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Journal of Pharmacological Sciences 128 (2015) 65e70

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Journal of Pharmacological Sciences

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Full paper The role of muscarinic receptor subtypes on -induced contraction of normal human detrusor and overactive detrusor associated with benign prostatic hyperplasia

* Tomonori Yamanishi a, , Kanya Kaga a, Miki Fuse a, Chiharu Shibata a, Takao Kamai a, Tomoyuki Uchiyama b

a Department of Urology, Continence Center, Dokkyo Medical University, Tochgi, Japan b Department of Neurology, Continence Center, Dokkyo Medical University, Tochgi, Japan

article info abstract

Article history: The aim of this study was to compare the effect of antimuscarinic antagonists on carbachol-induced Received 7 April 2015 contraction of normal human bladder and detrusor overactivity associated with benign prostatic hy- Received in revised form perplasia (DO/BPH). Samples of human bladder muscle were obtained from patients undergoing total 6 May 2015 cystectomy for bladder cancer (normal bladder), and those undergoing retropubic prostatectomy for Accepted 19 May 2015 BPH. All of the patients with DO/BPH had detrusor overactivity according to urodynamic studies. Available online 27 May 2015 Detrusor muscle strips were mounted in 10-ml organ baths containing Krebs solution, and concentration eresponse curves for carbachol were obtained in the presence of antimuscarinic antagonists (4-DAMP, Keywords: Human bladder , , , , trospium, propiverine, , and imidafe- e Muscarinic receptor subtype nacin) or vehicle. All antagonists competitively antagonized concentration response curves to carbachol Detrusor overactivity with high affinities in normal bladder. The rank order of mean pA2 values was as follows: trospium Benign prostatic hyperplasia (10.1) > 4-DAMP (9.87), (9.3) > solifenacin (8.8) > tolterodine (8.6) > oxybutynin (8.3) > propiverine (7.7) > pirenzepine (7.4) > methoctramine (6.6). The effects of these antimuscarinic antagonists did not change when tested with DO/BPH bladder, suggesting that each antimuscarinic antagonist has a similar effect in this condition. Schild plots showed a slope corresponding to unity, except for propiverine with DO/BPH detrusor. In conclusion, M3-receptors mainly mediate contractions in human bladder strips with normal state and DO/BPH. © 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

1. Introduction imidafenacin and were also developed, and were re- ported to be effective for OAB (4,6e8). For pharmacological treatment of overactive bladder (OAB), The smooth muscle of the urinary bladder, like the majority of antimuscarinic agents have long been the drugs of choice. However, smooth muscle in many species, expresses a heterogeneous pop- these agents can have unpleasant side effects, such as dry mouth, ulation of muscarinic receptors. Predominance of the M2 musca- constipation, headache, blurred vision, and tachycardia, while the rinic receptor subtype, with a minor population of M3 receptors, lower urinary tract symptoms of some patients are refractory has been reported in the bladder smooth muscle of several species. (1e4). Worldwide, six antimuscarinic drugs are currently marketed The proportion of muscarinic M2 and M3 receptors has been re- for the treatment of OAB, which are oxybutynin, tolterodine, pro- ported to be 70e80% and 20e30%, respectively, in the bladder piverine, trospium, , and solifenacin (2,5). Recently, smooth muscle of humans, guinea pigs, and rabbits (9e11). How- ever, pharmacological characterization of the muscarinic receptor mediating detrusor muscle contraction in pigs and humans has suggested that only the M3 receptor is involved (9,11,12). This * Corresponding author. Department of Urology, Continence Center, Dokkyo suggests that the M3 receptor is the predominant muscarinic re- Medical University, 880 Kitakobayashi, Mibu Tochigi, 321-0293, Japan. Tel.: þ81 282 86 1111; fax: þ81 282 86 5105. ceptor subtype responsible for detrusor muscle contraction in E-mail address: [email protected] (T. Yamanishi). response to muscarinic agonists, while the M2 receptor is not Peer review under responsibility of Japanese Pharmacological Society. directly involved (11,13).

