Effect of cell age and phenylhydrazine on the cation transport properties of rabbit erythrocytes.

We studied the effect of cell age on the cation transport systems of rabbit erythrocytes by increasing the proportion of circulating young erythrocytes with either repeated bleeding or with phenylhydrazine (PHZ) treatment. We found that when the reticulocyte content of rabbit blood is increased by bleeding (from 1 to 40-50% of the circulating red cells), the response of the various transport pathways differs. The largest increase (fivefold) was found in the activity of K-Cl cotransport, which peaked 3 days after the last bleeding. The Na-K pump activity peaked at a similar time, but the % increase was twofold less than the K-Cl cotransport. There was a very small increase in the activity of the Na-Li exchange, whereas the Na-H exchange reached peak values 10 days after the last bleeding (twofold increase), when activities of K-Cl cotransport and Na-K pump had returned to almost normal levels. In vivo PHZ treatment resulted in anemia and marked reticulocytosis (80-90% of circulating cells). Transport rates were markedly increased (Na-K pump 9.6-fold, Na-H exchange 6.8-fold, Na-Li exchange 2.75-fold; K-Cl cotransport: 10-20-fold). When blood from PHZ-treated rabbits was incubated in vitro for 24-48 hours, red cell volume and K content decreased. This process was associated with a 70% reduction in the activity of the K-Cl cotransport after 24 hours and a 90% reduction after 48 hours. The activity of the other systems also declined and approached baseline values after 48 hours. Loss of transport activity was not affected by 10 microM E-64, whereas 10 mM methylamine reduced the inactivation of the Na-H exchange and of the Na-Li exchange. PHZ treatment of rabbit red cells in vitro resulted in marked increase of the K-Cl cotransport and inhibition of Na-K pump, Na-H exchange, and Na-Li exchange. These effects were abolished by DTT, with the exception of the Na-K pump inhibition, which was DTT insensitive. Thus both cell age and oxidative damage are important determinants of cation transport in rabbit red cells.