共查询到20条相似文献,搜索用时 15 毫秒
1.
D J Stuehr N S Kwon C F Nathan 《Biochemical and biophysical research communications》1990,168(2):558-565
Following partial purification of macrophage nitric oxide (NO) synthase, enzyme activity requires L-arginine, NADPH, and constitutive cytosolic factors, one of which is tetrahydrobiopterin (BH4) (Kwon, N.S., Nathan, C.F. and Stuehr, D.J. [1989] J. Biol. Chem. 264, 20496). Here we identify FAD and GSH as two additional cofactors needed for full enzyme activity. With all defined cytosolic cofactors in excess, NO synthesis was linear over 3 h and was approximately 50% dependent on exogenous FAD, approximately 50% on glutathione (GSH), 84% on tetrahydrobiopterin (BH4), 95% on NADPH, and 98% on L-arginine. The concentrations of added FAD, GSH, and BH4 required for optimal activity were consistent with their levels in macrophage cytosol. Kinetic studies showed that GSH (or DTT) had little or no effect on the rate of NO generation over the first 20-30 min of the reaction, but prevented a subsequent dropoff in rate. This effect was distinct from thiol participation in BH4 regeneration. In contrast, exogenous FAD doubled the rate of NO synthesis throughout the assay period, consistent with a cofactor role. The role of NADPH was not to regenerate BH4, furnish NADP+, nor form reactive oxygen intermediates. These findings demonstrate NO synthesis by a partially purified enzyme in an otherwise defined system, and suggest that an NADPH-utilizing FAD flavoprotein may participate in the reaction. 相似文献
2.
Iodonium-class flavoprotein dehydrogenase inhibitors have been demonstrated to possess antiproliferative potential and to inhibit reactive oxygen production in human tumor cells, although the mechanism(s) that explains the relationship between altered cell growth and the generation of reactive oxygen species (ROS) remains an area of active investigation. Because of the ability of these compounds to inhibit the activity of flavoprotein-containing epithelial NADPH oxidases, we chose to examine the effects of several iodonium-class flavoprotein inhibitors on human colon cancer cell lines that express high, functional levels of a single such oxidase (NADPH oxidase 1, or Nox1). We found that diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodonium diphenyl inhibited the growth of Caco2, HT-29, and LS-174 T colon cancer cells at concentrations (10–250 nM for DPI, 0.5–2.5 μM for DTI, and 155 nM to 10 μM for iodonium diphenyl) substantially lower than needed for DU145 human prostate cancer cells, which do not possess functional NADPH oxidase activity. Drug treatment was associated with decreased H2O2 production and diminished intracellular ROS levels, lasting up to 24 h, after short-term (1-h) exposure to the iodonium analogs. Decreased tumor cell proliferation was caused, in part, by a profound block in cell cycle progression at the G1/S interface in both LS-174 T and HT-29 cells exposed to either DPI or DTI; and the G1 block was produced, for LS-174 T cells, by upregulation of p27 and a drug concentration-related decrease in the expression of cyclins D1, A, and E that was partially prevented by exogenous H2O2. Not only did DPI and DTI decrease intracellular ROS, they both also significantly decreased the mRNA expression levels of Nox1, potentially contributing to the prolonged reduction in tumor cell reactive oxygen levels. We also found that DPI and DTI significantly decreased the growth of both HT-29 and LS-174 T human tumor xenografts, at dose levels that produced peak plasma concentrations similar to those utilized for our in vitro experiments. These findings suggest that iodonium analogs have therapeutic potential for NADPH oxidase-containing human colon cancers in vivo and that at least part of their antineoplastic mechanism of action may be related to targeting Nox1. 相似文献
3.
4.
