Neuronal nNOS Research Articles

Betulinic acid protects against cerebral ischemia–reperfusion injury in mice by reducing oxidative and nitrosative stress

Increased production of reactive oxygen and nitrogen species following cerebral ischemia–reperfusion is a major cause for neuronal injury. In hypercholesterolemic apolipoprotein E knockout (ApoE-KO) mice, 2 h of middle cerebral artery (MCA) occlusion followed by 22 h of reperfusion led to an enhanced expression of NADPH oxidase subunits (NOX2, NOX4 and p22phox) and isoforms of nitric oxide synthase (neuronal nNOS and inducible iNOS) in the ischemic hemisphere compared with the non-ischemic contralateral hemisphere. This was associated with elevated levels of 3-nitrotyrosine, an indicator of peroxynitrite-mediated oxidative protein modification. Pre-treatment with betulinic acid (50 mg/kg/day for 7 days via gavage) prior MCA occlusion prevented the ischemia reperfusion-induced upregulation of NOX2, nNOS and iNOS. In parallel, betulinic acid reduced the levels of 3-nitrotyrosine. In addition, treatment with betulinic acid enhanced the expression of endothelial eNOS in the non-ischemic hemispheres. Finally, betulinic acid reduced infarct volume and ameliorated the neurological deficit in this mouse stroke model. In conclusion, betulinic acid protects against cerebral ischemia–reperfusion injury in mice. This is likely to result from a reduction of oxidative stress (by downregulation of NOX2) and nitrosative stress (by reduction of nNOS and iNOS), and an enhancement of blood flow (by upregulation of eNOS).

Activation of cortical interneurons during sleep: an anatomical link to homeostatic sleep regulation?

Although slow wave activity in the EEG has been linked to homeostatic sleep regulation, the neurobiological substrate of sleep homeostasis is not well understood. Whereas cortical neurons typically exhibit reduced discharge rates during slow wave sleep (SWS), a subpopulation of GABAergic interneurons, which express the enzyme neuronal nitric oxide synthase (nNOS), has recently been found to be activated during SWS. The extent of activation of these nNOS neurons is proportional to homeostatic sleep 'drive'. These cells are an exception among cortical interneurons in that they are projection neurons. We propose that cortical nNOS neurons are positioned to influence neuronal activity across widespread brain areas. They could thus provide a long-sought anatomical link for understanding homeostatic sleep regulation.

Leptin Induces Phosphorylation of Neuronal Nitric Oxide Synthase in Defined Hypothalamic Neurons

Studies have indicated that the neurotransmitter nitric oxide (NO) mediates leptin's effects in the neuroendocrine reproductive axis. However, the neurons involved in these effects and their regulation by leptin is still unknown. We aimed to determine whether NO neurons are direct targets of leptin and by which mechanisms leptin may influence neuronal NO synthase (nNOS) activity. Nicotinamide adenine dinucleotide phosphate diaphorase activity and leptin-induced phosphorylation of signal transducer and activator of transcription-3 immunoreactivity were coexpressed in subsets of neurons of the medial preoptic area, the paraventricular nucleus of the thalamus, the arcuate nucleus (Arc), the dorsomedial nucleus of the hypothalamus (DMH), the posterior hypothalamic area, the ventral premammillary nucleus (PMV), the parabrachial nucleus, and the dorsal motor nucleus of the vagus nerve. Fasting blunted nNOS mRNA expression in the medial preoptic area, Arc, DMH, PMV, and posterior hypothalamic area, and this effect was not restored by acute leptin administration. No difference in the number of neurons expressing nNOS immunoreactivity was noticed comparing hypothalamic sections of fed (wild type and ob/ob), fasted, and fasted leptin-treated mice. However, we found that in states of low leptin levels, as in fasting, or lack of leptin, as in ob/ob mice, the number of neurons expressing the phosphorylated form of nNOS is decreased in the Arc, DMH, and PMV. Notably, acute leptin administration to fasted wild-type mice restored the number of phosphorylated form of nNOS neurons to that observed in fed wild-type mice. Herein we identified the first-order neurons potentially involved in NO-mediated effects of leptin and demonstrate that leptin regulates nNOS activity predominantly through posttranslational mechanisms.

