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Department of Clinical Pharmacology, Lund University Hospital, Lund, Sweden (K.-E.A.); and Division of Urology, University of Pennsylvania Health System, Philadelphia, Pennsylvania (A.J.W.)
Abstract
Abstract I. Introduction II. Central Nervous System Targets A. Central Nervous Control B. Transmitter Systems III. Peripheral Targets A. Bladder B. Urethra IV. Effects of Sexual Hormones A. Estrogen and Progesterone B. Androgens C. Pregnancy V. Summary and Conclusions
The lower urinary tract constitutes a functional unit controlled by a complex interplay between the central and peripheral nervous systems and local regulatory factors. In the adult, micturition is controlled by a spinobulbospinal reflex, which is under suprapontine control. Several central nervous system transmitters can modulate voiding, as well as, potentially, drugs affecting voiding; for example, noradrenaline, GABA, or dopamine receptors and mechanisms may be therapeutically useful. Peripherally, lower urinary tract function is dependent on the concerted action of the smooth and striated muscles of the urinary bladder, urethra, and periurethral region. Various neurotransmitters, including acetylcholine, noradrenaline, adenosine triphosphate, nitric oxide, and neuropeptides, have been implicated in this neural regulation. Muscarinic receptors mediate normal bladder contraction as well as at least the main part of contraction in the overactive bladder. Disorders of micturition can roughly be classified as disturbances of storage or disturbances of emptying. Failure to store urine may lead to various forms of incontinence, the main forms of which are urge and stress incontinence. The etiology and pathophysiology of these disorders remain incompletely known, which is reflected in the fact that current drug treatment includes a relatively small number of more or less well-documented alternatives. Antimuscarinics are the main-stay of pharmacological treatment of the overactive bladder syndrome, which is characterized by urgency, frequency, and urge incontinence. Accepted drug treatments of stress incontinence are currently scarce, but new alternatives are emerging. New targets for control of micturition are being defined, but further research is needed to advance the pharmacological treatment of micturition disorders.
The pharmacological control of the lower urinary tract is exerted both in the central nervous system (CNS1) and peripherally (Andersson, 1993
, 1999b; de Groat et al., 1993, 1999; de Groat and Yoshimura, 2001). In the fetus and neonate, micturition is basically a spinal reflex, which during development becomes a spinobulbospinal reflex under suprapontine control (de Groat et al., 1999). However, information remains fragmentary on supraspinal (inter)connections and their function in the regulation of micturition, and there is a marked discrepancy between neuroanatomic knowledge and the functional description of the micturition cycle. Several CNS transmitters can modulate lower urinary tract storage and emptying, including glutamic acid, glycine, enkephalins, serotonin (5-HT), noradrenaline, dopamine, and GABA, but for many of them, a defined site of action in the micturition control has not yet been demonstrated (de Groat and Yoshimura, 2001; Andersson, 2002a).
The activity of the smooth and striated musculature in the urinary bladder, urethra, and periurethral sphincteric area is affected by various neurotransmitters, including acetylcholine, noradrenaline, ATP, nitric oxide, and neuropeptides (Table 1). Muscarinic receptors mediate normal and at least the main part of involuntary bladder contractions, but the role of other mechanisms for bladder control has not yet been established.
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Injuries or diseases of the nervous system, as well as drugs and disorders of the peripheral organs, can produce voiding dysfunctions, which roughly can be classified as disturbances of storage or disturbances of emptying (Wein, 1981, 2002). Failure to store urine may lead to various forms of incontinence (mainly urge and stress incontinence), and failure to empty can lead to urinary retention, which may result in overflow incontinence. Disturbances of bladder function leading to symptoms of urgency, frequency, and eventually incontinence have been termed overactive bladder (OAB) syndrome (Abrams et al., 2002), defined as the symptoms of urgency, with or without urge incontinence, usually with frequency and nocturia. OAB syndrome has been estimated to occur in nearly 17% of the population in the United States and Europe, including Scandinavia, and the prevalence of the syndrome increases with age (Milsom et al., 2001; Stewart et al., 2003).
Theoretically, a disturbed storage function can be improved by agents that decrease detrusor activity, increase bladder capacity, and/or increase outlet resistance. Many drugs have been tried, but the results are often disappointing, partly due to poor treatment efficacy and partly due to poor tolerability in the form of side effects (Kelleher et al., 1997). The development of pharmacological treatment has been slow, and use is based on results from controlled clinical trials for only a few drugs (Andersson, 1988; Andersson et al., 1999, 2002; Wein and Rovner, 2002; Yoshimura and Chancellor, 2002).
This review will cover recent advances in our understanding of the normal physiology of the lower urinary tract and of the pathophysiology of some voiding disorders as a pharmacological basis for current and emerging drug therapies. The terminology used to describe lower urinary tract function follows the recommendations of the International Continence Society (Abrams et al., 2002).
II. Central Nervous System Targets
The normal micturition reflex in the adult is mediated by a spinobulbospinal pathway, which passes through relay centers in the brain (Fig. 1). Micturition occurs in response to afferent signals from the lower urinary tract, and distension of the bladder wall is considered the primary stimulus (de Groat et al., 1999; de Groat and Yoshimura, 2001). However, signals generated in the urothelium and involving suburothelial nerves may be of importance both normally and in different disorders of micturition (Andersson, 2002b).
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During bladder filling (Fig. 2), once threshold tension is achieved, afferent impulses, conveyed mainly by the pelvic nerve, reach centers in the CNS. It has been proposed that the afferent neurons send information to the periaqueductal gray, which in turn communicates with the pontine tegmentum, where two different regions involved in micturition control have been described (Griffiths et al., 1990). One is a dorsomedially located M-region, corresponding to Barrington's nucleus or the pontine micturition center (PMC). A more laterally located L-region may serve as a pontine urine storage center, which has been suggested to suppress bladder contraction and to regulate the activity of the striated musculature of the bladder outlet during urine storage. The M- and L-regions may represent separate functional systems that act independently (Blok and Holstege, 1999a).
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Centers rostral to the pons determine the beginning of micturition. Thus, even if the forebrain is not essential for the basic micturition reflex, it plays a role in making the decision when and where micturition should take place (Blok and Holstege, 1999b). Recent positron emission tomography studies have given information on the brain structures involved in urine storage and voiding (Blok et al., 1997b; Nour et al., 2000; Athwal et al., 2001; Matsuura et al., 2002).
The micturition reflexes use several transmitters and transmitter systems that may be targets for drugs aimed at control of micturition (Fig. 3).
