Colocalization Of Nitric Oxide Synthase Research Articles

Nociceptin effect on intestinal motility depends on opioid-receptor like-1 receptors and nitric oxide synthase co-localization.

To study the effect of the opioid-receptor like-1 (ORL1) agonist nociceptin on gastrointestinal (GI) myenteric neurotransmission and motility. Reverse transcriptase - polymerase chain reaction and immunohistochemistry were used to localize nociceptin and ORL1 in mouse tissues. Intracellular electrophysiological recordings of excitatory and inhibitory junction potentials (EJP, IJP) were made in a chambered organ bath. Intestinal motility was measured in vivo. Nociceptin accelerated whole and upper GI transit, but slowed colonic expulsion in vivo in an ORL1-dependent manner, as shown using [Nphe(1)]NOC and AS ODN pretreatment. ORL1 and nociceptin immunoreactivity were found on enteric neurons. Nociceptin reduced the EJP and the nitric oxide-sensitive slow IJP in an ORL1-dependent manner, whereas the fast IJP was unchanged. Nociceptin further reduced the spatial spreading of the EJP up to 2 cm. Compounds acting at ORL1 are good candidates for the future treatment of disorders associated with increased colonic transit, such as diarrhea or diarrhea-predominant irritable bowel syndrome.

Colocalization of Nitric Oxide Synthase, Vasoactive Intestinal Polypeptide and Tyrosine Hydroxylase Immunoreactivities in Postganglionic Neurons of the Quail Superior Cervical Ganglion

The colocalization of immunoreactivity to nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP) and tyrosine hydroxylase (TH) in the superior cervical ganglion (SCG) was investigated in the quail. In this bird, a substantial amount of NOS-immunoreactive (IR) cells were consistently found in the SCG without colchicine treatment or nerve ligation. The finding worthy of pointing out was that three-fourths of these NOS-IR cells were positive for TH. VIP-IR cells appeared with markedly low frequency than NOS-IR cells. They were generally small in size and often located in the ganglion peripheral. There were no VIP-IR cells positive for TH or negative for NOS: VIP immunoreactivity always appears in NOS-IR cells negative for TH. Thus, the results of the present study clearly showed the existence of two distinct subpopulations of postganglionic NOS-IR neurons (one is catecholaminergic and negative for VIP, and the other is non-catecholaminergic and positive for VIP). This suggests that nitric oxide (NO) and possibly VIP act as postganglionic neurotransmitters or neuromodulators in the quail SCG. The predominant appearance of the former category of NOS-IR cells must be considered in relation to some specific NO-induced controlling mechanisms of SCG neurons.

The liver fluke Opisthorchis viverrini expresses nitric oxide synthase but not gelatinases

Host-parasite interaction during infection with the liver fluke Opisthorchis viverrini plays an important role in opisthorchiasis-associated cholangiocarcinoma via nitric oxide (NO) production. Host cells induce nitric oxide synthase (NOS)-dependent DNA damage and secrete Ras-related C3 botulinum toxin substrate (Rac)1, heme oxygenase (HO)-1, and gelatinases (matrix metalloproteinase (MMP)2 and MMP9). We evaluated whether these enzymes are expressed in O. viverrini. Colocalization of NOS and Rac1 was most prominently detected on day 30 post-infection (p.i.) in the gut, reproductive organ, eggs, acetabular and tegument. Expression of HO-1, an antioxidative enzyme, increased in a similar pattern to NOS, but was not present in the tegument. The levels of nitrate/nitrite, end products of NO, and ferric reducing antioxidant capacity, an indicator of antioxidant enzyme capacity, in parasite homogenates were highest on day 30 p.i. and then decreased on day 90 p.i. In contrast, zymography revealed that MMP2 and MMP9 were not present in parasite homogenates at all time points. In conclusion, O. viverrini induces NOS expression and NO production, but does not express gelatinases. The study may provide basic information and an insight into drug design for prevention and/or intervention approaches against O. viverrini infection.

