High-affinity bradykinin receptor-catalyzed G protein activation in rat myometrium

Binding of the labelled GTP analog, guanosine-5'-O-(3-thiotriphosphate ([35S]GTP[S] to G proteins was studied in rat myometrial membranes in the presence of GDP (1 microM). Binding was stimulated by bradykinin at subnanomolar concentrations. while oxotremorine increased binding of [35S]GTP[S] to myometrial membranes at micromolar concentrations. The bradykinin-induced stimulation was antagonized by the receptor antagonist, [D-Arg-(Hyp3, Thi5,8, D-Phe7)]bradykinin. Addition of NaCl (150 nM) decreased control binding and abolished the stimulatory effect of bradykinin. On the other hand, addition of CaCl2 (5 mM) had no effect on control binding but also prevented the bradykinin-induced increased in [35S]GTP[S] binding. Saturation experiments revealed that activation of the bradykinin receptor leads to about a three-fold increase in the apparent GTP[S] binding affinity of about 30% of the total GTP[S] binding sites measured in these membranes. The results provide evidence for a high-affinity bradykinin receptor in rat myometrial membranes which interacts with and activates G proteins. This receptor action, which appears to be under the control of both sodium and calcium ions, is catalytic and leads to a large signal amplification, in that one agonist-liganded bradykinin receptor can apparently activate up to 100 G proteins.