dimethyl-1-pyrroline-N-oxide/. OH adduct by a Fenton system was significantly inhibited by acetoxime, while O-acetylacetoxime and O-tert-butylacetoxime were inactive. These results suggest that the. OH-dependent oxidation of acetoxime initially proceeds via abstraction of a hydrogen atom from its hydroxyl group, as opposed to the oxidation of its >C=N- function. HepG2 cells with low levels of expression of P450 did not significantly produce NO2- from acetoxime, while HepG2 cells expressing CYP2E1 did, and this generation was blocked by a CYP2E1 inhibitor. Acetoxime was inactive either as a substrate or as an inhibitor of iNOS activity. These results indicate that reactive oxygen species play a key role in the oxidation of acetoxime to. NO by liver microsomes by a mechanism involving H abstraction from the OH moiety by hydroxyl radical-like oxidants and suggest the possibility that acetoxime may be an effective producer of. NO primarily in the liver by a pathway independent of NOS.     

Nitric Oxide Causes Glutamate Release from Brain Synaptosomes

Abstract: We determined the ability of pathological levels of nitric oxide (NO) to cause glutamate release from isolated rat brain nerve terminals using a fluorometric assay. It was found that NO (0.7 and 2 µ M ) produced (4 and 10 nmol/mg of synaptosomal protein) Ca²⁺-independent glutamate release from synaptosomes (after 1 min of exposure). Spermine/NO complex (spermine NONOate; a slow NO donor) and potassium cyanide (an inhibitor of cytochrome oxidase) also caused Ca²⁺-independent glutamate release. Preincubation of synaptosomes with 5 µ M 1 H -[1,2,4]oxadiazole[4,3- a ]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) had no effect on NO-induced Ca²⁺-independent glutamate release. Ca²⁺-independent glutamate release produced by NO was greater in a low-oxygen medium. NO, spermine NONOate, and potassium cyanide inhibited synaptosomal respiration with a similar order of potency with respect to their ability to cause glutamate release. Because NO has been shown previously to inhibit reversibly cytochrome oxidase in competition with oxygen, our findings in this study suggest that NO (and cyanide) causes glutamate release following inhibition of mitochondrial respiration at the level of cytochrome oxidase. Thus, elevated NO production leading to mitochondrial dysfunction, glutamate release, and excitotoxicity may contribute to neuronal death in neurological diseases.     

Subcellular Localization and Characterization of Neuronal Nitric Oxide Synthase

Abstract: In contrast to the predominantly participate, Ca²⁺/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca²⁺ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca²⁺/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC₅₀= 90 μ M ) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.     

Denitrosation of carcinostatic nitrosoureas by purified NADPH cytochrome P-450 reductase and rat liver microsomes to yield nitric oxide under anaerobic conditions

The nitrosoureas, CCNU (1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea) and BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) are representatives of a class of N-nitroso compounds which undergo denitrosation in the presence of NAD(P)H and deoxygenated hepatic microsomes from rats to yield nitric oxide (NO) and the denitrosated parent compound. Formation of NO during microsomal denitrosation of CCNU and BCNU was determined by three methods. With one procedure, NO was measured and concentration shown to increase over time in the head gas above microsomal incubations with BCNU. Two additional methods utilized NO binding to either ferrous cytochrome P-450 or hemoglobin to form distinct Soret maxima at 444 and 415 nm, respectively. Incubation of either BCNU or CCNU in the presence of NAD(P)H and deoxygenated microsomes resulted in the formation of identical cytochrome P-450 ferrous · NO optical difference spectra. Determination of the P-450 ferrous · NO extinction coefficient by the change in absorbance at 444 minus 500 nm allowed measurement of rates of denitrosation by monitoring the increase in absorbance at 444 nm. The rates of BCNU and CCNU denitrosation were determined to be 4.8 and 2.0 nmol NO/min/mg protein, respectively, for phenobarbital (PB) induced microsomes. For the purpose of comparison, the rate of [¹⁴C]CCNU (1-(2-[¹⁴C]chloroethyl)-3-(cyclohexyl)-1-nitrosourea turnover was examined by the isolation of [¹⁴C]CCU (1-(2-[¹⁴C] chloroethyl)-3-(cyclohexyl)-1-urea) from incubations that contained NADPH and deoxygenated PB-induced microsomes. These analyses showed stoichiometric amounts of NO and [¹⁴C]CCU being formed at a rate of 2.0 nmol/min/mg protein. Denitrosation catalysis by microsomes was enhanced by phenobarbital pretreatment and partially decreased by cytochrome P-450 inhibitors, SKF-525A, α-naphthoflavone (ANF), metyrapone, and CO, suggesting a cytochrome P-450-dependent denitrosation. However, in the presence of NADPH and purified NADPH cytochrome P-450 reductase reconstituted in dilauroylphosphatidylcholine, [¹⁴C]CCNU was shown to undergo denitrosation to [¹⁴C]CCU. Thus, NADPH cytochrome P-450 reductase could support denitrosation in the absence of cytochrome P-450.     

Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics

Abstract The filamentous fungus Cunninghamella elegans has the ability to metabolize xenobiotics, including polycyclic aromatic hydrocarbons and pharmaceutical drugs, by both phase I and II biotransformations. Cytosolic and microsomal fractions were assayed for activities of cytochrome P450 monooxygenase, aryl sulfotransferase, glutathione S -transferase, UDP-glucuronosyltransferase, UDP-glucosyltransferase, and N -acetyltransferase. The cytosolic preparations contained activities of an aryl sulfotransferase (15.0 nmol min⁻¹ mg⁻¹), UDP-glucosyltransferase (0.27 nmol min⁻¹ mg⁻¹) and glutathione 5-transferase (20.8 nmol min⁻¹ mg⁻¹). In contrast, the microsomal preparations contained cytochrome P450 monooxygenase activities for aromatic hydroxylation (0.15 nmol min⁻¹ mg⁻¹) and N -demethylation (0.17 nmol min⁻¹~' mg⁻¹) of cyclobenzaprine. UDP-glucuronosyltransferase activity was detected in both the cytosol (0.09 nmol min⁻¹ mg⁻¹) and the microsomes (0.13 nmol min⁻¹ mg⁻¹). N -Acetyltransferase was not detected. The results from these experiments provide enzymatic mechanism data to support earlier studies and further indicate that C. elegans has a broad physiological versatility in the metabolism of xenobiotics.     

Interactions between respiration and inorganic phosphate uptake in phosphate-limited cells of Chlamydomonas reinhardtii

Phosphate addition to P-limited cells of Chlamydomonas reinhardtii resulted in an immediate increase in the rate of respiratory O₂ consumption. The respiration rate continued to increase for several minutes after the addition of P₁. Similar patterns of P₁ stimulation of respiratory O₂ consumption were observed in the presence of cyanide (cytochrome oxidase inhibitor) and propyl gallate (alternative oxidase inhibitor). Stimulation of O₂ consumption was accompanied by rapid changes in levels of glycolytic intermediates. These changes were consistent with activation of ATP-dependent phosphofructokinase and pyruvate kinase. The adenylate pool exhibited only minor perturbations, P₁, uptake resulted in extracellular acidification, which continued for several minutes after the exhaustion of added P₁, whereas exhaustion of extracellular P₁ resulted in a rapid decline in the O₂ consumption rate. These results are consistent with control of respiration in P-limited cells occurring largely at the level of glycolysis.     

Nitric oxide modulates ozone-induced cell death, hormone biosynthesis and gene expression in Arabidopsis thaliana

Nitric oxide (NO) is involved together with reactive oxygen species (ROS) in the activation of various stress responses in plants. We have used ozone (O₃) as a tool to elicit ROS-activated stress responses, and to activate cell death in plant leaves. Here, we have investigated the roles and interactions of ROS and NO in the induction and regulation of O₃-induced cell death. Treatment with O₃ induced a rapid accumulation of NO, which started from guard cells, spread to adjacent epidermal cells and eventually moved to mesophyll cells. During the later time points, NO production coincided with the formation of hypersensitive response (HR)-like lesions. The NO donor sodium nitroprusside (SNP) and O₃ individually induced a large set of defence-related genes; however, in a combined treatment SNP attenuated the O₃ induction of salicylic acid (SA) biosynthesis and other defence-related genes. Consistent with this, SNP treatment also decreased O₃-induced SA accumulation. The O₃-sensitive mutant rcd1 was found to be an NO overproducer; in contrast, Atnoa1/rif1 ( Arabidopsis nitric oxide associated 1/resistant to inhibition by FSM1 ), a mutant with decreased production of NO, was also O₃ sensitive. This, together with experiments combining O₃ and the NO donor SNP suggested that NO can modify signalling, hormone biosynthesis and gene expression in plants during O₃ exposure, and that a functional NO production is needed for a proper O₃ response. In summary, NO is an important signalling molecule in the response to O₃.     

