Morphology

The individual smooth muscle cells in the bladder wall are small spindle-shaped cells with a central nucleus; fully relaxed, they are several hundred micrometers long with a 5- to 6-µm maximum diameter. Skeletal muscle fibers are some 20 times wider and thousands of times longer (Smet et al, 1996).

Smooth muscle has a unique range of physiologic properties and consists of sheets containing many small spindle-shaped cells linked together at specific junctions. Each cell has a single nucleus. No cross striations are visible under the microscope, but like skeletal and cardiac muscle, smooth muscle cells contain actin and myosin. In addition, they contain cytoskeletal intermediate filaments that assist in transmission of the force generated during contraction to the neighboring smooth muscle cells and connective tissue. Although there are no Z lines in smooth muscle, they have a functional counterpart in dense bodies that are distributed throughout the cytoplasm and that serve as attachments for both the thin and the intermediate filaments. The thin and thick filaments of smooth muscle fibers are arranged as myofibrils that cross the fibers obliquely in a lattice-like arrangement. The filaments of contractile proteins are attached to the plasma membrane at the junctional complexes between neighboring cells (Fig. 60–6). A comparison of some of the properties of smooth and skeletal muscle is listed in Table 60–1 (Chacko et al, 1999).

Figure 60–6 The organization of the contractile elements of smooth muscle fibers by a simple model of the contraction of smooth muscle. A, Relaxed smooth muscle cell. B, Contracted smooth muscle cell. Intermediate filaments, dense bodies, and dense bands of smooth muscle fibers harness the pull generated during myosin cross-bridge activity. Intermediate filaments attach to dense bodies scattered throughout the sarcoplasm and occasionally anchor to the dense bands situated between caveolae (invaginations of the sarcolemma). As the obliquely running contractile elements contract, the muscle shortens.

Table 60–1 Comparison of the Properties of Skeletal and Smooth Muscle

Smooth Muscle Cellular Mechanics

Detrusor smooth muscle contracts and shortens by interaction between thin and thick filaments. The thin actin filaments are anchored at the membranes on the dense bands, or in the cytoplasm on the dense bodies, and interact with the thick filaments through cross bridges formed from the heads of myosin molecules. In both cases, the contractile mechanism is a myosin-activated system. Cross-bridge cycling is initiated when the ATPase activity of the myosin heads is switched on. This is achieved by phosphorylation of two light chains on the cross bridge, through a specific enzyme, myosin light-chain kinase, that is activated by a rise in the intracellular calcium concentration (Hai and Murphy, 1989; Gunst et al, 1993; Andersson and Arner, 2004). Changes in the contractile proteins occur in developing bladders and also during bladder hypertrophy (Wang et al, 1995; Wu et al, 1995; Sjuve et al, 1996).

The bladder muscle has a broad length-tension relationship, allowing tension to be developed over a large range of resting muscle lengths (Uvelius and Gabella, 1980). The tissue shows viscoelasticity that influences muscle tension and is manifested as total bladder wall tension (Venegas, 1991). Isolated detrusor strips show spontaneous mechanical activity to a variable extent. It is more frequently seen in bladders from small mammals (Sibley, 1984) but can also be seen in muscle strips from human detrusor. However, spontaneous fused tetanic contractions, such as those commonly seen in smooth muscles from the gastrointestinal tract and uterus, are almost never seen in normal bladders.

Excitation-Contraction Coupling in Smooth Muscle

Like skeletal and cardiac muscle, smooth muscle contracts when intracellular calcium concentration rises. Because smooth muscle does not possess the tubules of the “T system,” the rise in calcium concentration occurs through calcium influx through voltage-gated Ca²⁺ channels in the plasma membrane or release from the sarcoplasmic reticulum after activation of receptors that increase the formation of IP₃. Although calcium serves the same triggering role in all muscle types, the mechanism of activation is different in smooth muscle. The contractile response is slower and longer lasting than that of skeletal and cardiac muscle. This can be explained by the slowness with which smooth muscle is able to hydrolyze ATP during the contractile process. A calcium-binding protein called calmodulin regulates the interaction between actin and myosin. The thin filaments lack troponin and are always ready for contraction. The slow and relatively steady generation of tension enables smooth muscle to generate and to maintain tension with relatively little expenditure of energy (Fig. 60–7 and Table 60–2).

Figure 60–7 Acetylcholine (ACh) and adenosine triphosphate (ATP) pathway in the bladder. Acetylcholine interacts with M₃ muscarinic receptors and activates phospholipase C (PLC) through a G protein. Phospholipase activation leads to the production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ elicits release of calcium from the sarcoplasmic reticulum through IP₃ receptors, and DAG may modulate voltage-sensitive Ca²⁺ channels in the plasma membrane. ATP acting through P2X purinergic receptors opens nonselective cation channels in the membrane, leading to depolarization that opens voltage-sensitive Ca²⁺ channels. Both lead to entry of calcium. This triggers release of more calcium from the stores through ryanodine receptors. The rise in intracellular free calcium concentration triggers contraction and may also open calcium-activated channels in the membrane, such as calcium-activated K⁺ channels, that can modulate the response.

(Redrawn from Brading A. Cellular biology. In: Abrams P, Khoury S, Wein A, editors. Incontinence. Plymouth [UK]: Health Publications; 1999. p. 73.)

Table 60–2 Detrusor Smooth Muscle Contraction Sequence

Ca2+ binds to calmodulin, activating it. Calmodulin activates the kinase enzyme. Kinase enzyme catalyzes phosphate transfer from adenosine triphosphate to myosin, allowing myosin to interact with actin of the thin filaments. Smooth muscle relaxes to widen intracellular decrease in Ca2+ levels.

The rise in cytoplasmic calcium concentration brought on by the action potential results in binding of calcium to calmodulin. Calcium-bound calmodulin is then capable of activating myosin light-chain kinase, permitting it to phosphorylate the myosin type II light chain. Phosphorylation of the light chain allows the myosin to interact with actin, leading to force generation (White et al, 1993; Chacko et al, 1994; Andersson and Arner, 2004). The actual geometric arrangement of the actin-myosin complex that permits force generation is unclear. Different isoforms of the myosin heavy chain have been defined by Chacko and associates (1994), as well as the components of the regulatory system linking cytoplasmic calcium levels to contraction (White et al, 1993). The smooth muscle myosin heavy chain is encoded by a single gene, and two heavy-chain variants formed by alternative splicing are identified (SM1 and SM2). Two additional myosin heavy-chain isoforms, SM-A and SM-B, are then formed by alternative splicing in the amino-terminal region, enabling four possible isoforms: SM1-A, SM1-B, SM2-A, and SM2-B (Babu et al, 2000). Because the SM-B isoform is shown to propel actin at a faster velocity (Lauzon et al, 1998), the relatively high expression of SM-B isoform in the bladder shown at the mRNA level (Arafat et al, 2001) would contribute to a comparatively fast smooth muscle type of the bladder (Andersson and Arner, 2004).

Interstitial Cells

Recent evidence suggests that the “normal” bladder may be spontaneously active and that exaggerated spontaneous contractions could contribute to the development of detrusor overactivity. In a rat model for detrusor overactivity, local areas of spontaneous contractions are increased and more coordinated in partial outlet-obstructed rat bladders (Drake et al, 2003). However, it is still not clear which cells generate spontaneous activity in the bladder. As mentioned before, detrusor myocytes could be spontaneously active, and electrical coupling through gap junctions could trigger spontaneous contractions (Brading, 1997b, 2006). Alternatively, another population of cells in the bladder known as interstitial cells, or myofibroblasts, has been proposed for a pacemaking role in spontaneous activity of the bladder (Andersson and Arner, 2004; Kumar et al, 2005). Interstitial cells have been identified in the human and guinea pig ureter, urethra, and bladder body (Kumar et al, 2005; Hashitani, 2006; Fry et al, 2007).

Suburothelial Interstitial Cells

In the human bladder, subepithelial interstitial cells, which are also called myofibroblasts, stain for vimentin and α–smooth muscle actin but not for desmin (Fry et al, 2004). These cells are linked by gap junctions consisting of connexin 43 proteins and make close appositions with C-fiber nerve endings in the submucosal layer of the bladder, suggesting that there is a network of functionally connected interstitial cells immediately below the urothelium that may be modulated by other nerve fibers (Fry et al, 2004) (Fig. 60–8). ATP can induce inward currents associated with elevated intracellular Ca²⁺ in isolated suburothelial interstitial cells (Fry et al, 2007). Immunohistochemical studies show the expression of P2Y receptors, most notably P2Y₆ receptors, and M₃ muscarinic receptors in suburothelial interstitial cells from guinea pigs (Fry et al, 2007; Grol et al, 2009). In the human bladder, increased expression of muscarinic M₂ and M₃ receptors in vimentin-stained suburothelial interstitial cells is found and correlates with the urgency score in humans with idiopathic detrusor overactivity (Mukerji et al, 2006). Because ATP or acetyl choline (ACh) is known to be released from the urothelium during bladder stretch, suburothelial interstitial cells are in an ideal position between the urothelium and nerve endings to modify a sensory feedback mechanism. Application of a nitric oxide donor sodium nitroprusside (SNP) also attenuates an increase in intracellular Ca²⁺ and current responses to ATP in guinea pig interstitial cells, suggesting the cGMP-dependent inhibition of cell activity (Sui et al, 2008).

Figure 60–8 Schematic representation of suburothelial interstitial cells (IC), which are also called myofibroblasts. Substances released from the basolateral surface during stretch, such as adenosine triphosphate (ATP) and acetylcholine (ACh), activate afferents in the suburothelial layer through the intermediation of suburothelially located interstitial cells, which express purinergic P2Y receptors, muscarinic M₂ and M₃ receptors, or capsaicin TRPV1 receptors, and are connected to each other by gap-junction proteins.

Intradetrusor Interstitial Cells

Interstitial cells are also found in the detrusor layer and shown to be spontaneously active (Kumar et al, 2005). These cells are stained for c-Kit and located along both boundaries of muscle bundles in the guinea pig bladder (McCloskey and Gurney, 2002; Hashitani et al, 2004; Hashitani, 2006). These cells can fire Ca²⁺ waves in response to cholinergic stimulation by M₃ muscarinic receptor activation and can be spontaneously active, suggesting that they could act as pacemakers or intermediaries in transmission of nerve signals to smooth muscle cells (McCloskey and Gurney, 2002; Johnston et al, 2008) (Fig. 60–9). However, Hashitani and colleagues (2004) have also suggested that interstitial cells in the detrusor may be more important for modulating the transmission of Ca²⁺ transients originating from smooth muscle cells rather than being the pacemaker of spontaneous activity because spontaneous Ca²⁺ transients occur independently in smooth muscles and interstitial cells. It has also been demonstrated that the c-Kit tyrosine kinase inhibitor Glivec decreased the amplitude of spontaneous contractions in the guinea pig bladder (Kubota et al, 2004, 2006) and in muscle strips from the overactive human bladder, in which c-KIT–positive cells were increased compared with normal subjects (Biers et al, 2006), suggesting that targeting these receptors expressed in intradetrusor interstitial cells may provide a new approach for treating overactive bladder. In addition, following application of SNP (a nitric oxide [NO] donor), interstitial cells throughout the bladder, but not detrusor muscle cells, demonstrate cyclic guanosine monophosphate (cGMP) immunoreactivity (Smet et al, 1996; Gillespie et al, 2004). Thus increased levels of cGMP found in interstitial cells by using phosphodiesterase-5 inhibitors, for example, may diminish synchronicity between detrusor muscle bundles (Hashitani, 2006). These cells are also a source of prostaglandin E₂ (PGE₂) because of their expression of cyclooxygenase and a reduction in spontaneous activity of bladder muscle strips by use of prostaglandin E₂ receptor (EP) antagonists in rabbits (Collins et al, 2009).

