Regulatory peptides in gut endocrine cells and nerves in the starfish Marthasterias glacialis

The endocrine cells of the starfish digestive tract are spindle-shaped, contacting both the lumen and the basiepithelial plexus. Silver impregnation labels the basiepithelial and subcoelomic plexuses as well as these cells. Twenty antisera have been tested using the avidinbiotin method, in order to identify the regulatory substances involved in this system. Endocrine cells and nerves immunoreactive to GFNSALMFamide- (S1), FMRFamide-, peptide tyrosine-tyrosine-(PYY), pancreatic polypeptide- (PP), melanocyte stimulating hormone- (MSH) and peptidylglycine alpha-amidating monooxygenase- (PAM) specific antisera have been found in the epithelium. The antibodies against S1, a peptide isolated from the nervous system of a starfish, and MSH, stain both the basiepithelial plexus and the subcoelomic plexus, but the others react only with nerves in the basiepithelial plexus. Absorption controls show that antibodies for S1 and FMRFamide totally crossreact recognizing the same molecule, possibly S1. The other antibodies do not show cross-reactivity to any of the rest, and thus we conclude that these regulatory peptides are present in starfish. This is the first report of the presence of FMRFamide, PYY, MSH and PAM in the Echinodermata. Under the electron microscope the endocrine cells exhibit secretory granules, microtubules and mitochondria. Direct contact with the subcoelomic plexus can be observed.     

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.     

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.     

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 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.     

Nitric oxide synthase sequences in the marine fish Stenotomus chrysops and the sea urchin Arbacia punctulata, and phylogenetic analysis of nitric oxide synthase calmodulin-binding domains

The phylogenetic distribution and structural diversity of the nitric oxide synthases (NOS) remain important and issues that are little understood. We present sequence information, as well as phylogenetic analysis, for three NOS cDNAs identified in two non-mammalian species: the vertebrate marine teleost fish Stenotomus chrysops (scup) and the invertebrate echinoderm Arbacia punctulata (sea urchin). Partial gene sequences containing the well-conserved calmodulin (CaM)-binding domain were amplified by RT-PCR. Identical 375-bp cDNAs were amplified from scup brain, heart, liver and spleen; this sequence shares 82% nucleic acid and 91% predicted amino acid identity with the corresponding region of human neuronal NOS. A 387-bp cDNA was amplified from sea urchin ovary and testes; this sequence shares 72% nucleic acid identity and 65% deduced amino acid identity with human neuronal NOS. A second cDNA of 381 bp was amplified from sea urchin ovary and it shares 66% nucleic acid and 57% deduced amino acid identity with the first sea urchin sequence. Together with earlier reports of neuronal and inducible NOS sequences in fish, these data indicate that multiple NOS isoforms exist in non-mammalian species. Phylogenetic analysis of these sequences confirms the conserved nature of NOS, particularly of the calmodulin-binding domains.     

Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.     

Acetylcholine inhibits nitric oxide (NO) synthesis in the gastropod nervous system

Acetylcholine (ACh) is one of the main signals regulating nitric oxide synthase (NOS) expression and nitric oxide (NO) biosynthesis in mammals. However, few comparative studies have been performed on the role of ACh on NOS activity in non-mammalian animals. We have therefore studied the cholinergic control of NOS in the snail Helix pomatia and compared the effects of ACh on NO synthesis in the enteric nervous system of the snail and rat. Analyses by the NADPH-diaphorase reaction, immunocytochemistry, purification with ion-exchange chromatography, Western-blot, and quantitative polymerase chain reaction have revealed the expression of neuronal NOS in the rat intestine and of a 60-kDa subunit of NOS in the enteric nerve plexus of H. pomatia. In H. pomatia, quantification of the NO-derived nitrite ions has established that NO formation is confined to the NOS-containing midintestine. Nitrite production can be elevated by L-arginine but inhibited by N_ω-nitro-L-arginine. In rats, ACh moderately elevates nitrite production, whereas ACh, the nicotinic receptor agonists (nicotine, acetyl thiocholine iodide, metacholine) and the cholinesterase inhibitor eserine reduce enteric nitrite formation in snails. The nicotinic receptor antagonist tubocurarine also provokes nitrite liberation, whereas the muscarinic receptor agonists or antagonists have no significant effect in snails. In the presence of EDTA or tetrodotoxin, ACh fails to inhibit nitrite production. In pharmacological studies, we have found that ACh contracts the midintestinal muscles and, in snails, simultaneously reduces the antagonistic muscle relaxant effect of L-arginine. Our experiments provide the first evidence for an inhibitory regulation of neuronal NO synthesis by ACh in an invertebrate species. This article is dedicated to Dr. Gábor Hollósi on the 50th anniversary of his graduation and being a teacher at the University of Debrecen.     

