Previous studies from this laboratory have demonstrated that chloride transport induced by forskolin, but not ionomycin, in T cells is highly dependent on an intact microtubular network. Using an antibody raised against a region of the R domain of CFTR, we now show by indirect immunofluorescence that forskolin causes relocation of CFTR to the apical domain of T cells. T cells grown on transparent filters were incubated with agonists and/or cytoskeletal inhibitors prior to fixation, permeabilization, and staining with the antibody. A 30 second stimulation with forskolin (10 μM) caused a twofold increase in relative fluorescence intensity at the apical surface. In contrast, a 30 second exposure to ionomycin (2 μM), had no effect on the distribution of CFTR-related fluorescence. Incubation of the cells with nocodazole (33 μM), a microtubule disrupting agent, prevented the forskolin-induced rise in CFTR fluorescence at the apical surface. However, incubation of the cells with cytochalasin D, an actin inhibitor, was without effect on forskolin-related re-distribution of CFTR-associated fluorescence. In double label experiments using antibodies against both β-tubulin and actin, CFTR-related fluorescence was found to co-localize with the microtubule network, but not with actin filaments. These observations are consistent with the microtubule-dependent acute recruitment of CFTR to the apical plasma membrane of T cells in response to elevations in intracellular cAMP. 84 84 84 84
