Caveolin-1 regulates nitric oxide-mediated matrix metalloproteinases activity and blood-brain barrier permeability in focal cerebral ischemia and reperfusion injury

The roles of caveolin-1 (cav-1) in regulating blood-brain barrier (BBB) permeability are unclear yet. We previously reported that cav-1 was down-regulated and the production of nitric oxide (NO) induced the loss of cav-1 in focal cerebral ischemia and reperfusion injury. The present study aims to address whether the loss of cav-1 impacts on BBB permeability and matrix metalloproteinases (MMPs) activity during cerebral ischemia-reperfusion injury. We found that focal cerebral ischemia-reperfusion down-regulated the expression of cav-1 in isolated cortex microvessels, hippocampus, and cortex of ischemic brain. The down-regulation of cav-1 was correlated with the increased MMP-2 and -9 activities, decreased tight junction (TJ) protein zonula occludens (ZO)-1 expression and enhanced BBB permeability. Treatment of N(G) -nitro-L-arginine methyl ester [L-NAME, a non-selective nitric oxide synthase (NOS) inhibitor] reserved the expression of cav-1, inhibited MMPs activity, and reduced BBB permeability. To elucidate the roles of cav-1 in regulating MMPs and BBB permeability, we used two approaches including cav-1 knockdown in cultured brain microvascular endothelial cells (BMECs) in vitro and cav-1 knockout (KO) mice in vivo. Cav-1 knockdown remarkably increased MMPs activity in BMECs. Meanwhile, with focal cerebral ischemia-reperfusion, cav-1 deficiency mice displayed higher MMPs activities and BBB permeability than wild-type mice. Interestingly, the effects of L-NAME on MMPs activity and BBB permeability was partly reversed in cav-1 deficiency mice. These results, when taken together, suggest that cav-1 plays important roles in regulating MMPs activity and BBB permeability in focal cerebral ischemia and reperfusion injury. The effects of L-NAME on MMPs activity and BBB permeability are partly mediated by preservation of cav-1.     

Transient focal cerebral ischemia down-regulates glutamate transporters GLT-1 and EAAC1 expression in rat brain

Transient focal cerebral ischemia leads to extensive excitotoxic neuronal damage in rat cerebral cortex. Efficient reuptake of the released glutamate is essential for preventing glutamate receptor over-stimulation and neuronal death. Present study evaluated the expression of the glial (GLT-1 and GLAST) and neuronal (EAAC1) subtypes of glutamate transporters after transient middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia in rats. Between 24h to 72h of reperfusion after transient MCAO, GLT-1 and EAAC1 protein levels decreased significantly (by 36% to 56%, p < 0.05) in the ipsilateral cortex compared with the contralateral cortex or sham control. GLT-1 and EAAC1 mRNA expression also decreased in the ipsilateral cortex of ischemic rats at both 24h and 72h of reperfusion, compared with the contralateral cortex or sham control. Glutamate transporter down-regulation may disrupt the normal clearance of the synaptically-released glutamate and may contribute to the ischemic neuronal death.     

大鼠局灶性脑缺血后一氧化氮合酶基因表达的变化

目的:观察大鼠局灶性脑缺血后3种类型一氧化氮合酶(NOS)mRNA表达的变化.方法:大鼠随机分为正常对照组、缺血后2、6、12、24 h组,以逆转录-聚合酶链反应(RT-PCR)法分别检测缺血脑组织NOS基因表达的变化.结果:脑缺血后eNOS、nNOSmRNA表达增强,分别于2、6 h达高峰;iNOS mRNA表达亦增高,但在缺血后12 h达高峰.结论:大鼠脑缺血早期eNOS和nNOS占主要地位,缺血后期iNOS占主要地位.     