Our findings show a significant increase of free radical production after exposure to 50 Hz electromagnetic fields at a flux density of 1 mT to mouse bone marrow-derived (MBM) promonocytes and macrophages, indicating the cell-activating capacity of extremely low frequency magnetic fields (ELF-MF). We demonstrate that after exposure to ELF-MF mainly superoxide anion radicals were produced, both in MBM macrophages (33%) and also in their precursor cells (24%). To elucidate whether NADPH- or NADH-oxidase functions are target proteins for MF interaction, the flavoprotein inhibitor diphenyleneiodonium chloride (DPI) was used. MF-induced free radical production was not inhibited by DPI, whereas tetradecanoylphorbolacetate (TPA)-induced free radical production was diminished by about 70%. TPA is known to induce a direct activation of NADPH-oxidase through the PKC pathway. Since DPI lacks an inhibitory effect in MF-exposed MBM cells, we suggest that 50 Hz MF stimulates the NADH-oxidase pathway to produce superoxide anion radicals, but not the NADPH pathway. Furthermore, we showed an oscillation (1-10 days) in superoxide anion radical release in mouse macrophages, indicating a cyclic pattern of NADH-oxidase activity. 相似文献
5.
AIMS: In this work, we studied the mechanisms by which diphenyleneiodonium chloride (DPI) inhibits nitric oxide (NO) synthesis induced by the proinflammatory cytokine interleukin-1beta (IL-1) in bovine articular chondrocytes. To achieve this, we evaluated the ability of DPI to inhibit the expression and activity of the inducible isoform of the NO synthase (iNOS) induced by IL-1. We also studied the ability of DPI to prevent IL-1-induced NF-kappaB activation and reactive oxygen species (ROS) production. RESULTS: Northern and Western blot analysis, respectively, showed that DPI dose-dependently inhibited IL-1-induced iNOS mRNA and protein synthesis in primary cultures of bovine articular chondrocytes. DPI effectively inhibited NO production (IC50=0.03+/-0.004 microM), as evaluated by the method of Griess. Nuclear factor-kappa B (NF-kappaB) activation, as evaluated by electrophoretic mobility shift assay, was inhibited by DPI (1-10 microM) in a dose-dependent manner. IL-1-induced ROS production, as evaluated by measurement of dichlorofluorescein fluorescence, was inhibited by DPI at concentrations that also prevented NF-kappaB activation and iNOS expression. CONCLUSIONS: DPI inhibits IL-1-induced NO production in chondrocytes by two distinct mechanisms: (i) by inhibiting NOS activity, and (ii) by preventing iNOS expression through the blockade of NF-kappaB activation. These results also support the involvement of reactive oxygen species in IL-1-induced NF-kappaB activation and expression of NF-kappaB-dependent genes, such as iNOS. 相似文献
6.
Lipopolysaccharide (LPS) induced a time-dependent synthesis of nitric oxide (NO) in EMT6 adenocarcinoma cells, assayed by accumulation of NO-derived nitrite in the medium. The induction of NO synthesis was inhibited in a concentration-dependent manner by the glucocorticoids dexamethasone (IC50 = 5 nM) and hydrocortisone (IC50 = 20 nM) and this effect was partially antagonized by progesterone and cortexolone. If addition of dexamethasone was delayed 6 h or more, inhibition of nitrite accumulation over 24 h was substantially reduced, indicating a lack of direct effect of glucocorticoids on the NO synthase. Nitrite accumulation was accompanied by cell damage, which was increased by L-arginine and inhibited by NG-monomethyl-L-arginine (L-NMMA) and dexamethasone. These data show that NO is a primary cytotoxic mediator and that suppression of its formation by glucocorticoids explains some of their anti-inflammatory and cytoprotective effects. 相似文献
7.
