- To elucidate the impact of the mucosa on detrusor muscle function by investigating force of contraction under various stimulatory conditions and during subsequent relaxation using catecholamines.
electric field stimulation
negative logarithm of the molar concentration producing 50% of the maximum relaxing effect
The urothelium of the urinary bladder not only serves as a barrier protecting the underlying smooth muscle against irritating urine constituents, but also provides significant inhibitory effects on detrusor contractions in response to various stimulations. In the literature, there is good evidence from the detrusor muscle of several species that contractions elicited by tachykinins or muscarinic agonists are strongly enhanced after removal of the urothelium [1-4]. Responses of feline and human detrusor to electric field stimulation (EFS) are enhanced in the absence of urothelium [5, 6]; however, contractions elicited by KCl depolarization appear not to be affected by the presence or absence of urothelium . Although it could be clearly shown that the relaxing effect of the urothelium is mediated by a diffusible factor, the chemical nature of this ‘urothelium-derived relaxing factor’  has not been resolved. Nevertheless, involvement of nitric oxide, cyclooxygenase products, purinergic P2Y receptors, TEA-sensitive K+ channels and small conductance Ca2+-activated K+ channels have been excluded [2, 5]. Urinary bladder smooth muscle relaxes in response to catecholamines via activation of β-adrenoceptors (ARs) [1, 7], and expression of all β-AR subtypes at the mRNA-level has been found in rat and human detrusor [8-10]; however, the specific β-AR subtype involved in detrusor relaxation seems to be quite variable between different species [11-13]. For instance, β2-ARs mediate relaxation of detrusor smooth muscle in rabbits , β2-ARs and β3-ARs in pigs , or even all three subtypes in rat detrusor , whereas in humans only β3-ARs are involved [14, 15, 17, 18].
In the presence of urothelium, relaxation mediated by β-ARs is impaired . Isoprenaline, for instance, is less effective in relaxing carbachol-precontracted intact detrusor strips than mucosa-denuded muscles. This finding would suggest that the urothelium plays a dual role: besides providing a urothelium-derived relaxing factor, it could also be associated with a ‘contracting factor’ antagonizing catecholamine-mediated relaxation [19, 20].
In the present study, we aimed to investigate which β-AR subtype is involved in blunting relaxation in intact precontracted human detrusor strips. According to our previous studies , catecholamine-induced relaxation is mediated by β3-ARs, however, β2-ARs are responsible for mucosa-induced impairment of relaxation.
All patients donating tissue gave fully informed consent in accordance with the Declaration of Helsinki and the regulation of the local ethics committee (approval No EK 194092004). Specimens of human detrusor muscle were obtained from the bladder dome of patients undergoing primary radical cystectomy for the treatment of carcinoma invading bladder muscle. Patients who had received chemotherapy or radiotherapy were excluded. The mean (sd) age of the 62 patients was 69 (2) years.
Care was taken to ensure that the muscle strips were obtained from tissue free of macroscopic tumour or inflammation. The tissue was transported in buffer solution to the laboratory within 15–30 min after excision. After careful removal of the serosa, four to eight longitudinal muscle strips (10–15 mm in length and 4–6 mm in width) were cut from the tissue. The mucosa was removed from half of the preparations, but remained intact in the other half. In some bladder strips the mucosa was removed and fixed close to the denuded strip within the organ bath.
