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Transient expression of NOS-II during development of the murine enteric nervous system
Abstract
In the enteric nervous system, nitric oxide (NO) is regarded as an important messenger for the non-adrenergic and non-cholinergic neurotransmission. Synthesized mainly by the constitutive nitric oxide synthase (NOS) isoforms NOS I and NOS III, this molecule exerts prejunctional inhibitory effects in the submucosal plexus as well as relaxation of enteric smooth muscles. In order to elucidate the role for NO during enteric development, we looked for the expression of all three NOS-isoforms in the enteric nervous system during mouse development from E8 to E20 using immunohistochemistry. Starting around midgestation, a transient expression of the NOS-II isoform during the very early development of enteric neurones was detected in parallel to that of HNK-1 exclusively in the myenteric plexus. Similar to findings for other neuronal systems, NOS-I and NOS III isoforms could be traced starting significantly later to increase toward the end of embryonic development when NOS II immunoreactivity faded and a strong expression of the vasointestinal peptide could be detected. In contrast to the NOSII expression, the constitutive isoforms can also be detected in the submucosal plexus. Altogether, these findings suggest NOS-II to be exclusively involved during early steps of enteric nervous system development. Absence of downstream signalling elements, such as sGC and cGMP both in neurons and in enteric muscle until the end of the second third of gestation, may indicate different effects executed by NO during development, expressed by Ca(2+) -dependent and Ca(2+) -independent NOS isoforms.
... nNOS is the enzyme that catalyses the synthesis of nitric oxide (NO), which is a cell-derived highly diffusible and unstable gas involved in intercellular and intracellular communication in the nervous system as well as an inhibitory transmitter of motor neurons in the gut (Arnhold et al., 2004). As an inhibitory transmitter of non-adrenergic, non-cholinergic neurons, NO is involved in muscle relaxation, vasodilatation, acid secretion, and mucus secretion (Brookes, 1993;Nakamura et al., 1998) and so nNOS plays an important role in the relaxation of smooth muscles. ...
... In rats, the axonal projections of nNOS-IR gastric myenteric neurons provide an extensive network of fibers running within the circular smooth muscle layer in a higher proportion than cholinergic ones (Berthoud, 1995;Jarvinen et al., 1999;Schemann et al., 2001). In addition, in the guinea pig, the gastric circular and longitudinal muscle layers receive ascending excitatory cholinergic innervation and descending inhibitory nitrergic innervation Michel et al., 2000;Schemann et al., 2001;Arnhold et al., 2004;Wang et al., 2005;Sarna, 2007;Harrington et al., 2007), whereas in the small intestine two types of neurons express NOS: motor neurons to the muscle and descending interneurons (Chiocchetti et al., 2003). Consistent with this, nNOS-IR neurons being inhibitory motor neurons, supply numerous terminals in the circular muscle of the rat small intestine (Aimi et al., 1993), but relatively few in the myenteric ganglia and are not observed in the perivascular plexuses and are very rare in the mucosa of mouse intestine (Sang and Young, 1996;Arnhold et al., 2004) and human gastric antrum and jejunum (Miller et al., 2001). ...
... In addition, in the guinea pig, the gastric circular and longitudinal muscle layers receive ascending excitatory cholinergic innervation and descending inhibitory nitrergic innervation Michel et al., 2000;Schemann et al., 2001;Arnhold et al., 2004;Wang et al., 2005;Sarna, 2007;Harrington et al., 2007), whereas in the small intestine two types of neurons express NOS: motor neurons to the muscle and descending interneurons (Chiocchetti et al., 2003). Consistent with this, nNOS-IR neurons being inhibitory motor neurons, supply numerous terminals in the circular muscle of the rat small intestine (Aimi et al., 1993), but relatively few in the myenteric ganglia and are not observed in the perivascular plexuses and are very rare in the mucosa of mouse intestine (Sang and Young, 1996;Arnhold et al., 2004) and human gastric antrum and jejunum (Miller et al., 2001). The activation of gastric myenteric neurons is mediated by vagal nicotinic pathways and includes cho l i n e r g i ca n dN O Ss y n t h e s i z i n g neurons, suggesting a central regulation of both excitatory and inhibitory myenteric pathways during acute cold exposure and may form an integral part of the neural alarm and protection system in the stomach as a protective mechanism against acid induced injury o ft h em u c o s a( Y u a ne ta l . ...
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... Importantly, it was found that in the 9-week-old fetuses, NOS expression stabilized and remained almost unchanged in 3-5-day-old individuals, except in the ileum, where more NOS-containing neurons were seen after birth. These observations are in agreement with the results obtained in mice (Arnhold et al. 2004). ...
The development of the enteric nervous system (ENS) is still a valid and intensely studied issue. However, literature in the field has no data on this topic in the dog. The present investigations were performed in three groups of fetuses from mongrel dogs – from the third, sixth-seventh, and ninth week of pregnancy – and in 3-5-day-old puppies (3 specimens for each age group). The tissues (the medial parts of the duodenum, jejunum, and ileum with the cecum and a small portion of the adjacent ascending colon) were cut using a cryostat and the sections were processed for single- and double-labeling immunohistochemistry using antisera against acetylated tubulin (AcTub), vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), galanin (GAL), neuropeptide Y (NPY), substance P (SP), and calcitonin gene-related peptide (CGRP). In the 3-week-old fetuses, some oval cells invading the gut wall were found. From the seventh week of pregnancy onwards, two different enteric ganglia were present: submucosal and myenteric. The estimated number of nerve elements in the 9-week-old fetuses was much higher than that observed in the 6-7-week-old individuals. There was no significant difference in the estimated number of nerve structures between the 9-week-old fetuses and the 3-5-day-old puppies. The colonization pattern and the development of the ENS in the canine small intestine are very similar to those observed in other mammals. However, a few exceptions have been confirmed, regarding the time of appearance of the VIP-, GAL-, and CGRP-immunoreactive neurons, and their distribution in different portions of the canine bowel during development.
... Studies on experimental models have shown that in the face of an infection by intestinal parasites, the infusion of anti-substance P antibodies induces an immuno-neutralization picture in the gastrointestinal tract. These data indicate that the bioavailability of substance P represents an important component in the neurochemical response to intestinal inflammation induced by parasites [217]. Another component of the NANC enteric system that presents an important modification in the face of parasitic infection is intrinsic nitric oxide (iNO), which is identified in the enteric nervous system by the enzyme responsible for its synthesis-nitric oxide synthase (iNOS). ...
Mammalian organisms form intimate interfaces with commensal and pathogenic gut microorganisms. Increasing evidence suggests a close interaction between gut microorganisms and the enteric nervous system (ENS), as the first interface to the central nervous system. Each microorganism can exert a different effect on the ENS, including phenotypical neuronal changes or the induction of chemical transmitters that interact with ENS neurons. Some pathogenic bacteria take advantage of the ENS to create a more suitable environment for their growth or to promote the effects of their toxins. In addition, some commensal bacteria can affect the central nervous system (CNS) by locally interacting with the ENS. From the current knowledge emerges an interesting field that may shape future concepts on the pathogen–host synergic interaction. The aim of this narrative review is to report the current findings regarding the inter-relationships between bacteria, viruses, and parasites and the ENS.
... During oxidative burst, activated phagocytes produce ROS including O 2 − @BULLET , H 2 O 2 and HOCl through activation of enzymes NADPH oxidase and myeloperoxidase. Intracellular and extracellular production of these ROS species can be detected using probes like luminol and lucigenin re- spectively [51][52][53][54]. Patuletin showed antioxidant activity, and during the in vitro studies found to inhibit both intra and extracellular ROS produced by the activated neutrophils and macrophages. ...
... ROS are released into the surrounding medium or into a membrane-enclosed subcellular organelle (37). A superoxide anion (O 2 •−) can either spontaneously or catalyzed by superoxide dismutase form hydrogen peroxide (H 2 O 2 ), a substrate for the myeloperoxidase system (MPO) which can be converted into hypochlorous acid (HOCl) (38,39). The emission of photons taking place during ROS production can be measured as chemiluminescence. ...
