Differential effects of glycolytic and oxidative metabolism blockers on the Na-K pump in erythrocytes of the frog, Rana temporaria.

This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content of frog erythrocytes. To eliminate K-Cl cotransport, the frog erythrocytes were incubated in nitrate media at 20 degrees C. Incubation of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K+ influx measured as 86Rb uptake for 60 min. The Na(+)-K(+)-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose or adenosine. Unexpectedly, the treatment of red cells with 1-2 mM glycolytic blocker iodoacetate produced a 2-fold increase in the ouabain-sensitive K+ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K+ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K+ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast, 0.01-0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation were without effect on K+ influx into frog erythrocytes. These results indicate that about one-half of the Na(+)-K(+)-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate "membrane-associated" ATP pool.