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PubMed1998

Inhibition of nitric oxide formation by neuronal nitric oxide synthase by quinones: nitric oxide synthase as a quinone reductase.

Inhibitory action of a variety of quinoid compounds on neuronal nitric oxide synthase (nNOS) activity was examined with a 20000g rat cerebellar supernatant preparation and purified nNOS. The inhibition of citrulline formation from l-arginine by quinones, which exhibit one-electron reduction potentials (E17) ranging between -240 and -100 mV, increased at a more positive one-electron reduction potential, suggesting that quinone appears to act as an electron acceptor for nNOS. Among the quinones tested, 9,10-phenanthraquinone (PQ), corresponding to an E17 value of -124 mV, exhibited the most potent inhibiton of citrulline formation (IC50 value = 10 microM). A kinetic study revealed that PQ is a competitive inhibitor with respect to NADPH, with a Ki value of 0.38 +/- 0.12 microM, and a noncompetitive inhibitor with respect to l-arginine, with a Ki value of 9.63 +/- 0.20 microM. Partial purification of the enzymes which are responsible for reducing PQ in 20000g supernatant of rat cerebellum by anion-exchange column chromatography indicated that one catalyst for PQ reduction was nNOS. Reductase activity of PQ by purified nNOS required CaCl2/calmodulin and was markedly suppressed by the flavoprotein inhibitor diphenyleneiodonium but not by l-nitroarginine which is a specific inhibitor for NO formation. nNOS effectively reduced the quinones as well as PQ causing a marked decrease in the production of NO from l-arginine, while 1, 4-benzoquinone, 9,10-anthraquinone, mitomycin C, and lapachol, which show negligible inhibitory action on nNOS activity, were poor substrates for the enzyme on reduction. These results indicate that PQ and other quinones used in the present study interact with the NADPH-cytochrome P450 reductase domain on nNOS and thus probably inhibit NO formation by shunting electrons away from the normal catalytic pathway. Therefore, our study suggests that quinones could possibly affect NO-dependent physiological and/or pathophysiological actions in vivo.

Animals↗

PubMed1996

Evidence that brain nitric oxide synthase is the major nitric oxide synthase isoform in the hypothalamus of the adult female rat and that nitric oxide potently regulates hypothalamic cGMP levels.

Recent studies suggest that nitric oxide (NO) may function as a neurotransmitter in the hypothalamus. In order to provide further evidence supporting this contention, we examined: (1) whether the hypothalamus displays significant NO synthase (NOS) activity and whether the activity is inhibited by an NOS inhibitor, (2) whether the different NOS isoforms [brain (b)-NOS, endothelial (e)-NOS and macrophage (m)-NOS] are expressed in the various nuclei of the hypothalamus of the random cycling adult female rat, (3) whether the NO donor molecule, sodium nitroprusside (SNP), regulates the heme-containing enzyme, guanylate cyclase in the preoptic area and medial basal hypothalamus of the random cycling adult female rat as well as the ovariectomized steroid (estradiol-17 beta)-treated rat. The results of the study showed that the preoptic area (POA) and medial basal hypothalamus (MBH) of the adult female rat displays significant NOS activity which can be dose-dependently inhibited by an NOS inhibitor. All three NOS isoform mRNA transcripts were present in the hypothalamus, with the order of expression being b-NOS > e-NOS > m-NOS. Immunohistochemical localization using monoclonal antibodies to the specific NOS isoform proteins revealed that b-NOS represented the major form of NOS in the hypothalamus based on density and distribution of immunostaining. b-NOS immunostaining was especially dense in the organum vasculosum laminae terminalis (OVLT), medial preoptic area (MPOA), supraoptic nucleus, and moderately dense in the arcuate nucleus/median eminence. The pattern and density of b-NOS staining closely mirrored our previously reported pattern of NADPH-diaphorase staining in the hypothalamus, and a polyclonal antibody to b-NOS yielded a similar staining pattern as that observed for the monoclonal antibody. In contrast to the dense staining observed for b-NOS in the hypothalamus, we observed no specific staining for m-NOS in the hypothalamus. e-NOS immunostaining, on the other hand, was present in the hypothalamus, but to a much lesser extent than b-NOS. Light e-NOS staining was observed in the OVLT, MPOA, supraoptic nucleus and arcuate nucleus/median eminence. That NO can regulate guanylate cyclase as a potential mediator of its effects was demonstrated using SNP which dose-dependently elevated cGMP levels in the POA and MBH of random cycling rats and estrogen-primed ovariectomized rats. The effect of SNP was due to its NO donor ability as it was blocked by the NO scavenger molecule, hemoglobin. Interestingly, hemoglobin alone caused a 50-60% reduction in basal cGMP levels, suggesting that endogenously produced NO regulates basal guanylate cyclase activity. Taken as a whole, the present study demonstrates that b-NOS is the major NOS isoform in the hypothalamus and it also provides evidence that cGMP may be a mediator of NO effects in the female hypothalamus as evidenced by the potent ability of SNP to elevate cGMP levels in the POA and MBH.