An immunohistochemical study of the distribution of nitric oxide synthase-immunoreactive neurons and fibers in the reticular groove of suckling lambs

The reticular groove (RG) is a specialized region of ruminant forestomach which, in suckling animals, via a vagovagal reflex, transforms itself into a tube to ensure the direct transport of milk from the esophagus to the abomasum. The nervous mechanism controlling the RG movement is not fully understood; however, at this level, the enteric nervous system (ENS) shows the highest neuronal density when compared with other forestomach compartments. Because nitric oxide is considered the putative major mediator of non-adrenergic non-cholinergic smooth muscle relaxation, the aim of the present study was to investigate the ENS of the RG of suckling lambs, both in the floor and in the lip, with particular regard to nitric oxide synthase (NOS)-immunoreactivity (-IR), by means of double immunohistochemical staining. NOS antiserum was used in association with some neurochemical markers which have been utilized by many authors in ENS. A rich innervation of fibers extended along the entire length of the RG. Proceeding distally, the number of neurons stained with a pan-neuronal marker increased; they were more numerous in the lips and lip-floor junction than in the floor itself. However, the percentage of NOS-IR neurons was the same in the proximal and distal parts. Many NOS-IR neurons often co-expressed galanin and dopamine β-hydroxylase. Neurochemical markers, such as calbindin, calcitonin gene-related peptide, IB4 and neurofilament 200 kDa, usually used to identify primary sensory neurons were not expressed in RG neurons, and the co-localization of NOS with tyrosine hydroxylase and substance P was rarely found. When compared with other districts, the RG showed some peculiar aspects, such as the lack of both neurons in the submucosal plexus and the lack of typical sensory neurons.

Co-localization of nitric oxide synthase and choline acetyltransferase in the brain of the goldfish ( Carassius auratus)

This work investigates the nitrergic and cholinergic systems in the brain and spinal cord of the goldfish ( Carassius auratus). We studied the immunohistochemical localization of antibodies against the neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT) by bright-field and confocal microscopy. Nitrergic and cholinergic cells were segregated within the telencephalon, in both dorsal and ventral areas, and co-distributed in some nuclei of the diencephalon, mesencephalon, rhombencephalon, and spinal cord. Double-labeling experiments revealed nNOS/ChAT-positive cells in (1) the diencephalon: the preoptic and suprachiasmatic nuclei, the habenula, the dorsal thalamus, and the nucleus of the medial longitudinal fasciculus; (2) the mesencephalon: the optic tectum, the mesencephalic portion of the trigeminal nucleus, the oculomotor and trochlear nuclei, and the Edinger-Westphal nucleus; and (3) the rhombencephalon: the secondary gustatory nucleus, the nucleus isthmi, the lateral lemniscus nucleus, the cerebellum, the reticular formation, different nuclei of the octaval column, the motor zone of the vagal lobe, and the trigeminal, facial, abducens, glosso-pharyngeal, vagal, and hypobranchial motor nuclei. Double-labeled cells were also observed in the spinal motor column. The percentage of double-labeled cells was different in each studied nucleus, indicating a selective distribution pattern. Because double-labeled cells were more abundant in those nuclei involved with sensory and motor physiological processes, we suggest the involvement of both nitric oxide and acetylcholine in these neural functions in fish.

Immunohistochemical and hodological characterization of calbindin‐D28k‐containing neurons in the spinal cord of the turtle, Pseudemys scripta elegans

Neurons and fibers containing the calcium-binding protein calbindin-D28k (CB) were studied by immunohistochemical techniques in the spinal cord of adult and juvenile turtles, Pseudemys scripta elegans. Abundant cell bodies and fibers immunoreactive for CB were widely and distinctly distributed throughout the spinal cord. Most neurons and fibers were labeled in the superficial dorsal horn, but numerous cells were also located in the intermediate gray and ventral horn. In the dorsal horn, most CB-containing cells were located in close relation to the synaptic fields formed by primary afferents, which were not labeled for CB. Double immunohistofluorescence demonstrated distinct cell populations in the dorsal horn labeled only for CB or nitric oxide synthase, whereas in the dorsal part of the ventral horn colocalization of nitric oxide synthase was found in about 6% of the CB-immunoreactive cells in this region. Choline acetyltransferase immunohistochemistry revealed that only about 2% of the neurons in the dorsal part of the ventral horn colocalized CB, whereas motoneurons were not CB-immunoreactive. The involvement of CB-containing neurons in ascending spinal projections to the thalamus, tegmentum, and reticular formation was demonstrated combining the retrograde transport of dextran amines and immunohistochemistry. Similar experiments demonstrated supraspinal projections from CB-containing cells mainly located in the reticular formation but also in the thalamus and the vestibular nucleus. The revealed organization of the neurons and fibers containing CB in the spinal cord of the turtle shares distribution and developmental features, colocalization with other neuronal markers, and connectivity with other tetrapods and, in particular with mammals.

