Drugs activating G proteins disturb cycling of ADH-dependent water channels in toad urinary bladder.

In the toad urinary bladder, antidiuretic hormone (ADH)-mediated changes in water permeability depend on exocytic insertion and endocytic retrieval of water channels into and from the apical membrane, respectively. Because GTP-binding proteins (G proteins) are well-recognized regulators of vesicular trafficking throughout the cell, we tested the hypothesis that drugs interfering with G protein would modify the hydrosmotic response to ADH and the ADH-regulated formation of endosomes, as assessed by luminal incorporation of a fluid-phase marker [fluorescein isothiocyanate (FITC)-dextran, 70 kDa]. Mastoparan (4 microM) and compound 48/80 (poly-p-methoxyphenylethylmethylamine; 50 micrograms/ml), added to the luminal side of the toad urinary bladder, as well as AlF3 added to the serosal side (400 microM), inhibited ADH- and 8-bromoadenosine 3',5'-cyclic monophosphate-induced transepithelial water flow by > 50% and simultaneously enhanced cellular incorporation of FITC-dextran by > 200%. The pattern of FITC-dextran uptake observed using fluorescence microscopy both in scraped cells and in the intact bladder was granular, suggesting fluid-phase endocytosis. Mastoparan and AlF3, which are both probes of G proteins, increased FITC-dextran uptake only in the presence of ADH and a transepithelial osmotic gradient, i.e., under conditions where water channel-carrying endosomes presumably cycle. Therefore, we suggest that the ADH-dependent cycling of water channels could be controlled by one or more G proteins associated with the apical membrane and/or the water channel-carrying vesicles.