Evidence for changing nerve growth factor signalling mechanisms during development, maturation and ageing in the rat molar pulp.

AIM: To examine rat molar pulp innervation and identify complex cellular signalling systems involving nerve growth factor (NGF) and its p75 receptors (NGFR) at different stages of development, maturation and ageing. METHODOLOGY: Decalcified mandibular first molar mesial cusps from Wistar rats of ages 0 day; 1, 2, 3, 4, 6, 9, 12 and 24 weeks (n = 5 per group) were sectioned (10 µm) and incubated with antibodies for NGF, NGFR, calcitonin gene-related peptide (CGRP) and neurofilament. Nerve densities in worn and intact regions of 3- to 24-week-old rats were compared by anova, Bonferroni and t-tests. RESULTS: During odontogenesis, differences in NGF and NGFR expression were observed, with no evidence of nerve fibres, suggesting a signalling mechanism controlling cellular differentiation and dentine formation. Tooth wear in 4-week rats was associated with reduced NGF expression and significantly decreased CGRP axons within affected odontoblast regions. The underlying subodontoblasts started expressing NGF which continued until 9 weeks. This may promote a significant increase in CGRP nerve density in affected regions. Nerve density in intact odontoblast regions increased gradually and reached significant levels in 12-week rats. Reduction in nerve densities within worn and intact regions of cusps was observed at 24 weeks. CONCLUSIONS: Age-related changes and responses to tooth wear may be controlled by the NGF signalling mechanism, with roles in odontoblast/subodontoblast communication and control of sensory innervation at different stages of tooth development, maturation and ageing. Greater understanding of cellular and nerve regulation in the injured pulp may promote therapeutic strategies for pulp survival.

Evidence for programmed odontoblast process retraction after dentine exposure in the rat incisor.

OBJECTIVE: To re-examine the morphology and potential functions of odontoblasts in intact rat incisors and after cavity preparation into dentine. DESIGN: Intact incisors were fixed, decalcified, snap frozen and sectioned (10µm), before staining with rhodamine phalloidin or antibodies for cyto-skeletal proteins: vimentin and actin, ion transporter: NaK-ATPase, and dendritic cell marker: OX6. Samples with cavity were processed similarly and stained for actin and vimentin before comparing the lengths of odontoblast processes (OP) at baseline, 3h and 24h (n=5 for each group). RESULTS: Actin was expressed through the full length of OP, while vimentin immunoreactivity was not uniform, with 4 distinct regions. OP showed morphological complexity with fine branches emanating within different regions of dentine. Novel actin-positive tree-like OP were identified within predentine which reduced in intensity and length toward the incisal portion of the tooth. Specimens with cavities showed time-dependant pulpal retraction of OP. CONCLUSIONS: Differences in structural antibody expression suggest functional variations in OP within different regions of dentine. The role of actin positive OP in predentine is not known, but could be related to dentine deposition, cellular stability or sensing mechanisms. Cavity preparation into dentine was followed by programmed retraction of OP which could be controlled either mechanically by the spatial limitation of the OP within dentinal tubules or structurally by the presence of vimentin, in addition to actin, in the mid-dentine.

Distribution and sub-types of afferent fibre in the mouse urinary bladder.

AIM: Increased afferent fibre activity contributes to pathological conditions such as the overactive bladder syndrome. Nerve fibres running near the urothelium are considered to be afferent as no efferent system has yet been described. The aim of this study was to identify sub-types of afferent nerve fibres in the mouse bladder wall based on morphological criteria and analyse regional differences. MATERIALS AND METHODS: 27 bladders of six month old C57BL/6 mice were removed and tissues were processed for immunohistochemistry. Cryostat sections were cut and stained for Protein Gene Product 9.5 (PGP), calcitonin gene related polypeptide (CGRP), neurofilament (NF), vesicular acetylcholine transporter (VAChT) and neuronal nitric oxide synthase (nNOS). RESULTS: In the sub-urothelium, different types of afferent nerve fibre were found, i.e. immunoreactive (IR) to; CGRP, NF, VAChT, and/or nNOS. At the bladder base, the sub-urothelium was more densely innervated by CGRP-IR and VAChT-IR nerve fibres, then at the lateral wall. NF- and nNOS nerves were sparsely distributed in the sub-urothelium throughout the bladder. At the lateral wall the inner muscle is densely innervated by CGRP-IR nerve fibres. NF, VAChT and nNOS nerves were evenly distributed in the different muscle layers throughout the bladder. Nerve fibre terminals expressing CGRP and NF were found within the extra-mural ganglia at the bladder base. CONCLUSIONS: Different types of afferent nerve fibres were identified in the sub-urothelium of the mouse bladder. At the bladder base the sub-urothelium is more densely innervated than the lateral wall by CGRP-IR and VAChT-IR afferent nerve fibres. CGRP and NF afferent nerve fibres in the muscle layer probably relay afferent input to external ganglia located near the bladder base. The identification of different afferent nerves in the sub-urothelium suggests a functional heterogeneity of the afferent nerve fibres in the urinary bladder.