Colocalization of nitric oxide synthase and monoamines in neurons of the amphibian brain

By means of double immunohistofluorescence techniques, we have investigated the colocalization of nitric oxide synthase and tyrosine hydroxylase (TH) or serotonin (5-HT) in the central nervous system of the anurans Rana perezi and Xenopus laevis and the urodele Pleurodeles waltl. A wide codistribution of neuronal populations, expressing these markers, was found throughout the brain and spinal cord. In contrast, colocalization of these markers was rather restricted. Only in the caudal portion of the brainstem raphe column in anurans, approximately 80% of the 5-HT-positive cells were also NOS-immunoreactive, whereas in the urodele brain, about 40% of the serotonergic cells at the level of the glossopharyngeal motor nucleus were simultaneously NOS-positive. In various brain regions, a wide codistribution of NOS- and TH-containing neurons was observed, but real colocalization of nitrergic and catecholaminergic cells was only found in a small neuron population in the posterior tubercle of anuran amphibians. Therefore, in amphibians, only a distinct and small cell population within the serotonergic raphe column (anurans and urodele) and in the catecholaminergic posterior tubercle (anurans) seem to produce simultaneously nitric oxide.

Colocalization of NO and VIP in neurons of the submucous plexus in the rat intestine

Since very few previous studies have carried out the quantitative analysis for the colocalization of nitric oxide (NO) and vasoactive intestinal peptide (VIP) in the submucous neurons in the rat digestive tract, we applied in vivo treatment of colchicine to enhance the immunoreactivity and examined the colocalization of NO synthase (nNOS) and VIP in neurons of the submucous plexus throughout the rat digestive tract. The density of nNOS-containing neurons in the submucous plexus in the stomach corpus (103±25 cells/cm 2, n=3) and that in the antrum (157±9 cells/cm 2, n=3) were significantly lower than those in small and large intestine. However no difference was detected in the cell density among duodenum (1967±188 cells/cm 2, n=3), jejunum (2640±140 cells/cm 2, n=3), ileum (2070±42 cells/cm 2, n=3), proximal colon (2243±138 cells/cm 2, n=3) and distal colon (2633±376 cells/cm 2, n=3). The proportion of nNOS-immunoreactive (IR), nNOS/VIP-IR and VIP-IR neurons to the total number of submucous neurons was examined. nNOS/VIP-IR neurons comprised 45–55% of total number of submucous neurons from the duodenum to the proximal colon, however those comprised 66.4±5.1% in the distal colon. The results showed that the dense distribution of nNOS-containing neurons was found in the submucous plexus throughout the small and large intestine, and large population of submucous neurons co-stored nNOS and VIP.

Innervation of guinea-pig stellate ganglia by nitric oxide synthase, cocaine- and amphetamine-regulated transcript protein- and pituitary adenylate cyclase activating polypeptide-immunoreactive fibers

The present study analyzed using immunohistochemical labeling the distribution and co-localization of nitric oxide synthase (NOS), cocaine- and amphetamine-regulated transcript peptide (CARTp) and pituitary adenylate cyclase activating polypeptide (PACAP) with choline acetyltransferase (ChAT)-immunoreactive fibers in the guinea-pig stellate ganglia. ChAT-immunoreactive fibers make pericellular baskets around virtually all stellate ganglia neurons. Pericellular baskets of NOS, CARTp and PACAP fibers were also present around numerous stellate ganglia neurons. Although all the NOS and PACAP fibers also exhibited ChAT immunoreactivity, only some of the CARTp fibers were ChAT-immunoreactive. No evidence of co-localization of NOS, PACAP and CARTp was obtained. These results indicate that NOS, PACAP and CARTp are present in distinct preganglionic axons innervating the guinea-pig stellate ganglia.

