NADPH-diaphorase and nitric oxide synthase in the canine superior cervical ganglion

By means of NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we demonstrate that considerable numbers of NADPH-d-positive neurons are distributed throughout the canine superior cervical ganglion (SCG). These neurons also show NOS immunoreactivity. This finding indicates that NADPH-d histochemistry, a simple and reliable technique, can be used as a reliable marker of NOS activity in the sympathetic innervation of canine head and neck. The present findings suggest that the participation of nitric oxide in the SCG differs greatly between species.     

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.     

Different distributions of nitric oxide synthase-containing neurons in the mouse and rat hypothalamus.

The distribution of nitric oxide synthase-containing neurons was studied in the rat and mouse hypothalamus by immunohistochemistry and NADPH-diaphorase histochemistry. Immunostaining and NADPH-diaphorase staining of hypothalamic neurons were comparable in all hypothalamic nuclei of either species except in the arcuate nucleus that stained positive for nitric oxide synthase immunoreactivity but negative for NADPH-diaphorase reactivity. The presence of nitric oxide synthase-immunopositive neurons in the arcuate nucleus was confirmed by nitric oxide synthase immunofluorescence viewed under the confocal microscope at 1 microm thickness. Cross-species comparison showed that, in general, the number and intensity of nitric oxide synthase-containing neurons were much higher in the rat than in the mouse hypothalamus. Differences in the distribution of nitric oxide synthase-containing neurons between these two rodents were found in most hypothalamic nuclei. In particular, two dense clusters of nitric oxide synthase-containing neurons were found in the paraventricular and supraoptic nuclei of the rat hypothalamus in contrast to their scarcity in the same nuclei of the mouse hypothalamus.     

Nitric oxide synthase in the rat carotid body and carotid sinus

The participation of nitric oxide synthase (NOS) in the innervation of the rat carotid body and carotid sinus was investigated by means of NADPH-diaphorase histochemistry and NOS immunohistochemistry using antisera raised against purified neuronal NOS and a synthetic tridecapeptide. NOS was detected in 23% of neurons at the periphery of the carotid bodies. Some negative neurons were surrounded by NOS-positive terminals. NOS-containing varicose nerve fibres innervated the arterial vascular bed and, to a lesser extent, the islands of glomus cells. These fibres persisted after transection of the carotid sinus nerve and are probably derived from intrinsic neurons. Large NOS-positive axonal swellings in the wall of the carotid sinus were absent after transection of the sinus nerve, indicating their sensory origin. The results suggest a neuronal nitrergic control of blood flow, neuronal activity and chemoreception in the carotid body, and an intrinsic role of NO in the process of arterial baroreception.     

Studies on the localization and expression of nitric oxide synthase using histochemical techniques

Summary This review provides an update on the variety of histochemical techniques available for the cellular localization and expression of nitric oxide synthase in formalin-fixed tissue sections. The techniques of immunohistochemistry and NADPH-diaphorase histochemistry are discussed and the suitability of various types of probes and reporters which are useful forin situ detection of nitric oxide synthase mRNA expression are assessed. Figures are also included which illustrate the techniques described and protocols forin situ hybridization and NADPH-diaphorase histochemistry.     

Neural and endothelial nitric oxide synthase activity in rat penile erectile tissue

NADPH-diaphorase (NADPH-D) activity and immunoreactivity for neural and endothelial nitric oxide synthase (nNOS and eNOS, respectively) were used to investigate nitric oxide (NO) regulation of penile vasculature. Both the histochemical and immunohistochemical techniques for NOS showed that all smooth muscles regions of the penis (dorsal penile artery and vein, deep penile vessels, and cavernosal muscles) were richly innervated. The endothelium of penile arteries, deep dorsal penile vein, and select veins in the crura and shaft were also stained for NADPH-D and eNOS. However, the endothelium of cavernous sinuses was unstained by both techniques. Fewer fibers were seen in the glans penis, those present being associated with small blood vessels and large nerve bundles near the trabecular walls. All penile neurons in the pelvic plexus, located by retrograde transport of a dye placed in the corpora cavernosa penis, were stained by the NADPH-D method. Essentially similar results were obtained with an antibody to nNOS. These data suggest that penile parasympathetic neurons comprise a uniform population, as all seem capable of forming nitric oxide. However, in contrast to the endothelium of penile vessels, the endothelium lining the cavernosal spaces may not be capable of nitric oxide synthesis.     

