Brain Nitric Oxide Synthase Research Articles

Increased expression of inducible nitric oxide synthase in psoriatic skin and cytokine-stimulated cultured keratinocytes.

Since nitric oxide (NO) has been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) were investigated in psoriatic skin by reverse transcriptase coupled to the polymerase chain reaction (PCR). The study showed that the mRNA expression of brain nitric oxide synthase (bNOS), one of two isoforms of cNOS, was weak in both psoriatic plaques lesions and uninvolved skin, while mRNA transcripts for the second isoform, endothelial nitric oxide synthase (eNOS), were not detectable using the present method. In contrast, the mRNA expression of iNOS was markedly increased in lesional skin as compared to uninvolved skin. Cultured human keratinocytes exposed to a combination of interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) for 4 h, showed strong gene expression of iNOS, while in 24 h, the expression had returned to baseline expression. In summary, the study demonstrates that mRNA for the inducible form of NOS is over-expressed in psoriatic lesions. The cause of this may be the local presence of inflammatory cytokines. These findings imply that iNOS may play an important part in local regulation of NO synthesis in psoriasis and other inflammatory dermatoses.

Sleep and nitric: oxide: effects of 7-nitro indazole, inhibitor of brain nitric oxide synthase

We examined the effect of 7-nitro indazole (7-NI, 2.5–50 mg/kg, i.p.), an inhibitor of central nitric oxide (NO) synthesis, on general behaviour and sleep. The results show that 7-NI induces ptosis, a loss of the righting reflex and decrease of the EEG amplitudes. Furthermore, a duration of slow wave sleep (SWS) and REM sleep decreased, while the latencies of SWS and REM sleep increased. The effects of 7-NI on general behaviour and sleep were partially antagonized by intraventricular administration of the NO precursor, l-arginine (600 μg). These findings indicate that 7-NI induces a state of prominent central depression associated with motor deficit and decrease in sleep stages and wakefulness. It further suggests that NO exerts a significant excitatory effect on the neuronal structure involved in the regulation of locomotion and vigilance.

Effect of hypoxia and reoxygenation on regional activity of nitric oxide synthase in brain of newborn piglets

The activity of nitric oxide synthase (NOS) was measured in homogenates from cortex, striatum, hippocampus, cerebellum, pons, thalamus and midbrain of the brain of newborn piglets and the effects of hypoxia and posthypoxic period on this activity was evaluated. The control activities were 19.7, 31.5, 26.8, 16.7, 33.6, 19.3 and 39.4 pmol/mg protein per min, respectively. A 1 h period of hypoxia (an Fi0 2 of 7%) resulted in statistically significant decreases in the activity of NOS in every region of the brain except for the cortex, where the activity was not significantly altered compared to control. By 2 h of reoxygenation following such a hypoxic episode, the NOS activities increased to above control levels in all regions of the brain, but this increase was statistically significant compared to control only in thalamus. Since hypoxia induced the greatest decrease in NOS activity in the cerebellum, the kinetic constants of the enzyme were measured in homogenates from this region of brain. The decreased activity following the hypoxic episode was associated with an approximately four-fold increase in the apparent affinity ( K M ) for arginine with no significant change in the maximal rate of reaction ( V ax). The decrease in NOS activity subsequent to a hypoxic episode may contribute to the disturbances in cellular metabolism in the immature brain induced by episodes of hypoxia-reoxygenation.

Nitric oxide release from kidneys of hypertensive rats treated with imidapril.

