AMERICAN JOURNAL 0F PI~Y~IOLOGY Vol. 228, No. 2, February 1975. Printed
Changes
in U.S.A.
of sodium
of the maturing
transport
in erythrocytes
rabbit
E. K. XL SMITH, Department
of
L. WEIHRAUCH, AND D. FARRIXGTON Medicine, McMaster University, Hamilton, Ontario, &nadu
M., L. WEIHRAUCH, AND ID. FARRINGTON. Changes in erythrocytes of the maturing rabbit. Am. J. Physiol. 228(Z) : 461-464. 1975.-The red blood cells of New Zealand white rabbits have a low sodium and high potassium content. As E. K.
SMITH,
of sodium
trcmsjmrt
the animals mature, the sodium concentration rises and the potassium content falls; studies of red cells from a group of five young and five mature animals revealed a highly significant increase of cell sodium with age that was associated with a significant fall in the rate of ouabain-inhibited active sodium efflux. This difference was still seen when the sodium concentration within the cells from old and young animals was equalized and elevated to saturating levels for active pump efflux. Total sodium efflux, however, increased significantly with age as did total sodium influx so that a steady state was reached. Ouabain-sensitive ATPase activity fell significantly in the cell membranes from older animals and ouabain-insensitive ATPase increased with age. The survival time of 51Cr-labeled red cells was significantly longer in old than in young animals and it is concluded that as the rabbit matures its red cells survive for a longer period and this is associated with the changes of sodium transport and ATPase activity that have been documented. membrane
L4TPase;
ouabain
CELLS of the rabbit, like those of man, have a low and a high potassium content. These are maintained by active transport mechanisms th at pump cations t their con centration gradients (1 1) . In man the red composi tion remains remarkably constant cell cation throughout life (Z), but in the rabbit there is a progressive rise in cell sodium and fall in cell potassium during the 1st yr of life (16). The experiments reported here were carried out in an attempt to pursue the extent of these and to de termin e their mechanism and their changes possible relevance to cation transpor *t in more complex cells. THE
RED
sodium
METHODS
Intracellular cation composition was measured by Aame photometry with red cells suspended at a known hematocrit after being washed in isotonic magnesium chloride (X6). Sodium efflux was measured by the method of Sachs and Welt (13) but with 22Na as a tracer (17). Efflux was expressed as the fraction of intracellular sodium extruded per hour (the rate constant); from this, total sodium efflux can be calculated from the relationship: sodium efflux (mM/h) = efflux rate constant X red cell sodium concentration.
Sodium influx was measured with 221Ya as a tracer and the technique of Smith and Samuel (17) was followed, where influx was expressed as millimoles per liter per hour. The standard incubation medium for both influx and efflux studies contained sodium chloride 140 mM and potassium chloride 5 mM. Phosphate buffer at pH 7.4 (A O-05) was present at a concentration of 1.4 mM. Dextrose was added (150 mg/lOO ml) and magnesium carbonate-glycylglycine buffer at pH 7.4 (A 0.05) made up 10 % of total volume. The final concentration of glycylglycine was 27 mM and magnesium carbonate 4.4 mM. Crystalline bovine serum albumin (BSA) was added to a concentration of 0.1 g/ 100 ml to prevent hemolysis. Some studies were made of cells whose intracellular sodium concentration was varied with p-chloromercuribenzene sulfonic acid (PCMBS) to alter cell permeability and dithiothreitol to reverse the effect. The method used was a modification described by Sachs (12) of the method of Garrahan and Rega (7). Adenosine triphosphatase activity was studied in membranes prepared from freshly drawn blood. After anticoagulation with heparin, membranes were prepared by the method of Dodge et al. (6). With this technique, a flocculant white precipitate of membranes was obtained with complete elution of visible hemoglobin and a remarkably uniform composition. The ATPase activity was measured (18) with a standard incubation medium containing NaCl 75 mM, KC1 25 mM, and MgClz 1 mM. Ethylenediaminetetraacetic acid (EDTA) was also present (0.25 mM) and the medium was buffered with Tris-HCl (25 mM) to pH 7.4 =t 0.05. The amount of inorganic phosphate (Pi) liberated from ATP ( 10B3 M) was measured by an automated method (5). Where relevant, the addition of ouabain was in aqueous solution at a final concentration of low3 M. Red cell survival was studied with chromium-labeled red cells with the technique described by Card and Weintraub (4). The separation of red blood cells in terms of their age was achieved by a modification of the technique of Borun et al. (3). This involves centrifugation of a column of red cells in a 4-mm-diam glass tube that can then be divided by sawing off segments of the glass tube and the column of blood it contains. In this way the cation content of blood cells at various points in the column can be measured, older cells being at the bottom and the younger cells at the topm
4,6I
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462
SMITH,
RESULTS
In an earlier study (16), rabbits were followed for up to 6 mo, during which time a progressive rise of cell sodium occurred. These studies have been extended for nearly 18 mo and, as can be seen in Fig. 1, the cell sodium concentration continued to rise for about 9 mo, then tended to level off. During this period the cell potassium fell with a similar time course and again reached a plateau (Fig* 2). Five rabbits, about a year of age, were then taken, together with five newly acquired young animals. The “old” rabbits had a mean (=t SD) sodium concentration of 17.6 & 2.2 mM of red cells, as indicated in Table 1; the “young” animals had a mean cell sodium of 9.1 Z& 1.15 mM of red cells. The difference in cell sodium between these two groups was highly significant (P < 0.00 1). We then studied the sodium transport mechanism in the two groups of animals designated old and young. The efflux rate constant (i.e., the proportion of intracellular sodium extruded in 1 h) for both these groups was then determined. Total sodium efflux rate constant was 0.68 4~ 0.08 (SD) in the old animals as compared with 1.02 & 0,05 in the young group. This difference was significant (P < *O l), as is shown in Table 1; it was largely due to a significant reduction of the ouabain-inhibited component of sodium efflux, which in old rabbits had a rate constant of 0.11 =t 0.07 whereas in young rabbits it was 0.35 & 0.05. This difference was highly significant (P < .Ol). There was, however, also a difference in rate constant for
WEIHRAUCH,
TABLE
1. &d
ouabain-inhibited --- -.
Old Young P
cell
sodium, sodium eflux, and sodium ej?ux
17.6 9.1
A 2.2 Al 1.15
0.68 1.02