TRP Channels and C-Fiber Pharmacotherapy

The superfamily of TRP (transient receptor potential) channels expressed in mammals are subdivided into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin) groups, which are Ca2+-permeable cation channels and activated by physical (depolarization, hot/cold temperature, mechanical stress) or chemical (pH, osmolality) stimuli and binding to specific ligands (vanilloids, menthol) (Krause et al, 2005; Everaerts et al, 2008). The available evidence suggests that TRP channels have a four-subunit combination, in either a homotetrameric or heterotetrameric complex, to form functional ion-permeation complexes (Krause et al, 2005).

TRPV1

TRPV1, the most extensively studied TRP channel, is expressed on capsaicin-sensitive afferent pathways, predominantly C-fiber nociceptors, and responds to increases in temperature to the noxious range (>43° C) and to protons, suggesting that it functions as a transducer of painful thermal stimuli and acidity in vivo. When it is activated, the channel opens, allowing an influx of Ca2+ and Na+ ions that depolarizes the nociceptive afferent terminals, initiating a nerve impulse that travels through afferent nerves into the central nervous system. Noxious temperature uses the same elements, which explains why the mouth feels hot when eating chili peppers (Clapham, 1997). In the lower urinary tract, TRPV1 is expressed in suburothelial afferent fibers, urothelium, detrusor smooth muscle, and other non-neuronal cells such as suburothelial interstitial cells (see Fig. 60–8). Studies using TRPV1 knockout mice showed that TRPV1 receptors are not essentially involved in conscious voiding but have a role in afferent sensitization due to cystitis because bladder overactivity induced by chemical cystitis, using cyclophosphamide or acrolein, was not observed in TRPV1 knockout mice (Birder et al, 2002; Charrua et al, 2007; Wang et al, 2008). In addition, TRPV1 expressed in the bladder urothelium may function as a stretch sensor because the release of ATP and NO from cultured urothelial cells during hypotonic stretch is reduced in TRPV1 knockout mice compared with the wild type (Birder et al, 2002).

Activation of TRPV1 by vanilloids, such as capsaicin and its ultrapotent analog resiniferatoxin (RTX), results in spikelike currents (Liu and Simon, 1996) and selectively excites and subsequently desensitizes TRPV1-expressing C fibers. Desensitization induced by capsaicin is defined as a long-lasting, reversible suppression of sensory neuron activity (Craft et al, 1995). Thus because of the unique ability to desensitize TRPV1-expressing C fibers, the vanilloids have been used for treatment of pain/overactivity of the bladder (Cheng et al, 1995; Chancellor and de Groat, 1999). The normal sensations of bladder filling appear to be mediated by small myelinated Aδ fibers. In the cat, Aδ fibers have pressure thresholds in the range of those at which humans report the first sensation of bladder filling (Janig and Morrison, 1986). C-fiber afferents, which are small and unmyelinated, have high mechanical thresholds and do not normally respond to even high levels of intravesical pressure in the cat (Häbler et al, 1990). C-fiber afferents are activated by noxious chemical irritation (Häbler et al, 1990) or by cold (Fall et al, 1990). In the irritated state, C-fiber afferents become responsive to low-pressure bladder distention such as mechanoreceptive Aδ fibers. C fibers, therefore, are normally “silent.” C-fibers have the specific function of signaling of inflammatory or noxious events in the bladder (Chancellor and de Groat, 1999) (Figs. 60-27 and 60-28).

RTX is the principal active ingredient in the drug euphorbium that is derived from the air-dried latex (resin) of the cactus-like plant Euphorbia resinifera. E. resinifera belongs to the Euphorbiaceae, commonly known as the spurge family, one of the most important families of medicinal plants. In 1975, the principal active ingredient in euphorbium was isolated and named resiniferatoxin (Hergenhahn et al, 1975). In 1989, RTX was recognized as an ultrapotent analog of capsaicin; however, it has unique pharmacologic effects as well (Szallasi and Blumberg, 1990), such as desensitization without prior excitation of the pulmonary chemoreflex pathway (Szolcsanyi, 1990). In patients with spinal cord injury–induced detrusor overactivity, clinical response to intravesical therapy with RTX led to a marked decrease of nerve fibers positively stained for PGP9.5, a neuronal marker, and TRPV1. Six of 17 patients in this investigation showed a satisfactory clinical response to RTX treatment, with marked improvements on cystometry and other parameters (Brady et al, 2004). Spinal-injury patients who did not respond to RTX showed no decrease in nerve fiber population, similar to controls. In addition, intravesical RTX administered to patients with idiopathic detrusor overactivity delayed or suppressed involuntary detrusor contractions during filling cystometry. The mean interval to the first involuntary contraction more than doubled versus baseline at 30 and 90 days; mean maximal cystometric capacity increased; the mean number of episodes of urinary incontinence daily fell to fewer than one; and mean daily frequency also decreased significantly (Silva et al 2002). It has also been reported that C-fiber desensitization induced by intravesical application of high-dose capsaicin and resiniferatoxin (RTX) is effective for treating painful symptoms in IC patients (Lazzeri et al, 1996, 2000) although a prospective, randomized clinical trial using intravesical RTX application showed no effect in patients with IC (Payne et al, 2005).

More recently, selective antagonists of TRPV1 channels are being evaluated as potential analgesics in clinical trials (Everaerts et al, 2008). GRC-6211, a new oral TRPV1 antagonist, has been shown to decrease bladder overactivity and noxious bladder input in cystitis animal models (Charrua et al, 2009), suggesting the possibility of TRPV1 antagonists for the treatment of bladder pain/overactivity.

TRPM8 and TRPA1

TRPM8 is a member of the temperature-sensitive TRP channels that responds to cold temperature of less than 23° C. Pharmacologic agents that evoke cool sensation, such as menthol and ilicin, can activate TRPM8. In sensory pathways, TRPM8 is expressed in DRG and trigeminal ganglion neurons that do not express TRPV1, isolectin-B4, or CGRP, which are usually markers of C-fiber afferents. Thus TRPM8 is considered to be expressed in a subpopulation of thermoceptive and nociceptive afferents that are different from the TRPV1-expressing subpopulation. In the human lower urinary tract, TRPM8 expression is found in the prostate, the testes, scrotal skin, and bladder (Stein et al, 2004). In addition, although the study by Stein and colleagues showed that expression in the human bladder was limited to the urothelium, Mukerji and colleagues (2006) showed TRPM8 immunoreactivity in the bladder urothelium, as well as in fine nerve fibers in the suburothelial layer, and that the number of TRPM8-positive C-fibers in the bladder suburothelium is increased in patients with idiopathic detrusor overactivity. In animal studies, activation of TRPM8 channels in the guinea pig bladder by intravesical application of menthol reduces volume threshold for micturition and increases sensitivity to bladder cooling (Tsukimi et al, 2005), while a TRPM8 antagonist, ammonium/methyl ammonium transport B (AMTB), decreases bladder contraction frequency without affecting contraction amplitude in cystometry, as well as the visceromotor reflex of abdominal muscle in response to noxious urinary bladder distention in rats (Lashinger et al, 2008). Therefore TRPM8 in bladder afferent pathways and urothelium could be involved in modulation of sensory function of the lower urinary tract.

TRPA1 is the only member of the ankyrin TRP channel and a receptor for several pungent chemicals that evoke pain, such as allyl-isothiocyanate (the pungent compound in mustard oil), allicin (garlic), cinnamaldehyde (in cinnamon), and acrolein (a metabolite of cyclophosphamide). TRPA1 also functions as a receptor-operated channel that can be activated by growth factors or proinflammatory peptides such as bradykinin, which increases intracellular Ca2+ levels using G protein–coupled receptors. TRPA1 is expressed in sensory neurons, in which it is coexpressed with TRPV1, but not with TRPM8. Although TRPA1 can be activated by cold (<17° C) by an increase in intracellular Ca2+ concentration when expressed in heterologous systems, its role as a cold sensor in native peripheral sensory neurons, including DRG cells, remains uncertain. In mice, cooling does not evoke unspecific rises in Ca2+ concentration in DRG neurons, while visceral sensory neurons in nodose ganglia exhibit a strong correlation between cold sensitivity and TRPA1 expression (Fajardo et al, 2008), suggesting that TRPA1 may contribute to cold transduction in visceral sensory neurons rather than somatic neurons (Caspani and Heppenstall, 2009). In the bladder, TRPA1 is expressed in the urothelium, TRPV1 and CGRP-positive suburothelial afferent nerves, and detrusor muscles in mice, rats, and humans (Nagata et al, 2005; Du et al, 2007, 2008; Streng et al, 2008). TRPA1 receptor activation by intravesical application of hydrogen sulfide, allyl isothiocyanate, and cinnamaldehyde induces frequent voiding as evidenced by a reduction in intercontraction intervals, which is suppressed by capsaicin-induced C-fiber desensitization in rats (Du et al, 2007; Streng et al, 2008). In addition, TRPA1 mRNA expression in the bladder mucosa from male patients with lower urinary tract symptoms due to bladder outlet obstruction is significantly increased compared with control subjects (Du et al, 2008). Thus TRPA1 expressed in the bladder urothelium and sensory pathways may have a role in sensory transduction in pathologic conditions, including overactive bladder.

In patients with various types of overactive bladder, the ice-water test, in which infusion of ice cold water in the bladder causes reflex detrusor contraction, has been used as a diagnostic tool to detect hyperexcitability of C-fiber bladder afferent pathways (Chancellor and de Groat, 1999). TRPM8 and TRPA1, both of which can be activated by cooling, may be responsible for activation of the bladder cooling reflex in the ice-water test, although the involvement of TRPA1 in cold transduction is still equivocal.

TRPV4

TRPV4 is a member of vanilloid TRPV channels and a nonselective cation channel activated by mechanical pressure, osmolality (hypotonicity), moderate warmth (>27° C), and chemical stimuli such as phorbol derivates. Its expression has been detected in urothelial cells and detrusor muscle, but not in the suburothelial layer, in the bladder of mice, rats, and guinea pigs (Birder, 2007; Gevaert et al, 2007; Thorneloe et al, 2008; Xu et al, 2009; Yamada et al, 2009). The TRPV4 agonist, 4α-phorbol 12,13-didecanoate, and hypotonic cell swelling promote Ca2+ influx and evoke ATP release in cultured urothelial cells from mice (Gevaert et al, 2007) or rats (Birder, 2007). In cultured urothelial cells from TRPV4 knockout mice, the intracellular Ca2+ increase and ATP release in response to stretch stimulation were significantly attenuated compared to the wild-type mice (Mochizuki et al, 2009). Cystometric experiments revealed that TRPV4 knockout mice exhibit a lower frequency of voiding contractions, as well as a higher frequency of nonvoiding contractions (Gevaert et al, 2007), and that intravesical application of TRPV4 agonists induces bladder overactivity as evidenced by increased micturition pressure in rats (Birder, 2007) or reduced contraction frequency in mice (Thorneloe et al, 2008). These results suggest that urothelial TRPV4 channels act as a pressure sensor to enhance bladder activity, predominantly through activation of bladder afferent pathways by urothelially released ATP.

