Expression of neuronal NO synthase α- and ß-isoforms in skeletal muscle of mice.

Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and ß-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-µ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/µ, α/non-µ, ß/µ and ß/non-µ. Furthermore, immunoblotting suggests that the transition between nNOS α- and ß-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS ß-isoforms that form homodimers or heterodimers with non-nNOS proteins.

Zymosan and lipopolysaccharide decrease gene expression of neuronal nitric oxide synthase in peripheral organs in chicks.

Nitric oxide (NO) is gaseous bioactive molecule that is synthesized by NO synthase (NOS). Inducible NOS (iNOS) expression occurs in response to pathogenic challenges, resulting in the production of large amounts of NO. However, there is a lack of knowledge regarding neuronal NOS (nNOS) and endothelial NOS (eNOS) in birds during pathogenic challenge. Therefore, the present study was conducted to determine the influence of intraperitoneal (IP) injection of zymosan (cell wall component of yeast) and lipopolysaccharide (LPS, a cell wall component of gram-negative bacteria) on NOS expression in chicks (Gallus gallus). Furthermore, the effect of NOS inhibitors on the corresponding behavioral and physiological parameters was investigated. Zymosan and LPS injections induced iNOS mRNA expression in several organs. Zymosan had no effect on eNOS mRNA expression in the organs investigated, whereas LPS increased its expression in the pancreas. Zymosan and LPS decreased nNOS mRNA expression in the lung, heart, kidney, and pancreas. The decreased nNOS mRNA expression in pancreas was probably associated with the NO from iNOS provided that such effect was reproduced by IP injection of sodium nitroprusside, which is a NO donor. Furthermore, pancreatic nNOS mRNA expression decreased following subcutaneous injection of corticosterone. Furthermore, IP injections of a nonspecific NOS inhibitor, NG-nitro-L-arginine methyl ester, and an nNOS-specific inhibitor, 7-nitroindazole, resulted in the significant decreases in food intake, cloacal temperature, and feed passage via the digestive tract in chicks. Collectively, the current findings imply the decreased nNOS expression because of fungal and bacterial infections, which affects food intake, body temperature, and the digestive function in birds.

Stress during pubertal development affects female sociosexual behavior in mice.

Puberty is a crucial phase for the development of female sexual behavior. Growing evidence suggests that stress during this period may interfere with the development of sexual behavior. However, the neural circuits involved in this alteration remain elusive. Here, we demonstrated in mice that pubertal stress permanently disrupted sexual performance without affecting sexual preference. This was associated with a reduced expression and activation of neuronal nitric oxide synthase (nNOS) in the ventrolateral part of the ventromedial hypothalamus (VMHvl). Fiber photometry revealed that VMHvl nNOS neurons are strongly responsive to male olfactory cues with this activation being substantially reduced in pubertally stressed females. Finally, treatment with a NO donor partially restored sexual performance in pubertally stressed females. This study provides insights into the involvement of VMHvl nNOS in the processing of olfactory cues important for the expression of female sexual behavior. In addition, exposure to stress during puberty disrupts the integration of male olfactory cues leading to reduced sexual behavior.

Cannabidiol induces systemic analgesia through activation of the PI3Kγ/nNOS/NO/KATP signaling pathway in neuropathic mice. A KATP channel S-nitrosylation-dependent mechanism.

BACKGROUND: Cannabidiol (CBD) is the second most abundant pharmacologically active component present in Cannabis sp. Unlike Δ-9-tetrahydrocannabinol (THC), it has no psychotomimetic effects and has recently received significant interest from the scientific community due to its potential to treat anxiety and epilepsy. CBD has excellent anti-inflammatory potential and can be used to treat some types of inflammatory and neuropathic pain. In this context, the present study aimed to evaluate the analgesic mechanism of cannabidiol administered systemically for the treatment of neuropathic pain and determine the endogenous mechanisms involved with this analgesia. METHODS: Neuropathic pain was induced by sciatic nerve constriction surgery, and the nociceptive threshold was measured using the paw compression test in mice. RESULTS: CBD produced dose-dependent antinociception after intraperitoneal injection. Selective inhibition of PI3Kγ dose-dependently reversed CBD-induced antinociception. Selective inhibition of nNOS enzymes reversed the antinociception induced by CBD, while selective inhibition of iNOS and eNOS did not alter this antinociception. However, the inhibition of cGMP production by guanylyl cyclase did not alter CBD-mediated antinociception, but selective blockade of ATP-sensitive K+ channels dose-dependently reversed CBD-induced antinociception. Inhibition of S-nitrosylation dose-dependently and completely reversed CBD-mediated antinociception. CONCLUSION: Cannabidiol has an antinociceptive effect when administered systemically and this effect is mediated by the activation of PI3Kγ as well as by nitric oxide and subsequent direct S-nitrosylation of KATP channels on peripheral nociceptors.