Inducible nitric oxide synthase expression in the ischemic core and penumbra after transient focal cerebral ischemia in mice

The present observations examined the hypothesis that the iNOS expression in the ischemic penumbra after a transient focal ischemic insult is involved in the recruitment of penumbra into infarction. The middle cerebral artery in mice was occluded for 2 h by an intraluminal filament and then recirculated. The measurement of iNOS activity, iNOS protein formation and NO concentration in the ischemic core and penumbra, and the determination of infarct volume were performed at 6, 12, 24 and 48 h after reperfusion. iNOS protein and iNOS enzymatic activity appeared at 6 h, peaked at 24 h, and declined at 48 h in the penumbra after reperfusion. iNOS protein was not detectable in contralateral area and in sham-operated brains. The time course of iNOS protein, enzymatic activity and NO concentration in the penumbra but not in the core matched the process of infarct maturation. Treatment with iNOS inhibitor aminoguanidine (100 mg.kg(-1), i.p.) at 6 and 12 h after reperfusion inhibited iNOS activity by 88.0 +/- 10.4% and reduced NO concentration by 48.5 +/- 8.3% in the penumbra, and lessened infarct size by 48.8 +/- 7.2%. The iNOS activity and NO level in the core were not affected by the administration of aminoguanidine. These results suggest that iNOS expression in the ischemic penumbra is involved in the recruitment of penumbra into infarction and thereby contributing to the enlargement of infarct.     

金雀异黄酮通过减少卵巢切除大鼠心肌小凹蛋白-1的表达而增加一氧化氮的生成

观察金雀异黄酮(genistein)替代治疗对卵巢切除大鼠心肌中一氧化氮(nitric oxide,NO)和内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)的影响.成年雌性Sprague-Dawley大鼠经双侧卵巢切除术,假手术组作为对照,术后三周将行卵巢切除术的大鼠随机分为低剂量genistein(0.5 mg/kg·d1)、高剂量genistein(5.0 mg/kg·d-1)、17-β雌二醇(0.1 mg/kg·d-1)和模型组(100μl/d芝麻油),各组均皮下注射给药并给予不含大豆的饲料喂养6周,测定大鼠尾动脉血压、心率,麻醉后放血处死大鼠称量子宫重量;放免法检测血浆中总雌二醇,亚硝酸还原酶法检测心肌匀浆中NO,Western blot检测心肌中eNOS的表达以及eNOS的调节蛋白小凹蛋白-1(caveolin-1)和钙调素(calmodulin)的表达情况.结果显示各组间大鼠血压无显著性差异,同17-β雌二醇一样,genistein能呈剂量依赖性地增加心肌组织中eNOS表达量和NO生成,同时genistein能明显降低内源性eNOS活性抑制物caveolin-1的表达,而不影响eNOS活性正性调节蛋白钙调素的表达.与溶媒对照组比较,0.5 mg/kg·d-1的genistein不增加子宫重量,5.0 mg/kg·d-1的genistein增加子宫重量3倍,但较17-β雌二醇(增加6倍)的作用小(P＜0.01).上述结果提示,植物雌激素genistein剂量依赖性地上调心肌组织eNOS的活性并增加NO的生成,减少抑制eNOS活性的小凹蛋白-1表达.     

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。     

左旋硝基精氨酸对肺缺血预处理的抗缺血/再灌注损伤作用的影响

目的:观察一氧化氮合酶(NOS)阻滞剂左旋硝基精氨酸(L-NNA)对肺缺血预处理的影响,探讨NOS在肺缺血预处理效应中的作用.方法:40只日本大耳兔随机分成假手术(S)组,缺血/再灌注组(I/R组),缺血预处理组(IPC组)和左旋硝基精氨酸组(LNNA组),观察各组血浆丙二醛(MDA)含量、超氧歧化酶(SOD)活力,肺组织湿干重比(W/D),肺损伤定量评价指标(IQA),肺组织NOS活力以及肺组织超微结构变化.结果:IPC组与I/R组比较,MDA、W/D、IQA下降(P＜0.05或P＜0.01),SOD活力上升(P＜0.05),总NOS(TNOS)、原生型NOS(cNOS)活性明显增高(P＜0.05),电镜下肺组织损伤较轻;而预先用L-NNA(LNNA组)后使上述改变发生逆转.结论:左旋硝基精氨酸可以拮抗肺缺血预处理的抗缺血再灌注损伤作用;NOS可能是介导肺IPC效应的重要酶蛋白.     