In stimulated murine macrophage, arginase and nitric oxide synthase (NOS) compete for their common substrate, l-arginine. The objectives of this study were (i) to test the new alpha-amino acid N(omega)-hydroxy-nor-l-arginine (nor-NOHA) as a new selective arginase inhibitor and (ii) to elucidate the effects of arginase inhibition on l-arginine utilization by an inducible NOS. Nor-NOHA is about 40-fold more potent than N(omega)-hydroxy-l-arginine (NOHA), an intermediate in the l-arginine/NO pathway, to inhibit the hydrolysis of l-arginine to l-ornithine catalyzed by unstimulated murine macrophages (IC(50) values 12 +/- 5 and 400 +/- 50 microM, respectively). Stimulation of murine macrophages with interferon-gamma and lipopolysaccharide (IFN-gamma + LPS) results in clear expression of an inducible NOS (iNOS) and to an increase in arginase activity. Nor-NOHA is also a potent inhibitor of arginase in IFN-gamma + LPS-stimulated macrophage (IC(50) value 10 +/- 3 microM). In contrast to NOHA, nor-NOHA is neither a substrate nor an inhibitor for iNOS and it appears as a useful tool to study the interplays between arginase and NOS. Inhibition of arginase by nor-NOHA increases nitrite and l-citrulline accumulation for incubation times higher than 12 h, under our conditions. Our results allow the determination of the kinetic parameters of the two competitive pathways and the proposal of a simple model which readily explains the differences observed between experiments. This model readily accounts for the observed effects and should be useful to predict the consequences of arginase inhibition in the presence of an active NOS on l-arginine availability. 相似文献
8.
Alpha 2-macroglobulin 'fast' forms inhibit superoxide production by activated macrophages 总被引:3,自引:0,他引:3
Mouse peritoneal macrophages activated by bacillus Calmette-Guerin (BCG) were incubated with human alpha 2-macroglobulin converted to its 'fast' form with either trypsin or methylamine before being stimulated with phorbol myrystate acetate. Both alpha 2-macroglobulin-trypsin and alpha 2-macroglobulin-methylamine inhibited macrophage production of superoxide anion (O2-) while native alpha 2-macroglobulin had little effect except at high concentration. The alpha 2-macroglobulin 'fast' forms, which bind with a Kd of about 8 nM, inhibited 50% generation of O2- (ID50) at a concentration of 7 nM while alpha 2-macroglobulin inhibited O2- production with an ID50 of 141 nM. The 'fast' forms of alpha 2-macroglobulin may play a role in the feedback regulation of inflammatory reactions. 相似文献
9.
Bartík P Chalupský K Vavruska L Muller B Stoclet JC Entlicher G 《Physiological research / Academia Scientiarum Bohemoslovaca》2004,53(6):615-620
Diaphorase was studied as a possible oxidoreductase participating in NO production from some vasorelaxants. In the presence of NADH or NADPH, diaphorase can convert selected NO donors, glycerol trinitrate (GTN) and formaldoxime (FAL) to nitrites and nitrates with NO as an intermediate. This activity of diaphorase was inhibited by diphenyleneiodonium (DPI) (inhibitor of some NADPH-dependent flavoprotein oxidoreductases), while it remained uninhibited by NG-nitro-L-arginine methyl ester (inhibitor of NO synthase) 7-Ethoxyresorufin (inhibitor of cytochrome P-450 1A1 and cytochrome P-450 NADPH-dependent reductase) inhibited the conversion of GTN only. Existence of NO as an intermediate of the reaction was supported by results of electron paramagnetic resonance spectroscopy. In addition to its ability to affect the above mentioned NO donors, diaphorase was able to reduce 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) and thus to eliminate its NO scavenging effect. This activity of diaphorase could also be inhibited by DPI. The reaction of diaphorase with GTN and PTIO was not affected by superoxide dismutase (SOD) or catalase. Reaction of FAL with diaphorase was lowered with SOD by 38 % indicating the partial participation of superoxide anion probably generated by the reaction of diaphorase with NADH or NADPH. Catalase had no effect. Diaphorase could apparently be one of the enzymes participating in the metabolism of studied NO donors to NO. The easy reduction and consequent elimination of PTIO by diaphorase could affect its use as an NO scavenger in biological tissues. 相似文献
10.
R Keller S Bassetti R Keist A Mülsch S Klauser 《Biochemical and biophysical research communications》1992,184(3):1364-1371
Various bacteria and bacterial products induce in pure, lymphocyte-free bone marrow-derived mononuclear phagocytes (BMM?) the generation of tumor necrosis factor, nitric oxide (NO) synthase, NO and nitrite (NO2-), the flow of L-arginine to citrulline, and tumoricidal activity. The flow of L-arginine to citrulline and formation of NO/NO2- on the one hand and expression of tumoricidal activity were not always closely related; however, these parameters were suppressed in a dose-dependent manner by the flavoprotein inhibitor, diphenyleneiodonium (DPI) and the L-arginine analogue, NG-monomethyl-L-arginine (NMMA). The findings support the concept of a central role of the NO synthase pathway in the generation of tumor necrosis factor-independent tumoricidal activity by activated macrophages but the exact conditions which enable the transfer of the lytic principle from the effector to the target cell remain to be elucidated. 相似文献
11.
