Non-competitive pharmacological antagonism at the rabbit B(1) receptor

Abstract

The B(1) receptor for kinins, stimulated by kinin metabolites without the C-terminal Arg residue (e.g., des-Arg(9)-bradykinin (BK) and Lys-des-Arg(9)-BK), is an increasingly recognized molecular target for the development of analgesic and anti-inflammatory drugs. Recently developed antagonists of this receptor were compared to a conventional antagonist, Ac-Lys-[Leu(8)]-des-Arg(9)-BK, in pharmacological assays based on the rabbit B(1) receptor. B-9858 (Lys-Lys-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]des-Arg(9)-BK) and three other analogues possessing the alpha-2-indanylglycine(5) (Igl(5)) residue (order of potency B-9858 approximately B-10146>B-10148>B-10050) were partially insurmountable antagonists of des-Arg(9)-BK in the contractility assay based on rabbit aortic rings. B-9858-induced depression of the maximal effect was more pronounced in tissues treated with the protein synthesis inhibitor cycloheximide to block the spontaneous increase of response attributed to the post-isolation formation of B(1) receptors, and only partly reversible on washing. By comparison, Ac-Lys-[Leu(8)]des-Arg(9)-BK was a surmountable antagonist (pA(2) 7. 5), even in cycloheximide-treated tissues. B-9958 (Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK) was also surmountable (pA(2) 8.5). The binding of [(3)H]-Lys-des-Arg(9)-BK to recombinant rabbit B(1) receptors expressed in COS-1 cells was influenced by two of the antagonists: while Ac-Lys-[Leu(8)]des-Arg(9)-BK competed for the radioligand binding without affecting the B(max), B-9858 decreased the B(max) in a time-dependent and washout-resistant manner. B-9858 and analogues possessing Igl(5) are the first reported non-competitive, non-equilibrium antagonists of the kinin B(1) receptor.

Figure 1

Change in the concentration-effect curve of the B₁ receptor agonist des-Arg⁹-BK as a function of the incubation time in rabbit aortic rings. Responses are expressed as per cent of the maximal effect of the peptide in a control recording established at time 3.5 h in each tissue. Values are the means, and vertical bars the s.e.mean of 36 determinations.

Figure 2

Effect of six B₁ receptor antagonists or their saline vehicle on des-Arg⁹-BK-induced contraction in the rabbit isolated aorta at time 5.5 h. Responses are expressed as a per cent of the maximal effect of des-Arg⁹-BK in a control recording established at time 3.5 h in each tissue.

Figure 3

Effect of protein synthesis inhibition applied to rabbit aortic rings after the construction of the initial concentration-effect curve of des-Arg⁹-BK, but before the subsequent curve recorded in the presence or absence of the antagonists B-9858 or Ac-Lys-[Leu⁸]des-Arg⁹-BK. Responses are expressed as in Figures 1 and 2.

Figure 4

Contractile effect of des-Arg⁹-BK (11.1 μM) on rabbit aortic rings, as modified by incubation time (control response recorded at 3.5 h), the presence of the antagonist B-9858 (54 nM in the time window 5–6 h in all tissues) and by the presence of cycloheximide (CHX, 71 μM, from the time point 4.5 h on in some tissues only). Values are expressed as per cent of the control contraction and are the means±s.e.mean of nine determinations. See text for statistics.

Figure 5

Binding of [³H]-Lys-des-Arg⁹-BK to recombinant rabbit B₁ receptors expressed in COS-1 cells as influenced by antagonist drugs (A). Saturation curves obtained without antagonist (Control) or in the presence of Ac-Lys-[Leu⁸]des-Arg⁹-BK (10 nM) or of B-9858 (0.54 nM). Antagonist drugs were introduced at the same time as the radioligand. In panel A, the ligand binding to untransfected cells is also shown (B). Saturation curves obtained in a different transfection with the same drugs; the cells were incubated with the antagonists in the culture medium for 30 min before initiating the binding assay. (C) Scatchard plots derived from the saturation data from panel B. (D) Effect of preincubation with antagonists followed by drug washout on the radioligand binding. See text for details and data analysis.