Methylene blue blocks cGMP production and disrupts directed migration of microglia to nerve lesions in the leech CNS

Migration and accumulation of microglial cells at sites of injury are important for nerve repair. Recent studies on the leech central nervous system (CNS), in which synapse regeneration is successful, have shown that nitric oxide (NO) generated immediately after injury by endothelial nitric oxide synthase (eNOS) stops migrating microglia at the lesion. The present study obtained results indicating that NO may act earlier, on microglia migration, and aimed to determine mechanisms underlying NO's effects. Injury induced cGMP immunoreactivity at the lesion in a pattern similar to that of eNOS activity, immunoreactivity, and microglial cell accumulation, which were all focused there. The soluble guanylate cyclase (sGC) inhibitor methylene blue (MB) at 60 microM abolished cGMP immunoreactivity at lesions and blocked microglial cell migration and accumulation without interfering with axon conduction. Time-lapse video microscopy of microglia in living nerve cords showed MB did not reduce cell movement but reduced directed movement, with significantly more cells moving away from the lesion or reversing direction and fewer cells moving toward the lesion. The results indicate a new role for NO, directing the microglial cell migration as well as stopping it, and show that NO's action may be mediated by cGMP.     

ATP and NO dually control migration of microglia to nerve lesions

Microglia migrate rapidly to lesions in the central nervous system (CNS), presumably in response to chemoattractants including ATP released directly or indirectly by the injury. Previous work on the leech has shown that nitric oxide (NO), generated at the lesion, is both a stop signal for microglia at the lesion and crucial for their directed migration from hundreds of micrometers away within the nerve cord, perhaps mediated by a soluble guanylate cyclase (sGC). In this study, application of 100 μM ATP caused maximal movement of microglia in leech nerve cords. The nucleotides ADP, UTP, and the nonhydrolyzable ATP analog AMP‐PNP (adenyl‐5′‐yl imidodiphosphate) also caused movement, whereas AMP, cAMP, and adenosine were without effect. Both movement in ATP and migration after injury were slowed by 50 μM reactive blue 2 (RB2), an antagonist of purinergic receptors, without influencing the direction of movement. This contrasted with the effect of the NO scavenger cPTIO (2‐(4‐carboxyphenyl)‐4,4,5,5‐teramethylimidazoline‐oxyl‐3‐oxide), which misdirected movement when applied at 1 mM. The cPTIO reduced cGMP immunoreactivity without changing the immunoreactivity of eNOS (endothelial nitric oxide synthase), which accompanies increased NOS activity after nerve cord injury, consistent with involvement of sGC. Moreover, the sGC‐specific inhibitor LY83583 applied at 50 μM had a similar effect, in agreement with previous results with methylene blue. Taken together, the experiments support the hypothesis that ATP released directly or indirectly by injury activates microglia to move, whereas NO that activates sGC directs migration of microglia to CNS lesions. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Repair and Regeneration of Functional Synaptic Connections: Cellular and Molecular Interactions in the Leech

A major problem for neuroscience has been to find a means to achieve reliable regeneration of synaptic connections following injury to the adult CNS. This problem has been solved by the leech, where identified neurons reconnect precisely with their usual targets following axotomy, re-establishing in the adult the connections formed during embryonic development.It cannot be assumed that once axons regenerate specific synapses, function will be restored. Recent work on the leech has shown following regeneration of the synapse between S-interneurons, which are required for sensitization of reflexive shortening, a form of non-associative learning, the capacity for sensitization is delayed.The steps in repair of synaptic connections in the leech are reviewed, with the aim of understanding general mechanisms that promote successful repair. New results are presented regarding the signals that regulate microglial migration to lesions, a first step in the repair process. In particular, microglia up to 900 m from the lesion respond within minutes by moving rapidly toward the injury, controlled in part by nitric oxide (NO), which is generated immediately at the lesion and acts via a soluble guanylate cyclase (sGC). The cGMP produced remains elevated for hours after injury. The relationship of microglial migration to axon outgrowth is discussed.     