All preparations were mounted in 5-mL organ baths containing carbogen-gassed Tyrode's solution at 37 °C. Isometric tension was measured with an isometric force transducer (GM2; Föhr Medical Instruments, Seeheim/Ober-Beerbach, Germany), amplified and recorded with Chart 4.0TM (AD Instruments, Sydney, Australia). Resting load was 10 mN. All experiments were carried out in the presence of the α-AR antagonists phentolamine (3 μM) and prazosin (1 μM) to exclude any α-AR-mediated processes. During an equilibration period of 60 min, the bath solution was changed three times. The detrusor strips were exposed to 40 mM KCl for 10 min, followed by washout. Muscle strips were precontracted with 1 μM carbachol, 40 mM KCl or EFS (5-s long stimuli at 30 Hz every 2 min) and responses became stable within 45 min. Detrusor contraction was measured as force and expressed as mN/mg wet weight of each muscle strip. Relaxation was then induced by a stepwise increase of the concentration of (−)-noradrenaline or (−)-isoprenaline, until a maximum was reached. All relaxations were expressed in percent of maximum relaxation achieved with 10 μM forskolin at the end of each experiment. Catecholamine-induced relaxation was either measured without any β-AR blocker in the bath (time-matched controls) or in the presence of either one of the subtype-selective β-AR antagonists, i.e. CGP 20712A (300 nM) for β1-ARs, ICI 118,551 (50 nM) for β2-ARs, and L-748,337 (100 nM–1 μM) for β3-ARs, added 45 min before catecholamine-induced relaxation.
The modified Ringer's solution contained: 149 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 0.1 mM NaH2HPO4 and 5.6 mM glucose. The Tyrode's solution contained: 127 mM NaCl, 5.4 mM KCl, 1.05 mM MgCl2, 1.8 mM CaCl2; 0.4 mM NaH2HPO4, 22 mM NaCO3, 0.04 mM EDTA, 0.2 mM ascorbic acid and 5.6 mM glucose, pH 7.4, when equilibrated with 95% O2 and 5% CO2. Drugs and chemicals were obtained from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK). All drugs were dissolved in Milli-Q water (Millipore, Billerica, MA, USA), with the exception of (−)-isoprenaline and (−)-noradrenaline, which were dissolved in water containing 200 mM ascorbic acid and 0.04 mM EDTA. L-748,337 and forskolin were dissolved in DMSO and stock solutions were further diluted with Milli-Q water.
Concentration–response curves for catecholamines were analysed by nonlinear regression of each individual experiment using GraphPad 4.0 (GraphPad Prism Software Inc., San Diego, CA, USA). The negative logarithm of the molar concentration producing 50% of the maximum relaxing effect (−logEC50) was calculated. Results are presented as mean −logEC50 (sem) values from n muscle strips. Statistical differences were evaluated using Student's t-test (paired or unpaired) and anova. A P value of <0.05 was considered to indicate statistical significance. Schild plot analysis was used to estimate apparent affinity values for L-748,337 .
Activation of urothelium-denuded and intact human detrusor strips, with 40 mM KCl, 1 μM carbachol or with EFS, yielded transient increases in force of contraction (Fig. 1) that turned into a constant steady-state force development within 30 min. Peak tension resulting from each mode of stimulation was significantly higher in urothelium-denuded than in intact preparations, but this difference was no longer significant at steady-state (Fig. 2). Interestingly, when detached urothelium was placed into the organ bath close to the denuded strip, contraction amplitudes in response to KCl or carbachol resembled those of intact strips (Fig. 2). These findings are consistent with previous reports of the existence of a urothelium-derived factor inhibiting contraction [2, 5]. Precontracted detrusor strips were then relaxed with cumulatively increasing concentrations of (−)-isoprenaline or (−)-noradrenaline (Fig. 3). Maximum relaxation obtained with either catecholamine was smaller than with forskolin. (−)-isoprenaline was equally potent and slightly more efficacious in denuded than in intact strips, although the difference was nonsignificant (Table 1). By contrast, (−)-noradrenaline was more potent and efficacious in denuded than in intact strips, irrespective of the precontracting agent used (Table 1). Denuded strips tested with the detached urothelium fixed closed to them exhibited intermediate responses to (−)-noradrenaline. After EFS, the presence or absence of urothelium did not significantly affect (−)-isoprenaline- or (−)-noradrenaline-induced relaxation because of the large scatter between values from individual strips; nevertheless, there was a trend for more relaxation in denuded than in intact strips (Table 1). Taken together, these results suggest that the intact urothelium may counteract relaxation induced by (−)-noradrenaline but not by (−)- isoprenaline.