Figure 60–9 Schematic representation of interstitial cells in the detrusor muscle layers. These cells are not contractile but may be pacemakers with spontaneous activity and propagate signals between detrusor muscles. They also express KIT receptors and muscarinic M₃ receptors, and can produce prostaglandins (PG), such as PGE₂, through activation of cyclooxygenase (COX).

Further research is definitely required to fully understand the role of suburothelial/intradetrusor interstitial cells and their contribution to spontaneous contractions or detrusor overactivity.

Bladder Wall Collagen

Most of the bladder wall collagen is found in the connective tissue outside the muscle bundles. Changes in the relative amounts of muscle and nonmuscle tissue in the bladder wall would therefore influence collagen concentration. A number of different collagen types have been identified. In the bladder, types I, III, and IV are the most common (Macarak et al, 1995; Andersson and Arner, 2004). Landau and coworkers (1994) developed morphometric and histochemical techniques to determine the percentage volume of connective tissue in the bladder wall and to measure the two major types (I and III) of collagen. These methods quantitate three parameters of bladder ultrastructure: percentage volume of connective tissue, ratio of connective tissue to smooth muscle, and ratio of type III to type I collagen. These parameters have been shown to be abnormally elevated in patients with bladder disease compared with normal patients. They further studied the ultrastructural changes that occur in the wall of dysfunctional bladders to determine the ability of new urodynamic techniques to reliably detect the clinical effect of these histologic changes. The study included 29 consecutive patients undergoing bladder augmentation. Preoperative urodynamic evaluation included measurement of the total bladder capacity, pressure-specific bladder volume, and dynamic analysis of bladder compliance. Full-thickness bladder biopsy specimens were obtained from the dome of the bladders during augmentation. The percentage of connective tissue and the ratio of connective tissue to smooth muscle were determined for all patients. These histologic results were compared with previously established normal values. All 29 patients had decreased bladder compliance, even though 9 had a normal bladder capacity. The ratio of connective tissue to smooth muscle was significantly increased in poorly compliant versus normal bladders. The ratio of type III to type I collagen was also significantly elevated. One can conclude that the poor storage function of poorly compliant bladders is secondary to an alteration in the connective tissue content of the bladder wall, especially increased collagen type III.

In the rat, infravesical obstruction or bladder denervation induces hypertrophy of the detrusor smooth muscle and, in turn, a decrease in the collagen concentration (Uvelius and Mattiasson, 1984, 1986). Aging is associated with a relative decrease in smooth muscle, in both men and women, relative to collagen content (Susset et al, 1978; Lepor et al, 1992). This could perhaps be related to the decreased packing density of submucosal collagen during aging (Levy and Wight, 1990).

Perhaps the most comprehensive work on bladder collagen has been performed by Macarak and Howard (1999), who have speculated that connections must exist between the tension-generating elements (i.e., the smooth muscle cells) and the other components of the bladder. In bladders that become noncompliant (e.g., from spinal cord injury), it is likely that there is some interference with the ability of the collagen fibers to alter their tortuosity. This, predictably, would reduce total bladder capacity. Further studies are required to establish the relationship between compliance changes and the passive mechanical elements of the bladder wall that make up its structural protein matrix.

Bladder Wall Elastin and Matrix

Elastic fibers are amorphous structures composed of elastin and a microfibrillar component located mainly around the periphery of the amorphous component (Rosenbloom et al, 1995). In the mature fiber, the amorphous component composes about 90%. The microfibrils contain a number of glycoproteins. Elastin fibers are sparse in the bladder, compared with collagen, but are found in all layers of the bladder wall (Murakumo et al, 1995). In spinal cord–injured rats, the elastin/collagen ratio first increased and is well correlated with increased bladder compliance due to bladder overdistention up to 6 weeks after injury, including a bladder areflexia phase. Then the ratio is reduced as bladder compliance is decreased due to the emergence of detrusor overactivity 10 weeks after injury, suggesting a potential role for elastin in the modulation of bladder compliance (Nagatomi et al, 2005; Toosi et al, 2008).

The nonfibrillar matrix in the stroma is largely composed of a gel of proteoglycans and water. Proteoglycans are glycoproteins with glycosaminoglycans covalently attached. The arrangement of the proteoglycans in the matrix creates a compartment of tissue water that has a viscous behavior when it is subjected to deformation.

Structure

The urothelium consists of a variable number of cell layers, depending on the species (Fawcett, 1984). There are generally three distinct layers. A layer of basal cells are germinal in nature and 5 to 10 µm in diameter. Intermediate cells are superficial to these and approximately 20 µm in diameter, and a layer of umbrella cells forms the luminal surface of the urothelium (Lewis, 2000; Apodaca, 2004). These umbrella cells are the largest epithelial cells in the body, measuring 100 to 200 µm in diameter; they are polyhedral, are generally hexagonal, and can flatten and have more surface area with stretching. They may also be multinucleate (Kelly, 1984). The surface of the umbrella cells is covered with a glycosaminoglycan layer (Fig. 60–10).

Figure 60–10 Diagrammatic representation of the bladder urothelium. The lumen of the bladder is on top and the lamina propria is below. The glycosaminoglycan layer is shown as discontinuous for illustrative purposes, enabling labeling of deeper structures.

Urothelial Permeability

Epithelial permeability, including that of the urothelium, depends on a number of factors. These are passive diffusion, osmotically driven diffusion, active transport, and inertness of the membrane to the solutes to which it is exposed.

Descriptions of finite passage of substances across the urothelium are well known. In 1856, Kaupp reported that the composition and volume of urine were altered with 12-hour voiding patterns instead of hourly voiding. These changes in volume have also been noted in rats during isovolumetric cystometrograms during 3-hour periods (Sugaya et al, 1997), and the rate of water loss has also been estimated by direct measurement of passive water diffusion in vitro in the rabbit (Negrete et al, 1996). There is a passive permeability to most substances in the blood or urine (Hicks, 1975).

The diffusive permeability of the mammalian bladder to water and urea has been directly measured in several species (Lavelle et al, 2000). The permeability of water in the bladder remains remarkably constant, ranging from 4.01 to 5.74 × 10⁻⁵ cm/sec, and that of urea, 1.5 to 4.51 × 10⁻⁶ cm/sec. Water permeability value in humans is similar at 6.5 × 10⁻⁵ cm/sec (Fellows and Marshall, 1972). This value was obtained by estimating the absorption of tritiated water into the plasma after instillation of the tritiated water into the bladder of volunteers. A direct measurement of urothelial diffusive permeability in the human has not yet been made.

Breakdown of the umbrella cells in animal models of cystitis has shown increased water and urea permeability. Presumably, leakage of urine into the detrusor is also responsible for the symptoms of cystitis (Lavelle et al, 1998, 2000). This increase in urothelial permeability with cystitis is increased further by distention of the bladder. The hypothesis is that with distention of the bladder, the weakened urothelium with denuded apical umbrella cells and no real barrier in the intermediate or basal cells is further disrupted, thus allowing further egress of urine constituents into the detrusor. Similar breakdown of the apical cells is thought to occur in most forms of infectious cystitis and also in radiation cystitis.

Direct measurements of the osmotic effect on permeability have not been performed on urothelium. However, the urothelium maintains an osmotic gradient between plasma (300 mOsm/kg approximately) and urine (100 to 1500 mOsm/kg), depending on the level of water balance and diuresis of the individual. In the normal bladder, the osmotic effects of the urine appear to go unnoticed, and the patients have few or no symptoms. However, once the bladder is inflamed, such as in bladder pain syndrome/interstitial cystitis (BPS/IC), the effects of osmotic gradients become important (Gao et al, 1994).

Patients with spinal cord injury or with myelodysplasia tend to have chronic cystitis with bacteriuria and inflamed urothelium. When detrusor activity was increased in the rat by instillation of hyperosmolar compounds, this was accompanied by neurogenic inflammation, including plasma extravasation of Evans blue that could be decreased by pretreatment with the C-fiber afferent neurotoxin capsaicin (Maggi et al, 1990) indicating that hyperosmolar solutions excite afferent nerves. With increased osmolality, detrusor contractions were much stronger and accompanied by blood pressure elevations. These effects were enhanced when the bladder was pretreated with dimethyl sulfoxide to simulate cystitis conditions (Hohlbrugger and Lentsch, 1985; Hohlbrugger, 1987).

Increased permeability of the urothelium in inflammatory conditions has become the basis of the potassium sensitivity test, during which the bladder is distended with 400 mM of potassium chloride solution. Potassium penetrates the urothelium in the inflamed bladder and causes pain by activation of bladder afferent nerves (Parsons, 1996). This does not occur in normal bladders. The validity and accuracy of the intravesical potassium chloride test has not yet been proven clinically by multicenter randomized double-blind studies.

The fact that changes in the osmolality, pH, and ionic composition of the urine can influence the activity of the detrusor is important in the performance of cystometry. The nature of the infusate could alter the outcome of the test, particularly in the neurogenic bladder, where there are usually some changes in the urothelium because of the chronic bacteriuria (Hohlbrugger, 1995).

Ionic Transport

The apical membrane of the urothelium has a high electrical resistance (Lavelle et al, 1998, 2000), whereas the basolateral membrane resistance is approximately 10-fold lower (Clausen et al, 1979). Active sodium transport across the urothelium has been demonstrated (Wickham, 1964; Lewis and Diamond, 1976). Na⁺ channels that exist on the apical surface of the umbrella cells and in the cytoplasmic vesicles below the apical surface are primarily amiloride sensitive (inhibition) and aldosterone responsive. However, amiloride-insensitive, cation-selective, as well as amiloride-insensitive, unstable cation channels have also been identified. Both of these channels were found to be degradation products of the amiloride-sensitive Na⁺ channel. The amiloride-sensitive Na⁺ channel is hydrolyzed by serine proteases such as kallikrein and urokinase and plasmin (normally found in the urine but produced by the kidney) (Lewis et al, 1995).

Sodium that is transported into the cell is removed at the basolateral membrane by a Na⁺–K⁺ exchanger. This leaves the cell with a negative intracellular charge. The basolateral membrane contains K⁺ and Cl⁻ channels, Na⁺–H⁺ exchangers, and Cl⁻–HCO₃⁻ exchangers. These channels and exchangers are important in recovery of cell volume during an increase in serosal osmolality (Donaldson and Lewis, 1990). Unfortunately, the precise role of the Na⁺ channel in the apical membrane of the umbrella cell is unknown. It is possible that the degradation of the channel might follow the filling of the bladder and that the changes in conductance of sodium may be a signaling factor for the bladder and micturition when it reaches capacity. Alternatively, it may be involved in the signaling pathway that allows insertion or removal of apical membrane on expansion of the bladder.

