Wortmannin inhibition of forskolin-stimulated chloride secretion by T84 cells

The time- and dose-dependent effects of wortmannin on transepithelial elect rical resistance (R-te) and forskolin-stimulated chloride secretion in TX4 monolayer cultures were studied. In both instances, maximal effects develop ed over 2 h and were stable thereafter. Inhibition of forskolin-stimulated chloride secretion, as measured by the short-circuit current (I,,) techniqu e, had an IC50 of 200-500 nM, which is 100-fold higher than for inhibition of phosphatidylinositol 3-kinase (PI3K), but similar to the IC50 for inhibi tion of myosin light chain kinase (MLCK) and mitogen-activated protein kina ses (MAPK). previous work demonstrated that 500 nM wortmannin did not inhib it the cAMP activation of apical membrane chloride channels. We show here t hat 500 nM wortmannin has no affect on basolateral Na/K/2Cl-cotransporter a ctivity, but inhibits basolateral membrane Na/K-ATPase activity significant ly. The MLCK inhibitors ML-7 and KT5926 were without affect on forskolin-st imulated I-sc. Similarly, the p38- and MEK-specific MAPK inhibitors SB20358 0 and PD98059 did not reduce forskolin-stimulated I-sc. In contrast, the no n-specific MAPK inhibitor apigenin reduced forskoiin-stimulated I-sc and ba solateral membrane Na/K-ATPase activity similar to wortmannin. Tn isolated membranes from T84 cells, wortmannin did not inhibit Na/K-ATPase enzymatic activity directly. We conclude that one or more MAPK may regulate the funct ional expression of basolateral membrane Na/K-ATPase by controlling the abu ndance of enzyme molecules in the plasma membrane. (C) 2000 Elsevier Scienc e B.V. All rights reserved.