Inhibition by amiloride of 22Na + transport into toad bladder microsomes

Abstract

The diuretic, amiloride, inhibited 22Na + accumulation by microsomes from toad ( Bufo marinus) bladder by between 40 and 70%. Accumulated 22Na + was separated from free isotope by ion-exchange chromatography. The amiloride-sensitive process was directly proportional to the protein concentration and a hyperbolic function of the Na + concentration. The inhibition was competitive. At least 9 times more activity was found in the microsomal fraction than the mitochondrial fraction, and none at all was found in the cytosol. Amiloride inhibition could not be reproduced by similar pyrazine and guanidine compounds, such as sulfaguanidine. Amiloride-sensitive Na + accumulation was totally reversed by nigericin. The intravesicular volume of the bladder microsomes as determined by the [ 3H]H 2O/[ 14C]inulin method was shown to be nearly equal to the volume occupied by the amiloride-sensitive portion of the 22Na + accumulation. The amiloride-sensitive Na + accumulation most likely represents transport into vesicles. Since amiloride does not affect gramicidin-mediated 22Na + uptake into phospholipid vesicles, the amiloride inhibition seen with bladder microsomes probably represents a specific channel blockade and not simply interference with cation movement that might be produced by any cationic hydrophobic molecule.