Differentiation in the immunocytochemical features of intrinsic and cortically projecting neurons in the rat claustrum — combined immunocytochemical and axonal transport study

Retrograde axonal transport method of the fluorescent tracer FluoroGold (FG) was combined with immunocytochemistry to investigate the occurrence of nitric oxide synthase (NOS), somatostatin (SOM), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) in both intrinsic and cortically projecting neurons of the rat claustrum. Only NOS was detected in both the scattered projecting neurons and internal neurons of the claustrum. Approximately 20% of NOS-immunoreactive neurons in the claustrum were also retrogradely labeled with FG after tracer injections into the frontal cortex. The other substances were exclusively confined to the population of interneurons, which mainly displayed an oval, round or fusiform shape and a medium size. Apart from the neuronal somata, the proximal parts of the dendritic arborization were clearly visible. The immunoreactive neurons were randomly distributed in the claustrum and their neuronal size and shape did not differ in the various parts of the studied structure. Co-localization of NOS and SOM or NOS and NPY was reported. In conclusion, SOM, VIP and NPY do not appear to play a significant role in the claustro-cortical projection but are most probably involved in modulation and information transfer in the claustrum. The appearance of NOS in both cortically projecting and intrinsic neurons of the claustrum may be indicative of a fundamentally different role in the functioning of the claustro-cortical loop.

Co-induction of nitric oxide synthase, Bcl-2 and growth-associated protein-43 in spinal motoneurons during axon regeneration in the lizard tail

In lizards, tail loss transects spinal nerves and the cut axons elongate in the regrowing tail, providing a natural paradigm of robust regenerative response of injured spinal motoneurons. We previously ascertained that these events involve nitric oxide synthase induction in the axotomized motoneurons, suggesting a correlation of this enzyme with regeneration-associated gene expression. Here we investigated, in lizards, whether the cell death repressor Bcl-2 protein and growth-associated protein-43 (GAP-43) were also induced in motoneurons that innervate the regenerated tail in the first month post-caudotomy. Single and multiple immunocytochemical techniques, and quantitative image analysis, were performed. Nitric oxide synthase, GAP-43 or Bcl-2 immunoreactivity was very low or absent in spinal motoneurons of control lizards with intact tail. Nitric oxide synthase and GAP-43 were induced during the first month post-caudotomy in more than 75% of motoneurons which innnervate the regenerate. Bcl-2 was induced in approximately 95% of these motoneurons at five and 15 days, and in about 35% at one month. The intensity of Bcl-2 and GAP-43 immunostaining peaked at five days, and nitric oxide synthase at 15 days; immunoreactivity to these proteins was still significantly high at one month. Immunofluorescence revealed co-localization of nitric oxide synthase, GAP-43 and Bcl-2 in the vast majority of motoneurons at five and 15 days post-caudotomy. These findings demonstrate that co-induction of nitric oxide synthase, Bcl-2 and GAP-43 may be part of the molecular repertoire of injured motoneurons committed to survival and axon regeneration, and strongly favor a role of nitric oxide synthase in motoneuron plasticity.

Differential effects of colchicine on the induction of nitric oxide synthase in neurons containing progesterone receptors of the guinea pig hypothalamus

Using nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPHd) histochemistry, we analyzed the effects of an intracerebroventricular injection of colchicine on the activity of neuronal nitric oxide synthase in the hypothalamic nuclei of intact and ovariectomized estradiol-primed guinea pigs. We also examined the effects of colchicine on the immunocytochemical colocalization of nitric oxide synthase with the progesterone receptor in the ventrolateral nucleus, a key region in the control of sexual behavior. Treatment with colchicine resulted in a significant increase in the number of NADPHd-positive neurons in the ventrolateral nucleus in intact as well as in ovariectomized estradiol-primed animals, whereas in the other hypothalamic regions analyzed (preoptic area, paraventricular nucleus and posterior arcuate nucleus), the enzymatic activity remained unchanged. Quantitative analysis showed a significantly greater number of NADPHd-positive cells in the medial and the posterior aspects of the ventrolateral nucleus of colchicine-treated guinea pigs compared to the control group. In the caudal subdivision of this nucleus, colchicine induced nitric oxide synthase in the target cells for progesterone. These results suggest that neuronal nitric oxide synthase activity in the hypothalamus is affected by colchicine in a region-specific manner and especially in the ventrolateral nucleus, which is involved in progesterone-facilitated lordosis.

