高压氧对急性脑创伤后皮层一氧化氮合酶mRNA表达的影响

目的:拟观察高压氧(HBO)治疗对急性创伤性颅脑损伤后皮层NOSmRNA表达的影响,探讨HBO治疗急性脑损伤的机理。方法:采用自由落体法打击模型制备SD大鼠急性脑创伤,伤后1 h、12 h采用0.25 MPaHBO治疗,伤后6 h、24 h取样皮层,应用半定量逆转录聚合酶链反应(RT-PCR)观察神经元型一氧化氮合酶(nNOS)、内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS)mRNA表达量变化。结果:0.25MPaHBO治疗各时间组nNOS、eNOS和iNOSmRNA较急性颅脑损伤各时间组显著下降(P<0.01),且HBO治疗24 h组较6 h组下降更明显(P<0.05,P<0.01),0.25 MPa常氧高氮各时间组与急性颅脑损伤各时间组NOSmRNA表达量无统计学意义。结论:HBO治疗可以下调nNOSmRNA、iNOSmRNA和eNOSmRNA的表达量,可能为HBO治疗脑创伤的机理之一。     

Sepiapterin enhances angiogenesis and functional recovery in mice after myocardial infarction

Uncoupling of nitric oxide synthase (NOS) has been implicated in left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). We hypothesized that inducible NOS (iNOS) plays a crucial role in LV remodeling after MI, depending on its coupling status. MI was created in wild-type, iNOS-knockout (iNOS(-/-)), endothelial NOS-knockout (eNOS(-/-)), and neuronal NOS-knockout (nNOS(-/-)) mice. iNOS and nNOS expressions were increased after MI associated with an increase in nitrotyrosine formation. The area of myocardial fibrosis and LV end-diastolic volume and ejection fraction were more deteriorated in eNOS(-/-) mice compared with other genotypes of mice 4 wk after MI. The expression of GTP cyclohydrolase was reduced, and tetrahydrobiopterin (BH(4)) was depleted in the heart after MI. Oral administration of sepiapterin after MI increased dihydrobiopterin (BH(2)), BH(4), and BH(4)-to-BH(2) ratio in the infarcted but not sham-operated heart. The increase in BH(4)-to-BH(2) ratio was associated with inhibition of nitrotyrosine formation and an increase in nitrite plus nitrate. However, this inhibition of NOS uncoupling was blunted in iNOS(-/-) mice. Sepiapterin increased capillary density and prevented LV remodeling and dysfunction after MI in wild-type, eNOS(-/-), and nNOS(-/-) but not iNOS(-/-) mice. N(ω)-nitro-L-arginine methyl ester abrogated sepiapterin-induced increase in nitrite plus nitrate and angiogenesis and blocked the beneficial effects of sepiapterin on LV remodeling and function. These results suggest that sepiapterin enhances angiogenesis and functional recovery after MI by activating the salvage pathway for BH(4) synthesis and increasing bioavailable nitric oxide predominantly derived from iNOS.     

Increased nitric oxide production accompanies blunted hypoxic pulmonary vasoconstriction in hyperoxic rat lung

Hyperoxia may affect lung physiology in different ways. We investigated the effect of hyperoxia on the protein expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, and hypoxic pulmonary vasoconstriction (HPV) in rat lung. Twenty-four male rats were divided into hyperoxic and normoxic groups. Hyperoxic rats were placed in > 90% F1O2 for 60 h prior to experiments. After baseline in vitro analysis, the rats underwent isolated, perfused lung experiments. Two consecutive hypoxic challenges (10 min each) were administered with the administration of a non-specific NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), in between. We measured intravascular NO production, pulmonary arterial pressure, and protein expression of eNOS and iNOS by immunohistochemistry. We found that hyperoxia rats exhibited increased baseline NO production (P < 0.001) and blunted HPV response (P < 0.001) during hypoxic challenges compared to normoxia rats. We also detected a temporal association between the attenuation in HPV and increased NO production level with a negative pre-L-NAME correlation between HPV and NO (R = 0.52, P < 0.05). After L-NAME administration, a second hypoxic challenge restored the HPV response in the hyperoxic group. There were increased protein expression of eNOS (12.6 +/- 3.1-fold, n = 3) (X200) and iNOS (8.1 +/- 2.6-fold, n = 3) (X200) in the hyperoxia group. We conclude that hyperoxia increases the protein expression of eNOS and iNOS with a subsequent increased release of endogenous NO, which attenuates the HPV response.     