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1. Glutamic Acid. It is well established that glutamate is a major excitatory transmitter in the mammalian CNS (Mayer and Westbrook, 1987), including pathways controlling the lower urinary tract (de Groat et al., 1999). However, glutamate is involved in many CNS functions, and drugs acting on the different glutamate receptors may affect not only micturition (Downie, 1999; Matsuura et al., 2000). Glutamate is involved in the afferent limb of the micturition reflex at the level of the lumbosacral spinal cord (Birder and de Groat, 1992; Kakizaki et al., 1996, 1998) and in the pathways connecting the PMC to the preganglionic bladder neurons in the spinal parasympathetic nucleus. L-Glutamate, injected at sites in the brain stem where electrical stimulation evoked bladder contraction, stimulated micturition in rats (Mallory et al., 1991; Matsumoto et al., 1995a,b; Matsuura et al., 2000). For example, coordinated micturition could be evoked by injections of L-glutamate in Barrington's nucleus (Matsuura et al., 2000). However, in cats, injection of L-glutamate into the medullary raphe nuclei, which are known to have an inhibitory function in voiding, elicited only inhibition (Chen et al., 1993), suggesting that glutamate can also activate inhibitory systems.
Glutamate has also been shown to be involved at interneuronal synapses on parasympathetic preganglionic neurons (Araki and De Groat, 1996, 1997). Both N-methyl-D-aspartate (NMDA) and non-NMDA glutamatergic [
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)] receptors are involved in micturition control (de Groat et al., 1999), but the receptors may serve different functions in the regulation of bladder and striated urethral sphincter (intramural and extramural or periurethral) activity. Intravenous or i.t. administration of the AMPA receptor antagonist, GYKI52466 [1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine hydrochloride], or the NMDA receptor antagonist, MK801 (5H-dibenzo[a,d]cyclohepten-5,10-imine), blocked the bladder contractions evoked by electrostimulation of the PMC, further supporting the view that AMPA and NMDA receptors both mediate excitatory transmission in the descending limb of the spinobulbospinal micturition reflex pathway (Matsumoto et al., 1995a,b).
Striated sphincter activities were also inhibited by the i.v. or i.t. administration of the AMPA receptor antagonist, LY215490 [±6-(2-(1H-tetrazol-5-yl)ethyl) decahydroisoquinoline-3-carboxylic acid], or the NMDA receptor antagonists, LY274614 (6-substituted decahydroisoquinoline-3-carboxylic acid) and MK801 (Yoshiyama et al., 1993, 1997). However, there were differences in the extent of inhibition by the NMDA receptor antagonists between bladder contraction and striated sphincter activity in the unanesthetized or spinalized rats. In freely moving, awake rats (Vera and Nadelhaft, 1994), unanesthetized decerebrate rats (Yoshiyama et al., 1994, 1997), or spinalized rats (Yoshiyama et al., 1997), NMDA receptor antagonists did not depress bladder reflexes but still depressed striated sphincter activity. These findings suggest that the NMDA receptor regulates bladder contraction through parasympathetic preganglionic neurons (intermediolateral nucleus), mainly at the supraspinal level, and it regulates striated sphincter activity through somatomotor neurons (dorsolateral nucleus neurons) at the spinal level.
Supporting this, Shibata et al. (1999) found that, in rats, sacral parasympathetic preganglionic neurons express high messenger RNA levels of GluR-A and GluR-B AMPA receptor subunits and NR1, but not NR2, NMDA receptor subunits. On the other hand, motoneurons in the urethral sphincter nucleus expressed all four AMPA receptor subunits (GluR-A, -B, -C, and -D) in conjunction with moderate amounts of NR2A and NR2B, as well as high levels of NR1 receptor subunits. The authors concluded that it seems likely that dorsolateral nucleus neurons, but not parasympathetic preganglionic neurons, are provided with functional NMDA receptors, which could induce activity-dependent changes in synaptic transmission in the efferent pathway for the lower urinary tract.
2. Glycine. Glycine can be found in neurons in the sacral dorsal gray commisure, which receives afferent input from the PMC (Sie et al., 2001). In large part, glycine is colocalized with GABA. This is in agreement with the finding that glycine, released from interneurons in the spinal cord in some instances, is co-released with GABA at synapses on parasympathetic preganglionic neurons (Araki, 1994). Relaxation of the striated sphincter during micturition is strongly inhibited by strychnine, which is considered to be a specific glycine receptor antagonist (Shefchyk et al., 1998; Downie, 1999). The interneurons in the sacral DCG are believed to inhibit the striated sphincter motoneurons during micturition (Sie et al., 2001).
Miyazato et al. (2003) studied the influence of lumbosacral glycinergic neurons on the spinobulbospinal and spinal micturition reflexes in different groups of female rats, intact animals, rats with acute injury to the lower thoracic spinal cord, and rats with chronic spinal cord injury. Their results suggested that glycinergic neurons may have an important inhibitory effect on the spinobulbospinal and spinal micturition reflexes at the level of the lumbosacral cord.
3. Enkephalins. Several lines of evidence suggest that enkephalinergic mechanisms in the brain and spinal cord have an important role in the regulation of both the storage and voiding phases of micturition. A rich occurrence of enkephalin-containing nerve terminals has been demonstrated in the region of the PMC and in the sacral parasympathetic and nuclei of Onuf in the spinal cord. These terminals exert an inhibitory control on the micturition reflex as shown by i.c.v. intrapontine or i.t. administration of enkephalins or opioids (de Groat et al., 1993, 1999; Downie, 1999). Intrathecal administration of morphine in conscious dogs increases the volume threshold for inducing micturition without altering voiding pressure, an effect blocked by naloxone (Bolam et al., 1986). Morphine and its more potent metabolite, morphine-6-glucoronide, also increased volume threshold when given i.t. to conscious rats (Igawa et al., 1993c). This suggests that opioid peptides can suppress the afferent limb of the micturition reflex at a spinal level (Downie, 1999).
Different types of opioid receptors are involved in micturition control. Four types of opioid receptors are recognized (Smith and Moran, 2001). The new designations for opioid-like receptors OP1, OP2, and OP3 correspond to the classic 
-, 
-, and µ-nomenclature, respectively. OP4 was previously known as ORL1, the receptor for the endogenous heptadecapeptide nociceptin/orphanin FQ (see Section III.A.7.x.). In the brain, µ-, -, and -opioid receptors mediate inhibitory effects on micturition, which are blocked by naloxone (Dray and Metsch, 1984a,b,c; Hisamitsu and de Groat, 1984; Dray and Nunan, 1987; Willette et al., 1988; Mallory et al., 1991; Downie, 1999). Naloxone administered alone i.c.v. or injected directly into the PMC facilitated the micturition reflex. In the cat spinal cord, -opioid receptors mediate inhibition of bladder activity, and receptors mediate inhibition of sphincter activity (Thor et al., 1989). In the rat spinal cord, and µ receptors, but not receptors, seemed to be involved in the suppression of bladder reflexes (de Groat et al., 1999; Downie, 1999). However, Gotoh et al. (2002) examined the effect of a receptor agonist on the bladder motility of anesthetized rats. They found that the receptor agonist could inhibit the micturition reflex as effectively as other opioids, and at least part of the inhibition was due to the diminished bladder sensation, based on the activation of the descending monoaminergic systems through the spinal -opioid receptors.
