The distribution of putative nitric oxide releasing neurones in the locust abdominal nervous system: a comparison of NADPHd histochemistry and NOS-immunocytochemistry

Nitric oxide is well established as a signalling molecule in the nervous system of vertebrates and invertebrates. In this study we evaluate the usefulness of NADPHdiaphorase histochemistry and immunocytochemistry for detecting the presence of nitric oxide synthase in locusts. We describe the distribution of putative nitric oxide releasing neurones and stained neuropiles in the locust ventral nerve cord, in particular the abdominal ganglia and abdominal neuromeres. NADPHdiaphorase histochemistry revealed prominent staining in all neuropilar regions and a specific distribution pattern of stained cell bodies in all examined ganglia. Nitric oxide synthase immunocytochemistry, using a commercially available universal antibody, labelled cells in corresponding positions within the ganglia. This was confirmed by double labelling of alternate sections. Western blot analysis demonstrated that in locusts this universal NOS-antibody binds to a protein of similar size to nitric oxide synthase identified in other insect species. The antibody also labelled axons in most peripheral nerves of all examined ganglia, whereas NADPHdiaphorase histochemistry only revealed such stained fibres within peripheral nerves in some preparations, because they may have been masked by intense background staining. We therefore conclude that nitric oxide synthase-immunocytochemistry and NADPHd histochemistry are both good markers for the presence of nitric oxide synthase in the locust ventral nerve cord, and that nitric oxide may be used as a signalling molecule by efferent neurones in locusts.     

神经型一氧化氮合酶的活性调控

一氧化氮是重要的信使分子,在生物体内参与众多生理及病理过程。生物体内存在着复杂的一氧化氮合酶活性调控机制以精确调控一氧化氮的生成。在神经系统中,一氧化氮主要由神经型一氧化氮合酶催化生成。神经型一氧化氮合酶的活性主要受到翻译后水平上钙离子和钙调蛋白的调控,其调控方式包括二聚化、多位点的磷酸化和去磷酸化,以及主要由PDZ结构域介导的蛋白质-蛋白质相互作用。一氧化氮本身对其合酶的活性具有负反馈调控作用。近年来的研究提示,细胞质膜上的脂筏微区在神经性一氧化氮合酶的活性调控中也起到重要的调节作用。     

Distribution of NADPH Diaphorase-Exhibiting Primary Afferent Neurons in the Trigeminal Ganglion and Mesencephalic Trigeminal Nucleus of the Rabbit

1. Nitric oxide (NO) is highly reactive gaseous molecule to which many physiological and pathological functions have been attributed in the central (CNS) and peripheral (PNS) nervous system. The present investigation was undertaken to map the distribution pattern of the enzyme responsible for the synthesis of NO, nitric oxide synthase (NOS), and especially its neuronal isoform (nNOS) in the population of primary afferent neurons of the trigeminal ganglion (TG) and mesencephalic trigeminal nucleus (MTN) of the rabbit.2. In order to identify neuronal structures expressing nNOS we applied histochemistry to its specific histochemical marker nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd).3. We found noticeable amount of NADPHd-exhibiting primary afferent neurons in TG of the rabbit under physiological conditions. The intensity of the histochemical reaction was highly variable reaching the maximum in the subpopulation of small-to-medium-sized neurons. The large-sized neurons were only weakly stained or actually did not posses any NADPHd-activity. In addition, NADPHd-positive nerve fibers were detected between clusters of the ganglionic cells and in the peripheral branches of the trigeminal nerve (TN). NADPHd-exhibiting MTN neurons were noticed in the whole rostrocaudal extent of the nucleus even though some differences were found concerning the ratio of NADPHd-positive versus NADPHd-negative cell bodies. Similarly, we observed striking diversity in the intensity of NADPHd histochemical reaction in the subpopulations of small-, medium-, and large-sized MTN neurons.4. The predominant localization of NADPHd in the subpopulation of small-to-medium-sized TG neurons which are generally considered to be nociceptive suggests that NO probably takes part in the modulation of nociceptive inputs from the head and face. Furthermore, we tentatively assume that NADPHd-exhibiting MTN neurons probably participate in transmission and modulation of the proprioceptive impulses from muscle spindles of the masticatory muscles and mechanoreceptors of the periodontal ligaments and thus provide sensory feedback of the masticatory reflex arc.     

