Characterization of cAMP-induced activation of epithelial sodium channels.

Incubating toad bladder with 10 mU/ml vasopressin increases the amiloride-blockable Na+ flux in membrane vesicles derived from the epithelial cells by about twofold. This stimulation is further enhanced by 3-isobutyl-1-methylxanthine and can be mimicked by 8-bromoadenosine 3', 5'-cyclic monophosphate. Thus the natriferic action of cAMP involves a sustained change of the apical membrane preserved by the isolated vesicles. The possibility that transport is modulated by direct phosphorylation/dephosphorylation of the Na+ channel was tested. Trapping purified cAMP-dependent protein kinase, cAMP, and ATP in apical vesicles failed to alter Na+ transport even though the enzyme proved active and could phosphorylate intravesicular proteins. Trapping several phosphatases partially purified from toad bladder in vesicles was ineffective as well. These data suggest that the cAMP-induced increase in Na+ conductance involves processes other than phosphorylation of the channel protein or direct channel-cAMP interaction.