Characterization of bradykinin receptors in canine cultured corneal epithelial cells: pharmacological and functional studies.
The pharmacological properties of bradykinin (BK) receptors were characterized in canine cultured corneal epithelial cells (CECs) using [(3)H]-BK as a radioligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant of 0.34 +/- 0.07 nM and a maximum receptor density of 179 +/- 23 fmol/mg protein. Neither a B(1) receptor-selective agonist (des-Arg(9)-BK) nor antagonist ([Leu(8), des-Arg(9)]-BK) significantly inhibited [(3)H]-BK binding to CECs, thus excluding the presence of B(1) receptors in canine CECs. The specific binding of [(3)H]-BK to CECs was inhibited by B(2) receptor-selective agonists (BK and kallidin) and antagonists (Hoe 140 and [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK), with a best fit using a one-binding-site model. The order of potency for the inhibition of [(3)H]-BK binding was BK = Hoe 140 > kallidin > [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK. Stimulation of CECs by BK produced a concentration-dependent accumulation of inositol phosphates (IP) and an initial transient peak of intracellular Ca(2+). B(2) receptor-selective antagonist ([D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK) significantly antagonized the BK-induced responses with dissociation constants of 6.0-6.1. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin did not alter the BK-induced IP accumulation. Incubation of CECs in the absence of external Ca(2+) led to a significant attenuation of the IP accumulation induced by BK. These results demonstrate that BK directly stimulates phospholipase C-mediated signal transduction through BK B(2) receptors via a PTX-insensitive G protein in canine CECs. This effect may function as the transducing mechanism for BK-mediated cellular responses.
Huang
Samuel C M
SC
Department of Ophthalmology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, ROC.
Hsiao
Li-Der
LD
Chien
Chin-Sung
CS
Wang
Chuan-Chwan
CC
Chiu
Chi-Tso
CT
Tsai
Ray J F
RJ
Yang
Chuen-Mao
CM
D000818:Animals; D001920:Bradykinin / Q000737:chemistry / Q000494:pharmacology; D065168:Bradykinin Receptor Antagonists; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D007609:Kallidin / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D011869:Radioligand Assay; D018002:Receptors, Bradykinin / Q000819:agonists / Q000378:metabolism*; D014316:Tritium / Q000737:chemistry / Q000378:metabolism
D065168:Bradykinin Receptor Antagonists; D018002:Receptors, Bradykinin; D014316:Tritium; D007609:Kallidin; D002772:Cholera Toxin; D037342:Pertussis Toxin; D001920:Bradykinin; D002118:Calcium