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) and membrane trafficking in many cells. To test the hypothesis that cystic fibrosis transmembrane conductance regulator (CFTR) traffics into and out of the plasma membrane during cAMP-stimulated epithelial Cl(-) secretion, we have studied the effects of wortmannin on forskolin-stimulated Cl(-) secretion by the human colonic cell line T84. At the PI3K inhibitory concentration of 100 nM, wortmannin did not affect significantly forskolin-stimulated Cl(-) secretion measured as short-circuit current (I(SC)). However, 500 nM wortmannin significantly inhibited forskolin-stimulated I(SC). cAMP activation of apical membrane CFTR Cl(-) channels in alpha-toxin-permeabilized monolayers was not reduced by 500 nM wortmannin, suggesting that inhibition of other transporters 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 absence of forskolin, wortmannin stimulated HRP endocytosis significantly. We conclude that, in T84 cells, apical fluid phase endocytosis is not dependent on PI3K activity and that CFTR does not recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.
Dickson
J L
JL
Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.
Conner
T D
TD
Ecay
T W
TW
D000730:Androstadienes / Q000494:pharmacology; D002451:Cell Compartmentation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D002867:Chromones / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D009025:Morpholines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019284:Thapsigargin / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; C505032:CFTR protein, human; D002712:Chlorides; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004815:Epidermal Growth Factor; D019284:Thapsigargin; D000242:Cyclic AMP; D000077191:Wortmannin