Introduction: This study was primarily designed to investigate the antioxidant, immunomodulatory and mitogenic properties of acacia honey. Materials and Methods: The antioxidant and immunomodulatory potential was evaluated by luminoland lucigenin-amplified chemiluminescence assays, whereas the mitogenic potential was evaluated using cytochalasin B-blocked micronucleus and mitotic index assays in vitro from whole blood, neutrophils, macrophages and peripheral lymphocytes. Superoxide anion and DPPH radical scavenging activity as well as reducing power were evaluated to further underscore the antioxidant property of acacia honey. Results: Dose-dependent (p<0.05) inhibition of the intensity of luminol- and lucigenin-amplified chemiluminescence was observed with different concentrations of honey versus control with IC50s of 0.75%, 0.2%, and 0.24% and <0.125% (v/v) for whole blood, neutrophils and macrophages, respectively. Mitotic index, however, decreased significantly (p<0.05) only at a low concentration but increased significantly at high doses compared with control. Similar results were obtained for both nuclear division and cytokinesis-block proliferation indices. The cytotoxicity index decreased significantly in a concentration-dependent manner. Furthermore, acacia honey induced significant dose-dependent inhibition of DPPH (IC50 of 1.58%) and superoxide anion (IC50 of 2.15%) radicals. The reducing power of honey increased with increasing concentrations. Conclusions: Our study demonstrates the antioxidant, mitogenic and immunomodulatory properties of acacia honey.
... Thus, the importance of endogenous NO as a regulator of embryonic development, specifically during the progression from two-cell stage to blastocyst and later [7] and limb development [8] has been established. NO-mediated actions later during organogenesis have been suggested to take place in different tissues and organs, as for instance in the placenta [9], the endothelium [10], the bone [11], the heart [12,13], the lung [14], and the nervous system [15][16][17][18][19][20][21][22][23]. A key downstream messenger of NO is cyclic GMP (cGMP). ...
... ROS are released into the surrounding medium or into a membrane-enclosed subcellular organelle [20] . Dismutation of O 2 @BULLET -, either spontaneously or catalyzed by superoxide dismutase, results in the formation hydrogen peroxide (H 2 O 2 ), which acts as a substrate for the myeloperoxidase system (MPO) and this H 2 O 2 converts to hypochlorous acid (HOCl) [21] . Emission of photons which takes place during ROS production can be measured easily as chemiluminescence. ...
Objective
To investigate the effects of fractionation of acacia honey on its antioxidant potential in contrast with the pure honey from whole blood, brain and liver in vitro.Methods
Honey was partitioned into three fractions (dichloromethane, ethyl acetate and aqueous). Their immuno-modulatory effect on whole blood was assayed using Luminol-amplified chemiluminescence technique. Their antioxidant activities on rat brain and hepatic tissues which covers for catalase, SOD activities and lipid peroxidation.ResultsFractions of the honey enhanced the production of radicals with no significant (P>0.05) antioxidant activity on whole blood where as pure honey does. Pure honey significantly (P<0.05) stimulates SOD and Catalase activity with no significant (P>0.05) effects on lipid peroxidation.Conclusions
Fractionation of acacia honey negatively affects its antioxidant potential thereby making it a radical generating agent in contrast with the unfractionated.
... The generation of highly reactive superoxide anion (O 2 •− ) by the NADPH-oxidase complex of phagocytes is the primary event of the oxidative burst. Dismutation of O 2 •− , either spontaneously or catalysed by superoxide dismutase, results in the formation hydrogen peroxide (H 2 O 2 ), which acts as a substrate for the myeloperoxidase system (MPO) and this H 2 O 2 converts to hypochlorous acid (HOCl) (Arnhold et al., 2004). Emission of photons which takes place during ROS production can be measured easily as chemiluminescence (CL). ...
Thrombin, hyperglycemia and reactive oxygen species (ROS) have been discovered to play a pivotal role in the pathogenesis of cardiovascular disease (CVD). The aim of the study was to evaluate the direct effect of bovine thrombin (BTh) on ROS production by human neutrophils and rodent macrophages and to investigate the effect of honey on BTh‐induced ROS production from phagocytes.
Professional phagocytes, i.e. neutrophils and macrophages, were stimulated by BTh and ROS production was measured in luminol/lucigenin enhanced chemiluminescence (CL) assays. In another experiment the effects of honey treatment on BTh‐induced ROS production by phagocytes was tested using a CL assay.
The results indicate that BTh directly activates phagocytes. A significant generation of ROS was noted with the luminol/lucigenin enhanced chemiluminescence (CL) system. Honey treatment of phagocytes activated by bovine thrombin showed effective suppression of oxidative respiratory burst monitored by the CL assay.
In conclusion, it can be assumed that this direct action of BTh on phagocytes causing ROS production might exaggerate the inflammatory response at the site of atheromatous plaques. The suppressive activity of honey towards thrombin‐induced ROS production by phagocytes could be beneficial in the interruption of the pathological progress of CVD and may play a cardioprotective role. Copyright © 2009 John Wiley & Sons, Ltd.
... More unusual is the discovery that the en- teric neurons continue to express NO- sensitive sGC activity throughout their differentiation, whereas most CNS neurons express sGCs only tran- siently ( Dawson et al., 1991;Roskams et al., 1994;Ward et al., 1994). In ver- tebrates, different isoforms of NOS have been detected in enteric neurons at various stages of development ( Balaskas et al., 1995;Van Ginneken et al., 1998;Arnhold et al., 2004), but a role for NO-dependent sGC activity in the formation of the ENS has not been systematically explored. ...
Like the vertebrate enteric nervous system (ENS), the insect ENS consists of interconnected ganglia and nerve plexuses that control gut motility. However, the insect ENS lies superficially on the gut musculature, and its component cells can be individually imaged and manipulated within cultured embryos. Enteric neurons and glial precursors arise via epithelial-to-mesenchymal transitions that resemble the generation of neural crest cells and sensory placodes in vertebrates; most cells then migrate extensive distances before differentiating. A balance of proneural and neurogenic genes regulates the morphogenetic programs that produce distinct structures within the insect ENS. In vivo studies have also begun to decipher the mechanisms by which enteric neurons integrate multiple guidance cues to select their pathways. Despite important differences between the ENS of vertebrates and invertebrates, common features in their programs of neurogenesis, migration, and differentiation suggest that these relatively simple preparations may provide insights into similar developmental processes in more complex systems.
... Ni 2+ was administered as chloride salt. Fura-2-acetoxymethylester (fura-2-AM), ODQ ( [1,2,4] Molecular Probes Europe, Leiden, The Netherlands) were dissolved as stock in dimethylsulphoxide (DMSO). If not indicated differently, chemicals were obtained from Sigma (Taufkirchen, Germany). ...
The aim of the present study was to characterize the action of the neurotransmitter NO on rat myenteric neurons. A NO donor such as GEA 3162 (10(-4) mol/l) induced an increase in the intracellular Ca2+ concentration as indicated by an increase in the fura 2 ratio in ganglia loaded with this Ca2+-sensitive fluorescent dye. The effect of GEA 3162 was strongly reduced in the absence of extracellular Ca2+, suggesting an influx of Ca2+ from the extracellular space evoked by NO. A similar nearly complete inhibition was observed in the presence of Ca2+ channel blockers such as Ni2+ (5 x 10(-4) mol/l) or nifedipine (10(-6) mol/l). Whole cell patch-clamp recordings confirmed the activation of voltage-dependent Ca2+ channels, measured as inward current carried by Ba2+, by the NO donor. The peak Ba2+-carried inward current increased from -100 +/- 19 to -185 +/- 34 pA in the presence of sodium nitroprusside (10(-4) mol/l). The consequence was a hyperpolarization of the membrane, which was blocked by intracellular Cs+ and thus most probably reflects the activation of Ca2+-dependent K+ channels. Furthermore, at least two subtypes of NO synthases, NOS-1 (neuronal form) and NOS-3 (endothelial form), were found as transcripts in mRNA isolated from the rat myenteric ganglia. The expression of these NO synthases was confirmed immunohistochemically. These observations suggest that NO, released from nitrergic neurons within the enteric nervous system, not only affects target organs such as smooth muscle cells in the gut but has in addition profound effects on the enteric neurons themselves, the key players in the regulation of many gastrointestinal functions.
This study was designed to investigate the alteration of redox status by commonly used antimalarials in Nigeria.
