Mucosal signaling in the bladder

AUTNEU-01792; No of Pages 8 Autonomic Neuroscience: Basic and Clinical xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu

Review

Mucosal signaling in the bladder Toby C. Chai a,b,⁎, Andrea Russo b, Shan Yu a, Ming Lu a a b

Department of Urology, United States Department of Obstetrics, Gynecology and Reproductive Science, Yale School of Medicine, New Haven, CT, United States

a r t i c l e

i n f o

Article history: Received 19 July 2015 Accepted 27 August 2015 Available online xxxx Keywords: Bladder Mucosa Urothelium Lamina propria Sensory Efferent Venules and arterioles

a b s t r a c t The bladder mucosa is comprised of the multilayered urothelium, lamina propria (LP), microvasculature, and smooth muscle fibers (muscularis mucosae). The muscularis mucosae is not always present in the mucosa, and its presence is related to the thickness of the LP. Since there are no mucus secreting cells, “mucosa” is an imprecise term. Nerve fibers are present in the LP of the mucosa. Efferent nerves mediate mucosal contractions which can be elicited by electrical field stimulation (EFS) and various agonists. The source of mucosal contractility is unknown, but may arise from the muscularis mucosae or myofibroblasts. EFS also increases frequency of mucosal venule contractions. Thus, efferent neural activity has multiple effects on the mucosa. Afferent activity has been measured when the mucosa is stimulated by mechanical and stretch stimuli from the luminal side. Nerve fibers have been shown to penetrate into the urothelium, allowing urothelial cells to interact with nerves. Myofibroblasts are specialized cells within the LP that generate spontaneous electrical activity which then can modulate both afferent and efferent neural activities. Thus mucosal signaling is defined as interactions between bladder autonomic nerves with non-neuronal cells within the mucosa. Mucosal signaling is likely to be involved in clinical functional hypersensory bladder disorders (e.g. overactive bladder, urgency, urgency incontinence, bladder pain syndrome) in which mechanisms are poorly understood despite high prevalence of these conditions. Targeting aberrant mucosal signaling could represent a new approach in treating these disorders. © 2015 Published by Elsevier B.V.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Can the mucosa be further dissected with separation of the urothelium apart from the LP? 1.2. Innervation of mucosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Mucosal contractility (efferent activity) . . . . . . . . . . . . . . . . . . . . . . . 1.4. Urothelial involvement in mucosal signaling . . . . . . . . . . . . . . . . . . . . . 1.5. Suburothelial myofibroblast (interstitial cells) . . . . . . . . . . . . . . . . . . . . 1.6. Mucosal vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The human bladder's main purpose is to store and expel urine, efficiently and without distraction or bother to the person. Bladder function can be analogized as a neuromuscular reflex: during storage of urine, the bladder smooth muscle is quiescent, and as bladder fills to a certain volume of filling, ever increasing afferent signals ultimately trigger ⁎ Corresponding author at: Department of Obstetrics, Gynecology and Reproductive Science, Yale School of Medicine, New Haven, CT, United States. E-mail address: [email protected] (T.C. Chai).

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

0 0 0 0 0 0 0 0 0

efferent signals to induce smooth muscles to contract. Furthermore psychological factors including cognition, emotion, stress, mood and behavior also modulate bladder function, thus increasing the complexity of bladder control. The bladder wall is organized into these histologic compartments: urothelium, lamina propria (LP), muscularis mucosae (smooth muscle bundles within the LP) and serosa, muscularis propria (main smooth muscle layer deep to the LP). A histologic image of the full thickness of the human bladder wall with these compartments is shown (Fig. 1). The LP contains microvasculature (capillaries, venules and arterioles), specialized “pacemaker cells”, or myofibroblasts, and nerve fibers

http://dx.doi.org/10.1016/j.autneu.2015.08.009 1566-0702/© 2015 Published by Elsevier B.V.

Please cite this article as: Chai, T.C., et al., Mucosal signaling in the bladder, Auton. Neurosci. (2015), http://dx.doi.org/10.1016/ j.autneu.2015.08.009

2

T.C. Chai et al. / Autonomic Neuroscience: Basic and Clinical xxx (2015) xxx–xxx

cystitis/painful bladder syndrome, benign prostatic hypertrophy), the common theme in these conditions is augmentation of bladder afferent pathways (Clemens, 2010). A better understanding of mucosal afferent signaling could lead to new treatment modalities of hypersensory symptoms. Bladder emptying dysfunction has been coined “detrusor underactivity” (or underactive bladder). While this condition could be an efferent problem, it can also be lack of afferent input (Osman and Chapple, 2014) to induce a motor response. Though speculative, augmentation of afferent signaling could trigger an underactive bladder to contract. This method could allow bladder contraction to be synchronized with bladder outlet relaxation which is less likely to occur if treatments focused only on augmenting the bladder motor outflow. This review will discuss issues related to dissecting mucosa and results obtained from these dissections. Results from published data in mucosal afferent and mucosal efferent signaling will be reviewed. The overarching goal for this review is to present a contemporary framework to understand how mucosal signaling can modulate urinary storage and emptying functions. 1.1. Can the mucosa be further dissected with separation of the urothelium apart from the LP?

Fig. 1. Human bladder full thickness bladder, H&E staining, 40 × magnification. 1 = urothelium, 2 = lamina propria, 3 = muscularis mucosae, 4 = muscularis propria, A = separation artifact, 1 + 2 + 3 = mucosa.