Pattern of distribution and co-localization of NOS and ATP in the myenteric plexus of human fetal stomach and intestine.

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.

NADPH-diaphorase and cytosolic urea cycle enzymes in the rat accessory olfactory bulb.

The nitric oxide cycle consists of nitric oxide synthase, argininosuccinate synthetase and argininosuccinate lyase to form nitric oxide. We have examined the colocalization of nitric oxide synthase and the cytosolic urea cycle enzymes (argininosuccinate synthetase, argininosuccinate lyase and arginase) in the accessory olfactory bulb of the rat by using a double labeling procedure combining reduced-nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) reaction with fluorescent immunocytochemistry. Each glomerulus showed a different NADPH-d activity, and those with the strongest NADPH-d activities were assembled in the caudomedial part of the accessory olfactory bulb. Argininosuccinate synthetase-like immunoreactive glomeruli were distributed in the caudomedial part of the accessory olfactory bulb, and most of them were also strongly NADPH-d positive. The mitral or tufted cells were argininosuccinate synthetase-, argininosuccinate lyase- and arginase-like immunoreactive, but were not NADPH-d positive. The granule cells were NADPH-d positive or argininosuccinate lyase-like immunoreactive, but were not argininosuccinate synthetase- or arginase-like immunoreactive. Some granule cells were both NADPH-d positive and argininosuccinate lyase-like immunoreactive. The results indicate the heterogeneity of glomeruli of the accessory olfactory bulb with respect to the distribution of these enzymes. The granule cells have nitric oxide synthase and argininosuccinate lyase, and thus may efficiently produce nitric oxide.

Quantitative studies of progesterone receptor and nitric oxide synthase colocalization with somatostatin, or neurotensin, or substance P in neurons of the guinea pig ventrolateral hypothalamic nucleus: an immunocytochemical triple-label analysis.

Previous double-label studies have demonstrated that progesterone receptors (PR) may coexist with nitric oxide synthase (NOS) and also with neuroactive peptides such as somatostatin (SOM), neurotensin (NT) and substance P (SP) in neurons of the ventrolateral hypothalamic nucleus (VL) of the guinea pig. In the present study, triple-label immunofluorescence histochemistry was used to determine whether neurons co-expressing both PR and NOS also contain one neuropeptide (SOM, NT or SP) and to quantify these colocalization relationships. Ovariectomized guinea pigs were primed with estradiol to induce PR immunoreactivity and treated with colchicine to visualize immunoreactive (IR) peptidergic neurons. Using three primary antibodies raised in different species and labeled with three different markers, it is possible to recognize which type(s) of immunoreactivity each cell contains on the same sections. We observed that PR, NOS and SOM co-occurred extensively, whereas PR, NOS and NT or PR, NOS and SP were colocalized infrequently in neurons of the VL. Combining our various quantitative observations, we found that SOM immunoreactivity appeared in 33% of the PR/NOS-IR neurons in the caudal aspect of the nucleus and that nearly all neurons containing SOM and NOS contained PR. The occasional localization between PR, NOS and SP seen in the rostral subdivision of the VL represented a very low proportion of the PR/NOS-IR neurons but 48% of NOS/SP-IR neurons. The relatively high proportion of PR/NOS-IR cells expressing SOM in the VL suggests that these neurons are part of a neural circuitry involved in a variety of steroid-dependent functions.

Nitrotyrosine formation with endotoxin-induced kidney injury detected by immunohistochemistry.