Chronic Intermittent Hypoxia Induces NMDA Receptor-Dependent Plasticity and Suppresses Nitric Oxide Signaling in the Mouse Hypothalamic Paraventricular Nucleus

Chronic intermittent hypoxia (CIH) is a concomitant of sleep apnea that produces a slowly developing chemosensory-dependent blood pressure elevation ascribed in part to NMDA receptor-dependent plasticity and reduced nitric oxide (NO) signaling in the carotid body. The hypothalamic paraventricular nucleus (PVN) is responsive to hypoxic stress and also contains neurons that express NMDA receptors and neuronal nitric oxide synthase (nNOS). We tested the hypothesis that extended (35 d) CIH results in a decrease in the surface/synaptic availability of the essential NMDA NR1 subunit in nNOS-containing neurons and NMDA-induced NO production in the PVN of mice. As compared with controls, the 35 d CIH-exposed mice showed a significant increase in blood pressure and an increased density of NR1 immunogold particles located in the cytoplasm of nNOS-containing dendrites. Neither of these between-group differences was seen after 14 d, even though there was already a reduction in the NR1 plasmalemmal density at this time point. Patch-clamp recording of PVN neurons in slices showed a significant reduction in NMDA currents after either 14 or 35 d exposure to CIH compared with sham controls. In contrast, NO production, as measured by the NO-sensitive fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein, was suppressed only in the 35 d CIH group. We conclude that CIH produces a reduction in the surface/synaptic targeting of NR1 in nNOS neurons and decreases NMDA receptor-mediated currents in the PVN before the emergence of hypertension, the development of which may be enabled by suppression of NO signaling in this brain region.

The effects of glucagon-like peptide 2 on enteric neurons in intestinal inflammation

Intestinal inflammation alters the structure and function of the enteric nervous system (ENS). Glucagon-like peptide 2 (GLP-2) reduces intestinal inflammation and has trophic effects on isolated neurons. This study examined the effects of GLP-2 treatment on the submucosal plexus of rat colon in the trinitrobenzene sulfonic acid (TNBS) model of colitis. After administration of TNBS or saline/ethanol for controls, animals were allocated to treatment with GLP-2 (50 μg kg⁻¹ day⁻¹, s.c.) or sham injection of vehicle, twice daily. Animals were monitored, following clinical parameters, and killed on day 5. The number of neuronal cell bodies per ganglion was quantified using immunohistochemistry on submucosal whole mount preparations, with further characterization of specific subpopulations using antibodies against vasoactive intestinal polypeptide (VIP), neuronal nitric oxide synthase (nNOS), and enteric glial cells with glial fibrillary acid protein and S100. Glucagon-like peptide 2 treatment was associated with a significant amelioration of weight loss, and reduced neutrophil infiltration and microscopic colitis scores in the TNBS animals. Inflammation resulted in a loss of enteric neurons in submucosal ganglia; GLP-2 treatment restored the enteric neuronal populations to normal. In control, non-inflamed animals, GLP-2 treatment increased the number of VIP expressing neurons per ganglion; in TNBS-treated animals, GLP-2 prevented an inflammation-induced reduction in the numbers of VIP expressing neurons per ganglion. Glucagon-like peptide 2 did not change the numbers of nNOS neurons or enteric glial cells in either the control, or inflamed state. These findings show that GLP-2 increased the number of VIP expressing neurons in normal animals, and prevents the inflammation-induced loss of neurons in the colonic submucosal ganglia, with an increase in the proportion of VIP expressing neurons. They suggest that GLP-2 may have a role in protecting or regulating the circuitry of the ENS under basal and inflamed states.

Damage of the interstitial cells of Cajal and myenteric neurons causing ileus in acute necrotizing pancreatitis rats

