Transcellular chloride pathways in Ambystoma proximal tubule

The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl- absorption linked to Na+ were investigated in isolated perfused tubules using Cl--selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl- (a(i)(Cl)) is 11.3 +/- 0.5 mM, the basolateral (V-1), apical (V-2), and transepithelial (V-3) potential differences are -68 +/- 1.2 mV, +62 +/- 1.2 mV and -6.4 +/- 0.3 mV, respectively. When Na+ absorption is decreased by removal of organic substrates from the lumen, a(i)(Cl) falls by 1.3 +/- 0.3 mM and V-2 hyperpolarizes by +11.4 +/- 1.7 mV. Subsequent removal of Na+ from the lumen causes a(i)(Cl) to fall further by 2.3 +/- 0.4 mM and V-2 to hyperpolarize further by +15.3 +/- 2.4 mV. The contribution of transporters and channels to the observed changes of a(i)(Cl) was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on a(i)(Cl). The effects of luminal HCO3- removal and/ or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO3 or Cl-OH. The effects of basolateral HCO3- removal, of basolateral Na+ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO3 exchange. Several lines of evidence favor conductive Cl- transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the a(i)(Cl) by 2.4 +/- 0.6 mM or 2.9 +/- 1.0 mM respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO3--free Ringer, the equilibrium potential for Cl- does not differ from the membrane potential V-2. Finally, the logarithmic changes in a(i)(Cl) in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl- channels at the apical and basolateral cell membranes of the proximal tubule.