Differential effects of B2 receptor antagonists upon bradykinin‐stimulated phospholipase C and D in guinea‐pig cultured tracheal smooth muscle

Abstract

1 Guinea-pig tracheal smooth muscle cells were isolated and maintained in culture for 14–21 days prior to the study of the effect of a selective bradykinin B₁ agonist and B₂ antagonists upon bradykinin-stimulated phospholipase C and D activities. 2 Bradykinin-stimulated phospholipase C activity was determined by mass measurement of inositol (1,4,5)trisphosphate (Ins(1,4,5)P₃) in unlabelled cells, whereas phospholipase D activity was assayed by the accumulation of [³H]-phosphatidylbutanol ([³H]-PtdBut) in [³H]-palmitate-labelled cells, which were stimulated in the presence of butan-1-ol (0.3%, v/v). 3 Bradykinin elicited the rapid and transient formation of Ins(1,4,5)P₃, in a concentration-dependent manner (log EC₅₀ = −7.55 ± 0.1 m, n = 3). Bradykinin also rapidly activated the concentration-dependent (log EC₅₀ = −8.3 ± 0.4 m, n = 3) phospholipase D-catalysed accumulation of [³H]-PtdBut; the accumulation of [³H]-PtdBut was sustained. These effects were not inhibited by pretreatment of the cells with indomethacin (1 μm). 4 The bradykinin B₁ agonist, desArg⁹-bradykinin (1 μm) was without effect upon phospholipase C or phospholipase D activity. Bradykinin-stimulated (10 nm, EC₄₀) Ins(1,4,5)P₃ formation was inhibited by B₂ receptor antagonists, d-Arg-[Hyp³,d-Phe⁷]-bradykinin (NPC 567) and d-Arg-[Hyp³,Thi^5,8,d-Phe⁷]-bradykinin (NPC 349), with log IC₅₀ values of −6.3 ± 0.5 m and −6.3 ± 0.4 m, respectively. However, bradykinin-stimulated (10 nm, EC₁₀₀) [³H]-PtdBut accumulation was poorly inhibited and with low potency by each B₂ receptor antagonist and bradykinin-stimulated phospholipase D activity persisted at concentrations of antagonist that completely blocked bradykinin-stimulated Ins(1,4,5)P₃ formation (30 μm). 5 These observations suggest that the activation of phospholipase C by bradykinin may be mediated through a bradykinin B₂ receptor population, whereas bradykinin-stimulated phospholipase D may be activated via a distinct population of bradykinin receptors that do not appear to be either B₁ or B₂ receptor types, based upon pharmacological specificity. The mechanism of the activation of phospholipase D by bradykinin and the role of the putative B₃ bradykinin receptor are discussed.