Effects of (−)‐RO363 at human atrial β ‐adrenoceptor subtypes, the human cloned β 3 ‐adrenoceptor and rodent intestinal β 3 ‐adrenoceptors

Chronic treatment of patients with β‐blockers causes atrial inotropic hyperresponsiveness through β 2 ‐adrenoceptors, 5‐HT 4 receptors and H 2 ‐receptors but apparently not through β 1 ‐adrenoceptors despite data claiming an increased β 1 ‐adrenoceptor density from homogenate binding studies. We have addressed the question of β 1 ‐adrenoceptor sensitivity by determining the inotropic potency and intrinsic activity of the β 1 ‐adrenoceptor selective partial agonist (−)‐RO363 and by carrying out both homogenate binding and quantitative β‐adrenoceptor autoradiography in atria obtained from patients treated or not treated with β‐blockers. In the course of the experiments it became apparent that (−)‐RO363 also may cause agonistic effects through the third atrial β‐adrenoceptor. To assess whether (−)‐RO363 also caused agonistic effects through β 3 ‐adrenoceptors we studied its relaxant effects in rat colon and guinea‐pig ileum, as well as receptor binding and adenylyl cyclase stimulation of chinese hamster ovary (CHO) cells expressing human β 3 ‐adrenoceptors. β‐Adrenoceptors were labelled with (−)‐[ 125 I]‐cyanopindolol. The density of both β 1 ‐ and β 2 ‐adrenoceptors was unchanged in the 2 groups, as assessed with both quantitative receptor autoradiography and homogenate binding. The affinities of (−)‐RO363 for β 1 ‐adrenoceptors (p K i =8.0–7.7) and β 2 ‐adrenoceptors (p K i =6.1–5.8) were not significantly different in the two groups. (−)‐RO363 increased atrial force with a pEC 50 of 8.2 (β‐blocker treated) and 8.0 (non‐β‐blocker treated) and intrinsic activity with respect to (−)‐isoprenaline of 0.80 (β‐blocker treated) and 0.54 (non‐β‐blocker treated) ( P <0.001) and with respect to Ca 2+ (7 m m ) of 0.65 (β‐blocker treated) and 0.45 (non‐β‐blocker treated) ( P <0.01). The effects of (−)‐RO363 were resistant to antagonism by the β 2 ‐adrenoceptor antagonist, ICI 118,551 (50 n m ). The effects of 0.3–10 n m (−)‐RO363 were antagonized by 3–10 n m of the β 1 ‐adrenoceptor selective antagonist CGP 20712A. The effects of 20–1000 n m (−)‐RO363 were partially resistant to antagonism by 30–300 n m CGP 20712A. (−)‐RO363 relaxed the rat colon, partially precontracted by 30 m m 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 n m ) with pEC 50 =8.5 and fraction 0.66, the other resistant to (−)‐propranolol (200 n m ) with pEC 50 =5.6 and fraction 0.34 of maximal relaxation. (−)‐RO363 relaxed the longitudinal muscle of guinea‐pig ileum, precontracted by 0.5 μ m histamine, with intrinsic activity of 1.0 compared to (−)‐isoprenaline and through 2 components, one antagonized by (−)‐propranolol (200 n m ) with pEC 50 =8.7 and fraction 0.67, the other resistant to (−)‐propranolol with pEC 50 =4.9 and fraction 0.33 of maximal relaxation. (−)‐RO363 stimulated the adenylyl cyclase of CHO cells expressing human β 3 ‐adrenoceptors with pEC 50 =5.5 and intrinsic activity 0.74 with respect to (−)‐isoprenaline (pEC 50 =5.9). (−)‐RO363 competed for binding with [ 125 I]‐cyanopindolol at human β 3 ‐adrenoceptors transfected into CHO cells with p K i =4.5. (−)‐Isoprenaline (p K i =5.2) and (−)‐CGP 12177A (p K i =6.1) also competed for binding at human β 3 ‐adrenoceptors. We conclude that under conditions used in this study, (−)‐RO363 is a potent partial agonist for human β 1 ‐ and β 3 ‐adrenoceptors and appears also to activate the third human atrial β‐adrenoceptor. (−)‐RO363 relaxes mammalian gut through both β 1 ‐ and β 3 ‐adrenoceptors. (−)‐RO363, used as a β 1 ‐adrenoceptor selective tool, confirms previous findings with (−)‐noradrenaline that β 1 ‐adrenoceptor‐mediated atrial effects are only slightly enhanced by chronic treatment of patients with β‐blockers. Chronic treatment with β 1 ‐adrenoceptor‐selective blockers does not significantly increase the density of human atrial β 1 ‐ and β 2 ‐adrenoceptors.British Journal of Pharmacology (1997) 120 , 165–176; doi: 10.1038/sj.bjp.0700850