In vivo inhibition of inducible nitric oxide and evaluation of the brain tissue damage induced by Toxocara canis larvae in experimentally infected mice

Nitric oxide (NO) is known to be produced by macrophages, endothelial cells and neurons and synthesized by an enzyme called nitric oxide synthase (NOS). Various effector mechanisms and infections can affect the NO production. Excessive amount of NO will lead to biochemical reactions, which cause toxic effects. In this study the role of NO has been evaluated in larval toxocarosis, which is a systemic parasite infection caused by T. canis larvae. Infection was established in the Balb/c mice with or without inducible NOS (iNOS) inhibition and the effects of infection and NOS inhibition were observed according to the results of SOD and LPx measurements in brain tissue and NADPH-diaphorase (NADP-d) histochemistry. Results of NADPH-d histochemistry indicate that iNOS inhibition has protective effect on the brains of infected mice and that larval T. canis infection could be related to oxidative stress, and NO production and iNOS inhibition can protect the tissue from damage in this infection.     

Nitric oxide decreases ammonium release in tadpoles of the clawed frog, <Emphasis Type="Italic">Xenopus laevis</Emphasis>, Daudin

In the present study, we quantified the physiological consequences of nitric oxide (NO) on ammonium release in tadpoles of Xenopus laevis. Tadpoles exposed to S-nitro-N-acetylpenicillamine (SNAP), an NO-donor, or l-arginine, the substrate of NO synthase (NOS), showed a reversible decrease, whereas animals exposed to the NOS inhibitor Nω-methyl-l-arginine (l-NMMA) exhibited an increase in ammonium release. Release of ammonium may be of physiological relevance during stress response of the animal. Handling of tadpoles as well as exposure to hyposmotic environments increased ammonium release. To localize NO synthesizing cells, we used diaminofluorescein-diacetate (DAF-2DA), an NO-sensitive fluorescent dye, and NADPH-diaphorase histochemistry, an indicator for NOS activity. We observed a fluorescence signal as well as NADPH-diaphorase activity in small, solitary cells in the epidermis. Similarly to NADPH-diaphorase histochemistry, silver nitrate staining and rhodamine labelling, markers for mitochondria-rich cells, showed a strong reaction in these cells. These observations indicate that NO (1) inhibits ammonium release, and (2) is endogenously synthesized in mitochondria-rich cells in Xenopus tadpoles. Based on our histochemical results, we speculate that gill epithelium and epidermis work in parallel to release ammonium as epidermal tissue contains mitochondria-rich and NADPH-diaphorase positive cells.     

Seasonality in expression and distribution of nitric oxide synthase isoforms in the testis of the catfish, Clarias batrachus: Role of nitric oxide in testosterone production

Nitric oxide (NO) is a well-recognized versatile signaling molecule. It is produced by catalytic action of nitric oxide synthase (NOS) on L-arginine in a variety of animal tissues. Existence of different isoforms of NOS has been shown in mammalian testis, but report on their presence in the testis of ectothermic vertebrates is non-existent. This study demonstrates the differential expressions of two isoforms of nitric oxide synthase (neuronal-nNOS and inducible-iNOS) like molecules in different cell types in the testis of seasonally breeding catfish, Clarias batrachus through immunohistochemistry. Positive immunoprecipitation of nNOS and iNOS like molecules were detected in germ cells as well as interstitial cells only in the recrudescing and fully mature fish. The immunoreactions differed in intensity and varied with changing reproductive status. Treatment of adult male fish with NO donor, sodium nitroprusside, and a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME) increased and decreased the total nitrate and nitrite concentration in the testis, respectively. Sodium nitroprusside and L-NAME also induced simultaneous decline and rise in the testicular testosterone level, respectively. These findings, thus, suggest that NOS isoforms are expressed variedly in different cell types in the testis of reproductively active fish. This investigation also suggests that NO inhibits testosterone production in the testis.     

Nitric oxide synthase in photoreceptive pineal organs of fish

Photoreceptor cells in the fish pineal gland transduce light-dark information differentially into a neuroendocrine melatonin message; distinguishing features are the presence or absence of endogenous oscillators that drive these rhythms. In the present study, we have analysed the presence and distribution of nitric oxide (NO) synthase in both pineal types by NADPH-diaphorase (NADPHd) histochemistry and determined the effects of NO donors on cGMP formation and melatonin production. NADPHd staining was confined to photoreceptor cells in clock-driven pineal organs of zebrafish and goldfish as evidenced by a codistribution with S-antigen-immunoreactivity (-ir) or cyclic GMP-ir and, in the pineal of the trout, to cells that are S-antigen negative. In the trout pineal, but not in the other species, NADPHd staining was clearly codistributed with growth associated protein-43 (GAP-43) immunoreactivity, an antibody that recognizes developing and regenerating neurons in the fish brain. The presence of a functional NO system in photosensory pineal organs is supported by the fact that NO donors like S-nitroso N-acetylpenicillamine (SNAP) elevate intracellular cGMP levels. However, despite the significant rise in cGMP levels nitric oxide donors did neither affect acute light-dependent melatonin formation in the trout pineal nor the rhythmic production of melatonin in the zebrafish pineal.     