Botulinum Toxin

In recent years, there has been increasing evidence for the therapeutic efficacy of botulinum neurotoxin (BoNT) for the treatment of various urethral and bladder dysfunctions (Smith and Chancellor, 2004; Apostolidis and Fowler, 2008).

Botulinum toxins act by inhibiting acetylcholine release at the presynaptic cholinergic nerve terminal, thereby inhibiting striated and smooth muscle contractions. The toxins are synthesized as single-chain polypeptides with a molecular weight of about 150 kD (DasGupta, 1994). Initially, the parent chain is cleaved into its active dichain polypeptide form, consisting of a heavy chain (approximately 100 kD) connected by a disulfide bond to a light chain (approximately 50 kD) with an associated zinc atom (Schiavo et al, 1992). Four steps are required for toxin-induced paralysis: binding of the toxin heavy chain to an as yet unidentified nerve terminal receptor, internalization of the toxin within the nerve terminal, translocation of the light chain into the cytosol, and inhibition of neurotransmitter release. Neurotransmitter release involves the ATP-dependent transport of the vesicle from the cytosol to the plasma membrane (Barinaga, 1993). Vesicle docking requires the interaction of various cytoplasm, vesicle, and target membrane proteins (i.e., synaptosome-associated membrane receptor [SNARE] proteins), some of which are specifically targeted with clostridial neurotoxins (Fig. 60–29). For example, BoNT-A cleaves the cytosolic translocation protein SNAP-25, thus preventing vesicle fusion with the plasma membrane (Fig. 60–30) (Schiavo et al, 1993).

Seven immunologically distinct neurotoxins are designated types A, B, C, D, E, F, and G. Clinically, the urologic community has used commercial preparations of BTX-A to treat patients with neurogenic and idiopathic detrusor overactivity (Dykstra et al, 1988; Dykstra and Sidi, 1990; Schurch et al, 1996; Petit et al, 1998; Schurch et al, 2000; Apostolidis et al, 2009). Although acetyl choline (ACh) release from bladder parasympathetic efferent terminals is the primary target of BoNT treatment, suppression of bladder afferent activity with BoNT treatment is also evident because the reduction of urgency symptom in patients with neurogenic and idiopathic detrusor overactivity is associated with reduced expression of the capsaicin receptor (TRPV1) and the ATP receptor (P2X3) in C-fibers (Apostolidis et al, 2005). In addition, in basic research, botulinum toxins are shown to suppress not only efferent nerve activity by inhibition of the release of acetylcholine but also afferent nerve activity by release of inhibition of neurotransmitters, such as substance P and CGRP, from sensory terminals (Chuang et al, 2004; Dressler et al, 2005). There is also evidence that the toxin can reduce the release of ATP from urothelial cells in spinalized rats (Khera et al, 2004; Smith et al, 2005; Smith et al, 2008). Thus the use of the toxins has been expanded to treat women with pelvic floor spasticity, as well as patients with non-neurogenic overactive bladder and even bladder pain syndrome (Smith et al, 2003; Smith and Chancellor, 2004; Smith et al, 2005; Apostolidis and Fowler, 2008). The efficacy of botulinum toxins was also recently identified in patients with benign prostatic hyperplasia, in whom BoNT-A injection into the prostate induced an atrophy of the prostate by inducing apoptosis, inhibiting proliferation, and downregulating α1A-adrenergic receptors (Chuang et al, 2006).

Actions of Drugs on Bladder Smooth Muscle

Two major classes of pharmacologic agents that are used clinically to inhibit spontaneous activity of the bladder are calcium channel blockers, such as diltiazem and verapamil, and potassium channel openers, such as cromakalim and pinacidil (see Table 60–5) (Andersson, 1993, 1997; Andersson et al, 1999). Also, the direct effect of tricyclic antidepressants on the bladder is briefly discussed.

Calcium Channel Blockers

In the bladder smooth muscle, both spontaneous and evoked contractile activities are mediated by membrane depolarization and the movement of calcium into the smooth muscle cell through L-type Ca2+ channels (Brading, 1997b; Fry and Wu, 1997; Brading, 2006). In addition, Ca2+ channels and nonselective stretch-activated divalent ion channels have been demonstrated in detrusor smooth muscle (Brading, 1997b; Fry and Wu, 1997; Martin et al, 1997). The one thing in common is that all contractile activity involves the entrance of extracellular calcium. Some forms of contractile responses are mediated by both the entrance of extracellular calcium and the release of intracellular calcium from the sarcoplasmic reticulum (Levin and Wein, 1995; Zderic et al, 1996; Brading, 1997b; Fry and Wu, 1997; Martin et al, 1997).

The relationship between various forms of contractile response and calcium translocation and release has been the subject of a large number of studies. Inhibition of the entrance of calcium can inhibit spontaneous and evoked contractile activity (Levin et al, 1991). L-type Ca2+-blocking agents, such as nifedipine, can reduce the level and severity of spontaneous myogenic contractile activity, which depends primarily on the entrance of extracellular calcium through L-type Ca2+ channels. Ca2+ channel blockers are less effective in suppressing nerve-mediated contractions, which are dependent on both the entrance of extracellular calcium and the stimulated release of intracellular calcium from the sarcoplasmic reticulum through IP3-mediated mechanisms (Levin and Wein, 1995; Zderic et al, 1996; Brading, 1997b; Andersson and Arner, 2004). This leads to the possibility of developing selective Ca2+-channel blocking agents that eliminate spontaneous contractions without affecting micturition contractions.

Potassium Channel Openers

K+ channel openers, such as cromakalim and pinacidil (see Table 60–5), stimulate the movement of potassium out of the cell, resulting in membrane hyperpolarization and a reduction in spontaneous contractile activity (Andersson, 1993; Andersson, 1997; Andersson et al, 1999). In the detrusor, various types of K+ channels have been identified, including ATP-sensitive (KATP), calcium-dependent small conductance (SKCa), and calcium-dependent large conductance (BKCa) channels (Andersson, 1992; Trivedi et al, 1995; Andersson and Arner, 2004; Gopalakrishnan and Shieh, 2004). It is currently still under investigation what the relationship is between each of these K+ channels and the different forms of contractile activity (myogenic contractions, neurogenic contractions, and micturition contractions). However, it is apparent that pharmacologic manipulation of both Ca2+ and K+ channels can result in reduced spontaneous contractile activity (Levin et al, 1991; Andersson, 1992; Trivedi et al, 1995; Brading, 1997b; Fry and Wu, 1997; Martin et al, 1997; Andersson and Arner, 2004). It has been documented that intravesical instillation of the bladder-selective KATP channel opener ZD6169 significantly reduced detrusor overactivity in bladder outlet–obstructed rats (Pandita and Andersson, 1999). The mechanisms of action may be both direct smooth muscle action and action on the afferent nerve endings near the urothelium (Hu and Kim, 1997; de Groat et al, 1999). Another KATP channel opener ZD0947 caused detrusor muscle relaxation through activation of glibenclamide-sensitive KATP channels in human and guinea pig urinary bladders (Aishima et al, 2006; Yunoki et al, 2008). Moreover, mice with the gene deletion of mSlo1 for the pore-forming subunit of the BKCa channel exhibit increased urinary bladder spontaneous and nerve-evoked contractions, as well as a marked elevation in urination frequency (Meredith et al, 2004). It has also been demonstrated that local injection of hSlo cDNA into the bladder lumen to increase BKCa channel activity eliminated detrusor overactivity in rats with bladder outlet obstruction (Christ et al, 2001), suggesting an important role of BKCa channels in urinary bladder function and overactive bladder. Thus there is considerable interest in the development of specific K+ channel openers for therapeutic use in the treatment of bladder overactivity (Pandita and Andersson, 1999; Andersson and Arner, 2004).

Tricyclic Antidepressants

Tricyclic antidepressants, such as imipramine and amitriptyline, have been shown to be clinically effective in reducing the level of detrusor overactivity under specific conditions (Wein, 1998). Their effectiveness has been linked to antimuscarinic activity, inhibition of calcium translocation, and direct smooth muscle relaxant properties, and possibly to an action on the central nervous system (Maggi et al, 1989). Each of these actions would relax the bladder and reduce the level of spontaneous activity. Because these are nonselective agents with multiple mechanisms of action, there is little research in the development of more bladder-selective tricyclic agents.

Spinal Ascending and Descending Pathways

It is possible to conjecture about the sites of action of drugs in the spinal cord on the basis of certain experimental designs. For example, alterations in the volume threshold for inducing micturition (in the absence of other changes of bladder activity) are interpreted as indicating an effect on the ascending limb of the micturition reflex. Alterations in the magnitude of bladder contractions (or pelvic nerve discharge) produced by PMC stimulation (Matsumoto et al, 1995b) may be taken to indicate an action on the descending limb of the micturition reflex.

Glutamatergic Mechanisms

Intrathecal or intravenous administration of glutamatergic NMDA or α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) antagonists in urethane-anesthetized rats depressed reflex bladder contractions and electromyographic activity of the external urethral sphincter in animals with an intact spinal cord, as well as in animals with chronic spinal injury (Yoshiyama et al, 1994, 1997). These results indicate that spinal reflex pathways controlling bladder and sphincter functions use NMDA and AMPA glutamatergic transmitter mechanisms. In spinal cord–injured rats, external sphincter muscle activity was more sensitive than bladder reflexes to glutamatergic antagonists, raising the possibility that the two reflex pathways might have different glutamatergic receptors (Yoshiyama et al, 1994). This was confirmed with in-situ hybridization techniques, which revealed that sacral parasympathetic preganglionic neurons in the rat express high mRNA levels of GluR-A and GluR-B AMPA receptor subunits and NR1, but not NR2 NMDA receptor subunits (Shibata et al, 1999). Conversely, motoneurons in the urethral sphincter nucleus express all four AMPA receptor subunits (GluR-A, GluR-B, GluR-C, and GluR-D) in conjunction with moderate amounts of NR2A and NR2B, as well as high levels of NR1 receptor subunits. It seems likely that this difference in expression accounts for the different sensitivity of bladder and sphincter reflexes to glutamatergic antagonists. In addition, intrathecal application of an mGluR agonist inhibits isovolumetric bladder contractions (Tanaka et al, 2003), although intrathecal application of an mGluR antagonist has no effects on bladder activity in rats (Yoshiyama and de Groat, 2007), suggesting that spinal mGluR are not tonically active, but can inhibit the micturition reflex when stimulated.