http://dx.doi.org/10.1016/j.jphs.2015.05.005 1347-8613/© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 66 T. Yamanishi et al. / Journal of Pharmacological Sciences 128 (2015) 65e70

However, it has been reported that M2 receptors participate in 2.1. Statistical analysis the contractile response of both rat and human bladder smooth muscle under pathologic conditions. M2 receptors have been re- The agonist potency and maximum response were expressed as ported to mediate detrusor contraction in rats with pelvic nerve the mean pEC50 ± SEM (logarithm of the molar concentration of denervation or spinal cord transection, and in patients with agonist resulting in 50% of the maximum response) and the mean neurogenic bladder dysfunction (14e16). An increase of total maximum contraction ± SEM, respectively. The pA2 value for a muscarinic receptor density and M2 receptor density along with a muscarinic receptor antagonist (determined as the x-intercept on reduction of M3 receptor density have been reported in obstructed, the Schild regression plot) was only measured when the Schild plot hypertrophic rat bladders (17). In contrast, the bladder contractile slope was unity. The dissociation constant (apparent KB value) for response to carbachol was reported to be mediated by the M3 re- each antagonist was calculated from the following equation: 1 ceptor in rats with partial bladder outlet obstruction and in humans KB ¼ antagonist concentration (molar) (concentration ratio 1) with idiopathic or neurogenic OAB (18,19). The present study was where the concentration ratio is the ratio of EC50 values obtained in performed to investigate whether the antagonist effect of various the presence and absence of the antagonist. Data are normalized for antimuscarinic agents on carbachol-induced contraction differed the maximal response in the first (control) curve, and are expressed between normal human bladder muscle and detrusor overactivity as the mean ± SEM. The unpaired Student's t-test was used for (DO) in patients with benign prostatic hyperplasia (BPH). statistical comparison of normal bladder and DO/BPH bladder tissues. 2. Materials and methods 2.2. Drugs and chemicals The procedures were approved by the local ethics committee of Dokkyo Medical University, and written informed consent was Carbachol (carbamylcholine chloride), 4-diphenyl acetoxy- obtained from each patient before enrollment. Tissue samples methyl piperidine methiodide (4-DAMP), methoctramine, pir- (8 2 mm) were obtained from the dome of the bladder in patients enzepine, oxybutynin, and propiverine were purchased from Sigma without lower urinary tract symptoms undergoing total cystectomy Chemical (St Louis, MO., USA). Tolterodine, trospium, solifenacin for bladder cancer (normal bladder), and in patients undergoing and imidafenacin were kind gifts from Pfizer Inc (New York, USA), retropubic prostatectomy for BPH whose bladders showed DO (DO/ Nihon Kemifa Ltd. (Tokyo, Japan), Asteras Ltd. (Tokyo, Japan), and BPH). The serosa and urothelium were removed. A section of Kyorin Ltd. (Tokyo, Japan), respectively. bladder dome tissue was dissected from an area free of cancer for use in the study. All of the patients with DO/BPH were proven to 3. Results have DO and bladder outlet obstruction by video-urodynamic studies or ambulatory urodynamic studies before surgery. Muscle Carbachol induced concentration-dependent contraction of strips were mounted in 10 ml organ baths containing Krebs solu- normal bladder strips, with a mean pEC50 value and maximum tion (in mM: NaCl 118.4, KCl 4.7, CaCl2 1.9, NaHCO3 24.9, MgSO4 1.15, response of 6.5 ± 0.1 and 9.6 ± 0.7 g, respectively. Carbachol also KH2PO4 1.15, and glucose 11.7), which was maintained at 37 C and induced concentration-dependent contraction of DO/BPH bladder aerated