Small intestinal motility is impaired in acute necrotizing pancreatitis (ANP). The present study was designed to detect the impairment in small intestinal motility and to assess the role of interstitial cells of Cajal (ICC), myenteric neurons and the associated mechanism in the pathogenesis of ileus during experimentally induced acute pancreatitis. ANP was induced by intraperitoneal injections of 30% L-ornithine at a dose of 3 g/kg at hourly intervals. The alterations of small intestine electrical activity--migrating myoelectric complexes (MMCs), and slow waves--were measured 24 hr after ANP induction. The spontaneous mechanical activity and the contractile response to ACh, KCl, tetrodotoxin (TTX) and the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) were evaluated by organ bath technique, and the morphologic alterations of the network of ICC, myenteric neurons and neuronal nitric oxide synthase (nNOS) immunoreactive cells were evaluated using the markers of c-Kit, PGP9.5, and nNOS, respectively. To demonstrate the deficiencies in enteric neuronal origin, we also measured nNOS expression in the muscular layer of ileum. L-ornithine-induced necrotizing pancreatitis manifests with multiple symptoms, including decreased amplitude of spontaneous contractions in small intestinal smooth muscle, declined contractile response to ACh, TTX, and L-NNA in vitro, disrupted MMC cycle length, decreased dominant frequency and dominant power of slow waves in vivo. Furthermore, the morphologic studies demonstrated the damage of ICC (ANP group versus control; P = .000), myenteric neurons (ANP group versus control; P = .001) and nNOS immunoreactive neurons (ANP group versus control; P = .000). We also observed a substantial loss in the expression of nNOS protein in muscular layer of the small intestine (ANP group versus control; P = .032). Our results suggest that the pathogenesis of the small intestinal paralysis in ANP may be related to the deficiencies in ICC and nNOS neurons.

Further characterization of sleep-active neuronal nitric oxide synthase neurons in the mouse brain

We recently demonstrated that Fos is induced in a subpopulation of cortical neuronal nitric oxide synthase (nNOS)-immunoreactive neurons in three rodent species both during spontaneous sleep (SS) and recovery sleep (RS) after a period of sleep deprivation (SD); the proportion of cortical Fos +/nNOS neurons was significantly correlated with non-REM (NREM) sleep delta energy. The present study was undertaken to evaluate the specificity of this state-dependent activation of cortical nNOS cells. The percentage of nNOS neurons that expressed Fos during SD and RS was determined in nine subcortical brain regions and the cortex of the mouse brain; a significantly greater proportion of Fos +/nNOS neurons was observed during RS only in the cortex and in none of the nine subcortical regions. The proportion of calretinin-, calbindin- and parvalbumin-immunoreactive cortical interneurons that expressed Fos during SD and RS was also determined. In contrast to cortical nNOS neurons, a higher percentage of Fos +/calbindin neurons was found during SD than RS; there were no differences in the proportions of Fos-expressing parvalbumin or calretinin neurons between these conditions. Since the nNOS and calretinin cortical interneuron populations overlap extensively in the mouse brain, triple-labeling with these two phenotypic markers and Fos was undertaken in mice from the RS group to determine which combination of markers could best identify the rare “sleep-active” cortical interneuron population. The proportions of both Fos +/nNOS neurons and Fos +/nNOS/calretinin neurons far exceeded the proportion of Fos +/calretinin neurons during RS, but the proportions of these two cell types were not significantly different during RS. Thus, functional activation of nNOS neurons during sleep appears to be restricted to the cerebral cortex and cortical nNOS cells and nNOS/calretinin cells collectively define a cortical interneuron population that is activated during sleep.

10 Altered Gastrointestinal Motility and Reduced nNOS Neurons in TLR4 Mutant-Mice

10 Altered Gastrointestinal Motility and Reduced nNOS Neurons in TLR4 Mutant-Mice

Perinatal food restriction impaired spatial learning and memory behavior and decreased the density of nitric oxide synthase neurons in the hippocampus of adult male rat offspring

Perinatal undernutrition has adverse effects on brain physiology as well as learning and memory activity. However, the mechanism is still incompletely understood. Nitric oxide (NO) synthesized by neuronal nitric oxide synthase (nNOS) has important roles in neuronal survival and synaptic plasticity as well as contributes to the learning and memory task. The aims of the present study were to investigate whether 50% perinatal food restriction (FR50) produced deleterious effects on the population of nNOS neurons in CA1 and CA3 and the dentate gyrus (DG) region of the hippocampus using ABC immunohistochemical method. The results showed FR50 reduced body weight of offspring on postnatal day (PD)1, PD7, PD10, PD14 and PD21, and this type of food restriction impaired learning and memory of adult male offspring rats (postnatal day 70) and decreased the density of nNOS-positive cells in the CA1, CA3 and DG region of the hippocampus. These findings suggest that perinatal undernutrition affects the activity of nNOS in hippocampus. Thus, these changes in the density of nNOS neurons may partly explain learning and memory disturbances commonly observed in undernourished rats and provide clues to the knowledge of malnutrition effects upon the brain.