Parasympathetic Pathways

Parasympathetic preganglionic neurons innervating the lower urinary tract are located in the lateral part of the sacral intermediate gray matter in a region termed the sacral parasympathetic nucleus (Nadelhaft et al, 1980; Morgan et al, 1981; de Groat et al, 1993a; Morgan et al, 1993; de Groat et al, 1996). Parasympathetic preganglionic neurons send axons through the ventral roots to peripheral ganglia, where they release the excitatory transmitter acetylcholine (de Groat and Booth, 1993). Parasympathetic postganglionic neurons in humans are located in the detrusor wall layer as well as in the pelvic plexus. This is an important fact to remember because patients with cauda equina or pelvic plexus injury are neurologically decentralized but may not be completely denervated. Cauda equina injury allows possible afferent and efferent neuron interconnection at the level of the intramural ganglia (de Groat et al, 1993a; de Groat et al, 1996).

Sympathetic Pathways

Sympathetic outflow from the rostral lumbar spinal cord provides a noradrenergic excitatory and inhibitory input to the bladder and urethra (Andersson, 1993). Activation of sympathetic nerves induces relaxation of the bladder body and contraction of the bladder outlet and urethra, which contribute to urine storage in the bladder. The peripheral sympathetic pathways follow a complex route that passes through the sympathetic chain ganglia to the inferior mesenteric ganglia and then through the hypogastric nerves to the pelvic ganglia.

Somatic Pathways

The external urethral sphincter motoneurons are located along the lateral border of the ventral horn, commonly referred to as the Onuf nucleus (Fig. 60–13) (Thor et al, 1989). Sphincter motoneurons also exhibit transversely oriented dendritic bundles that project laterally into the lateral funiculus, dorsally into the intermediate gray matter, and dorsomedially toward the central canal.

Figure 60–13 Cross section of sacral spinal cord; neuroanatomic distribution of primary afferent and efferent components of storage and micturition reflexes. For purposes of clarity, afferent components are shown only on the left, and efferent components are shown only on the right. Both components are, of course, distributed bilaterally and thus overlap extensively. Visceral afferent components represent bladder, urethral, and genital (glans penis or clitoris) afferent fibers contained in the pelvic and pudendal nerves. Cutaneous perineal afferent components represent afferent fibers that innervate the perineal skin contained in the pudendal nerve. Muscle spindle afferent components represent Ia/b afferent fibers contained in the levator ani nerve that innervate muscle spindles in the levator ani muscle. EUS, external urethral sphincter; LCP, lateral collateral projection; MCP, medial collateral projection; SPN, sacral parasympathetic nucleus.

Afferent Pathways

Afferent axons in the pelvic, hypogastric, and pudendal nerves transmit information from the lower urinary tract to the lumbosacral spinal cord (de Groat, 1986; Janig and Morrison, 1986; Yoshimura et al, 2008). The primary afferent neurons of the pelvic and pudendal nerves are contained in sacral dorsal root ganglia (DRG), whereas afferent innervation in the hypogastric nerves arises in the rostral lumbar DRG. The central axons of the DRG neurons carry the sensory information from the lower urinary tract to second-order neurons in the spinal cord (Morgan et al, 1981; de Groat, 1986; Thor et al, 1989; de Groat et al, 1996) (see Fig. 60–13). Visceral afferent fibers of the pelvic (Morgan et al, 1981) and pudendal (Thor et al, 1989) nerves enter the cord and travel rostrocaudally within the Lissauer tract.

Pelvic nerve afferents, which monitor the volume of the bladder and the amplitude of the bladder contraction, consist of myelinated (Aδ) and unmyelinated (C) axons (Table 60–3). During neuropathic conditions and possibly inflammatory conditions, there is recruitment of C fibers that form a new functional afferent pathway that can cause urgency incontinence and possibly bladder pain.

Table 60–3 Bladder Afferent Properties

Sensing bladder volume is of particular relevance during urine storage. On the other hand, afferent discharges that occur during a bladder contraction have an important reflex function and appear to reinforce the central drive that maintains the detrusor contraction. Afferent nerves that respond to both distention and contraction, that is, “in-series tension receptors,” have been identified in the pelvic and hypogastric nerves of cats and rats (see Table 60–3) (Iggo, 1955; Floyd et al, 1976; Morrison, 1997). Afferents that respond only to bladder filling have been identified in the rat bladder (Morrison, 1998) and appear to be volume receptors, possibly sensitive to stretch of the mucosa. In the cat bladder, some in-series tension receptors may also respond to bladder stretch (Downie and Armour, 1992). In the rat, there is evidence that many C bladder afferents are volume receptors that do not respond to bladder contractions, a property that distinguishes them from in-series tension receptors (Morrison, 1998).

In the mouse pelvic nerve, four classes of bladder afferents (serosal, muscular, muscular/urothelial, and urothelial) have been identified based on responses to receptive field stimulation with different mechanical stimuli, including probing, stretch, and stroking the urothelium. Both low-threshold, representing 65% to 80% of the total population, and high-threshold stretch-sensitive muscular afferents are present (Daly et al, 2007; Xu and Gebhart, 2008). The muscular afferents can be sensitized by application of a combination of inflammatory mediators (bradykinin, serotonin, prostaglandin, and histamine at pH 6.0) (Xu and Gebhart, 2008).

In the guinea pig bladder, four classes of C-fiber afferents have also been detected (Zagorodnyuk et al, 2006, 2007). The first class, muscle mechanoreceptors, are activated by stretch but not by mucosal stroking with light (0.05 to 0.1 mN) von Frey hairs or by hypertonic saline, α,β-methylene ATP or capsaicin. Removal of the urothelium did not affect their stretch-induced firing. The second class, muscle-mucosal mechanoreceptors, are activated by both stretch and mucosal stroking, by hypertonic solution, α,β-methylene-ATP but not by capsaicin. Stroking- and stretch-induced firing is significantly reduced by removal of the urothelium. The third class of afferents, mucosal high-responding mechanoreceptors, are stretch insensitive but can be activated by mucosal stroking, hypertonic solution, α,β-methylene-ATP, and capsaicin. Stroking-induced activity is reduced by removal of the urothelium. The fourth class of afferents, mucosal low-responding mechanoreceptors, are stretch insensitive but can be weakly activated by mucosal stroking but not by hypertonic solution, α,β-methylene-ATP, or capsaicin. Removal of the urothelium reduces stroking-induced firing. A recent study by Zagorodnyuk and colleagues (2009) also showed that mechanotransduction during stretch or mucosal stroking in the second and third classes of afferents, stretch-sensitive muscle-mucosal mechanoreceptors, and stretch-insensitive, mucosal high-responding afferents, are not dependent upon Ca²⁺-dependent exocytotic release of mediators or ATP, but are likely to be induced by benzamil-sensitive, stretch-activated ion channels on nerve endings. This is because stretch- or stroking-induced firing of these afferents are not affected by the nonselective P2 purinoreceptor antagonist or Ca²⁺-free solution, but are inhibited by benzamil, a mechanogated channel blocker. These results suggest that substances released from the urothelium during mechanical stimulation, such as ATP, may not be involved in the modulation of the activity of bladder afferents located in the vicinity of the urothelium, at least in the normal condition, which is against the current theory of the urothelium–afferent interaction by urothelially released substances during bladder stretch.

Species differences, as well as differences of nomenclature, might account for some of the variations in reported properties of bladder afferents. For example, the conduction velocity that differentiates Aδ and C fibers is 2 m/sec in the cat, whereas it is 1.3 m/sec in the rat (Waddell et al, 1989). In the cat, Aδ bladder afferents appear to be low-threshold mechanoreceptors (Häbler et al, 1993), whereas C-bladder afferents (Häbler et al, 1990) are generally mechanoinsensitive (“silent C fibers”) (see Table 60–3). Some of the latter may be nociceptive and have been found to be sensitized by intravesical administration of chemicals (such as high potassium), low pH, high osmolality, and irritants such as capsaicin (Fig. 60–14) (Maggi et al, 1987; McMahon and Abel, 1987; Häbler et al, 1990; Wen et al, 1994; Wen and Morrison, 1994, 1996; Zagorodnyuk et al, 2009). After exposure to these substances, the sensitivity of bladder mechanoreceptors to distention increases, and some “silent” afferents become mechanoreceptive.

Figure 60–14 Actions of chemical mediators that may sensitize mechanosensory nerve endings in the bladder mucosa. Adenosine triphosphate (ATP) can be released from the urothelium and may sensitize the mechanoreceptors, which respond to stretch of the mucosa during bladder distention. Neuropeptides transported to the sensory ending by axoplasmic transport may be released during distention and chemical stimulation, and neurokinin A can act on NK₂ autoreceptors, which sensitize the mechanosensitive endings. This mechanism can be induced by high urinary potassium concentrations and possibly by other sensitizing solutions within the bladder lumen, such as those with high osmolality or low pH; the presence in the tissues of inflammatory mediators may also sensitize the endings. The smooth muscle can generate force that may influence some mucosal endings, and the production of nerve growth factor is another mechanism that can influence the mechanosensitivity of the sensory ending through the TrkA receptor.

In the urethra, afferent nerves are distributed between the muscle fibers, around blood vessels, in the urothelium, and in a dense suburothelial plexus (Crowe et al, 1986; Tainio, 1993; Fahrenkrug and Hannibal, 1998). In some species, afferent nerves extend to the luminal surface of the urothelium. The striated sphincter muscle that surrounds the urethra receives a very sparse afferent innervation that is localized primarily to nerve bundles passing between the muscle bundles. Specialized tension receptors (muscle spindles) that are innervated by large-diameter myelinated group IA afferents and that are prominent in most striated muscles are absent (Gosling et al, 1981) or are present in low density (Lassmann, 1984) in striated sphincter muscles.

The Storage Phase of the Bladder

Intravesical pressure measurements during bladder filling in both humans and animals reveal low and relatively constant bladder pressures when bladder volume is below the threshold for inducing voiding (Fig. 60–17). The accommodation of the bladder to increasing volumes of urine is primarily a passive phenomenon dependent on the intrinsic properties of the vesical smooth muscle and stroma, as well as the quiescence of the parasympathetic efferent pathway (Torrens and Morrison, 1987; de Groat et al, 1993a; Yoshimura et al, 2008). The bladder to sympathetic reflex also contributes as a negative feedback or urine storage mechanism that promotes closure of the urethral outlet and inhibits neurally mediated contractions of the bladder during bladder filling (de Groat and Theobald, 1976) (see Table 60–4). Reflex activation of the sympathetic outflow to the lower urinary tract can be triggered by afferent activity induced by distention of the urinary bladder (de Groat and Theobald, 1976; de Groat et al, 1993a). This reflex response is organized in the lumbosacral spinal cord and persists after transection of the spinal cord at the thoracic levels (Fig. 60–18). However, this bladder to sympathetic mechanism to suppress bladder contractions during urine storage may be weak in humans, given that bilateral retroperitoneal lymph node dissection, in which the sympathetic chains are destroyed, has no discernible alteration of filling or storage function in humans.

