Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl− absorption

Abstract

Cellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage,V _e , which attends net Cl⁻ absorption,J _Cl ^net , and to assess how ADH might augment bothJ _Cl ^met andV _e in the mouse medullary thick ascending limb of Henle microperfusedin vitro. Substituting luminal 5mm Ba⁺⁺ for 5mm K⁺ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio,R _c /R _bl , and increasing luminal K⁺ from 5 to 50mm in the presence of luminal 10⁻⁴ m furosemide resulted in a 53-mV depolarization of apical membrane voltage,V _a . Thus K⁺ accounted for at least 85% of apical membrane conductance. Either with or without ADH. 10⁻⁴ m luminal furosemide reducedV _e andJ _Cl ^net to near zero values and hyperpolarized bothV _a andV _bl , the voltage across basolateral membranes; however, the depolarization ofV _bl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization ofV _a , Thus ADH-dependent increases inV _b were referable to greater depolarizations ofV _bl in the presence of ADH than in the absence of ADH 68% of the furosemide-induced hyperpolarization ofV _a was referable to a decrease in the K⁺ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization ofV _bl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl⁻ current. Thus an increase in intracellular Cl⁻ activity may have contributed to the depolarization ofV _bl during net Cl⁻ absorption, and the intracellular Cl⁻ activity was likely greater with ADH than without hormone. Since ADH increases apical K⁺ conductance and since the chemical driving force for electroneutral Na⁺,K⁺,2Cl⁻ cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba⁺⁺-sensitive conductance K⁺ channels and electroneutral Na⁺,K⁺,2Cl⁻ cotransport units in apical plasma membranes.