Amiloride analog stimulation of short-circuit current in larval frog skin epithelium

The skin of the bullfrog Rana catesbeiana tadpole contains an apical non-selective cation channel that is activated by amiloride. This is in contrast to the adult skin, which has a highly Na+-selective channel that is blocked by amiloride. The purpose of the present study was to characterize further the nature of the tadpole channel using amiloride and its analogs benzamil, dimethyl amiloride (DMA), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and methyl isobutyl amiloride (MIBA). Tadpole skins were mounted in modified Ussing chambers with Ca2+-free KCl or NaCl Ringer on the apical side and standard NaCl Ringer (containing 2 mmol l-1 Ca2+) on the basolateral side. Drugs were added to the apical solution at concentrations between 0.1 and 1000 micromol l-1. Amiloride caused the short-circuit current (Isc) to increase rapidly from near zero to a peak of approximately 30-50 microA and then to decline back towards zero over several seconds. The peak response was largest at 100 micromol l-1. The rate of decline was noticeably faster at the higher concentrations. Benzamil and DMA had similar time courses to amiloride, but with smaller effects on Isc. The largest peak responses occurred at 5-50 micromol l-1. EIPA and MIBA gave small responses at 1-10 micromol l-1 and, at higher concentrations (50-500 micromol l-1), the responses consisted of rapid, small increases in Isc followed by rapid decreases. The largest peak response occurred at 10 micromol l-1 for both drugs. After apical membrane resistance had been reduced by nystatin, addition of analogs to the apical solution caused no change in Isc or transepithelial resistance. This suggests that the decline in Isc after amiloride analog treatment was not due to increases in the resistance of the basolateral membrane. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) blocked stimulation by all of the analogs. These data are consistent with amiloride analogs acting as both activators and inhibitors of short-circuit current in tadpole skin and extend the list of ligands that activate these channels.