O2-acetoxymethyl-protected diazeniumdiolate-based NSAIDs (NONO-NSAIDs): synthesis, nitric oxide release, and biological evaluation studies

A novel group of O2-acetoxymethyl-protected diazeniumdiolate-based non-steroidal anti-inflammatory prodrugs (NONO-NSAIDs) were synthesized by esterifying the carboxylate group of aspirin, ibuprofen, or indomethacin with O2-acetoxymethyl 1-[N-(2-hydroxyethyl)-N-methylamino]diazeniumdiolate. The resulting nitric oxide (*NO)-releasing prodrugs (7-9) did not exhibit in vitro cyclooxygenase (COX) inhibitory activity against the COX-1 and COX-2 isozymes (IC50s>100 microM). In contrast, prodrugs 7 and 8 significantly decreased carrageenan-induced rat paw edema showing enhanced in vivo anti-inflammatory activities (ID50's=552 and 174 micromol/kg, respectively) relative to those of the parent NSAIDs aspirin (ID50=714 micromol/kg) and ibuprofen (ID50=326 micromol/kg). The rate of porcine liver esterase-mediated *NO release from prodrugs 7-9 (2 mol of *NO/mol of test compound in 0.6-6.5 min) was substantially higher compared to that observed without enzymatic catalysis (about 1 mol of *NO/mol of test compound in 40-48 h). These incubation studies suggest that both *NO and the parent NSAID would be released upon in vivo activation (hydrolysis) by esterases. Data acquired in an in vivo ulcer index (UI) assay showed that NONO-aspirin (UI=0.8), NONO-indomethacin (UI=1.3), and particularly NONO-ibuprofen (UI=0) were significantly less ulcerogenic compared to the parent drugs aspirin (UI=57), ibuprofen (UI=46) or indomethacin (UI=34) at equimolar doses. The release of aspirin and *NO from the NONO-aspirin (7) prodrug constitutes a potentially beneficial property for the prophylactic prevention of thrombus formation and adverse cardiovascular events such as stroke and myocardial infarction.     

Contributions of endothelium-derived relaxing factors to control of hindlimb blood flow in the mouse in vivo

We determined the contributions of various endothelium-derived relaxing factors to control of basal vascular tone and endothelium-dependent vasodilation in the mouse hindlimb in vivo. Under anesthesia, catheters were placed in a carotid artery, jugular vein, and femoral artery (for local hindlimb circulation injections). Hindlimb blood flow (HBF) was measured by transit-time ultrasound flowmetry. N(omega)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg plus 10 mg x kg(-1) x h(-1)), to block nitric oxide (NO) production, altered basal hemodynamics, increasing mean arterial pressure (30 +/- 3%) and reducing HBF (-30 +/- 12%). Basal hemodynamics were not significantly altered by indomethacin (10 mg x kg(-1) x h(-1)), charybdotoxin (ChTx, 3 x 10(-8) mol/l), apamin (2.5 x 10(-7) mol/l), or ChTx plus apamin (to block endothelium-derived hyperpolarizing factor; EDHF). Hyperemic responses to local injection of acetylcholine (2.4 microg/kg) were reproducible in vehicle-treated mice and were not significantly attenuated by L-NAME alone, indomethacin alone, L-NAME plus indomethacin with or without co-infusion of diethlyamine NONOate to restore resting NO levels, ChTx alone, or apamin alone. Hyperemic responses evoked by acetylcholine were reduced by 29 +/- 11% after combined treatment with apamin plus charybdotoxin, and the remainder was virtually abolished by additional treatment with L-NAME but not indomethacin. None of the treatments altered the hyperemic response to sodium nitroprusside (5 microg/kg). We conclude that endothelium-dependent vasodilation in the mouse hindlimb in vivo is mediated by both NO and EDHF. EDHF can fully compensate for the loss of NO, but this cannot be explained by tonic inhibition of EDHF by NO. Control of basal vasodilator tone in the mouse hindlimb is dominated by NO.     

