Localization Of Nitric Oxide Synthase Research Articles

Nitric Oxide as a Switching Mechanism between Axon Degeneration and Regrowth during Developmental Remodeling

During development, neurons switch among growth states, such as initial axon outgrowth, axon pruning, and regrowth. By studying the stereotypic remodeling of the Drosophila mushroom body (MB), we found that the heme-binding nuclear receptor E75 is dispensable for initial axon outgrowth of MB γ neurons but is required for their developmental regrowth. Genetic experiments and pharmacological manipulations on ex-vivo-cultured brains indicate that neuronally generated nitric oxide (NO) promotes pruning but inhibits regrowth. We found that high NO levels inhibit the physical interaction between the E75 and UNF nuclear receptors, likely accounting for its repression of regrowth. Additionally, NO synthase (NOS) activity is downregulated at the onset of regrowth, at least partially, by short inhibitory NOS isoforms encoded within the NOS locus, indicating how NO production could be developmentally regulated. Taken together, these results suggest that NO signaling provides a switching mechanism between the degenerative and regenerative states of neuronal remodeling.

Lymphangiogenesis and NOS Localization in Healing Process after Tooth Extraction in Akita Mouse.

Type I diabetes, an autoimmune disease, induces insulin deficiency, which then disrupts vascular endothelial cell function, affecting blood and lymphatic vessels. Nitric oxide (NO) is an immune-induced destructive mediator in type I diabetes, and inhibition of its production promotes arteriosclerosis. In this study, lymphangiogenesis and expression of NO synthase (NOS) during the healing process after tooth extraction were investigated immunohistochemically in control (C57BL) and Akita mice as a diabetes model. Between 1, 4, and 10 days after extraction, expression of NOS, vascular endothelial growth factor-C (VEGF-C), VEGF receptor-3 (VEGFR-3), and von Willebrand factor was strongest during the granulation tissue phase. This suggests that severe inflammation triggers regulation of NOS and these other angiogenic and lymphangiogenic factors. During the callus phase, a few days after extraction, induced osteoblasts were positive for VEGF-C and VEGFR-3 in both the control and Akita mice, suggesting that bone formation is active in this period. Bone formation in the Akita group exceeded that in the controls. Bone tissue formation was disrupted under hyperglycemic conditions, however, suggesting that such activity would be insufficient to produce new bone.

The multifaceted role of nitric oxide synthases in mitochondrial biogenesis and cell differentiation

Nitric oxide (NO) is physiologically synthetized by a family of enzymes called NO synthases (NOSs). NO is a pleiotropic second messenger having a fundamental role in several cellular processes including cell differentiation. Being a high reactive molecule, NO must be synthetized in close proximity to the effector/target. For this reason, the subcellular localization of NOSs is tightly regulated by different post-translation mechanisms. Recently, in murine C2C12 myoblasts, we have demonstrated that mitochondrial biogenesis, an essential event for cell differentiation, can be effective only if the site of NO production is located at nuclear level, where NO favors the CREB-dependent expression of PGC-1α gene. The increase of NO flux in nuclei is elicited by the up-regulation and redistribution of neuronal NOS (nNOS) toward nuclei.Herein we show that an upregulation of endothelial NOS (eNOS) occurs during adipocyte differentiation in 3T3-L1 cells. However, differently to differentiating myocytes, a concomitant redistribution of eNOS toward nuclei was not detected. We also observed that, upon treatment with the NO synthesis inhibitor L-NAME, mitochondrial biogenesis as well as triglyceride accumulation that normally occurs during adipogenesis were not impeded. The absence of eNOS in nuclei together with the ineffectiveness of L-NAME suggest that, at least during 3T3-L1 differentiation, NO is not fundamental for the induction of mitochondrial biogenesis and adipogenesis.

