Extracellular superoxide dismutase overexpression protects against aging-induced cognitive impairment in mice

Extracellular superoxide dismutase (EC-SOD) controls the availability of extracellular superoxide and appears to play a role in controlling oxidative stress and intercellular signaling. Whether EC-SOD overexpression would help or hinder neurobehavioral function appears to depend on the age of the individual. In young adult mice, we have found that EC-SOD overexpression can interfere with learning on the radial-arm maze, possibly by reducing control over nitric oxide neurotransmission. In aged mice, we found, in the current study, that EC-SOD overexpression greatly improves learning on the radial-arm maze. Control (N = 17) and EC-SOD overexpressing mice (N = 13) acquired the 8-arm radial maze over 21 sessions of training. The EC-SOD overexpressing mice had significantly better choice accuracy than the control mice (p < 0.005). The EC-SOD overexpressing mice averaged 6.34 ± 0.22 correct arm entries before an error (entries to repeat) during the acquisition phase, while the control mice averaged 5.18 ± 0.22 entries to repeat. EC-SOD genotype did not cause a main effect on response latency. The advantage held by the EC-SOD overexpressing mice persisted during the eight-session post-acquisition phase of testing (p < 0.01). When there was a shift from high to low levels of motivation by reducing the period of food restriction before testing, the EC-SOD overexpression-induced improvement was reduced slightly, but it was still significant compared with the wild-type controls (p < 0.025). Then, after 4 months of no testing, the mice were tested for retention and reacquisition of performance on the radial-arm maze. The EC-SOD overexpressing mice maintained their significantly better choice accuracy (p < 0.05). Enhancement of EC-SOD activity appears to improve learning and memory performance, specifically in aging mice. EC-SOD mimetic treatment during the course of aging may hold promise for aging-induced cognitive impairment.     

Molecular Overexpression of Extracellular Superoxide Dismutase Increases the Dependency of Learning and Memory Performance on Motivational State

Extracellular superoxide dismutase (EC-SOD) controls the availability of extracellular superoxide and appears to play a role in controlling intercellular signaling. In this role EC-SOD can have potent effects on neurobehavioral function. In previous studies, we have found that either over- or under-expression of EC-SOD in mice significantly impairs spatial learning on the radial-arm maze. In the current study, the neurobehavioral nature of the EC-SOD role in cognitive function was determined. EC-SOD overexpression altered the relationship between both learning and memory with motivational state. Mice were tested in the radial-arm maze under a high motivational state (22–24 hours of food restriction) or a low motivational state (4–6 hours of food restriction). Under a high motivational state, the EC-SOD overexpressing mice were able to learn in the radial-arm maze, albeit at a slightly lower rate than wild-type controls. This contrasts with the failure to learn by EC-SOD overexpressing mice in our previous study conducted with the low motivational state. The change in motivational state did not significantly alter the learning rate of controls. Similarly, during postacquisition memory phase of testing, the EC-SOD overexpressing mice were significantly worse than controls when tested in a low motivational state but not under a high motivation state. As with learning, motivational state did not significantly affect memory performance in controls. This study shows that mice with EC-SOD overexpression are not incapable of learning and memory in the radial-arm maze, but that the mechanisms which allow control animals to perform this task well under low motivational states are deficient in the mice with EC-SOD overexpression.     

Memory Decline of Aging Reduced by Extracellular Superoxide Dismutase Overexpression

Extracellular superoxide dismutase (EC-SOD) plays an important role in controlling oxidative stress as well as intercellular signaling. In the current study, we tested the effect of EC-SOD overexpression over the lifespan of a set of mice and their wild-type controls to determine the time scale over which EC-SOD overexpression might attenuate aging-induced memory impairment. Mice with overexpression of EC-SOD and wild-type controls were initially trained on the radial-arm maze as young adults (3–5 months) and then retrained during middle age (12–14 months) and retested in old age at 27 and 30 months. There was little EC-SOD effect during the young adult middle age periods. EC-SOD overexpression prevented the decline in choice accuracy when the mice were 27–30 months of age. The EC-SOD overexpressing mice maintained their performance, while the wild-type mice declined to naïve levels of performance by 30 months of age. Enhancement of EC-SOD activity appears to improve memory performance specifically in aging mice. EC-SOD mimetic treatment during the course of aging may hold promise for aging-induced cognitive impairment.     

