Identification of [3H]prazosin binding sites in crude membranes and isolated cells of brown adipose tissue as alpha 1-adrenergic receptors.

The alpha 1-receptor selective adrenergic antagonist [3H]prazosin was used to study adrenergic binding sites in crude membranes and isolated cells from hamster brown adipose tissue. The antagonist labelled a site which fulfilled the criteria for being the alpha 1-receptor which participates in mediation of a part of the norepinephrine-induced respiration (thermogenesis) in intact cells. The similarity between the characteristics of the binding site in crude membrane fractions and in isolated brown fat cells suggested that the site is of postsynaptic origin. In equilibrium binding studies [3H]prazosin bound with very high affinity (Kd = 0.4 nM), and the maximal binding capacity was 72 fmol/mg protein and 207 fmol/10(6) cells, equal to 120 000 receptors per cell. The kinetically calculated Kd had a value of 0.17 nM, in good agreement with that determined in the equilibrium binding experiments. The relative potencies of adrenergic agents to displace [3H]prazosin revealed a typical alpha 1-specificity: WB-4101 = prazosin greater than phentolamine greater than dihydroergocryptine much greater than yohimbine greater than propranolol for antagonists, and L-phenylephrine = L-norepinephrine = L-epinephrine greater than L-isoprenaline greater than D-norepinephrine for agonists. Thus stereoselectivity was also shown. The actual Ki values for antagonists were closely similar in crude membranes and isolated cells whereas the alpha 1-receptor showed a 10-20 times higher affinity for agonists in the cellular preparation than in the crude membranes. The Ki values for the different antagonists and agonists derived from binding studies in isolated cells were compared with the IC50 and EC50 values for these agents obtained from studies on alpha 1-mediated cellular effects. It is suggested that tight coupling exists between alpha 1-receptor occupancy and alpha 1-mediated effects.