Binding of agonists and antagonists to β-adrenoceptors in rat vas deferens: relationship to functional response

The properties of β-adrenoceptors in rat vas deferens were examined using radioligand binding assays of ¹²⁵I-pindolol (¹²⁵IPIN) and inhibition of electrically-evoked contractions of vas deferens in vitro. ¹²⁵IPIN labelled a single class of high affinity binding sites with apparently mass action kinetics in membrane preparations of vas deferens with properties consistent with an essentially homogeneous population of β₂-adrenoceptors. Isoprenaline inhibited electrically evoked (60 V, 1.0 ms, 0.1 Hz) contractions of vas deferens with an EC₅₀ of 18.0±2.1 nM. K_B values for antagonists in competitively antagonizing this response correlated well (r²=0.99) with the K_D values for inhibition of ¹²⁵IPIN binding. Inhibition of ¹²⁵IPIN binding by isoprenaline, adrenaline, noradrenaline and salbutamol was determined under conditions designed to produce high and low affinity agonist binding. In the presence of 10 mM MgCl₂, agonists inhibited specific ¹²⁵IPIN binding with a relatively high potency and low Hill slope, while in the presence of 154 mM NaCl and 300 μM guanosine-5′-triphosphate, agonists inhibited specific ¹²⁵IPIN binding with a lower potency and an apparent Hill slope closer to 1. To determine which affinity state was relevant to functional receptor stimulation, receptor density was decreased with bromoacetylalprenololmenthane (BAAM). Treatment of membrane preparations with 0.3 μM BAAM produced a 45% decrease in the B_max for ¹²⁵IPIN with no change in the apparent K_D. Treatment of intact vasa deferentia with increasing concentrations of BAAM resulted in a progressive rightward shift in the dose-response curve to isoprenaline or salbutamol folowed by a decreased maximum response. K_A values for isoprenaline and salbutamol in activating the functional β-adrenoceptors were compared with K_I values for agonist inhibition of specific ¹²⁵IPIN binding. The K_A values for both agonists were not significantly different from the low affinity K_I values, but were significantly different from the high affinity K_I values. These data suggest that 1) a homogeneous population of β₂-adrenoceptors inhibiting contraction of rat vas deferens can be labelled with ¹²⁵IPIN, 2) there is a substantial β-adrenoceptor reserve in rat vas deferens; and 3) the initial event in signal transduction by β-adrenoceptors in rat vas deferens is the binding of agonists to the low affinity form of the receptor which is not complexed with the guanine nucleotide binding protein.