J. De Vente

Distribution of nitric oxide synthase-immunoreactive nerves and identification of the cellular targets of nitric oxide in guinea-pig and human urinary bladder by cGMP immunohistochemistry.

The distribution of nerves with the potential to synthesize nitric oxide was examined within the urinary bladder and proximal urethra of humans and guinea-pigs, using an antibody to nitric oxide synthase. Further experiments identified cells in which cGMP-immunoreactivity was induced following exposure to the nitric oxide donor, sodium nitroprusside. These cells represent the potential physiological targets of neuronally released nitric oxide, since activation of soluble guanylate cyclase, and a consequent rise in intracellular cGMP, mediate many of the effects of this transmitter. Nitric oxide synthase-immunoreactivity was widely distributed in the lower urinary tract. In guinea-pigs, 50-68% of all intrinsic vesical neurons expressed nitric oxide synthase-immunoreactivity, while in humans 72-96% of neurons in the wall of the bladder contained nitric oxide synthase. In both humans and guinea-pigs, varicose nitric oxide synthase-immunoreactive nerve terminals provided a moderate innervation to the detrusor muscle of the bladder body, and a denser innervation to the urethral muscle. Immunoreactive nerves also projected to the subepithelium and around blood vessels, but were rarely observed encircling intramural vesical ganglia. Following stimulation with sodium nitroprusside, smooth muscle cells of the urethra expressed strong cGMP-immunoreactivity, but detrusor muscle cells remained uniformly negative. Although the detrusor muscle fibres did not express cGMP, numerous interstitial cells throughout the bladder body demonstrated an intense induction of cGMP-immunoreactivity by sodium nitroprusside. These cells had long dendritic processes extending parallel to the smooth muscle fibres, and contained vimentin, an intermediate filament expressed by cells of mesenchymal origin. Other cell types in which sodium nitroprusside exposure induced cGMP-immunoreactivity were the uroepithelial cells, vascular smooth muscle cells and pericytes, and a small number of varicose nerve terminals. In the guinea-pig, a minor proportion (less than 10%) of intrinsic neurons in the wall of the bladder also expressed cGMP. No intrinsic neurons were observed in specimens of human bladder processed for cGMP immunohistochemistry. The results provide anatomical evidence that nitric oxide may function as a neurotransmitter in the lower urinary tract. Although nerves with the capacity to produce nitric oxide supply both the detrusor muscle and the urethra, distinct regional differences exist in the effects of nitric oxide on the induction of cGMP. If the nitric oxide-mediated induction of cGMP is a reliable indicator of the physiological responsiveness of a cell to nitric oxide, then smooth muscle cells appear to be the predominant targets of nitric oxide in the urethra, while in the bladder body, interstitial cells may serve this role. These findings support previous studies which have implicated nitric oxide as an inhibitory transmitter involved in the relaxation of the bladder neck. Our experiments further indicate that a number of cell types within the lower urinary tract could potentially mediate the effects of endogenously released nitric oxide.

Localization of cGMP in the cerebellum of the adult rat: an immunohistochemical study

The localization of cGMP in the cerebellum of the adult rat after fixation with formaldehyde was studied with an antibody raised against a cGMP-formaldehyde-thyroglobulin conjugate. Three different protocols were used: (1) in vitro incubation of 300 microns cerebellar slices followed by fixation and cryostat sectioning; (2) in vitro incubation of 100 microns cerebellar slices followed by fixation with no further sectioning; (3) perfusion fixation of the anesthetized rat followed by vibratome sectioning. All 3 protocols gave essentially the same results: cGMP-immunoreactivity was found predominantly in Bergmann fibers in the molecular layer, in Bergmann cell bodies in the Purkinje cell layer (but not in Purkinje cells), and in astroglial cells in the granular layer.

