Uncoupling of bradykinin‐induced phosphoinositide hydrolysis and Ca 2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells

1 Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca 2+ concentration ([Ca 2+ ] i by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca 2+ ] i peak in a concentration‐dependent manner. 2 Pretreatment of TECs with phorbol 12‐myristate 13‐acetate (PMA, 1 μ M ) for 30 min attenuated the BK‐induced IPs formation and Ca 2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3 The concentrations of PMA that gave half‐maximal (pEC 50 ) inhibition of BK‐induced IPs accumulation and an increase in [Ca 2+ ] i were 7.07  M and 7.11  M , respectively. Inactive phorbol ester, 4α‐phorbol 12,13‐didecanoate at 1 μ M , did not inhibit these responses. Prior treatment of TECs with staurosporine (1 μ M ), a PKC inhibitor, inhibited the ability of PMA to attenuate BK‐induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 4 In parallel with the effect of PMA on the BK‐induced IPs formation and Ca 2+ mobilization, the translocation and down‐regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC‐α, βI, βII, γ, δ, ε, Θ and ζ. With PMA treatment of the cells for various times, translocation of PKC‐α, βI, βII, γ, δ, ε and Θ from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down‐regulation of these PKC isozymes. PKC‐ζ was not significantly translocated and down‐regulated at any of the times tested. 5 Treatment of TECs with 1 μ M PMA for either 30 min or 6 h did not significantly change the K D and B max receptor for BK binding (control: K D =1.7±0.3 n M ; B max =50.5±4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK‐induced responses. 6 In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca 2+ ] i increase or inhibit independently both responses to BK. The translocation of pKC‐α, βI, βII, δ, ε, γ, and Θ induced by PMA caused an attenuation of BK‐induced IPs accumulation and Ca 2+ mobilization in TECs.British Journal of Pharmacology (1998) 125 , 627–636; doi: 10.1038/sj.bjp.0702094