cAMP-sensitive endocytic trafficking in A6 epithelia

Blocker-induced noise analysis and laser scanning confocalmicroscopy were used to test the idea that cAMP-mediated vesicle exocytosis/endocytosis may be a mechanism for regulation of functional epithelial Na⁺ channels (ENaCs) at apical membranes of A6epithelia. After forskolin stimulation of Na⁺ transport andlabeling apical membranes with the fluorescent dyeN-(3-triethylammoniumpropyl)4-(6-4 diethylaminophenyl)hexatrienyl pyridinium dibromide (FM 4-64), ENaC densities(N_T) decreased exponentially (time constant~20 min) from mean values of 320 to 98 channels/cell within 55 minduring washout of forskolin. Two populations of apical membrane-labeledvesicles appeared in the cytosol within 55 min, reaching mean valuesnear 18 vesicles/cell, compared with five vesicles per cell in control,unstimulated tissues. The majority of cAMP-dependent endocytosedvesicles remained within a few micrometers of the apical membranes forthe duration of the experiments. A minority of vesicles migrated to >5µm below the apical membrane. Because steady states require identicalrates of endocytosis and exocytosis, and because forskolin increased endocytic rates by fivefold or more, cAMP/protein kinase A acts kinetically not only to increase rates of cycling of vesicles at theapical membranes, but also principally to increase exocytic rates.These observations are consistent with and support, but do not prove,that vesicle trafficking is a mechanism for cAMP-mediated regulation ofapical membrane channel densities in A6 epithelia.