Chapter 1
|Chapter 1 |
|Introduction |
|An overview of the anatomy and physiology of the |
|bladder |
1.1 Anatomy of the urinary bladder
Regions of the bladder
The urinary bladder is a hollow, muscular and distensible organ located posteriorly to the pubic symphysis bone in the pelvis when empty, but extends into the lower part of the abdominal space when filled. The bladder is separated from the pubic symphysis by the retropubic space of Retzius, and is enveloped by connective tissue and a layer of extra-peritoneal fat. It is roughly pyramidal in shape when empty, becoming ovoid when filled.
The human bladder has an average capacity of 300ml-600ml (Hole, 1981), although a completely full bladder is capable of holding approximately 1 litre of fluid (Irons-Georges, 1998). The desire to urinate is usually experienced when the bladder contains approximately 150ml-200ml of urine (Hole, 1981; Irons-Georges, 1998).
Urine is delivered from the kidneys via the ureters to the bladder, whose main function is to store and, when appropriate, periodically void urine, via the urethra, at low pressure (figure 1.1). The bladder is comprised of a number of functionally and histologically distinct regions;
1. The dome – comprised of the detrusor smooth muscle, a powerful muscle which is histologically and functionally distinct from the trigone and urethra
2. The trigone – a smooth, triangular region of tissue inside the bladder whose angles are formed by the two ureteral entrances and the urethral orifice.
3. The bladder neck – the lower region of the bladder which leads into the urethra
Layers of the bladder wall
The bladder wall is comprised of many layers as shown in figure 1.2. These are described below from outermost, to innermost:
1. Adventitia /peritoneum – the outer, serous membrane layer of the bladder
2. Muscular layer comprising of:-
i. outer longitudinal layer
ii. circular muscle layer
iii. inner longitudinal layer
3. Mucosal layer comprising of:-
i. submucosal layer /lamina propria – contains lymphatic and blood vessels and connects the muscle layer to the urothelial layer
ii. urothelium – protective, transitional epithelial barrier layer, with neuron-like properties (see section 1.2).
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Figure 1.1: Diagrammatic representation of the regions and layers of the bladder (Mc Graw Hill anatomy). The urinary bladder consists of functionally and histologically distinct regions: - dome, trigone and bladder neck. A cross section of the bladder wall reveals several distinct layers, the adventitia, muscle and mucosal layers.
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Figure 1.2: Layers of the bladder wall. A, Micrograph from human bladder indicating the 3 layers of the bladder wall. B, Increased magnification allows the identification of the 5-7 cell thick arrangement of the urothelial layer, taken from – ‘The Urinary System’. C, Diagrammatic representation of the cross section of the bladder wall, detailing distinct components of the mucosal and detrusor muscle layers (Martini, 2001).
The urethra
The urethra is a tube that extends from the neck of the bladder connecting the urinary bladder to the genitals and serving as the route in which urine is expelled from the body (figure 1.3). In the female bladder, the urethra measures approximately 1.5 inches, and urine exits the body through the external urethral orifice which lies between the clitoris and the vagina. The female urethra consists of three layers, including, from outermost to innermost:-
1. muscular layer– the outermost layer of the urethra, continuous with the muscle layer of the bladder consisting of circular fibres and extending the whole length of the urethra
2. erectile layer – containing muscle fibres and a large plexus of veins
3. mucous layer – lined by stratified squamous epithelia near the external urethral orifice, and transitional epithelial cells as it exits the bladder neck
In the human male, the urethra measures approximately 8 inches long, and as well as allowing the expulsion of urine provides a reproductive function in providing the route for the delivery of semen during ejaculation.
The male urethra is divided into four regions:-
1. pre-prostatic/intra-mural urethra – exits the bladder and enters the prostate gland, lined by transitional epithelial layer. This region varies in length (between 0.5cm and 1.5cm), depending on the stage of the micturition cycle, i.e. whether the bladder is filling or emptying.
2. prostatic urethra – passes through the prostate, and lined by a transitional epithelial layer which consists of 3 openings:-
i. the ejaculatory duct – which receives ejaculate fluid from the seminal vesicle, and sperm from the vas deferens
ii. prostatic ducts – allows entry of fluid from the prostate that contributes to the ejaculatory fluid
iii. prostatic utricle – ‘blind’ and doesn’t appear to be attached to any other structures
3. membranous urethra – passes through the external urethral sphincter, measuring approximately 1-2cm and lined by pseudo-stratified columnar cells
4. spongy/penile urethra – measures 15cm-16cm in length, and runs through the corpus spongiosum along the length of the ventral side of the penis. The ducts of the urethral gland open into this portion of the urethra. Proximally the penile urethra is lined with pseudo-stratified columnar cells, and distally, lined by stratified squamous cells.
The urethral sphincters
Two urethral sphincters are important in the control of the exit of urine from the bladder via the urethra. Firstly, the internal urethral sphincter which is located at the junction of the urethra with the bladder neck is the primary muscle in inhibiting the expulsion of urine. This sphincter is a continuation of the detrusor muscle of the bladder and therefore is under autonomic control (figure 1.3).
Conversely, the second sphincter involved in controlling the flow of urine through the urethra, known as the external urethral sphincter, is located at the distal inferior end of the female bladder and inferior to the prostate gland in the human male, and is comprised of skeletal muscle under voluntary control. The external urethral sphincter is innervated by branches of the pudendal nerve (figure 1.3).
The urethra is controlled by autonomic and somatic nerves, and serves two major functions. Firstly it must generate sustained tone at greater pressure than the intraluminal pressure of the bladder to prevent leakage of urine during the filling/storage phase of micturition, and also when the intra-abdominal pressure increases for example when laughing, sneezing or coughing. During the storage phase, to accommodate bladder filling, sympathetic stimulation causes and maintains contraction of the internal urethral sphincter, and relaxation of the detrusor smooth muscle of the bladder. Secondly, during the voiding phase of the micturition reflex, the urethra must relax, thereby increasing the lumen of the urethra and facilitating the flow of urine. This is achieved via parasympathetic nerve signalling, which causes simultaneous contraction of the detrusor smooth muscle and relaxation of the internal urethral sphincter.
The external urethral sphincter is under somatic control, therefore is consciously contracted during the storage phase, and relaxed during voiding.
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Figure 1.3: The male and female urethra. Diagrammatic representation depicting the differences between the male and female urethra, and location of the internal and external urethral sphincters (Cummings, 2001).
1.2 The urothelium
Structure
A layer of specialised epithelial cells, known as the urothelium, lies on top of the lamina propria and lines the inside surface of the bladder. The luminal surface of the urothelium is covered with a glycosaminoglycan (GAG) layer which is immediately in contact with the urine contents stored in the bladder and has been shown to possess antibacterial-like properties.
Typically, the urothelium in the contracted bladder measures 5-7 cells thick, and in the dilated bladder reduces to 2-4 cells thick and consists of three distinct cell layers (figure 1.4) including, from innermost (luminal surface) to outermost:-
1. Umbrella cell layer – large, hexagonal-shaped, cells measuring 25-250µm in diameter depending on the degree of bladder stretch (Birder et al., 2007).
2. Intermediate cell layer – smaller cells measuring 10-25 µm in diameter
3. Basal cell layer – basal cells are attached to a basement membrane and the lamina propria lies immediately beneath. Basal cells are the smallest cell type of the urothelium measuring 5-10 µm in diameter (Lewis, 2000) and are germinal in nature. Cell replacement is initiated in the basal cell layer, with fusion of basal cells to form intermediate cells, and subsequent fusion of intermediate cells to form umbrella cells.
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Figure 1.4: The urothelium. A, A schematic of the unstretched urothelial layer of the bladder identifying the principal cell types that constituent the urothelium (Lewis, 2000). B, Bladder histology showing the 3 principal cell types of the urothelium, umbrella cells, intermediate cells and basal cells (Histology resources for urologists, American Urological Association (AUA))
Function
The urothelium has traditionally been viewed as a barrier layer, with the primary function of preventing harmful substances contained in the urine from permeating into the underlying layers of the bladder wall thereby protecting the underlying muscle layers from damage or irritation. The bladder has an important function in storing urine at a similar composition to that delivered by the kidneys, often for extended periods of time therefore the urothelium must be able to accommodate stretch. More recently, studies have highlighted neuron-like properties of the urothelium and demonstrated that the urothelium is capable of releasing and responding to neuromediators in order to modulate bladder function. The urothelium must therefore possess a number of mechanisms in order to perform these functions, as discussed below.
The urothelium as a barrier
The urothelium offers a minimal surface area to urine volume ratio, thereby reducing the surface area for transport of substances across the urothelial layer into underlying muscle layers and aside from active transport of physiologically important substances between the lumen and the blood, the healthy urothelium is impermeable to all substances present in the urine. The umbrella cells possess several unique features which enable them to maintain barrier function despite large alterations in volume and increases in intraluminal pressure during the filling phase of the micturition reflex. Firstly, tight-junctions composed of multiple proteins, for example claudins, reduce the movement of ions and solutes between the umbrella cells. Approximately 70-80% of the apical surface of the umbrella cell is covered by uroplakins, proteins which reduce the permeability of the cells to molecules such as urea, water and protons. Additionally, a glycosaminoglycan (GAG) layer covers the entire apical surface of this layer, and has been demonstrated to act as a defence mechanism against infection with anti-bacterial, anti-adherence properties (Parsons et al., 1979; Parsons et al., 1988).
The urothelium as a sensory structure
Beyond serving purely as a barrier thereby preventing harmful substances in the urine from crossing into the underlying muscle layers, there is rapidly growing evidence that the urothelium exhibits sensory properties that play a critical role in the detection and transmission of both physiological and noxious sensory signals. Traditionally urothelial cells were viewed as passive bystanders, and their contribution to the mediation of visceral sensation was unappreciated, however recent growing evidence has supported the hypothesis that urothelial cells also function as primary transducers of specific chemical and mechanical stimuli with the ability to communicate with the underlying nerves, smooth muscle, inflammatory cells, and myofibroblasts. A hypothetical model of this, described as the ‘sensory web’(Apodaca et al., 2007), is depicted in figure 1.5, (Birder et al., 2007).
At least 3 lines of evidence exist in support of the ability of the bladder urothelial cells to participate in the detection of chemical and physical stimuli in the ‘sensory web’, as discussed in the following pages.
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Figure 1.5: Hypothetical model depicting possible interactions between the urothelium, myofibroblasts, the detrusor muscle and the underlying nerve supply.
Stimulation of the receptors and ion channels on urothelial cells either via autocrine (i.e. release of mediators from urothelial cells) or paracrine mechanisms (i.e. release from nearby nerve terminals), initiates mediator release from the urothelium. These mediators then target bladder nerves, detrusor muscle cells, myofibroblasts and other cell types for example inflammatory cells (not shown). Abbreviations: NO, nitric oxide; PG, prostaglandins; SP, substance P; Ach, acetylcholine; NGF, nerve growth factor; AdR, adrenergic receptor; NicR, nicotinic receptor; MR, muscarinic receptor; P2X/P2Y, purinergic receptors; P2R, purinergic 2 unidentified subtype; Trk-A, receptor tyrosine kinase A; TRPs, transient receptor potential channels (Birder et al., 2007).
(1)Localisation of afferent and efferent nerves
Bladder afferent and efferent nerve terminals lie in close proximity to the urothelium, and indeed some of the nerve terminals themselves reside in the urothelial layer, (Birder et al., 2007; Kunze et al., 2006; Zagorodnyuk et al., 2009). Studies in human bladder samples discovered that only unmyelinated nerve fibres are localised in the urothelial and suburothelial layers (Wiseman et al., 2002), whereas peptidergic, P2X and TRPV1 immunoreactive nerve fibres have been observed in the bladder musculature as well as in the suburothelial plexus, beneath and extending into the urothelial layer itself (Birder et al., 2007; Birder et al., 2002). Further evidence suggests that these nerve fibres in the suburothelial plexus are sensory nerve fibres, with the ability to release neurotransmitters. Incidentally in human tissue samples from patients with neurogenic detrusor overactivity, intraluminal perfusion of c-fibre afferent neurotoxin resiniferatoxin, reduced the density of immunoreactivity of TRPV1 and P2X3 suburothelial nerves, indicating that these nerves are sensory nerves (Apostolidis et al., 2005; Brady et al., 2004). Immunohistochemical studies have also identified that both adrenergic (tyrosine hydroxylase positive) and cholinergic (ChAT positive) nerve fibres lie in close proximity to the urothelium (Jen et al., 1995). Whilst this predominantly suggests the presence of efferent nerve terminals, another study has identified ChAT immunoreactivity present in sensory nerves as well as in efferent nerves (Gillespie et al., 2006).
In addition, morphologically and characteristically similar cells to the properties of myofibroblasts/interstitial cells, have been observed in the suburothelial space in animal models and in humans (Brading et al., 2005; Sui et al., 2004). Myofibroblasts, linked by gap junctions, and residing within close proximity to underlying nerve fibres, have been shown to be responsive to mediators released from the urothelial cells or from nerve terminals, suggesting they have an intermediary or amplification role in signal transduction (Brading et al., 2005). Therefore, anatomically, the apparatus for bi-directional urothelial-neuronal communication exists within the bladder.
The presence of myofibroblasts/interstitial cells (IC) in the bladder was first described in the detrusor (Smet et al., 1996), and has since been shown to be widely distributed throughout the lower urinary tract of a variety of species, including in the mouse bladder (Lagou et al., 2006). The positioning of suburothelial myofibroblasts/ICs located in close proximity to afferent nerve terminals suggests that these cells may be involved in the amplification and modification of signalling between the afferent nerves and the urothelium thereby contributing to the efficiency of the ‘sensory web’(Sui et al., 2004).
(2)Expression of receptors and ion channels on urothelial cells similar to those found on nociceptors and mechanoreceptors.
Expression of various receptors on the urothelial cells further suggests a role for urothelial cells in sensory signal transduction. Receptors and ion channels that have been identified on urothelial cells include receptors for; ATP,(P2X1-7/P2Y1,2,4 ), adenosine (A1, A2a, A2b, A3), adrenaline (α and ß), ACh (muscarinic and nicotinic receptors), proteases (PARs), bradykinin (B1 and B2), neurotrophins (p75, trk-A), growth factors (VEGF), oestrogens (ER α and ERß), cannabinoids (CB1 and CB2) prostaglandins, and various TRP channels (TRPV1, TRPV2, TRPV4, TRPM8, TRPA1), [pic](Beckel et al., 2006; Birder et al., 2003; Birder et al., 2007; Birder et al., 2002; Burnstock, 2001; Chess-Williams, 2002; Chopra et al., 2005; Chopra et al., 2008; Ferguson et al., 1997; Kullmann et al., 2009; Saban et al., 2008; Yu et al., 2006).
The expression of many receptors and ion channels on urothelial cells enables the urothelium to respond to a number of sensory signals from a range of sources, including inflammatory cells and nerve fibres
(3) Secretion of mediators from the urothelium.
Finally, urothelial cells possess the ability to secrete a number of chemical mediators which in turn, are capable of altering excitability of afferent nerves. The range of chemical mediators shown to be released from the urothelium is vast, and includes ATP, ACh, neurotrophins, peptides, prostaglandins, cannabinoids, prostacyclin, cytokines, and nitric oxide (Apodaca et al., 2007). Release of these various mediators from the urothelium can modulate sensory nerve activity as well as the spontaneous activity of the detrusor smooth muscle (Ikeda et al., 2007). Release of mediators from the urothelium can be stimulated by both mechanical and chemical stimuli (Birder et al., 2007).
In summary, a number of studies have shown the ability of the urothelium and afferent nerve terminals to communicate with each other via complimentary release and binding of mediators, as discussed in detail in chapter 6. The urothelium is capable of responding to mediators released from the afferent nerve terminals and in releasing mediators that modulate afferent nerve activity. Defects in the urothelial/neuronal sensory web, or aberrant signalling from either or both structures is likely to contribute to the pathophysiology of certain bladder disorders, for example following spinal cord injury[pic](Apodaca et al., 2003; de Groat et al., 1990; Drake et al., 2000). Additionally, bladders with chemically induced cystitis are associated with augmented release of ATP from the urothelium (Birder et al., 2003), which has then been shown to augment afferent nerve sensitivity, and cause changes in bladder reflexes induced by excitation of purinergic receptors on nearby afferent nerve fibres, (Sun et al., 2006), as well as acting in an autocrine manner, facilitating its own release from urothelial cells (Sun et al., 2001). Enhanced release of ATP from the urothelium has also been observed clinically in patients suffering from painful bladder syndrome (Kumar et al., 2007), and detrusor overactivity (Kumar et al., 2010).
These transmitters and receptors/ion channel signalling pathways, and specifically their effect on afferent nerve firing and the detrusor smooth muscle are discussed below.
Cannabinoids
Since reports that multiple sclerosis patients find ameliorating effects of the smoking of cannabis on symptoms associated with urinary dysfunction, for example urinary urgency and urge incontinence as well as other disease symptoms, interest and research has grown into the beneficial effects of the endocannabinoid signalling pathway. As part of the ‘Cannabinoids in Multiple Sclerosis’ (CAMS) study, randomised patients who received oral administration of cannabis extract cannabidiol showed reduced urge incontinence episode rate in comparison to placebo treated controls (Freeman et al., 2006). Similarly, cannabinoid and vanilloid administration into the bladder of rats with turpentine induced urothelial damage, dose-dependently attenuated the referred hyperalgesia in the hind limb (Farquhar-Smith et al., 2001), thereby supporting a role for cannabinoids (CB’s) in afferent sensitivity and pain, as was shown in an earlier study where anandamide attenuated the turpentine induced viscera-visceral hyper-reflexia in the rat bladder (Jagger et al., 1998). In a pilot study, urinary urgency, frequency, nocturia and the number and volume of incontinence episodes were reduced in patients administered with cannabidiol (Brady et al., 2004), with few troublesome side-effects. These data suggest that cannabis-based medicinal extracts offer an effective and safe therapeutic avenue for urinary dysfunction.