Drugs used were artemisinin, artesunate, chloroquine, coartem and quinine at the final concentrations of
0.5–8.0 mg/mL. Blood samples were collected from malarial patients and apparently healthy humans for
comparison. Reduced glutathione, catalase, superoxide dismutase (SOD) activities, protein content and lipid
peroxidation were determined. All drugs significantly (p < 0.05) increases the protein level relative to control
in normal blood, whereas in the infected, a significant (p < 0.05) reduction was observed. In normal blood, the
antimalarials dose dependently decreased (p < 0.05) SOD and catalase activities with significant (p < 0.05)
increase in the infected. The level of glutathione in normal blood significantly (p < 0.05) increases as compared
with control, whereas in the infected, similar observation was made except that the levels were less, relative to
control sample. Malondialdehyde level significantly (p < 0.05) increases with increase in drugs concentration
even though less than the level in the control with few exceptions. These effects were dose dependent and
more pronounced in non-malarial conditions. Commonly used antimalarials might alter the redox status in
both healthy and non-healthy subjects thereby inducing oxidative stress.
Nitric oxide (NO) is a diffusible second messenger with a great variety of functions in the brain. NO is produced by three isoforms of NO synthase (NOS), NOS1, NOS2, and NOS3. Although broad agreement exists regarding the expression of NOS1 and NOS3 in neurons and endothelial cells, the pattern of NOS2 expression is still controversial and remains elusive. We have now generated a novel transgenic mouse that expresses the fluorescent reporter tdTomato and the CRE recombinase under the control of the Nos2 gene regulatory regions. Such tool allows the reliable tracking of NOS2 expression in tissue and further unravels episodes of transient NOS2 expression. Using this transgenic mouse, we show that in the healthy brain, NOS2 is only transiently expressed in neurons scattered in the piriform and entorhinal cortex, the amygdaloid nuclei, the medial part of the thalamus, the hypothalamus, the dentate gyrus, and the cerebellum. NOS2 expression was rarely detected in microglia. We further show that inflammation, induced by intracerebral injection of LPS and IFNγ, triggers transient expression of NOS2 in microglia but not in neurons. This novel transgenic tool has thus allowed us to clarify the NOS2 expression pattern and its differential profile in neurons and microglia in healthy and inflammatory conditions. GLIA 2014.
With the success of Web applications, most of our data is now stored on
various third-party servers where they are processed to deliver personalized
services. Naturally we must be authenticated to access this personal
information, but the use of personalized services only restricted by
identification could indirectly and silently leak sensitive data. We analyzed
Google Web Search access mechanisms and found that the current policy applied
to session cookies could be used to retrieve users' personal data. We describe
an attack scheme leveraging the search personalization (based on the same SID
cookie) to retrieve a part of the victim's click history and even some of her
contacts. We implemented a proof of concept of this attack on Firefox and
Chrome Web browsers and conducted an experiment with ten volunteers. Thanks to
this prototype we were able to recover up to 80% of the user's search click
history.
The effects of natural honey and its major sugar constituents (i.e. D-fructose, D-glucose, maltose and sucrose) on phagocytic respiratory burst have been studied. Pre-incubated whole blood and isolated leukocytes with honey samples and sugars were induced for phagocytosis and the level of reactive oxygen species (ROS) was monitored by using chemiluminescence assays. Honey samples were found to decrease the luminol-enhanced chemiluminescence in opsonized zymosan-stimulated whole blood and isolated leukocytes with statistically significant differences; indicating inhibition of ROS production including hydrogen peroxide, hydroxyl free radical and hypochlorous acid. Thus honey appears to modify the oxidative burst process by inhibiting phagocytic myeloperoxidase activity. Chemiluminescence assays further showed that among the major sugar constituents of honey, D-fructose in high concentration exerted an inhibitory effect on exocytosis-associated myeloperoxidase catalyzed ROS formation. These results pointed out an immuno-modulatory potential of honey in the course of phagocytosis.
We have previously reported that supernatant derived from LPS-activated BV-2 cells, an immortalized microglial cell line, induces death of NSC-34 cells (a motor neuron hybridoma) through a TNFalpha and nitric oxide synthase (NOS) dependant mechanism. In this study, we have observed that LPS-activated BV-2 supernatant induces NSC-34 cell death in association with an upregulation of the TNF receptor 1 (TNFR1) expression on NSC-34 cells, both at the transcription level and at the cell surface protein level. The upregulation of TNFR1 receptor was independent of TNFalpha, and could be partly inhibited by the inhibition of iNOS activation in the BV-2 cells. The TNFR2 receptor was not involved. These observations have important implications in understanding the mechanism by which microglial activation contributes to the motor neuron degeneration.
In order to clarify the developmental regulation of the eNOS activity in intestine by phosphorylation, we examined the immunohistochemical localizations of the eNOS phosphorylation sites at Ser(1177), Ser(116) and at Thr(495) in cells of the mouse enteric mucosa and myenteric plexus at E13.5, E14.5, E16.5, E18.5, E20.5 and P3. In addition, in cells of the E16.5 stage the protein levels of eNOS and the phosphorylation sites of eNOS at Ser(1177), Ser(116) and at Thr(495) were investigated by immunoblot. From E14.5 to P3, phosphorylation residues of eNOS at Ser(1177) and at Ser(116) were detected with different staining intensities in the enteric mucosa epithelium. In ganglion cells of the myenteric plexus Ser(116) was identified at E18.5 to P3. The absence of phosphorylated Thr(495) in cells of intestine during all developmental stages, was confirmed by immunoblot at E16.5. The immunoblot levels of eNOS and eNOS phosphorylated at Ser(1177) and at Ser(116) were comparable with the immunohistochemical results of E16.5 mouse intestine. It was concluded that development of epithelial cells of the enteric mucosa may be modulated by phosphorylation of eNOS at Ser(1177) and at Ser(116). The phosphorylation of eNOS in cells of the myenteric plexus is modulated at Ser(116). These data suggest that there is a developmental stage and cell type dependent phosphorylation of eNOS.
NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.
The pattern of distribution and co-localization of nitric oxide synthase (NOS) and quinacrine fluorescence (indicative of vesicular adenosine 5'-triphosphate, ATP), and co-localization of NADPH-diaphorase (NADPH-d) activity and NOS-immunoreactivity in the myenteric plexus of pre-term human fetal (6-17 weeks of gestation) stomach and small intestine was examined using immunohistochemical and histochemical techniques. In all stages of gestation investigated, NOS-immunoreactive and NADPH-d-reactive myenteric neurons and nerve fibres were seen in the fetal intestine and stomach. However, in fetuses of 6-10 weeks of gestation, only 15% of the NADPH-d-positive myenteric neurons were NOS-immunoreactive, whereas a 100% co-localization was found in samples of 12-17 weeks of gestation. Quinacrine fluorescent myenteric neurons and nerve fibres were found only in the fetal intestine of 12-17 weeks of gestation, of which 25% of the NADPH-d-positive myenteric neurons in these samples were quinacrine fluorescent. These findings demonstrate the presence and co-localization of markers for nitric oxide (NO)- and ATP-utilizing myenteric neurons and nerve fibres in the early stages of gestation, suggesting possible co-transmitter and/or trophic roles of ATP and NO in the process of development and maturity of human myenteric neurons. In addition, the fact that only a small percentage of NADPH-d-reactive myenteric neurons express NOS immunoreactivity at 6-10 weeks of gestation confirms that NADPH-d-reactivity does not always represent NOS activity.
Mechanisms controlling the transition of a neural precursor cell from proliferation to differentiation during brain development determine the distinct anatomical features of the brain. Nitric oxide (NO) may mediate such a transition, because it can suppress DNA synthesis and cell proliferation. We cloned the gene encoding the neuronal isoform of Xenopus NO synthase (XNOS) and found that in the developing brain of Xenopus tadpoles, a zone of XNOS-expressing cells lies adjacent to the zone of dividing neuronal precursors. Exogenous NO, supplied to the tadpole brain in vivo, decreased the number of proliferating cells and the total number of cells in the optic tectum. Conversely, inhibition of NOS activity in vivo increased the number of proliferating cells and the total number of cells in the optic tectum. NOS inhibition yielded larger brains with grossly perturbed organization. Our results indicate that NO is an essential negative regulator of neuronal precursor proliferation during vertebrate brain development.