(motor and sensory). Because of multiple specialized cells within the LP, some have proposed to analogize the LP as the “functional center” of the bladder (Andersson and McCloskey, 2014). The term “mucosa” refers to the tissue that is easily dissected off the muscularis propria. The mucosa contains urothelium, LP and muscularis mucosae (though smooth muscle is inconsistently present in the mucosa). Because there are no specialized mucous secreting cells in the mucosa, the term “mucosa” is inaccurate. But in order to be consistent with published literature, and to be able to denote to the compartments of urothelium, LP and muscularis mucosae unambiguously as one tissue, we maintain the use of the term “mucosa” throughout this review. Since the mucosa contains both sensory and motor nerves, the autonomic nervous system must play a role in mucosal function. However, what function can we ascribe to the mucosa? Traditionally, the working model for bladder mucosal function (and more specifically, urothelial function) is providing an impermeable layer to protect the bladder from urinary waste, toxins and microbes. However, there is a growing body of literature suggesting that the mucosa can modulate nonbarrier functions such as urinary storage and emptying, which requires nervous system input. In this review, we will define mucosal signaling as any interactions between autonomic nerves and other mucosal specialized cells (e.g. myofibroblasts, urothelial, endothelial, detrusor and vascular smooth muscle cells). Because of interactions between multiple cell types, mucosal signaling is complex, but this complexity offers increased or finer control of urinary storage and emptying functions that has been under appreciated. A common clinical problem of urinary storage dysfunction is characterized by hypersensory symptoms (urinary urgency, urgency incontinence, frequency, and nocturia) also called lower urinary tract symptoms (LUTS). While clinical “labels” have been used as diagnostic terms for patients with LUTS (e.g. overactive bladder, interstitial

The two major components of the mucosa are the urothelium and the LP. Ideally, one needs to be able to separate these compartments apart from each other to understand each compartment's unique contribution to overall mucosal signaling. However, is this possible? An example of confusion within the literature is the apparent interchangeable use of the terms “mucosa” and “urothelium” (Zagorodnyuk et al., 2007). In this paper, the investigators performed two different procedures: “removal of urothelium” and “mucosal stroking”. No histologic images were shown of the “urothelium” obtained during “removal of urothelium”. It is likely that the “urothelium” removed is the “mucosa”. The ability to remove a pure urothelium without the underlying LP has been demonstrated in mice (Lu and Chai, 2014), but it is uncertain whether pure urothelial tissue can be dissected off other species such as guinea pigs or pig. Photomicrographs obtained after muscle organ bath experiments of porcine mucosal strips showed the presence of smooth muscle, but interestingly, loss of urothelium (Sadananda et al., 2008). Pig mucosal strips dissected off the luminal surface of the bladder were histologically examined and shown in Fig. 2a and b (two separate strips). The pig mucosa strip contained urothelium, LP, and smooth muscle. A possible source for mucosal contractility is the presence of smooth muscle in the mucosal dissections. Because of the presence of muscularis mucosae in porcine mucosa, mucosal contractions could be due to the presence of smooth muscle. The mouse bladder wall is much thinner and the LP is much less prominent without evidence of muscularis mucosae (Fig. 3). A technique was recently described to dissect only the urothelium off the underlying LP in mice bladders (Lu and Chai, 2014). Fig. 4 shows the histology of a pure sheet of mouse urothelium dissected off the LP. Furthermore, investigators were able to patch clamp urothelial cells in situ from the dissected urothelial sheet, preserving the location of the cell within the stratified urothelium. This technique could be used to help clarify unique and separate functional roles between the urothelium and the underlying LP. 1.2. Innervation of mucosa In the classic paper by Gosling and Dixon (Gosling and Dixon, 1974), mucosa innervation was described using technology available at that time. It should be noted that these authors stated “In the present paper, attention had been focused on the subepithelial connective tissue, termed ‘submucosa’ for brevity, between the base of the epithelium and the inner aspect of the muscular coat.” Thus, ‘submucosa’ was synonymous to LP and their entire study focused on studying innervation

Please cite this article as: Chai, T.C., et al., Mucosal signaling in the bladder, Auton. Neurosci. (2015), http://dx.doi.org/10.1016/ j.autneu.2015.08.009

T.C. Chai et al. / Autonomic Neuroscience: Basic and Clinical xxx (2015) xxx–xxx

3

Fig. 3. Mouse bladder full thickness. 1 = mucosa, 2 = muscularis propria. Note that the mouse mucosa is much thinner and lacks smooth muscle.

the c-fibers that likely mediate mucosal signaling, especially in bladder disease. They are abundant in the suburothelium at the bladder neck and trigone, and the plexus becomes less prominent moving toward the dome (Birder et al., 2014; Andersson, 2002). c-Fibers respond to stretch at a higher threshold than Aδ fibers, and respond more readily to sensory input such as chemical irritants or cold exposure (Andersson, 2002). Bladder sensory afferents have also been divided into categories based upon their responses: mechanoreceptors (stretch-sensitive), chemoreceptors (stretch-insensitive) and silent afferents. Mechanoreceptors afferent fibers express mechano-gated channels (e.g. stretch activated channels such as DEG/ENaC and TRP superfamilies' of proteins). Chemoreceptors express receptors (e.g. P2X purinergic receptors) that respond to mediators released by non-neuronal cells (e.g. urothelial cells, suburothelial myofibroblasts) such as ATP, though these nonneuronal cells may also release mediators in response to mechanical (e.g. stretch) stimuli. Thus it is possible for chemoreceptive afferent fibers to be able to respond to stretch indirectly because of the ability of non-neuronal cells to release mediators in response to stretch. Using an in vitro guinea pig bladder preparation, afferent nerves close to the exterior of the bladder were studied electrophysiologically during various stimuli to the bladder including bladder stretch and mucosal stimulation with light von Frey's hair (Zagorodnyuk et al., 2006; Zagorodnyuk et al., 2007). The technique of “close-to target” afferent electrophysiologic recording was used. This technique involved removal of the bladder from the guinea pig, splitting and pinning the bladder open with

Fig. 2. Two different porcine mucosal strips showing inconsistent presence of smooth muscle.

within the LP of the mucosa. They concluded that there were two types of nerves in the mucosa: noradrenergic nerves innervating blood vessels (vascular smooth muscles) and acetylcholinesterase nerves functioning as sensory nerves. Bladder afferent fibers can be further categorized into unmyelinated c-fibers and myelinated Aδ fibers. The Aδ fibers form a myogenic afferent signaling pathway and reside primarily within the muscularis propria to respond to stretch of this muscle during filling (Kanai and Andersson, 2010; Yoshimura, 2007; Andersson, 2002). However, it is

Fig. 4. H&E staining of pure mouse urothelium without LP.