Analysis of Changes in Nitric Oxide Synthase Signaling in BB/WOR Diabetic Prostate

Purpose: Diabetes is associated with increased lower urinary tract symptom (LUTS) severity. An underlying cause of LUTS is increased prostate smooth muscle tone. A critical regulator of prostate innervation and tone is nitric oxide (NO), produced by nitric oxide synthase (NOS). Since NO regulates proliferation and relaxation, and NOS signaling is altered in patients with LUTS, we propose that decreased NO in diabetic patients leads to increased LUTS. We examined this hypothesis by quantifying changes in NOS signaling in the BB/WOR diabetic rat prostate. Materials and Methods:Protein and RNA abundance and localization of NOS I, -II and -III were examined in control and diabetic BB rat prostate by Real time RT-PCR, Western, immunohistochemical analysis and in situ.Morphological changes were examined by electron microscopy (EM), and TUNEL. Results:NosIII is the most abundant isoform in ventral and dorsal prostate. NOS I, -II and -III protein and RNA localize to ductal epithelium. NOS III protein and RNA were significantly decreased in diabetic prostate. Apoptosis was increased in diabetic dorsal prostate. EM of the diabetic dorsal prostate showed abundant protein filled vacuoles and abnormal cytoplasmic morphology indicative of apoptosis. Conclusions:Since NOS III is the most abundant form of NOS in the prostate diabetes may contribute to LUTS severity by down regulating NO, which may lead to increased proliferation in ductal epithelium

Nitric oxide synthase in the olfactory center of the snail, Helix pomatia. A light- and electronmicroscopic histochemical study

Event Abstract Back to Event Nitric oxide synthase in the olfactory center of the snail, Helix pomatia. A light- and electronmicroscopic histochemical study K. Nacsa1, K. Elekes1 and Z. Serfőző1* 1 Balaton Limnological Research Institute, Hungarian Academy of Sciences, Department of Experimental Zoology, Hungary Nitric oxide (NO) contributes to the generation of oscillatory waves which are related to the process of memory formation in the procerebrum (PC), the olfactory center of terrestrial snails. The anatomy of NO synthase (NOS) containing neurons is known very little in the PC. Therefore in this study we localized NOS using both NADPH-diaphorase (NADPH-d) histochemical reaction (NADPH-d) and NOS immunohistochemistry in the PC of Helix pomatia. Labeling alternate cryostat sections obtained from the PC revealed an identical NADPH-d and NOS localization; NOS was detected both in the globuli cell bodies and axons, and the different neuropil areas. A gradual from top to base appearance of NOS was seen in the terminal neuropil, whereas it was evenly and strongly expressed in the internal neuropil. A lateral neuropil region receiving NOS positive axon terminals both from the upper and lower tentacles was also intensively labeled. At ultrastructural level NADPH-d cytochemistry revealed that NOS was bound to the nuclear envelope and endoplasmic reticulum elements in the globuli cells, whereas it was attached to the membrane of agranular synaptic vesicles and medium electron dense granular vesicles in the neuropil. These observations refer to the synthesis of NO in globuli cells, and support the involvement of NO both in the process of transmitter release and intercellular signaling in the different neuropil regions. Acknowledgements Support: OTKA, Nos. 78224 (K.E.), PD75276 (Z.S.) Keywords: Sensory and motor activity, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Sensory and motor activity Citation: Nacsa K, Elekes K and Serfőző Z (2011). Nitric oxide synthase in the olfactory center of the snail, Helix pomatia. A light- and electronmicroscopic histochemical study. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00003 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. Z. Serfőző, Balaton Limnological Research Institute, Hungarian Academy of Sciences, Department of Experimental Zoology, Tihany, Hungary, serfozo@tres.blki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers K. Nacsa K. Elekes Z. Serfőző Google K. Nacsa K. Elekes Z. Serfőző Google Scholar K. Nacsa K. Elekes Z. Serfőző PubMed K. Nacsa K. Elekes Z. Serfőző Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Enzyme-Independent NO Stores in Human Skin: Quantification and Influence of UV Radiation

