Inhibition of phosphatidylinositol 3-kinase does not alter forskolin-stimulated Cl(-) secretion by T84 cells

Wortmannin is a potent inhibitor ofphosphatidylinositol 3-kinase (PI3K) and membrane trafficking in manycells. To test the hypothesis that cystic fibrosis transmembraneconductance regulator (CFTR) traffics into and out of the plasmamembrane during cAMP-stimulated epithelial Clsecretion, we have studied the effects of wortmannin onforskolin-stimulated Cl secretion by the humancolonic cell line T84. At the PI3K inhibitory concentration of 100 nM,wortmannin did not affect significantly forskolin-stimulatedCl secretion measured as short-circuit current(I_SC). However, 500 nM wortmannin significantlyinhibited forskolin-stimulated I_SC. cAMP activationof apical membrane CFTR Cl channels in-toxin-permeabilized monolayers was not reduced by 500 nMwortmannin, suggesting that inhibition of other transporters accountsfor the observed reduction in T84 Cl secretion.Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP),but wortmannin did not alter forskolin inhibition of apical HRPendocytosis. In the absence of forskolin, wortmannin stimulated HRPendocytosis significantly. We conclude that, in T84 cells, apical fluidphase endocytosis is not dependent on PI3K activity and that CFTR doesnot recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.