Determination of adrenoceptors of the alpha2-subtype on isolated human fat cells

Binding state for the selective alpha2-adrenergic antagonist (3H)-yohimbine on isolated intact human subcutaneous abdominal fat cells is described in the present study. Specific binding was rapid, reversible, saturable and stereospecific. Adrenergic antagonists competed with (3H)-yohimbine binding in the order indicative of alpha-receptors. Competition studies with adrenergic antagonists revealed that (3H)-yohimbine binding sites displayed properties consistent with those of alpha2-subtype. Saturation experiments demonstrated that (3H)-yohimbine specific binding was of high affinity with a dissociation constant (Kd) of 3.7 ± 0.4 nmol/l and a maximum occupancy (B0) of 10.5 ± 2.1 pmol/107 cells. The latter corresponded to approximately 600,000 alpha2-receptors/fat cell. A significant (P < 0.002) linear relationship was found between the fat cell volume and B0. A complete saturation experiment for the determination of Kd and B0 could be performed using 0.5 g adipose tissue (0.5 ml isolated fat cells), which is 30 times less required when adipocyte membranes are used for these studies. It is concluded that the method can be used for the study of alpha2-receptor properties and regulation in subcutaneous adipose tissue in a given individual.