1. Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+](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 [Ca2+](i) peak in a concentration-dependent manner. 2. Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 μM) for 30 min attenuated the BK-induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3. The concentrations of PMA that gave half-maximal (pEC50) inhibition of BK-induced IPs accumulation and an increase in [Ca2+](i) were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4α-phorbol 12,13-didecanoate at 1 μM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 μM), 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 Ca2+ 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, PI, 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 μM 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 nM; 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 [Ca2+](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 Ca2+ mobilization in TECs.
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