Pharmacologic perspective on the physiology of the lower urinary tract

PHARMACOLOGIC PERSPECTIVE ON THE PHYSIOLOGY OF THE LOWER URINARY TRACT KARL-ERIK ANDERSSON

AND

PETTER HEDLUND

ABSTRACT Myogenic activity, distention of the detrusor, and signals from the urothelium may initiate voiding. In the bladder, afferent nerves have been identified not only in the detrusor, but also suburothelially, where they form a plexus that lies immediately beneath the epithelial lining. Extracellular adenosine triphosphate (ATP) has been found to mediate excitation of small-diameter sensory neurons via P2X3 receptors, and it has been shown that bladder distention causes release of ATP from the urothelium. In turn, ATP can activate P2X3 receptors on suburothelial afferent nerve terminals to evoke a neural discharge. However, most probably, not only ATP but also a cascade of inhibitory and stimulatory transmitters and mediators are involved in the transduction mechanisms underlying the activation of afferent fibers during bladder filling. These mechanisms may be targets for future drugs. The central nervous control of micturition involves many transmitter systems, which may be suitable targets for pharmacologic intervention. ␥-Aminobutyric acid, dopamine, enkephalin, serotonin, and noradrenaline receptors and mechanisms are known to influence micturition, and potentially, drugs that affect these systems could be developed for clinical use. However, a selective action on the lower urinary tract may be difficult to obtain. Most drugs currently used for treatment of detrusor overactivity have a peripheral site of action, mainly the efferent (cholinergic) neurotransmission and/or the detrusor muscle itself. In the normal bladder, muscarinic receptor stimulation produces the main part of detrusor contraction, but evidence is accumulating that in disease states, such as neurogenic bladders, outflow obstruction, idiopathic detrusor instability, and interstitial cystitis, as well as in the aging bladder, a noncholinergic activation via purinergic receptors may occur. If this component of activation is responsible not only for part of the bladder contractions, but also for the symptoms of the overactive bladder, it should be considered an important target for therapeutic interventions. UROLOGY 60 (Suppl 5A): 13–21, 2002. © 2002, Elsevier Science Inc.

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he limitations of current drugs for treatment of the overactive bladder (OAB) have focused interest on new targets for pharmacologic intervention and on the physiologic basis for lower urinary tract (LUT) function and dysfunction. New information on potential future targets is continuously added,1 and special interest has recently been given to possibilities of influencing the sensory/afferent mechanisms that regulate the micturition reflex.2 Adequate sensory input is the prerequisite for conscious bladder control, and changes in sensory/ afferent mechanisms may give rise to disturbances in bladder function (eg, LUT symptoms and OAB). Central nervous mechanisms involved in micturiFrom the Department of Clinical Pharmacology, Lund University Hospital, Lund, Sweden. Reprint requests: Karl-Erik Andersson, MD, PhD, Department of Clinical Pharmacology, Institute for Laboratory Medicine, Lund University Hospital, S-221 85 Lund, Sweden. E-mail: [email protected] © 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED

tion control3 and purinergic signaling in the LUT have also attracted attention.4 AFFERENT NERVES IN THE BLADDER All components of the LUT, including the detrusor, trigone, bladder neck, urethral smooth and striated muscle, and the pelvic floor, have to be coordinated to allow normal storage and evacuation of urine from the bladder. This coordination is achieved by a complex neural control system, in which afferent activity from the LUT plays an important role. Afferent nerves have been identified in both the LUT smooth muscles and the lamina propria. Suburothelially, these nerves form a plexus that lies immediately beneath the urothelial lining, with some terminals reaching into the basal parts of the urothelium.5,6 In the dome of the bladder, the suburothelial plexus is relatively sparse. However, it becomes progressively denser near the bladder neck and is particularly prominent in the 0090-4295/02/$22.00 PII S0090-4295(02)01786-7 13

