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The presence of NOS coincides with that of NADPH diaphorase activity; therefore, NADPH diaphorase staining is widely used to detect NOS-containing cells in neural and non-neural tissues. With the appropriate fixation procedure, this method is capable of detecting cells containing any of the NOS isoforms.

Nitric oxide has been recognized for years as a toxic, reactive, free radical gas, but in recent years it has been identified as having functions in a variety of metabolic and signaling pathways. NO is synthesized, by nitric oxide synthase, on demand and diffuses to the site of action where it forms noncovalent and covalent linkages with target molecules. This overview presents the pathway of NO formation and also discusses the functions of NO in the nervous, immune and vascular systems.

Genetically engineered mice with targeted disruption of the neuronal nitric oxide synthase (nNOS) gene established the inhibitory role of nitric oxide (NO) in male impulsive aggressive behavior. This was later confirmed by using selective nNOS inhibitors in male wild-type mice. The molecular mechanisms accounting for the aggressive behavior caused by the lack of neuronally derived NO is not known. Recent studies suggest that central serotonergic neuronal circuits and particularly 5-HT(1A) and 5-HT(1B) receptors play a prominent role in the regulation of aggression. Accordingly, we investigated whether the aggressiveness caused by the lack of nNOS might be because of alterations in serotonergic function. We now demonstrate that the excessive aggressiveness and impulsiveness of nNOS knockout mice is caused by selective decrements in serotonin (5-HT) turnover and deficient 5-HT(1A) and 5-HT(1B) receptor function in brain regions regulating emotion. These results indicate an important role for NO in normal brain 5-HT function and may have significant implications for the treatment of psychiatric disorders characterized by aggressiveness and impulsivity.

Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in response to a variety of physiologic and pathologic stimuli. Although the dynamic regulation of nNOS is well established, the molecular mechanisms by which such diverse stimuli regulate nNOS expression have not yet been identified. We describe experiments demonstrating that Ca(2+) entry through voltage-sensitive Ca(2+) channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca(2+). Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca(2+) response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade of CREB function results in a dramatic loss of nNOS transcription. These findings suggest that nNOS is a Ca(2+)-regulated gene through the interactions of CREB on the CREs within the nNOS exon 2 promoter and that these interactions are likely to be centrally involved in the regulation of nNOS in response to neuronal injury and activity-dependent plasticity.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) damages dopaminergic neurons as seen in Parkinson disease. Here we show that after administration of MPTP to mice, there was a robust gliosis in the substantia nigra pars compacta associated with significant upregulation of inducible nitric oxide synthase (iNOS). These changes preceded or paralleled MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking the iNOS gene were significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that iNOS is important in the MPTP neurotoxic process and indicates that inhibitors of iNOS may provide protective benefit in the treatment of Parkinson disease.

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.

Nitric oxide (NO) is an endogenous gas that functions as a neurotransmitter. Because NO is very labile with a half-life of less than 5 sec, most functional studies of NO have manipulated its synthetic enzyme, NO synthase (NOS). Three isoforms of NOS have been identified: (1) in the endothelial lining of blood vessels (eNOS), (2) an inducible form found in macrophages (iNOS), and (3) in neurons (nNOS). Most pharmacological studies to date have blocked all three isoforms of NOS. Previous studies using such agents have revealed that NO might be necessary for photic entrainment of circadian rhythms; general NOS inhibitors attenuate phase shifts of free-running behavior, light-induced c-fos expression in the suprachiasmatic nucleus (SCN), and phase shifts of neural firing activity in SCN maintained in vitro. To assess the specific role of nNOS in mediating entrainment of circadian rhythms, mice with targeted deletion of the gene encoding the neuronal isoform of NOS (nNOS-/-) were used. Wild-type (WT) and nNOS-/- mice initially were entrained to a 14:10 light:dark (LD) cycle. After 3 weeks, the LD cycle was either phase advanced or phase delayed. After an additional 3 weeks, animals were held in either constant dim light or constant dark. WT and nNOS-/- animals did not differ in their ability to entrain to the LD cycle, phase shift locomotor activity, or free run in constant conditions. Animals held in constant dark were killed after light exposure during either the subjective day or subjective night to assess c-fos induction in the SCN. Light exposure during the subjective night increased c-fos expression in the SCN of both WT and nNOS-/- mice relative to animals killed after light exposure during the subjective day. Taken together, these findings suggest that NO from neurons might not be necessary for photic entrainment.

