The morphological and possible functional interactions between the connective tissue and enamel organ cells were examined during the maturation phase of enamel formation, using immunohistochemical techniques. Decalcified mandibular sections (10 µm) including incisors were used from Wistar rats ages 10–12 weeks. Sections were incubated with one or two primary antibodies targeting cell cytoskeleton (vimentin, α-actin, α-tubulin), dendritic marker (OX6), gap junctions (cx-43), enzymes (nitric-oxide synthase (nos1) and cyclooxygenase (cox1)), and the ion transporters (Na + /H + exchanger (NHE1) and Na + /Ca 2+ exchanger (NCX)) for 24 h, before incubation with the appropriate conjugated fluorescent secondary antibodies. Sections were examined by fluorescence microscopy. Haematoxylin–eosin slides were also employed. Cellular heterogeneity and morphological modulations were identified within enamel organ cells and connective tissue covering suggesting complex cellular interactions and indicating a new functional concept and possible complementary role during enamel maturation. Also, some ion transportation activity, and nos1 and cox1 signalling pathways have been identified, indicating intercellular communication between these regions. A hypothesis is suggested, to explain the morphological modulation of ameloblasts and papillary cells during enamel maturation which functions to increase the transporting membrane surface area to accomplish faster and bulker ion transportation to achieve controlled pH and to direct Ca 2+ towards enamel.

AimTo examine rat molar pulp innervation and identify complex cellular signalling systems involving nerve growth factor (NGF) and its p(75) receptors (NGFR) at different stages of development, maturation and ageing. MethodologyDecalcified mandibular first molar mesial cusps from Wistar rats of ages 0day; 1, 2, 3, 4, 6, 9, 12 and 24weeks (n=5 per group) were sectioned (10m) 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. ResultsDuring 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 9weeks. 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 24weeks. ConclusionsAge-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.

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 (10 pm), 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, 3 h and 24 h (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.

Objectives: To describe the process of combining Analysis Grid for Environments Linked to Obesity (ANGELO) with community engagement, qualitative and co-production methods to promote local strategies around child healthy weight (CHW) and to highlight steps taken to engage local people in developing a community CHW action plan around two school communities in Dundee, Scotland. Study design: The Eat, Play, Learn Well (Learn Well) approach applied an action-oriented research approach, using qualitative methods. Methods: Focus group discussions (FGDs), a co-production approach, and ANGELO were linked by applying a novel three-step process. FGDs were recorded by scribe and following face-to-face interview's key themes were identified using a novel, predefined five-step process, and ANGELO grids were populated. Prioritization events allowed local people to rank most important health statements, with community conversations offering further insights to help create a local CHW action plan. Results: Three FGDs were conducted with parents (n = 24) and two with workers (n = 15). Eighty-seven attended a prioritization event at school B (41 adults), 59 attended at school A (35 adults), where each school community chose its top four priorities from 11 health statements developed. Two further community conversations then took place and led to the creation of a CHW action plan with five overarching themes. Conclusions: The Learn Well test approach helped gain important insights into local environments linked to obesity and production of a pragmatic, step-by-step process suitable for real-life public health practice that can enable local people to identify key early intervention and prevention priorities, in a tangible way. (C) 2018 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.

ABSTRACT This study characterizes the complex structural and functional elements of the female rat urethra that may be involved in controlling urethral closure and continence. Urethras were dissected from female Sprague‐Dawley rats (N = 12) euthanized by pentobarbital overdose. Tissues were fixed (4% paraformaldehyde), frozen, and sectioned (8 μm) for light microscopy and immunohistochemistry. Antibodies were used to detect immunoreactivity to calcitonin gene related peptide, nitric oxide synthase, vesicular acetylcholine transporter, and tyrosine hydroxylase. Measurements of urethral wall compliance were taken along its length and in different axes using a closed ended catheter with a circular aperture. The bladder neck and proximal urethra are characterized by a highly folded epithelium and lamina propria. A smooth muscle layer is apparent but not pronounced. Distal to this region the smooth muscle layer thickens and forms the body of the internal sphincter, which has a complex innervation. In the mid urethra, the smooth muscle is thickened resulting in a luminal protrusion, producing an occlusion of the lumen. The structure of the distal urethra is different. The epithelium has few folds and, immediately below the lamina propria large thin walled vascular lacunae can be found. Measurements of the urethral wall compliance demonstrate distinct regional differences with proximal and distal specialisations. These variations, which correlate with muscular and vascular elements, suggest the operation of discrete systems, hence effecting urethral closure during filling. An understanding of these systems may yield insights into urethral pathology and direct approaches to develop pharmacological interventions to promote continence. Anat Rec, 301:1276–1289, 2018. © 2018 Wiley Periodicals, Inc.

