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.
D'Allaire
F
F
Department of Physiology, Faculty of Medicine, Laval University, Québec, PQ, Canada.
Atgié
C
C
Mauriège
P
P
Simard
P M
PM
Bukowiecki
L J
LJ
D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D002001:Adipose Tissue, Brown / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D011412:Propanolamines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism*; D018342:Receptors, Adrenergic, beta-1 / Q000187:drug effects / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000187:drug effects / Q000378:metabolism; D022702:Receptors, Adrenergic, beta-3
D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D011412:Propanolamines; D011943:Receptors, Adrenergic, beta; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D022702:Receptors, Adrenergic, beta-3; C033394:CGP 12177