Effect of chronic ethanol administration on hepatic eNOS activity and its association with caveolin-1 and calmodulin in female rats

Although chronic and excessive alcohol consumption is associated with liver disease, the mechanism of alcoholic liver injury is still not clear. Whether reduced hepatic production of nitric oxide, which is evident in models of liver injury, is associated with alcohol-induced liver injury has not been investigated. We measured nitric oxide synthase (NOS) activity in the liver of pair-fed rats receiving liquid diet with or without alcohol [3% (vol/vol)] for 12 wk. Compared with control rats, hepatic NOS activity was significantly reduced in alcohol-treated rats along with the evidence of liver injury. Interestingly, there was no difference in the hepatic expression of endothelial NOS (eNOS) between ethanol-fed and pair-fed rats. We then tested the hypothesis that an imbalance between the binding of eNOS with inhibitory and stimulatory proteins may underlie the reduced activity of eNOS because eNOS catalytic activity is regulated partly through dynamic interactions with the inhibitory protein caveolin-1 and the stimulatory protein calmodulin. We found that hepatic caveolin-1 was markedly increased in alcohol-treated rats compared with control rats, whereas calmodulin remained unaltered. The binding of caveolin-1 and calmodulin with eNOS was increased and decreased, respectively, in alcohol-treated rats. Our results suggest that chronic alcohol intake attenuates hepatic eNOS activity by increasing the expression of the inhibitory protein caveolin-1 and enhancing its binding with eNOS.     

NO and NOS isoforms in the development of apoptosis in renal ischemia/reperfusion

Nitric oxide (NO) and the expression of endothelial (eNOS) and inducible (iNOS) isoforms of nitric oxide synthase (NOS) are recognized as important mediators of physiological and pathological processes of renal ischemia/reperfusion (I/R) injury, but little is known about their role in apoptosis. The ability of the eNOS/NO system to regulate the iNOS/NO system and thus promote apoptosis was assessed during experimental renal I/R. Renal caspase-3 activity and the number of TUNEL-positive cells increased with I/R, but decreased when NOS/NO systems were blocked with L-NIO (eNOS), 1400W (iNOS), and N-nitro-l-arginine methyl ester (L-NAME; a nonselective NOS inhibitor). I/R increased renal eNOS and iNOS expression as well as NO production. The NO increase was eNOS- and iNOS-dependent. Blockage of NOS/NO systems with L-NIO or L-NAME also resulted in a lower renal expression of iNOS and iNOS mRNA; in contrast, eNOS expression was not affected by iNOS-specific blockage. In conclusion, two pathways define the role of NOS/NO systems in the development of apoptosis during experimental renal I/R: a direct route, through eNOS overexpression and NO production, and an indirect route, through expression/activation of the iNOS/NO system, induced by eNOS.     

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish ( Carassius auratus ). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5–40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5–40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo . None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.     

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.     

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.     

机械刺激诱导的烟草悬浮细胞一氧化氮产生途径及其与C矿和钙调素的关系

通过提高摇床转速对烟草细胞施加机械刺激（Ms）可诱导其胞内一氧化氮（No）的快速产生和一氧化氮合酶（Nos）活性的提高,这种MS诱导的NO产生可被N0清除剂cPTIO和NOS抑制剂L-NMMA显著抑制。此外,Ca2＋螯合剂EGTA、质膜Ca＋通道阻断剂La3＋、胞内Ca2＋通道拮抗剂钌红,以及钙调素抑制剂CPZ和TFP预处理均不同程度地抑制了机械刺激诱导的烟草细胞NO的产生,而机械刺激过程中钙调素活性显著上升并与NOS活性和NO含量的变化相一致。这些结果暗示着（类）Nos酶催化的精氨酸依赖途径可能是机械刺激诱发烟草细胞NO产生的主要途径,Ca2＋／CAM可能通过调节（类）NOS活性来调控No的产生。     

Estrogen modulation of eNOS activity and its association with caveolin-3 and calmodulin in rat hearts

Previous studies have shown that estrogen modulation of endothelial nitric oxide (NO) synthase (eNOS) may confer protection against heart disease. Here, we demonstrate an association between reductions in baroreflex-mediated bradycardia and in cardiac NOS activity in ovariectomized (Ovx) rats compared with controls. The latter resulted, at least in part, from a reduction in cardiac eNOS protein. eNOS-derived NO and its biological effects are determined by the levels of eNOS protein and by eNOS catalytic activity; the latter is regulated partly through the dynamic interaction with an inhibitory protein (caveolin) and a stimulatory protein (calmodulin). The association of eNOS immunoprecipitated with caveolin-3 and calmodulin was examined. Caveolin-3 and calmodulin binding with eNOS was increased and decreased, respectively, in Ovx rats. 17 beta-Estradiol replacement restored, to within normal levels, the baroreflex-mediated bradycardic responses along with eNOS activity, eNOS expression, and the association of eNOS with caveolin-3 and calmodulin. Our findings may help to elucidate the molecular mechanism underlying the favorable effects of estrogen on cardiac responses to baroreflex activation.     