To examine whether endothelial dysfunction in hypertension is reversible or not, we studied the effects of imidapril, an angiotensin-converting enzyme inhibitor, on nitric oxide release in stroke-prone spontaneously hypertensive rats (SHR) and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. After a 4-week treatment with imidapril (1 or 10 mg/d SC) or vehicle, acetylcholine-induced vasodilation and nitric oxide release in the isolated kidneys were determined. Nitric oxide release was measured by a chemiluminescense assay. Imidapril lowered blood pressure in stroke-prone SHR in a dose-dependent manner. Untreated stroke-prone SHR exhibited significantly attenuated responses to acetylcholine (10(-8) mol/L) of both renal perfusion pressure (stroke-prone SHE 42 +/- 4% versus Wistar-Kyoto rats [WKY] 58 +/- 4% [mean +/- SE], P < .01) and nitric oxide release (stroke-prone SHR +7.6 +/- 2.1 versus WKY +29.7 +/- 9.7 fmol/min per gram of kidney wt, P < .01). Imidapril at 10 mg/d significantly increased acetylcholine-induced renal vasodilation and nitric oxide release in stroke-prone SHR (renal perfusion pressure, 56 +/- 3%; nitric oxide release, +27.1 +/- 6.4 fmol/min per gram of kidney wt; both P < .01 versus stroke-prone SHR treated with vehicle). On the other hand, imidapril neither decreased blood pressure nor changed nitric oxide release induced by acetylcholine in DOCA-salt hypertensive rats. Staining for endothelial nitric oxide synthase and brain nitric oxide synthase was clearly detected in the kidneys of both stroke-prone SHR and WKY, whereas staining intensity was weaker in DOCA-salt hypertensive rats. Inducible nitric oxide synthase immunoreactivity was barely noticeable in any type of rat. Thus, imidapril restored endothelial damage by pressure-dependent mechanisms. Most of the nitric oxide detected in the perfusate seemed to be derived from constitutive nitric oxide synthase.

Human seminal plasma inhibits brain nitric oxide synthase activity

Nitric oxide is a chemical messenger which functions as a neurotransmitter or as a cytotoxic agent. Nitric oxide synthase (NOS) has been isolated from various mammalian reproductive tissues. The presence or absence of NOS in spermatozoa has not yet been reported. We therefore tested human and murine spermatozoa for NOS activity by measuring the conversion of arginine to citrulline. No activity was found either in human or in murine spermatozoa. Human native semen and human seminal plasma exerted an inhibition on brain NOS activity, as assayed on rat brain cytosolic fractions. This inhibitory effect was dependent on the amount of protein present in the human seminal plasma. No inhibitory effect was observed when homogenates of washed spermatozoa were tested. The human seminal plasma did not affect the Michaelis constant (Km) of NOS for L-arginine (endogenous NOS substrate) whereas the maximal velocity (Vmax) was reduced, suggesting that it contains a non-competitive inhibitor of brain NOS. This inhibitory component was virtually insensitive to heat; a 10 min treatment to 95 degrees C only slightly reduced its ability to inhibit brain NOS. The physiological relevance of our observations remains to be elucidated. Human seminal plasma may exert an inhibition of nitric oxide synthesis on cells other than spermatozoa or on cells from the male or female genital tract, modulating directly or indirectly (via modulation of reactive oxygen species formation) the functional state of the spermatozoa.

Identification of the domains of neuronal nitric oxide synthase by limited proteolysis

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147-15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.

Roles of IL-1 and TNF-alpha in endotoxin-induced activation of nitric oxide synthase in cultured rat brain cells.

In astrocytes and microglia, bacterial lipopolysaccharide (LPS) stimulates production and release of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO). Although IL-1 beta and TNF-alpha are themselves capable of inducing NO synthase (NOS) in glia, the specific factors mediating LPS induction of NOS in brain have not been identified. To determine whether LPS induction of NOS in brain cells is mediated by IL-1 or TNF-alpha, acting alone or in concert, the effects of IL-1-receptor antagonist (IL-1Ra) and of TNF-soluble receptor (TNFsRp55), presented individually and in combination, on LPS-induced NOS activity were tested. In glial-enriched mixed primary cultures of neonatal rat telencephalic cells, LPS (0.1-100 ng/ml), IL-1 beta (0.01-10 nM), and TNF-alpha (0.1-100 nM) each concentration dependently stimulated accumulation of nitrite, an indicator of NO production. Induction of nitrite accumulation by LPS and by IL-1 was blocked by N omega-nitro-L-arginine methyl ester and N omega-monomethyl-L-arginine, indicating that it was mediated by NOS. TNF-alpha alone induced NO production weakly as compared with IL-1, but combined submaximal concentrations of IL-1 beta (1 nM) and TNF-alpha (10 nM) induced NOS synergistically. Furthermore, TNFsRp55 and IL-1Ra each produced a dose-dependent partial inhibition of the NO response to LPS, and the effect of TNFsRp55 was equal to or greater than that of IL-1Ra. TNFsRp55 and IL-1Ra in combination were not significantly more effective than TNF-sRp55 alone. The results indicate that LPS induction of NOS activity in brain cells is mediated in part by both IL-1 beta and TNF-alpha.