Nitric oxide (NO) has many functions in the skin, including the mediation of inflammation and antimicrobial defense, wound healing, regulation of keratinocyte homeostasis, and regulation of apoptosis following UV radiation. NO is synthesized by a family of NO synthase enzymes, but its rapid release following UV exposure suggests the existence of preformed stores. NO can be converted into nitrite or nitrosothiols that are stable until cleaved by UV to release NO. Using dermal microdialysis, suction blister epidermal samples, and sweat collection, we demonstrated cutaneous concentrations of total NO-related products of 12+/-5.97 microM, 0.03+/-0.03 micromol mg(-1) epidermal protein, and 22+/-9.34 microM, respectively. The predominant oxyanion was nitrate (60-75%) followed by nitrite. S-Nitrosothiols were barely detectable. Serum total NO-related products correlated directly with those of the upper dermis and sweat (R(2)=0.62 and 0.3, respectively). UVA irradiation (10 mW cm(-2)) increased the yield of NO-related products by microdialysis, peaking after 30 minutes. Dialysis with noradrenaline abrogated this rise. Both the skin and the dermal vasculature contain biologically significant stores of NO, particularly nitrite, which can be directly mobilized by UVA irradiation. The level of circulating NO-related products probably determines skin-bound stores.

Expression of nitric oxide synthases and endogenous NO metabolism in bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD), a multifactorial disease of preterm neonates of complex etiology, is a significant problem within very low birth weight infants. Nitric oxide (NO) has been implicated in both the pathogenesis and as a potential therapeutic of this disease. At this time, there is little direct evidence of the changes in NO production and metabolism that occur within BPD in humans. Animal models have implied that reduced nitric oxide synthase (NOS) expression and NO production in the early stages of the disease may be critical factors. However, inflammation and hence iNOS expression, is also thought to play a role. In the present study we have utilized pathological samples to determine changes in the expression of NOS and NO metabolites within late stage BPD. It is our contention that within these samples iNOS expression is increased and associated with increased NO metabolite production. Mild immunostaining of all three nitric oxide synthase (NOS) enzymes (neuronal, inducible and endothelial) is observed in control lung with tight localization to the endothelium and epithelial airway. This tight localization was lost in samples from subjects with BPD. There was also a marked increase in iNOS expression throughout the lung tissue with strong coexistence with an epithelial cell marker cytokeratin. NO reaction products are altered with BPD as evidenced by increased S-nitrosothiol (SNO) and strong nitrotyrosine (NO(2)Y) imunoreactivity. This study demonstrates a strong correlation between products of NO reactivity and NOS localization in BPD.

CONSTITUTIVE NITRIC OXIDE SYNTHASES IN THE HEART FROM HYPERTROPHY TO FAILURE

1. Endogenous myocardial nitric oxide (NO) may modulate the transition from adaptive to maladaptive hypertrophy leading to heart failure. This review summarizes the information on the interrelations between the precise localization of NO synthases (NOS) and their regulatory functions within different compartments of the heart. 2. In rodent models of pressure overload or myocardial infarction, the three NOS isoforms (NOS1, NOS2, NOS3) were shown to play a neutral, protective, or even adverse role in myocardial remodelling, depending on the NOS activity, the location of each NOS and their regulators. 3. The analysis of conditions that modulate the expression of NOS1 and NOS3 in the heart according to physiopathological situations, indicated that, beside the level of total NOS activity, unique changes in NO compartmentation secondary to NOS1 or NOS3 subcellular location might be involved in the development of cardiac hypertrophy and failure. 4. Thus, different circuits in NO-signalling pathways in myocardium might be activated and this principle is a key to understand contradictions existing in NO biology in the heart. Unravelling the mechanisms behind the NO, NOS and cardiac function is still an ongoing challenge.