Immunocytochemical mapping of the novel echinoderm neuropeptide SALMFamide 1 (S1) in the starfish Asterias rubens

The recent isolation and characterization of the SALMFamide neuropeptides S1 and S2 from the starfish Asterias rubens has initiated a series of studies on their distribution. Specific antisera have been raised against S1 and used in light-microscopical immunocytochemistry. The results of this study reveal for the first time a possible hyponeural innervation of the visceral musculature of the gut and the widespread neuronal distribution of S1, (i) in axons and cell bodies of both ectoneural and hyponeral regions of the radial nerve cord and circumoral nerve ring, (ii) in the nerve ring and nerve plexus of the tube feet, (iii) in the apical muscle, (iv) in skin, and (v) extensively throughout the digestive system. These discoveries are of particular interest in terms of the possible functional roles for S1 in Asterias rubens.Part of this work has been previously reported as an abstract: (Moore et al.1990, 1991)     

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.     

Phe-met-arg-phe (FMRF)-amide is a substrate source of NO synthase in the gastropod nervous system

The possible involvement of the L-arginine-containing Phe-met-arg-phe (FMRF)-amide (FMRFa) in neuronal nitric oxide (NO) biosynthesis was studied in a gastropod species. We found NADPH-diaphorase-positive neurons and FMRFa-containing fibers in close proximity in the enteric nervous system. Administration of L-arginine and FMRFa induced quantitatively similar nitrite production in both intact intestinal tissues and tissue homogenates. These changes could be prevented by the presence of NOARG (an NO synthase inhibitor). Neither chemically modified FMRFa (D-arginine instead of L-arginine) nor amino acid constituents of FMRFa (methionine, phenylalanine) affected basal nitrite production. FMRFa-induced alterations were reduced in the presence of Na⁺ channel blockers (tetrodotoxin, amiloride, lidocaine), the Na⁺/K⁺ATPase inhibitor ouabain, or protease inhibitors (leupeptine, pepstatine-a). FMRFa and its amino acid constituents were analyzed by paper chromatography. When FMRFa was added to tissue homogenates, the peptide was eliminated within 1–2 min, whereas methionine, phenylalanine, arginine, and citrulline levels were elevated simultaneously. We tested the effects of FMRFa, L-arginine, and NOARG on intestinal contractile activity. FMRFa relaxed the intestine for 1–2 min and then induced contractions for 20–40 min. In the presence of NOARG, no relaxant effect of FMRFa was recorded. As administration of L-arginine strongly inhibits the mechanical activity of the intestinal muscle, NO production presumably plays a substantial role in the action of FMRFa, at least in the initial phase. Our biochemical data indicate a direct involvement of FMRFa in NO biosynthesis. FMRFa might be hydrolyzed by extracellular peptidases and then the locally released arginine might be transported into the cells and broken-down to produce NO. Depolarization-induced NO production attributable to the activation of amiloride-sensitive Na⁺ channels might also be involved.     

Localisation of gamma aminobutyric acid (GABA)-like immunoreactivity in the echinoderm Asterias rubens

Gamma amino butyric acid (GABA) is believed to be the principal inhibitory neurotransmitter in the mammalian central nervous system, a function that has been extended to a number of invertebrate systems. We have used a specific antiserum raised against GABA to demonstrate GABA-like immunoreactivity in the radial nerve cord (RNC), tube feet and the digestive system of the asteroid Asterias rubens. In the RNC, immunoreactivity was restricted to ectoneural fibres and cell bodies while in the tube feet fibres were revealed in the basal nerve ring and longitudinal nerve. In the gut, extensive labelling was apparent in the basi-epithelial plexus as well as in mucosal perikarya.     

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.     

Nitric oxide synthase inhibitors influence dynorphin A (1-17) immunoreactivity in the rat brain following hyperthermia

Summary.  The possibility that nitric oxide synthase (NOS) inhibitors influence dynorphin immunoreactivity following hyperthermia was examined in a rat model using a pharmacological approach. Previous reports from our laboratory show that hyperthermia induces an upregulation of NOS in several brain regions that seems to be instrumental in causing cell injury. Recent reports suggest that nitric oxide (NO) can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. Since dynorphin is neurotoxic in different animal models of brain or spinal cord injury, it may be that the peptide will contribute to the cell injury in hyperthermia. The present investigation was carried out to determine whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of NOS will influence the peptide distribution in the brain following heat stress. Rats subjected to hyperthermia at 38°C for 4 h in a biological oxygen demand incubator (BOD) resulted in a marked upregulation of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. Pretreatment of rats with two potent NOS inhibitors, L-NAME (30 mg/kg/day, i.p. for 7 days) or L-NMMA (35 mg/kg/day, i.p. for 7 days) significantly attenuated the dynorphin immunoreactivity in the brain. These drugs were also able to reduce hyperthermia induced blood-brain barrier (BBB) permeability, brain edema formation and cell injury. Taken together, our results suggest that (i) hyperthermia has the capacity to upregulate dynorphin immunoreactivity in the brain, (ii) inhibition of NOS considerably attenuates the dynorphin immunoreaction following heat stress and (iii) upregulation of dynorphin is somehow contributing to hyperthermia induced brain damage, not reported earlier. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