trigone.6 Afferent nerves can be identified by, for example, their content of substance P (SP) and calcitonin gene-related peptide (CGRP). In the mucosa of the human urinary bladder, numerous SPimmunoreactive varicose nerve fibers were seen in the lamina propria, and most of them ran freely in the connective tissue. In addition, SP-immunoreactive nerve fibers were observed beneath the urothelium and perivascularly.7 The afferent innervation of the musculature was diffuse and appeared uniform throughout the bladder. Smet et al.8 found that nerves containing CGRP and tachykinins were typically present within the suburothelial region, encircling intramural ganglia and around blood vessels. These nerves were sparsely distributed and only very rarely projected to the smooth muscle bundles of the detrusor. In contrast, vasoactive intestinal polypeptide– containing nerves formed a dense suburothelial plexus and also projected to the detrusor muscle bundles.8 In the guinea pig, the number and distribution pattern of CGRP-immunoreactive nerves appeared to be identical to that of SP-containing nerves, whereas in the rat, the former predominated.9 INITIATION OF THE MICTURITION REFLEX There is an ongoing afferent signaling during storage. Distention of the bladder has long been considered to evoke afferent activity via myelinated A␦-fibers connected in series with the smooth muscle fibers,10,11 reacting to distention or contraction of the cells (Figure 1). Detrusor smooth muscle cells have a tone and can be myogenically active.12 Myogenic contractions, which are not coordinated, may contribute to the afferent signaling. These contractions may become more coordinated with increasing stretch and may thus generate an increasing afferent activity, which eventually initiates the micturition reflex. However, both the detrusor and the lamina propria (see below) contain unmyelinated C fibers, whose role in bladder activation in the adult is unclear. In the cat, they seem to be mechanoinsensitive and “silent” and may not participate in normal micturition,13 whereas in rats, some C fibers may be volume receptors and may not respond to bladder contraction.11 THE CONCEPT OF UROTHELIAL MECHANOAFFERENT TRANSDUCTION The urothelium is not only a barrier for toxic substances in the urine but also is a metabolically highly active tissue that may take an active part in both the storage and voiding phases of the micturition cycle.14 Ferguson et al.15 provided the first evidence that adenosine triphosphate (ATP) may be involved in sensory signaling in the urinary 14

bladder. They showed ATP release from the serosal (but not luminal) side of rabbit urothelium in response to stretch, a process that was dependent on changes in the transepithelial potential. They also postulated that the released ATP could activate suburothelial sensory nerves. Burnstock16 put forward the hypothesis that “distention of tubes (including ureter, vagina, salivary and bile ducts, gut) and sacs (including urinary and gall bladders, and lung) leads to release of ATP from the lining epithelium, which then acts on P2X2/3 receptors on subepithelial sensory nerves to convey information to the CNS [central nervous system].” In the bladder, this would mean that urothelially generated ATP by action of suburothelial nerves would be able to initiate bladder voiding (Figure 2). The ATP receptor P2X3 is expressed on smalldiameter primary sensory neurons in various species.17,18 In the bladder, P2X2/3 receptors have been demonstrated not only on suburothelial nerves, but also in the urothelium of humans,19,20 rats,19 and mice.21,22 ATP can be produced by and released from the urothelium.15,22,23 Namasivayam et al.24 showed, using an in vitro pelvic nerve–afferent rat mode, that afferent activity induced by bladder distention was reduced by up to 75% after desensitization with ␣,␤-methylene ATP. These results were consistent with the view that ATP is released from the bladder urothelium by distention and activates pelvic nerve afferents. Further supporting this series of events is that cystometry in P2X3-deficient mice revealed decreased voiding frequency and increased bladder capacity and voiding volume, but it also revealed normal bladder pressures.21 Taken together, these findings suggest that P2X3 receptors are involved in the normal physiologic regulation of afferent pathways that control volume reflexes in the urinary bladder and thus that P2X3 receptor– containing neurons may serve as volume receptors. Vlaskovska et al.,22 using a mouse bladder–pelvic nerve preparation, detected a release of ATP proportional to the extent of bladder distention in both P2X3⫹/⫹ and P2X3⫺/⫺ mice, despite the fact that the P2X3⫺/⫺ bladder had an increased capacity compared with that of the P2X3⫹/⫹ bladder. During gradual bladder distention, they found that the activity of multifiber pelvic afferents increased progressively. However, the bladder afferents from P2X3⫺/⫺ mice showed an attenuated response to bladder distention. Intravesical injections of P2X agonists (ATP or ␣,␤-methylene ATP) caused a rapid activation of bladder afferents of P2X3⫹/⫹, but not P2X3⫺/⫺, mice. By contrast, P2X antagonists attenuated distention-induced discharges in bladder afferents, further suggesting UROLOGY 60 (Supplement 5A), November 2002

FIGURE 1. Initiation of the micturition reflex.