The enteric nervous system arises from the neural crest. In embryonic mice, vagal neural crest cells enter the developing foregut at approximately embryonic day 9.5 (E9.5) and then migrate rostrocaudally to colonize the entire gastrointestinal tract by E14.5. This study showed that a subpopulation of vagal crest-derived cells, very close to the migratory wavefront, starts to differentiate into neurons early, as shown by the expression of neuron-specific proteins and the absence of Sox10. Many of the early differentiating neurons transiently exhibited tyrosine hydroxylase (TH) immunoreactivity. The TH cells were demonstrated to be the progenitors of nitric oxide synthase (NOS) neurons. Immunohistochemistry, lesions, and DiI tracing were used to examine the projections of developing enteric neurons. The axons of first neurons in the gut (the TH-NOS neurons) projected in the same direction (caudally), and traversed the same pathways through the mesenchyme, as the migrating, undifferentiated, vagal crest-derived cells. To examine if the direction of migration and direction of axon projection are linked, coculture experiments were set up in which vagal crest-derived cells migrated either rostrocaudally (as they do in vivo), or caudorostrally (which they do not normally do), to colonize explants of embryonic aneural hindgut. The direction in which neurons projected was correlated with the direction of cell migration, but migration direction appears to be not the only mechanism influencing axon projection. Peristaltic reflexes involve both orally (rostrally) projecting neurons and anally (caudally) projecting neurons. Because few rostrally projecting neurons could be detected before birth, the full circuitry for peristaltic reflexes appears to develop after birth.
Inflammation, the reaction of vascularized tissue to local injury, not only limits the effects of injury; it may also be the underlying pathological process which initiates or sustains disease. In this paper, the evidence is reviewed for a role for nitric oxide (NO) as a chemical indicator of inflammation and inflammatory diseases.
Part of the regulation of gastrointestinal (GI) smooth muscles is provided by nonadrenergic noncholinergic (NANC) nerves. Stimulation of these nerves, either by field stimulation or via neural reflex pathways, elicits hyperpolarization of postjunctional smooth muscle membranes referred to as inhibitory junction potentials and relaxation. The transmitter(s) that mediate NANC inhibitory neural transmission have been a controversial topic for nearly 30 years. Recent evidence suggests that nitric oxide (NO) may serve as a NANC inhibitory transmitter in the GI tract. This hypothesis is supported by the following. 1) Immunohistochemical studies have shown that the enzyme necessary for NO synthesis is expressed in enteric neurons. In vitro studies of muscles from nearly all levels of GI tract have also shown that arginine analogues, which inhibit NO synthesis, reduce inhibitory effects of NANC neurotransmission. Effects of arginine analogues can be restored by addition of excess L-arginine, the substrate for NO synthesis. These data suggest that NO can be synthesized by enteric nerves. 2) Bioassays have demonstrated nerve-evoked release of a substance that has been identified as NO during NANC nerve stimulation. Oxyhemoglobin, known to bind to and sequester NO, also blocks NANC responses. These data suggest that NO is released into extracellular fluid during nerve stimulation. 3) Addition of NO causes rapid hyperpolarization of GI smooth muscle cells and relaxes muscles strips. These effects are similar to NANC nerve responses. NO and electrical field stimulation also increase tissue guanosine 3',5'-cyclic monophosphate, which may be the second messenger involved in NANC responses. 4) Removal of NO is easily accomplished by its rapid spontaneous breakdown in physiological solutions. 5) The pharmacology of NO and the NANC neurotransmitter in many preparations is similar, e.g., oxyhemoglobin blocks responses to NANC nerve stimulation and to exogenous NO. In summary, it would appear that many of the criteria necessary for NO to be considered a neurotransmitter have been satisfied.
The nitric oxide (NO) signaling pathway is a major nonadrenergic-noncholinergic transmitter mechanism in the enteric nervous system. Our aim was to localize the enzymes in question, i.e., neuronal nitric oxide synthase (nNOS), soluble guanylate cyclase (sGC), and cGMP-dependent kinase type I (cGK-I) in rat small intestine by indirect immunofluorescence. nNOS staining was found in neurons of the myenteric plexus and in varicose nerve fibers mainly in the circular muscle layer. The cells positive for neurokinin-1 (NK-1) receptor and c-kit (interstitial cells of Cajal, ICC) in the deep muscular plexus (DMP) did not show nNOS reactivity, but nNOS-positive nerve fibers were directly adjacent to them. sGC was found in flattened cells surrounding myenteric ganglia (periganglionic cells, PGC), in ICC of the DMP, faintly in smooth muscle cells (SMC), and in cells perivascularly scattered throughout the circular muscle layer. cGK-I immunoreactivity was found abundantly in PGC (which presumably are ICC), in ICC of DMP, in SMC of the innermost circular and longitudinal muscle layers, but less intensively in the outer circular layer. Weak cGK-I staining occurred in nerve cells within the myenteric and submucosal plexus. Conclusively the key enzymes of the NO signaling pathway are differentially distributed: Occurrence of nNOS exclusively in neurons and the presence of sGC and cGK-I predominantly in ICC suggest a sequence of neuronal NO release, activation of ICC, and consecutive smooth muscle relaxation. ICC of the DMP seem to be the primary targets for neurally released NO.
Reactive oxygen species (ROS) and nitric oxide (NO) are important participants in signal transduction that could provide the cellular basis for activity-dependent regulation of neuronal excitability. In young rat cortical brain slices and undifferentiated PC12 cells, paired application of depolarization/agonist stimulation and oxidation induces long-lasting potentiation of subsequent Ca(2+) signaling that is reversed by hypoxia. This potentiation critically depends on NO production and involves cellular ROS utilization. The ability to develop the Ca(2+) signal potentiation is regulated by the developmental stage of nerve tissue, decreasing markedly in adult rat cortical neurons and differentiated PC12 cells.
The distribution of nitric oxide synthase (NOS) immunoreactivity was investigated in the guinea-pig small intestine. There were many immunoreactive nerve cell bodies in the myenteric plexus but very few in submucous ganglia. NOS immunoreactivity was not found in non-neuronal cells except for rare mucosal endocrine cells. Abundant immunoreactive nerve fibres in both myenteric and submucous ganglia, and in the circular muscle, arose from myenteric nerve cells whose axons projected anally along the intestine. NOS immunoreactivity coexisted with VIP-immunoreactivity, but not with substance P immunoreactivity. We conclude that nitric oxide synthase is located in a sub-population of enteric neurons, amongst which are inhibitory motor neurons that supply the circular muscle layer.
Part of the regulation of gastrointestinal (GI) smooth muscles is provided by nonadrenergic noncholinergic (NANC) nerves. Stimulation of these nerves, either by field stimulation or via neural reflex pathways, elicits hyperpolarization of postjunctional smooth muscle membranes referred to as inhibitory junction potentials and relaxation. The transmitter(s) that mediate NANC inhibitory neural transmission have been a controversial topic for nearly 30 years. Recent evidence suggests that nitric oxide (NO) may serve as a NANC inhibitory transmitter in the GI tract. This hypothesis is supported by the following. 1) Immunohistochemical studies have shown that the enzyme necessary for NO synthesis is expressed in enteric neurons. In vitro studies of muscles from nearly all levels of GI tract have also shown that arginine analogues, which inhibit NO synthesis, reduce inhibitory effects of NANC neurotransmission. Effects of arginine analogues can be restored by addition of excess L-arginine, the substrate for NO synthesis. These data suggest that NO can be synthesized by enteric nerves. 2) Bioassays have demonstrated nerve-evoked release of a substance that has been identified as NO during NANC nerve stimulation. Oxyhemoglobin, known to bind to and sequester NO, also blocks NANC responses. These data suggest that NO is released into extracellular fluid during nerve stimulation. 3) Addition of NO causes rapid hyperpolarization of GI smooth muscle cells and relaxes muscles strips. These effects are similar to NANC nerve responses. NO and electrical field stimulation also increase tissue guanosine 3',5'-cyclic monophosphate, which may be the second messenger involved in NANC responses. 4) Removal of NO is easily accomplished by its rapid spontaneous breakdown in physiological solutions. 5) The pharmacology of NO and the NANC neurotransmitter in many preparations is similar, e.g., oxyhemoglobin blocks responses to NANC nerve stimulation and to exogenous NO. In summary, it would appear that many of the criteria necessary for NO to be considered a neurotransmitter have been satisfied.