Cannabinoids: Localisation and expression in the bladder
Currently the cannabinoid receptor family consists of 2 distinct receptors, CB1 and CB2, and belong to the GPCR superfamily, with 7 transmembrane spanning domains (Demuth et al., 2006).
CB1 is primarily expressed in the central nervous system whereas CB2 receptors are mainly localised in immune tissues at low levels in microglia and some neurons of the central nervous system (Mukerji et al., 2010; Pertwee et al., 1996).
In the bladder CB1 has been shown to be localised in the rat urothelium (Hayn et al., 2008) and in pre-functional neurons of the mouse (Pertwee et al., 1996). As well as evidence to support the localisation of CB1 in urothelial cells in the mouse (Walczak et al., 2009), CB1 expression has also been shown in the afferent nerve fibres which terminate in both the suburothelial and muscular layers. CB1 receptors were also found to be co-expressed with P2X3 receptors, especially in the umbrella cells and muscular layer, supporting the hypothesis of an interaction between cannabinoid and purinergic systems, (Walczak et al., 2009). In the human bladder, CB1 receptor expression was higher in the urothelium and detrusor muscle than CB2 receptor, and both CB1 and CB2 expression were 2 fold higher in the urothelium than the detrusor (Tyagi et al., 2009). Whilst the expression of CB1 in the bladder has been shown in a number of studies, contradictory findings have reported no expression of CB1 in the urothelium or in afferent nerve fibres, and only identified expression in immuno-competent cells in the suburothelium and between smooth muscle cells in rat, monkey and human bladders (Gratzke et al., 2009). Furthermore, in a later study, CB2 staining was only weakly expressed in the urothelium, but more strongly expressed in the suburothelium and the detrusor muscle of human bladders (Mukerji et al., 2010). Interestingly, in bladder strips from patients with painful bladder syndrome and idiopathic detrusor overactivity, suburothelial nerve fibre expression of CB1 was significantly increased relative to controls. Detrusor CB1 nerve fibre density was also increased in bladders from patients with idiopathic detrusor overactivity, but not in bladder strips from patients with painful bladder syndrome (Mukerji et al., 2010).
CB2 receptor expression has also been demonstrated in the bladders of humans and laboratory animals. Western blot analysis revealed that the density of CB2 receptors in the mucosa was 72.8% higher than in the detrusor layer of human bladder tissue samples (Gratzke et al., 2009). In the same study, strong CB2 immunoreactivity was identified in the urothelial and suburothelial cell layers. Additionally, CB2 immunoreactive nerve fibres were observed in the suburothelial regions as well as, with lower expression, in the muscle wall. Immunoreactivity for CB2 was also observed on nerve fibres that extended into the urothelium that coexpressed TRPV1 immunoreactivity, suggesting an interaction between TRPV1 and CB2 receptors (Gratzke et al., 2009).
Other studies have also shown the localisation of CB2 receptors in the rat and human urothelium respectively [pic](Hayn et al., 2008; Tyagi et al., 2009), although not as highly expressed as the expression of CB1 (Tyagi et al., 2009).
Cannabinoids: Signal transduction
Activation of cannabinoid receptors causes the activation of multiple different intracellular effectors, thereby enabling the diversity and selectivity of the responses regulated by cannabinoid receptors. Figure 1.6, indicates the diverse effects of cannabinoid receptor activation in the bladder (Bosier et al., 2010).
Initially cannabinoid receptors 1 and 2 were believed to regulate a variety of central and peripheral physiological functions, as well as modulating cell death, proliferation, etc. via activation of heterotrimeric Gi/Go type G-proteins (Howlett, 1995). However, evidence suggests that following treatment with pertussis toxin, a potent inactivator of Gi/Go type G-proteins, cannabinoids can interact and continue to signal via Gs and Gq G-proteins in cultured striatal neurons [pic](Bosier et al., 2010; Glass et al., 1997; Lauckner et al., 2005). However, the cannabinoid receptor signalling web is becoming increasingly complex as new methods of signal transduction are being described in various systems.
Both CB1 and CB2 receptors are associated with the activation of the alpha subunit of Gi/o leading to the inhibition of adenylyl cyclase activity, and the activation of the MAP kinase pathway via activation of the ß subunit of Gi/o. Inhibition of adenylyl cyclase activity consequently inhibits the production of 2nd messenger cAMP (as adenylyl cyclase activity catalyses the conversion of ATP to cAMP), and thereby inhibits protein kinase A (PKA) signalling. Reduced PKA activity consequently is related to reduced gene expression via non-activation of the cyclic AMP response element binding protein (CREB). The classical signalling cascade, i.e. adenylyl cyclase catalyses the production of cAMP, which activate PKA, causing the phosphorylation of CREB and the initiation of gene transcription is therefore reduced following cannabinoid receptor binding.
The MAP kinase pathway, a key regulator of cellular functions such as growth and apoptosis, is also altered by cannabinoid signalling. Usually activation of the MAP kinase pathway is associated with the activation of tyrosine kinase linked receptors and consequent activation of RAS proteins. However, cannabinoid receptors have been shown to link to MAP kinase in a variety of cells. At present, the exact mechanism for the activation of the MAP kinase pathway by cannabinoid receptors has not been fully elucidated, however it is possible that cannabinoids can activate PI-3 kinase, which mediates the activation of Raf, and thereby the MAP kinase signalling cascades, as demonstrated in human prostate epithelia (Sánchez et al., 2003).
In addition, both CB1 and CB2 receptors can modulate intracellular Ca2+ concentration. Administration of anandamide (an endogenous cannabinoid), in cultured arterial endothelial cells evoked an increase in the intracellular concentration of Ca2+ (Fimiani et al., 1999). This release of Ca2+ from the internal intracellular stores of excitable cells can then lead to the inhibition of neuronal activity via the activation of Ca2+ dependent K+ channels, leading to hyperpolarisation and decreased nerve activity.
Aside from the conserved signalling pathways of CB1 and CB2 receptors, CB1 receptors have been reported to cause further effects via other signalling mechanisms. CB1 receptors have been reported to couple to L, N, P/Q-type voltage-gated Ca2+ channels and regulate inwardly rectifying K+ channels. The modulation of N, P/Q type Ca2+ channels and various K+ channels is proposed to underlie the cannabinoid-induced inhibition of neurotransmitter release on presynaptic terminals.
The complexity and diversity of the signalling processes involved in cannabinoid signalling is vast, and although evidence supports cannabinoid activation in cells and tissues, as described, the pathways involved in cannabinoid signalling in the bladder are relatively unclear.
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Figure 1.6: The complexity of cannabinoid receptor signalling contributing to the diversity and selectivity of responses regulated by cannabinoid receptors. A) Both CB1 and CB2 receptors are associated with the Gαi/o mediated inhibition of adenylyl cyclase (AC) activity and the Gßγ dependent activation of various MAP kinase signalling cascades. Additionally, CB1 receptors inhibit voltage gated Ca2+ channels and activate inwardly rectifying K+ channels (Kir). CB1 receptor signalling also induces the elevation of intracellular Ca2+ via Gßγ activation of phospholipase C (PLC). Cannabinoid mediated inhibition of protein kinase A (PKA) activity is related to inhibition of gene expression via the inactivation of cAMP response element (CRE) activity. Reduced PKA activity also leads to a decrease in constitutive inhibitory phosphorylation of c-Raf and a consecutive activation of the ERK1/2 pathway. B) In addition, it has recently been demonstrated that the activation of CB1 receptors not only activates Gi and Go proteins, but also leads to the activation of Gs and Gq proteins, and the activation of non-G-protein partners such as the adaptor protein FAN.
Cannabinoids: Modulation of bladder activity
Although the exact downstream signalling pathways following cannabinoid receptor activation are unknown, cannabinoids have been demonstrated to affect both detrusor muscle contraction and afferent nerve sensitivity in the bladder. Electrically evoked contractions of the isolated bladder detrusor of the mouse were inhibited by application of cannabinoid, or cannabinoid-like agonists, including anandamide, and delta-9-tetrahydrocannabinol (∆9-THC) (Pertwee et al., 1996) and the inhibitory effect was found to be dose-related. The inhibition of contractile activity was interestingly not present in contractions elicited by acetylcholine application (Pertwee et al., 1996). This finding, along with the observation that bladder contractions evoked by electrical stimulation were almost completely attenuated in the presence of tetradotoxin ( a Na+ channel inhibitor) suggests that the main effect of the electrical stimulation was to provoke prejunctional nerve terminal release of various neurotransmitters involved in contraction, and that the ability of cannabinoids to inhibit electrically evoked contraction in the bladder relies on the ability of cannabinoids to inhibit or suppress the release of these contractile transmitters, rather than an ability to interact directly with the detrusor smooth muscle (Pertwee et al., 1996). In addition, following application of relatively selective CB1 receptor antagonist SR141716A, small, but significant, increases in the amplitude of electrically evoked contractions were observed, further supporting the hypothesis that cannabinoid receptors in the mouse bladder inhibit muscle contraction.
A later study has also demonstrated the inhibitory effect of various cannabinoid receptor agonists on electrically evoked contractions in the isolated bladder of the rat, where application of SR141716A potentiated contractions following electrical stimulation (Martin et al., 2000). Further to these findings, the study by Martin and colleagues showed that CB1 antagonist (SR141716A) and CB2 antagonist (SR144528) were sufficient to reverse the inhibitory effect of cannabinoid agonist WIN 55212-2(Martin et al., 2000).
Conversely in muscle strips isolated from the bladder of the rat, anandamide produced concentration dependent contractions of the muscle strips. This response was partially attenuated by the application of a prostaglandin EP1 receptor antagonist (ONO8130) (Saiotoh et al., 2007). Previous studies have shown that anandamide can be metabolised by COX-2 to prostaglandin E2 ethanolamine, which was shown to cause contraction in the guinea pig trachea via EP1 receptors (Ross et al., 2002). This suggests that the contractile effect of anandamide may not be specific to cannabinoid signalling. Similarly, addition of cannabinoid antagonists AM251 and AM630 was unable to block the anandamide induced contraction of the bladder muscle strips, indicating that there was no involvement of CB1 or CB2 receptors in this study.
Afferent nerve activity has been shown to be altered by cannabinoid agonists and antagonists in the bladder. Following perfusion of the bladder with cannabinoid receptor agonist AZ12646915, mechanically evoked afferent nerve firing or the pelvic nerve was reduced (Walczak et al., 2009). The reduction in afferent nerve firing in response to cannabinoid receptor stimulation has also been inferred indirectly from various experiments, where sensory pain perception of patients with bladder dysfunction was reduced following administration of cannabinoid agonists (Farquhar-Smith et al., 2001), and CB1 immunoreactivity was increased (Mukerji et al., 2010). Another study also concluded that in bladder overactivity evoked by irritation of the rat bladder with acetic acid, addition of CB1 and CB2 agonist IP-751, suppressed urinary frequency induced by bladder nociceptive stimuli, and concluded that this was probably via suppression of bladder afferent nerve activity, rather than via efferent mechanisms as bladder pressure was unaffected (Hiragata et al., 2007). Similarly, application of anandamide in an established rat model of visceral pain (turpentine treatment of the bladder), decreased the viscera-visceral hyper-reflexia response, again providing further evidence for a role of cannabinoids in regulating bladder afferent sensitivity (Farquhar-Smith et al., 2001).
Prostaglandins
Prostaglandins (PGs) are found in virtually all tissues of the body where they exert a wide variety of functions, the bladder being no exception. Many studies have shown that the bladder in many species can synthesise several prostaglandins from the prostaglandin-F (PGF) and prostaglandin-E (PGE) ranges, and has the ability to synthesise prostacyclin.
Synthesis of PGE2 in the bladder of the rat has been shown to occur by both the epithelial and muscular layers of the bladder. This synthesis was increased by arachidonic acid and inhibited by PG antagonist indomethacin. PG G-protein coupled receptors have also been demonstrated throughout the bladder, and have been identified and quantified in the urothelium. Studies performed previously have shown that antagonists of EP1 receptor inhibit the overactivity of the bladder induced by spinal cord injury (Lee et al., 2007). Furthermore, another study has demonstrated the facilitation of afferent nerve activity by PGs via EP1 receptors during inflammation of the rat bladder, and shown that EP1 receptor antagonists inhibit the increase in neural activity induced by inflammation (Ikeda et al., 2006). The EP3 PG receptor has also been shown to have an important role in bladder function. Following perfusion of the mouse bladder with EP3 receptor agonist GR63799X, bladder capacity was reduced. This effect was shown to be specific to EP3 receptors, as in EP3 receptor knockout mice there was a time dependent increase in bladder capacity. Furthermore, perfusion of PGE2 into the bladder of a wildtype mouse induced bladder overactivity, an effect that was significantly blunted in EP3 knockout mice, thereby implicating EP3 as a contributor to the progression of bladder dysfunction in the pathological bladder (McCafferty et al., 2008). The role of EP3 receptors in the modulation of bladder afferent nerve function has also been studied where application of an EP3 receptor antagonist (DH-041) attenuated the afferent nerve mechanosensitivity response to bladder distension, as well as significantly reduced the contractions of the detrusor smooth muscle (Su et al., 2008).
Cyclophosphamide induced overactivity in the rat bladder has been demonstrated to upregulate the expression of EP4 receptors in the bladder urothelium, by 100%, and was consequently accompanied by overactivity of the detrusor muscle (Chuang et al., 2010). These findings implicated the EP4 receptor as a potential target for therapy of bladder overactivity in human patients.
Prostaglandins: Localisation and expression
Prostaglandin receptors type 1 and 2 (EP1/EP2) have been identified in both the urothelial and suburothelial cells, and in the lamina propria of the guinea pig bladder (Rahnama'i et al., 2010). In addition, cyclooxygenase 1 (COX-1) enzyme was identified in the basal urothelial cells, suggesting this as the site of prostaglandin synthesis in the urothelium (Rahnama'i et al., 2010). Synthesis of PGs has been shown to occur as a result of stretch of the detrusor, mucosal damage, inflammation, and following stimulation of the bladder nerves. The study also suggested that the PGs synthesised by COX-1 in the urothelium, may target EP1 and EP2 receptors in the urothelium and suburothelium (Rahnama'i et al., 2010).
Prostaglandins: Synthesis and signal transduction
PG synthesis has been demonstrated to increase in response to ATP stimulation in the rat bladder detrusor muscle, suggesting that ATP has the ability to activate cyclooxygenase enzymes, resulting in an increase in the level of PG synthesis (Kasakov et al., 1985). PG synthesis occurs from the precursor arachidonic acid, which is transformed to prostaglandin H2 (PGH2) by the action of COX-1 and COX-2 enzymes. PGH2 is then further converted by prostaglandin synthases, generating the production of prostaglandins as shown in figure 1.7.
In vivo, 5 principal PGs are synthesised, PGE2, PGF2a, PGD2, PGI2 and TXA2. These prostaglandins have varying contributions to overall bladder function in both physiology and pathology (Wibberley, 2005).
2 different forms of COX enzymes have been identified and described in the bladder. COX-1 is constitutively expressed and is proposed to mediate physiological bladder responses, whereas COX-2 is primarily responsible for the synthesis of PGs in acute and chronic inflammatory bladder conditions (Hata et al., 2004). Cyclooxygenase inhibitors have been shown to inhibit the micturition reflex in rats , emphasising the importance of their action to normal bladder function (Angelico et al., 2006). These experiments showed that in rats with both normal, and inflamed bladders (following chemical irritation), the micturition reflex was attenuated following perfusion of COX-2 inhibitors, although the magnitude of inhibition was greater in rats with bladder inflammation than in ‘normal’ rats (Angelico et al., 2006). However, in another study, application of indomethacin has been demonstrated to suppress the metabolism and urinary expression of GAGs, thereby increasing mucosal permeability and causing degeneration of the urothelial layer (Cetinel et al., 2003). It is hypothesised that the deterioration of the GAG layer by indomethacin could occur via the inhibition of PG synthesis, as PGs have previously been shown to exert a protective effect on the mucous layer of the gastric mucosa (Holmäng et al., 1991).
[pic]
Figure 1.7: The production of multiple prostaglandins from arachidonic acid. Phospholipases (PLA2) first catalytically hydrolyse the bond of phospholipids and subsequently causes the release of arachidonic acid from the plasma membrane. Cyclooxygenase enzymes (COX) then catalyse the formation of prostaglandin H2 (PGH2) from liberated arachidonic acid. Prostaglandin synthases are then responsible for the conversion of PGH2 to one of a variety of prostanoids including PGF2, PGI2, PGE2, PGD2 and TXA2.
Prostaglandins: Modulation of bladder activity
The contractile response of the bladder detrusor smooth muscle to ACh and ATP has also been shown to rely on prostaglandins to some extent. Application of cyclooxygenase inhibitors, for example indomethacin, exerts an inhibitory effect on electrically evoked contractions of the mammalian detrusor muscle (Maggi et al., 1984), whilst application of exogenous PGs increases the nerve mediated contraction of the smooth muscle of the bladder (Downie et al., 1981). Another study showed that administration of indomethacin, or other non-steroidal anti-inflammatory (NSAID) drugs caused attenuation of spontaneous muscle contractions and a reduction in muscle tone (Bultitude et al., 1976). The contraction of the detrusor muscle elicited by PG agonists was also demonstrated in a more recent study, where agonists for PGE1, PGE2, PGD2, PGF2a and TXA2 caused contraction of isolated bladder strips from human patients (Palea et al., 1998).
