1 The binding properties of beta(1)-, beta(2)- and beta(3)-adrenoceptors were determined in isolated brown adipocytes of the rat rather than in membrane preparations from tissue homogenates, because typical brown adipocytes represent only about 40% of the various cells present in brown adipose tissue. Binding characteristics were assessed with the hydrophilic beta-adrenoceptor radioligand, (-)-[H-3]-CGP 12177. The potent beta-antagonist, bupranolol (100 mu M) was used to determine nonspecific binding. Characterization was essentially performed by saturation and competition studies. 2 The saturation curve of(-)[H-3]-CGP 12177 was clearly biphasic (Hill coefficient, nH = 0.57 +/- 0.11, P<0.01) indicating the presence of two different beta-adrenoceptor populations of high (K-D = 0.24 +/- 0.04 nM) and low (K-D = 80 +/- 7 nM) affinity. The low affinity sites were more numerous (B-max = 121 000 +/- 30 000 sites/cell) than the high affinity sites (B-max = 12 000 +/- 1 000 sites/cell). 3 (-)[H-3]-CGP 12177 (25 nM) was displaced by adrenaline (Ad), noradrenaline (NA), isoprenaline (Iso), phenylephrine (Phe) and by the new beta(3) agonist, CL 316 243 (CL) in a biphasic pattern. The order of potency for (-)-[H-3]-CGP 12177 displacement from the small population of high affinity sites (Iso>> NA> Ad>> CL >> Phe was in agreement with a beta(1)/beta(2)-classification. In contrast, the potencies of the same agonists for displacing the radioligand from the low affinity binding sites (CL>> Iso> NA> Ad>> Phe) revealed the presence of a distinct population of adrenoceptors obeying a BB classification. 5-HT did not displace (-)[H-3]-CGP 12177 (25 nM) when used at concentrations as high as 0.1 mM. 4 The beta-adrenoceptor antagonist, (-)-bupranolol, was more effective than (-)-propranolol for displacing (-)[H-3]-CGP 12177 (25 nM) from the high (K-i = 0.029 +/- 0.011 and 0.19 +/- 0.07 nM, respectively and low (K-i = 0.27 +/- 0.04 mu M and 1.6 +/- 0.2 mu M, respectively) affinity binding sites. The selective beta(1)-antagonist CGP 20712A efficiently displaced the radioligand from a small population (K-i = 65 +/- 19 pM) of binding sites, confirming the presence of beta(1)-adrenoceptors. 5 To evaluate whether beta(2)-adrenoceptors could be identified in the population of high affinity binding sites, displacement studies were performed at a low concentration of (-)-[3H]-CGP 12177 (4 nM) that mainly labelled beta(1)/beta(2)-adrenoceptors. ICI 118 551 (a selective beta(2)-antagonist) and procaterol (a selective beta(2)-agonist) displaced (-)-[H-3]-CGP 12177 from its binding sites with very low affinity (K-i = 0.17 +/- 0.02 CIM and K-i = 11 +/- 2 mu M respectively). 6 From these observations, we conclude that: (1) two kinds of binding sites with low and high affinities for (-)-[H-3]-CGP 12177 can be detected in intact brown adipocytes, (2) there are 10 times more low than high affinity B-adrenoceptors, as determined by saturation or competition curve analysis, (3) the high affinity binding sites mainly correspond to beta(1)-adrenoceptors, whereas the low affinity sites represent beta(3)-adrenoceptors, and (4) beta(2)-adrenoceptors are undetectable. 7 It is suggested that the low affinity beta(3)-adrenoceptors represent the physiological receptors for noradrenaline secreted from sympathetic nerve endings when the concentration of the neurohormone in the synaptic cleft is very high and/or when the high affinity beta(1)-adrenoceptors are desensitized by prolonged sympathetic stimulation such as chronic cold exposure.