Background: Fifteen to thirty percent of AIDS patients develop some type of neurologic disorder during the course of their illness and the vast majority of these neurologic disorders will be HIV-associated dementia (HAD). These patients can exhibit varying degrees of severity and rates of progression of HAD. Neuropathologic variables that are associated with the rate of progression of HAD are not known., Materials and Methods: Tissue was collected at autopsy from the Johns Hopkins University HIV Neurology Program. Seventy-one AIDS patients of this prospectively characterized population were followed until death to obtain information on dementia severity and the rate of neurological progression. Immunoblot analysis of immunological nitric oxide synthase (iNOS), HAM56, gp41, p24, gp120, and beta-tubulin was performed and the levels of iNOS, HAM56, gp41, and p24 were normalized to beta-tubulin and analyzed for significance by means of the Kruskal-Wallis test for multiple groups., Results: We have identified unique groups within this spectrum and designated them slow, moderate, and rapid progressors. Slow and moderate progressors' neurological progression occurs over a course of months to years, whereas the rapid progressors' disease shows rapid increases in severity over weeks to months. In the present study we demonstrate that the severity and rate of progression of HAD correlates significantly with levels of the HIV-1 coat protein, gp41, iNOS, and HAM56, a marker of microglial/macrophage activation., Conclusion: The severity and rate of progression of HAD correlates with indices of immune activation as well as levels of iNOS and gp41. There appears to be a threshold effect in which high levels of gp41, iNOS, and immune activation are particularly associated with severe (Memorial Sloan-Kettering score 3 to 4) and rapidly progressive HAD.

Of the individuals with human immunodeficiency virus type 1 (HIV-1) infection, 20-30% will develop the neurological complication of HIV-associated dementia (HAD). The mechanisms underlying HAD are unknown; however, indirect immunologically mediated mechanisms are theorized to play a role. Recently, the HIV-1 coat protein gp41 has been implicated as a major mediator of HAD through induction of neurocytokines and subsequent neuronal cell death. Using primary mixed cortical cultures from neuronal nitric oxide synthase (NOS) null (nNOS-/-) mice and immunological NOS null (iNOS-/-) mice, we establish iNOS-derived NO as a major mediator of gp41 neurotoxicity. Neurotoxicity elicited by gp41 is markedly attenuated in iNOS-/- cultures compared with wild-type and nNOS-/- cultures. The NOS inhibitor L-nitroarginine methyl ester is neuroprotective in wild-type and nNOS-/- cultures, confirming the role of iNOS-derived NO in gp41 neurotoxicity. Confirming that iNOS-/- cultures lack iNOS, gp41 did not induce iNOS in iNOS-/- cultures, but it markedly induced iNOS in wild-type and nNOS-/- cultures. We elucidate the region of gp41 that is critical for iNOS induction and neuronal cell death by monitoring iNOS induction with overlapping peptides spanning gp41. We show that the N-terminal region of gp41, which we designate as the neurotoxic domain, induces iNOS protein activity and iNOS-dependent neurotoxicity at picomolar concentrations in a manner similar to recombinant gp41 protein. Our experiments suggest that gp41 is eliciting the induction of iNOS through potential cell surface receptors or binding sites because the induction of iNOS is dose dependent and saturable and occurs at physiologically relevant concentrations. These data confirm that the induction of iNOS by gp41 and the production of NO are primary mediators of neuronal damage and identify a neurotoxic domain of gp41 that may play an important role in HAD.

