ADH increases apical Na+, K+, 2Cl- entry in mouse medullary thick ascending limbs of Henle

These studies were designed to evaluate the mechanism for the ADH-dependent increase in transcellular conductance (Gc, mS X cm-2), which accompanies hormone-dependent increases in the spontaneous transepithelial voltage (Ve, mV) and in the net rate of Cl- absorption in single medullary thick ascending limbs of Henle (mTALH) isolated from mouse kidney. The total transepithelial conductance (Ge, mS X cm-2) was measured with perfusing solutions containing 5 mM K+, zero Ba2+; Gc was that component of Ge blocked by luminal 20 mM Ba2+, zero K+. In paired experiments, antidiuretic hormone (ADH) increased Gc from 44.5 +/- 5.6 to 58.9 +/- 8.9 mS X cm-2 (delta = 14.3 +/- 5.5; P less than 0.02); however, in the presence of 10(-4) M luminal furosemide, ADH had no significant effect on Gc (delta = 5.0 +/- 4.3; NS). A set of similarly paired measurements together with paired observations on the effects of bath Cl- deletion, permitted an assessment of the effect of ADH on the magnitude of the fall in Gc on bath Cl- removal (delta GClc, mS X cm-2). delta GClc was clearly larger with ADH, 29.6 +/- 4.3, than without ADH, 19.2 +/- 1.0 (delta = 10.4 +/- 4.9; P less than 0.05). However, with luminal furosemide, ADH had no significant effect on delta GClc (delta = 1.7 +/- 4.5; NS). These results indicate that the ADH-dependent increase in Gc is secondary to increased salt entry across the apical membrane. We computed apical (ga, mS X cm-2) and basolateral (gb, mS X cm-2) membrane conductances from the Gc measurements and apical-to-basolateral membrane resistance ratios (Ra/Rb) obtained from cell impalement: the ADH-dependent Gc increase was due to an increase in gb, which was blocked entirely by luminal furosemide. We propose that ADH increases the number of functioning apical membrane Na+,K+,2Cl- transport units, and that gb increases because cell Cl- activity rises and depolarizes the basolateral membrane. Thus the calculated cellular Cl- activity was 16.3 mM without ADH, and 25 mM with ADH.