Jpn. J. Pharmacol. 86, 262 – 264 (2001) Short Communication

Endothelium-Dependent Relaxation in Response to Low Concentrations of Bradykinin Is Enhanced by Phosphoramidon, Bosentan and BQ-123 in Bovine Coronary Arteries In Vitro

Shigeru Ishiguro, Yutaka Nakamura, June Kawase, Atsushi Miyamoto and Akira Nishio*

Department of Veterinary Pharmacology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan

Received January 26, 2001 Accepted April 11, 2001

ABSTRACT—An endothelin (ET)-converting enzyme inhibitor phosphoramidon (10 mM), an ETAB-receptor antagonist bosentan (10 mM) and an ETA-receptor antagonist BQ-123 (1 mM) potentiated endothelium- dependent relaxation of bovine coronary arteries in response to bradykinin (BK) at femtomolar to picomolar

concentrations, but not at nanomolar concentrations. BQ-788 (3 mM), an ETB-receptor antagonist, showed no significant effects on fM – nM BK-induced relaxation. These results suggest that the endothelium- dependent relaxation of isolated bovine coronary arteries induced by very low concentrations of BK is partly

regulated by a complex mechanism involving the ETA-receptor antagonism.

Keywords: Bradykinin, Endothelin, Endothelium-dependent relaxation

It is well known that endothelial cells play a key role and cleaned of adhering connective tissue, and two rings in the regulation of vascular tone through release of vaso- were cut about 3 mm. Each ring was mounted horizontally dilating and vasoconstricting substances such as NO and in an organ bath filled with 15 ml of Krebs-Ringer bicar- endothelin (ET) in response to alterations in hemodynamic bonate solution of the following composition: 118 mM factors (1). Antagonism between NO and ET-1 has been NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 1.2 mM KH2PO4, demonstrated in human arteries (2). 2.5 mM CaCl2,25mMNaHCO3 and 10 mM glucose. The It has been reported that bradykinin (BK) induces endo- bath solution was maintained at 37°C and gassed with 95% thelium-dependent relaxation of arteries isolated from a O2 and 5% CO2. The coronary artery ring was attached to variety of species by releasing endothelium-derived relax- a force transducer (TB-611T; Nihon Kohden Kogyo Co., ing factors (1). Our previous study showed that very low Tokyo), and isometric force was recorded on a pen recorder concentrations of BK induced relaxation which was medi- (WI-641G, Nihon Kohden Kogyo Co.). Each ring was ated by NO through stimulation of B2-receptors on the endo- stretched to an optimal tension of 20 milliNewtons, as deter- thelium in isolated bovine coronary arteries (3). Recently mined by repeated stimulation with 60 mM KCl. Each ring we found that phosphoramidon (4, 5), an ET-converting was allowed to equilibrate for 90 – 120 min before starting enzyme inhibitor, enhanced the endothelium-dependent the experiment. All experiments were performed in the relaxation evoked by very low concentrations of BK (6). presence of 10 mM indomethacin to eliminate the effects Moreover it has been reported that BK suppresses ET- of prostanoids (8). The ring was contracted with 3 mM induced contraction of arteries through its B2-receptor on prostaglandin F2= (PGF2=) before the addition of BK. Cumu- the endothelium (7). The present study was done to deter- lative concentration-response curves were obtained by in- mine whether or not ET-1 participates in the relaxant re- creasing the concentration of BK in a cumulative manner; sponse to very low concentrations of BK using ET-1 antag- the addition was made as soon as a steady response had onists. been obtained with the preceding concentration. When an Experiments were performed on circumflex coronary ET converting enzyme inhibitor or ET-antagonists were arteries isolated from bovine hearts obtained from a slaugh- used, each drug was added to the organ bath 30 min before terhouse. Each coronary artery was carefully dissected out the concentration-response curves for BK were obtained. In

coronary rings contracted with PGF2=, the responses were *Corresponding author. FAX: +81-99-285-8717 expressed as a percentage of the relaxation induced by E-mail: [email protected] 100 mM sodium nitroprusside (SNP). Unless stated other-

262 Short Communication 263 wise, n refers to the number of animals from which the Pharmaceutical Co., Ltd. (Kamakura) and Banyu Pharma- coronary rings were isolated. The results were expressed as ceutical Co., Ltd. (Tsukuba), respectively. mean values ± S.E.M. Statistical evaluation of the data was Endothelium-dependent relaxant responses to very low performed using Student’s paired t-test. Significance was concentrations (10 – 100 pM) of BK were significantly established when the probability level was less than 5%. enhanced by phosphoramidon (Fig. 1a). It has been report- BK (Sigma Chemical Co., St. Louis, MO, USA), indo- ed that phosphoramidon, a metalloproteinase inhibitor (4), methacin and SNP (Nacalai Tesque, Kyoto), PGF2= (Ono has inhibitory effects on a neutral endopeptidase (9) that Pharmaceutical Co., Ltd., Osaka), ET-1 and phosphora- catalyzes the degradation of BK and on an ET-converting midon (Peptide Institute Inc., Osaka) were obtained com- enzyme (5) that catalyzes the conversion of big ET-1 to mercially. Bosentan, BQ-123 (cyclo-(D-Trp-D-Asp-Pro-D- ET-1 (5). In this experiment, we examined one possibility Val-Leu-)-Na) and BQ-788 (N-cis-2,6-dimethylpiperidino- that ET-converting enzyme inhibition might contribute to carbonyl-L-g-methylleucyl-D-Nin-methoxycarbonyltrypto- the potentiating effect of phosphoramidon on the relaxation phanyl-D-norleucine) were donated by Nippon Roche induced by very low concentrations of BK. Firstly, the ef-