A subset of familial cases of amyotrophic lateral sclerosis are linked to missense mutations in copper/zinc superoxide dismutase type 1. Patients with missense mutations in copper/zinc superoxide dismutase type 1 develop a paralytic disease indistinguishable from sporadic amyotrophic lateral sclerosis through an unknown toxic gain of function. Nitric oxide reacts with the superoxide anion to form the strong oxidant, peroxynitrite, which participates in neuronal injury in a variety of model systems. Peroxynitrite is an alternate substrate for copper/zinc superoxide dismutase type 1, causing catalytic nitration of tyrosine residues in other proteins. Mutations in copper/zinc superoxide dismutase type 1 may disrupt the active site of the enzyme and permit greater access of peroxynitrite to copper, leading to increased nitration by peroxynitrite of critical cellular targets. To investigate whether neuronal-derived nitric oxide plays a role in the pathogenesis of familial amyotrophic lateral sclerosis, we examined the effects of three different nitric oxide synthase inhibitors: a non-selective nitric oxide synthase inhibitor, nitro-L-arginine methyl ester; a relatively selective inhibitor of neuronal nitric oxide synthase, 7-nitroindazole; and a novel highly selective neuronal nitric oxide synthase inhibitor, AR-R 17,477, in transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 (Gly-->Ala at position 93; G93A) containing a high transgene copy number and a low transgene copy number. AR-R 17,477, but not nitro-L-arginine methyl ester or 7-nitroindazole, significantly prolonged survival in both the high and low transgene transgenic mice. To determine whether neuronal nitric oxide synthase is involved in the pathogenesis resulting from the familial amyotrophic lateral sclerosis copper/zinc superoxide dismutase type 1 mutation, we produced mice with the copper/zinc superoxide dismutase type 1 mutation which lack the neuronal nitric oxide synthase gene. The transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 on neuronal nitric oxide synthase null background do not live significantly longer than transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1. Western blot analysis indicates the presence of two neuronal nitric oxide synthase-like immunoreactive bands in spinal cord homogenates of the neuronal nitric oxide synthase null mice, and residual neuronal nitric oxide synthase catalytic activity ( > 7%) is detected in the spinal cord of the transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 on neuronal nitric oxide synthase null background. This amount of residual activity probably does not account for lack of protection afforded by the disrupted neuronal nitric oxide synthase gene in the familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 mice. Immunological nitric oxide synthase is not detected in the copper/zinc superoxide dismutase type 1 mutant mice at several different ages, thus excluding immunological nitric oxide synthase as a contributor to the pathogenesis of familial amyotrophic lateral sclerosis. Levels of neuronal nitric oxide synthase as well as Ca2+-dependent nitric oxide synthase catalytic activity in the copper/zinc superoxide dismutase type 1 mutant mice do not differ from wild type mice. Endothelial nitric oxide synthase levels may be decreased in the copper/zinc superoxide dismutase type 1 mutant mice. Together, these results do not support a significant role for neuronal-derived nitric oxide in the pathogenesis of familial amyotrophic lateral sclerosis transgenic mice.

Nitric oxide (NO) synthesized within mammalian sinoatrial cells has been shown to participate in cholinergic control of heart rate (HR). However, it is not known whether NO synthesized within neurons plays a role in HR regulation. HR dynamics were measured in 24 wild-type (WT) mice and 24 mice in which the gene for neuronal NO synthase (nNOS) was absent (nNOS-/- mice). Mean HR and HR variability were compared in subsets of these animals at baseline, after parasympathetic blockade with atropine (0.5 mg/kg i.p.), after beta-adrenergic blockade with propranolol (1 mg/kg i.p.), and after combined autonomic blockade. Other animals underwent pressor challenge with phenylephrine (3 mg/kg i.p.) after beta-adrenergic blockade to test for a baroreflex-mediated cardioinhibitory response. The latter experiments were then repeated after inactivation of inhibitory G proteins with pertussis toxin (PTX) (30 microgram/kg i.p.). At baseline, nNOS-/- mice had higher mean HR (711+/-8 vs. 650+/-8 bpm, P = 0.0004) and lower HR variance (424+/-70 vs. 1,112+/-174 bpm2, P = 0.001) compared with WT mice. In nNOS-/- mice, atropine administration led to a much smaller change in mean HR (-2+/-9 vs. 49+/-5 bpm, P = 0.0008) and in HR variance (64+/-24 vs. -903+/-295 bpm2, P = 0.02) than in WT mice. In contrast, propranolol administration and combined autonomic blockade led to similar changes in mean HR between the two groups. After beta-adrenergic blockade, phenylephrine injection elicited a fall in mean HR and rise in HR variance in WT mice that was partially attenuated after treatment with PTX. The response to pressor challenge in nNOS-/- mice before PTX administration was similar to that in WT mice. However, PTX-treated nNOS-/- mice had a dramatically attenuated response to phenylephrine. These findings suggest that the absence of nNOS activity leads to reduced baseline parasympathetic tone, but does not prevent baroreflex-mediated cardioinhibition unless inhibitory G proteins are also inactivated. Thus, neuronally derived NO and cardiac inhibitory G protein activity serve as parallel pathways to mediate autonomic slowing of heart rate in the mouse.

