Thymocyte K+, Na+ and Water Balance During Dexamethasone- and Etoposide-Induced Apoptosis

Abstract

The mechanism of apoptotic cell volume decrease was studied in rat thymocytes treated with dexamethasone (Dex) or etoposide (Eto). Cell shrinkage, i.e. dehydration, was quantified by using buoyant density of the thymocytes in a continuous Percoll gradient. The K⁺ and Na⁺ content of cells from different density fractions were assayed by flame emission analysis. Apoptosis was tested by microscopy and flow cytometry of acridine orange stained cells as well as by flow DNA cytometry. Treatment of the thymocytes with 1 µM Dex for 4-5.5 h or 50 µM Eto for 5 h resulted in the appearance of a new distinct high-density cell subpopulation. The cells from this heavy subpopulation but not those with normal buoyant density had typical features of apoptosis. Apoptotic increase of cell density was accompanied by a decrease in cellular K⁺ content, which exceeded the simultaneous increase in cellular Na⁺ content. Cellular loss of K⁺ contributed to most of the estimated loss of cellular osmolytes, but owing to the parallel loss of cell water, the decrease in cytosolic K⁺ concentration was less than one third. Due to gain of Na⁺ and loss of cell water the cytosolic Na⁺ concentration in thymocytes rose following treatment with Dex (5.5 h) or Eto (5 h) by a factor of about 3.6 and 3.1, respectively.