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Neuronal Nitric Oxide Synthase (neuronal + nitric_oxide_synthase)
Selected Abstracts4-Substituted Indazoles as New Inhibitors of Neuronal Nitric Oxide Synthase.CHEMINFORM, Issue 40 2007Michel Boulouard Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Substituted 6-Phenyl-pyridin-2-ylamines: Selective and Potent Inhibitors of Neuronal Nitric Oxide Synthase.CHEMINFORM, Issue 52 2004Deane M. Nason Abstract For Abstract see ChemInform Abstract in Full Text. [source] ChemInform Abstract: Inhibition of Neuronal Nitric Oxide Synthase by 7-Methoxyindazole and Related Substituted Indazoles.CHEMINFORM, Issue 32 2001Pascale Schumann Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Neuronal nitric oxide synthase (nNOS) mRNA is down-regulated, and constitutive NOS enzymatic activity decreased, in thoracic dorsal root ganglia and spinal cord of the rat by a substance P N-terminal metaboliteEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2001Katalin J. Kovacs Abstract Nitric oxide (NO) in the spinal cord plays a role in sensory and autonomic activity. Pain induced by acetic acid in the abdominal stretch (writhing) assay and hyperalgesia associated with chronic pain are highly sensitive to NO synthase (NOS) inhibitors. Because substance P (SP) is released and up-regulated in some models of chronic pain, we hypothesized that an accumulation of SP metabolites may influence NOS expression and activity. To test this hypothesis, we examined the effect of intrathecally (i.t.) injected substance P (1-7) [SP(1-7)], the major metabolite of SP in the rat, on neuronal NOS (nNOS) mRNA in the thoracic and lumbar spinal cord, dorsal root ganglia (DRG) and on the corresponding constitutive NOS (cNOS) enzyme activity. Detected using quantitative RT-PCR, nNOS mRNA content in the thoracic spinal cord was decreased 6 h after injection of 5 µmol of SP(1-7) and returned to control 2 days later. In thoracic DRG, nNOS mRNA was reduced 48 h after SP(1-7). The cNOS enzymatic activity in thoracic spinal tissue was gradually decreased to a minimum at 72 h. Down-regulation of NOS by SP(1-7) in the thoracic area appears to be highly associated with capsaicin-sensitive primary afferent neurons. No similar changes in either parameter were measured in the lumbar area after SP(1-7). These data suggest that N-terminal SP fragments, which are known to cause long-term antinociception in the writhing assay, may do so by their ability to down-regulate NO synthesis along nociceptive pathways. [source] Neuronal nitric oxide synthase does not contribute to the modulation of pulmonary vascular tone in fetal lambs with congenital diaphragmatic hernia (nNOS in CDH lambs),PEDIATRIC PULMONOLOGY, Issue 4 2008Anthony S. de Buys Roessingh MD Abstract Aim The aim of this study was to determine the presence of the neuronal nitric oxide synthase (nNOS) in near full-term lambs with congenital diaphragmatic hernia (CDH) and its role in the modulation of pulmonary vascular basal tone. Methods We surgically created diaphragmatic hernia on the 85th day of gestation. On the 135th, catheters were used to measure pulmonary pressure and blood flow. We tested the effects of 7-nitroindazole (7-NINA), a specific nNOS antagonist and of N -nitro- l -arginine (l -NNA), a nonspecific nitric oxide synthase antagonist. In vitro, we tested the effects of the same drugs on isolated pulmonary vessels. The presence of nNOS protein in the lungs was detected by Western blot analysis. Results Neither 7-NINA nor l -NNA modified pulmonary vascular basal tone in vivo. After l -NNA injection, acetylcholine (ACh) did not decrease significantly pulmonary vascular resistance (PVR). In vitro, l -NNA increased the cholinergic contractile-response elicited by electric field stimulation (EFS) of vascular rings from lambs with diaphragmatic hernia. Conclusion We conclude that nNOS protein is present in the lungs and pulmonary artery of near full-term lamb fetuses with diaphragmatic hernia, but that it does not contribute to the reduction of pulmonary vascular tone at birth. Pediatr Pulmonol. 2008; 43:313,321. © 2008 Wiley-Liss, Inc. [source] The Effect of Intracavernous Injection of Adipose Tissue-Derived Stem Cells on Hyperlipidemia-Associated Erectile Dysfunction in a Rat ModelTHE JOURNAL OF SEXUAL MEDICINE, Issue 4pt1 2010Yun-Ching Huang MD ABSTRACT Introduction., Hyperlipidemia has been associated with erectile dysfunction (ED) via damage to the cavernous endothelium and nerves. Adipose tissue-derived stem cells (ADSC) have been shown to differentiate into endothelial cells and secrete vasculotrophic and neurotrophic factors. Aim., To assess whether ADSC have therapeutic effects on hyperlipidemia-associated ED. Methods., Twenty-eight male rats were induced to develop hyperlipidemia with a high-fat diet (hyperlipidemic rats, HR). Ten additional male rats were fed a normal diet to serve as controls (normal rats, NR). Five months later, all rats were subjected to ADSC isolation from paragonadal fat. The cells were cultured for 1 week, labeled with 5-ethynyl-2,-deoxyuridine (EdU), and then injected autologously into the corpus cavernosum of 18 HR. The remaining 10 HR rats were injected with phosphate buffered saline (PBS). At 2 and 14 days post-transplantation, four rats in the HR + ADSC group were sacrificed for tracking of the transplanted cells. At 28 days post-transplantation, all remaining rats were analyzed for serum biochemistry, erectile function, and penile histology. Main Outcome Measures., Erectile function was assessed by intracavernous pressure (ICP) measurement during electrostimulation of the cavernous nerve. Cavernous nerves, endothelium, and smooth muscle were assessed by immunohistochemistry. Results., Serum total cholesterol and low-density lipoprotein levels were significantly higher in HR than in NR. High-density lipoprotein level was significantly lower in HR than in NR. Mean ICP/mean arterial pressure ratio was