Distribution Of Nitric Oxide Synthase Research Articles

Distinct effects of tetragastrin, histamine, and CCh on rat gastric mucin synthesis and contribution of NO.

Although gastrin, histamine, and carbachol (CCh) accelerate gastric mucin metabolism, information about their target cells of mucin production is lacking. To clarify this, we examined the effects of these stimulants, including the possible participation of nitric oxide (NO), on mucin biosynthesis in distinct sites and layers of rat gastric mucosa. Pieces of tissue obtained from the corpus and antrum were incubated in a medium containing radioactive precursors and each stimulant, with or without NO synthase (NOS) inhibitor. Distribution of NOS was compared with that of the specific mucins by immunostaining using specific antiserum and monoclonal antibodies. In the full-thickness corpus mucosa, tetragastrin enhanced [3H]glucosamine incorporation into mucin but had no effect on [14C]threonine incorporation. Both histamine and CCh dose dependently increased 3H- and 14C-labeled corpus mucin. Only CCh stimulated antral mucin biosynthesis. CCh stimulation was noted in the corpus mucosa after removal of surface mucous cells, but stimulation by tetragastrin or histamine disappeared as a result of this pretreatment. Only tetragastrin-induced activation was completely blocked by the NOS inhibitor. NOS immunoreactivity was limited to surface mucous cells. Mucus-producing cells present in the different sites and layers of the gastric mucosa have distinct mechanisms for regulation of mucin biosynthesis. Gastrin-stimulated mucin biosynthesis mediated by NO is limited to surface mucous cells of rat gastric oxyntic mucosa.

Chapter 4 A web-accessible digital atlas of the distribution of nitric oxide synthase in the mouse brain

We have produced a digital atlas of the distribution of nitric oxide synthase (NOS) in the mouse brain as a reference source for our studies on the roles of nitric oxide in brain development and plasticity. NOS was labeled using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In addition, choline acetyltransferase (ChAT) immunocytochemistry was used to identify cholinergic cells because many of the NADPHd positive cells were thought to colocalize acetylcholine. Some sections were also labeled with antibodies to either the neuronal (nNOS) or endothelial (eNOS) isoforms of NOS. Series of sections from 11 C57/BL6 mice were collected and labeled for NADPHd and/or ChAT. We collected two types of data from this material: color digital photographs illustrating the density of cell and fiber labeling, and computer/microscope plots of the locations of all the labeled cells in selected sections. The data can be viewed as either a series of single-section maps produced by combining the plots with the digital images, or as 3-D views derived from the cell plots. The atlas of labeled cell maps, together with selected color photographs and 3-D views, is available for viewing via the World Wide Web (http:@nadph.anatomy.lsumc.edu). Examination of the atlas data has revealed several points about the distribution of NOS throughout the mouse brain. Firstly, different populations of NADPHd-positive neurons can be distinguished by different patterns of staining. In some brain areas neurons are intensely stained by the NADPHd technique where label fills the cell bodies and much of the dendritic trees. In other brain regions labeling is much lighter, is principally confined to the cytoplasm of the cell soma, and extends only a short distance within proximal dendrites. Intense labeling is typical of neurons in the caudate/putamen and mesopontine tegmental nuclei. Most of the labeled neurons in the cortex also stain this way. Lighter, "granular" label is found in many other nuclei, including the medial septum, hippocampus, and cerebellum. In addition to staining pattern, we have also noted that different subpopulations of NOS-neurons can be distinguished on the basis of colocalization with ChAT. Substantial overlap of the distributions of these two substances was observed although very little colocalization was found in most cholinergic cell groups except the mesopontine tegmental nuclei. Other points of interest arising from this project include the apparent lack of NADPHd labeling in the CA1 pyramidal cells of the hippocampus or the Purkinje neurons in the cerebellum. This observation is especially relevant given that synaptic plasticity in these regions is reported to be nitric-oxide dependent.

Eye enucleation alters intracellular distribution of NO synthase in the superior colliculus.