Fig. 1. Effects of phosphoramidon (a), bosentan (b), BQ-123 (c) and BQ-788 (d) on bradykinin (BK)-induced endothelium- dependent relaxation of isolated bovine coronary arteries precontracted with PGF2= (3 mM) in the presence of indomethacin (10 mM). Relaxation induced by sodium nitroprusside (100 mM) was taken as 100%. Each point represents the mean ± S.E.M. of eight coronary rings obtained from different animals. Closed circle, control; open circle, drug-treated. *Significantly different from the corresponding control values (*P<0.05, **P<0.01). 264 Short Communication fects of some ET-1 antagonists on the relaxation induced by Boulanger C, Siebenmann R, Turina M and Bühler FR: Inter-

BK were examined. An ETAB-receptor antagonist, bosentan action between endothelin-1 and endothelium-derived relaxing factor in human arteries and veins. Circ Res 66,1088– 1094 (10), and an ETA-receptor antagonist, BQ-123 (11), en- (1990) hanced the relaxation induced by very low concentrations 3 Nakamura Y, Kawagoe K, Obi T, Miyamoto A, Ishiguro S and of BK (Fig. 1: b and c). However, the ETB-receptor antag- Nishio A: Femtomolar bradykinin-induced relaxation of isolated onist BQ-788 (12) had no significant effect on the relax- bovine coronary arteries, mediated by endothelium-derived ation induced by very low concentrations of BK (Fig. 1d). nitric oxide. J Vet Pharmacol Ther 21,304– 308 (1998) Phosphoramidon (10 mM), bosentan (10 mM), BQ-123 4 Matsumura Y, Hisaki K, Takaoka M and Morimoto S: Phos- (1 mM) and BQ-788 (3 mM) showed no significant effects phoramidon, a metalloproteinase inhibitor, suppresses the hyper- tensive effect of big endothelin-1. Eur J Pharmacol 185,103– on vascular tone under both resting and PGF2=-induced conditions. 106 (1990) 5Télémaque S, Gratton JP, Claing A and D’Orléans-Juste P: This is the first report to show that BK-induced relax- Pharmacologic evidence for the specificity of the phosphora- ation is enhanced by an ET-converting enzyme inhibitor midon-sensitive endothelin-converting enzyme for big endo- and by ET-antagonists. These results suggest that the relax- thelin-1. J Cardiovasc Pharmacol 22, Suppl 8,S85– S89 (1993) ation induced by low concentration of BK in bovine coro- 6 NishioA,NakamuraY,MiyamotoAandIshiguroS:Potentia- nary arteries is regulated by NO and ET-1 from endothelial tion by phosphoramidon of endothelium-dependent relaxation cells (1) and that the released ET-1 contracts the smooth induced by bradykinin in isolated bovine coronary artery. Jpn J Pharmacol 73, Suppl I, 166P (1997) muscle cells upon stimulation of ETA-receptors (S. Ishiguro 7 Ohde H, Morimoto S, Ohnishi K, Yamaoka E, Fukuo K, Yasuda et al., unpublished data). Our preliminary data showed that O and Ogihara T: Bradykinin suppresses endothelin-induced the bovine coronary rings with endothelium released spon- contraction of coronary, renal and femoral arteries through its taneously NO (pM level) and ET-1 (fM level) and that BK B2-receptor on the endothelium. Agents Actions 38, Suppl pt 3, (10–12 and 10–9 M) concentration-dependently enhanced the 14 – 22 (1992) release of NO and inhibited the release of ET-1 from these 8 Rosolowsky M and Campbell WB: Role of PGI2 and epoxyeico- rings (A. Nishio et al., unpublished data). Further studies satrienoic acids in relaxation of bovine coronary arteries to are needed to clarify that ET-1 at fM level affects the BK- arachidonic acid. Am J Physiol 264, H327 – H335 (1993) 9 Graf K, Grafe M, Auch-Schwelk W, Baumgarten CR, Bossaller induced relaxation in this artery. The relaxation induced by C and Fleck E: Bradykinin degrading activity in cultured human relatively high concentrations of BK (above 100 nM) was endothelial cells. J Cardiovasc Pharmacol 20, Suppl 9,S16– not enhanced by phosphoramidon, bosentan or BQ-123. S20 (1992) The precise reasons for this are unclear. One possibility is 10 Clozel M, Breu V, Gray GA, Kalina B, Loffler BM, Burri K, that a relatively high concentration of BK releases much CassalJM,HirthG,MullerM,NeidhartWandRamuzH: more NO from endothelial cells, and that NO inhibits the Pharmacological characterization of bosentan, a new potent formation or release of ET from endothelial cells (13, 14). orally active nonpeptide endothelin receptor antagonist. 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