The selective PDE5 inhibitor, sildenafil, improves object memory in Swiss mice and increases cGMP levels in hippocampal slices

Previous studies have shown memory enhancing effects of phosphodiesterase type 5 (PDE5) inhibitors in rats. However, differences in nitric oxide (NO)-mediated cyclic GMP (cGMP) signaling in the hippocampus have been described between rats and mice. In the present study we investigated the memory enhancing effects of the PDE5 inhibitor, sildenafil on memory performance in Swiss mice using the object recognition task. Sildenafil (0.3, 1 and 3 mg/kg) was administered orally directly after the first trial. The memory for the objects was retested 24 h later when mice show no memory for the familiar object. Sildenafil improved the object discrimination performance of Swiss mice at a dose of 1 mg/kg. Hippocampal slices of Swiss mice incubated with sildenafil (10 microM) increased cGMP levels in varicosities in the CA3 region of the hippocampus and a number of short, thin fibers. Addition of DEA/NO, an NO donor (10 microM), in the presence of sildenafil (10 microM) strongly increased cGMP immunoreactivity of varicosities in the CA3 region. Double immunostaining of cGMP with the presynaptic marker synaptophysin did not reveal any co-localization of these markers under any circumstance. Taken together, inhibition of PDE5 improves object recognition memory in mice. Furthermore, a postsynaptic role of cGMP could be involved in this respect.

Nitric oxide targets in the guinea-pig intestine identified by induction of cyclic GMP immunoreactivity

The immunohistochemical localization of cyclic GMP was used to determine potential physiological sites of action of nitric oxide in the guinea-pig small intestine and colon. In control tissue, cyclic GMP-immunoreactivity was observed only in macrophages, whose identity was confirmed by double-label experiments using either F4/80, a macrophage-specific antibody, or fluorescein isothiocyanate-labelled dextran injected intravenously. Following exposure to the nitric oxide donor, sodium nitroprusside, cyclic GMP-immunoreactivity was induced in subpopulations of neurons in the myenteric and submucosal plexuses of the ileum and colon. In the colon, cyclic GMP-immunoreactivity was induced in 5-10% of myenteric neurons. The cyclic GMP-immunoreactive neurons did not contain nitric oxide synthase. In the ileum, cyclic GMP-immunoreactive neurons comprised about 2% of myenteric neurons and 40% of submucosal neurons; these cyclic GMP-immunoreactive neurons were also immunoreactive for vasoactive intestinal peptide, but they did not contain nitric oxide synthase. Interstitial cells between the mesothelium and the longitudinal muscle layer, vascular smooth muscle and vascular pericytes also showed sodium nitroprusside-induced cyclic GMP-immunoreactivity. The interstitial cells of Cajal at the inner surface of the circular muscle layer and the smooth muscle cells of the circular and longitudinal muscle layers showed increases in cyclic GMP-immunoreactivity that varied in extent from animal to animal. The results suggest that nitric oxide could act at several sites in the intestine through the stimulation of guanylyl cyclase.

NO-mediated cGMP synthesis in cholinergic neurons in the rat forebrain: effects of lesioning dopaminergic or serotonergic pathways on nNOS and cGMP synthesis

Nitric oxide synthase (NOS) activity and NO‐mediated cGMP synthesis were studied in the rat forebrain of control animals and animals which had received a unilateral lesioning of dopaminergic or serotonergic pathways. Lesioning of the dopaminergic innervation using 6‐hydroxydopamine resulted in a 50% decrease in NOS activity in the lesioned frontal cortex and caudate putamen. Lesioning of the serotonergic innervation using 5,7‐dihydroxytryptamine had no effect on NOS activity. NO‐mediated cGMP accumulation in rat forebrain slices was not affected by 6‐hydroxydopamine or 5,7,‐dihydroxytryptamine lesioning. Using cGMP immunocytochemistry, it was demonstrated that NO‐mediated cGMP synthesis was absent from dopaminergic, serotonergic, GABA‐ergic and neuronal NOS‐containing nerve fibres. A minor colocalization of cGMP immunoreactivity was found in parvalbumin‐containing fibres in the cortex. Extensive colocalization between cGMP immunoreactivity and the acetylcholine transporter was found in all cortical areas and in the caudate putamen. There was no effect of the lesions on this colocalization. These results demonstrate NO‐mediated cGMP accumulation in cholinergic fibres in the forebrain of the rat and suggest an anterograde signalling function of NO in cholinergic neuronal systems in the cortex and caudate putamen of the rat.

Expression of neuronal nitric oxide synthase (nNOS) and nitric-oxide-induced changes in cGMP in the urothelial layer of the guinea pig bladder.