Vascular Stiffness and Genetic Variation at the Endothelial Nitric Oxide Synthase Locus

Arterial stiffness is a moderately heritable trait that is affected by alterations in the bioavailability of NO. Previous studies have found associations between variants in the gene for endothelial NO synthase (NOS3) and arterial properties. We previously identified a linkage peak for forward pressure wave amplitude in the immediate vicinity of NOS3. Therefore, we evaluated relations between arterial stiffness measures and common genetic variants at this locus. Eighteen single nucleotide polymorphisms capturing approximately 90% of underlying common variation in NOS3 were genotyped in unrelated Framingham Heart Study participants (N=1157; 52.2% women; mean age: 62 years) with routinely ascertained tonometry data that provided 5 arterial phenotypes (forward and reflected pressure wave amplitude, central pulse pressure, carotid-femoral pulse wave velocity, and mean arterial pressure). In women but not men, the genotype for the common NOS3 missense mutation (Glu298Asp, rs1799983) was related to central pulse pressure (women: GG=53+/-0.9, GT=54+/-0.9, and TT=47+/-2.0 mm Hg, P=0.0047; men: GG=50+/-1.0, GT=49+/-0.9, and TT=47+/-1.8 mm Hg, P=0.30) and forward wave amplitude (women: GG=41+/-0.7, GT=42+/-0.7, and TT=38+/-1.6 mm Hg, P=0.029; men: GG=42+/-0.9, GT=41+/-0.8, and TT=39+/-1.5 mm Hg, P=0.47). The only other nominally significant sex-specific association in men but not women was between an intronic polymorphism (rs1800781) and reflected wave amplitude (women: AA=10.4+/-0.4, AG=11.1+/-0.6, and GG=8.9+/-2.2 mm Hg, P=0.50; men: AA=6.1+/-0.3, AG=7.3+/-0.5, and GG=11.3+/-2.3 mm Hg, P=0.014). After adjusting for multiple testing (18 polymorphisms and 5 phenotypes), these nominal associations were no longer significant. The present study was suggestive of modest relations between common genetic variants at the NOS3 locus and arterial stiffness.

Ubiquitous presence of argininosuccinate at millimolar levels in the central nervous system of Aplysia californica

Endogenous nitric oxide (NO) is generated by nitric oxide synthases (NOSs), which convert arginine (Arg) and oxygen to citrulline (Cit) and NO. Cit can be enzymatically transformed back to Arg by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) via a pathway involving argininosuccinate (ArgSuc). Arg, Cit, and ArgSuc levels have been measured in single neurons, neuronal clusters, and neuropil from the nervous system of the common neurobiological model Aplysia californica. Using capillary electrophoresis with laser-induced fluorescence detection, ArgSuc was found to be present in the nervous system in millimolar concentrations at levels significantly exceeding Cit levels (p<0.01). ArgSuc levels are proportional to Arg concentrations in single neurons, whereas they have no clear correlation to the Cit or Arg/Cit ratio. NOS-expressing neurons often exhibit fixative-resistant nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining. Incubation of ganglia with Arg results in an increase in Cit and ArgSuc levels in the NADPH-d-positive neuropil with no effect on ArgSuc levels in NADPH-d-negative neurons, suggesting NOS activity in the neuropil. Similar incubation with Cit leads to decreased ArgSuc levels in NADPH-d-negative neurons. These results can be explained by localization of NOS and ASS in different neurons; therefore, the complete Arg-Cit-NO cycle may not be present in the same neuron. The surprisingly high intracellular ArgSuc concentration suggests alternative sources of ArgSuc and that at least a portion may be formed by the reverse reaction of ASL (catalyzing the conversion of Arg to ArgSuc), which can be inhibited by Cit.