红花黄色素对新生鼠缺氧后一氧化氮合酶表达的影响

目的:观察红花黄色素对缺氧后脑内诱生型一氧化氮合酶(iNOS)、神经原型一氧化氮合酶(nNOS)及内皮型一氧化氮合酶(eNOS)基因表达的影响,探讨红花黄色素抗缺氧脑损伤的作用.方法:采用SD新生鼠缺氧模型,于缺氧前30 min腹腔注射红花黄色素生药7g/kg,缺氧40 min后复氧48 h,提取脑组织总RNA,应用RT-PCR技术检测三种NOS mRNA的表达量.结果:新生鼠缺氧再复氧48 h,脑内iNOS、nNOS基因表达上升(P＜0.05),预先给予红花黄色素能抑制iNOS、nNOS基因的表达(P＜0.05),但eNOS基因表达不受影响.结论:红花黄色素对缺氧脑损伤的保护作用与NOS基因表达有关.     

1,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

A series of 1,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase. A variety of flexible and restricted basic amine side chain substitutions was explored at the 1-position of the indole ring, while keeping the amidine group fixed at the 5-position. Compounds having N-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)- (12, (R)-12, (S)-12 and 13) and N-(1-(1-methylazepan-4-yl)- side chains (14, 15, (-)-15 and (+)-15) showed increased inhibitory activity for the human nNOS isoform and selectivity over eNOS and iNOS isoforms. The most potent compound of the series for human nNOS (IC(50)=0.02 μM) (S)-12 showed very good selectivity over the eNOS (eNOS/nNOS=96-fold) and iNOS (iNOS/nNOS=850-fold) isoforms.     

一氧化氮对心肌的抑制性保护作用

内皮型与神经型一氧化氮合酶(eNOS,nNOS)在心肌细胞内持续表达,而细胞应激可引起诱导型NOS(iNOS)表达.心肌细胞结构型eNOS与nNOS源性NO,在生理条件下对心肌主要发挥4方面的抑制作用:减缓心肌细胞搏动频率,轻度抑制心肌细胞收缩功能,加速心肌细胞舒张并增加顺应性,以及轻度抑制线粒体电子传递而增强氧利用效...     

Nitric oxide synthases in infants and children with pulmonary hypertension and congenital heart disease

Rationale

Nitric oxide is an important regulator of vascular tone in the pulmonary circulation. Surgical correction of congenital heart disease limits pulmonary hypertension to a brief period.

Objectives

The study has measured expression of endothelial (eNOS), inducible (iNOS), and neuronal nitric oxide synthase (nNOS) in the lungs from biopsies of infants with pulmonary hypertension secondary to cardiac abnormalities (n = 26), compared to a control group who did not have pulmonary or cardiac disease (n = 8).

Methods

eNOS, iNOS and nNOS were identified by immunohistochemistry and quantified in specific cell types.

Measurements and main results

Significant increases of eNOS and iNOS staining were found in pulmonary vascular endothelial cells of patients with congenital heart disease compared to control infants. These changes were confined to endothelial cells and not present in other cell types. Patients who strongly expressed eNOS also had strong expression of iNOS.

Conclusion

Upregulation of eNOS and iNOS occurs at an early stage of pulmonary hypertension, and may be a compensatory mechanism limiting the rise in pulmonary artery pressure.     