Background: Nitric oxide (NO) plays an important role in numerous reproductive processes. To date, most studies have assessed the role of NO by using nonspecific pharmacological inhibitors of the precursor to NO, nitric oxide synthase (NOS). These pharmacological NOS inhibitors suppress all isoforms of NOS; thus, the precise contribution of each isoform to female reproductive physiology is unknown. The purpose of this study was to determine the specific role of neuronal NOS (nNOS) in the regulation of ovulation in female mice lacking the gene that encodes for nNOS (nNOS-/-)., Materials and Methods: Ovulation was assessed in wild-type (WT) and nNOS-/- female mice by examining the number of ovarian rupture sites and number of oocytes recovered from the oviducts following mating or exposure to exogenous gonadotropins (i.e., 5 IU pregnant mares serum gonadotropin [PMSG] and 5 IU human chorionic gonadotropin [hCG]). Ovulatory efficiency was determined as the number of ovulated oocytes per number of ovarian rupture sites. To examine whether ovulatory deficits in nNOS-/- mice were due to alternations in central mechanisms, plasma luteinizing hormone (LH) concentrations were assessed in WT and nNOS-/- mice that were challenged with 25 ng of gonadotropin-releasing hormone (GnRH). To determine whether ovulatory deficits in nNOS-/- mice were due to local ovulation processes, nerves innervating the reproductive tract of WT and nNOS-/- females were examined for the presence of nNOS protein., Results: There were substantial fertility deficits in nNOS-/- female mice; the nNOS-/- mice had fewer oocytes in their oviducts following spontaneous and gonadotropin-stimulated ovulation. Pituitary responsiveness to exogenous GnRH challenge was intact in nNOS-/- mice. Dense nNOS protein staining was observed in nerves innervating the reproductive tracts of WT mice., Conclusions: The reproductive deficits in nNOS-/- females are most likely due to alternations in the transfer of oocytes from the ovaries to the oviducts during ovulation. These results suggest that defects in neuronally derived NO production may contribute to female infertility.

In skeletal muscle, neuronal nitric oxide synthase (nNOS) is anchored to the sarcolemma via the dystrophin-glycoprotein complex. When dystrophin is absent, as in Duchenne muscular dystrophy patients and in mdx mice, nNOS is mislocalized to the interior of the muscle fiber where it continues to produce nitric oxide. This has led to the hypothesis that free radical toxicity from mislocalized nNOS may contribute to mdx muscle pathology. To test this hypothesis directly, we generated mice devoid of both nNOS and dystrophin. Overall, the nNOS-dystrophin null mice maintained the dystrophic characteristics of mdx mice. We evaluated the mice for several features of the dystrophic phenotype, including membrane damage and muscle morphology. Removal of nNOS did not alter the extent of sarcolemma damage, which is a hallmark of the dystrophic phenotype. Furthermore, muscle from nNOS-dystrophin null mice maintain the histological features of mdx pathology. Our results demonstrate that relocalization of nNOS to the cytosol does not contribute significantly to mdx pathogenesis.

Neuronal nitric oxide synthase (nNOS) neurons kill adjacent neurons through the action of NMDA-glutamate receptor activation, although they remain relatively resistant to the toxic effects of NMDA and NO. The molecular basis of the resistance of nNOS neurons to toxic insults is unknown. To begin to understand the molecular mechanisms of the resistance of nNOS neurons, we developed a pheochromacytoma-derived cell line (PC12) that is resistant to the toxic effects of NO. We found through serial analysis of gene expression (SAGE) that manganese superoxide dismutase (MnSOD) is enriched in the NO-resistant PC12 cell-derived line (PC12-R). Antisense MnSOD renders PC12-R cells sensitive to NO toxicity and increases the sensitivity to NO in the parental, NO-sensitive PC12 line (PC12-S). Adenoviral transfer of MnSOD protects PC12-S cells against NO toxicity. We extended these studies to cortical cultures and showed that MnSOD is enriched in nNOS neurons and that antisense MnSOD renders nNOS neurons susceptible to NMDA neurotoxicity, although it has little effect on the overall susceptibility of cortical neurons to NMDA toxicity. Overexpression of MnSOD provides dramatic protection against NMDA and NO toxicity in cortical cultures, but not against kainate or AMPA neurotoxicity. Furthermore, nNOS neurons from MnSOD -/- mice are markedly sensitive to NMDA toxicity. Adenoviral transfer of MnSOD to MnSOD-/- cultures restores resistance of nNOS neurons to NMDA toxicity. Thus, MnSOD is a major protective protein that appears to be essential for the resistance of nNOS neurons in cortical cultures to NMDA mediated neurotoxicity.