Animals↗

PubMed1995

Role of nitric oxide in the regulation of coronary vascular tone in hearts from hypertensive rats. Maintenance of nitric oxide-forming capacity and increased basal production of nitric oxide.

In arterial hypertension, coronary flow reserve, expressed by the difference between autoregulated and maximal coronary flow, is frequently impaired. Previous experimental and clinical investigations using acetylcholine as a stimulus for the production of endothelium-derived relaxing factor suggested that an impaired endothelium-dependent vasodilation, presumably caused by a decreased formation of nitric oxide (NO), may account for this microvascular dysfunction. However, so far no study has been performed that quantifies the formation of NO within the coronary circulation of hypertensive hearts to assess its role in setting coronary vascular tone in the hypertensive heart. We therefore quantified NO formation within the coronary circulation of constant flow-perfused, isolated hearts from spontaneously hypertensive rats (SHR, 16th to 26th week), as a model for hypertensive heart disease, and from the normotensive control strain (Wistar-Kyoto, WKY) using the oxyhemoglobin technique. Coronary perfusion pressure and vascular resistance were almost 30% higher in SHR compared with WKY hearts. Intracoronarily applied NO decreased coronary vascular resistance by maximally 45% of resting values in a concentration-dependent manner in both groups. The bradykinin-induced decrease in coronary vascular resistance and the parallel increase in NO release were comparable in SHR and WKY hearts and fell within the vasodilator range of exogenously applied NO. Moreover, basal release of NO normalized to heart wet weight was 50% higher in SHR compared with WKY hearts. Rates of basal NO release were correlated inversely with changes in coronary perfusion pressure and vascular resistance in both groups (r = -.85 and -.84, respectively, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Cobalt(II) Oxidation by the Marine Manganese(II)-Oxidizing Bacillus sp. Strain SG-1.

The geochemical cycling of cobalt (Co) has often been considered to be controlled by the scavenging and oxidation of Co(II) on the surface of manganese [Mn(III,IV)] oxides or manganates. Because Mn(II) oxidation in the environment is often catalyzed by bacteria, we have investigated the ability of Mn(II)-oxidizing bacteria to bind and oxidize Co(II) in the absence of Mn(II) to determine whether some Mn(II)-oxidizing bacteria also oxidize Co(II) independently of Mn oxidation. We used the marine Bacillus sp. strain SG-1, which produces mature spores that oxidize Mn(II), apparently due to a protein in their spore coats (R.A. Rosson and K. H. Nealson, J. Bacteriol. 151:1027-1034, 1982; J. P. M. de Vrind et al., Appl. Environ. Microbiol. 52:1096-1100, 1986). A method to measure Co(II) oxidation using radioactive Co as a tracer and treatments with nonradioactive (cold) Co(II) and ascorbate to discriminate bound Co from oxidized Co was developed. SG-1 spores were found to oxidize Co(II) over a wide range of pH, temperature, and Co(II) concentration. Leucoberbelin blue, a reagent that reacts with Mn(III,IV) oxides forming a blue color, was found to also react with Co(III) oxides and was used to verify the presence of oxidized Co in the absence of added Mn(II). Co(II) oxidation occurred optimally around pH 8 and between 55 and 65 degrees C. SG-1 spores oxidized Co(II) at all Co(II) concentrations tested from the trace levels found in seawater to 100 mM. Co(II) oxidation was found to follow Michaelis-Menten kinetics. An Eadie-Hofstee plot of the data suggests that SG-1 spores have two oxidation systems, a high-affinity-low-rate system (K(m), 3.3 x 10 M; V(max), 1.7 x 10 M . spore . h) and a low-affinity-high-rate system (K(m), 5.2 x 10 M; V(max), 8.9 x 10 M . spore . h). SG-1 spores did not oxidize Co(II) in the absence of oxygen, also indicating that oxidation was not due to abiological Co(II) oxidation on the surface of preformed Mn(III,IV) oxides. These results suggest that some microorganisms may directly oxidize Co(II) and such biological activities may exert some control on the behavior of Co in nature. SG-1 spores may also have useful applications in metal removal, recovery, and immobilization processes.

Journal Article↗

PubMed2005

The role of dietary oxidized cholesterol and oxidized fatty acids in the development of atherosclerosis.