The fact that the spinal opioid modulation of the external urethral sphincter is different from that regulating the bladder suggests that this mechanism should be a promising target for selective reduction of sphincter activity (Downie, 1999).
The potent inhibitory effect of opioids on micturition does not seem to have been used therapeutically in voiding disorders, with few exceptions. Tramadol, which is widely used as an analgesic, is by itself a very weak µ receptor agonist, but it is metabolized to several different compounds, some of them almost as effective as morphine at the µ receptor. The drug combines the effects on µ-opioid receptors with inhibition of the uptake of noradrenaline and 5-HT. Pandita et al. (2003) analyzed the combination of these mechanisms by studying the effects of tramadol and its enantiomers on micturition in unanesthetized rats. The most conspicuous effect of i.v. (±)- and (+)-tramadol (0.1-10 mg/kg) was a dose-dependent increase in threshold pressure and an increase in the bladder contraction interval, eventually resulting in dribbling incontinence. The activity seemed to be produced mainly by the opioid component, which is carried together with the 5-HT uptake inhibition by the (+)-enantiomer, whereas the (-)-enantiomer comprises the noradrenaline inhibitory activity.
(±)-Tramadol, given i.v. at doses below or similar to those shown to be analgesic in rats, abolished apomorphine-induced detrusor overactivity by increasing bladder capacity and abolishing nonvoiding contractions (Pehrson and Andersson, 2003). In rats, tramadol abolished experimentally induced detrusor overactivity caused by cerebral infarction (Pehrson et al., 2003). These data suggest that tramadol may have a clinically useful effect on detrusor overactivity.
4. Serotonin. The major source of 5-HT-containing terminals in the spinal cord is the raphe nuclei. The lumbosacral autonomic and the sphincter motor nuclei receive a dense serotonergic input (de Groat et al., 1993), and the descending bulbospinal pathway to the urinary bladder is essentially an inhibitory circuit, with 5-HT as a key neurotransmitter (de Groat et al., 1993; de Groat, 2002). Electrical stimulation of 5-HT-containing neurons in the caudal raphe and activation of postsynaptic 5-HT receptors in the spinal cord of cats cause marked inhibition of bladder contractions (McMahon and Spillane, 1982; Espey and Downie, 1995).
Multiple 5-HT receptors have been characterized in mammalian species and divided into different families (5-HT1-7) based upon structural diversity, transduction mechanism, and pharmacology (Gerhardt and van Heerikhuizen, 1997). Additionally, some 5-HT receptors have been subdivided into subtypes (e.g., the 5-HT1 receptor was further subdivided into 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F subtypes), each of which exhibits a distinct pharmacological profile (Hoyer et al., 2002). Many drugs acting on 5-HT receptors can influence micturition. 5-HT1A Receptors, which have been discussed as an interesting drug target (de Groat, 2002), act as somatodendritic and presynaptic receptors on nerve cells, modulating neural firing, and at the postsynaptic level, where they mediate inhibitory functions. There seem to be multiple sites of serotonergic modulation of micturition in the spinal cord. However, the exact sites of the modulation have not been determined, and the roles of the different 5-HT receptor subtypes have not been established (Downie, 1999; de Groat, 2002).
It has been demonstrated (Lecci et al., 1992) that the selective 5-HT1A agonist, 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), injected i.v. or i.c.v., activates the micturition reflex, inducing an increase in the frequency of isovolumic bladder contractions in anesthetized rats. Testa et al. (1999) synthesized several novel N-arylpiperazine derivatives and studied their ability to affect the micturition reflex in anesthetized and conscious rats. Neutral antagonists potently inhibited volume-induced voiding contractions in anesthetized rats. Also, in conscious rats during continuous transvesical cystometry, the neutral antagonists increased bladder capacity, whereas micturition pressure was only slightly, and not dose-dependently, reduced. Based on these and further studies, Testa et al. (1999, 2001) concluded that neutral 5-HT1A receptor antagonists have favorable effects on the bladder, inducing an increase in bladder capacity with no derangement of bladder contractility. This conclusion has been supported by the results of other investigators (Kakizaki et al., 2001; Pehrson et al., 2002b). Kakizaki et al. (2001) showed that rhythmic isovolumetric bladder contractions, evoked by bladder distension, were abolished by i.t. administration of the 5-HT1A antagonist, WAY100635 [[O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride], in a dose-dependent manner. The drug was effective only if injected at the L6-S1 spinal cord level, but not at the thoracic or cervical cord levels. WAY100635 significantly reduced the amplitude of bladder contractions evoked by electrical stimulation of the PMC. However, the field potentials in the rostral pons evoked by electrical stimulation of pelvic nerve were not affected by i.t. or i.v. injection of WAY100635. These results suggest that the 5-HT1A receptors at the L6-S1 level of the spinal cord have an important role in the tonic control of the descending limb of the micturition reflex pathway in the rat.
Read et al. (2003) injected the selective 5-HT7 receptor antagonist SB-269970 [(R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)pyrrolidine-1-sulfonyl)phenol] i.c.v. in urethane-anesthetized female rats. Based on their experiments, Read et al. suggested that 5-HT7 receptors located supraspinally in the rat are involved in the control of micturition. Whether this receptor can be a useful target for drugs meant for micturition control remains to be established.
Drugs interfering with serotonin or with serotonin receptors (for example, the selective serotonin reuptake inhibitors, or SSRIs) have not been systematically tested as a treatment for voiding disorders in humans. Whether or not imipramine, which blocks the reuptake of serotonin among other effects, depresses detrusor overactivity by this mechanism (Maggi et al., 1989b) has not been established. It has been suggested that there may be a deficiency of serotonin behind both depression and overactive bladder (Zorn et al., 1999; Steers and Lee, 2001; Littlejohn and Kaplan, 2002). If this is the case, the question is if SSRIs are effective for micturition control only in depressed patients, or if selective serotonin uptake inhibition is a general principle that can be used for treatment of the overactive bladder. A model suggesting that serotonin deficiency and urinary incontinence are linked has recently been reported (Na et al., 2003). Clomipramine was given to female rat pups. After 15 weeks, treated rats had developed increased voiding frequency, low bladder capacity, and detrusor overactivity demonstrable at cystometry. If the rats were treated with fluoxetine, micturition was normalized. The authors suggested that this supported the hypothesis that depression was linked to OAB syndrome and idiopathic urge incontinence. This would imply that SSRIs may be useful for treatment of OAB syndrome. On the other hand, there are reports suggesting that the SSRIs in patients without incontinence actually can cause incontinence, particularly in the elderly (Movig et al., 2002). This causes some doubts over the anecdotal reports that SSRIs may be used as a general treatment for OAB syndrome.
