Discrimination between putative bradykinin B2 receptor subtypes in guinea pig ileum smooth muscle membranes with a selective, iodinatable, bradykinin analogue
Liebmann, C.; Bossé, R.; Escher, E.
Molecular Pharmacology 46(5): 949-956
1994
ISSN/ISBN: 0026-895X PMID: 7969085 Document Number: 422881
We have synthesized a potent, selective, radioiodinated bradykinin (BK) analogue with high specific radioactivity (1000-1500 Ci/mmol). The new tracer, 125I-(p-Phe-5)BK, was prepared carrier-free from the corresponding nitro precursor, (p-NO-2-Phe-5)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 (B-max = 22 fmol/mg of protein) and a low affinity dissociation constant of 192 pm (B-max = 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-NO-2-Phe-5)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-Phe-5)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-Phe-5)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 B-2 receptor antagonists D-Arg-0-(Hyp-3,Thi-5,D-Tic-7,Oic-8)BK and DArg-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 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.