Discrimination between putative bradykinin B2 receptor subtypes in guinea pig ileum smooth muscle membranes with a selective, iodinatable, bradykinin analogue.

We have synthesized a potent, selective, radioiodinated bradykinin (BK) analogue with high specific radioactivity (1000-1500 Ci/mmol). The new tracer, 125I-[p-Phe5]BK, was prepared carrier-free from the corresponding nitro precursor, [p-NO2-Phe5]BK, via catalytic hydrogenation and halodediazotation. This peptide bound to guinea pig ileum membranes in a biphasic pattern, with a high affinity dissociation constant of 3 pM (Bmax = 22 fmol/mg of protein) and a low affinity dissociation constant of 192 pM (Bmax = 245 fmol/mg of protein). The kinetically determined Kd values were 2 pM and 910 pM, respectively. The properties of the new tracer and of the peptide analogues [p-iodo-Phe5]BK and [p-NO2-Phe5]BK were compared with those of [3,4-3H(N)] [2,3-prolyl]BK as label in both saturation and inhibition studies. The results indicated that [p-iodo-Phe5]BK possessed increased affinity for the high affinity site and decreased affinity for the low affinity site, relative to BK. In rat myometrial membranes, in contrast, [p-iodo-Phe5]BK failed to reveal a high affinity site and displayed reduced affinity for the low affinity site, compared with BK. The nitro precursor was a nonselective ligand with nanomolar affinity for all labeled binding sites in both membrane preparations. Measuring the influence of BK and its analogues on guanosine-5'-O-(3-[35S]thio)triphosphate binding to guinea pig ileum membranes, we showed that G proteins were separately activated via both binding sites, qualifying these sites as constituents of signal transduction pathways and, therefore, real membrane receptors. With the new tracer as label, the B2 receptor antagonists D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]BK and D-Arg0-[Hyp3,Thi5,8,D-Phe7]BK recognized both binding sites with very high affinity in guinea pig ileum membranes, classifying these sites as B2 receptors. The BK-induced contraction in guinea pig ileum is obviously mediated via the receptor with nanomolar affinity, but the physiological role of the high affinity receptor is still unknown.