Duloxetine, a combined noradrenaline and 5-HT reuptake inhibitor has been shown, in animal experiments, to increase the neural activity to the striated urethral sphincter, and to increase bladder capacity through effects on the CNS (Thor and Katofiasc, 1995; Thor and Donatucci, 2004). Promising clinical experiences with the drug in the treatment of stress incontinence have been reported (Norton et al., 2002; Dmochowski et al., 2003; Millard et al., 2004; van Kerrebroeck et al., 2004). Another dual noradrenaline and 5-HT reuptake inhibitor, venlafaxine, increased intraurethral pressure in rats, and was suggested to be useful for treatment of stress incontinence (Bae et al., 2001). However, no clinical data for this drug are available.
5. Noradrenaline. The role of central nervous noradrenergic pathways in micturition control remains unclear. Noradrenergic neurons originating in the locus coeruleus react to bladder filling (Elam et al., 1986) and project to the autonomic and somatic nuclei in the lumbosacral spinal cord (de Groat et al., 1999). Bladder control through these bulbospinal pathways may involve both 1- and 2-adrenoceptors (ARs).
a. 1-Adrenoceptors. The 1-ARs seem to be tonically active in both the sympathetic and somatic neural control of the lower urinary tract (Danuser and Thor, 1995; Ramage and Wyllie, 1995). Thus, doxazosin, given i.t., decreased micturition pressure, both in normal rats and in rats with postobstruction bladder hypertrophy (Ishizuka et al., 1996b), the effect being much more pronounced in the animals with hypertrophied/overactive bladders. Doxazosin did not markedly affect the frequency or amplitude of the unstable contractions observed in obstructed rats. On this basis, it was suggested that doxazosin may have an action at the level of the spinal cord and ganglia, thereby reducing activity in the parasympathetic nerves to the bladder and that this effect was more pronounced in rats with bladder hypertrophy than in normal rats.
Urodynamic studies revealed that spontaneously hypertensive rats (SHR) have pronounced detrusor overactivity (Persson et al., 1998b). These animals also have an increased voiding frequency (Steers et al., 1999). Although the control rats (Wistar-Kyoto rats) have a regular contraction frequency during continuous cystometry, the SHR show both micturition and nonmicturition contractions as well as a decreased bladder capacity. In SHR treated with 6-hydroxydopamine to chemically destroy the peripheral noradrenergic nerves, detrusor overactivity was maintained, as demonstrated by continuous cystometry (Andersson and Pandita, unpublished results). Furthermore, 1-AR antagonists, injected i.a. near the bladder, did not abolish the detrusor overactivity. On the other hand, when given i.t., the 1-AR antagonists normalized micturition. This suggested that the effect on detrusor overactivity was exerted within the CNS.
Yoshiyama et al. (2000) studied the role of spinal 1-AR mechanisms in the control of urinary bladder function using cystometry in anesthetized and decerebrate, unanesthetized female rats. Their results suggested that two types of spinal 1-AR mechanisms are involved in reflex bladder activity. First, there seems to exist an inhibitory control of the frequency of voiding reflexes, presumably by 1-ARs regulating afferent processing in the spinal cord. Second, 1-ARs may mediate a facilitatory modulation of the descending limb of the micturition reflex pathway.
The contribution of different subtypes of 1-ARs (1A, 1B, 1D) in the lumbosacral spinal cord to the control of the urinary bladder was examined in urethane-anesthetized rats by Yoshiyama and de Groat (2001). They suggested that different 1-AR subtypes were involved in the modulation of reflex bladder activity. Via 1A- or 1B-ARs, an inhibitory control of the frequency of voiding reflexes is exerted, presumably by an alteration in the processing of bladder afferent input. 1A-ARs mediate facilitatory modulation of the descending efferent limb of the micturition reflex pathway. Yoshiyama and de Groat (2001) also concluded that spinal 1D-ARs did not appear to have a significant role at either site. Sugaya et al. (2002) investigated the effects of i.t. tamsulosin (selective for 1A/D-receptors) and naftopidil (selective for 1D-receptors) on isovolumetric bladder contractions in urethane-anesthetized rats. They found that both tamsulosin and naftopidil transiently abolished isovolumetric rhythmic bladder contractions, and that the effects were reversible. The amplitude of bladder contraction was decreased by naftopidil, but not by tamsulosin. The authors suggested that the noradrenergic projections from the brainstem to the spinal cord promote the afferent limb rather than the efferent limb of the micturition reflex pathway, and that the main -AR in the afferent limb of this reflex pathway may be 1D-receptors. This finding is of particular interest considering the findings of Smith et al. (1999), who investigated the neuronal localization of 1-AR subtypes in the human spinal cord. Although all three 1-AR subtypes were found to be present throughout the human spinal cord, 1D-AR mRNA predominated overall. However, it should be noted that the distribution of 1-AR mRNA subtypes in the rat spinal cord is not necessarily the same as that in humans (Day et al., 1997).
It has been claimed that although both tamsulosin and naftopidil improve both emptying and storage disorders in benign prostatic hyperplasia patients, tamsulosin is superior for emptying disorders, and naftopidil is superior for collecting disorders (Hayashi et al., 2002). It was speculated that, in addition to an antagonistic action on the -ARs of the smooth muscle of the lower urinary tract, both drugs (especially naftopidil) may also act on the lumbosacral cord to improve storage disorders.
Clinically, 1-AR antagonists have been observed occasionally to abolish detrusor overactivity in patients with benign prostatic hyperplasia. 1-AR antagonists have also been used to treat patients with neurogenic detrusor overactivity, however, with moderate success (Andersson et al., 2002). Whether the site of action is within the CNS or peripherally has not been established.
b. 2-Adrenoceptors. Several studies have suggested that spinal and/or supraspinal 2-ARs can modulate lower urinary tract function (de Groat et al., 1993). Smith et al. (1995) found that in the human spinal cord, 2-AR mRNA was present predominantly in the sacral region. The thoracic, lumbar, and sacral spinal regions showed an increasing predominance of 2B-AR mRNA. This was different from findings in the rat, where 2A-AR and 2C-AR predominated (Stone et al., 1998; Shi et al., 1999).