STUDIES ON NICOTINIC ACETYLCHOLINE RECEPTORS IN MAMMALIAN BRAIN. CHARACTERIZATION OF A MICROSOMAL SUBFRACTION ENRICHED IN RECEPTOR FUNCTION FOR DIFFERENT NEUROTRANSMITTERS

Abstract— A subfraction, derived from the microsomal fraction of rat cerebral cortex, with a buoyant density of 1.112 g μ ml⁻¹ appears to be enriched in receptor sites for a number of potential neurotrans-mitters. These include the cholinergic (nicotinic and muscarinic) and ß-adrenergic receptors. This microsomal subfraction (P₃B₂) has been isolated on a preparative scale by two sequential isopycnic sedimentations in discontinuous sucrose gradients.
We have studied the morphology, enzymatic markers and protein composition of this fraction and have compared them with the properties of other subcellular fractions from the same source. Synaptic plasma membranes resembled P₃B₂ by exhibiting the same high extent of enrichment in receptors. However, the synaptic membranes appear to contain more mitochondrial and presynaptic (axonal and cell surface) membranes than does P₃B₂, and the postsynaptic membranes in the two fractions appear morphologically distinct since P₃B₂ does not contain the characteristic postsynaptic densities. Thus these membranes may be derived from Gray's type II synapses.     

Nitric oxide, induced by wounding, mediates redox regulation in pelargonium leaves

The subject of this study was the participation of nitric oxide (NO) in plant responses to wounding, promoted by nicking of pelargonium ( Pelargonium peltatum L.) leaves. Bio-imaging with the fluorochrome 4,5-diaminofluorescein diacetate (DAF-2DA) and electrochemical in situ measurement of NO showed early (within minutes) and transient (2 h) NO generation after wounding restricted to the site of injury. In order to clarify the functional role of NO in relation to modulation of the redox balance during wounding, a pharmacological approach was used. A positive correlation was found between NO generation and regulation of the redox state. NO caused a slight restriction of post-wounded O₂⁻ production, in contrast to the periodic and marked increase in H₂O₂ level. The observed changes were accompanied by time-dependent inhibition of catalase (CAT) and ascorbate peroxidase (APX) activity. The effect was specific to NO, since the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) reversed the inhibition of CAT and APX, as well as temporarily enhancing H₂O₂ synthesis. Finally, cooperation of NO/H₂O₂ restricted the depletion of the low-molecular weight antioxidant pool ( i.e . ascorbic acid and thiols) was positively correlated with sealing and reconstruction changes in injured pelargonium leaves ( i.e . lignin formation and callose deposition). The above results clearly suggest that NO may promote restoration of wounded tissue through stabilisation of the cell redox state and stimulation of the wound scarring processes.     

A common enzyme may be responsible for the conversion of organic nitrates to nitric oxide in vascular microsomes.

We compared the nitric oxide (NO)-generating behavior of nitroglycerin (NTG), pentaerythritol trinitrate (PEtriN) and isosorbide dinitrate (ISDN), in the microsomal preparation of bovine coronary artery smooth muscle cells. The comparative NO generating activities among these nitrates were consistent with their relative reported vasodilating activities. Consistent with our previous observations with NTG, 400 microM bromosulfophthalein did not affect NO generation from PEtriN and ISDN in vascular microsomes while 400 microM 1-chloro-2,4-dinitrobenzene completely inhibited NO generation from these nitrates. Gel filtration chromatography with solubilized microsomes of bovine aortic smooth muscle cells showed the primary activity of NO generation from all three nitrates to be eluted at about 200 kD, consistent with that found with solubilized microsomes from the bovine coronary artery microsomes. These results suggest that organic nitrates may be converted to NO by one common enzyme in vascular microsomes.     

