Volume regulation by human lymphocytes: Characterization of the ionic basis for regulatory volume decrease

Abstract

The mechanism of volume regulation in hypotonic media was analysed in human peripheral blood mononuclear (PBM) cells. Electronic cell sizing showed that hypotonic swelling is followed by a regulatory volume decrease (RVD) phase. This was confirmed by both electron microscopy and by cellular water determinations. The rate of regulatory shrinking was proportional to the degree of hypotonicity in the 0.5-0.9 X isotonic range. Cell viability was only marginally affected in this range. The content of cellular K+ decreased during RVD, while Na+ content remained unchanged. Similarly, the efflux of 86Rb (used as a K+ analog) increased upon dilution, whereas 22Na efflux was not altered. 86Rb uptake was enhanced by hypotonic stress and both ouabain-sensitive and -insensitive components were affected. A ouabain-sensitive stimulation was also seen in Na+- free media. Ouabain partially inhibited RVD only if added to the cells hours before hypotonic challenge. A normal shrinking response was observed in K+-free media, and also in Na+-free media when Li+, choline+, or Tris+ were the substitutes. In high K+ or RB+ hypotonic media shrinking was absent and a second swelling phase was observed. Cs+ displayed an intermediate behavior, with shrinking observed at lower dilutions and secondary swelling at higher ones. The direction and magnitude of the response also changed when the external K+ concentration was varied and, with 50 mM K+, no regulatory volume change occurred following hypotonic stress. These findings suggest that RVD occurs largely by a passive loss of cellular K+, resulting from a selective increase in permeability to this ion. In addition, the (Na-K) pump appears to be activated upon cell swelling by a mechanism other than Na+ entry into the cell, but this activation is not essential for RVD.

Volume regulation by human lymphocytes. Identification of differences between the two major lymphocyte subpopulations.

“…As expected, 1 min after dilution, before the regulatory mechanism becomes apparent, the increase in relative volume was essentially the same for PBM and HTC, with the increment dictated by the degree of dilution of the medium. (8), the rapid initial swelling of PBM (Fig. 2C) was followed by a slower secondary swelling.…”

“…We have studied volume regulation by peripheral blood mononuclear cells (PBM) and confirmed the central role of potassium ions (K+) in this process (8). During RVD, intracellular K+ content decreased while Na+ content remained essentially unchanged.…”

“…When subjected to hypotonic challenge, many cell types have the ability to restore their volume after an initial phase of passive swelling (1)(2)(3)(4)(5)(6)(7)(8)(9). This regulation of cell volume is mediated through shifts in cellular ion and water content (1,3,7).…”

“…This regulation of cell volume is mediated through shifts in cellular ion and water content (1,3,7). The shrinking phase or regulatory volume decrease (RVD)l occurs very rapidly after initial swelling in human lymphocytes (4,8). Indeed, in contrast to erythrocytes (9), peripheral blood lymphocytes can readjust their volume very quickly, despite a much lower anion permeability, suggesting that different mechanisms may be operative in different cell types.…”

Characterization of ion channels seen in subconfluent human dermal fibroblasts.

“…The ability to volume regulate has been studied in lymphocytes and is probably common to all mammalian cells. In lymphocytes, the regulatory volume decrease seen in response to hypotonic solutions is dependent on a rise in both Cl-and K+ permeability (Cheung, Grinstein, Dosch & Gelfand, 1982). As both of these ions exit the cell down their concentration gradients, the loss of solutes causes a loss of intracellular water.…”