|Precontraction, KCl 40 mM Mean ± sem||Precontraction, carbachol 1 μM Mean ± sem||EFS Mean ± sem|
|β-AR agonist||β-AR blocker||Urothelium||−logEC50, M||Emax, %||n||−logEC50, M||Emax, %||n||−logEC50, M||Emax, %||n|
|Isoprenaline||None||Intact||6.73 ± 0.25||50 ± 5||7||6.73 ± 0.12||51 ± 5||7||6.27 ± 0.34||44 ± 8||5|
|None||Denuded||6.74 ± 0.17||64 ± 5||11||6.67 ± 0.07||64 ± 2||9||5.95 ± 0.28||64 ± 6||5|
|Noradrenaline||None||Detached||6.19 ± 0.01**||6.19 ± 0.01**||9||6.19 ± 0.01**||68 ± 7||6||n.d.||n.d.|
|None||Intact||6.20 ± 0.07**||6.20 ± 0.07**||8||6.20 ± 0.07**||55 ± 3***||12||6.07 ± 0.96||30 ± 10||6|
|None||Denuded||6.57 ± 0.08||84 ± 3||9||6.41 ± 0.09||70 ± 3||24||6.48 ± 0.31||55 ± 8||7|
|CGP20712A; 300 nM||Intact||5.80 ± 0.39||69 ± 1*||4||6.01 ± 0.12||60 ± 4*||4|
|Denuded||6.33 ± 0.25||80 ± 4||3||6.46 ± 0.39||80 ± 5||4|
|ICI118,551; 50 nM||Intact||6.52 ± 0.03||73 ± 7||3||6.39 ± 0.16||72 ± 1||4|
|Denuded||6.80 ± 0.16||88 ± 3||3||6.60 ± 0.23||79 ± 5||4|
|L-748,337; 100 nM||Intact||5.81 ± 0.12||78 ± 2||7||5.15 ± 0.20||68 ± 2||8|
|Denuded||5.57 ± 0.18||66 ± 4||11||5.44 ± 0.12||73 ± 2||6|
|ICI 118.551, 50 nM and L-748,337, 100 nM||Intact||5.14 ± 0.10||58 ± 4**||6|
|Denuded||5.20 ± 0.11||77 ± 5||8|
Next, we examined whether activation of specific β-AR subtypes might contribute to the differences in (−)-noradrenaline responses between denuded and intact detrusor strips. For this purpose, concentration–response curves were repeated in the presence of subtype-selective β-AR antagonists. The β1-AR blocker CGP 20712A (300 nM) did not alter mean -logEC50 or maximum effect of (−)-noradrenaline in intact or denuded strips (Fig. 4A, Table 1). The β2-AR blocker ICI 118,557 (50 nM) enhanced the potency and efficacy of (−)-noradrenaline in intact but not in denuded detrusor strips, so that the difference between denuded and intact muscle was abolished (Fig. 4B, Table 1). This finding suggests that urothelial β2-ARs are involved in mediating the effect that opposes (−)-noradrenaline-induced detrusor relaxation. It should be noted that ICI 118,551 does not antagonize the effects of (−)-noradrenaline in denuded strips (Fig. 4, left and middle bottom panels), ruling out a participation of β2-ARs in (−)-noradrenaline-evoked relaxation. When stimulating KCl- and carbachol-precontracted, denuded and intact detrusor strips with the β2-AR selective agonist salbutamol, relaxation was observed only at very high concentrations, at which β3-ARs are also activated (Fig. 4C).
The selective β3-AR antagonist L-748,337 at 100 nM shifted significantly more to the right the concentration–response curve of noradrenaline in denuded than intact strips (Fig. 5, Table 1). The joint administration of 50 nM ICI 118,551 plus 100 nM L-748,337 increased the antagonism compared with L-748,337 alone (Fig. 5). Higher concentrations of L-748,337 (300 nM–1 μM) shifted the concentration–response curve for (−)-noradrenaline-induced relaxation in carbachol-precontracted detrusor strips systematically to higher concentrations without affecting maximum relaxation regardless of the presence or absence of urothelium (Fig. 5).
