Development and persistence of long-lasting behavioral sensitization to cocaine in female mice: Role of the nNOS gene

Our recent studies have shown that the neuronal nitric oxide synthase (nNOS) gene is required for the development and persistence of psychomotor sensitization to cocaine in adult but not adolescent male mice (Balda, M.A., Anderson, K.L., Itzhak, Y., 2008. Differential role of the nNOS gene in the development of behavioral sensitization to cocaine in adolescent and adult B6;129S mice. Psychopharmacology (Berlin) 200, 509-519.). The aim of the present study was to investigate the contribution of the nNOS gene to cocaine-induced behavioral sensitization in adolescent and adult female mice. Adolescent and adult wild type (WT) and nNOS knockout (KO) mice received saline or cocaine (20 mg/kg) for 5 days and then were challenged with cocaine (20 mg/kg) after a drug-free period of either 10, 30, or 90 days. Context-dependent sensitization was determined by measuring saline-induced locomotor activity in the previously cocaine-paired environment. Results show that adolescent females of both genotypes, like their adult counterparts, developed long-lasting behavioral sensitization to cocaine (a three-month period), suggesting high vulnerability of females to cocaine regardless of age. An effect of genotype was observed in the initiation of sensitization, e.g., delayed onset in the absence of the nNOS gene. The only age-dependent difference observed was that adult, but not adolescent mice developed context-dependent sensitization. The present study suggests that long-term expression of cocaine-induced behavioral sensitization in females (adolescent and adult) is nNOS-independent, unlike our previous findings in adult males.     

Differential role of the nNOS gene in the development of behavioral sensitization to cocaine in adolescent and adult B6;129S mice

Rationale  Previous studies have suggested the involvement of neuronal nitric oxide synthase (nNOS) in the development of sensitization to psychostimulants. Ontogeny-dependent differences in the response to psychostimulants have been reported. Objective  The objectives were to investigate (a) the short- and long-term consequences of adolescent and adult cocaine exposure on behavioral sensitization and (b) the role of the nNOS gene in behavioral sensitization in adolescent and adult mice. Materials and methods  Adolescent and adult wild type (WT) and nNOS knockout (KO) mice received saline or cocaine (20 mg/kg) for 5 days and then were challenged with cocaine (20 mg/kg) after a drug-free period of 10 or 30 days. Locomotor activity was recorded by infrared beam interruptions. nNOS immunoreactive (ir) neurons in the dorsal and ventral striatum were quantified 24 h after repeated administration of cocaine to adolescent and adult WT mice. Results  Repeated administration of cocaine to either WT or nNOS KO mice during adolescence resulted in locomotor sensitization, which persisted into adulthood. WT but not KO adult mice developed long-term sensitization to cocaine. Repeated cocaine administration resulted in a 96% increase in the expression of nNOS-ir neurons in the dorsal striatum of adult but not adolescent WT mice. Conclusions  The nNOS gene is essential for the induction of behavioral sensitization to cocaine in adulthood but not in adolescence. The increased expression of nNOS-ir neurons in the dorsal striatum may underlie the induction of behavioral sensitization in adulthood. Thus, the NO-signaling pathway has an ontogeny-dependent role in the neuroplasticity underlying cocaine behavioral sensitization.     

神经元型一氧化氮合酶在心脏功能调节中的作用

神经元型一氧化氮合酶(nNOS)不仅在神经细胞中表达,也存在于心肌细胞。生理状况下,nNOS源性的一氧化氮(NO)通过自分泌以负反馈的调节方式维持细胞内Ca2+浓度稳态,防止细胞内Ca2+浓度超载,对于维持正常的心功能具有重要意义。病理状况下,nNOS的活性发生明显变化,nNOS源性的NO可能参与了心脏疾病的发生和发展。     