NADPH-diaphorase histochemistry in the terminal abdominal ganglion of the crayfish

Nitric oxide (NO) has an important modulatory role on the processing of sensory signals in vertebrates and invertebrates. In this investigation we studied the potential sources of NO in the terminal abdominal ganglion of the crayfish, Pacifastacus leniusculus, using NADPH-diaphorase (NADPHd) histochemistry, with NADPHd acting as a marker for NO synthase (NOS). In the terminal ganglion a mean of 27 strongly labelled NADPHd-positive cell bodies were found, and of these 80% [of stained cell bodies] [corrected] occurred in three regions located in antero-lateral, central and posterior parts of the ganglion. Ventral and antero-ventral commissures as well as specific dorsal and ventral areas of the dendritic neuropil showed positive staining. Intense labelling was seen in the ventro-medial tract, and in the connective between the terminal ganglion and the 5th abdominal ganglion. In addition, some motor neurones and neurones with branches in the sensory commissures were NADPHd positive. Our finding that NADPHd-positive cells occur in consistent patterns in the terminal abdominal ganglion implies that NO may have a role in mechanosensory processing in the crayfish.     

Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase.

Nitric oxide is a free radical that has been recently recognized as a neural messenger molecule. Nitric oxide synthase has now been purified and molecularly cloned from brain. Using specific antibodies and oligonucleotide probes, we have localized brain nitric oxide synthase to discrete neuronal populations in the rat and primate brain. Nitric oxide synthase is exclusively neuronal, and its localization is absolutely coincident with NADPH diaphorase staining in both rat and primate.     

Nitric oxide and ATP-sensitive potassium channels mediate lipopolysaccharide-induced depression of central respiratory-like activity in brain slices

Infection may result in early abnormalities in respiratory movement, and the mechanism may involve central and peripheral factors. Peripheral mechanisms include lung injury and alterations in electrolytes and body temperature, but the central mechanisms remain unclear. In the present study, brainstem slices harvested from rats were stimulated with lipopolysaccharide at different doses. Central respiratory activities as demonstrated by electrophysiological activity of the hypoglossal rootlets were examined and the mechanisms were investigated by inhibiting nitric oxide synthase and ATP-sensitive potassium channels. As a result, 0.5 μg/ml lipopolysaccharide mainly caused inhibitory responses in both the frequency and the output intensity, while 5 μg/ml lipopolysaccharide caused an early frequency increase followed by delayed decreases in both the frequency and the output intensity. At both concentrations the inhibitory responses were fully reversed by inhibition of nitric oxide synthase with Nω-nitro-L-arginine methyl ester hydrochloride (20 μM), and by inhibition of ATP- sensitive potassium channels with glybenclamide (100 μM). These results show that direct lipopolysaccharide challenge altered central respiratory activity in dose- and time- related manners. Nitric oxide synthase and ATP-sensitive potassium channels may be involved in the respiratory changes.     

Glutathione depletion is accompanied by increased neuronal nitric oxide synthase activity

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

Interplay of KNDy and nNOS neurons: A new possible mechanism of GnRH secretion in the adult brain

Reproduction in mammals is favoured when there is sufficient energy available to permit the survival of offspring. Neuronal nitric oxide synthase expressing neurons produce nitric oxide in the proximity of the gonadotropin-releasing hormone neurons in the preoptic region. nNOS neurons are an integral part of the neuronal network controlling ovarian cyclicity and ovulation. Nitric oxide can directly regulate the activity of the GnRH neurons and play a vital role neuroendocrine axis. Kisspeptin neurons are essential for the GnRH pulse and surge generation. The anteroventral periventricular nucleus (AVPV), kisspeptin neurons are essential for GnRH surge generation. KNDy neurons are present in the hypothalamus's arcuate nucleus (ARC), co-express NKB and dynorphin, essential for GnRH pulse generation. Kisspeptin-neurokinin B-dynorphin (KNDy) neuroendocrine molecules of the hypothalamus are key components in the central control of GnRH secretion. The hypothalamic neurons kisspeptin, KNDy, nitric oxide synthase (NOS), and other mediators such as leptin, adiponectin, and ghrelin, play an active role in attaining puberty. Kisspeptin signalling is mediated by NOS, which further results in the secretion of GnRH. Neuronal nitric oxide is critical for attaining puberty, but its direct role in adult GnRH secretion is poorly understood. This review mainly focuses on the role of nNOS and its interplay with KNDy neurons in the hormonal regulation of reproduction.     