Nitric oxide (NO), an unconventional and diffusible neurotransmitter, is synthesized by nitric oxide synthase (NOS). NMDA glutamate receptors are potent regulators of NO synthesis. We have used dual-label immunofluorescence and confocal microscopy to examine forebrain neurons in the rat that contain high levels of neuronal NOS (nNOS) for the presence of the NMDAR1 receptor subunit protein and regions of this protein encoded by three alternative spliced segments of the NMDAR1 mRNA: N1, C1, and C2. In the neostriatum, neocortex, and hippocampus, nNOS-labeled neurons exhibit strong NMDAR1 immunoreactivity (-ir). In all three of these regions, nNOS-positive neurons are characterized by the absence of immunoreactivity for the C1 segment of NMDAR1, whereas C1-ir is abundant in most nNOS-negative neurons. In addition, nNOS-ir neurons exhibit selective staining for the alternative C2' terminus of NMDAR1 that is produced when the C2 segment is absent. These results demonstrate directly that neurons with abundant nNOS-ir contain NMDAR1 receptor subunit proteins and that the NMDAR1 isoforms present in these cells differ from those of most other neurons in these regions. The distinct NMDA receptor phenotype of these nNOS-positive neurons is likely to contribute to both the physiological regulation of NO release by glutamate as well as to NO-mediated excitotoxic injury.

Neuronal nitric oxide synthase (nNOS) participate in a variety of physiologic and pathologic processes in the nervous system. nNOS was originally felt to be a constitutively expressed enzyme, but recent observations suggest that its levels are dynamically controlled in response to neuronal development, plasticity and injury. nNOS expression is regulated through alternative promoter usage through alternative mRNA splicing and it is likely that this plays an important role in the inducibility of gene expression in response to extracellular stimuli. Emerging data also suggests that NO may be the key mediator linking activity to gene expression and long-lasting neuronal responses through NO activating p21Ras through redox-sensitive modulation.

Nitric oxide acts as a neural messenger in both the central and peripheral nervous systems. Mice with targeted disruption of the neuronal isoform of nitric oxide synthase (nNOS - / -) are extremely aggressive relative to wild-type (WT) mice. Male nNOS - / - mice exhibit an increase in the number and duration of aggressive encounters compared to WT animals when tested in a variety of paradigms used to test rodent aggression. This inappropriate aggressive behavior has only been observed in male nNOS - /- mice; nNOS - /- females, like female WT mice, exhibit little or no aggression. The present study sought to test the dependence of increased aggressive behavior in nNOS - / - males on testosterone. Intact nNOS - / - males exhibited elevated levels of aggression relative to intact WT males. Castration reduced aggression in both WT and nNOS - /- males to equivalent low levels. Testosterone replacement restored aggression to precastration levels in both genotypes. These data provide evidence that increased aggressive behavior of nNOS - /- mice, like aggression in WT mice, is testosterone-dependent.

