AJP - Cell Physiology, Vol 258, Issue 4 C599-C609, Copyright © 1990 by American Physiological Society
Kinetic mechanism of ATP action in Na(+)-K(+)-Cl- cotransport of HeLa cells determined by Rb+ influx studies
T. Ikehara, H. Yamaguchi, K. Hosokawa and H. Miyamoto
Department of Physiology, School of Medicine, University of Tokushima, Japan.
The kinetics of Na(+)-K(+)-Cl- cotransport were studied by measuring
ouabain-insensitive furosemide-sensitive Rb+ influx (JRb) into HeLa cells
while varying the cellular ATP and the extracellular Rb+ and Na+
concentrations. Results reveal that ATP stimulates JRb by increasing the
affinity of the cotransporter for Rb+ (K+), and the apparent Michaelis
constant (Km) for ATP was 0.95 +/- 0.03 mmol/l cell water. Two ATP
molecules may relate to the uptake of one Rb+ by the cotransport pathway,
as examined by the nonlinear least-squares method for goodness-of-fit and a
Hill plot, JRb was strengthened by an increase in the inward chemical
gradient associated with cell swelling on preincubation in a low-Na+
high-K+ medium, accompanying an increase in the affinity of the transporter
for ATP. JRb was apparently activated by extracellular Na+, and the
activation was enhanced by an increase in the cellular ATP concentration.
Lactate production stimulated by 2 microM carbonylcyanide m-chlorophenyl
hydrazone (CCCP) was reduced by 10 microM ouabain but not altered by
further addition of 0.1 mM furosemide. Increases in cellular adenosine
3',5'-cyclic monophosphate (cAMP) caused by treatment with 0.1 mM
isoproterenol plus 0.5 mM 3-isobutyl-1-methylxanthine or with 0.1 mM
dibutyryl cAMP did not influence JRb. From this and previous studies, we
propose a general and a specific model of Na(+)-K(+)-Cl- cotransport, which
elucidate the order of binding of extracellular ions and reaction of
cellular ATP.