Role of neuronal nitric oxide synthase in the regulation of the neuroendocrine stress response in rodents: insights from mutant mice

Nitric oxide (NO) is a free radical gas synthesised from arginine and oxygen by enzymes of the family of the nitric oxide synthase. In particular, the neuronal nitric oxide synthase (nNOS) is highly expressed by cells of the hypothalamic paraventricular nucleus, where the sympatho-adrenal system, the hypothalamic-pituitary-adrenal axis and the hypothalamic-neurohypophyseal system originate. These structures are deputed to regulate the neuroendocrine stress response. In the past years, evidence has been accumulated to suggest that NO of nNOS origin plays a significant role in modulating the activity of the above mentioned systems under acute stressor exposure. The availability of nNOS knock-out mice allowed to investigate not only the physiological consequences of a constitutive lack of NO of nNOS origin at the hormonal and molecular level, but also to examine possible behavioural alterations. In this review, we shall discuss and confront the current trends of research in this area, especially focusing on the latest findings gained from genetically modified mice.     

Amyloid-beta (25-35) increases activity of neuronal NO-synthase in rat brain

Nitric oxide (NO) is a free radical with multiple functions in the nervous system. NO plays an important role in the mechanisms of neurodegenerative diseases including Alzheimer's disease. The main source of NO in the brain is an enzymatic activity of nitric oxide synthase (NOS). The aim of the present study was to analyze the expression and activity of both neuronal (nNOS) and inducible (iNOS) isoenzymes in the cerebral cortex and hippocampus of rats after intracerebroventricular administration of amyloid-beta (A beta) peptide fragment A beta(25-35). NADPHd histochemistry as well as immunohistochemistry were also used to investigate nNOS and iNOS expression in rat brain. The data presented here show that A beta(25-35) did not influence levels of nNOS or iNOS mRNA or protein expression in both structures studied. A beta(25-35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity. A beta(25-35) decreased the number of NADPHd-expressing neurons in the neocortex, but it did not significantly influence the number NADPHd-positive cells in the hippocampus. The peptide had no effect on the number of nNOS containing cells. We hypothesize that increased synthesis of NO induced by A beta(25-35) is related to qualitative alterations of nNOS molecule, but not to changes in NOS protein expression.     

Immunohistochemical, Histochemical and Radioassay Analysis of Nitric Oxide Synthase Immunoreactivity in the Lumbar and Sacral Dorsal Root Ganglia of the Dog