Long-range inhibitory neurons mediate cortical neurovascular coupling.

To meet the high energy demands of brain function, cerebral blood flow (CBF) parallels changes in neuronal activity by a mechanism known as neurovascular coupling (NVC). However, which neurons play a role in mediating NVC is not well understood. Here, we identify in mice and humans a specific population of cortical GABAergic neurons that co-express neuronal nitric oxide synthase and tachykinin receptor 1 (Tacr1). Through whole-tissue clearing, we demonstrate that Tacr1 neurons extend local and long-range projections across functionally connected cortical areas. We show that whisker stimulation elicited Tacr1 neuron activity in the barrel cortex through feedforward excitatory pathways. Additionally, through optogenetic experiments, we demonstrate that Tacr1 neurons are instrumental in mediating CBF through the relaxation of mural cells in a similar fashion to whisker stimulation. Finally, by electron microscopy, we observe that Tacr1 processes contact astrocytic endfeet. These findings suggest that Tacr1 neurons integrate cortical activity to mediate NVC.

Further proof on the role of accumbal nNOS in cocaine-seeking behavior in rats.

BACKGROUND: Cocaine use disorder (CUD) remains a severe health problem with no effective pharmacological therapy. One of the potential pharmacological strategies for CUD pharmacotherapy includes manipulations of the brain glutamatergic (Glu) system which is particularly involved in drug withdrawal and relapse. Previous research indicated a pivotal role of ionotropic N-methyl-D-aspartate (NMDA) receptors or metabotropic receptors' type 5 (mGlu5) receptors in controlling the reinstatement of cocaine. Stimulation of the above molecules results in the activation of the downstream signaling targets such as neuronal nitric oxide synthase (nNOS) and the release of nitric oxide. METHODS: In this paper, we investigated the molecular changes in nNOS in the prefrontal cortex and nucleus accumbens following 3 and 10 days of cocaine abstinence as well as the effectiveness of nNOS blockade with the selective enzyme inhibitor N-ω-propyl-L-arginine hydrochloride (L-NPA) on cocaine seeking in male rats. The effect of L-NPA on locomotor activity in drug-naïve animals was investigated. RESULTS: Ten-day (but not 3-day) cocaine abstinence from cocaine self-administration increased nNOS gene and protein expression in the nucleus accumbens, but not in the prefrontal cortex. L-NPA (0.5-5 mg/kg) administered peripherally did not change locomotor activity but attenuated the reinstatement induced with cocaine priming or the drug-associated conditioned cue. CONCLUSIONS: Our findings support accumbal nNOS as an important molecular player for cocaine seeking while its inhibitors could be considered as anti-cocaine pharmacological tools in male rats.

Modulatory effect of olanzapine on neuronal nitric oxide synthase (nNOS) expression in the rat striatum.

Nitric oxide (NO) has been thought to be a novel factor involved in the mechanisms of mental disorders pathogenesis for quite some time. However, little is known about potential crosstalk between neuronal NO signaling and neuroleptics action. The present work was, therefore, focused on gene expression of neuronal NO synthase (nNOS) in the brains of rats chronically treated with olanzapine, an atypical antipsychotic drug. Studies were carried out on adult, male Sprague-Dawley rats that were divided into 2 groups: control and experimental animals treated with olanzapine (28-day-long intraperitoneal injection, at dose 5 mg/kg daily). All individuals were killed under anesthesia and the whole brains excised. Immunohistochemical procedure was used for histological assessment of the whole brain, and for both descriptive and quantitative analysis of nNOS protein distribution in selected brain structures. Long-term treatment with olanzapine is reflected in different changes in the number of enzyme-expressing cells in the rat brain. Olanzapine decreased the number of nNOS-expressing cells and possibly reduced NO synthesis in the rat striatum. Olanzapine can be taken into account as a potential inhibitor of NO synthesis in the rat striatum.

Stroke Alters the Function of Enteric Neurons to Impair Smooth Muscle Relaxation and Dysregulates Gut Transit.

