Direct assessment of β‐adrenergic receptors in intact rat adipocytes by binding of [3H]CGP 12177

Open Access
Publisher Website
Google Scholar
Abstract
The characteristics of the binding of the hydrophilic β-adrenergic antagonist [3H]CGP 12177 to intact rat adipocytes were studied at 37°C and 6°C. At both temperatures and at 90% saturation, the non-specific binding was less than 30% of the total binding. At 37°C, specific [3H]CGP 12177 binding was rapid, reversible of high affinity (1.8 ± 0.4 nM) and saturable. The number of specific binding sites per adipocyte increased with the fat cell size (about 35000 and 115000 sites per cell in adipocytes with diameters of 60 μm and 88 μm, respectively) but remained constant when expressed per unit fat cell surface area. Displacement of [3H]CGP 12177 bound to adipocytes by unselective and selective β-antagonists was stereospecific, had the same characteristics as those found in adipocyte membranes and showed a heterogeneous specificity for β1 and β2 adrenergic subtypes. In contrast, β agonist competition curves, Which modeled to two affinity-states of binding, showed high-affinity-state Kd values for agonists 10–25-times higher than those found in membranes under the same experimental conditions. At 6°C, although the number and affinity of the specific binding sites for [3H]CGP 12177 were the same as those found at 37°C, the Kd value for (–)-isoproterenol binding to the high affinity state of these sites (3.0±0.5 nM) was 25-times lower than at 37°C and similar to the value found in membrane preparations (1.5–4 nM). These results show that the [3H]CGP 12177 specific binding sites detected on intact adipocytes represent the physiological β-adrenergic receptors. Moreover, this study extends to the adipocyte the validity of the model recently proposed for other cell lines, according to which in intact cells, but not in membranes, agonist-binding promotes a rapid and temperature-dependent conformational change of the β-adrenergic receptors, leading to a progressive loss of capacity of agonists to form a high-affinity complex.