AJP - Cell Physiology, Vol 264, Issue 5 C1270-C1277, Copyright © 1993 by American Physiological Society
Inhibition of Na-K-2Cl cotransport and bumetanide binding by ethacrynic acid, its analogues, and adducts
H. C. Palfrey and S. Leung
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637.
The inhibitory effect of ethacrynic acid (EA) and a variety of its
derivatives on Na-K-2Cl cotransport in avian erythrocytes was investigated.
The most potent compound tested was the adduct of EA with L-cysteine, with
an IC50 of 7.2 x 10(-7) M. EA itself, dihydro-EA, EA-D-cysteine, and
adducts of EA with other sulfhydryl (-SH) compounds were much less potent.
The mechanism of action of EA and EA-L-cysteine differed in several
respects: 1) EA-L-cysteine acted more rapidly than EA (half times of < 1
and 4 min, respectively, at 37 degrees C); 2) the action of EA-L-cysteine
was reversible by washing, whereas that of EA was not; and 3) the degree of
inhibition by EA-L-cysteine varied with medium [K], whereas that of EA did
not. The inhibitory effects of both EA-L-cysteine and EA were affected by
medium [Na] and [Cl]. We conclude that EA-L-cysteine does not "deliver" EA
to transport-related -SH residues or act as an alkylating agent but has
some stereospecific effect on cotransport that is a property of the entire
molecule. EA does appear to inhibit cotransport by alkylating -SH residues,
as closely related compounds lacking the ability to covalently react with
such groups were reversible, and other -SH reagents (e.g.,
N-ethylmaleimide) also inhibited cotransport. EA, EA-L-cysteine, and
EA-D-cysteine all inhibited [3H]bumetanide binding to membranes from
activated avian erythrocytes at concentrations similar to those that
inhibited cotransport. It is possible that the EA and bumetanide types of
diuretics interact with closely apposed sites on the Na-K-2Cl
cotransporter.
