Thiazide-sensitive Na-Cl cotransport mediates NaCl absorption in amphibian distal tubule

To find out the mechanism(s) underlying NaCl absorption in the distal tubule of Necturus, we devised a variant of the split-drop technique. Following injection an oil column, subsequently split by a NaCl solution isotonic to plasma, a double-barrelled microelectrode (conventional/selective to Na+ or to Cl- ions) recorded Na+ (alpha Na) or Cl- (alpha Cl) activity and transepithelial potential (Vte). Paired control/low-Na+ solutions yielded reabsorptive half-times (t1/2) of 0.68 +/- 0.11 min and 7.6 +/- 1.8 min respectively; corresponding Vte values were -22.2 +/- 4.0 mV and -7.6 +/- 1.9 mV. t1/2 values of control versus low-Cl- solutions were 0.77 +/- 0.32 min and 6.5 +/- 1.7 min respectively, whereas respective Vte values were not different from one another: -23.8 +/- 4.3 mV versus -18.8 +/- 5.5 mV. Nominally K(+)-free solutions or bumetanide, 10 mumol/l, did not alter t1/2 or Vte, with regard to the paired control. Amiloride, 5 mumol/l or 2 mmol/l, failed to decrease t1/2 or to lower Vte; apparently, the role of a Na+/H+ antiport does not contribute significantly to NaCl absorption. Furosemide, 0.1 mmol/l, reduced t1/2 by 54% with regard to the control state. Determination of t1/2 as a function of increasing hydrochlorothiazide concentrations revealed apical high- and low-affinity sites, estimated at 0.56 mumol/l and 0.115 mmol/l respectively. Taken together these observations indicate that NaCl absorption is predominantly carried out by an electroneutral Na(+)-Cl- cotransport.