Changes in the volume of marginal cells induced by isotonic 'Cl^- depletion/restoration': Involvement of the Cl^- channel and Na⁺-K⁺-Cl^- cotransporter

Marginal cells constitute the endolymph-facing epithelium responsible for the secretion of endolymph by the stria vascularis in the inner ear. We have studied the possible involvement of Cl^- conductance and Na⁺-K⁺-Cl^- cotransport in the mechanism of changes in cell volume upon isotonic Cl^- depletion/restoration. Changes in cell volume were estimated from video- microscopic images with the aid of an image processor. Marginal cells shrank to ~80% of their original volume in 30 s and to 65-70% in 90 s upon total replacement of [Cl](o) (~ 150 mM) by gluconate^-, and the original volume of the shrunken cells was restored within 2 min after restoration of Cl^-. The order of potency of anions to induce isotonic shrinkage was gluconate^- > I^- > F^- > Br^-. The cell shrinkage caused by Cl^- depletion was partially inhibited by 5-Nitro-2-(3-phenyl-propylamino)-benzoic acid (NPPB, 0.2 mM), but not by either 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS, 0.5 mM), bumetanide (10 μM) or ouabain (1 mM). The cell shrinkage caused by a reduction of [Cl](o) from ~ 150 mM to 7.5 mM was not affected by [K](o) in the range of 3.6 mM to 72 mM. These results suggest that the main efflux pathway(s) responsible for the 'Cl removal'-induced shrinkage depends on volume-correlated Cl^- conductance (Takeuchi and Irimajiri, J. Membrane Biol. 150, 47-62, 1996) and that this pathway(s) is essentially independent of the Na⁺-K⁺-Cl^- cotransporter, the Na⁺,K⁺-ATPase, and the K⁺-Cl^- cotransporter. With regard to volume recovery after isotonic shrinkage, its critical dependence on the simultaneous presence of Na⁺, K⁺ and Cl^- in the bath and its substantial inhibition by bumetanide (10 μM) both indicate a major role for Na⁺-K⁺-Cl^- cotransport. The strong influence on cell volume of solute fluxes working through the Cl^- channel and the Na⁺-K⁺-Cl^- cotransporter implies an essential role for these pathways in the ion transport mechanism(s) of the marginal cell.