Complex cellular responses to tooth wear in rodent molar.

The arrangement and roles of the odontoblast and its process in sensing and responding to injuries such as tooth wear are incompletely understood. Evidence is presented that dentine exposure by tooth wear triggers structural and functional changes that aim to maintain tooth integrity. Mandibular first molars from freshly culled 8 week Wistar rats were prepared for light microscopy ground-sections (n=6), or fixed in 4% paraformaldehyde, decalcified in 17% EDTA, sectioned and stained with antibodies to cyto-skeletal proteins (vimentin (vim), α-tubulin (tub) and α-actin), cellular homeostatic elements (sodium potassium ATPase (NaK-ATPase) and sodium hydrogen exchanger (NHE-1)), and sensory nerve fibres (CGRP) (n=10) for fluorescence microscopy of worn and unworn regions of the mesial cusp. Immunoreactivity (IR) to vim, actin, NaK-ATPase and CGRP was confined to the pulpal third of odontoblast processes (OPs). IR to tub and nhe-1 was expressed by OPs in full dentine thickness. In areas associated with dentine exposure, the tubules contained no OPs. In regions with intact dentine, odontoblasts were arranged in a single cell layer and easily distinguished from the sub-odontoblast cells. In regions with open tubules, the odontoblasts were in stratified or pseudo-stratified in arrangement. Differences in structural antibody expression suggest a previously unreported heterogeneity of the odontoblast population and variations in different regions of the OP. This combined with differences in OPs extension and pulp cellular arrangement in worn and unworn regions suggests active and dynamic cellular responses to the opening of dentinal tubules by tooth wear.

Beta adrenergic modulation of spontaneous microcontractions and electrical field-stimulated contractions in isolated strips of rat urinary bladder from normal animals and animals with partial bladder outflow obstruction.

Spontaneous microcontractions and electrical field stimulation (EFS)-evoked contractions in isolated rat bladder strips from normal and from 6 weeks partial bladder outflow obstruction (pBOO) animals were studied to identify the potential site of action for the ß3-adrenoceptor (AR) agonist mirabegron in detrusor overactivity in rats. For this, effects of the ß-AR agonist isoprenaline and mirabegron were tested in presence or absence of selective antagonists for ß-AR subtypes, namely CGP-20712A for ß1-AR, ICI-118,551 for ß2-AR, and L-748,337 for ß3-AR. In detrusor strips from both normal and obstructed animals, EFS-induced contractions were weakly affected by isoprenaline and even less so by mirabegron. In contrast, microcontraction activity was more potently reduced by isoprenaline (pIC50 7.3; Emax ±85 %), whereas mirabegron showed a small effect. In pBOO strips, concentration response curves for isoprenaline and mirabegron at inhibition of EFS and spontaneous microcontractions were similar to those in normal strips. Isoprenaline-induced inhibition of microcontractions and EFS was antagonized by the ß1-AR antagonist, but not by the ß2- and ß3-AR antagonists. In the context of ß3-AR-mediated bladder functions for mirabegron in other experiments, the current data question a role for effects at spontaneous microcontractions, or neurogenic detrusor stimulation in the mode of action for mirabegron in vivo, since functional bladder effects for mirabegron are reported to occur at much lower concentrations.

Prostaglandin E2 excitatory effects on rat urinary bladder: a comparison between the ß-adrenoceptor modulation of non-voiding activity in vivo and micro-contractile activity in vitro.