Please cite this article as: Chai, T.C., et al., Mucosal signaling in the bladder, Auton. Neurosci. (2015), http://dx.doi.org/10.1016/ j.autneu.2015.08.009

4

T.C. Chai et al. / Autonomic Neuroscience: Basic and Clinical xxx (2015) xxx–xxx

urothelium face up, and then dissecting out and recording from “axons in fine nerve trunks entering the bladder trigone”. Different stimuli were applied to the bladder including: bladder stretch (which stretched the entire thickness of the bladder, including muscularis propria and mucosa), urothelial exposure to hypertonic saline, urothelial exposure to chemical mediators (α,β-methylene-ATP or capsaicin), and stroking of urothelium with light von Frey hairs. Four different categories of afferent mechanoreceptor activities were detected: “muscle”, “musclemucosal”, “mucosal high-responding” and “mucosal low responding”. These categories implied that the sensory fibers terminated at different levels of the bladder wall from the muscle to the mucosa. Therefore, mucosal signaling was mediated by two types of mucosal afferent fibers, high- and low-responding. The high-responding fibers were stretchinsensitive, but responded to urothelial stroking, hypertonic saline and chemical mediators. Removal of “urothelium” (probably mucosa, rather than just the urothelium) reduced stroke-induced firing of these afferents. The low-responding fibers were stretch-insensitive, lacked response to hypertonic saline and chemical mediators, but weakly responded to urothelial stroking. The percentages of afferent fibers measured that were in each category were: 15% “muscle”, 39% “muscle-mucosal”, 12% “mucosal high-responding” and 28% “mucosal low responding”. These investigators further studied the physiologic mechanisms underlying mucosal signaling in “muscle-mucosal” and “mucosal high-responding” mechanoreceptors (Zagorodnyuk et al., 2009). It was found that activities in these two types of afferents do not require calcium-dependent exocytosis or endogenous ATP. Other investigators, using an ex vivo dissected mice preparation of bladder and pelvic nerves, studied bladder afferent signaling by instilling purinergic agonists and antagonists intravesically and measuring afferent nerve activity (Rong et al., 2002). These investigators concluded that purinergic signaling plays an important role in bladder afferent mechanisms. Presumably, the intravesical purinergic agents acted primarily on the mucosa as the ability for ATP to penetrate beneath the mucosa and to interact with bladder compartment deeper than the mucosa is unlikely. Using antibodies to putative neuronal markers, immunohistology and immunohistofluorescence have been used to study the neuroanatomy/neuropharmacology of mucosal innervation. A list of a variety of markers, the corresponding references, and the type of nerve fibers identified in the mucosa are listed in Table 1. However, newer techniques to anatomically localize nerve fibers that do not require antibodies have been developed. For example, neuronal reporter mice can allow investigators to localize and visualize nerve fibers in any tissue. The sodium voltage gated channel 1.8 (NaV 1.8) is a specific marker for sensory afferents that detect mechanical, cold and inflammatory pain, but not neuropathic pain or heat sensing (Abrahamsen et al., 2008). NaV1.8 is commonly found on afferent c-fibers (Amaya et al., 2000). Nav1.8 was shown to be important in a rat model of visceral pain (Yoshimura et al., 2001). More recently, a transgenic mouse with

fluorescent protein tdTomato (red fluorescence) tagged to NaV1.8 was created (Shields et al., 2012). This study found that Nav1.8 was also expressed in non-nociceptive peripheral nerves. An immunofluorescent image from the bladder of a tdTomato-NaV1.8 mouse is shown in Fig. 5. The Nav1.8 nerve fibers course within the LP and also are found within the urothelium (pink arrows). There is a dense plexus of NaV1.8 nerve fibers within the LP of the mucosa and these nerve fibers play a role in mucosal signaling mediating bladder sensory function. Some of the sensory fibers penetrate the urothelium.

1.3. Mucosal contractility (efferent activity) There are two ways to consider how the mucosa affects efferent motor function of the bladder: 1. The mucosa modulates underlying detrusor smooth muscle (muscularis propria) function and 2. The mucosa contracts independently from muscularis propria. The effect of mucosa modulating contractility of the guinea pig bladder smooth muscle was reported almost 20 years ago (Maggi et al., 1987). This study compared contractility of bladder strips, with and without mucosa, to substance P, EFS, histamine and KCl. These investigators found that contractility to substance P of bladder strips without mucosa was greater than full thickness bladder strips (with mucosa). However, there were no differences in responses to EFS, histamine or KCl between strips with and without mucosa. It was hypothesized that the mucosa provided barrier function and prevented substance P from accessing the muscularis propria. Other investigators examined bradykinin induced contractions of rat bladder strips with and without mucosa (termed “epithelium” in the paper) (Pinna et al., 1992). These investigators found that the presence of mucosa did not make a difference in non-diabetic animals. However, in diabetic animals, removal of the mucosa decreased bladder contractility to bradykinin, suggesting that diabetes affected mucosaldetrusor smooth muscle interactions. In another study, investigators found that the feline mucosa inhibited the detrusor smooth muscle contractions (Levin et al., 1995). The “urothelium” of pig bladders was found to also relax smooth muscle (Hawthorn et al., 2000), although the use of the term “urothelium” in this paper is referring to the mucosa. So while the relaxing factor was hypothesized to come from the “urothelium”, it could be any of the other cell types within the mucosa. The contractile responses of the mucosa itself to various stimuli have been explored. The first study to describe mucosal contractions found that (Sadananda et al., 2008) mucosa contracted when stimulated by neurokinin A (NKA) and carbachol. These investigators also stated that these contractions were not due to detrusor smooth muscle present within the mucosa as “removal of smooth muscle remnants from mucosal strips did not alter the responses to NKA”. These authors theorized that mucosal contractions were due to suburothelial

Table 1 Summary of immunohistochemical studies of bladder mucosa. Neuronal marker

Presumed type of nerve fibers

Associations within mucosa

PGP9.5 (protein gene product 9.5) Tyrosine hydroxylase Neurofilament Choline acetyltransferase (ChAT) CGRP (calcitonin gene related peptide) SP (substance P) TRPV1

Non-specific Sympathetic Non-specific Parasympathetic Afferent Afferent Afferent

P2X3 (ATP receptor)

Afferent

Venules Ganglia Close proximity to CGRP expressing nerves, Arteriole Cose proximity to ChAT expressing nerves, Venules Venules Urothelial cells, myofibroblasts, Co-localizes with P2X3, CB-1 Co-localization with CB-1, TRPV1

NK-1 (tachykinin receptor) CB-1 (cannabinoid receptor 1) NADPH diaphorase

Afferent Afferent Nitrergic

Venules Co-localization with TRPV1, P2X3 Blood vessels, “close to the epithelium”