BACKGROUND: Gut dysmotility is common after ischemic stroke, but the mechanism underlying this response is unknown. Under homeostasis, gut motility is regulated by the neurons of the enteric nervous system that control contractile/relaxation activity of muscle cells in the gut wall. More recently, studies of gut inflammation revealed interactions of macrophages with enteric neurons are also involved in modulating gut motility. However, whether poststroke gut dysmotility is mediated by direct signaling to the enteric nervous system or indirectly via inflammatory macrophages is unknown. METHODS AND RESULTS: We examined these hypotheses by using a clinically relevant permanent intraluminal midcerebral artery occlusion experimental model of stroke. At 24 hours after stroke, we performed in vivo and ex vivo gut motility assays, flow cytometry, immunofluorescence, and transcriptomic analysis. Stroke-induced gut dysmotility was associated with recruitment of muscularis macrophages into the gastrointestinal tract and redistribution of muscularis macrophages away from myenteric ganglia. The permanent intraluminal midcerebral artery occlusion model caused changes in gene expression in muscularis macrophages consistent with an altered phenotype. While the size of myenteric ganglia after stroke was not altered, myenteric neurons from post-permanent intraluminal midcerebral artery occlusion mice showed a reduction in neuronal nitric oxide synthase expression, and this response was associated with enhanced intestinal smooth muscle contraction ex vivo. Finally, chemical sympathectomy with 6-hydroxydopamine prevented the loss of myenteric neuronal nitric oxide synthase expression and stroke-induced slowed gut transit. CONCLUSIONS: Our findings demonstrate that activation of the sympathetic nervous system after stroke is associated with reduced neuronal nitric oxide synthase expression in myenteric neurons, resulting in impaired smooth muscle relaxation and dysregulation of gut transit.

Why is early-onset atrial fibrillation uncommon in patients with Duchenne muscular dystrophy? Insights from the mdx mouse.

AIMS: A reduction in both dystrophin and neuronal nitric oxide synthase (NOS1) secondary to microRNA-31 (miR-31) up-regulation contributes to the atrial electrical remodelling that underpins human and experimental atrial fibrillation (AF). In contrast, patients with Duchenne muscular dystrophy (DMD), who lack dystrophin and NOS1 and, at least in the skeletal muscle, have raised miR-31 expression, do not have increase susceptibility to AF in the absence of left ventricular (LV) dysfunction. Here, we investigated whether dystrophin deficiency is also associated with atrial up-regulation of miR-31, loss of NOS1 protein, and increased AF susceptibility in young mdx mice. METHODS AND RESULTS: Echocardiography showed normal cardiac structure and function in 12-13 weeks mdx mice, with no indication by assay of hydroxyproline that atrial fibrosis had developed. The absence of dystrophin in mdx mice was accompanied by an overall reduction in syntrophin and a lower NOS1 protein content in the skeletal muscle and in the left atrial and ventricular myocardium, with the latter occurring alongside reduced Nos1 transcript levels (exons 1-2 by quantitative polymerase chain reaction) and an increase in NOS1 polyubiquitination [assessed using tandem polyubiquitination pulldowns; P < 0.05 vs. wild type (WT)]. Neither the up-regulation of miR-31 nor the substantial reduction in NOS activity observed in the skeletal muscle was present in the atrial tissue of mdx mice. At difference with the skeletal muscle, the mdx atrial myocardium showed a reduction in the constitutive NOS inhibitor, caveolin-1, coupled with an increase in NOS3 serine1177 phosphorylation, in the absence of differences in the protein content of other NOS isoforms or in the relative expression NOS1 splice variants. In line with these findings, transoesophageal atrial burst pacing revealed no difference in AF susceptibility between mdx mice and their WT littermates. CONCLUSION: Dystrophin depletion is not associated with atrial miR-31 up-regulation, reduced NOS activity, or increased AF susceptibility in the mdx mouse. Compared with the skeletal muscle, the milder atrial biochemical phenotype may explain why patients with DMD do not exhibit a higher prevalence of atrial arrhythmias despite a reduction in NOS1 content.

Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry.

Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN-containing reductase domain, and the heme-containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved. Interestingly, hydrogen-deuterium exchange and mass spectrometry studies revealed interactions of the FMN domain and CaM with the oxygenase domain for iNOS, but not nNOS. This finding prompted us to utilize covalent crosslinking and mass spectrometry to clarify interactions of CaM with nNOS. Specifically, MS-cleavable bifunctional crosslinker disuccinimidyl dibutyric urea was used to identify thirteen unique crosslinks between CaM and nNOS as well as 61 crosslinks within the nNOS. The crosslinks provided evidence for CaM interaction with the oxygenase and reductase domain residues as well as interactions of the FMN domain with the oxygenase dimer. Cryo-EM studies, which gave a high-resolution model of the oxygenase domain, along with crosslink-guided docking provided a model of nNOS that brings the FMN within 15 Å of the heme in support for a more compact conformation than previously observed. These studies also point to the utility of covalent crosslinking and mass spectrometry in capturing transient dynamic conformations that may not be captured by hydrogen-deuterium exchange and mass spectrometry experiments.

The neuronal nitric oxide synthase expression increases during satellite cell-derived primary myoblasts differentiation.

The neuronal nitric oxide synthase (nNOS; encoded by NOS1)-derived nitric oxide (NO) plays an important role in maintaining skeletal muscle mass. In adult skeletal muscle, nNOS localizes to the cell membrane, cytosol, and nucleus, and regulates muscle hypertrophy and atrophy in various subcellular fractions. However, its role in muscle stem cells (also known as muscle satellite cells), which provide myonuclei for postnatal muscle growth, maintenance, and regeneration, remains unclear. The present study aimed to determine nNOS expression in muscle satellite cell-derived primary myoblasts during differentiation and its DNA methylation levels, an epigenetic modification that controls gene expression. Undifferentiated and differentiated satellite cell-derived primary myoblasts were found to express nNOS. Immunohistochemical analysis revealed that nNOS colocalized with Pax7 (satellite cell marker) only in the undifferentiated myoblasts. Furthermore, nNOS immunoreactivity spread to the cytosol of Pax7-negative differentiated myotube-like cells. The level of Nos1µ mRNA, the main isoform of skeletal muscle nNOS, was increased in differentiated satellite cell-derived primary myoblasts compared to that in the undifferentiated cells. However, Nos1 methylation levels remained unchanged during differentiation. These findings suggest that nNOS induction and the appropriate transition of its subcellular localization may contribute to muscle differentiation.

The Physiological Function of nNOS-Associated CAPON Proteins and the Roles of CAPON in Diseases.

In this review, the structure, isoform, and physiological role of the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) are summarized. There are three isoforms of CAPON in humans, including long CAPON protein (CAPON-L), short CAPON protein (CAPON-S), and CAPON-S' protein. CAPON-L includes three functional regions: a C-terminal PDZ-binding motif, carboxypeptidase (CPE)-binding region, and N-terminal phosphotyrosine (PTB) structural domain. Both CAPON-S and CAPON-S' only contain the C-terminal PDZ-binding motif. The C-terminal PDZ-binding motif of CAPON can bind with neuronal nitric oxide synthase (nNOS) and participates in regulating NO production and neuronal development. An overview is given on the relationship between CAPON and heart diseases, diabetes, psychiatric disorders, and tumors. This review will clarify future research directions on the signal pathways related to CAPON, which will be helpful for studying the regulatory mechanism of CAPON. CAPON may be used as a drug target, which will provide new ideas and solutions for treating human diseases.

Discovery of benzyloxy benzamide derivatives as potent neuroprotective agents against ischemic stroke.

Aberrant activation of N-methyl-d-aspartate receptors (NMDAR) and the resulting neuronal nitric oxide synthase (nNOS) excessive activation play crucial pathogenic roles in neuronal damage caused by stroke. Disrupting postsynaptic density protein 95 (PSD95)-nNOS protein-protein interaction (PPI) has been proposed as a potential therapeutic strategy for ischemic stroke without incurring the unwanted side effects of direct NMDAR antagonism. Based on a specific PSD95-nNOS PPI inhibitor (SCR4026), we conducted a detailed study on structure-activity relationship (SAR) to discover a series of novel benzyloxy benzamide derivatives. Here, our efforts resulted in the best 29 (LY836) with improved neuroprotective activities in primary cortical neurons from glutamate-induced damage and drug-like properties. Whereafter, co-immunoprecipitation experiment demonstrated that 29 significantly blocked PSD95-nNOS association in cultured cortical neurons. Furthermore, 29 displayed good pharmacokinetic properties (T1/2 = 4.26 and 4.08 h after oral and intravenous administration, respectively) and exhibited powerful therapeutic effects in rats subjected to middle cerebral artery occlusion (MCAO) by reducing infarct size and neurological deficit score. These findings suggested that compound 29 may be a promising neuroprotection agent for the treatment of ischemic stroke.