continuously with 95% O2 and 5% CO2. The strips were strips, with a mean pEC50 value and maximum response of 6.4 ± 0.1 placed under a resting tension of 1 g (9.8 mN) and allowed to and 9.8 ± 1.0 g, respectively. Both the mean pEC50 value and the equilibrate for 60 min, during which time they were washed every maximum response did not differ significantly between the two 10 min and the resting tension was adjusted. The isometric tension tissues. generated by each muscle specimen was measured by a Power Lab 4-DAMP (a selective M3-muscarinic receptor antagonist) and data acquisition system (Analog Digital Instruments, Sydney, New methoctramine (a selective M2-muscarinic receptor antagonist) South Wales, Australia). produced parallel, rightward displacement of the CRCs to carbachol Cumulative concentrationeresponse curves (CRCs) for carba- without affecting maximum responses and yielded mean (±SEM) chol were obtained. Then each muscle strip was washed for about pA2 values of 9.87±0.09 and 6.64±0.05, respectively in bladder 45 min until a stable resting tension was attained, followed by strips from normal bladder. The pA2 values for 4-DAMP and equilibration for 30 min with Krebs solution containing the methoctramine decreased significantly (P ¼ 0.0004 and P ¼ 0.0347, appropriate concentration of antagonist or vehicle (time control). respectively) in bladder strips from DO/BPH compared with the After incubation for 30 min, a second CRC for carbachol was ob- normal bladder (Table 1). Schild slopes for 4-DAMP and tained in the continued presence of the antagonist or vehicle. An- methoctramine did not change significantly (Figs. 1 and 2). tagonists were added at increasing concentrations after washing Pirenzepine (a selective M1-muscarinic receptor antagonist) for 45 min. In this way, 4 CRCs for carbachol were obtained from also produced parallel, rightward displacement of the CRCs to each muscle strip, with three being obtained in the presence of carbachol without affecting maximum responses and yielded mean increasing concentrations of a {(4-DAMP (3, (±SEM) pA2 values of 7.38±0.09 and Schild slopes not significantly 10, 30 nM), methoctramine (1, 3 and 10 mM), pirenzepine (1, 3 and different from unity. The pA2 values and Schild slopes for pir- 10 mM), tolterodine (3, 10, 30 nM), solifenacin (3, 10, 30 nM), enzepine did not change significantly in bladder strips from DO/ trospium (3, 10, 30 nM), propiverine (10, 30, 100 nM), oxybutynin BPH (Table 1). (3,10,30 nM), or imidafenacin (3,10,30 nM)} or in the presence of Tolterodine, trospium and propiverine (nonselective anti- the vehicle. Control experiments were performed with addition of muscarinic antagonists), and oxybutynin, solifenacin and imidafe- the vehicle instead of an antagonist and the data were used to nacin (M1- and M3-selective antimuscarinic antagonists) also correct for any tachyphylaxis or time-dependent changes of tissue caused parallel, rightward displacement of the CRCs for carbachol sensitivity and responsiveness. The correction factors for EC50 and without affecting the maximum response for strips of normal and the maximum response between the first and second curves were DO/BPH bladder, respectively. The mean (±SEM) pA2 value and both 1.0, those between the first and third curves were 1.5, and Schild plot slope for the effect of each muscarinic antagonist on the 0.85, respectively, and those between the first and fourth curves CRCs for carbachol are summarized in Table 1. The pA2 values of were 2.8 and 0.67, respectively. These correction factors were these muscarinic antagonists did not differ significantly between applied in all experiments (9,11e13,18). the normal bladder and DO/BPH bladder. In addition, the slope of T. Yamanishi et al. / Journal of Pharmacological Sciences 128 (2015) 65e70 67