Tolerance to the cataleptic effect that follows repeated nitric oxide synthase inhibition may be related to functional enzymatic recovery

Systemic or intra-striatal acute administration of nitric oxide synthase (NOS) inhibitors causes catalepsy in rodents. This effect disappears after sub-chronic treatment. The aim of the present study was to investigate if this tolerance is related to changes in the expression of NOS or dopamine-2 (D2) receptor or to a recovery of NOS activity. Male albino Swiss mice (25-30 g) received single or sub-chronic (once a day for 4 days) i.p. injections of saline or L-nitro-arginine (L-NOARG, 40 mg/kg), a non-selective inhibitor of neuronal nitric oxide synthase (nNOS). Twenty-four hours after the last injection, the animals were killed and their brains were removed for immunohistochemistry assay to detect the presence of nNOS or for 'in-situ' hybridisation study using (35)S-labeled oligonucleotide probe complementary to D2 receptor mRNA. The results were analysed by computerised densitometry. Independent groups of animals received the same treatment, but were submitted to the catalepsy test and had their brain removed to measure nitrite and nitrate (NOx) concentrations in the striatum. Acute administration of L-NOARG caused catalepsy that disappeared after sub-chronic treatment. The levels of NOx were significantly reduced after acute L-NOARG treatment. The decrease in NOx after drug injection suffered a partial tolerance after sub-chronic treatment. The catalepsy time after acute or sub-chronic treatment with L-NOARG was negatively (r = -0.717) correlated with NOx levels. Animals that received repeated L-NOARG injections also showed an increase in the number of nNOS-positive neurons in the striatum. No change in D2 receptor mRNA expression was found in the dorsal striatum, nucleus accumbens and substantia nigra. Together, these results suggest that tolerance to L-NOARG cataleptic effects do not depend on changes in D2 receptors. They may depend, however, on plastic changes in nNOS neurons resulting in partial recovery of NO formation in the striatum.

Differentiation of the zebrafish enteric nervous system and intestinal smooth muscle

Development of the enteric nervous system is critical for normal functioning of the digestive system. In vertebrates, enteric precursors originate from the neural crest and migrate into the digestive system. Enteric neurons enable the digestive system to sense and respond to local conditions without the need for central nervous system input. Here we describe major steps in differentiation of the zebrafish enteric nervous system. During migration and neural differentiation of enteric precursors, we identify regions of the enteric nervous system in different phases of differentiation. Early in migration, a small group of anterior enteric neurons are first to form. This is followed by an anterior to posterior wave of enteric neural differentiation later in the migratory phase. Enteric precursors continue proliferating and differentiating into the third day of embryogenesis. nNOS neurons form early while serotonin neurons form late toward the end of enteric neural differentiation. Numbers of enteric neurons increase gradually except during periods of circular and longitudinal intestinal smooth muscle differentiation.

Neuronal nitric oxide synthase immunopositive neurons in cat claustrum—a light and electron microscopic study

Nitric oxide is a unique neurotransmitter, which participates in many physiological and pathological processes in the organism. Nevertheless there are little data about the neuronal Nitric Oxide Synthase immunoreactive (nNOS-ir) neurons and fibers in the dorsal claustrum (DC) of a cat. In this respect the aims of this study were: (1) to demonstrate nNOS-ir in the neurons and fibers of the DC; (2) to describe their light microscopic morphology and distribution; (3) to investigate and analyze the ultrastructure of the nNOS-ir neurons, fibers and synaptic terminals; (4) to verify whether the nNOS-ir neurons consist a specific subpopulation of claustral neurons; (5) to verify whether the nNOS-ir neurons have a specific pattern of organization throughout the DC. For demonstration of the nNOS-ir the Avidin-Biotin-Peroxidase Complex method was applied. Immunopositive for nNOS neurons and fibers were present in all parts of DC. On the light microscope level nNOS-ir neurons were different in shape and size. According to the latter they were divided into three groups-small (with diameter under 15 microm), medium-sized (with diameter from 16 to 20 microm) and large (with diameter over 21 microm). Some of nNOS-ir neurons were lightly-stained while others were darkly-stained. On the electron microscope level the immunoproduct was observed in neurons, dendrites and terminal boutons. Different types of nNOS-ir neurons differ according to their ultrastructural features. Three types of nNOS-ir synaptic boutons were found. As a conclusion we hope that the present study will contribute to a better understanding of the functioning of the DC in cat and that some of the data presented could be extrapolated to other mammals, including human.

