Biochemical Analysis and Solubilization of Central ₁-Adrenoceptors in the Rat

¹ From the Département de Pharmacologie, Hôpital Necker, Paris, France

To study the structure and the molecular mechanisms of action of brain -adrenoceptors, their solubilization was undertaken. ₁-Adrenoceptors were first successfully solubilized from fresh rat brain membranes by treatment with 0.3 percent deoxycholate, after prelabeling of the binding site by the highly specific tritiated antagonist ³H-prazosin. The complex thus solubilized underwent a rapid loss of activity at 25°C. Direct solubilization of brain ₁-adrenoceptors was obtained by treatment with a new zwitterionic derivative of cholic acid (CHAPS) at a concentration of 5 to 10 mM. The soluble complex was detected by precipitation by polyethylene glycol 6,000 with gamma globulin as a carrier. Binding of ³H-prazosin at 25°C was rapid; at 4°C the steady state was obtained within two hours and remained unchanged for at least six hours. The affinity of the soluble binding site, determined by Scatchard analysis (0.6 nM), varied with the concentration of detergent. Specificity of the membrane-bound receptor was preserved as demonstrated by incubation in the presence of ₁- and ₂-antagonists at various concentrations (by order of potency: prazosin > phentolamine > yohimbine). Stereoselectivity was also retained in the solubilized binding protein. The solubilization of an active brain ₁-adrenoceptor will allow further investigation at the molecular level.