DRUGS ACTIVATING G-PROTEINS DISTURB CYCLING OF ADH-DEPENDENT WATER CHANNELS IN TOAD URINARY-BLADDER

In the toad urinary bladder, antidiuretic hormone (ADH)-mediated chang es in water permeability depend on exocytic insertion and endocytic re trieval of water channels into and from the apical membrane, respectiv ely. Because GTP-binding proteins (G proteins) are well-recognized reg ulators of vesicular trafficking throughout the cell, we tested the hy pothesis that drugs interfering with G protein would modify the hydros motic response to ADH and the ADH-regulated formation of endosomes, as assessed by luminal incorporation of a fluid-phase marker [fluoreseci n isothiocyanate (FITC)dextran, 70 kDa]. Mastoparan (4 mu M) and compo und 48/80 (poly-p-methoxyphenylethylmethylamine; 50 mu g/ml), added to the luminal side of the toad urinary bladder, as well as AlF3 added t o the serosal side (400 mu M), inhibited ADH- and 8-bromoadenosine 3', 5'-cyclic monophosphate-induced transepithelial water flow by > 50% an d simultaneously enhanced cellular incorporation of FITC-dextran by > 200%. The pattern of FITC-dextran uptake observed using fluorescence m icroscopy both in scraped cells and in the intact bladder was granular , suggesting fluid-phase endocytosis. Mastoparan and AlF3, which are b oth probes of G proteins, increased FITC-dextran uptake only in the pr esence of ADH and a transepithelial osmotic gradient, i.e., under cond itions where water channel-carrying endosomes presumably cycle. Theref ore, we suggest that the ADH-dependent cycling of water channels could be controlled by one or more G proteins associated with the apical me mbrane and/or the water channel-carrying vesicles.