Modification of intracellular level of free radicals and apoptosis in cultured human endotheliocytes and carcinoma cells

The intracellular levels of superoxide \textO₂^- {\text{O}}_2^{-} and nitric oxide NO were directly measured under intravital conditions in cultured human endotheliocytes ECV 304 and carcinoma cells HeLa G-63. Comparative analysis of changes in the intracellular levels of superoxide and NO induced by ascorbic acid revealed a negative correlation between NO and \textO₂^- {\text{O}}_2^{-} levels, whose strength depended on concentration of the acid. In pharmacological concentrations, ascorbic acid induced apoptotic death of carcinoma cells, but did not trigger apoptosis of endotheliocytes. The study demonstrated a possible way of cytotoxic action of ascorbic acid in pharmacological concentrations.     

Nitric Oxide, Superoxide, and Peroxynitrite Effects on the Insulin Secretion and Viability of βTC3 Cells

The onset of insulin-dependent diabetes mellitus (IDDM) is often associated with the infiltration of pancreatic cells by macrophages. Upon activation, macrophages release nitric oxide (NO) and superoxide (O ₂ ^- ). These species or their reactive intermediates can be cytoxic, mutagenic, or carcinogenic. Previous studies have reported both positive and negative effects of extracellularly generated NO on insulin secretion and viability of pancreatic cells. Inherent problems of several previous studies assessing the effects of NO on insulin secretion include unsteady state NO concentration exposures and the generation of other potentially damaging species. In this study, these problems were eliminated by using a modified experimental system in which NO delivery was achieved via diffusion across a gas-permeable tube and O ₂ ^- delivery was maintained using an enzymatic reaction. The delivery rates were constant, leading to steady state concentrations of NO and O ₂ ^- in the experimental system. Based on reaction kinetics, a model was developed to predict NO, O ₂ ^- and peroxynitrite ONOO^- concentrations during the experiment. This study showed that NO, O ₂ ^- and ONOO^- at predicted concentrations as high as 2.8 M, 0.25 M, and 0.1 nM, respectively, do not affect the insulin secretion rates of TC3 pancreatic cells over short times. © 2000 Biomedical Engineering Society. PAC00: 8717-d     

Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome

Microparticles are membrane vesicles that are released during cell activation and apoptosis. Elevated levels of microparticles occur in many cardiovascular diseases; therefore, we characterized circulating microparticles from both metabolic syndrome (MS) patients and healthy patients. We evaluated microparticle effects on endothelial function; however, links between circulating microparticles and endothelial dysfunction have not yet been demonstrated. Circulating microparticles and their cellular origins were examined by flow cytometry of blood samples from patients and healthy subjects. Microparticles were used either to treat human endothelial cells in vitro or to assess endothelium function in mice after intravenous injection. MS patients had increased circulating levels of microparticles compared with healthy patients, including microparticles from platelet, endothelial, erythrocyte, and procoagulant origins. In vitro treatment of endothelial cells with microparticles from MS patients reduced both nitric oxide (NO) and superoxide anion production, resulting in protein tyrosine nitration. These effects were associated with enhanced phosphorylation of endothelial NO synthase at the site of inhibition. The reduction of O₂⁻ was linked to both reduced expression of p47^phox of NADPH oxidase and overexpression of extracellular superoxide dismutase. The decrease in NO production was triggered by nonplatelet-derived microparticles. In vivo injection of MS microparticles into mice impaired endothelium-dependent relaxation and decreased endothelial NO synthase expression. These data provide evidence that circulating microparticles from MS patients influence endothelial dysfunction.     

Overexpression of Alpha2C-adrenoceptors impairs water maze navigation

We investigated the role of overexpression of alpha2C-adrenoceptors in water maze navigation in mice transgenically manipulated to have a threefold overexpression of the alpha2C-adrenoreceptors. Alpha2C-adrenoreceptors overexpressing mice swam more in the peripheral annulus of the pool and did not find the hidden escape platform as well as the wild type control mice. A subtype-nonselective alpha2-adrenoreceptor antagonist, atipamezole (ATI, 1000 μg/kg, s.c.), fully reversed the deficit in platform finding and search strategy in overexpressing mice. Noradrenaline depletion (−95%) induced by N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) did not impair platform finding of wild type or overexpressing mice. The DSP-4 lesion slightly increased swimming in the peripheral annulus in wild type mice, but not in overexpressing mice. The DSP-4 lesion produced a dissociable effect on the action of atipamezole to improve platform finding and search strategy in overexpressing mice: atipamezole did not alleviate the platform finding deficit in DSP-4 lesioned overexpressing mice, but normalized their abnormal search strategy.These results suggest that the abnormal search pattern and deficit in the accuracy of platform finding are mediated by constitutive activity of overexpressed alpha2C-adrenoreceptors.     