The dopamine beta-hydroxylase promoter has been shown to direct expression of the reporter gene product, beta-galactosidase, to enteric neurons and putative embryonic neuroblasts in transgenic mice (Mercer et al., 1991; Kapur et al., 1991). In this paper, expression of the transgene, D beta H-nlacZ, in the gastrointestinal tract is characterized in more detail in wild-type mice and mice which are also homozygous for the lethal spotted allele (ls). Expression of the transgene in wild-type embryos was first detected in scattered mesenchymal cells in the proximal foregut on embryonic day 9.5, and progressed distally until embryonic day 13.5 when the entire length of the gut was colonized by such cells. Several observations suggest that the mesenchymal cells which express the transgene (MCET) are, in fact, enteric neuroblasts, probably derived from the vagal neural crest. (1) The presence of MCET in progressively more caudal portions of the embryonic gut correlated with the neurogenic potential of isolated gastrointestinal segments grafted under the renal capsule. (2) Mitotic activity of MCET was demonstrated by incorporation of [3H]thymidine in utero. (3) The migratory behavior of MCET and/or their precursors was revealed in anastomotic subcapsular grafts of gut from transgenic and non-transgenic embryos; enteric ganglia of the latter were populated by MCET from the former. (4) Enteric expression of the transgene postnatally was restricted to intrinsic neurons that coexpressed other phenotypic markers of neuronal differentiation. The pattern of transgene expression in ls/ls mice was identical to that seen in ls/+ and +/+ mice until embryonic day 12.5.(ABSTRACT TRUNCATED AT 250 WORDS)
The hypothesis was tested that developing enteric neurons withdraw from the cell cycle in a sequence related to their phenotype. The birthdays of immunocytochemically identified myenteric and submucosal neurons were determined in the murine duodenum and jejunum. [3H]thymidine ([3H]TdR) was injected into timed pregnant mice or pups at 4-8 hour intervals over a 24 hour period. Pups were killed on postnatal day 30 (P30). [3H]TdR incorporation was detected by radioautography in enteric neurons, which were phenotypically identified by the simultaneous detection of the immunoreactivities of 5-hydroxytryptamine (5-HT), choline acetyl transferase (ChAT), neuropeptide Y (NPY), enkephalin (ENK), calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP). The dates of the earliest withdrawal from the cell cycle of neurons containing these markers were determined, as well as the length of time during which the identified neurons continued to be born, and the date on which their rate of birth was maximal. The birthdates of myenteric neurons that contained 5-HT (E8-E14, peak at E10) or ChAT (E8-E15, peak at E12) tended to be earlier than those that contained ENK (E10-E18, peak at E14), NPY (E10-E18, peak at E15), VIP (E10-P5, peak at E15), or CGRP (E10-P3, peak at E17). For any given immunocytochemically defined neuronal phenotype, submucosal neurons tended to be born later than their myenteric counterparts and submucosal neurons that contained neuropeptides were born later than those that contained only ChAT immunoreactivity. The day (E8) on which the first 5-HT- and ChAT-immunoreactive neurons became postmitotic is earlier than the day (E9) on which the colonization of the bowel by crest-derived cells has been detected. The population of neural precursors that colonizes the gut, therefore, is heterogeneous; many cells are proliferating, but a specific subset, which will ultimately give rise to serotoninergic or cholinergic neurons, is already postmitotic. Neurons continued to be born throughout fetal life and even after birth. Consequently, terminally differentiated neurons coexist in the developing enteric nervous system with dividing neural precursor cells. This observation is consistent with the idea that early developing neurons could affect the development of enteric neural precursors; moreover, they also demonstrate that it is possible to add neurons to the enteric plexuses even after the neural circuits on which the bowel depends have become functional.
Motility of the gastrointestinal tract is directly controlled by enteric inhibitory and excitatory motor neurons that innervate the layers of smooth muscle. Inhibitory motor neurons mediate receptive and accommodative relaxations and control the opening of sphincters, thus playing an important role in normal gut motility. Recent studies have demonstrated that nitric oxide (NO) is an important neurotransmitter released by inhibitory motor neurons in animal and human gut. Antagonists of nitric oxide synthase (NOS), the synthetic enzyme for NO, reduce the effectiveness of transmission from inhibitory motor neurons. Exogenous NO mimics inhibitory nerve activation, and a variety of compounds that affect the availability of endogenously produced NO modulate relaxations of gastrointestinal smooth muscle. It is clear, however, that NO is unlikely to be the only transmitter released by enteric inhibitory motor neurons: several other substances such as vasoactive intestinal polypeptide (VIP), or related peptides, and adenosine triphosphate (ATP) are also likely to contribute to nerve-mediated inhibition. The identification of NO as a major inhibitory neurotransmitter to gastrointestinal smooth muscle fills an important gap in our understanding of the physiological control of motility and opens up a wide range of new experimental possibilities. It may eventually lead to the development of new drugs for motility disorders. It should be noted, however, that NO is important in the brain, in cardiovascular control, in blood cell function and in many other organ systems, suggesting that it may be difficult to achieve specific pharmacological intervention targeted on inhibitory neurotransmission in the gut, without undesirable side effects.
Neuronal nitric oxide synthase (NOS), visualized immunohistochemically or with NADPH diaphorase histochemistry, is transiently expressed in discrete areas of the developing rat nervous system. In the brain transient NOS expression occurs in the cerebral cortical plate. At E15-E19, the majority of cells in the plate stain, with their processes extending through the corpus striatum to the thalamus. This staining decreases after birth and vanishes by the 15th postnatal day. Neurons in olfactory epithelium also express NOS from E15 till early postnatal life. In embryonic sensory ganglia virtually all neuronal cells are NOS positive, whereas by early adulthood only 1% express NOS. By contrast to these areas of transient NOS expression, in other neuronal sites NOS staining appears after cell bodies cease dividing and cells extend processes, and the staining persists in adult life. The transient expression of neuronal NOS may reflect a role in developmental processes such as programmed cell death.
Nitric oxide (NO) is a diffusible free radical that functions as a second messenger and neurotransmitter. NO synthase (NOS) is highly and transiently expressed in neurons of the developing olfactory epithelium during migration and establishment of primary synapses in the olfactory bulb. NOS is first expressed at E11 in cells of the presumptive nervous layer of the olfactory placode. NOS immunoreactivity persists in the descendants of these cells that differentiate into embryonic olfactory receptor neurons (ORNs). Olfactory NOS expression in the ORN and in its afferents rapidly declines after birth and is undetectable by P7. Following bulbectomy, NOS expression is rapidly induced in the regenerating ORN and is particularly enriched in their outgrowing axons. Immunoblot and Northern blot analyses similarly demonstrate an induction of NOS protein and mRNA expression, respectively, the highest levels of which coincide with peaks of ORN regeneration. These data argue against a role for NO in odorant-sensitive signal transduction, but suggest a prominent function for NO in activity-dependent establishment of connections in both developing and regenerating olfactory neurons.
The distribution and abundance of nitric oxide synthase (NOS)-containing neurons and their terminals in the gastrointestinal tract of the guinea-pig were examined in detail using NADPH diaphorase histochemistry and NOS immunohistochemistry. NOS-containing cell bodies were found in the myenteric plexus throughout the gastrointestinal tract and in the submucous plexus of the stomach, colon and rectum. NOS-containing neurons comprised between 12% (in the duodenum) and 54% (in the esophagus) of total myenteric neurons. In the ileum, NOS neurons represented 19% of total myenteric neurons. Most of the NOS neurons throughout the gastrointestinal tract possessed lamellar dendrites and a single axon. NOS-containing terminals were abundant in the circular muscle, including that of the sphincters, but were rare in the longitudinal muscle, except for the taeniae of the caecum. The muscularis mucosae of the esophagus, stomach, colon and rectum received a medium to dense innervation by NOS terminals. Within myenteric ganglia, NOS-containing terminals were extremely sparse in the esophagus, stomach and duodenum, common in the ileum and distal colon and extremely dense in the proximal colon and rectum. The submucous plexus in the ileum and large intestine contained a sparse plexus of NOS-containing terminals. NOS terminals were not observed in the mucosa of any region. We conclude that throughout the gastrointestinal tract of the guinea-pig, NOS neurons are inhibitory motor neurons to the circular muscle; in the ileum and large intestine, NOS neurons may also function as interneurons.
Arrest of cell division is a prerequisite for cells to enter a program of terminal differentiation. Mitogenesis and cytostasis of neuronal cell precursors can be induced by the same or by different growth or trophic factors. Response of PC12 cells to nerve growth factor (NGF) involves a proliferative phase that is followed by growth arrest and differentiation. Here we present evidence that the cytostatic effect of NGF is mediated by nitric oxide (NO), a second messenger molecule with both para- and autocrine properties that can diffuse freely and act within a restricted volume. We show that NGF induces different forms of nitric oxide synthase (NOS) in neuronal cells, that nitric oxide (NO) acts as a cytostatic agent in these cells, that inhibition of NOS leads to reversal of NGF-induced cytostasis and thereby prevents full differentiation, and that capacity of a mutant cell line to differentiate can be rescued by exogenous NO. We suggest that induction of NOS is an important step in the commitment of neuronal precursors and that NOS serves as a growth arrest gene, initiating the switch to cytostasis during differentiation.