Table 1 Antagonist affinities and Schild slopes of muscarinic antagonists against concentrationeresponse curves to carbachol in the muscle strips of normal human detrusor and overactive detrusor associated with benign prostatic hyperplasia (DO/BPH).

Antagonist 4-DAMP Methoctramine Pirenzepine

Normal bladder N ¼ 9N¼ 6N¼ 4

pA2 ± SEM 9.87±0.09 6.64 ± 0.05 7.38 ± 0.09 Schild slope 1.09 ± 0.37 0.69 ± 0.01 0.77 ± 0.17

DO/BPHb N ¼ 5N¼ 5N¼ 5 c c pA2 ± SEM 9.01 ± 0.12 6.36 ± 0.09 7.33 ± 0.11 Schild slope 0.93 ± 0.15 0.76 ± 0.18 0.89 ± 0.42

Antagonist Tolterodine Trospium Propiverine Oxybutynin Solifenacin Imidafenacin

Normal bladder N ¼ 6N¼ 5N¼ 6N¼ 4N¼ 8N¼ 6

pA2 ± SEM 8.63 ± 0.17 10.12 ± 0.11 7.74 ± 0.20 8.33 ± 0.16 8.80 ± 0.10 9.29 ± 0.26 Schild slope 1.29 ± 0.01 0.91 ± 0.29 1.06 ± 0.20 1.01 ± 0.12 1.06 ± 0.20 0.93 ± 0.12

DO/BPHb N ¼ 5N¼ 7N¼ 5N¼ 4N¼ 5N¼ 6

pA2 ± SEM 8.74 ± 0.16 10.14 ± 0.64 7.60 ± 0.11 8.41 ± 0.12 8.73 ± 0.09 9.24 ± 0.09 Schild slope 1.23 ± 0.06 1.04 ± 0.44 0.49 ± 0.14a 1.16 ± 0.11 0.98 ± 0.04 1.22 ± 0.17

