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 [³H]-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 B₁ receptor-selective agonist (des-Arg ⁹-BK) nor antagonist ([Leu⁸, des-Arg⁹]-BK) significantly inhibited [³H]-BK binding to CECs, thus excluding the presence of B₁ receptors in canine CECs. The specific binding of [³H]- BK to CECs was inhibited by B₂ receptor-selective agonists (BK and kallidin) and antagonists (Hoe 140 and [D-Arg⁰, Hyp³, Thi^5,8, D-Phe⁷]-BK), with a best fit using a one-binding-site model. The order of potency for the inhibition of [³H]-BK binding was BK = Hoe 140 > kallidin > [D-Arg⁰, Hyp³, Thi^5,8, D-Phe⁷]-BK. Stimulation of CECs by BK produced a concentration-dependent accumulation of inositol phosphates (IP) and an initial transient peak of intracellular Ca²⁺. B₂ receptor-selective antagonist ([D- Arg⁰, Hyp³, Thi^5,8, D-Phe⁷]-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 Ca²⁺ 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 B₂ receptors via a PTX-insensitive G protein in canine CECs. This effect may function as the transducing mechanism for BK-mediated cellular responses.