Afferent nerve firing in response to distension of the bladder has also been shown to be attenuated by PG antagonists (Su et al., 2008). In this study, a selective EP3 antagonist (DGH-041) selectively inhibited the afferent nerve response to distension of the rat bladder. In particular, firing of the slow conductance, high threshold afferent nerve fibres was attenuated (Su et al., 2008). In another similar study in the rat bladder, intraluminal instillation of PGE2 augmented C-fibre afferent nerve activity, but interestingly had no effect on Aδ-fibre afferent nerve activity (Aizawa et al., 2010).
Previous studies have also suggested that some effects of PGE2 in the bladder are mediated via EP1 receptors. This was demonstrated in a study by Ikeda et al, who reported that administration of loxoprofen (an NSAID) reduced afferent nerve firing to 40% of control firing rate. Furthermore, following acetic acid infusion of the rat bladder in order to cause surface damage and an inflammatory response, perfusion of loxoprofen or a selective EP1 receptor antagonist (ONO-8711) inhibited the inflammation related increase in afferent nerve activity, suggesting the EP1 receptor as a possible site for the development of novel therapeutics for bladder dysfunction (Ikeda et al., 2006). The EP3 receptor has also been implicated in afferent nerve sensitivity in the bladder, and research has shown that application of DG-041 ( a potent, selective EP3 receptor antagonist) inhibited the visceromotor reflex that is used in the detection of nociceptive stimuli (Su et al., 2008). Incidentally, each of the receptors for PGE2 has been demonstrated to be localised on afferent sensory nerve fibre terminals, thereby further providing evidence for a role of prostaglandins in determining excitability and sensory signalling in the bladder (Breyer et al., 2003).
In the clinic, PGE2 levels are enhanced in the urine of both male and female patients with overactive bladder disorders (Kim J.C. et al., 2006; Kim et al., 2005), and it has been proposed that the levels of PGs as wells as NGF can be used clinically in the diagnosis of bladder disorders. This has also been observed in isolated bladder strips from the spinal cord injured rat where excretion of urinary PGE2 was higher in spinal cord injured rats than in control animals (Masunaga et al., 2006). Furthermore, removal of the urothelium significantly attenuated the PGE2 released in both control and spinal cord injured animals (Masunaga et al., 2006). Whilst PGE2 has been shown to be increased in patients with overactive bladder and urinary tract infection, another study has suggested that PGE2 release is impaired in urothelial cells in bladders with interstitial cystitis, and since PGE2 is widely accepted to by cryoprotective and have a role in wound healing, it has been suggested that this PG may be beneficial in the treatment of interstitial cystitis (Rastogi et al., 2008). However, in a study in the bladder of rats with chronic cyclophosphamide-induced cystitis, the level of PGE2 was increased 2.6 fold (Hu et al., 2003).
Despite the conflicting reports, there is a widely accepted view in the literature that prostaglandin signalling in the bladder is crucial in bladder function, and alterations in synthesis (via COX enzymes) or in the receptor distribution itself, can contribute to certain bladder pathologies, thereby providing another potential target for development of therapeutics for the treatment of bladder dysfunction.
ATP
Aside from its unpopular beginning in the 1970’s, adenosine 5’-triphosphate (ATP) is now recognised as a neurotransmitter responsible not only for non-adrenergic, non-cholinergic neurotransmission in the gut and bladder, but also in all nerves of both the peripheral and central nervous systems. The purinergic signalling hypothesis became accepted in the early 1990’s following successful cloning of P1 (adenosine) and P2 (ATP receptors). Since then, a vast amount of research has taken place and provided evidence for the mechanisms of ATP release and breakdown by ectonucleotidases, and more recently clinical strategies for purinergic therapy of disease of the gut, kidney and bladder, and in the maintenance of pain and cancer have been studied.
In recent years, a purinergic hypothesis for the development of interstitial cystitis (IC), painful bladder syndrome (PBS) and overactive bladder (OAB) have been postulated and investigated.
ATP: Localisation and expression
The purinergic signalling system has become increasingly more appreciated for its involvement in functions of the bladder, both motor and sensory. P2X receptors for ATP have been identified in the bladder of humans as well as other mammalian species. These receptors form a family of ligand gated cation channels, and to date seven isoforms of the receptor have been identified (P2X1-P2X7), (Burnstock, 2001). Multimeric assembly of channel subunits arranged as both homo-multimers and hetero-multimers form functional P2X receptors, (Torres et al., 1999).
In the rat bladder, immunohistochemical studies have identified the distribution of P2X receptors in the bladder, providing evidence of P2X1-P2X6 receptors in the detrusor muscle cells, in both homomeric and P2X receptor cluster formations (Dutton et al., 1999). However, a more recent study identified P2X1 immunoreactivity with nerve bundles in the detrusor muscle, and non-membrane associated staining of P2X2, P2X5 and P2X6 receptors (Lee et al., 2000).
P2X3 receptors have also been identified on the suburothelial sensory nerves and evidence has accumulated to suggest a sensory role for ATP in bladder function. Incidentally, a range of P2X (P2X1-P2X7) and P2Y (P2Y1, P2Y2 and P2Y4) receptors have been demonstrated to exist in the bladder urothelium (Birder et al., 2004; Lee et al., 2000). ATP has been shown to be released from urothelial cells of the rabbit bladder in response to hydrostatic pressure changes (Ferguson et al., 1997). This study also showed that ATP release in the rabbit bladder predominantly occurs from the urothelium, as in urothelial denuded bladder strips there was minimal ATP release from the muscle layer even following electrical stimulation, yet in the isolated urothelial bladder strips, ATP release in response to the same electrical stimulation was of the same magnitude as the release of ATP measured in intact tissue (Ferguson et al., 1997). Interestingly, in this study, investigators also performed Ca2+ free experiments to elucidate the role of Ca2+ in release of ATP following electrical stimulation. In the absence of Ca2+ ATP release increased. This study concluded that this response was due to both decreased ectonucleotidase activity due to Ca2+ depletion, therefore reduced ATP breakdown, and also that ATP release itself was not via a Ca2+ dependent vesicular release mechanism (Ferguson et al., 1997).
The urothelium has since been concluded to be the predominant site of ATP release in both human and porcine bladders (Kumar et al., 2004), again supporting a sensory role for ATP in normal bladder function.
Animal studies in P2X3 knockout mice have supported the evidence to suggest a major sensory role for urothelially released ATP in the bladder. The pelvic afferent nerve response to bladder distension in P2X3 knockout mice was attenuated relative to P2X3 wildtype controls suggesting that urothelially released ATP activates P2X3 receptors on pelvic afferent nerve fibres in the normal response to distension of the bladder (Vlaskovska et al., 2001). This supported previous data that demonstrated that P2X3 knockout mice had reduced voiding frequency and increased voiding volume (Cockayne et al., 2005).
ATP: Modulation of bladder activity
Appreciation and evidence supportive of a mechanosensory role of ATP and purinergic signalling in bladder function has led to the hypothesis that aberrant alterations in either P2X or P2Y receptor expression or in ATP release from the urothelium, could provide an alternative explanation and therapeutic potential for various bladder disorders.
In cats with IC, ATP release from the urothelium is increased relative to ATP release in the normal cat bladder and there is a reduction in P2X1 and complete loss of P2Y2 receptor staining (Birder et al., 2004). Augmented extracellular ATP release was also reported in urothelial cells from patients with IC, (Sun et al., 2006), and was also enhanced in response to stretch in human bladders biopsies of patients suffering from painful bladder syndrome (PBS) compared to healthy control bladders (Kumar et al., 2007).
Cyclophosphamide induced cystitis in rats has been used as a model of chronic bladder inflammation., Following hypo-osmotic shock of these damaged bladders, evoked ATP release was increased by 94% relative to control animals, but following BOTOX-A treatment urothelial ATP release was significantly reduced (Smith et al., 2005), again highlighting the importance of the regulation of ATP for normal bladder function.
The presence of P2X receptors on bladder sensory nerves, and recent studies investigating the effects of on afferent nerve firing in P2X3 knockout mice (Vlaskovska et al., 2001) have provided evidence for a role of urothelially released ATP as a sensory mediator conveying signals regarding the degree of distension of the bladder (Ferguson et al., 1997). In a later in vitro study in the rat bladder, distension of the bladder with saline in the presence of purinergic antagonist suramin produced a 50% attenuation of afferent nerve firing in response to distension (Namasivayam et al., 1999). Prolonged exposure of the rat bladder to purinergic agonist α-ß-methyleneATP, which is known to desensitise P2X receptors following prolonged exposure, confirmed these observations, as afferent nerve activity in response to distension was reduced by 75% compared with control (Namasivayam et al., 1999).
Evidence has also accumulated to support the hypothesis that P2X3 receptor mediated activity contributes to both nociceptive and physiological mechanosensory transduction in the bladder. This was shown in an in vitro mouse preparation where application of the selective P2 purinoceptor agonist α-ß-methyleneATP into the lumen of the bladder activated a large proportion of both low threshold (65.6%) and high threshold (56%) fibres, and also activated 11 ‘silent’ bladder afferent nerves (Rong et al., 2002). Further to this, intraluminal administration of TNP-ATP (a selective P2X receptor antagonist) effectively blocked the excitatory effect of α-ß-methyleneATP on the afferent nerves of the bladder,(Vlaskovska et al., 2001) and when applied alone, TNP-ATP perfusion resulted in inhibition of multifibre afferent nerve firing (Rong et al., 2002).
P2X3 receptors have been shown to be critical in regulating the excitability of the micturition reflex in a P2X3 deficient mouse model. These P2X3 deficient mice showed decreased pain-related behaviour following injection of ATP and exhibited a decrease in voiding frequency and increased capacity of the bladder, despite normal bladder pressures (Cockayne et al., 2000). P2X2 knockout mice were later investigated, and also shown to display reduced urinary bladder reflexes and decreased afferent nerve firing in response to distension of the bladder (Cockayne et al., 2005). In fact, P2X2/P2X3 double knockout mice produced minimal to no afferent nerve response following stimulation with ATP (Cockayne et al., 2005). These conclusions were supported by another study in spinal cord injured rats, who displayed increased levels of ATP in the bladder and increased activation of P2X3 and P2X2 receptors, strongly suggesting that these receptors may be involved in intensified sensory signals, the generation of non-voiding contractions of the bladder muscle, and the development of bladder pathologies (Munoz et al., 2012). Furthermore, other studies have also identified the importance of individual purinergic receptors in normal bladder function, such as P2Y2 receptors facilitating purinergic currents and contributing to neuronal excitability and hypersensitivity in the mouse bladder (Chen et al., 2010).
Aside from a mechanosensory role, neuronal and urothelial ATP release also stimulate purinergic receptors on the detrusor muscle, consequently mediating contractions and relaxations. P2X receptors, which are classified traditionally as contraction mediating, and P2Y receptors (relaxation mediating) have been identified in the bladder detrusor muscle (Dutton et al., 1999). Application of ATP on to muscle strips from the marmoset bladder caused a rapid and transient contraction of the muscle (mediated by P2X receptors), and a following prolonged period of muscle relaxation (P2Y receptor mediated), confirming the importance of ATP signalling in bladder muscle function (McMurray et al., 1998). The urothelium has been implicated in the maintenance of bladder contractility by releasing mediators that activate receptors on the detrusor muscle. Experiments in bladder strips have produced data in support of both an excitatory and inhibitory role of urothelially released ATP, providing data to show that high concentrations of ATP suppress carbachol induced contraction in both urothelial intact and urothelial denuded bladder strips in the rat (Santoso et al., 2010), and elicited relaxation of the detrusor in intact muscle strips of the mouse bladder (Boland et al., 1993).
P2X1 receptors have been identified in the bladder smooth muscle membrane of the mouse, alongside P2X3, P2X4 that have also been identified yet not at the membrane. Some staining for P2X7 has also been observed in the smooth muscle, although again not at the membrane, yet immunoreactivity for P2X3, P2X5 and P2X6 was not detected anywhere in the smooth muscle of the mouse in one particular study (Vial et al., 2000). Further experiments showed the critical requirement of P2X1 receptors for contractile activity of the bladder muscle, as in P2X1 receptor deficient mice, P2X receptor mediated contractions of the bladder were abolished, as P2X2 and P2X4 receptors are not functionally expressed at the detrusor smooth muscle membrane therefore offered no compensation for P2X1 in these animals (Vial et al., 2000).
Despite the obvious importance of P2X1 receptor signalling in contractile responses of the bladder, the residual contractile response appears to be mediated by muscarinic acetylcholine receptors. Following nerve stimulation in the wildtype mouse bladder, contractions with both P2X and muscarinic ACh receptor mediated components were observed, however in P2X1 deficient mice, bladder contractions induced by nerve stimulation were mediated solely by muscarinic ACh receptors suggesting that the residual muscarinic acetylcholine receptor mediated response is sufficient to maintain normal bladder function again highlighting the dual purinergic and cholinergic systems acting as ‘failsafe’ mechanisms to preserve bladder contractile function (Vial et al., 2000).
It is worth noting the species differences in the relative contributions of P2X receptors to neurogenic contractions between humans and rodents, and between the physiological and pathological states of the human bladder In the physiological human bladder, neurally evoked contraction of the bladder is abolished following perfusion of atropine (a muscarinic receptor antagonist), suggesting that muscarinic receptors provide the main signalling transduction pathway for bladder contraction,(Kinder et al., 1985), despite the expression of P2X receptors throughout the human bladder and their ability to mediate contractions following exogenous application of P2X agonists (Palea et al., 1994). However, in the pathological bladder, for example in IC, a 40% residual P2X mediated contractile component has been measured, suggesting an up-regulation of this pathway in disease states (Vial et al., 2000).
Enhanced ATP release from the urothelium has also been observed in bladder tissue biopsies from patients with painful bladder syndrome in comparison to normal bladder, further suggesting the importance of ATP in bladder function in both health and disease mechanism (Kumar et al., 2007). An increase in ATP release from the urothelium was also observed in human bladders with both neurogenic and idiopathic detrusor overactivity (Kumar et al., 2010), and in bladder urothelial cells from patients with interstitial cystitis (Sun et al., 2006). In animal models of chronic inflammation and spinal cord injury, the urothelial release of ATP was greatly increased over normal values compared to control animals (Khera et al., 2004; Smith et al., 2005). These findings correlate with another study in diabetic rat bladders that demonstrated an increased release of ATP from the urothelium (Munoz et al., 2011).
The extensive studies in the literature relating to the role of ATP in bladder physiology and pathology highlight the importance of purinergic signalling, and suggest that alterations or an imbalance in ATP release and signalling can lead to the development and progression of some bladder disorders.
ATP breakdown/hydrolysis by ATPases in the bladder
As critical as the presence of ATP and functional purinergic receptors has been demonstrated to be in healthy bladder function, so is the elimination of signalling by ATPase mediated ATP hydrolysis. Furthermore, ATP activity has been shown to be more potent in detrusor muscle strips from patients with bladder disorders compared with bladder strips from healthy bladders (Kumar et al., 2007).
Many reasonable hypotheses have been put forward in an attempt to elucidate the mechanism preceding enhanced potency of ATP in pathological bladders, for example augmented ATP release, and alterations in receptor expression. Outcomes of various studies have also suggested that alterations in ATPases, the enzymes responsible for the breakdown of ATP, could be responsible for elevated ATP signalling in bladder disorders. In the detrusor muscle of stable bladders, ATP is completely hydrolysed and eliminated by ATPases in the synaptic cleft, and therefore cannot activate purinergic receptors in the detrusor muscle nor on the afferent nerve terminals, thereby providing a method of control of purinergic signalling (Fry et al., 2002). However, in pathological bladders, hydrolysis of ATP remains incomplete, therefore prolonging the effects of ATP on bladder muscle contractility and on sensory nerve mechanotransduction. The decrease in ATPase activity provides another possible explanation for the increased potency of ATP and the atropine resistant contractions that remain in the unstable bladder (Fry et al., 2002).
ATP is inactivated by breakdown via ecto-nucleotidases, which degrade ATP to ADP (adenosine 5’-diphosphate) and adenosine. These enzymes have been discovered in many tissues of the body (Ziganshin et al., 1994) as well as in the smooth muscle cells of the detrusor muscle of the bladder (Cusack et al., 1984). The initial hydrolysis of ATP to ADP is the most crucial step of degradation of ATP, as ADP has a lower potency, therefore is less active at purinoceptors, enabling quick cessation of the most active pathway of purinergic signalling.
Identification of and experimentation with an ecto-ATPase inhibitor (ARL67156) applied to guinea pig bladder strips, provided evidence for the importance of ectoATPase activity in normal bladder function. Electrical field stimulation of the parasympathetic nerve fibres of guinea pig bladder muscle strips produced biphasic contractions of the muscle, yet in the presence of ARL67156, the magnitude of these contractions was increased. Similarly the contractions elicited by exogenous ATP application were enhanced following application of ARL67156, again demonstrating the importance of enzymatic degradation by ectoATPase regulation of ATP signalling in the bladder (Westfall et al., 1997). Furthermore, total ATPase activity in unstable bladder strips was about 50% of that measured in stable patient bladder biopsies, again supporting the hypothesis that the greater potency of ATP and the generation of spontaneous detrusor muscle contractions in the unstable bladder, may be due to decreased extracellular ATP degradation by ATPases, thereby leaving increased levels of ATP in the bladder and allowing greater access to purinergic receptors on sensory nerve terminals and smooth muscle (Harvey et al., 2002).