The NO donor molsidomine reduces endothelin-1 gene expression in chronic hypoxic rat lungs

We investigated the effects of the nitric oxide (NO) donor molsidomine and the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) on pulmonary endothelin (ET)-1 gene expression and ET-1 plasma levels in chronic hypoxic rats. Two and four weeks of hypoxia (10% O2) significantly increased right ventricular systolic pressure, the medial cross-sectional vascular wall area of the pulmonary arteries, and pulmonary ET-1 mRNA expression (2-fold and 3.2-fold, respectively). ET-1 plasma levels were elevated after 4 wk of hypoxia. In rats exposed to 4 wk of hypoxia, molsidomine (15 mg x kg(-1) x day(-1)) given either from the beginning or after 2 wk of hypoxia significantly reduced pulmonary hypertension, pulmonary vascular remodeling, pulmonary ET-1 gene expression, and ET-1 plasma levels. L-NAME administration (45 mg x kg(-1) x day(-1)) in rats subjected to 2 wk of hypoxia did not modify these parameters. Our findings suggest that in chronic hypoxic rats, exogenously administered NO acts in part by suppressing the formation of ET-1. In contrast, inhibition of endogenous NO production exerts only minor effects on the pulmonary circulation and pulmonary ET-1 synthesis in these animals.     

一氧化氮的释放对海马脑片CA1区痫样放电的影响

用自制的一氧化氮（ＮＯ）敏感电极－Ｎａｆｉｏｎ－壳聚糖合镍修饰铂电极（Ｎａｆｉｏｎ－ＣＴＳ（Ｎｉ）－Ｐｔ）连续测定了青霉素致痫海马脑片ＣＡ１区锥体层神经元ＮＯ的释放,并同时观察了ＮＯ合酶抑制剂７－ｎｉｔｒｏ－ｉｎｄａｚｏｌｅ（７－ＮＩ）及Ｎ＾ω－ｎｉｔｒｏ－Ｌ－ａｒｇｉｎｉｎｅ（Ｌ－ＮＮＡ）对诱发痫波及ＮＯ释放量的影响。研究观察到：（１）在青霉素致痫脑片模型上,诱发的痫波随青霉素浓度的增加而增多,     

Investigation of the vasorelaxant effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) and diethylamine/nitric oxide (DEA/NO) on the human radial artery used as coronary bypass graft

The radial artery (RA) is used as a spastic coronary bypass graft. This study was designed to investigate the mechanism of vasorelaxant effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO (diethylamine/nitric oxide), a NO-nucleophile adduct, on the human RA. RA segments (n = 25) were obtained from coronary artery bypass grafting patients and were divided into 3-4 mm vascular rings.Using the isolated tissue bath technique, the endothelium-independent vasodilatation function was tested in vitro by the addition of cumulative concentrations of YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) following vasocontraction by phenylephrine in the presence or absence of 10-5 mol/L ODQ (1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one), the selective sGC inhibitor, 10-7 mol/L iberiotoxin, a blocker of Ca2+-activated K+ channels, or 10-5 mol/L ODQ plus 10-7 mol/L iberiotoxin. We also evaluated the effect of YC-1 and DEA/NO on the cGMP levels in vascular rings obtained from human radial artery (n = 6 for each drug). YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) caused the concentration-dependent vasorelaxation in RA rings precontracted with phenylephrine (10-5 mol/L) (n = 20 for each drug). Pre-incubation of RA rings with ODQ, iberiotoxin, or ODQ plus iberiotoxin significantly inhibited the vasorelaxant effect of YC-1, but the inhibitor effect of ODQ plus iberiotoxin was significantly more than that of ODQ and iberiotoxin alone (p < 0.05). The vasorelaxant effect of DEA/NO almost completely abolished in the presence of ODQ and iberiotoxin plus ODQ, but did not significantly change in the presence of iberiotoxin alone (p > 0.05). The pEC50 value of DEA/NO was significantly lower than those for YC-1 (p < 0.01), with no change Emax values in RA rings. In addition, YC-1-stimulated RA rings showed more elevation in cGMP than that of DEA/NO (p < 0.05). These findings indicate that YC-1 is a more potent relaxant than DEA/NO in the human RA. The relaxant effects of YC-1 could be due to the stimulation of the sGC and Ca2+-sensitive K+channels, whereas the relaxant effects of DEA/NO could be completely due to the stimulation of the sGC. YC-1 and DEA/NO may be effective as vasodilator for the short-term treatment of perioperative spasm of coronary bypass grafts.     