Ishizuka et al. (1996a) performed continuous cystometry in normal, conscious rats in the presence of 2-AR stimulation and blockade. Given i.t., the selective 2-AR agonist, dexmedetomidine, stimulated bladder activity and eventually caused total incontinence. Given i.a., dexmedetomidine decreased micturition pressure, bladder capacity, micturition volume, residual urine, and basal pressure. The selective 2-AR antagonist, atipamezole, given i.t., increased micturition pressure, bladder capacity, residual urine, and decreased micturition volume. Similar effects were obtained when atipamezole was given i.a.
Kontani et al. (2000) administered the 2-AR agonists clonidine and oxymetazoline i.t. and i.c.v. to conscious rats and demonstrated that both drugs induced detrusor overactivity, which could be prevented by the selective 2-AR antagonist, idazoxan. They suggested that this overactivity could be produced via 2A-AR stimulation both at spinal and supraspinal sites.
Collectively, available information suggests that both 1- and 2-ARs are involved in central micturition control. The role of 2-ARs and the possibility that these receptors can be targets for drugs aiming at micturition control remain to be established.
6. Acetylcholine. There is evidence that cholinergic pathways in the cerebral cortex play an important role in the regulation of the micturition reflex, and studies in animals have indicated that drugs acting on muscarinic receptors may have both excitatory (Sugaya et al., 1987; O'Donnell, 1990; Ishiura et al., 2001; Ishizuka et al., 2002) and inhibitory (Matsuzaki, 1990; Ishiura et al., 2001) effects on these pathways. Yokoyama et al. (2001) suggested, based on their results in rats, that the M1 receptor is involved in a forebrain inhibitory mechanism controlling the micturition reflex and that muscarinic receptors in the dorsal pontine tegmentum contribute to excitatory control. Since M1 receptor mRNA does not seem to exist in the pons, it may well be that different muscarinic receptor subtypes mediate inhibition and excitation. In rats, muscarinic receptor mechanisms may mediate a tonic excitatory influence on voiding (Ishiura et al., 2001; Ishizuka et al., 2002), whereas the inhibitory receptors do not appear to be tonically active. Masuda et al. (2001) suggested that excitation of a central muscarinic cholinergic pathway at a supraspinal or spinal site promotes proximal urethral relaxation during the voiding phase through the activation of efferent pathways in the pelvic nerves.
Muscarinic and nicotinic receptors may be involved in the control of voiding function. In the rat, stimulation of nicotinic receptors in the brain increased bladder capacity, suggesting that nicotinic agonists can activate mechanisms that inhibit voiding reflexes (Lee et al., 2003).
The bladder effects of antimuscarinics used for treatment of OAB syndrome are generally considered to be exerted peripherally. However, for those drugs passing into the CNS, effects on central mechanisms controlling lower urinary tract function cannot be excluded. Whether or not such effects are beneficial or not, however, remains to be established.
7. Dopamine. Central dopaminergic pathways can have both facilitatory and inhibitory effects on micturition by actions through D1-like (D1 or D5) and D2-like (D2, D3, or D4) dopaminergic receptors. Patients with Parkinson's disease often have neurogenic detrusor overactivity and voiding dysfunction (Berger et al., 1987), possibly as a consequence of nigrostriatal dopamine depletion and failure to activate inhibitory D1-like receptors (Yoshimura et al., 1993). However, through other dopaminergic pathways, micturition can be activated via D2-like receptors. Facilitation of the micturition reflex mediated via D2-like receptors may involve actions on brainstem and spinal cord. Thus, microinjection of dopamine into the pontine micturition center reduced bladder capacity and facilitated the micturition reflex in cats (de Groat et al., 1993).
Sillén et al. (1981) showed that apomorphine, which stimulates both D1- and D2-like receptors, induced bladder overactivity in anesthetized rats. In female rats, the role of dopamine D1 and D2 receptors in the volumeinduced micturition reflex, was investigated cystometrically before and after i.v. administration of SKF38393(2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; a selective D1 receptor agonist), SCH 23390 (a selective D1 receptor antagonist), quinpirole (a selective D2 receptor agonist), and remoxipride (a selective D2 receptor antagonist) (Seki et al., 2001). The results, which are in agreement with previously obtained data (de Groat and Yoshimura, 2001), suggested that D1 receptors tonically inhibit the micturition reflex, and that D2 receptors are involved in its facilitation. Thus, central dopaminergic pathways exhibit different effects on micturition via actions on multiple receptors at different sites in the central nervous system.
Experimental cerebral infarction yields damage to the basal ganglia and motor cortex, which may be areas suppressing D2-like receptor-mediated actions in the normal rat. Antagonism of D2-like receptors was without effect on bladder capacity in normal, conscious rats, but it increased bladder capacity in cerebral infarcted rats (Yokoyama et al., 1999). Thus, selective blockade of D2-like receptors, or a specific D1-like receptor agonist, might be helpful in stroke patients with detrusor overactivity.
8. GABA. GABA (
-amino butyric acid) has been identified as an inhibitory transmitter at both spinal and supraspinal synapses in the mammalian CNS. At least in some species, the supraspinal micturition reflex pathway is under a tonic GABAergic inhibitory control (de Groat et al., 1993, 1999). GABA functions appear to be triggered by binding of GABA to its ionotropic receptors, GABAA and GABAC, which are ligand-gated chloride channels, and its metabotropic receptor, GABAB (Chebib and Johnston, 1999). Since blockade of GABAA and GABAB receptors in the spinal cord (Igawa et al., 1993a; Pehrson et al., 2002a) and brain (Maggi et al., 1987a; Pehrson et al., 2002a) stimulated rat micturition, an endogenous activation of GABAA+B receptors may be responsible for continuous inhibition of the micturition reflex within the CNS. In the spinal cord, GABAA receptors are more numerous than GABAB receptors, except for the dorsal horn where GABAB receptors predominate (Malcangio and Bowery, 1996; Coggeshall and Carlton, 1997).
GABA transporters, present on neuronal and glial cells in the brain, brainstem, and spinal cord (Jursky et al., 1994), are presumed to provide an inactivation mechanism (Malcangio and Bowery, 1996). Four different GABA transporters (GATs) have been described (Bowery, 1993). Tiagabine is a selective inhibitor of one of these GABA transporters, GAT1 (Borden et al., 1994), and is able to increase extracellular levels of GABA (Fink-Jensen et al., 1992). Intravenous administration of tiagabine to rats decreased micturition pressure and decreased voided volume. Given i.t., tiagabine reduced micturition pressure and increased bladder capacity (Pehrson and Andersson, 2002), suggesting that increasing endogenous levels of GABA in the CNS may improve micturition control.