Summary In this study, immunohistochemistry for neuronal nitric oxide synthase (bNOS-IR), nicotinamide adenine dinucleotide phosphate diaphorase histochemistry (NADPHd) and nitric oxide synthase radioassay were used to study the occurrence, number and distribution pattern of nitric oxide synthesizing neurons in the lumbar (L1–L7) and sacral (S1–S3) dorsal root ganglia of the dog. Nitric oxide synthase immunolabelling was present in a large number of small- (area <1000 μm²) and medium-sized (area 1000–2000 μm²) as well as in a limited number of large-sized (area >2000 μm²) neurons. Although neuronal nitric oxide synthase immunolabelling and histochemical staining provided intense staining of multiple small- and medium-sized neurons in all lumbar and sacral dorsal root ganglia, immunolabelled or histochemically stained somata exhibited little topographic distribution in individual dorsal root ganglia. Great heterogeneity was noticed in the immunolabelling of medium-sized nitric oxide synthase immunopositive neurons ranging from lightly immunolabelled somata to heavily immunoreactive ones with completely obscured nuclei. Both staining procedures proved to be highly effective in visualizing intraganglionic fibers of various diameters. In general, the largest fibers revealed at the peripheral end of lumbar and sacral dorsal root ganglia were larger, 6.49–9.35 μm in diameter, while those running centrally and proceeding into the dorsal roots were about 30% reduced, ranging between 5.32 and 8.67 μm in diameter. Peripherally, the occurrence of nitric oxide synthase detected in axonal profiles, and confirmed histochemically, in the specimens of the femoral and sciatic nerves, is the first indication of the presence of nitric oxide synthase in the peripheral processes of somata located in L4–S2 dorsal root ganglia. Large and thin central nitric oxide synthase immunoreactive processes of L1–S3 dorsal root ganglion neurons segregate shortly before entering the spinal cord, the former making a massive medial bundle in the dorsal root accompanied by a slim lateral bundle penetrating Lissauer's tract. Quantitative assessment of the distribution of bNOS-IR and/or NADPHd-stained neurons showed a peculiar pattern in relation to spinal levels. Apparent incongruity was found in the total number of NADPHd-stained versus bNOS-IR neurons, demonstrating a clear prevalence of small bNOS-IR somata in all lumbar ganglia, while medium-sized NADPHd-stained somata clearly prevailed all along the rostrocaudal axis with a peak in L5 ganglion. While the number of small bNOS-IR neurons clearly outnumbered NADPHd-stained and NADPHd-unstained somata in S1–S3 ganglia, an inverse relation appeared comparing the total number of medium-sized NADPHd-stained and NADPHd-unstained somata compared with the number of moderate and intense bNOS-IR neurons. Densitometry of bNOS-IR and NADPHd-stained neurons in lumbar and sacral ganglia revealed two distinct subsets of densitometric profiles, one relating to more often found medium-sized bNOS immunolabelled and the other, characteristic for moderately bNOS immunoreactive somata of the same cell size. Considerable differences in catalytic nitric oxide synthase activity, determined by conversion of [³H]arginine to [³H]citrulline were obtained in lumbosacral dorsal root ganglia all along the lumbosacral intumescence, the lowest (0.898± 0.2 dpm/min/μg protein) being in the L4 dorsal root ganglion and the highest (4.194± 0.2 dpm/min/μg protein) in the S2 dorsal root ganglion.     

Ultrastructural localization of NADPH-diaphorase and colocalization of nitric oxide synthase in endothelial cells of the rabbit aorta

This is the first report on the ultrastructural pattern of distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in endothelial cells, using the rabbit aorta, and its colocalization with the neuronal isoform (type I) of nitric oxide synthase. About 30% of the endothelial cells showed a positive reaction for NADPH-d compared to about 6% for nitric oxide synthase immunoreactivity. Simultaneous double histochemical-immunocytochemical labelling procedures indicate that all of the cells displaying nitric oxide synthase-positive reactivity also contained NADPH-d; the remainder of NADPH-d-positive endothelial cells were negative for this isoform of nitric oxide synthase. Nitric oxide synthase-immunogold labelling was mostly associated with free ribosomes, while NADPH-d activity was distributed largely in patches in the cytoplasm and in association with the cell membrane.     

Neurotransmitter release regulated by nitric oxide in PC-12 cells and brain synaptosomes