We investigated the role of retinotectal projections on the distribution of nitric oxide synthase (NOS) in the retinoceptive layers of the rat superior colliculus (SC) using histochemical methods. Rats enucleated at birth showed no alteration in the temporal pattern of NOS expression. There was, however, a dramatic change in the NADPH-diaphorase staining pattern of NOS-positive cells. NOS was absent from the distal portions of the dendritic trees of the deafferented SC. Nevertheless, staining the dendritic trees of these cells with Lucifer yellow showed that they were not morphologically different from those of the ipsilateral SC of monoenucleated animals. The same results were obtained when enucleation was performed in adult rats. We conclude that NOS intracellular distribution in the SC cells can be regulated by retinotectal projections in both developing and adult animals.

Nitric oxide synthase activity in human lung cancer.

Nitric oxide synthases (NOS) exist in human tumor cell lines and solid tumor tissues, and it has been suggested that NO may play important roles in growth, progression or metastasis of tumors. We investigated the activity and distribution of NOS in a series of human lung cancer and normal lung tissues. Seventy‐two primary lung cancer samples (44 cases of adenocarcinoma, 18 of squamous cell carcinoma, 4 of large cell carcinoma, 2 of small cell carcinoma, 2 of adenosquamous carcinoma, and 2 of carcinoids) and corresponding normal lung samples were obtained from surgically treated patients. In normal lung tissues, little NOS activity was observed with no correlation between the patients' age and NOS activity. The total NOS activities in lung adenocarcinoma samples were significantly higher than those in other types of lung cancers or normal lung samples (P<0.05), Analysis by tumor grade of the adenocarcinoma samples revealed no significant difference of NOS activity between grades. TNM classification showed that, although T stage did not correlate with NOS activity, cancer tissues from patients with N2 disease tended to have lower activity than those from patients with NO or Nl disease. Immunohistochemical studies revealed that the intensity of NOS immunoreactivity correlated with NOS activity. These results suggest that NO may play an important role in the metabolism and behavior of lung cancers, especially adenocarcinoma.

Nitric oxide synthase activity in tissues of the bovine eye.

Nitric oxide synthase (NOS) is present in many ocular tissues where it may have different physiological functions. This warrants a thorough characterization of NOS activity in the eye. NOS distribution and its biochemical properties were determined in the retina, choroid, ciliary processes (CP), and trabecular meshwork (TM). Retinal NOS required NADPH (diphenylene-iodonium, a flavoprotein inhibitor, which inhibited enzyme activity with an IC50 of 0.36 microM, FAD (40 microM), FMN (40 microM), and BH4 (4 microM) as cofactors for optimal activity. Ocular NOS appeared to be regulated by free divalent cations, since its activity was inhibited by EDTA (slopes > 3.0 and IC50 values of 12.8, 19.7, and 53 microM, respectively). Ocular NOS required calmodulin, since NOS activity was inhibited by trifluoperazine (calmodulin inhibitor, IC50 = 41 microM). NOS activity is widely distributed in the eye, (choroid > retina > CP > TM) and is mainly cytosolic (70-95%). L-Arginine analogs inhibited NOS in the retina, choroid, and TM. In all three tissues, NG-methyl-L-arginine displayed the highest affinity for inhibition (IC50 = 0.2-0.7 microM) followed by canavanine (IC50 = 13-33 microM), while aminoguanidine only weakly inhibited NOS (IC50 = 93-179 microM). In all tissues, the order of potency of inhibition points to the presence of constitutive rather than inducible NOS. Moreover, it is possible that TM contains more than a single form of NOS.

Pattern of distribution of constitutive isoforms of NO synthase in the normal prostate and obstructive prostatic hyperplasia

Nitric oxide (NO) is suggested as an important mediator for the regulation of biological processes. In the present study we tried to determine histochemically and immunohistochemically the localization and distribution of the constitutive NO-synthase Isoforms (bNOS and eNOS) of 14 normal non-obstructive and 12 hyperplastic obstructive human prostates. Differentiated nitrinergic innervation was shown for the prostate glands, fibromuscular stroma and blood vessels by NADPH-diaphorase staining and immunohistochemically with specific NOS antibodies. In the specimens with benign prostatic hyperplasia nitrinergic innervation seems to be distinctly reduced. The vascular distribution of NOS provides evidence for segmental differentiation of NO-mediated vascular regulation. The NADPH-diaphorase reaction was not confirmed immunohistochemically by the specific NOS antibody in the glandular epithelium. The distribution of NO synthase shows the importance of nitric oxide in the regulation of smooth muscle tone, blood flow and secretory function in the normal and hyperplastic human prostate.

