gramicidin-a and 5-dimethylamiloride

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 microM), 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.

Topics: Amiloride; Animals; Anions; Bicarbonates; Bumetanide; Chlorides; Colforsin; Glyburide; Gramicidin; In Vitro Techniques; Male; Membrane Potentials; Methazolamide; Parotid Gland; Patch-Clamp Techniques; Rats; Rats, Wistar