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

Wortmannin is a potent inhibitor of phosphatidylinositol 3-kinase (PI3K) an d membrane trafficking in many cells. To test the hypothesis that cystic fi brosis transmembrane conductance regulator (CFTR) traffics into and out of the plasma membrane during cAMP-stimulated epithelial Cl- secretion, we hav e studied the effects of wortmannin on forskolin-stimulated Cl- secretion b y the human colonic cell line T84. At the PI3K inhibitory concentration of 100 nM, wortmannin did not affect significantly forskolin-stimulated Cl- se cretion measured as short-circuit current (I-SC) However, 500 nM wortmannin significantly inhibited forskolin-stimulated I-SC. cAMP activation of apic al membrane CFTR Cl- channels in alpha-toxin-permeabilized monolayers was n ot reduced by 500 nM wortmannin, suggesting that inhibition of other transp orters accounts for the observed reduction in T84 Cl- secretion. Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP), but wortmannin did not alter forskolin inhibition of apical HRP endocytosis. In the absen ce of forskolin, wortmannin stimulated HRP endocytosis significantly. We co nclude that, in T84 cells, apical fluid phase endocytosis is not dependent on PI3K activity and that CFTR does not recycle through a PI3K-dependent an d wortmannin-sensitive membrane compartment.