Presence of endothelial calcium-dependent nitric oxide synthase in breast apocrine metaplasia

Endothelial calcium-dependent nitric oxide (NO) synthase has been shown to be expressed in human malignant breast tumours, and its presence correlates with tumour grade. Moreover, NO, being synthesised in breast tumour cells, may increase tumour blood flow and promote angiogenesis. In view of these aspects, we have assessed the distribution of NO synthase within a series of benign breast tumours using a monoclonal antibody against human endothelial calcium-dependent NO synthase. Activity was predominantly localised in apocrine metaplastic cells of fibrocystic disease, as well as in endothelia throughout all tissue sections. Consistent with previous reports, no endothelial calcium-dependent NO synthase immunoreactivity was observed in poorly differentiated infiltrating duct carcinoma cells. In conclusion, expression of endothelial calcium-dependent NO synthase in human breast apocrine metaplasia may be of significance in view of the NO's vascular effects in benign breast disease.

Nitric oxide synthase I immunoreactivity and NOS-associated NADPHd histochemistry in the visceral epithelial cells of the intraplacental mouse yolk sac

In the course of our studies on the local blood flow modulation in the NMRI-mouse placenta we have focussed on regulatory pathways involving recently appreciated gaseous messenger molecules nitric oxide (NO) and carbon monoxide (CO), which are generated by NO synthase (NOS) and heme oxygenase (HO)-2, respectively. The distribution of NOS was investigated by immunohistochemistry using an antiserum to the neuronal isoform (NOS-I) and by NADPH diaphorase (NADPHd) histochemistry, supplemented with procedures (permanganate and formaldehyde method) serving to enhance the specificity of the enzyme histochemical method for NOS visualization. HO-2 was demonstrated immunohistochemically. In addition, cyclic guanosine monophosphate (cGMP)-forming soluble guanylate cyclase (sGC) and dehydrogenases generating the NOS co-substrate NADPH were analysed either by immunohistochemistry or enzyme histochemistry. NOS-I immunostaining was observed in the intraplacental visceral yolk sac epithelial cells but not in the placenta and extraplacental visceral epithelial yolk sac cells. Co-localization of NOS-I immunolabeling and NOS-associated NADPHd was exclusively found in the intraplacental visceral epithelial cells, while NADPHd activity not associated to NOS was present in other placental and extraplacental cells additionally analysed for control reasons. HO-2 and sGC immunoreactivity could not be detected in the placenta including the intraplacental visceral epithelial cells but were expressed in several extraplacental cells. Dehydrogenases producing the NOS co-substrate NADPH were present in the intraplacental visceral epithelium as well as in other placental and extraplacental cells. Since the intraplacental visceral epithelial yolk sac layer closely accompanies large fetal blood vessels entering the placental labyrinth from the chorionic plate it may be assumed that NO, generated by the NADPH-consuming NOS-I in the intraplacental yolk sac epithelium, acts to regulate the blood flow by relaxing smooth muscle cells in the wall of these fetal vessels. The lack of immunoreactivity to the NO-effector molecule sGC may be due to methodological reasons. The absence of the HO-2/CO system suggests its insignificant role as a potential gas signaling pathway in the vascular smooth muscle system of the intraplacental visceral yolk sac of mice.