significantly lower in HR + PBS than in NR + PBS or HR + ADSC. Neuronal nitric oxide synthase (nNOS)-positive nerve fibers and endothelial cells were fewer in HR + PBS than in HR + ADSC. Smooth muscle content was significantly higher in both HR groups than in NR. Conclusions., Hyperlipidemia is associated with abnormalities in both the nerves and endothelium. Treatment with ADSC ameliorates these adverse effects and holds promise as a potential new therapy for ED. Huang Y-C, Ning H, Shindel AW, Fandel TM, Lin G, Harraz AM, Lue TF, and Lin C-S. The effect of intracavernous injection of adipose tissue-derived stem cells on hyperlipidemia-associated erectile dysfunction in a rat model. J Sex Med 2010;7:1391,1400. [source] The Breakdown of Preformed Advanced Glycation End Products Reverses Erectile Dysfunction in Streptozotocin-Induced Diabetic Rats: Preventive Versus Curative TreatmentTHE JOURNAL OF SEXUAL MEDICINE, Issue 2 2006Mustafa F. Usta MD ABSTRACT Objectives., Accumulation of advanced glycation end products (AGEs) has been linked to many of the complications of diabetes mellitus, including erectile dysfunction (ED). Furthermore, it has been demonstrated that inhibitors of AGE formation, such as aminoguanidine, can prevent ED in diabetic animals. However, it is unknown whether late administration of a putative cross-link breaker, ALT-711, can reverse diabetic ED. We therefore compared ALT-711 and aminoguanidine in their ability to reverse ED in diabetic rats. Materials and Methods., Male Sprague,Dawley rats were randomly divided into four groups: (i) age-matched controls; (ii) streptozotocin (STZ)-induced diabetic rats (60 mg/kg; intraperitoneal injection); (iii) STZ diabetic rats treated with ALT-711 (3 mg/kg/day, intraperitoneal injection); and (iv) STZ diabetic rats treated with aminoguanidine (1 gm/L in drinking water) during the final 6 weeks of 12 weeks of induced diabetes. At the end of 12 weeks, erectile response to cavernous nerve stimulation (CNS) was determined. Neuronal nitric oxide synthase (nNOS) contents were measured in all penises, and AGE levels were determined both in penile tissues and in serum samples. Results., Erectile responses to CNS and penile nNOS protein content were significantly reduced, while AGE levels were elevated in the penises and serum of untreated diabetic animals. Treatment with ALT-711, but not with aminoguanidine, reversed ED and nNOS depletion and reduced serum and penile tissue AGE levels. Conclusions., These results suggest that cross-link breakers, such as ALT-711, are the optimal therapeutic approach, compared with treatment with inhibitors of AGE formation, in the reversal of diabetes-related ED. Usta MF, Kendirci M, Gur S, Foxwell NA, Bivalacqua TJ, Cellek S, and Hellstrom WJG. The breakdown of preformed advanced glycation end products reverses erectile dysfunction in streptozotocin-induced diabetic rats: Preventive versus curative treatment. J Sex Med 2006;3:242,252. [source] Role of neuronal nitric oxide synthase in response to hypertonic saline loading in ratsACTA PHYSIOLOGICA, Issue 4 2004R. Wangensteen Abstract Aims:, This study analyses the influence of neuronal nitric oxide synthase (nNOS) blockade with 7-nitroindazole (7NI) on the haemodynamic and renal response to a hypertonic saline load (HSL). We also evaluated the effects of non-specific NOS inhibitor N, -nitro- l -arginine methyl ester (l -NAME). Methods:, The following groups were used: controls, rats treated with 7NI at 0.5 or 5 mg kg,1, and rats treated with l -NAME at 0.5 or 5 mg kg,1. A further five groups received an isotonic saline load (ISL). Results:, Mean arterial pressure (MAP) was significantly increased in control rats after HSL. MAP was further increased in both 7NI-treated groups, and the l -NAME groups showed marked dose-related pressor responses. During ISL, MAP was only significantly increased in the group treated with 5 mg kg,1 of l -NAME. The pressure,natriuresis relationship during the experimental period after the HSL was reduced in the 7NI group treated with 5 mg kg,1 and severely attenuated in both l -NAME groups. The increase in plasma sodium was significantly greater after the HSL in both 7NI groups and both l -NAME groups compared with controls. Conclusions:, The present results suggest that nNOS and other NOS isozymes play a counter-regulatory role in the pressor response to HSL. Moreover, the blockade of nNOS with the higher dose of 7NI produces a blunted pressure,natriuresis relationship in response to the HSL. Finally, it is concluded that nNOS participates in the homeostatic cardiovascular and renal response to hypertonic saline loading by attenuating the blood pressure increase and hypernatremia, and facilitating natriuresis. [source] Splice-isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ-complex-building nNOS, ,-finger is largely exposed to antibodiesDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2007Kristina Langnaese Abstract Knock out mice deficient for the splice-isoform ,, of neuronal nitric oxide synthase (nNOS,,) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, ,, and ,,, we generated isoform-specific anti-peptide antibodies against the nNOS,, specific ,,-finger motif involved in PDZ domain scaffolding and the nNOS,, specific N-terminus. The nNOS,, ,,-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOS,, on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the ,,-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOS,, ,,-finger antibody in pull-down assays. By contrast, nNOS,, ,,-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOS,, knock out mice, nNOS,, was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting ,,/,,-isoforms in these cells. The nNOS,, antibody clearly detected bacterial expressed nNOS,, fusion protein and nNOS,, in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOS,, in nNOS,, deficient animals. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] Expression of a dominant negative form of Daxx in vivo rescues motoneurons from Fas (CD95)-induced cell deathDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2005Cedric Raoul Abstract Fas-induced death of motoneurons in vitro has been shown to involve two signaling cascades that act together to execute the death program: a Fas-Daxx-ASK-1-p38 kinase-nNOS branch, which controls transcriptional and post-translational events, and the second classical Fas-FADD-caspase-8 branch. To analyze the role of Daxx in the developmental motoneuron cell death, we studied Fas-dependent cell death in motoneurons from transgenic mice that overexpress a dominant-negative form of Daxx. Motoneurons purified from these transgenic mice are resistant to Fas-induced death. This protective effect is specific to Fas because ultraviolet irradiation-triggered death is not affected by the transgene. The Daxx and the FADD pathways work in parallel because only Daxx, but not FADD, is involved in the transcriptional control of neuronal nitric oxide synthase and nitric oxide production. Nevertheless, we do not observe involvement of Daxx in developmental motoneuronal cell death, as the pattern of naturally occurring programmed cell death in vivo is normal in transgenic mice overexpressing the dominant negative form of Daxx, suggesting that Daxx-independent pathways are used during development. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005 [source] Role of peroxynitrite in methamphetamine-induced dopaminergic neurotoxicity and sensitization in miceADDICTION BIOLOGY, Issue 3 2000Syed F. Ali Methamphetamine (METH)-induced dopaminergic neurotoxicity is thought to be associated with the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Recently, we have reported that copper/zinc(CuZn)-superoxide dismutase transgenic mice are resistant to METH-induced neurotoxicity. In the present study, we examined the role of the neuronal nitric oxide synthase (nNOS), susceptibility of nNOS knockout (KO) mice and sensitization to psychostimulants after neurotoxic doses of METH. Male SwissWebster mice were treated with or without 7-nitroindazole (7-NI) along with METH (5 mg/kg,ip,q 3h × 3) and were sacrificed 72 h after the last METH injection. Dopamine (DA) and dopamine transporter (DAT) binding sites were determined in striatum from saline and METH-treated animals. 7-NI completely protected against the depletion of DA, and DAT in striatum. In follow-up experiments nNOS KO mice along with appropriate control (C57BL/6N, SV129 and B6JSV129) mice were treated with METH (5 mg/kg,ip, q 3h × 3) and were sacrificed 72 h after dosing. This schedule of METH administrations resulted in only 10,20% decrease in tissue content of DA and no apparent change in the number of DAT binding sites in nNOS KO mice. However, this regime of METH resulted in a significant decrease in the content of DA as well as DAT binding sites in the wild-type animals. Pre-exposure to single or multiple doses of METH resulted in a marked locomotion sensitization in response to METH. However, the nNOS KO mice show no sensitization in response to METH after single or multiple injections of METH. Therefore, these studies strongly suggest the role of peroxynitrite, nNOS and DA system in METH-induced neurotoxicity and behavioral sensitization. [source] Impairment of CaMKII activation and attenuation of neuropathic pain in mice lacking NR2B phosphorylated at Tyr1472EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2010Shinji Matsumura Abstract Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of long-term potentiation (LTP), which can be triggered by N -methyl- d -aspartate (NMDA) receptor-mediated Ca2+ influx. We previously demonstrated that Fyn kinase-mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F-KI mice with a knock-in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F-KI mice among the major phosphorylation sites. There was no difference in the Ca2+ response to glutamate and sensitivity to NMDA receptor antagonists between naive wild-type and Y1472F-KI mice, and the Ca2+ response to glutamate was attenuated in the Y1472F-KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F-KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase C, at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild-type and Y1472F-KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor-mediated CaMKII signaling in Y1472F-KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain. [source] Demonstration of long-range GABAergic connections distributed throughout the mouse neocortexEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2005Ryohei Tomioka Abstract ,-Aminobutyric acid (GABA)ergic neurons in the neocortex have been mainly regarded as interneurons and thought to provide local interactions. Recently, however, glutamate decarboxylase (GAD) immunocytochemistry combined with retrograde labeling experiments revealed the existence of GABAergic projection neurons in the neocortex. We further studied the network of GABAergic projection neurons in the neocortex by using GAD67-green fluorescent protein (GFP) knock-in mice for retrograde labeling and a novel neocortical GABAergic neuron labeling method for axon tracing. Many GFP-positive neurons were retrogradely labeled after Fast Blue injection into the primary somatosensory, motor and visual cortices. These neurons were labeled not only around the injection site, but also at a long distance from the injection site. Of the retrogradely labeled GABAergic neurons remote from the injection sites, the vast majority (91%) exhibited somatostatin immunoreactivity, and were preferentially distributed in layer II, layer VI and in the white matter. In addition, most of GABAergic projection neurons were positive for neuropeptide Y (82%) and neuronal nitric oxide synthase (71%). We confirmed the long-range projections by tracing GFP-labeled GABAergic neurons with axon branches traveled rostro-caudally and medio-laterally. Axon branches could be traced up to 2 mm. Some (n = 2 