Smooth muscle NOS, colocalized with caveolin-1, modulates contraction in mouse small intestine

Neuronal nitric oxide synthase (nNOS) in myenteric neurons is activated during peristalsis to produce nitric oxide which relaxes intestinal smooth muscle. A putative nNOS is also found in the membrane of intestinal smooth muscle cells in mouse and dog. In this study we studied the possible functions of this nNOS expressed in mouse small intestinal smooth muscle colocalized with caveolin-1(Cav-1). Cav-1 knockout mice lacked nNOS in smooth muscle and provided control tissues. 60 mM KCl was used to increase intracellular [Ca(2+)] through L-type Ca(2+) channel opening and stimulate smooth muscle NOS activity in intestinal tissue segments. An additional contractile response to LNNA (100 muM, NOS inhibitor) was observed in KCl-contracted tissues from control mice and was almost absent in tissues from Cav-1 knockout mice. Disruption of caveolae with 40 mM methyl-beta cyclodextrin in tissues from control mice led to the loss of Cav-1 and nNOS immunoreactivity from smooth muscle as shown by immunohistochemistry and a reduction in the response of these tissues to N-omega-nitro-L-arginine (LNNA). Reconstitution of membrane cholesterol using water soluble cholesterol in the depleted segments restored the immunoreactivity and the response to LNNA added after KCl. Nicardipine (1 muM) blocked the responses to KCl and LNNA confirming the role of L-type Ca(2+) channels. ODQ (1 muM, soluble guanylate cyclase inhibitor) had the same effect as inhibition of NOS following KCl. We conclude that the activation of nNOS, localized in smooth muscle caveolae, by calcium entering through L-type calcium channels triggers nitric oxide production which modulates muscle contraction by a cGMP-dependent mechanism.     

Endothelial nitric oxide synthase is segregated from caveolin-1 and localizes to the leading edge of migrating cells

The enzyme endothelial Nitric Oxide Synthase (eNOS) is involved in key physiological and pathological processes, including cell motility and apoptosis. It is widely believed that at the cell surface eNOS is localized in caveolae, where caveolin-1 negatively regulates its activity, however, there are still uncertainties on its intracellular distribution. Here, we applied high resolution confocal microscopy to investigate the surface distribution of eNOS in transfected HeLa cells and in human umbilical vein endothelial cells (HUVEC) endogenously expressing the enzyme. In confluent and non-confluent HUVEC and HeLa cells, we failed to detect substantial colocalization between eNOS and caveolin-1 at the cell surface. Instead, in non-confluent cells, eNOS was concentrated in ruffles and at the leading edge of migrating cells, colocalizing with actin filaments and with the raft marker ganglioside G(M1), and well segregated from caveolin-1, which was restricted to the posterior region of the cells. Treatments that disrupted microfilaments caused loss of eNOS from the cell surface and decreased Ca(2+)-stimulated activity, suggesting a role of the cytoskeleton in the localization and function of the enzyme. Our results provide a morphological correlate for the role of eNOS in cell migration and raise questions on the site of interaction between eNOS and caveolin-1.     

Role of nitric oxide in the reperfusion induced injury in hyperthyroid rat hearts

We recently reported that hyperthyroidism affects the heart response to ischemia/reperfusion. A significant tachycardia during reperfusion was associated with an increase in the oxidative stress of hearts from T₃-treated animals. In the present study we checked the possible role of nitric oxide (NO) in this major stress induced by the hyperthyroid state. We compared the functional recovery from ischemia/reperfusion of Langendorff preparations from euthyroid (E) and hyperthyroid (H, ten daily intraperitoneal injections of T₃, 10 μg/100 g body weight) rats, in the presence and in the absence of 0.2 mM N^ω-nitro-L-arginine (L-NNA). At the end of the ischemia/reperfusion protocol (10 min preischemic perfusion, 20 min global ischemia, 30 min reperfusion) lipid peroxidation, antioxidant capacity (C_A) and susceptibility to in vitro oxidative stress were determined on heart homogenates. The main effect of hyperthyroidism on the reperfusion functional response was confirmed to be a strong tachycardic response (154% recovery at 25 min reperfusion) accompanied by a low recovery in both left ventricular diastolic pressure (LVDP) and left ventricular dP/dt_max. This functional response was associated with a reduction in C_A and an increase in both lipid peroxidation and susceptibility to oxidative stress. Perfusion of hearts with L-NNA per se had small but significant negative chronotropic and positive inotropic effects on preischemic performance of euthyroid rat hearts only. More importantly, L-NNA perfusion completely blocked the reperfusion tachycardic response in the hyperthyroid rats. Concomitantly, myocardium oxidative state (lipid peroxidation, C_A and in vitro susceptibility to oxidative stress) of L-NNA perfused hearts was similar to that of E animals. These results suggest that the higher reperfusion-induced injury occurring in hyperthyroid animals is associated with overproduction of nitric oxide.     

