TANNIN INHIBITION OF PROTEIN-KINASE-C IN AIRWAY EPITHELIUM

Tannin, a polydisperse polyphenol extracted from cotton bracts (CBE), has been implicated in the pathogenesis of byssinosis, a lung disease of mill workers. CBE tannin inhibits chloride secretion in airway epithelial cells by means of an unknown mechanism(s). Activation of protein kinase C (PKC) by PMA (phorbol 12-myristate 13-acetate) in airway cells increases chloride secretion. The effect of tannin on this PKC pathway was examined, using canine tracheal epithelium mounted in Ussing chambers. PMA addition (10 nM) to the mucosal bath resulted in a 0.36 +/- 0.07 mu Eq/cm(2) . h (mean +/- SEM, n = 20) increase in short-circuit current (I-sc) and a 0.38 +/- 0.17 mu Eq/cm(2) . h increase in net chloride secretion (J(net)). The inactive 4 alpha-phorbol had no effect. Tannin addition to the mucosal bath produced a dose-dependent decrease in I-sc and J(net). In tissues pretreated with 2-50 mu g/ml tannin, and subsequently stimulated with PMA, tannin inhibited PMA stimulation of chloride secretion beginning at a tannin concentration of 10 mu g/ml (0.09 +/- 0.05 mu Eq/cm(2) . h [n = 10] increase in I-sc and 0.08 +/- 0.03 mu Eq/cm(2) . h increase in J(net) with PMA after tannin pretreatment). At 50 mu g/ml tannin, the stimulatory effect of PMA was completely abolished. The known PKC inhibitor, H-7 (20 mu M), inhibited PMA stimulation, while chelerythrine (2 mu M) had not effect on PMA-stimulated I-sc and J(net), and calphostin C was toxic to the airway epithelium. In membrane fragments, 2.5 mu g/ml tannin inhibited the rate of histone III phosphorylation by PMA from 32.1 +/- 4.4 nmol/mg protein per min to 20.1 +/- 2.7 nmol/mg protein per min (n = 7). In bovine airway cells, tannin pretreatment (2.5 mu g/ml) decreased the cytosolic activity of PKC but had no effect on PKC translocation to the membrane. We conclude that tannin inhibits chloride secretion in airway epithelial cells in part by inhibiting PKC.