of 4) were shown to cross the areal boundaries. The GABAergic projection neurons preferentially received neocortical inputs. From these results, we conclude that GABAergic projection neurons are distributed throughout the neocortex and are part of a corticocortical network. [source] Glial-derived arginine, the nitric oxide precursor, protects neurons from NMDA-induced excitotoxicityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2001Gilbert Grima Abstract Excitotoxic neuronal cell death is characterized by an overactivation of glutamate receptors, in particular of the NMDA subtype, and the stimulation of the neuronal nitric oxide synthase (nNOS), which catalyses the formation of nitric oxide (NO) from l -arginine (L-Arg). At low L-Arg concentrations, nNOS generates NO and superoxide (O2,,), favouring the production of the toxin peroxynitrite (ONOO,). Here we report that NMDA application for five minutes in the absence of added L-Arg induces neuronal cell death, and that the presence of L-Arg during NMDA application prevents cell loss by blocking O2,, and ONOO, formation and by inhibiting mitochondrial depolarization. Because L-Arg is transferred from glial cells to neurons upon activation of glial glutamate receptors, we hypothesized that glial cells play an important modulator role in excitotoxicity by releasing L-Arg. Indeed, as we further show, glial-derived L-Arg inhibits NMDA-induced toxic radical formation, mitochondrial dysfunction and cell death. Glial cells thus may protect neurons from excitotoxicity by supplying L-Arg. This potential neuroprotective mechanism may lead to an alternative approach for the treatment of neurodegenerative diseases involving excitotoxic processes, such as ischemia. [source] Dual effects of NMDA receptor activation on polysialylated neural cell adhesion molecule expression during brainstem postnatal developmentEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2001Farima Bouzioukh Abstract Here we show a dual role of N -methyl- d -aspartate receptor (NMDAR) activation in controlling polysialylated neural cell adhesion molecule (PSA-NCAM) dynamic expression in the dorsal vagal complex (DVC), a gateway for many primary afferent fibres. In this structure the overall expression of PSA-NCAM decreases during the first 2 weeks after birth to persist only at synapses in the adult. Electrical stimulation of the vagal afferents causes a rapid increase of PSA-NCAM expression both in vivo and in acute slices before postnatal day (P) 14 whereas a similar stimulation induces a decrease after P15. Inhibition of NMDAR activity in vitro completely prevented these changes. These regulations depend on calmodulin activation and cGMP production at all stages. By contrast, blockade of neuronal nitric oxide synthase (nNOS) prevented these changes only after P10 in agreement with its late expression in the DVC. The pivotal role of NMDAR is also supported by the observation that chronic blockade induces a dramatic decrease in PSA-NCAM expression. [source] The emerging role of neuronal nitric oxide synthase in the regulation of myocardial functionEXPERIMENTAL PHYSIOLOGY, Issue 6 2006Barbara Casadei The recent discovery of a NOS1 gene product (i.e. a neuronal-like isoform of nitric oxide synthase or nNOS) in the mammalian left ventricular (LV) myocardium has provided a new key for the interpretation of the complex experimental evidence supporting a role for myocardial constitutive nitric oxide (NO) production in the regulation of basal and ,-badrenergic cardiac function. Importantly, nNOS gene deletion has been associated with more severe LV remodelling and functional deterioration in murine models of myocardial infarction, suggesting that nNOS-derived NO may also be involved in the myocardial response to injury. To date, the mechanisms by which nNOS influences myocardial pathophysiology remain incompletely understood. In particular, it seems over simplistic to assume that all aspects of the myocardial phenotype of nNOS knockout (nNOS,/,) mice are a direct consequence of lack of NO production from this source. Emerging data showing co-localisation of xanthine oxidoreductase (XOR) and nNOS in the sarcoplasmic reticulum of rodents, and increased XOR activity in the nNOS,/, myocardium, suggest that nNOS gene deletion may have wider implications on the myocardial redox state. Similarly, the mechanisms regulating the targeting of myocardial nNOS to different subcellular compartments and the functional consequences of intracellular nNOS trafficking have not been fully established. Whether this information could be translated into a better understanding and management of human heart failure remains the most important challenge for future investigations. [source] Thermodynamic and kinetic analysis of the isolated FAD domain of rat neuronal nitric oxide synthase altered in the region of the FAD shielding residue Phe1395FEBS JOURNAL, Issue 12 2004Adrian J. Dunford In rat neuronal nitric oxide synthase, Phe1395 is positioned over the FAD isoalloxazine ring. This is replaced by Trp676 in human cytochrome P450 reductase, a tryptophan in related diflavin reductases (e.g. methionine synthase reductase and novel reductase 1), and tyrosine in plant ferredoxin-NADP+ reductase. Trp676 in human cytochrome P450 reductase is conformationally mobile, and plays a key role in enzyme reduction. Mutagenesis of Trp676 to alanine results in a functional NADH-dependent reductase. Herein, we describe studies of rat neuronal nitric oxide synthase FAD domains, in which the aromatic shielding residue Phe1395 is replaced by tryptophan, alanine and serine. In steady-state assays the F1395A and F1395S domains have a greater preference for NADH compared with F1395W and wild-type. Stopped-flow studies indicate flavin reduction by NADH is significantly faster with F1395S and F1395A domains, suggesting that this contributes to altered preference in coenzyme specificity. Unlike cytochrome P450 reductase, the switch in