人补体C5b~9复合物对大鼠肾系膜 细胞一氧化氮生成的影响

目的:探讨人补体 C_5b~9复合物对大鼠肾小球系膜细胞(MC)合成一氧化氮(NO)的影响。方法:首先提取人补体C_5b~9复合物,同时培养出大鼠肾小球MC。然后用C_5b~9复合物刺激MC,观察刺激后6 h、24 h、和48 h培养上清液中NO代谢产物-硝酸根(NO₃^-)和亚硝酸根(NO₂^-)量的变化,并测定刺激后24 h、48 h时MC内环鸟苷酸(cGMP)的水平。结果:用人C_5b~9复合物刺激大鼠MC后可致培养上清中NO₃^-/NO₂^-含量增加,细胞内cGMP水平上升。NO合成增多能够被NO合酶(NOS)抑制剂—─-N^G-硝基-L-精氨酸甲基酯(L-NAME)所抑制。结论:人C_5b~9复合物能够增加大鼠肾MC 的NO合成。     

荷S180小鼠血浆ET、NO水平与瘤重变化及相关研究

目的和方法:复制荷S₁₈₀小鼠模型,采用放射免疫均相竞争法和硝酸还原酶法测定不同病期(荷瘤5d、10d和15d)的小鼠血浆中ET和NO₂^-/NO ₃^-水平,称取瘤重,在观察三者的变化规律基础上,探讨不同病期荷S₁₈₀小鼠血浆ET、NO水平的变化,及其与肿瘤发展的关系。结果:不同病期荷瘤鼠血浆ET、NO₂^-/NO₃^-均显著高于对照组(P＜0.05)。随病期延长,瘤重及NO₂^-/NO₃^-水平逐渐升高(P＜0.05),ET水平也有上升趋势。NO₂^-/NO₃^-水平与瘤重呈正相关(r=0.955,P＜0.05)。NO₂^-/NO₃^-与ET的比值呈先下降后上升的变化。结论:ET、NO与S₁₈₀肉瘤的发生发展有关,二者在肿瘤发展中可能有相互促进作用。     

Loss of tau elicits axonal degeneration in a mouse model of Alzheimer's disease

A central issue in the pathogenesis of tauopathy is the question of how tau protein dysfunction leads to neurodegeneration. We have previously demonstrated that the absence of tau protein is associated with destabilization of microtubules and impaired neurite outgrowth (13 and 64). We now hypothesize that the absence of functional tau protein may render the central nervous system more vulnerable to secondary insults such as the overexpression of mutated beta amyloid precursor protein (APP) and traumatic brain injury. We therefore crossed tau knockout mice (Dawson et al., 2001) to mice overexpressing a mutated human APP (APP_670,671, A_sw) (Hsiao et al., 1996) and created a mouse model (A_sw/mTau^−/−) that provides evidence that the loss of tau function causes degeneration of neuronal processes. The overexpression of APP_670,671 in tau knockout mice, elicits the extensive formation of axonal spheroids. While spheroids are only found associated with Aβ plaques in mice expressing APP_670,671 on an endogenous mouse tau background (Irizarry et al., 1997), A_sw/mTau^−/− mice have spheroids not only surrounding Aβ plaques but also in white matter tracks and in the neuropil. Plaque associated and neuropil dystrophic neurites and spheroids are prominent features of Alzheimer's disease (52, 74 and 72), and our current data suggests that loss of tau function may lead to neurodegeneration.     

Extracellular superoxide dismutase in macrophages augments bacterial killing by promoting phagocytosis

Extracellular superoxide dismutase (EC-SOD) is abundant in the lung and limits inflammation and injury in response to many pulmonary insults. To test the hypothesis that EC-SOD has an important role in bacterial infections, wild-type and EC-SOD knockout (KO) mice were infected with Escherichia coli to induce pneumonia. Although mice in the EC-SOD KO group demonstrated greater pulmonary inflammation than did wild-type mice, there was less clearance of bacteria from their lungs after infection. Macrophages and neutrophils express EC-SOD; however, its function and subcellular localization in these inflammatory cells is unclear. In the present study, immunogold electron microscopy revealed EC-SOD in membrane-bound vesicles of phagocytes. These findings suggest that inflammatory cell EC-SOD may have a role in antibacterial defense. To test this hypothesis, phagocytes from wild-type and EC-SOD KO mice were evaluated. Although macrophages lacking EC-SOD produced more reactive oxygen species than did cells expressing EC-SOD after stimulation, they demonstrated significantly impaired phagocytosis and killing of bacteria. Overall, this suggests that EC-SOD facilitates clearance of bacteria and limits inflammation in response to infection by promoting bacterial phagocytosis.     