Expression of CAPON after Spinal Cord Injury in Rats

The adaptor protein, carboxy-terminal PDZ ligand of nNOS (CAPON), regulates the distribution of neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI) and produces the key signaling molecule nitric oxide (NO). But little is known about the role of CAPON in the pathological process of SCI. The main objective of the present study was to investigate expression of CAPON and nNOS in a spinal cord contusion model in adult rats. Real time-polymerase chain reaction (PCR) and Western blot analysis revealed that mRNA and protein for CAPON increased at 2 h after SCI and reached the peak at 8 h, gradually recovered to the baseline level at 14 days. The expression of nNOS mRNA and protein was similar to that of CAPON. During the peak expression, CAPON mRNA was found in the ventral horn, mediate zone, dorsal horn, and white matter by in situ hybridization. Immunofluorescence showed that CAPON was colocalized with nNOS in neurons, oligodendrocytes, and some astrocytes of spinal cord tissues within 5 mm from the epicenter. Interaction between CAPON and nNOS was also detected by co-immunoprecipitation. Thus, the transient expression of high levels of CAPON may provide new insight into the secondary response after SCI.

Simvastatin Upregulates the Expression of nNOS and eNOS in Neuronal Cells

In a previous study we showed that simvastatin (SIM) therapy normalized sympathetic outflow and cardiovascular reflex regulation in chronic heart failure (CHF). However, the precise neural and cellular pathways for these effects are still unknown. Some studies indicate that statins are neuroprotective, in part, by a nitric oxide (NO)- dependent mechanism. It is also well known that NO is sympathoinhibitory. Therefore, we hypothesized that SIM exerts its beneficial effect on sympathetic outflow in CHF via upregulation of neuronal nNOS and eNOS mechanisms. The aims of this study were to determine the effects of SIM on the expression of nNOS and eNOS in a neuronal cell line (CATH.a) and the expression of nNOS in RVLM and PVN in normal rabbits. The results indicate that SIM (1 umol/L) upregulated nNOS expression (mRNA: 1.21±0.14 to 2.12±0.21; ratio of protein: 0.26±0.02 to 0.37±0.03 P<0.05) and eNOS expression (mRNA: 1.43±0.11 to 3.23±0.51, P<0.05) in CATHa cells by real time PCR and Western blotting. The upregulation the expression of nNOS and eNOS were depressed by AngII (100 nmol/L). We also found a similar effect in the RVLM and PVN in normal rabbits fed SIM (3.0 mg/kg/day) for 3 weeks. These data provide implications for the mechanism by which SIM (and probably other statins) normalize sympathetic outflow and cardiovascular reflex regulation in CHF and hypertension. Supported by NIH grant HL038690.

Neuronal nitric oxide synthase immunopositive neurons in cat vestibular complex: a light and electron microscopic study

Nitric oxide is a unique neurotransmitter, which participates in many physiological and pathological processes in the organism. Nevertheless, there are little data about the neuronal nitric oxide synthase immunoreactivity (nNOS-ir) in the vestibular complex of a cat. In this respect, the aims of this study were to: (1) demonstrate nNOS-ir in the neurons and fibers, from all major and accessory vestibular nuclei; (2) describe their light microscopic morphology and distribution; (3) investigate and analyze the ultrastructure of the NOS I-immunopositive neurons, fibers, and synaptic boutons. For demonstration of the nNOS-ir, the peroxidase-antiperoxidase-diaminobenzidin method was applied. Immunopositive for nNOS neurons and fibers were present in all major and accessory vestibular nuclei. On the light microscope level, the immunopositive neurons were different in shape and size. According to the latter, they were divided into four groups--small (with diameter less than 15 microm), medium-sized (with diameter from 15 to 30 microm), large type I (with diameter from 30 to 40 microm), and large type II (with diameter greater than 40 microm). On the electron microscope level, the immunoproduct was observed in neurons, dendrites, and terminal boutons. According to the ultrastructural features, the neurons were divided into three groups--small (with diameter less than 15 microm), medium-sized (with diameter from 15 to 30 microm), and large (with diameter greater than 30 microm). At least two types of nNOS-ir synaptic boutons were easily distinguished. As a conclusion, we hope that this study will contribute to a better understanding of the functioning of the vestibular complex in cat and that some of the data presented could be extrapolated to other mammals, including human.