Nitric oxide regulates axonal regeneration in an insect embryonic CNS

In higher vertebrates, the central nervous system (CNS) is unable to regenerate after injury, at least partially because of growth-inhibiting factors. Invertebrates lack many of these negative regulators, allowing us to study the positive factors in isolation. One possible molecular player in neuronal regeneration is the nitric oxide (NO)-cyclic guanosine-monophosphate (cGMP) transduction pathway which is known to regulate axonal growth and neural migration. Here, we present an experimental model in which we study the effect of NO on CNS regeneration in flat-fillet locust embryo preparations in culture after crushing the connectives between abdominal ganglia. Using whole-mount immunofluorescence, we examine the morphology of identified serotonergic neurons, which send a total of four axons through these connectives. After injury, these axons grow out again and reach the neighboring ganglion within 4 days in culture. We quantify the number of regenerating axons within this period and test the effect of drugs that interfere with NO action. Application of exogenous NO or cGMP promotes axonal regeneration, whereas scavenging NO or inhibition of soluble guanylyl cyclase delays regeneration, an effect that can be rescued by application of external cGMP. NO-induced cGMP immunostaining confirms the serotonergic neurons as direct targets for NO. Putative sources of NO are resolved using the NADPH-diaphorase technique. We conclude that NO/cGMP promotes outgrowth of regenerating axons in an insect embryo, and that such embryo-culture systems are useful tools for studying CNS regeneration.     

Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation

Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179D eNOS) increases nitric oxide (NO) production constitutively, in the absence of agonist challenge. Here, we examine the potential mechanism of how aspartate at 1179 increases eNOS activity using purified proteins. Examination of NO production and cytochrome c reduction resulted in no substantial changes in the K(m)/EC(50) for L-arginine, calmodulin, and calcium, whereas there was a 2-fold increase in the rate of NO production for S1179D and a 2-4-fold increase in reductase activity (based on cytochrome c reduction). The observed increase in activity for both assays of NOS function indicates that a faster rate of electron flux through the reductase domain is likely the rate-limiting step in NO formation from eNOS. In addition, S1179D eNOS did show an increased resistance to inactivation by EGTA compared with wild type eNOS. These results suggest that a negative charge imposed at serine 1179, either by phosphorylation or by replacement with aspartate, increases eNOS catalytic activity by increasing electron flux at the reductase domain and by reducing calmodulin dissociation from activated eNOS when calcium levels are low.     

一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS

Recent studies indicated that there is a distinct mitochondrial nitric oxide synthase (mtNOS) enzyme, which may be identical to the other known NOS isoforms. We investigated the possible involvement of the endothelial, the neuronal, and the inducible NOS isoforms (eNOS, nNOS, iNOS, respectively) in mitochondrial NO production. Mouse liver mitochondria were prepared by Percoll gradient purification from wild-type and NOS knockout animals. NOS activity was measured by the arginine conversion assay, NO production of live mitochondria was visualized by the fluorescent probe DAF-FM with confocal microscopy and measured with flow cytometry. Western blotting or immunoprecipitation was performed with 12 different anti-NOS antibodies. Mitochondrial NOS was purified by arginine, 2,5 ADP and calmodulin affinity columns. We observed NO production and NOS activity in mitochondria, which was not attenuated by classic NOS inhibitors. We also detected low amounts of eNOS protein in the mitochondria, however, NO production and NOS activity were intact in eNOS knockout animals. Neither nNOS nor iNOS were present in the mitochondria. Furthermore, we could not find mitochondrial targeting signals in the sequences of either NOS proteins. Taken together, the presented data do not support the hypothesis that any of the known NOS enzymes are present in the mitochondria in physiologically relevant levels.     

Nitric oxide mediates the interleukin-1beta- and nicotine-induced hypothalamic-pituitary-adrenocortical response during social stress.

