Agonist-induced modulation of agonist binding to α₁-adrenoceptors in bovine aorta

Prolonged exposure of tissues to hormone agonists results in a subsequent reduction in the sensitivity of the tissue through a process known as desensitization. The desensitization response, either homologous or heterologous, has been shown to be correlated with receptor phosphorylation. Recently we have provided evidence that protein kinase C, when activated by a phorbol ester, regulates α1-adrenoceptor coupling to a G-protein. In the present study, the effects of epinephrine (10 μM) pretreatment on the binding behavior of the α1-adrenoceptor were determined from radioligand binding assays at 25° and 2°C. Pretreatment of tissues with epinephrine for 25 min moderately decreased [3H]prazosin binding by 12% (Bmax 121.5 fmol/mg) in comparison to control (139.3 fmol/mg) with no change in its affinity. The second consequence of desensiization by epinephrine is a decrease in the affinity of agonist binding to α1-adrenoceptors associated with uncoupling of the receptors from the G-protein. In control membranes, at 25°C, epinephrine defined two different affinity states of the receptor, viz. high affinity (KDH 16.5 nM, % RH 21) and low affinity (KDL 710 nM, % RL 79). The high affinity state formed at 25°C is stabilized by forming a ternary complex with a G-protein. Addition of guanylylimidodiphosphate (Gpp(NH)p) reduced the stability of this complex resulting in a loss of high affinity sites in control membranes. On the other hand, epinephrine treated membranes exhibited only a single class of low affinity agonist binding (KDH 659 nM) and further, Gpp(NH)p had no significant effect on binding. In low-temperature binding studies, both treated and untreated preparations exhibited a large proportion of sites (% RH 88 and 87) in the high affinity state indicating that receptors retain the ability to achieve a high affinity conformation, as established by the reduced temperature. Our results indicate that epinephrine desensitization includes a selective loss in the ability of G-proteins to stabilize a high affinity state of the α1-adrenoceptor. This effect of agonist preexposure, thus, resembles receptor/G-protein uncoupling described previously following protein kinase C activation.