Nitric oxide signaling in invertebrates

Nitric oxide (NO) is an unconventional neurotransmitter and neuromodulator molecule that is increasingly found to have important signaling functions in animals from nematodes to mammals. NO signaling mechanisms in the past were identified largely through experiments on mammals, after the discovery of NO's vasodilatory functions. The use of gene knock out mice has been particularly important in revealing the functions of the several isoforms of nitric oxide synthase (NOS), the enzyme that produces NO. Recent studies have revealed rich diversity in NO signaling. In addition to the well-established pathway in which NO activates guanylyl cyclase and cGMP production, redox mechanisms involving protein nitrosylation are important contributors to modulation of neurotransmitter release and reception. NO signaling studies in invertebrates are now generating a wealth of comparative information. Invertebrate NOS isoforms have been identified in insects and molluscs, and the conserved and variable amino acid sequences evaluated. Calcium-calmodulin dependence and cofactor requirements are conserved. NADPH diaphorase studies show that NOS is found in echinoderms, coelenterates, nematodes, annelids, insects, crustaceans and molluscs. Accumulating evidence reveals that NO is used as an orthograde transmitter and cotransmitter, and as a modulator of conventional transmitter release. NO appears to be used in diverse animals for certain neuronal functions, such as chemosensory signalin, learning, and development, suggesting that these NO functions have been conserved during evolution. The discovery of NO's diverse and unconventional signaling functions has stimulated a plethora of enthusiastic investigations into its uses. We can anticipate the discovery of many more interesting and some surprising NO signaling functions.     

The olfactory responses of the antenna and maxillary palp of the fleshfly,Neobellieria bullata (Diptera: Sarcophagidae), and their sensitivity to blockage of nitric oxide synthase

The relative sensitivities of the olfactory receptors in the antenna and maxillary palp of the fleshfly, Neobellieria bullata, were assessed using simultaneous electroantennograms (EAGs) and electropalpograms (EPGs). In general, the antennae and maxillary palps were more sensitive to odors related to animals (blood extract and saturated carboxylic acid) than to odors that were plant-derived (citral, hexenol, hexenal). In addition, the maxillary palps were relatively less sensitive to plant-derived odorants than the antennae, perhaps related to their anatomical position. Scanning electron microscopy was also used to assess the types of sensilla found on the two organs. In addition, NADPH-diaphorase histochemistry was used in an attempt to localize the enzyme nitric oxide synthase (NOS) in the antenna and the maxillary palps. We found evidence of NADPH-diaphorase staining in both organs, with localized staining in the antennal cells and more general staining in the maxillary palps. When NOS was selectively blocked using the antagonist L-NAME, the amplitude of the EAGs and EPGs to odorants fell by 30-50%. In contrast, application of the inactive enantiomer, D-NAME, did not change the amplitude of the EAGs or the EPGs. Our results indicate that NOS is involved in the function of olfactory receptor cells in the fleshfly.     

Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [³H]arginine to [³H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor N^G-methyl- -arginine ( -NMA), but not the arginase inhibitors -valine and -ornithine. NOS activity was predominantly cytosolic, and was characterised by a K_m for arginine of 19.05 μM and a V_max of 2.96 pmol/min per μg protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.     

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.     

Nitric oxide and nitric oxide synthase activity in plants

Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signalling molecule in different intracellular processes in plants. The NO emission from plants is known since the 1970s, and now there is abundant information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defence-related genes against pathogens and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and the induction of ethylene emission. NO can be produced in plants by non-enzymatic and enzymatic systems. The NO-producing enzymes identified in plants are nitrate reductase, and several nitric oxide synthase-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time, and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different evidence available indicate that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450, and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought.     

Nitric oxide synthase: models and mechanisms

The overproduction or underproduction of nitric oxide has been implicated in pathological symptoms such as endotoxic shock, diabetes, allograft rejection, and myocardial ischimia/reperfusion injury. A thorough understanding of the biosynthesis of nitric oxide is necessary to probe and manipulate these signaling events. There is also considerable pharmacological interest in developing selective inhibitors of the several isoforms of nitric oxide synthase. The recently determined crystal structures of complexes between nitric oxide synthase and substrate, the mechanisms of the enzymatic reaction that generate nitric oxide and chemical precedents and models for these reactions are now coming into focus, but there are still numerous fascinating and unanswered questions regarding nitric oxide biosynthesis.     

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.     

Localization and distribution of nitric oxide synthase and other neuronal markers in the podia of Holothuria arguinensis

The organization of the nervous system of the holothurian podia—the tentacles, papillae, and tube feet—is still poorly understood, which limits the development of functional studies. Knowledge of nitric oxide (NO) signaling in sea cucumbers is nonexistent, although it is known to play an important role in many essential biological functions, including neurotransmission, throughout the animal kingdom. The objective of this study was to characterize the holothurian podia in Holothuria arguinensis. To this end, we used classical histology, nitric oxide synthase (NOS) distribution, using NADPH‐diaphorase histochemistry and NOS immunostaining, and neuronal immunohistochemistry. Our results revealed an abundant distribution of NO in the nervous components of the holothurian podia, suggesting an important role for NO as a neuronal messenger in these structures. Nitrergic fibers were intensely labeled in the longitudinal nerve and the nerve plexus surrounding the stem, but were more weakly labeled in the mesothelium. NOS was also found in scattered cell bodies and abundant fibers in the podia terminal end (i.e., the discs in tentacles and tube feet, and the pointed conical structures in the papillae), with evident neuronal projections to the bud surface, especially in the tentacles. The podia terminal end was the most specialized area and was characterized by a specific nervous arrangement, consisting of a distinct nerve plate, rich in cells and fibers containing potential sensory cells staining positively for neuronal markers, which makes this the most likely candidate to be a chemosensory region and an important candidate for future exploration.     