Nitric oxide synthesis in the CNS endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues. 总被引:9,自引:0,他引:9
L E Lambert J P Whitten B M Baron H C Cheng N S Doherty I A McDonald 《Life sciences》1991,48(1):69-75
Inhibition of nitric oxide production by arginine analogues was examined in three cell systems; macrophages, CNS tissue and endothelial cells. Nitric oxide production was assessed indirectly using in vitro assays measuring nitrite production (macrophages), cGMP elevation (CNS) and acetylcholine-induced relaxation of aortic ring segments (endothelium). NG-monomethyl-L-arginine and NG-amino-L-arginine possessed similar inhibitory activity in all three assays, while NG-nitro-L-arginine displayed a striking selectivity for inhibition of brain and endothelial cell nitric oxide synthesis, with IC50 values of 0.05 microM in the CNS versus 200 microM in macrophages. These results suggest that distinct enzymes are responsible for nitric oxide synthesis in different cell types, and indicate that it may be possible to selectively modulate nitric oxide production in vivo. 相似文献
12.
Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis. 总被引:8,自引:0,他引:8
D Amin S A Cornell S K Gustafson S J Needle J W Ullrich G E Bilder M H Perrone 《Journal of lipid research》1992,33(11):1657-1663
Some bisphosphonates used for the treatment of bone disorders are also potent inhibitors of squalene synthase, a critical enzyme for sterol biosynthesis. Among seven drugs tested, YM 175 (cycloheptylaminomethylene-1,1-bisphosphonic acid) was the most potent inhibitor of rat liver microsomal squalene synthase (Ki = 57 nM) and sterol biosynthesis from [14C]mevalonate in rat liver homogenate (IC50 = 17 nM). EB 1053 (3-(1-pyrolidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) and PHPBP (3-(1-piperidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) were less potent inhibitors in both these assays. Pamidronate and alendronate were poor inhibitors of squalene synthase (IC50 > 10 microM) but were potent inhibitors of sterol biosynthesis from mevalonate (IC50 = 420 and 168 nM, respectively), suggesting that the latter two agents may have inhibited other enzymes involved in the synthesis of farnesyl pyrophosphate from mevalonate. Etidronate and clodronate were inactive in both these assays. YM 175 also inhibited sterol biosynthesis in mouse macrophage J774 cells (IC50 = 64 microM) and in rats, when administered acutely, it inhibited cholesterol biosynthesis in the liver (ED50 = 30 mg/kg, s.c.). Structural modifications on YM 175 to enhance cell permeability may result in a new class of cholesterol-lowering agents. 相似文献
13.
H Yoneyama H Kosaka T Ohnishi T Kawazoe K Mizoguchi Y Ichikawa 《European journal of biochemistry》1999,266(3):771-777
A water-soluble iron complex with N-dithiocarboxysarcosine (Fe-DTCS) has been developed as an ESR spin-trapping agent for NO and successfully applied to ESR imaging of endogenous NO production in mice. We attempted to measure NO produced by purified neuronal NO synthase (nNOS) by this method, but could not detect NO. We speculated that Fe-DTCS inhibits NOS activity. In fact, it markedly inhibited NOS activity with an IC50 value of 9.7 +/- 0.7 microM in the citrulline-formation assay. DTCS alone did not inhibit the activity. An iron complex with N-methyl-D-glucamine dithiocarbamate, a similar spin-trapping agent for NO, also inhibited the activity, with an IC50 value of 25.1 +/- 2.9 microM. Fe-DTCS suppressed cytochrome c and ferricyanide reductase activities of nNOS, and markedly increased nNOS-mediated NADPH oxidation. Concomitantly, it accelerated oxygen consumption caused by activated nNOS. These results suggest that the ESR spin-trapping agent Fe-DTCS inhibits NO synthesis by interfering with the physiological electron flow from NADPH to nNOS heme iron. 相似文献
14.