FIGURE 2. Principle for mechanoafferent transduction. ATP ⫽ adenosine triphosphate. (Data from J Anat.16)

that urothelially released ATP acts via P2X3 receptors on a subpopulation of pelvic afferent fibers. Further supporting a role for ATP in urothelial signaling, Pandita and Andersson25,26 found that UROLOGY 60 (Supplement 5A), November 2002

intravesical instillation of ATP could induce bladder overactivity in unanesthetized, freely moving rats. The ATP-induced effects were effectively counteracted by the P2X3 receptor antagonist, 15

2⬘,3⬘-O-trinitrophenyl (TNP)-ATP, which by itself caused an increase in bladder capacity. Interestingly, the effects of ATP could be prevented by pretreatment with L-arginine and by the neurokinin-2 (NK-2) receptor antagonist, SR 48,968,26 suggesting that both nitric oxide (NO) and tachykinins could interfere with the actions of ATP. However, neither TNP-ATP nor L-arginine could prevent the effects of intravesical capsaicin, raising the possibility that ATP and capsaicin may not affect the same subpopulation of afferents. NO can be produced by and released from the urothelium23,27 and may be involved in the regulation of afferent nerves. In support of such a view, oxyhemoglobin, a scavenger of NO that does not penetrate the urothelium, was found to induce bladder overactivity in unanesthetized rats when given intravesically, probably by interfering with NO generated in the urothelium or suburothelium.28 The effect of oxyhemoglobin could be prevented by pretreatment with L-arginine and also by intravesical administration of a potassium channel opener. A role for tachykinins in urothelial signaling has been suggested by investigations showing that detrusor overactivity induced by chemical irritation of the urothelium can be inhibited or prevented by NK receptor antagonists.29 NK-1 receptors have been demonstrated over the endothelium of arterial blood vessels within the human detrusor muscle and lamina propria and over small vessels in the suburothelium. NK-1 receptors were not observed over the detrusor muscle. NK-2 receptors were seen over the detrusor muscle and very sparsely over blood vessels.30 When instilled intravesically in normal, unanesthetized rats, NKA (but not SP or NKB) stimulated micturition, and it was suggested that NKA may initiate micturition by stimulation of the NK-2 receptors in the urothelium or suburothelium.31 Nakatsuka et al.32 found that treatment of dorsal root ganglion neurons with ATP significantly depleted SP immunoreactivity on the neurites and somata of the neurons, and they suggested that activation of P2X receptors may result in release of SP from primary afferent neurons. It is reasonable to assume that urothelially derived ATP can cause release not of only SP but also of NKA from suburothelial afferent nerves, thus explaining why the effects of intravesical ATP were prevented by NK-2 receptor antagonism. It has been suggested that a number of additional receptors, including nicotinic, muscarinic, adrenergic, prostanoid, and vanilloid receptors (VRs), are involved in urothelial and suburothelial signaling.27 Most probably, a cascade of mediators, released from the urothelium and the suburothelial plexus of nerves in response to bladder distention, can 16