P-ATPases
In the bladder, various subtypes of ATPase have been identified, all of which are proposed to contribute to the degradation of ATP in the healthy bladder. P-type ATPases, including plasma membrane Ca2+-ATPases (PMCA), and sarcoplasmic reticulum Ca2+-ATPases (SERCA), function to transport specific ions across a membrane using the hydrolysis of ATP for energy. Both PMCA and SERCA are responsible for lowering intracellular Ca2+ concentration which consequently leads to decreased ATP concentration inside the cell. For every ATP hydrolysed, SERCA transports 2 Ca2+ ions, whereas PMCA has a 1:1 ratio of Ca2+ transport to ATP hydrolysis. P-ATPases have been identified in myocytes of the guinea pig bladder, and following administration of cyclopiazonic acid (CPA), a potent blocker of SERCA, the resting Ca2+ concentration was increased. The rise in Ca2+ concentration was continually augmented for the duration of CPA administration, suggesting a leakage of Ca2+ unmasked by inhibition of SERCA, highlighting the importance of P-ATPases in buffering and control of Ca2+ influx in the bladder (Yoshikawa et al., 1996).
In a later study, PMCA was also proven to play a significant role in Ca2+ maintenance and ATP hydrolysis in the bladder, and was calculated to contribute 25-30% to bladder relaxation, whilst SERCA, 20%, and the Na+/Ca2+ exchanger as much as 70% (Liu et al., 2005).
E-ATPases
Extracellular ectoATPases are membrane bound cell surface enzymes, with a broad substrate specificity and the ability to hydrolyse ATP, ADP and UTP. The 4 cell surface localised enzymes specifically involved in the hydrolysis of ATP in the mammalian bladder are NTPD-1, NTPD-2, NTPD-3 and NTPD-8. NTPD-1 is the major ectonucleotidase critical for the degradation of ATP, and has been prominently observed within urothelial cells of the bladder (Yu et al., 2011). NTPD-2 positive cells have been identified in the deeper layers of the lamina propria of the bladder, whilst NTPD-3 is uniquely localised in the urothelium but not in cell membranes of the bladder basal cell layer surface. In contrast, immunostaining for NTPD-8 was diffuse, evident through several regions of the bladder, but was most highly abundant within umbrella cells (Yu et al., 2011).
The important role of ectonucleotidases in the bladder is only just beginning to emerge, as previous experimental energy has been preoccupied with the regulation of purinergic receptors and ATP signalling itself. However, the presence and coordinated activity of ectonucleotidases in the bladder enables the rapid conversion of ATP to adenosine, consequently leading to cessation of P2X mediated contraction of the detrusor, and therefore muscle relaxation – an essential pre-requisite for accommodation of urine during the successive filling cycle. A secondary role for the hydrolysis of ATP to adenosine is in the ability of adenosine to facilitate the relaxation of the detrusor via adenosine receptor A2b. Adenosine receptors are expressed abundantly in the detrusor muscle of the bladder (Stehle et al., 1992) and studies have shown that contraction of the bladder as a consequence of carbachol, high K+, electrical field stimulation or ACh, is inhibited by adenosine occupancy of A2b receptors (Brown et al., 1979), (King et al., 1997). These data suggest that ectonucleotidases may have a dual role in normal bladder function, firstly in cessation of contraction via ATP breakdown, and secondly in the activity of breakdown product adenosine, promoting muscle relaxation via A2b receptors.
Inhibition of ectoATPase activity in the bladder of the guinea pig by cyclopiazonic acid (CPA), potentiated purinergic mediated contractions of the detrusor muscle 15 fold, (Ziganshin et al., 1994), however, authors also suggested that it was unlikely that the inhibition of ectoATPases was entirely responsible for the potentiation of bladder contractions as CPA also potentiated bladder contractions elicited by other stimuli, such as carbachol, histamine, K+ solution and electrical field stimulation. These data suggest that the effects of CPA are non-specific, and as previous studies have shown the ability of CPA also to suppress the Ca2+ dependent K+ current (Suzuki et al., 1992), as well as ectoATPase activity these data should be interpreted cautiously.
Mg2+ and Ca2+ have been shown to play a significant role in the activity of ectoATPases, hence the name often used - Ca2+/ Mg2+ ATPase. Whilst other divalent cations (for example, Cu2+, Co2+, Zn2+, Mn2+) can, albeit with lowered potency, substitute for Ca2+ or Mg2+ there is substantial evidence to suggest the critical role of Ca2+ or Mg2+ in optimal ectoATPase activity. Following omission of Ca2+ from the external HEPES buffer in a guinea pig muscle strip preparation, ATPase activity was reduced by 15%, whilst the absence of both Ca2+ and Mg2+ from the external solution reduced ATP hydrolysis by 30% [pic](Ziganshin A.U. et al., 1995; Ziganshin et al., 1994). Incidentally, when Ca2+ chelator EDTA was perfused with the Ca2+ and Mg2+ free buffer, ATPase activity was drastically reduced by 88%, again demonstrating the critical role of divalent ions to ectoATPase activity [pic](Ziganshin A.U. et al., 1995). Other studies have also shown that the activity of ecto-nucleotidases is inhibited by chelators of divalent metal cations, (Zimmermann, 2000). Alterations in purinergic signalling are often associated with bladder pathologies, but at present the role of ectonucleotidases in the regulation of purinergic signalling both in physiological and pathological bladders is, in comparison, understudied and its contribution remains relatively unknown. However, increasing evidence from various studies suggests ectoenzymes as a tempting arena for scientific study for the development of novel therapeutics for bladder pathologies such as overactive bladder and painful bladder syndrome.
Nitric Oxide (NO)
NO is considered to be involved in many functions of the bladder, from the modulation of bladder afferent nerve sensitivity to inhibition of neurotransmission in the urethra and relaxation of the detrusor smooth muscle (Mumtaz et al., 2000). Furthermore, the expression of multiple isoforms of NOS enzyme including inducible/inflammatory nitric oxide synthase (iNOS), and the constitutively active epithelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) have been discovered throughout the bladder, in various studies, further supporting the hypothesis that NO has a major role in the micturition reflex pathway (Mumtaz et al., 2000). Alterations in the level of NO could affect the sensitivity of the afferent nerve fibres and lead to bladder abnormalities and urinary tract pathologies. For example, spinal cord injury or chronic inflammation have been described to alter the expression of NOS, leading to the possibility that the neurotransmitter function of NO could be altered in pathological conditions, and contribute to bladder hypersensitivity and pain sensation (de Groat, 1997). This has also been shown in studies using intraluminal administration of oxyhaemoglobin (a NO scavenger), which resulted in bladder hyperactivity, again highlighting an important inhibitory role of NO in bladder function (Pandita et al., 2000). Importantly, from a clinical perspective, it has been reported that NO concentration in the bladders of patients with interstitial cystitis is reduced (Hosseini et al., 2004), and addition of NO donors in animals pre-treated with cyclophosphamide, suppressed the associated hyperactivity (Ozawa et al., 1999). It is also possible that alterations in NO synthesis may contribute to the loss of urothelial barrier integrity in disease states, as this has been shown in the gut, where changes in NO production in intestinal epithelial cells increased the permeability to hydrophilic macromolecules in inflammatory bowel disease (Kolios et al., 2004).
NO: Synthesis and signalling
Nitric oxide synthase (NOS) enzymes synthesise the production of the free radical NO. 3 types of NOS have been identified, neuronal NOS (nNOS) and epithelial NOS (eNOS) which together make up the constitutively expressed calcium-dependent isoforms, and finally the calcium independent isoform, inducible/inflammatory NOS (iNOS). Each NOS isoform varies considerably in location, structure, regulation and function. Endogenous NO is formed via the hydroxylation of L-arginine to citrulline, a reaction catalysed by one of the 3 NOS isoforms. Although NO can exert its effects on many proteins, the best categorised is its action on soluble guanylate cyclase which produces guanosine 3’, 5’- cyclic monophosphate (cGMP) and therefore mediates relaxation of muscle, amongst other effects.
NO is not stored in vesicles, thereby requiring efficient production on demand. Although eNOS and nNOS are dependent on Ca2+ and calmodulin for activation of NO synthesis, and iNOS is produced independently of Ca2+ elevation, all 3 isoforms are heme-containing enzymes that catalyse NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) and O2 oxidation of L-arginine to NO and citrulline. Synthesis of iNOS can be induced by γ-interferon (IFN-γ), interleukins (IL1, IL2) and tumour necrosis factor (TNF), and transcription can be inhibited by transforming growth factor ß (TGF-ß), interleukins 4 and 10 (IL4, IL10) and macrophage differentiation factors (MDF). The control of iNOS activity is also likely to be regulated by alterations in the subcellular concentration of arginine, altering the production of NADP (nicotinamide adenine dinucleotide phosphate) and the availability of calmodulin (CAM).
NO: Distribution of NOS in the bladder
Immunocytochemistry and NADPH histochemistry has identified NO synthesis sites throughout the bladder, in the detrusor muscle blood vessels, urothelium and in the nerves supplying the bladder. NADPH-diaphorase activity and NOS immunoreactivity have shown the localisation of NOS enzymes in the pelvic ganglia of the bladder (Vizzard et al., 1994), and following removal of the pelvic ganglia in the rat, the NO dependent relaxation of the smooth muscle in the urethra was abolished (Persson et al., 1998). Afferent nerves with terminals in the suburothelial and urothelial layers display NADPH-diaphorase activity, and are therefore considered as a potential source of NO (Ehren et al., 1994). Bladder afferent neurons in L6 and S1 dorsal root ganglia also display NADPH-diaphorase activity, although NOS immunoreactivity in the spinal cord and dorsal root ganglia has not been detected in the healthy, physiological bladder (Vizzard et al., 1993). However, following chronic irritation of the bladder, NOS immunoreactivity in the spinal cord and dorsal root ganglia can be detected (Vizzard et al., 1996).
In agreement with other studies, NADPH diaphorase reactivity has been detected in the intramural ganglion cells, predominantly in the base of the bladder. Furthermore, the study showed that 70% of the NADPH diaphorase positive neurons in the bladder expressed immunoreactivity for NOS enzymes (Zhou et al., 1998).
In another study, NOS immunoreactive nerve fibres were identified throughout the bladder in both the body, neck and urethra (Smet et al., 1996). Interestingly, a greater concentration of NOS immunoreactive nerve fibres was shown to supply the urethra rather than the bladder body itself, suggesting that NO has a more predominant role in modulation of urethral activity compared with the role in the bladder (Smet et al., 1996). In the porcine bladder, almost all neuronal structures displaying NOS immunolabelling were also co-stained for NADPH diaphorase. The presence of NOS enzymes in nerve fibres of the bladder further suggest the importance of NO in inhibitory neurotransmission (Persson et al., 1993). Capsaicin has been shown to initiate the release of NO from the nervous tissues in the bladder, again highlighting the importance of NO signalling in the maintenance of physiological bladder function (Birder et al., 1998).
NOS enzymes and the synthesis of NO has also now more recently been described in the urothelial layer of the bladder, where both eNOS and iNOS isoforms have been identified. The urothelium of the bladder in the pig (Persson et al., 1993), human (Smet et al., 1994), and rat (Alm et al., 1995), amongst other species have been shown to display NADPH-diaphorase labelling, and both eNOS and iNOS isoforms have been found within the urothelium (Burnett et al., 1997; Lemack et al., 1999). RT-PCR in isolated rat bladders has also confirmed the presence of eNOS and iNOS in urothelial cells (Birder et al., 2002).
The role of NO in bladder detrusor muscle has only within the last few years begun to be established. Studies have previously demonstrated a prominent role of cGMP by NO in the urethral muscle, but this was not shown in the bladder detrusor muscle (Smet et al., 1996), and it was initially believed that NO generated by nerve stimulation had an important role in the relaxation of the bladder neck and urethra, but was unlikely to majorly contribute to detrusor muscle relaxation. However, more recently, the detrusor muscle in human bladder biopsies was recognised as an important location of eNOS, with the urothelium also showing eNOS expression, although less prominent in comparison to the detrusor muscle (Fathian-Sabet et al., 2001). Furthermore, the detrusor and vascular smooth muscle were identified as targets for NO, shown by the expression of guanylyl cyclase. The study concluded that the expression of eNOS and other NOS isoforms and the expression of guanylyl cyclase provided evidence to suggest nitric oxide-cyclic guanosine monophosphate mediated relaxation of the detrusor smooth muscle (Fathian-Sabet et al., 2001). Similarly, activity has been demonstrated on nerve fibres which penetrate the detrusor muscle (Persson et al., 1993), and low levels of NOS activity were identified in the rabbit detrusor muscle (Masuda et al., 2002).
The interstitial cells in the detrusor muscle express cGMP immunoreactivity and one study has suggested these cells are the predominant target of NO in the bladder detrusor (Smet et al., 1996). The findings in the literature implicate NO as an important inhibitory mediator involved in the relaxation of the bladder muscle and the inhibition of sensory nerve activity, and studies have indicated that a number of cell types in the bladder and urethra can mediate the effects of endogenously synthesised and released NO.
Relaxations of the smooth muscle of the lower urinary tract can occur in response to NO release in the bladder. Both strips from the pig trigone and detrusor pre-contracted by noradrenaline, carbachol or endothelin-1 showed concentration dependent relaxation following exogenous administration of NO or an NO donor, for example SIN-1 (Persson et al., 1992). In another study, in wildtype mice, nNOS inhibitor 7-NI reduced the amplitude of electrical field stimulation evoked contractions of the detrusor muscle (Meng et al., 2012), however a study in nNOS knockout mice showed that mice voided normally and displayed no significant changes in muscle contractility, suggesting that the contribution of nNOS to normal detrusor muscle function is minimal, or at least compensated for by other mechanisms (Sutherland et al., 1997).
Detrusor muscle strips pre-contracted by carbachol or K+ solution were partially relaxed by NO administration, however NO administration in urethral preparations caused a 93-100% relaxation, suggesting that the NO pathway is critically important in the mediation of relaxation in the urethral outlet region, but the role of NO in the detrusor is in comparison less important (Persson et al., 1992).
In the rat bladder, urothelial denuded bladder strips exhibited larger carbachol induced contractions than urothelial intact preparations, again providing evidence of an inhibitory role of the urothelium in mediating contractility of the detrusor muscle (Santoso et al., 2011). Furthermore, in urothelial intact bladders, transverse muscle strips treated with L-NAME (an NO inhibitor) displayed increased contractility in response to carbachol administration. However in transverse urothelial denuded and longitudinal urothelial intact bladders, contractility was unaffected by L-NAME administration, suggesting differences in the regulation of directional detrusor contractility (Santoso et al., 2011).
In rat bladders with capsaicin induced detrusor overactivity, characterised by decreased inter-contraction intervals, administration of NO inhibitor L-NAME further decreased the inter-contraction interval suggesting that locally released NO can suppress the detrusor overactivity induced by capsaicin treatment in the rat bladder (Masuda et al., 2007). These findings had also been previously described in the rat detrusor where intra-arterial perfusion of L-NAME decreased micturition volume and bladder capacity, and increased the spontaneous contractions of the detrusor (Persson et al., 1992). Similarly, rat detrusor strips were reversibly relaxed following photo induced generation of NO, and during this period, the tissue level of cGMP was significantly increased compared to control (Chung et al., 1996). As detrusor overactivity and generation of spontaneous muscle contractions are associated with bladder dysfunction, muscle contractility regulation by NO offers a direction for the development of therapeutics for bladder disorders.
In the rat bladder, compounds which activated the NO/cGMP signalling cascade, for example NO donors and sodium nitroprusside (SNP) inhibited the hyperactivity of the bladder associated with C fibre afferent nerve activation (Caremel et al., 2010). Similarly, compounds which inhibited the NO/cGMP pathway, for example L-NAME, and guanylate cyclase inhibitors, increased bladder hyperactivity (Caremel et al., 2010).
In the gut, the inhibition of mechanosensitivity by exogenous NO has been demonstrated by direct afferent nerve recordings (Page et al., 2009), however only recently has the effect of NO on mechanosensitivity been demonstrated in the bladder. Administration of L-NAME (a NO inhibitor) increased the activity of both Aδ and C fibre afferent nerve fibres. Additionally, administration of L-arginine (a NO donor) attenuated the afferent nerve activities of both Aδ and C fibres during saline instillation, following protamine sulphate exposure (Aizawa et al., 2011).
Evidence in the literature suggests that NO has an important role in normal bladder physiology. Dysfunction of NO therefore has been extensively studied as a cause and drug target for various bladder disorders.
NO: Bladder pathology
The role of NO in bladder physiology has become increasingly well-established over recent years, and research has investigated the potential of NO signalling interventions for maintenance and treatment of various bladder disorders.
NO has been implicated in a range of bladder pathologies, including bladder outlet obstruction (Hu et al., 2004; Zvara et al., 2004), bladder cancer (Wolf et al., 2000), bladder inflammation (Kang et al., 2004) and overactivity of the bladder associated with spinal cord injury (de Groat et al., 1990).
Interstitial cystitis has also been associated with disrupted NO signalling. In feline interstitial cystitis, researchers observed an increase in baseline NO release (Birder et al., 2005). Although increased NO in IC may serve as a protective mechanism in order to try to compensate for the loss of NO due to urothelial damage, excessive NO release over a prolonged period of time is considered to cause cellular damage, thereby contributing to a compromised urothelial barrier, resulting in increased excitability of nearby afferent nerve fibres (Birder et al., 2005). Additionally, in studies in rat bladders with cyclophosphamide-induced interstitial cystitis characterised by bladder hyperactivity, intraluminal administration of NO donors suppressed the cyclophosphamide induced bladder hyperactivity, and hypothesised that this effect was due to inhibition of afferent nerve sensory pathways (Ozawa et al., 1999).