Figure 60–17 Combined cystometrogram and sphincter electromyogram (EMG) comparing reflex voiding responses in an infant (A) and in a paraplegic patient (C) with a voluntary voiding response in an adult (B). The x-axis in all records represents bladder volume in milliliters, and the y-axis represents bladder pressure in centimeters of water and electrical activity of the electromyographic recording. On the left side of each trace, the arrows indicate the start of a slow infusion of fluid into the bladder (bladder filling). Vertical dashed lines indicate the start of sphincter relaxation that precedes by a few seconds the bladder contraction in A and B. In B, note that a voluntary cessation of voiding (stop) is associated with an initial increase in sphincter electromyographic activity followed by a reciprocal relaxation of the bladder. A resumption of voiding is again associated with sphincter relaxation and a delayed increase in bladder pressure. On the other hand, in the paraplegic patient (C), the reciprocal relationship between bladder and sphincter is abolished. During bladder filling, transient uninhibited bladder contractions occur in association with sphincter activity. Further filling leads to more prolonged and simultaneous contractions of the bladder and sphincter (bladder-sphincter dyssynergia). Loss of the reciprocal relationship between bladder and sphincter in paraplegic patients interferes with bladder emptying.

(From de Groat WC. Basic neurophysiology and neuropharmacology. In: Abrams P, Khoury S, Wein A, editors. Incontinence. Plymouth [UK]: Health Publications; 1999. p. 112.)

Figure 60–18 Diagram showing bladder to urethra reflex pathways. Afferent pathway (dashed line) from the detrusor activates spinal reflex mechanisms that induce firing in somatic cholinergic nerves to the external urethral sphincter, sympathetic adrenergic nerves to the urethral smooth muscle, and cholinergic and nitrergic nerves to the urethral smooth muscle. Bulbospinal pathways from the brain can modulate these spinal reflex mechanisms. ACh, acetylcholine; NEPI, norepinephrine; NO, nitric oxide; excitatory (+) and inhibitory (−) mechanisms.

During bladder filling, the activity of the sphincter electromyogram also increases (see Fig. 60–17), reflecting an increase in efferent firing in the pudendal nerve and an increase in outlet resistance that contributes to the maintenance of urinary continence. Pudendal motoneurons are activated by bladder afferent input (the guarding reflex) (Park et al, 1997), whereas during micturition the motoneurons are reciprocally inhibited (de Groat et al, 1993a). External urethral sphincter motoneurons are also activated by urethral or perineal afferents in the pudendal nerve (Fedirchuk et al, 1992). This reflex may represent, in part, a continence mechanism that is activated by proprioceptive afferent input from the urethra or pelvic floor and that induces closure of the urethral outlet. These excitatory sphincter reflexes are organized in the spinal cord. Inhibition of external urethral sphincter reflex activity during micturition is dependent, in part, on supraspinal mechanisms, because it is weak or absent in chronic spinal animals and humans, resulting in simultaneous contractions of bladder and sphincter (i.e., detrusor-sphincter dyssynergia) (Rossier and Ott, 1976; Blaivas, 1982).

Sphincter to Bladder Reflexes

It is well known that stimulation of somatic afferent pathways projecting in the pudendal nerve to the caudal lumbosacral spinal cord can inhibit voiding function. The inhibition can be induced by activation of afferent input from various sites, including the penis, vagina, rectum, perineum, urethral sphincter, and anal sphincter (de Groat et al, 1979, 1993a, 2001). Electrophysiologic studies in cats showed that the inhibition was mediated by suppression of interneuronal pathways in the sacral spinal cord and also by direct inhibitory input to the parasympathetic preganglionic neurons (de Groat et al, 1982).

On the basis of experiments in our laboratory and the review of medical literature, we believe that contractions of the external urethral sphincter, and possibly other pelvic floor striated muscles, stimulate firing in muscle proprioceptive afferents, which then activate central inhibitory mechanisms to suppress the micturition reflex (Fig. 60–19). A similar inhibitory mechanism has been identified in monkeys by directly stimulating the anal sphincter muscle (McGuire et al, 1983). In monkeys, at least part of the inhibitory mechanism must be localized in the spinal cord, because it persisted in T4 chronic paraplegic animals.

Figure 60–19 Urethra to bladder reflexes. Activity in afferent nerves (dashed lines) from the urethra can facilitate parasympathetic efferent outflow to the detrusor by means of a supraspinal pathway passing through the pontine micturition center (PMC), as well as a spinal reflex pathway. Afferent input from the external urethral sphincter (EUS) can inhibit parasympathetic outflow to the detrusor through a spinal reflex circuit. Electrical stimulation of motor axons in the S1 ventral root elicits an EUS contraction and EUS afferent firing that, in turn, inhibits reflex bladder activity, excitatory (+) and inhibitory (−) mechanisms.

The Emptying Phase of the Bladder

The storage phase of the bladder can be switched to the voiding phase either involuntarily (reflexively) or voluntarily. The former is readily demonstrated in the human infant or in patients with neuropathic bladder when the bladder wall tension due to increased volume of urine exceeds the micturition threshold. At this point, increased afferent firing from tension receptors in the bladder reverses the pattern of efferent outflow, producing firing in the sacral parasympathetic pathways and inhibition of sympathetic and somatic pathways. The expulsion phase consists of an initial relaxation of the urethral sphincter (see Fig. 60–17) followed in a few seconds by a contraction of the bladder, an increase in bladder pressure, and the flow of urine. Relaxation of the urethral smooth muscle during micturition is mediated by activation of a parasympathetic pathway to the urethra that triggers the release of nitric oxide, an inhibitory transmitter (Andersson, 1993; Bennett et al, 1995), and by removal of excitatory inputs to the urethra. Secondary reflexes elicited by flow of urine through the urethra facilitate bladder emptying (Torrens and Morrison, 1987; de Groat et al, 1993a; Jung et al, 1999). These reflexes require the integrative action of neuronal populations at various levels of the neuraxis (see Fig. 60–16A). The parasympathetic outflow to the detrusor and urethra has a more complicated central organization involving spinal and spinobulbospinal pathways passing through a micturition center in the pons (pontine micturition center) (see Fig. 60–16B).

Urethra to Bladder Reflexes

A landmark in the historical progress of neurourology is the contribution of Barrington. Using his keen observational skills, Barrington (1931, 1941) reported that urine flow or mechanical stimulation of the urethra with a catheter could excite afferent nerves that, in turn, facilitated reflex bladder contractions in the anesthetized cat (see Fig. 60–19). He proposed that this facilitatory urethra to bladder reflex could promote complete bladder emptying. Barrington identified two components of this reflex. One component was activated by a somatic afferent pathway in the pudendal nerve and produced facilitation by a supraspinal mechanism involving the pontine micturition center (Barrington, 1931) (see Fig. 60–19). Studies have confirmed the existence of this type of reflex by the pudendal nerve because low-frequency electrical stimulation of afferent axons in the pudendal nerve in humans, or the deep perineal nerve (a caudal branch of the pudendal nerve) in cats, can initiate reflex bladder contractions and voiding (Shefchyk and Buss, 1998; Boggs et al, 2005). The other component was activated by a visceral afferent pathway in the pelvic nerve and produced facilitation by a spinal reflex mechanism (Barrington, 1941).

Studies (Jung et al, 1999) in the anesthetized rat have also provided additional support for Barrington’s findings (Dokita et al, 1991). Measurements of reflex bladder contractions, under isovolumetric conditions during continuous urethral perfusion (0.075 mL/min), revealed that the frequency of micturition reflexes was significantly reduced when urethral perfusion was stopped or after infusion of lidocaine (1%) into the urethra. Intraurethral infusion of nitric oxide donors (S-nitroso-N-acetylpenicillamine [SNAP], or nitroprusside, 1 to 2 mM) markedly decreased urethral perfusion pressure (approximately 30%) and decreased the frequency of reflex bladder contractions (45% to 75%) but did not change the amplitude of bladder contractions (Fig. 60–20). Desensitization of the urethral afferent with intraurethral capsaicin also dramatically altered the micturition reflex (Fig. 60–21). It was concluded that activation of urethral afferents during urethral perfusion could modulate the micturition reflex in the rat. This may be an explanation of why stress incontinence and urgency incontinence often occur together in women. Jung and colleagues (1999) speculated that in women with mixed incontinence, leakage of urine into the urethra can stimulate afferents and induce or increase detrusor overactivity. The theory is that stress incontinence can induce urgency incontinence (Lavelle et al, 2000). Surgical cure of the stress incontinence of women with mixed incontinence has resolved the urgency incontinence in up to half of the patients.

Figure 60–20 Effects of intraurethral S-nitroso-N-acetylpenicillamine (SNAP) on the bladder pressure (Pves) and urethral pressure (Pura) in normal female rats. A, Before treatment. B, After intraurethral administration of SNAP (2 mM). Urethral perfusion pressure immediately decreased. In addition, bladder contraction frequency was significantly decreased. The duration of reflex urethral relaxation was increased.

(From Jung SY, Fraser MO, Ozawa H, et al. Urethral afferent nerve activity affects the micturition reflex: implication for the relationship between stress incontinence and detrusor instability. J Urol 1999;162:204–12.)

Figure 60–21 Effects of intraurethral capsaicin on the bladder pressure (Pves) and urethral pressure (Pura) in normal female rats. A, Before treatment. B, After intraurethral administration of capsaicin (100 µM). Initially, intraurethral capsaicin instillation increased the bladder contraction frequency, but 30 minutes after continuous infusion, the activity was blocked.

(From Jung SY, Fraser MO, Ozawa H, et al: Urethral afferent nerve activity affects the micturition reflex: implication for the relationship between stress incontinence and detrusor instability. J Urol 1999;162:204–12.)

Spinal Cord

In the spinal cord, afferent pathways terminate on second-order interneurons that relay information to the brain or to other regions of the spinal cord, including the preganglionic and motor nuclei. Because disynaptic or polysynaptic pathways, but not monosynaptic pathways, mediate bladder, urethral, and sphincter reflex, interneuronal mechanisms must play an essential role in the regulation of lower urinary tract function. Electrophysiologic (de Groat et al, 1981; Araki and de Groat, 1997) and neuroanatomic (Birder and de Groat, 1993; Vizzard et al, 1995; Nadelhaft and Vera, 1996; Sugaya et al, 1997) techniques have identified lower urinary tract interneurons in the same regions of the cord that receive afferent input from the bladder.

As shown in Figure 60–13, horseradish peroxidase labeling techniques in the cat revealed that afferent projections from the external urethral sphincter and levator ani muscles (i.e., pelvic floor) project into different regions of the sacral spinal cord. The external urethral sphincter afferent terminals are located in the superficial layers of the dorsal horn and at the base of the dorsal horn (laminae V to VII and lamina X), whereas the levator ani afferents project into a region just lateral to the central canal and extend into the medial ventral horn. The external urethral sphincter afferents overlap closely with the central projections of visceral afferents in pelvic nerve that innervate the bladder and urethra (Morgan et al, 1981). Intracellular labeling experiments also showed that the dendritic patterns of external urethral sphincter motoneurons (Sasaki, 1994) and parasympathetic preganglionic neurons (Morgan et al, 1993) are similar. Pharmacologic experiments revealed that glutamic acid is the excitatory transmitter in these pathways. In addition, approximately 15% of interneurons medial to the sacral parasympathetic nucleus in laminae V through VII make inhibitory synaptic connections with the preganglionic neurons (de Groat et al, 1996). These inhibitory neurons release γ-aminobutyric acid (GABA) and glycine. Reflex pathways that control the external sphincter muscles also use glutamatergic excitatory and GABAergic and glycinergic inhibitory interneuronal mechanisms.