Ascorbic acid and N-acetyl cysteine prevent uncoupling of nitric oxide synthase and increase tolerance to ischemia/reperfusion injury in diabetic rat heart

Abstract

Oxidative stress may cause a loss of tetrahydrobiopterin (BH4), a co-factor of nitric oxide synthase (NOS), decrease the bioavailability of NO and aggravate ischemia/reperfusion (I/R) injury in diabetic heart. We hypothesized that ascorbic acid (AA) and N-acetyl cysteine (NAC) protect the diabetic heart from I/R injury by increasing BH4/dihydrobiopterin (BH2) ratio and inhibiting uncoupling of NOS. Diabetes mellitus was induced in rats by streptozotocin treatment, and the hearts were isolated and perfused. BH4 and BH4/BH2 ratio decreased in the diabetic heart associated with increased production of superoxide and nitrotyrosine (NT). Treatment with AA or NAC significantly increased BH4/BH2 ratio in the diabetic heart associated with decreased production of superoxide and NT and increased generation of nitrate plus nitrite (NOx). Pre-treatment with AA or NAC before 30 min ischemia followed by 120 min reperfusion improved left ventricular (LV) function and reduced infarct size in the diabetic but not non-diabetic hearts. The NOS inhibitor, L-NAME, inhibited the increase in the generation of superoxide, NT and NOx, but aggravated LV function and increased infarct size in the diabetic heart. L-NAME also abrogated the increase in NOx and improvement of LV function and the infarct size-limiting effect induced by AA or NAC in the diabetic heart. These results suggest that AA and NAC increase BH4/BH2 ratio and prevent NOS uncoupling in the diabetic heart. Resultant increase in the bioavailability of NO renders the diabetic heart toleratant to I/R injury.     

Nitric oxide impairs mitochondrial movement in cortical neurons during hypoxia

Cortical nitric oxide (NO) production increases during hypoxia/ischemia in the immature brain and is associated with both neurotoxicity and mitochondrial dysfunction. Mitochondrial redistribution within the cell is critical to normal neuronal function, however, the effects of hypoxia on mitochondrial dynamics are not known. This study tested the hypothesis that hypoxia impairs mitochondrial movement via NO-mediated pathways. Fluorescently labeled mitochondria were studied using time-lapse digital video microscopy in cultured cortical neurons exposed either to hypoxia/re-oxygenation or to diethyleneamine/nitric oxide adduct, DETA-NO (100-500 microm). Two NO synthase inhibitors, were used to determine NO specificity. Mitochondrial mean velocity, the percentage of movement (i.e. the time spent moving) and mitochondrial morphology were analyzed. Exposure to hypoxia reduced mitochondrial movement to 10.4 +/- 1.3% at 0 h and 7.4 +/- 1.7% at 1 h of re-oxygenation, versus 25.6 +/- 1.4% in controls (p < 0.05). Mean mitochondrial velocity (microm s(-1)) decreased from 0.374 +/- 0.01 in controls to 0.146 +/- 0.01 at 0 h and 0.177 +/- 0.02 at 1 h of re-oxygenation (p < 0.001). Exposure to DETA-NO resulted in a significant decrease in mean mitochondrial velocity at all tested time points. Treatment with NG-nitro-L-arginine methyl ester (L-NAME) prevented the hypoxia-induced decrease in mitochondrial movement at 0 h (30.1 +/- 1.6%) and at 1 h (26.1 +/- 9%) of re-oxygenation. Exposure to either hypoxia/re-oxygenation or NO also resulted in the rapid decrease in mitochondrial size. Both hypoxia and NO exposure result in impaired mitochondrial movement and morphology in cultured cortical neurons. As the effect of hypoxia on mitochondrial movement and morphology can be partially prevented by a nitric oxide synthase (NOS) inhibitor, these data suggest that an NO-mediated pathway is at least partially involved.     