Selective pharmacological inhibition of distinct nitric oxide synthase isoforms

Nitric oxide (NO) is produced in physiological and pathophysiological conditions by three distinct isoforms of NO synthase (NOS): endothelial NOS (ecNOS), inducible NOS (iNOS), and brain NOS (bNOS). Selective inhibition of iNOS may be beneficial in various forms of shock and inflammation, whereas inhibition of bNOS may protect against neuroinjury. This article surveys the enzymatic mechanism of NO production, lists the strategies and pharmacological tools for selective inhibition of distinct NOS isoforms, and considers the side-effects of the various approaches. Selective inhibition of NOS isoforms is achieved by: (a) targeting the differential co-factor (calmodulin or tetrahydrobiopterin) requirement of various NOS isoforms of NOS; (b) targeting the differential substrate requirements of cells expressing various isoforms of NOS ( l-arginine uptake blockers or arginase); (c) the use of pharmacological agents that are selectively taken up by cells expressing various isoforms of NOS (7-nitroindazole); or (d) developing pharmacological NOS inhibitors with isoform specificity. The amino acid-based NOS inhibitor, N G-nitro-L-arginine, shows a preference for ecNOS and bNOS over iNOS, whereas Ll- N 6-(1-iminoethyl)lysine is selective for iNOS over bNOS. Certain non-amino acid-based small molecules, such as aminoguanidine and certain S-alkylated isothioureas, also express selectivity towards iNOS and have anti-inflammatory and anti-shock properties. 7-Nitroindazole, a bNOS-selective inhibitor, protects in central nervous system injury. Clearly, there are a number of distinct approaches that are worthy of further research efforts in order to achieve even more selective targeting of various NOS isoforms.

Correlation between brain nitric oxide synthase activity and opiate withdrawal.

The opiate withdrawal induced by administration of naloxone to morphine-dependent mice correlates with an increment of calcium- dependent nitric oxide synthase (NOS) activity in the cerebellum. L-NAME, an irreversible competitive inhibitor of NOS (0.5, 5, 25, 50 mg/kg) injected sc. 45 min. prior to naloxone significantly reduced the number of escape jumps and other motor symptoms of abstinence. In addition, L-NAME also decreased NOS activity in cerebellum. L-arginine, but not D-arginine, when coadministered with L-NAME, prevented both the inhibition of NOS activity and the reduction of withdrawal symptoms induced by L-NAME in morphine-withdrawn animals. These results demonstrate a hyperactivity of the L-arginine: NO pathway in opiate withdrawal and suggests the possibility of a therapeutic use of NOS inhibitors in this state.

Increase in brain nitric oxide synthase activity in daunorubicin-treated rats.

Anthracyclines such as daunorubicin are very effective anticancer agents. These drugs are known to cause side effects including cardiotoxicity. Anthracyclines are neurotoxic to laboratory animals. Nitric oxide is a novel and very important chemical messenger in the brain. However, at higher levels, nitric oxide causes well defined neurotoxicity. Therefore, we determined nitric oxide synthase activity in rat brain after daunorubicin treatment in an effort to explain the neurotoxicity produced by anthracyclines. Male Sprague-Dawley rats were treated with different subcutaneous doses of daunorubicin (0.1-4.0 mg/kg/week for five weeks) while control animals were injected with phosphate buffered saline. There was a significant increase (80%) of nitric oxide synthase activity in daunorubicin-treated animals as compared to controls. This activity was inhibited by N-monomethyl-L-arginine (NMMA), nitroarginine, N-6-aminohexyl-5-chloro-1-napthalene sulfonamide (W-7), a calmodulin antagonist, suggesting that the nitric oxide synthase activity is calmodulin dependent. Further, our in vitro studies demonstrated that daunorubicin interacted with calmodulin as measured by N-phenyl-1-napthylamine (NPN) fluorescence. These results indicate that daunorubicin increases nitric oxide synthase activity in rat brain which may increase the levels of nitric oxide. The increased levels of nitric oxide may cause neurotoxicity. Our results further indicate that daunorubicin interacts with calmodulin and enhances nitric oxide synthase activity which is dependent on calmodulin.

