title: Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells.
abstract: The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.
authors.affiliation: Department of Pharmacology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan. chuenmao@mail.cgu.edu.tw
authors.forename: C M
authors.initials: CM
authors.lastname: Yang
authors.forename: Y J
authors.initials: YJ
authors.lastname: Tsai
authors.forename: S L
authors.initials: SL
authors.lastname: Pan
authors.forename: W B
authors.initials: WB
authors.lastname: Wu
authors.forename: C C
authors.initials: CC
authors.lastname: Wang
authors.forename: Y S
authors.initials: YS
authors.lastname: Lee
authors.forename: C C
authors.initials: CC
authors.lastname: Lin
authors.forename: S C
authors.initials: SC
authors.lastname: Huang
authors.forename: C T
authors.initials: CT
authors.lastname: Chiu
mesh_terms: D000818:Animals; D001011:Aorta; D001920:Bradykinin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling; D002478:Cells, Cultured; D004789:Enzyme Activation; D007295:Inositol Phosphates / Q000378:metabolism; D007609:Kallidin / Q000494:pharmacology; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000187:drug effects* / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009543:Nifedipine / Q000494:pharmacology; D037342:Pertussis Toxin; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000502:physiology; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000187:drug effects* / Q000502:physiology; D010738:Type C Phospholipases / Q000378:metabolism; D014700:Verapamil / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D002121:Calcium Channel Blockers; D007295:Inositol Phosphates; D009124:Muscle Proteins; D010716:Phosphatidylinositols; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D010566:Virulence Factors, Bordetella; C053748:B 4162; D007609:Kallidin; C065679:icatibant; D014700:Verapamil; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D009543:Nifedipine; D001920:Bradykinin; D002118:Calcium