References Brady et al. (2004a), Andersson (2002) Mitsui and Hashitani (2013), Shimizu et al. (2014) Ost et al. (2002), Gillespie et al. (2006) Gillespie et al. (2006), Mitsui and Hashitani (2013) Gillespie et al. (2006), Shimizu et al. (2014) Shimizu et al. (2014) Birder et al. (2001), Ost et al. (2002), Brady et al. (2004b), Apostolidis et al. (2005), Gillespie et al. (2006) Brady et al. (2004b), Apostolidis et al., (2005), Walczak et al. (2009) Sugasi et al. (2000) Walczak et al. (2009), Walczak and Cervero (2011) Keast and Kawatani (1994)

Please cite this article as: Chai, T.C., et al., Mucosal signaling in the bladder, Auton. Neurosci. (2015), http://dx.doi.org/10.1016/ j.autneu.2015.08.009

T.C. Chai et al. / Autonomic Neuroscience: Basic and Clinical xxx (2015) xxx–xxx

Fig. 5. Immunofluorescence of full thickness bladder of tdTomato-NaV1.8 mouse. NaV1.8 expression is in red and represent sensory nerve fibers which are present in mucosa (LP) and muscularis propria. Note that sensory fibers can penetrate into urothelium (pink arrows). U = urothelium, LP = lamina propria. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

myofibroblasts (see section below) and that these contractions are necessary for matching luminal surface area to bladder volume. More recently, the roles of EFS, muscarinic agonists, adrenergic agonists, KCl, non-adrenergic/non-adrenergic/non-purinergic agent in mediating contractions of the isolated pig bladder mucosa were described (Moro et al., 2013; Moro and Chess-Williams, 2012a; Moro et al., 2012b; Moro et al., 2011). These investigators used the term urothelium/lamina propria rather than the word “mucosa”, though these two terms would be synonymous. These series of studies of pig mucosa did not control for the possibility of detrusor smooth muscle within the LP. As shown in Fig. 2a and b, there is variable presence of smooth muscle within the LP which could contribute to mucosal contractions. Future studies in mucosal efferent function should account for the presence/content of smooth muscle within the mucosal preparations. 1.4. Urothelial involvement in mucosal signaling The urothelium of the mucosa is a multilayered epithelium comprised of apical (umbrella), intermediate and basal urothelial cells. The apical urothelial cells contact the urine and the basal urothelial cells contact the basement membrane adjacent to the LP. The urothelium is highly impermeable, allowing the mucosa to resist absorption/infiltration of urinary substances (solutes, toxins, bacteria, etc.). The impermeability function is thought to be the primary function for the urothelium, and is mediated by expression of tight junction proteins and uroplakins by apical urothelial cells (Acharya et al., 2004; Hu et al., 2002). Thus, apical urothelial cells are the prime contributors to urothelial impermeability function. In the current paradigm of urothelial cell differentiation, basal urothelial cells, which neither express tight junctions nor uroplakins, terminally differentiate into apical urothelial cells. However, in addition to impermeability function, there is a growing body of literature to show that urothelial cells also play an important role in mucosal signaling. Bladder urothelial cells have been shown to have “sensor-transducer” function in which they can release and respond to different molecules (Birder and Andersson, 2013). Urothelial cells can be active in sensing chemical, mechanical, and thermal stimuli, thus generating signals in response to these stimuli. Urothelial cells release a number of signaling

5

molecules such as ATP, acetylcholine, substance P, nerve growth factor, nitric oxide, antiproliferative factor, and cytokines (Sui et al., 2014; Birder et al., 2010; de Groat, 2013; Apodaca et al., 2007; Birder and de Groat, 2007; Yoshimura, 2007). Urothelial cells also express multiple different classes of transducer receptors including: cholinergic (nicotinic and muscarinic), adrenergic (α and β), transient receptor potential (TRPV1, TRPV2, TRPV4, TRPM8), purinergic (P2X, P2Y), adenosine (A2a, A2b, and A3), and estrogen (ERα, ERβ) receptors. (de Groat, 2013; de Groat and Yoshimura, 2009; Birder et al., 2010; Birder and de Groat, 2007; Birder et al., 2002). Mechanosensitive proteins expressed by urothelial cells include amiloride sensitive ion channels (Du et al., 2007; Olsen et al., 2011) and the new discovery of Piezo1 (Miyamoto et al., 2014). These mechanosensitive receptors respond to stretch with increased intracellular calcium with subsequent release of ATP. The ATP released by urothelial cells can signal other urothelial cells (autocrine function, Sun and Chai, 2006), or interact with other cells in LP such as myofibroblasts or nerve fibers (de Groat and Yoshimura, 2009). Much of the published data on urothelial cellular function have come from cell culture experiments. The use of cell cultures provides a method to study urothelial cellular function in vitro. Urothelial cells have been successfully cultured from the bladders of different species including rat, mouse, cat, guinea pig, pig and human. Primary urothelial cell cultures maintain epithelial phenotype and express cytokeratins and tight junction proteins (Smith et al., 2015). However, there are issues regarding using cultured monolayer of cells for functional studies. Cultured urothelial cells may lose phenotypic uniqueness inherent in a multilayered urothelium such as basal cells, intermediate cells and apical cells having different functions. Investigators have been able to get urothelial cell cultures to grow in a stratified manner, though stratification did not correspond with differentiation/maturation of urothelial cells (Southgate et al., 1994; Sugasi et al., 2000). A new technique, not involving cell cultures, was developed to isolate a pure sheet of bladder urothelium devoid of all underlying LP (Lu and Chai, 2014). The dissected urothelial sheet was used to perform single urothelial cell patch-clamp electrophysiology in situ within the tissue allowing denotation of whether the cells patched were apical, intermediate or basal in location within the urothelium. Using this technique, phenotypic differences in potassium membrane conductivities were measureable between apical versus intermediate/basal cells. The importance of urothelium in modulating bladder function has been studied in transgenic mice with nerve growth factor (NGF) overexpression restricted to the urothelium. Investigators leveraged the uroplakin II promoter sequence to limit NGF overexpression only to the urothelium (Schnegelsberg et al., 2010). These animals were found to have marked expansion of nerve fiber tissue in the suburothelium (essentially the LP). Their voiding behavior was overactive voiding with frequent small volume voids. They also had evidence of somatic pelvic hypersensitivity on von Frey filament testing. This transgenic animal showed urothelial upregulation of NGF that led to mucosal abnormalities and ultimately, to altered bladder function. The urothelium is the most studied component within the mucosa. While the urothelium's main purpose is forming the impermeability layer for the mucosa, the additional abilities of the urothelium in modulating mucosal signaling with interactions with mucosal nerves, myofibroblasts and blood vessels are likely to be important. The relative ease of accessing the urothelium in a patient population makes the urothelium an attractive target for bladder sensory conditions. 1.5. Suburothelial myofibroblast (interstitial cells) The term “myofibroblast” was first used to describe fibroblasts with structural and functional features of smooth muscle that developed during wound healing (Gabbiani et al., 1971; Ryan et al., 1974). During an immunohistologic study of bladder nerves using cGMP as a marker, investigators described a type of cell “present in large numbers

