Vol. 43, No. 3 Biol. Pharm. Bull. 43, 493–502 (2020) 493 Regular Article Effects of Catecholamine Metabolites on Beta-Adrenoceptor-Mediated Relaxation of Smooth Muscle: Evaluation in Mouse and Guinea-Pig Trachea and Rat Aorta Fumiko Yamaki, Anna Koike, Hikari Kono, Xiaoyue Zhang, Kento Yoshioka, Keisuke Obara,* and Yoshio Tanaka Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan. Received October 3, 2019; accepted December 17, 2019 The β-adrenoceptor (β-AR)-mediated pharmacological effects of catecholamine (CA) metabolites are not well known. We examined the effects of seven CA metabolites on smooth muscle relaxation in mouse and guinea pig (GP) tracheas and rat thoracic aorta. Among them, metadrenaline (MA) significantly re- laxed GP trachea (β2-AR dominant), even in the presence of clorgiline, a monoamine oxidase-A inhibitor. 4 In mouse trachea (β1-AR dominant), normetadrenaline (NMA) and MA (10 M each) apparently did not affect isoprenaline (ISO)-induced relaxation, but significantly inhibited it in the presence of clorgiline. ISO- induced relaxation was also unaffected by 3,4-dihydroxyphenylglycol (DHPG) (104 M), but significant sup- pression was observed with the addition of 3,5-dinitrocatechol, a catechol-O-methyltransferase inhibitor. In GP trachea, NMA, MA, 3,4-dihydroxymandelic acid (DOMA), and DHPG (104 M each) significantly aug- mented ISO-induced relaxation. However, in the presence of clorgiline plus 3,5-dinitrocatechol, both NMA and MA (104 M) significantly suppressed ISO-induced relaxation. DHPG (104 M) also significantly sup- pressed ISO-induced relaxation in the presence of 3,5-dinitrocatechol. In rat thoracic aorta, DHPG (104 M) significantly suppressed relaxation induced by CGP-12177 A (a β3-AR partial agonist) in the presence of 3,5-dinitrocatechol plus propranolol. Our findings indicate that 1) MA may possess β2-AR agonistic action; 2) NMA and MA augment β2-AR-mediated tracheal relaxation in the absence of CA metabolic inhibitors, though themselves possessing β1-, β2-AR antagonistic action (β2 > β1); 3) DHPG exhibits β1-, β2-, β3-AR an- tagonistic action, and this is particularly marked for β3-AR. Our observations may help explain some of the pathologies associated with pheochromocytoma, which is characterized by increased CA metabolite levels. Key words metadrenaline; normetadrenaline; 3,4-dihydroxyphenylglycol; β-adrenoceptor; tracheal relax- ation; aortic relaxation INTRODUCTION mediated pharmacological actions requires further study. In order to clarify whether various CA metabolites exert Catecholamine (CA) metabolites are generated through their pharmacological actions through β-ARs, we investi- oxidative deamination of noradrenaline (NA) and adrenaline gated their possible agonistic/antagonistic effects on β-AR (AD) by monoamine oxidase (MAO) and their O-methylation using smooth muscle preparations in which functional β1-AR 1,2) by catechol-O-methyltransferase (COMT) (Fig. 1). It is gen- or β2-AR was dominantly expressed. The smooth muscle erally recognized that CA metabolites do not show pharmaco- preparations used were mouse and GP trachea in which 2) logical activity. However, normetadrenaline (NMA) and me- β1-AR-/β2-AR-mediated relaxations were exclusively and tadrenaline (MA), COMT metabolites of NA/AD, can act as easily detected.5,6) In addition, rat thoracic aorta (TA) that 7,8) agonists of adrenoceptors (ARs). For example, NMA and MA produces β3-AR-mediated relaxation was also used. CA have been shown to contract the nictitating membrane smooth metabolites examined in this study were NMA, MA, DOMA, muscle via α-AR in cats, and to possess positive chronotropic DHPG, VMA, MHPG, and MHPG sulfate (MHPG-S), which 3) action in guinea pig (GP) atrial muscle through β (β1)-AR. have been reported to increase in the blood and urine of Extending from this work, we recently found that NMA/MA patients with pheochromocytoma.1,9,10) The present pharmaco- contracted rat prostate and thoracic aorta through activation of logical approaches are clinically significant, since the informa- α1A-AR/α1D-AR (our unpublished observation). tion about CA metabolites’ β-AR-mediated effects on smooth In contrast, four CA metabolites, 3,4-dihydromandelic acid muscle relaxation may help further understanding of the pa- (DOMA), 3,4-dihydroxyphenylglycol (DHPG), vanillylman- thology of pheochromocytoma. delic acid (VMA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) were reported not to show β (β1)-AR stimulatory MATERIALS AND METHODS or antagonistic actions in GP atrial muscle.3) In addition, we found that five CA metabolites (NMA, DOMA, DHPG, VMA, Drugs The following drugs were used: DL-normetanephrine and MHPG) did not show β3-AR agonistic activity, based on (DL-normetadrenaline, or