Schild plot analysis revealed that slopes were not significantly steeper than those with data from intact and denuded strips. The affinity estimate for L-748,337 was larger for β3-ARs of denuded than intact detrusor with mean (sem) apparent affinity values of 8.01 (0.05) and 7.61 (0.08) [Fig. 5].
The aim of the present study was to characterize the influence of the urothelium on human detrusor smooth muscle function. The major novel findings were: (i) the presence of an intact urothelium reduced the potency and efficacy of (−)-noradrenaline-induced relaxation; (ii) blockade of β1-ARs did not affect (−)-noradrenaline-induced relaxation of denuded and intact strips, whereas blockade of β2-ARs abolished the difference so that intact strips responded like denuded strips; and (iii) the β3-AR blocker L-748,337 antagonized the (−)-noradrenaline-evoked relaxation more in urothelium-denuded than in intact strips.
Using a large number of detrusor strips we have confirmed our previous data, showing that the presence of urothelium significantly attenuated contractile responses to high KCl and carbachol , although lower KCl concentrations (40 mM) were used in the present study, and also reduced responses to EFS. Assuming that the urothelium accounts for the differences in responses to contracting agents between intact and denuded strips, it could decrease drug responses by reducing access for the drug to smooth muscle , by causing drug degradation  or by releasing a urothelium-derived inhibitory factor . Detached mucosa placed close to the smooth muscle suppressed contractile responses almost as effectively as in intact urothelium strips. In addition, differences in force of contraction with and without urothelium faded away with time (45 min). These findings support the idea that the urothelium releases a diffusible factor upon muscarinic stimulation, either directly or by depolarization, and that this urothelium-derived inhibitory factor may not be stable over time. It has been proposed that the factor may modulate pressure changes during filling of the bladder . In addition, the effects caused by the urothelium-derived inhibitory factor are not influenced by any of the following factors: inhibition of nitric oxide synthase, cyclooxygenase products, adenosine, purinergic receptors or potassium channels [2, 5].
The situation was reversed when detrusor strips were relaxed by stimulation of β-ARs. Urothelium-denuded strips relaxed more completely than intact muscle strips and required lower concentrations of (−)-noradrenaline for relaxation, but there were no differences for (−)-isoprenaline. Others have reported that passively stretched human detrusor was relaxed more potently by isoprenaline in the absence than in the presence of urothelium . In other species, such as pig, relaxation responses attributable to isoprenaline appeared not to be affected by the urothelium , although the former study used different carbachol concentrations to induce stable and similar size precontractions in intact and denuded strips. Because of these conflicting results, the role of the urothelium in the relaxant effect of (−)-isoprenaline cannot be resolved at present.
All three β-AR subtypes are expressed at the mRNA level in detrusor tissue [12, 24] and also in urothelium [10, 19] from various species. Nomiya and Yamaguchi  reported that the mRNA of β3-AR would represent ∼94% of mRNA from all AR subtypes detected in the human bladder. Also, in functional studies, β3-ARs have been shown to be the predominant subtype for the relaxation responses in human urinary bladder with minor [12, 14, 15, 17, 24, 25] or no involvement of β1-ARs or β2-ARs .
As the presence of urothelium significantly inhibited relaxation attributable to (−)-noradrenaline, we investigated which β-AR subtype might be involved in the catecholamine-stimulated urothelium-mediated modulation of relaxation with the natural neurotransmitter (−)-noradrenaline rather than with (−)- isoprenaline. Selective β1-AR blockade with CGP 20712A did not influence (−)-noradrenaline-induced detrusor relaxation, irrespective of the presence or absence of mucosa. Interestingly, the β2-AR antagonist ICI 118,551 abolished the differences in potency and efficacy of (−)-noradrenaline between denuded and intact strips, by shifting the concentration–response curve for intact strips to that for denuded strips; therefore, activation of β2-AR in the urothelium appears to be responsible for the reduced efficacy and potency of (−)-noradrenaline in intact strips. In analogy to the urothelium-derived relaxing factor released by contractile stimuli such as carbachol and high KCl, impairment of relaxation could be mediated by a urothelium-derived contractile factor released by stimulation of β2-ARs.