The membrane-bound NADPH:O2 oxidoreductase of human neutrophils has been solubilized in approximately 70% yield and purified on concanavalin A-Sepharose and gel sieving columns of varying bed volumes and sieving ranges. The half-life of the solubilized oxidoreductase stored at 2-4 degrees C in the presence of 25% glycerol at pH 8.6 is approximately 30 h. The oxidoreductase contains a flavoprotein identifiable by its fluorescence spectrum for FAD which binds weakly to concanavalin A-Sepharose and elutes from gel sieving columns at a molecular weight range of approximately 51,000. This flavoprotein accounts for approximately 70% of the total FAD content found in granular membrane fractions recovered from activated neutrophils. Recovery of oxidoreductase activity from both concanavalin A-Sepharose affinity and gel sieving columns is affected by the resolution of the flavoprotein free of the cytochrome b component of the oxidoreductase. The resolved flavoprotein and cytochrome b appear unable to catalyze either NADH nor NADPH oxidase activities with O2, ferricyanide, or nitroblue tetrazolium salt serving as electron acceptors. 相似文献
15.
Nitric oxide synthase (NOS: EC 1.14.13.39) catalyzes L-arginine oxidation to generate nitric oxide (NO) and L-citrulline. Recently, 7-ethoxyresorufin (7-ER), a specific substrate of cytochrome P-4501A1, was used as a cytochrome P-450 inhibitor to study the mechanism underlying the vasodilatation caused by some drugs, and was suggested to inhibit nitric oxide-mediated relaxation. Herein we demonstrate that 7-ER inhibits NO synthesis by uncoupling neuronal nitric oxide synthase (nNOS). 7-ER is a noncompetitive inhibitor of nNOS with respect to L-arginine with a Ki value of 0.76 +/- 0.06 microM. The decrease in NO formation is inversely correlated with an increase in NADPH oxidation. 7-ER binds to nNOS with a Km value of 0.68 +/- 0.07 microM, as calculated from the nNOS-dependent NADPH oxidation in the absence of L-arginine. nNOS catalyzes the reduction of 7-ER at the expense of NADPH. The flavoprotein inhibitor, diphenyleneiodonium chloride (100 microM), completely inhibited nNOS-dependent 7-ER reduction. While nitro-L-arginine (1 mM) and N(G)-nitro-L-arginine methyl ester (1 mM), specific inhibitors of nNOS, and phenylisocyanide (0.1 mM), a specific heme iron ligand, did not affect the reduction of 7-ER. These results indicate that the reductase domain, but not the oxygenase domain, of nNOS is involved in the reduction of 7-ER. 7-ER uncouples nNOS, shunting electrons from the reductase domain to the oxygenase domain of the enzyme. As a consequence, NO synthesis is inhibited. 相似文献
16.
The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages. 总被引:13,自引:3,他引:10
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Peritoneal macrophages were elicited in rats by using casein as a stimulus; when stimulated with phorbol 12-myristate 13-acetate (PMA) they produced O2.-. Nearly 60% of the total cytochrome b had a low Em,7.0 of -247 mV, typical of the cytochrome b component found in the NADPH-dependent O2(.-)-generating oxidase of neutrophils. The rate of O2.- generation by macrophages was 1.23 mol of O2.-/s per mol of cytochrome b. Treatment of intact macrophages with diphenyleniodonium (DPI) at 0.9 microM caused 50% inhibition of PMA-induced O2.- generation, with little effect on mitochondrial respiratory activity; KCN inhibited respiratory activity without affecting PMA-induced O2.- generation. A similar specificity of inhibition was found for di-2-thienyliodonium (50% inhibition of O2.- generation at 0.5 microM) and, at higher concentrations, for diphenyl iodonium. When macrophage suspensions were incubated with [125I]DPI followed by autoradiography of SDS/polyacrylamide-gel-electrophoresis-separated polypeptides, radioactivity was most strongly associated with a band of Mr 45,000, similar to that found in neutrophils [Cross & Jones (1986) Biochem. J. 237, 111-116]. The O2(.-)-generating oxidase of macrophages appears to have components in common with the NADPH oxidase of neutrophils, despite differences in activity. Its sensitivity to DPI suggests that selective prevention of radical generation by macrophages in vivo is possible. 相似文献
17.