914 Distribution of Bax-like immunoreactivity in the rat nervous system

Nitric oxide (NO) production in the sensory neurons of the rat nodose ganglion was studied by examining the distribution of NO synthase (NOS) by use of NADPH diaphorase (NADPHd) histochemistry and immunohistochemistry for the presence of isoforms of NOS: neuronal (nNOS), endothelial (eNOS) and the inducible isoform (iNOS). Distribution and changes in NO production during acute hypoxia were studied in vital vibratome sections with the fluorescent marker for NO, diaminotriazolofluorescein (DAF-2T). Furthermore, changes in reactive oxygen species (ROS) in vibratome slices were examined utilizing 2′,7′-dichlorofluorescein (DCF). By use of these histochemical methods, a positive NADPH reaction and positive immunoreactivity for eNOS were noted in all neurons observed. While for nNOS immunoreactivity, both strongly positive cells but also many negative cells are seen., no iNOS immunoreactive cells were observed. In vital vibratome slices, a dot-like distribution of fluorescence for DAF-2T, indicating production of NO, was observed in the nodose ganglion cells. Neurons exposed to hypoxia showed stronger DAF-2T fluorescence than cells exposed to normoxia, indicating an increased production of NO during hypoxia. When Ca2+ was removed from the incubation buffer, the intensity of fluorescence for DAF-2T decreased but did not disappear completely. Using a photoconversion technique, DAF-2T was localized in the inner membrane of mitochondria in the ganglion cells by electron microscopy. The level of DCF signals for detection of ROS was higher in neurons incubated in the normoxic medium than those incubated under conditions of hypoxia. Nerve cells exposed to hypoxia followed by reoxygenation (3 min in normoxic conditions) showed higher fluorescence for DCF than those exposed to normoxia. The results of the present study demonstrate clearly that the basal production of NO in viscerosensory neurons is increased during hypoxia and is due to the isoform eNOS rather than nNOS, moreover, that ROS is augmented by reoxygenation but not during hypoxia.

912 Cloning and characterization of a novel vesicle transport related factor from cultured rat astrocytes

Nitric oxide (NO) production in the sensory neurons of the rat nodose ganglion was studied by examining the distribution of NO synthase (NOS) by use of NADPH diaphorase (NADPHd) histochemistry and immunohistochemistry for the presence of isoforms of NOS: neuronal (nNOS), endothelial (eNOS) and the inducible isoform (iNOS). Distribution and changes in NO production during acute hypoxia were studied in vital vibratome sections with the fluorescent marker for NO, diaminotriazolofluorescein (DAF-2T). Furthermore, changes in reactive oxygen species (ROS) in vibratome slices were examined utilizing 2′,7′-dichlorofluorescein (DCF). By use of these histochemical methods, a positive NADPH reaction and positive immunoreactivity for eNOS were noted in all neurons observed. While for nNOS immunoreactivity, both strongly positive cells but also many negative cells are seen., no iNOS immunoreactive cells were observed. In vital vibratome slices, a dot-like distribution of fluorescence for DAF-2T, indicating production of NO, was observed in the nodose ganglion cells. Neurons exposed to hypoxia showed stronger DAF-2T fluorescence than cells exposed to normoxia, indicating an increased production of NO during hypoxia. When Ca2+ was removed from the incubation buffer, the intensity of fluorescence for DAF-2T decreased but did not disappear completely. Using a photoconversion technique, DAF-2T was localized in the inner membrane of mitochondria in the ganglion cells by electron microscopy. The level of DCF signals for detection of ROS was higher in neurons incubated in the normoxic medium than those incubated under conditions of hypoxia. Nerve cells exposed to hypoxia followed by reoxygenation (3 min in normoxic conditions) showed higher fluorescence for DCF than those exposed to normoxia. The results of the present study demonstrate clearly that the basal production of NO in viscerosensory neurons is increased during hypoxia and is due to the isoform eNOS rather than nNOS, moreover, that ROS is augmented by reoxygenation but not during hypoxia.

913 Characterization and cloning of a novel stress protein, the 150 kDa Oxygen Regulated Protein (ORP150) from cultured astrocytes and its expression in ischmaic mouse brain