Please cite this article as: Chai, T.C., et al., Mucosal signaling in the bladder, Auton. Neurosci. (2015), http://dx.doi.org/10.1016/ j.autneu.2015.08.009

6

T.C. Chai et al. / Autonomic Neuroscience: Basic and Clinical xxx (2015) xxx–xxx

throughout the muscle bundles and within the interstitium of the bladder, but were particularly concentrated in the outer fibromuscular coat [sic muscularis propria], where the processes formed a dense interconnecting network” (Smet et al., 1996). These cells were described as having appearance of “myofibroblasts” and were reminiscent of “interstitial cells of Cajal” described in smooth muscle of the gastrointestinal tract (Thuneberg et al., 1982). However, these “myofibroblasts” were most predominant in the muscularis propria (and therefore probably these cells should be denoted as muscularis propria myofibroblasts to differentiate from myofibroblasts found in the LP — see below). There is another type of cell localized to the LP, just beneath the urothelial basal cell, that has also been called “myofibroblast” (and should be denoted as suburothelial myofibroblasts or suburothelial interstitial cells). These suburothelial myofibroblasts were first described in human bladders by (Sui et al., 2002). These myofibroblasts contact each other and express connexin-43, which is a gap junction protein allowing for ion passage between cells, thus electrically coupling the cells. Detailed single cell electrophysiologic studies on guinea pig bladder suburothelial myofibroblasts were performed (Wu et al., 2004). After mucosal cells were dissociated, suburothelial myofibroblasts were identified based on microscopic morphology without the need for special staining. This technique was reported thusly: “urothelium was separated from underlying detrusor layer by blunt dissection”. After this, the “urothelium” was digested by collagenase and the investigators could separate out urothelial cells (“large round”) from suburothelial myofibroblasts (“ovoid or spindle-shaped cells”) under light microscopy. While the histology of the dissected “urothelium” was not shown, it is probable that the “urothelium” necessarily included LP cells, and thus probably should be more accurately denoted as “mucosa” rather than “urothelium”. In another publication studying the human bladder mucosa, investigators found two types of interstitial cells, one type in the “upper” LP (ULP, closer to the urothelium) and another type in the “deep” LP (DLP, closer to the muscularis propria) (Gevaert et al., 2014). The ULP interstitial cells expressed vimentin, α-smooth muscle actin, caveolin1 and 2, PDGFRα, phosphorylated and non-phosphorylated connexin 43, but did not express CD34 and c-kit. The DLP interstitial cells expressed vimentin, CD34 and non-phosphorylated connexin 43, but did not express α-smooth actin, caveolin-1 and 2, PDGFRα, phosphorylated connexin 43 and c-kit. Electron microscopy revealed that ULP interstitial cells were fibroblasts with myoid features and sparse myofibroblasts while DLP interstitial cells were interstitial cell of Cajal-like cells. However, the functional differences between the ULP versus DLP interstitial cells remain unknown as they were not studied in this publication. The location of the myofibroblasts within the mucosa theoretically allows bidirectional communication with both urothelium and afferent nerves, thus making some to speculate that myofibroblasts represent an important mediator of bladder afferent function. It is thought that suburothelial myofibroblasts act as an amplifier of the sensory response to mucosal stretch during bladder filling (Fry et al., 2007; Wang et al., 2010). Other functional studies such as calcium microfluorimetry and patch clamp electrophysiology have been performed on suburothelial myofibroblasts. However, the actual interactions between myofibroblasts-urothelial cells, myofibroblasts-neurons, or myofibroblasts-smooth muscle cell have yet to be demonstrated. Augmentation of the spontaneous electrical activity of the suburothelial myofibroblasts has been hypothesized to underlie the etiologic mechanism of the clinical syndrome of idiopathic overactive bladder.

extensive vascular network beneath the transparent urothelium (Fig. 6). Anatomic study of the mouse bladder blood vessels has been performed using vascular corrosion casting technique (Hossler et al., 2013). There is a rich plexus of capillaries, supplied by arterioles and drained by venules, within the LP. The capillaries have been shown to physically contact the basal urothelial cells with scanning electron microscopy. It is likely that the blood vessels seen cystoscopically are either venules or arterioles as capillaries are microscopic. The arterioles and venules are highly coiled and the capillaries have high degree of interconnection with other capillaries. Furthermore, sphincteric structures were visualized on the arterioles suggesting that mucosal blood inflow is neurally controlled. This organization allows the mucosa to receive adequate blood flow during stretch and contraction which occur during bladder filling and emptying. The mouse mucosal vascular microanatomy is similar to what has been seen in human, dog and rabbit (Hossler and Monson, 1995; Hossler, 1997; Hossler and Kao, 2007). Immunohistochemical studies revealed varicose nerve bundles immunoreactive for tyrosine hydroxylase (sympathetic nerves), choline acetyltransferase (cholinergic nerves) or substance P (primary afferent nerves) alongside both suburothelial arterioles and venules (Mitsui and Hashitani, 2013). Mucosal venule responses to neural signaling have been studied in rat mucosa (Shimizu et al., 2014). Investigators dissected mucosa away from detrusor, then the urothelium was removed from mucosa exposing the suburothelial vessels (though no histology of the “urothelium” removed was shown). The contractility (as visualized microscopically) of these venules was studied in response to EFS and various agonists/antagonists. These investigators concluded that the venules responded to sympathetic neural activity with constriction and increased frequency of contractions. These venule activities were blocked with α-blockers and β3-agonists, whereas acetylcholine increased venule contractile activities. What is interesting is that agents used to treat overactive bladder such as α-blockers, β3-agonists and anticholinergics all correspond to diminishing venule activities (decreased constriction and decreased frequency of contractions). It was theorized that these agents, which are typically thought to work at the bladder smooth muscle, might interact with mucosal vascularity leading to changes in mucosal signaling. Studies addressing bladder ischemia have been conducted by common iliac arterial intimal injury coupled with high fat diet (Azadzoi et al., 1999). However, this model involves obstruction of proximal blood inflow, and not a primary alteration of mucosal vascularity (arterioles, venules and capillaries). It is unknown how a proximal arterial