However other studies have shown that the overproduction of peroxynitrate , a strong oxidant and nitrating species as a consequence of NO production, are suggested to be involved in the damage to the bladder caused by cyclophosphamide treatment (Korkmaz et al., 2005), suggesting that peroxynitrate and NO (via iNOS) may contribute to the pathogenesis of cyclophosphamide induced cystitis (Korkmaz et al., 2005). The expression of iNOS was reported to be increased in both the mucosal and submucosal layers of the dome of the rat bladder following cyclophosphamide treatment (Cho et al., 2010), and it has been suggested that the enhanced activity of NO during cystitis may cause the bladder barrier function of the urothelium to be disrupted (Andersson et al., 2008). It has also been suggested that NO can be used as a marker for measuring response to treatment in patients with interstitial cystitis, as patients who reported a decrease in symptom score following oral treatment for 8 weeks with prednisone (a steroid), also displayed reduced NO concentration ion the bladder (Hosseini et al., 2004). This data suggests that whilst an increase in iNOS activity may contribute or be a marker for bladder pathology in interstitial cystitis, manipulation of NO signalling may be important in the treatment of IC and other bladder disorders, for example in urine from patients with interstitial cystitis there is an elevation of cytokine interleukin 6, an inhibitor of NOS activity, suggesting that up regulation of NOS may provide beneficial treatment in some patients (Lotz et al., 1994). It has also been reported that nitric oxide therapy reduces bladder hyperactivity in cyclophosphamide treated rats (Ozawa et al., 1999), and shown that administration of DMSO, used for symptomatic relief of interstitial cystitis in patients, when applied in rats, directly caused the release of NO from both isolated rat dorsal root ganglia cells, and from isolated strips of rat bladder, suggesting that enhanced NO release and consequent effects on bladder detrusor muscle and afferent nerves may explain the amelioration of symptoms in IC patients treated with DMSO (Birder et al., 1997).
Whilst anomalies exist between different studies, as yet, inadequate knowledge, and indeed the complexity of bladder dysfunction itself, complicates the role of NO in bladder function and pathology. There is a strong possibility that alterations in NO signalling lead to various bladder pathologies. This suggests another potential avenue for therapy of bladder disorders. The complexity of symptoms, various manifestations and severities also contribute to the complexity of the role of NO signalling in the treatment and understanding of the role of NO in bladder physiology and disorders.
Acetylcholine (ACh) and muscarinic receptors
At present, if suitable, the majority of patients with overactive bladder (OAB) are treated with muscarinic receptor antagonists. These drugs prevent the activation of post-junctional muscarinic receptors by efferent nerve release of ACh, resulting in increased bladder capacity.
ACh antagonists also target muscarinic receptors on the detrusor smooth muscle, and evidence now exists to suggest that the full complement of muscarinic receptors (M1, M2, M3, M4 and M5) are expressed on the urothelium, thereby fuelling interest in urothelial muscarinic receptors as a target for bladder hyperactivity.
ACh/muscarinic receptors: Localisation and expression
Like other receptors and mediator release sites, muscarinic receptors are differentially expressed throughout various regions of the bladder and differentially expressed across different species. In the bladder muscarinic receptors have been identified in 3 locations:
1. The detrusor muscle, where they are involved in the mediation of contraction
2. The urothelium, where they initiate the release of an inhibitory factor that inhibits the contraction of the detrusor
3. On sympathetic and parasympathetic nerve terminals, where they influence the release of neurotransmitters.
In the detrusor muscle, M2 and M3 receptors are expressed, with the expression of M2 receptors exceeding that of M3 receptors between 3-9 times (Wang et al., 1995), however the M3 receptors are predominantly responsible for physiological micturition and smooth muscle contraction (Chess-Williams, 2002; Fry et al., 2010). This has been demonstrated in M3 receptor knockout mice, where although M2 receptors were capable of mediating carbachol induced contractions, contraction amplitude of the carbachol induced contractions of the detrusor muscle strips was reduced to only 5% of the contraction amplitude in wildtype mice (Matsui et al., 2000). Furthermore, these mice had relatively normal micturition and displayed little change in cystometric parameters, highlighting the importance of ACh and muscarinic mechanisms for the micturition reflex in mice (Matsui et al., 2000).
Whilst the M2 and M3 receptor subtypes dominate the muscarinic receptors in the detrusor muscle, M1, M4 and M5 receptor subtypes have been identified in the human detrusor, but as yet, their role in bladder function is incompletely understood (Tyagi et al., 2006).
Recently, muscarinic receptors have also been identified on urothelial cells in the bladder, with one study indicating the presence of M2 in umbrella cells, whereas subtypes M3, M4 and M5 were detected in the intermediate and basal cell layers, and M1 immunoreactivity was distinct in the basal cells at the plasma membrane [pic](Andersson et al., 2012; Tyagi et al., 2006; Zarghooni et al., 2007). Interestingly, in cyclophosphamide induced cystitis (one of the most frequently used models of interstitial cystitis), in the rat, muscarinic M5 receptor expression was up-regulated, particularly in the urothelium (Giglio et al., 2005).
Muscarinic receptors have also been identified on presynaptic nerve terminals, where they participate in the regulation of neurotransmitter release, both with inhibitory and excitatory roles. In animals, the M2 receptor has been classified as the inhibitory pre-junctional receptor (Somogyi et al., 1992; Tobin et al., 1998), whereas in the human bladder, the M4 receptor performs this role (D'Agostino et al., 2000). M1 receptors mediate the facilitatory effects in both animals and human bladders (Somogyi et al., 1999).
In addition, nicotinic ACh receptors (nAChR) α subunits have been identified in the mouse urothelium. Nicotinic receptors α2, α4, α5, α6, α7, α9 and α10, have all been identified in the urothelium, whereas the expression of the α3 subunits has not yet been identified (Zarghooni et al., 2007).
ACh: Modulation of bladder activity
It is generally accepted that the M3 receptor is responsible for mediating the contractile response of the bladder detrusor muscle in all species of animal examined, thereby heavily influencing pharmaceutical research. M3 receptors are coupled to Gq/11 proteins. Binding of ACh to M3 receptors in the bladder detrusor, causes the activation of phospholipase-C (PLC), thus generating second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 binding to receptors on intracellular Ca2+ stores initiates Ca2+ release from the sarcoplasmic reticulum (SR). The rise in intracellular Ca2+ binds to calmodulin and activates myosin light chain kinase (MLC kinase), which phosphorylates and activates the binding of contractile proteins myosin and actin, thereby initiating bladder contraction (Fry et al., 2010). The rise in intracellular Ca2+ also causes further Ca2+ release from Ca2+ stores via ryanodine receptors (RyR), thereby accelerating the increase of intracellular Ca2+ concentration. ACh stimulation of M3 has also been suggested to activate the rho kinase pathway. Rho kinase phosphorylates and attenuates the activity of myosin light chain phosphatase (MLC phosphatase), thereby preventing inactivation of the myosin/actin contractile protein complex. This was suggested following the finding that some inhibitors of PLC activity are ineffective in completely inhibiting contractions mediated by carbachol, a muscarinic agonist, suggesting alternative pathways for muscarinic receptors mediated contraction of the detrusor muscle. In this case, the rise in intracellular Ca2+ concentration was attributed to activation of cation channels, for example L-type Ca2+ channels, which lead to activation of calmodulin and activate the binding of myosin to actin. In this instance, M3 activation of the Rho pathway is hypothesised to enhance the sensitivity of the contractile proteins to Ca2+ entry via L-type Ca2+ cation channels. DAG has also been identified as playing a supplementary role in M3 mediated contraction via the direct activation of protein kinase C (PKC), which in turn phosphorylates, thereby reducing the activity of, MLC phosphatase, again increasing the Ca2+ sensitivity of contractile proteins actin and myosin. A diagram further illustrating the role of the muscarinic receptor in contraction of the bladder is shown in chapter 4, figure 4.1 of this thesis.
It is however surprising that M3 receptors dominate the mediation of the contractile complex in the bladder, despite an overwhelming pre-dominance (90%) of M2 receptors in the bladder (Wang et al., 1995). However, studies have since suggested that whilst M2 and M3 cause contraction of the detrusor muscle, M3 activation is proposed to cause direct contraction of th bladder, whilst M2 receptors cause contraction of the bladder by reversing sympathetic (ß-adrenoceptor) mediated relaxation as observed following selective M3 receptor depletion by 4-DAMP mustard (Hedge et al., 1997). In a study in pig bladders, isolated strips of urothelium with intact lamina propria developed spontaneous contractions, which were enhanced following carbachol administration, and decreased following administration of muscarinic antagonist 4-DAMP (Moro et al., 2011). This contractile activity was increased during stretch, suggesting that this mechanism could drive contractions of the detrusor either via ACh mediator release from the urothelium or via a direct mechanism on cells initiating phasic contraction (Moro et al., 2011).
A basal release of ACh has been detected in the bladder, and interestingly, following removal of the urothelium ACh release was significantly decreased, further supporting a role of the urothelium in the release of ACh (Yoshida et al., 2006; Yoshida et al., 2004). Urothelial release of ACh could therefore result in activation of cholinergic receptors on bladder afferent nerves, myofibroblasts and smooth muscle cells, of function in an autocrine fashion to activate cholinergic receptors on the urothelium (Hedge, 2006).
Release of ACh from the urothelium and detrusor muscle activates cholinergic receptors ion afferent nerve terminals, thereby modulating afferent sensitivity. In the rat, intraluminal administration of muscarinic receptor agonist oxotremorine-M (oxo-M) enhanced afferent nerve firing, suggesting an excitatory role for ACh on afferent nerve sensitivity in the bladder (Yu et al., 2010), a finding hypothesised by other studies demonstrating that intraluminal application of oxo-M decreased intercontraction intervals and bladder capacity, characteristics of bladder overactivity (Kullmann et al., 2008; Matsumoto et al., 2010). Furthermore, intraluminal application of the rat bladder with Darifenacin, a widely used anti-muscarinic drug for treatment of OAB with high selectivity for M3 receptors, reduced afferent nerve activity in both Aδ and C fibres, with a more pronounced decrease in C fibres than Aδ fibres, suggesting that the efficacy of Darifenacin in ameliorating OAB symptoms is in part due to desensitisation of afferent nerves (IIjima et al., 2007). However, in contrast, a recent study provided evidence for an inhibitory role of muscarinic receptor pathways, as stimulation of muscarinic receptors in the mouse bladder depressed sensory nerve transduction independently of changes in detrusor muscle tone (Daly et al., 2010).
ACh release from various regions of the bladder, including the urothelium, has a major role in a number of bladder pathologies. Administration of antimuscarinic drugs, a long standing, well-established treatment for OAB, exhibits efficacy during the storage phase of the micturition reflex, when efferent nerves of the bladder are ‘silent’, thereby suggesting that ACh release from non-neuronal sources plays an important role in bladder pathologies. In support of this hypothesis, studies in biopsies from elderly patients who demonstrated a greater incidence of overactive bladder, also showed increased ACh release from the urothelium (Yoshida et al., 2004). Studies have also indicated that muscarinic receptor expression is altered in cyclophosphamide induced cystitis in rats, particularly enhanced M5 expression in the urothelium (Giglio et al., 2005). In biopsies from bladders of patients undergoing surgery for bladder cancer, the non-neuronal ACh release was shown to be increased with age, and it was demonstrated that stretch of bladder muscle strips caused an increase in non-neuronal ACh release, and this was higher in strips with intact urothelium in comparison to denuded specimens (Yoshida et al., 2008). Another study has suggested that intrathecal application of ACh esterase inhibitor neostigmine, increased the intercontraction interval in both cystitis and control rats. This effect was inhibited by atropine, suggesting that the increase in ACh levels in the rat spinal cord increases the threshold for initiation of the micturition reflex via muscarinic ACh receptors (Masuda et al., 2009).
Antimuscarinic treatment effectively enhances the storage phase of micturition in patients with OAB. During the storage phase , parasympathetic nerve fibres are silent, therefore it is postulated that ACh release from the urothelium may contribute to an overactive detrusor (Yoshida et al., 2006). It has also been proposed that ACh release from neighbouring efferent nerve terminals could activate urothelial muscarinic receptors, or activate the release of mediators such as ATP, that consequently alters bladder sensation following stimulation of nearby afferent nerves (Birder et al., 2003). The many signalling pathways and effector sites of ACh has attracted intense basic science and pharmaceutical research. In recent years, the use of botulinum toxin to decrease hyperactivity in then overactive bladder detrusor has been investigated, as well as in other disorders such as interstitial cystitis and detrusor sphincter dyssynergia [pic](Chancellor, 2005; Tiwari et al., 2006; Toft et al., 2006). Bladder application of botulinum toxin causes relaxation of the detrusor muscle via the inhibition of exocytotic release of various mediators from afferent nerves and the urothelium (Apostolidis et al., 2006; Smith et al., 2005).
Data in the literature highlights the crucial role of ACh in bladder processes and the clinical use of antimuscarinics as the major therapeutic option for treatment of OAB suggests a major role for cholinergic pathways in the bladder.
Urothelial derived inhibitory factor (UDIF)
Aside from the contribution of the previously described transmitters and receptors and ion channels, other signalling mechanisms have also been identified between the urothelium, detrusor, nerve terminals and myofibroblasts, including substance P, nerve growth factor (NGF), and the elusive urothelial derived inhibitory factor (UDIF).
Contractile responses of the detrusor are depressed in the presence of an intact urothelium, an effect that is not inhibited following antagonism of known mediator pathways (Templeman et al., 2002). The UDIF has not yet been identified, as numerous avenues explored with the objective of identification of UDIF have failed to identify this inhibitory factor, although carbachol is known to elicit its release (Templeman et al., 2002).
Following stimulation of the muscarinic receptors on the urothelium in both human and pig bladders, the inhibitory effect by UDIF was not prevented in the presence of inhibitors of the nitric oxide or cyclooxygenase pathways, or in the presence of inhibitors of purinergic receptors or ß-adrenoceptors (Hawthorn et al., 2000). Interestingly, in strips of urothelial denuded pig bladder, the inhibition of smooth muscle contraction was induced by the presence of a second bladder strip with a functional urothelium, suggesting the release of a diffusible urothelial inhibitory mediator (Hawthorn et al., 2000).
Interactions between various signalling processes in the bladder
Various studies have reported the effects of individual signalling processes in modulation of afferent nerve sensitivity and detrusor contraction of the bladder. However, it seems unlikely that such a simple system exists in which only one urothelial signal is released to activate various effectors, without the input or crosstalk of other signalling mediators. It is now becoming clear that complex interactions exist amongst the various signalling molecules of the bladder urothelium, and it is hypothesised that understanding these interactions may enable the ‘fine tuning’ of bladder activity in bladder pathologies. For example, it has been demonstrated that one function of ATP is to promote the release of prostaglandin PGE2 in unstretched urothelium/lamina propria strips, and that nitric oxide donors can inhibit the production of PGE2 (Nile et al., 2012). It has been shown previously that ATP is co-released alongside ACh following nerve stimulation, and is responsible for the contractile component mediated by P2X1 receptors (Burnstock, 2001), and whilst ATP induces a fast, yet transient increase in intracellular Ca2+ concentration, ACh initiates a comparatively prolonged, delayed increase in intracellular Ca2+. It has therefore been suggested that the orchestration of the 2 transmitters together is essential for the maintenance of normal bladder function, with ATP mediating fast, transient contractions, thereby initiating micturition, whilst ACh induces a slow, prolonged contraction that is essential for maintaining bladder emptying (Chancellor et al., 1992; Theobald, 1995).
The balance of various inhibitory and excitatory signalling molecules has also been proposed to be important in maintaining normal bladder function, and it has been shown that the ATP/NO ratio may act as a useful biomarker to characterise the severity of bladder dysfunction (Munoz et al., 2011). An interaction between NO and prostaglandins has also been identified in the rabbit bladder, where results showed that iNOS induced NO synthesis increased prostaglandin synthesis via COX-2, suggesting that both mechanisms may work simultaneously (Masuda et al., 2009). Complex interactions have also been observed in the urothelium involving ATP, NO, ACh and prostaglandins. ATP and PGE2 stimulate the release of ACh from the guinea pig urothelium, and similarly ACH can also modulate the release of PGE2 (Nile et al., 2012). In the rat bladder, ACh was found to increase ATP release and NO has been shown to inhibit ACh and ATP dependent synthesis of PGE2. The findings in the literature also suggest that COX enzymes can be activated by ACh and ATP to induce PGE2 expression, and show that NO has the ability to inhibit COX enzymes, thereby downregulate PGE2 and sensory signalling (Nile et al., 2012).
Ca2+ dependent synthesis/ release of urothelial mediators.
Although initially only appreciated for its role as an impermeable barrier, increasing evidence from various studies suggests that the urothelium is capable of releasing and responding to various mediators, and as such communicating with underlying afferent nerve terminals by complementary release and binding of mediators between the two structures in a proposed ‘sensory web’ (Apodaca et al., 2007).
Ca2+ dependent exocytosis is likely to play a major role in the release of certain mediators from the urothelium, including ATP and ACh (Apodaca et al., 2007). A mechanism was previously proposed to explain the upregulation of ATP release observed in the urothelium in feline bladders with interstitial cystitis where the release of ATP was concluded to be mediated, at least in part, by a Ca2+ dependent exocytotic process as ATP release was reduced following removal of Ca2+ from the external solution and was blocked by incubation with various agents that interfere with release of Ca2+ from internal stores for example caffeine and heparin (Birder et al., 2003). It is thought that the combination of extracellular Ca2+ influx, combined with M3 muscarinic-receptor mediated increase in intracellular calcium may facilitate the exocytosis and subsequent release of ATP from the urothelium (de Groat, 2004).
More recently, in the case of distension-induced ATP release it was discovered that in the presence of either Ca2+ channel blocker nifedipine or 2-APB, an inhibitor of store operated Ca2+ influx and IP3 receptors, distension induced ATP release in the rabbit bladder was attenuated (Dunning-Davies et al., 2012), suggesting that Ca2+ is required for the release of ATP in the bladder.
Aside from ATP, a rise in intracellular Ca2+ has been shown to stimulate the release of prostaglandins in the bladder as well as in several other tissues (Arruda, 1982).
Aside from its role in regulating release of mediators, a rise in intracellular Ca2+ concentration is also essential in the synthesis of nitric oxide by endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS), although synthesis of inducible nitric oxide synthase (iNOS) is Ca2+ independent (Davies et al., 1995; Mumtaz et al., 2000).