Glutamate also plays a role as an excitatory transmitter in the afferent limb of the micturition reflex. The c-FOS expression induced in spinal interneurons by activation of bladder afferents is suppressed by the administration of both NMDA and non-NMDA glutamatergic receptor antagonists (Birder and de Groat, 1992; Kakizaki et al, 1996).

Inhibitory Amino Acids

Intrathecal injection of either GABAA or GABAB agonists increases bladder capacity and decreases voiding pressure and efficiency in rats (Igawa et al, 1993). Glycine and GABA inhibitory mechanisms have also been identified in the neonatal rat spinal cord in local interneuronal inhibitory pathways projecting directly to the preganglionic neurons (Araki, 1994). Clinical studies have revealed that intrathecal administration of a GABAB receptor agonist (baclofen) increased the volume threshold for inducing the micturition reflex (Bushman et al, 1993). Intrathecally administered baclofen also produced a phaclofen-sensitive inhibition of distention-evoked micturition in conscious rats that appears to be resistant to capsaicin (substance P depletion) or parachlorophenylalanine (5-HT depletion) pretreatment. Because baclofen also inhibits field stimulation–evoked release of CGRP from primary afferent terminals in dorsal horn slices, one possible site for this action is suppression of transmitter release from primary afferent terminals in the spinal cord. In addition, studies by Miyazato and colleagues (2003, 2005) have revealed that the level of glycine in the spinal cord is decreased by approximately 50% in rats with detrusor overactivity induced by chronic spinal cord injury compared with spinal intact rats, and that dietary supplement with glycine can restore bladder function, along with an increase in the serum level of glycine in spinal cord–injured rats. The level of glutamic acid decarboxylase, a GABA synthesis enzyme, is also reduced in lumbosacral DRG and the spinal cord in spinal cord–injured rats, and intrathecal application of GABAA or GABAB receptor agonists suppresses detrusor overactivity and sphincter-detrusor dyssynergia (Miyazato et al, 2008). These results suggest that downregulation of the spinal glycinergic and GABAergic mechanisms may contribute to the emergence of neurogenic detrusor overactivity associated with spinal cord injury.

Adrenergic Mechanisms

In the spinal cord, α adrenoceptors can mediate excitatory and inhibitory influences on the lower urinary tract. In anesthetized cats, α1 adrenoceptors were implicated in a bulbospinal noradrenergic excitatory pathway from the locus ceruleus to the sacral parasympathetic outflow to bladder (Yoshimura et al, 1988, 1990), although subsequent studies could not confirm these findings in conscious cats (Espey et al, 1992).

Experiments in conscious or anesthetized rats (Ishizuka et al, 1996; de Groat et al, 1999) revealed that intrathecal administration of an α1-adrenergic antagonist (doxazosin) decreased the amplitude of bladder contractions (Ishizuka et al, 1996, 1997). The bladder inhibitory effect of the intrathecal α1-adrenergic antagonist was more prominent in animals with chronic outlet obstruction (Ishizuka et al, 1996). Also, intrathecal administration of doxazosin suppressed detrusor overactivity (unstable bladder contractions) in spontaneously hypertensive rats (Persson et al, 1997). Although intrathecal injection of doxazosin suppressed the amplitude of reflex bladder contractions in anesthetized rats, it increased the frequency of isovolumetric contractions, indicating the presence of a tonic adrenergic inhibitory mechanism (de Groat et al, 1999). This was supported by the finding that phenylephrine, an α1-adrenergic agonist, applied intrathecally, decreased the frequency of bladder contractions without changing contraction amplitude (de Groat et al, 1999). Thus it appears that efferent and afferent limbs of the micturition reflex receive excitatory and inhibitory input, respectively, from spinal noradrenergic systems.

Evidence for a modulatory role of α2 adrenoceptors in micturition is conflicting because both facilitatory and inhibitory roles of α2 adrenoceptors have been documented (Ishizuka et al, 1996; de Groat et al, 1999). Atipamezole, an α2-adrenergic antagonist given intrathecally, can increase micturition pressure in the conscious rat, implying that there is a tonic inhibitory adrenergic control (Ishizuka et al, 1996). However, yohimbine, an α2-adrenergic antagonist, inhibits micturition in rats anesthetized with chloralose-urethane (Kontani et al, 1992). In paraplegic patients, intrathecal injection of clonidine suppressed detrusor overactivity (Denys et al, 1998). Conversely, in conscious spinal cats, clonidine, an α2-adrenergic agonist, increased bladder pressures and facilitated voiding (Galeano et al, 1986).

Pharmacologic experiments showed that the bladder-sympathetic reflex pathway is modulated by central noradrenergic mechanisms (Danuser and Thor, 1995; de Groat et al, 1999; de Groat and Yoshimura, 2001). In the chloralose-anesthetized cat, prazosin or doxazosin, α1- adrenoceptor antagonists, suppressed spontaneous firing (Ramage and Wyllie, 1995) or the reflex discharge recorded on the hypogastric nerve in response to pelvic nerve afferent stimulation (Danuser and Thor, 1995). Administration of α2-adrenergic agonists also suppresses reflex sympathetic activity (Danuser and Thor, 1995). These observations suggest that bulbospinal noradrenergic pathways provide a tonic α1-excitatory control of the bladder-sympathetic reflex in the spinal cord. α2-Adrenergic inhibitory mechanisms are not active under control conditions in anesthetized animals but can be upregulated by elevating endogenous norepinephrine levels with an inhibitor (tomoxetine) of norepinephrine reuptake (Danuser and Thor, 1995). These results suggest that the lumbar sympathetic outflow is controlled by α1-excitatory and α2-inhibitory mechanisms.

The activation of urethral sphincter motoneurons by stimulation of bladder (pelvic nerve) or urethral or perineal (pudendal nerve) afferents is part of a continence-maintaining mechanism. These reflexes, recorded as efferent discharges on the pudendal nerve in chloralose-anesthetized cats, were suppressed by the α1-adrenoceptor antagonist prazosin (Gajewski et al, 1984; Danuser and Thor, 1995; Downie, 1999), but not by the α2 blocker idazoxan (Danuser and Thor, 1995). Conversely, clonidine, an α2-adrenoceptor agonist, suppressed the reflex in anesthetized cats (Downie and Bialik, 1988). The norepinephrine uptake blocker tomoxetine produced a slight inhibition alone and only a slightly greater inhibition after prazosin. However, it greatly facilitated the reflex when given after idazoxan (Danuser et al, 1995). These data indicate the existence of α2 adrenoceptor–mediated inhibition and α1 adrenoceptor–mediated tonic facilitation of sphincter function, and that the α2 adrenoceptor–dependent inhibitory mechanism is the dominant adrenergic modulator of the pudendal nerve reflex (Thor and Donatucci, 2004). These excitatory α1 and inhibitory α2 adrenoceptor–mediated modulations of external sphincter function are also involved in the urethral continence reflexes during passive intravesical pressure rises and sneezing. Nisoxetine, a norepinephrine uptake blocker, induces an increase in pressure responses at the middle urethra measured by a microtransducer-tipped catheter during sneezing, which is antagonized by prazosin, an α1 adrenoceptor antagonist, in normal rats and rats with simulated birth trauma induced by vaginal distention (Kaiho et al, 2007). On the contrary, external urethral sphincter–electromyographic (EUS-EMG) activity that increased during lower abdominal wall compression was reduced by medetomidine, an α2-adrenoceptor antagonist, in rats (Furuta et al, 2009).

Serotonergic Mechanisms

Neurons containing 5-HT in the raphe nucleus of the caudal brainstem send projections to the dorsal horn, as well as to the autonomic and sphincter motor nuclei in the lumbosacral spinal cord. In cats, activation of raphe neurons or 5-HT receptors in the spinal cord inhibits reflex bladder contractions and firing of the sacral efferent pathways to the bladder (McMahon and Spillane, 1982; Chen et al, 1993; de Groat et al, 1993a; de Groat, 2002) and also inhibits firing of spinal dorsal horn neurons elicited by stimulation of pelvic nerve afferents (Fukuda and Koga, 1991). In rats, administration of 5-HT2C receptors agonists, such as m-chlorophenylpiperazine, Ro 60-0175, and WAY 161503, suppresses efferent activity on bladder nerves and/or the micturition reflex (Steers and de Groat, 1989; Mbaki and Ramage, 2008). These effects are blocked by mesulergine, a 5-HT2 receptor antagonist (Steers and de Groat, 1989; Guarneri et al, 1996), or by SB 242084, a 5-HT2C antagonist (Mbaki and Ramage, 2008). Intrathecal administration of methysergide, a 5-HT1/2 antagonist, or zatosetron, a 5-HT3 antagonist, decreases the micturition volume threshold in cats (Espey et al, 1998), implying that descending serotonergic pathways tonically depress the afferent limb of the micturition reflex.

8-OH-DPAT, a 5-HT1A agonist, administered intrathecally, facilitated bladder activity in both normal and spinal cord–injured rats but not in rats in which bladder afferents were damaged by treatment with capsaicin at birth (Lecci et al, 1992). Conversely, administration of the 5-HT1A receptor antagonist WAY 100635, which increases the firing rate of raphe neurons by blocking 5-HT1A inhibitory autoreceptors, inhibits reflex bladder contractions (Testa et al, 1999). The inhibition is antagonized by pretreatment with mesulergine, a 5-HT2 receptor antagonist, indicating that 5-HT2 receptors are involved in descending raphe and spinal inhibitory mechanisms (Testa et al, 1999). When the effects of intrathecal administration of WAY 100635 on the ascending and descending limbs of the micturition reflex pathway were examined in anesthetized rats, WAY 100635 depressed bladder contractions evoked by electrical stimulation of the PMC but did not alter the evoked field potentials in the region during electrical stimulation of afferent axons in the pelvic nerve, indicating that the drug suppresses the pathway from the brainstem to the spinal cord but does not alter the afferent pathway from the bladder to the PMC (Kakizaki et al, 2001; de Groat, 2002). However, in contrast to 5-HT1A receptor-mediated facilitatory effects on bladder activity in rats, 5-HT1A receptor activation exerts inhibitory effects in cats, especially when C-fiber afferent function is unregulated because 8-OH-DPAT induces dose-dependent increases in threshold volume and bladder capacity in cats with acetic acid–induced cystitis (Thor et al, 2002) or spinal cord injury (Gu et al, 2004; Gu et al, 2007). Thus micturition in the rat is facilitated by stimulation of 5HT1A inhibitory autoreceptors, whereas in the cat 5HT1A receptor activation appears to act primarily through postsynaptic mechanisms to suppress bladder activity.