Tracing with neurotropic viruses, such as pseudorabies virus, has been particularly useful. Pseudorabies virus can be injected into a target organ and then move intra-axonally from the periphery to the central nervous system. In the nervous system, the virus can replicate and then pass retrogradely across synapses to infect second- and third-order neurons in the neural pathways (Vizzard et al, 1995; Nadelhaft and Vera, 1996; Sugaya et al, 1997). Because pseudorabies virus can be transported across many synapses, it could sequentially infect all the neurons that connect directly or indirectly to the lower urinary tract. Interneurons identified by retrograde transport of pseudorabies virus injected into the urinary bladder are located in the region of the sacral parasympathetic nucleus, the dorsal commissure, and the superficial laminae of the dorsal horn (de Groat et al, 1996; Nadelhaft and Vera, 1996; Sugaya et al, 1997). A similar distribution of labeled interneurons has been noted after injection of virus into the urethra (Vizzard et al, 1995) or the external urethral sphincter (Nadelhaft and Vera, 1996), indicating a prominent overlap of the interneuronal pathways controlling the various target organs of the lower urinary tract.

The micturition reflex can be modulated at the level of the spinal cord by interneuronal mechanisms activated by afferent input from cutaneous and striated muscle targets. Micturition reflex can also be modulated by inputs from visceral organs (de Groat et al, 1975; McGuire, 1977; de Groat, 1978; de Groat et al, 1981; McMahon and Morrison, 1982; Torrens and Morrison, 1987; de Groat et al, 1993a; Morrison et al, 1995; Yoshimura et al, 2008). Stimulation of afferent fibers from various regions (anus, colon-rectum, vagina, uterine cervix, penis, perineum, pudendal nerve) can inhibit the firing of sacral interneurons evoked by bladder distention (de Groat et al, 1981). This inhibition may be a result of presynaptic inhibition at primary afferent terminals or be due to direct postsynaptic inhibition of the second-order neurons. Direct postsynaptic inhibition of bladder preganglionic neurons can also be elicited by stimulation of somatic afferent axons in the pudendal nerve or visceral afferents from the distal bowel (de Groat and Ryall, 1969; de Groat, 1978). Suppression of detrusor overactivity in patients by sacral root stimulation may reflect, in part, activation of the afferent limb of these visceral-bladder and somatic-bladder inhibitory reflexes (Wheeler et al, 1992; Bosch and Groen, 1995; Chancellor and Chartier-Kastler, 2000).

Pontine Micturition Center and Brainstem Modulatory Mechanisms

Various studies indicate that the micturition reflex is normally mediated by a spinobulbospinal reflex pathway passing through relay centers in the brain (see Fig. 60–16B) (de Groat, 1975; Torrens and Morrison, 1987; de Groat et al, 1993a; Yoshimura et al, 2008). Studies in animals by use of brain-lesioning techniques revealed that neurons in the brainstem at the level of the inferior colliculus have an essential role in the control of the parasympathetic component of micturition (Torrens and Morrison, 1987; de Groat et al, 1993a; Yoshimura and de Groat, 1997; Yoshimura et al, 2008). Removal of areas of brain above the colliculus by intercollicular decerebration usually facilitates micturition by elimination of inhibitory inputs from more rostral centers. However, transections at any point below the colliculi abolish micturition.

The dorsal pontine tegmentum has been firmly established as an essential control center for micturition in normal subjects. First described by Barrington (1921), it has subsequently been called the Barrington nucleus, the pontine micturition center (Blok and Holstege, 1997), or the M region (Blok and Holstege, 1996; Holstege et al, 1996) because of its medial location.

In addition to providing axonal inputs to the locus ceruleus and the sacral spinal cord (Ding et al, 1995; Otake and Nakamura, 1996; Valentino et al, 1996), neurons in the pontine micturition center (PMC) also send axon collaterals to the paraventricular thalamic nucleus, which is thought to be involved in the limbic system modulation of visceral behavior (Otake and Nakamura, 1996). Some neurons in the PMC also project to the periaqueductal gray region (PAG) (Blok et al, 1998), which regulates many visceral activities as well as pain pathways (Valentino et al, 1995). Thus neurons in the PMC communicate with multiple supraspinal neuronal populations that may coordinate micturition with other functions of the organism. Although the circuitry in humans is uncertain, brain imaging studies have revealed increases in blood flow in this region of the pons during micturition (Blok et al, 1997b). This change presumably reflects increases in neuronal activity. Thus the PMC appears critical for micturition across species.

Neurons in the PMC provide direct synaptic inputs to sacral preganglionic neurons (Blok and Holstege, 1997) as well as to GABAergic neurons in the sacral dorsal commissure region (Blok et al, 1997a). The former neurons carry the excitatory outflow to the bladder, whereas the latter neurons are thought to be important in mediating an inhibitory influence on external urethral sphincter motoneurons (Blok et al, 1998). Because of these reciprocal connections, the PMC can promote bladder–sphincter synergy. Studies in rats indicate that activation of bladder preganglionic neurons by input from the PMC can be blocked by inotropic glutamate receptor antagonists, suggesting that neurons in the PMC use glutamate as a neurotransmitter (Matsumoto et al, 1995a).

Besides the PMC region, transneuronal virus tracing methods have identified many populations of neurons in the brainstem that are involved in the control of bladder, urethra, and the urethral sphincter, including medullary raphe nuclei, which contain serotonergic neurons; the locus coeruleus, which contains noradrenergic neurons; PAG; and the A5 noradrenergic cell group (Nadelhaft and Vera, 1992; Vizzard et al, 1995; Sugaya et al, 1997). Other anatomical studies in which anterograde tracer substances were injected into brain areas and then identified in terminals in the spinal cord are consistent with the virus tracing data. Projections from neurons in the lateral pons (L-region or nucleus locus subcoeruleus) terminate rather selectively in the sphincter motor nucleus to enhance the contraction of pelvic floor muscle and increased urethral pressure during the storage phase in cats, providing evidence that this area in the lateral pons functions as “the pontine continence center” (Blok, 2002; Sugaya et al, 2003). The parabrachial nuclei (PBN) at the dorsolateral pons, which send projections to the sacral spinal cord neurons, are also known to be involved in the control of micturition (Lumb and Morrison, 1987; de Groat et al, 1993b). A recent study in rats has shown that firing of PBN neurons corresponds to bladder contraction during cystometry and that the medial subnuclei of PBN plays an important role in the mediation of normal bladder contractions (Liu et al, 2007).

In the midbrain, there is increasing evidence that the mesencephalic PAG, which directly receives information of bladder fullness by ascending spinal tract neurons and sends projections to the PMC, seems to play an important role in the integration of the micturition reflex (Blok, 2002; Holstege, 2005). Stimulation of the pelvic nerve results in short latency potentials in the caudal periaqueductal gray (Noto et al, 1991). Electrical and chemical stimulation of the ventrolateral PAG induces micturition in rats and cats (Matsuura et al, 2000; Taniguchi et al, 2002), and this PAG-mediated micturition reflex seems to be induced by subsequent activation of the PMC following PAG stimulation (Matsuura et al, 2000). In cats, there are different patterns of neuronal firing in response to storage/micturition cycles in the PAG, and stimulation of the rostral PAG can inhibit the micturition reflex (Liu et al, 2004; Numata et al, 2008). Thus the PAG appears to function as a relay center between the PMC and spinal cord neurons that are critically involved in the excitatory and inhibitory control of the micturition reflex.

Human Brain Imaging Studies

In the last decade, the areas of the brain involved in the control of micturition have been examined in human brain imaging studies using single photon emission–computed tomography (SPECT), positron emission tomography (PET), and functional magnetic resonance imaging (fMRI) (DasGupta et al, 2007; Griffiths and Tadic, 2008) (Fig. 60–22). Some studies evaluated the brain areas responsible for the perception of bladder fullness and the sensation of the desire to void during bladder filling, whereas others examined brain activity during micturition, voluntary contractions of the pelvic floor during urine withholding, or during cold stimulation of the bladder. PET scan studies in normal men and women revealed that during voiding two cortical areas (the dorsolateral prefrontal cortex and anterior cingulate gyrus) were active (i.e., exhibited increased blood flow). The hypothalamus, including the preoptic area, as well as the pons and the PAG, also showed activity in concert with voluntary micturition (Blok et al, 1997c, 1998). Another PET study during voiding also confirmed that micturition was associated with increased activity in the pons, inferior frontal gyrus, hypothalamus, and PAG, while also showing activity in several other cortical areas (postcentral gyrus, superior frontal gyrus, thalamus, insula, and globus pallidus) and the cerebellar vermis (Nour et al, 2000).

Figure 60–22 A, Areas of brain activation during the urinary storage phase based on human imaging studies using PET and fMRI. SMA, supplementary motor area. PAG, periaqueductal gray. B, Simplified model of supraspinal control system. Ascending afferent fibers (afferents) carrying the information from the bladder synapse in the PAG and are relayed to the right insula (RI), forming the substrate for sensation. The anterior cingulate gyrus (ACG) is responsible for monitoring, arousal, and efferent output to the PAG and pontine micturition center (PMC). The prefrontal cortex (PFC) is involved in voluntary decision about voiding and generates efferent signals to control ACG and ultimately PMC. The PMC then provides motor output to cause voiding.

(A, Modified from Fowler A, Griffiths CJD, et al. The neural control of micturition. Nat Rev Neurosci 2008;9:453–66; B, modified from Griffiths D, Tadic SD. Bladder control, urgency, and urge incontinence: evidence from functional brain imaging. Neurourol Urodyn 2008; 27:466–74.)

Other PET studies that examined the changes in brain activity during filling of the bladder revealed that increased activity occurred in the PAG, the midline pons, the anterior and midcingulate gyrus, anterior insula, and bilaterally in the frontal lobes (Athwal et al, 1999; Athwal et al, 2001; Matsuura et al, 2002). The results were consistent with the notion that the PAG receives information about bladder fullness and then relays this information to other brain areas involved in the control of bladder storage. In fMRI studies in men and women, activation during urinary storage is found in the supplementary motor area, midcingulate cortex, insula, and right prefrontal cortex, and the right anterior insula and midbrain PAG were more active at higher than at lower bladder volumes (Kuhtz-Buschbeck et al, 2005, 2009). Other fMRI studies also addressed the effect of pelvic floor contraction with a full bladder, and reported activation of the parietal cortex, cerebellum, putamen and supplementary motor area (Zhang et al, 2005), or the frontal cortex, basal ganglia, and cerebellum (Seseke et al, 2006).