Hyperoxic Vasoconstriction in the Brain Is Mediated by Inactivation of Nitric Oxide by Superoxide Anions

The hypothesis that decreases in brain blood flow during respiration of hyperbaric oxygen result from inactivation of nitric oxide (NO) by superoxide anions (O_{2 –}) is proposed. Changes in brain blood flow were assessed in conscious rats during respiration of atmospheric air or oxygen at a pressure of 4 atm after dismutation of O_{2 –} with superoxide dismutase or suppression of NO synthesis with the NO synthase inhibitor L-NAME. I.v. administration of superoxide dismutase increased brain blood flow in rats breathing air but was ineffective after previous inhibition of NO synthase. Hyperbaric oxygenation at 4 atm induced decreases in brain blood flow, though prior superoxide dismutase prevented hyperoxic vasoconstriction and increased brain blood flow in rats breathing hyperbaric oxygen. The vasodilatory effect of superoxide dismutase in hyperbaric oxygenation was not seen in animals given prior doses of the NO synthase inhibitor. These results provide evidence that one mechanism for hyperoxic vasoconstriction in the brain consists of inactivation of NO by superoxide anions, decreasing its basal vasorelaxing action.     

Gene transfer of extracellular superoxide dismutase to atherosclerotic mice

Clinical and epidemiological studies have provided circumstantial evidence that oxidized low-density lipoprotein (LDL) and antioxidants are involved in the pathogenesis of atherosclerosis. Superoxide dismutases (SODs) have been shown in vitro to protect LDL from deleterious effects of superoxide anions. In the present study, we have used adenoviral gene transfer to determine effect of extracellular SOD (EC-SOD) on atherogenesis in LDL receptor -/- mice. Intravenous administration of EC-SOD adenovirus (2 x 10(9) plaque forming units) into tail vein targeted transgene mainly to liver and induced a 3.5- to sevenfold increase in plasma total SOD activity. EC-SOD was secreted into circulation for 2-3 weeks mostly in a truncated B-form, suggesting that endogenous proteolytic mechanisms control the level and distribution of the enzyme. Therapeutic potential was determined by measuring plasma resistance against copper oxidation and analyzing atherosclerotic lesion areas in aortas of LDL receptor -/- mice. Mice were kept on a cholesterol diet for 10 weeks before gene transfer and 3 or 6 weeks after the gene transfer. Results showed a tendency for a reduction in the overall lesion area after EC-SOD gene transfer as compared with LacZ transduced control mice, but the difference did not reach statistical significance. It is concluded that short-term overexpression of EC-SOD in vivo does not affect atherogenesis in LDL receptor -/- mice.     

Aged SOD overexpressing mice exhibit enhanced spatial memory while lacking hippocampal neurogenesis

The recent finding that hippocampal slices from aged mice overexpressing the gene for superoxide dismutase (SOD) exhibit long-term potentiation (LTP) of reactivity to afferent stimulation that is significantly larger than that produced in aged wild-type (wt) mice has encouraged the exploration of the effects of reactive oxygen species (ROS) on learning in aged mice. In addition, young-adult and aged wt and SOD transgenic mice were used in an attempt to correlate adult neurogenesis with spatial learning. Aged wt and SOD mice exhibited a 90% reduction in doublecortin-labeled new dentate gyrus neurons as compared to young mice, with no significant difference between genotypes. In addition, aged SOD mice exhibited better performance than wt controls in both reference and working memory tasks in a water maze. These findings provide a behavioral measure to demonstrate that excessive production of H2O2 is beneficial in aged mice.     