The urothelium plays a sensory role responding to deformation of the bladder wall; this involves the release of adenosine trisphosphate (ATP) and nitric oxide (NO), which affect afferent nerve discharge and bladder sensation. The urothelial cells responsible for producing ATP and NO and the cellular targets, other than afferent nerves, for ATP and NO remain largely unexplored. Sub-urothelial interstitial cells (SU-ICs) lie immediately below the urothelium and respond to NO with a rise in cGMP. To determine which cells might target SU-ICs by producing NO, areas of dome, lateral wall and base wall were treated with isobutyl-methyl-xanthine, exposed to the NO donor diethylamino NONOate and then fixed for immunohistochemistry. Surface urothelial cells (SUCs) in the base and dome expressed neuronal nitric oxide synthase (nNOS), whereas those in the lateral wall did not. Distinct populations of SUCs were present in the bladder base. SUCs with significant amounts of nNOS lay adjacent to cells with low levels of nNOS. In specific base regions, the few SUCs present contained nNOS within discrete intracellular particles. In the basal urothelial cell (BUC) layer of the lateral wall, nNOS-positive (NOS+) BUCs neither showed an elevation in cGMP in response to NO, nor expressed the β1 sub-unit of soluble guanylate cyclase, protein kinase I or protein kinase II. Thus, they produced but did not respond to NO. The BUC layer also stained for the stem cell factor c-Kit suggesting its involvement in urothelial cell development. No NOS+ BUCs were present in the SUC-sparse region in the bladder base. Exogenous NO produced an elevation in cGMP in SUCs and SU-ICs. The distribution and proportion of these target cells varied between the dome, lateral wall and base. cGMP+ SU-ICs were present as a dense layer in the bladder base but were rarely seen in the lateral wall, which contained nNOS+ BUCs. No nNOS+ BUCs and cGMP+ SU-ICs were apparent in the dome. The degree of complexity in nNOS distribution and NO target cells is therefore greater than has previously been described and may reflect distinct physiological functions that have yet to be identified.

Sensory collaterals, intramural ganglia and motor nerves in the guinea-pig bladder: evidence for intramural neural circuits

The afferent output from the bladder is important for triggering micturition. This study identifies different types of afferent nerve and explores the connections of their collateral fibres on intramural ganglia and potential ganglionic targets. The experiments were performed on tissues from male guinea-pigs (n=16). Fibres positive for choline acetyl transferase (ChAT+) were found to originate close to the urothelium, to transit the sub-urothelial interstitial cell layer and to pass into the lamina propria. A different population of fibres, immunopositive for calcitonin gene-related peptide (CGRP), capsaicin receptors or neurofilament protein (NF), were seen to intertwine with the ChAT+ fibres in the lamina propria. The ChAT+ fibres did not express NF. Ganglia with ChAT+ and NF+ neurones were found in the lamina propria and muscle. ChAT+ fibres, with pronounced terminal varicosities, were present on the nerve cell bodies. Two types were noted: NF+ terminals and those with little or no NF (NF−) suggesting that their origins were the ChAT+ afferent collaterals and the adjacent ganglia. Fibres containing CGRP or substance P were seen on the ganglionic cells. α1B adrenergic receptors were also found on the neurones indicative of adrenergic synapses. Thus, the ganglia had multiple inputs. Different types of ChAT+ nerves were seen in the muscle: NF+ and NF−. The ChAT+/NF+ nerves may represent a ganglionic output to the muscle. This complex neuronal network may therefore represent the elements generating and modulating bladder sensations. The role of such a scheme in bladder pathology and the therapeutic sites of action of anticholinergic and sympathomimetic drugs are discussed.

The effects of nitric oxide on magnocellular neurons could involve multiple indirect cyclic GMP-dependent pathways.