The etiology of atherosclerosis is complex and multifactorial but there is extensive evidence indicating that oxidized lipoproteins may play a key role. At present, the site and mechanism by which lipoproteins are oxidized are not resolved, and it is not clear if oxidized lipoproteins form locally in the artery wall and/or are sequestered in atherosclerotic lesions following the uptake of circulating oxidized lipoproteins. We have been focusing our studies on demonstrating that such potentially atherogenic oxidized lipoproteins in the circulation are at least partially derived from oxidized lipids in the diet. Thus, the purpose of our work has been to determine in humans whether oxidized dietary oxidized fats such as oxidized fatty acids and oxidized cholesterol are absorbed and contribute to the pool of oxidized lipids in circulating lipoproteins. When a meal containing oxidized linoleic acid was fed to normal subjects, oxidized fatty acids were found only in the postprandial chylomicron/chylomicron remnants (CM/RM) which were cleared from circulation within 8 h. No oxidized fatty acids were detected in low density lipoprotein (LDL) or high density lipoprotein (HDL) fractions at any time. However, when alpha-epoxy cholesterol was fed to human subjects, alpha-epoxy cholesterol in serum was found in CM/RM and also in endogenous very low density lipoprotein, LDL, and HDL and remained in the circulation for 72 h. In vitro incubation of the CM/RM fraction containing alpha-epoxy cholesterol with human LDL and HDL that did not contain alpha-epoxy cholesterol resulted in a rapid transfer of oxidized cholesterol from CM/RM to both LDL and HDL. We have suggested that cholesteryl ester transfer protein is mediating the transfer. Thus, alpha-epoxy cholesterol in the diet is incorporated into CM/RM fraction and then transferred to LDL and HDL contributing to lipoprotein oxidation. We hypothesize that diet-derived oxidized fatty acids in chylomicron remnants and oxidized cholesterol in remnants and LDL accelerate atherosclerosis by increasing oxidized lipid levels in circulating LDL and chylomicron remnants. This hypothesis is supported by our feeding experiments in animals. When rabbits were fed oxidized fatty acids or oxidized cholesterol, the fatty streak lesions in the aorta were increased by 100%. Moreover, dietary oxidized cholesterol significantly increased aortic lesions in apo-E and LDL receptor-deficient mice. A typical Western diet is rich in oxidized fats and therefore could contribute to the increased arterial atherosclerosis in our population.

Animals↗

PubMed2000

Single-step nitrification models erroneously describe batch ammonia oxidation profiles when nitrite oxidation becomes rate limiting.

Nitrification involves the sequential biological oxidation of reduced nitrogen species such as ammonium-nitrogen (NH(4)(+)-N) to nitrite-nitrogen (NO(2)(-)-N) and nitrate-nitrogen (NO(3)(-)-N). The adequacy of modeling NH(4)(+)-N to NO(3)(-)-N oxidation as one composite biochemical reaction was examined at different relative dynamics of NH(4)(+)-N to NO(2)(-)-N and NO(2)(-)-N to NO(3)(-)-N oxidation. NH(4)(+)-N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation by a mixed nitrifying consortium were uncoupled using selective inhibitors allylthiourea and sodium azide. The kinetic parameters of NH(4)(+)-N to NO(2)(-)-N oxidation (q(max,ns) and K(S,ns)) and NO(2)(-)-N to NO(3)(-)-N oxidation (q(max,nb) and K(S,nb)) were determined by a rapid extant respirometric technique. The stoichiometric coefficients relating nitrogen removal, oxygen uptake and biomass synthesis were derived from an electron balanced equation. NH(4)(+)-N to NO(2)(-)-N oxidation was not affected by NO(2)(-)-N concentrations up to 100 mg NO(2)(-)-N L(-1). NO(2)(-)-N to NO(3)(-)-N oxidation was noncompetitively inhibited by NH(4)(+)-N but was not inhibited by NO(3)(-)-N concentrations up to 250 mg NO(3)(-)-N L(-1). When NH(4)(+)-N to NO(2)(-)-N oxidation was the sole rate-limiting step, complete NH(4)(+)-N to NO(3)(-)-N oxidation was adequately modeled as one composite process. However, when NH(4)(+)-N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation were both rate limiting, the estimated lumped kinetic parameter estimates describing NH(4)(+)-N to NO(3)(-)-N oxidation were unrealistically high and correlated. These findings indicate that the use of single-step models to describe batch NH(4)(+) oxidation yields erroneous kinetic parameters when NH(4)(+)-to-NO(2)(-) oxidation is not the sole rate-limiting process throughout the assay. Under such circumstances, it is necessary to quantify NH(4)(+)-N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation, independently.

Ammonia↗

PubMed1975

Breakage of a DNA-protein complex induced by 4-nitroquinoline 1-oxide, 4-nitropyridine 1-oxide, and their derivatives in cultured mouse fibroblasts.