Expression profiles of NOS isoforms in gingiva of nNOS knockout mice

Nitric oxide is a gaseous molecule associated with many distinct physiological functions, and is derived from l-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide synthase has 3 isoforms: nNOS, iNOS and eNOS. Although these NOS isoforms are believed to play an important role in gingival tissue, little information is available on their morphological dynamics. The aim of this study was to investigate the profiles of NOS isoforms in deficiency of nNOS in gingiva of mice. Twelve male (6 normal (C57BL/6) and 6 nNOS knockout) mice were used. All mice were 5-week-old, weighing approximately 20–25 g each. After sacrifice, the jaws of the mice were removed by mechanical means and specimens analyzed by histology, in situ hybridization and immunohistochemistry. Immunohistochemical observation revealed positive staining for iNOS and eNOS, especially in lamina propria. Similar results in the mRNA expression levels were shown by in situ hybridization analysis. It may suggest that iNOS and eNOS compensated nNOS deficiency in the gingiva of nNOS knockout mice.     

Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase

Peroxynitrite (ONOO(-)), a nitric oxide-derived oxidant, uncouples endothelial nitric oxide synthase (eNOS) and increases enzymatic production of superoxide anions (O(2)()) (Zou, M. H., Shi, C., and Cohen, R. A. (2002) J. Clin. Invest. 109, 817-826). Here we studied how ONOO(-) influences eNOS activity. In cultured bovine aortic endothelial cells (BAEC), ONOO(-) increased basal and agonist-stimulated Ser(1179) phosphorylation of eNOS, whereas it decreased nitric oxide production and bioactivity. However, ONOO(-) strongly inhibited the phosphorylation and activity of Akt, which is known to phosphorylate eNOS-Ser(1179). Moreover, expression of an Akt dominant-negative mutant did not prevent ONOO(-)-enhanced eNOS-Ser(1179) phosphorylation. In contrast to Akt, ONOO(-) significantly activated 5'-AMP-activated kinase (AMPK), as evidenced by its increased Thr(172) phosphorylation as well as increased Ser(92) phosphorylation of acetyl-coenzyme A carboxylase, a downstream target of AMPK. Associated with the increased release of O(2)(), ONOO(-) significantly increased the co-immunoprecipitation of eNOS with AMPK. Further, overexpression of the AMPK-constitutive active adenovirus significantly enhanced ONOO(-) up-regulated eNOS-Ser(P)(1179). In contrast, overexpression of a dominant-negative AMPK mutant attenuated the ONOO(-)-enhanced eNOS-Ser(1179) phosphorylation as well as O(2)() release. We conclude that ONOO(-) inhibits Akt and increases AMPK-dependent Ser(1179) phosphorylation of eNOS resulting in enhanced O(2)() release.     

Stimulation of nitric oxide synthase in cerebral cortex due to elevated partial pressures of oxygen: an oxidative stress response

The purpose of this investigation was to determine the impact of elevated partial pressures of O(2) on the steady state concentration of nitric oxide ((*)NO) in the cerebral cortex. Rodents with implanted O(2)- and (*)NO-specific microelectrodes were exposed to O(2) at partial pressures from 0.2 to 2.8 atmospheres absolute (ATA) for up to 45 min. Elevations in (*)NO concentration occurred with all partial pressures above that of ambient air. In rats exposed to 2.8 ATA O(2) the increase was 692 +/- 73 nM (S.E., n = 5) over control. Changes were not associated with alterations in concentrations of nitric oxide synthase (NOS) enzymes. Based on studies with knock-out mice lacking genes for neuronal NOS (nNOS) or endothelial NOS (eNOS), nNOS activity contributed over 90% to total (*)NO elevation due to hyperoxia. Immunoprecipitation studies indicated that hyperoxia doubles the amount of nNOS associated with the molecular chaperone, heat shock protein 90 (Hsp90). Both (*)NO elevations and the association between nNOS and Hsp90 were inhibited in rats infused with superoxide dismutase. Elevations of (*)NO were also inhibited by treatment with the relatively specific nNOS inhibitor, 7 nitroindazole, by the ansamycin antibiotics herbimycin and geldanamycin, by the antioxidant N-acetylcysteine, by the calcium channel blocker nimodipine, and by the N-methyl-D-aspartate inhibitor, MK 801. Hyperoxia did not alter eNOS association with Hsp90, nor did it modify nNOS or eNOS associations with calmodulin, the magnitude of eNOS tyrosine phosphorylation, or nNOS phosphorylation via calmodulin kinase. Cerebral cortex blood flow, measured by laser Doppler flow probe, increased during hyperoxia and may be causally related to elevations of steady state (*)NO concentration. We conclude that hyperoxia causes an increase in (*)NO synthesis as part of a response to oxidative stress. Mechanisms for nNOS activation include augmentation in the association with Hsp90 and intracellular entry of calcium.     