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.     

Nitric oxide synthase in human salivary glands

The distribution of the three nitric oxide synthase (NOS) isoforms was determined immunohistochemically in the human minor and major salivary glands with comparison to that of rat salivary glands. In contrast to rat glands, which contained a dense plexus of neuronal NOS-immunoreactive nerve fibers, only a minority of the nerve fibers in human glands showed neuronal NOS immunoreactivity. Human labial and submandibular glands contained sparse NOS-immunoreactive fibers, while only occasional nerve fibers in the parotid or sublingual glands were stained. Furthermore, in contrast to the animal glands, most duct epithelial cells in all human salivary glands were immunoreactive for neuronal NOS. No specific immunoreactivity for inducible or endothelial NOS were observed in the nerve fibers or duct epithelium. We provide evidence to suggest that the role of nitric oxide in the regulation of salivary gland function is different in human as compared to experimental animals. Nitricergic innervation in human tissue is very sparse and thus nitric oxide is probably of minor importance as a neural regulator of salivary glands. Instead, NOS localized in duct epithelial cells suggests that nitric oxide might directly regulate saliva secretion and it is a putative source of nitrates previously reportedly secreted into the saliva.     

Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

Nitric oxide synthase is up-regulated in muscle fibers in muscular dystrophy

Nitric oxide (NO) mediates fundamental physiological actions on skeletal muscle. The neuronal NO synthase isoform (NOS1) was reported to be located exclusively in the sarcolemma. Its loss from the sarcolemma was associated with development of Duchenne muscular dystrophy (DMD). However, new studies evidence that all three NOS isoforms-NOS1, NOS2, and NOS3-are co-expressed in the sarcoplasm both in normal and in DMD skeletal muscles. To address this controversy, we assayed NOS expression in DMD myofibers in situ cytophotometrically and found NOS expression in DMD myofibers up-regulated. These results support the hypothesis that NO deficiency with consequent muscle degeneration in DMD results from NO scavenging by superoxides rather than from reduced NOS expression.     

Species-independent expression of nitric oxide synthase in the sarcolemma region of visceral and somatic striated muscle fibers

The expression and distribution of nitric oxide synthase (NOS) was studied by use of the newly designed specific histochemical NADPH diaphorase staining method and the indirect immunofluorescence technique employing an antiserum to brain NOS in visceral and somatic striated muscles of several mammalian species. Histochemical activity and immunoreactivity were located in the sarcolemma region of type I and II fibers of all muscles investigated. Visceral muscles were more strongly stained than somatic muscles. Furthermore, type II fibers, identified by staining of myosin adenosine triphosphatase activity after pre-incubation at alkaline pH, were more intensely labeled than type I fibers. In addition, NOS activity was detected in the area of the sarcolemma of intrafusal fibers. No obvious differences between species were observed. It was concluded that NOS of striated muscles probably makes up the richest and most important nitric oxide source in mammals.     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

Detection of nitrous oxide in the neuronal nitric oxide synthase reaction by gas chromatography-mass spectrometry

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.     

Seasonal periodicity of enteric nitric oxide synthesis and its regulation in the snail, Helix lucorum

Abstract. The snail Helix lucorum has been used as a model to study the adaptation of a nitric oxide (NO)‐forming enteric neural network to the long‐term resting period of summer estivation or winter hibernation. Quantification of the NO‐derived nitrite established that NO formation is confined to the nitric oxide synthase (NOS)‐containing myenteric network of the mid‐intestine. In active snails but not in resting snails, NO production could be enhanced by the NOS substrate l ‐arginine (l ‐ARG, 1 mM). We followed the enteric NO synthesis in a snail population kept at natural conditions for 1 year. Our findings indicate that NO synthesis was depressed in July during entry to the estivation, had a peak in autumn before hibernation, and finally was reduced during hibernation. Monoamines (histamine, serotonin, and adrenalin) could inhibit the NO liberation in active snails. Cofactors of NOS (β‐NADPH, β‐NAD, FAD, FMN, Ca²⁺, TH4) did not alter the low nitrite production in hibernating snails. We conclude that enteric NO synthesis in H. lucorum has a regular seasonal periodicity following the annual physiological cycles of terrestrial snails. During estivation or hibernation, NOS activity is blocked. Monoamines, the levels of which are elevated during hibernation, can trigger decreased NOS activity. The reduced activity of NOS cannot be restored by the administration of NOS cofactors; therefore, their absence cannot be the cause of the temporarily blocked L‐ARG/NO conversion ability of NOS.