Experiments using conscious and anesthetized rats demonstrated that exogenous GABA, muscimol (GABAA receptor agonist) and baclofen (GABAB receptor agonist), given i.v., i.t., or i.c.v., inhibit micturition (Maggi et al., 1987a,c; Pehrson et al., 2002a). Similar effects were obtained in nonanesthetized mice (Zhu et al., 2002).
Baclofen, given i.t., attenuated oxyhemoglobin-induced detrusor overactivity, suggesting that the inhibitory actions of GABAB receptor agonists in the spinal cord may be useful for controlling micturition disorders caused by C-fiber activation in the urothelium and/or suburothelium (Pehrson et al., 2002a). In mice, where detrusor overactivity was produced by intravesical citric acid, baclofen given subcutaneously had an inhibitory effect which was blocked by the selective GABAB receptor antagonist CGP55845[(2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenyl-methyl)phosphinic acid] (Zhu et al., 2002).
Stimulation of the PMC results in an immediate relaxation of the external striated sphincter and a contraction of the detrusor muscle of the bladder. Blok et al. (1997) demonstrated in cats a direct pathway from the PMC to the dorsal gray commissure of the sacral cord. It was suggested that the pathway produced relaxation of the external striated sphincter during micturition via inhibitory modulation by GABA neurons of the motoneurons in the sphincter of Onuf (Blok et al., 1997a). In rats, i.t. baclofen and muscimol ultimately produced dribbling urinary incontinence (Igawa et al., 1993a; Pehrson et al., 2002a), and this was also found in conscious mice given muscimol and diazepam subcutaneously (Zhu et al., 2002). Thus, normal relaxation of the striated urethral sphincter is probably mediated via GABAA receptors (Pehrson and Andersson, 2002; Pehrson et al., 2002a), and GABAB receptors have a minor influence on motoneuron excitability (Rekling et al., 2000).
a. Gabapentin. Gabapentin was originally designed as an anticonvulsant GABA mimetic capable of crossing the blood-brain barrier (Maneuf et al., 2003). However, its effects do not appear to be mediated through interaction with GABA receptors, and its mechanism of action is still controversial (Maneuf et al., 2003). Gabapentin is also widely used not only for seizures and neuropathic pain, but also for many other indications such as anxiety and sleep disorders, due to its apparent lack of toxicity.
In a pilot study, Carbone et al. (2003) reported on the effect of gabapentin on neurogenic detrusor activity. They found a positive effect on symptoms and a significant improvement of urodynamic parameters after treatment and suggested that the effects of the drug should be explored in further controlled studies in both neurogenic and non-neurogenic detrusor overactivity.
9. Tachykinins. The main endogenous tachykinins, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB), and their preferred receptors, NK1, NK2, and NK3, respectively, have been demonstrated in various CNS regions, including those involved in micturition control (Lecci and Maggi, 2001; Saffroy et al., 2001, 2003; Covenas et al., 2003). NK1 receptor expressing neurons in the dorsal horn of the spinal cord may play an important role in detrusor overactivity. Thus, Ishizuka et al. (1994) found that at the spinal level, there was a tachykinin involvement via NK1 receptors in the micturition reflex induced by bladder filling. This was demonstrated in normal rats, and more clearly, in rats with bladder hypertrophy secondary to bladder outflow obstruction. Seki et al. (2002) demonstrated that NK1 receptor-expressing neurons in the spinal cord could be eliminated by using i.t. substance P-saponin conjugate (SSP-SAP). They found that SSP-SAP capsaicin-induced detrusor overactivity was reduced, and they suggested that SSP-SAP could be effective to treat overactivity induced by bladder irritation without affecting normal bladder function.
Spinal NK1 receptor blockade could suppress detrusor activity induced by dopamine receptor (L-DOPA) stimulation (Ishizuka et al., 1995a).
Intracerebroventricular injection of SP, a selective NK1 receptor agonist, but not selective NK2 and NK3 receptor agonists, was found to inhibit isovolumetric contractions in urethane-anesthetized rats (Palea et al., 1993b). In conscious rats, however, i.c.v. SP stimulated micturition (Dib et al., 1998). Furthermore, in conscious rats undergoing continuous cystometry, antagonists of both NK1 and NK2 receptors inhibited micturition, decreasing micturition pressure and increasing bladder capacity at low doses, and inducing dribbling incontinence at high doses. This was most conspicuous in animals with outflow obstruction (Gu et al., 2000). Intracerebroventricular administration of NK1 and NK2 receptor antagonists to awake rats suppressed detrusor activity induced by L-DOPA stimulation (Ishizuka et al., 2000). The differences between the results of Palea et al. (1993b) and later investigators probably can be attributed to differences in experimental conditions (including anesthesia).
Taken together, available information suggests that spinal and supraspinal NK1 and NK2 receptors may be involved in micturition control. Involvement of NK3 receptors does not seem to have been demonstrated (Lecci and Maggi, 2001). Whether or not spinal and/or supraspinal NK receptors can be useful targets for drugs aiming at control of micturition disturbances remains to be established.
1. Urothelium and Interstitial Cells. The uroepithelium (urothelium) was long regarded as a protecting barrier that allowed for urine storage. However, recent investigations indicate that urothelial cells sense and respond to mechanical stimuli such as pressure, and that they may communicate mechanical stimuli to the nervous system (Apodaca, 2004). Thus, the urothelium may serve as a mechanosensor which, by producing nitric oxide, ATP, acetylcholine, and other mediators, can control the activity in afferent nerves, and thereby the initiation of the micturition reflex (Andersson, 2002b). Low pH, high K+, increased osmolality, and low temperatures can all influence afferent nerves, possibly via effects on the vanilloid receptor (capsaicin-gated ion channel TRPV1), which is expressed both in afferent nerve terminals and in the urothelial cells (Birder et al., 2001, 2002) A network of interstitial cells, extensively linked by Cx43-containing gap junctions, was found to be located beneath the urothelium in human bladder by Sui et al. (2002, 2004) This interstitial cellular network was suggested to operate as a functional syncytium, integrating signals and responses in the bladder wall, or as an electrical network acting as a control step in bladder sensory function (Sui et al., 2004).
Interstitial cells can also be found within the detrusor (McCloskey and Gurney, 2002; Hashitani et al., 2004). Hashitani et al. (2004) suggested that these cells modulated signal transmission within the bladder. Interestingly, these cells reacted to muscarinic receptor stimulation by initiating calcium transients.
If the bladder interstitial cells are important for the generation of OAB syndrome, they may be an interesting target for drugs meant for treatment of this disorder.
2. Afferent Nerves. From the dorsal root ganglia, afferent nerve cell bodies project both to the bladder, where information is received, and to the spinal cord, where connections with other neurons are established. Retrograde tracing studies have shown that most of the afferent innervation of the bladder and urethra originates in the dorsal root ganglia of the sacral region and travels via the pelvic nerve (Morrison et al., 2002). In addition, some afferents originating in ganglia at the level of the sympathetic outflow project via the hypogastric nerve. The afferent nerves of the striated muscle in the external striated sphincter (rhabdosphincter) travel in the pudendal nerve to the sacral region of the spinal cord. Sacral afferent nerve terminals are uniformly distributed to all areas of the detrusor and urethra, whereas lumbar afferent nerve endings are most frequently found in the trigone and are scarce in the bladder body (Lincoln and Burnstock, 1993). The afferent hypogastric and pelvic pathways mediate the sensations associated with normal bladder filling and with bladder pain. The pelvic and pudendal pathways are concerned also with the sensation that micturition is imminent and with thermal sensations of the urethra.
In both humans and animals, afferent nerves have been identified suburothelially as well as in the detrusor muscle (Gosling and Dixon, 1974; Maggi, 1993, 1995; Wakabayashi et al., 1993; Gabella and Davis, 1998). Suburothelially, they form a nerve plexus, which lies immediately beneath the epithelial lining. Some terminals may even be located within the basal parts of the urothelium. This suburothelial plexus is relatively sparse in the dome of the bladder, but becomes progressively denser near the bladder neck, and it is particularly prominent in the trigone (Gabella and Davis, 1998). Gabella and Davis (1998) studied in detail the distribution of afferent axons in the bladder of rats, using immunohistochemistry for calcitonin gene-related peptide (CGRP). They found that the afferent axons were distributed over four distinct targets: at the base of the epithelium, inside the epithelium, on blood vessels (both arteries and veins), and along muscle bundles. The afferent innervation of the musculature was diffuse, and appeared uniform throughout the bladder. There was a bilateral innervation of many regions of the lamina propria and the musculature, including individual muscle bundles. However, the epithelium and lamina propria of the dome of the bladder had no afferent axons.
Wakabayashi et al. (1993) studied the ultrastructure of SP-containing axon terminals in the lamina propria of the human urinary bladder. Numerous SP-immunoreactive varicose nerve fibers were observed, and most of them ran freely in the connective tissue. Many SP-immunoreactive nerve fibers were found beneath the epithelium, and perivascular SP-immunoreactive nerves were also seen in the lamina propria. The density of suburothelial, presumptive sensory nerves in the bladder wall was assessed in women with idiopathic detrusor overactivity and compared with the density of these nerves in asymptomatic women (Moore et al., 1992; Smet et al., 1997). The results suggested that a relative abundance of suburothelial sensory nerves may serve to increase the appreciation of bladder filling, giving rise to the frequency and urgency of micturition, which are characteristic of patients with detrusor overactivity.
The most important afferents for the micturition process are myelinated A-fibers and unmyelinated C-fibers traveling in the pelvic nerve, conveying information from receptors in the bladder wall to the spinal cord. The A-fibers respond to passive distension and active contraction, thus conveying information about bladder filling (Janig and Morrison, 1986). The activation threshold for A-fibers is 5 to 15 mm H2O, which is the intravesical pressure at which humans report the first sensation of bladder filling (de Groat et al., 1993). Afferents, which respond only to bladder filling, have been identified in the rat bladder and appear to be volume receptors, possibly sensitive to stretching of the mucosa. Experiments in rats suggested that ATP may play a significant role in driving volume-evoked micturition reflexes mediated by A-fibers (Smith et al., 2002a).
C-fibers have a high mechanical threshold and respond primarily to chemical irritation of the bladder mucosa (Habler et al., 1990) or cold (Fall et al., 1990). Following chemical irritation, the C-fiber afferents exhibit spontaneous firing when the bladder is empty and increased firing during bladder distension (Habler et al., 1990). These fibers are normally inactive and are therefore termed "silent fibers." Some of these C-fibers may be nociceptive, and glutamate may be the predominant transmitter (Smith et al., 2002a). They are sensitive to chemical stimulation and may then become mechano-sensitive. Both high-threshold fibers (>15 mm Hg; known to be associated with nociception) and low-threshold fibers (<15 mm Hg; probably associated with non-nociceptive events) could be induced to discharge by intravesical ,
-methylene ATP, suggesting that purinergic mechanisms contribute to both nociceptive and non-nociceptive (physiological) mechanosensory transduction in the urinary bladder (Rong et al., 2002). There is evidence that epithelial and suburothelial afferents may respond to changes in the chemical composition of the urine or to chemicals [e.g., nitric oxide (NO), prostaglandins, and ATP] released from the urothelial cells (Andersson, 2002b).
3. Efferent Nerves. Bladder emptying and urine storage involve a complex pattern of efferent and afferent signaling in parasympathetic, sympathetic, and somatic nerves. These nerves are parts of reflex pathways that either maintain the bladder in a relaxed state, enabling urine storage at low intravesical pressure, or facilitate micturition by relaxing the outflow region and mediating a coordinated contraction of the bladder smooth muscle. Contraction of the detrusor smooth muscle and relaxation of the outflow region result from activation of parasympathetic neurons located in the sacral parasympathetic nucleus in the spinal cord at the level of S2-S4 (de Groat et al., 1993). The axons pass through the pelvic nerve and synapse with the postganglionic nerves in either the pelvic plexus, in ganglia on the surface of the bladder (vesical ganglia), or within the walls of the bladder and urethra (intramural ganglia) (Lincoln and Burnstock, 1993). The preganglionic neurotransmission is predominantly mediated by acetylcholine acting on nicotinic receptors, although the transmission can be modulated by adrenergic, muscarinic, purinergic, and peptidergic presynaptic receptors (de Groat et al., 1993). The postganglionic neurons in the pelvic nerve mediate the excitatory input to the human detrusor smooth muscle by releasing acetylcholine, acting on muscarinic receptors. However, an atropine-resistant component has been demonstrated, particularly in functionally and morphologically altered human bladder tissue (see Section III.A.7.). The pelvic nerve also conveys parasympathetic fibers to the outflow region and the urethra. These fibers exert an inhibitory effect and therefore relax the outflow region. This is mediated partly by NO (Andersson and Persson, 1993), although other transmitters might be involved (Bridgewater and Brading, 1993; Hashimoto et al., 1993; Werkström et al., 1995).
Most of the sympathetic innervation of the bladder and urethra originates from the intermediolateral nuclei in the thoraco-lumbar region (T10-L2) of the spinal cord. The axons travel either through the inferior mesenteric ganglia and the hypogastric nerve, or they pass through the paravertebral chain and enter the pelvic nerve. Thus, sympathetic signals are conveyed in both the hypogastric and pelvic nerves (Lincoln and Burnstock, 1993).
