Na/Na exchange through the Na/K pump of HK sheep erythrocytes

Dear Sir : In their classic paper of 1960, Tosteson and Hoffman (13) provided the physiological basis for the HK/LK polymorphism of sheep erythrocytes (see Lauf (11 ] for a recent review). In their results, the function of the Na/K pump in HK sheep cells differed in one important respect from pumps in human red cells. Garrahan and Glynn (5) showed that human red cell pumps, in the absence of extracellular K, exchange Na for Na, 1 :1. Tosteson and Hoffman (13) reported that there was no Na/Na exchange through the pumps in normal HK sheep cells. They did report a substantial Na/Na exchange independent of the pumps, i .e., Na/Na exchange insensitive to ouabain, similar to a mode ofNa/Na exchange observed more recently in human red cells (12). If Na/K pumps differ in this respect between sheep and human red cells, there may be important implications for our understanding of the mechanism of the pump, much of which is based upon the demonstration and characterization of various modes of its operation in human red cells (7). ATP/ADP exchange, first demonstrated in Na,K-ATPase from eel electric organ (4), and characterized more recently in human red cell ghosts (8), can be understood in terms of its relationship to Na/Na exchange (7, 9). HK sheep red cell membranes catalyze ouabain-sensitive ATP/ADP exchange (14) ; if HK red cell Na/K pumps cannot mediate Na/Na exchange, then the significance of ATP/ADP exchange should be reconsidered. We have reexamined the function of the Na/K pump of HK sheep red cells in the hope of determining if Na/Na exchange through it is possible. Mindful that both ADP and ATP are required for Na/Na exchange in human cells (6), and that nucleotide concentrations are lower in sheep than in human cells (1), we incubated the cells with appropriate metabolic substrates in order to increase the nucleotide concentrations. We also raised intracellular Na concentrations by treatment with nystatin. We were able to demonstrate Na/Na exchange through the pumps of the HK sheep cells. Blood was drawn into heparin from the jugular vein of adult Suffolk HK sheep. The cells were then washed twice by centrifugation and resuspension in an isotonic medium containing (mM): 150 NaCl, 10 Tris-HCI, 5 glucose, pH 7.4. To raise the intracellular nucleotide concentrations, aliquots of cells were incu-bated for 1 h at 37°C, 5% hematocrit, in a medium containing (mM): 150 NaCl, Address reprint …