The role of endogenous nitric oxide (NO) as a modulator of enteric neurotransmission was investigated in longitudinal muscle myenteric plexus (LMMP) preparations of guinea‐pig isolated ileum.
In tissues previously incubated with [ ³ H]‐choline, exogenous NO inhibited electrically‐evoked [ ³ H]‐choline overflow as well as responses to exogenous agonists, indicating that NO has the potential of neuromodulation both pre‐ and postjunctionally.
A series of NO synthase inhibitors enhanced contractile responses to nerve stimulation indicating inhibitory neuromodulation by endogenous NO.
The potency order of the NO synthase inhibitors and their consistent effects after dexamethasone, on responses to nerve stimulation, indicate action on a constitutive NO synthase.
Responses enhanced by NO synthase inhibitors were inhibited by the substance P receptor antagonist, spantide, suggesting a neuromodulatory influence on substance P‐like neurotransmission by the endogenous NO.
NO synthase inhibition did not modify contractile responses to application of acetylcholine or substance P, or [ ³ H]‐choline overflow, indicating that endogenous NO mainly has a prejunctional inhibitory action on substance P‐like neurotransmission. Nor did it modify responses to direct electrical muscle stimulation in the presence of tetrodotoxin. This suggests a prejunctional enhancing effect by NO synthesis inhibition.
Evidence for endogenous NO modulation of acetylcholine release was obtained when NO synthase inhibition modified atropine‐sensitive, nerve‐mediated contractile responses. However, [ ³ H]‐choline overflow was unaltered by NO synthase inhibition.
NO synthase inhibition did not modify responses to inhibitory neurotransmission.
The findings suggest that endogenous NO inhibits substance P‐like motor neurotransmission, probably via prejunctional mechanisms. Cholinergic transmission may also be reduced by endogenous NO, acting prejunctionally.
Inflammation, the reaction of vascularized tissue to local injury, not only limits the effects of injury; it may also be the underlying pathological process which initiates or sustains disease. In this paper, the evidence is reviewed for a role for nitric oxide (NO) as a chemical indicator of inflammation and inflammatory diseases.
Unlabelled:
Mice lacking neuronal nitric oxide synthase gene (ncNOS) were used to determine the enzymatic source of nitric oxide (NO) and its relationship with other putative inhibitory neurotransmitters. Inhibitory junction potentials (IJP) of circular smooth muscle of gastric fundus were studied. The IJP in the wild-type mice consists of overlapping components, the fast and slow IJPs. NOS inhibitor L-NA or VIP receptor antagonist VIP(10-28), blocks the slow IJP but not the fast IJP. The fast UP is blocked by alpha-beta methylene ATP tachyphylaxis, by reactive blue 2, and by apamin. The IJP in the ncNOS-deficient [ncNOS(-)] mutant is of short duration and is abolished by blockers of the fast IJP, but is unaffected by blockers of the slow UP. Exogenous VIP produces membrane hyperpolarization in strips from wild-type but not ncNOS(-) mice. The hyperpolarizing action of VIP is resistant to nifedipine but is sensitive to omega-conotoxin GVIA.
In conclusion:
(a) NO derived from ncNOS is an inhibitory neurotransmitter rather than a postjunctional mediator; (b) VIP is a prejunctional neurotransmitter that causes release of evanescent NO; and (c) ATP acts in parallel with the VIP/NO pathway.
Vasoactive intestinal peptide (VIP) release, nitric oxide (NO) formation, and relaxation induced by nerve stimulation were examined in rabbit and rat gastric muscle. VIP stimulated NO formation in muscle strips, whereas NO stimulated VIP release. Nerve stimulation (0.025-16 Hz or 2-940 pulses) elicited frequency-dependent stimulation of VIP release, NO formation, and relaxation, all significant at two to three pulses. NG-nitro-L-arginine (L-NMA) abolished NO formation, abolished VIP release and relaxation at low frequencies, and partly inhibited them at higher frequencies. Oxyhemoglobin (oxy-Hb) inhibited VIP release and relaxation by 80% at low frequencies and 20-30% at higher frequencies. VIP-(10-28) abolished NO formation and relaxation at lower frequencies and partly inhibited them at higher frequencies; in contrast, VIP-(10-28) augmented VIP release in both species. The pattern of inhibition was similar in both species. Inhibition of maximal NO formation by VIP-(10-28) (82% in rabbit; 48% in rat) implied that a major component of NO is formed in muscle cells by the action of VIP. Thus 1) inhibition of relaxation by L-NNA reflects suppression of NO and VIP release from nerve terminals and NO formation in muscle cells, 2) inhibition by VIP-(10-28) partly reflects suppression of NO formation in muscle cells, and 3) inhibition by oxy-Hb reflects neutralization of extracellular NO and suppression of VIP release. The study demonstrates the dual origin of NO from nerves and muscle and its interplay with VIP in regulating relaxation.
Cell division and subsequent programmed cell death in imaginal discs of Drosophila larvae determine the final size of organs and structures of the adult fly. We show here that nitric oxide (NO) is involved in controlling the size of body structures during Drosophila development. We have found that NO synthase (NOS) is expressed at high levels in developing imaginal discs. Inhibition of NOS in larvae causes hypertrophy of organs and their segments in adult flies, whereas ectopic expression of NOS in larvae has the opposite effect. Blocking apoptosis in eye imaginal discs unmasks surplus cell proliferation and results in an increase in the number of ommatidia and component cells of individual ommatidia. These results argue that NO acts as an antiproliferative agent during Drosophila development, controlling the balance between cell proliferation and cell differentiation.
The projections of different subpopulations of myenteric neurons in the mouse small and large intestine were examined by combining immunohistological techniques with myotomy and myectomy operations. The myotomies were used to examine the polarity of neurons projecting within the myenteric plexus and showed that neurons containing immunoreactivity for nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), calbindin and 5-HT projected anally, while neurons with substance P (SP)-immunoreactivity projected orally, in both the small and large intestine. Neurons containing neuropeptide Y (NPY)- and calretinin-immunoreactivity projected locally. In the large intestine, GABA-immunoreactive neurons projected both orally and anally, with more axons tending to project anally. Myectomy operations revealed that circular muscle motor neurons containing NOS/VIP/ +/-NPY and calretinin neurons projected anally both in the small and large intestine, while SP-immunoreactive circular muscle motor neurons projected orally. In the large intestine, GABA-IR circular muscle motor neurons projected both orally and anally. This study showed that although some neurons, such as the NOS/VP inhibitory motor neurons and interneurons, SP excitatory motor neurons and 5-HT interneurons had similar projections to those in other species, the projections of other chemical classes of neurons in the mouse intestine differed from those reported in other species.
Among three NO synthase (NOS) isoforms only the inducible NOS-II was localized in developing olfactory receptor neurons of the mouse. First NOS-II immunoreactive receptor cells including their processes were detected by embryonic day 11 when the olfactory pit starts to invaginate. Cellular staining lasted until embryonic day 16, and was reduced during the next few days. At embryonic day 20 no reactivity was found in the olfactory epithelium, whereas centripetal nerve fibers remained positive. This transient expression of NOS-II implies a role for the differentiation of early olfactory receptor neurons and synaptic plasticity.
Although neurons containing neuronal nitric oxide synthase (NOS) are abundant in the myenteric plexus of the small intestine of all mammalian species examined to date, NOS-containing neurons are sparse in the submucous plexus, and there does not appear to be an innervation of the mucosa by nerve fibres containing NOS. In this study, we used immunohistochemical techniques to examine the presence of neuronal NOS in the mouse intestine during development. At embryonic day 18 and postnatal day 0 (P0), about 50% of the neurons in the submucous plexus of the small intestine showed strong immunoreactivity to NOS, and NOS-immunoreactive nerve fibres were present in the mucosa. By P7, there was a gradation in the intensity of NOS immunostaining exhibited by submucosal neurons, varying from intense to extremely weak. During subsequent development, the proportion of submucous neurons showing NOS immunoreactivity decreased, and immunoreactive nerve fibres were no longer observed in the mucosa. In adult mice, NOS neurons comprised only 3% of neurons in the submucous plexus, which is significantly less than at P0. In contrast to the submucous plexus, the percentage of neurons that showed NOS immunoreactivity in the myenteric plexus did not change significantly during development.