a The Schild slope was different from unity in tissues from DO/BPH patients, and decreased significantly compared to that of normal bladder (P ¼ 0.0476). Apparent pKB values were calculated because of non-competitive antagonism. b Detrusor overactivity associated with benign prostatic hyperplasia. c The pA2 values for 4-DAMP and methoctramine decreased significantly (P ¼ 0.0004 and P ¼ 0.0347, respectively) in bladder strips from DO/BPH compared with those from the normal bladder. the Schild plot corresponded to unity for all antagonists and did not The affinity values of 4-DAMP and methoctramine decreased change significantly, except for propiverine with DO/BPH muscle significantly in pathologic states in DO/BPH. However the affinity strips. The Schild slope for propiverine were different from unity in value of pirenzepine and the rank order of these muscarinic an- tissues from DO/BPH patients, and decreased significantly tagonists did not change in this pathologic condition indicating that compared to that of normal bladder (P ¼ 0.0476). M3-receptor still predominantly mediated contraction in this The rank order of pA2 value of muscarinic antagonists in the pathological condition. The antagonist activity may not be the same normal bladder was, trospium (10.1) > 4-DAMP (9.9) > imidafena- between in the normal and in the pathologic state. We cannot cin (9.3) > solifenacin (8.8) > tolterodine (8.6) > oxybutynin explain the reasons, but one of the reasons may be that the uro- (8.3) > propiverine (7.7) > pirenzepine (7.4) > methoctramine (6.6). thelium was removed in the present study because the presence of That in DO/BPH was, trospium (10.1) > imidafenacin (9.2) > 4- urothelium may influence contraction of the detrusor. The purpose DAMP (9.0) > solifenacin (8.7) ¼ tolterodine (8.7) > oxybutynin of this study was to investigate whether the antagonist affinity of (8.4) > propiverine (7.6) > pirenzepine (7.3) > methoctramine (6.4). bladder smooth muscle changed between the normal and the pathological states. Thus we have removed the urothelium to study 4. Discussion solely the affinity of muscarinic receptor subtypes for the bladder smooth muscle. The affinity of these muscarinic antagonists may Detrusor overactivity (DO) has been reported to be the cause of have changed when the urothelium was intact. The further study overactive bladder symptoms. It has been reported that DO is noted remains to be necessary to investigate the interaction of urothelium in 40e60% of patients with BPH (20). Bladder outlet obstruction in and detrusor. BPH can be a factor contributing to DO, possibly through cholin- Tolterodine is a nonselective antimuscarinic agent that is widely ergic denervation of the detrusor and hypersensitivity of musca- used for the treatment of OAB, and it has been reported to be safe rinic receptors to , although the prevalence of OAB is and effective for overactive bladder in men with BPH (21,22). The similar in men and women across the age groups (2). Anti- affinity (pA2) value of tolterodine against CRC to carbachol in our muscarinic agents are the most widely used treatment for OAB, study in the human bladder from normal and DO/BPH appeared to including urgency incontinence (1). Each of the antimuscarinic be similar to the previous reports in the normal human bladder drugs has demonstrated efficacy against OAB symptoms, but their (23). pharmacokinetics and adverse event profiles differ somewhat due Solifenacin and imidafenacin are antimuscarinic drugs that are to differences of structure (tertiary vs. quaternary amines), widely used for the treatment of OAB with selectivity for M3 and M1 muscarinic receptor subtype selectivity, and organ selectivity (1,2). muscarinic receptor subtypes (3,4,24), and the affinity values for Among the various antimuscarinic agents, trospium, propiverine normal human bladder in the previous report were also similar to and tolterodine have a non-selective antimuscarinic action, while our result for bladder muscle strips from normal states and DO/BPH oxybutynin, solifenacin, and imidafenacin show selectivity for the (7,25). M3 and M1 muscarinic receptor subtypes. Propiverine has a nonselective antimuscarinic action and is also The muscarinic antagonists tested in the present study a calcium antagonist; it has been widely used in Japan and Europe demonstrated competitive antagonism with Schild slopes not (26), and the affinity values for normal human bladder in the different from unity in the normal bladder. Affinity values were the previous report were also similar to our result for bladder muscle highest for 4-DAMP (a selective M3-muscarinic receptor antago- strips from normal states and DO/BPH (7,25). However, the slope for nist), followed by pirenzepine (a selective M1-muscarinic receptor Schild plot was less than unity in propiverine with DO/BPO muscle ± antagonist) and methoctramine (a selective M2-muscarinic recep- strips (0.49 0.14). The reason may be that propiverine also shows tor antagonist). These affinity values were comparable with the activity as a calcium antagonist, so both its antimuscarinic and previous results in pig and human, indicating that M3-muscarinic calcium antagonist effects may have operated in DO/BPO. receptor predominantly mediates contraction in human detrusor in The present study showed that all of the muscarinic antagonists normal state (11,13,18). tested, including clinical available antimuscarinic agents 68 T. Yamanishi et al. / Journal of Pharmacological Sciences 128 (2015) 65e70

Fig. 1. Effects of 4-DAMP on concentrationeresponse curves to carbachol and Schild plot in normal bladder (upper panel) and detrusor overactivity due to benign prostatic hy- perplasia (DO/BPH; lower panel).