Expression of Inflammatory Cytokines and Inducible Nitric Oxide Synthase in Brains of SIV-Infected Rhesus Monkeys: Applications to HIV-Induced Central Nervous System Disease

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) can lead to severe impairments in cognition, behavior, and motor skills. The mechanism(s) by which HIV-1 induces CNS disease are not well understood. Recent evidence suggests that expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) may contribute to HIV-1-induced neurologic disease. We sought to determine if these factors were present in the CNS of rhesus monkeys with simian immunodeficiency virus (SIV)-induced CNS disease. Total NO production in cerebral spinal fluid (CSF) from infected monkeys was determined by measuring nitrite (NO2-) and nitrate (NO3-) (stable NO degradation products) utilizing Greiss reagents. In situ hybridization revealed iNOS, interferon-gamma (IFNgamma), and interleukin 1 beta (IL-1 beta) mRNA in the brains of SIV-infected monkeys. Microglia were isolated from animals infected with SIV. Following stimulation with LPS, induction of iNOS mRNA in isolated microglia was analyzed by reverse transcriptase-polymerase chain reaction. Serial CSF samples from an SIV-infected monkey reveal increased levels of NO2-/NO3-. In situ hybridization demonstrated iNOS, IFN gamma, and IL-1 beta mRNAs in post-mortem brain tissue of SIV-infected monkeys. Furthermore, stimulated microglia from an SIV-infected monkey could produce iNOS mRNA. The presence of iNOS in the brain and NO2-/NO3- in the CSF indicates that NO is produced in the CNS of SIV-infected monkeys. The data suggest that iNOS and NO may be contributing to SIV-induced CNS disease.

Evidence that brain nitric oxide synthase is the major nitric oxide synthase isoform in the hypothalamus of the adult female rat and that nitric oxide potently regulates hypothalamic cGMP levels.

Recent studies suggest that nitric oxide (NO) may function as a neurotransmitter in the hypothalamus. In order to provide further evidence supporting this contention, we examined: (1) whether the hypothalamus displays significant NO synthase (NOS) activity and whether the activity is inhibited by an NOS inhibitor, (2) whether the different NOS isoforms [brain (b)-NOS, endothelial (e)-NOS and macrophage (m)-NOS] are expressed in the various nuclei of the hypothalamus of the random cycling adult female rat, (3) whether the NO donor molecule, sodium nitroprusside (SNP), regulates the heme-containing enzyme, guanylate cyclase in the preoptic area and medial basal hypothalamus of the random cycling adult female rat as well as the ovariectomized steroid (estradiol-17 beta)-treated rat. The results of the study showed that the preoptic area (POA) and medial basal hypothalamus (MBH) of the adult female rat displays significant NOS activity which can be dose-dependently inhibited by an NOS inhibitor. All three NOS isoform mRNA transcripts were present in the hypothalamus, with the order of expression being b-NOS > e-NOS > m-NOS. Immunohistochemical localization using monoclonal antibodies to the specific NOS isoform proteins revealed that b-NOS represented the major form of NOS in the hypothalamus based on density and distribution of immunostaining. b-NOS immunostaining was especially dense in the organum vasculosum laminae terminalis (OVLT), medial preoptic area (MPOA), supraoptic nucleus, and moderately dense in the arcuate nucleus/median eminence. The pattern and density of b-NOS staining closely mirrored our previously reported pattern of NADPH-diaphorase staining in the hypothalamus, and a polyclonal antibody to b-NOS yielded a similar staining pattern as that observed for the monoclonal antibody. In contrast to the dense staining observed for b-NOS in the hypothalamus, we observed no specific staining for m-NOS in the hypothalamus. e-NOS immunostaining, on the other hand, was present in the hypothalamus, but to a much lesser extent than b-NOS. Light e-NOS staining was observed in the OVLT, MPOA, supraoptic nucleus and arcuate nucleus/median eminence. That NO can regulate guanylate cyclase as a potential mediator of its effects was demonstrated using SNP which dose-dependently elevated cGMP levels in the POA and MBH of random cycling rats and estrogen-primed ovariectomized rats. The effect of SNP was due to its NO donor ability as it was blocked by the NO scavenger molecule, hemoglobin. Interestingly, hemoglobin alone caused a 50-60% reduction in basal cGMP levels, suggesting that endogenously produced NO regulates basal guanylate cyclase activity. Taken as a whole, the present study demonstrates that b-NOS is the major NOS isoform in the hypothalamus and it also provides evidence that cGMP may be a mediator of NO effects in the female hypothalamus as evidenced by the potent ability of SNP to elevate cGMP levels in the POA and MBH.