脂多糖对离体培养大鼠血管平滑肌细胞增殖的影响

本研究观察到１０－７～１０－５ｋｇ／Ｌ脂多糖（ｌｉｐｏｐｏｌｙｓａｃｈａｒｉｄｅ,ＬＰＳ）可显著促进血管平滑肌细胞（ＶＳＭＣ）的增殖及ＤＮＡ的合成（Ｐ＜００５）。５×１０－４～１０－３ｋｇ／ＬＬＰＳ却抑制ＶＳＭＣ的增殖及ＤＮＡ的合成,降低其活力（Ｐ＜００１）,并呈时间依赖效应。一氧化氮合酶抑制剂ＮＮｉｔｒｏＬＡｒｇｉｎｉｎｅ（ＬＮＮＡ）可拮抗ＬＰＳ的抑制作用。大剂量ＬＰＳ作用组ＶＳＭＣ上清液中一氧化氮（ＮＯ）代谢产物ＮＯ－３和ＮＯ－２的含量与对照组相比显著增加（Ｐ＜００１）,４８ｈ组比２４ｈ组增加９１％,７２ｈ组比４８ｈ组增加４５％;同时,诱导性一氧化氮合酶（ｉｎｄｕｃｔｉｖｅｎｉｔｒｉｃｏｘｉｄｅｓｙｎｔｈａｓｅ,ｉＮＯＳ）免疫组化染色呈阳性。结果表明,低浓度ＬＰＳ促进ＶＳＭＣ增殖和ＤＮＡ合成,而高浓度ＬＰＳ却明显抑制ＶＳＭＣ增殖和ＤＮＡ合成,降低其活力。这种抑制作用可能与ＬＰＳ诱导ＶＳＭＣ产生的ＮＯ有关。     

A novel nitric oxide biosensor based on electropolymerization poly(toluidine blue) film electrode and its application to nitric oxide released in liver homogenate

A novel poly(toluidine blue)-modified electrode has been constructed for the determination of nitric oxide in biological sample. The electrochemical behavior of poly(toluidine blue) film electrode and its electrocatalytic activity toward NO were studied in detail by cyclic voltammetry. Possible interferences were tested and evaluated after further coated with Nafion. The poly(toluidine blue) and Nafion composite film-modified electrode exhibits a good linear relationship over a NO concentration of 1.8 x 10(-7) to 8.6 x 10(-5)mol/L, and the detection limit is 1.8 x 10(-8)mol/L (S/N=3). NO release from the rat liver homogenate stimulated by l-arginine was studied, and the responses were decreased by the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine.     

一氧化氮在17-β雌二醇抑制血管平滑肌细胞增殖和原癌基因c -fos表达中的作用

实验利用大鼠血管平滑肌细胞（vascular smooth muscle cells,VSMC)作为模型,观察17-β雌二醇（E2）对VSMC增殖和原癌基因c-fos表达的影响,并探讨VSMC源性一氧化氮（NO）在基中的作用,检测指标包括NO释放的测定,细胞计数、^3H-Tdr掺入,噻唑蓝（MTT）测定和c-fosmRNA表达,结果显示,E2（10^-12-10^-8mol/L)呈浓度依赖性地促进VSMC中NO的释放;10^-8mol/LE2能明显抑制10%小牛血清（FCS）和10^-7mol/L内皮素-1（ET-1）诱导的细胞增殖和DNA合成,E2的抑制作用均可被雌激素受体（ER）拮抗剂tamoxifen(10^-7mol/L)和一氧化氮合酶抑制剂L-NAME（10^-6mol/L)明显减轻;E2（10^-8mol/L)可明显抑制10^-7mol/LET-1诱导的VSMCc-fos表达,这种抑制作用可被L-NAME（10^-6mol/L)明显减轻,这些结果提示E2能抑制VSMC增殖和原癌基因c-fos表达,这种促进VSMC的NO释放密切相关,而且该作用至少部分通过ER介导。     

Wine polyphenols improve cardiovascular remodeling and vascular function in NO-deficient hypertension

