FUNCTIONAL WATER CHANNELS AND PROTON PUMPS ARE IN SEPARATE POPULATIONS OF ENDOCYTIC VESICLES IN TOAD BLADDER ANTIGRANULOCYTES CELLS

Functional water channels are retrieved by endocytosis from the apical membrane of toad bladder granular cells in response to vasopressin [Shi, L.-B., & Verkman, A. S. (1989) J. Gen. Physiol. 94, 1101-1115]. To examine whether endocytic vesicles which contain the vasopressin-sensitive water channel fuse with acidic vesicles for entry into a lysosomal pathway, ATP-dependent acidification and osmotic water permeability were measured in endosomes from control bladders and bladders treated with vasopressin (VP) and/or phorbol myristate acetate (PMA). Endosomes were labeled with the fluid-phase markers 6-carboxyfluorescein or fluorescein-dextran. Osmotic water permeability (P(f)) was measured by stopped-flow fluorescence quenching and proton ATPase activity by ATP-dependent, N-ethylmaleimide-inhibitable acidification. In a microsomal pellet, P(f) was low (< 0.002 cm/s, 20-degrees-C) in labeled endocytic vesicles from control bladders but high (0.05-0.1 cm/s) in a subpopulation (50-70%) of vesicles from VP- and PMA-treated bladders. Following ATP addition, the average drop in pH was 0.1 (control), 0.3 (VP), and 0.2 (PMA) unit. Measurement of pH in individual endocytic vesicles by quantitative image analysis showed that < 20% of vesicles from VP-treated bladders acidified by > 0.5 pH unit. To examine whether water channels and proton pumps were present in the same endocytic vesicles, the pH of endosomes with high and low water permeability was measured from the effect of ATP on the amplitude of the fluorescence quenching signal in response to an osmotic gradient. ATP did not alter the pH of endosomes with high P(f) but decreased by > 0.3 unit the average pH of endosomes with low P(f). We conclude that endocytic vesicles from toad bladder granular cells that contain the vasopressin-sensitive water channel selectively do not contain functional proton pumps. The data suggest that trafficking of the vasopressin-sensitive water channel retrieved by endocytosis does not involve fusion with acidic intracellular vesicles.