Pharmacological and functional characterization of bradykinin receptors inrat cultured vascular smooth muscle cells

The pharmacological properties of bradykinin receptors were characterized i n rat cultured vascular smooth muscle cells (VSMCs) using [H-3]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dis sociation constant (K-D) of 1.2 +/- 0.2 nM and a maximum receptor density ( B-max) of 47.3 +/-: 4.4 fmol/mg protein. The specific binding of [H-3]-brad ykinin to VSMCs was inhibited by the B-2 receptor-selective agonists (brady kinin and kallidin) and antagonists ([D-Arg(0), Hyp(3), Thi(5), D-Tic(7), O ic(8)]-bradykinin (Hoe 140) and [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-brad ykinin) with an order of potency as kallidin = bradykinin = Hoe 140 >, [D-A rg(0), Hyp(3), Thi(5,8), D-Phe(7)]-bradykinin, but not by a Bi receptor-sel ective agonist (des-Arg(9)-bradykinin) and antagonist ([Leu(8), des-Arg(9)] -bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-d ependent inositol phosphate (IP) accumulation, and initial transient peak o f [Ca2+](i) with half-maximal responses (pECj(50)) were 7.53 and 7.69, resp ectively. B-2 receptor-selective antagonists (Hoe 140 and [D-Arg(0), Hyp(3) , Thi(5,8) D-Phe(7)]-bradykinin) significantly antagonized the bradykinin-i nduced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pr etreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not. alter t he bradykinin-induced inositol phosphate accumulation and [Ca2+](i) changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of res ponses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, vera pamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulate s phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-i nsensitive G-protein in rat VSMCs. Bradykinin B-2 receptors may be predomin antly mediating IP accumulation and subsequently induction of Ca2+ mobiliza tion may function as the transducing mechanism for bradykinin-stimulated co ntraction of vascular smooth muscle.