The activity of both nNOS and eNOS is initiated by increases in intracellular Ca2+, which binds to calmodulin to form a complex that is crucial for enzyme activity (Davies et al., 1995; Forstermann et al., 1994).
The exact mechanisms involved in urothelial release of mediators is unknown, however it is generally accepted in the literature that mediator release results from a rise in intracellular Ca2+, therefore suggesting that a similar mechanism is important in urothelial mediator release in the bladder.
Clinical and experimental stimulation of the urothelium to promote release of mediators.
Many different mechanisms can be employed in order to experimentally stimulate release of mediators from the urothelium, including electrical field stimulation and use of high K+ solutions.
The potassium (K+) sensitivity test
In the clinic, the potassium sensitivity test has been used in the diagnosis of interstitial cystitis. In the examination, following catheterisation of the bladder of the patient, the bladder is first instilled with water, to examine the degree of volume sensitivity. This is done prior to K+ instillation as opposed to following, as the response to water instillation following K+ instillation would be abnormal, therefore it would be difficult to diagnose those patients who were extremely volume sensitive. Following instillation of water, the bladder is drained and then instilled with the K+ solution (16mEq K+) for 5 minutes (Parsons et al., 1998). The patient is then asked to rate symptoms of urgency and frequency.
Patients with healthy bladders do not experience pain during the potassium sensitivity test, yet patients with interstitial cystitis report sensations of pain and urgency. The results of the examination are considered indicative of the state of damage and permeability of the urothelium. Most patients suffering from interstitial cystitis feel pain during the examination because the urothelium is damaged or permeabilised, thereby enabling the passage of K+ ions across the urothelium, and into underlying muscle layers and also directly stimulating afferent nerve terminals, whereas patients with healthy bladders, with intact, impermeable urothelial layers report no sensation of pain, because the urothelial barrier prevents sensitisation of the afferent nerve terminals. However, if the bladder of a healthy patient is infused with protamine, the urothelial layer is temporarily compromised and damaged, therefore when the K+ sensitivity test is repeated, the patient reports sensations of pain and urgency, similar to the response of patients suffering from interstitial cystitis (Parsons et al., 1998). Therefore, clinically, the K+ sensitivity test can be used as an indicator of urothelial integrity.
Damage of the urothelium
Damage to the urothelium sensitises afferent nerve firing, and causes spontaneous contractions of the detrusor muscle. As highlighted previously, (Parsons et al., 2001; Parsons et al., 1998), damage of the human bladder urothelium with protamine, significantly increased afferent nerve firing in response to high K+ perfusion of the bladder lumen. Protamine sulphate (PS) has been used in various studies in animal models of disease, to attempt to provide evidence to correlate the degree of urothelial damage to the severity of pain. In lab animals, protamine sulphate has been used to understand the contribution of urothelial cells to overall physiological bladder function, and in order to test the efficacy of certain pharmacological drugs. PS causes an increase in permeability, and destroys the GAG layer, leading to a leaky urothelium, and consequently influx of noxious ions and substances across the urothelium and into underlying tissue layers. In the rat, injection of protamine sulphate into the bladder, resulted in histologically verified ulceration and irregularity of the urothelial cell layer [pic](Cetinel et al., 2003; Zeybek et al., 2006), however in the presence of melatonin, a strong antioxidant, a relatively normal urothelial morphology was observed (Cetinel et al., 2003). Taurine has also been shown to have a protective effect on protamine sulphate induced damage of the bladder (Zeybek et al., 2006), as has leukotriene D4 receptor antagonist (Cetinel et al., 2010).
Aside from protamine sulphate, other chemical agents have been utilised to permeabilise and damage the urothelium of various animal models in order to investigate the contribution of the urothelium in signal transduction and ultimately to understand the physiological and diseased manifestations of the urothelium.
Cyclophosphamide is used clinically in the treatment of cancers and various autoimmune diseases, and it was initially from urinary tract symptoms these patients reported as a side effect that the damaging role of cyclophosphamide on urothelial integrity was discovered. It was discovered that the toxic metabolite of cyclophosphamide, acrolein, accumulates in the bladder and causes acute inflammation and chronic symptoms of cystitis in cancer patients (Cox, 1979).
Repeated treatment of the rat bladder with cyclophosphamide was reported to cause severe damage to the urothelium and the development of symptoms of interstitial cystitis (Cox, 1979; Maggi et al., 1993).
In rat bladders with cyclophosphamide induced inflammation, P2X receptor function in afferent nerve terminals was shown to be enhanced, again supporting evidence that the urothelium and afferent nerve terminals are able to communicate with each other via release and binding of neuromodulators (Dang et al., 2008). Similarly, in another study in cyclophosphamide treated rats, both muscarinic and purinergic signalling pathways were down-regulated following cyclophosphamide treatment (Kageyama et al., 2008), further supporting evidence that muscarinic and purinergic receptors are located on urothelial cells. In mice, histological evidence has highlighted the changes in the urothelium that occur following cyclophosphamide treatment. Sections of the bladder following cyclophosphamide treatment revealed inflammation, urothelial cell loss (with proportionate increased severity of inflammation with urothelial cell loss), ulceration of the urothelium, and infiltration of inflammatory cells in the underlying muscle layers (Starkman et al., 2008). This histological evidence supports the use of cyclophosphamide in inducing a damaged urothelial preparation. Fluorescein has also been used to demonstrate damage to the bladder urothelium following exposure to cyclophosphamide (Eichel et al., 2001). Due to the causative role of the cyclophosphamide metabolite acrolein in urothelial damage, and because the administration of cyclophosphamide caused life-limiting side-effects in animal models, acrolein or protamine sulphate is now more commonly used in experimental investigations. Treatment of the bladder, in mice, has been shown to cause extensive structural damage of the bladder, inflammation and loss of urothelium, along with loss of uroplakins in some areas of the bladder sections (Bjorling et al., 2007). More recently, acrolein has been used as a method of performing urothelial damage in a study in rats, where as a consequence of a damaged urothelial layer, the activity of Aδ and C fibre afferent nerve fibres was drastically increased, further supporting the proposed second function of the urothelium in the regulation of sensory responses (Aizawa et al., 2011).
The ability of the urothelium to accommodate stretch
The urothelium must accommodate the storage of urine and the subsequent increase in intraluminal pressure and bladder size, ideally maintaining a minimum surface area to volume ratio, whilst maintaining barrier function. This is achieved by folding and unfolding of the apical membrane, enabling the urothelium to accommodate the empty, filling and full bladder. Studies have shown an unfolding of the apical membrane and transformation of the umbrella cells from a roughly hexagonal shape, to a more flat and squamous morphology during the initial phase of bladder filling, and re-orientation of cytoplasmic filaments from a position parallel to the lateral membrane, to a parallel position to the apical membrane, thereby bringing the basal membrane towards the apical membrane. During the later stages of filling there is insertion of cytoplasmic vesicles which requires an intact microfilament system and there is evidence to suggest that ATP is necessary for vesical insertion during filling, but has a less dominant role in vesical removal during emptying and collapse (Sarikas et al., 1986). During voiding, there is further organisation of the urothelial cells, and endocytotic trafficking of inserted vesicals which enables the urothelium to reduce its surface area. Evidence of this phenomenon was shown by capacitance studies in the rabbit bladder, which measured the increased surface area of the urothelium during bladder filling (Truschel et al., 2002).
The role of the urothelium in maintaining bladder tone
In addition to the barrier and sensory roles of the urothelium, a number of studies have also demonstrated that the urothelium can influence bladder smooth muscle tone and contractility via the release of neuromediators which then diffuse into underlying smooth muscle layers, most notably ATP and ACh. Both these mediators, amongst others are released from the urothelium in response to stretch (Ferguson et al., 1997), and have been shown to modulate detrusor smooth muscle contractility, for example ATP mediated contractions in the marmoset bladder (McMurray et al., 1998), and the modulation of detrusor smooth muscle tone by ACh (Chess-Williams, 2002; Fry et al., 2010). Evidence of the contribution of a variety of urothelially released mediators to bladder contractility is discussed in detail in chapter 6.
1.3 Interstitial Cells
An extensive network of interstitial cells (ICs) reside in the sub urothelial region, at the boundary of smooth muscles in the detrusor muscle and in the connective tissue spaces between the muscle bundles, as identified by c-kit immunolabelling, and diagrammatically displayed in figure 1.8 (Davidson et al., 2005).
[pic]
Figure 1.8: A diagrammatic representation of the location of interstitial cells. C-kit immunolabelling in the guinea pig bladder identified the presence of ICs (c-kit positive cells) throughout the suburothelial region, at the boundary of smooth muscle and within the connective tissue spaces between smooth muscle bundles (Davidson et al., 2005).
The varied locations of ICs in the bladder suggest that these cells have different functional roles dependent on their geography. For example, as the ICs residing in the urothelium were found to have close contact with nerve cells it is hypothesised that they relay nerve signals, or indeed alter nerve signalling directly. Furthermore, ICs lying immediately below the urothelium are ideally placed to have a role in responding to mediators released from the urothelium, for example NO and ATP (Davidson et al., 2005), (Ferguson et al., 1997), (Smet et al., 1996) .
Confocal imaging revealed that in the guinea pig bladder, ICs are uniformly distributed, extending through the lamina propria and into the underlying detrusor (as shown in figure 1.8), thereby, in effect, connecting the urothelium to the detrusor muscle and providing a means of communication throughout the layers of the bladder wall (Davidson et al., 2005). This could be an essential mechanism in the maintenance of bladder compliance, whereby an increased volume of urine causes mechanical stimulation of urothelial cells causing the release of mediators which then subsequently stimulates ICs to communicate with the detrusor muscle thereby modifying the activity of smooth muscle cells.
Finally, ICs located in the detrusor muscle layer itself are reported to have a ‘pacemaker’ function, influencing the activity of neighbouring smooth muscle cells to propagate signals and to orchestrate coordinated, smooth contractions of the bladder
1.4 Innervation of the bladder
The integration of somatic and autonomic afferent and efferent mechanisms allows coordination and control of the lower urinary tract, enabling the storage and release of urine in a controlled manner. The storage and voiding of urine is dependent on the activity of two components:
1. The reservoir – the urinary bladder itself, which stores urine
2. The outlet – the bladder neck, urethra and internal and external urethral sphincters.
Three sets of peripheral nerves control these structures (figure 1.9),
1. Pelvic nerves – sacral parasympathetic
2. Hypogastric nerves – thoracolumbar sympathetic nerves
3. Pudendal nerves – sacral somatic nerves
Efferent supply of the urinary bladder
Sacral parasympathetic pathways
The major excitatory input to the bladder is provided by the sacral parasympathetic input. Cholinergic preganglionic neurones are located in the sacral region of the spinal cord (S2-S4), and send axons via the pelvic nerve to the ganglion cells located in the pelvic plexus or in the wall of the detrusor. Transmission in the bladder ganglia is mediated by the activation of various receptors, including purinergic, peptidergic, adrenergic and muscarinic (de Groat et al., 1993).
The release of cholinergic and non-adrenergic-noncholinergic transmitters from the parasympathetic ganglion cells excites the detrusor smooth muscle, most notably, ACh release from these cells provides the main excitatory input to the bladder resulting in contraction of the detrusor muscle via activation of muscarinic receptors (Chess-Williams, 2002).
Thoracolumbar sympathetic pathways
Sympathetic pathways to the bladder originate in the lumbosacral region of the spinal cord (T11-L2) as well as in the prevertebral inferior mesenteric ganglia. Input to the bladder from the sacral chain ganglia travels to the bladder in conjunction with the parasympathetic neurones of the pelvic plexus via the pelvic nerves. Fibres from the inferior mesenteric ganglia and the rostral lumbar region of the spinal cord travel to the bladder via the hypogastric nerves (figure 1.9).
Sympathetic post-ganglionic efferent pathways in the pelvic and hypogastric nerves elicit similar effects in the bladder, for example inhibition of the detrusor smooth muscle via release of noradrenaline from nerve terminals causing activation of ß-adrenoceptors, and secondly, excitation of the urethra via noradrenaline release and activation of urethral α1-adrenoceptors (de Groat, 2006).
Somatic efferent supply to the urethra
The efferent innervation of the urethra originates from cells in the lateral ventral horn, called Onuf’s nucleus (de Groat et al., 2001). Motorneurones to the muscles of the external urethral sphincter project their axons via the pudendal nerve and excite the sphincter muscles via release of ACh, stimulating post-junctional nicotinic receptors (de Groat et al., 2001).
Afferent pathways in the urinary bladder
The afferent nerves of the bladder convey information regarding visceral sensation to the central nervous system (CNS), and also play a crucial role in spinal voiding reflexes.
Sensory information from the lower urinary tract is carried in afferent axons in the pelvic, hypogastric and pudendal nerves. In humans, the somata of the pudendal and pelvic nerves are located in the dorsal root ganglia in the sacral segments S2-S4, whilst the soma of the hypogastric nerve is located in dorsal root ganglia in the thoracolumbar segment, T11-L2, of the spinal cord. Upon entry into the spinal cord pudendal and pelvic primary afferent nerve fibres travel rostral in Lissuaer’s tract and sensory information is transmitted to second order neurons in the spinal cord.
Afferents travelling via the pelvic nerves to the sacral spinal cord monitor bladder volume during the storage phase, and the amplitude of the bladder contractions during voiding, and are considered the most important afferents involved in the initiation of micturition. Both pelvic and hypogastric afferents consist of both small myelinated Aδ fibres and unmyelinated C-fibres, which convey sensory information from the bladder wall to second order neurones in the spinal cord (figure 1.9). Aδ fibres have a predominant role in conveying information about bladder filling, as they respond to contraction of the bladder and to passive distension (Janig et al., 1986). Conversely, C fibre afferents are insensitive to bladder distension under physiological conditions and primarily are involved in the afferent nerve response to noxious stimuli (Habler et al., 1990). The cell bodies of Aδ fibres and c fibre afferent nerves are located in the dorsal root ganglia (DRG) at spinal segments S2-S4 and T11-L2. These axons then synapse with interneurons and spinal tract neurones which project to and convey information to higher brain regions involved in bladder control. Denervation studies have identified a bilateral formation of the afferent innervation in the bladder, with an intermingling of axons at the level of the bladder supplying both contra-lateral and ipsilateral regions (Gabella et al., 1998).
Afferent nerve axons in the bladder have been demonstrated to be distributed in the base and inside the urothelium, diffusely and with uniform density along muscle bundles, and on blood vessels (Su et al., 1986).
The highest density of axons are found within the mucosal layer of the bladder. Here they form a plexus lying in the lamina propria, at the base of the urothelial layer (Gabella, 1990), however the role of these varicosities remains unclear. In the efferent innervation system, these varicosities are packed with vesicles and are the positions from which neurotransmitter release occurs, regulating nerve excitability and detrusor smooth muscle activity by mediator release. It has been postulated that the afferent varicosities are capable of a similar mechanism, performing a local regulatory role in bladder function (Smet et al., 1997).
A dense plexus of afferent nerves has also been identified in the suburothelial layer of the urinary bladder (Gabella, 1995; Gosling et al., 1974), with some nerve fibres terminals projecting into the urothelial layer itself [pic](Birder, 2001; Gabella et al., 1998; Smet et al., 1996; Zagorodnyuk et al., 2006). This plexus of afferent nerves is most prominent at the bladder neck and first part of the urethra, with a more sparse presence at the bladder dome (Gabella et al., 1998).
[pic]
Figure 1.9: A schematic diagram of the neural circuits controlling continence and micturition. The pelvic nerves consist of the majority of Aδ and C fibre afferents that innervate the bladder. The remaining afferent nerve supply to the bladder is carried via the hypogastric nerves, which also contain sympathetic efferent nerve fibres which originate in the thoracolumbar region of the spinal cord. The pudendal nerves carry the sacral somatic afferent and efferent innervation to the urethral sphincter. In healthy patients, the micturition reflex is controlled predominantly by the signalling of Aδ afferent nerve fibres which communicate with the spinal cord to higher regions in the pons and cortex. In the pathological bladder C fibre afferent nerve signalling to the spinal cord may become dominant (Ford et al., 2006).
Classification of afferent nerve fibres in the bladder
Afferent nerve fibres of the bladder have been classified in to different groups based on different properties, for example based on receptive field site, and response to various chemical and mechanical stimuli. Different methods of classifying bladder afferents are described below.
Activation threshold
One way in which the afferent nerves supplying the bladder can be classified is based on activation threshold (Rong et al., 2002). Electrophysiological recordings in the feline and rodent bladders demonstrated that bladder afferent nerves respond in a graded manner to distension of the bladder, or to active contraction [pic](Habler et al., 1990; Iggo, 1955; Sengupta et al., 1994). Two main types of response were observed, a low threshold afferent nerve response, where afferents were activated at low intraluminal pressures, and a high threshold response, where afferent nerve fibres were activated at higher intraluminal pressures, as outlined below.
Aδ afferent nerve fibres, with a conduction velocity of ~11.0m/s, are located primarily in the detrusor smooth muscle layer, and respond in a graded manner to passive distension and contractions, and exhibit pressure activation thresholds in the range of 5-15mmHg, and so are often described as ‘low threshold’ afferent nerve fibres. This pressure range in humans is equivalent to the pressure range at which the sensation of bladder filling first occurs, therefore these afferent nerve fibres are associated with physiological sensation (Sengupta et al., 1994; Shea et al., 2000).
Unmyelinated C fibre afferents, with a conduction velocity of ~1.7m/s, have a more widespread location, with terminals in the detrusor muscle, lamina propria and adjacent to urothelial cells These fibres exhibit high pressure activation thresholds in the noxious range (above 15mmHg), and are activated by chemical irritation and inflammation of the urothelium (Habler et al., 1990). As such, these afferent fibres are often described as ‘high threshold’ afferent nerve fibres, and are associated with pathophysiological sensory signalling (Habler et al., 1990; Zagorodnyuk et al., 2006).