The effects of the red wine polyphenolic compounds (Provinol) on hypertension, left ventricular hypertrophy, myocardial fibrosis, and vascular remodeling were investigated after chronic inhibition of nitric oxide (NO) synthase by administration of N(G)-nitro-L-arginine methyl ester (L-NAME) to rats. Rats were divided into four groups: a control group, a group treated for 4 wk with L-NAME (40 mg x kg(-1) x day(-1)), and two groups treated with L-NAME followed by 3 wk of either spontaneous recovery or recovery with Provinol treatment (40 mg x kg(-1) x day(-1)). Administration of Provinol produced a greater readiness of the decrease in blood pressure than that in the spontaneous recovery group. Provinol significantly depressed myocardial fibrosis and expedited the decrease in aortic cross-sectional area, the increase in endothelium-dependent relaxation, and the decrease in contraction of the aorta. These effects of Provinol were associated with a greater increase of NO synthase activity in the left ventricle and the aorta. The present study provides evidence that Provinol accelerates the regression of blood pressure and improves structural and functional cardiovascular changes produced by chronic inhibition of NO synthesis.     

Participation of the components of L-arginine/nitric oxide/cGMP cascade by chemically-induced abdominal constriction in the mouse

The purpose of this study was to investigate the role of the L-arginine/nitric oxide (NO)/cGMP pathway in p-benzoquinone-induced writhing model in mouse. L-arginine, a NO precursor, displayed antinociceptive effects at the doses of 0.125-1.0 mg/kg. When the doses of L-arginine were increased gradually to 10-100 mg/kg, a dose-dependent triphasic pattern of nociception-antinociception-nociception was obtained. The NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) (18.7515 mg/kg), possessed antinociceptive activity. Methylene blue (MB), a guanylyl cyclase and/or NOS inhibitor, (5-160 mg/kg) also produced a dose-dependent triphasic response. When L-arginine (50 mg/ kg) was combined with L-NAME (75 mg/kg). L-arginine-induced antinociception did not change significantly. Cotreatment of L-arginine with 5 mg/kg MB significantly decreased MB-induced antinociception and reversed the nociception induced by 40 mg/kg MB to antinociception. It is concluded that the components of L-arginine/nitric oxide/cGMP cascade may participate in nociceptive processes both peripherally and centrally by a direct effect on nociceptors or by the involvement of other related pathways of nociceptive processes induced by NO.     

A nitric oxide biosensor based on the multi-assembly of hemoglobin/montmorillonite/polyvinyl alcohol at a pyrolytic graphite electrode

Direct electron transfer of hemoglobin (Hb) can be achieved in a Hb/montmorillonite (MMT)/polyvinyl alcohol multi-assembly at a pyrolytic graphite (PG) electrode. Accordingly, a novel nitric oxide (NO) biosensor is proposed. The reduction of NO is observed at a potential of approximately -783 mV (vs. SCE) at pH 5.5. At optimum pH, this biosensor shows a wide linear range of 1.0x10(-6)-2.5x10(-4) mol/l with a detection limit of 5.0x10(-7) mol/l. The sensor-to-sensor reproducibility is good consideringmacr; a relative standard deviation of 3.5% in five independent determinations at 5.0x10(-5) mol/l NO. The modified electrode is conveniently constructed and durable in long-term operations.     

N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine.

