1 The pharmacological features of rat white adipocyte beta-adrenoceptor subtypes were investigated by saturation and beta-agonist competition studies with [H-3]-CGP 12177 and by lipolysis induced by beta-agonists as well as their inhibition by CGP 20712A (selective beta(1)-antagonist) and ICI 118551 (selective beta(2)-antagonist) in an attempt to establish a relationship between the functionality and binding capacity of beta-adrenoceptor subtypes. 2 Two populations of binding sites were identified on adipocyte membranes, one with high affinity (0.22+/-0.07 nM) and the other with low affinity (23+/-7 nM). The low affinity binding sites constituted 90% of the total binding sites. 3 The competition curves, with 15 nM [H-3]-CGP 12177, for the beta-agonists, isoprenaline (Iso), noradrenaline (NA) and adrenaline (Ad), and the selective beta(3)-agonist, BRL 37344 (BRL), were clearly biphasic (P<0.001). The rank orders of agonist potency (pK(i)) in competing for [H-3]-CGP 12177 high affinity and low affinity binding sites, respectively, were Iso (9.28+/-0.24)> NA (8.90+/-0.12)> Ad (8.65+/-0.12)> > BRL (4.53+/-0.17) and BRL (7.38+/-0.19)> > Iso (2.96+/-0.26)greater than or equal to NA (2.80+/-0.17)> Ad (210+/-0.11) indicating the expression of beta(1)- and beta(3)-adrenoceptor subtypes on rat white adipocytes, respectively. Inversely, competition studies with the selective beta(1)-agonist, xamoterol (Xam), provided evidence for a single homogeneous population of binding sites with low density (81+/-9 fmol mg(-1)) and high pK(i) value (7.23+/-0.26) confirming the presence of beta(1)-adrenoceptors. 4 To assess a possible contribution of the beta(2)-subtype, procaterol (Proc), a selective beta(2)-agonist, was used to compete with 2 nM [H-3]-CGP 12177. A single low affinity (4.61+/-0.07) population of binding sites was identified. The density of these sites (71+/-12 fmol mg(-1)) was similar to the one obtained with Xam, suggesting that Proc displaced [H-3]-CGP 12177 from the beta(1)-subtype. 5 The functional potency (pD(2)) order with BRL (9.07+/-0.20) and catecholamines (Iso: 7.26+/-0.06, NA: 6.89+/-0.02 and Ad: 6.32+/-0.07) was the same as that found for the low affinity binding sites in competition studies. Xam induced lipolysis with greater potency than dobutamine (Dob), 6.31+/-0.06 and 5.66+/-0.10, respectively. Proc stimulated lipolysis with a low potency (5.59+/-0.21). 6 The lipolytic response to 0.001 mu M BRL was inhibited by both, selective beta(1)- and beta(2)-antagonist, in a monophasic manner with low potencies (CGP 20712A pK(i): < 4.5 and ICI 118551 pK(i): 5.57+/-0.13). Similar monophasic profiles were obtained for inhibition of Xam- and Dob-induced lipolysis. In this case, CGP 20712A was more potent (> 10 times) than ICI 118551. The monophasic inhibition was also observed with ICI 118551 in the presence of 0.05 mu M Iso or 0.13 mu M NA. In contrast, two populations of sites were identified with CGP 20712A in the presence of Iso as well as NA. The pK(i) values for the first sites were 8.41+/-0.09 and 8.58+/-0.17, respectively, and for the second population of sites 4.73+/-0.22 and 4.27+/-0.27, respectively. The proportion of the first sites was low: 19+/-4 and 22+/-5%, respectively. Biphasic curves were obtained with both antagonists using 2.5 mu M Proc (CGP 20712A: pK(i)1: 8.17+/-0.08, site 1: 23+/-6%, pK(i)2: 4.77+/-0.14; ICI 118551: pK(i)1: 7.78+/-0.03, site 1: 37+/-2%, pK(i)2: 5.35+/-0.25). 7 Our results show that the radioligand [H-3]-CGP 12177 allows the characterization of beta(1)- and beta(3)-adrenoceptor subtypes on rat white adipocytes. Lipolysis is highly dependent on beta(1)- and beta(3)-adrenoceptors. Finally, binding and functional studies confirm that lipolysis is mainly driven by the beta(3)-subtype.