The predominant effects of the sympathetic innervation of the lower urinary tract in humans are inhibition of the parasympathetic pathways at spinal and ganglion levels and mediation of contraction of the bladder base and the urethra. However, in several animals, the adrenergic innervation of the detrusor is believed to contribute to relaxation of the detrusor by releasing noradrenaline (Andersson, 1993). The normal response of isolated bladder body tissue to noradrenaline, released by electrical stimulation of nerves, or added exogenously, is relaxation (Andersson, 1993; Nomiya and Yamaguchi, 2003).
4. Neurotransmission: Cholinergic Mechanisms.
a. Cholinergic Nerves. Although histochemical methods that stain for acetylcholine esterase (AChE) are not specific for acetylcholine-containing nerves (Lincoln and Burnstock, 1993), they have been used as an indirect indicator of cholinergic nerves. The vesicular acetylcholine transporter (VAChT) is considered a specific marker for cholinergic nerve terminals (Arvidsson et al., 1997). For example, in rats, bladder smooth muscle bundles were supplied with a very rich number of VAChT-positive terminals also containing neuropeptide Y (NPY), NOS, and vasoactive intestinal polypeptide (VIP) (Persson et al., 1997a). Similar findings have been reported in human bladders of neonates and children (Dixon et al., 2000). The muscle coat of the bladder showed a rich cholinergic innervation, and small VAChT-immunoreactive neurons were found scattered throughout the detrusor muscle. VAChT-immunoreactive nerves were also observed in a suburothelial location in the bladder. The function of these nerves is unclear, but an afferent function or a neurotrophic role with respect to the urothelium cannot be excluded (Dixon et al., 2000).
b. Muscarinic Receptors. As described in detail elsewhere (Caulfield and Birdsall, 1998), muscarinic receptors comprise five subtypes, encoded by five distinct genes. The five gene products correspond to pharmacologically defined receptors, and M1 through M5 are used to describe both the molecular and pharmacological subtypes. Muscarinic receptors are coupled to G-proteins, but the signal transduction systems vary. M1, M3, and M5 receptors couple preferentially to Gq/11, activating phosphoinositide hydrolysis, in turn leading to mobilization of intracellular calcium. M2 and M4 receptors couple to pertussis toxin-sensitive Gi/o, resulting in inhibition of adenylyl cyclase activity.
In the human bladder, the mRNAs for all muscarinic receptor subtypes have been demonstrated (Sigala et al., 2002), with a predominance of mRNAs encoding M2 and M3 receptors (Yamaguchi et al., 1996; Sigala et al., 2002). These receptors are also functionally coupled (Eglen et al., 1996; Hegde and Eglen, 1999; Chess-Williams, 2002). Also, in most animal species, detrusor smooth muscle contains muscarinic receptors of the M2 and M3 subtypes (Eglen et al., 1996; Hegde and Eglen, 1999; Chess-Williams, 2002).
The M3 receptors in the human bladder are believed to be the most important for detrusor contraction (Fig. 4) and to cause contraction through phosphoinositide hydrolysis (Andersson et al., 1991b; Harriss et al., 1995). In cat detrusor muscle, contraction induced by acetylcholine was found to be mediated via M3 receptor-dependent activation of Gq/11 and phospholipase C-1 and inositol tris phosphate (IP3)-dependent Ca2+ release (An et al., 2002). Jezior et al. (2001) found that bethanechol-induced contractions in the rabbit detrusor were practically abolished by inhibitors of Rho-kinase [Y27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexane carboxamide), HA 1077 (fasudil)] in combination with a nonselective cation channel inhibitor, LOE-908 [(R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate]. They suggested that muscarinic receptor activation of detrusor muscle includes both nonselective cation channels and activation of Rho-kinase. Supporting a role of Rho-kinase in the regulation of rat detrusor contraction and tone, Wibberley et al. (2003) found that Rho-kinase inhibitors (Y27632, HA 1077) inhibited contractions evoked by carbachol without affecting the contraction response to KCl. They also demonstrated high levels of Rho-kinase isoforms (I and II) in the bladder. Supporting a role for Rho-kinase in detrusor contraction, Fleichman et al. (2004) demonstrated in the rat bladder that carbachol-induced contraction did not involve protein kinase C, phosphatidylinositol-3-kinase, tyrosine kinases, or extracellular signal-regulated kinases, but that Rho-associated kinases were involved. In the human detrusor, Schneider et al. (2004) confirmed that the muscarinic receptor subtype mediating carbachol-induced contraction was the M3 receptor: They also demonstrated that the phospholipase C inhibitor U-73122 [1-[6-[[17-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione] did not significantly affect carbachol-stimulated bladder contraction, despite blocking IP3 generation. A phospholipase D inhibitor caused only a small inhibition of the contraction. However, the L-type calcium channel blocker, nifedipine, almost completely inhibited carbachol-induced detrusor contraction, whereas an inhibitor of store-operated Ca2+ channels, caused little inhibition. Protein kinase C inhibition did not significantly affect carbachol-induced contraction, but in contrast, the Rho-kinase inhibitor, Y27632, concentration-dependently and effectively attenuated the carbachol-induced responses. Schneider et al. (2004) concluded that carbachol-induced contraction of human urinary bladder is mediated via M3 receptors and largely depends on Ca2+ entry through nifedipine-sensitive channels and activation of the Rho-kinase pathway.
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Previous studies have indicated an important role for extracellular calcium in muscarinic receptor activation of the human bladder (Fovaeus et al., 1987), but the sources of calcium used for contractile activation have been a matter of debate (Andersson 1993). As pointed out by Hashitani et al. (2000), in most studies of the contribution of IP3 production to muscarinic receptor-mediated contractions in the detrusor, relatively high concentrations of muscarinic receptor agonists have been used. Hashitani et al. (2000) speculated that the concentration of neurally released acetylcholine, which acts on the muscarinic receptors of the detrusor, is not always sufficiently high to stimulate IP3 production. They suggested that M3 receptors, which on stimulation produce IP3, operate at high concentrations of muscarinic agonists, whereas M2 receptors, which do not trigger the formation of IP3, may be activated by lower concentrations.
Thus, the main pathway for muscarinic receptor activation of the detrusor via M3 receptors may be calcium influx via L-type calcium channels, and increased sensitivity to calcium of the contractile machinery produced via inhibition of myosin light-chain phosphatase through activation of Rho-kinase (Fig. 4).
The functional role for the M2 receptors has not been clarified, but it has been suggested that M2 receptors may oppose sympathetically mediated smooth muscle relaxation, mediated by -ARs (Hegde et al., 1997). M2 receptor stimulation may also activate nonspecific cation channels (Kotlikoff et al., 1999) and inhibit KATP channels through activation of protein kinase C (Bonev and Nelson, 1993b