Authentic N omega-hydroxy-L-arginine was synthesized and used to determine whether it is an intermediate in nitric oxide (.NO) synthesis from L-arginine by macrophage .NO synthase. The apparent Km (6.6 microM) and Vmax (99 nmol x min-1 x mg-1) observed with N omega-hydroxy-L-arginine were similar to those observed with L-arginine (Km = 2.3 microM; Vmax = 54 mumol x min-1 x mg-1). N omega-Hydroxy-D-arginine was not a substrate. Stable isotope studies showed that .NO synthase exclusively oxidized the hydroxylated nitrogen of N omega-hydroxy-L-arginine, forming .NO and L-citrulline. As with L-arginine, O2 was the source of the ureido oxygen in L-citrulline from N omega-hydroxy-L-arginine. In the presence of excess N omega-hydroxy-L-arginine, .NO synthase generated a metabolite of L-[14C]arginine that cochromatographed with authentic N omega-hydroxy-L-arginine. The labeled metabolite exhibited identical chromatographic behavior in three solvent systems and generated the same product (L-citrulline) upon alkaline hydrolysis as authentic N omega-hydroxy-L-arginine. Experiments were then run to identify which redox cofactor (NADPH or tetrahydrobiopterin) participated in the enzymatic synthesis of N omega-hydroxy-L-arginine. Both cofactors were required for synthesis of .NO from either N omega-hydroxy-L-arginine or L-arginine. However, with L-arginine, the synthesis of 1 mol of .NO was coupled to the oxidation of 1.52 +/- 0.02 mol of NADPH; whereas with N omega-hydroxy-L-arginine, only 0.53 +/- 0.04 mol of NADPH was oxidized per mol of .NO formed. These results support a mechanism in which N omega-hydroxy-L-arginine is generated as an intermediate in .NO synthesis through an NADPH-dependent hydroxylation of L-arginine.     

Nitric oxide synthase and cGMP activity in the salivary glands of the American dog tick Dermacentor variabilis

We colocalized nitric oxide synthase (NOS) activity in epithelial cells that surround the salivary gland duct in female Dermacentor variabilis with NADPH diaphorase histochemistry and immunohistochemistry using a polyclonal anti-endothelial NOS. Using size-exclusion chromatography, a fraction with a molecular mass of about 185 kDa that had diaphorase activity was eluted from tick salivary gland homogenate. This fraction converted arginine to citrulline with the production of nitric oxide (NO), which was detected by using electron spin resonance spectroscopy. The complete activity of the diaphorase fraction was dependent on NADPH, FAD, tetrahydrobiopterin, calmodulin, (CaM), and Ca(2+), but was not dependent on dithiothreitol. The arginine analog N(G)-monomethyl-L-arginine inhibited the activity of this fraction. NO and arginine activated soluble guanylate cyclase to produce cGMP in dopamine-stimulated isolated salivary glands. Dopamine-stimulated isolated salivary glands treated with tick saline containing either EDTA, the NOS inhibitor N(G)-nitro-L-arginine methyl ester, or the calcium/CaM binding inhibitor W-7 showed no increase in cGMP. The NO donor sodium nitroprusside significantly increased cGMP levels in unstimulated isolated salivary glands. A possible function for NO in salivation by this ixodid tick is discussed.     

Nitric oxide synthase-1 is enriched in fast-twitch oxidative myofibers

Nitric oxide synthase-1 (NOS-1) is found in high concentrations in skeletal muscles, where its synthesis product nitric oxide (NO) is reported to be involved in a number of processes, including the modulation of the oxidative metabolism of myofibers. Performing immunoblot analysis and quantification of formazan produced by its specific NADPH diaphorase activity, we found NOS-1 to be enriched in rat skeletal muscles with a high proportion of fast-twitch myofibers. Since these myofibers represent a metabolically heterogeneous subpopulation, we extended our investigation to the level of individual myofibers. Using serial sections we combined myosin heavy chain-based fiber-typing with quantitative succinate dehydrogenase histochemistry to determine three groups of fiber-types, comprising fast-oxidative, fast-glycolytic and slow-oxidative myofibers. Image analysis showed that NOS-1 diaphorase activity is significantly enriched in fast-oxidative myofibers compared with fast-glycolytic and slow-oxidative ones. In order to characterize potential biological effects of the fiber-type-specific enrichment of NOS-1, we performed cytochrome oxidase histochemistry in the presence of the NO donors NOC-9 and SNAP. Both NO donors reduced cytochrome oxidase activity in all myofibers investigated with almost identical semi-maximal inhibition rates, although fast-oxidative and slow-oxidative myofibers contained twice as much basal catalytic activity than fast-glycolytic ones. In summary, we suggest that the NOS-1/NO system of skeletal muscles exerts its biological role especially in fast-oxidative myofibers, since these myofibers express more NOS-1 than fast-glycolytic or slow-oxidative ones and also contain the highest concentrations of cytochrome oxidases as potential target molecules of NO.     

