CYTOPLASMIC DILUTION INDUCES ANTIDIURETIC-HORMONE WATER CHANNEL RETRIEVAL IN TOAD URINARY-BLADDER

Antidiuretic hormone (ADH) increases the osmotic water permeability (P(f)) of the toad urinary bladder by insertion of water channels into the apical cell membrane. Transepithelial water flow (J(v)) reduces P(f) by inducing endocytosis of apical water channels despite continuous ADH stimulation. This phenomenon is termed flux inhibition. We wished to determine whether cytoplasmic dilution or transcellular J(v) causes flux inhibition because both have been proposed previously as a primary regulatory mechanism for this process. Apical membrane endocytosis was quantified by monitoring the uptake of the fluid phase marker fluorescein isothiocyanate dextran (FITC-dextran). FITC-dextran fluorescence was monitored in Triton X-100 extracts of epithelial cells as the ratio of total tissue fluorescence compared with background fluorescence. The background was defined as cellular autofluorescence and nonspecific tissue staining due to the presence of small amounts of free fluorescein contaminating the FITC-dextran. FITC-dextran uptake measured under symmetric isotonic (220 mosmol/kgH2O) conditions in either the absence (1.0 +/- 0.4 SD; n = 14) or presence (1.3 +/- 0.3; n = 4) of ADH was not statistically different from that of background. In contrast, flux inhibition induced by a 180 mosmol/kgH2O apical-to-basolateral osmotic gradient increased FITC-dextran uptake to 3.4 +/- 1.3 (n = 7). FITC-dextran uptake was identical in bladders exposed to symmetric hypotonic (150 mosmol/kgH2O) solutions during ADH (3.6 +/- 0.9; n = 6) or adenosine 3',5'-cyclic monophosphate (3.1 +/- 0.4 fold; n = 3) stimulation. Exposure of nonstimulated bladders to symmetric hypotonicity had no effect on FITC-dextran uptake (0.9 +/- 0.3, n = 4). ADH-stimulated bladders exposed to symmetric hypotonic conditions exhibited a significantly (P < 0.001) lower P(f) (1.12 +/- 0.07 x 10(-2) cm/s; n = 3) compared with paired bladders exposed to symmetric isotonicity (2.76 +/- 0.31 x 10(-2) cm/s; n = 3). In bladders exposed to either symmetric hypotonic or an apical-to-basolateral osmotic gradient and examined by epifluorescence microscopy, FITC-dextran was observed to be present in large numbers of cytoplasmic vesicles. Vesicles from symmetric hypotonicity bladders exhibited a P(f) (2.4 +/- 0.5 x 10(-1) cm/s; n = 3) equal to or greater than that possessed by water channel-containing vesicles from bladders with transepithelial J(v) (1.5 +/- 0.2 x 10(-1) cm/s; n = 14). These data demonstrate that cell swelling and/or cytoplasmic dilution induce the apical membrane retrieval of water channels. Cell swelling during J(v) is likely an important mediator of flux inhibition.