1.6. Mucosal vasculature A potentially important, but understudied component of the mucosa, is the vasculature. When cystoscopy is performed in clinics, what is interesting is that the most prominent feature visualized is the

Fig. 6. Image from cystoscopy of human bladder showing network of blood vessels in the lamina propria. The human urothelium, despite being multilayered, is translucent.

Please cite this article as: Chai, T.C., et al., Mucosal signaling in the bladder, Auton. Neurosci. (2015), http://dx.doi.org/10.1016/ j.autneu.2015.08.009

T.C. Chai et al. / Autonomic Neuroscience: Basic and Clinical xxx (2015) xxx–xxx

obstruction in the common iliac artery might affect downstream vascular changes in the mucosa. Studies of mucosal vascular function have been recently initiated. Because the autonomic nerves within the LP of the mucosa regulate mucosal vascular function, mucosal signaling involves not just afferent and efferent functions, but also blood flow modulation. 2. Conclusions The mucosa acts as a sentinel compartment of the bladder relaying sensory information to higher cortical centers. Within the mucosa, there are numerous unique cells including urothelial cells, suburothelial myofibroblasts, afferent and efferent nerves, endothelial cells (venules, arterioles, capillaries), and smooth muscle cells. Each of these cells has specialized functions, and because sensory nerves can interact with these other cells in the mucosa, there is complexity to mucosal signaling. Many of the common clinical bladder disorders are defined by augmented bladder sensation. Therefore, the ability to translate mucosal signaling abnormalities to these disorders would represent a significant advance in the physiologic understanding of bladder function. References Abrahamsen, B., Zhao, J., Asante, C.O., Cendan, C.M., Marsh, S., Martinez-Barbera, J.P., Nassar, M.A., Dickenson, A.H., Wood, J.N., 2008. The cell and molecular basis of mechanical, cold, and inflammatory pain. Science 321, 702–705. Acharya, P., Beckel, J., Ruiz, W.G., Wang, E., Rojas, R., Birder, L., Apodaca, G., 2004. Distribution of the tight junction proteins ZO-1, occludin, and claudin-4, −8, and −12 in bladder epithelium. Am. J. Physiol. Ren. Physiol. 287, F305–F318. Amaya, F., Decosterd, I., Samad, T.A., Plumpton, C., Tate, S., Mannion, R.J., Costigan, M., Woolf, C.J., 2000. Diversity of expression of the sensory neuron-specific TTXresistant voltage-gated sodium ion channels SNS and SNS2. Mol. Cell. Neurosci. 15, 331–342. Andersson, K.E., 2002. Bladder activation: afferent mechanisms. Urology 59 (5 Suppl. 1), 43–50. Andersson, K.E., McCloskey, K.D., 2014. Lamina propria: the functional center of the bladder? Neurourol. Urodyn. 33, 9–16. Apodaca, G., Balestreire, E., Birder, L.A., 2007. The uroepithelial-associated sensory web. Kidney Int. 72, 1057–1064. Apostolidis, A., Popat, R., Yiangou, Y., Cockayne, D., Ford, A.P., Davis, J.B., Dasgupta, P., Fowler, C.J., Anand, P., 2005. Decreased sensory receptors P2X3 and TRPV1 in suburothelial nerve fibers following intradetrusor injections of botulinum toxin for human detrusor overactivity. J. Urol. 174, 977–982. Azadzoi, K.M., Tarcan, T., Siroky, M.B., Krane, R.J., 1999. Atherosclerosis-induced chronic ischemia causes bladder fibrosis and non-compliance in the rabbit. J. Urol. 161, 1626–1635. Birder, L., Andersson, K.E., 2013. Urothelial signaling. Physiol. Rev. 93, 653–680. Birder, L.A., de Groat, W.C., 2007. Mechanisms of disease: involvement of the urothelium in bladder dysfunction. Nat. Clin. Pract. Urol. 4, 46–54. Birder, L.A., Kanai, A.J., de Groat, W.C., Kiss, S., Nealen, M.L., Burke, N.E., Dineley, K.E., Watkins, S., Reynolds, I.J., Caterina, M.J., 2001. Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc. Natl. Acad. Sci. U. S. A. 98, 13396–13401. Birder, L.A., Nealen, M.L., Kiss, S., de Groat, W.C., Caterina, M.J., Wang, E., Apodaca, G., Kanai, A.J., 2002. Beta-adrenoceptor agonists stimulate endothelial nitric oxide synthase in rat urinary bladder urothelial cells. J. Neurosci. 22, 8063–8070. Birder, L.A., Wolf-Johnston, A.S., Chib, M.K., Buffington, C.A., Roppolo, J.R., Hanna-Mitchell, A.T., 2010. Beyond neurons: involvement of urothelial and glial cells in bladder function. Neurourol. Urodyn. 29, 88–96. Birder, L.A., Andersson, K.E., Kanai, A.J., Hanna-Mitchell, A.T., Fry, C.H., 2014. Urothelial mucosal signaling and the overactive bladder-ICI-RS 2013. Neurourol. Urodyn. 33, 597–601. Brady, C.M., Apostolidis, A.N., Harper, M., Yiangou, Y., Beckett, A., Jacques, T.S., Freeman, A., Scaravilli, F., Fowler, C.J., Anand, P., 2004a. Parallel changes in bladder suburothelial vanilloid receptor TRPV1 and pan-neuronal marker PGP9.5 immunoreactivity in patients with neurogenic detrusor overactivity after intravesical resiniferatoxin treatment. BJ. Int. 93, 770–776. Brady, C.M., Apostolidis, A., Yiangou, Y., Baecker, P.A., Ford, A.P., Freeman, A., Jacques, T.S., Fowler, C.J., Anand, P., 2004b. P2X3-immunoreactive nerve fibres in neurogenic detrusor overactivity and the effect of intravesical resiniferatoxin. Eur. Urol. 46, 247–253. Clemens, J.Q., 2010. Afferent neurourology: an epidemiological perspective. J. Urol. 184, 432–439. de Groat, W.C., 2013. Highlights in basic autonomic neuroscience: contribution of the urothelium to sensory mechanisms in the urinary bladder. Auton. Neurosci. 177, 67–71. de Groat, W.C., Yoshimura, N., 2009. Afferent nerve regulation of bladder function in health and disease. Handb. Exp. Pharmacol. 194, 91–138.

