Characterization of beta 1- and beta 3-adrenoceptors in intact brown adipocytes of the rat.

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, (-)-[3H]-CGP 12177. The potent beta-antagonist, bupranolol (100 microM) was used to determine nonspecific binding. Characterization was essentially performed by saturation and competition studies. 2. The saturation curve of (-)-[3H]-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 (KD = 0.24 +/- 0.04 nM) and low (KD = 80 +/- 7 nM) affinity. The low affinity sites were more numerous (Bmax = 121,000 +/- 30,000 sites/cell) than the high affinity sites (Bmax = 12,000 +/- 1,000 sites/cell). 3. (-)-[3H]-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 (-)-[3H]-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 beta 3-classification. 5-HT did not displace (-)-[3H]-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(- )-[3H]-CGP 12177 (25 nM) from the high (Ki= 0.029 =/- 0.011 and 0.19 +/- 0.07 nM, respectively and low (Ki= 0.27 +/- 0.04 microM and 1.6 +/- 0.2 lM, respectively) affinity binding sites. The selective beta 1 antagonist CGP 20712A efficiently displaced the radioligand from a small population (Ki = 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 (- )-[3H]-CGP 12177 from its binding sites with very low affinity (Ki = 0.17 +/- 0.02 micro M and Ki = 11 +/- 2 micro M respectively).6. From these observations, we conclude that: (1) two kinds of binding sites with low and high affinities for (-)-[H]-CGP 12177 can be detected in intact brown adipocytes, (2) there are 10 times more low than high affinity beta-adrenoceptors, as determined by saturation or competition curve analysis, (3) the high affinity binding sites mainly correspond to beta1-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.