coenzyme specificity is not attributed to differential binding of NADPH and NADH, but probably results from improved geometry for hydride transfer in the F1395S, and F1395A,NADH complexes. Potentiometry indicates that the substitutions do not significantly perturb thermodynamic properties of the FAD, although considerable changes in electronic absorption properties are observed in oxidized F1395A and F1395S, consistent with changes in hydrophobicity of the flavin environment. In wild-type and F1395W FAD domains, prolonged incubation with NADPH results in development of the neutral blue semiquinone FAD species. This reaction is suppressed in the mutant FAD domains lacking the shielding aromatic residue. [source] Alterations of postsynaptic density proteins in the hippocampus of rat offspring from the morphine-addicted mother: Beneficial effect of dextromethorphanHIPPOCAMPUS, Issue 6 2006San Nan Yang Abstract Infants passively exposed to morphine or heroin through their addicted mothers usually develop characteristic withdrawal syndrome of morphine after birth. In such early life, the central nervous system exhibits significant plasticity and can be altered by various prenatal influences, including prenatal morphine exposure. Here we studied the effects of prenatal morphine exposure on postsynaptic density protein 95 (PSD-95), an important cytoskeletal specialization involved in the anchoring of the NMDAR and neuronal nitric oxide synthase (nNOS), of the hippocampal CA1 subregion from young offspring at postnatal day 14 (P14). We also evaluated the therapeutic efficacy of dextromethorphan, a widely used antitussive drug with noncompetitive antagonistic effects on NMDARs, for such offspring. The results revealed that prenatal morphine exposure caused a maximal decrease in PSD-95 expression at P14 followed by an age-dependent improvement. In addition, prenatal morphine exposure reduced not only the expression of nNOS and the phosphorylation of cAMP responsive element-binding protein at serine 133 (CREBSerine-133), but also the magnitude of long-term depression (LTD) at P14. Subsequently, the morphine-treated offspring exhibited impaired performance in long-term learning and memory at later ages (P28,29). Prenatal coadministration of dextromethorphan with morphine during pregnancy and throughout lactation could significantly attenuate the adverse effects as described above. Collectively, the study demonstrates that maternal exposure to morphine decreases the magnitude of PSD-95, nNOS, the phosphorylation of CREBSerine-133, and LTD expression in hippocampal CA1 subregion of young offspring (e.g., P14). Such alterations within the developing brain may play a role for subsequent neurological impairments (e.g., impaired performance of long-term learning and memory). The results raise a possibility that postsynaptic density proteins could serve an important role, at least in part, for the neurobiological pathogenesis in offspring from the morphine-addicted mother and provide tentative therapeutic strategy. © 2006 Wiley-Liss, Inc. [source] Sex differences in cerebral injury after severe haemorrhage and ventricular fibrillation in pigsACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 3 2010E. SEMENAS Background: Experimental studies of haemorrhagic shock have documented a superior haemodynamic response and a better outcome in female animals as compared with male controls. Such sexual dimorphism has, nevertheless, not been reported after circulatory arrest that follows exsanguination and shock. We aimed to study differences in cerebral injury markers after exsanguination cardiac arrest in pre-pubertal piglets. The hypothesis was that cerebral injury is less extensive in female animals, and that this difference is independent of sexual hormones or choice of resuscitative fluid. Methods: Thirty-two sexually immature piglets (14 males and 18 females) were subjected to 5 min of haemorrhagic shock followed by 2 min of ventricular fibrillation and 8 min of cardiopulmonary resuscitation, using three resuscitation fluid regimens (whole blood, hypertonic saline and dextran, or acetated Ringers' solution plus whole blood and methylene blue). Haemodynamic values, cellular markers of brain injury and brain histology were studied. Results: After successful resuscitation, female piglets had significantly greater cerebral cortical blood flow, tended to have lower S-100, values and a lower cerebral oxygen extraction ratio. Besides, in female animals, systemic and cerebral venous acidosis were mitigated. Female piglets exhibited a significantly smaller increase in neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) expression in their cerebral cortex, smaller blood,brain-barrier (BBB) disruption and significantly smaller neuronal injury. Conclusion: After resuscitation from haemorrhagic circulatory arrest, cerebral reperfusion is greater, and BBB permeability and neuronal injury is smaller in female piglets. An increased cerebral cortical iNOS and nNOS expression in males implies a mechanistic relationship with post-resuscitation neuronal injury and warrants further investigation. [source] Delayed pre-conditioning by 3-nitropropionic acid prevents 3,4-methylenedioxymetamphetamine-induced 5-HT deficitsJOURNAL OF NEUROCHEMISTRY, Issue 3 2010Elena Puerta J. Neurochem. (2010) 114, 843,852. Abstract The aim of the present study was to investigate whether late pre-conditioning using 3-nitropropionic acid (3NP) prevents the 5-hydroxytryptamine (5-HT) deficits caused by the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA) in the rat. For this purpose we administered 3NP 24 h before MDMA (3 × 5 mg/kg i.p., every 2 h) and rats were killed 7 days later. Pre-treatment of 3NP afforded complete protection against MDMA-induced 5-HT deficits independent of any effect on MDMA-induced hyperthermia or 5-HT transporter activity. To identify the transductional mechanisms responsible for the neuroprotective effect of 3NP, we first examined the involvement of nitric oxide (NO) by using selective inhibitors of all three nitric oxide synthase isoforms. Inhibition of endothelial and neuronal nitric oxide synthase, but not inducible nitric oxide synthase, reversed 3NP-induced pre-conditioning. The NO donor S -Nitroso- N -acetylpenicilamine mimicked 3NP effects further suggesting the involvement of NO in mediating 3NP protection. To investigate the involvement of NOS/soluble guanylate cyclase (sGC)/protein kinase G/mitochondrial ATP-sensitive potassium channels (mitoKATP) signaling pathway we examined the effect of 5-hydroxydecanoate (5-HD), a selective mitoKATP blocker, and 1H -(1,2,4)oxadiazolo[4,3- a]quinoxaline-1-one, a potent inhibitor of sGC, on 3NP-induced tolerance. 