Prostaglandin E2 (PGE2) is well known to modulate urinary bladder functions, but it is also thought to be involved in the pathophysiology of lower urinary tract dysfunctions, since high levels of PGE2 have been found in overactive bladder (OAB) patients. ß-Adrenoceptors are major players in detrusor muscle relaxation, and the selective ß3-adrenoceptor (AR) agonist mirabegron was recently approved for the treatment of overactive bladder (OAB). ß-Adrenoceptor modulation of PGE2 excitatory effects on bladder detrusor muscle was investigated by i.v. mirabegron after intravesical PGE2 infusion in conscious rats. Non-voiding activity (NVA) was assessed under isovolumetric conditions. In addition, mirabegron and isoprenaline (0.01-10 µM) were studied on PGE2-increased micro-contractile activity during isometric tension recordings of intact isolated bladder muscle strips. Our investigations showed that PGE2 dramatically increased NVA in vivo and spontaneous micro-contractions in vitro. In vivo administration of mirabegron (0.1, 0.3 and 3 mg/kg) reduced PGE2-augmented NVA in dose-dependent manner, while the PGE2-increased micro-contractions in isolated bladder strips were poorly inhibited. Isoprenaline inhibited PGE2-augmented micro-contractions in a concentration-dependent manner and had a higher potency compared to mirabegron. The apparent pKB of 7.25 for metoprolol at the isoprenaline concentration-response curve for PGE2-augmented micro-contractions suggests a ß1-AR-mediated.

The characteristics of intrinsic complex micro-contractile activity in isolated strips of the rat bladder.

In the resting and un-stimulated state, the bladder wall is not quiescent and discrete contractile events, microcontractions, can be recorded in almost all species. This activity contributes to the active element of compliance and to the basal resting tension. This intrinsic activity underpins the more complex phasic activity, non-voiding activity (NVA) that can be seen to increase progressively as the bladder is filled. The NVA represents the motor component of a motor sensory system that relays information to the CNS on bladder volume. Despite the importance of this intrinsic motor activity, little is known about the mechanisms involved in its generation and modulation. The present experiments were done on isolated hemi-bladders from normal rats and measurements made of the intrinsic motor activity. Detailed analysis of the resting state reveals the presence of discrete phasic contractile events, micro-contractions that range in amplitude from 0.1-0.6 mN. These events seem to occur randomly and the basal activity has the appearance of 'noise'. An analysis of the frequency amplitude distribution of the contractile events, reveals that the total activity appears to be the sum of a number of discrete contractile units, each generating a phasic contraction about a specific mean value and with characteristic frequency. In a hemi-bladder, there are between 20-30 units generating the activity at rest. Using the timed integral of the activity (product of amplitude and frequency), it was noted that the activity was increased by the muscarinic agonist carbachol, but it was decreased by the ß-adrenergic agonist isoprenaline. Stretching the preparations also increased the activity. Using these observations, a simple model of the structural and functional organisation of the isolated rat bladder is proposed: the wall appears to be arranged into a number of discrete motor units acting independently. However, the activity can be stimulated or inhibited by pharmacological agents and mechanically (stretch). The possible relevance of this activity, its relationship to NVA and in relation to the mode of action of drugs are discussed. [Corrected]

The expression of ß3-adrenoceptor and muscarinic type 3 receptor immuno-reactivity in the major pelvic ganglion of the rat.

Bladder afferent outflow, linked to sensation, plays a critical role in bladder pathology: abnormal outflow results in altered sensation, leading to increased voiding frequency, urge and often incontinence. ß3-adrenoceptor agonists have been suggested to be beneficial in treating these symptoms. However, the absence of a significant sympathetic innervation of the detrusor and only a modest relaxation of bladder muscle by ß3 agonists has questioned the therapeutic site of action of ß3 agonists in the bladder. The present study was done to explore the possibility that ß3-adrenoceptors might be located in the pelvic plexus. Using the rat, where the pelvic plexus is located primarily within a single ganglion, the major pelvic ganglion (MPG), immuno-histochemical approaches were used to identify structures expressing ß3-adrenoceptor immuno-reactivity (ß3AR-IR). The only structures found to express ß3AR-IR were small-diameter tyrosine hydroxylase and vesicular mono-amine transporter immuno-reactive (TH-IR and vmat-IR) neurones. These neurones, found in clusters or singly on the periphery of the ganglion, or dispersed in smaller clumps throughout the MPG, are similar to the small intensely fluorescent (SIF) cells described previously. Not all small cells expressed ß3AR-IR. A population of the small cells were also immuno-reactive to the type 3 muscarinic receptor (M3R-IR) and the P2X3 purinergic receptor (P2X3-IR). Clumps of small cells were associated with calcitonin gene-related peptide immuno-reactive (CGRP-IR) nerve fibres (putative sensory fibres) and a small number were contacted by putative cholinergic nerves expressing immuno-reactivity to vesicular acetylcholine transporter (vacht-IR). These observations are consistent with the idea that small cells are interneurons and one of the components making up complex neural circuits within the MPG. The precise physiological role of these neural elements in the MPG is unknown. However, as one therapeutic action of ß3-adrenoceptor agonists is to modulate sensation, it is possible that these neural circuits may be involved in the regulation of afferent outflow and sensation.