The colonization of the rodent gastrointestinal tract by enteric neuron precursors is controversial due to the lack of specific cellular markers at early stages. The transcription factor, Phox2b, is expressed by enteric neuron precursors (Pattyn et al. Development 124, 4065-4075, 1997). In this study, we have used an antiserum to Phox2b to characterize in detail the spatiotemporal expression of Phox2b in the gastrointestinal tract of adult mice and embryonic mice and rats. In adult mice, all enteric neurons (labeled with neuron-specific enolase antibodies), and a subpopulation of glial cells (labeled with GFAP antibodies), showed immunoreactivity to Phox2b. In embryonic mice, the appearance of Phox2b-immunoreactive cells was mapped during development of the gastrointestinal tract. At Embryonic Days 9.5-10 (E9.5-10), Phox2b-labeled cells were present only in the stomach, and during subsequent development, labeled cells appeared as a single rostrocaudal wave along the gastrointestinal tract; at E14 Phox2b-labeled cells were present along the entire length of the gastrointestinal tract. Ret and p75 have also been reported to label migratory-stage enteric neuron precursors. A unidirectional, rostral-to-caudal colonization of the gastrointestinal tract of embryonic mice by Ret- and p75-immunoreactive cells was also observed, and the locations of Ret- and p75-positive cells within the gut were very similar to that of Phox2b-positive cells. To verify the location of enteric neuron precursors within the gut, explants from spatiotemporally defined regions of embryonic intestine, 0.3-3 mm long, were grown in the kidney subcapsular space, or in catenary organ culture, and examined for the presence of neurons. The location and sequence of appearance of enteric neuron precursors deduced from the explants grown under the kidney capsule or in organ culture was very similar to that seen with the Phox2b, Ret, and p75 antisera. Previous studies have mapped the rostrocaudal colonization of the rat intestine by enteric neuron precursors using HNK-1 as a marker. In the current study, all HNK-1-labeled cells in the gastrointestinal tract of rat embryos showed immunoreactivity to Phox2b, but HNK-1 cells comprised only a small subpopulation of the Phox2b-labeled cells. In addition, in rats, Phox2b-labeled cells were present in advance of (more caudal to) the most caudal HNK-1-labeled cells by 600-700 microm in the hindgut at E15. We conclude that the neural crest cell population that arises from the vagal level of the neural axis and that populates the stomach, midgut, and hindgut expresses Phox2b, Ret, and p75. In contrast, the sacral-level neural crest cells that populate the hindgut either do not express, or show a delayed expression of, all of the known markers of vagal- and trunk-level neural crest cells.
PC12 cells are used as a model system to study neuronal differentiation. Nerve growth factor (NGF) triggers a differentiation pathway in PC12 cells. Neurite outgrowth (a morphological marker of differentiation) in PC12 cells is significantly reduced in the presence of the NOS inhibitor l-NAME, but not d-NAME, implicating NOS in the differentiation process. Previously we have shown that the neuronal NO synthase (nNOS) isoform is induced in PC12 cells in the presence of NGF. Thus, we wished to further evaluate the role of nNOS and NO in PC12 cell differentiation. When a dominant negative mutant nNOS expression vector was transiently transfected into NGF-treated PC12 cells, it significantly reduced PC12 cell neurite outgrowth. Thus, we concluded that the NO required for PC12 cell differentiation, in response to NGF, is produced by nNOS. NO alone was insufficient to induce differentiation as cells treated with the NO donor, sodium nitroprusside did not produce neurites. Treatment of PC12 cells with oxyhemoglobin (an NO scavenger) was also found to significantly reduce the number of neurites produced by PC12 cells treated with NGF. Thus, NO appears to be necessary, but not sufficient, to induce differentiation, and its mode of action appears to be extracellular. A well documented action of NO is to activate soluble guanylate cyclase. Thus, we determined the role of soluble guanylate cyclase activation as a means by which NO induces PC12 cell differentiation. However, in the presence of NGF (to prime PC12 cells for differentiation) and l-NAME (to specifically remove the NO component), 8Br-cGMP (a cGMP analog) failed to induce PC12 cell differentiation. In addition, blockade of sGC activity with specific inhibitors failed to block NGF-induced PC12 cell differentiation. We conclude that the NO required for PC12 cell differentiation is produced by nNOS and that the NO exerts its effects on surrounding PC12 cells in a sGC/cGMP independent manner.
Based on in vitro studies, nitric oxide (NO) is reported to be involved in initial neuronal differentiation. In order to compare this finding with the situation in vivo, we have looked for the expression of the three NO synthase isoforms in the developing mouse vestibulocochlear system. From these isoforms only the inducible NOS II is expressed during inner ear development. Examination of a series of embryonic and early postnatal animals, up to postnatal day 6, reveals a maturation-dependent, monophasic expression of this isoform. Initial expression is observed by day 10 of gestation in nerve cells of the vestibolocochlear ganglion and on their fibres. By day 14 of gestation, these afferent fibres penetrate the epithelium of the prospective receptor fields making contact with early, differentiating immunoreactive cochlear hair cells and receptor cells of the macula and crista ampullaris. This receptor-cell-derived immunoreactivity vanished in differentiated sensory hair cells by postnatal day 6, when both the constitutive isoforms and subsequent activated members of the down stream second messenger cascade (guanylate cyclase/cGMP) of the adult mouse were not then detectable. The strict phasic expression of NOS-II, independent of the second messenger system mentioned above, implies that there is a unique role for the inducible NOS isoform in nerve cell differentiation, independent of the NO/guanylate cyclase/cGMP pathway.
To identify the enzymatic source of nitric oxide (NO) in the lower esophageal sphincter (LES), studies were performed in wild-type and genetically engineered endothelial nitric oxide synthase [eNOS(-)] and neuronal NOS [nNOS(-)] mice. Under nonadrenergic noncholinergic (NANC) conditions, LES ring preparations developed spontaneous tone in all animals. In the wild-type mice, electrical field stimulation produced frequency-dependent intrastimulus relaxation and a poststimulus rebound contraction. NOS inhibitor N(omega)-nitro-L-arginine methyl ester (100 microM) abolished intrastimulus relaxation and rebound contraction. In nNOS(-) mice, both the intrastimulus relaxation and rebound contraction were absent. However, in eNOS(-) mice there was no significant difference in either the relaxation or rebound contraction from the wild-type animal. Both nNOS(-) and eNOS(-) tissues showed concentration-dependent relaxation to NO donor diethylenetriamine-NO and there was no difference in the sensitivity to the NO donor in nNOS(-), eNOS(-), or wild-type animals. These results indicate that in mouse LES, nNOS rather than eNOS is the enzymatic source of the NO that mediates NANC relaxation and rebound contraction.
Functional expression of the rat colonic H(+)-K(+)-ATPase was obtained by coexpressing its catalytic alpha-subunit and the beta(1)-subunit of the Na(+)-K(+)-ATPase in Xenopus laevis oocytes. We observed that, in oocytes expressing the rat colonic H(+)-K(+)-ATPase but not in control oocytes (expressing beta(1) alone), NH(4)Cl induced a decrease in (86)Rb uptake and the initial rate of intracellular acidification induced by extracellular NH(4)Cl was enhanced, consistent with NH(+)(4) influx via the colonic H(+)-K(+)-ATPase. In the absence of extracellular K(+), only oocytes expressing the colonic H(+)-K(+)-ATPase were able to acidify an extracellular medium supplemented with NH(4)Cl. In the absence of extracellular K(+) and in the presence of extracellular NH(+)(4), intracellular Na(+) activity in oocytes expressing the colonic H(+)-K(+)-ATPase was lower than that in control oocytes. A kinetic analysis of (86)Rb uptake suggests that NH(+)(4) acts as a competitive inhibitor of the pump. Taken together, these results are consistent with NH(+)(4) competition for K(+) on the external site of the colonic H(+)-K(+)-ATPase and with NH(+)(4) transport mediated by this pump.
Nitric oxide is a ubiquitous molecule involved in a variety of biological processes. The specific action of NO depends on its enzymatic sources namely neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) and all three isoforms have been localized in the gastrointestinal tract. Constitutive synthesis of NO by nNOS or eNOS isoforms is involved in the maintaining of the gastrointestinal mucosal integrity through modulation of gastric mucosal blood flow, epithelial secretion and barrier function. However, large amounts of NO synthesized from the inducible isoform have been implicated in tissue injury in the gut during inflammatory reactions. In this review we provide an overview of the dual role of nitric oxide in modulating gastrointestinal mucosal defense and injury. In addition, we highlight the therapeutic potential of NO modulation.