The effects of a number of 4-nitroquinoline 1-oxide and 4-nitropyridine 1-oxide derivatives, with varying carcinogenic potencies, on the scission of proteins linking DNA were studied in cultured mouse fibroblasts, strain L-P-3. With twenty-two 4-nitroquinoline 1-oxide derivatives and twelve-4-nitroquinoline 1-oxide derivatives tested, an excellent correlation was found between the scission effect of each compound and its carcinogenicity. All carcinogens, whether strong or weak, showed positive results in the scission test. Strong carcinogens such as 4-nitroquinoline 1-oxide, 2-methyl-4-nitroquinoline 1-oxide, 6-methyl-4-nitroquinoline 1-oxide, 6-chloro-4-nitroquinoline 1-oxide,and 4-hydroxyaminoquinoline 1-oxide induced the scission at a low concentration of 1 x 10-5 M, while weak carcinogens such as 3-methyl-4-nitroquinoline 2-oxide, 6-n-butyl-4-nitroquinoline 1-oxide, 6-tert-butyl-4-nitroquinoline 1-oxide, 6-n-hexyl-4-nitroquinoline 1-oxide, and 6-carboxy-4-nitroquinoline 1-oxide only produced the same effect a dose levels higher than 5 x10-5 M. On the other hand, some noncarcinogenic derivatives such as 8-nitroquinoline 1-oxide, 4-hydoxy-quinoline 1-oxide, 4-aminoquinoline 1-oxide, and 6-nitroquinoline could not induce the scission, while other noncarcinogens such as 3-nitroquinoline 1-oxide, 5-nitroquinoline 1-oxide, and 5-nitroquinoline did induce scission at concentrations higher than 1 x 10-4 M. Throughout these tests the effective concentrations of active compounds were generally much lower than the concentration at which the compounds were cytotoxic. The implication of the results and the feasibility of the present method of analysis as a screening procedure for potential carcinogens and muagens are discussed.

Animals↗

PubMed2001

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.

Animals↗

PubMed1997

Contrasting effects of diethylenetriamine-nitric oxide, a spontaneously releasing nitric oxide donor, on pregnant rat uterine contractility in vitro versus in vivo.

OBJECTIVE: The aim of the study was to investigate the in vitro (on tension) and in vivo (on intrauterine pressure) effects of a spontaneously releasing nitric oxide adduct, diethylenetriamine-nitric oxide, on rat uterine contractility. STUDY DESIGN: Contractile responses to the nitric oxide donor diethylenetriamine-nitric oxide on isometric tension of rat uterine strips (in vitro) and on intrauterine pressure (in vivo) in anesthetized and conscious animals were quantified at late gestation and during preterm and term labor. Preterm labor was induced with the administration of a single injection of the antiprogestin onapristone (10 mg). All control animals were injected with diethylenetriamine, the parent compound, without nitric oxide. RESULTS: The nitric oxide donor diethylenetriamine-nitric oxide relaxes rat uterine tissues when given in vitro during gestation (median effective dose 0.30 +/- 0.09 mmol/L) but fails to have an effect on uterine tissues from laboring term and preterm animals in the muscle bath. Intraperitoneal injection of the nitric oxide adduct in doses of 5 mg and 50 mg produced a significant and sustained decrease in intrauterine pressure in both delivering and nondelivering animals compared with intrauterine pressure values before administration. Analysis of intrauterine pressure levels at different time frames (before injection, immediately after injection, and after 30, 60, and 90 minutes) in anesthetized rats between days 19 and 21 of gestation shows that the dose of 5 mg diethylenetriamine-nitric oxide significantly (p < 0.05) decreased contractility starling at 30 minutes after administration. Similarly, during labor diethylenetriamine-nitric oxide (5 mg intraperitoneally) decreased contractility (p < 0.05) but only starting at 60 minutes after injection. However, with the dose of 50 mg diethylenetriamine-nitric oxide contractility was reduced 30 minutes (p < 0.05) after treatment during pregnancy (days 19 to 21). The same dose of diethylenetriamine-nitric oxide produced a greater relaxation immediately after intraperitoneal injection (p < 0.05) in the laboring group. Diethylenetriamine-nitric oxide (50 mg) also induced an earlier onset and greater relaxation in animals delivering preterm compared with nondelivering animals at the same stage of gestation. The intrauterine pressure maintained constant contractility levels throughout the 100-minute recording period in the control groups of anesthetized and conscious animals that were injected with diethylenetriamine at the equivalent doses. In the preterm laboring group the effects were similar in both anesthetized and conscious animals. CONCLUSION: Although in vitro studies of uterine muscle show decreased inhibitory responses to nitric oxide donors during spontaneous term labor and preterm labor compared with tissues collected during late pregnancy (day 19), studies in vivo demonstrate that nitric oxide donors can decrease uterine contractility even more effectively during delivery. The results suggest that nitric oxide donors may act indirectly or that the fetal-placental unit increases the availability of nitric oxide in vivo. Therefore, despite what in vitro studies alone suggest, nitric oxide donor drugs may be very effective in suppressing either term or preterm labor.

Animals↗

PubMed1996

Mechanism of uptake of copper-oxidized low density lipoprotein in macrophages is dependent on its extent of oxidation.