Nitric oxide (NO) production in the sensory neurons of the rat nodose ganglion was studied by examining the distribution of NO synthase (NOS) by use of NADPH diaphorase (NADPHd) histochemistry and immunohistochemistry for the presence of isoforms of NOS: neuronal (nNOS), endothelial (eNOS) and the inducible isoform (iNOS). Distribution and changes in NO production during acute hypoxia were studied in vital vibratome sections with the fluorescent marker for NO, diaminotriazolofluorescein (DAF-2T). Furthermore, changes in reactive oxygen species (ROS) in vibratome slices were examined utilizing 2′,7′-dichlorofluorescein (DCF). By use of these histochemical methods, a positive NADPH reaction and positive immunoreactivity for eNOS were noted in all neurons observed. While for nNOS immunoreactivity, both strongly positive cells but also many negative cells are seen., no iNOS immunoreactive cells were observed. In vital vibratome slices, a dot-like distribution of fluorescence for DAF-2T, indicating production of NO, was observed in the nodose ganglion cells. Neurons exposed to hypoxia showed stronger DAF-2T fluorescence than cells exposed to normoxia, indicating an increased production of NO during hypoxia. When Ca2+ was removed from the incubation buffer, the intensity of fluorescence for DAF-2T decreased but did not disappear completely. Using a photoconversion technique, DAF-2T was localized in the inner membrane of mitochondria in the ganglion cells by electron microscopy. The level of DCF signals for detection of ROS was higher in neurons incubated in the normoxic medium than those incubated under conditions of hypoxia. Nerve cells exposed to hypoxia followed by reoxygenation (3 min in normoxic conditions) showed higher fluorescence for DCF than those exposed to normoxia. The results of the present study demonstrate clearly that the basal production of NO in viscerosensory neurons is increased during hypoxia and is due to the isoform eNOS rather than nNOS, moreover, that ROS is augmented by reoxygenation but not during hypoxia.

Application of [ 3H] l- NG-nitro-arginine labelling to measure cerebellar nitric oxide synthase in patients with schizophrenia

Brains from patients with schizophrenia have been reported to contain deficient and dysplastic forebrain neurons containing nitric oxide synthase (NOS). As part of a study of NOS in schizophrenia, we decided to investigate the cerebellum, which has particularly high levels of NOS. We used an autoradiographic method to measure the density and distribution of NOS. Sections of frozen cerebellum removed at autopsy were labelled with the selective NOS inhibitor [ 3H] l- N G-nitro-arginine. NOS levels were visualized in sagittal sections of vermis from 16 control subjects and 21 schizophrenia patients, and measurements were taken from the three groups of developmentally-distinct lobules I-V, VI-VII and VIII-X. The highest NOS density was in the Purkinje/molecular layer of cerebellar cortex, although there was some NOS in the granule cell layer. There were no differences in Purkinje/molecular or granule cell layer NOS levels between the two groups of subjects. The mild structural faults in cerebellar vermis observed in some patients with schizophrenia probably do not involve reductions in NOS-containing cells.

Development of nerves containing nitric oxide synthase in the human male urogenital organs.

To determine the spatial and temporal distribution of nitric oxide synthase (NOS) in the urogenital organs of a series of human male fetuses, using an immunohistochemical technique. Thirteen pre-natal specimens ranging in gestational age from 13 to 30 weeks were acquired following abortion or miscarriage. The distribution of NOS, which catalyses the production of nitric oxide (NO), was revealed using an indirect immunolabelling technique and compared with the overall innervation of each specimen visualized using the general nerve-marker protein gene product 9.5 (PGP). At 13 weeks of gestation the majority of nerves supplying the developing prostate gland expressed NOS while similar nerves formed a very minor proportion of the total innervation to the urinary bladder and intramural ureters. With increasing gestational age, NOS-containing nerves became more numerous in the lower urinary tract, the majority occurring at the bladder neck and around the prostatic urethra. In contrast, NOS-containing nerves were not detected in the muscle coat of the vas deferens and seminal vesicle until 23 weeks of gestation and at 30 weeks still only formed a small proportion of the intramuscular nerves. From 23 weeks onwards NOS-containing nerves were present occasionally in the dense subepithelial nerve plexuses which developed in the bladder, prostate, vas deferens and seminal vesicle. Also from 23 weeks onwards, many of the epithelial cells lining the vas deferens, seminal vesicle and ejaculatory ducts showed immunoreactivity to NOS but no immunoreactivity was observed in the epithelial lining of the urinary bladder and the intramural ureters. Based on the comparative density of NOS-containing nerves and the difference in their temporal development among the various urogenital organs it is apparent that NO plays an increasingly important role in the autonomic control of the lower urinary tract during fetal development but that its involvement in the functional control of the vas deferens and seminal vesicle is relatively minor before birth.

Nitric oxide synthase activity in human breast cancer.

