A Robust Bioassay of the Human Bradykinin B2 Receptor that Extends Molecular and Cellular Studies: The Isolated Umbilical Vein

Abstract

Bradykinin (BK) has various physiological and pathological roles. Medicinal chemistry efforts targeted toward the widely expressed BK B₂ receptor (B₂R), a G-protein-coupled receptor, were primarily aimed at developing antagonists. The only B₂R 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. Furthermore, the B₂R antagonists notoriously exhibit species-specific pharmacological profiles. Classical smooth muscle contractility assays are exploited over a time scale of several hours and support determining potency, competitiveness, residual agonist activity, specificity, and reversibility of pharmacological agents. The contractility assay based on the isolated human umbilical vein, expressing B₂R at physiological density, was introduced when investigating the first non-peptide B₂R antagonist (WIN 64338). Small ligand molecules characterized using the assay include the exquisitely potent competitive antagonist, Pharvaris Compound 3 or the partial agonist Fujisawa Compound 47a. The umbilical vein assay is also useful to verify pharmacologic properties of special peptide B₂R ligands, such as the carboxypeptidase-activated latent agonists and fluorescent probes. Furthermore, the proposed agonist effect of tissue kallikrein on the B₂R has been disproved using the vein. This assay stands in between cellular and molecular pharmacology and in vivo studies.

Keywords: B2 receptor; bradykinin; human umbilical vein; kallikrein-kinin system.

Figure 1

(A) Immunohistochemistry for angiotensin-I converting enzyme (ACE) and cell markers (monoclonal anti-α-actin for the smooth muscle cells, polyclonal anti-von Willebrand factor (vWF) for the endothelium) in paraffin sections of the human umbilical vein (100×, approximate width of rectangular fields 700 µm). The dark precipitates indicate positive cells. The position of tunicae intima, media and adventitia are indicated (the intima by arrows). Modified from [63] with permission. (B) Left: Construction of a cumulative concentration–effect curve for BK-induced contraction by using a ring of human umbilical vein pre-equilibrated for 3 h. Abscissa scale: time; ordinate scale: isometric contraction, grams. △ indicates the application of BK (cumulative nanomolar concentration indicated) and ▼ the first of a series of washouts. Right tracing recorded in the same tissue at time 5 h: the antagonist icatibant at a high concentration reduced the sensitivity to BK, but not its maximal effect. This experiment was part of a project approved by the local ethics committee (CHU de Québec-Université Laval, File no. 2017-3720). Umbilical cords were obtained following elective caesarean sections. Informed consent was obtained from the mothers.

Figure 2

Structure of selected nonpeptide ligands of the B₂R. Modified from [65] and [36] with permission.

Figure 3

(A) Effect of the nonpeptide antagonist of the B₂R, Compound 19c, on bradykinin (BK)-induced contraction in the human isolated umbilical vein. Each tissue was subjected to the construction of two full cumulative concentration-effect curves, in the absence of antagonist (3 h, not shown) and in the presence of an antagonist or its DMSO vehicle applied 30 min earlier (5 h). Values are the means ± s.e.m. of the number determinations indicated by n. For the highest concentration of the antagonist, the maximal effect of BK was evaluated from the separate curve constructed at 3 h. (B) Schild plot analysis for Compounds 19c; the x-axis intercepts of the regressions for 2 other nonpeptide antagonists that span the whole potency range (Table 1) are also shown for comparison. Modified from [45] with permission.

Figure 4

Effect of low-molecular-weight kininogen (LK) replenishment (9.4 nM applied between the second and third stimulation) on tissue kallikrein (KLK-1)-induced contraction in the isolated human umbilical vein. LK restored the effect of the protease in desensitized tissues. Presentation as in Figure 1B. Modified from [81] with permission.

Figure 5

Bradykinin C-terminally prolonged by Arg (BK-Arg), an example of a peptidase-activated latent B₂R agonist. (A) BK-Arg is designed to be activated by widely distributed arginine carboxypeptidases (Arg-CP); Plummer’s inhibitor is an arginine analog that inhibits these peptidases with selectivity. (B) BK-Arg has a low affinity for the B₂R, as evidenced by the competition of the [³H]BK binding to a recombinant form of the B₂R in an assay conducted at 0 °C. (C,D) Plummer’s inhibitor has no significant effect on the potency of BK in the umbilical vein contractility assay, whereas it reduces that of BK-Arg 15-fold, supporting its metabolism into BK in the tissue. E. In anesthetized rats, the hypotensive effect of BK-Arg (measured as the maximal drop of mean arterial pressure, ΔMAP) is inhibited by pretreatment with either Plummer’s inhibitor or the B₂R antagonist icatibant. Plummer’s inhibitor had no effect on the hypotensive effect of BK [73] or on the contractile effect of BK (C) because Arg-CPs are a minor metabolic pathway for BK inactivation. Red arrows (D,E) emphasize the effect of Arg-CP blockade. Modified from [73] (A,E) and [89] (B–D). Values are means ± s.e.m. of the numbers of replicates indicated between parentheses.