Involvement of nitric oxide in endothelium-dependent arterial relaxation by leptin

Leptin is a polypeptide, mainly produced in white adipose tissue, and increases sympathetic nerve activity. A few studies investigated leptin's effect on peripheral vessels. We examined the vasorelaxant effects of human leptin on rat arteries. Arterial rings were precontracted with 1 x 10(-6) mol/l of phenylephrine, and leptin was superfused. Leptin relaxed phenylephrine-precontracted arterial rings in a dose-dependent manner. ED50 was calculated to 8.4 microg/ml. Removal of endothelium abolished the effects of leptin. Indomethacin (1 x 10(-5) mol/l) did not affect the vasorelaxation by leptin, whereas 1 x 10(-4) mol/l of N(omega)-nitro-L-arginine methyl ester (L-NAME) completely suppressed it. The inhibition was antagonized by 1 x 10(-4) mol/l of L-arginine. Leptin normally relaxed arterial rings during superfusion of K channel blockers, including 3 x 10(-5) mol/l of glibenclamide, 1 x 10(-6) of mol/l apamin, and 5 x 10(-7) mol/l of charybdotoxin. Low Cl(-) solution (8. 3 mmol/l) inhibited leptin-induced relaxation, but endothelium-independent vasodilatation by nitroprusside was not impaired at low Cl(-) solution. These results suggest that arterial relaxation by leptin is mediated by nitric oxide released from endothelium, and Cl(-) plays an important role in leptin-induced nitric oxide release.     

Effect of combination of nitric oxide synthase and cyclooxygenase inhibitors on carrageenan-induced pleurisy in rats

In the present study, we examined the effects of L-nitroarginine methylester (L-NAME), a non-selective nitric oxide synthase (NOS) inhibitor, indomethacin (IND), a non-selective COX inhibitor and a combination of these agents (L-NAME+IND) on carrageenan-induced pleurisy in rats. Exudate volume, albumin leakage, leukocyte influx, exudate and plasma nitrite/nitrate (NO(x)) levels and exudate PGE(2) levels increased markedly 6 h after an intrapleural injection of 2% carrageenan. First, the effects of L-NAME and IND alone were investigated. L-NAME non-significantly reduced exudate volume by 26% at 10 mg/kg (i.p.), and significantly by 45% at 30 mg/kg. IND dose-dependently decreased the exudate volume at 0.3-10 mg/kg (p.o.) and the effect reached the maximal level at 1 mg/kg (33%). Second, the effects of L-NAME (10 mg/kg, i.p.), IND (1 mg/kg, p.o.) and L-NAME+IND were examined. L-NAME and IND alone at the dose employed significantly reduced the exudate volume and albumin levels by 21-26%. L-NAME but not IND tended to reduce the increased exudate and plasma NO(x) by 18% and 19%, respectively. IND but not L-NAME significantly reduced leukocyte numbers and PGE(2) levels in the exudates by 25% and 77%, respectively. L-NAME+IND significantly reduced exudate volume, albumin leakage, leukocyte number, PGE(2) and NO(x) by 43%, 41%, 31%, 80% and 37%, respectively. The inhibitory effects of L-NAME+IND on exudate volume, albumin leakage and NO(x) levels were greater than those of L-NAME and IND alone. In conclusion, a non-selective NOS inhibitor and COX inhibitor showed anti-inflammatory effects at the early phase of carrageenan-induced pleurisy, and a combination of both inhibitors had a greater effect than each alone probably via the potentiation of NOS inhibition. The simultaneous inhibition of NOS and COX could be a useful approach in therapy for acute inflammation.     

Orexin-A-induced feeding is dependent on nitric oxide

Orexin-A is a peptide produced in the lateral hypothalamus/perifornical area, which stimulates feeding. The production of orexin-A is determined by the metabolic state of the animal. We have previously shown that nitric oxide (NO) plays an important role as a mediator of feeding induced by a variety of neuropeptides. This raises the question of whether orexin-A's effects are NO dependent. Here, we first determined that intracerebroventricular administration of 25 ng of orexin-A significantly increased food intake in satiated mice. We next examined the effects of Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, on orexin-A-induced increase in food intake. L-NAME (50 mg/kg; SC) significantly blocked the orexin-A-induced increase in food intake. Orexin-A administration increased the levels of nitric oxide synthase in the hypothalamus. To further verify the importance of NO in the orexin-A-induced increase in food intake, we compared the ability of orexin-A to increase food intake in neuronal nitric oxide synthase knockout (NOS-KO) mice and their wild-type controls. Orexin-A failed to increase food intake in the NOS-KO mice, whereas it did increase food intake in the wild-type controls. This supports the hypothesis that nitric oxide is a central regulator of food consumption.     