甲醛炎性痛增强大鼠海马NOS表达和NO生成

目的：观察甲醛炎性痛过程中大鼠痛行为、海马一氧化氮合酶（NOS）活性及一氧化氮（NO）含量的变化以及变化的时程及区域特征。方法：采用辐射热甩尾法测定大鼠痛阈变化;采用NADPH—d组织化学法和硝酸还原酶法分别测定大鼠海马NOS表达和No含量。结果：皮下注射甲醛溶液后,大鼠出现伤害性感受反应及痛阈降低。注射甲醛后6h,海马CA1、CA2～3区及DG区NOS阳性细胞数目、阳性细胞染色深度均显著增加。海马NO含量亦显著增加;注射甲醛后12h时这些改变最为显著,48h时恢复至对照组水平。结论：甲醛炎性痛可诱导海马NOS活性增强及NO生成增多．这种改变可发生在海马各区．并具有一定的时程特征。     

Exogenous nitric oxide negatively regulates c-Jun N-terminal kinase activation via inhibiting endogenous NO-induced S-nitrosylation during cerebral ischemia and reperfusion in rat hippocampus

Nitric oxide (NO), synthesized from l -arginine by NO synthases, is a small endogenous free radical with multiple functions. The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in mediating apoptosis in cerebral ischemia and reperfusion. In this study, we found that the NO donor sodium nitroprusside (SNP) can decrease the damage of hippocampal neurons induced by cerebral ischemia and reperfusion. Our current study demonstrates that SNP can suppress the phosphorylation of JNK3 by suppressing the increased S-nitrosylation of JNK3 induced by cerebral ischemia and reperfusion. In contrast, dithiothreitol reversed the effect of SNP on S-nitrosylation of JNK3. Furthermore, the inhibitor of nNOS (7-NI) and the inhibitor of iNOS (AMT) can decrease JNK3 phosphorylation through decreasing S-nitrosylation of JNK3. Our data suggest that endogenous NO synthesized by NO synthases can increase JNK3 phosphorylation by means of S-nitrosylation during global ischemia/reperfusion in rat hippocampus. However, the exogenous NO (SNP) can reverse the effect of endogenous NO by inhibiting S-nitrosylation of JNK3. Together, these results suggest that the exogenous NO may provide a new clue for stroke therapy.     

Fibroblast growth factors in myocardial ischemia / reperfusion injury and ischemic preconditioning

Angiogenic growth factors such as fibroblast growth factors (FGFs) are currently in clinical trials for accelerating blood vessel formation in myocardial and limb ischemic conditions. However, recent experimental evidence suggests that FGFs can also participate as endogenous cardioprotective agents. In this report, the current knowledge for FGFs implication in myocardial ischemic tolerance will be summarized. Pharmacologic preconditioning with drugs as FGFs that mimic the beneficial effects of ischemic preconditioning could lead to novel therapeutic approaches for the treatment of ischemic disorders including myocardial infarction and stroke.     

大鼠脑内caveolin-1蛋白的表达及其在分辨学习中的作用

Caveolin-1（Cav—1）蛋白作为细胞质膜结构小窝（caveolae）的标志蛋白,在胆固醇运输、膜组装、信号转导和细胞转化过程中扮演重要的角色。为了探讨Cav-1蛋白在中枢神经系统可塑性及学习记忆中的作用,本文以Sprague—Dawley大鼠为实验对象,利用蛋白质免疫印迹杂交方法观察了Car-1蛋白在不同年龄大鼠脑内表达的特征,并研究了Y-迷宫训练前后Cav-1蛋白表达的变化。结果表明：（1）大鼠不同脑区Cav-1蛋白表达的年龄特征不同。海马内的表达属青年鼠最高,其次是老年鼠和幼年鼠;皮层内的表达属幼年鼠最高,其次是老年鼠,青年鼠最低;小脑内的表达无明显年龄差异。（2）Y-迷宫训练引起青年鼠海马和前额叶皮层内Cav-1蛋白的表达显著增加。结果提示,Cav-1蛋白与动物脑发育和学习记忆有密切关系,可能参与中枢可塑性的调节。     