Response to stimuli
Afferent nerves of the bladder are also classified based on their responses to various stimuli. 4 main classes of bladder afferents have been defined by their activation characteristics using different stimuli including stretch, stroking of the receptive fields, Von Frey hair probing, and application of chemical stimuli to the mucosa. These classes of afferents were:-
1. Muscle mechanoreceptors – encode the degree of bladder distension by distortion of the receptive field acting as ‘in-series’ tension receptors (Iggo, 1955). Afferent activity of these fibres maintains a precise relationship with the change in dimension of the receptive field (Downie et al., 1992). There is also the suggestion of ‘in parallel’ like responses to changes in bladder volume (Morrison, 1997; Morrison, 1999). Another study has indicated that removal of the mucosal layer did not affect mechanotransduction of these afferents suggesting that these nerve fibres have their receptive fields located in the muscle layer (Zagorodnyuk et al., 2006).
2. Tension-mucosal receptors – detect fine mucosal stroking and stretch of the detrusor wall, suggesting that these afferents may have receptive fields interspersed between the muscle and mucosal layers (Zagorodnyuk et al., 2006). The physiological importance of these sensory receptors remains unclear.
3. Mucosal mechanoreceptors – insensitive to stretch and show no resting activity yet respond to chemical stimulation, and spontaneous afferent nerve firing following exposure to an irritant (Zagorodnyuk et al., 2006).
4. Chemoreceptors – sensitive to endogenous mediators or exogenous application of chemical stimuli for example KCl solution (Moss et al., 1997), or acid (Zagorodnyuk et al., 2006). Furthermore, application of acid or KCl solution to the mucosal layer of the bladder excited stretch-insensitive pelvic afferents in the guinea pig bladder.
5. ‘Silent’ nociceptors - A further class of bladder afferents were also identified and described as ‘silent nociceptors’, as they responded to stimulation only following chemical irritation or inflammation of the bladder (Zagorodnyuk et al., 2006). A previous study showed that 42% of bladder afferents were unresponsive to distension of the bladder (Sengupta et al., 1994) but could be sensitised by irritation of the bladder with mustard oil (Habler et al., 1990).
Location of receptive field
Most recently, afferent nerve fibres of the bladder have been classified based on their response to receptive field stimulation with different mechanical stimuli. In this manner, four classes of afferent were identified:-
1. Serosal
2. Muscle
3. Muscle/urothelial
4. Urothelial
Lumbar splanchnic nerves principally contain fibres with receptive fields in the serosal and muscular layers (97% of sample), whereas the pelvic nerve contains all four classes of afferent nerve fibre, with the majority (63% of sample) with their receptive field in the muscular layer as shown in figure 1.10, (Xu et al., 2008).
[pic]
Figure 1.10: Location of pelvic afferent nerve receptive fields. The pelvic afferent nerves have receptive fields in all four layers of the bladder wall, including the muscle layer, muscle/urothelial layer, urothelial layer. and the serosal layer (Andersson, 2002) identified in electrophysiological studies in the guinea pig bladder (Zagorodnyuk et al., 2006; Zagorodnyuk et al., 2007).
Mechanosensitivity – direct and indirect mechanotransduction
Mechanosensitivity, defined as the ability of an afferent nerve to sense and respond to mechanical change, is a crucial determining factor in mediating the onset of micturition. Via the afferent nerves, the bladder constantly sends information to the central nervous system regarding the degree of distension of the bladder. Incidentally, these signals are used to generate voiding reflexes.
The exact mechanism governing mechanosensitivity in the bladder is unclear. Both direct mechanotransduction, by stress to ion channels and changes in membrane tension, or indirect mechanisms involving the release of mediators from various structures, provide two possible explanations.
Direct mechanotransduction
Mechanical distension of cell membranes caused by stretch or distension can alter the opening and closing rate of stretch activated/mechanosensitive ion channels by distortion of ion channel structure. This distortion in structure can result from changes in the tension of the membrane bilayer and a ‘tugging’ of the ion channel, resulting in the physical opening of the pore region, or via distortion of the cytoskeletal tethers (Sachs, 1991). Furthermore, stretch of cytoskeletal proteins, cell-cell adhesion molecules and the extracellular matrix, can cause unfolding of the structure, thereby altering biochemical properties. This mechanism has been demonstrated in the viscera where 40% of colonic dorsal root ganglia were shown to respond directly to direct mechanical probing, suggesting a direct mechanism of mechanotransduction (Raybould et al., 1999).
Indirect mechanotransduction
As is the main focus of this thesis, it is also postulated that there is an indirect mechanism for mechanotransduction, involving the release of mediators via interaction with other structures, such as the urothelium. The role of urothelially released mediators in regulating mechanosensitivity is discussed in section 1.2. However it is important to highlight ATP and ACh as the main candidates for mediators of mechanosensitivity. ATP has been demonstrated to be released from the urothelium in response to stretch (Ferguson et al., 1997), acting on urothelial afferent nerve terminals to regulate mechanosensitivity in the bladder (Cockayne et al., 2005; Namasivayam et al., 1999; Rong et al., 2002).
The theory of mechanotransduction via the release of secondary mediators from the urothelium is disputed by some following observations that pharmacological blockade of P2X receptors by the use of a calcium free Krebs solution to prevent exocytotic release of mediators had no effect on mechanotransduction in the guinea pig bladder, oesophagus or rectum (Zagorodnyuk et al., 2006; Zagorodnyuk et al., 2005), and suggested that the onset of stretch-induced afferent nerve firing occurs too rapidly(~5ms) for it to be as a result of indirect mechanotransduction (Zagorodnyuk et al., 2005).
1.5 Detrusor muscle contraction.
The regulation of bladder muscle contraction and relaxation is essential for the maintenance of the micturition reflex.
An increase in intracellular Ca2+ ([Ca2+]i), is an essential process in the initiation of contraction of the detrusor muscle, however, the source of this influx is uncertain. Studies have shown that Ca2+ influx can occur from the extracellular space ([Ca2+]e) with the movement of Ca2+ into the cytoplasm via Ca2+ channels in the cell membrane, or be released from the sarcoplasmic reticulum (Damaser et al., 1997).
Ca2+ release from the SR is an important process for the activation of contraction of the detrusor muscle. Following blockade of the uptake and release of Ca2+ from the SR with thapsigargin and ryanodine (SR function inhibitors), bladder pressure generation and emptying were inhibited in the rabbit (Damaser et al., 1997).
Molecular and pharmacological studies have identified 5 subtypes of muscarinic receptor in the bladder. In the detrusor muscle, both M2 and M3 subtypes are predominantly expressed, with the expression of M2 receptors 3-9 times greater than the expression of M3 subtypes (Wang et al., 1995). However, despite the predominant expression of the M2 receptor subtype in the detrusor muscle, pharmacological studies using selective muscarinic receptor subtype antagonists has revealed that it is the less numerous M3 receptor that is responsible for the activation of contraction (Hedge, 2006) whilst the M2 receptor is postulated to serve an important role in some pathological bladders, or during M3 receptor desensitisation (Braverman et al., 2006a).
[pic]
Figure 1.11: The role of Ca2+ in detrusor muscle contraction (Fry et al., 2010).
Binding of Ach to the M3 muscarinic receptor subtype initiates the synthesis of secondary messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) from membrane bound phospholipids (PIP2) via phospholipase C (PLC). Intracellular Ca2+ is elevated following release of Ca2+ from intracellular stores, via the binding of IP3 to IP3 receptors (IP3R). The elevated Ca2+ binds to calmodulin (CaM), activating myosin light chain kinase (MLC kinase), which initiates bladder contraction via the activation of binding of contractile proteins, actin and myosin. The elevated concentration of intracellular Ca2+ is accelerated via further release of Ca2+ via ryanodine receptors (RyR) to promote sustainable contraction. ACh stimulation of M3 also activates the Rho kinase pathway which inhibits the breakdown of the myosin/actin contractile apparatus by MLC phosphatase, and also enhances the sensitivity of the contractile proteins to Ca2+ entry via L-type Ca2+ cation channels thereby increasing the concentration of intracellular Ca2+.
Following binding of acetylcholine (ACh) to the Gq/11-protein coupled M3 receptor, the enzyme phospholipase C (PLC) is activated, resulting in the breakdown of membrane phospholipids (PIP2) to intracellular secondary messengers inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 binding to IP3 receptors (IP3R) on intracellular Ca2+ stores initiates Ca2+ release from the sarcoplasmic reticulum (SR). The rise in intracellular Ca2+ binds to soluble protein, calmodulin (CaM), and activates myosin light chain kinase (MLC kinase), which phosphorylates and activates the binding of contractile proteins, myosin and actin, thus activating bladder contraction as shown in figure 1.11 (Fry et al., 2010). The rise in intracellular Ca2+ also causes further Ca2+ release from Ca2+ stores via ryanodine receptors (RyR), thereby accelerating the increase of intracellular Ca2+.
The elevation in Ca2+ concentration in this manner within the smooth muscle cells is transient, therefore other mechanisms in order to maintain the contractile response must exist. This is achieved via a Ca2+ sensitising mechanism, whereby ACh stimulation of M3 also activates the Rho kinase pathway. Following activation, Rho kinase phosphorylates and attenuates the activity of myosin light chain phosphatase (MLC phosphatase), thereby preventing inactivation of the myosin/actin contractile protein apparatus. This was suggested following the finding that some inhibitors of PLC activity are ineffective in completely inhibiting contractions mediated by muscarinic agonist carbachol, suggesting the presence of alternative pathways for muscarinic receptor mediated contraction of the detrusor muscle. In this case, the rise in intracellular Ca2+ was attributed to activation of cation channels, for example L-type Ca2+ channels which lead to activation of CaM and initiates the binding of myosin to actin. Via this mechanism, M3 activation of the Rho pathway is hypothesised to enhance the sensitivity of the contractile proteins to Ca2+ entry via L-type Ca2+ cation channels (Frazier et al., 2007b). In the rat bladder, Ca2+ entry blocker nifedipine almost completely abolished the carbachol induced contraction of the detrusor. In comparison, 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl, an antagonist of store operated Ca2+ channels, had little effect on carbachol induced contractions. This study demonstrated that carbachol-induced contraction of the rat bladder predominantly depends on Ca2+ entry via L-type Ca2+ channels (Schneider et al., 2004). This was also shown in the human bladder, where carbachol-induced contraction of the bladder was shown to be largely dependent on Ca2+ entry via L-type Ca2+ channels and activation of Rho kinase, and the contribution of store-operated Ca2+ channels and PLC was shown to contribute only in a minor way (Schneider et al., 2004b).
DAG has also been identified as playing a supplementary role in M3 mediated contraction via the direct activation of protein kinase C, which in turn phosphorylates and inactivates MLC phosphatase, again increasing the Ca2+ sensitivity of contractile proteins actin and myosin.
Relaxation of the detrusor occurs when the Ca2+ concentration in the sarcoplasm is reduced to resting levels following reuptake into the sarcoplasmic reticulum via an ATP consuming Ca2+ pump (SERCA). Some Ca2+ is lost across the cell, and whilst exact mechanisms in the detrusor muscle remain unclear, it is postulated that it occurs via either a Ca2+ pump energised by ATP hydrolysis, or a Na+ and Ca2+ counter exchanger. Ca2+/Mg2+ ectoATPases are also present in the sarcoplasmic reticulum and plasma membrane and remove Ca2+ from the cytosol, thereby aiding muscle relaxation. Furthermore, during relaxation, receptor and voltage operated Ca2+ channels are closed in the plasma membrane to reduce further Ca2+ entry into the cell. Replenishment of intracellular Ca2+ also occurs during the relaxation phase of the bladder, via voltage activated Ca2+ channels and Na+/Ca2+ exchange (de Groat et al., 2001). Myocytes in the detrusor muscle contain a large proportion of L-type Ca2+ channels and it has been postulated that although they are not necessarily the main participant in the generation of contraction, they may be important in the replenishment of intracellular stores between bladder contractions. This was shown in single isolated cells from the guinea pig, where addition of 10µM nifedipine (a potent L-type Ca2+ channel blocker) applied during the inter-contraction interval between repeated applications of carbachol decreased the carbachol induced force of detrusor contraction to 14% of the control response, suggesting that there was insufficient replenishment of Ca2+ ready for successive contractions due to L-type Ca2+ channel inhibition (Wu et al., 2002). T-type Ca2+ channels have also been identified in detrusor myocytes, and have been demonstrated to have their active range over a m ore negative range of potentials compared to L-type Ca2+ channels, thereby ensuring that some Ca2+ channels remain open over the range of resting potentials recorded in the bladder for replenishment of Ca2+ stores and Ca2+ activated contraction (Sui et al., 2001).
Ca2+ antagonists have been shown to have a potent effect on detrusor muscle activity in the porcine bladder, causing concentration dependent inhibition. The most potent antagonist identified in one particular study was the L-type Ca2+ channel blocker nifedipine, which completely abolished the contractions of the detrusor and was also sufficient to almost completely suppress the muscle response of the bladder following treatment with carbachol (Badawi et al., 2006). Importantly, from a pharmacological perspective, nifedipine was sufficient to almost completely reverse the enhanced contractions of the detrusor smooth muscle in the bladders of diabetic mice (Leiria et al., 2011).
It has been proposed that properties of the detrusor muscle itself are a cause of detrusor overactivity, therefore as a potential pharmacological target, research has focussed on understanding the process of contraction and relaxation of the detrusor muscle. Donated bladder samples from humans for scientific experimentation have yielded evidence to support the myogenic hypothesis of overactive bladder and bladder instability, where results concluded that spontaneous cellular activity of human detrusor smooth muscle cells was mediated by influx of extracellular Ca2+ and intracellular release of Ca2+ from internal stores. This finding supports the hypothesis that defective activation of Ca2+ contributes to the up-regulation of contractile responses in the overactive bladder (Sui et al., 2009).
In experiments in the L-type Cav1.2 channel knockout mouse, also known as SMACKO mice, spontaneous contractile activity of the detrusor muscle was absent, and K+ and carbachol induced contractions were drastically reduced (Wegener et al., 2004), again providing evidence of the importance of extracellular Ca2+ influx for muscle contraction. Whilst clearly both extracellular and intracellular influx of Ca2+ is important for the generation of muscle contraction, species differences between pig, mouse, and human bladders have been identified, for example 1 µM of L-type Ca2+ channel blocker nifedipine reduced carbachol induced contractions to 18% of control in porcine bladders, 27% of control in the mouse bladder, and 74% of pre-nifedipine control in human bladder (Wuest et al., 2007), thereby demonstrating that the potency of nifedipine in reducing muscarinic receptor mediated contraction of the bladder is potentially lower in humans than in other species. Furthermore, inhibition of IP3 induced Ca2+ release by administration of 2-aminoethoxyphenyl borate (2-APB), reduced carbachol induced contractions in human detrusor to 60% of control, to 35% of control in porcine control, and had greatest potency in the mouse bladder, where contraction was reduced to 20% of control. Incidentally, inhibition of Ca2+ release with ryanodine had no significant effect on carbachol induced contractions in all 3 species examined (Wuest et al., 2007). This study suggests that species differences may exist in the contribution of a variety of Ca2+concentration increasing mechanisms, and therefore suggests that animal models of disease only have a certain level of reliability in extrapolation of finding to the clinic.
In another study in guinea pig bladder smooth muscle strips, removal of Ca2+ from the external solution almost completely inhibited the carbachol induced contractions (Rivera et al., 2006). In separate experiments, administration of L-type Ca2+ channel blocker nifedipine greatly inhibited carbachol induced contractions of both pig and guinea pig muscle strips (Rivera et al., 2006). The contribution of intracellular Ca2+ release in the generation of small contractions of the detrusor was investigated in muscle strips from human patients. In the presence of low doses of carbachol (to induce contraction), administration of thapsigargin (a potent irreversible inhibitor of the sarcoplasmic reticulum Ca2+ pump), inhibited the generation of muscle contractions, yet as progressively greater doses of carbachol were administered, the effect of thapsigargin was reduced, and contractions of the bladder detrusor muscle strips were only reduced by 8% relative to pre-treatment control (Masters et al., 1999). These findings suggest that the detrusor smooth muscle relies predominantly on release of Ca2+ via intracellular mechanisms to activate small contractions of the bladder but to a lesser extent for larger contractions of the detrusor, therefore leaving extracellular Ca2+ influx to contribute the majority of Ca2+ required to activate the contractile response (Masters et al., 1999).
Taken together, the evidence from the literature suggests that there are multiple sources of Ca2+ required for contraction of the detrusor muscle in both humans and other mammalian species as previously suggested (Maggi et al., 1989), and that an increase in the concentration of intracellular Ca2+ is essential for the activation of the contractile machinery in bladder detrusor muscle and consequently muscle contraction (Rivera et al., 2006).
However, despite this evidence, a study in muscle strips from the human and rabbit bladders provided evidence that contraction of the bladder was still activated in a Ca2+ free+ EGTA containing external environment, suggesting that neither stored Ca2+ nor the Ca2+ /calmodulin MLC kinase system is involved in some bladder contractions. It has been postulated that a Ca2+ independent mechanism exists which enables contraction of the bladder possibly via a PKC or muscarinic receptor mediated mechanism (Yoshimura et al., 1997).
1.6 The micturition reflex.
The function of the lower urinary tract is to store and periodically release urine at low pressure in a controlled fashion, a process known as micturition. The micturition reflex consists of two phases, the storage or filling phase, and the voiding phase.