BACKGROUND: Nitric oxide is a messenger molecule of the nervous system, which is produced by the enzyme nitric oxide synthase, which may regulate cyclic guanosine monophosphate levels and which has been implicated in the control of neurotransmitter release. PC-12 pheochromocytoma cells differentiate to form neuronal cells in culture when they are exposed to nerve growth factor. The levels of cyclic guanosine monophosphate in the cells and their ability to release acetylcholine in response to K(+)-depolarization are both maximal after eight days of treatment with nerve growth factor. We set out to assess a possible role for nitric oxide in the processes that occur in differentiating PC-12 cells. RESULTS: Nitric oxide synthase is first evident in differentiating PC-12 cells eight days after beginning treatment with nerve growth factor, coinciding with the marked increase in K(+)-depolarization-induced release of acetylcholine. The release of both acetylcholine and dopamine in response to K(+)-depolarization is blocked by inhibitors of nitric oxide synthase and by hemoglobin, which binds nitric oxide. Providing l-arginine, a precursor required for nitric oxide synthesis, reverses the effects of the inhibitors. In synaptosomal preparations from the corpus striatum, inhibitors of nitric oxide synthase prevent the release of glutamate in response to the glutamate derivative N-methyl-d-aspartate but not in response to K(+)-depolarization. CONCLUSION: Nitric oxide may mediate the release of acetylcholine and dopamine in response to K(+)-depolarization in PC-12 cells and the release of glutamate in response to N-methyl-d-aspartate in striatal synaptosomes. Nitric oxide synthase expression is induced after eight days of treating PC-12 cells with nerve growth factor, coinciding with a marked enhancement of the release of neurotransmitters in response to K(+)-depolarization.     

一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

Nitrergic Proprioceptive Afferents Originating from Quadriceps Femoris Muscle are Related to Monosynaptic Ia-Motoneuron Stretch Reflex Circuit in the Dog

1. The aim of the present study was to examine the occurrence of the neuronal nitric oxide synthase immunoreactivity in the stretch reflex circuit pertaining to the quadriceps femoris muscle in the dog.2. Immunohistochemical processing for neuronal nitric oxide synthase and histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase were used to demonstrate the presence of neuronal nitric oxide synthase in the proprioceptive afferents issuing in the quadriceps femoris muscle. The retrograde tracer Fluorogold injected into the quadriceps femoris muscle was used to detect the proprioceptive afferents and their entry into the L5 and L6 dorsal root ganglia.3. A noticeable number of medium-sized intensely nitric oxide synthase immunolabelled somata (1000–2000 μm² square area) was found in control animals in the dorsolateral part of L5 and L6 dorsal root ganglia along with large-caliber intraganglionic nitric oxide synthase immunolabelled fibers, presumed to be Ia axons. Before entering the dorsal funiculus the large-caliber nitric oxide synthase immunolabelled fibers of the L5 and L6 dorsal roots formed a massive medial bundle, which upon entering the dorsal root entry zone reached the dorsolateral part of the dorsal funiculus and were distributed here in a funnel-shaped fashion. The largest nitric oxide synthase immunolabelled fibers, 8.0–9.2 μm in diameter, remained close to the dorsal horn, while medium-sized fibers were seen dispersed across the medial portion of the dorsal funiculus. Single, considerably tapered nitric oxide synthase immunolabelled fibers, 2.2–4.6 μm in diameter, were seen to proceed in ventrolateral direction until they reached the mediobasal portion of the dorsal horn and the medial part of lamina VII. In lamina IX, only short fragments of nitric oxide synthase immunoreactive fibers and their terminal ramifications could be seen. Nitric oxide synthase immunolabelled terminals varying greatly in size were identified in control material at the base of the dorsal horn, in the vicinity of motoneurons ventrally and ventrolaterally in L5 and L6 segments and in Clarke’s column of L3 and L4 segments. Injections of the retrograde tracer Fluorogold into the quadriceps femoris muscle and cut femoral nerve, combined with nitric oxide synthase immunohistochemistry of the L5 and L6 dorsal root ganglia, confirmed the existence of a number of medium-sized nitric oxide synthase immunoreactive and Fluorogold-fluorescent somata presumed to be proprioceptive Ia neurons (1000–2000 μm² square area) in the dorsolateral part of both dorsal root ganglia. L5 and L6 dorsal rhizotomy caused a marked depletion of nitric oxide synthase immunoreactivity in the medial bundle of the L5 and L6 dorsal roots and in the dorsal funiculus of L5 and L6 segments.4. The analysis of control material and the degeneration of the large- and medium-caliber nitric oxide synthase immunoreactive Ia fibers in the dorsal funiculus of L5 and L6 segments confirmed the presence of nitric oxide synthase in the afferent limb of the monosynaptic Ia-motoneuron stretch reflex circuit related to the quadriceps femoris muscle. Abbreviations ABC, avidin–biotin complex; bNOS, neuronal nitric oxide synthase; bNOS-IR, neuronal nitric oxide synthase immunoreactive; bNOS-IRB_s, neuronal nitric oxide synthase immunoreactive boutons; cNOS, catalytic nitric oxide synthase; DAB, diaminobenzidine; DF, dorsal funiculus; DH, dorsal horn; DREZ-one, dorsal root entry zone; DRG_s, dorsal root ganglia; eNOS, endothelial nitric oxide synthase; FG, Fluorogold; FN, femoral nerve; mNOS, macrophage nitric oxide synthase; NADPHd, nicotinamide adenine dinucleotide phosphate diaphorase; NBT, nitroblue tetrazolium; NO, nitric oxide; NOS, nitric oxide synthase; NOS-IR, nitric oxide synthase immunoreactive; PBS, phosphate-buffered saline; VGLUT1 and VGLUT2, vesicular glutamate transporters     