The presence of nitrotyrosine in the kidney has been associated with several pathological conditions. In the present study, we investigated nitrotyrosine formation in rat kidney after animals received endotoxin for 24 h. With lipopolysaccharide (LPS) treatment, immunohistochemical data demonstrated intense nitrotyrosine staining throughout the kidney. In spite of marked nitrotyrosine formation, the architectural appearance of tubules, glomeruli, and capillaries remained intact when examined by reticulin staining. Our data suggested that the marked staining of nitrotyrosine in proximal tubular epithelial cells was in the subapical compartment where the endocytic lysosomal apparatus is located. Thus a large portion of nitrotyrosine may come from the hydrolysis of nitrated proteins that are reabsorbed by the proximal tubule during the LPS treatment. We also found the colocalization of nitric oxide synthase (NOS-1) and nitrotyrosine within the macula densa of LPS-treated rats by using a double fluorescence staining method. In renal arterial vessels, vascular endothelial cells were more strongly stained for nitrotyrosine than vascular smooth muscle cells. Control animals without LPS treatment showed much less renal staining for nitrotyrosine. The general distribution of nitrotyrosine staining in control rat renal cortex is in the proximal and convoluted tubules, whereas the endothelial cells of vasa recta are major areas of nitrotyrosine staining in inner medulla. The renal distribution of nitrotyrosine in control and LPS-treated animals suggests that protein nitration may participate in renal regulation and injury in ways that are yet to be defined.

NADPH-diaphorase and NOS enzymatic activities in some neurons of reptilian gut and their relationships with two neuropeptides.

The distribution of neurons containing the enzymes NADPH-diaphorase (NADPH-d) and nitric oxide synthase (NOS) has been studied in the gastrointestinal tract of lizard (Podarcis s. sicula) and snake (Thamnophis sirtalis). The techniques employed were the NADPH-d/nitroblue tetrazolium histochemical method, and the indirect immunofluorescence applied to cryostat sections and to whole-mount preparations. The colocalization of NADPH-d with NOS, with vasoactive intestinal polypeptide (VIP) and with galanin (Gal) was also studied, and a Western blot analysis using an antibody directed against mammalian Gal was performed on lizard stomach extracts. NADPH-d positive nerve cell bodies and fibres were found in the myenteric and submucous plexuses throughout the gastrointestinal tract of both reptiles. These nerve structures were also present in the other intramural nerve plexuses, although in smaller quantities. Both in lizard and snake, the stomach revealed a positive nerve population that was more dense than elsewhere in the gut. The population of the NADPH-d-positive neurons observed in the lizard was larger than that observed in the snake. The distribution of both populations was similar to those that have been described in the gut of several mammalian and non-mammalian vertebrates. Both in lizard and snake, a one-to-one correspondence was noted between NOS- and NADPH-d-containing nerve cell bodies, and the nitrergic neurons containing Gal appeared to be more numerous than those containing VIP. Western blot analysis recognised a single band with a molecular weight (3.4 kDa) very similar to that of porcine Gal. It is hypothesised that at least some of the nitrergic neurons of the lizard and snake gut are inhibitory motor neurons innervating the circular smooth musculature. In addition, the colocalization of NOS and VIP in neurons enhances their inhibitory action. The role of the neurons containing both NOS and Gal remains unknown.

Distribution and colocalization of nitric oxide synthase and calretinin in myenteric neurons of developing, aging, and Crohn's disease human small intestine.

The pattern of distribution and colocalization of nitric oxide synthase and the calcium-binding protein calretinin in myenteric neurons and nerve fibers were examined in the human small intestine from preterm fetuses (14-17 weeks of gestation), normal adults (mean age 50 years old), old age (mean age 80 years old), and Crohn's disease patients (mean age 30 years old) using NADPH-diaphorase histochemistry and immunohistochemical techniques. In all age groups investigated, NADPH-diaphorase-reactive and calretinin-immunoreactive neurons and nerve fibers were seen throughout the myenteric plexus. The highest proportion of NADPH-diaphorase-reactive neurons was found in the myenteric ganglia of old age intestines (56% of protein gene product-immunoreactive neurons) followed by fetal intestines (41%) and Crohn's intestine (30%) compared with intestines of control adults (20%). A similar trend was observed for calretinin-immunoreactive neurons where the highest proportion of immunoreactive neurons was found in the myenteric ganglia of old age intestines (28% of protein gene product-immunoreactive neurons), followed by fetal intestines (22%), and Crohn's intestines (18%) compared with intestines of control adults (9%). A colocalization of NADPH-diaphorase activity and calretinin immunoreactivity was only seen in the myenteric neurons of fetal intestines (2% of NADPH-diaphorase-reactive neurons were also calretinin-immunoreactive). The pattern of distribution of NADPH-reactive and calretinin-immunoreactive neurons in the myenteric ganglia of fetal intestine differs from that of the other age groups. In the fetal intestine, the myenteric neurons containing either calretinin or NADPH-diaphorase are distributed through out the myenteric ganglia with no specific orientation to one another. In the intestines of control adult, Crohn's, and old age patients, single large calretinin-immunoreactive neurons are surrounded by a number of small NADPH-diaphorase-positive neurons, with this feature being more prominent in intestines of old-age and Crohn's disease patients. In summary, a high number of both NADPH-diaphorase-reactive and calretinin-immunoreactive neurons were seen in the myenteric ganglia of fetal, old age, and Crohn's intestines; we discuss that there may be a role for nitric oxide and calretinin in the process of development, aging, and pathological changes in the human intestine associated with alteration in the calcium homeostasis in the myenteric neurons.