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.     

Nitric oxide synthase in pulmonary hypertension: lessons from knockout mice

Nitric oxide (NO) is implicated in a wide variety of biological roles. NO is generated from three nitric oxide synthase (NOS) isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) all of which are found in the lung. While there are no isoform-specific inhibitors of NOS, the recent development and characterization of mice deficient in each of the NOS isoforms has allowed for more comprehensive study of the importance of NO in the lung circulation. Studies in the mouse have identified the role of NO from eNOS in modulating pulmonary vascular tone and in attenuating the development of chronic hypoxic pulmonary hypertension.     

Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

不同亚型一氧化氮合酶在脑缺血/再灌注早期的表达变化

目的:观察脑缺血/再灌注(CI/R)早期缺血区脑组织的内皮型一氧化氮合酶(eNOS)与神经型一氧化氮合酶(nNOS)表达的变化。方法:健康wistar大鼠60只,体重200～280g,由中国医科大学动物中心提供,雌雄各半。随机分为6组(n=10):假手术组、缺血1h组、缺血2h组、再灌注0.5h组、再灌注1h组、再灌注2h组。采用线栓法制作大鼠CI/R模型,免疫组化方法检测缺血区脑组织的eNOS与nNOS蛋白表达情况。结果:与假手术组比较,CI/R模型大鼠脑组织血管内皮细胞内eNOS表达在缺血1h内升高,之后到再灌注2h内持续降低。而nNOS的表达在缺血到再灌注2h内持续上升。结论:CI/R模型中缺血区脑组织的eNOS与nNOS的变化趋势不同,表明一氧化氮在缺血性脑损伤病理过程的作用与一氧化氮合酶亚型的变化有关。     

Structural basis for dipeptide amide isoform-selective inhibition of neuronal nitric oxide synthase

Three nitric oxide synthase (NOS) isoforms, eNOS, nNOS and iNOS, generate nitric oxide (NO) crucial to the cardiovascular, nervous and host defense systems, respectively. Development of isoform-selective NOS inhibitors is of considerable therapeutic importance. Crystal structures of nNOS-selective dipeptide inhibitors in complex with both nNOS and eNOS were solved and the inhibitors were found to adopt a curled conformation in nNOS but an extended conformation in eNOS. We hypothesized that a single-residue difference in the active site, Asp597 (nNOS) versus Asn368 (eNOS), is responsible for the favored binding in nNOS. In the D597N nNOS mutant crystal structure, a bound inhibitor switches to the extended conformation and its inhibition of nNOS decreases >200-fold. Therefore, a single-residue difference is responsible for more than two orders of magnitude selectivity in inhibition of nNOS over eNOS by L-N(omega)-nitroarginine-containing dipeptide inhibitors.     

Expression of nitric oxide synthase isoforms in the dorsal horn of monoarthritic rats: effects of competitive and uncompetitive <Emphasis Type="Italic">N</Emphasis>-methyl-D-aspartate antagonists