initiate or depress activation of the bladder. For example, Yiangou et al.20 found that VR1 and P2X3-immunoreactive fine nerve terminals were scattered throughout the urothelium of the normal human bladder and had similar distributions. Avelino et al.33 found colocalization of VR1, and SP and CGRP immunoreactivities in unmyelinated axons and varicosities beneath or among epithelial cells in the LUT of the rat. Capsaicin and structurally related molecules, such as resiniferatoxin, are known to bind to VR1 receptors on the peripheral terminals of nociceptive neurons.34 Both capsaicin and resiniferatoxin have been used successfully to treat bladder function disturbances, but the role of VRs in the pathogenesis of detrusor overactivity has not been established, and an endogenous agonist has not yet been found. VR1 is expressed by primary sensory neurons of the “pain” pathway and can be activated not only by vanilloid compounds but also by protons or heat.35 However, VR1s were found to be confined not only to primary afferent neurons, but also in the urothelium.27 VR1-positive nerve fibers were found in close association with basal urothelial cells, which opens the possibility that capsaicin and resiniferatoxin act not only on suburothelial nerves but also directly on the urothelium. Prostanoids have been shown to be released by distention of the bladder36 and by mechanical trauma and inflammation of the bladder mucosa. It has been suggested that prostanoids are involved in the regulation of normal bladder function and the pathophysiology of, for example, OAB.37,38 Different types of prostanoid receptors may be involved. Autoradiographic and in situ hybridization studies have demonstrated prostacyclin receptors on sensory neurons. A high density of binding sites for [3H]iloprost was observed in rat dorsal root ganglia and the dorsal horn of the spinal cord.39 The latter sites were thought to represent prostacyclin receptors on the terminals of primary afferents, because binding-site density decreased after dorsal rhizotomy. Prostanoids probably do not act as true effector messengers along the efferent arm of the micturition reflex but rather as neuromodulators of efferent and afferent neurotransmission. In normal conscious rats, intravesically instilled prostaglandin E2 facilitated micturition and increased basal intravesical pressure.40 The effect was attenuated by both the NK-1 and NK-2 receptor antagonists. It was suggested that intravesically administered prostaglandin E2 can stimulate micturition by releasing tachykinins from nerves in and/or immediately below the urothelium and that these tachykinins, in turn, initiate a micturition reflex by stimulating NK-1 and NK-2 receptors on afferent nerves and detrusor smooth muscle. UROLOGY 60 (Supplement 5A), November 2002

Thus, the urothelium may serve as a mechanosensor that— by producing NO, ATP, and other mediators— can control the activity in afferent nerves and, thereby, the initiation of the micturition reflex. The firing of suburothelial afferent nerves and the threshold for bladder activation may be modified by both inhibitory (eg, NO) and stimulatory (eg, ATP, tachykinins, prostanoids) mediators. These mechanisms can be involved in the generation of bladder overactivity, causing urge, frequency, and incontinence, and thus seem to be interesting targets for pharmacologic intervention. AFFERENT FUNCTIONS Projections from cell bodies in the dorsal root ganglia reach the bladder and the spinal cord. Most of the sensory/afferent innervation of the bladder and urethra originates in the thoracolumbar region and travels via the pelvic nerve.41 Some afferents, originating in ganglia at the thoracolumbar level of the sympathetic outflow, project via the hypogastric nerve. The afferent nerves of the striated muscle in the rhabdosphincter travel in the pudendal nerve to the sacral region of the spinal cord. Sacral sensory nerve terminals are uniformly distributed to all areas of the detrusor and urethra, whereas lumbar sensory nerve endings are most frequent in the trigone and scarce in the bladder body. The hypogastric and pelvic pathways are implicated not only in the sensations associated with normal bladder filling but also with bladder pain. The pelvic and pudendal pathways are involved with the sensation that micturition is imminent and with thermal sensations from the urethra.42 The incoming information also controls the micturition reflex and the activity in the parasympathetic, sympathetic, and somatic efferent nerves to the LUT. At least 2 different populations of spinal dorsal horn neurons were suggested, both encoding for a stimulus of urinary bladder distention.43

