The exposure of rat and human lymphoid cells to mitogenic concentrations of phytohemagglutinin resulted in an apparent decrease in cellular K+ without a significant change in cellular Na+ when the cells were washed with isotonic Hepes buffered choline chloride prior to cation determination. The apparent reduction in total cellular Na+ plus K+ concentration, however, was not accompanied by a change in cell volume. We inferred that the constant cell volume could occur only if the lost intracellular K+ was exchanged for an external cation during the washing procedure used to prepare cells for Na+ and K+ measurement. This inference was supported by the quantitative recovery of lost cellular K+ in the choline chloride washing solution and the demonstration that a comparable proportion of 86Rb+ (K+ analogue) 42K+ was lost from prelabelled cells during choline chloride washing. Use of medium 199 with Hanks salts, 150 mM NaCl, or 100 mM MgCl2 as the washing solution did not prevent K+ exchange although exchange was less in the presence of MgCl2. These findings indicate that phytohemagglutinin produces a rapid alteration in lymphocyte plasma membranes so as to allow abnormal K+ exchange. This observation is of importance because investigators who measure intracellular solutes in phytohemagglutinin-treated lymphocytes must consider the possibility of lossduring preparative washes. Also, changes in membrane permeability following phytohemagglutinin treatment may modulate mitogenesis and/or permit the transmission of chemical messages between cells.
The K+ content of human lymphocytes has been examined during the initial 24 hours after exposure of cells to phytohemagglutinin (PHA). We have reconfirmed that lymphocyte K+ exchanges rapidly for extracellular counterions during preparative washing if cells are exposed to PHA. By using a technique to measure cation content which does not require removal of cells from their culture medium, we have shown that K+ does not change for 24 hours following PHA treatment. Previous reports have demonstrated that an enhanced uptake of K+ occurs in lymphocytes treated with PHA. This increased uptake may be a compensatory change for an increased exodus, explaining the failure of K+ to change following lectin treatment.
We have found that PHA produces an alteration in the lymphocyte membrane which allows 86Rb+ or 42K+ in prelabeled lymphocytes to exchange for cations present in washing solutions. These observations suggested that PHA might induce an increase in the exodus of intracellular potassium during incubation in physiologic media. We, therefore, examined 86Rb+ and 42K+ efflux from rat and human lymphocytes during incubation in tissue culture medium. The rate constant for efflux, Ke, was significantly increased by PHA. 86Rb+ efflux was increased by 27% in rat thymic lymphocytes and by 78% in human blood lymphocytes following PHA treatment.
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