Colocalization of nitric oxide synthase and some neurotransmitters in the intramural ganglia of the guinea pig urinary bladder

The distribution of nitrergic neurons was investigated by using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry in wholemount preparations of the urinary bladder in guinea pigs. Both NADPH-d+ and NOS+ neurons were located predominantly in the bladder base. Double staining showed that 70.9% of the NADPH-d+ neurons coexpressed NOS. Acetylcholinesterase histochemistry revealed that a majority of the intramural neurons were reactive, and about half of them (51.4%) were double labelled for NOS. Tyrosine hydroxylase-positive neurons were also distributed mainly in the bladder base but in a neuronal population that was separate from the preponderant NADPH-d+ neurons. Vasoactive intestinal polypeptide immunoreactivity was also detected in the some of intramural ganglion cells, in which 21.3% of them coexpressed NADPH-d. Calcitonin gene-related peptide and substance P immunoreactivities were confined to nerve fibers, often in close association with NADPH-d+ cells or extended along the blood vessels. These results have demonstrated the colocalization of NADPH-d and NOS in the majority of intramural ganglion cells. Many of the nitrergic neurons are apparently cholinergic, indicating that they are parasympathetic postganglionic neurons, and this underscores NO as the major neuromodulator in the parasympathetic nerves in the bladder walls. The localization of vasoactive intestinal polypeptide in nitrergic neurons suggests that the peptide may complement NO for regulation of micturition reflex. The close relationship of NADPH-d-reactive intramural neurons with calcitonin gene-related peptide and substance P fibers, most probably derived from dorsal root ganglion cells, suggests that NO released from the local neurons may exert its influence on the sensory neural pathways in the urinary bladder. J. Comp. Neurol. 394:496–505, 1998. © 1998 Wiley-Liss, Inc.

Colocalization of nitric oxide synthase and some neurotransmitters in the intramural ganglia of the guinea pig urinary bladder

The distribution of nitrergic neurons was investigated by using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry in wholemount preparations of the urinary bladder in guinea pigs. Both NADPH-d+ and NOS+ neurons were located predominantly in the bladder base. Double staining showed that 70.9% of the NADPH-d+ neurons coexpressed NOS. Acetylcholinesterase histochemistry revealed that a majority of the intramural neurons were reactive, and about half of them (51.4%) were double labelled for NOS. Tyrosine hydroxylase-positive neurons were also distributed mainly in the bladder base but in a neuronal population that was separate from the preponderant NADPH-d+ neurons. Vasoactive intestinal polypeptide immunoreactivity was also detected in the some of intramural ganglion cells, in which 21.3% of them coexpressed NADPH-d. Calcitonin gene-related peptide and substance P immunoreactivities were confined to nerve fibers, often in close association with NADPH-d+ cells or extended along the blood vessels. These results have demonstrated the colocalization of NADPH-d and NOS in the majority of intramural ganglion cells. Many of the nitrergic neurons are apparently cholinergic, indicating that they are parasympathetic postganglionic neurons, and this underscores NO as the major neuromodulator in the parasympathetic nerves in the bladder walls. The localization of vasoactive intestinal polypeptide in nitrergic neurons suggests that the peptide may complement NO for regulation of micturition reflex. The close relationship of NADPH-d-reactive intramural neurons with calcitonin gene-related peptide and substance P fibers, most probably derived from dorsal root ganglion cells, suggests that NO released from the local neurons may exert its influence on the sensory neural pathways in the urinary bladder.