UNCOUPLING OF BRADYKININ-INDUCED PHOSPHOINOSITIDE HYDROLYSIS AND CA2-CELLS( MOBILIZATION BY PHORBOL ESTER IN CANINE CULTURED TRACHEAL EPITHELIAL)

1 Regulation of the increase in inositol phosphates (IPs) production a nd 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 caus ed an initial transient [Ca2+](i) peak in a concentration-dependent ma nner. 2 Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA , 1 mu M) for 30 min attenuated the BK-induced IPs formation and Ca2mobilization. The maximal inhibition occurred after incubating the cel ls with PMA for 2 h. 3 The concentrations of PMA that gave half-maxima l (pEC(50)) inhibition of BK-induced IPs accumulation and an increase in [Ca2+](i) were 7.07 M and 7.11 M, respectively. Inactive phorbol es ter, 4 alpha-phorbol 12,13-didecanoate at 1 mu M, did not inhibit thes e responses. Prior treatment of TECs with staurosporine (1 mu M), a PK C inhibitor, inhibited the ability of PMA to attenuate BK-induced resp onses, suggesting that the inhibitory effect of PMA is mediated throug h the activation of PKC. 4 In parallel with the effect of PMA on the B K-induced IPs formation and Ca2+ mobilization, the translocation and d own-regulation of PKC isozymes were determined. Analysis of cell extra cts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-alpha, beta I, beta II, gamma, delta , epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, beta I, beta II, gamma, delta, eps ilon and theta from cytosol to the membrane was seen after 5 min, 30 m in, 2 h, and 4 h treatment. However, 6h treatment caused a partial dow n-regulation of these PKC isozymes. PKC-I was not significantly transl ocated and down-regulated at any of the times tested. 5 Treatment of T ECs with 1 mu M PMA for either 30 min or 6 h did not significantly cha nge 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 recept ors are not a site for the inhibitory effect of PMA on BK-induced resp onses. 6 In conclusion, these results suggest that activation of PKC m ay inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+](i) increase or inhibit independently both responses to BK. The translocation of pKC-alpha, beta I, beta II, delta, epsilon, gamma , and theta induced by PMA caused an attenuation of BK-induced IPs acc umulation and Ca2+ mobilization in TECs.