Gramicidin-Perforated Patch Analysis on HCO₃ ⁻ Secretion Through a Forskolin-Activated Anion Channel in Rat Parotid Intralobular Duct Cells

Abstract.

Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO₃ ⁻ secretion in the intralobular duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K⁺, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO₃ ⁻. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO₃ ⁻ significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl⁻ channel located in luminal membranes) and production of cytosolic HCO₃ ⁻ induce the inward anion current and resulting depolarization. Inhibition of the Na⁺-K⁺-2Cl⁻ cotransporter and the Cl⁻-HCO₃ ⁻ exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl⁻ via the Na⁺-K⁺-2Cl⁻ cotransporter or the Cl⁻-HCO₃ ⁻ exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO₃ ⁻ produced intracellularly, but not of Cl⁻ due to lack of active Cl⁻ transport in parotid duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.