雪莲提取物对脑缺血再灌注损伤小鼠皮层区一氧化氮合酶亚型表达的影响

目的 探讨雪莲提取物对脑缺血再灌注损伤后小鼠皮层3种一氧化氮合酶(NOS)亚型表达的影响.方法 32只健康雄性ICR小鼠完全随机分为假手术组、生理盐水组、雪莲注射液组和依达拉奉组,每组8只,分别给予生理盐水、雪莲注射液或依达拉奉7d后,制作大脑中动脉阻塞模型,缺血60 min再灌注24h后应用免疫组化染色观察计算缺血皮层区每个高倍镜视野下3种NOS亚型的表达.结果 脑缺血再灌注损伤后24h,生理盐水组小鼠皮层神经元型一氧化氮合酶(nNOS)、诱导型一氧化氮合酶(iNOS)及内皮型一氧化氮合酶(eNOS)表达分别为(14.0±4.8)个/HP、(15.0±5.5)个/HP、(18.2±5.5)个/HP,较假手术组[(2.1±0.8)个/HP,(1.3±0.6)个/HP,(3.3±1.9)个/HP]均明显上调(P均为0.000).雪莲注射液组模型制作后皮层nNOS及iNOS阳性细胞表达分别为(7.5±3.8)个/HP、(7.1±3.7)个/HP,较生理盐水组明显减少(P均为0.000);eNOS阳性细胞表达为(22.3±2.3)个/HP,与生理盐水组比较差异无统计学意义(P=0.072).结论 雪莲提取物通过抑制脑缺血再灌注损伤后nNOS及iNOS表达,从而发挥神经保护作用.     

脑复合剂对创伤性脑损伤模型大鼠脑内NO、nNOS活性及表达的影响

目的:探讨脑复合剂治疗对创伤性脑损伤(traumatic brain injury,TBI)模型大鼠脑保护作用可能的分子机制.方法:用自由落体打击(Feeney法)建立TBI模型,分别设立假手术组、TBI模型组及中药治疗组.中药治疗组给予脑复合剂10 g· kg-1·d-1,假手术组及TBI模型组给予同等剂量的等渗盐水...     

Associative learning is enhanced by selective neuronal nitric oxide synthase inhibitors and retarded by a nitric oxide donor in the rabbit

RATIONALE: Previous studies had reported that the nitric oxide (NO) donor, sodium nitroprusside (SNP), retarded and the non-specific NO synthase (NOS) inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME), enhanced acquisition of classically conditioned responses (CRs). These effects of IV SNP and IP L-NAME on CR acquisition occurred in the absence of any effect on non-associative processes or performance variables and at a time when there were no alterations in blood pressure or heart rate. OBJECTIVES: In this study, we examined whether the changes in associative learning produced by L-NAME and SNP were due to their central effects on NO content of brain. To this end, we examined the effects of the selective neuronal NOS inhibitors 7-nitroindazole (7-NI) and AR-R 17477 and the effects of central (ICV) administration of the NO donor SNP on learning. METHODS: Effects of drugs on CR acquisition were determined during classical conditioning of the rabbit's nictitating membrane (NM) response. Explicitly unpaired presentations of conditioned stimuli (CSs) and unconditioned stimuli (USs) were employed to measure non-associative levels of responding and unconditioned response (UR) topography. RESULTS: The SC injection of 7-NI and AR-R 17477 significantly enhanced associative learning while ICV administration of SNP significantly retarded learning. CONCLUSION: Production of NO within the brain by neuronal NOS normally acts to retard associative learning presumably by decreasing excitability within neuronal circuits involved in the acquisition of the classically conditioned NM reflex.     

Inhibition of nitric oxide formation and superoxide generation during reduction of LY83583 by neuronal nitric oxide synthase