Pretreatment of Astrocytes with Interferon-α/β Prevents Neuronal Mitochondrial Respiratory Chain Damage

Abstract: Excessive nitric oxide/peroxynitrite generation has been implicated in the pathogenesis of multiple sclerosis, and the demonstration of increased astrocytic nitric oxide synthase activity in the postmortem brain of multiple sclerosis patients supports this hypothesis. Interferon-β is used for the treatment of multiple sclerosis, but currently little is known regarding its mode of action. Exposure of astrocytes in culture to interferon-γ plus lipopolysaccharide results in stimulation of nitric oxide release. Using a coculture system, we have been able to use astrocytes as a source of nitric oxide/peroxynitrite in an attempt to "model" the effects of raised cytokine levels observed in multiple sclerosis and to monitor the effect on neurones. Our results indicate that stimulation of astrocytic nitric oxide synthase activity causes significant damage to the mitochondrial activities of complexes II/III and IV of neighbouring neurones. This damage was prevented by a nitric oxide synthase inhibitor, suggesting that the damage was nitric oxide-mediated. Furthermore, interferon-α/β also prevented this damage. In view of these results, we suggest that a possible mechanism of action of interferon-β in the treatment of multiple sclerosis is that it prevents astrocytic nitric oxide production, thereby limiting damage to neighbouring cells, such as neurones.     

Spatiotemporal Alterations of the NO/NOS Neuronal Pools Following Transient Abdominal Aorta Occlusion: Morphological and Biochemical Studies in the Rabbit

1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusion may lead to degeneration of specific spinal cord neurons and to irreversible loss of neurological function. The alteration of nitric oxide/nitric oxide synthase (NO/NOS) pool occurring after ischemic insult may play a protective or destructive role in neuronal survival of affected spinal cord segments.2. In the present study, the spatiotemporal changes of NOS following transient ischemia were evaluated by investigating neuronal NOS immunoreactivity (nNOS-IR), reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, and calcium-dependent NOS (cNOS) conversion of [³H] l-arginine to [³H] l-citrulline.3. The greatest levels of these enzymes and activities were detected in the dorsal horn, which appeared to be most resistant to ischemia. In that area, the first significant increase in NADPHd staining and cNOS catalytic activity was found immediately after a 15-min ischemic insult.4. Increases in the ventral horn were observed later (i.e., after a 24-h reperfusion period). While the most intense increase in nNOS-IR was detected in surviving motoneurons of animals with a shorter ischemic insult (13 min), the greatest increase of cNOS catalytic activity and NADPHd staining of the endothelial cells was found after stronger insult (15 min).5. Given that the highest levels of nNOS, NADPHd, and cNOS were found in the ischemia-resistant dorsal horn, and nNOS-IR in surviving motoneurons, it is possible that NO production may play a neuroprotective role in ischemic/reperfusion injury.     