Formation and reactions of the heme-dioxygen intermediate in the first and second steps of nitric oxide synthesis as studied by stopped-flow spectroscopy under single-turnover conditions

To better understand the mechanism of nitric oxide (NO) synthesis, we studied conversion of N-hydroxy-L-arginine (NOHA) or L-arginine (Arg) to citrulline and NO under single-turnover conditions using the oxygenase domain of neuronal nitric oxide synthase (nNOSoxy) and rapid scanning stopped-flow spectroscopy. When anaerobic nNOSoxy saturated with H(4)B and NOHA was provided with 0.5 or 1 electron per heme and then exposed to air at 25 degrees C, it formed 0.5 or 1 mol of citrulline/mol of heme, respectively, indicating that NOHA conversion had 1:1 stoichiometry with respect to electrons added. Identical experiments with Arg produced substoichiometric amounts of NOHA or citrulline even when up to 3 electrons were provided per heme. Transient spectral intermediates were investigated at 10 degrees C. For NOHA, four species were observed in the following sequence: starting ferrous nNOSoxy, a transient ferrous-dioxygen complex, a transient ferric-NO complex, and ferric nNOSoxy. For Arg, transient intermediates other than the ferrous-dioxygen species were not apparent during the reaction. Our results provide a kinetic framework for formation and reactions of the ferrous-dioxygen complex in each step of NO synthesis and establish that (1) the ferrous-dioxy enzyme reacts quantitatively with NOHA but not with Arg and (2) its reaction with NOHA forms 1 NO/heme, which immediately binds to form a ferric heme-NO complex.     

17β-雌二醇诱导血管内皮细胞一氧化氮释放及其与细胞内钙的关系

利用小牛胸主动脉内皮细胞（ＢＡＥＣｓ）作为模型,观察１７β－雌二醇（Ｅ２）ＢＡＥＣｓ一氧化氮（ＮＯ）释放、一氧化氮合酶（ｅＮＯＳ）ｍＲＮＡ表达和细胞内钙（〔Ｃａ＾２＋〕ｉ）的影响,以及雌激素受体（ＥＲ）拮抗剂ｔａｍｏｘｉｆｅｎ和ＮＯＳ抑制剂（Ｌ－ＮＡＭＥ）的作用。结果显示,Ｅ２（１０＾－１２￣１０＾－８ｍｏｌ／Ｌ）呈尝试依赖性促进ＢＡＥＣｓ中ＮＯ的释放,以１０＾－８ｍｏｌ／Ｌ浓度Ｅ２处理ＢＡＥＣｓ     

The contradictory effects of nitric oxide in caerulein-induced acute pancreatitis in rats

This study was planned to observe the effects of nitric oxide synthesis on the antioxidative defense enzymes and pancreatic tissue histology in caerulein-induced acute pancreatitis. Acute pancreatitis was induced by intraperitoneal injections of 50 microg/kg caerulein, L-arginine used for NO induction and N(omega)-nitro-L-arginine methyl ester (L-NAME) used for NO inhibition. In the caerulein group acinar cell degeneration, interstitial inflammation, oedema and haemorrhage were detected. Pancreatic damage scores were decreased with both NO induction and inhibition (p<0.05). MDA, GSH-Px, CAT, GSH and SOD activities were significantly changed in the caerulein group and indicated increased oxidative stress. Both NO induction and inhibition decreased this oxidative stress. It is concluded that both nitric oxide induction and inhibition ameliorated caerulein-induced acute pancreatitis. The findings indicate that a certain amount of NO production has beneficial effects in experimental acute pancreatitis, but uncontrolled over-production of NO may be detrimental.