Subcellular targeting and trafficking of nitric oxide synthases

Unlike most other endogenous messengers that are deposited in vesicles, processed on demand and/or secreted in a regulated fashion, NO (nitric oxide) is a highly active molecule that readily diffuses through cell membranes and thus cannot be stored inside the producing cell. Rather, its signalling capacity must be controlled at the levels of biosynthesis and local availability. The importance of temporal and spatial control of NO production is highlighted by the finding that differential localization of NO synthases in cardiomyocytes translates into distinct effects of NO in the heart. Thus NO synthases belong to the most tightly controlled enzymes, being regulated at transcriptional and translational levels, through co- and post-translational modifications, by substrate availability and not least via specific sorting to subcellular compartments, where they are in close proximity to their target proteins. Considerable efforts have been made to elucidate the molecular mechanisms that underlie the intracellular targeting and trafficking of NO synthases, to ultimately understand the cellular pathways controlling the formation and function of this powerful signalling molecule. In the present review, we discuss the mechanisms and triggers for subcellular routing and dynamic redistribution of NO synthases and the ensuing consequences for NO production and action.

Novel Pathogenetic Mechanisms in Myocarditis: Nitric Oxide Signaling

The nitric oxide (NO) signaling pathway plays important roles in the regulation of most organ systems, participating in physiologic regulation and pathophysiologic organ dysfunction. Physiologic NO signaling is mediated by the precise subcellular localization of NO synthases (NOS) in proximity to target effector molecules [1–4]. Organ dysfunction can occur by NOS downregulation, loss of spatial localization, or the induction of high-output NOS isoforms, such as inducible or calcium-independent NOS (iNOS or NOS2), leading to nitrosative stress. Myocarditis represents a prototypic clinical scenario for the dysregulation of NOS isoforms within the heart. This article reviews the physiologic roles for neuronal NOS (nNOS or NOS1) and endothelial NOS (eNOS or NOS3) in cardiac function, and the various consequences of spatial regulation of NOS

Common Genetic Variation at the Endothelial Nitric Oxide Synthase Locus and Relations to Brachial Artery Vasodilator Function in the Community

Sequence variants at the endothelial nitric oxide synthase (NOS3) locus have been associated with endothelial function measures, but replication has been limited. In reference pedigrees, we characterized linkage disequilibrium structure at the NOS3 locus using 33 common single nucleotide polymorphisms (SNPs). Eighteen SNPs that capture underlying common variation were genotyped in unrelated Framingham Heart Study participants (49.5% women; mean age, 62 years) with measured brachial artery flow-mediated dilation (n=1446) or hyperemic flow velocity (n=1043). Within 3 defined blocks of strong linkage disequilibrium that spanned NOS3, 11 SNPs captured >80% of common haplotypic variation. Among men, there were nominally significant associations between 8 NOS3 SNPs (minimum P=0.002) and between haplotypes (minimum P=0.002) and either flow-mediated dilation or hyperemic flow velocity. In women, we did not observe significant associations between NOS3 SNPs or haplotypes and endothelial function measures. To correct for multiple testing, we constructed 1000 bootstrapped null data sets and found that empirical probability values exceeded 0.05 for both phenotypes. A parsimonious set of SNPs captures common genetic variation at the NOS3 locus. A conservative interpretation of our results is that, accounting for multiple testing, we did not observe statistically significant relations between NOS3 sequence variants and endothelial function measures in either sex. The nominal associations of select NOS3 variants with endothelial function in men (unadjusted for multiple testing) should be viewed as hypothesis-generating observations and may merit testing in other cohorts and experimental designs.

Roles of nitric oxide and prostaglandins in pathogenesis of delayed colonic transit after burn injury in rats