NMA), hydrochloride, DL- observations of relaxant responses in rat urinary bladder prep- metanephrine (DL-metadrenaline, or MA), hydrochloride, DL- 4) arations. However, whether CA metabolites exhibit β-AR- 3,4-dihydroxymandelic acid (DOMA), DL-3,4-dihydroxyphenyl * To whom correspondence should be addressed. e-mail: [email protected] © 2020 The Pharmaceutical Society of Japan 494 Biol. Pharm. Bull. Vol. 43, No. 3 (2020) Fig. 1. Metabolic Pathways of Noradrenaline (NA) and Adrenaline (AD) by Monoamine Oxidase (MAO) and Catechol-O-Methyltransferase (COMT) NMA: normetadrenaline; MA: metadrenaline; DOMA: 3,4-dihydroxymandelic acid; DHPG; 3,4-dihydroxyphenylglycol; VMA: vanillylmandelic acid; MHPG: 3-methoxy-4-hydroxyphenylglycol; MHPG-S; MHPG sulfuric acid; DHMA: 3,4-dihydroxymandelaldehyde; MHMA: 3-methoxy-4-hydroxymandelaldehyde; AO: aldehyde oxidase; AR: aldehyde reductase. glycol (DHPG), DL-4-hydroxy-3-methoxymandelic acid (VMA), on May 17, 2017, 18-54-294, accredited on May 7, 2018, and 4-hydroxy-3-methoxyphenylglycol (MHPG) hemipiperazinium 19-55-294, accredited on April 10, 2019) and was conducted salt, 4-hydroxy-3-methoxyphenylglycol sulfate (MHPG-S) in accordance with the User’s Guidelines for the Laboratory potassium salt, (−)-adrenaline (AD) (+)-bitartrate salt, car- Animal Center of the Faculty of Pharmaceutical Sciences, bamyl choline chloride, (−)-isoproterenol (isoprenaline; ISO) Toho University. hydrochloride, N-methyl-N-propargyl-3-(2,4-dichlorophenoxy) Preparation of Trachea and TA Tissues The mice and propylamine (clorgiline) hydrochloride, 3,5-dinitrocatechol, GPs were anesthetized with isoflurane by inhalation and eu- (−)-phenylephrine hydrochloride, salbutamol hemisulfate salt, thanized by exsanguination from the carotid arteries. There- (±)-CGP-12177 A hydrochloride, (±)-propranolol hydrochloride, after, the tracheal tissues were quickly removed and immersed indomethacin (all from Sigma-Aldrich, St. Louis, MO, U.S.A.), in Locke-Ringer solution of the following composition (mM): (−)-(R)-noradrenaline (NA) hydrogen tartrate monohydrate, NaCl, 154; KCl 5.6; CaCl2, 2.2; MgCl2, 2.1; NaHCO3, 5.9; D- silodosin (FUJIFILM Wako Pure Chemical Corporation, Osaka, (+)-glucose, 2.8. The tracheal tissues were cleaned of unnec- Japan), (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl) oxy]-3- essary adipose and connective tissues under a dissecting mi- [(1-methylethyl) amino]-2-butanol hydrochloride (ICI-118551) croscope. Subsequently, tracheal cartilage containing smooth (Tocris Bioscience, Ellisville, MO, U.S.A.), acetylcholine muscles was cut into pieces about 2 mm long. In this series of chloride (Daiichi Sankyo Co., Ltd., Tokyo, Japan), and (±)- experiments, the intimal surface of tracheal tissue was gently phentolamine mesylate (Novartis Pharma K.K., Tokyo, Japan). rubbed with moistened filter paper to remove tracheal epithe- All other chemicals used were commercially available and of lium as much as possible. reagent grade. The rats were anesthetized with isoflurane by inhalation Indomethacin was dissolved in 100% ethanol as a stock and euthanized by exsanguination from the carotid arteries. solution at 10−2 M, and 3,5-dinitrocatechol was dissolved in Thereafter, the TA was quickly removed and immersed in dimethyl sulfoxide (DMSO) as a stock solution at 5 × 10−3 M. normal Tyrode’s solution of the following composition (mM): All other drugs were prepared as aqueous stock solutions. All NaCl, 158.3; KCl, 4.0; CaCl2, 2.0; MgCl2, 1.05; NaH2PO4, stock solutions were diluted with distilled water to obtain de- 0.42; NaHCO3, 10.0; D-(+)-glucose, 5.6. After removing the sired concentrations. surrounding adipose and connective tissue under a dissecting Animals Male Wistar rats (8–11 weeks old; weight microscope, the isolated TA was then cut into spiral segments 150–280 g; Sankyo Labo Service Co., Tokyo, Japan), male (approximately 2 mm in width and 20 mm in length), and the ddY mice (8–12 weeks old; weight 35–46 g; Sankyo Labo endothelium was removed by gentle rubbing of the intimal Service Co.), and male Hartley GP (5–8 weeks old; weight surface with filter paper. 290–465 g; Kyudo Co., Ltd., Saga, Japan) were housed under Recordings of Isometric Tension Changes of Smooth controlled conditions at a temperature of 21–22°C, relative Muscle Tissues Tracheal tissues were suspended with stain- air humidity of 50 ± 5%, and a fixed 12 h light-dark cycle less steel hooks (outer diameter 200 µm) in a 5-mL organ (08 : 00–20:00), with food and water available ad libitum. This bath (UC-5; UFER Medical Instrument, Kyoto, Japan) filled study was approved by the Toho University Animal Care with Locke-Ringer solution. Rat TAs were suspended with and User Committee (approval numbers