The similarities between heme oxygenase-2 (HO-2) and nitric oxide synthase (nNOS) and the transient expression of nNOS during development led us to investigate whether both systems are similarly affected by changes that occur during development and by regional differences along the small intestine. By combining NADPH diaphorase histochemistry and HO-2 immunohistochemistry on whole-mount preparations and by using stereologic methods, a qualitative and quantitative description of HO-2 and nNOS expression was obtained. Examinations were carried out on the small intestine of fetal, 1-2-day and 5-6-week-old pigs. In all age groups, three enteric plexuses were distinguished. The presence of HO-2-immunoreactive (HO-2-IR) and NADPH diaphorase-positive neurons corresponded to earlier morphological and physiological reports. Nevertheless, the total number of nitrergic neurons remained constant or decreased in the enteric plexuses, whereas the total number of HO-2-IR neurons displayed an overall increase. Changing concentrations of glucocorticoids, target-derived signals, presynaptic input, and an effect of HO-2 activity on nNOS synthesis are likely to play roles in the observed developmental changes. The numerical density of HO-2-IR neurons remained relatively constant along the intestinal tract; in contrast, the nitrergic neurons were most numerous in the inner submucous and myenteric plexus in the duodenum and ileum, respectively. It is believed that the duodenal nitrergic neurons in the inner submucous plexus could be involved in the regulation of duodenal secretion processes, whereas the region-dependent density in the myenteric plexus possibly forms the morphological basis for a regionally different participation of NO in the relaxation of the small intestine.
The nitric oxide (NO) signaling pathway is a major nonadrenergic-noncholinergic transmitter mechanism in the enteric nervous system. Our aim was to localize the enzymes in question, i.e., neuronal nitric oxide synthase (nNOS), soluble guanylate cyclase (sGC), and cGMP-dependent kinase type I (cGK-I) in rat small intestine by indirect immunofluorescence. nNOS staining was found in neurons of the myenteric plexus and in varicose nerve fibers mainly in the circular muscle layer. The cells positive for neurokinin-1 (NK-1) receptor and c-kit (interstitial cells of Cajal, ICC) in the deep muscular plexus (DMP) did not show nNOS reactivity, but nNOS-positive nerve fibers were directly adjacent to them. sGC was found in flattened cells surrounding myenteric ganglia (periganglionic cells, PGC), in ICC of the DMP, faintly in smooth muscle cells (SMC), and in cells perivascularly scattered throughout the circular muscle layer. cGK-I immunoreactivity was found abundantly in PGC (which presumably are ICC), in ICC of DMP, in SMC of the innermost circular and longitudinal muscle layers, but less intensively in the outer circular layer. Weak cGK-I staining occurred in nerve cells within the myenteric and submucosal plexus. Conclusively the key enzymes of the NO signaling pathway are differentially distributed: Occurrence of nNOS exclusively in neurons and the presence of sGC and cGK-I predominantly in ICC suggest a sequence of neuronal NO release, activation of ICC, and consecutive smooth muscle relaxation. ICC of the DMP seem to be the primary targets for neurally released NO.
Nitric oxide (NO), a cell-derived highly diffusible and unstable gas is regarded to be involved in inter- and intracellular communication in the nervous system. Based on findings about the expression of the inducible NO synthase (NOS) isoform during development of early mouse olfactory as well as vestibulocochlear receptor neurons, we intended to prove a general role of this isoform for neuronal differentiation. Using immunohistochemical techniques, an exclusive expression of the inducible NOS-II isoform in early post-mitotic neurons of the developing mouse cortex and retina can be detected. In a pharmacological approach using cultures of the mouse cortex as well as embryonic stem cell-derived neural precursor cells, we investigated the functional role of NO on initial neuronal differentiation. Effects of NOS inhibitors and NO donors on the morphological differentiation were correlated with developmentally regulated calcium current densities, focusing on the effects of the specific NOS-II inhibitor GW 274150. Furthermore, involvement of the soluble guanylate cyclase (sGC)/cGMP signaling cascade was pharmacologically investigated. Our data indicate that while a specific block of NOS-II provokes a clear inhibition of neurite outgrowth formation as well as a decrease of calcium current densities, the inverse is true for exogenous NO donation. In line with lacking immunoreactivity for the sGC and cGMP there are only minor effects of compounds manipulating the sGC/cGMP pathway, suggesting the downstream sGC/cGMP pathway not to be essential in these early differentiation steps.
Information on equipment and subcellular distribution of nitric oxide synthase (NOS) isoforms in myenteric neurons and pacemaker cells (ICC) might help to identify nitric oxide (NO) pathway(s) acting on gastrointestinal motility. In sections of mouse colon labelled with neuronal (n)NOS, endothelial (e)NOS and inducible (i)NOS antibodies, all myenteric neurons co-expressed eNOS and iNOS and a subpopulation of them co-expressed nNOS. ICC co-expressed nNOS and eNOS. In the neurons, nNOS-labeling was intracytoplasmatic, in the ICC at cell periphery. In both cell types, eNOS-labeling was on intracytoplasmatic granules, likely mitochondria. In conclusion, myenteric neurons and ICC co-express several NOS isoforms with specific subcellular distribution. Different nNOS splice variants are presumably present: intracytoplasmatic nNOSbeta and nNOSalpha producing neurogenic NO, plasma membrane-bound nNOSalpha producing ICCgenic NO. eNOS might be implicated in mitochondrial respiration and, in ICC, also in pacemaker activity. Neurons express iNOS also in basal condition.
Nitric oxide (NO) inhibits the release of acetylcholine and cholinergic contractions in the small intestine of several species, but no information is available about the mouse ileum. This study examines the effects of NO on the electrically evoked release of [3H]acetylcholine and smooth muscle contraction in myenteric plexus-longitudinal muscle preparations of wild-type mice and of neuronal NO synthase (nNOS) and endothelial NOS (eNOS) knockout mice. The NOS inhibitor N(G)-nitro-L-arginine (L-NNA) and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) concentration dependently increased the evoked [3H]acetylcholine release and cholinergic contractions in preparations from wild-type mice and from eNOS knockout mice. Effects of L-NNA were specifically antagonized by L-arginine. In contrast, L-NNA and ODQ did not modify the release and contractions in preparations from nNOS knockout mice. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited the electrically evoked release of [3H]acetylcholine and longitudinal muscle contractions in a quantitatively similar manner in wild-type preparations as well as in nNOS and eNOS knockout preparations. We conclude that endogenous NO released by electrical field stimulation tonically inhibits the release of acetylcholine. Furthermore, data suggest that nNOS and not eNOS is the enzymatic source of NO-mediating inhibition of cholinergic neurotransmission in mouse ileum.
Hirschsprung disease is the result of aganglionosis of a variable length of the terminal bowel, which arises from the incomplete colonisation of the embryonic gut by vagal neural crest-derived cells (NCC) that migrate caudally from the pharyngeal gut to the rectum. We have previously shown that a very small group of NCC, at the leading edge of this wave of migration, can proliferate and differentiate to innervate the entire distal gut. It remains unknown if this capability is unique to those cells at the leading edge of NCC migration. The hypothesis tested was that NCC capable of acting as stem cells are found throughout the developing enteric nervous system (ENS). Gut was taken from mice at embryonic day 11.5 as the leading edge of NCC migration enters the colon. Terminal colon was separated as aganglionic recipient gut and its rostral end juxtaposed to the caudal end of the small intestine or caecum. The explants were cultured on nitrocellulose filters for up to 120 h, after which time the apposed segments had fused. The gut was then fixed and examined by immunohistochemistry to detect the neuronal markers PGP9.5 and nitric-oxide synthase (NOS) to assess development of enteric ganglia. NCC migrated from the proximal gut into the terminal colon, colonising it along its entire length. The pattern of NCC colonisation and differentiation of NOS-positive neurons was the same, regardless of whether the NCC were derived from the leading edge of migration in the caecum or from more proximal regions of the small intestine. Vagal NCC have the capacity to migrate into separated aganglionic terminal colon and differentiate into neurons. NCC at the leading edge of migration and those located more proximally within the gut demonstrate equivalent ability to migrate to and differentiate in the terminal rectum. Further studies are required to confirm which of these migrating NCC have the properties of ENS stem cells.
Time of origin of neu-rons in the murine enteric nervous system: sequence in relation to phenotype
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