Several investigators have reported nonreciprocal cross-competition between unlabeled acetyl low density lipoprotein (LDL) and oxidized LDL for the degradation of the corresponding labeled LDLs. The failure of acetyl LDL to compete fully for the degradation of oxidized LDL has been interpreted as evidence for additional receptor(s) specific for oxidized LDL. In the present study, it is demonstrated that the ability of oxidized LDL to compete for the degradation of acetyl LDL is determined largely by its extent of oxidation. Extensively oxidized LDL competed for 90% of acetyl LDL degradation in murine macrophages, and hence there appears to be no pathway in these cells that is specific for acetyl LDL but not oxidized LDL. The reciprocal situation (competition by acetyl LDL for uptake and degradation of oxidized LDL) proved to be more complicated. Oxidized LDL is known to be susceptible to aggregation, and less than half of the aggregates found in the present experiments were large enough to be removed by filtration or centrifugation at 10,000 x g. When oxidized LDL was prepared under conditions that resulted in minimal aggregation, acetyl LDL competed for greater than 80% of oxidized LDL degradation. With more extensive oxidation and aggregation of LDL, acetyl LDL only competed for about 45% of oxidized LDL degradation, while polyinosinic acid remained an effective competitor. Individual preparations of oxidized LDL that differed in degree of oxidation were separated into aggregated and nonaggregated fractions, and it was shown that both fractions were competed to a similar degree by acetyl LDL in mouse peritoneal macrophages and in Chinese hamster ovary cells transfected with human scavenger receptor type I cDNA. Hence, aggregation by itself did not alter the apparent rate of uptake by the scavenger receptor pathway. These results indicate that the extent of oxidation of LDL affects its mechanism of uptake and that about half of the uptake of very extensively oxidized LDL appears to be via a pathway distinct from the scavenger receptor type I/II. The uptake of very extensively oxidized LDL was not affected by cytochalasin D, an inhibitor of phagocytosis. As well, it was not affected by an antibody to CD36 in human monocyte-derived macrophages or in THP-1 cells, suggesting that this alternate pathway does not involve CD36.

Animals↗

PubMed1997

Pretreatment with inhaled nitric oxide inhibits neutrophil migration and oxidative activity resulting in attenuated sepsis-induced acute lung injury.

OBJECTIVE: To determine if, and by what mechanisms, inhaled nitric oxide attenuates acute lung injury in a porcine model of adult respiratory distress syndrome induced by Gram-negative sepsis. DESIGN: Nonrandomized, controlled study. SETTING: Laboratory at a university medical center. SUBJECTS: Thirty pathogen-free Yorkshire swine (15 to 20 kg). INTERVENTIONS: Four groups of swine were anesthetized, mechanically ventilated, and studied for 5 hrs. Both control-nitric oxide and septic-nitric oxide animals received inhaled nitric oxide at 20 parts per million throughout the study. Control (n = 10) and control-nitric oxide (n = 5) animals received a 1-hr infusion of sterile saline. Sepsis was induced in septic (n = 10) and septic-nitric oxide (n = 5) animals with a 1-hr intravenous infusion of live Pseudomonas aeruginosa. MEASUREMENTS AND MAIN RESULTS: Untreated septic animals developed a progressive decrease in Pao2 that was prevented in septic-nitric oxide animals (73 +/- 4 vs. 214 +/- 23 torr [9.7 +/- 0.5 vs. 28.5 +/- 3.1 kPa], respectively, at 5 hrs, p < .05). Untreated septic animals showed a significant increase in bronchoalveolar lavage protein and neutrophil count at 5 hrs, compared with the baseline value, indicating acute lung injury. Septic-nitric oxide animals showed no significant increase in these parameters. Peripheral blood neutrophils from untreated septic animals and septic-nitric oxide animals exhibited significant (p < .05) up-regulation of CD18 receptor expression and oxidant activity (10.5 +/- 0.9 and 5.0 +/- 0.9 nmol of superoxide anion/10(6) neutrophils/10 mins, respectively) compared with both control and control-nitric oxide animals (3.0 +/- 0.6 and 2.6 +/- 0.2 nmol of superoxide anion/10(6) neutrophils/10 mins, respectively). Also, priming for the oxidant burst at 5 hrs was decreased by 50% in septic-nitric oxide animals compared with untreated septic animals. Both untreated septic and septic-nitric oxide animals showed a significant increase in pulmonary arterial pressure at 30 mins (47.5 +/- 2.4 and 51.0 +/- 3.0 mm Hg, respectively), followed by a progressive decrease (32.8 +/- 2.6 and 31.3 +/- 5.4 mm Hg, respectively, at 5 hrs). Both of these changes were significant (p < .05) compared with baseline values and compared with the control groups. There was no significant difference in pulmonary arterial pressure or systemic arterial pressure at any time between untreated septic and septic-nitric oxide animals. CONCLUSIONS: These results demonstrate that inhaled nitric oxide attenuates alveolar-capillary membrane injury in this porcine model of Gram-negative sepsis but does not adversely affect systemic hemodynamics. The data suggest that inhaled nitric oxide preserves alveolar-capillary membrane integrity by the following means: a) inhibiting transendothelial migration of activated, tightly adherent neutrophils; and b) possibly by attenuating the neutrophil oxidant burst.