(tolterodine, solifenacin, propiverine, trospium, oxybutynin and was increased) in hypertrophic bladders after bladder outlet imidafenacin) competitively antagonized carbachol responses with obstruction. There was a shift in the affinity of muscarinic antag- high affinity in normal human detrusor. The rank order of their pA2 onists towards M2-mediated contraction in addition to muscarinic value was as follows: trospium (10.1) > 4-DAMP (9.9) > imidafena- supersensitivity in rat models of neurogenic bladder (denervation cin (9.3) > solifenacin (8.8) > tolterodine (8.6) > oxybutynin and spinal cord injury) (14,15). On the contrary, Krichevsky et al. (8.3) > propiverine (7.7) > pirenzepine (7.4) > methoctramine (6.6). (19), found no change in the affinity of muscarinic antagonists The pA2 values did not differ significantly between the normal state between the normal bladder and partial bladder outlet obstruction and detrusor overactivity (DO/BPH). The pA2 values for these in rats. In humans, Pontari et al. (16), reported that the M2 receptor antimuscarinic agents and their rank order seemed to be similar to played a role in the contraction of overactive detrusor muscle in those in previous reports. Chess-Williams et al. (9), and Sellers et al. patients with neurogenic bladder. However, Stevens et al. (18), re- (12), reported that the antagonistic affinity of various muscarinic ported no change in the affinity of muscarinic antagonists between antagonists was best correlated with the affinity for cloned M3 normal and overactive (idiopathic and neurogenic) detrusor muscle muscarinic receptor subtype. These observations suggest that the in humans. M3 receptor is the predominant muscarinic receptor subtype Limitation of this study is that it is difficult to get human mediating detrusor contraction in response to muscarinic agonists, detrusor tissues, similar to other studies (7,9e13). As a control, we while the M2 receptor is not directly involved in contraction of the collected bladder tissues from patients undergoing total cys- normal bladder (1,2,9,12). Reports about the role of the M2 receptor tectomy for bladder cancer. We selected patients without lower in animal models of detrusor overactivity have been conflicting. urinary tract symptoms, but we could not exclude patients with DO Braverman and Ruggieri (17) found a significant increase of M2 because we could not perform urodynamic study in these patients receptor mRNA transcript and protein levels in hypertrophic uri- for ethical reasons. It was more difficult and limited to obtain hu- nary bladders after major pelvic ganglion electrocautery and partial man bladder tissues from patients undergoing retropubic prosta- bladder outlet obstruction. However, the M3 receptor protein level tectomy because transurethral resection of prostate is the mainstay was reported to be decreased (although M3-receptor transcription of surgery for BPH. T. Yamanishi et al. / Journal of Pharmacological Sciences 128 (2015) 65e70 69

Fig. 2. Effects of methoctramine on concentrationeresponse curves to carbachol and Schild plot in normal bladder (upper panel) and detrusor overactivity due to benign prostatic hyperplasia (DO/BPH; lower panel).

In the present study, carbachol evoked concentration- antagonists, and trospium, tolterodine and propiverine, non- dependent contraction with a similar potency (pEC50) and selective muscarinic receptor antagonists antagonized CRCs to maximal effect in strips of both normal and DO/BPO detrusor carbachol with high affinities. Methoctramine, a selective M2-re- muscle. This result was different from that reported by Stevens et al. ceptor antagonist, and pirenzepine, a selective M1-receptor antag- (18), who found an increase of sensitivity to muscarinic agonists onist antagonized carbachol-induced contractions with relatively (pEC50), highlighting a change of receptor-mediated contraction in low affinities. These results suggest that M3-receptors mainly idiopathic and neurogenic DO compared with the normal detrusor. mediate contractions in normal human bladder. The rank order of The reasons for the discrepancy are unknown, but may be related to the affinity values of these antimuscarinic antagonists did not differences of the underlying disease (idiopathic or neurogenic OAB change when tested with DO/BPH bladder, suggesting that each vs. BPH). antimuscarinic antagonist has a similar effect in this condition. The above results suggest that antimuscarinic agents are effec- Schild plots showed a slope corresponding to unity, except for tive for DO/BPO, and that the M3 receptor mainly mediates detrusor propiverine with DO/BPH detrusor. In conclusion, M3-receptors contraction in this condition. mainly mediate contractions in human bladder strips with normal Although the role of M2 receptor could not be demonstrated in state and DO/BPH. this present study, it may have effects when M3 receptor was inactivated (11,13). M2 receptor in the urothelium may also have a Conflicts of interest role in activating afferent c-fibers in the pathological conditions (27). The role of M2 receptor should be elucidated in the future The authors have no conflict of interest. studies.

Acknowledgments 5. Conclusions This work was supported by a Grant-in-Aid for Scientific 4-DAMP, a selective M3-receptor antagonist, oxybutynin, sol- Research (C) (22591801) from the Ministry of Education, and a ifenacin and imidafenacin, selective M1- and M3-receptor grant from Pfizer Inc (New York, USA). 70 T. Yamanishi et al. / Journal of Pharmacological Sciences 128 (2015) 65e70

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