5-hydroxydecanoate, but not 1H -(1,2,4)oxadiazolo[4,3- a]quinoxaline-1-one, suppressed 3NP-mediated protection suggesting that mitoKATP opening, but not NO-mediated activation of sGC, participates in the mechanism underlying tolerance to MDMA. Our data also showed that the protective effect of 3NP was abolished by cycloheximide, supporting the involvement of de novo protein synthesis. In conclusion, 3NP-induced delayed tolerance against 5-HT deficits caused by MDMA occurs via NO production. [source] Characterization of signaling pathway for the translocation of neuronal nitric oxide synthase to the plasma membrane by PACAPJOURNAL OF NEUROCHEMISTRY, Issue 6 2008Takayuki Ohnishi Abstract In the central nervous system, the activation of neuronal nitric oxide synthase (nNOS) is closely associated with activation of NMDA receptor, and trafficking of nNOS may be a prerequisite for efficient NO production at synapses. We recently demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) and NMDA synergistically caused the translocation of nNOS to the membrane and stimulated NO production in PC12 (pheochromocytoma) cells. However, the mechanisms responsible for trafficking and activation of nNOS are largely unknown. To address these issues, here we constructed a yellow fluorescent protein (YFP)-tagged nNOS N-terminal (1,299 a.a.) mutant, nNOSNT-YFP, and visualized its translocation in PC12 cells stably expressing it. PACAP enhanced the translocation synergistically with NMDA in a time- and concentration-dependent manner. The translocation was blocked by inhibitors of protein kinase A (PKA), protein kinase C (PKC), and Src kinase; and the effect of PACAP could be replaced with PKA and PKC activators. The ,-finger region in the PSD-95/disc large/zonula occludens-1 domain of nNOS was required for the translocation of nNOS and its interaction with post-synaptic density-95 (PSD-95), and NO formation was attenuated by dominant negative nNOSNT-YFP. These results demonstrate that PACAP stimulated nNOS translocation mediated by PKA and PKC via PAC1 -receptor (a PACAP receptor) and suggest cross-talk between PACAP and NMDA for nNOS activation by Src-dependent phosphorylation of NMDA receptors. [source] Fenfluramine-induced serotonergic neurotoxicity in mice: lack of neuroprotection by inhibition/ablation of nNOSJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Yossef Itzhak Abstract Previous studies have implicated a role for nitric oxide (NO) and peroxynitrite in methamphetamine-induced dopaminergic neurotoxicity. The present study was undertaken to investigate whether NO is involved in serotonergic neurotoxicity caused by fenfluramine. In the first experiment, the effect of the neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI; 25 mg/kg × 4) on fenfluramine (25 mg/kg × 4)-induced serotonergic neurotoxicity in Swiss Webster mice was investigated. In the second experiment, the effect of fenfluramine (25 mg/kg × 4) on nNOS (,/,) and wild-type (WT) mice was investigated. Fenfluramine induced hypothermia in all three mouse strains, and 7-NI had no thermoregulatory effect. Selective depletion of 5-HT and 5-HT transporter binding sites in the striatum, frontal cortex and hippocampus in all three mouse strains was observed, with no evidence of dopaminergic neurotoxicity. In the first experiment, 7-NI did not attenuate serotonergic neurotoxicity in Swiss Webster mice. In the second experiment, nNOS(,/,) and WT mice were equally sensitive to serotonergic neurotoxicity. These findings suggest that NO and peroxynitrite do not mediate fenfluramine-induced serotonergic neurotoxicity, and that NO is a selective mediator of amphetamines-induced dopaminergic neurotoxicity. [source] L -NAME reverses quinolinic acid-induced toxicity in rat corticostriatal slices: Involvement of src family kinasesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2007Cinzia Mallozzi Abstract Quinolinic acid (QA) is an endogenous excitotoxin acting on N -methyl- d -aspartate receptors (NMDARs) that leads to the pathologic and neurochemical features similar to those observed in Huntington's disease (HD). The mechanism of QA toxicity also involves free radicals formation and oxidative stress. NMDARs are particularly vulnerable to the action of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can act as modulators of the activity of protein tyrosine kinases (PTKs) and phosphotyrosine phosphatases (PTPs). Because QA is able to activate neuronal nitric oxide synthase (nNOS) as well as to stimulate the NMDARs, we evaluated the effect of N,-Nitro- l -arginine-methyl ester (l -NAME), a selective nNOS inhibitor, on QA-induced neurotoxicity in rat corticostriatal slices. In electrophysiologic experiments we observed that slice perfusion with QA induced a strong reduction of field potential (FP) amplitude, followed by a partial recovery at the end of the QA washout. In the presence of l -NAME the recovery of FP amplitude was significantly increased with respect to QA alone. In synaptosomes, prepared from corticostriatal slices after the electrophysiologic recordings, we observed that l -NAME pre-incubation reversed the QA-mediated inhibitory effects on protein tyrosine phosphorylation pattern, c-src, lyn, and