Administration, Inhalation↗

PubMed1999

Evidence for nitric oxide and nitric oxide synthase activity in proximal stumps of transected peripheral nerves.

Nitric oxide may be liberated as an inflammatory mediator within injured peripheral nerve trunks. We evaluated the proximal stumps of injured peripheral nerve stumps that later form neuromas or regenerative nerve sprouts, for evidence of local nitric oxide elaboration and activity. Proximal stumps were created in male Sprague-Dawley rats by sectioning of the sciatic nerve and resection of its distal portions and branches. There was striking physiological evidence of nitric oxide activity at the tips of 48-h and 14-day-old proximal nerve stumps. We detected local nitric oxide-mediated hyperemia of both extrinsic plexus and endoneurial microvessels that was reversible, in a dose-dependent stereospecific fashion, by the broad-spectrum nitric oxide synthase inhibitors, Nomega-nitro-L-arginine-methyl ester or Nomega-nitro-L-arginine, but not by 7-nitroindazole, an inhibitor with relative selectivity for neuronal nitric oxide. Immunohistochemical studies provided evidence for the localization of nitric oxide generators at the same sites. In 48-h but not 14-day stumps increased expression of two isoforms of nitric oxide synthase was detected: endothelial nitric oxide and to a much lesser extent neuronal nitric oxide synthase. Both isoforms appeared in axonal endbulb-like profiles that co-localized with neurofilament immunostaining. Western immunoblots identified a band consistent with endothelial nitric oxide synthase expression. In 14-day stumps with early neuroma formation, but not 48-h stumps, there was staining for immunological nitric oxide synthase in some endoneurial and epineurial macrophages. Total nitric oxide synthase biochemical enzymatic activity, measured by labelled arginine to citrulline conversion, was increased in 14-day but not 48-h stumps. Injured peripheral nerves have evidence of early nitric oxide action, nitric oxide synthase expression and nitric oxide activity in proximal nerve stumps. Nitric oxide may have an important impact on the regenerative milieu.

Animals↗

PubMed1989

Oxidation of vitamin E in red cell membranes by fatty acids, hydroperoxides and selected oxidants.

Our laboratory previously reported that vitamin E (tocopherol) in human blood platelets was oxidized in vitro by various oxidants. This paper shows that diamide, superoxide, hydroperoxides and polyunsaturated fatty acids induce oxidation of tocopherols in red cell membranes. In contrast to platelets, red cell membrane tocopherol was oxidized by hydrogen peroxide and tertiary butyl hydroperoxide. Alpha tocopherolquinone was one of the products of oxidation. Among the fatty acids, the cis polyunsaturated acids were the most potent oxidizing agents with monounsaturated and trans compounds relatively ineffective. The oxidation is not a detergent effect of the fatty acids since neither the detergents Brij and Lubrol, when present in concentrations under 0.5 mM, nor sodium arachidonate (1.25 mM), could oxidize the membrane tocopherol. When red cell membrane samples were incubated with 0.5 mM arachidonate, 47 +/- 11% (S.D.) of the tocopherol lost was converted to tocopherolquinone. Unlike arachidonate, oxidants such as diamide, hydrogen peroxide and tertiary butylhydroperoxide are unable to oxidize all of the membrane tocopherol and produce less tocopherolquinone from oxidation (10-15%) under the experimental conditions of this study. Linoleic acid hydroperoxide is a much more potent oxidant and produces less quinone than arachidonate. The mechanisms of tocopherol oxidations induced by the various compounds seem to be different since the yields of quinone during oxidation vary with the nature of the oxidant. Tocopherol is consumed by oxidation as it protects the membrane from oxidant damage induced by compounds such as unsaturated fatty acids and hydroperoxides.

Adult↗

PubMed1989

Protein oxidation and proteolysis during aging and oxidative stress.

Previous studies in this laboratory have shown that glutamine synthetase (GS) and other key metabolic enzymes are inactivated by metal-catalyzed oxidation reactions in vitro. Oxidative inactivation renders these proteins highly susceptible to proteolysis, especially to a class of newly identified alkaline proteases which exhibit little or no activity against the native enzymes. These studies have suggested that oxidative inactivation may be an important marking step for intracellular protein degradation. Because many of the enzymes which have been shown to accumulate as inactive or less active forms during aging are readily inactivated by metal-catalyzed oxidation reactions in vitro, we have investigated the possible relationship between protein oxidation and proteolysis during aging and oxidative stress in vivo. Oxidized proteins accumulate in hepatocytes of rats exposed to 100% oxygen during the first 48 h of oxygen treatment. In the interval between 48 and 54 h the levels of oxidized proteins decline sharply. The specific activities of at least two liver enzymes, glutamine synthetase and glucose-6-phosphate dehydrogenase (G-6-PDH), decrease during the 54-h experiment. GS and G-6-PDH specific immunological cross-reactivity remains high during the first 48 h of oxygen treatment and then declines in the interval between 48 and 54 h. During this same interval the levels of alkaline proteases which degrade oxidized proteins increase, indicating that these activities are induced or activated in response to oxidative stress and subsequently degrade the proteins which have become oxidized during the initial phase of oxygen treatment. Oxidized proteins accumulate progressively during aging in hepatocytes from rats 3 to 26 months old, with the largest incremental increase between 20 and 26 months. The increase in protein oxidation is correlated with a loss of specific activity of GS and G-6-PDH without a concomitant loss of immunological cross-reactivity. The levels of alkaline proteases which degrade oxidized proteins in hepatocytes from 26-month-old rats is only 20% that of 3-month-old rats, suggesting that oxidized proteins accumulate in hepatocytes from old rats, in part, because the proteases which degrade them are deficient or defective. moreover, when old rats are subjected to treatment with 100% oxygen, the levels of oxidized proteins continue to increase and the alkaline protease activity remains low, indicating that these protease activities are not increased in response to oxidative stress in old rats.