Dysfunctional and Dysregulated Nitric Oxide Synthases in Cardiovascular Disease: Mechanisms and Therapeutic Potential.

Nitric oxide (NO) plays an important and diverse signalling role in the cardiovascular system, contributing to the regulation of vascular tone, endothelial function, myocardial function, haemostasis, and thrombosis, amongst many other roles. NO is synthesised through the nitric oxide synthase (NOS)-dependent L-arginine-NO pathway, as well as the nitrate-nitrite-NO pathway. The three isoforms of NOS, namely neuronal (NOS1), inducible (NOS2), and endothelial (NOS3), have different localisation and functions in the human body, and are consequently thought to have differing pathophysiological roles. Furthermore, as we continue to develop a deepened understanding of the different roles of NOS isoforms in disease, the possibility of therapeutically modulating NOS activity has emerged. Indeed, impaired (or dysfunctional), as well as overactive (or dysregulated) NOS activity are attractive therapeutic targets in cardiovascular disease. This review aims to describe recent advances in elucidating the physiological role of NOS isoforms within the cardiovascular system, as well as mechanisms of dysfunctional and dysregulated NOS in cardiovascular disease. We then discuss the modulation of NO and NOS activity as a target in the development of novel cardiovascular therapeutics.

Nitric Oxide Synthase Inhibition Prevents Cell Proliferation in Glioblastoma.

Glioblastoma multiforme (GBM) is a prevalent and aggressive primary brain tumor, presenting substantial treatment challenges and high relapse rates. GBM is characterized by alterations in molecular signaling and enzyme expression within malignant cells. This tumor exhibits elevated nitric oxide (NO.) levels. NO. is a crucial signaling molecule involved in the regulation of neuronal functions, synaptic transmission, and cell proliferation. It is primarily synthesized from L-arginine by nitric oxide synthase (NOS) enzymes. The increased levels of NO. in GBM stem from dysregulated activity and expression of clinically relevant NOS isoforms, particularly inducible NOS (iNOS) and neuronal NOS (nNOS). Based on this knowledge, we hypothesize that targeted pharmacological intervention with N6-(1-iminoethyl)-L-lysine (L-NIL), an iNOS inhibitor, and 7-Nitroindazole (7-NI), an nNOS inhibitor, may suggest a promising therapeutic strategy for the treatment of GBM. To test our hypothesis, we utilized the U87-MG cell line as an in vitro model of GBM. Our results showed that treatment with L-NIL and 7-NI led to a reduction in NO. levels, NOS activity, and clonogenic proliferation in U87-MG cells. These findings suggest that NO. and NOS enzymes might be prospective therapeutic targets for GBM.

ARL-17477 is a dual inhibitor of NOS1 and the autophagic-lysosomal system that prevents tumor growth in vitro and in vivo.

ARL-17477 is a selective neuronal nitric oxide synthase (NOS1) inhibitor that has been used in many preclinical studies since its initial discovery in the 1990s. In the present study, we demonstrate that ARL-17477 exhibits a NOS1-independent pharmacological activity that involves inhibition of the autophagy-lysosomal system and prevents cancer growth in vitro and in vivo. Initially, we screened a chemical compound library for potential anticancer agents, and identified ARL-17477 with micromolar anticancer activity against a wide spectrum of cancers, preferentially affecting cancer stem-like cells and KRAS-mutant cancer cells. Interestingly, ARL-17477 also affected NOS1-knockout cells, suggesting the existence of a NOS1-independent anticancer mechanism. Analysis of cell signals and death markers revealed that LC3B-II, p62, and GABARAP-II protein levels were significantly increased by ARL-17477. Furthermore, ARL-17477 had a chemical structure similar to that of chloroquine, suggesting the inhibition of autophagic flux at the level of lysosomal fusion as an underlying anticancer mechanism. Consistently, ARL-17477 induced lysosomal membrane permeabilization, impaired protein aggregate clearance, and activated transcription factor EB and lysosomal biogenesis. Furthermore, in vivo ARL-17477 inhibited the tumor growth of KRAS-mutant cancer. Thus, ARL-17477 is a dual inhibitor of NOS1 and the autophagy-lysosomal system that could potentially be used as a cancer therapeutic.

Neuroprotective Effects of Nanowired Delivery of Cerebrolysin with Mesenchymal Stem Cells and Monoclonal Antibodies to Neuronal Nitric Oxide Synthase in Brain Pathology Following Alzheimer's Disease Exacerbated by Concussive Head Injury.