Background: Mice with targeted disruption of the gene for the neuronal isoform of nitric oxide synthase (nNOS) display exaggerated aggression. Behavioral studies of mice with targeted gene deletions suffer from the criticism that the gene product is missing not only during the assessment period but also throughout development when critical processes, including activation of compensatory mechanisms, may be affected. To address this criticism, we have assessed aggressive behavior in mice treated with a specific pharmacological inhibitor of nNOS., Materials and Methods: Aggressive behavior, as well as brain citrulline levels, were monitored in adult male mice after treatment with a specific nNOS inhibitor, 7-nitroindazole (7-NI) (50 mg/kg i.p.), which is known to reduce NOS activity in brain homogenates by > 90%. As controls, animals were treated with a related indazole, 3-indazolinone (3-I) (50 mg/kg i.p.) that does not affect nNOS or with on oil vehicle., Results: Mice treated with 7-NI displayed substantially increased aggression as compared with oil- or 3-I-injected animals when tested in two different models of aggression. Drug treatment did not affect nonspecific locomotor activities or body temperature. Immunohistochemical staining for citrulline in the brain revealed a dramatic reduction in 7-NI-treated animals., Conclusions: 7-NI augmented aggression in WT mice to levels displayed by nNOS- mice, strongly implying that nNOS is a major mediator of aggression. NOS inhibitors may have therapeutic roles in inflammatory, cardiovascular, and neurologic diseases. The substantial aggressive behavior soon after administration of an nNOS inhibitor raises concerns about adverse behavioral sequelae of such pharmacological agents.

NMDA neurotoxicity, which is mediated, in part, by formation of nitric oxide (NO) via activation of neuronal NO synthase (nNOS), is modulated by neurotrophins. nNOS expression in rat and mouse primary neuronal cultures grown on a glial feeder layer is significantly less than that of neurons grown on a polyornithine (Poly-O) matrix. Neurotrophins markedly increase the number of nNOS neurons, nNOS protein, and NOS catalytic activity and enhance NMDA neurotoxicity via NO-dependent mechanisms when neurons are grown on glial feeder layers. In contrast, when rat or mouse primary cortical neurons are grown on a Poly-O matrix, neurotrophins have no influence on nNOS neuronal number or NOS catalytic activity and reduce NMDA neurotoxicity. Primary neuronal cultures from mice lacking nNOS grown on a glial feeder layer fail to respond to neurotrophin-mediated enhancement of neurotoxicity. Together, these results indicate that nNOS expression and NMDA NO-mediated neurotoxicity are dependent, in part, on the culture paradigm, and neurotrophins regulate the susceptibility to NMDA neurotoxicity via modulation of nNOS. Furthermore, these results support the idea that NMDA neurotoxicity in culture is critically dependent on the developmental state of the neurons being assessed and suggest that, when cortical neurons are cultured on a glial feeder layer, they do not reach nearly as mature a phenotype as when grown on a Poly-O matrix.

Background and Purpose: Cessation of blood flow to the brain, for even a few minutes, sets in motion a potential reversible cascade of events resulting in neuronal cell death. Oxygen free radicals and oxidants appear to play an important role in central nervous system injury after cerebral ischemia and reperfusion. Recently, divergent roles for the newly identified neuronal messenger molecule and oxygen radical, nitric oxide (NO), have been identified in various models of cerebral ischemia. Because of the chemical and physical properties of NO, the numerous physiological activities it mediates, and the lack of specific agents to modulate the activity of the different isoforms of NO synthase (NOS), reports regarding the role of NO in focal cerebral ischemia have been confounding and often conflicting. Recent advances in pharmacology and the development of transgenic knockout mice specific for the different isoforms of NOS have advanced our knowledge and clarified the role of NO in cerebral ischemia., Methods: Animal models of focal ischemia employ occlusion of nutrient cerebral vessels, most commonly the middle cerebral artery. Primary cortical cultures are exposed to excitotoxic or ischemic conditions, and the activities of NOS isoforms or NO production are evaluated. Transgenic mice lacking expression of either the neuronal isoform of NOS (nNOS), the endothelial isoform of NOS (eNOS), or the immunologic isoform of NOS (iNOS) have been examined in models of excitotoxic injury and ischemia., Results: Excitotoxic or ischemic conditions excessively activate nNOS, resulting in concentrations of NO that are toxic to surrounding neurons. Conversely, NO generated from eNOS is critical in maintaining cerebral blood flow and reducing infarct volume. iNOS, which is not normally present in healthy tissue, is induced shortly after ischemia and contributes to secondary late-phase damage., Conclusions: Pharmacological and genetic approaches have significantly advanced our knowledge regarding the role of NO and the different NOS isoforms in focal cerebral ischemia. nNOS and iNOS play key roles in neurodegeneration, while eNOS plays a prominent role in maintaining cerebral blood flow and preventing neuronal injury.