tegmentum, the periaqueductal gray, and the preoptic area of the hypothalamus.45 Blok et al.,45 using positron emission tomography (PET), were able to confirm the importance of these areas for micturition in humans. They further showed that in women and men, the same specific nuclei exist in the pontine tegmentum responsible for the control of micturition and that the cortical and pontine micturition sites are more active on the right than on the left side.46 Nour et al.47 investigated micturition in normal men by PET and concluded that the onset and maintenance of micturition are associated with activity in a vast network of cortical and subcortical regions. It has been proposed that the afferent neurons send information to the periaqueductal gray, which, in turn, communicates with the pontine tegmentum, where 2 different regions involved in micturition control have been described.48 Recent evidence obtained by PET scanning in humans supported the hypothesis that the periaqueductal gray receives information about bladder fullness and relays this information to areas involved in the control of bladder storage.49 An example is found in the pons in a laterally located L region, which may serve as a urine storage center. It has been suggested that a dorsomedially located M region, corresponding to the Barrington nucleus or the pontine micturition center, suppresses bladder contraction and regulates the external sphincter muscle activity during urine storage. The M and L regions may represent separate functional systems, acting independently.50 The micturition reflexes use several transmitters and transmitter systems that may be targets for drugs aimed at controlling micturition, including ␥-aminobutyric acid, opioid, serotonin, noradrenaline, dopamine, or glutamatergic receptors and mechanisms.1,3 Still, no drug with a clearly defined central mode of action has been developed for OAB treatment.

CENTRAL CONTROL OF AFFERENT INFORMATION

EFFERENT PATHWAYS AND ACTIVATION OF THE DETRUSOR

Myogenic activity, distention of the detrusor, and signals from the urothelium may generate afferent activity. In the adult human, the normal micturition reflex is mediated by a spinobulbospinal pathway, passing through relay centers in the brain. Distention of the bladder wall is considered the primary stimulus.1,44 During bladder filling, afferent impulses conveyed by the pelvic nerve reach centers in the central nervous system. In the cat, it has been suggested that 3 areas in the brainstem and diencephalon are specifically implicated in the control of micturition: the dorsomedial pontine

MUSCARINIC RECEPTORS Normal bladder contraction in humans is mediated mainly through stimulation of muscarinic receptors in the detrusor muscle.12,51 Human detrusor smooth muscle contains muscarinic receptors of the M2- and M3-subtype,52,53 with M2-receptors (66%) dominating quantitatively over M3-receptors (33%). However, the M3-receptors are believed to cause a direct smooth muscle contraction through phosphoinositide hydrolysis54 and are mainly responsible for the normal micturition contraction.52,53,55 Even in the obstructed rat bladder, M3-receptors were found to play a predominant

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role in mediating detrusor contraction.56 The role for the M2-receptors in bladder activation has not been clarified. It has been suggested that M2-receptors may oppose sympathetically (via ␤-adrenoceptors) mediated smooth muscle relaxation, because activation of M2-receptors results in an inhibition of adenylyl cyclase.55,57 Stimulation of M2-receptors may, in addition, activate a nonspecific cationic channel and inactivate potassium channels. In certain disease states, M2-receptors may contribute to bladder contraction. Thus, in the denervated rat bladder, M2-receptors or a combination of M2- and M3-receptors mediated contractile responses.58,59 Also, in patients with neurogenic bladder dysfunction, detrusor contraction can be mediated by M2-receptors.59 In M3-receptor knockout mice, carbachol-induced contractions were mediated by M2-receptors.60 However, the contractile amplitude of the contractions of isolated detrusor strips was only 5% of that found in normal bladders. Muscarinic receptors may also be located on the presynaptic nerve terminals and participate in the regulation of transmitter release. The inhibitory prejunctional muscarinic receptors have been classified as M2-receptors in the animal bladder61,62 and as M4-receptors in the human bladder.63 Prejunctional facilitatory muscarinic receptors appear to be of the M1-receptor subtype in animal and probably also in human bladders.61,62,64 The muscarinic facilitatory mechanism seems to be upregulated in OABs from long-term spinal cord– transected rats. The facilitation in these preparations is primarily mediated by M3-muscarinic receptors.64 NONCHOLINERGIC MECHANISMS Atropine-resistant (nonadrenergic, noncholinergic [NANC]) contractions have been reported to occur in normal human detrusor,65– 68 even if it represents only a small percentage of the total contraction in response to nerve stimulation. However, a significant degree of atropine resistance may exist in morphologically and/or functionally changed bladders. It has been reported to occur in hypertrophic bladders,65,69 –71 idiopathic detrusor instability,70,72 interstitial cystitis bladders,73 neurogenic bladders,74 and aging bladders.75 In some of these bladders, the NANC component of the nerve-induced response may be responsible for up to 40% to 50% of the total bladder contraction. There is good evidence that the transmitter responsible for the NANC component is ATP,70,72 acting on P2X receptors found in the detrusor smooth muscle membranes of rats76 and humans.77 The receptor subtype predominating in both species seemed to be the P2X1 subtype. Moore et al.78 reported that detrusors from patients with idio18