Concussive head injury (CHI) is one of the major risk factors in developing Alzheimer's disease (AD) in military personnel at later stages of life. Breakdown of the blood-brain barrier (BBB) in CHI leads to extravasation of plasma amyloid beta protein (ΑßP) into the brain fluid compartments precipitating AD brain pathology. Oxidative stress in CHI or AD is likely to enhance production of nitric oxide indicating a role of its synthesizing enzyme neuronal nitric oxide synthase (NOS) in brain pathology. Thus, exploration of the novel roles of nanomedicine in AD or CHI reducing NOS upregulation for neuroprotection are emerging. Recent research shows that stem cells and neurotrophic factors play key roles in CHI-induced aggravation of AD brain pathologies. Previous studies in our laboratory demonstrated that CHI exacerbates AD brain pathology in model experiments. Accordingly, it is quite likely that nanodelivery of NOS antibodies together with cerebrolysin and mesenchymal stem cells (MSCs) will induce superior neuroprotection in AD associated with CHI. In this review, co-administration of TiO2 nanowired cerebrolysin - a balanced composition of several neurotrophic factors and active peptide fragments, together with MSCs and monoclonal antibodies (mAb) to neuronal NOS is investigated for superior neuroprotection following exacerbation of brain pathology in AD exacerbated by CHI based on our own investigations. Our observations show that nanowired delivery of cerebrolysin, MSCs and neuronal NOS in combination induces superior neuroprotective in brain pathology in AD exacerbated by CHI, not reported earlier.

The importance of the endothelial nitric oxide synthase on the release of 6-nitrodopamine from mouse isolated atria and ventricles and their role on chronotropism.

6-nitrodopamine (6-ND) is released from rat isolated atria, where it acts as a potent positive chronotropic agent. The release of 6-ND from rat isolated atria and ventricles is significantly reduced when pre-incubated with l-NAME, and the release was not affected by tetrodotoxin pre-treatment, indicating that in the heart, the origin of 6-ND is not neurogenic. Since l-NAME inhibits all three isoforms of NO synthase, it was investigated the basal release of 6-ND from isolated atria and ventricles from nNOS-/-, iNOS-/- and eNOS-/- mice of either sex. The release of 6-ND was measured by LC-MS/MS. There were no significant differences in the 6-ND basal release from isolated atria and ventricles from male control mice, as compared to female control mice. The 6-ND release from atria obtained from eNOS-/- mice was significantly reduced when compared to atria obtained from control mice. The 6-ND release in nNOS-/- mice was not significantly different compared to control animals whereas the 6-ND release from atria obtained from iNOS-/- mice was significantly higher when compared to control group. Incubation of the isolated atria with l-NAME caused a significant decrease in the basal atrial rate of control, nNOS-/-, and iNOS-/- mice, but not in eNOS-/- mice. The results clearly indicate that eNOS is the isoform responsible for the synthesis of 6-ND in the mice isolated atria and ventricles and supports the concept that 6-ND is the major mechanism by which endogenous NO modulates heart rate.

Calcium transients in nNOS neurons underlie distinct phases of the neurovascular response to barrel cortex activation in awake mice.

Neuronal nitric oxide (NO) synthase (nNOS), a Ca2+ dependent enzyme, is expressed by distinct populations of neocortical neurons. Although neuronal NO is well known to contribute to the blood flow increase evoked by neural activity, the relationships between nNOS neurons activity and vascular responses in the awake state remain unclear. We imaged the barrel cortex in awake, head-fixed mice through a chronically implanted cranial window. The Ca2+ indicator GCaMP7f was expressed selectively in nNOS neurons using adenoviral gene transfer in nNOScre mice. Air-puffs directed at the contralateral whiskers or spontaneous motion induced Ca2+ transients in 30.2 ± 2.2% or 51.6 ± 3.3% of nNOS neurons, respectively, and evoked local arteriolar dilation. The greatest dilatation (14.8 ± 1.1%) occurred when whisking and motion occurred simultaneously. Ca2+ transients in individual nNOS neurons and local arteriolar dilation showed various degrees of correlation, which was strongest when the activity of whole nNOS neuron ensemble was examined. We also found that some nNOS neurons became active immediately prior to arteriolar dilation, while others were activated gradually after arteriolar dilatation. Discrete nNOS neuron subsets may contribute either to the initiation or to the maintenance of the vascular response, suggesting a previously unappreciated temporal specificity to the role of NO in neurovascular coupling.