Brain imaging studies have also been performed to identify changes in cerebral perception of detrusor overactivity. A PET study in patients with Parkinson disease reported activation of the PAG, supplementary motor area, basal ganglia, and cerebellum during detrusor overactivity, (Kitta et al, 2006). When comparing healthy control subjects and those with confirmed overactivity using fMRI, infusion into the bladder is associated with activity in the PAG, thalamus, insula, and anterior cingulated gyrus; however, weak responses or deactivation in the prefrontal cortex or the limbic system, as well as exaggerated responses in the anterior cingulate gyrus at large bladder volumes, are observed in urgency-incontinent patients (Griffiths et al, 2005, 2007). Based on the results in human imaging studies, Griffiths proposes that (1) during urine storage in healthy subjects, bladder and urethral afferents received in the PAG and mapped in the insula form normal sensations of desire to void, which is monitored by the anterior cingulate gyrus, and the voiding reflex is continuously inhibited until the decision to void is made in the prefrontal cortex; and (2) urgency-incontinent patients have weak responses or deactivation observed in the prefrontal cortex or the limbic system, leading to a defect of supraspinal bladder control, which may cause urgency incontinence, and enhanced responses in the anterior cingulate gyrus, which may be due to abnormal bladder afferents or related to a loss of control in other brain areas, and could be correlated with the urgency sensation (Griffiths and Tadic, 2008) (see Fig. 60–22). Radiographic imaging of the central nervous system (CNS), with control and dysregulation of the lower urinary tract, is an exciting area of research, but the results are preliminary. Subjects are conscious and thinking about their bladder during the test and may have an indwelling catheter that may alter what parts of the brain are activated naturally or artificially.

Muscarinic Mechanisms

Detrusor strips from normal human bladders are contracted by cholinergic muscarinic receptor agonists and by electrical stimulation of intrinsic cholinergic nerves. Contractile responses can be completely abolished by atropine (Sibley, 1984). There are at least five receptor subtypes based on molecular cloning and four different receptor subtypes based on pharmacology (M₁ to M₅) (Somogyi et al, 1994; Wang et al, 1995; Eglen et al, 1996; Yamaguchi, 1996; Hegde et al, 1997).

Pharmacologically, M₁, M₂, and M₃ receptor subtypes have been found in the human bladder by receptor binding assays (Kondo et al, 1995); all M₁ to M₅ receptor mRNAs are detected by reverse transcription–polymerase chain reaction assays (Andersson and Wein, 2004; Mansfield et al, 2005). Although ligand receptor binding studies revealed that M₂ receptors predominate, M₃ receptors mediate cholinergic contractions (Eglen et al, 1994; Harriss et al, 1995; Yamaguchi, 1996; Hegde et al, 1997; Lai et al, 1998). Stimulation of M₃ receptors by acetylcholine leads to IP₃ hydrolysis due to phospholipase C activation and then to the release of intracellular calcium and a smooth muscle contraction (Harriss et al, 1995; Fry et al, 2002) (Fig. 60–25). The involvement of transmembrane flux of calcium ions through nifedipine-sensitive L-type Ca²⁺ channels has also been indicated in M₃ receptor–mediated detrusor muscle contractions because the L-type Ca²⁺ channel inhibitor nifedipine strongly suppressed carbachol-induced detrusor contractions, whereas the phospholipase C inhibitor or the store-operated Ca²⁺ channel inhibitor caused little inhibition in rats and humans (Andersson and Arner, 2004; Andersson and Wein 2004; Schneider et al, 2004a, 2004b; Frazier et al, 2008) (see Fig. 60–25). However, other studies have indicated the major contribution of the phospholipase C–mediated mechanism to M₃ receptor–induced detrusor contractions, because phospholipase C inhibitors significantly suppressed carbachol-induced detrusor contractions in rats (Braverman et al, 2006a, 2006b), and intracellular calcium elevation after carbachol application was observed without membrane depolarization in human bladders, which is required for the opening of L-type Ca²⁺ channels (Fry et al, 2002). Hashitani and colleagues (2000) have reported that the stimulation of muscarinic receptors activates both calcium influx through L-type Ca²⁺ channels and calcium release from intracellular calcium stores in guinea pig bladders.

Figure 60–25 Transmitter signal pathways involved in activation of detrusor contractions through muscarinic M₃ receptors. There seem to be differences between species in the contribution of the different pathways in contractile activation. ACh, acetylcholine; CIC, calcium-induced calcium release; DAG, diacylglycerol; IP₃, inositol 1.4.5-trisphosphate; MLC, myosin light chain; PKC, protein kinase C; PLC, phospholipase C; SR, sarcoplasmic reticulum.

(From 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.)

It has also been proposed (Hegde et al, 1997; Ehler et al, 2005) that coactivation of M₂ receptors could enhance the response to M₃ stimulation by (1) inhibition of adenylate cyclase, thereby suppressing sympathetically mediated depression of detrusor muscle; (2) inactivation of K⁺ channels; or (3) activation of nonspecific cation channels. In addition, because the specific Rho-kinase inhibitor Y-27632 reportedly suppresses carbachol-induced detrusor contractions in rats and humans, muscarinic receptor activation in detrusor smooth muscles is likely to stimulate the Rho-kinase pathway, leading to a direct inhibition of myosin phosphatase that induces calcium sensitization to enhance the ability of the muscle to generate the same contractile force with lower levels of intracellular calcium (Andersson and Wein, 2004; Schneider et al, 2004a, 2004b; Frazier et al, 2008). Although the involvement of M₃ receptors for the Rho-kinase activation has been suggested (Andersson and Arner, 2004; Andersson and Wein, 2004; Schneider et al, 2004a) (see Fig. 60–25), a study has also suggested the participation of M₂ receptors in this mechanism because Y-27632 not only suppressed carbachol-induced muscle contractions but also increased the affinity of darifenacin, an M₃ receptor antagonist with approximately a 30-fold selectivity for M₃ over M₂ receptors, for inhibiting carbachol-induced contractions of rat bladders (Braverman et al, 2006a, 2006b). It has also been reported that the muscarinic receptor subtype–mediated detrusor contractions shift from M₃ to M₂ receptor subtype in certain pathologic conditions, such as obstructed or denervated hypertrophied bladders in rats (Braverman and Ruggieri, 2003; Braverman et al, 2006a, 2006b), as well as in bladder muscle specimens from patients with neurogenic bladder dysfunction (Pontari et al, 2004).

Studies using constructed mutant mice lacking the M₃ receptor or the M₂ and M₃ receptors have demonstrated that this subtype plays key roles in salivary secretion, pupillary constriction, and detrusor contractions (Matsui et al, 2000, 2002; Igawa et al, 2004). However, M₃-mediated signals in digestive and reproductive organs are dispensable, probably because of redundant mechanisms through other muscarinic acetylcholine receptor subtypes or other mediators (Matsui et al, 2000). In addition, it has also been found that male M₃ knockout mice had a distended bladder and larger bladder capacity compared with females, indicating a considerable sex difference in the micturition mechanism (Matsui et al, 2002; Igawa et al, 2004). Thus M₃ or M₂ and M₃ double-knockout mice should provide a useful animal model for the detrusor overactivity pathophysiology and pharmacology.

Muscarinic receptor antagonists tolterodine and oxybutynin (Table 60–5) are the most widely prescribed drugs for urinary incontinence. Oxybutynin is a nonspecific muscarinic antagonist with additional smooth muscle relaxant properties. The smooth muscle relaxation properties of oxybutynin may be clinically relevant only with intravesical instillation of the drug. Because new antimuscarinic drugs are such a hot topic for pharmaceutical development, all urologists should be aware of the existence of muscarinic receptor subtypes and their distribution in the lower urinary tract and other organs.

Table 60–5 Drugs with Bladder Action

We will briefly present two additional issues regarding the effect of antimuscarinic drugs on the bladder and salivary glands that have clinical relevancy. First, antimuscarinic drugs are metabolized, and their metabolites have pharmacologic effects. It has been shown that oxybutynin has less of a dry mouth effect than does its metabolite desethyoxybutynin (Gupta and Sathyan, 1999). Therefore the controlled-release formulation of oxybutynin maintains the efficacy of immediate-release oxybutynin but with significantly fewer side effects. Tolterodine and solifenacin have been shown in cats and rats, respectively, to have less activity on the salivary gland muscarinic receptors than on the bladder muscarinic receptors (Nilvebrant et al, 1997; Ohtake et al, 2004). Second, the site and speed of antimuscarinic metabolism appear to have profound effects in terms of clinical efficacy and side effects.

Muscarinic receptors are also located prejunctionally on cholinergic nerve terminals in the bladder (D’Agostino et al, 1986; Somogyi and de Groat, 1992; Somogyi et al, 1996; D’Agostino et al, 1997; Braverman et al, 1998; D’Agostino et al, 2000). Activation of M₁ prejunctional receptors facilitates acetylcholine release (Somogyi and de Groat, 1992; Somogyi et al, 1996), whereas activation of M₂ to M₄ receptors inhibits the release (D’Agostino et al, 1997; Braverman et al, 1998). It has been proposed that inhibitory M₂ to M₄ receptors are preferentially activated by autofeedback mechanisms during short periods of low-frequency nerve activity and thereby suppress cholinergic transmission during urine storage (Somogyi and de Groat, 1992). Conversely, M₁ receptors are activated during more prolonged, high-frequency nerve firing that would occur during voiding and thus participate in an amplification mechanism to promote complete bladder emptying. M₁-mediated facilitation of transmitter release involves the activation of a phospholipase C–protein kinase C signaling cascade that appears to facilitate the opening of L-type Ca²⁺ channels that are necessary for prejunctional facilitation of acetylcholine release from parasympathetic nerve terminals (Somogyi et al, 1996, 1997). Inhibitory and facilitatory muscarinic receptors are also present in the central nervous system (de Groat et al, 1993a; Yoshimura and de Groat, 1997; Ishiura et al, 2001) and in bladder parasympathetic ganglia, where they modulate nicotinic transmission (de Groat and Booth, 1993). The ability to modulate neurotransmission in the bladder through the selective activation and inhibition of specific presynaptic receptors may lead, in the future, to novel forms of pharmacologic therapy for specific forms of lower urinary tract dysfunction.

Purinergic Mechanisms

Purinergic contribution to parasympathetic stimulation in vivo, or field stimulation in vitro, has been proved to exist in a variety of species including rat, rabbit, and guinea pig (Burnstock et al, 1972; Chancellor et al, 1992; Burnstock, 1996). However, there is less evidence that purinergic neurotransmission exists in humans, at least regarding normal responses to stimulation, but it may play a role in pathologic conditions such as detrusor overactivity or bladder outlet obstruction (Palea et al, 1993; Burnstock, 2000; O’Reilly et al, 2001a).

ATP acts on two families of purinergic receptors: an ion channel family (P2X) and a G protein–coupled receptor family (P2Y) (Inoue and Brading, 1990; Inoue and Gabella, 1991; McMurray et al, 1997). Seven P2X subtypes and eight P2Y subtypes have been identified. Analysis of the structure-activity relationships of a series of excitatory purinergic agonists on the guinea pig bladder revealed an order of potency consistent with P2X₁ or P2X₂ receptors (Burnstock, 2001; Zhong et al, 2001). In other species (rabbit, cat, and rat), various studies suggested that multiple purinergic excitatory receptors are present in the bladder (Burnstock, 2000). Immunohistochemical experiments with specific antibodies for different P2X receptors showed that P2X₁ receptors are the dominant subtype in membranes of rat detrusor muscle and vascular smooth muscle in the bladder (Lee et al, 2000). Clusters of P2X₁ receptors were detected on rat bladder smooth muscle cells, some of which were closely related to nerve varicosities. Northern blotting and in-situ hybridization revealed the presence of P2X₁ and P2X₄ mRNA in the bladder (Valera et al, 1995). The predominant expression of P2X₁ receptors has also been confirmed in the human bladder (O’Reilly et al, 2001a, 2001b). Investigators also found that the amount of P2X₁ receptors was increased in the obstructed bladder compared with the control bladder, suggesting upregulated purinergic mechanisms in the overactive bladder due to bladder outlet obstruction (O’Reilly et al, 2001a). In addition, ATP also seems to act through P2Y receptors in the smooth muscle to suppress cholinergic and purinergic contractions (Lee et al, 2000; Burnstock, 2001).