Distribution of calbindin-D28k, neuronal nitric oxide synthase, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in the lateral nucleus of the sheep amygdaloid complex

This study describes calbindin-D28k (CB), neuronal nitric oxide synthase (nNOS), and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) expression in the lateral nucleus of the sheep amygdaloid complex. Double immunofluorescence protocol was used in order to determine whether there is colocalization of CB and nNOS. The CB-immunoreactive (IR) neuronal population was composed especially of non-pyramidal neurons, but a few pyramidal cells were also present. The non-pyramidal neurons showed a multipolar and, occasionally, a fusiform morphology. The comparison between single-labeled CB-IR non-pyramidal neurons and cells belonging to CB-IR neuronal population showed they were identical for morphology, mean size, and distribution. The single-labeled CB-IR non-pyramidal neurons were only the 17.8% of the total non-pyramidal neurons counted. The nNOS-IR neuronal population was represented by non-pyramidal multipolar and fusiform neurons. Single-labeled nNOS-IR non-pyramidal neurons had the same morphology, mean area, and distribution as cells belonging to nNOS-IR neuronal population. Single-labeled nNOS-IR non-pyramidal neurons were more numerous than single-labeled CB-IR, and represented the 73.7% of total non-pyramidal neurons counted. NADPH-d-positive cells had the same morphology and distribution as the nNOS-IR neurons. Double immunolabeling (CB/nNOS) was found mostly in non-pyramidal multipolar neurons and only in a few non-pyramidal fusiform cells. These neurons had a mean perikaryal area significantly higher and significantly smaller than that of single-labeled nNOS and single-labeled CB-IR non-pyramidal neurons, respectively. CB and nNOS coexist only in a minority of non-pyramidal neurons (8.5%). The 32.4% of all CB-IR non-pyramidal neurons were nNOS-positive; only 10.4% of nNOS-IR non-pyramidal neurons were CB-positive. These results indicate that CB and nNOS are expressed by selective neurons and that the majority of nNOS-IR non-pyramidal neurons are lacking in CB.

Participation of catecholamines and NO in regulation of apoptosis of nonapeptidergic neurons of neonatal rat pups

Effects of catecholamines (CA) and the character of interaction of CA and NO in regulation of apoptosis were studied in vasopressinergic (VP-ergic) neurons of supraoptic (SON) and paraventricular (PVN) nuclei of rat pups in early postnatal ontogenesis. To study role of CA in regulation of programmed cell death in SON and PVN in the course of embryonal development, pregnant female rats were intraperitoneally injected daily from the 13th to the 20th day with αMPT—a blocker of CA synthesis. The second group of pregnant rats was injected from the 13th to the 20th day with the same volume of saline. The third group was composed of intact animals. The born rat pups were sacrificed at the 3rd and 15th days of life. Caspase 9, Bcl-2, tyrosine hydroxylase, and neuronal NO-synthase (nNOS) in SON and PVN neurons were revealed immunohistochemically, and the amount of immunoreactive substance in neuronal bodies was estimated using the computerized digital analyzer of TV image and Video Test software. Caspase-9 was shown to play an important role in postnatal formation of cellular composition of hypothalamic nonapeptidergic centers by leading to initiation of apoptosis and rejection of “useless” postmitotic SON and PVN neurons. Survival of “useless” nonapeptidergic neurons in early postnatal ontogenesis seems to be connected with antiapoptotic action of Bcl-2. Death of postmitotic neurons, and therefore formation of cellular composition begins earlier and, accordingly, is completed earlier in SON, in which neurons were noted to have a considerable decrease of the caspase-9 expression and, therefore, also a decrease of intensity of neuronal death via caspase-9-dependent pathway. In PVN, neurons continue to die also at the 15th day of rat life, i.e., almost two weeks later than in SON. The observed high correlation between the content of nNOS, caspase-9, and Bcl-2 in the SON and PVN neurons of intact rats of both age groups allows suggesting participation of NO in realization of apoptosis in the course of early postnatal development. The increase of nNOS expression in hypothalamic neurons as a result of disturbances in CA-ergic innervation in embryogenesis might be a possible cause of the long preserved enhancement of expression of apoptosis signal proteins. It can be suggested that CA participate in morphogenesis of hypothalamic neurons by increasing expression of nNOS in neurons and thereby affecting expression of apoptotic proteins.

