Importance of anion in hypotonic volume regulation of rabbit proximal straight tubule

Rabbit proximal straight tubules rapidly swell to a maximum volume when abruptly immersed into hypotonic medium. However, in a second, slower phase, termed volume regulatory decrease (VRD), tubules shrink toward their basal volume due to the efflux of K, accompanying anion and water. In the present study, we investigated the nature of the anion during hypotonic volume regulation. We removed Cl and/or the HCO₃ buffer to assess their relative importance in VRD, and we used furosemide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and barium to investigate the nature of the transmembrane anion pathway in VRD. Isometric replacement of peritubular Cl with gluconate had no effect on either tubule volume in isotonic medium or the initial osmometric swelling response in hypotonic medium. However, the VRD in such Cl-depleted tubules was significantly inhibited. Control tubules regulated 81% below their maximal volume in dilute medium. By contrast, Cl-depleted tubules regulated only 39%. This inhibitory effect could not be attributed to the absence of peritubular Cl or to the presence of gluconate. The absence of the HCO₃ buffer or the presence of SITS (0.5 mM) had no inhibitory effect on the rate or extent of VRD. Furosemide alone (1 mM) also had no inhibitory effect on VRD. However, whereas barium alone delays VRD, addition of furosemide to barium-treated tubules further slowed their maximal rate of fluid efflux and delayed VRD even more. Notwithstanding the limitations of ion-substitution and inhibitor experiments, these findings demonstrate that Cl is a principal anion involved in VRD and are consistent with the operation of parallel KCl cotransport and basolateral Cl conductance in the volume regulatory efflux of Cl.