pathic detrusor instability had a selective absence of P2X3 and P2X5 receptors, and they suggested that this specific lack might impair control of detrusor contractility and contribute to the pathophysiology of urge incontinence. On the other hand, O’Reilly et al.72 found that in patients with idiopathic detrusor instability, P2X2 receptors were significantly elevated, whereas other P2X receptor subtypes were significantly decreased. They were unable to detect a purinergic component of nerve-mediated contractions in control (normal) bladder specimens, but there was a significant component in unstable bladder specimens, where the purinergic component was approximately 50%. They concluded that this abnormal purinergic transmission in the bladder might explain symptoms in these patients and suggested that the purinergic pathway could be a novel target for the pharmacologic treatment of OAB. Using real-time quantitative reverse transcriptase–polymerase chain reaction for detecting and analyzing RNA, O’Reilly et al.79 confirmed that the P2X1 receptor was the predominant purinoceptor subtype in the human male bladder. They also found that the amount of P2X1 receptor per smooth muscle cell was greater in the obstructed than in the control bladder, suggesting an increase in purinergic function in the unstable bladder arising from bladder outlet obstruction. If abnormalities in the purinergic transmission in the bladder can explain OAB symptoms both in idiopathic detrusor instability in women and men with bladder outflow obstruction, it is obvious that P2X receptors, both P2X3 and P2X1, might be targets for pharmacologic intervention. Abnormal purinergic activation of the detrusor may also explain why antimuscarinic treatment fails in a number of patients. The clinical implications of signaling both via P2X3 receptors on suburothelial nerves and via P2X1 receptors on detrusor smooth muscle remain to be established. REFERENCES 1. de Groat WC, and Yoshimura N: Pharmacology of the lower urinary tract. Annu Rev Pharmacol Toxicol 41: 691– 721, 2001. 2. Andersson K-E: Bladder activation: afferent mechanisms. Urology 59(suppl 5A): 43–50, 2002. 3. Andersson K-E: Treatment of the overactive bladder: possible central nervous system drug targets. Urology 59(suppl 5A): 18 –24, 2002. 4. Burnstock G: Purinergic signalling in lower urinary tract, in Abbracchio MP, and Williams M (Eds), Purinergic and Pyrimidinergic Signalling I: Molecular, Nervous and Urogenitary System Function. Berlin, Springer-Verlag, 2001, pp 423–515. 5. Dixon JS, and Gilpin CJ: Presumptive sensory axons of the human urinary bladder: a fine structural study. J Anat 151: 199 –207, 1987. 6. Gabella G, and Davis C: Distribution of afferent axons in the bladder of rats. J Neurocytol 27: 141–155, 1998. UROLOGY 60 (Supplement 5A), November 2002

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DISCUSSION FOLLOWING DR. ANDERSSON’S PRESENTATION Alan J. Wein, MD (Philadelphia, PA): If the myogenic theory is at all true, then what role do neurotransmitters have in producing the symptoms or findings that you would see in such a state? Also, do you put much stock in that theory in regard to pathophysiology? Karl-Erik Andersson, MD, PhD (Lund, Sweden): To have a coordinated bladder contraction, you have to have the nervous system involved. A myogenic contraction, even if it involves a region within the bladder, can increase the signal. How20

ever, it cannot, by itself, contract the whole bladder. The detrusor muscle does not work as a syncytium. There are strands of smooth muscle that are also working as units. However, these units are isolated from each other by connective tissue. Therefore, even if you have increased myogenic activity in different parts, the bladder would rather be in the state of fibrillation, and there will be no coordinated contractions. The myogenic activity just starts the afferent activity and can change the threshold for initiating the micturition reflex. UROLOGY 60 (Supplement 5A), November 2002