The sympathetic autonomic nuclei, as well as the sphincter motor nuclei, also receive a serotonergic input from the raphe nucleus (de Groat et al, 1979; Downie, 1999; Thor and Donatucci, 2004). Serotonergic activity mediated by 5-HT2 and 5-HT3 receptors enhances urine storage by facilitating sphincter reflexes in acts and rats (Danuser and Thor, 1996; Espey et al, 1998; Mbaki and Ramage, 2008). The role of 5-HT1A receptors in the control of EUS activity may be different depending on animal species or experimental conditions. Activation of 5-HT1A receptors with 8-OH-DPAT increased external sphincter activity during stimulation of the pelvic nerve in normal and spinalized rats, and this effect was reversed by WAY-100635 (Chang et al, 2006, 2007). In cats, both 8-OH-DPAT and 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), 5-HT1A receptor agonists, cause dose-dependent decreases in bladder activity and increases in EUS-EMG activity especially when acetic acid was infused into the bladder to induce bladder irritation (Thor et al, 2002). However, another study in urethane-anesthetized chronic spinal cord injured rats has shown that 8-OH-DPAT induces periodic external urethral sphincter relaxation during voiding, suggesting the 5-HT1A-mediated suppression of sphincter activity (Dolber et al, 2007).

Duloxetine, a combined norepinephrine and 5-HT reuptake inhibitor (Sharma et al, 2000), has been shown, in a bladder-irritated model, to increase the neural activity of both the urethral sphincter and the bladder (Thor and Katofiasc, 1995; Thor and Donatucci, 2004). Duloxetine appears to have effects on both the bladder and the sphincter and has been proposed for treatment of both stress incontinence and urgency incontinence (Cannon et al, 2003; Thor and Donatucci, 2004). Duloxetine increases the neural activity to the external urethral sphincter and decreases bladder activity through effects on the central nervous system in cats (Thor and Donatucci, 2004). In a rat study, duloxetine also enhances the urethral continence reflex during sneezing as evidenced by an increase in sneeze-induced pressure responses at the middle urethra, although the effect appears to be mainly mediated by α1 adrenoceptors (Miyazato et al, 2008). Clinical trials have also shown the efficacy of duloxetine for the treatment of stress urinary incontinence; the drug has been approved in Europe and is already available in several countries (Castro-Diaz and Amoros, 2005), although it has been withdrawn from the Food and Drug Administration approval process in the United States by the manufacturer.

Opioid Peptides

Opioid peptides have an inhibitory action on reflex pathways in the spinal cord. In the cat spinal cord, inhibition of reflex bladder activity is mediated by δ receptors, whereas inhibition of sphincter activity is mediated by κ receptors (de Groat et al, 1993a; Yoshimura and de Groat, 1997; de Groat and Yoshimura, 2001; Gu et al, 2004). In the rat, both µ and δ receptors mediate bladder inhibition (Dray and Metsch, 1984b). Enkephalin gene transfer to bladder afferent pathways using herpes simplex virus vectors reduces pain behavior and bladder overactivity induced by capsaicin injection into the bladder by activation of spinal opioid receptors in rats (Yokoyama et al, 2008).

Pontine Micturition Center and Supraspinal Mechanisms

Glutamatergic Mechanisms

Glutamic acid also has a role in excitatory transmission at supraspinal sites in the micturition reflex pathway (See Fig. 60–31 on the Expert Consult website). imageExogenous L-glutamate, or its analog, were injected at sites (locus ceruleus or parabrachial nucleus) in the brainstem of supracollicular decerebrate cats, where electrical stimulation–evoked bladder contractions elicited voiding or increased frequency and amplitude of rhythmic bladder contractions (Kruse et al, 1990; Mallory et al, 1991).

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Figure 60–31 Diagram of the central reflex pathways that regulate micturition in the cat. Normally, micturition is initiated by a supraspinal reflex pathway passing through the pontine micturition center (PMC) in the brainstem. The pathway is triggered by myelinated afferents (Aδ) connected to tension receptors in the bladder wall (detrusor). Spinal tract neurons carry information to the brain. During micturition, pathways from the PMC activate the parasympathetic outflow to the bladder and inhibit the somatic outflow to the urethral sphincter. Transmission in the PMC is modulated by cortical-diencephalic mechanisms. Interruption of these mechanisms leads to bladder instability. In spinal cord–transected animals, connections between the brainstem and the sacral spinal cord are interrupted and micturition is initially blocked. In animals with chronic spinal cord injury, a spinal micturition reflex emerges that is triggered by unmyelinated (C-fiber) bladder afferents. The C-fiber reflex pathway is usually weak or undetectable in animals with an intact nervous system. Stimulation of the C-fiber bladder afferents by instillation of ice water into the bladder (cold stimulation) activates voiding reflexes in patients with spinal cord injury. Capsaicin (20 to 30 mg/kg subcutaneously) blocks the C-fiber reflexes in cats with chronic spinal cord injury but does not block micturition reflexes in intact cats. Intravesical capsaicin also suppresses bladder instability and cold-evoked reflexes in patients with neurogenic bladder dysfunction. Glutamic acid is the principal excitatory transmitter in the ascending and descending limbs of the micturition reflex pathway, as well as in the reflex pathway controlling sphincter function. Glutamate acts on both N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamatergic receptors. Other neurotransmitters that regulate transmission in the micturition reflex pathway include γ-aminobutyric acid (GABA), enkephalins (Enk), acetylcholine (ACh), and dopamine (DA). Acetylcholine and dopamine have both excitatory and inhibitory effects on the pathway; excitatory (+) and inhibitory (−) synapse.

Administration of glutamatergic agonists into the region of the PMC in cats and rats elicits voiding or increases frequency and amplitude of bladder contractions (Mallory et al, 1991; de Groat et al, 1999), whereas injection of agonists at other brainstem nuclei known to have inhibitory functions in micturition elicits inhibitory effects (Chen et al, 1993). Intracerebroventricular injection of AMPA or NMDA receptor antagonists blocks reflex bladder contractions in anesthetized rats, indicating that glutamatergic transmission in the brain is essential for voiding function (Yoshiyama and De Groat, 1996; Yoshiyama and de Groat, 2005). Similarly, Yokoyama and colleagues (1999) have shown that glutamate plays an important role, especially through NMDA receptors, in the rat middle cerebral artery occlusion model of detrusor overactivity.

Cholinergic Mechanisms

Excitatory and inhibitory cholinergic influences on the micturition pathway have been identified at the supraspinal level. A decreased volume threshold and increased micturition pressure were detected after administration of bethanechol, a muscarinic agonist, into the central circulation of the cross-perfused dog (O’Donnell, 1990). One site of action can be localized to the midbrain-pons region, because cholinergic agonists are effective after supracollicular decerebration in rats (Sillen et al, 1982). In the rat brain, muscarinic receptor–mediated cholinergic mechanisms may be involved in both inhibitory and facilitatory modulation of the micturition reflex (Ishiura et al, 2001; Yokoyama et al, 2001; Ishizuka et al, 2002), and the muscarinic inhibitory mechanism seems to involve an activation of M1 muscarinic receptors (Yokoyama et al, 2001; Masuda et al, 2009) and protein kinase C (Nakamura et al, 2003). In the brainstem, microinjection of acetylcholine to the PMC in cats increased or decreased the threshold volume for inducing a reflex contraction of the bladder (Sugaya et al, 1987; Yoshimura and de Groat, 1997). These effects were blocked by atropine, indicating a role for muscarinic receptors. Nicotinic receptors are also involved in the control of voiding function because a nicotinic receptor agonist, epibatidine, injected into the lateral ventricle suppressed the micturition reflex in the rat (Lee et al, 2003).

GABAergic Mechanisms

GABA has been implicated as an inhibitory transmitter at supraspinal sites where it can act on both GABAA and GABAB receptors (de Groat and Booth, 1993; Yoshimura and de Groat, 1997; de Groat et al, 1999; Kanie et al, 2000; de Groat and Yoshimura, 2001). Injection of GABA or muscimol, a GABAA receptor agonist, into the PMC of decerebrate cats suppressed reflex bladder activity and increased the volume threshold for inducing micturition (Mallory et al, 1991). These effects were reversed by bicuculline, a GABAA receptor antagonist. Because bicuculline alone stimulated bladder activity and lowered the volume threshold for micturition, the micturition reflex pathway in the PMC must be tonically inhibited by a GABAergic mechanism. Intracerebroventricular baclofen, a GABAB agonist, suppressed distention-evoked micturition in urethane-anesthetized rats, but the effect was not blocked by phaclofen, a GABAB receptor antagonist (de Groat and Yoshimura, 2001; de Groat et al, 1993a).

Dopaminergic Mechanisms

In the central nervous system, dopaminergic pathways exert inhibitory and facilitatory effects, respectively, on the micturition reflex through D1-like (D1 or D5 subtype) and D2-like (D2, D3, or D4 subtype) dopaminergic receptors (Albanese et al, 1988; Kontani et al, 1990; Yoshimura et al, 1992; Yoshimura et al, 1993, 1998, 2003; Yokoyama et al, 1999; Seki et al, 2001; Hashimoto et al, 2003) (see Fig. 60–31 on the Expert Consult website). imageIn anesthetized cats, activation of dopaminergic neurons in the substantia nigra inhibits reflex bladder contractions through D1-like receptors (Yoshimura et al, 1992). A study also revealed that a D1 dopaminergic antagonist (SCH 23390) facilitated the micturition reflex, whereas a D1 agonist (SKF 38393) had no effect on the reflex bladder contractions in awake rats, suggesting that D1 receptor–mediated suppression of bladder activity is tonically active in the normal awake state (Seki et al, 2001). Disruption of this tonic dopaminergic inhibition by destroying the nigrostriatal pathway with the neurotoxin methyphenyltetrahydropyridine (MPTP) produces Parkinson-like motor symptoms in monkeys accompanied by detrusor overactivity (Albanese et al, 1988), as reported in patients with Parkinson disease (Albanese et al, 1988; Yoshimura et al, 1993; Steers et al, 1996). Similarly, a rat model of Parkinson disease induced by a unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway also exhibits detrusor overactivity. In these parkinsonian animals, detrusor overactivity was suppressed by stimulation of D1-like receptors with SKF 38393 or pergolide (Yoshimura et al, 1993, 1998, 2003).