Developmental changes in the response of murine cerebellar granule cells to nitric oxide

Nitric oxide is a diffusible messenger that plays a multitude of roles within the nervous system including modulation of cell viability. However, its role in regulating neuronal survival during a defined period of neurodevelopment has never been investigated. We discovered that expression of the messenger RNA for both neuronal and endothelial nitric oxide synthase increased in the early postnatal period in the cerebellum in vivo, whilst the expression of inducible nitric oxide synthase remained constant throughout this time in development. Whilst scavenging of nitric oxide was deleterious to the survival of early postnatal cerebellar granule neurons in vitro, this effect was lost in cultures derived at increasing postnatal ages. Conversely, sensitivity to exogenous nitric oxide increased with advancing postnatal age. Thus, we have shown that as postnatal development proceeds, cerebellar granule cells alter their in vitro survival responses to both nitric oxide inhibition and donation, revealing that the nitric oxide's effects on developing neurons vary with the stage of development studied. These findings have important consequences for our understanding of the role of nitric oxide during neuronal development.     

Regulation of bone resorption and osteoclast survival by nitric oxide: possible involvement of NMDA-receptor

Nitric oxide has been shown to play an important role in regulation of bone resorption. However, the role of endogenous nitric oxide on osteoclast activity remains still controversial. In this work, using RT-PCR amplification, we demonstrated that rabbit mature osteoclasts express mRNA encoding for neuronal nitric oxide synthase suggesting that this enzyme could be involved in basal nitric oxide production in these cells. Then we assessed the effect of carboxy-PTIO, a nitric oxide scavenger, on in vitro bone resorption and osteoclast survival. Carboxy-PTIO (10-100 microM) inhibited osteoclastic bone resorption in a dose dependent manner and induced osteoclast apoptosis by a mechanism involving caspase 3 activation. These results suggest that basal concentration of endogenous nitric oxide may be essential for normal bone resorption by supporting osteoclast survival. Because osteoclasts express N-methyl-d-aspartate-receptor (NMDA-R), we hypothesized that in osteoclasts NMDA-R may be involved in nitric oxide production as in neuronal cells. We confirmed that blockade of NMDA-R with specific non-competitive antagonists, MK801 and DEP, strongly inhibited bone resorption. As for carboxy-PTIO, we showed that blockade of NMDA-R by both antagonists induced osteoclast apoptosis in a dose dependent manner by a mechanism dependent on caspase 3 activation. Intracellular calcium concentration in osteoclasts decreased within minutes in the presence of both antagonists. Finally, MK801-induced osteoclast apoptosis was partially reversed in the presence of small amount of SNAP (100 nM), a nitric oxide donor, suggesting that the effect of NMDA-R on osteoclast apoptotic cell death could be due to a decrease in nitric oxide production. Taken together, our results are consistent with the hypothesis that NMDA-R on osteoclasts could have a similar function as those in neuronal cells, i.e., to allow a calcium influx, which in turn activates a constitutive neuronal nitric oxide synthase. Nitric oxide generated by this pathway may be essential for osteoclast survival and hence for normal bone resorption.     

Histochemical differentiation between nitric oxide synthase-related and -unrelated diaphorase activity in the rat olfactory bulb

The widely used NADPH-diaphorase reaction for demonstrating neuronal nitric oxide synthase is not as specific as previously thought, as it visualizes both a nitric oxide synthase-related activity and a nitric oxide synthase-unrelated diaphorase. In the present study, we used the rat olfactory bulb as a model to characterize the NADPH-diaphorase activity of neuronal nitric oxide synthase histochemically in comparison with neuronal nitric oxide-unrelated diaphorase activity. The NADPH-diaphorase activity of nitric oxide synthase peaked at pH 8 and at Triton X-100 concentrations of 1--2.5%. It was stable in an acidic environment but was reduced in the presence of Triton X-100 and was inactivated by the flavoprotein inhibitor, diphenyleneiodonium. It preferred beta-NADPH as the co-substrate to alpha-NADPH and alpha-NADH. In contrast, nitric oxide synthase-unrelated diaphorase peaked at pH 10, displayed a Triton X-100 optimum at a concentration of 1%, was unstable in an acidic environment and used beta-NADPH, alpha-NADPH and alpha-NADH to similar extents. Differences in the characteristics between neuronal nitric oxide synthase-related and nitric oxide synthase-unrelated NADPH-diaphorase can be used to increase the specificity of the histochemical nitric oxide synthase marker reaction. © Chapman & Hall