Anthracene-9-carboxylic acid inhibits an apical membrane, chloride conductance in canine tracheal epithelium

Abstract

Canine tracheal epithelium secretes Cl from the submucosal to the mucosal surface via an electrogenic transport process that appears to apply to a wide variety of secretory epithelia. Cl exit across the apical membrane is thought to be a passive, electrically conductive process. To examine the cellular mechanism of Cl secretion we studied the effect of anthracene-9-carboxylic acid (9-AC), an agent known to inhibit the Cl conductance of muscle membrane. When added to the mucosal solution, 9-AC rapidly and reversibly decreases short-circuit current and transepithelial conductance, reflecting a reduction in electrogenic Cl secretion. The inhibition is concentration-dependent and 9-AC does not appear to compete with Cl for the transport process. The decrease in current and conductance results from a decrease in the net and both unidirectional transepithelial Cl fluxes without substantial alterations of Na fluxes. Furthermore, 9-AC specifically inhibits a Cl conductance: tissues bathed in Cl-free solutions showed no response to 9-AC. Likewise, when the rate of secretion and Cl conductance were minimized with indomethacin, addition of 9-AC did not alter transepithelial conductance. In contrast, neither removal of Na from the media nor blockade of the apical Na conductance with amiloride prevented a 9-AC-induced decrease in transepithelial conductance. We also found that the effect of 9-AC is independent of transepithelial transport: 9-AC decreases transepithelial conductance despite inhibition of Cl secretion with ouabain or furosemide. Intracellular electrophysiologic techniques were used to localize the effect of 9-AC to a reduction of the electrical conductance of the apical cell membrane: 9-AC hyperpolarizes the electrical potential difference across the apical membrane and decreases its relative conductance. 9-AC also prevents the characteristic changes in the cellular electrical potential profile, transepithelial conductance, and the ratio of membrane conductances produced by a reduction in mucosal bathing solution Cl concentration. These results indicate that 9-AC inhibits Cl secretion in tracheal epithelium by blocking an electrically conductive Cl exit step in the apical cell membrane. Thus, they support a cellular model of Cl secretion in which Cl leaves the cell across a Cl permeable apical membrane driven by its electrochemical gradient.