Purinergic nerves are likely to have other functions in the lower urinary tract, because excitatory receptors for ATP are present in parasympathetic ganglia (Theobald and de Groat, 1989; Nishimura and Tokimasa, 1996; Zhong et al, 1998, 2001), afferent nerve terminals (Dmitrieva et al, 1998; Namasivayam et al, 1999; Lee et al, 2000; Burnstock, 2001), and urothelial cells (Buffington et al, 1999; Vlaskovska et al, 2001). Excitatory purinergic receptors in pelvic ganglia have been demonstrated in the cat (Theobald and de Groat, 1989), rabbit (Nishimura and Tokimasa, 1996), and rat (Zhong et al, 1998, 2001).

P2X₃ receptors that have been identified in small-diameter afferent neurons in DRG have also been detected immunohistochemically in the wall of the bladder and ureter in a suburothelial plexus of afferent nerves (Lee et al, 2000). Studies using patch clamp recordings in rats have also demonstrated that the majority (90%) of bladder afferent neurons projecting by pelvic nerves responded to ATP and α,β-methylene ATP with persistent currents, suggesting that bladder afferent pathways in the pelvic nerve express predominantly P2X_2/3 heteromeric receptors rather than P2X₃ homomeric receptors (Zhong et al, 2003; Dang et al, 2005). Intravesical or intra-arterial administration of ATP- or 2-methylthio-ATP–activated bladder afferent fibers and enhanced reflex bladder activity (Dmitrieva et al, 1998; Namasivayam et al, 1999; Pandita and Andersson, 2002; Zhang et al, 2003; Nishiguchi et al, 2005). On the other hand, desensitization of purinergic receptors by intravesical administration of α,β-methylene ATP, a purinergic receptor agonist, or administration of a receptor antagonist, suramin, depressed bladder afferent activity (Dmitrieva et al, 1998; Namasivayam et al, 1999). In P2X₃ knockout mice, afferent activity induced by bladder distention was significantly reduced (Burnstock, 2000; Cockayne et al, 2000; Cook and McCleskey, 2000; Souslova et al, 2000). Furthermore, deletion of the gene encoding P2X₂ and loss of heteromeric P2X_2/3 receptors also result in a marked urinary bladder hyporeflexia, as evidenced by increased thresholds for bladder contractions during bladder filling (Cockayne et al, 2005). These data indicate that purinergic receptors are involved in mechanosensory as well as nociceptive signaling in the bladder.

ATP is also released by afferent nerves and might act in an autofeedback manner to regulate afferent excitability. Two studies (Dmitrieva et al, 1998; Morrison, 1998) presented evidence for purinergic sensitization of bladder afferent nerve endings. Intra-arterial injection of ATP can activate bladder afferent nerves (Dmitrieva et al, 1998), whereas suramin, an antagonist of certain types of ATP receptors (P2X purinergic receptors), given intravesically reduced by 50% the firing of bladder mechanoreceptors induced by bladder distention (Morrison, 1998). Intravesical administration of α,β-methylene ATP also reduced the firing of mechanosensitive afferents, presumably by desensitizing purinergic receptors on the afferent terminals.

In addition, adenosine, which can be formed by the metabolism of ATP, can depress parasympathetic nerve-evoked bladder contractions by activating P1 inhibitory receptors in parasympathetic ganglia (Akasu et al, 1984), in postganglionic nerve terminals, and in the bladder muscle (Cockayne et al, 2000; Burnstock, 2001). Adenosine P1 receptors have been further classified into a number of subtypes (i.e., A₁, A_2A, A_2B, and A₃) (Olah et al, 1995). A study has demonstrated that adenosine reduces the force of nerve-mediated contractions by acting predominantly at presynaptic sites at the nerve-muscle junction through a subtype of an adenosine receptor (the A₁ receptor in guinea pigs), although these actions of adenosine are less evident in human detrusor muscles (Fry et al, 2004).

Adrenergic Mechanisms

α-Adrenergic

Although α-adrenergic stimulation is not prominent in the normal bladder, recent evidence indicates that under pathologic conditions, such as detrusor overactivity associated with bladder outlet obstruction, the α-adrenergic receptor density, especially the α_1D-receptor subtype, can increase to such an extent that the norepinephrine-induced responses in the bladder are converted from relaxation to contraction (Andersson and Arner, 2004). In rats with outflow obstruction, the proportion of α_1D-receptor subtype in the total α₁-receptor mRNA in the bladder is increased to 70% from 25% in normal rat bladders (Hampel et al, 2002), and urinary frequency is suppressed by an inhibition of α_1D and α_1A receptors by tamsulosin, whereas α_1A-receptor suppression by 5-methyl-urapidil has no effect. Moreover, α_1D-receptor knockout mice have larger bladder capacity and voided volumes than do their wild-type controls, which supports an important role of α_1D receptors in the control of bladder function (Chen et al, 2005). However, in humans, there is the predominant expression of α_1D receptors already in the normal bladder (Malloy et al, 1998), and the level of expression of α-adrenoceptor mRNA, which is considerably low compared with β₃ adrenoceptors in normal bladders, was not increased in the bladder with outflow obstruction (Nomiya and Yamaguchi, 2003). Thus the contribution of α_1D receptors to detrusor overactivity observed in a variety of pathologic conditions, including obstructive uropathy and incontinence, still needs to be established (Andersson and Arner, 2004).

α-Adrenergic mechanisms are more important in urethral function. Substantial pharmacologic and physiologic evidence indicates that urethral tone and intraurethral pressure are influenced by α-adrenergic receptors. The presence of α₁ and α₂ adrenoceptors has been shown in the urethra of various species including humans. Among α₁ adrenoceptors, the α_1A adrenoceptor is the major subtype expressed in urethral smooth muscle at the mRNA and protein levels (Yono et al, 2004; Michel and Vrydag, 2006). Isolated human urethral smooth muscle contracts in response to α-adrenergic agonists (Yalla et al, 1977; Awad et al, 1978; Nordling, 1983; Mattiasson et al, 1984). It is also reported in the rabbit that the urethral contraction is mediated by the α_1A-adrenoceptor subtype (Testa et al, 1993; Michel and Vrydag, 2006). Likewise, hypogastric nerve stimulation and α-adrenergic agonists raise intraurethral pressure, which is blocked by α₁-adrenergic antagonists (Awad et al, 1976; Yalla et al, 1977). These findings provide the rationale for use of α-adrenergic agonists to promote urine storage by increasing urethral resistance.

Conversely, α-adrenergic receptor antagonists facilitate urine release in conditions of functionally increased urethral resistance, such as benign prostatic hyperplasia. Although the α_1A adrenoceptor is the major subtype in the prostate and urethra, highly selective α_1A- adrenoceptor antagonists (e.g., RS-17053) do not alter lower urinary tract symptom (LUTS) scores in men with BPH, but these agents are effective at relaxing prostate smooth muscle and increasing urine flow in men (Schwinn and Roehrborn, 2008). In contrast, α₁-adrenoceptor antagonists that contain α_1D-adrenoceptor blocking activity improve bladder-based symptoms in humans (Nishino et al, 2006), suggesting the important role of the α_1D adrenoceptors for storage symptoms associated with bladder outlet obstruction, receptors potentially located at the bladder or the spinal cord (Schwinn and Roehrborn, 2008).

α₂-Adrenergic antagonists increase the release of norepinephrine from urethral tissues through a presynaptic mechanism, but this does not affect the contractility of urethral smooth muscle in vitro (Mattiasson et al, 1984; Willette et al, 1990; Michel and Vrydag, 2006). The human urethra lacks postjunctional α₂-adrenergic receptors, although in-vitro prejunctional activation of these receptors produces a feedback inhibition of norepinephrine release. Pharmacologic and electrophysiologic data suggest that adrenergic nerves influence excitatory cholinergic transmission in pelvic ganglia. de Groat and Booth (1993) have shown, in the cat, that hypogastric nerves inhibit excitatory cholinergic transmission in vesical ganglia by activation of α₂-adrenergic receptors (Fig. 60–26). Conversely, β-adrenergic agonists facilitate transmission in vesical ganglia.

Figure 60–26 Diagram of possible transmitters in an adrenergic terminal supplying the bladder or urethra. Norepinephrine (NE) release can activate α₁-adrenergic receptors and produce contraction (+) or β receptors and cause relaxation (−) of the detrusor. Feedback inhibition of NE release through α₂ receptors can also occur. Neuropeptide Y (NPY) can produce smooth muscle contraction (+) or inhibit acetylcholine (ACh) release (not shown), or feedback can inhibit NE release. Adenosine triphosphate (ATP) can activate P2 receptors in the detrusor, which elicit contraction (+) or inhibit (−) further ATP release through P1 prejunctional receptors. ACh release from terminals in synaptic contact with an adrenergic varicosity can inhibit firing of adrenergic axons by activation of M₁ receptors.

Nitric Oxide

Nitric oxide (NO) has been identified as a major inhibitory transmitter mediating relaxation of the urethral smooth muscle during micturition (Andersson et al, 1992; Andersson, 1993; Andersson and Persson, 1995; Bennett et al, 1995). In 1931, Barrington hypothesized that stimulation of the pelvic nerve, in addition to evoking a bladder contraction, produces urethral relaxation. Indeed, urodynamic studies document a reduction in urethral pressure just before a bladder contraction (Scott et al, 1964; van Waalwijk van Doorn et al, 1991). In support of the contention that parasympathetic nerves mediate this urethral relaxation, sacral root stimulation reduces urethral pressure (McGuire, 1978; Torrens, 1978). Cholinergic innervation to the urethra has been supported by histochemical identification of acetylcholinesterase-staining fibers and ultrastructural data showing varicosities containing small clear vesicles in the human urethra (Ek, 1977; Gosling et al, 1977). Exogenous cholinergic agonists do not relax but rather contract urethral smooth muscle in vivo (Nergardh and Boreus, 1972; Ek et al, 1978) and in vitro (Ek et al, 1978). Therefore parasympathetic pathways mediating urethral relaxation must rely on a noncholinergic transmitter.

In the rat, NO is released by postganglionic nerves arising from neurons in the major pelvic ganglia (Fraser et al, 1995) (see Fig. 60–18). These neurons contain nitric oxide synthase (NOS), the enzyme that synthesizes NO, as well as nicotinamide adenine dinucleotide phosphate diaphorase, a marker for NOS (Vizzard et al, 1994). Electrophysiologic studies in female rats showed that electrical stimulation of the lumbosacral (L6-S1) spinal roots elicits simultaneous bladder contractions and urethral relaxation (Fraser et al, 1995). The urethral relaxation was inhibited by NOS inhibitors, which did not alter the bladder responses. The inhibition was reversed by administration of L-arginine, a precursor of NO. The electrically evoked urethral relaxation was abolished by ganglionic blocking agents, indicating that it was mediated by stimulation of preganglionic parasympathetic axons in the lumbosacral roots.