Nitric oxide (NO) is generated by a family of isoenzymes (NO synthases) expressed in a wide range of mammalian cells. We have recently reported NO synthase expression in human gynaecological cancers. In this study we have assessed the activity and distribution of NO synthase in a series of human breast tumours and in normal breast tissue. Calcium-dependent (constitutive) and -independent (inducible) NO synthase activity, as well as NO biosynthesis, was high in invasive tumours compared with benign or normal tissue. Furthermore, for invasive ductal carcinomas, NO biosynthesis was significantly greater for grade III compared with grade II tumours. Immunohistochemical investigations revealed immunolabelling with a monoclonal antibody to murine inducible NO synthase predominantly within tumour-associated macrophages. Immunolabelling with a polyclonal antiserum raised against rat brain NO synthase was also observed in vascular endothelial and myoepithelial cells. Thus NO synthase is expressed in human breast tumours, where its presence correlates with tumour grade.

An Assay Method for Nitric Oxide Synthase in Crude Samples by Determining Product NADP +

An assay method for nitric oxide synthase (NOS) was developed based on fluorometric or enzymatic determination of NADP +. An aliquot (≤ 2 μ1) of crude enzyme sample, homogenate or supernatant of rat cerebellum, was added to a reaction mixture containing arginine, NADPH, and O 2 and incubated at 25°C for 30 min. A strongly fluorescent substance was formed from a product, NADP +, and measured (fluorometric assay). When rat cerebellar layers were assayed, freeze-dried sections of layers (0.2 to 2 μg dry wt) were added directly into 1.24 μl of NOS reaction mixture and the total NADP + formed in picomole amounts was specifically amplified up to 4000-fold and determined, using an enzymatic NADP cycling amplification reaction (enzymatic-cycling assay). NOS activity was calculated as the difference in NADP +-forming activity in the absence and presence in the reaction mixture of N G-nitro-L-arginine, a specific inhibitor of NOS. Enzymatic activity was analyzed in rat cerebellar supernatants by two procedures. With supernatants (and purified macrophage NOS), the ratio between the specific activities on a protein basis using the present NADP + formation assay and using [ 3H]citrulline formation from [ 3H]arginine as substrate was 2. The distribution of NOS activity was shown between the particulate and supernatant fractions of rat cerebellum. The molecular and granular layers of rat cerebellum contained similar NOS activities, while the activity in the white matter was negligibly low. NOS distribution is also reported among rat organs.

Co-existence of nitrergic, peptidergic and acetylcholine esterase-positive nerves in the pig lower urinary tract

The distribution of NO synthase (NOS) immunoreactive nerves and the possible co-existence with other neurotransmitters were investigated in the pig lower urinary tract. NOS immunoreactive nerves were found in the muscle layer, in the lamina propria and around blood vessels. The density of NOS immunoreactive nerves was more prominent in the trigone and urethra than in the detrusor. All parts of the lower urinary tract were supplied by numerous acetylcholine esterase (AChE) positive nerves. The number of adrenergic nerves in the trigone and urethra was moderate to rich, whereas only very few adrenergic nerves were demonstrated in the detrusor. A low to moderate number of nerve fibres containing neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) were observed in the trigone and urethra, while very few were found in the detrusor. A small number of nerves, confined to the trigone and urethra, were stained for calcitonin-gene-related peptide, somatostatin and leu-enkephalin. Nerve fibres exhibiting immunoreactivity to bombesin/gastrin releasing peptide, gastrin/cholecystokinin, substance P or neurokinin A were virtually absent. Co-localization studies revealed that some NOS-immunoreactive nerves also stained for NPY, VIP or AChE. The present study shows that nitrergic nerves are present in the pig lower urinary tract in a density lower than the cholinergic, but higher than any of the studied peptidergic nerves. Coinciding localization of NOS-positive nerves with nerves expressing AChE, VIP and NPY suggests that NO may have a role as a messenger in the lower urinary tract directly and by interaction with other transmitters.

