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This Article Full Text Full Text (PDF) All Versions of this Article: 568/1/123    most recent jphysiol.2005.093526v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hamann, S. Articles by Zeuthen, T. Search for Related Content PubMed PubMed Citation Articles by Hamann, S. Articles by Zeuthen, T.

¹ Nordic Centre for Water Imbalance Related Disorders, Department of Medical Physiology, The Panum Institute, University of Copenhagen, Denmark
² Departamento de Neurobiología, Instituto Nacional de Psiquiatría, Mexico DF, Mexico
³ Department of Pharmacology and Toxicology, Wright State University, Dayton, OH, USA

Water transport properties of the Na⁺–K⁺–2Cl^– cotransporter (NKCC) were studied in cultures of pigmented epithelial cells (PE) from the ciliary body of the eye. Here, the membrane that faces upwards contains NKCCs and can be subjected to rapid changes in bathing solution composition and osmolarity. The anatomy of the cultured cell layer was investigated by light and electron microscopy. The transport rate of the cotransporter was determined from the bumetanide-sensitive component of ⁸⁶Rb⁺ uptake, and volume changes were derived from quenching of the fluorescent dye calcein. The water permeability (L_p) of the membrane was halved by the specific inhibitor bumetanide. The bumetanide-sensitive component of the water transport exhibited apparent saturation at osmotic gradients higher than 200 mosmol l^–1. Cell shrinkages produced by NaCl or KCl were smaller than those elicited by equi-osmolar applications of mannitol, indicating reflection coefficients for these salts close to zero. The activation energy of the bumetanide-sensitive component of the L_p was 21 kcal mol^–1, which is four times higher than that of an aqueous pore. The data suggest that osmotic transport via the cotransporter involves conformational changes of the cotransporter and interaction with Na⁺, K⁺ and Cl^–. Similar measurements were performed on immortalized cell cultures from the thick ascending limb of the loop of Henle (TALH). Given similar overall transport rates of bumetanide-sensitive ⁸⁶Rb⁺, the NKCCs of this tissue did not contribute any bumetanide-sensitive L_p. This suggests that the cotransporters of the two tissues are either different isoforms or the same cotransporter but in two different transport modes.

(Received 24 June 2005; accepted after revision 7 July 2005; first published online 14 July 2005)
Corresponding author S. Hamann: Nordic Centre for Water Imbalance Related Disorders, Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Email: shamann{at}mfi.ku.dk

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