Guanine Nucleotide Regulation of B2 Kinin Receptors

We have examined the binding of [3H]bradykinin to bovine myometrial membranes and assessed its sensitivity to guanine nucleotides. Total binding displayed a typical B2 kinin receptor specificity. However, saturation binding isotherms were resolved into at least two components with Ko values of 8 pM (45%) and 378 PM (55%). Low affinity binding exhibited relatively rapid rates of association (12,bs = 1.40 X lo-’ s-‘) and dissociation (kel = 3.82 x 10e3 s-l), while high affinity binding exhibited considerably slower rates (Iz,b = 9.52 x 10m4 s-l and k1 = 4.43 x lo-’ s-l). Pre-equilibrium dissociation kinetics revealed that formation of high affinity binding was characterized as a time-dependent accumulation of the slow dissociation rate at the expense of at least one other more rapid dissociation rate. In the presence of 10 pM guanyl-5’-yl imidodiphosphate (Gpp(NH)p), at least two binding components were resolved with KD values of 37 pM (12%) and 444 PM (88%). Gpp(NH)p apparently specifically perturbed high affinity binding by completely preventing the accumulation of the slow dissociation phase. Instead, two more rapid dissociation rates (km1 = 8.53 x 10m3 s-’ and 4.43 x lo-’ s-‘) were observed. These results suggest that [3H]bradykinin interacts with at least two B2 kinin receptor-like binding sites in bovine myometrial membranes. A three-state model for the guanine nucleotide-sensitive agonist interaction with the high affinity binding sites is proposed.