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Flow cytometry and sorting of amphibian bladder endocytic vesicles containing ADH-sensitive water channels

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Summary

The water permeability of ADH target epithelial cells is believed to be regulated by a cycle of exo-endocytosis of vesicles containing functional water channels. These vesicles were selectively labeled in intact frog urinary bladders with an impermeant fluorescent marker, 6-carboxyfluorescein. Vesicle suspensions containing the labeled endosomes were obtained by homogenization and differential centrifugation of bladder epithelial cells. The osmotic permeability of the endocytic vesicles was measured, using a stopped-flow fluorescence technique, in the absence or in the presence of HgCl2. This permeability was found very high (500 μm/sec) and inhibited by 1 mm HgCl2 (90%), thus confirming the presence of water channels. The labeled endosomes were then separated from the other membrane vesicles by flow cytometry and sorting. Their protein content was analyzed by electrophoresis on ultrathin polyacrylamide gels. Two double bands were found at 71 and 55 kDa as well as a small band at 43 kDa. They respectively correspond to 31, 38 and 10% of the total amount of silver-stained proteins present in the sorted endosomes, while they only represent 2, 4, and less than 1% of the proteins contained in the vesicle suspension, before sorting. These highly enriched proteins (or at least one of them) are likely to be involved in the mechanism of water transport. Associated to their partial purification by differential centrifugation, the sorting of the endosomes by flow cytometry seems a good way to further characterize the water channel.

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References

  • Bradford, M.M. 1976. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brown, D., Grosso, A., De Sousa, R. 1983. Correlation between water flow and intramembrane particle aggregates in toad urinary epidermis. Am. J. Physiol. 245:334–342

    Google Scholar 

  • Chevalier, J., Bourguet, J., Hugon, J.S. 1974. Membrane associated particles: Distribution in frog urinary bladder epithelium at rest and after oxytocin treatment. Cell Tissue Res. 152:129–140

    Google Scholar 

  • Coleman, R.A., Harris, H.W., Jr., Wade, J.B. 1987. Visualization of endocytosed markers in freeze-fracture studies of toad urinary bladder. J. Histochem. Cytochem. 35:1405–1414

    Google Scholar 

  • Courtoy, J.P., Quintart, J., Bauduin, P. 1984. Shift of equilibrium density induced by 3,3′-diaminobenzidine cytochemistry: A new procedure for the analysis and purification of peroxidase-containing organelles. J. Cell Biol. 98:870–876

    Google Scholar 

  • Gaucher, J.-C., Grünwald, D., Frelat, G. 1988. Cytametry 9:557–565

    Google Scholar 

  • Grossman, E.B., Hammond, T., Harris, H.W., Zeidel, M.L. 1991. Purification of ADH regulated water channel containing vesicles from toad urinary bladder using flow cytometry. J. Am. Soc. Nephrol. 1:674A

    Google Scholar 

  • Handler, J.S. 1988. Antidiuretic hormone moves membranes. Am. J. Physiol. 255:F375-F382

    Google Scholar 

  • Harris, H.W., Jr., Handler, J.S., Blumenthal, R. 1990. Apical membrane vesicles of ADH-stimulated toad bladder are highly water permeable. Am. J. Physiol. 258:F237-F243

    Google Scholar 

  • Harris, H.W., Jr., Murphy, H.R., Willingham, M.C., Handler, J.S. 1987. Isolation and characterization of specialized regions of toad urinary bladder apical plasma membrane involved in the water permeability response to antidiuretic horone. J. Membrane Biol. 96:175–186

    Google Scholar 

  • Harris, H.W., Jr., Wade, J.B., Handler, J.S. 1986. Fluorescent markers to study membrane retrieval in antidiuretic hormone-treated toad urinary bladder. Am. J. Physiol. 251:C274-C284

    Google Scholar 

  • Hays, R.M., Franki, N. 1970. The role of water diffusion in the action of vasopressin. J. Membrane Biol. 2:263–276

    Google Scholar 

  • Hays, R.M., Leaf, A. 1962. Studies on the movement of water through the isolated toad bladder and its modification by vasopressin. J. Gen. Physiol. 45:905–919

    Google Scholar 

  • Hoch, B.S., Gorfien, P.C., Linzer, D., Fusco, M.J., Levine, S.D. 1989. Mercurial reagents inhibit flow through ADH-induced water channels in toad bladder. Am. J. Physiol. 256:F948-F953

    Google Scholar 

  • Humbert, F., Montesano, R., Grosso, A., De Sousa, R., Orci, L. 1977. Particle aggregates in plasma and intracellular membranes of toad bladder granular cells. Experientia 33:1364–1367

    Google Scholar 

  • Ibarra, C., Ripoche, P., Bourguet, J. 1989. Effect of mercurial compounds on net water transport and intramembrane particle aggregates in ADH-treated frog urinary bladder. J. Membrane Biol. 110:115–126

    Google Scholar 

  • Kachadorian, W.A., Casey, C., DiScala, V.A. 1978. Time course of ADH-induced intramembranous particle aggregation in toad urinary bladder. Am. J. Physiol. 234:F461-F465

    Google Scholar 

  • Kessler, M., Acuto, O., Storelli, C., Murer, H., Müller, M., Semenza, G. 1978. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush-border membranes. Biochim. Biophys. Acta 506:136–154

    Google Scholar 

  • Macey, R.I. 1984. Transport of water and urea in red blood cells. Am. J. Physiol. 246:C195-C203

    Google Scholar 

  • Macey, R.I., Farmer, R.E. 1970. Inhibition of water and solute permeability in human red cells. Biochim. Biophys. Acta 211:104–106