Attenuation of cocaine kindling by 7-nitroindazole, an inhibitor of brain nitric oxide synthase

Recent studies suggest the involvement of the N-methyl-D-aspartate (NMDA) type of glutamate receptors and nitric oxide synthase (NOS) in the process of increased sensitivity to the convulsive effect of cocaine (“cocaine kindling”). The present study was undertaken to analyze the various behavioral stages in the development of cocaine kindling and to investigate the effect of 7-nitroindazole (7-NI), a relatively selective inhibitor of the neuronal NOS isoform, on the induction and expression of sensitization to the convulsive effect of cocaine. Also, the effect of 7-NI on responses produced by acute systemic administration of cocaine or N-methyl-D-aspartate (NMDLA) was investigated. Cocaine kindling was assessed on a five-stage scale following the administration of a sub-convulsant dose of the drug (35 mg/kg/day; i.p.) to Swiss Webster mice for 10 days. Stage 5 seizures developed following the 9th day of cocaine administration. Pre-treatment with 7-NI (25 mg/kg/day; i.p.) 15 min before cocaine for 10 days completely prevented the appearance of stage 4 and 5 seizures, and it significantly attenuated stage 3 behavior in response to a challenge cocaine dose (35 mg/kg) given either 24 hr or 10 days after 7-NI/cocaine administration was stopped. A single injection of 7-NI (25 mg/kg; i.p.) completely prevented the expression of cocaine kindled seizures. Whereas 7-NI had no effect on the responses elicited by acute cocaine administration (60 mg/kg; i.p.), this agent partially attenuated the effects induced by systemic administration of the NMDA receptor agonist NMDLA (250 mg/kg; i.p.). The present study indicates that 7-NI attenuates both the induction and expression of sensitization to the convulsive effect of cocaine. The findings that 7-NI attenuated cocaine kindling and partially blocked the effects produced by activation of the NMDA receptor, but not the effects induced by acute cocaine administration, support the role of the NMDA receptor and brain NOS in the development of cocaine kindling rather than in the acute effects of the drug.

Synthesis and effects on arginase and nitric oxide synthase of two novel analogues of Nω-hydroxyarginine, Nω-hydroxyindospicine and p-hydroxyamidinophenylalanine

Two novel amino acids, Nω-hydroxy-D,L-indospicine and p-hydroxyamidino-D,L-phenylalanine, have been synthesized in four steps from tert-butoxycarbonylglycine. Both compounds act as good inhibitors of arginase, Nω-hydroxyindospicine being one of the best inhibitors of this enzyme known so far (IC50= 50 µmol dm–3). In contrast, with brain NO synthase the two compounds are almost without effect.

P-114 - Effect of ethanol on the activity of NO synthase in the mouse brain.

P-114 - Effect of ethanol on the activity of NO synthase in the mouse brain.

Different Mechanisms for Ca2+ Dissociation from Complexes of Calmodulin with Nitric Oxide Synthase or Myosin Light Chain Kinase

We have determined the stoichiometry and rate constants for the dissociation of Ca2+ ion from calmodulin (CaM) complexes with rabbit skeletal muscle myosin light chain kinase (skMLCK), rat brain nitric oxide synthase (nNOS) or with the respective peptides (skPEP and nPEP) representing the CaM-binding domains in these enzymes. Ca2+ dissociation kinetics determined by stopped-flow fluorescence using the Ca2+ chelator quin-2 MF are as follows. 1) Two sites in the CaM-nNOS and CaM-nPEP complexes have a rate constant of 1 s-1. 2) The remaining two sites have a rate constant of 18 s-1 for CaM-nPEP and > 1000 s-1 for CaM-nNOS. 3) Three sites have a rate constant of 1.6 s-1 for CaM-skMLCK and 0.15 s-1 for CaM-skPEP. 4) The remaining site has a rate constant of 2 s-1 for CaM-skPEP and > 1000 s-1 for CaM-skMLCK. Comparison of these rate constants with those determined for complexes between the peptides and tryptic fragments representing the C- or N-terminal lobes of CaM indicate a mechanism for Ca2+ dissociation from CaM-nNOS of 2C slow + 2N fast and from CaM-skMLCK of (2C + 1N) slow + 1N fast. Ca2+ removal inactivates CaM-nNOS and CaM-skMLCK activities with respective rate constants of > 10 s-1 and 1 s-1. CaM-nNOS inactivation is fit by a model in which rapid Ca2+ dissociation from the N-terminal lobe of CaM is coupled to enzyme inactivation and slower Ca2+ dissociation from the C-terminal lobe is coupled to dissociation of the CaM-nNOS complex. CaM-skMLCK inactivation is fit by a model in which the three slowly dissociating Ca(2+)-binding sites are coupled to both dissociation of the complex and enzyme inactivation.