Chronic pain is associated with N-methyl-D-aspartate (NMDA) receptor activation and downstream production of nitric oxide, which has a pivotal role in multisynaptic local circuit nociceptive processing in the spinal cord. The formation of nitric oxide is catalyzed by three major nitric oxide synthase (NOS) isoforms (neuronal, nNOS; inducible, iNOS; endothelial, eNOS), which are increased in the spinal cord of rodents subjected to some tonic and chronic forms of experimental pain. Despite the important role of NOS in spinal cord nociceptive transmission, there have been no studies exploring the effect of NMDA receptor blockade on NOS expression in the dorsal horn during chronic pain. Furthermore, NOS isoforms have not been fully characterized in the dorsal horn of animals subjected to arthritic pain. The aim of this work was therefore to study the expression of nNOS, iNOS and eNOS in the dorsal horns of monoarthritic rats, and the modifications in NOS expression induced by pharmacological blockade of spinal cord NMDA receptors. Monoarthritis was produced by intra-articular injection of complete Freund's adjuvant into the right tibio-tarsal joint. At week 4, monoarthritic rats were given either the competitive NMDA antagonist (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) or the uncompetitive NMDA antagonist ketamine. After 6 and 24 hours, animals were killed and posterior quadrants of the lumbar spinal cord were dissected. Sample tissues were homogenized and subjected to immunoblotting with anti-nNOS, anti-iNOS or anti-eNOS monoclonal antibodies. The nNOS isoform, but not the iNOS and eNOS isoforms, were detected in the dorsal horns of control rats. Monoarthritis increased the expression of nNOS, iNOS and eNOS in the dorsal horns ipsilateral and contralateral to the inflamed hindpaw. Intrathecal administration of CPP and ketamine reduced nNOS expression in monoarthritic rats but increased the expression of iNOS and eNOS. Results suggest that blockade of spinal cord NMDA receptors produces complex regulatory changes in the expression of NOS isoforms in monoarthritic rats that may be relevant for nitridergic neuronal/glial mechanisms involved in the pathophysiology of monoarthritis and in the pharmacological response to drugs interacting with NMDA receptors.     

Resveratrol protects myocardial ischemia-reperfusion injury through both NO-dependent and NO-independent mechanisms

We previously showed that resveratrol (3,4',5-trihydroxystilbene) stimulates NO production and is cardioprotective in rat heart subjected to ischemia-reperfusion (I/R rat heart). We now show that in I/R rat heart, inducible nitric oxide synthase (iNOS) expression is markedly induced, while expression of endothelial nitric oxide synthase (eNOS) and nueronal nitric oxide synthase (nNOS) is unchanged. In animals preconditioned with resveratrol (0.5 to 1 mg/kg body wt), I/R-induced iNOS induction is abrogated; however, expression of eNOS and nNOS is greatly upregulated. The protective effects of resveratrol on I/R rat heart include reduced rhythm disturbances, reduced cardiac infarct size, and decreased plasma levels of lactate dehydrogenase (LDH) and creatine kinase (CK). Among these, the reductions in LDH/CK levels and infarct size are NO-dependent as the coadministration of N(omega)-nitro-L-arginine methyl ester (L-NAME, 1 mg/kg body wt) with resveratrol abolishes the resveratrol effect. In contrast, the reductions in the severity of ventricular arrhythmia and mortality rate are not affected by L-NAME coadministration, suggesting that a NO-independent mechanism is involved.     

Obesity, insulin resistance, and skeletal muscle nitric oxide synthase

The molecular mechanisms responsible for impaired insulin action have yet to be fully identified. Rodent models demonstrate a strong relationship between insulin resistance and an elevation in skeletal muscle inducible nitric oxide synthase (iNOS) expression; the purpose of this investigation was to explore this potential relationship in humans. Sedentary men and women were recruited to participate (means ± SE: nonobese, body mass index = 25.5 ± 0.3 kg/m(2), n = 13; obese, body mass index = 36.6 ± 0.4 kg/m(2), n = 14). Insulin sensitivity was measured using an intravenous glucose tolerance test with the subsequent modeling of an insulin sensitivity index (S(I)). Skeletal muscle was obtained from the vastus lateralis, and iNOS, endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) content were determined by Western blot. S(I) was significantly lower in the obese compared with the nonobese group (～43%; P < 0.05), yet skeletal muscle iNOS protein expression was not different between nonobese and obese groups. Skeletal muscle eNOS protein was significantly higher in the nonobese than the obese group, and skeletal muscle nNOS protein tended to be higher (P = 0.054) in the obese compared with the nonobese group. Alternative analysis based on S(I) (high and low tertile) indicated that the most insulin-resistant group did not have significantly more skeletal muscle iNOS protein than the most insulin-sensitive group. In conclusion, human insulin resistance does not appear to be associated with an elevation in skeletal muscle iNOS protein in middle-aged individuals under fasting conditions.