Nitric oxide synthase immunoreactivity in the developing and adult human retina

Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) from L-arginine. In this study, the cellular localization of neuronal NOS (nNOS) activity in the human retina since fetal development was examined by immunohistochemistry. No detectable staining in the fetal retina was present at 14 weeks of gestation (wg), the earliest age group examined. A centro-peripheral gradient of development of nNOS immunoreactivity was evident at 16–17 wg, with the midperipheral retina showing nNOS immunoreactivity in most of the cell types and the inner plexiform layer while the peripheral part demonstrated moderate immunoreactivity only in the ganglion cell layer and photoreceptor precursors. A transient increase in nNOS immunoreactivity in the ganglion cells and Müller cell endfeet between 18–19 and 24–25 wg was observed at the time when programmed cell death in the ganglion cell layer, loss of optic nerve fibres as well as increase in glutamate immunoreactivity and parvalbumin (a calcium binding protein) immunoreactivity in the ganglion cells was reported. These observations indicate that programmed cell death of ganglion cells in the retina may be linked to glutamate toxicity and NO activity, as also suggested by others in the retina and cerebral cortex. The presence of nNOS immunoreactivity in the photoreceptors from 16–17 weeks of fetal life to adulthood indicates other functions, besides their involvement in photoreceptor function of transduction and information processing.     

Correlation between NADPH-diaphorase and iNOS in bank vole Leydig cells in vitro and in testicular sections with the use of histochemistry and immunocytochemistry

Histochemistry for NADPH-diaphorase detects an enzymatic activity associated with nitric oxide synthase while immunohistochemistry detects the nitric oxide synthase molecule. NADPH-diaphorase and inducible isoform of nitric oxide synthase in Leydig cells in vitro and in testis sections of the bank vole were demonstrated histochemically and immunocytochemically. Histochemical studies revealed localization of NADPH-diaphorase reaction product in the cytoplasm of cultured Leydig cells as well as in the interstitial area, mainly in Leydig cells and in vascular endothelium. Distribution pattern of NADPH-diaphorase was different in Leydig cell cytoplasm of individual cells. Using immunocytochemistry, the immunoreactivity for nitric oxide synthase was observed both in cultured Leydig cells and testis sections. Moreover, a co-localization of positively immunostained cells with those histochemically detected was noticed. Addition of hCG to the cultured medium or injections in vivo resulted in a small decrease in reaction intensity in Leydig cells. Treatment with N omega-nitro-L-arginine methyl ester resulted in distinctly weaker reactivity of the enzymes studied which was correlated with a higher testosterone and estradiol levels in Leydig cells measured radioimmunologically. The results have indicated that nitric oxide synthase is able to act directly within the male gonad regulating androgen secretion by Leydig cells.     

Nitrergic innervation and nitrergic cells in arteriovenous anastomoses

Nitrergic innervation and nitrergic epithelioid cells were studied in arteriovenous anastomoses of the tongue, ear, eye, and glomus organ of the finger in different species (rat, rabbit, dog, and man), by means of immunohistochemistry for nitric oxide synthase and enzyme histochemistry utilizing the catalytic activity of this enzyme (the NADPH-diaphorase reaction). Nitrergic perivascular fibers of the tongue were concentrated along the arterial tree and were maximal at the arteriovenous anastomoses in all species. Generally, fewer fibers were located around comparable segments of the episcleral eye vasculature. Only a few nitrergic fibers were found in the canine and rabbit ear, and in the glomus organ of the human finger; however, epithelioid cells in the tunica media of arteriovenous anastomoses of these organs were NADPH-diaphorase-positive and were moderately immunoreactive for nitric oxide synthase. In the epithelioid cells, the reaction product of the NADPH-diaphorase could also be demonstrated by transmission electron microscopy. The epithelioid cells were negative for the panneural and neuroendocrine marker PGP 9.5 confirming the myocytotic nature of these nitrergic cells. Thus, nitric oxide might play a role in mediating the vessel tone of arteriovenous anastomoses via nitrergic nerves or epithelioid cells.     

Nitric oxide (NO) synthase immunoreactivity in the starfish Marthasterias glacialis

The neuroendocrine system of the starfish Marthasterias glacialis was investigated immunocytochemically using antisera specific for rat neuronal, bovine aortic endothelial, and mouse macrophage, nitric oxide (NO) synthases. Immunoreactivity was detected only with the antibodies specific for the neural enzyme, in the ectoneural and hyponeural tissues of the radial nerve cords and in the basiepithelial plexus and endocrine cells of the digestive tract. The pyloric stomach showed more immunoreactive structures than the other digestive organs, with the rectal caeca showing the least activity. Immunoreactive endocrine cells were located in the cardiac and pyloric stomachs and in the pyloric caeca. Co-localization of the enzyme immunoreactivity, and the staining for NADPH-diaphorase, demonstrate the presence of NO synthase in echinoderms. These results provide further evidence that NO is a neuronal messenger of early phylogenetic origin which has been conserved throughout evolution.     