Idiopathic voiding disorders affect up to 10-15% of men and women. We describe bladder abnormalities in mice with targeted deletion of the gene for neuronal nitric oxide synthase which model the clinical disorders. The mice possess hypertrophic dilated bladders and dysfunctional urinary outlets which do not relax in response to electrical field stimulation or L-arginine. The mice also display increased urinary frequency.

Nitric oxide subserves diverse physiologic roles in the nervous system. NO is produced from at least three different NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS, and immunologic NOS (iNOS). We show that nNOS is the predominant isoform constitutively expressed in glia. NO derived from nNOS in glia inhibits the transcription factor nuclear factor kappaB (NF kappaB) as NOS inhibitors enhance basal NF kappaB activation. Pyrrolidine dithiocarbamate (PDTC) is an inhibitor of NF kappaB in most cells; however, we show that PDTC is also a potent scavenger of NO through formation of mononitrosyl iron complexes with PDTC. In Jurkat cells, a human T-cell lymphoma cell line, tumor necrosis factor-alpha (TNF-alpha) induces NF kappaB activation that is inhibited by PDTC. Contrary to the results in Jurkat cells, PDTC did not inhibit tumor necrosis factor-alpha-induced NF kappaB activation in astrocytes; instead PDTC itself induces NF kappaB activation in astrocytes, and this may be related to scavenging of endogenously produced NO by the PDTC iron complex. In astrocytes PDTC also dramatically induces the NF kappaB-dependent enzyme, iNOS, supporting the physiologic relevance of endogenous NO regulation of NF kappaB. NF kappaB activation in glia from mice lacking nNOS responds more rapidly to PDTC compared with astrocytes from wild-type mice. Our data suggest that nNOS in astrocytes regulates NF kappaB activity and iNOS expression, and indicate a novel regulatory role for nNOS in tonically suppressing central nervous system, NF kappaB-regulated genes.

Indirect mechanisms are implicated in the pathogenesis of the dementia associated with human immunodeficiency virus-type 1 (HIV-1) infection. Proinflammatory molecules such as tumor necrosis factor alpha and eicosanoids are elevated in the central nervous system of patients with HIV-1-related dementia. Nitric oxide (NO) is a potential mediator of neuronal injury, because cytokines may activate the immunologic (type II) isoform of NO synthase (iNOS). The levels of iNOS in severe HIV-1-associated dementia coincided with increased expression of the HIV-1 coat protein gp41. Furthermore, gp41 induced iNOS in primary cultures of mixed rat neuronal and glial cells and killed neurons through a NO-dependent mechanism. Thus, gp41-induced NO formation may contribute to the severe cognitive dysfunction associated with HIV-1 infection.

Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide (.O2-) at lower L-Arg concentrations. By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and .O2- formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the Ca2+ ionophore A23187 triggered NO generation but no .O2- was seen. With cells in L-Arg-free medium, we observed .O2- formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined. .O2- formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells. Activation by A23187 was cytotoxic to L-Arg-depleted, but not to control cells, with marked lactate dehydrogenase release. The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating .O2- and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving .O2-/NO-mediated cellular injury.

Background: Nitric oxide (NO) has been implicated as a mediator of penile erection, because the neuronal isoform of NO synthase (NOS) is localized to the penile innervation and NOS inhibitors selectively block erections. NO can also be formed by two other NOS isoforms derived from distinct genes, inducible NOS (iNOS) and endothelial NOS (eNOS). To clarify the source of NO in penile function, we have examined mice with targeted deletion of the nNOS gene (nNOS- mice)., Materials and Methods: Mating behavior, electrophysiologically induced penile erection, isolated erectile tissue isometric tension, and eNOS localization by immunohistochemistry and Western blot were performed on nNOS- mice and wild-type controls., Results: Both intact animal penile erections and isolated erectile tissue function are maintained in nNOS mice, in agreement with demonstrated normal sexual behaviors, but is stereospecifically blocked by the NOS inhibitor, L-nitroarginine methyl ester (L-NAME). eNOS is abundantly present in endothelium of penile vasculature and sinusoidal endothelium within the corpora cavemosa, with levels that are significantly higher in nNOS- mice than in wild-type controls., Conclusions: eNOS mediates NO-dependent penile erection in nNOS- animals and normal penile erection. These data clarify the role of nitric oxide in penile erection and may have implications for therapeutic agents with selective effects on NOS isoforms.

