EFFECTS OF (-)-RO363 AT HUMAN ATRIAL BETA-ADRENOCEPTOR SUBTYPES, THE HUMAN CLONED BETA(3)-ADRENOCEPTOR AND RODENT INTESTINAL BETA(3)-ADRENOCEPTORS

1 Chronic treatment of patients with beta-blockers causes atrial inotr opic hyperresponsiveness through beta(2)-adrenoceptors, 5-HT4 receptor s and H-2-receptors but apparently not through beta(1)-adrenoceptors d espite data claiming an increased beta(1)-adrenoceptor density from ho mogenate binding studies. We have addressed the question of beta(1)-ad renoceptor sensitivity by determining the inotropic potency and intrin sic activity of the beta(1)-adrenoceptor selective partial agonist (-) -RO363 and by carrying out both homogenate binding and quantitative be ta-adrenoceptor autoradiography in atria obtained from patients treate d or not treated with beta-blockers. In the course of the experiments it became apparent that (-)-RO363 also may cause agonistic effects thr ough the third atrial beta-adrenoceptor. To assess whether (-)-RO363 a lso caused agonistic effects through beta(3)-adrenoceptors we studied its relaxant effects in rat colon and guinea-pig ileum, as well as rec eptor binding and adenylyl cyclase stimulation of chinese hamster ovar y (CHO) cells expressing human beta(3)-adrenoceptors. 2 beta-Adrenocep tors were labelled with (-)-[I-125]-cyanopindolol. The density of both beta(1)- and beta(2)-adrenoceptors was unchanged in the 2 groups, as assessed with both quantitative receptor autoradiography and homogenat e binding. The affinities of (-)-RO363 for beta(1)-adrenoceptors (pK(i ) = 8.0-7.7) and beta(2)-adrenoceptors (pK(i) = 6.1-5.8) were not sign ificantly different in the two groups. 3 (-)-RO363 increased atrial fo rce with a pEC(50) of 8.2 (beta-blocker treated) and 8.0 (non-beta-blo cker treated) and intrinsic activity with respect to (-)-isoprenaline of 0.80 (beta-blocker treated) and 0.54 (non-beta-blocker treated) (P< 0.001) and with respect to Ca2+ (7 mM) of 0.65 (beta-blocker treated) and 0.45 (non-beta-blocker treated) (P<0.01). The effects of (-)-RO363 were resistant to antagonism by the beta(2)-adrenoceptor antagonist, ICI 118,551 (50 nM). The effects of 0.3-10 nM (-)-RO363 were antagoniz ed by 3-10 nM of the beta(1)-adrenoceptor selective antagonist CGP 207 12A. The effects of 20-1000 nM (-)-RO363 were partially resistant to a ntagonism by 30-300 nM CGP 20712A. 4 (-)-RO363 relaxed the rat colon, partially precontracted by 30 mM KCl, with an intrinsic activity of 0. 97 compared to (-)-isoprenaline. The concentration-effect curve to (-) -RO363 revealed 2 components, one antagonized by (-)-propranolol (200 nM) with pEC(50)=8.5 and fraction 0.66, the other resistant to (-)-pro pranolol (200 nM) with pEC(50)=5.6 and fraction 0.34 of maximal relaxa tion.5 (-)-RO363 relaxed the longitudinal muscle of guinea-pig ileum, precontracted by 0.5 mu M histamine, with intrinsic activity of 1.0 co mpared to (-)-isoprenaline and through 2 components, one antagonized b y (-)-propranolol (200 nM) with pEC(50)=8.7 and fraction 0.67, the oth er resistant to (-)-propranolol with pEC(50)=4.9 and fraction 0.33 of maximal relaxation. 6 (-)-RO363 stimulated the adenylyl cyclase of CHO cells expressing human beta(3)-adrenoceptors with pEC(50)=5.5 and int rinsic activity 0.74 with respect to (-)-isoprenaline (pEC(50)=5.9). ( -)-RO363 competed for binding with [I-125]cyanopindolol at human beta( 3)-adrenoceptors transfected into CHO cells with pK(i)=4.5. (-)-Isopre naline (pk(i)=5.2) and (-)-CGP 12177A (pK(i)=6.1) also competed for bi nding at human beta(2)-adrenoceptors. 7 We conclude that under conditi ons used in this study, (-)-RO363 is a potent partial agonist for huma n beta(1)- and beta(3)-adrenoceptors and appears also to activate the third human atrial beta-adrenoceptor. (-)-RO363 relaxes mammalian gut through both beta(1)- and beta(3)-adrenoceptors. (-)-RO363, used as a beta(1)-adrenoceptor selective tool, confirms previous findings with ( -)-noradrenaline that beta(1)-adrenoceptor mediated atrial effects are only slightly enhanced by chronic treatment of patients with beta-blo ckers. Chronic treatment with beta(1)-adrenoceptor-selective blockers does not significantly increase the density of human atrial beta(1)- a nd beta(2)-adrenoceptors.