Mode of inhibition of active chloride transport in the frog cornea by furosemide

The mechanism of inhibition of active Cl^- secretion by 1 mM furosemide and 0.1 mM bumetanide was characterized in the isolated frog corneal epithelium. Transepithelial and transmembrane cell electrical parameters as well as transmembrane Cl^- electrochemical potential difference were measured with conventional glass microelectrodes and Cl^- selective microelectrodes. Furosemide caused the potential difference across the apical membrane to hyperpolarize by 20 mV while the transepithelial potential difference declined by 13 mV. The apical-to-basolateral membrane resistance ratio increased 3-4 times after furosemide or bumetanide addition. Preincubation with furosemide prevented a 30-mV depolarization of the apical membrane potential difference normally observed when Cl^- was removed from the tear side bathing solution. In control conditions, intracellular Cl^- activity was above equilibrium. Bumetanide further increased the Cl^- electrochemical gradient between the cell compartment and the bathing solutions even though intracellular Cl^- activity fell from 18 to 12 mM. In contrast, perfusion with Cl^--free Ringer in the stroma side bathing solution decreased the Cl^- electrochemical gradient across the apical membrane to zero, indicating an equilibrium distribution. Adenosine, which selectively increases Cl^- permeability of the apical membrane, also decreased the Cl^- electrochemical gradient across the apical membrane. These results suggest that the diuretics inhibit active Cl^- transport primarily by decreasing the Cl^- permeability of the apical membrane.