Functional demonstration of Na+-K+-2Cl−cotransporter activity in isolated, polarized choroid plexus cells

The function of the apical Na+-K+-2Cl- cotransporter in mammalian choroid plexus (CP) is uncertain and controversial. To investigate cotransporter function, we developed a novel dissociated rat CP cell preparation in which single, isolated cells maintain normal polarized morphology. Immunofluorescence demonstrated that in isolated cells the Na+-K+-ATPase, Na+-K+-2Cl- cotransporter, and aquaporin 1 water channel remained localized to the brush border, whereas the Cl-/HCO-3 (anion) exchanger type 2 was confined to the basolateral membrane. We utilized video-enhanced microscopy and cell volume measurement techniques to investigate cotransporter function. Application of 100 microM bumetanide caused CP cells to shrink rapidly. Elevation of extracellular K+ from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling was blocked completely by Na+ removal or by addition of 100 microM bumetanide. Exposure of CP cells to 5 mM BaCl2 induced rapid swelling that was inhibited by 100 microM bumetanide. We conclude that the CP cotransporter is constitutively active and propose that it functions in series with Ba2+-sensitive K+ channels to reabsorb K+ from cerebrospinal fluid to blood.