Interactions between cholinergic and prostaglandin signaling elements in the urothelium: role for muscarinic type 2 receptors.

OBJECTIVE: To characterize the interactions between the cholinergic and prostaglandin signaling systems within the urothelium-lamina propria of the guinea pig and elucidate the role of muscarinic receptors in these interactions. METHODS: The urothelium-lamina propria was isolated from guinea pig bladders, cut into strips (5×10 mm), and maintained in vitro. The tissue was either stretched or left unstretched but exposed to 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate tri(triethylammonium) salt, arecaidine, and prostaglandin E2 (PGE2). Acetylcholine and PGE2 release was measured using a GeneBLAzer M3 CHO-K1-bla cell reporter assay and an enzyme immunoassay, respectively. The role of the muscarinic type 2 and 3 (M2 and M3, respectively) receptors and nitric oxide in mediating PGE2 release was determined in the presence of the muscarinic antagonists 11-[(2-[(diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4] benzodiazepin-6-one and darafenicin and a nitric oxide donor (NONOate). RESULTS: Acetylcholine release was detected in response to stretch and in the unstretched preparations exposed to PGE2 or the adenosine triphosphate analog 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate tri(triethylammonium) salt. The cholinergic agonist arecaidine induced a concentration-dependent production of PGE2 (half-maximal concentration 75 nM). The arecaidine stimulation of PGE2 production was inhibited in a dose-dependent manner by the antagonist AFDX-116 (M2>M3; half-maximal inhibition 110 nM) but not darifenacin (M3>>M2). Finally, in the presence of the nitric oxide donor, NONOate, arecaidine-stimulated PGE2 production was inhibited. CONCLUSION: These observations demonstrate that complex signal interactions occur within the urothelium involving acetylcholine, adenosine triphosphate, nitric oxide, and PGE2. In addition, the data have demonstrated a role for muscarinic M2 receptors and nitric oxide in the cholinergic regulation of PGE2 production in the bladder wall.

On the nature of bladder sensation: the concept of sensory modulation.

AIMS: Going to the toilet is an essential everyday event. Normally, we do not give much thought to the sensations and factors that trigger voiding behavior: we just go. For many people, this apparently simple task is complicated and dominates their life. They have strong sensations and sudden desires to void, often resulting in incontinence. It is therefore important that we understand the origins for this functional change and identify means to alleviate it. METHODS: Literature survey. RESULTS: A considerable body of work has focused on this problem and ideas and concepts on the nature of bladder sensations are embedded in the literature. In this paper we argue the necessity to return to first principles and a re-examination of the problem. We explore the use of focus groups to identify relevant bladder sensation and what triggers 'bladder' behavior. We argue that there are differences in what can be described as 'introspective bladder sensations' and the sensations reported immediately before a void, 'void sensations'. Finally, we propose an alternative model describing how peripheral information generating 'introspective sensations' and 'void sensations' might be different but interrelated sensations. By exploring such ideas and identifying such complexity it is our intention to stimulate debate and generate further research in the field in order to understand better the physiology of bladder sensation and the pathology of increased urge, frequency and incontinence. CONCLUSIONS: Review of the literature on bladder sensation and the established ideas suggests that we might be missing something and the problem of normal and increased sensation and of urgency may be much more complex.

Changes in bladder innervation in a mouse model of Alzheimer's disease.

