Effects of (−)‐RO363 at human atrial β‐adrenoceptor subtypes, the human cloned β3‐adrenoceptor and rodent intestinal β3‐adrenoceptors
Authors: Molenaar Peter , Sarsero Doreen , Arch Jonathan R S , Kelly John , Henson Sian M , Kaumann Alberto J 
Published: August 2006

Chronic treatment of patients with β‐blockers causes atrial inotropic hyperresponsiveness through β2‐adrenoceptors, 5‐HT4 receptors and H2‐receptors but apparently not through β1‐adrenoceptors despite data claiming an increased β1‐adrenoceptor density from homogenate binding studies. We have addressed the question of β1‐adrenoceptor sensitivity by determining the inotropic potency and intrinsic activity of the β1‐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 β3‐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 β3‐adrenoceptors.
β‐Adrenoceptors were labelled with (−)‐[125I]‐cyanopindolol. The density of both β1‐ and β2‐adrenoceptors was unchanged in the 2 groups, as assessed with both quantitative receptor autoradiography and homogenate binding. The affinities of (−)‐RO363 for β1‐adrenoceptors (pKi=8.0–7.7) and β2‐adrenoceptors (pKi=6.1–5.8) were not significantly different in the two groups.
(−)‐RO363 increased atrial force with a pEC50 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 Ca2+ (7 mm) 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 β2‐adrenoceptor antagonist, ICI 118,551 (50 nm). The effects of 0.3–10 nm (−)‐RO363 were antagonized by 3–10 nm of the β1‐adrenoceptor selective antagonist CGP 20712A. The effects of 20–1000 nm (−)‐RO363 were partially resistant to antagonism by 30–300 nm CGP 20712A.
(−)‐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 pEC50=8.5 and fraction 0.66, the other resistant to (−)‐propranolol (200 nm) with pEC50=5.6 and fraction 0.34 of maximal relaxation.
(−)‐RO363 relaxed the longitudinal muscle of guinea‐pig ileum, precontracted by 0.5 μm histamine, with intrinsic activity of 1.0 compared to (−)‐isoprenaline and through 2 components, one antagonized by (−)‐propranolol (200 nm) with pEC50=8.7 and fraction 0.67, the other resistant to (−)‐propranolol with pEC50=4.9 and fraction 0.33 of maximal relaxation.
(−)‐RO363 stimulated the adenylyl cyclase of CHO cells expressing human β3‐adrenoceptors with pEC50=5.5 and intrinsic activity 0.74 with respect to (−)‐isoprenaline (pEC50=5.9). (−)‐RO363 competed for binding with [125I]‐cyanopindolol at human β3‐adrenoceptors transfected into CHO cells with pKi=4.5. (−)‐Isoprenaline (pKi=5.2) and (−)‐CGP 12177A (pKi=6.1) also competed for binding at human β3‐adrenoceptors.
We conclude that under conditions used in this study, (−)‐RO363 is a potent partial agonist for human β1‐ and β3‐adrenoceptors and appears also to activate the third human atrial β‐adrenoceptor. (−)‐RO363 relaxes mammalian gut through both β1‐ and β3‐adrenoceptors. (−)‐RO363, used as a β1‐adrenoceptor selective tool, confirms previous findings with (−)‐noradrenaline that β1‐adrenoceptor‐mediated atrial effects are only slightly enhanced by chronic treatment of patients with β‐blockers. Chronic treatment with β1‐adrenoceptor‐selective blockers does not significantly increase the density of human atrial β1‐ and β2‐adrenoceptors.

British Journal of Pharmacology (1997) 120, 165–176; doi:10.1038/sj.bjp.0700850