6-Anilino-5,8-quinolinedione (LY83583) has been widely used as an agent to reduce levels of nitric oxide (NO)-dependent cGMP in tissues. We report here that suppression of NO formation and production of superoxide during enzymatic reduction of LY83583 by neuronal NO synthase appeared to be potentially involved in the pharmacological action caused by LY83583. LY83583 suppressed neuronal NO synthase activity of 20,000×g rat cerebellar supernatant preparation in a concentration-dependent manner (IC₅₀ value=12.9 μM). A kinetic study revealed that LY83583 is a competitive inhibitor with respect to NADPH, with a K_i value of 2.57 μM. With purified neuronal NO synthase it was found that LY83583 was a potent inhibitor of NO formation by the enzyme and served as efficient substrate for reduction with a specific activity of 173 nmol of NADPH oxidized per mg of protein per minute. The reductase activity was stimulated about 19.8-fold by addition of CaCl₂/calmodulin, indicating that the presence of CaCl₂/calmodulin is essential to express maximal activity of LY83583 reduction. Although LY83583 was a good substrate for both NADPH-cytochrome P450 reductase (P450 reductase) and DT-diaphorase, these flavin enzymes-catalyzed reductions of LY83583 were less than the neuronal NO synthase-mediated reduction in the presence of CaCl₂/calmodulin. Enzymatic generation of superoxide during reduction of LY83583 by neuronal NO synthase, P450 reductase or DT-diaphorase was confirmed by electron spin resonance (ESR) experiments. Thus the present results indicate that a benzoquinone derivative LY83583 appears to interact with the P450 reductase domain on neuronal NO synthase, resulting in inhibition of NO formation and superoxide generation, which is involved in suppression of intracellular cGMP content.     

ACE活性和eNOS含量与妊高征相关性研究

目的:探讨胎盘组织一氧化氮合酶含量和血清中血管紧张素转化酶(ACE)活性改变在妊高征发生、发展中的作用。方法:选择轻、中、重度妊高征病人各10例为实验组,正常妊娠妇女30例为对照组,用马尿酸微量比色法检测血清中ACE活性,采用免疫组化ABC法分析妊高征胎盘组织中内皮型一氧化氮合酶(eNOS)含量的变化情况。结果:1妊高征组ACE活性明显高于对照组;2妊高征患者胎盘中eNOS含量显著低于正常足月孕者(P<0.05),且轻度妊高征患者胎盘eNOS含量显著高于中度及重度患者(P<0.05)。结论:血清ACE活性升高与妊高征的发生、发展密切相关。正常足月孕胎盘和妊高征患者胎盘绒毛血管内皮细胞中均存在eNOS抗原,妊高征患者胎盘中含量的减少与其发病有关。     

内皮型一氧化氮合酶基因G894T多态性与成人过敏性紫癜易感性的关系

目的探讨内皮型一氧化氮合酶基因G894T多态性与成人过敏性紫癜易感性的关系。方法将邯郸市第一医院治疗的98例成人过敏性紫癜患者作为观察组,同时随机选取同一时间进行体检的91例健康成人作为对照组。首先从两组所有人体内提取DNA,然后在PCR扩增仪上完成对内皮型一氧化氮合酶基因进行目的片段的扩增,接着进行酶切。最后观察酶切产物。结果观察组患者体内G基因明显多于对照组的健康群体（P〈0．05）。结论机体内皮型一氧化氮合酶基因G894T增多会增加成人过敏性紫癜的发生,其易感性与内皮型一氧化氮合酶基因G894T多态性有很大的关系。     

戊四唑癫痫大鼠脑组织一氧化氮含量和一氧化氮合酶活性变化

探讨一氧化氮在戊四唑癫病发机制中的作用。方法每天注射戊四唑建立在鼠癫痫模型,测定癫病发作后大鼠大脑皮质,海马一氧化氮和一氧化氮合酶活性变化,结果癫痫发作后海马ＮＯ含量和ＮＯＳ活性显著升高,结 戊四唑诱导的癫痫中具有致痫性。     

大鼠对氟西泮抗痫耐受和依赖时海马中一氧化氮合酶的变化

目的通过测定大鼠对氟西泮抗痫耐受性和依赖性时海马中一氧化氮合酶(NOS)蛋白表达及活性的变化,探讨一氧化氮(NO)在此过程中可能的作用。方法建立大鼠对氟西泮抗痫耐受性和依赖性的模型。运用免疫印迹及免疫组化法检测海马中NOS蛋白表达,以及比色法检测NOS活性的变化。结果大鼠对氟西泮抗痫耐受组的海马中NOS蛋白表达明显高于对照组;而对氟西泮抗痫依赖组则与对照组差别无统计学意义。两组NOS活性均显著高于各自的对照组。结论NO可能是介导氟西泮抗痫耐受性和依赖性的因素之一。     