ABSTRACTThe aim of this study was to characterize the number, type and distribution of immunochemically identified nerves in epithelium and lamina propria of the female rat urethra. Urethras from female Sprague–Dawley rats (n = 12) were fixed, frozen and sectioned (8 μm). Standard immunohistochemical techniques were used to identify putative nerves using the following antibodies: calcitonin gene related peptide (cgrp), neuronal nitric oxide synthase (nNos), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (vacht). The number, distribution and characteristics of all immunoreactive (IR) structures adjacent to the urethral epithelium and in the lamina propria was assessed. In the bladder, few cgrp‐IR and vacht‐IR fibers were associated with the urothelium or suburothelium of the lateral wall. In contrast, large numbers of vacht‐IR, nNos‐IR and cgrp‐IR fibers were found close to the epithelium and subepithelium of the bladder neck and throughout the urethra. The number of cgrp‐IR fibers was significantly higher in the urethra in comparison with the bladder neck. A population of undescribed cgrp‐IR cells associated with the bladder neck and proximal urethra has been characterized. Each of these cells appears to be associated with a nerve fiber. In the distal urethra, the number of peptidergic fibers penetrating the epithelium was significantly higher than the rest of the urethra. Clearly, this study has revealed a highly complex and heterogeneous network of putative afferent nerves fibers along the length of the urethra. These structural specializations need to be taken into account when probing the different functions of the urethra. Anat Rec, 302:201–214, 2019. © 2018 Wiley Periodicals, Inc.

The process of tooth formation and development is complex involving many signalling pathways and molecules. The enamel formation is a process controlled entirely by the enamel organ with many cell-cell interaction and signalling. Although this process was studied extensively, the full understanding is still to be achieved. Twenty dental pulps from rat mandibular incisor were dissected, fixed, frozen, sectioned, stained with specific antibodies then carefully examined using fluorescence microscope. The basic findings were cellular heterogeneity, presence of spherical vacuoles which may be blood vessels, and striking differential expression of some very important signalling molecules antigen throughout the enamel organ at different stages of development. This paper revealed some of the complexity associated with enamelogenesis and proved that the previous description of enamel organ is very simplistic.

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. (C) 2016 Elsevier B.V. All rights reserved.

A growing body of work is describing the absence of a significant sympathetic innervation of the detrusor implying little sympathetic regulation of bladder contractility. However, low doses of adrenergic agonists are capable of relaxing the bladder smooth muscle. If these effects underpin a physiological response then the cellular nature and operation of this system are currently unknown. The present immunohistochemistry study was done to explore the existence of alternative adrenergic signaling elements in the rat bladder wall. Using antibodies to tyrosine hydroxylase (TH) and vesicular mono-amine transporter (vmat), few adrenergic nerves were found in the detrusor although TH immunoreactive (IR) nerves were apparent in the bladder neck. TH-IR and vmat-IR nerves were however abundant surrounding blood vessels. A population of vmat-IR cells was found within the network of interstitial cells that surround the detrusor muscle bundles. These vmat-IR cells were not or only weakly TH-IR. This suggests that these interstitial cells have the capacity to store and release catecholamines that may involve noradrenaline. Cells expressing the β1-adrenoceptor (β1AR-IR) were also detected within the interstitial cell network. Double staining with antibodies to β1AR and vmat suggests that the majority of vmat-IR interstitial cells show β1AR-IR indicative of an autocrine signaling system. In conclusion, a population of interstitial cells has the machinery to store, release and respond to catecholamines. Thus, there might exist a non-neuronal β-adrenergic system operating in the bladder wall possibly linked to one component of motor activity, micro-contractions, a system that may be involved in mechanisms underpinning bladder sensation.

β3-adrenoceptor agonists influence overactive bladder in humans and animal models. However, data is emerging that the mode of action of these drugs is complex. The present study explored the actions of the β3-adrenergic agonist mirabegron and the non-selective agonist isoprenaline on the contractile systems in the rat and guinea pig bladder. Intravesical pressure was measured in isolated whole bladders from female adult animals. In both species spontaneous contractile activity was observed. The muscarinic agonist arecaidine produced complex responses consisting of an initial transient pressure rise followed by complex phasic activity. Three contractile elements were identified: intrinsic micro-contractile activity, initial transient response and steady state phasic activity. The intrinsic and steady state activity could be further divided into a baseline pressure with superimposed phasic activity. The effects of isoprenaline and mirabegron were investigated on these elements. In the rat, the micro-contractile activity could be completely inhibited by isoprenaline (full agonist). The arecaidine-induced initial and steady state baseline pressures were partially reduced, while the phasic activity was little affected. In the guinea pig, both the arecaidine-induced baseline pressure and the phasic activity were affected by isoprenaline. Mirabegron didn't produce significant inhibitory effects in any of the contractile elements in either species. These results show that complex contractile systems operate in the rat and guinea pig bladder that can be modulated by β1/β2-adrenoceptor mechanisms. No evidence was obtained for any β3-dependent regulation of contraction. These data support similar data in humans. Therefore the primary site of therapeutic action of β3-adrenergic agonists remains unknown.