By contrast to (−)-noradrenaline, (−)-isoprenaline and salbutamol did not facilitate detrusor relaxation in urothelium-denuded preparations. This is puzzling because the affinity of (−)-noradrenaline for recombinant β2-ARs is seventeenfold and fourfold lower than the affinity of (−)-isoprenaline and salbutamol, respectively, and its efficacy is only half that of these agonists . We would have expected at least a similar potency difference between intact and denuded detrusor with (−)-isoprenaline and salbutamol, as found with (−)-noradrenaline but there was no difference at all. It would appear, therefore, that the β2-ARs of the urothelium, when activated by (−)-noradrenaline, but not by (−)-isoprenaline or salbutamol, selectively blunt the (−)-noradrenaline-evoked detrusor relaxation mediated through β3-ARs.
Interestingly, blockade of β2-ARs with ICI 118,551 sensitized the intact detrusor to the relaxant effects of (−)-noradrenaline (Fig. 4). In addition ICI118.551-induced blockade of β2-ARs also appeared to increase the antagonism by 100 nM L-748,337 of the relaxant effects of (−)-noradrenaline at β3-ARs in intact strips (precontracted with 40 mM KCl), compared with antagonism in the absence of ICI 118551 (Fig. 5A,B). L-748,337 100 nM, and to a smaller extent the higher concentrations of 300 nM and 1 μM, antagonize less the detrusor relaxation caused by (−)-noradrenaline in intact than denuded strips. L-748,337 has also a moderate affinity for human β2-ARs with pKD of 6.69 . Thus, it is plausible that high concentrations of L-748,337 could block also urothelial β2-ARs, as did ICI 118,551, thereby enhancing the blockade of β3-ARs, as observed with ICI 118,551 (Fig. 4C). The relaxation elicited by (−)-isoprenaline and salbutamol is consistent with mediation through β3-ARs. At recombinant β3-ARs the affinity of (−)-isoprenaline and (−)-noradrenaline is similar and the affinity of salbutamol approximately thirtyfold lower, but the efficacy of the three agonists is similar . These properties are consistent with the lower relaxant potency of salbutamol, compared with (−)-isoprenaline and (−)-noradrenaline.
Since ICI 118,551 only enhances both the relaxation to (−)-noradrenaline and blockade by 100 nM L-748,337 in intact but not denuded strips, we assume that urothelial β2-ARs blunt the interaction of (−)-noradrenaline and L-748,337 with the detrusor β3-AR system. The mechanism of the control of detrusor β3-AR function by urothelial β2-ARs is still unknown. It may be speculated that urothelial β2-ARs release a factor from the urothelium that reduces the affinity of (−)-noradrenaline and L-748,337 for β3-ARs. The different β-AR subtypes involved in the relaxing effect of catecholamines in human detrusor could also be altered by pathology. β3-ARs might be overexpressed in obstructed or diseased human bladder ; however, this issue cannot be resolved because detrusor tissue from healthy human probands is not available.
Our findings could also have some clinical implications. It may be speculated, that highly selective β3-AR agonists, such as the recently approved mirabegron , may exhibit their good clinical efficacy in overactive bladder because they do not blunt relaxation via urothelial β2-AR stimulation as do catecholamines. Conversely, adding a selective β2-AR antagonist might even further improve β3-AR mediated relaxation.
In conclusion, the presence of urothelium in human detrusor has dual effects: the mucosa impairs force development upon stimulation with KCl, carbachol or EFS, but also blunts the relaxing effects of catecholamines. Whilst β3-ARs mediate human detrusor relaxation, the blunting effect of the urothelium on this relaxation involves activation of β2-ARs. These findings may explain the good clinical efficacy of selective β3-AR agonist as compared with non-selective β-AR agonists.
We thank Maria Feilmeier for her excellent technical help.