7

Du, S., Araki, I., Mikami, Y., Zakoji, H., Beppu, M., Yoshiyama, M., Takeda, M., 2007. Amiloride-sensitive ion channels in urinary bladder epithelium involved in mechanosensory transduction by modulating stretch-evoked adenosine triphosphate release. Urology 69, 590–595. Fry, C.H., Sui, G.P., Kanai, A.J., Wu, C., 2007. The function of suburothelial myofibroblasts in the bladder. Neurourol. Urodyn. 26 (6 Suppl.), 914–919. Gabbiani, G., Ryan, G.B., Majne, G., 1971. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 27, 549–550. Gevaert, T., Vanstreels, E., Daelemans, D., Franken, J., Van Der Aa, F., Roskams, T., De Ridder, D., 2014. Identification of different phenotypes of interstitial cells in the upper and deep lamina propria of the human bladder dome. J. Urol. 192, 1555–1563. Gillespie, J.I., Markerink-van Ittersum, M., de Vente, J., 2006. Sensory collaterals, intramural ganglia and motor nerves in the guinea-pig bladder: evidence for intramural neural circuits. Cell Tissue Res. 325, 33–45. Gosling, J.A., Dixon, J.S., 1974. Sensory nerves in the mammalian urinary tract. An evaluation using light and electron microscopy. J. Anat. 117, 133–144. Hawthorn, M.H., Chapple, C.R., Cock, M., Chess-Williams, R., 2000. Urothelium-derived inhibitory factor(s) influences on detrusor muscle contractility in vitro. Br. J. Pharmacol. 129, 416–419. Hossler, F., 1997. Unusual features of the microvasculature of the urinary bladder revealed by vascular corrosion casting. In: Motta, P.M. (Ed.), Recent Advances in Microscopy of Cells, Tissues and Organs. Antonio Delfino Editore, Rome, pp. 501–506. Hossler, F., Kao, R., 2007. Microvasculature of the urinary bladder of the dog: a study using vascular corrosion casting. Microsc. Microanal. 13, 220–227. Hossler, F.E., Monson, F.C., 1995. Microvasculature of the rabbit urinary bladder. Anat. Rec. 243, 438–448. Hossler, F.E., Lametschwandtner, A., Kao, R., Finsterbusch, F., 2013. Microvascular architecture of mouse urinary bladder described with vascular corrosion casting, light microscopy, SEM, and TEM. Microsc. Microanal. 19, 1428–1435. Hu, P., Meyers, S., Liang, F.X., Deng, F.M., Kachar, B., Zeidel, M.L., Sun, T.T., 2002. Role of membrane proteins in permeability barrier function: uroplakin ablation elevates urothelial permeability. Am. J. Physiol. Ren. Physiol. 283, F1200–F1207. Kanai, A., Andersson, K.E., 2010. Bladder afferent signaling: recent findings. J. Urol. 183, 1288–1295. Keast, J.R., Kawatani, M., 1994. Extensive distribution of NADPH diaphorase activity in the nerve supply of the cat lower urinary tract. J. Auton. Nerv. Syst. 50, 161–169. Levin, R.M., Wein, A.J., Krasnopolsky, L., Atta, M.A., Ghoniem, G.M., 1995. Effect of mucosal removal on the response of the feline bladder to pharmacological stimulation. J. Urol. 153, 1291–1294. Lu, M., Chai, T.C., 2014. A method to study bladder urothelial cellular function in situ. Bladder 1 (1), e3 (open access at www.bladderj.org). Maggi, C.A., Santicioli, P., Parlani, M., Astolfi, M., Patacchini, R., Meli, A., 1987. The presence of mucosa reduces the contractile response of the guinea-pig urinary bladder to substance P. J. Pharm. Pharmacol. 39, 653–655. Mitsui, R., Hashitani, H., 2013. Immunohistochemical characteristics of suburothelial microvasculature in the mouse bladder. Histochem. Cell Biol. 140, 189–200. Miyamoto, T., Mochizuki, T., Nakagomi, H., Kira, S., Watanabe, M., Takayama, Y., Suzuki, Y., Koizumi, S., Takeda, M., Tominaga, M., 2014. Functional role for Piezo1 in stretchevoked Ca2 ⁺ influx and ATP release in urothelial cell cultures. J. Biol. Chem. 289, 16565–16575. Moro, C., Chess-Williams, R., 2012a. Non-adrenergic, non-cholinergic, non-purinergic contractions of the urothelium/lamina propria of the pig bladder. Auton. Autocoid Pharmacol. 32, 53–59. Moro, C., Uchiyama, J., Chess-Williams, R., 2011. Urothelial/lamina propria spontaneous activity and the role of M3 muscarinic receptors in mediating rate responses to stretch and carbachol. Urology 78 (1442), e9–15. Moro, C., Leeds, C., Chess-williams, R., 2012b. Contractile activity of the bladder urothelium/lamina propria and its regulation by nitric oxide. Eur. J. Pharmacol. 674, 445–449. Moro, C., Tajouri, L., Chess-Williams, R., 2013. Adrenoceptor function and expression in bladder urothelium and lamina propria. Urology 81 (211), e1–e7. Olsen, S.M., Stover, J.D., Nagatomi, J., 2011. Examining the role of mechanosensitive ion channels in pressure mechanotransduction in rat bladder urothelial cells. Ann. Biomed. Eng. 39, 688–697. Osman, N.I., Chapple, C.R., 2014. Contemporary concepts in the aetiopathogenesis of detrusor underactivity. Nat. Rev. Urol. 11, 639–648. Ost, D., Roskams, T., Van Der Aa, F., De Ridder, D., 2002. Topography of the vanilloid receptor in the human bladder: more than just the nerve fibers. J. Urol. 168, 293–297. Pinna, C., Caratozzolo, O., Puglisi, L., 1992. A possible role for urinary bladder epithelium in bradykinin-induced contraction in diabetic rats. Eur. J. Pharmacol. 214, 143–148. Rong, W., Spyer, K.M., Burnstock, G., 2002. Activation and sensitisation of low and high threshold afferent fibres mediated by P2X receptors in the mouse urinary bladder. J. Physiol. 541, 591–600. Ryan, G.B., Cliff, W.J., Gabbiani, G., Irlé, C., Montandon, D., Statkov, P.R., Majno, G., 1974. Myofibroblasts in human granulation tissue. Hum. Pathol. 5, 55–67. Sadananda, P., Chess-Williams, R., Burcher, E., 2008. Contractile properties of the pig bladder mucosa in response to neurokinin A: a role for myofibroblasts? Br. J. Pharmacol. 153, 1465–14673. Schnegelsberg, B., Sun, T.T., Cain, G., Bhattacharya, A., Nunn, P.A., Ford, A.P., Vizzard, M.A., Cockayne, D.A., 2010. Overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity, and changes in urinary bladder function. Am. J. Physiol. Regul. Integr. Comp. Physiol. 298, R534–R547. Shields, S.D., Ahn, H.S., Yang, Y., Han, C., Seal, R.P., Wood, J.N., Waxman, S.G., Dib-Hajj, S.D., 2012. Nav1.8 expression is not restricted to nociceptors in mouse peripheral nervous system. Pain 153, 2017–2030.

