cAMP-sensitive endocytic trafficking in A6 epithelia

Blocker-induced noise analysis and laser scanning confocal microscopy 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 A6 epithelia. After forskolin stimulation of Na+ t ransport and labeling apical membranes with the fluorescent dye N-(3-trieth ylammoniumpropyl)4-(6-4 diethylaminophenyl) hexatrienyl pyridinium dibromid e (FM 4-64), ENaC densities (N-T) decreased exponentially (time constant si milar to 20 min) from mean values of 320 to 98 channels/cell within 55 min during washout of forskolin. Two populations of apical membrane-labeled ves icles appeared in the cytosol within 55 min, reaching mean values near 18 v esicles/cell, compared with five vesicles per cell in control, unstimulated tissues. The majority of cAMP-dependent endocytosed vesicles remained with in a few micrometers of the apical membranes for the duration of the experi ments. A minority of vesicles migrated to >5 mum below the apical membrane. Because steady states require identical rates of endocytosis and exocytosi s, and because forskolin increased endocytic rates by fivefold or more, cAM P/protein kinase A acts kinetically not only to increase rates of cycling o f vesicles at the apical membranes, but also principally to increase exocyt ic rates. These observations are consistent with and support, but do not pr ove, that vesicle trafficking is a mechanism for cAMP-mediated regulation o f apical membrane channel densities in A6 epithelia.