香菇多糖对巨噬细胞一氧化氮和一氧化氮合酶活性的影响

目的研究香菇多糖(LTN)诱导巨噬细胞的一氧化氮(NO)生成和一氧化氮合酶(iNOS)的活性,探讨LTN的免疫调节作用机理.方法采用Griess反应和荧光法测定不同剂量的LTN作用小鼠腹腔巨噬细胞后NO的生成量和iNOS活性.观察mRNA转录抑制剂、蛋白质合成抑制剂和iNOS抑制剂对巨噬细胞NO的生成和iNOS活性的影响.结果LTN能使小鼠腹腔巨噬细胞NO生成增加,iNOS活性增高,并呈作用剂量依赖关系.3种抑制剂均能抑制LTN诱导的小鼠腹腔巨噬细胞N0的生成和iNOS活性.结论LTN能刺激小鼠腹腔巨噬细胞提高iNOS活性和NO的生成.提示LTN的免疫调节作用机制可能与LTN刺激巨噬细胞NO生成有关.     

Effects of nitric oxide synthase inhibitors on the discriminative stimulus effects of cocaine in rats

RATIONALE: Nitric oxide synthase (NOS) inhibitors may modulate the discriminative stimulus effects of cocaine because they alter dopamine (DA) release. OBJECTIVES: The effects of the NOS inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitro-indazole (7-NI) were examined in experiments designed to better understand the mechanisms that may underlie the interactions between NOS inhibitors and cocaine. METHODS: Rats were trained to discriminate 10 mg/kg cocaine from saline, and then substitution and pretreatment tests with L-NAME and 7-NI were conducted. To determine if the combined effects of NOS inhibitors and cocaine might be related to DA mechanisms and/or to N-methyl-D-aspartate (NMDA) receptor mechanisms, substitution tests with other indirect DA agonists and NMDA antagonists were carried out in the presence and absence of L-NAME. In addition, the roles of the D1 and D2 families of DA receptors in mediating the cocaine-altering effects of L-NAME and 7-NI were examined in antagonism tests using SCH 23390 and haloperidol, respectively. RESULTS: The results demonstrated that neither NOS inhibitor alone substituted for the 10 mg/kg cocaine training dose, but when given as a pretreatment, 100 mg/kg L-NAME as well as 10 mg/kg 7-NI enhanced the discriminative stimulus and rate-decreasing effects of cocaine. L-NAME pretreatment also enhanced the potency of (+)-amphetamine and GBR 12909, but not MK-801, phencyclidine, or NPC 17742, for producing discriminative stimulus and rate-decreasing effects in substitution tests. Further testing showed that the cocaine-enhancing effects of L-NAME and 7-NI were attenuated by doses of haloperidol and SCH 23390 that minimally altered the effects of cocaine alone. CONCLUSIONS: These findings suggest that L-NAME and 7-NI may increase the potency of cocaine and other indirect DA agonists through a central mechanism whereby DA neurotransmission is directly enhanced by NOS inhibition.     

高压氧对脑梗死后血清一氧化氮和一氧化氮合酶含量的影响

目的 研究高压氧对脑梗死后血清一氧化氮、一氧化氮合酶含量的影响。方法 将脑梗死患者分为高压氧治疗组和非高压氧治疗组,观察不同病期血清一氧化氮、一氧化氮合酶含量的变化,并与正常对照组进行比较。结果 高压氧治疗组一氧化氮含量较非高压氧治疗组上升快,在治疗后15天恢复正常,但在一疗程高压氧治疗结束后,却又有所下降;一氧化氮合酶含量在治疗后均有上升,未能恢复到正常水平,两组之间无差异。结论 高压氧通过提高NO的含量,减轻缺血区脑组织的损伤,改善脑血循环;高压氧对NOS有一定影响,但作用不大;应适当延长高压氧疗程,尽可能缓解因NOS含量下降所造成的NO释放量不足。     