慢性低氧性肺动脉高压大鼠内源性一氧化氮代谢及其外源性吸入的研究

目的:研究一氧化氮(NO)在大鼠慢性低氧性肺动脉高压发生发展中的代谢变化,观察外源性吸入NO对肺血流动力学的疗效。方法:67只SD雄性成年大鼠,随机分为7组:常氧对照组(n=9),慢性间断性低氧(6h/d,7d/周)1周组(n=7),低氧2周组(n=11),低氧3周组(n=11),L-NAME(NO合成酶抑制剂,30mg/kg,灌饲)处理组(n=10),L-Arg(NO合成前质,10mg/kg,灌饲)处理组(n=9),NO吸入(0.0004%,20min)组(n=10)。插管测肺动脉平均压(MPAP),分离右心室(R)、室间膈+左心室(S+L),计算R/(S+L)(g/g)和R/Wt(Wt:体重,g/kg)。结果:①低氧1周组MPAP显著高于对照组,低氧2周时更高,R/(S+L)和R/Wt也显著高于对照组;②血浆NO₂^-/NO₃^-含量在低氧2周组显著高于对照组,而在低氧3周组显著低于低氧2周组和低氧1周组;低氧1周,血浆ET-1含量显著高于对照组,血浆ET-1含量与MPAP和R/(S+L)均呈显著正相关,r分别为0.43和0.46,P值均＜0.01;③L-NAME组大鼠血浆NO₂^-/NO₃^-含量降低33.2%,R/(S+L)显著增加15.2%,P＜0.05;④L-Arg组大鼠血浆NO₂^-/NO₃^-含量和PAPM无显著改变,但R/(S+L)降低8.7%,P＜0.05;⑤吸入NO,MPAP降低17.8%,P＜0.01。结论:内源性NO在慢性低氧早期(1-2周)继发性增加,但进一步低氧时则减少,血浆ET-1含量的增加在低氧性心肺血管重建中可能具有重要意义,吸入低浓度NO对以肺血管增生和右心室肥大为主要基础的肺动脉高压仍具有明显降低作用。     

一氧化氮在失血性休克再灌注损伤中的作用及牛磺酸的影响

目的:探讨一氧化氮(NO)在失血性休克再灌注损伤中的作用及牛磺酸的影响。方法:新西兰种兔24只随机分为3组(n=8):对照组、休克组、牛磺酸治疗组。采用失血性休克-再灌注损伤模型。连续观察休克前、休克1.5h、再灌注1h、2h、3h时血浆一氧化氮合酶(NOS)活性、一氧化氮代谢产物(NO^-₂／NO^-₃)含量、超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量、乳酸脱氢酶(LDH)活性的动态变化。结果:①休克组再灌注各时限血浆NOS活性、NO^-₂／NO^-₃含量、MDA含量、LDH活性显著高于休克前及休克1.5h;SOD活性显著低于休克前及休克1.5h。②休克组再灌注3h时心、肺组织NOS活性、NO^-₂／NO^-₃含量、MDA含量显著高于对照组;SOD活性显著低于对照组。③牛磺酸(40mg·kg^-1, iv)可减轻再灌注各时限上述指标的变化。④血浆、心肺组织中NO^-₂／NO^-₃含量与MDA含量均呈正相关。结论:NO介导了休克再灌注损伤, 大量释放的NO参与休克再灌注损伤的脂质过氧化反应, 牛磺酸的拮抗作用可能与减少NO的生成、抗脂质过氧化有关。     

一氧化氮对大鼠学习记忆和胆碱能系统的影响

目的:探讨一氧化氮(nitric oxide,NO)在大鼠空间学习和记忆过程中的作用及其对胆碱能受体作用机制。方法:大鼠侧脑室分别注射NO前体左旋精氨酸(L-arginine,L-Arg,L-Arg组)、α7烟碱型乙酰胆碱受体(α7nicotinic acetylcholine receptor,α7nAChR)拮抗剂甲基牛扁亭(methyllycaconitine,MLA,MLA组)、α7nAChR激动剂氯化胆碱(choline chloride,CC组)、一氧化氮合酶抑制剂Nω-硝基-L-精氨酸甲酯(nω-nitro-L-arginine methylester,L-NAME,L-NAME组)以及先注射MLA再注射L-Arg(ML组)、先注射L-NAME再注射氯化胆碱(NC组),并以等量生理盐水(NS组)作为对照。用Y型迷宫刺激器、硝酸还原酶法、免疫组织化学以及Western-Blot等技术分别检测大鼠空间学习和记忆行为能力、大脑皮质和海马NO含量和α7nAChR的表达。结果:与对照组比较,Y迷宫空间学习能力达标次数和24 h后30次测试记忆行为中错误反应次数在L-Arg组和CC组均减少,而在MLA组和L-NAME组均增多;大脑前额叶皮质和海马NO含量和α7nAChR阳性细胞数以及蛋白含量在L-Arg组和CC组均明显增多,而在MLA组和L-NAME组均明显减少。ML组和NC组分别与L-Arg和CC组相比较,大鼠学习和记忆行为能力均明显减弱,并且大脑前额叶皮质和海马NO含量以及α7nAChR的表达均减少。结论:侧脑室应用MLA或L-NAME可减弱L-Arg或氯化胆碱对大鼠空间学习和记忆行为能力的促进作用;NO通过α7nAChR促进大鼠空间学习和记忆能力。     

