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.