AIM: The aims of this study were to compare the structure of bladders from a transgenic mouse model of Alzheimer's disease with age matched control animals and to explore the idea that any structural differences might be related to functional bladder changes associated with the condition. MATERIALS AND METHODS: Two groups of mice were used. Transgenic animals in which the murine Amyloid Precursor Protein (APP) gene has been partly replaced by the human APP including both the Swedish and London mutations and that overexpress a mutant of the human Presenilin 1 gene (PS1M146L) driven by the PDGF promoter. The transgenic mice (App(SL)/PS1(M146L)) aged 24+/-3 months were used. The second group was an age matched control group of C57 black mice. The bladders from each group were isolated, fixed in 4% paraformaldehyde and prepared for immunohistochemistry. Antibodies to the vesicular acetylcholine transporter (VAChT) and neuronal nitric oxide synthase (nNOS) were used to identify neural structures. RESULTS: Cholinergic nerves (VAChT(+)) were observed in the inner and outer muscle bundles of App(SL)/PS1(M146L) and control mice. No major differences were noted in the distribution of these fibres. In contrast, there was a distinct difference in the innervation of the sub-urothelial layer. In App1(SL)/PS1(M146L) mice there were numerous VAChT and nNOS positive fibres in sharp contrast to the paucity of similar nerves in control animals. VAChT and nNOS did not appear to co-localise in the same nerve fibres within the lamina propria. Pairs of nerve fibres, nNOS(+) and VAChT(+), were observed to be intertwined and run in close proximity. A particularly unusual feature of the App(SL)/PS1(M146L) mouse bladder was the presence of neurones within the bladder wall. These nerve cell bodies were seen in all App(SL)/PS1(M146L) mouse bladders. The neurones could be found singly or in small ganglion like groups of cells and were located in all layers of the bladder wall (sub-urothelium, in the lamina propria adjacent to the inner muscle and within the inner muscle and outer muscle layers). No nerve cells or small ganglia were noted in any of the control bladders studied. CONCLUSIONS: There are structural differences in the bladders of App(SL)/PS1(M146L) mice compared to control animals. These differences are associated with sub-urothelial nerves which, because of their location, are likely to be sensory fibres. This may lead to a changed sensory processing from the App(SL)/PS1(M146L) bladders. The physiological role of the intra-mural neurones and ganglia is not known. It is speculated that they may be associated with peripheral motor/sensory mechanisms linked to the generation and modulation of sensation.

The localization of cyclo-oxygenase immuno-reactivity (COX I-IR) to the urothelium and to interstitial cells in the bladder wall.

Localized phasic contractions in the bladder wall (autonomous activity) have been hypothesized to be an integral part of a motor/sensory system contributing to bladder sensation. The sites responsible for generating this activity, the mechanisms involved in its propagation and modulation remain unknown. This phasic motor activity is modulated by exogenous prostaglandins. Therefore, analysis of the sites of prostaglandin production and action within the bladder wall may shed light on the mechanisms of generation and modulation of this phasic activity. In this paper we report the localization of immuno-reactivity indicative of the expression of cyclo-oxygenase enzyme type I (COX I-IR) within the bladder wall. Basically, three types of COX I-IR cell were identified: epithelial cells in the basal and intermediate layers of the urothelium, complex vimentin-positive and COX I-IR cells in the lamina propria and vimentin-negative COX I-IR cells in the lamina propria and on the surface of the inner muscle bundles. These vimentin-negative/COX I-IR cells appear to be in close apposition to a continuous network of vimentin-positive cells, which extends from the lamina propria into the inner muscle layers and subsequently into the outer muscle layers. However, the interstitial cells in this region might form a distinctly different sub-type. First, the interstitial cells in this region differ from those in the inner layer by their responsiveness to NO with a rise in cGMP. Two subtypes have been identified: cells on the surface of the muscle bundles and within the muscle bundles. Second, COX I-IR cells are not associated with the interstitial cells in the outer layers. The physiological significance for these apparent differences in the interstitial cell network is not clear. However, such differences are likely to reflect differences in the processes involved in their activation, modulation and control.

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.

A comparison of CFU-GM, BFU-E and endothelial progenitor cells using ex vivo expansion of selected cord blood CD133(+) and CD34(+) cells.

BACKGROUND: CD133 is a newly developed hematopoietic stem cell marker but little is known about its function. Whether CD133(+) cell selection provides any advantage over CD34(+) selection for hematopoietic stem cell isolation and transplantation is unclear. The present study compared colony formation and endothelial cell differentiation of these two cell types from umbilical cord blood (UCB). METHODS: Mononuclear cells from the same UCB samples were used for both CD133(+) and CD34(+) cell selection. Cells with 97.1% purity were incubated in semi-solid culture medium containing stem cell growth factor (SCGF) and G-CSF or erythropoietin (EPO). Purified cells were also cultured in M199 containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor-1 (IGF-1). RESULTS: CD34(+) and CD133(+) cells produced similar numbers of CFU-GM colonies (median 43.25 and 30.5, respectively; P>0.2). However, a greater than four-fold difference in BFU-E colony formation was observed from CD34(+) cells compared with CD133(+) cells (median 35 and 8, respectively; P<0.04). CD34(+) cells gave rise to endothelial-like cells when stimulated with VEGF, bFGF and IGF-1. CD133(+) cells were unable produce this cell type under the same conditions. DISCUSSION: CD133(+) cells produced smaller BFU-E colonies and were unable to differentiate into mature endothelial cells. CD34(+) cells contained endothelial progenitors that could differentiate into mature cells of this lineage. Based on these data, it appears that CD133 offers no distinct advantage over CD34 as a selective marker for immunoaffinity-based isolation of hematopoietic stem cells and endothelial progenitor cells.