Free radical profiles in an encapsulated pancreatic cell matrix model

The survival of encapsulated pancreatic cells or islets is often limited because of nutrient deficiency, fibrotic overgrowth, and immune attack. Activated immune cells, such as macrophages, release nitric oxide (NO) and superoxide O ₂ ^- These species or their reactive intermediates, such as peroxynitrite, can be cytotoxic, mutagenic, and/or carcinogenic. The transport of these free radicals to encapsulated pancreatic cells cannot be impeded by the present immunoisolation technology. A model has been developed simulating free radical profiles within an encapsulation matrix due to macrophage immune cells attached to the surface of an encapsulation matrix. The model incorporates the transport and reactions of NO,O ₂ ^- O² and total peroxynitrite (PER). The model predictions of NO, O ₂ ^- and PER concentrations to which pancreatic cells are potentially exposed are in the range of 8–42 M, 0.5–8 nM, and 0.1–0.8 M, respectively, for a 100–500 m radius encapsulation matrix. The results demonstrate that the potential exists for free radical damage of encapsulated pancreatic cells and also demonstrates that additional exposure studies may be necessary for assessing free radical effects on pancreatic cell function. Also, care must be taken in assuming that encapsulated cell systems are completely protected from immunological action. © 2002 Biomedical Engineering Society. PAC2002: 8716Ac, 8239Rt, 8716Uv     

A novel missense mutant inactivates GTP cyclohydrolase I in dopa-responsive dystonia

To clarify the biological function of neurotrophin-3 (NT-3) at the postnatal stage, we created a line of transgenic mice overexpressing NT-3 under the control of the mouse nerve growth factor gene promoter. Transgenic mice showed high-level NT-3 expression in the hippocampus and several tissues. We performed behavioral tests in young-adult (7-months-old) and aged (25-months-old) mice. Although aged non-transgenic mice exhibited spatial learning impairments in the Morris water maze, overexpression of NT-3 protected against these age-dependent spatial learning impairments in mice.     

Oxygen or low concentrations of nitric oxide reverse pulmonary vasoconstriction induced by nitric oxide synthesis inhibition in rabbits

The objective of this study was to investigate the role of nitric oxide and oxygen in the regulation of pulmonary vascular resistance, especially by means of substitution with nitric oxide after inhibition of endogenous nitric oxide formation. In artificially ventilated open-chest rabbits pulmonary vascular resistance at normoxic ventilation (F₁0₂= 21%) was 56±6cmH₂O ml^-1 mim-¹ 1000^-1 (mRU_L). N^w-nitro-L-arginine methyl ester (l-NAME, 30 mg kg^-1), an inhibitor of NO synthase, increased pulmonary vascular resistance to 122±17 mRU_L at normoxic ventilation. In response to l-NAME there was also an increase in mean arterial blood pressure. Exogenous nitric oxide (0.014-9 p.p.m. in the inhaled air) dose-dependently and reversibly counteracted the effect of l-NAME on pulmonary vascular resistance at normoxic ventilation, without affecting systemic blood pressure. In addition, the L-NAME-induced vasoconstriction was critically dependent on oxygen. Thus, during hypoxic ventilation (F₁O₂= 10%) the pulmonary vascular resistance was increased approximately four-fold by the presence of L-NAME (30 mg kg^-1), and increments in F,0₂ (21–100%) dose-dependently and reversibly counteracted the effect of L-NAME on pulmonary vascular resistance. Taken together these findings demonstrate that inhalation of low doses of NO may act as a replacement when endogenous NO synthesis is inhibited, and that pulmonary vasoconstriction induced by NO synthesis inhibition is likely to be the result of interference with oxygen-dependent regulatory mechanisms. Endogenous NO cooperates with oxygen to evoke a vasodilator component of the pulmonary hypoxic pressor response, balancing a hitherto unknown constrictor mechanism.