Aims To study bladder sensation during a forced diuresis protocol and to assess differences in sensation perceived by different ethnic groups and after drinking artificially sweetened water. Methods Female Caucasian and south Indian Asian volunteers performed the diuresis protocol drinking water, or water sweetened with saccharin (5 mg/kg body weight). Participants recorded filling sensation every 5 min while drinking 250‐350 mL/15 min. They were asked to record the strongest sensation before voiding as maximum sensation, before voiding. The void was measured and sensation immediately recorded as minimum. The process was repeated. Voided volume and time required to achieve maximum sensation during cycle 2 were compared by water and sweetener, ethnic group, and age. Results Twenty Asian and 20 Caucasian volunteers participated. No differences in maximum voided volume or diuresis rate was seen by ethnicity. Median diuresis with sweetener was 16.7 mL/min (8.6‐35) compared to 13.2 (7.1‐25) with water ( P = 0.008), a difference accounted for by 16 women with >5 mL/min difference in diuresis rate. These were excluded to leave 24 women with similar diuresis rates with both sweetener and water (14.8 mL/min (8.6‐28.0) and 13.2 mL/min (7.1‐25.0). In these women, time to achieve maximum sensation was lower with sweetener than water: 37.5 min (20‐85) versus 50.0 min (20‐80), P = 0.002, with no difference in voided volume. Conclusions Water sweetened with saccharin produced an increased diuresis rate in some women. After controlling for this, time to recording maximum sensation was decreased with sweetened water, suggesting saccharin has an effect upon perceived sensation.

Background and PurposeThe aims of the present study were to characterize the role of PAR1 in rat bladder under inflammatory conditions and determine whether a selective PAR1 antagonist, F16357, can prevent the pathophysiological symptoms of cyclophosphamide‐induced interstitial cystitis (IC).Experimental ApproachImmunohistochemistry, contractile activity in isolated bladder and urodynamics were determined before and after cyclophosphamide treatment. F16357 was administered intravesically during the acute phase of inflammation, and effects on PAR1 and PAR1‐related bladder contraction evaluated 24 h after cyclophosphamide injection. Urodynamics and associated voided volumes were recorded 7 and 24 h after cyclophosphamide.Key ResultsIn control conditions, PAR1 was present only in some umbrella cells. Cyclophosphamide disrupted the urothelium and expression of PAR1 by all remaining urothelial cells. After F16357 treatment, urothelial damage was absent and PAR1 immunoreactivity similar to control tissues. Thrombin and TFLLR‐NH2 induced bladder contractions. These were increased in inflammatory conditions and antagonized by F16357 in a concentration‐dependent manner. In telemetric experiments, furosemide increased urine production and voiding frequency for 60 min, 7 h after cyclophosphamide injection. Intravesical administration of F16357 blocked these changes with a return to a physiological profile; 24 h after cyclophosphamide, the volume of micturition was still lower with no increase in number of micturitions. F16357 30 μM reduced the number of micturitions and improved bladder capacity, but did not affect diuresis. Under similar experimental conditions, lidocaine 2% induced comparable effects.Conclusions and ImplicationsPAR1 is expressed in rat bladder, overactivated in inflammatory conditions and involved in bladder function and sensation. F16357 could represent an interesting candidate for IC treatment.

