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1 Department of Anatomy and Cell Biology, Faculty of Health Sciences, University of Cape Town, Observatory 7925, Cape Town, South Africa; and 2 Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
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+ transport and labeling apical membranes with the fluorescent dye N-(3-triethylammoniumpropyl)4-(6-4 diethylaminophenyl) hexatrienyl pyridinium dibromide (FM 4-64), ENaC densities (NT) decreased exponentially (time constant ~20 min) from mean values of 320 to 98 channels/cell within 55 min during washout of forskolin. Two populations of apical membrane-labeled vesicles appeared in the cytosol within 55 min, reaching mean values near 18 vesicles/cell, compared with five vesicles per cell in control, unstimulated tissues. The majority of cAMP-dependent endocytosed vesicles remained within a few micrometers of the apical membranes for the duration of the experiments. A minority of vesicles migrated to >5 µm below the apical membrane. Because steady states require identical rates 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 the apical 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 of apical membrane channel densities in A6 epithelia.
epithelial sodium channel; noise analysis; confocal microscopy; channel trafficking; vesicles; forskolin; kidney; cortical collecting ducts; endocytosis
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