Conversely, activation of central D2-like dopaminergic receptors with quinpirole or bromocriptine facilitates the micturition reflex pathway in rats, cats, and monkeys (Kontani et al, 1990; Yoshimura et al, 1993, 1998, 2003; Yokoyama et al, 1999). D2-like receptor-mediated facilitation of the micturition reflex may involve actions on brainstem because microinjection of dopamine to the PMC reduced bladder capacity and facilitated the micturition reflex in cats (de Groat et al, 1993a) D2-like receptors are also involved in detrusor overactivity induced by middle cerebral artery occlusion in rats (Yokoyama et al, 1999). Thus central dopaminergic pathways exhibit different effects on micturition through actions on multiple receptors at different sites in the brain.

It is also known in cats that neurons in the substantia nigra pars compacta and the ventral tegmental area, which are the major dopamine-containing nuclei in the midbrain, respond to the storage/micturition cycles of isovolumetric cystometry (Sakakibara et al, 2002), and that the dopamine level in the striatum, where nigrostriatal dopaminergic nerves terminals are found, is shown to increase during the urine storage phase (Yamamoto et al, 2005). For the control of urethral function, activation of D2-like receptors at a supraspinal site suppresses the activity of the striated sphincter muscle to reduce urethral pressure. Inhibition of dopamine D1- or D2-like receptors has a minimal effect on urethral function in anesthetized rats, suggesting the dopaminergic control of urethral function is minimally active in the normal condition (Ogawa et al, 2006).

Opioid Peptides

Intracerebroventricularly administered morphine suppressed isovolumic bladder contractions, and this effect was blocked by naloxone (Dray and Metsch 1984a). Naloxone administered intracerebroventricularly also reversed the effects of systemically administered morphine. Naloxone administered alone intracerebroventricularly or injected directly into the PMC facilitates the micturition reflex (Hisamitsu and de Groat, 1984). Both µ and δ opioid receptors mediate inhibitory effects that are blocked by naloxone (Hisamitsu and de Groat, 1984; Mallory et al, 1991; Downie, 1999). In a recent study, activation of µ and δ1, but not δ2, opioid receptors in the brain increases bladder capacity in both normal rats and rats with cerebral infarction that exhibit frequent voiding; however, κ receptor activation increases bladder capacity only in cerebral infarct rats (Nagasaka et al, 2007).

Mechanisms of Detrusor Overactivity

A variety of models have been used to explore the pathogenesis of detrusor overactivity and to formulate treatments for overactive bladder and urgency incontinence. Models for detrusor overactivity in several species have been developed that are relevant to spinal cord injury, obstruction, denervation, Parkinson disease, interstitial cystitis, diabetes, multiple sclerosis, and aging (de Groat et al, 1993a; Dupont et al, 1994). In addition, the spontaneously hypertensive rat has provided a useful genetic model for detrusor overactivity (Steers et al, 1999). A common feature of many of these models is that changes in smooth muscle function can elicit long-term changes in nerves. Investigators are accustomed to examining short-term effects. However, there is now a greater appreciation that long-term events involving growth factors lead to plasticity in neural pathways with implications for disorders of micturition.

Neurotransmitters, prostaglandins, and neurotrophic factors, such as nerve growth factor (NGF), are substances that provide mechanisms for communication between muscle and nerve. Disturbances in these mechanisms can cause detrusor overactivity by alterations in autonomic reflex pathways. This detrusor overactivity can, in turn, lead to urgency incontinence. Cystometry and urinary frequency are commonly used to define detrusor overactivity and can be used to monitor responses to drugs or other therapies. A multidisciplinary approach incorporating biochemical, molecular, pharmacologic, physiologic, and behavioral methods can provide insight into the pathogenesis of detrusor overactivity. In addition, recent advances in constructing mutant mice lacking specific genes provide a useful tool to study the contribution of specific molecules to the lower urinary tract function or the emergence of detrusor overactivity. For example, bladder function has been investigated in knockout mice lacking muscarinic receptors (M1 to M5) (Matsui et al, 2002; Igawa et al, 2004), purinergic receptors (P2X2, P2X3) (Cockayne et al, 2000, 2005), TRPV1 (Birder et al, 2002), hSlo for BKCa channels (Meredith et al, 2004), serotonin reuptake transporter (Cornelissen et al, 2005), or TRP channels (Birder et al, 2002; Mochizuki et al, 2009).

Please see the Expert Consult website for additional discussion of Spinal Cord Injury and Neurogenic Detrusor Activity, Bladder Outlet Obstruction, Inflammation, Aging, and Neurogenic Mechanisms Underlying Detrusor Activity, as well as Figures 60–31 and 60–32. image

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Figure 60–32 Possible mechanisms underlying plasticity in bladder reflex pathways induced by various pathologic conditions. Bladders from rats with chronic spinal cord injury, urethral obstruction, chronic inflammation, and bladder denervation and those that are spontaneously hypertensive exhibit increased level of neurotrophic factors (NTF), such as nerve growth factor. NTFs can increase the excitability of C-fiber bladder afferent neurons and alter reflex mechanisms in parasympathetic excitatory pathways in the pelvic nerve (PN), as well as in sympathetic pathways in the hypogastric nerve (HGN). These reflex circuits are organized in the spinal cord as positive-feedback loops that induce involuntary bladder activity. In certain situations, such as the spontaneously hypertensive rat, peripheral efferent mechanisms are also altered: Excitatory α1-adrenoceptor mechanisms are upregulated, providing an additional excitatory input to the bladder.

Spinal Cord Injury and Neurogenic Detrusor Overactivity

Damage to the spinal cord above the sacral level results in detrusor overactivity (Kaplan et al, 1991; Chancellor, 1997). Acute spinal cord injury disrupts normal supraspinal circuits that control urine storage and release. After the spinal shock period of urinary retention that generally lasts a few weeks, hyper-reflexic voiding develops. Electrophysiologic data reveal that this detrusor overactivity is mediated by a spinal micturition reflex that emerges in response to a reorganization of synaptic connections in the spinal cord (de Groat, 1975; de Groat et al, 1981, 1990; Araki and de Groat, 1997; Yoshimura, 1999). In addition, bladder afferents that are normally unresponsive to low intravesical pressures become more mechanosensitive, leading to the development of detrusor overactivity.

Normal micturition is associated with a spinobulbospinal reflex mediated by lightly myelinated Aδ afferents (de Groat et al, 1975, 1993a). These fibers represent only 30% of bladder afferents in some species. Compared with Aδ fibers, the more prevalent unmyelinated C fibers are relatively insensitive to gradual distention of the urinary bladder, at least in the cat (Häbler et al, 1990). Most C fibers in this species remain silent during normal filling of the bladder, although in the rat, some studies indicate that C fibers can fire at low pressures (Sengupta and Gebhart, 1994), whereas other studies (Morrison, 1998) showed firing at higher intravesical pressures of approximately 30 mm Hg. After spinal cord injury, a capsaicin-sensitive C-fiber–mediated spinal reflex develops (Fig. 60–31). These C-fiber afferents are thought to play a role in the development of detrusor overactivity after spinal cord injury. Capsaicin-sensitive C fibers have also been implicated in detrusor overactivity after upper motoneuron diseases, such as spinal cord injury and multiple sclerosis (Fowler et al, 1992, 1994; Geirsson et al, 1995; Szallasi and Fowler, 2002). Studies in humans have also revealed an increased density of TRPV1 and P2X3 immunoreactivity, as well as immunoreactivity to a pan-neuronal marker (PGP9.5) in suburothelial nerves and increased TRPV1 immunoreactivity in the basal layer of the urothelium in patients with neurogenic detrusor overactivity (NDO) resulting from spinal cord lesions (spinal cord injury or multiple sclerosis) (Brady et al, 2004; Apostolidis et al, 2005). Treatment of NDO patients with intravesical capsaicin or another C-fiber neurotoxin, resiniferatoxin, produces symptomatic improvement in a subpopulation of these patients and reduces the density of TRPV1, P2X3, and PGP9.5 immunoreactive nerve fibers and urothelial TRPV1-immunoreactivity (Brady et al, 2004).

Insight into the mechanism underlying the increased mechanosensitivity of C fibers after spinal cord injury has been gained by examination of the DRG cells supplying the bladder. Plasticity in these afferents is manifested by enlargement of these cells (Kruse et al, 1995) and increased electrical excitability (Yoshimura and de Groat, 1997; Yoshimura, 1999). Upregulation of tetrodotoxin (TTX)-sensitive Na+ channels and downregulation of TTX-resistant Na+ channels, as well as low-threshold A-type K+ channels occurs after spinal cord injury (Yoshimura and de Groat, 1997; Yoshimura, 1999).

Plasticity in bladder afferents after spinal cord injury and upper motoneuron lesions may involve the retrograde transport of substances from either the spinal cord or the bladder to the DRG neuron. NGF has been implicated as a chemical mediator of disease-induced changes in C-fiber afferent nerve excitability and reflex bladder activity (Yoshimura, 1999; Vizzard, 2000). Chronic administration of NGF into bladder afferent pathways induced bladder overactivity and increased the firing frequency of dissociated bladder afferent neurons in rats (Yoshimura et al, 2006), and the production of neurotrophic factors, including NGF, increased in the bladder after spinal cord injury (Vizzard, 2000). Thus it seems that target organ–neural interactions mediated by neurotrophic factors, such as NGF, produced in the bladder may contribute to changes in C-fiber bladder afferent pathways that induce detrusor overactivity and detrusor-sphincter dyssynergia after spinal cord injury. In addition, increased NGF in the spinal cord after spinal cord injury is also responsible for inducing hyperexcitability of C-fiber bladder afferent pathways, and intrathecal application of NGF antibodies, which neutralizes NGF in the lumbosacral spinal cord and DRG, suppresses detrusor overactivity and detrusor-sphincter dyssynergia in spinal cord–injured rats (Seki et al, 2002, 2004). Intrathecal administration of NGF antibodies also reportedly blocks autonomic dysreflexia in paraplegic rats (Krenz et al, 1999). Thus NGF and its receptors in the bladder or the spinal cord are potential targets for new therapies to suppress detrusor overactivity and detrusor-sphincter dyssynergia after spinal cord injury.

Other neurogenic disorders associated with urgency incontinence respond to intravesical therapy with capsaicin or resiniferatoxin, suggesting that plasticity in C-fiber afferents could form the neurogenic basis for detrusor overactivity (Geirsson, 1993; Fowler et al, 1994; Szallasi and Fowler, 2002). The emergence of a spinal reflex circuit activated by C-fiber bladder afferents represents a positive feedback mechanism (Fig. 60–32) that may be unresponsive to voluntary control by higher brain centers and thereby be able to trigger involuntary voiding. The bladder ice-water urodynamic test has been suggested as a method to assess the C fiber–mediated micturition reflex. Although the ice-water test is consistent in a strictly controlled research environment, it has not been adequately sensitive or specific in routine clinical use (Chai et al, 1998; Chancellor et al, 1998).

