DISCRIMINATION BETWEEN PUTATIVE BRADYKININ B-2 RECEPTOR SUBTYPES IN GUINEA-PIG ILEUM SMOOTH-MUSCLE MEMBRANES WITH A SELECTIVE, IODINATABLE,BRADYKININ ANALOG

We have synthesized a potent, selective, radioiodinated bradykinin (BK ) analogue with high specific radioactivity (1000-1500 Ci/mmol). The n ew tracer, I-125-[p-Phe(5)]BK, was prepared carrier-free from the corr esponding nitro precursor, to-NO(2)Phe(5)]BK, via catalytic hydrogenat ion and halodediazotation. This peptide bound to guinea pig ileum memb ranes in a biphasic pattern, with a high affinity dissociation constan t of 3 pM (B-max = 22 fmol/mg of protein) and a low affinity dissociat ion constant of 192 pM (B-max = 245 fmol/mg of protein). The kinetical ly determined K-d values were 2 pM and 910 pM, respectively. The prope rties of the new tracer and of the peptide analogues [p-iodo-Phe(5)]BK and [p-NO(2)Phe(5)]BK were compared with those of [3,4-H-3(N)] [2,3-p rolyl]BK as label in both saturation and inhibition studies. The resul ts indicated that [p-iodo-Phe(5)]BK possessed increased affinity for t he high affinity site and decreased affinity for the low affinity site , relative to BK. In rat myometrial membranes, in contrast, [p-iodo-Ph e(5)]BK failed to reveal a high affinity site and displayed reduced af finity for the low affinity site, compared with BK. The nitro precurso r was a nonselective ligand with nanomolar affinity for all labeled bi nding sites in both membrane preparations. Measuring the influence of BK and its analogues on guanosine-5'-O-(3-[S-35]thio)triphosphate bind ing to guinea pig ileum membranes, we showed that G proteins were sepa rately activated via both binding sites, qualifying these sites as con stituents of signal transduction pathways and, therefore, real membran e receptors. With the new tracer as label, the B-2 receptor antagonist s D-Arg(0)-[Hyp(3),Thi(5),D-TiC7,OiC(8)]BK and D- Arg(0)]-[Hyp(3),Thi( 5.8),D-Phe(7)]BK recognized both binding sites with very high affinity in guinea pig ileum membranes, classifying these sites as B-2 recepto rs. The BK-induced contraction in guinea pig ileum is obviously mediat ed via the receptor with nanomolar affinity, but the physiological rol e of the high affinity receptor is still unknown.