Biol. Pharm. Bull. 42(5): 736-743 (2019)

736 Biol. Pharm. Bull. 42, 736–743 (2019) Vol. 42, No. 5 Regular Article Noradrenaline-Induced Relaxation of Urinary Bladder Smooth Muscle Is Primarily Triggered through the β3-Adrenoceptor in Rats Keisuke Obara,a Serena Suzuki,a Hiroko Shibata,a Naoki Yoneyama, a Shoko Hamamatsu,a Fumiko Yamaki,a Koji Higai,b and Yoshio Tanaka*,a a Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan: and b Laboratory of Medical Biochemistry, Faculty of Pharmaceutical Sciences, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan. Received November 18, 2018; accepted January 25, 2019 β-Adrenoceptors are subclassified into 3 subtypes (β1–β3). Among these, β3-adrenoceptors are present in various types of smooth muscle and are believed to play a role in relaxation responses of these muscles. β3-Adrenoceptors are also present in urinary bladder smooth muscle (UBSM), although their expression varies depending on the animal species. To date, there has been little information available about the endogenous ligand that stimulates β3-adrenoceptors to produce relaxation responses in UBSM. In this study, to determine whether noradrenaline is a ligand of UBSM β3-adrenoceptors, noradrenaline-induced relaxation was analyzed pharmacologically using rat UBSM. We also assessed whether noradrenaline metabolites were ligands in UBSM. In isolated rat urinary bladder tissues, mRNAs for β1-, β2-, and β3-adrenoceptors were detected using RT-PCR. In UBSM preparations contracted with methacholine (3 105 M), noradrenaline- 6 induced relaxation was not inhibited by the following antagonists: atenolol (10 M; selective β1-adrenoceptor 8 7 antagonist), ICI-118,551 (3 10 M; selective β2-adrenoceptor antagonist), propranolol (10 M; non-selective β-adrenoceptor antagonist), and bupranolol (107 M; non-selective β-adrenoceptor antagonist). In the presence of propranolol (106 M), noradrenaline-induced relaxation was competitively inhibited by bupranolol 7 6 7 6 (3 10 –3 10 M) or SR59230A (10 –10 M; selective β3-adrenoceptor antagonist), with their pA2 values calculated to be 6.64 and 7.27, respectively. None of the six noradrenaline metabolites produced significant relaxation of methacholine-contracted UBSM. These findings suggest that noradrenaline, but not its metabolites, is a ligand for β3-adrenoceptors to produce relaxation responses of UBSM in rats. Key words rat urinary bladder smooth muscle; noradrenaline; β-adrenoceptor INTRODUCTION regulation of the UBSM relaxation response and, thus, urine storage. However, whether noradrenaline acts as an endo- The urinary bladder (UB) is an organ that stores urine and genous ligand for the β3-adrenoceptor to induce UBSM relax- discharges it outside the body. These physiological functions ation has not been convincingly established. This is because are controlled by the relaxation and contraction responses of almost all pharmacological studies on UBSM β-adrenoceptors UB smooth muscle (UBSM), and both responses are affected were designed using synthetic β-adrenoceptor agonists (iso- strongly by autonomic nerves. In particular, urine discharge is prenaline or selective β3-adrenoceptor agonists), but not endo- associated with UBSM contraction. This contraction is prin- genous catecholamines such as noradrenaline. cipally triggered by acetylcholine that is released from para- The purpose of this study was to investigate whether sympathetic nerve endings, the activity of which predominates noradrenaline is a ligand for the β3-adrenoceptor using in the micturition phase. In contrast, urine storage (retention) rat UBSM tissue. This tissue expresses all subtypes of is associated with UBSM relaxation. This relaxation response β-adrenoceptors,10,11) allowing the determination of whether is generally considered to be triggered by noradrenaline that noradrenaline can stimulate β1 and β2 subtypes in addition is released from sympathetic nerve endings, the activity of to β3. In this study, we also examined six metabolites of nor- which predominates in the UB filling phase.1–3) However, how adrenaline in order to verify whether they can induce UBSM sympathetic nerves contribute to the relaxation of UBSM and relaxation via stimulation of β3-adrenoceptors. urine storage is not completely understood. If noradrenaline is a key molecule to trigger UBSM re- MATERIALS AND METHODS laxation, it is reasonable to postulate that it would target β-adrenoceptors in UBSM, as it does in other smooth muscles. Drugs The followings drugs were used: (±)-ateno- 4) β-Adrenoceptors are classified into 3 subtypes (β1–β3), and lol, desipramine hydrochloride, 3,4-dihydroxymandelic acid 5) UBSM subtype expression is species-dependent. In humans, (DOMA), DL-3,4-dihydroxyphenylglycol (DHPG), 3,5-dini- the main β-adrenoceptor subtype in UBSM has been identi- trocatechol, DL-4-hydroxy-3-methoxymandelic acid (VMA), 5,6) fied as β3 at the mRNA level. In addition, a selective β3- 4-hydroxy-3-methoxyphenyl glycol (MHPG) hemipiperazin- adrenoceptor agonist, mirabegron, has been reported to induce