Purpose: It is well known that afferent input from the urethra can modulate bladder function. Nevertheless, little is known about the functional properties of urethral afferents. In the current study we investigated the effect of urethral distension on single fiber afferent activities of the lower urinary tract in the female rat.Materials and Methods: Female Sprague Dawley (R) rats were anesthetized. Single fiber afferent activities were recorded from the left L6 dorsal root and classified by conduction velocity. The response of pelvic and pudendal units on urethral distension (60 seconds) was measured. Two distension diameters were measured in the proximal and the distal urethra.Results: A total of 93 pelvic and 72 pudendal units were isolated in 15 rats. Of the units 20 (8 pelvic and 12 pudendal) were responsive to urethral distension. Three patterns of response could be distinguished, including a fast adapting and 2 groups of slow adapting afferents. The largest grade of distension resulted in the greatest response in both nerves. Five pelvic and 3 pudendal units responded exclusively to proximal distension, 2 pelvic and 5 pudendal units responded to distal distension, and 1 pelvic and 4 pudendal units responded to both types of distension. The responses were reproducible. No association was found between the type of nerve and the location of the response to distension.Conclusions: This electrophysiological study demonstrates the presence of urethral distension evoked afferents in the pelvic and pudendal nerves, and describes their response to distension. Differences in sensory signaling in type and in location were demonstrated. The current technique can be used for further investigation of urethral afferents.

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. β3-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 β3 agonists has questioned the therapeutic site of action of β3 agonists in the bladder. The present study was done to explore the possibility that β3-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 β3-adrenoceptor immuno-reactivity (β3AR-IR). The only structures found to express β3AR-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 β3AR-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 β3-adrenoceptor agonists is to modulate sensation, it is possible that these neural circuits may be involved in the regulation of afferent outflow and sensation.

The present study was done to explore the cholinergic systems operating in the wall of the isolated rat bladder. In a first set of experiments, bladder strips in vitro were subjected to cumulative concentration-response curve (CRC) to non-selective muscarine agonist carbachol or the partially M2 > M3 selective agonist arecaidine to establish optimal concentration to be used thereafter. In a second set of experiments, the effects of drugs (solifenacin, isoproterenol and mirabegron) were tested on urinary bladder contraction induced by the non-selective muscarinergic agonist carbachol. For both agonists, the contractile responses are qualitatively similar: an initial transient rise in tension followed by complex bursts of high-frequency small ‘micro’-contractions superposed on a tonic contraction, with immediate transient ‘rebound’ contraction after the agonist is washed from the preparation. This rebound contraction is greater with carbachol than arecaidine. Components of the responses to cholinergic stimulation, notably the micro-contractions, were found to be differently stimulated and inhibited by the M3 > M2 selective antagonist solifenacin and by the β-adrenoceptor agonists isoprenaline and mirabegron. A physiological role for the muscarinic dependent phasic contractions and the micro-anatomical elements that might be involved are not known but may be related to non-voiding activity observed during filling cystometry in conscious animals related to afferent discharge and possibly sensation. Furthermore, suggestions for the potential impact of these findings and design of further studies in relation to bladder physiology, pharmacology and pathology are discussed.

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.

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. β-Adrenoceptors are major players in detrusor muscle relaxation, and the selective β3-adrenoceptor (AR) agonist mirabegron was recently approved for the treatment of overactive bladder (OAB). β-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 β1-AR-mediated.

The present study was done to explore the cholinergic systems operating in the wall of the isolated rat bladder. In a first set of experiments, bladder strips in vitro were subjected to cumulative concentration-response curve (CRC) to non-selective muscarine agonist carbachol or the partially M2>M3 selective agonist arecaidine to establish optimal concentration to be used thereafter. In a second set of experiments, the effects of drugs (solifenacin, isoproterenol, and mirabegron) were tested on urinary bladder contraction induced by the non-selective muscarinergic agonist carbachol. For both agonists, the contractile responses are qualitatively similar: an initial transient rise in tension followed by complex bursts of high-frequency small 'micro'-contractions superposed on a tonic contraction, with immediate transient 'rebound' contraction after the agonist is washed from the preparation. This rebound contraction is greater with carbachol than arecaidine. Components of the responses to cholinergic stimulation, notably the micro-contractions, were found to be differently stimulated and inhibited by the M3>M2 selective antagonist solifenacin and by the β-adrenoceptor agonists isoprenaline and mirabegron. A physiological role for the muscarinic dependent phasic contractions and the micro-anatomical elements that might be involved are not known but may be related to non-voiding activity observed during filling cystometry in conscious animals related to afferent discharge and possibly sensation. Furthermore, suggestions for the potential impact of these findings and design of further studies in relation to bladder physiology, pharmacology, and pathology are discussed.

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 β3-adrenoceptor (AR) agonist mirabegron in detrusor overactivity in rats. For this, effects of the β-AR agonist isoprenaline and mirabegron were tested in presence or absence of selective antagonists for β-AR subtypes, namely CGP-20712A for β1-AR, ICI-118,551 for β2-AR, and L-748,337 for β3-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 β1-AR antagonist, but not by the β2- and β3-AR antagonists. In the context of β3-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.