Large-scale purification of rat brain nitric oxide synthase from baculovirus overexpression system

Large-scale purification of rat brain nitric oxide synthase from baculovirus overexpression system

Glutathione depletion is accompanied by increased neuronal nitric oxide synthase activity.

The effect of glutathione depletion, in vivo, on rat brain nitric oxide synthase activity has been investigated and compared to the effect observed in vitro with cultured neurones. Using L-buthionine sulfoximine rat brain glutathione was depleted by 62%. This loss of glutathione was accompanied by a significant increase in brain nitric oxide synthase activity by up to 55%. Depletion of glutathione in cultured neurones, by approximately 90%, led to a significant 67% increase in nitric oxide synthase activity, as judged by nitrite formation, and cell death. It is concluded that depletion of neuronal glutathione results in increased nitric oxide synthase activity. These findings may have implications for our understanding of the pathogenesis of neurodegenerative disorders in which loss of brain glutathione is considered to be an early event.

Calcium-activated release of nitric oxide potentiates excitatory synaptic potentials in immature rat sympathetic preganglionic neurons.

1. Whole cell patch recordings were made from sympathetic preganglionic neurons (SPNs), the majority of which contain brain nitric oxide synthase (bNOS), in transverse spinal cord slices of 12- to 16-day-old rats. 2. Repetitive discharge of SPNs induced by a train of depolarizing current pulses (40 Hz. 10 s) was followed by a long-lasting increase (140 +/- 22%, mean +/- SD) of the amplitude of excitatory postsynaptic potentials (EPSPs) evoked by stimulation of lateral funiculus in 50 of 75 SPNs. 3. In slices pretreated with the nitric oxide synthase inhibitors, NG-monomethyl-L-arginine (L-NMA: 100 microM) or Nw-nitro-L-arginine (L-NARG; 30 microM) or with bovine hemoglobin (100 microM), repetitive discharge of SPNs was not followed by a significant increase of EPSPs. 4. Superfusing the slices with L-arginine (L-Arg, 300 microM) but not D-Arg reversibly increased the EPSPs by an average of 140 +/- 19%. 5. Inclusion of the Ca2+ chelator 1.2-bis(2-aminophenoxy)- ethane-N,N,N',N'-tetraacetic acid (BAPTA, 1 mM) in the patch electrodes resulted in no significant increase of EPSPs after repetitive discharge in all cells studied. 6. It is concluded that during repetitive discharge of SPNs, Ca2+ influx via voltage-gated channels activates bNOS, resulting in a release of nitric oxide and potentiation of EPSPs.

In vivo metabolites of [formula omitted] methyl ester: methanol and [formula omitted

N ω- nitro- L-arginine methyl ester ( L-NAME) is commonly used as a selective inhibitor for in vivo studies of brain nitric oxide (NO) synthase. We aimed to study the fate of N ω- nitro- L-arginine [ 11C]methyl ester ([ 11C] L-NAME) using position emission tomography in monkey and high performance liquid chromatography methods in dogs and rats. We found that [ 11C] L-NAME was rapidly t 1 2 = 2 min ) metabolized into N ω- nitro- L-arginine ( L-NA) and [ 11C]methanol which both had a slow rate of elimination. Although, in vivo L-NAME administration leads to long-lasting NO synthase inhibition by L-NA, methanol which is a potent neurotoxin in primate may produce detrimental effects unrelated to NO synthase inhibition.