一氧化氮合酶基因转移在心血管疾病治疗中的研究进展

基因治疗包括功能基因转移至宿主细胞 ,用于校正特定基因的功能缺陷或减轻疾病的症状。对于心血管疾病的基因转移 ,腺病毒载体最有效。该文从方法学上回顾了血管NOS基因转移领域的最新进展以及NOS基因转移对重要心血管疾病的潜在应用。NOS基因转移方法在动物模型中具有可行性 ,但是 ,当前可用的载体还有许多技术和安全限制。在用于人类心血管疾病NOS基因治疗前必须给予克服     

铅对海马神经元型一氧化氮合酶及其基因表达的影响

目的　观察实验性铅中毒对小鼠海马神经元型一氧化氮合酶 (nNOS)及其基因表达的影响 ,探讨铅致学习记忆损害的分子毒理学机制。方法　小鼠用 5g L醋酸铅溶液染毒。采用逆转录 聚合酶链反应 (RT PCR)方法 ,半定量分析染毒 2、4和 8周小鼠海马nNOS基因表达的变化。采用尼克酰胺腺嘌呤二核苷酸磷酸黄递酶 (NADPH d)组织化学染色法分析铅染毒对小鼠海马NOS阳性神经元的影响。结果　nNOSmRNA存在于正常小鼠海马组织中 ;铅染毒后仅 2周 ,海马nNOSmRNA的含量显著减少 ;随着染毒时间的增加 ,海马nNOSmRNA的含量逐渐下降。镜下观察 ,视野中正常小鼠海马平均NOS阳性细胞数为 ( 5 7 63± 11 5 )个 ,而铅染毒组仅为 ( 2 9 3 5± 9 10 )个。结论　铅可使小鼠海马nNOS及其基因表达下降     

小鼠内皮型一氧化氮合酶表达的变化与肾间质纤维化的关系

目的探讨内皮型一氧化氮合酶(eNOS)在小鼠单侧输尿管梗阻(UUO)模型肾组织中的变化及其与肾间质纤维化的关系。方法 48只小鼠随机均分为UUO模型组和假手术组,每组术后第1、3、7、14天分别处死6只小鼠,观察肾组织的病理改变,检测eNOS和结缔组织生长因子(CTGF)表达,计算管周毛细血管(PTC)密度。结果模型组术后第7天部分肾小管萎缩,纤维化形成;术后第14天肾小管间质纤维化更加明显。假手术组eNOS表达较多,CTGF低表达。与假手术组相比,模型组第7、14天eNOS表达减少(P<0.05),CTGF表达增多(P<0.05),第3-14天PTC密度下降(P<0.05)。结论小鼠UUO肾组织eNOS表达下降参与了肾间质纤维化的进展。     

Pharmacokinetics and protein binding of the selective neuronal nitric oxide synthase inhibitor 7-nitroindazole

Utilization of nitric oxide (NO) synthase (NOS) inhibitors to probe the role of NO in various central nervous system processes requires use of an inhibitor selective for neuronal NOS, and is facilitated by knowledge of the pharmacokinetics of the inhibitor. The present project was undertaken to elucidate the disposition of the selective neuronal NOS inhibitor 7-nitroindazole (7-NI). A simple, specific HPLC assay was developed with requisite sensitivity to quantitate 7-NI in serum after administration of pharmacologically relevant doses. Further experiments were performed to assess the effects of administered dose on 7-NI disposition. 7-NI displayed marked nonlinearity, consistent with saturable elimination, when administered by ip injection in peanut oil. The nonlinearity was related to total dose, but not to the concentration of 7-NI in the vehicle. Binding of 7-NI in rat serum was concentration-independent and does not contribute to the nonlinearity. Various formulations for iv administration of this water-insoluble compound were evaluated; the optimal vehicle, from the standpoint of 7-NI solubility, appeared to inhibit the clearance of 7-NI from the systemic circulation. Considering the nonlinear disposition of 7-NI, knowledge of the pharmacokinetics of this inhibitor is requisite to designing administration protocols to achieve the desired magnitude and duration of NOS inhibition.     