Aging↗

PubMed1999

Metabolism of oxidized phosphatidylcholines formed in oxidized low density lipoprotein by lecithin-cholesterol acyltransferase.

The possible involvement of lecithin-cholesterol acyltransferase (LCAT) in the metabolism of oxidized phosphatidylcholine (PC) in plasma was investigated. A variety of oxidized products are formed from PC following oxidation of low density lipoproteins (LDL). A significant increase in LDL oxidation levels in patients with familial LCAT deficiency (FLD) has been previously demonstrated by a sensitive sandwich ELISA for oxidized LDL using the monoclonal antibody DLH3 which recognizes oxidized products of PC. In the present study, we found that LCAT produces various metabolites from oxidized PC and that oxidized PC molecules in LDL particles serve as substrates. When the neutral lipid fraction was separated by TLC after the incubation of oxidized 1-palmitoyl-2-[1-14C]linoleoyl PC with human plasma, a number of radioactive bands were formed in addition to cholesteryl ester. These products were not formed from native 1-palmitoyl-2-[1-14C]linoleoyl PC. Plasma from FLD patients also failed to form the additional products from oxidized PC. The addition of dithio-bis(nitrobenzoate) (DTNB), an LCAT inhibitor, or the inactivation of LCAT activity by treating the plasma at 56 degrees C for 30 min abolished the generation of these products from oxidized PC. The activity was recovered in the high density lipoprotein (HDL) fraction but not in the LDL fraction separated from normal plasma. When 1-palmitoyl-2-[1-14C](9-oxononanoyl) PC and 1-stearoyl-2-[1-14C](5-oxovaleroyl)PC, PC oxidation products that contain short chain aldehydes, were incubated with human plasma, radioactive products in the neutral lipid fraction were observed on TLC. LDL containing oxidized PC was measured by sandwich ELISA using an anti-apolipoprotein B antibody and DLH3. The reconstituted oxidized PC-LDL particles were found to have lost their ability to bind DLH3 upon incubation with HDL, while the reactivity of the reconstituted oxidized PC-LDL remained unchanged in the presence of DTNB. These results suggest that LCAT is capable of metabolizing a variety of oxidized products of PC and preventing the accumulation of oxidized PC in circulating LDL particles.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2005

Lack of alteration of endogenous nitric oxide pathway during prolonged nitric oxide inhalation in intensive care unit patients.

OBJECTIVE: To compare hemodynamic and gasometric variables and the plasma concentrations of nitric oxide metabolites (cyclic guanosine monophosphate and nitrate and nitrite), endothelin-1, and renin-angiotensin metabolites before and after the start of nitric oxide inhalation, after prolonged nitric oxide inhalation, and before and after nitric oxide withdrawal. DESIGN: Prospective study. SETTING: Surgical intensive care unit, university hospital. SUBJECTS: Patients with acute lung injury and right ventricular failure. INTERVENTIONS: Nitric oxide inhalation (10-12 ppm) during a median of 2.9 days (12 hrs to 6.5 days). MEASUREMENTS AND MAIN RESULTS: The pulmonary vasodilator effects of inhaled nitric oxide improved arterial oxygenation in patients with acute lung injury (p < .05) and reduced right atrial pressure in patients with right ventricular dysfunction (p < .01). These beneficial effects lasted the whole period of prolonged inhaled nitric oxide therapy up to 6.5 days. However, when inhaled nitric oxide was withdrawn, pulmonary vasodilator effects rapidly disappeared, and Pao2/Fio2 ratio markedly deteriorated in all studied patients to return to pre-inhaled nitric oxide levels. Changes in plasma cyclic guanosine monophosphate and nitrate and nitrite paralleled those of pulmonary vasodilatory effects. An immediate increase in plasma cyclic guanosine monophosphate with a slightly delayed increase in plasma nitrate and nitrite was observed at inhaled nitric oxide start with no attenuation during the prolonged inhaled nitric oxide therapy. A marked decrease toward pre-inhaled nitric oxide levels was seen within hours of inhaled nitric oxide withdrawal. In addition, no alteration of plasma endothelin-1 or renin-angiotensin mediators was observed during or after inhaled nitric oxide therapy. CONCLUSIONS: Our study showed a lack of attenuation in the beneficial effects of inhaled nitric oxide and a lack of alteration of endogenous nitric oxide, endothelin-1, and renin-angiotensin pathways during prolonged nitric oxide inhalation.

