A robust bioassay of the human bradykinin B₂ receptor that extends molecular/cellular studies: The isolated umbilical vein

Bradykinin (BK) and kallidin (Lys-BK) are small peptides cleaved from circulating kininogens by proteases called kallikreins. BK-related peptides are vasodilators, increase microvascular permeability and stimulate nociceptors, reproducing the cardinal signs of inflammation. Medicinal chemistry efforts targeted at their widely expressed B₂ receptor (B₂R), a GPCR, mainly aimed at producing antagonists. The only BK antagonist in clinical use is the peptide icatibant, approved to abort attacks of hereditary angioedema. However, the anti-inflammatory applications of B₂R antagonists are potentially wider. While the screening of new compounds relies on in vitro assays, like radioligand binding competition, classical smooth muscle contractility supports pharmacological evaluation in a time scale of several hours and allows determining potency, surmountability, residual agonist activity, specificity and reversibility. Further, the BK B₂R antagonists notoriously exhibit species-specific pharmacological profiles. When investigating the first non-peptide B₂R antagonist (WIN 64338), I have introduced the contractility assay based on the isolated human umbilical vein to evaluate ligands of the naturally expressed human B₂R. Small molecule ligands characterized using the assay include the partial agonist Fujisawa compound 47a or the exquisitely potent competitive antagonist, Pharvaris compound 3. The umbilical vein assay is also useful to verify pharmacologic properties of peptide B₂R ligands, such as the carboxypeptidase-activated latent agonist BK-Arg and agonist or antagonist fluorescent probes. Further, the proposed agonist effect of tissue kallikrein on the B₂R has been disproved using the vein. This assay has an intermediate level of complexity between cellular and molecular pharmacology on one hand, and in vivo studies on the other.