Bladder Outlet Obstruction

It is important to understand that the bothersome symptoms of patients with urethral obstruction are in most cases caused by the bladder. Bladder outlet obstruction, such as that in patients with benign prostatic hyperplasia, often produces detrusor hypertrophy and detrusor overactivity (Gosling et al, 2000; Andersson and Wein, 2004). After chronic partial obstruction of the urethra in rats, the bladder enlarges and is about 15 times heavier, but it has the same shape as in control subjects; the growth is mainly accounted for by muscle hypertrophy. The outer surface of the hypertrophic bladder is increased sixfold over that of the controls; the muscle is increased threefold in thickness and is more compact. Mitoses are not found, but there is a massive increase in muscle cell size (Gabella and Uvelius, 1990). Obstruction-induced detrusor overactivity with irritative voiding symptoms has been attributed to denervation supersensitivity, because increased contractile responses of the bladder smooth muscle to cholinergic agonists have been observed (Speakman et al, 1987; Andersson and Wein, 2004). Alterations in detrusor contractility may also result from changes in contractile proteins (Uvelius et al, 1989; Cher et al, 1990; Chacko et al, 1999, 2004).

Brading and Turner (1994) proposed that all cases of detrusor overactivity have a common feature—detrusor smooth muscle change that predisposes it for unstable contraction. They have demonstrated that detrusor overactivity, as shown in a pig model of obstruction, may occur without participation of a micturition reflex. Mills and coworkers (2000) have also implicated abnormalities in the detrusor muscle and its pattern of innervation in idiopathic detrusor overactivity. Compared with the bladder wall in control subjects, there was evidence in the detrusor smooth muscle of altered spontaneous contractile activity consistent with increased electrical coupling of cells, patchy denervation of the detrusor, and potassium supersensitivity (Mills et al, 2000). One of the manifestations of this abnormality is a partial denervation of the detrusor smooth muscle. In rats with bladder outlet obstruction induced by partial urethral ligation, acetylcholine release during electrical stimulation of obstructed bladder muscle strips was significantly decreased 3 to 6 months after obstruction, along with a reduction in the number of nerve fibers in the obstructed bladder compared with control rats (Murakami et al, 2008). Partial denervation in obstructed bladder leads to various functional changes in smooth muscles including denervation supersensitivity of cholinergic (muscarinic) receptors (Speakman et al, 1987) and increases in purinergic receptor–mediated contractile responses as well as expression of purinergic receptors such as P2X1 (Boselli et al, 2001; O’Reilly et al, 2001). Changes in the cell-to-cell communication in detrusor muscles have also been indicated as a mechanism inducing detrusor overactivity, because there is an upregulation of connexin 43, a gap-junction protein, in rats with detrusor overactivity induced by bladder outlet obstruction (Christ et al, 2003; Mori et al, 2005; Li et al, 2007; Imamura et al, 2009; Miyazato et al, 2009). Increased expression of connexin 43 is also identified in the bladders from patients with neurogenic detrusor overactivity (Haferkamp et al, 2004) or with urgency symptoms (Neuhaus et al, 2005). Thus increases in receptor-mediated muscle contractility and interaction between smooth muscle cells can result in coordinated myogenic contraction of the entire bladder and detrusor overactivity (DO).

In addition, another population of cells in the bladder, known as interstitial cells, has been proposed for a pacemaking role in spontaneous activity of the bladder (Andersson and Arner, 2004; Yoshimura and Chancellor, 2007). Because it has been reported that the number of interstitial cells is increased in a guinea pig model of bladder outlet obstruction (Kubota et al, 2008) and that c-Kit tyrosine kinase inhibitors, which inhibit interstitial cell activity, decreased the amplitude of spontaneous contractions in the guinea pig and human bladder, (Biers et al, 2006; Kubota et al, 2006) interstitial cells may also be involved in the emergence of detrusor overactivity due to enhanced autonomous detrusor muscle activity.

Alterations also occur in neural networks in the central nervous system after obstruction of the lower urinary tract. Bladder outlet obstruction in rats causes enhancement of a spinal reflex (Steers and de Groat, 1988). Similarly, in obstructed humans, a capsaicin-sensitive spinal reflex can be detected by the ice-water test (Chai et al, 1998; Hirayama et al, 2003, 2005). Within the spinal cord, obstruction stimulates an increased expression of growth-associated protein 43 that has been associated with axonal sprouting after injury (Steers and Tuttle, 1997). These observations suggest an enhancement or de novo development of new spinal circuits after obstruction. Similar to spinal cord injury, obstruction causes hypertrophy of bladder afferent and efferent neurons (Steers et al, 1990, 1991). Conversely, relief of obstruction is associated with the reduction of urinary frequency and reversal of these neural changes (Steers and Tuttle, 1997). In animals that fail to revert to a normal voiding pattern after relief of obstruction, this neuroplasticity persists. An immunohistochemical analysis of the distribution and density of growth associated protein–43 (GAP-43) showed that this protein was increased in the spinal cord in the region of the sacral parasympathetic nucleus in bladder outlet obstruction (BOO) rats (Steers and Tuttle, 2006). Because this protein is a marker for axonal sprouting, its upregulation provides further indirect support for morphologic plasticity in afferent pathways after BOO. Nevertheless, these findings are not mutually exclusive of changes in the bladder smooth muscle, which are also likely to participate in the development of detrusor overactivity (Turner and Brading, 1997).

Bladder outlet obstruction appears to initiate the morphologic and electrophysiologic afferent plasticity through a mechanism involving NGF (see Fig. 60–32). NGF is responsible for the growth and maintenance of sympathetic and sensory neurons and has been shown to be responsible for neuronal regrowth after injury. NGF content is increased in obstructed bladders in animals and in humans (Steers et al, 1991). This increase in NGF content precedes the enlargement of bladder neurons and the developmental of urinary frequency (Steers et al, 1990, 1991). Moreover, blockade of NGF action with autoantibodies prevents the neural plasticity and urinary frequency after obstruction (Steers et al, 1991). In animals with persistent urinary frequency after relief of obstruction, NGF remains elevated in the bladder. These findings suggest a cause-and-effect relationship between NGF-mediated changes in bladder afferents and an enhanced spinal micturition reflex and urinary frequency associated with obstruction. Increased levels of urinary NGF have also been detected in BOO patients exhibiting overactive bladder (OAB) symptoms. Total urinary NGF levels were low in controls (0.5 pg/mL) and in patients with BOO without OAB symptoms (1 pg/mL), but considerably higher in patients with BOO and OAB symptoms (41 pg/mL) or BOO and detrusor overactivity (50 pg/mL).

Inflammation

Cystitis, which is an inflammatory condition of the urinary bladder that can occur as a result of infection, radiation-induced damage, irritant chemicals in the urine, or unknown causes (bladder pain syndrome/interstitial cystitis, BPS/IC), is accompanied by pain, unusual hypersensitivity to bladder distention, edema, and accumulation of large numbers of inflammatory cells in the bladder mucosa and musculature.

Histologic analysis of bladders from patients with BPS/IC revealed marked edema, vasodilation, proliferation of nerve fibers, and infiltration of mast cells (Johansson and Fall, 1997). Chemically-induced cystitis in animals using cyclophosphamide, mustard oil, turpentine oil, low pH solutions, or acrolein, which increase urinary frequency, is initiated by sensitizing mechanosensitive afferents and/or recruitment of afferents normally unresponsive to mechanical stimulation (i.e., silent C fibers) (Häbler et al, 1990; Sengupta and Gebhart, 1994; Dmitrieva and McMahon, 1996; Dmitrieva et al, 1997). Proinflammatory agents, such as prostaglandin E2 (PGE2), serotonin (5-HT), histamine, bradykinin, and adenosine, as well as neurotrophic factors such as nerve growth factor (NGF), which are released during chemical irritation can induce bladder hyperactivity, as well as functional and chemical changes in C-fiber afferents that can lead to hyperexcitability (Dmitrieva and McMahon, 1996; Gold et al, 1996). For example, chronic chemical irritation of the bladder changes ion channel function in bladder afferent neurons and also increases the expression of various markers, including nitric oxide synthase (NOS) (Vizzard et al, 1996), GAP-43 (Vizzard and Boyle, 1999), PACAP, substance P (Vizzard, 2001), and protease-activated receptors (Dattilio and Vizzard, 2005). The density of peptidergic afferent nerves also increases in the bladder mucosa and detrusor muscle (Dickson et al, 2006), and afferent peptidergic axons and parasympathetic efferent axons/varicosities are commonly observed in close contact, suggesting that sprouting of peripheral nerves occurs during chronic cystitis.

NGF has attracted considerable attention as a key player in the link between inflammation and altered pain signaling. NGF is expressed widely in various cells, including urothelial cells, smooth muscle cells, and mast cells, and can activate mast cells to degranulate and proliferate. In patients with BPS/IC, neurotrophins, including nerve growth factor, neurotrophin-3 (NT-3), and glial cell–derived neurotrophic factor (GDNF), have been detected in the urine (Okragly et al, 1999). Increased expression of NGF is also present in bladder biopsies from women with IC (Lowe et al, 1997). In the cyclophosphamide-induced chronic cystitis model in rats, increased expression of neurotrophic growth factors, such as NGF, brain-derived neurotrophic factor (BDNF), and CTNF in the bladder, as well as phosphorylation of tyrosine kinase receptors (TrkA, TrkB) in bladder afferent neurons, has also been presented as direct evidence for increased neurotrophin-mediated signaling in chronic bladder inflammation (Vizzard, 2000; Qiao and Vizzard, 2002). The enhanced neurotrophic factor mechanisms are also associated with increased phosphorylated cyclic AMP response-element binding protein (CREB) in bladder afferent neurons. Phosphorylated CREB, which is a transcription factor in the neurotrophin intracellular signaling pathway, is coexpressed with phosphorylated TrkA in a subpopulation of bladder afferent neurons (Qiao and Vizzard, 2004). Resiniferatoxin, a C-fiber neurotoxin, reduced cyclophosphamide-induced upregulation of phosphorylated CREB in DRG cells, suggesting that cystitis is linked with altered CREB phosphorylation in capsaicin-sensitive C-fiber bladder afferents (Qiao and Vizzard, 2004). These results suggest that upregulation of phosphorylated CREB may be mediated by a neurotrophin/TrkA signaling pathway, and that CREB phosphorylation may play a role as a transcription factor in lower urinary tract plasticity induced by cystitis.