Immunodetection and localization of nitric oxide synthase in the olfactory center of the terrestrial snail, Helix pomatia

The procerebrum of stylommatophoran snails produces nitric oxide (NO)-modulated oscillatory local field potentials which are considered the basis of olfactory information processing. Although the function of NO is well characterized in the PC, the identification and distribution of NO synthase (NOS) has not known completely. In the present study, applying a mammalian anti-NOS antibody, a 170 kDa molecular weight NOS-like protein was demonstrated in the procerebrum homogenate of Helix pomatia. NOS-like immunolabeling of the globuli cells, the internal and terminal neuropils displayed an identical distribution compared to that of NADPH-diaphorase reactive material, confirming the specificity of immunohistochemistry. The detailed characteristics of the immunostaining (different intensity of the neural perikarya, a gradual appearance in the terminal neuropil and in the axon bundles of the tentacular nerve, as well as an intense, homogeneous distribution of NOS-like immunoreactivity in the internal neuropil) suggest that NOS is expressed constitutively, maintaining a high level of the enzyme in neuropil areas. NOS accumulation in the internal neuropil suggests that NO plays an important role in delivering olfactory signals extrinsic to the procerebrum, and integrating them with other sensory modalities, respectively. Our results are the first, demonstrating unequivocally the presence of NOS and resolving its differential distribution in the Helix procerebrum.     

Detection of NADPH-diaphorase activity in Paramecium primaurelia

Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell-cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for beta-NADPH and alpha-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized. The non-denaturing electrophoresis, followed by exposure to beta-NADPH or alpha-NADH co-substrates, revealed the presence of a band of apparent molecular mass of about 124 kDa or a band of apparent molecular mass of about 175 kDa, respectively. In immunoblot experiments, the bNOS antibody recognized a single band of apparent molecular mass of about 123 kDa.     

Rho-kinase mediates spinal nitric oxide formation by prostaglandin E2 via EP3 subtype

Prostaglandin E2 (PGE2), the principal pro-inflammatory prostanoid, is known to play versatile roles in pain transmission via four PGE receptor subtypes, EP1-EP4. We recently demonstrated that continuous production of nitric oxide (NO) by neuronal NO synthase (nNOS) following phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) and NMDA receptor NR2B subunits is essential for neuropathic pain. These phosphorylation and nNOS activity visualized by NADPH-diaphorase histochemistry were blocked by indomethacin, a PG synthesis inhibitor. To clarify the interaction between cyclooxygenase and nNOS pathways in the spinal cord, we examined the effect of EP subtype-selective agonists on NO production. NO formation was stimulated in the spinal superficial layer by EP1, EP3, and EP4 agonists. While the EP1- and the EP4-stimulated NO formation was markedly blocked by MK-801, an NMDA receptor antagonist, the EP3-stimulated one was completely inhibited by H-1152, a Rho-kinase inhibitor. Phosphorylation of MARCKS and NADPH-diaphorase activity stimulated by the EP3 agonist were also blocked by H-1152. These results suggest that PGE2 stimulates NO formation by Rho-kinase via EP3, a mechanism(s) different from EP1 and EP4.     

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.     

Perivascular nerve fibres and endothelial cells of the rat basilar artery: immuno-gold labelling of antigenic sites for type I and type III nitric oxide synthase

Ultrastructural localisation of type I (neuronal) and type III (endothelial) isoforms of nitric oxide synthase in perivascular nerve fibres (axons) and endothelial cells was studied in the Wistar rat cerebral basilar artery, using monoclonal antibodies either to type I or type III nitric oxide synthase and post-embedding colloidal-gold immunocytochemistry. Labelling signal (gold particles) for type I and type III nitric oxide synthase was localised both in axons and endothelial cells. In the axon profiles, labelling for either type I or type III nitric oxide synthase was localised in the axoplasm and the lumen and/or membrane of small agranular synaptic vesicles. In the endothelial cells, labelling for either type-I or type-III nitric oxide synthase was predominantly in the cytoplasm. The present qualitative data extends our previous study of cerebrovascular nerve fibres and endothelial cells employing monoclonal antibodies; the localisation of nitric oxide synthase in a subpopulation of synaptic vesicles in nitric oxide synthase-positive cerebrovascular nerves suggests that vesicular mechanisms may be involved in the production/release of nitric oxide. © 1998 Chapman and Hall