The possible involvement of different effector systems (nitric oxide synthase, guanylate cyclase, beta-adrenergic and muscarinic cholinergic receptors, cyclooxygenase and lipoxygenase, and Na(+),K(+)-ATPase) was evaluated in a histamine H(3) receptor agonist-induced ((R)alpha-methylhistamine, (R)alpha-MeHA) endothelium-dependent rat aorta relaxation assay. (R)alpha-MeHA (0.1 nM - 0.01 mM) relaxed endothelium-dependent rat aorta, with a pD(2) value of 8.22 +/- 0.06, compared with a pD(2) value of 7.98 +/- 0.02 caused by histamine (50% and 70% relaxation, respectively). The effect of (R)alpha-MeHA (0.1 nM - 0.01 mM) was competitively antagonized by thioperamide (1, 10 and 30 nM) (pA(2) = 9.21 +/- 0.40; slope = 1.03 +/- 0.35) but it was unaffected by pyrilamine (100 nM), cimetidine (1 muM), atropine (10 muM), propranolol (1 muM), indomethacin (10 muM) or nordthydroguaiaretic acid (0.1 mM). Inhibitors of nitric oxide synthase, L-N(G)-monomethylarginine (L-NMMA, 10 muM) and N(G)-nitro-L-arginine methylester (L-NOARG, 10 muM) inhibited the relaxation effect of (R)alpha-MeHA, by approximately 52% and 70%, respectively). This inhibitory effect of L-NMMA was partially reversed by L-arginine (10 muM). Methylene blue (10 muM) and ouabain (10 muM) inhibited relaxation (R)alpha-MeHA-induced by approximately 50% and 90%, respectively. The products of cyclooxygenase and lipoxygenase are not involved in (R)alpha-MeHA-induced endothelium-dependent rat aorta relaxation nor are the muscarinic cholinergic and beta-adrenergic receptors. The results also suggest the involvement of NO synthase, guanylate cyclase and Na(+),K(+)-ATPase in (R)alpha-MeHA-induced endothelium-dependent rat aorta relaxation. 相似文献
18.
Role of p38 mitogen-activated protein kinase phosphorylation and Fas-Fas ligand interaction in morphine-induced macrophage apoptosis 总被引:5,自引:0,他引:5
Singhal PC Bhaskaran M Patel J Patel K Kasinath BS Duraisamy S Franki N Reddy K Kapasi AA 《Journal of immunology (Baltimore, Md. : 1950)》2002,168(8):4025-4033
In this study, we evaluated the molecular mechanisms involved in morphine-induced macrophage apoptosis. Both morphine and TGF-beta promoted P38 mitogen-activated protein kinase (MAPK) phosphorylation, and this phosphorylation was inhibited by SB 202190 as well as by SB 203580. Anti-TGF-beta Ab as well as naltrexone (an opiate receptor antagonist) inhibited morphine-induced macrophage P38 MAPK phosphorylation. Anti-TGF-beta Ab also attenuated morphine-induced p53 as well as inducible NO synthase expression; in contrast, N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase, inhibited morphine-induced P38 MAPK phosphorylation and Bax expression. Morphine also enhanced the expression of both Fas and Fas ligand (FasL), whereas anti-FasL Ab prevented morphine-induced macrophage apoptosis. Moreover, naltrexone inhibited morphine-induced FasL expression. In addition, macrophages either deficient in FasL or lacking p53 showed resistance to the effect of morphine. Inhibitors of both caspase-8 and caspase-9 partially prevented the apoptotic effect of morphine on macrophages. In addition, caspase-3 inhibitor prevented morphine-induced macrophage apoptosis. These findings suggest that morphine-induced macrophage apoptosis proceeds through opiate receptors via P38 MAPK phosphorylation. Both TGF-beta and inducible NO synthase play an important role in morphine-induced downstream signaling, which seems to activate proteins involved in both extrinsic (Fas and FasL) and intrinsic (p53 and Bax) cell death pathways. 相似文献
19.