ium salt, 4-hydroxy-3-methoxyphenylglycol sulfate (MHPG- UBSM relaxation and improve overactive bladder (OAB) S) potassium salt, indomethacin, ICI-118,551 hydrochloride, 7–9) symptoms by increasing bladder capacity. These findings (−)-isoproterenol (ISO) hydrochloride, DL-normetanephrine suggest that β3-adrenoceptors play a significant role in the (NMN) hydrochloride, N-methyl-N-propargyl-3-(2,4-dichlo- * To whom correspondence should be addressed. e-mail: [email protected] © 2019 The Pharmaceutical Society of Japan Vol. 42, No. 5 (2019) Biol. Pharm. Bull. 737 rophenoxy) propylamine (clorgiline) hydrochloride, DL-propran- GGC AGA GTT GGC ATA GC-3′), and β-actin (forward, 5′-ATG olol hydrochloride, SR 59230A (all from Sigma-Aldrich Co., GTG GGT ATG GGT CAG AA-3′ and reverse, 5′-ACC CTC St. Louis, MO, U.S.A.); (±)-phentolamine mesylate (Novartis ATA GAT GGG CAC AG-3′) were synthesized at Eurofins Pharma, Basel, Switzerland); acetyl-β-methylcholine (metha- Genomics (Tokyo, Japan). The PCR samples were heated for choline) chloride, (R)-(−)-norepinephrine (noradrenaline) hy- 2 min at 95°C, and then amplified by 35 cycles at 95°C for drogen tartrate monohydrate (both from Wako Pure Chemical 20 s, 60°C for 5 s, and 72°C for 30 s (β-adrenoceptors) or by Industries, Ltd., Osaka, Japan); and (±)-bupranolol hydrochlo- 25 cycles at 95°C for 20 s, 60°C for 5 s, and 72°C for 30 s ride (Kaken Pharmaceutical Co., Ltd., Tokyo, Japan). All other (β-actin), followed by a 5-min extension at 72°C. The PCR chemicals were commercially available and reagent grade. products were separated via 1.5% agarose gel (containing Atenolol was dissolved in 0.1 N hydrochloric acid (HCl) as ethidium bromide) electrophoresis and visualized under UV a stock solution at 2 × 10−2 M and diluted with distilled water. illumination. Indomethacin was dissolved in pure ethanol as a stock solu- Preparation of UB Strips and Recording of Isotonic tion at 10−2 M. 3,5-Dinitrocatechol was dissolved in dimethyl Tension Changes The isolated rat UB, after removing the sulfoxide (DMSO) as a stock solution at 4 × 10−3 M and dilut- surrounding adipose tissue, connective tissue, and bladder ed with distilled water. SR 59230A was dissolved in DMSO as trigone, was opened with a longitudinal incision and UB strips a stock solution at 2 × 10−2 M and diluted with distilled water. (approximately 2 mm in width ×25 mm in length) were pre- All other drugs were prepared as aqueous stock solutions and pared in normal Tyrode’s solution. The UB strips were mount- diluted with distilled water. ed under an optimal resting tension of 0.5 g in a 20-mL organ Animals Male Wistar rats (8–10 weeks old; weight bath containing normal Tyrode’s solution, aerated with 95% 165–265 g, Sankyo Labo Service Corporation, Tokyo, Japan) O2 and 5% CO2, and maintained at 32 ± 1°C. Tension changes were housed under controlled conditions (21–22°C, relative air were recorded isotonically via a kymograph and lever. The humidity 50 ± 5%, fixed 12 h light–dark cycle (08:00–20:00)) UB preparations were equilibrated for 60 min prior to the with food and water available ad libitum. This study was first methacholine-induced contraction, during which time the approved by the Toho University Animal Care and User normal Tyrode’s solution was replaced every 20 min with fresh Committee (approval number: 16-52-294, accredited on May solution. After the initial 60-min incubation, the UB prepara- 16, 2016; approval number: 17-53-294, accredited on May tion was contracted with methacholine (3 × 10−5 M). When the 17, 2017) and was conducted in accordance with the User’s contraction reached steady-state, the preparation was relaxed Guideline to the Laboratory Animal Center of Faculty of by applying isoprenaline. This procedure was performed Pharmaceutical Sciences, Toho University. twice before starting the experiment (performed 3 times in RT-PCR Analysis of β-Adrenoceptor Subtype mRNA total). All experiments were carried out in the presence of Expression The rats were anaesthetized with isoflurane indomethacin (3 × 10−6 M) to prevent any possible effects of (inhalation) and euthanized by exsanguination from a carotid endogenous prostaglandins. artery. The UB, atrium (both right and left atria), and ileum Evaluation of Effects of β-Adrenoceptor Antagonists on were immediately removed and placed in normal Tyrode’s Noradrenaline-Induced Relaxation After conducting the solution of the following composition (mM): NaCl, 158.3; KCl, preliminary procedures described in the previous section, the −5 4.0; CaCl2, 2.0; MgCl2, 1.05; NaH2PO4, 0.42; NaHCO3, 10.0; UB preparations were treated with methacholine (3 × 10 M). and glucose, 5.6. All tissues were stripped of surrounding When the contraction reached a steady-state level, noradrena- adipose and connective tissue, and the bladder trigone from line was cumulatively applied to the bath solution in order to the UB. After washing out debris, and inserting an acrylic obtain a concentration-response curve.

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