Natriuretic peptide responsive, cyclic guanosine monophosphate producing structures in the guinea pig bladder.

PURPOSE: We examined the localization of natriuretic peptide responsive, cyclic guanosine monophosphate producing cells in the guinea pig bladder. MATERIALS AND METHODS: The bladder was removed from male guinea pigs sacrificed by cervical dislocation. The lateral wall of the bladder was cut into strips 2 mm thick. The tissue pieces were incubated in the presence of human atrial natriuretic peptide, rat brain natriuretic peptide and C-type natriuretic peptide or the nitric oxide donor DEANO (diethylamine NONOate or 1,1-diethyl-2-hydroxy-2-nitrosohydrazine) (Sigma). Cyclic guanosine monophosphate immunoreactivity was localized using an antibody against formaldehyde fixed cyclic guanosine monophosphate. RESULTS: Atrial natriuretic peptide and brain natriuretic peptide stimulated cyclic guanosine monophosphate synthesis in suburothelial interstitial cells, whereas C-type natriuretic peptide was not effective. In contrast, DEANO stimulated cyclic guanosine monophosphate synthesis in urothelial umbrella cells, suburothelial interstitial cells, muscle interstitial cells and neurons. The effect of atrial natriuretic peptide and brain natriuretic peptide was not inhibited by ODQ (1H-[1, 2, 4]oxadiazolo[4-3a]quinoxalin-1-one), an inhibitor of nitric oxide responsive soluble guanylyl cyclase. CONCLUSIONS: To our knowledge our findings show for the first time a localized effect of atrial natriuretic peptide and brain natriuretic peptide to the suburothelial cells of the guinea pig bladder. These cells express the soluble guanylyl cyclase and particulate guanylyl cyclase-A isoforms. The specific physiological role of these cells is not known but it was suggested that they may be involved in the generation or modulation of sensation. The results imply a role for natriuretic peptide-cyclic guanosine monophosphate signaling in the processing of sensory information in the bladder.

Endogenous nitric oxide/cGMP signalling in the guinea pig bladder: evidence for distinct populations of sub-urothelial interstitial cells.

We have examined structures that may operate by using nitric oxide (NO)/soluble guanylyl cyclase (sGC) signalling in the lamina propria of the guinea pig bladder. Cells on the luminal surface of the urothelium and sub-urothelial interstitial cells (SU-ICs) responded to NO with a rise in cGMP. The distribution of these different cells varied between the base, lateral wall and dome. In the base, two regions were identified: areas with sparse surface urothelial cells and areas with a complete covering. A layer of cGMP-positive (cGMP(+)) cells (up to 10 cells deep) was found in the base. cGMP(+)/SU-ICs were also observed in the lateral wall. However, here, the cGMP(+) cells were confined to a layer of only 1-2 cells immediately below the basal urothelial layer (basal cGMP(+)/SU-ICs). Below these cGMP(+)/SU-ICs lay cells that had a similar structure but that showed little cGMP accumulation (deep cGMP(-)/SU-ICs). Both basal and deep SU-ICs expressed the beta1 subunit of sGC and the cGMP-dependent protein kinase I (cGKI), suggesting that the deep SU-ICs can sense NO and signal via cGMP. By using BAY 41-2272, a sensor of endogenous NO production, NO-dependent cGMP synthesis was observed primarily in the basal SU-ICs. A third population of cGKI(+)/cGMP(-) cells was seen to lie immediately below the basal urothelial layer. These cells ("necklace" cells) were less numerous than SU-ICs and extended linking processes suggesting a network. The specific functions of these structures are not known but they may contribute to the emerging multiple roles of the urothelium associated with the generation of bladder sensation.

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. alpha1B 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.

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 beta1 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.