Calcium and ATP regulation of ion transport in larval frog skin

Abstract

Ion transport measured as short circuit current (Isc) across the skin of larval frogs is activated by amiloride, acetylcholine, and ATP. In many epithelia, ATP stimulation of Isc involves an increase in intracellular calcium. To define the role of changes in intracellular calcium in ATP stimulation of Isc in larval frog skin, epithelial cells were loaded with calcium by adding 5 μM ionomycin to a 2 mM calcium apical Ringer's solution. Calcium loading had no observable effect on baseline Isc or on stimulation by ATP. Minimizing changes in intracellular calcium by loading the cell with the calcium chelator BAPTA also had no measurable effect on ATP stimulation of Isc. When the apical side was bathed with Ca²⁺-free Ringer's solution, ionomycin increased Isc up to 15 μA. This increase was partially blocked by 2 mM Ca²⁺, 2 mM Mg²⁺, and 10 μM W-7. Other experiments showed that baseline-stimulated and ATP-stimulated Isc were always larger in 2 mM Mg²⁺ Ringer's compared to 2 mM Ca²⁺. In dissociated cells bathed in 2 mM Ca²⁺ Ringer's, ATP had no effect on intracellular calcium as measured by Fluo-LR fluorescence changes. In conclusion, ATP apparently stimulates Isc without concomitant changes in intracellular calcium. This is consistent with a directly ligand-gated receptor at the apical membrane with P2X-like characteristics.