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

Experiments were designed to differentiate the mechanisms of bradykinin receptors mediating the changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in canine cultured corneal epithelial cells (CECs). Bradykinin and Lys-bradykinin caused an initial transient peak of [Ca²⁺]_i in a concentration-dependent manner, with half-maximal stimulation (pEC₅₀) obtained at 6.9 and 7.1, respectively. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin (CTX) for 24 h did not affect the bradykinin-induced [Ca²⁺]_i changes. 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 increased [Ca²⁺]_i in Ca²⁺ -free buffer, and subsequently induced Ca²⁺ influx when Ca²⁺ was readded to this buffer. Pretreatment of CECs with TG completely abolished bradykinin-induced initial transient [Ca²⁺]_i, but had slight effect on bradykinin-induced Ca²⁺ influx. Pretreatment of CECs with 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF96365) and 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H- pyrrole-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 that bradykinin directly stimulates B₂ receptors and subsequently Ca²⁺ mobilization via a PTX-insensitive G protein in canine CECs. These results suggest that bradykinin-induced Ca²⁺ influx into the cells is not due to depletion of these Ca²⁺ stores, as prior depletion of these pools by TG has no effect on the bradykinin-induced Ca²⁺ influx that is dependent on extracellular Ca²⁺ in CECs.