Amiloride blockage of Na⁺ channels in amphibian epithelia does not require external Ca²⁺

Abstract

Noise analysis was used to study the influence of external Ca²⁺ on the blockage of Na⁺ transport by amiloride. Experiments were done using frog skin (Rana temporaria and Rana catesbeiana), toad urinary bladder (Bufo marinus) and epithelia of A6 cells. In non-depolarized skins and bladders, removal of Ca²⁺ from the mucosal bath diminished markedly the inhibitory effect of amiloride. Ca²⁺ depletion also gave rise to the appearance of an additional noise component related to cation movement through the poorly selective cation channel in the apical membrane [Aelvoet I, Erlij D, Van Driessche W (1988) J Physiol (Lond) 398:555–574; Van Driessche W, Desmedt L, Simaels J (1991) Pflügers Arch 418:193–203]. The amplitude of this Ca²⁺-blockable noise component was elevated by amiloride and markedly exceeded the amiloride-induced Lorentzian noise levels as recorded in the presence of Ca²⁺. On the other hand, in K⁺-depolarized skins and bladders as well as in non-depolarized epithelia of A6 cells, the Ca²⁺-blockable noise was absent or of much smaller amplitude. Depolarization of frog skin and toad urinary bladder apparently inactivated the poorly selective channels, whereas in A6 cells they were not observed. Under these conditions the typical amiloride-induced blocker noise could also be analysed in the absence of Ca²⁺ and demonstrated that the on and off rates for amiloride binding were not significantly altered by external Ca²⁺. We conclude that (a) external Ca²⁺ per se does not affect the inhibitory potency of amiloride, and (b) that the observed differences between frog skin, toad urinary bladder and A6 cells originate from the presence or absence of a poorly selective cation channel rather than from a different amiloride receptor structure.