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Increase in Intracellular Na⁺: Transmembrane Signal for Rejoining of DNA Strand Breaks in Proliferating Lymphocytes¹

Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Two early events in the mitogen-induced entry of murine splenocytes into proliferation are (a) a rapid rise in influx of Na⁺, causing its total internal concentration to increase by 42 ± 7% within 2 h of culture with concanavalin A (Con A), and (b) rejoining of some 3000 DNA strand breaks per diploid genome within the same period. Con A did not induce rejoining in low Na⁺ (<9 mM) medium, but the process began directly when Na⁺ was added, at its usual concentration, to the growth medium. Incubation of cells with ouabain, an inhibitor of the Na⁺K⁺-ATPase, or monensin, a Na⁺ ionophore, caused an increase in the internal Na⁺ concentration in normal, but not in low, Na⁺ medium. In the former (but not in the latter) medium, both ouabain and monensin caused rejoining of the DNA strand breaks to occur in resting lymphocytes, i.e., in the absence of mitogen. Stimulation of splenocytes with Con A also resulted in a rapid but transient increase in the level of intracellular free Ca²⁺. This effect was also observed in the absence of extracellular Na⁺; however, deprivation of extracellular Ca²⁺ completely abolished this effect. Moreover, the intracellular free Ca²⁺ level was significantly higher in cells suspended in Na⁺-free buffer or medium. Since the Con A-induced rejoining of DNA strand breaks occurred in the absence of extracellular Ca²⁺ and removal of extracellular Na⁺ had no inhibitory effect on the Con A-induced increase in the level of intracellular free Ca²⁺, the Con A-stimulated repair could not have been mediated by the initial increase in Ca²⁺ influx. The early mitogen-induced increase in the internal Na⁺ concentration is a necessary and sufficient signal for the rejoining of breaks, an event that must occur before the proliferating lymphocytes can replicate their DNA.

¹ This research was supported by grants from the Natural Sciences and Engineering and Medical Research Councils of Canada and equipment grants from the Alberta Heritage Foundation for Medical Research.

² Postdoctoral fellows of the Alberta Heritage Foundation for Medical Research.

³ To whom requests for reprints should be addressed.

Received 3/ 3/87. Revised 6/10/87. Accepted 7/16/87.

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