fyn kinase activities and tyrosine phosphorylation of NMDAR subunit NR2B, whereas the PTP activity was not recovered in the presence of l -NAME. These findings suggest that NO plays a key role in the molecular mechanisms of QA-mediated excitotoxicity in experimental model of HD. © 2007 Wiley-Liss, Inc. [source] Facilitation of Myocardial PI3K/Akt/nNOS Signaling Contributes to Ethanol-Evoked Hypotension in Female RatsALCOHOLISM, Issue 7 2009Mahmoud M. El-Mas Background:, The mechanism by which ethanol reduces cardiac output (CO) and blood pressure (BP) in female rats remains unclear. We tested the hypothesis that enhancement of myocardial phosphatidylinositol 3-kinase (PI3K)/Akt signaling and related neuronal nitric oxide synthase (nNOS) and/or endothelial nitric oxide synthase (eNOS) activity constitutes a cellular mechanism for the hemodynamic effects of ethanol. Methods:, We measured the level of phosphorylated eNOS (p-eNOS) and p-nNOS in the myocardium of ethanol (1 g/kg intragastric, i.g.) treated female rats along with hemodynamic responses [BP, CO, stroke volume, (SV), total peripheral resistance, (TPR)], and myocardial nitrate/nitrite levels (NOx) levels. Further, we investigated the effect of selective pharmacological inhibition of nNOS with N, -propyl- l -arginine (NPLA) or eNOS with N5 -(1-iminoethyl)- l -ornithine (l -NIO) on cellular, hemodynamic, and biochemical effects of ethanol. The effects of PI3K inhibition by wortmannin on the cardiovascular actions of ethanol and myocardial Akt phosphorylation were also investigated. Results:, The hemodynamic effects of ethanol (reductions in BP, CO, and SV) were associated with significant increases in myocardial NOx and myocardial p-nNOS and p-Akt expressions while myocardial p-eNOS remained unchanged. Prior nNOS inhibition by NPLA (2.5 or 12.5 ,g/kg) attenuated hemodynamic effects of ethanol and abrogated associated increases in myocardial NOx and cardiac p-nNOS contents. The hemodynamic effects of ethanol and increases in myocardial p-Akt phosphorylation were reduced by wortmannin (15 ,g/kg). On the other hand, although eNOS inhibition by l -NIO (4 or 20 mg/kg) in a dose-dependent manner attenuated ethanol-evoked hypotension, the concomitant reductions in CO and SV remained unaltered. Also, selective eNOS inhibition uncovered dramatic increases in TPR in response to ethanol, which appeared to have offset the reduction in CO. Neither NPLA nor l -NIO altered plasma ethanol levels. Conclusions:, These findings implicate the myocardial PI3K/Akt/nNOS signaling in the reductions in BP and CO produced by ethanol in female rats. [source] Mutation analysis of 12 candidate genes for distal hereditary motor neuropathy type II (distal HMN II) linked to 12q24.3JOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 2 2002Joy Irobi Abstract Distal hereditary motor neuropathies (distal HMNs) are characterized by degeneration of anterior horn cells of the spinal cord resulting in muscle weakness and atrophy. Distal HMN type II is genetically linked to chromosome 12q24.3 and located within a 13 cM region flanked by D12S86 and D12S340. We previously excluded 5 positional and functional candidate genes for distal HMN II. Here, we report the exclusion of 12 additional candidate genes localized within the distal HMN II region; the genes include musashi (Drosophila) homolog 1 (MSI1), protein inhibitor of neuronal nitric oxide synthase (PIN), peripherin (PRPH), tubulin alpha ubiquitous (K-ALPHA-1), tubulin alpha 3 (TUBA3), tubulin alpha 6 (TUBA6), splicing factor arginine/serine-rich 9 (SFRS9), U5 snRNP 100 kd (U5-100K), putative chemokine receptor, GTP-binding protein (HM74), MondoA, cut (Drosophila)-like homeobox 2 (CUX2) and ADP-ribosylation factor 3 (ARF3). [source] Ascorbate Inhibits Reduced Arteriolar Conducted Vasoconstriction in Septic Mouse Cremaster MuscleMICROCIRCULATION, Issue 7 2007REBECCA L. MCKINNON ABSTRACT Objective: The mechanism of neuronal nitric oxide synthase (nNOS)-dependent reduction in arteriolar conducted vasoconstriction in sepsis, and the possible protection by antioxidants, are unknown. The authors hypothesized that ascorbate inhibits the conduction deficit by reducing nNOS-derived NO production. Methods: Using intravital microscopy and the cecal ligation and perforation (CLP) model of sepsis (24 h), arterioles in the cremaster muscle of male C57BL/6 wild-type mice were locally stimulated with KCl to initiate conducted vasoconstriction. The authors used the ratio of conducted constriction (500 , m upstream) to local constriction as an index of conduction (CR500). Cremaster muscle NOS enzymatic activity and protein expression, and plasma nitrite/nitrate levels were determined in control and septic mice. Intravenous ascorbate bolus (200 mg/kg in 0.1 ml of saline) was given early (0 h) or delayed at 23 h post CLP. Results: Sepsis reduced CR500 from 0.73 ± 0.03 to 0.21 ± 0.03, increased nNOS activity from 87 ± 9 to 220 ± 29 pmol/mg/h and nitrite/nitrate from 16 ± 1 to 39 ± 3 , M, without affecting nNOS protein expression. Ascorbate at 0 and 23 h prevented/reversed the conduction deficit and the increases in nNOS activity and nitrite/nitrate level. NO donor SNAP (S -nitroso- N -acetylpenicillamine) reestablished the conduction deficit in ascorbate-treated septic mice. Superoxide scavenger MnTBAP (Mn(III)tetrakis(4-benzoic acid)porphyrin chloride) did not affect this deficit. Conclusion: These data indicate that early and delayed intravenous boluses of ascorbate prevent/reverse sepsis-induced deficit in arteriolar conducted vasoconstriction in the cremaster muscle by inhibiting nNOS-derived NO production. [source] NADPH-diaphorase activity in the superficial layers of the superior colliculus of rats during agingMICROSCOPY RESEARCH AND TECHNIQUE, Issue 1 2006Florentina Díaz Abstract Neurons in the superficial layers of the superior colliculus are key elements in the visual system of rodents since they receive extensive afferent projections