Storage/filling phase
Urine is delivered from the kidneys to the bladder via the ureters without a significant change in intraluminal pressure because of the highly compliant nature of the bladder, a manifestation of the law of Laplace which states that the pressure in a sphere is equal to twice the wall tension divided by the radius. In the bladder as the bladder fills both the tension of the bladder wall and the radius of the bladder sphere increases, therefore maintaining a low intraluminal pressure until the bladder is relatively full (Morrison, 1997; Morrison et al., 2005).
Stretch receptors in the wall of the bladder project sensory information regarding the degree of bladder distension via pelvic afferent nerve fibres to the spinal cord and convey this information to the pontine micturition centre (PMC) and periaqueductal grey (PAG). As the stretch of the bladder wall is low during the initial part of the filling phase, afferent nerve firing is low thereby inhibiting the sacral parasympathetic nerves and exciting lumbar sympathetic preganglionic neurons (figure 1.12A). Stimulation of sympathetic neurons initiates the release of noradrenaline which acts on ß-adrenoceptors in the bladder detrusor muscle to cause relaxation of the muscle, and acts on α-adrenoceptors on the urethral sphincter causing contraction of the sphincter muscle and thereby restricting leakage of urine during bladder filling (de Groat, 2006). As the bladder becomes increasingly more filled, afferent nerve firing increases, and when this reaches threshold there is the desire to void, however in the healthy, continent adult, the switch from the storage to voiding phases is voluntary, therefore the voiding of urine can begin only when appropriate.
Voiding phase
When the volume of the bladder nears, reaches and exceeds micturition threshold, afferent nerve signals produce nerve firing in the sacral parasympathetic pathways and inhibit the sympathetic and somatic pathways (figure 1.12B). Voiding of urine consists of two phases, firstly parasympathetic mediated relaxation of the urethral sphincter by nitric oxide (Persson et al., 1992) as well as cessation of excitatory adrenergic and somatic inputs to the urethra. Secondly, parasympathetic nerves of the bladder release ACh which acts on muscarinic receptors on detrusor smooth muscle cells and elicits bladder contraction, mediated by muscarinic receptors, and an increase in intraluminal pressure, and flow of urine through the urethra (de Groat, 2006).
[pic]
Figure 1.12: Neural circuits controlling continence and micturition. A, The effective storage of urine, without leakage, relies on distension of the bladder stimulating low-level afferent nerve firing which stimulates the sympathetic nerve outflow to the bladder base and urethra, and the pudendal nerve outflow to the external urethral sphincter, thereby contacting the bladder outlet region and preventing leakage of urine. Simultaneously, sympathetic afferent nerve firing also inhibits the detrusor smooth muscle, thereby preventing contraction of the detrusor. These spinal reflex pathways promote continence. B, During the voiding reflex, augmented afferent nerve firing from the bladder activates the spinobulbospinal reflex pathways which pass via the spinal cord, through the pontine micturition centre, stimulating the parasympathetic outflow to the bladder and urethral smooth muscle. Simultaneously, the sympathetic and pudendal nerve outflow to the urethral outlet is inhibited. The detrusor muscle contacts and the outlet region relaxes, allowing the voiding of urine.
1.7 Bladder pathology and clinical implications
Overactive bladder syndrome is a debilitating and bothersome medical condition affecting significant numbers of people. Due to the embarrassing nature of the symptoms, and an acceptance of a gradual increased frequency associated with aging there is a reluctance to seek medical attention, suggesting that epidemiology figures are in fact probably much lower than the true figures. Nonetheless, using the standardised International Continence Society (ICS) figures, the overall prevalence is estimated at 11.8%, with a clear increase in aging. Urinary incontinence is predicted to affect approximately 200 million people worldwide (Vulker, 1998). Traditionally, more women were reported to be affected than men, due to pregnancy and childbirth, however more recently studies have suggested that there is a somewhat similar prevalence in males and females (Irwin et al., 2008).
Bladder disorders, such as urinary incontinence, overactive bladder and interstitial cystitis vary between patients, for example the differences between ‘OAB wet’, where there is urgency incontinence, and ‘OAB dry’, where there is no associated urgency incontinence.
Before discussing specific symptoms associated with individual disorders, it is important to understand the definitions of some key terms used clinically to describe bladder symptoms as provided by the International Continence Society (Abrams et al., 2002):-
1. Urgency – the complaint of a sudden compelling desire to pass urine, which is difficult to defer.
2. Detrusor overactivity (DO): a urodynamic observation characterised by involuntary detrusor contractions during the filling phase, which may be spontaneous or provoked.
3. Increased daytime frequency: a complaint by the patient who considers that he/she voids too often by day.
4. Nocturia: the complaint that the individual has to wake at night one or more times to void.
Overactive bladder syndrome (OAB)
Overactive bladder syndrome (OAB) is a symptom complex disorder associated with alterations in the storage function of the urinary bladder. OAB is characterised as ‘urgency, with or without urge incontinence, usually with frequency and nocturia’ (Wein et al., 2002). OAB can then be further characterised by the observation of the presence or absence or urge incontinence, and described as either ‘OAB wet’ which is associated with urgency incontinence, or ‘OAB dry’ where there is no associated urgency incontinence (Stewart et al., 2003). It is also important to consider urinary tract infections or bladder cancer as possible causes of these symptoms, before final diagnosis.
OAB can be classified according to its aetiology:-
1. Idiopathic overactive bladder – bladder overactivity with no association to a relevant neurological condition, and with no definable cause
2. Neurogenic bladder dysfunction – bladder overactivity associated with a relevant neurological condition, such as degeneration of neural pathways for example in syringomyelia, or following injury to the spinal cord
The control of urination requires the intact signalling between the brain and the lower urinary tract, therefore when this is disrupted, at any level, the brain is no longer able to send signals to the spinal cord and communicate with organs below the level of injury. In neurogenic bladder dysfunction, the physiological signals to inhibit bladder activity (during the filling phase) or to initiate contraction (during voiding) are not conveyed, and dependent on the site of the lesion on the spinal cord the cause of neurogenic bladder dysfunction can be defined. Neurogenic bladder dysfunction can therefore be classified by the site of spinal cord lesion:-
1. Supraspinal lesions – cerebral diseases such as cerebral haemorrhage, tumours, dementia and Parkinson’s disease can cause bladder overactivity. When the bladder fills there is an automatic trigger to initiate voiding due to the absence of cerebral inhibition of micturition. However, if the pons and pontine micturition centre are unaffected by the lesion, the external urethral sphincter still functions in a coordinated manner with detrusor contractions.
2. Suprasacral lesions – refers to all spinal cord lesions occurring above the sacrum, and is the most common cause of spinal cord injury related neurogenic OAB. Following spinal cord injury there is a period of ‘spinal shock’ in which there is little neurological activity and an inability of the bladder detrusor muscle to contract, thereby causing urinary retention. This period can last several months following spinal cord injury. Reflex activity gradually recovers and bladder contractions return but are often uninhibited and uncoordinated due to loss of control from higher brain centres, resulting in a hyperactive bladder and incomplete and involuntary voiding. Additionally, in the majority of suprasacral spinal cord injuries detrusor sphincter dyssynergia develops, where coordination between the urethral sphincter and bladder is disrupted, therefore instead of sphincter relaxation to aid voiding, the urethral sphincter remains contracted and the detrusor muscle contracts against a closed urethral output. This results in urinary retention and vesicoureteral reflux, where the bladder is less compliant, and the bladder sphincters impair the exit of urine, leading to reflux of urine into the ureter and kidney, resulting in renal impairment.
3. Infrasacral lesions – refers to all lesions occurring to motor sensory neurones outside the spinal canal. The sensation of the fullness of the bladder is lost, and there is no urge to urinate, leading to overdistension of the bladder and urinary retention.
Types of urinary incontinence
Urinary incontinence is a medical condition which affects approximately 14 million people, of all ages, in the UK alone. Incontinence may or may not be associated with OAB depending on patient symptoms and diagnosis of ‘OAB wet’ or ‘OAB dry’(Stewart et al., 2003). Urinary incontinence is described as the ‘involuntary loss of urine that is a social or hygienic problem’ (Abrams et al., 2002). There are several types of urinary incontinence:-
1. Stress incontinence – involuntary leakage of urine when intra-abdominal pressure is increased during physical activities such as coughing, sneezing and laughing.
2. Urge incontinence – involuntary leakage of urine immediately preceded by the sensation of urgency
3. Mixed incontinence – involuntary leakage of urine associated with urgency and as a result of sneezing, coughing, laughing, etc.
4. Overflow incontinence – loss of urine from an overfilled bladder with no feeling or urge to pass urine
5. Nocturnal enuresis – involuntary loss of urine during sleep
6. Post micturition dribble – occurs in men, who experience an involuntary loss of urine immediately after they finish passing urine, usually after they have left the toilet.
Causes of detrusor overactivity
The mechanisms underlying the pathogenesis of detrusor overactivity are unclear but are likely due to dysfunction of the detrusor muscle (myogenic), nervous system control (neurogenic) or urothelium (urotheliogenic) of the bladder.
A myogenic basis for detrusor overactivity.
The healthy bladder is required to be highly compliant as it must be able to store an increasing volume of urine, as the bladder fills, whilst keeping intraluminal pressure low, yet expel the contents rapidly during voiding by stimulating synchronous contractions and rapid elevation of intraluminal pressure. In cell recordings from smooth muscle cells of the detrusor, it has been shown that the cells are spontaneously active, therefore ready to respond to stimulation enabling synchronised contraction. However, during the filling phase of the micturition reflex, there is poor electrical coupling of smooth muscle cells, and the activity of these cells is not synchronous therefore large sustained contractions of the detrusor smooth muscle (as during voiding) are avoided (Brading, 1997).
However, detrusor muscle strips dissected from unstable bladders show abnormal spontaneous activity of the muscle strips associated with increased electrical coupling between muscle cells and therefore the generation of synchronised, coordinated contractions of the detrusor (Brading, 1997). In addition, spontaneous activity of individual isolated cells in overactive bladder tissue samples is increased (Sui et al., 2009), and this has been hypothesised to be as a result of aberrant Ca2+ regulation . L-type and T-type Ca2+ currents can be measured in detrusor smooth muscle cells, however in bladders showing detrusor overactivity the proportion of T-type current is increased (Sui et al., 2003), and as T type channels are opened at more negative membrane potentials, increased T-type channel density would increase the spontaneous electrical activity of detrusor muscle cells (Yanai et al., 2006).
In addition, in samples of OAB detrusor muscle from the human and porcine bladders, there is hypersensitivity to applied agonists such as muscarinic receptor agonists and depolarisation by intraluminal perfusion of potassium chloride (KCl) solution (Sibley, 1987; Yokoyama et al., 1991). Strips from human bladders from patients with unstable, obstructed bladders have also been shown to develop atropine and TTX-resistant contractions following direct electrical stimulation of the detrusor muscle, that are absent in normal, healthy tissue samples (Brading, 1997) suggesting that increased sensitivity of detrusor smooth muscle cells could contribute to the generation of bladder overactivity.
The integrative hypothesis of detrusor overactivity
The integrative hypothesis suggests that localised, spontaneous contractions in the detrusor muscle, known as micro-motions, are a normal, physiological phenomena which indicate the state of bladder filling. These micro-motions are purported to be determined by interactions of the various cell types in the bladder wall, e.g. interstitial cells (Drake, 2007). Altered properties or signalling properties in these cells has been shown to result in exaggerated micro-contractions and therefore overactive bladder and detrusor overactivity symptoms (Drake et al., 2001).
A neurogenic basis for detrusor overactivity
Bladder overactivity could also been induced by changes in peripheral and central nervous control, as shown by lesions of the spinal cord in humans and in animal models. The cerebral cortex has an important role in inhibiting voiding; therefore a lesion in the suprapontine region reduces the central nervous system inhibition of muscle activity, thereby inducing overactivity of the muscle. This mechanism is also supported by the observation that patients with Parkinson’s disease develop overactivity of the bladder, and also suggests a role of dopaminergic neurons in the control of micturition (de Groat, 1997; Yoshimura et al., 1993).
Spinal cord injury also induces changes in the afferent nerve system of the bladder. In the healthy bladder, the afferent limb of the micturition reflex is mediated by myelinated Aδ fibres, whereas in cats with chronic spinal cord injury there is considerable reorganisation of the afferent signalling, and signalling via unmyelinated C fibre afferents is facilitated whilst signalling via Aδ fibres is inhibited, consistent with the idea of afferent axonal sprouting following spinal cord injury (de Groat, 1997; de Groat et al., 1990).
Studies in the rat bladder have implicated a change in muscarinic receptor subtype expression following injury of the spinal cord in the mechanism by which activity of the detrusor muscle is increased. Firstly, the frequency-response characteristics of muscarinic receptor signalling was shown to be shifted, so that lower frequencies of stimulation were required to elicit ACh release thereby enhancing the parasympathetic input to the bladder detrusor and contributing to hyperreflexia in spinal cord injured animals (de Groat, 1997). It has also been shown that in the neurogenic bladder, the predominant cholinergic receptor subtype on the detrusor muscle differs from control bladders, and the M2 receptor population becomes predominant in mediating detrusor muscle contractions, rather than the M3 receptor population as in healthy controls, however how this alteration influences overactivity of the detrusor remains unclear (Braverman et al., 2006a; Braverman et al., 2003)
A urotheliogenic basis for detrusor overactivity
More recently, the urothelium has been shown to influence detrusor muscle activity (Ikeda et al., 2008), both increasing and decreasing detrusor muscle activity. In the isolated porcine bladder, the magnitude of carbachol induced and nerve mediated contractions of the detrusor was inhibited if the urothelium and suburothelium were left intact, or indeed if a urothelial strip was placed into the same organ bath recording chamber, suggesting the release of an diffusible inhibitory mediator that modulates detrusor muscle contractility (Hawthorn et al., 2000). However, as yet, the identity of this mediator remains a mystery.
Other studies have shown that spontaneous contractile activity of the detrusor is increased if the urothelium is left intact, showing that spontaneous activity of the detrusor muscle often originates in the suburothelial and urothelial layers and spreads to the detrusor muscle layer (Kanai et al., 2007). Consistent with this finding, another study demonstrated an increase in the number of suburothelial interstitial cells in bladders with detrusor overactivity (Ikeda et al., 2007) suggesting a mechanism of generating increased activity in detrusor overactivity.
Clinical treatment of OAB
At present, if suitable, anti-muscarinic drugs, including fesoterodine and oxybutynin are used in the treatment of overactive bladder. These drugs prevent neuromuscular transmission by blockade of muscarinic receptor signalling, thereby prevent leakage of urine. However, patients report bothersome side effects from taking these drugs, including, dry mouth and difficulty swallowing, thirst, blurred vision and difficulty accommodating to and sensitivity towards light, dry, hot flushed skin, heart palpations and arrhythmias and ironically, urinary retention due to a difficulty in voiding urine.
As such, the area for drug development is still evolving, with new therapies under research such as sacral neuromodulation, tibial nerve stimulation and botulinum neurotoxin-A injections, which prevent the synaptic release of neurotransmitters, especially from cholinergic terminals thereby reducing bladder contractions and afferent nerve fibre activity.
Interstitial cystitis (IC) and painful bladder syndrome (PBS)
Interstitial cystitis (IC) is a disorder in which there is a complaint of suprapubic pain related to bladder filling accompanied by symptoms such as increased daytime (>8 times) and night-time (>1 times) voiding frequency, and histological evidence of urothelial damage, including presence of inflammatory cells and mast cell infiltration, in the absence of infection or other pathology. Painful bladder syndrome (PBS) has a similar presentation as IC, but without the histological evidence of urothelial damage (Warren et al., 2006).
Damage of the urothelium in patients with IC can be diagnosed by the sensation of pain during intraluminal infusion of potassium chloride solution into the bladder of the patient, suggesting that the barrier function of the urothelium has been comprised and permeability of the urothelium has been increased, therefore there is direct activation of sensory nerve fibres causing the painful sensation (Parsons et al., 1998).
Treatment of IC includes oral administration of analgesics and anti-inflammatory medicines in order to reduce the painful sensation alongside pentosan polysulfate sodium (Elmiron), and antihistamines. Intraluminal administration of dimethyl sulfoxide (DMSO) has also been FDA approved (US Food and Drug Administration), Laboratory experiments in animals have suggested a down regulation of afferent nerve firing via nitric oxide signalling following DMSO treatment, suggesting this is the mechanism by which DMSO provides amelioration of symptoms (Birder et al., 1997). Transcutaneous electrical nerve stimulation (TENS) is also effective in some patients in preventing the symptoms of IC.
1.8 Aim of this thesis
The overall aim of this thesis is ‘to investigate the role of the urothelium in mediating sensory signalling from the bladder’ and provide direct evidence to support the hypothesis that the urothelium and afferent nerve fibres are capable of communicating with each other and via modulation of urothelial mediator release the activity of afferent nerve fibres can be altered .
Understanding the influence of the urothelium on afferent nerve sensitivity both during and between distensions of the bladder could reveal future targets for therapeutic intervention for treatment of lower urinary tract disorders, but equally importantly, contribute to our understanding of normal bladder function and the importance of a functioning urothelium in mediating afferent sensitivity.
Experiments in this thesis have explored the effect of urothelial signalling on afferent nerve sensitivity by several methods:-
1. Inhibition of urothelial mediator release – see chapter 4
2. Stimulation of urothelial mediator release – see chapter 5
3. Damage to the urothelium and the effect of K+ stimulation – see chapter 5
4. Pharmacological attenuation of urothelial signalling pathways – see chapter 6
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-----------------------
B.
A.
360 X
17 X
C.
Urothelium
Lamina propria
Inner longitudinal muscle
Circular muscle
Outer longitudinal muscle
Mucosa
Detrusor
Lamina propria
B
Urothelium
Lamina propria
A.
B.
muscle
muscle/urothelial
urothelial
serosal
pelvic nerve
hypogastric nerve
pudendal nerve
................
................
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