Characterization of β1‐ and β3‐adrenoceptors in intact brown adipocytes of the rat
Authors: D'Allaire François , Atgié Claude , Mauriège Pascale , Simard Pierre‐Michel , Bukowiecki Ludwik Jan 
Published: July 2012

The binding properties of β1‐, β2‐ and β3‐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 β‐adrenoceptor radioligand, (−)‐[3H]‐CGP 12177. The potent β‐antagonist, bupranolol (100 μm) was used to determine nonspecific binding. Characterization was essentially performed by saturation and competition studies.

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 β‐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).

(−)‐[3H]‐CGP 12177 (25 nm) was displaced by adrenaline (Ad), noradrenaline (NA), isoprenaline (Iso), phenylephrine (Phe) and by the new β3 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 β1/β2‐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 β3‐classification. 5‐HT did not displace (−)‐[3H]‐CGP 12177 (25 nm) when used at concentrations as high as 0.1 mm.

The β‐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 μm and 1.6 ± 0.2 μm, respectively) affinity binding sites. The selective β1‐antagonist CGP 20712A efficiently displaced the radioligand from a small population (Ki = 65 ± 19 pm) of binding sites, confirming the presence of β1‐adrenoceptors.

To evaluate whether β2‐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 β1/β2‐adrenoceptors. ICI 118 551 (a selective β2‐antagonist) and procaterol (a selective β2‐agonist) displaced (−)‐[3H]‐CGP 12177 from its binding sites with very low affinity (Ki = 0.17 ± 0.02 μm and Ki = 11 ± 2 μm respectively).

From these observations, we conclude that: (1) two kinds of binding sites with low and high affinities for (−)‐[3H]‐CGP 12177 can be detected in intact brown adipocytes, (2) there are 10 times more low than high affinity β‐adrenoceptors, as determined by saturation or competition curve analysis, (3) the high affinity binding sites mainly correspond to β1‐adrenoceptors, whereas the low affinity sites represent β3‐adrenoceptors, and (4) β2‐adrenoceptors are undetectable.

It is suggested that the low affinity β3‐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 β1‐adrenoceptors are desensitized by prolonged sympathetic stimulation such as chronic cold exposure.