from retinal ganglion cells. The NADPH-diaphorase histochemical technique was used to detect differences in neuronal nitric oxide synthase (nNOS) in the superficial layers of the superior colliculus (sSC) of young adult (3 months) and aged (24 and 26 months) rats. The orientation of the dendritic processes of NADPH-diaphorase-positive neurons, cross-sectional area, and number of neurons per mm2 were analyzed. NADPH-d histochemistry revealed a high number of NADPH-d-positive cells in the stratum zonale and stratum griseum superficiale in adult and aged animals. NADPH-d-positive neurons were classified into the following morphological types: marginal, horizontal, pyriform, narrow-field vertical, wide-field vertical, and stellate. During aging, narrow field vertical and wide field vertical neurons present somatic atrophy and an increase in dendritic processes with dorsoventral orientation, whereas wide field vertical neurons show a decrease in those with lateromedial orientation. Marginal neurons undergo somatic hypertrophy at 26 months when compared with those at 3 months. The remaining types of neurons do not undergo size changes. Finally, the number of NADPH-d-positive neurons per mm2 in the various types of morphology does not significantly change with age. It is suggested to be likely that the aging process in the nitrergic neurons of the sSC does not lead to significant changes in the synthesis of NO from the constitutive NOS isoforms. Microsc. Res. Tech. 69:21,28, 2006. © 2006 Wiley-Liss, Inc. [source] Localization of sarcoglycan, neuronal nitric oxide synthase, ,-dystroglycan, and dystrophin molecules in normal skeletal myofiber: Triple immunogold labeling electron microscopyMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2001Yoshihiro Wakayama Abstract In order to investigate the mode of existence of the sarcoglycan complex, neuronal nitric oxide synthase (nNOS), ,-dystroglycan, and dystrophin in the normal skeletal myofiber, we examined the ultrastructural localization and mutual spatial relationship of nNOS, ,-dystroglycan, dystrophin, and the individual components of the sarcoglycan complex by using triple immunogold labeling electron microscopy. Each molecule of ,-, ,-, ,- and ,-sarcoglycans is located intracellularly or extracellularly near the muscle plasma membrane mostly in accordance with the sarcoglycan antigenic sites against which the antibodies were generated. The association of different two and/or three sarcoglycan molecules out of ,-, ,-, ,- and ,-sarcoglycan molecules was frequently observed. Each molecule of nNOS, ,-dystroglycan, and dystrophin was ultrastructurally noted along the cell surface of normal skeletal myofibers. Moreover, the close relation of a sarcoglycan molecule with ,-dystroglycan and dystrophin, and the association of nNOS with dystrophin were also confirmed ultrastructurally. Thus, this study demonstrated that the constituting molecules of the sarcoglycan complex, nNOS, ,-dystroglycan, and dystrophin existed in the form of a cluster at the normal muscle plasma membrane. The association of nNOS with dystrophin and its associated glycoproteins may form a macromolecular signaling complex at the muscle plasma membrane. Microsc. Res. Tech. 55:154,163, 2001. © 2001 Wiley-Liss, Inc. [source] Association of neuronal nitric oxide synthase (nNOS) with ,1-syntrophin at the sarcolemmaMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2001Yuko Miyagoe-Suzuki Abstract ,1-syntrophin is a PDZ-containing dystrophin-associated protein, expressed predominantly in striated muscle and brain. ,1-syntrophin null mice generated by gene targeting technique showed no overt muscular dystrophic phenotype. Though other dystrophin-associated proteins were localized at the sarcolemma, neuronal nitric oxide synthase (nNOS) was selectively lost from the membrane fraction but remained in the cytoplasm. Thus, the ,1-syntrophin null mice are useful in the elucidation of the functional importance of nNOS targeting at the sarcolemma. In addition, the mice would facilitate identification of other signaling molecules, which are targeted to dystrophin complex via interaction with ,1-syntrophin. Microsc. Res. Tech. 55:164,170, 2001. © 2001 Wiley-Liss, Inc. [source] Effect of age on the enteric nervous system of the human colonNEUROGASTROENTEROLOGY & MOTILITY, Issue 7 2009C. E. Bernard Abstract, The effect of age on the anatomy and function of the human colon is incompletely understood. The prevalence of disorders in adults such as constipation increase with age but it is unclear if this is due to confounding factors or age-related structural defects. The aim of this study was to determine number and subtypes of enteric neurons and neuronal volumes in the human colon of different ages. Normal colon (descending and sigmoid) from 16 patients (nine male) was studied; ages 33,99. Antibodies to HuC/D, choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), and protein gene product 9.5 were used. Effect of age was determined by testing for linear trends using regression analysis. In the myenteric plexus, number of Hu-positive neurons declined with age (slope = ,1.3 neurons/mm/10 years, P = 0.03). The number of ChAT-positive neurons also declined with age (slope = ,1.1 neurons/mm/10 years of age, P = 0.02). The number of nNOS-positive neurons did not decline with age. As a result, the ratio of nNOS to Hu increased (slope = 0.03 per 10 years of age, P = 0.01). In the submucosal plexus, the number of neurons did not decline with age (slope = ,0.3 neurons/mm/10 years, P = 0.09). Volume of nerve fibres in the circular muscle and volume of neuronal structures in the myenteric plexus did not change with age. In conclusion, the number of neurons in the human colon declines with age with sparing of nNOS-positive neurons. This change was not accompanied by changes in total volume of neuronal structures suggesting compensatory changes in the remaining neurons. [source] | |||||||||||||||||||||||||