Exogenous NGF can induce bladder nociceptive responses and bladder overactivity in rats when applied acutely into the bladder lumen (Dmitrieva et al, 1997; Chuang et al, 2001), or chronically to the bladder wall or intrathecal space (Lamb et al, 2004; Yoshimura et al, 2006; Zvara and Vizzard, 2007). Conversely, application of NGF-sequestering molecules (TrkA-IgG or REN1820) can reduce referred thermal hyperalgesia elicited by bladder inflammation induced by intravesically applied turpentine oil (Jaggar et al, 1999) or bladder overactivity elicited by cyclophosphamide-induced cystitis (Hu et al, 2005), suggesting that increased NGF expression is directly involved in the emergence of bladder-related nociceptive responses in cystitis.

Purinergic mechanisms may also contribute to the bladder dysfunction following chronic inflammation. ATP release from the urothelium is enhanced in patients and cats with BPS/IC (Birder et al, 2008). In conscious rats with cyclophosphamide-induced cystitis, purinergic receptor antagonists (pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid [PPADS] and A-317491) reduce nonvoiding contractions and decrease voiding frequency (Ito, Iwami et al. 2008). In in-vitro whole-bladder pelvic afferent nerve preparations from rats with cyclophosphamide-induced cystitis, afferent nerve firing induced by bladder distention or by direct electrical stimulation is markedly increased compared with firing in normal rats (Yu and de Groat, 2008). Exogenous purinergic agonists mimic the facilitatory effects of cyclophosphamide treatment, and P2X purinergic receptor antagonists suppress the effects of purinergic agonists and cystitis. These results suggest that endogenous purinergic agonists released in the inflamed bladder can enhance the excitability of bladder afferent nerves by activating P2X receptors. Patch clamp studies on bladder afferent neurons from rats revealed that chronic cyclophosphamide treatment increases the currents induced by purinergic agonists in both thoracolumbar and lumbosacral neurons (Dang et al, 2008). Analysis of the kinetics of the currents indicated that increased receptor expression and/or properties of homomeric P2X3 in thoracolumbar neurons and P2X2/3 in lumbosacral neurons contributes to the enhanced responses during cystitis.

Cystitis also induces chemical changes in the spinal cord. Acute or chronic bladder irritation increases immediate early gene expression (c-FOS) in spinal neurons (Birder and de Groat, 1993), as well as increasing in GFRα1-IR in the spinal dorsal horn and in areas associated with autonomic neurons (Forrest and Keast, 2008). There was a much smaller increase in GFRα3-IR and no change in GFRα2-IR. Changes in spinal cord mitogen–activated protein (MAP) kinases (extracellular signal-related kinases 1 and 2 [ERK 1 and 2]) may also play a role in the facilitation of reflex voiding after bladder inflammation. Immunohistochemical studies revealed that, in noninflamed rat bladders, noxious but not non-noxious stimulation significantly increased phospho-ERK immunoreactivity (Cruz et al, 2007). After bladder inflammation, innocuous and noxious bladder distention increased the number of spinal neurons exhibiting phosphor-ERK-immunoreactivity. ERK inhibition with intrathecal injection of PD98059 decreases reflex bladder activity and spinal c-FOS expression in animals with inflamed bladders but not in normal animals (Cruz et al, 2007). The results suggest that activation of spinal cord ERK contributes to acute and chronic inflammatory pain perception and mediates reflex bladder overactivity accompanying chronic bladder inflammation.

Direct evidence linking chronic bladder inflammation with functional changes in C-fiber afferents has been obtained in rat chronic cystitis models induced by cyclophosphamide or hydrochloric acid. In these models, the electrical properties of bladder afferent neurons (dissociated from L6 and S1 dorsal root ganglia [DRG]), as well as the activity of the inflamed bladder, were measured. The majority of bladder afferent neurons from both control and cyclophosphamide-treated rats are capsaicin-sensitive and exhibit high-threshold tetrodotoxin-resistant action potentials and Na+ currents. However, neurons from rats with cystitis exhibit significantly lower thresholds for spike activation and show tonic rather than phasic firing characteristics (Hayashi et al, 2009). Other significant changes in bladder afferent neurons from cystitis rats include increased somal diameter, increased input capacitance, and decreased density of slowly inactivating A-type K+ (KA) currents (Yoshimura and de Groat, 1999; Hayashi et al, 2009). In addition, the reduction in KA currents in the hydrochloric acid–induced cystitis model was associated with reduced expression of the Kv1.4 α–subunit protein (which can form KA channels) in bladder afferent neurons (Hayashi et al, 2009), suggesting that the Kv1.4 subunit may be a molecule responsible for reduced KA currents and increased excitability of bladder afferent neurons after cystitis. Previous experiments using cats with naturally occurring feline-type IC have also demonstrated that capsaicin-sensitive dorsal root ganglion neurons exhibit an increase in cell size and increase in firing rates of depolarizing current pulses due to a reduction in low-threshold K+ currents (Sculptoreanu et al, 2005). Taken together, chronic inflammation in BPS/IC could induce both cell hypertrophy and hyperexcitability of C-fiber bladder afferent neurons. If these changes in neuronal cell bodies also occur at C-fiber afferent terminals in the bladder wall, such hyperexcitability may represent an important mechanism for inducing pain in the inflamed bladder. This is supported by some clinical studies showing that C-fiber desensitization induced by intravesical application of capsaicin or resiniferatoxin is effective for treating painful symptoms in patients with BPS/IC (Lazzeri et al, 1996; Lazzeri et al, 2000), although a previous prospective, randomized clinical trial using intravesical resiniferatoxin (RTX) application was not effective in patients with BPS/IC (Payne et al, 2005).

There is little information available about the neuroplasticity of Aδ-fiber bladder afferents in BPS/IC. However, a previous study (Roppolo et al, 2005) using single nerve fiber recordings has documented that Aδ-fiber bladder afferents in cats with feline-type IC are more sensitive to bladder pressure changes than afferents in normal cats, suggesting that, in addition to neuroplasticity of C-fiber afferents, Aδ-fiber bladder afferents might also undergo functional changes in BPS/IC.

Chronic bladder inflammation can also induce changes in functional properties of chemosensitive receptors such as TRPV1 in sensory neurons. Sculptoreanu and colleagues (2005) reported that DRG neurons obtained from cats with feline IC exhibit capsaicin-induced responses that are larger in amplitude and desensitize more slowly compared with those obtained from normal cats, and that altered TRPV1 receptor activity in feline IC cats is reversed by an application of an inhibitor of protein kinase C (PKC), suggesting that BPS/IC can alter TRPV1 activity due to enhanced endogenous PKC activity. Because TRPV1 receptors are reportedly responsible, at least in part, for bladder overactivity elicited by cyclophosphamide-induced cystitis (Dinis et al, 2004), enhanced activity of TRPV1 receptors could contribute to bladder pain in BPS/IC. Studies in mice have also demonstrated a role of TRPV1 in cystitis. Systemic treatment with cyclophosphamide or intravesical administration of acrolein (the irritant metabolite of cyclophosphamide) produces not only bladder hyperactivity but also a sensitization of the paw withdrawal responses to mechanical stimulation of the paw (mechanical hyperalgesia). These responses do not occur in TRPV1 knockout mice (Charrua et al, 2007; Wang et al, 2008). In addition, GRC-6211, a new oral-specific TRPV1 antagonist, has been shown to decrease bladder overactivity and noxious bladder input in cystitis animal models (Charrua et al, 2009).

Overall, therapies targeting bladder afferent pathways, including TRPV1 agonist/antagonists, are being considered for bladder inflammatory conditions, including BPS/IC. However, they would be useful only if C-fiber afferent pathways are involved in bladder inflammatory events. There is no definite proof that chronic bladder inflammation (or pain) is mediated by C-fibers in the human, although there is some experimental evidence in some models that says otherwise. The concept of using “sensory drugs” to treat bladder pain and inflammation is appealing but remains to be tested.

Aging

Lower urinary tract symptoms, such as increased voiding frequency, urgency, urgency incontinence, and poor bladder emptying are common and troublesome problems in older men and women (Resnick, 1995; Naughton and Wyman, 1997; Nuotio et al, 2002). Previous studies have reported various changes in lower urinary tract function, including a reduction in bladder capacity, increased bladder sensation, and detrusor overactivity (DO) (Diokno et al, 1986; Homma et al, 1994; Hald and Horn, 1998; Madersbacher et al, 1998, 1999; Nuotio et al, 2002). However, there are few studies that address the normal changes in the lower urinary tract (LUT) that occur with aging. Studies by Pfisterer and colleagues (2006) have examined age-related changes in bladder function among 85 community-dwelling female volunteers and demonstrated that detrusor contractility, bladder sensation, and urethral pressure decline with age and that a reduction in bladder capacity associated with age may be related to DO rather than to aging itself, because bladder capacity did not decrease with age, but was smaller in subjects with DO (Pfisterer et al, 2006). Thus aging appears to induce hypofunction of the bladder and urethra in humans.

In animal studies, impaired bladder function, as evidenced by increased voided volume per micturition associated with a high micturition-pressure threshold, has also been demonstrated in aged rats compared with the young counterpart (Chun et al, 1988; Chai et al, 2000). In addition, aged rats exhibit reduced sensitivity of pelvic nerve afferents in response to increased bladder volume, but not pressure, and a reduction in the maximal bladder pressure generated during pelvic nerve stimulation (Hotta et al, 1995). A significant linear reduction in the amount of acetylcholinesterase-positive nerve was observed with increasing age in the human bladder (Gilpin et al, 1986), suggesting reduced parasympathetic innervation of the aged bladder. It was also shown that expression of neuropeptides, such as CGRP and SP in lumbosacral dorsal root ganglion (DRG) neurons, decreases with age (Mohammed and Santer, 2002), and that there is a marked reduction in the density of PACAP innervation of the subepithelial plexus and of the muscle layer of the bladder base, as well as slight reductions in CGRP and SP innervation of the muscle layer in old rats (Mohammed et al, 2002). Taken together, these results suggest that impaired activity of the aged bladder is likely, at least in part, due to reduced activity of efferent and afferent nerves innervating the bladder.

Changes in the central nervous system in relation to lower urinary tract function have also been demonstrated in aged animals. Immunohistochemical analyses in aged rats revealed significant age-associated declines in the serotonergic (5-HT) and adrenergic innervation of various spinal cord regions, including the intermediolateral cell nucleus, sacral parasympathetic nucleus, dorsal grey commissure, and in the ventral horn nucleus that contains the Onuf nucleus. However, 5-HT innervation of the sacral parasympathetic nucleus and tyrosine hydroxylase-like immunoreactivity in the ventral horn nucleus were maintained (Ranson et al, 2003). It was also shown that sympathetic preganglionic neurons in the L1-L2 spinal cord that project to the major pelvic ganglion exhibit a number of degenerative changes, such as reductions in the cell number, the length of their dendrites, and the synaptic contact made by glutamate-immunoreactive boutons onto the dendrites in aged rats, although these changes are not seen in parasympathetic preganglionic neurons in the L6-S1 spinal cord (Santer et al, 2002). Chai and colleagues (2000) also reported that frequent voiding produced by apomorphine-induced dopamine receptor activation is more pronounced in aged rats compared with young rats, suggesting that aged rats are more susceptible to altered central processing to induce bladder overactivity despite decline of baseline bladder function with aging (Chai et al, 2000). Hypoactivity of the bladder or the underactive bladder represent an unmet medical need, moving forward in light of the aging populations in developed countries (Chancellor and Kaufman, 2008).

