Water permeability of Na⁺–K⁺–2Cl⁻ cotransporters in mammalian epithelial cells

Abstract

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 86 Rb + 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 86 Rb + , 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.

Na+–K+–2Cl− Cotransport Inhibitor Attenuates Cerebral Edema Following Experimental Stroke via the Perivascular Pool of Aquaporin-4

“…We have previously demonstrated a temporal profile of changes in AQP4 expression following experimental stroke [43]. Our study cannot rule out the direct possible effect of bumetanide on inhibiting water transport through Na + -K + -2Cl -cotransporter itself or other cotransporters as demonstrated by previous studies [44,45].…”

Analysis of Solution Exchange in Flow Chambers with Applications to Cell Membrane Permeability Measurement

“…Reports of membrane permeability values that appear to decrease with increasing osmolality are not uncommon in the published literature. 1,8,9,28,34,37 Whereas the authors of such studies have attributed the observed trends to some property of the cell membrane that changes with osmolality or with direction of water flow, a more likely explanation may be that the apparent dependence of L p on P e is caused by artifacts due to solute transport limitations in the extracellular solution.…”

“…Although a similar strategy has previously been proposed by Farinas et al, 6 this approach is seldom utilized in the literature. For example, both Maran˜on et al 21 and Hamann et al 9 have reported that slow solution exchange kinetics limited the accuracy of their membrane permeability estimates, but both groups measured the transient cell volume change only after exposure to hypertonic solutions; had reduction of the perfusate osmolality been attempted in these studies, the accuracy of the permeability estimates may have improved. Despite the evident benefits of reducing perfusate osmolality, this strategy has some limitations.…”

“…Therefore, most flow chambers used for membrane permeability measurement are designed for rapid solution exchange. 2,[4][5][6][7]9,10,27,30 Nonetheless, there is no consensus in the literature as to the optimal flow chamber design. For example, various chamber thicknesses have been used, ranging from 100 lm to 2.5 mm.…”

“…Parallel-plate flow chambers are commonly used for this purpose. [2][3][4][5][6][7][9][10][11]21,27,29,30,32 In such devices, cells are immobilized on one of the interior chamber surfaces parallel to the direction of fluid flow; cell volume measurements can be made via an optical path transverse to the flow direction. The extracellular osmotic pressure is modified by altering the solute concentration in the aqueous liquid used to perfuse the chamber; typically, this is done by rapidly switching between two reservoirs containing solutions of differing composition.…”