Characterization and localization of nitric oxide synthase in the human prostate

Objectives To characterize nitric oxide synthase (NOS), which catalyzes nitric oxide (NO) production, in the human prostate using biochemical and immunohistochemical techniques. Methods NOS catalytic assay and NOS immunohistochemistry were performed on histologically verified nonmalignant prostate tissue obtained from the peripheral and transition zones of seven radical prostatectomy specimens. Results Biochemical analysis revealed NOS activity in the human prostate, with a greater amount in the peripheral zone than in the transition zone ( P < 0.01). In both prostate zones, NOS was immunohistochemically localized to nerve fibers and ganglia coursing throughout the smooth musculature of the stroma and to subepithelial nerve plexuses. NOS immunoreactivity was also localized to glandular epithelium. Conclusions The presence, activity, and distribution of NOS were described in two regions of the human prostate. The present evidence implicates NO in the autonomic innervation and physiology of the human prostate. It is proposed that NO may modulate smooth muscle tone and secretory functions in the human prostate, although functional studies are needed to support these hypotheses.

Location of nitric oxide synthase in the developing avian ciliary ganglion

A study has been made of the distribution of nitric oxide synthase (NOS) in the developing avian ciliary ganglion. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity first appeared in ciliary neurones at embryonic day 10 (E10). The number of NADPH-d positive neurones appeared maximal at this age and thereafter declined; at post hatched day 4 (P4) these neurones were found predominately in the periphery of the ganglion. At the light microscope level the NADPH-d stain appeared throughout the cell soma of the ciliary neurones. This was confirmed using tissue culture techniques. Ultrastructural delineation of horseradish peroxidase-labelled NOS antibodies was also found in the calyx where it was bound to the membranes of the endoplasmic reticulum as well as to the outer membranes of mitochondria. This distribution of NOS in the soma and calyx is consistent with the physiological role of NO as a co-transmitter and retrograde messenger that regulates the quantal secretion of the principal transmitter, acetylcholine, from the calyx.

Nitric oxide pathway in cat esophagus: localization of nitric oxide synthase and functional effects.

In the cat lower esophageal sphincter (LES) and esophageal body, nitric oxide synthase (NOS) immunoreactive nerves were abundant in the circular smooth muscle layer, especially in the LES region. NADPH diaphorase staining showed an identical pattern. The ability to form L-citrulline from L-arginine corresponded roughly to the distribution of NOS. Confocal microscopic analysis indicated colocalization within neurons of vasoactive intestinal peptide (VIP) in 65% of NOS-positive nerves. In LES circular smooth muscle preparations, electrically induced relaxations (single train stimuli) were generally abolished by NG-nitro-L-arginine (L-NNA). Continuous electrical stimulation for 2 min evoked a relaxation in the presence of L-NNA. This relaxation was inhibited by VIP antiserum and followed by a decrease in guanosine 3',5'-cyclic monophosphate, but not by any consistent change in adenosine 3',5'-cyclic monophosphate levels. K+ (124 mM) induced a biphasic relaxation, with L-NNA inhibiting the first phase but not the second. We conclude that nitric oxide (NO) has a major role as the mediator responsible for relaxation in the cat esophagus. NO seems also to initiate the release and enhance the effect of another transmitter.

Immunohistochemical demonstration of a paracrine role of nitric oxide in bronchial function.

We addressed the controversial role of nitric oxide (NO) in bronchial function by an immunohistochemical study of the localization of NO synthase (NOS) and its effector protein, soluble guanylate cyclase, in rat bronchus. For this study, a monoclonal antibody to the bovine constitutive neuronal NOS was developed and characterized. In Western blot analysis, this monoclonal antibody (anti-NOS antibody) reacted with bovine cerebellum NOS (150 kDa) as well as with structurally different NOSs from cultured bovine aortic endothelial cells (130 kDa) and cultured RAW 264.7 macrophages (130 kDa). The reactivity of anti-NOS antibody was confirmed by immunohistochemical staining of rat cerebellum, arterial endothelial cells, and cultured stimulated macrophages. When the distribution of NOS in rat airway was characterized, the anti-NOS antibody showed immunoreactivity within respiratory epithelium but not in the bronchial smooth muscle. The NADPH-diaphorase staining correlated with the immunostaining. In contrast, a monoclonal antibody to the rat lung-soluble guanylate cyclase immunostained respiratory smooth muscle but not epithelium. This study suggests a paracrine role for NO in bronchial function analogous to the function of the NOS-soluble guanylate cyclase pathway in blood vessels.