Plasmid-mediated virulence genes in non-typhoid Salmonella serovars

Abstract Among aerobic prokaryotes, many different terminal oxidase complexes have been described. Sequence comparison has revealed that the aa ₃-type cytochrome c oxidase and the bo ₃-type quinol oxidase are variations on the same theme: the heme-copper oxidase. A third member of this family has recently been recognized: the cbb ₃-type cytochrome c oxidase. Here we give an overview, and report that nitric oxide (NO) reductase, a bc -type cytochrome involved in denitrification, shares important features with these terminal oxidases as well. Tentative structural, functional and evolutionary implications are discussed.     

Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: involvement of cytochrome P-450

Glyceryl trinitrate was denitrated by rat liver microsomes in the presence of NADPH with formation of a mixture of glyceryl dinitrates and glyceryl mononitrates. The highest activity was obtained under anaerobic conditions and the reaction was inhibited by O2 indicating that it is a reductive denitration. It was also inhibited by CO, metyrapone and miconazole showing that it was catalyzed by cytochrome P-450. Finally the formation of the cytochrome P-450-Fe(II)-NO complex during this reaction was shown by visible spectroscopy. These data demonstrate that microsomal reductive denitration of glyceryl trinitrate is catalyzed by cytochrome P-450 and can be involved in the formation of the endothelium-derived relaxing factor (EDRF = nitric oxide).     

Evidence for cytochrome P-450 and P-450-mediated benzo(a) pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium

Abstract The presence of cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylase activity in both microsomal and soluble fractions of the white rot fungus Phanerochaete chrysosporium was shown. The reduced carbon monoxide difference spectrum showed maxima at 448–450 and 452–454 nm for microsomal and cytosolic fractions, respectively. Both P-450 fractions produced a Type I substrate binding spectrum on addition of benzo(a)pyrene. Activity for benzo(a)pyrene hydroxylation was NADPH-dependent and inhibited by carbon monoxide. K _m values for activity showed a difference between the cellular fractions with a K _m of 89 μM for microsomal P-450 and 400 μM for cytosolic P-450. The V _max values observed were 0.83 nmol min⁻ (nmol microsomal P-450) ⁻¹ and 0.4 nmol min⁻¹ (nmol cytosolic P-450)⁻¹. The results indicate that P-450-mediated benzo(a)pyrene hydroxylase activity could play a role in xenobiotic transformation by this fungus beside the known ligninolytic exocellular enzymes.     

Cardiovascular responses of eggs, embryos and alevins of Atlantic salmon and rainbow trout to nitric oxide donors, sodium nitroprusside and isosorbide dinitrate, and inhibitors of nitric oxide synthase, N-nitro-l-arginine methyl ester and aminoguanidine, following short- and long-term exposure

Atlantic salmon embryos and alevins Salmo salar that had been exposed to isosorbide dinitrate (ISDN) for 4 weeks, on transfer to fresh water, showed an increase in heart rate. Unexposed embryos and alevins showed a decrease in heart rate following transfer to 100 μmol l⁻¹ ISDN for 4 h. This is in contrast to adult rainbow trout and higher vertebrates where tachycardia occurred in response to nitric oxide (NO) donors. The decreased heart rate in response to ISDN was inhibited by 2 mg 1⁻¹ methylene blue, indicating that NO activates cardiovascular events via guanylyl cyclase and cyclic guanidine monophosphate. Heart rate of rainbow trout alevins Oncorhynchus mykiss exposed to 100 μmol l⁻¹ aminoguanidine responded with a slowly developed but significant bradycardia over 10 min as did those reared in aminoguanidine for 4 weeks then transferred to fresh water. A potentiated increase in heart rate on exposure to the NO donor sodium nitroprusside (SNP), occurred within 1 min in salmon alevins reared in l -nitro-arginine methyl ester ( l -NAME) for 4 weeks, indicating up-regulation of NO receptors. The evidence for down-regulation of SNP-reared alevins exposed to l -NAME was less well defined. The results suggest that both salmonid embryos and alevins have a functional l -arginine-NO pathway and that NO has a physiological role in control of cardiovascular events.     