Kumagai Y Hayashi T Miyauchi T Endo A Iguchi A Kiriya-Sakai M Sakai S Yuki K Kikushima M Shimojo N 《American journal of physiology. Regulatory, integrative and comparative physiology》2001,281(1):R25-R30
Diesel exhaust particles cause an impairment of endothelium-dependent vasorelaxation and are associated with cardiopulmonary-related diseases and mortality, but the mechanistic details are poorly understood. Since we reported previously that phenanthraquinone, an environmental chemical contained in diesel exhaust particles, suppresses neuronal nitric oxide synthase (nNOS) activity by shunting electrons away from the normal catalytic pathway, it was hypothesized that phenanthraquinone inhibits endothelial NOS (eNOS) activity and affects vascular tone. Therefore, the effects of phenanthraquinone on eNOS activity, endothelium-dependent relaxation, and blood pressure were examined in the present study. Phenanthraquinone inhibited NO formation evaluated by citrulline formed by total membrane fraction of bovine aortic endothelial cells with an IC(50) value of 0.6 microM. A kinetic study revealed that phenanthraquinone is a competitive inhibitor with respect to NADPH and a noncompetitive inhibitor with respect to L-arginine. Endothelium-dependent relaxation of rat aortic rings by ACh was significantly inhibited by phenanthraquinone (5 microM), whereas the endothelium-independent relaxation by nitroglycerin was not. Furthermore, an intraperitoneal injection of phenanthraquinone (0.36 mmol/kg) to rats resulted in an elevation of blood pressure (1.4-fold, P < 0.01); under this condition, plasma levels of stable NO metabolites, nitrite/nitrate, in phenanthraquinone-treated rats was reduced to 68% of control levels. The present findings suggest that phenanthraquinone has a potent inhibitory action on eNOS activity via a similar mechanism reported for nNOS, thereby causing the suppression of NO-mediated vasorelaxation and elevation of blood pressure. 相似文献
20.
Michael August Oliver Wingerter Matthias Oelze Philip Wenzel Andrei L Kleschyov Andreas Daiber Alexander Mülsch Thomas Münzel Nikolaos Tsilimingas 《Nitric oxide》2006,15(3):241-251
In the present study we compared the vascular reactivity and integrity of the nitric oxide (NO)-cyclic 3',5'-guanosine monophopsphate (cGMP) pathway in carotid arteries of hyper- and normolipidemic rabbits. Vasodilation to acetylcholine, nitroglycerin, and sodium nitroprusside was desensitized in hyperlipidemia, but the nitroprusside-induced relaxation was normalized by an NO synthase inhibitor in endothelium-intact and -denuded vessels. Hyperlipidemic carotid arteries exhibited increased basal NO (detected by EPR spin-trapping) and reactive oxygen species formation (detected by chemiluminescence), whereas acetylcholine-induced NO formation was nearly abolished. Hyperlipidemia increased NADPH-dependent superoxide formation in carotid membranes, and carotid cryosections stained with the fluorescent dye dihydroethidium revealed increased endothelial and medial reactive oxygen species formation. Hyperlipidemia elicited macrophage invasion into the carotid wall, as detected by a dot-immunoblot. The basal activity of cGMP-dependent proteinkinase, the nitroprusside-stimulated activity of soluble guanylyl cyclase, and its protein expression were decreased by hyperlipidemia. The cGMP phosphodiesterase activity was marginally increased by hyperlipidemia, such that the ratio of cGMP-forming vs. -degrading capacity was decreased by 2-fold. Hyperlipidemia triggers infiltration of macrophages into the carotid wall and endothelial as well as smooth muscle superoxide formation. Consequently, relaxation of the carotid arteries are impaired due to smooth muscle and endothelial dysfunction. 相似文献