Non classical, multiple-site interaction of [3H]-prazosin with the alpha 1-adrenoceptor of intact BC3H1 cells.

1. In intact BC3H1 cells the EC50 of noradrenaline (NA) for the inositol phosphate response measured at 37 degrees C (EC50 = 193 nM) was much lower than its apparent dissociation constant (Ki37 degrees C = 83.211 microM) determined at this temperature by [3H]-prazosin binding. 2. After pretreatment of the cells with NA at 37 degrees C for 45 min, the time used in binding assays at this temperature, this difference between EC50 and Ki37 degrees C did not decrease significantly. An agonist-induced reduction in alpha 1-adrenoceptor affinity can therefore not explain the very high Ki37 degrees C value. 3. NA pretreatment at 37 degrees C decreased the number of [3H]-prazosin binding sites (assessed by whole cell binding at 2 degrees C) by only 49%; not by 100%, the value expected if agonist-induced receptor internalization were the origin of the very low Ki37 degrees C. 4. The EC50 of NA for the inositol phosphate response in the presence of 156 pM [3H]-prazosin was 1.841 microM but the IC50 of NA for the inhibition of [3H]-prazosin binding (126 pM) was 316 microM. As there is no alpha 1-adrenoceptor reserve in these cells we propose that at 37 degrees C [3H]-prazosin interacts, not only with the catecholamine recognition site (site 1) of the receptor, but also reacts weakly with another site from which it cannot be directly displaced by catecholamine-like substances (site 2).