Electron-transport cytochrome P-450 system is involved in the microsomal metabolism of the carcinogen chromate

The kinetics of chromate reduction by liver microsomes isolated from rats pretreated with phenobarbital or 3-methylcholanthrene with NADPH or NADH cofactor have been followed. Induction of cytochrome P-450 and NADPH-cytochrome P-450 reductase activity in microsomes by phenobarbital pretreatment caused a decrease in the apparent chromate-enzyme dissociation constant, Km, and an increase in the apparent second-order rate constant, kcat/Km, but did not affect the kcat of NADPH-mediated microsomal metabolism of chromate. Induction of cytochrome P-448 in microsomes by 3-methylcholanthrene pretreatment did not affect the kinetics of NADPH-mediated reduction of chromate by microsomes. The kinetics of NADH-mediated microsomal chromate reduction were unaffected by the drug treatments. The effects of specific enzyme inhibitors on the kinetics of microsomal chromate reduction have been determined. 2'-AMP and 3-pyridinealdehyde-NAD, inhibitors of NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase, inhibited the rate of microsomal reduction of chromate with NADPH and NADH. Metyrapone and carbon monoxide, specific inhibitors of cytochrome P-450, inhibited the rate of NADPH-mediated microsomal reduction of chromate, whereas high concentrations of dimethyl-sulfoxide (0.5 M) enhanced the rate. These results suggest that the electron-transport cytochrome P-450 system is involved in the reduction of chromate by microsomal systems. The NADPH and NADH cofactors supply reducing equivalents ultimately to cytochrome P-450 which functions as a reductase in chromate metabolism. The lower oxidation state(s) produced upon chromate reduction may represent the ultimate carcinogenic form(s) of chromium. These studies provide evidence for the role of cytochrome P-450 in the activation of inorganic carcinogens.     

Energy conservation in Nitrobacter

Abstract The generation of ATP and NADH in total cells of Nitrobacter was measured under aerobic and anaerobic conditions. NADH synthesis was driven by an ATP independent reaction with nitrite or nitric oxide as electron donors. The rate of NADH formation was about 200 times higher, if nitric oxide instead of nitrite served as electron donor. Approximately 2 mol nitric oxide were needed for reduction of 1 mol NAD⁺. Nitrite caused an end-product inhibition of the nitric oxide induced NADH synthesis. ATP was synthesized by NADH oxidation with oxygen and nitrate as terminal electron acceptors.     

Nitric oxide decreases renal medullary Na+, K+-ATPase activity through cyclic GMP-protein kinase G dependent mechanism.

The aim of this study was to investigate the effect of nitric oxide on renal Na+,K(+)-ATPase and ouabain-sensitive H+,K(+)-ATPase activities. The study was performed in male Wistar rats. The investigated substances were infused under general anaesthesia into abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. NO donor, S-nitroso-N-acetylpenicillamine (SNAP), infused at doses of 10(-7) and 10(-6)mol/kg/min decreased medullary Na+,K(+)-ATPase activity by 29.4% and 45.2%, respectively. Another NO donor, spermine NONOate, administered at the same doses reduced Na+,K(+)-ATPase activity in the renal medulla by 31.7% and 46.5%, respectively. Neither of NO releasers had any effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase. Infusion of NO precursor, L-arginine (100 micromol/kg/min), decreased medullary Na+,K(+)-ATPase activity by 32.2%, whereas inhibitor of nitric oxide synthase, L-NAME (10 nmol/kg/min), increased this activity by 20.7%. The effect of synthetic NO donors was mimicked by 8-bromo-cGMP and blocked by inhibitors of soluble guanylate cyclase, ODQ or methylene blue, as well as by specific inhibitor of protein kinase G, KT5823. In addition, inhibitory effect of either SNAP or 8-bromo-cGMP on medullary Na+,K(+)-ATPase was abolished by 17-octadecynoic acid (17-ODYA), which inhibits cytochrome P450-dependent metabolism of arachidonic acid. These data suggest that NO decreases Na+,K(+)-ATPase activity in the renal medulla through the mechanism involving cGMP, protein kinase G, and cytochrome P450-dependent arachidonate metabolites. In contrast, NO has no effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase.