The identification of β-adrenoceptors at the protein level is typically based upon binding studies with radioligands such as [125I]iodocyanopindolol, [125I]iodopindolol, [3H]CGP 12,177 or [3H]dihydroalprenolol. [125I]iodocyanopindolol and [3H]CGP 12,177 have much lower affinity for β3- than for β1- or β2-adrenoceptors (Hoffmann et al., 2004; Baker, 2005). Data from our lab confirm this and further demonstrate that [3H]dihydroalprenolol yields a similarly poor labelling of β3-adrenoceptors (Niclauss et al., unpublished observations), a finding that is entirely consistent with the low β3-adrenoceptor affinity of unlabelled alprenolol (Hoffmann et al., 2004). While high concentrations of [125I]iodocyanopindolol and [3H]CGP 12,177 have successfully been used to label β3-adrenoceptors in transfected cells, the use of similarly high concentrations in tissues yields very high nonspecific binding and will saturate β1- and β2-adrenoceptors. Both problems make the detection of β3-adrenoceptors in tissues expressing mixed β-adrenoceptor subtype populations virtually impossible. A potential alternative would be the use of a β3-adrenoceptor-selective radioligand such as [3H]SB 206,606. However, this ligand has only high nanomolar affinity for β3-adrenoceptors (Kd values of 200–500 nM) (Muzzin et al., 1994; Klaus et al., 1995). Therefore, [3H]SB 206,606 is also a poor choice for the labelling of β3-adrenoceptors in tissues. These technical limitations must be considered when interpreting existing radioligand-binding data in the bladder and other tissues.
Radioligand-binding studies on bladder β-adrenoceptors have been reported for rats, rabbits, pigs and humans. Saturation-binding studies with various radioligands have reported 6–42 fmol mg−1 protein in rats (Nishimoto et al., 1995; Ma et al., 2002), 60–92 fmol mg−1 protein in rabbits (Levin et al., 1988; Latifpour et al., 1990; Morita et al., 1998), 30–154 fmol mg−1 protein in pigs (Goepel et al., 1997; Yamanishi et al., 2002b, 2002c) and 22–60 fmol mg−1 protein in humans (Levin et al., 1988; Goepel et al., 1997; Morita et al., 2000; Li et al., 2003). Limited attempts have been made to identify the receptor subtypes in the bladder by radioligand binding. Based upon competition studies with the β2-selective antagonist ICI 118,551 and a β1-selective antagonist, sites in the rabbit (Latifpour et al., 1990) and human bladder (Goepel et al., 1997) were reported to largely belong to the β2-subtype. On the other hand, three studies in the porcine bladder detected few, if any, high-affinity sites for ICI 118,551, and the β1-selective antagonist CGP 20,712A recognized largely low-affinity sites in those studies (Goepel et al., 1997; Yamanishi et al., 2002b, 2002c). Two of the studies additionally report about 60% high-affinity sites for SR 59,230A (Yamanishi et al., 2002b, 2002c); the latter authors interpreted these findings as evidence in favour of the presence of a population of largely β3-adrenoceptors. However, three reasons argue against this interpretation: Firstly, ICI 118,551 may not be β2-selective in pigs (Goepel et al., 1996), which make the low affinity of this compound in the porcine bladder difficult to interpret. Secondly, while SR 59,230A can be used to functionally block β3-adrenoceptors, it is not selective for this subtype and, at least in humans, has even slightly lower affinity for β3- than for β1- and β2-adrenoceptors (Hoffmann et al., 2004). Thirdly, the radioligands used in all of the above studies are unlikely to label a major fraction of possibly present β3-adrenoceptors due to their low affinity for this subtype (at least in humans; see above). Therefore, we consider the presently available pig data to be inconclusive. This does not exclude the presence of β3-adrenoceptors at the protein level in any of these species, but the currently available radioligand-binding techniques are probably inadequate to detect their presence. Hence, the reported densities of β-adrenoceptors in the bladder may represent an underestimation if the additional presence of β3-adrenoceptors is taken into account.
In vitro function
Since activation of adenylyl cyclase is the prototypical signalling pathway of β-adrenoceptors, it is not surprising that an isoprenaline-stimulated, propranolol-sensitive elevation of cAMP content has also been reported in rat bladder (Derweesh et al., 2000; Ma et al., 2002; Uchida et al., 2005). However, various recent studies have questioned whether this can sufficiently explain β-adrenoceptor-mediated smooth muscle relaxation (Horinouchi et al., 2003; Peters & Michel, 2003; Tanaka et al., 2003). One study in rat bladder demonstrated that the concentration–response relationships for isoprenaline, clenbuterol and FR 165,101 for relaxation and cAMP elevations were largely superimposable in noncontracted muscle; however, no such relationship was observed during KCl-induced contraction (Uchida et al., 2005). Accordingly, the adenylyl cyclase inhibitor SQ 22,536, in a concentration where it fully suppressed cAMP formation, inhibited rat bladder relaxation by all three agonists in the absence of pre-contraction, but not in its presence (Uchida et al., 2005). Similarly, SQ 22,536 and the protein kinase A inhibitors H7, H89 and Rp-cAMPs, if anything, inhibited isoprenaline-induced relaxation of rat bladder only against passive tension, but not against KCl-induced tension in another study (Frazier et al., 2005a). These data demonstrate that, at least in rats, elevation of cAMP is relevant for the regulation of bladder smooth muscle tone against passive tension, but not in the presence of a depolarizing stimulus such as KCl. Interestingly, a combination of adenylyl and guanylyl cyclase inhibitors (SQ 22,536 and ODQ) caused the strongest inhibition of relaxation against passive tension, but was also inactive against KCl-induced tension (Frazier et al., 2005a).
A possible modulation of membrane potential, ion-channel activity and intracellular ion concentrations has been studied as an alternative means of β-adrenoceptor control of bladder function. In guinea-pig bladder smooth muscle bundles exhibiting spontaneous action potentials, isoprenaline was found to hyperpolarize the cells, prevent action potentials and reduce the associated Ca2+ transients; the elevation of membrane potential was blocked by protein kinase A inhibitors and by high extracellular K+ concentrations, but not by K+ channel inhibitors (Nakahira et al., 2001). In other studies, both isoprenaline and the receptor-independent adenylyl cyclase activator forskolin were shown to increase iberiotoxin-sensitive K+ currents in guinea-pig bladder smooth muscle cells, and such stimulation was sensitive to a peptidergic inhibitor of protein kinase A (Kobayashi et al., 2000). In a later study, these investigators also demonstrated propranolol-sensitive isoprenaline inhibition of Ba2+ current through L-type Ca2+ channels due to a shift of steady-state for inactivation by 11 mV; this effect was apparently mediated by protein kinase A, but did not involve protein kinase G (Kobayashi et al., 2003). Other investigators reported that isoprenaline caused marginal increases in Ca2+ currents after large conditioning depolarizations (but not in their absence) in the guinea-pig bladder, and that this effect was not mimicked by forskolin (Smith et al., 1999). On the other hand, a third group found that isoprenaline causes intracellular Ca2+ sparks and activates voltage-dependent Ca2+ channels in guinea-pig bladder, and proposed that this may underlie the activation of large-conductance, iberiotoxin-sensitive K+ channel (Petkov & Nelson, 2005). Differences in the electrophysiological procedures used by the two groups may have contributed to this apparent controversy. Activation of iberiotoxin-sensitive K+ channels can relax the urinary bladder (Malysz et al., 2004). Several studies have assessed the functional relevance of ion channel modulation by β-adrenoceptor stimulation. Studies using KCl-precontracted bladder strips from guinea-pigs (Kobayashi et al., 2000) or rats (Frazier et al., 2005a; Uchida et al., 2005) have consistently found that K+ channel blockers such as iberiotoxin or charybdotoxin inhibit isoprenaline-induced bladder relaxation. Interestingly, the latter two studies also report that relaxation against passive tension is not sensitive to those toxins.
Prostaglandins may play a role in bladder contraction by several agents such as protease-activated receptors or bradykinin (Nakahara et al., 2003; 2004; Chopra et al., 2005). Therefore, it is surprising that prostaglandins were also postulated to play a permissive role for β-adrenoceptor-mediated relaxation of the urinary bladder (Bolle et al., 1999).
The key function of β-adrenoceptors in the bladder is smooth muscle relaxation and an increase in bladder compliance during the filling phase of the micturition cycle. The interpretation of in vitro bladder relaxation experiments has to take into account that the results are sensitive to the experimental conditions. Thus, it has been found that the β-adrenoceptor agonist isoprenaline was approximately six times more potent when tested against passive tension than when tested against KCl-induced bladder tone in rats (Frazier et al., 2005a; Uchida et al., 2005). This is consistent with indirect comparisons in the published literature, where a pEC50 for isoprenaline of 8.3 (Yamazaki et al., 1998) vs 7.2 (Longhurst & Levendusky, 1999) and of 9.1 (Yamazaki et al., 1998) vs 7.3 (Oshita et al., 1997) were reported in rats and rabbits, respectively, for passive tension vs pre-contraction. In a comparison between KCl-induced and carbachol-induced tension in rat isolated detrusor, isoprenaline was significantly less potent and effective against the latter (Longhurst & Levendusky, 1999). Moreover, the choice of passive tension vs pre-contraction for relaxation experiments may also affect the underlying signal transduction of the β-adrenoceptor response (Frazier et al., 2005a; Uchida et al., 2005). A second methodological consideration relates to the use of muscarinic receptor agonists to induce bladder pre-contraction in combination with β-adrenoceptor agonists such as BRL 37,344 to induce relaxation. This drug has affinity for muscarinic acetylcholine receptors in the same concentration range where it acts as a β-adrenoceptor agonist (Kubota et al., 2002); hence, data using this combination may at least partly reflect direct muscarinic receptor antagonism rather than β-adrenoceptor agonism (see below).
A relaxation of bladder smooth muscle by β-adrenoceptor agonists has been demonstrated against passive tension (Igawa et al., 2001; Takeda et al., 2002a), endothelin receptor-mediated (Takeda et al., 2003), muscarinic receptor-mediated (Seguchi et al., 1998; Nomiya & Yamaguchi, 2003) and KCl-induced pre-contraction (Nishimoto et al., 1995; Yamanishi et al., 2003a) or against field stimulation-induced tone (Nishimoto et al., 1995; Hudman et al., 2001). Moreover, relaxation responses have been demonstrated in the detrusor of various species, including rats (Kolta et al., 1984; Nishimoto et al., 1995; Oshita et al., 1997; Seguchi et al., 1998; Yamazaki et al., 1998; Fujimura et al., 1999; Longhurst & Levendusky, 1999; Lluel et al., 2000; Morita et al., 2000; Woods et al., 2001; Matsubara et al., 2002; Inci et al., 2003; Malysz et al., 2004; Uchida et al., 2005; Frazier et al., 2005a), mouse (Matsui et al., 2003), rabbits (Oshita et al., 1997; Morita et al., 1998; 2000; Yamazaki et al., 1998; Bing et al., 2003), guinea-pigs (Li et al., 1992; Gopalakrishnan et al., 1999; Kobayashi et al., 2000; Malysz et al., 2004), ferrets (Takeda et al., 2000a), cats (Nergardh et al., 1977), dogs (Yamazaki et al., 1998), pigs (Yamanishi et al., 2002b, 2002c; 2003a), monkeys (Takeda et al., 2002a) and humans (Nergardh et al., 1977; Fujimura et al., 1999; Igawa et al., 1999; 2001; Takeda et al., 1999; Morita et al., 2000; Nomiya & Yamaguchi, 2003). In contrast, β-adrenoceptor stimulation did not consistently relax the basal tone of the human bladder neck (Caine et al., 1975).
Some studies have performed direct inter-species comparisons regarding the ability of β-adrenoceptor agonists to induce bladder relaxation. Such comparisons of, for example, rat vs dog (Takeda et al., 2003), rat vs rabbit (Oshita et al., 1997) or rat vs rabbit vs dog (Yamazaki et al., 1998) have consistently reported that the maximum effects of an agonist without subtype selectivity, such as isoprenaline, were similar in various species. However, within the same study, the rank order of isoprenaline potency consistently was rabbit>rat>dog, suggesting that rabbits may have the largest and dogs the smallest receptor reserve for this response, respectively. Similar inter-species comparisons with subtype-selective β-adrenoceptor agonists are more difficult to interpret, since the subtype being involved may differ between species.
Functional studies into the β-adrenoceptor subtypes mediating bladder relaxation have been hampered by several problems. Firstly, some drugs proposed to be β3-adrenoceptor-selective agonists may have effects independent of β-adrenoceptors. For example, it was reported that both BRL 37,344 and SR 58,611 can cause vasodilatation, which is insensitive to β-adrenoceptor antagonists (Brahmadevara et al., 2003). Moreover, BRL 37,344 was reported to be a direct muscarinic receptor antagonist (Kubota et al., 2002) and α1-adrenoceptor antagonist (Leblais et al., 2005). Secondly, no truly β3-adrenoceptor-selective antagonist has been described. Thus, SR 59,230, the most frequently used drug to antagonize β3-adrenoceptors, does not discriminate human β-adrenoceptor subtypes (Hoffmann et al., 2004) and, similarly to the chemically related bupranolol, may also be an α1-adrenoceptor antagonist (Leblais et al., 2005). When binding to β3-adrenoceptors, SR 59,230 may exhibit agonist rather than antagonist properties in some tissues (Horinouchi & Koike, 2001). Such limitations should be taken into account when interpreting the functional data presented below.
Studies in various species have used agonist and antagonist potency to identify the functional involvement of β-adrenoceptor subtypes in bladder relaxation. Since absolute agonist potency may differ between species even for nonsubtype-selective agonists (see above), the former approach has used either rank orders of potency of various agonists or the potency of highly subtype-selective agonists to classify the receptor subtype being involved. Most studies have been reported from rats. Based upon a high potency of β3-selective agonists such as CL 316,243 (Woods et al., 2001) and FK175 (Fujimura et al., 1999), it has been proposed that rat bladder relaxation predominantly occurs via this subtype. However, studies assessing the rank order of potency of multiple subtype-selective agonists have proposed a mixed involvement of β2- and β3-adrenoceptors in rat bladder relaxation in most cases. These were based upon rank orders such as isoprenaline=procaterol (β2-selective)>CL 316,243>dobutamine (β1-selective) (Takeda et al., 2003), CL 316,243isoprenalineprocaterol (Takeda et al., 2000b), isoprenalineCL 316,243procaterol>dobutamine (Yamazaki et al., 1998), BRL 37,344isoprenaline (Oshita et al., 1997), isoprenaline=GS-332 (β3-selective)clenbuterol (β2-selective) (Morita et al., 2000) or isoprenaline>FR 165101 (β3-selective)clenbuterol≫dobutamine (Uchida et al., 2005). One study, based upon a rank order of agonist potency of isoprenaline>BRL 37,344T-0509 (β1-selective)>terbutaline (β2-selective)SR 58,611 (β3-selective), has even proposed a mixed involvement of β1-, β2- and β3-adrenoceptors in rat bladder relaxation (Longhurst & Levendusky, 1999). Antagonist studies have reported that ICI 118,551 inhibits the effects of clenbuterol against low-, but not high-frequency field stimulation (Hudman et al., 2000). Relaxant effects of the β3-agonist FK175 were moderately inhibited by the nonselective bupranolol, but not by even high concentrations of the β1-selective CGP 20,712 or the β2-selective ICI 118,551 (Fujimura et al., 1999). Similarly, relaxation induced by BRL 37,344 was not inhibited by low propranolol concentrations, but by CGP 12,177 or SR 59,230 when added atop of propranolol; in the same study, relaxation by CGP 12,177 was not affected even by high propranolol concentrations (Longhurst & Levendusky, 1999). These data indicate that β2- and β3-selective agonists may indeed cause rat bladder relaxation via their cognate receptor subtypes. With regard to nonsubtype-selective agonists such as isoprenaline or noradrenaline, several studies report relatively poor antagonism by propranolol, metoprolol, butoxamine or ICI 118,551 (Oshita et al., 1997; Seguchi et al., 1998; Longhurst & Levendusky, 1999). However, SR 59,230, which should inhibit the cloned β3-adrenoceptor, also caused only poor isoprenaline antagonism (Longhurst & Levendusky, 1999). Taken together, these data argue against a strong involvement of β1- and β2-adrenoceptors, but also fail to provide clear evidence for a β3-adrenoceptor. Interestingly, the isoprenaline-induced cAMP response in rat bladder was fully sensitive to propranolol (Ma et al., 2002), which is in line with the proposal that β-adrenoceptor-mediated bladder relaxation occurs largely cAMP-independent (Frazier et al., 2005a; Uchida et al., 2005).
In vitro relaxation studies in rabbit bladder have reported agonist rank orders of potency of isoprenalineadrenaline>noradrenalineBRL 37,344 (Oshita et al., 1997), procaterol>isoprenaline>adrenalineCGP 12,177>noradrenalinedobutamine>CL 316,243 (Yamazaki et al., 1998) or clenbuterol≫GS-332 (Morita et al., 2000). Propranolol, bupranolol and ICI 118,551 antagonized the isoprenaline-induced relaxation with high potency, whereas CGP 20,712, in concentrations up to 100 nM, had no effect (Oshita et al., 1997; Yamazaki et al., 1998). Taken together, these data demonstrate that relaxation of the rabbit detrusor is predominantly mediated by a β2-adrenoceptor.
In the porcine detrusor, there was a rank order of potency of salbutamol (β2-agonist)>noradrenaline>BRL 37,344>CGP 12,177 (the latter two being partial agonists only); while the BRL 37,344 response was antagonized by SR 59,230, the corresponding Schild slope was significantly smaller than unity (Yamanishi et al., 2002a). The same investigators also reported a low potency of BRL 37,344 sensitive to SR 59,233 in the porcine bladder base (Yamanishi et al., 2002c). More recently, these authors also reported porcine bladder base relaxation by isoprenaline and salbutamol (Yamanishi et al., 2003a). CGP 20,712 did not inhibit the isoprenaline responses, whereas propranolol and ICI 118,551 caused inhibition, but with a Schild slope of less than unity; in contrast, ICI 118,551 inhibited the salbutamol responses with high potency and a Schild slope close to unity. Another group of investigators found an order of potency of isoprenaline=adrenalineprocaterolBRL 37,344>CGP 12,177salbutamol>CL 316,243noradrenaline; in this regard, BRL 37,344, CL 316,243 and, surprisingly, noradrenaline were reported to be partial agonists and CGP 12,177 was found to be a weak partial agonist (Badawi et al., 2005). Taken together, these findings suggest that both β2-adrenoceptors and an additional subtype, possibly β3-adrenoceptors, mediate porcine bladder relaxation.
Data from several other animal species are too limited or controversial to allow definitive conclusions. In guinea-pigs, a predominant role of β1-adrenoceptors was proposed based upon relaxation by dobutamine, but not by BRL 37,344, salbutamol or clenbuterol, and antagonism of the isoprenaline, noradrenaline and adrenaline responses by atenolol (Yamamoto et al., 1998). Another study also proposed an involvement of β1-adrenoceptors based upon partial antagonism of the isoprenaline response by metoprolol, but reported an even greater role of β2-adrenoceptors based upon partial agonism by salbutamol and terbutaline and antagonism of the isoprenaline response by ICI 118,551 (Li et al., 1992). A more recent study based upon whole bladder contraction reported relaxation by noradrenaline and BRL 37,344, but not by formoterol (β2-selective) (Gillespie, 2004). Limited data from one study in cats have suggested a predominant involvement of β1-adrenoceptors (Nergardh et al., 1977). One study in ferrets has proposed a primary involvement of β3-adrenoceptors based upon an agonist rank order of potency of BRL 37,344>CGP 12,177isoprenalineCL 316,243>dobutamineprocaterol and upon antagonism of the isoprenaline response by SR 58,894, but not by CGP 20,712 or ICI 118,551 (Takeda et al., 2000a). One study in dogs reported an agonist rank order of potency of CL 316,243>isoprenalineCGP 12,177>noradrenalinedobutamineprocateroladrenaline, and that the isoprenaline-induced relaxation was inhibited with high potency by bupranolol, but not by CGP 20,712 or ICI 118,551 (Yamazaki et al., 1998); the same group later confirmed the rank order of CL 316,243>dobutamine∼procaterol (Takeda et al., 2003), suggesting predominantly an involvement of β3-adrenoceptors. A study in Cynomolgus monkeys found an agonist rank order of potency of isoprenaline>noradrenalineCGP 12,177>BRL 37,344adrenaline>dobutaminesalbutamolprocaterol, with the β1-selective xamoterol being a very weak partial agonist; the effects of isoprenaline were inhibited by bupranolol, but not by CGP 20,712 or ICI 118,551 (Takeda et al., 2002a), suggesting a predominant involvement of a β3-adrenoceptor.
Early reports on human bladder relaxation already proposed that this does not occur via a β1- or β2-adrenoceptor (Nergardh et al., 1977). Several more recent studies suggest that it indeed occurs via a β3-adrenoceptor. Igawa et al. (1998) originally reported relaxation of the human bladder (inhibited by bupranolol), whereas dobutamine, procaterol and CGP 12,177 caused much smaller if any relaxation. Thereafter, they reported an agonist order of potency of BRL 37,344isoprenalinenoradrenalineadrenalineCGP 12,177CL 316,243; in that study, isoprenaline responses were inhibited by SR 58,894, but only poorly by ICI 118,551 and not at all by CGP 20,712 (Figure 2) (Igawa et al., 1999). Another study from the same group reported an order of BRL 37,344isoprenaline>CGP 12,177CL 316,243, with all but isoprenaline being partial agonists (Igawa et al., 2001). Another study reported a rank order of potency of BRL 37,344>CGP 12,177>isoprenaline, with the former two being partial agonists only, and the β3-adrenoceptor agonist ZD 7114 being a very poor partial agonist; the isoprenaline responses were inhibited by SR 59,230, but not by butoxamine and atenolol (Takeda et al., 1999). In another study, isoprenaline and the β3-adrenoceptor selective agonist L 755,507, but not dobutamine or clenbuterol, relaxed carbachol-contracted human bladder strips (Nomiya & Yamaguchi, 2003). A very recent study reported a rank order of potency of isoprenaline>procaterol=CL 316,243=salbutamol, with the latter three compounds being considerably less effective than isoprenaline (Badawi et al., 2005). Finally, GS 332 was found to be more potent in the human bladder than clenbuterol in another study (Morita et al., 2000). In agreement with the predominant expression of β3-adrenoceptor mRNA in the human bladder (see above), these data demonstrate that this subtype is also most important for bladder relaxation in vitro. With the possible exception of ferrets and monkeys, the role of this subtype in other animal species is less prominent.
Figure 2. Inhibition of isoprenaline-induced relaxation of human bladder detrusor by the β1-antagonist CGP 20,712, the β2-antagonist ICI 118,551 and the nonselective antagonist SR 58,894. Taken with permission from Igawa et al. (1999).
Download figure to PowerPoint
In vivo function
Functional in vivo effects on bladder function can be assessed in several ways. Noninvasive studies frequently look at micturition frequency, which is a key symptom of OAB (see Abrams et al., 2002). Invasive studies are based upon the insertion of a catheter coupled to a pressure transducer into the bladder and subsequent filling of the bladder endogenously or by installation of fluid. This allows various types of measurements, including the frequency of bladder contractions, maximum detrusor pressure, filling volume at first contraction or bladder compliance, all of which are typically also assessed in urodynamic studies in humans (see Abrams et al., 2002). Moreover, it should be considered that the effects of systemically administered drugs on bladder function are not necessarily mediated by drug targets located in the bladder (see the above section on bladder α1-adrenoceptors). Finally, the use of anaesthetized vs conscious animals may differentially affect the endogenous sympathetic tone.
Studies in rats (Lecci et al., 1998; Takeda et al., 2000b; 2003; Kaidoh et al., 2002; Tucci et al., 2002), ferrets (Takeda et al., 2000a) and monkeys (Takeda et al., 2002a) demonstrate that β-adrenoceptor agonists such as isoprenaline can reduce intra-vesical pressure, indicating that this is a consistent feature in biology. On the other hand, propranolol had little, if any, effects on bladder function on its own (Durant et al., 1988), indicating that either there is little endogenous β-adrenergic tone under the chosen experimental conditions and/or that the receptor mediating the bladder effects is propranolol-insensitive, that is, distinct from β1- and β2-adrenoceptors. Consistent with the latter possibility, neither i.v. terbutaline nor oral propranolol affected intra-vesical pressure in healthy women (Thind et al., 1993b), but both drugs caused small increases in bladder volume in another study in healthy women (Norlen et al., 1978).
In a model of distension-induced bladder activity under isovolumetric conditions in urethane-anaesthetized rats, the reduction of intra-vesical pressure by i.v. isoprenaline decreased with increasing intra-vesical volumes (Lecci et al., 1998), possibly reflecting a physiological increase in endogenous β-adrenergic tone with increased bladder filling. In pentobarbital-anaesthetized rats, isoprenaline-induced reduction of bladder tone was attenuated by the cyclooxygenase inhibitor indomethacin, the Ca2+ flux blocker ruthenium red and the neurokinin A receptor antagonist MEN-10376, whereas the phosphodiesterase inhibitor papaverine did not affect them (Tucci et al., 2002). These data were interpreted to suggest that β-adrenoceptor-mediated bladder relaxations in vivo involve prostaglandins, neurokinin A and capsaicin-sensitive nerve fibres. Moreover, the lack of effect of papaverine is consistent with a cAMP-independent relaxation that has been demonstrated in vitro (Frazier et al., 2005a; Uchida et al., 2005).
In urethane-anaesthetized rats, isoprenaline, the β2-agonist procaterol and the β3-agonist CL 316,243 dose-dependently lowered intra-vesical pressure; CL 316,243 also increased bladder capacity and micturition intervals and reduced micturition pressure, whereas procaterol only increased bladder capacity and residual volume (Takeda et al., 2000b). Neither drug altered the total micturition volume, but their combination had somewhat greater effects on micturition interval, bladder capacity and residual volume than either drug alone. In conscious, unrestrained rats, i.v. procaterol did not affect voiding pressure relative to vehicle, and had little effect on bladder capacity, whereas CL 316,243 had no effect on bladder capacity but reduced voiding pressure (Kaidoh et al., 2002). Both procaterol and CL 316,243 reduced intra-vesical pressure in another study in urethane-anaesthetized rats; the procaterol effect was inhibited by ICI 118,551 and the CL 316,243 effect by the β3-adrenoceptor antagonist L 748,337, whereas neither antagonist affected the response to the other agonist (Takeda et al., 2003). In pentobarbital-anaesthetized ferrets, isoprenaline and CL 316,243 dose-dependently reduced bladder pressure, whereas dobutamine and procaterol had little effect (Takeda et al., 2000a). Taken together, these data suggest that both β2- and β3-adrenoceptors contribute to bladder relaxation in rats in vivo, whereas only β3-adrenoceptors are involved in ferrets. Both conclusions are consistent with the available in vitro data (see above).
In this context, it is interesting to note that the β3-adrenoceptor agonists (in contrast to nonsubtype-selective or β2-selective agonists) consistently had only small, if any, cardiovascular effects in the above studies (Takeda et al., 2000b; 2003; Kaidoh et al., 2002), indicating a possible safety advantage. On the other hand, two β3-adrenoceptor agonists, ZD 7114 and ZD 2079, were reported to induce cystitis and renal tubular necrosis upon chronic dosing in male and female rats (Waghe et al., 1999), but it remains unclear whether this is a specific effect of these two compounds or related to their mechanism of action; moreover, it is unclear whether this is limited to rats or can be extrapolated to other species.
Regulation of receptor expression and function
A possible gender effect on β-adrenoceptor-mediated regulation of bladder function has been studied in rabbits and rats. Radioligand-binding studies with [3H]dihydroalprenolol as the radioligand have found a significantly greater receptor number in young female as compared to young male rabbits in the bladder base, but no such differences were seen in the bladder base of older rabbits or in the bladder dome of either age group (Latifpour et al., 1990). Another study with the same radioligand confirmed a greater receptor number in the trigonal part (but not the detrusor) of young adult female as compared to male rabbits (Morita et al., 1998). Within the same study, this was confirmed functionally by a greater isoprenaline-induced relaxation in female than in male trigonal, but not detrusor muscle. Similarly, a study on ovariectomized Wistar rats reported a reduced potency for BRL 37,344 in relaxing bladder strips as compared to control- or oestrogen-treated ovariectomized rats; similar differences for isoprenaline did not reach statistical significance (Matsubara et al., 2002). On the other hand, relaxant responses for the weak partial agonist CGP 12,177 were reduced in female relative to male Wistar rats, but no such alterations was seen for the agonists BRL 37,344, isoprenaline and noradrenaline (Frazier et al., 2005b).
A regulation of β-adrenoceptor responsiveness with age has been demonstrated in several species. A binding study with [3H]dihydroalprenolol reported that the number of β-adrenoceptors increased in rabbit bladder dome and base with age (Latifpour et al., 1990). In contrast, a study using [125I]iodopindolol in 1- vs 3- vs 22-month-old male Fischer 344 rats reported an age-related decrease in receptor density (Nishimoto et al., 1995). A similar decrease in [3H]dihydroalprenolol-binding sites was also reported for human bladder (Li et al., 2003). Consistent with these findings, an age-related reduction of isoprenaline-stimulated cAMP formation has been found in a comparison of bladders from 3-, 6- and 24-month-old male Fischer 344 rats (Derweesh et al., 2000). The latter was accompanied by an increase in the expression of α-subunits of Gs, Go and Gi proteins, with the latter two increasing more than Gs and hence shifting the overall balance towards inhibition rather than stimulation of adenylyl cyclase. In line with these biochemical findings, it was reported from a comparison of 1- vs 3- vs 22-month-old male Fischer 344 rats that bladder relaxation by noradrenaline or isoprenaline against KCl-induced tone was attenuated, involving a reduction in agonist potency and maximum effects; isoprenaline effects against field stimulation-induced tone were similarly reduced (Nishimoto et al., 1995). Within that study, relaxation responses to forskolin, but not those to dibutyryl-cAMP, were also reduced with age, indicating that an alteration prior to cAMP formation rather than in cAMP responsiveness is involved. While these biochemical and functional studies in male Fischer 344 rats are consistent with a reduced β-adrenoceptor function with age, not all studies have confirmed that. Thus, one study in 7- vs 17- vs 29-month-old male Fischer 344 rats detected no alteration in the potency or efficacy of isoprenaline to relax isolated bladder strips (Kolta et al., 1984). Similarly, a study in 3- vs 23-month-old male Wistar rats reported similar concentration-dependent bladder strip relaxation by isoprenaline, noradrenaline, BRL 37,344 and CGP 12,177 in both age groups (Frazier et al., 2005b). A study in 10- vs 30-month-old female Wistar/Rij rats also found similar isoprenaline-induced bladder strip relaxation in both age groups (Lluel et al., 2000). Finally, a study comparing newborn, 1- and 4-month-old Sprague–Dawley rats also did not detect alterations of β-adrenoceptor-mediated bladder relaxation (Tugay et al., 2003), a finding probably more related to development than to ageing. In this context, it should be noted that studies on age-related differences of muscarinic receptor responsiveness in the bladder have found major strain differences, with Wistar rats most closely resembling the situation in humans (Schneider et al., 2005). A single and limited study in humans has reported that bladder relaxation responses to isoprenaline and BRL 37,344 and also receptor-independently to forskolin and dibutyryl-cAMP are lower in a group of subjects in their mid-60s than in those in their late 20s (Li et al., 2003), indicating that the observed difference may at least partly relate to an overall reduced ability to relax rather than a specific β-adrenoceptor desensitization.