Burn injury has been shown to impair gut transit, but the exact mechanism remains unknown. The present study investigated whether nitric oxide synthase (NOS) and cyclooxygenase (COX) mediated changes in burn-induced colonic transit. After rats underwent 30% total body surface area burn injury, they were injected with S-methylisothiourea (SMT, selective inducible NOS inhibitor), 7-nitronidazole (7-NI, selective neuronal NOS inhibitor), and nimesulide (NIM, selective COX-2 inhibitor), respectively. The protein and mRNA of NOS and COX-2 were measured by Western blot analysis and real-time RT-RCR, and localization of NOS and COX-2 protein was determined by immunohistochemistry. Our results showed that colonic transit assessed by the geometric center was delayed from 3.47+/-0.28 in controls to 2.21+/-0.18 after burn (P<0.009). SMT and NIM significantly improved colonic transit in burned rats but had no effect in sham-operated rats. 7-NI failed to modify delayed transit in burned rats but significantly delayed colonic transit in sham-operated rats. Both protein and mRNA of inducible NOS and COX-2 increased significantly but not neuronal NOS in burned rats. Inducible NOS protein expression was noted not only in epithelial cells but also in neurons of the myenteric ganglia in burned rats. These findings suggest that nitric oxide (NO) produced by neuronal NOS plays an important role in mediating colonic transit under the physiological condition. NO produced by inducible NOS and prostaglandins synthesized by COX-2 are both involved in the pathogenesis of delayed colonic transit after burn injury. Inducible NOS expression in neurons of the myenteric ganglia may contribute to dysmotility with burn injury.

The Roles of Human-Inducible Nitric Oxide Synthase and Xanthine Oxidase in Regulating Chronic Inflammation in Radicular Cysts

The immunohistochemical activities of nitric oxide synthase (NOS), xanthine oxidase(XO) and nitrotyrosine(NT) in human radicular cysts were measured. The molecular interactions of nitric oxide (NO) or peroxynitrate(ONOO-) and superoxide(O2- •) generated in the xanthine/XO system were kinetically investigated in vitro to clarify the mechanism of tissue injury. To investigate the interaction between NO • and O2- • generated in radicular cysts, the localization of NOS and XO was evaluated by an immunohistochemical staining method. Furthermore, the presence of ONOO- was immunohistochemically estimated. The molecular interactions of O2- •, NO • and ONOO- were analyzed by using the electron spin resonance spin-trapping method. In an area of inflammatory reaction, human-inducible NOS (iNOS), XO and NT were positive compared with a normal area, although only iNOS production was confirmed an area of fibrous reaction. Kinetic studies showed that 1-hydroxy-2-oxo-3 (N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7) reacted directly with O2- •, whereas ONOO- did not react with O2- • but inhibited XO directly. The close involvement of iNOS production in the inflammatory region and the influence of the inflammatory reaction with both NO • production from iNOS and O2- • production from XO were suggested. The presence of iNOS in cysts suggests that NO • produced in periapical lesions plays an important role in the regulation of chronic lesions via the reaction with O2- • produced catalyzed by XO. From the reaction with O2- •, and NO • generated by the iNOS system, the formation of ONOO- and its direct inhibition of XO activity decreased the amount of O2- • produced and as a result inhibited the continued formation of ONOO-.

Probable Function of Boettcher Cells based on results of Morphological Study: Localization of Nitric Oxide Synthase

Boettcher cells lie on the basilar membrane beneath Claudius cells. The cells are considered supporting cells for the organ of Corti, and present only in the lower turn of the cochlea, which responds to high-frequency sound. Boettcher cells interdigitate with each other, and project microvilli into the intercellular space. Their structural specialization suggests that Boettcher cells may play a significant role in the function of the cochlea. Nitric oxide synthase (NOS) has previously been detected in substructures of the cochlea. In the cochlea, it is believed that nitric oxide plays an important role in neurotransmission, blood flow regulation, and induction of cytotoxicity under pathological conditions. Findings concerning detection of NOS on Boettcher cells are rare. We demonstrated here the localization of NOS on Boettcher cells of the rat by immunohistochemistry using polyclonal antibody to NOS. On observation with the light microscope using DAB staining, positive immunostaining to NOS was observed in Boettcher cells. In immunoelectron micrographs, NOS was detected abundantly in the cytoplasm of the interdigitations. This suggests that the interdigitations may play significant roles by using NOS. It follows from this that the nitric oxide (NO) on Boettcher cells may influences neighboring Boettcher cells. The ultrastructure of Boettcher cells suggests that they may be active cells, which perform both secretory and absorptive functions.

To NO or not to NO: 'where?' is the question.