Please cite this article as: Chai, T.C., et al., Mucosal signaling in the bladder, Auton. Neurosci. (2015), http://dx.doi.org/10.1016/ j.autneu.2015.08.009

8

T.C. Chai et al. / Autonomic Neuroscience: Basic and Clinical xxx (2015) xxx–xxx

Shimizu, Y., Mochizuki, S., Mitsui, R., Hashitani, H., 2014. Neurohumoral regulation of spontaneous constrictions in suburothelial venules of the rat urinary bladder. Vasc. Pharmacol. 60, 84–94. Smet, P.J., Jonavicius, J., Marshall, V.R., de Vente, J., 1996. 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. Neuroscience 71, 337–348. Smith, N.J., Hinley, J., Varley, C.L., Eardley, I., Trejdosiewicz, L.K., Southgate, J., 2015. The human urothelial tight junction: claudin 3 and the ZO-1α + switch. Bladder 2, e9 (open access www.bladderj.org). Southgate, J., Hutton, K.A., Thomas, D.F., Trejdosiewicz, L.K., 1994. Normal human urothelial cells in vitro: proliferation and induction of stratification. Lab. Investig. 71, 583–594. Sugasi, S., Lesbros, Y., Bisson, I., Zhang, Y.Y., Kucera, P., Frey, P., 2000. In vitro engineering of human stratified urothelium: analysis of its morphology and function. J. Urol. 164, 951–957. Sui, G.P., Rothery, S., Dupont, E., Fry, C.H., Severs, N.J., 2002. Gap junctions and connexin expression in human suburothelial interstitial cells. BJ. Int. 90, 118–129. Sui, G., Fry, C.H., Montgomery, B., Roberts, M., Wu, R., Wu, C., 2014. Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions. Am. J. Physiol. Ren. Physiol. 306, F286–F298. Sun, Y., Chai, T.C., 2006. Augmented extracellular ATP signaling in bladder urothelial cells from patients with interstitial cystitis. Am. J. Physiol. Cell Physiol. 290, C27–C34. Thuneberg, L., Rumessen, J.J., Mikkelsen, H.B., 1982. Interstitial cells of Cajal — an intestinal impulse generation and conduction system? Scand. J. Gastroenterol. Suppl. 71, 143–144.

Walczak, J.S., Cervero, F., 2011. Local activation of cannabinoid CB₁ receptors in the urinary bladder reduces the inflammation-induced sensitization of bladder afferents. Mol. Pain 9 (7), 31. Walczak, J.S., Price, T.J., Cervero, F., 2009. Cannabinoid CB1 receptors are expressed in the mouse urinary bladder and their activation modulates afferent bladder activity. Neuroscience 159, 1154–1163. Wang, Y., Fang, Q., Lu, Y., Song, B., Li, W., Li, L., 2010. Effects of mechanical stretch on interstitial cells of Cajal in guinea pig bladder. J. Surg. Res. 164, e213–e219. Wu, C., Sui, G.P., Fry, C.H., 2004. Purinergic regulation of guinea pig suburothelial myofibroblasts. J. Physiol. 559, 231–243. Yoshimura, N., 2007. Lower urinary tract symptoms (LUTS) and bladder afferent activity. Neurourol. Urodyn. 26 (6 Suppl), 908–913. Yoshimura, N., Seki, S., Novakovic, S.D., Tzoumaka, E., Erickson, V.L., Erickson, K.A., Chancellor, M.B., de Groat, W.C., 2001. The involvement of the tetrodotoxinresistant sodium channel Na(v)1.8 (PN3/SNS) in a rat model of visceral pain. J. Neurosci. 21, 8690–8696. Zagorodnyuk, V.P., Costa, M., Brookes, S.J., 2006. Major classes of sensory neurons to the urinary bladder. Auton. Neurosci. 126-127, 390–397. Zagorodnyuk, V.P., Gibbins, I.L., Costa, M., Brookes, S.J., Gregory, S.J., 2007. Properties of the major classes of mechanoreceptors in the guinea pig bladder. J. Physiol. 585, 147–163. Zagorodnyuk, V.P., Brookes, S.J., Spencer, N.J., Gregory, S., 2009. Mechanotransduction and chemosensitivity of two major classes of bladder afferents with endings in the vicinity to the urothelium. J. Physiol. 587, 3523–3538.

Please cite this article as: Chai, T.C., et al., Mucosal signaling in the bladder, Auton. Neurosci. (2015), http://dx.doi.org/10.1016/ j.autneu.2015.08.009