Several studies have investigated the effects of β-adrenoceptor agonists in animal models of bladder dysfunction. Some of them compared such effects with those in healthy animals to test the possible alterations by disease, whereas other studies looked at the pathological condition only to determine whether β-adrenoceptor agonists might be effective therapeutics in such settings. Spontaneously hypertensive rats are a genetic animal model, which exhibits several features of OAB, including increased urinary frequency and reduced bladder capacity. A comparison of male spontaneously hypertensive with normotensive Wistar Kyoto rats detected a reduced bladder relaxation in response to noradrenaline and isoprenaline, but not to the partial agonist BRL 37,344 (Frazier et al., 2005b). OAB-like symptoms can also occur secondarily to bladder outlet obstruction. CL 316,243 dose-dependently inhibited spontaneous bladder contraction in obstructed rats, but a direct comparison with healthy rats (who have much less if any such spontaneous contractions) was not reported (Woods et al., 2001). When bladder hyper-reflexia was induced by intra-vesical installation of acetic acid, CL 316,243 also concentration-dependently reduced bladder contractions; comparison to the obstruction data from the same study indicates that the hyper-reflexic model may be more sensitive to this agonist (Woods et al., 2001). Bladder hyperactivity can also be induced by intra-vesical installation of prostaglandin E2. In this model, CL 316,243 dose-dependently increased micturition interval and micturition volume and decreased basal pressure, whereas threshold pressure and micturition pressure were not affected; on the other hand, procaterol reduced threshold pressure, but did not significantly affect the other parameters (Takeda et al., 2002b). Bladder hyper-reflexia can also be induced by cerebral infarction, which impairs some of the central nervous control of the bladder. While CL 316,243 had little effect on bladder capacity in control animals, it dose-dependently restored the reduced bladder capacity in cerebral infarction rats, and a similar restoration of bladder capacity was seen with procaterol; neither drug normalized voiding pressures within the tested dose range (Kaidoh et al., 2002).
Few studies have looked into alterations of β-adrenoceptor responsiveness in the bladder of patients. A limiting factor of all these studies is the problem of obtaining tissue from matched healthy controls. Apparently tumour-free tissue from cancer patients is most frequently used as control; while this appears the only feasible option, it remains unclear how representative such tissue is for healthy subjects. One study compared the relaxation of isolated bladder strips without pre-contraction by the β-adrenoceptor agonists isoprenaline, BRL 37,344, CL 316,243 and CGP 12,177 in patients with low bladder compliance, hyperreflexic bladders and controls; agonist potency was similar in all three groups for each agonist, and maximum effects were also similar across groups for the agonists, except for an increased effect of CGP 12,177 in low-compliance bladders (Igawa et al., 2001). Another study has reported on the relaxation of field stimulation-contracted bladder strips from patients with urodynamically confirmed urge incontinence with those from continent patients without a history of incontinence; clenbuterol caused only weak relaxation in control subjects at both 1 and 40 Hz stimulation, but significantly greater relaxation in strips from incontinent patients (Hudman et al., 2001). A comparison of bladder from males with and without bladder outlet obstruction detected statistically significant differences in the abundance of β1-, β2- or β3-adrenoceptor mRNA between groups; similarly, potency and maximum effects of isoprenaline and the β3-selective L 755,507 were similar in both groups (Nomiya & Yamaguchi, 2003). Taken together, the limited available animal and human data do not provide conclusive evidence for an alteration of β-adrenoceptor function in states of bladder dysfunction.