Nitric oxide (NO) is a gaseous radical with unique biological functions essential for the cardiovascular system, host defense and neuro-transmission. For two decades it was thought that NO was able to diffuse freely across relatively long distances and to traverse major parts of the cell, if not multiple cell layers. However, NO has been proven to be extremely reactive: it reacts with other reactive oxygen species, heavy metals, as well as with cysteine and tyrosine residues in proteins. In accordance, it is now widely accepted that once NO is generated, it is very short-lived and diffuses only over a short distance. This urges for the local production of NO and the localization of NO synthases in the proximity of their downstream targets. This review discusses the highly organized localization of NO synthases, with the endothelial isoform (eNOS) as its main focus, since from this synthase most is known about its subcellular localization and regulation.

Lymphodynamic properties governing sentinel lymph nodes.

Biological properties of lymph microvessels include characteristics of spontaneous contractions of lymph vessels, nitric oxide (NO)-mediated modulation of active lymph pump activity, flow-induced production and release of NO from lymphatic endothelial cells, and localization of NO synthase in cultured lymphatic endothelial cells. B16-BL6 melanoma cells release factors that inhibit active pump activity in isolated lymph microvessels. We have found that basic fibroblast growth factor (bFGF) induces significant proliferation and migration of canine cultured lymphatic endothelial cells. This growth factor facilitates the formation of capillary-like tubes by the cultured endothelial cells.

Morphological basis of nitric oxide production and its correlation with the polysialylated precursor cells in the dentate gyrus of the adult guinea pig hippocampus.

Neurogenesis in the hippocampus persist throughout life and precursors of neurons reside in the granule cell layer of the dentate gyrus. Until now, the role of nitric oxide (NO) in the phenomenon has been unclear. By using specific antibodies and a confocal laser scanning microscope, the localization of NO synthase (NOS) was examined in the dentate gyrus of the adult guinea pig in relation with the neuronal precursor marker highly polysialylated neural cell adhesion molecule (PSA-N-CAM). Observation of single immunolabeled sections has revealed that both the PSA-N-CAM- and most NOS-positive cells were localized in the granule cell layer of the dentate gyrus. The former were small in size and showed a punctate, clustered immunoreaction with an irregular cellular margin, whereas the latter showed somewhat diverse cellular profiles. Some NOS-positive neurons had elliptical-like morphology with elongated dendrites, whereas others were small, irregularly shaped and mostly lacking dendritic spines. Double immunolabeling has revealed that NOS-immunoreactivity intermingled, as well as colocalized, with that of PSA-N-CAM, particulary in the granule cell layer. The doubly stained cells were morphologically indistinguishable from PSA-N-CAM single positive cells. These results not only suggest the role of NO production in adult hippocampal neurogenesis, but also indicate that some PSA-N-CAM-expressing neuronal precursors produce NO.

Relocalization of neuronal nitric oxide synthase (nNOS) as a marker for complete restoration of the dystrophin associated protein complex in skeletal muscle

A lack of effective treatments for Duchenne muscular dystrophy, a fatal X-linked myopathy, has focused attention on the possibility of gene therapy. The aim of the gene therapy approach is the restoration of the dystrophin associated complex of proteins, one member of which is neuronal nitric oxide synthase, an important enzyme in signal transduction. Transgenic mdx mice and plasmid gene transfer of both human and murine recombinant dystrophins was used to assess whether nNOS could be restored to the sarcolemma following dystrophin gene transfer at a variety of levels of expression. Murine revertant fibres and human patients with different dystrophin deletions were used to assess the relationship between exon deletion and loss of neuronal nitric oxide synthase localization to the sarcolemma. We demonstrate that the domain encoded by exons 45–48 is required for localization of neuronal nitric oxide synthase to the sarcolemma. On the basis of these observations we suggest that neuronal nitric oxide synthase is a useful marker for complete restoration of the dystrophin associated complex and should be used as one of the criteria for selecting the recombinant molecule to be used for gene therapy in Duchenne muscular dystrophy.