Mutual regulation between serine and nitric oxide metabolism in human glioblastoma cells

d-Serine indirectly caused dose- and time-dependent inhibition of neuronal nitric oxide synthase (nNOS) without affecting endothelial nitric oxide synthase (eNOS) in human glioblastoma cell line U87. Activity of d-amino acid oxidase (DAAO), catalyzing the oxidative deamination of d-amino acid, was enhanced by NO in a dose-dependent manner. Recently, we have reported that serine racemase (SR) is inhibited by NO and activated by d-serine through nitrosylation and denitrosylation, respectively [K. Shoji, S. Mariotto, A.R. Ciampa, H. Suzuki, Regulation of serine racemase activity by d-serine and nitric oxide in human glioblastoma cells, Neurosci. Lett., in press]. Thus, the metabolism of both d-serine and NO in U87 cells is functionally correlated in a complex manner. Suppression of NO production by d-serine in U87 cells contrasts its known action in enhancing nNOS in neurons.

Brain neuronal nitric oxide synthase neuron-mediated sympathoinhibition is enhanced in hypertensive Dahl rats

To elucidate the role of central neurons containing neuronal nitric oxide synthase (nNOS neurons) in the sympathetic nervous system in hypertensive Dahl salt-sensitive (DS) rats. Dahl rats were fed either a regular-salt (0.4% NaCl) or high-salt (8% NaCl) diet for 4 weeks. The effect of intracerebroventricular administration of S-methyl-L-thiocitrulline, a selective nNOS inhibitor, on renal sympathetic nerve activity was examined in chronically instrumented conscious DS rats. The activity and protein amount of brain nNOS was evaluated by enzyme assay and western blot analysis. The distribution and number of nNOS neurons in the brainstem were examined immunohistochemically in hypertensive and normotensive DS rats. S-methyl-L-thiocitrulline induced a larger increase in tonic renal sympathetic nerve activity generated before baroreflex-mediated inhibition in hypertensive DS rats than normotensive DS rats. Hypertensive DS rats showed increased nNOS activity in the brainstem, but not in the diencephalon or cerebellum. High nNOS activity was confirmed by an increase in the amount of nNOS protein. nNOS Neurons were localized in several nuclei throughout the brainstem; the dorsolateral periaqueductal gray, pedunculopontine tegmental nucleus, dorsal raphe nucleus, laterodorsal tegmental nucleus, lateral parabrachial nucleus, rostral ventrolateral medulla, nucleus tractus solitarius and raphe magnus. The number of nNOS neurons in these nuclei, except for the two raphes, was significantly greater in hypertensive than in normotensive DS rats. These findings suggest that central nNOS-mediated sympathoinhibition may be enhanced in salt-sensitive hypertensive Dahl rats. The upregulated nNOS-mediated inhibition may occur in the central sympathetic control system generated before baroreflex-mediated inhibition.

A Shocking Effect of eNOS

Nitric oxide synthase (NOS) exists in constitutively expressed isoforms (neuronal nNOS and endothelial eNOS) and an inducible isoform (iNOS) that is not apparent under basal conditions. Proinflammatory cytokines and lipopolysaccharides (LPS) stimulate the transcription of iNOS and thereby the production of nitric oxide (NO), which is involved in antibacterial defenses. Overproduction of NO, however, has been implicated in the systemic hypotension and organ failure seen in septic shock. Connelly et al. , who had previously shown that eNOS facilitated macrophage production of iNOS in response to LPS, used eNOS-knockout mice to investigate the role of eNOS in a mouse model of sepsis. The hypotensive effect of LPS on mean arterial blood pressure was markedly attenuated in the eNOS knockout mice, as was the LPS-mediated increase in the plasma concentration of NO − 2 and NO − 3 and in the abundance of iNOS protein. LPS transiently stimulated the phosphorylation of the serine-threonine protein kinase Akt and of eNOS in cultured human umbilical vein endothelial cells (HUVECs). Similarly, LPS transiently stimulated Akt phosphorylation in bone marrow-derived macrophages (BMDM∅s) from both wild-type and knockout mice. Pharmacological analysis in wild-type and knockout BMDM∅s indicated that the phosphoinositide 3-kinase (PI3K)-Akt pathway was involved in LPS-mediated activation of eNOS and the subsequent increases in guanosine 3′,5′-monophosphate (cGMP) accumulation and iNOS expression. Thus, activation of eNOS appears to play an important role in stimulating iNOS transcription in vivo and thereby in the pathogenesis of septic shock. L. Connelly, M. Madhani, A. J. Hobbs, Resistance to endotoxic shock in endothelial nitric-oxide synthase (eNOS) knock-out mice. J. Biol. Chem. 280 , 10040-10046 (2005). [Abstract] [Full Text]