Bradykinin-induced phosphoinositide hydrolysis and Ca²⁺ mobilization in canine cultured tracheal epithelial cells

1. Experiments were designed to differentiate the mechanisms and subtype of kinin receptors mediating the changes in intracellular Ca²⁺ concentration ([Ca²⁺](i)) induced by bradykinin (BK) in canine cultured tracheal epithelial cells (TECs). 2. BK and Lys-BK caused an initial transient peak of [Ca²⁺](i) in a concentration-dependent manner, with half-maximal stimulation (pEC₅₀) obtained at 7.70 and 7.23, respectively. 3. Kinin B₂ antagonists Hoe 140 (10 nM) and [D-Arg⁰, Hyp³, Thi^5.8, D-Phe⁷]-BK (1 μM) had high affinity in antagonizing BK-induced Ca²⁺ response with pK(B) values of 8.90 and 6.99, respectively. 4. Pretreatment of TECs with pertussis toxin (100 ng ml^-1) or cholera toxin (10 μg ml^-1) for 24 h did not affect the BK-induced IP accumulation and [Ca²⁺](i) changes in TECs. 5. Removal of Ca²⁺ by the addition of EGTA or application of Ca²⁺-channel blockers, verapamil, diltiazem, and Ni²⁺, inhibited the BK-induced IP accumulation and Ca²⁺ mobilization, indicating that Ca²⁺ influx was required for the BK-induced responses. 6. 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 re-added to this buffer. Pretreatment of TECs with TG completely abolished BK-induced initial transient [Ca²⁺](i), but had slight effect on BK-induced Ca²⁺ influx. 7. Pretreatment of TECs with SKF96365 and U73122 inhibited the BK-induced Ca²⁺ influx and Ca²⁺ release, consistent with the inhibition of receptor-gated Ca²⁺-channels and phospholipase C in TECs, respectively. 8. These results demonstrate that BK directly stimulates kinin B₂ receptors and subsequently phospholipase C-mediated IP accumulation and Ca²⁺ mobilization via a pertussis toxin-insensitive G protein in canine TECs. These results also suggest that BK-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 BK-induced Ca²⁺ influx that is dependent on extracellular Ca²⁺ in TECs.