Mechanisms of bradykinin-mediated Ca2+ signalling in canine cultured corneal epithelial cells

Experiments were designed to differentiate the mechanisms of bradykinin rec eptors mediating the changes in intracellular Ca2+ concentration ([Ca2+](i) ) in canine cultured corneal epithelial cells (CECs). Bradykinin and Lys-br adykinin caused an initial transient peak of [Ca2+](i) in a concentration-d ependent manner, with half-maximal stimulation (pEC(50)) obtained at 6.9 an d 7.1, respectively. Pretreatment of CECs with pertussis toxin (PTX) or cho lera toxin (CTX) for 24 h did not affect the bradykinin-induced [Ca2+](i) c hanges. Application of Ca2+ channel blockers, diltiazem and Ni2+, inhibited the bradykinin-induced Ca2+ mobilization, indicating that Ca2+ influx was required for the bradykinin-induced responses. Addition of thapsigargin (TG ), which is known to deplete intracellular Ca2+ stores, transiently increas ed [Ca2+](i) in Ca2+-free buffer, and subsequently induced Ca2+ influx when Ca2+ was readded to this buffer. Pretreatment of CECs with TG completely a bolished bradykinin-induced initial transient [Ca2+](i), but had slight eff ect on bradykinin-induced Ca2+ influx. Pretreatment of CECs with 1-[beta-[3 -(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF96365) and 1-(6-((17 beta -3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrol e-2,5-dione (U73122) inhibited the bradykinin-induced Ca2+ release and Ca2 influx, consistent with the inhibition of receptor-gated Ca2+ channels and phospholipase C (PLC) in CECs, respectively. These results demonstrate tha t bradykinin directly stimulates B-2 receptors and subsequently Ca2+ mobili zation via a PTX-insensitive G protein in canine CECs. These results sugges t that bradykinin-induced Ca2+ influx into the cells is not due to depletio n of these Ca2+ stores, as prior depletion of these pools by TG has no effe ct on the bradykinin-induced Ca2+ influx that is dependent on extracellular Ca2+ in CECs. (C) 2001 Elsevier Science Inc. All rights reserved.