In contrast to altered nerve activity, there appears to be no significant change in detrusor contractile responses to cholinergic or electrical stimulation between young and old animals (Chun et al, 1989; Longhurst et al, 1992; Yu et al, 1996; Lieu et al, 1997; Lin et al, 1997; Schneider et al, 2004b), although old rats have a reduced density of muscarinic receptors in the bladder (Schneider et al, 2004b). In contrast, there are some reports of age-related changes of the detrusor response to adrenergic stimulation (Latifpour et al, 1990). Most studies showed that detrusor contractile responses to α-adrenergic stimulation increased in old male and female rats (Saito et al, 1991, 1993; Lin et al, 1992; Nishimoto et al, 1995) in association with upregulation of α1D-receptor expression in the bladder (Dmitrieva et al, 2008). However, another study showed no age-dependent changes in α1-adrenoceptor properties, such as phenylephrine-induced contractile responses, total receptor density, and mRNA expression of α1-adrenoceptor subtypes (α1A, α1B and α1D) in the rat bladder base and dome (Yono et al, 2006). The detrusor response to β-adrenergic stimulation is reduced in old male rats (Lin et al, 1992; Nishimoto et al, 1995), along with a reduction in the density of β-adrenergic receptors and decreased cAMP production (Nishimoto et al, 1995) in response to β-adrenergic stimulation. The combination of increased α-adrenergic excitatory response and decreased β-adrenergic inhibitory response results in a net contracting effect of norepinephrine on the aged bladder, in contrast to the relaxing effect of norepinephrine in the young bladder (Lin et al, 1992). However, the contribution of these changes in adrenoceptor properties to age-related alterations in lower urinary tract function is still to be determined.

Neurogenic Mechanisms Underlying Detrusor Overactivity

Changes in bladder innervation orchestrated by neurotrophins manufactured by detrusor smooth muscle are temporally linked with detrusor overactivity (see Fig. 60–32). The ability of local anesthetics, intravesical afferent neurotoxins, and destruction of afferent nerves in the bladder neck and prostate to reduce urgency, frequency, and urgency incontinence indicates an important role for afferent-evoked reflexes (Chalfin and Bradley, 1982). The development of a spinal reflex (ice-water test response) in patients with neurogenic bladders (Geirsson et al, 1999), as well as in patients with bladder outlet obstruction (Chai et al, 1998; Hirayama et al, 2003, 2005), suggests a common underlying plasticity in nerves supplying the bladder. Moreover, the association between elevated blood pressure and lower urinary tract symptoms in patients with benign prostatic hyperplasia (Pool, 1994; Sugaya et al, 2003) provides a link between changes in sympathetic tone and voiding complaints.

Thus the unifying story that is present in each of the pathologic conditions discussed is neuroplasticity. Urinary frequency and possibly urgency incontinence are often associated with elevated neurotrophin production, enhanced C-fiber afferent-evoked bladder reflex, and noradrenergic function. The realization that nerves supplying the lower urinary tract can undergo long-term changes that lead to detrusor overactivity offers a novel avenue for therapeutic intervention.

Summary

This chapter began with the premise that the lower urinary tract has two main functions: storage and periodic elimination of urine. These functions are regulated by the unique biomechanics of bladder and urethral muscles, as well as by a complex neural control system located in the brain and spinal cord. The neural control system performs like a simple switching circuit to maintain a reciprocal relationship between the reservoir (urinary bladder) and the outlet components (urethra and urethral sphincter) of the urinary tract. The switching circuit is modulated by various neurotransmitters and is sensitive to a variety of drugs. In infants, the switching circuits function in a purely reflex manner to produce involuntary voiding; however, in adults urine storage and release are subject to voluntary control (see Fig. 60–1).

Injuries or diseases of the nervous system in adults can disrupt the voluntary control of micturition, causing the reemergence of reflex micturition, resulting in detrusor overactivity and incontinence. Because of the complexity of the central nervous control of the lower urinary tract, incontinence can result from a variety of neurologic disorders. Experimental studies indicate that detrusor overactivity occurs after a wide range of neurologic diseases, including interruption of cortical inhibitory circuits, disruption of basal ganglia function in models of Parkinson disease, damage to pathways from the brain to the spinal cord (multiple sclerosis, spinal cord injury), and sensitization of bladder afferents. Various mechanisms contribute to the emergence of bladder dysfunction, including reorganization of synaptic connections in the spinal cord, changes in the expression of neurotransmitters and receptors, alterations in neural target organ interactions mediated by neurotrophic factors, and changes in smooth muscle function. An understanding of the physiologic events mediating micturition and continence provides a rational basis for the management of lower urinary tract dysfunction.

Future Research

During the past few years, research in neurourology and female urology has led to the development of new concepts regarding the etiology of unstable bladder dysfunction and urinary incontinence. The breakthroughs outlined in this chapter have stimulated the development of new therapeutic approaches for incontinence, including the local administration of botulinum toxin and afferent neurotoxins, such as capsaicin and RTX. What are the research priorities for the future? It will be important to focus on the development of neuropharmacologic agents that can suppress the unique components of abnormal bladder reflex mechanisms and thereby act selectively to diminish symptoms without altering normal voiding function.

Neurourologic research with a clinical application is intensifying at major medical centers and also in the pharmaceutical industry. With the aging population across all the developed nations and public education that is demystifying the stigma of bladder control problems and urinary incontinence, more patients are seeking help. The dramatically expanding commercial markets and need for improved medications for the lower urinary tract have been recognized and publicized. We need better drugs and approaches to help the 85% of people with detrusor overactivity and urinary incontinence who have not yet sought medical help.

To end this chapter, we would like to look into our crystal ball and speculate about a few areas of research that we think may pay off within the next 5 years with new and better treatments of urinary incontinence.

1. NGF has been implicated as one of the key factors inducing bladder overactivity and pain conditions. Can the tissue or urinary level of NGF be a biomarker for the diagnosis and/or evaluation of therapeutic efficacy of bladder dysfunction?
2. Can the drug companies develop a truly bladder-specific and effective anticholinergic drug with no dry mouth? β3-Agonists are currently in phase 3 studies internationally.
3. Tachykinin or TRP channel antagonists are appealing, because they may be effective for unstable and painful bladder conditions without increasing residual urine volumes. Can clinically useful and safe oral antagonists be developed?
4. We need an effective drug for stress incontinence, such as one based on urethral smooth muscle– or skeletal muscle–specific agonists/antagonists or norepinephrine/5-HT reuptake inhibitors.
5. Although not as clinically obvious as the ideas just listed, we believe that drugs that can affect urothelial trafficking and permeability may be tremendously helpful. These types of drugs not only can aid the overactive and painful bladder but may have indications for bladder cancer and even urinary tract infection.
6. In addition, given that spontaneous activity in the detrusor muscle and interstitial cells in the urothelium and bladder wall has an important role in the regulation of bladder activity, targeting detrusor muscles or interstitial cells could be effective for treating detrusor overactivity, especially that of myogenic origin.

Beyond the horizon of near-term advances, we predict a brave new paradigm in neurourology. What has already started is the evolution of unstoppable forces of change in medicine that include pharmacogenomics, tissue engineering, and gene therapy. These will change how we practice urology.

Stem Cells and Tissue Engineering

Rapid advances are being made in tissue engineering (Atala, 2004) and organ reconstruction with autologous tissue and stem cells (Chancellor et al, 2000). We envision a day, in the not too distant future, when stress incontinence is cured not with a cadaver ligament and metal screws into the bones but rather with minimally invasive injection of muscle stem cells that will not only bulk up the deficient sphincter but actually improve the deficient sphincter’s contractility.

Gene Therapy

Benign prostatic hyperplasia (Marcelli et al, 2000), diabetic neurogenic bladder (Phelan et al, 2000; Goins et al, 2001; Sasaki et al, 2004), neurogenic detrusor overactivity (Miyazato et al, 2009), and even BPS/IC (Yokoyama et al, 2008) may be cured with one or more treatments with a gene vector that the urologist will inject into the bladder or prostate. The injection of a viral vector expression apoptotic factor can direct the hypertrophied prostate to shrink. Injection of NGF through a herpes simplex virus vector into the bladder of a diabetic person may promote the recovery of bladder sensation and innervation (Fig. 60–33). Finally, what about the single condition most troubling to just about every urologist, BPS/IC? Can the introduction of a viral vector that is targeted to nerves, and that carries a gene for an endogenous opioid peptide that blocks pain pathways, be used to help alleviate pain, regardless of the cause of BPS/IC?

Acknowledgments

We want to thank Dr. William C. (Chet) de Groat for reviewing this chapter. Around the world, Dr. de Groat is warmly regarded as the godfather of neurourology, but to the two of us, Dr. de Groat is much more. Chet is the father of our laboratory and our research. His intellectual brilliance is matched only by his decency of heart. Chet is always there to help us in developing research ideas and writing papers and grants. Chet is there when we run into trouble and need guidance. We dedicate our research in honor of our teacher, Dr. Chet de Groat. We would also like to thank Dr. John Lavelle at the University of North Carolina for his assistance with the section on bladder urothelium and Dr. Michael Sacks at the University of Pittsburgh for helping us with the bladder biomechanics section.

Suggested Readings

Andersson KE, Wein AJ. Pharmacology of the lower urinary tract: basis for current and future treatments of urinary incontinence. Pharmacol Rev. 2004;56:581-631.

Birder LA. More than just a barrier: urothelium as a drug target for urinary bladder pain. Am J Physiol Renal Physiol. 2005;289:F489-F495.

Brading AF. Spontaneous activity of lower urinary tract smooth muscles: correlation between ion channels and tissue function. J Physiol. 2006;570(Pt 1):13-22.

de Groat WC, Yoshimura N. Pharmacology of the lower urinary tract. Annu Rev Pharmacol Toxicol. 2001;41:691-721.

Fowler CJ, Griffiths D, de Groat WC. The neural control of micturition. Nat Rev Neurosci. 2008;9:453-466.

Yoshimura N, Chancellor MB. Current and future pharmacological treatment for overactive bladder. J Urol. 2002;168:1897-1913.

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