Abstract

Unlike the widely distributed and preformed B₂ receptors, the bradykinin B₁ receptors exhibit a highly regulated expression and minimal agonist-induced endocytosis. To evaluate the potential usefulness of fluorescent B₁ receptor probes applicable to live cell microscopy and cytofluorometry, combined chemical synthesis and pharmacologic evaluation have been conducted on novel 5(6)-carboxyfluorescein [5(6)CF]-containing peptides. Representative agents are the antagonist B-10376 [5(6)CF-ϵ-aminocaproyl-Lys-Lys-[Hyp³, CpG⁵, d-Tic⁷, CpG⁸]des-Arg⁹-bradykinin] and the agonist B-10378 [5(6)CF-ϵ-aminocaproyl-Lys-des-Arg⁹-bradykinin]. B-10376 has a K_i of 10 to 20 nM to displace [³H]Lys-des-Arg⁹-bradykinin from rabbit or human recombinant B₁ receptors expressed in human embryonic kidney (HEK) 293 cells and is a surmountable antagonist in the rabbit aorta contractility assay (pA₂, 7.49). B-10378 was a full agonist at the naturally expressed B₁ receptor (rabbit aorta contraction, calcium transients in human smooth muscle cells) and had a binding competition K_i of 19 or 89 nM at the recombinant rabbit or human receptor, respectively. Both fluorescent probes can label with specificity human or rabbit B₁ receptors expressed in HEK 293 cells (epifluorescence or confocal microscopy), but the agonist was associated with discontinuous plasma membrane labeling, which coincided with that of a red-emitting caveolin-1 conjugate. Cytofluorometry with B-10376 was applied to recombinant and, in human vascular smooth muscle cells, to naturally expressed B₁ receptors. In all fluorescent applications, the specific labeling was reduced by an excess of a B₁ receptor nonpeptide antagonist. Despite the loss of affinity determined by the introduction of a fluorophore in B₁ receptor agonist or antagonist peptides, the resulting agents allow original applications (imaging in live cells, cytofluorometry).