    Google Scholar 

  • Muller, J., Kachadorian, W.A. 1984. Aggregate-carrying membranes during ADH stimulation and washout in toad bladder. Am. J. Physiol. 247:C90-C98

    Google Scholar 

  • Muller, J., Kachadorian, W.A., DiScala, V.A. 1980. Evidence that ADH-stimulated intramembrane particle aggregates are transferred from cytoplasmic to luminal membrane in toad bladder epithelial cells. J. Cell Biol. 85:83–85

    Google Scholar 

  • Olsson, I., Axio-Fredriksson, U.-B., Degerman, M., Olsson, B. 1988a. Fast horizontal electrophoresis. I. Isoelectric focusing and polyacrylamide gel electrophoresis using PhastSystem. Electrophoresis 9:16–22

    Google Scholar 

  • Olsson, I. Wheeler, R., Johansson, C., Ekstrom, B., Stafstrom, N., Bikhabhai, R., Jacobson, G. 1988b. Fast horizontal electrophoresis. II. Development of fast automated staining procedures using PhastSystem. Electrophoresis 9:22–27

    Google Scholar 

  • Pinkel, D., Stovel, R. 1985. Flow chambers and sample handling. In: Flow cytometry: Instrumentation and data analysis. M. Van Dilla, P. Dean, O. Laerum and M. Melamed, editors, pp. 77–128. Academic, London.

    Google Scholar 

  • Pratz, J., Ripoche, P., Corman, B. 1986. Evidence for proteic water pathways in the luminal membrane of kidney proximal tubule. Biochim. Biophys. Acta 856:259–266

    Google Scholar 

  • Rambourg, A., Clermont, Y., Pisam, M., Ripoche, P. 1991. Effects of low temperature on the formation of secretion granules in the Golgi apparatus of granular cells of the frog urinary bladder. Biol. Cell 73:139–149

    Google Scholar 

  • Shi, L.-B., Brown, D., Verkman, A.S. 1990. Water, proton and urea transport in toad urinary bladder endosomes that contain the vasopressin-sensitive water channel. J. Gen. Physiol. 95:941–960

    Google Scholar 

  • Shi, L.-B., Verkman, A.S. 1989. Very high water permeability in vasopressin-induced endocytic vesicles from toad urinary bladder. J. Gen. Physiol. 94:1101–1115

    Google Scholar 

  • Solomon, A.K., Chasan, B., Dix, J.A., Lukacovic, M.F., Toon, M.R., Verkman, A.S. 1983. The aqueous pore in the red cell membrane: Band 3 as a channel for anions, cations, nonelectrolytes, and water. Ann. N.Y. Acad. Sci. 414:97–124

    Google Scholar 

  • Van der Goot, F., Corman, B., Ripoche, P. 1991. Evidence for permanent water channels in the basolateral membrane of an ADH-sensitive epithelium. J. Membrane Biol. 120:59–65

    Google Scholar 

  • Van der Goot, F.G., Podevin, R.-A., _Corman, B. 1989. Water permeability and salt reflexion coefficients of luminal, basolateral and intracellular membrane vesicles isolated from rabbit kidney proximal tubule. Biochim. Biophys. Acta 979:272–274

    Google Scholar 

  • Van Heeswijk, M.P.E., van Os, C.H. 1986. Osmotic water permeability of brush-border and basolateral membrane vesicles from rat renal cortex and small intestine. J. Membrane Biol. 92:183–193

    Google Scholar 

  • Van Hoek, A.N., de Jong, M.D., van Os, C.H. 1990. Effects of dimethylsulfoxide and mercurial sulfhydryl reagents on water and solute permeability of rat kidney brush border membranes. Biochim. Biophys. Acta 1030:203–210

    Google Scholar 

  • Verkman, A.S., Ives, H.E. 1986. Water permeability and fluidity of renal basolateral membranes. Am. J. Physiol. 250:F633-F643

    Google Scholar 

  • Wade, J.B., Stetson, D.L., Lewis, S.A. 1981. ADH action: Evidence for a membrane shuttle mechanism. Ann. N. Y. Acad. Sci. 372:106–117

    Google Scholar 

  • Wang, Y.-X., Shi, L.-B., Verkman, A.S. 1991. Functional water channels and proton pumps are in separate populations of endocytic vesicles in toad bladder granular cells. Biochemistry 30:2888–2894

    Google Scholar 

  • Whittembury, G., Carpi-Medina, P., Gonzales, E., Linares, H. 1984. Effect of para-chloromercuribenzensulfonic acid and temperature on cell water osmotic permeability of proximal straight tubules. Biochim. Biophys. Acta 775:365–373

    Google Scholar 

  • Wilson, R.B., Murphy, R.F. 1989. Flow-cytometric analysis of endocytic compartments. Meth. Cell Biol. 31:293–317

    Google Scholar 

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Authors and Affiliations

  1. European Molecular Biology Laboratory, D-6900, Heidelberg, Germany

    F. G. van der Goot

  2. ‡DEIN/SIR, C.E.N. Saclay, 91191, Gif-sur-Yvette, France

    A. Seigneur & J. -C. Gaucher

  3. Département de Biologie Cellulaire et Moléculaire, C.E.N. Saclay, 91191, Gif-sur-Yvette, France

    P. Ripoche

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  1. F. G. van der Goot
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  2. A. Seigneur
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  3. J. -C. Gaucher
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  4. P. Ripoche
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van der Goot, F.G., Seigneur, A., Gaucher, J.C. et al. Flow cytometry and sorting of amphibian bladder endocytic vesicles containing ADH-sensitive water channels. J. Membarin Biol. 128, 133–139 (1992). https://doi.org/10.1007/BF00231886

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  • DOI: https://doi.org/10.1007/BF00231886

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