In addition to its functions as a neuronal messenger molecule, nitric oxide (NO) has also been implicated in playing a major role in ischemic damage and glutamate neurotoxicity. Using primary cortical cultures from transgenic neuronal NO synthase (NOS) null (nNOS-) mice, we definitively establish NO as a mediator of NMDA and hypoxic neurotoxicity. Neurotoxicity elicited by NMDA is markedly attenuated in nNOS- cortical cultures compared with wild-type cultures. The NOS inhibitor nitro-L-arginine is neuroprotective in wild-type but not nNOS-cultures, confirming the role of nNOS-derived NO in glutamate neurotoxicity. Confirming that the nNOS- cultures lack NMDA-stimulated nNOS activity, NMDA did not stimulate the formation of cGMP in nNOS- cultures, but markedly elevates cGMP in wild-type cultures. Both wild-type and nNOS- cultures are sensitive to toxicity induced by NO donors, indicating that pathways stimulated by NO that result in neuronal cell death are still intact in the transgenic mice. Superoxide dismutase is neuroprotective against NMDA and NO neurotoxicity in both wild-type and nNOS- cultures, highlighting the importance of superoxide anion in subsequent neuronal damage. The unknown cellular factors that endow differential resistance to NMDA neurotoxicity and differential susceptibility to quisqualate neurotoxicity remain intact in the nNOS- cultures, because the response of somatostatin-immunopositive neurons in nNOS- cultures to high-dose NMDA and low-dose quisqualate is identical to the response of NOS-immunopositive neurons in the wild-type cultures. There is no difference in susceptibility to kainate neurotoxicity between nNOS- and wild-type cultures and only a modest resistance to quisqualate neurotoxicity, confirming observations that NO-mediated neurotoxicity is associated primarily with activation of the NMDA receptor. The nNOS- cultures are markedly protected from 60 min of combined oxygen-glucose deprivation neurotoxicity compared with wild-type cultures. Wild-type cultures are protected from neuronal cell death by the NMDA receptor antagonist MK-801 and the NOS inhibitor L-nitroarginine methyl ester, but not its inactive stereoisomer D-nitroarginine methyl ester. nNOS- cultures were not additionally protected. These data confirm that activation of NMDA receptors and production of NO are primary mediators of neuronal damage after ischemic insult.

Nitric oxide is a ubiquitous and unique biological messenger molecule. It mediates blood vessel relaxation by endothelium, immune function of macrophages, and neurotransmission of central and peripheral nervous systems. Endothelial and neuronal nitric oxide synthases are constitutively expressed and activated by calcium entry into cells, whereas the macrophage nitric oxide synthase is inducible with new RNA and protein synthesis upon immune stimulation. Nitric oxide may play a role in the neurotransmitter release, neural development, synaptic plasticity, and regulation of gene expression. Excessive production of nitric oxide is neurotoxic and is implicated in a variety of neurological disorders.

Nitric oxide is a unique biological messenger molecule. It is produced by endothelial cells to mediate blood vessel relaxation; it mediates, in part, the immune functions of activated macrophages; and in the central and peripheral nervous system it serves as a neurotransmitter. In the nervous system, nitric oxide may regulate neurotransmitter release, it may play a key role in synaptic plasticity and morphogenesis, and it may regulate sexual and aggressive behavior. Under conditions of excessive formation, nitric oxide is emerging as an important neurotoxin.

In addition to its role in blood vessel and macrophage function, nitric oxide (NO) is a neurotransmitter found in high densities in emotion-regulating brain regions. Mice with targeted disruption of neuronal NO synthase (nNOS) display grossly normal appearance, locomotor activity, breeding, long-term potentiation and long-term depression. The nNOS- mice are resistant to neural stroke damage following middle cerebral artery ligation. Although CO2-induced cerebral vasodilatation in wild-type mice is NO-dependent, in nNOS- mice this vasodilation is unaffected by NOS inhibitors. Establishing a behavioural role for NO has, until now, not been feasible, as NOS inhibitor drugs can only be administered acutely and because their pronounced effects on blood pressure and other body functions obfuscate behavioural interpretations. We now report a large increase in aggressive behaviour and excess, inappropriate sexual behaviour in nNOS- mice.