Administration, Inhalation↗

PubMed2000

The antianginal drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase.

Trimetazidine is a clinically effective antianginal agent that has no negative inotropic or vasodilator properties. Although it is thought to have direct cytoprotective actions on the myocardium, the mechanism(s) by which this occurs is as yet undefined. In this study, we determined what effects trimetazidine has on both fatty acid and glucose metabolism in isolated working rat hearts and on the activities of various enzymes involved in fatty acid oxidation. Hearts were perfused with Krebs-Henseleit solution containing 100 microU/mL insulin, 3% albumin, 5 mmol/L glucose, and fatty acids of different chain lengths. Both glucose and fatty acids were appropriately radiolabeled with either (3)H or (14)C for measurement of glycolysis, glucose oxidation, and fatty acid oxidation. Trimetazidine had no effect on myocardial oxygen consumption or cardiac work under any aerobic perfusion condition used. In hearts perfused with 5 mmol/L glucose and 0.4 mmol/L palmitate, trimetazidine decreased the rate of palmitate oxidation from 488+/-24 to 408+/-15 nmol x g dry weight(-1) x minute(-1) (P<0.05), whereas it increased rates of glucose oxidation from 1889+/-119 to 2378+/-166 nmol x g dry weight(-1) x minute(-1) (P<0.05). In hearts subjected to low-flow ischemia, trimetazidine resulted in a 210% increase in glucose oxidation rates. In both aerobic and ischemic hearts, glycolytic rates were unaltered by trimetazidine. The effects of trimetazidine on glucose oxidation were accompanied by a 37% increase in the active form of pyruvate dehydrogenase, the rate-limiting enzyme for glucose oxidation. No effect of trimetazidine was observed on glycolysis, glucose oxidation, fatty acid oxidation, or active pyruvate dehydrogenase when palmitate was substituted with 0.8 mmol/L octanoate or 1.6 mmol/L butyrate, suggesting that trimetazidine directly inhibits long-chain fatty acid oxidation. This reduction in fatty acid oxidation was accompanied by a significant decrease in the activity of the long-chain isoform of the last enzyme involved in fatty acid beta-oxidation, 3-ketoacyl coenzyme A (CoA) thiolase activity (IC(50) of 75 nmol/L). In contrast, concentrations of trimetazidine in excess of 10 and 100 micromol/L were needed to inhibit the medium- and short-chain forms of 3-ketoacyl CoA thiolase, respectively. Previous studies have shown that inhibition of fatty acid oxidation and stimulation of glucose oxidation can protect the ischemic heart. Therefore, our data suggest that the antianginal effects of trimetazidine may occur because of an inhibition of long-chain 3-ketoacyl CoA thiolase activity, which results in a reduction in fatty acid oxidation and a stimulation of glucose oxidation.

Angina Pectoris↗

PubMed1983

Soluble guanylate cyclase activation by nitric oxide and its reversal. Involvement of sulfhydryl group oxidation and reduction.

Pre-incubation of either crude or purified nitric oxide-stimulated soluble lung guanylate cyclase resulted in a temperature-dependent decay of enzyme activity. The decay of nitric oxide-stimulated activity during pre-incubation was associated with a reduced responsiveness of the enzyme to reactivation by a second exposure to nitric oxide. This loss of enzyme responsiveness to reactivation by nitric oxide was greater with purified guanylate cyclase than with the crude enzyme and was highly dependent upon the nitric oxide dose. The addition of dithiothreitol or other thiols to nitric oxide-stimulated enzyme markedly accelerated the decay of activity in a dose-dependent manner. In addition, thiols prevented the loss of responsiveness of guanylate cyclase to reactivation by nitric oxide. Nitric oxide-stimulated enzyme activity was, therefore, reversed by the addition of thiol reducing agents. The addition of the thiol oxidizing agents, diamide or oxidized glutathione, to nitric oxide-stimulated guanylate cyclase caused a rapid and irreversible loss of activity. The effects of diamide or oxidized glutathione on the crude enzyme were prevented by excess dithiothreitol. Dithiothreitol did not prevent the destruction of purified nitric oxide-stimulated guanylate cyclase activity by diamide or oxidized glutathione, however. The results suggest that nitric oxide activation and its reversal are linked to the reversible oxidation and reduction, respectively, of sulfhydryl groups on guanylate cyclase which are involved in enzyme activation. The results further suggest the existence of a second class of sulfhydryl groups involved in the maintenance of enzyme activity.

Animals↗