NO is also involved in controlling bladder afferent nerve activity. Inhibitors of NOS, given systemically or intrathecally, do not affect normal micturition in conscious or anesthetized rats. However, detrusor overactivity that accompanies irritation with turpentine, acetic acid, or cyclophosphamide is ameliorated by spinal application of NOS inhibitors (Rice, 1995; Kakizaki and de Groat, 1996; Lagos and Ballejo, 2004). However, intravesically administered capsaicin induces detrusor overactivity that is not influenced by an intrathecally applied NOS inhibitor, although the behavioral effects of the irritation are reduced (Pandita et al, 1997). It is believed that NO is involved in mediating N-methyl-D-aspartate (NMDA) receptor–dependent effects but not those involving neurokinin 2 (NK₂) receptors. Overall, the NO mechanism at the spinal level has an excitatory effect on the micturition reflex.

However, NO seems to have an inhibitory effect in the bladder. Intravesical application of NO can suppress bladder overactivity due to cyclophosphamide-induced bladder irritation in rats (Ozawa et al, 1999). Intravesical oxyhemoglobin, an NO scavenger, also induces bladder overactivity as evidenced by reductions in bladder capacity and micturition volume, which is prevented by L-arginine or enhanced by the guanylate cyclase inhibitor in rats (Pandita et al, 2000). Masuda and colleagues (2007) also showed that bladder overactivity induced by intravesical capsaicin instillation was enhanced by a NOS inhibitor (L-NAME) administered intravenously or intravesically, and that these L-NAME–induced excitatory effects were significantly suppressed by desensitization of C-fiber afferent pathways by capsaicin pretreatment. Thus NO released locally in the bladder can mediate inhibitory effects by modulation of bladder afferent activity (Yoshimura et al, 2001).

Afferent Neuropeptides

Afferent neurons innervating the lower urinary tract exhibit immunoreactivity for various neuropeptides, such as substance P (SP), calcitonin gene–related peptide (CGRP), pituitary adenylate cyclase–activating polypeptide (PACAP), leucine enkephalin, corticotropin-releasing factor, and vasoactive intestinal polypeptide (VIP) (de Groat, 1986; de Groat, 1989; Keast and de Groat, 1992; Maggi, 1993; Vizzard, 2001, 2006), as well as growth-associated protein-43 (GAP43), nitric oxide synthase (NOS) (Vizzard et al, 1996), glutamic acid, and aspartic acid (Keast and Stephensen, 2000). These substances have been identified in many species and at one or more locations in the afferent pathways including (1) afferent neurons in lumbosacral dorsal root ganglia, (2) afferent nerves in the peripheral organs, and (3) afferent axons and terminals in the lumbosacral spinal cord (Kawatani et al, 1985, 1986, 1996; Morrison et al, 2005). The majority (>70%) of bladder DRG neurons in rats appear to contain multiple neuropeptides—CGRP, substance P, or PACAP being the most common. In cats, VIP is also contained in a large percentage of bladder DRG neurons (de Groat, 1989).

Many of these peptides, which are contained in capsaicin-sensitive, C-fiber bladder afferents, are released in the bladder by noxious stimulation and contribute to inflammatory responses by triggering plasma extravasation, vasodilation, and alterations in bladder smooth muscle activity (Maggi, 1993; Ishizuka et al, 1994, 1995b). These agents also function as transmitters at afferent terminals in the spinal cord.

Prostanoids, Endothelins, and Hormones

“Transducer” Function of the Urothelium

Whereas the urothelium has historically been viewed primarily as a “barrier,” there is increasing evidence that urothelial cells display a number of properties similar to sensory neurons (nociceptors and mechanoreceptors) and that both types of cells use diverse signal-transduction mechanisms to detect physiologic stimuli. Examples of “sensor molecules” (i.e., receptors and ion channels) associated with neurons that have been identified in urothelium include receptors for bradykinin (Chopra et al, 2005), neurotrophins (TrkA and p75) (Murray et al, 2004), purines (P2X and P2Y) (Lee et al, 2000; Hu et al, 2002; Birder et al, 2004; Tempest et al, 2004; Chopra et al, 2008), norepinephrine (α and β) (Birder et al, 1998; Birder et al, 2002), acetylcholine (nicotinic and muscarinic) (Chess-Williams, 2002; Beckel et al, 2006; Kullmann et al, 2008a), protease-activated receptors, amiloride-/mechanosensitive Na⁺ channels such as ENaC (Smith et al, 1998; Wang et al, 2003; Araki et al, 2004), and a number of TRP channels (TRPV1, TRPV2, TRPV4, TRPM8) (Birder and de Groat, 1998; Birder et al, 2001, 2002, 2007; Stein et al, 2004; Gevaert et al, 2007).

When urothelial cells are activated through these receptors and ion channels in response to mechanical as well as chemical stimuli, they can, in turn, release chemical mediators such as NO, ATP, acetylcholine, and substance P (Ferguson et al, 1997; Birder et al, 1998; Burnstock, 2001; Birder et al, 2003; Chess-Williams, 2004). These agents are known to have excitatory and inhibitory actions on afferent nerves that are close to or in the urothelium (Bean et al, 1990; Dmitrieva et al, 1998; Birder et al, 2001; Yoshimura et al, 2008). Chemicals released from urothelial cells may act directly on afferent nerves or indirectly through an action on suburothelial interstitial cells (also referred to as myofibroblasts) that lie in close proximity to afferent nerves. Myofibroblasts are extensively linked by gap junctions and can respond to chemicals that in turn modulate afferent nerves (Fowler et al, 2008). Thus it is believed that urothelial cells and myofibroblasts can participate in sensory mechanisms in the urinary tract by chemical coupling to the adjacent sensory nerves.

NO can be released by the urothelium, particularly during inflammation (Birder et al, 1998). The release of NO may be evoked by the calcium ionophore A-23187, norepinephrine, and capsaicin. Substance P also acts on receptors on urothelial cells to release NO. The adrenergic release of NO from bladder strips was reduced by 85% after removal of the urothelium. Denervation of the bladder did not completely block the release of capsaicin-induced NO production, suggesting other sites of production. This is consistent with the observations that capsaicin released NO from cultured rat, cat, rabbit, and human urothelial cells and that the TRPV1 capsaicin receptor is expressed in cultured urothelial cells. NOS expression in afferent neurons is also increased in chronic bladder inflammation. Given that NO does not have much effect on the detrusor muscle but does inhibit Ca²⁺ channels in rat bladder afferent neurons (Yoshimura et al, 2001), the role of NO in the urothelium has still to be clarified. However, NO released locally in the bladder appears to have an inhibitory effect on afferent activity in the bladder because suppression of endogenous NO by intravesical oxyhemoglobin, an NO scavenger, or L-NAME, a NOS inhibitor, enhances bladder activity in rats (Pandita et al, 2000; Masuda et al, 2007).

ATP released from urothelial cells during stretch can activate a population of suburothelial bladder afferents expressing P2X₃ receptors, signaling changes in bladder fullness and pain (Ferguson et al, 1997; Burnstock, 2001). Accordingly, P2X₃ null mice exhibit a urinary bladder hyporeflexia, suggesting that this receptor, as well as neural–epithelial interactions, are essential for normal bladder function (Cockayne et al, 2000). This type of regulation may be similar to epithelium-dependent secretion of mediators in airway epithelial cells, which are thought to modulate submucosal nerves and bronchial smooth muscle tone and may play an important role in inflammation (Homolya et al, 2000; Jallat-Daloz et al, 2001). Thus it is possible that activation of bladder nerves and urothelial cells can modulate bladder function directly or indirectly by the release of chemical factors in the urothelial layer. ATP released from the urothelium or surrounding tissues may also play a role in the regulation of membrane trafficking. This is supported by studies in the urinary bladder in which urothelium-derived ATP release purportedly acts as a trigger for exocytosis, in part by autocrine activation of urothelial purinergic (P2X, P2Y) receptors (Wang et al, 2005). These findings suggest a mechanism whereby urothelial cells sense or respond to ATP and thereby translate extracellular stimuli into functional processes.

Prostaglandins are also released from the urothelium. These are assigned two possible functions: regulation of detrusor muscle activity and cytoprotection of the urothelium, based on effective treatment of hemorrhagic cystitis by prostaglandins (Jeremy et al, 1987). The predominant forms found in human urothelium from biopsy specimens are 6-oxo-PGF_2α > PFE₂ > PGF_2α > thromboxane B₂. PGI₂ (prostacyclin) is also produced. These findings were confirmed and further developed in the guinea pig, in which it was found that the major production of prostaglandins occurred in the urothelium. The production of prostaglandins also increased greatly with inflammation (Saban et al, 1994). Prostaglandin synthesis also occurs in the ureter, where it is speculated to be important in the regulation of ureteral peristalsis and also in reducing the development of blood clots in the lumen of the ureter (Ali et al, 1998).

Evidence also suggests that the involvement of the muscarinic receptor in bladder function extends beyond detrusor contractility and into afferent sensory functioning. Muscarinic receptors are found on the urothelium at high density (Hawthorn et al, 2000), and there is a basal release of acetylcholine from the urothelium that is increased by stretch and aging (Yoshida et al, 2006). Thus activation of the muscarinic receptors in the urothelium releases substances that modulate afferent nerves and smooth muscle activity (Hawthorn et al, 2000; de Groat, 2004; Kullmann et al, 2008b).

The urothelium also releases substances called urothelium-derived inhibitory factors, which decrease the force of detrusor muscle contraction in response to muscarinic stimulation (Hawthorn et al, 2000; Kumar et al, 2005). The molecular identity of this factor is not known; however, pharmacologic studies suggest that it is not NO, a prostaglandin, prostacyclin, adenosine, catecholamine, GABA, or one that acts through apamine-sensitive, small-conductance K⁺ channels. It has been shown that an inhibitory response through this factor is attenuated in a fetal model of bladder outlet obstruction (Thiruchelvam et al, 2003). Further studies are required to clarify the identity of this substance and its role in bladder function.

Serotonin

Serotonin (5-HT) has been found in neuroendocrine cells along the urethra and in the prostate ((Hanyu et al, 1987). In animals, 5-HT₂ and, possibly, 5-HT₃ agonists contract the urethra. If inflammatory conditions promote release of serotonin from paraurethral cells, irritative symptoms, such as the urethral syndrome, may arise because of serotonergic mechanisms. The 5-HT₂ antagonist ketanserin has been shown to reduce urethral pressure in humans (Horby-Petersen et al, 1985). However, the reduction in urethral pressure after ketanserin administration can also be the result of blockade of α-adrenergic receptors (Thor and Katofiasc, 1995).

5-HT also has several pharmacologic effects on mammalian urinary bladders, both in vitro and in vivo. Human and pig isolated detrusor muscles are known to contract in a concentration-dependent manner in response to 5-HT (Klarskov and Horby-Petersen, 1986). In human isolated urinary bladder, there was potentiation of the contractions induced by electrical field stimulation, mediated by the 5-HT₄ receptor subtype (Candura et al, 1996; Darblade et al, 2005). A similar response is present on guinea pig detrusor muscle through 5-HT_2A and 5-HT₄ receptors, whereas in the rabbit and the rat, the receptors involved are the 5-HT₃ and 5-HT₇ subtypes, respectively (Palea et al, 2004).