Gender-specific vascular effects elicited by chronic ethanol consumption in rats: a role for inducible nitric oxide synthase

BACKGROUND AND PURPOSE: Epidemiological data suggest that the risk of ethanol-associated cardiovascular disease is greater in men than in women. This study investigates the mechanisms underlying gender-specific vascular effects elicited by chronic ethanol consumption in rats. EXPERIMENTAL APPROACH: Vascular reactivity experiments using standard muscle bath procedures were performed on isolated thoracic aortae from rats. mRNA and protein for inducible NO synthase (iNOS) and for endothelial NOS (eNOS) was assessed by RT-PCR or western blotting, respectively. KEY RESULTS: In male rats, chronic ethanol consumption enhanced phenylephrine-induced contraction in both endothelium-intact and denuded aortic rings. However, in female rats, chronic ethanol consumption enhanced phenylephrine-induced contraction only in endothelium denuded aortic rings. After pre-incubation of endothelium-intact rings with L-NAME, both male and female ethanol-treated rats showed larger phenylephrine-induced contractions in aortic rings, compared to the control group. Acetylcholine-induced relaxation was not affected by ethanol consumption. The effects of ethanol on responses to phenylephrine were similar in ovariectomized (OVX) and intact (non-OVX) female rats. In the presence of aminoguanidine, but not 7-nitroindazole, the contractions to phenylephrine in rings from ethanol-treated female rats were greater than that found in control tissues in the presence of the inhibitors. mRNA levels for eNOS and iNOS were not altered by ethanol consumption. Ethanol intake reduced eNOS protein levels and increased iNOS protein levels in aorta from female rats. CONCLUSIONS AND IMPLICATIONS: Gender differences in the vascular effects elicited by chronic ethanol consumption were not related to ovarian hormones but seemed to involve the upregulation of iNOS.     

Altered L-arginine/nitric oxide synthase/nitric oxide pathway in the vascular adventitia of rats with sepsis

1. In recent studies, the vascular adventitia has been established as an important source of inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) production, even more powerful than the media in response to certain inflammatory factors, such as lipopolysaccharide (LPS). The adventitia has an independent L-arginine (L-Arg)/NOS/NO pathway and is involved in the regulation of vascular function. In the present study, we explored the changes in and the pathophysiological significance of the L-Arg/NOS/NO pathway in the adventitia of rats with sepsis. 2. Sepsis was induced by caecal ligation and puncture in order to observe changes in L-Arg transport, NOS gene expression and activity and NO generation in the vascular adventitia to determine the mechanism of activation of the L-Arg/NOS/NO pathway. 3. Severe sepsis resulted in severe disturbance of haemodynamic features, with decreased mean arterial blood pressure, brachycardia and inhibited cardiac function (decreased left ventricular +/-dP/dt(max)). Left ventricular end-diastolic pressure was elevated threefold (P < 0.01) under anaesthesia. Rats with sepsis showed severe glucopenia and lacticaemia. Plasma levels of the inflammatory factors macrophage chemoattractant protein-1 and interleukin-8 were increased five- and 29-fold, respectively (P < 0.01). 4. In the adventitia of the thoracic and abdominal aortas, the L-Arg/NO pathway was similarly characterized: the uptake of [(3)H]-L-Arg was Na(+) independent, with the peak occurring at approximately 40 min incubation. Total NOS activity was largely calcium independent (> 90%). The V(max) of L-Arg transport in the sepsis group was increased by 83.5% (P < 0.01), but the K(m) value was not significantly different compared with controls. 5. The mRNA levels of cationic amino acid transporter (CAT)-1 and CAT-2B in the sepsis group were increased by 86 and 62%, respectively (both P < 0.01). Inducible NOS activity was increased 2.8-fold compared with controls (P < 0.01) and iNOS mRNA levels were elevated approximately sixfold (P < 0.01). The NO levels in the plasma and incubation media (incubation for 40 min) in the sepsis group were increased by 144 and 273%, respectively (both P < 0.01). 6. The Arg/NOS/NO pathway was activated in the vascular adventitia of rats with sepsis shock. The L-Arg/NOS/NO pathway in the aortic adventitia may play an important role in the pathogenesis of sepsis and septic shock.