We investigated the role of nitric oxide (NO) in the interleukin 1beta (IL-1beta) and nicotine induced hypothalamic-pituitary-adrenal axis (HPA) responses, and a possible significance of CRH and vasopressin in these responses under basal and social stress conditions. Male Wistar rats were crowded in cages for 7 days prior to treatment. All compounds were injected i.p., nitric oxide synthase (NOS) inhibitors, alpha-helical CRH antagonist and vasopressin receptor antagonist 15 min before IL-1beta or nicotine. Identical treatment received control non-stressed rats. Plasma ACTH and serum corticosterone levels were measured 1 h after IL-1beta or nicotine injection. L-NAME (2 mg/kg), a general nitric oxide synthase (NOS) inhibitor, considerably reduced the ACTH and corticosterone response to IL-1beta (0.5 microg/rat) the same extent in control and crowded rats. CRH antagonist almost abolished the nicotine-induced hormone responses and vasopressin antagonist reduced ACTH secretion. Constitutive endothelial eNOS and neuronal nNOS inhibitors substantially enhanced the nicotine-elicited ACTH and corticosterone response and inducible iNOS inhibitor, aminoguanidine, did not affect these responses in non-stressed rats. Social stress significantly attenuated the nicotine-induced ACTH and corticosterone response. In crowded rats L-NAME significantly deepened the stress-induced decrease in the nicotine-evoked ACTH and corticosterone response. In stressed rats neuronal NOS antagonist did not alter the nicotine-evoked hormone responses and inducible NOS inhibitor partly reversed the stress-induced decrease in ACTH response to nicotine. These results indicate that NO plays crucial role in the IL-1beta-induced HPA axis stimulation under basal and social stress conditions. CRH and vasopressin of the hypothalamic paraventricular nucleus may be involved in the nicotine induced alterations of HPA axis activity. NO generated by eNOS, but not nNOS, is involved in the stress-induced alterations of HPA axis activity by nicotine.     

肢体缺血预适应的肝保护作用与一氧化氮／内皮素-1系统关系的研究

目的：观察肢体缺血／再灌注（I／R）后一氧化氮／内皮素-1（NO／ET-1）失衡与肝损伤的关系以及缺血预适应（1pc）对NO／ET-1系统的调节作用。方法：实验用雄性Wistar大鼠18只,随机分为3组（n=6）：对照组（control）、缺血／再灌注组（I／R）和缺血预适应组（IPC＋I／R）,分别测定血浆谷草转氨酶（ALT）、谷丙转氨酶（AST）;血浆和肝组织一氧化氮（NO）、内皮素-1（ET-I）的含量变化,一氧化氮／内皮素-1（NO／ET-1）比值及肝组织的总一氧化氮合酶（tNOS）、诱导型一氧化氮合酶（iNOS）、结构型一氧化氮合酶（cNOS）的水平;免疫组化法检测肝组织的诱导型一氧化氮舍酶（iNOS）、内皮型一氧化氮合酶（eNOS）的表达;HE染色,在光学显微镜下观察肝组织的形态学改变。结果：发现肢体再灌注期血浆和肝组织NO、ET-1均明显增加,而NO／ET-1的比值却明显降低,同时血浆ALT、AST升高,光学显微镜下肝细胞、内皮细胞肿胀,肝细胞变性及肝窦淤血,炎性细胞浸润,肝损伤加重,肢体I／R后肝组织iNOS的表达增强,而eNOS（主要为eNOS）的表达减少,伴有总NOS活性增强。说明肢体缺血再灌注后肝组织内皮源的NO产生减少,而非内皮源的NO产生增多;IPC减轻了肢体I／R后引起的NO／ET-1失衡。结论：肢体I／R后肝组织损伤与NO／ET-1失衡有关,IPC对肢体I／R继发的肝组织损伤的保护作用可能是通过对NO／ET-1系统的调节作用而介导的,此时内皮源的NO产生增加,非内皮源的NO产生减少。     

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.     

尾加压素对新生大鼠心肌细胞一氧化氮合成的影响

应用半定量逆转录－多聚酶链反应法,观察尾加压素（urotensin Ⅱ,UⅡ）对培养的新生SD大鼠心肌细胞内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS)mRNA表达的影响,并测定UⅡ对心肌细胞内一氧化氮合酶（nitric oxide synthase,NOS)活性和一氧化氮（nitric oxide,NO)释放的影响。结果显示：UⅡ抑制培养的新生大鼠心肌细胞eNOS mRNA表达、抑制NOS的活性及NO释放;0.1μmol/L浓度的UⅡ呈时间依赖性抑制心肌细胞NOS的活性及NO生成。上述实验结果提示UⅡ的心血管作用可能与NO合成系统有关。     

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.