缺血后适应对局灶性脑缺血/再灌注大鼠p38表达的影响

目的：探讨缺血后适应对大鼠局灶性脑缺血/再灌注损伤后p38表达的影响。方法：将30只雄性SD大鼠随机分为3组（n=10）：假手术组（sham组）、缺血/再灌注（I/R）组和缺血后适应（IP）组。利用TUNEL法观察神经细胞凋亡的变化,应用Westernblot检测大鼠局灶性脑I/R损伤后p38蛋白表达水平的变化。结果：大鼠脑缺血/再灌注后凋亡细胞数量和p38蛋白表达水平均显著升高,而IP组凋亡细胞数量和p38蛋白表达水平均显著低于IR组（P〈0.01）。结论：缺血后适应可抑制大鼠脑缺血/再灌注后细胞凋亡的发生,此作用可能与下调p38蛋白表达有关。     

大鼠心脏缺血-再灌注损伤对心肌L-Arg/NO途径的影响

为探讨大鼠心脏缺血-再灌注损伤(IRI)期间一氧化氮(NO)生成增加的环节和过程。本实验用离体灌流大鼠心脏,预灌流15 min,停灌45 min,取30 ml KH 液循环灌流15 min,观察冠脉流出液中细胞胞浆酶(LDH)、蛋白质、肌红蛋白漏出量和NO     

诱生型一氧化氮合酶与脑缺血后期损伤

脑缺血后期敏感细胞受缺血局部产生的某些细胞因子以及血管切变力、氧化还原状态的改变等因素所诱导,表达活性诱生型一氧化氮合酶（ｉｎｄｕｃｉｂｌｅｎｉｔｒｉｃｏｘｉｄｅｓｙｎｔｈａｓｅ,ｉＮＯＳ）并持续催化大量的ＮＯ。后者通过干扰细胞正常代谢、形成自由基等途径产生强大的神经细胞毒性。脑缺血损伤后２４ｈ应用选择性的ｉＮＯＳ抑制剂仍能明显减少实验动物的脑缺血梗死区面积,提示ｉＮＯＳ选择性抑制剂有望成为极有价值的脑缺血损伤治疗药物。     

Neuropeptide Y and its receptor analogs differentially modulate the immunoreactivity for neuronal or endothelial nitric oxide synthase in the rat brain following focal ischemia with reperfusion

Summary An intracerebroventricular (icv) injection of neuropeptide Y (NPY) or [Leu³¹, Pro³⁴]-NPY (non-Y2 receptor agonist) given during middle cerebral artery occlusion (MCAO) increases the infarct volume and nitric oxide (NO) overproduction in the rat brain. An icv injection of NPY3-36 (non-Y1 receptor agonist) has no effects, while BIBP3226 (selective Y1 receptor antagonist) reduces the infarct volume and NO overproduction. This study examined the effects of NPY or its receptor analog on the immunoreactivity (ir) for three isoforms of NO synthase (NOS) following 1h of MCAO and 3h of reperfusion. Focal ischemia/reperfusion led to increased ir for neuronal NOS (nNOS) within the ipsilateral caudate putamen and insular cortex. NPY or [Leu³¹, Pro³⁴]-NPY enhanced but BIBP3226 suppressed such increase in the nNOS-ir. Focal ischemia/reperfusion also led to an ipsilateral increase in extent and/or intensity of the ir for endothelial NOS (eNOS) in the caudate putamen and/or parietal cortex. NPY or [Leu³¹, Pro³⁴]-NPY suppressed but BIBP3226 enhanced such change in the eNOS-ir. NPY3-36 did not consistently influence the nNOS-ir or eNOS-ir following MCAO. Specific ir for inducible NOS was undetectable. These opposing effects of NPY-Y1 receptor activation or inhibition on nNOS and eNOS may lead to harmful or beneficial consequences following ischemia/reperfusion.