The distribution of glutamate-like immunoreactivity in the thoracic and abdominal ganglia of the locust (Schistocerca gregaria)

The distribution of glutamate-like immunore-activity in the thoracic and abdominal ganglia of the locust was studied using two polyclonal antibodies against glutamate. Because glutamate is a precursor of the inhibitory transmitter -amino butyric acid (GABA) the distribution of immunostaining by antibodies against glutamate and GABA was closely compared in adjacent serial sections. When the antibodies were used at optimal dilutions there was no overlap in the distribution of immunostaining for glutamate and GABA. In the pro- and mesothoracic ganglia 360–400 somata are immunoreactive for glutamate, while in the metathoracic ganglion about 600 somata were stained. These range in diameter from 10–100 m in diameter and include the majority of the large somata in these ganglia. Bundles of primary neurites emerging from these large somata can be traced through the neuropile. Most of the bundles correspond to the known paths of motor neurone primary neurites. In addition the T-tract is also immunolabelled. The free abdominal ganglia each contain 80–100 somata ranging in size from 10–45 m while the terminal ganglion contains about 250 somata, 10–60 m in diameter.     

Nitric oxide biosynthesis by Leishmania amazonensis promastigotes containing a high percentage of metacyclic forms

Due to the diversity of its physiological and pathophysiological functions and general ubiquity, the study of nitric oxide (NO) has become of great interest. In this work, it was demonstrated that Leishmania amazonensis promastigotes produces NO, a free radical synthesized from l-arginine by nitric oxide synthase (NOS). A soluble NOS was purified from L. amazonensis promastigotes by affinity chromatography (2′, 5′-ADP-agarose) and on SDS-PAGE the enzyme migrates as a single protein band of 116.2 (±6) kDa. Furthermore, the presence of a constitutive NOS was detected through indirect immunofluorescence using anti-cNOS and in NADPH consumption assays. The present work show that NO production, detected as nitrite in culture supernatant, is prominent in promastigotes preparations with high number of metacyclic forms, suggesting an association with the differentiation and the infectivity of the parasite.     

Effect of zinc on lysozyme-like activity of the seastar Marthasterias glacialis (Echinodermata, Asteroidea) mucus

Lysozyme represents the best characterized enzyme involved in the self-defense from bacteria. In this study we analysed the effects of zinc on the lysozyme-like activity of the seastar Marthasterias glacialis mucus. This activity, detected by measuring the cleared lysis area of dried Micrococcus lysodeikticus cell walls on Petri dishes, was significantly reduced in presence of zinc. The results are discussed in the light of elucidating the possible relationship between environmental contaminants and increased disease susceptibility in seastars due to the decrease of antibacterial protection. The benefits of using the test of lysozyme activity to monitoring environmental pollution are highlighted.     

Ontogeny of some endocrine cells of the digestive tract in sea bass (Dicentrarchus labrax): An immunocytochemical study

Serotonin- and ten peptide-immunoreactive (IR) cell types were identified in the digestive tract of sea bass (Dicentrarchus labrax L.) larvae of four morphofunctional phases ranging in age from hatching to 61 days. The sequence of appearance and location of endocrine cells during ontogenetic development of the larvae was determined. The differentiation of endocrine cells followed a distal-proximal gradient in the gut which paralleled the morphofunctional differentiation. Serotonin-IR cells were identified in the last portion of the digestive tract from phase I onwards and in the gastric region from phase III, before these regions were morphofunctionally differentiated; met-enkephalin-IR cells were identified from phase II onwards in both the differentiated rectum and the undifferentiated intestine; cholecystokinin (CCK)- and synthetic human gastrin-34-IR cells were located only in the intestine and first found in the undifferentiated intestine of phase II; human gastrin-17-, peptide YY (PYY)- and neuropeptide Y (NPY)-IR cells appeared in the intestine from phase II and in stomach in phase IV, when it showed gastric glands; pancreatic polypeptide (PP)- and glucagon-IR cells were observed in both intestine and stomach, but insulin- and somatostatin-IR cells only in stomach, from phase III, during which the intestine but not the stomach was differentiated. PP- and PYY-, PP- and glucagon-, and PYY- and glucagon-like immunoreactivities coexisted from their first appearance in some cells of the gut.