Nitric oxide (NO) is known to regulate the release of arginine‐vasopressin (AVP) and oxytocin (OT) by the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). The aim of the current study was to identify in these nuclei the NO‐producing neurons and the NO‐receptive cells in mice. The determination of NO‐synthesizing neurons was performed by double immunohistochemistry for the neuronal form of NO synthase (NOS), and AVP or OT. Besides, we visualized the NO‐receptive cells by detecting cyclic GMP (cGMP), the major second messenger for NO, by immunohistochemistry on hypothalamus slices. Neuronal NOS was exclusively colocalized with OT in the PVN and the SON, suggesting that NO is mainly synthesized by oxytocinergic neurons in mice. By contrast, cGMP was not observed in magnocellular neurons, but in GABA‐, tyrosine hydroxylase‐ and glutamate‐positive fibers, as well as in GFAP‐stained cells. The cGMP‐immunostaining was abolished by incubating brain slices with a NOS inhibitor (L‐NAME). Consequently, we provide the first evidence that NO could regulate the release of AVP and OT indirectly by modulating the activity of the main afferents to magnocellular neurons rather than by acting directly on magnocellular neurons. Moreover, both the NADPH‐diaphorase activity and the mean intensity of cGMP‐immunofluorescence were increased in monoamine oxidase A knock‐out mice (Tg8) compared to control mice (C3H) in both nuclei. This suggests that monoamines could enhance the production of NO, contributing by this way to the fine regulation of AVP and OT release and synthesis.

Alterations to network of NO/cGMP-responsive interstitial cells induced by outlet obstruction in guinea-pig bladder

Interstitial cells (ICs) play a role in regulating normal bladder activity. This study explores the possibility that the sub-urothelial and muscle networks of NO/cGMP-responsive ICs are altered in animals with surgically induced outflow obstruction. In sham-operated animals, the urothelium comprised NO-stimulated cGMP-positive (cGMP+) umbrella cells, an intermediate layer and a basal layer that stained for nNOS. cGMP+ sub-urothelial interstitial cells (su-ICs) were found below the urothelium. cGMP+ cells were also associated with the outer muscle layers: on the serosal surface, on the surface of the muscle bundles and within the muscle bundles. Several differences were noted in tissues from obstructed animals: (1) the number of cGMP+ umbrella cells and intensity of staining was reduced; (2) the intermediate layer of the urothelium consisted of multiple cell layers; (3) the su-IC layer was increased, with cells dispersed being throughout the lamina propria; (4) cGMP+ cells were found within the inner muscle layer forming nodes between the muscle bundles; (5) the number of cells forming the muscle coat (serosa) was increased; (6) an extensive network of cGMP+ cells penetrated the muscle bundles; (7) cGMP+ cells surrounded the muscle bundles and nodes of ICs were apparent, these nodes being associated with nerve fibres; (8) nerves were found in the lamina propria but rarely associated with the urothelium. Thus, changes occur in the networks of ICs following bladder outflow obstruction. These changes must have functional consequences, some of which are discussed.

Localization and age-related changes of nitric oxide- and ANP-mediated cyclic-GMP synthesis in rat cervical spinal cord: an immunocytochemical study

An immunocytochemical technique was used to study the localization and developmental aspects of cyclic GMP (cGMP)-synthesizing structures in the cervical spinal cord of 2-week and 3-month-old Lewis rats in response to the nitric oxide (NO) donor sodium nitroprusside (SNP) and/or atrial natriuretic peptide (ANP). By using cell-specific markers, the cell structures involved were investigated. To visualize cGMP, a combined technique of low- and high-power magnification, using a confocal laser scanning microscope was used. NOS-mediated cGMP synthesis was observed in the cervical spinal cord in laminae I, II and III in 14-day-old rats, which activity was mainly absent at the age of 3 months. The involvement of NO in the NMDA-mediated increase in cGMP immunostaining (cGMP-IS) was demonstrated by the absence of cGMP-IS in slices incubated in the presence of NMDA together with the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). This NO-mediated effect of NMDA on cGMP-IS was completely absent in the 3-month-old rats. ANP-mediated cGMP synthesis resulted in an increase in cGMP in laminae I and II, which was generally similar at both ages. Astrocytes in both white and gray matter were found to be cGMP-IS in the basal, NO- and ANP-stimulated conditions. Using confocal laser microscopy, NO-mediated cGMP synthesis was observed in large cholinergic terminals nearby motor neurons in the ventral horn. An extensive colocalization between NO-stimulated cGMP synthesis and parvalbumin-positive (GABAergic) neurons and fibers was observed in all laminae. In the ANP-stimulated condition, a colocalization with parvalbumin structures was found in laminae II and III. No NO- or ANP-mediated cGMP synthesis was found in fibers immunopositive for the presynaptic glutamate transporter, serotonin, or tyrosine hydroxylase.