Selective inhibition by bis(2-chloroethyl)methylamine (nitrogen mustard) of the Na/K/Cl cotransporter of murine L1210 leukemia cells

Incubation of L1210 murine leukemia cells in vitro with 10 μM of the bifunctional alkylating agent bis(2-chloroethyl)methylamine (nitrogen mustard, HN2) for 10 min brought about a fall of more than 99.9% in their ability to form colonies when the cells were suspended in 0.5% nutrient agar. Incubation with HN2 also inhibited the influx of the potassium congener ⁸⁶Rb⁺ to exponentially proliferating L1210 cells in a concentration-dependent manner. This inhibition was specific and was accounted for by a reduction of a diuretic-sensitive component of ⁸⁶Rb⁺ influx, identified in the preceding paper (Wilcock, C. and Hickman, J.A. (1988) Biochim. Biophys. Acta 946, 359–367) as being mediated by a Na⁺/K⁺/Cl⁻ cotransporter. Inhibition by 10 μM HN2 was complete after a 3-h incubation. There was no inhibition at this time of the ouabain-sensitive component of ⁸⁶Rb⁺ influx, mediated by Na⁺/K⁺-ATPase. After 3 h of incubation with 10 μM HN2 there was also no change in the membrane potential of the treated cells as measured by the distribution of the ³H]TPMP⁺, no decrease in cellular ATP concentration and no change in intracellular pH, and the ability of the cells to exclude the vital dye Trypan blue was not significantly different from control values. These effects of HN2, therefore, appeared to follow lethal damage, but precede cell death. In the stationary phase of L1210 cell growth, the component of HN2 and diuretic-sensitive K⁺ influx to L1210 cells was reduced, whilst the component constituting the HN2-insensitive ouabain-sensitive sodium pump was increased. The monofunctional alkylating agent MeHN1 (2-chloroethyldimethylamine) which cannot cross-link cellular targets and has no antitumour activity, did not inhibit ⁸⁶Rb⁺ influx to L1210 cells when incubated at equimolar or equitoxic concentrations to HN2. Intracellular potassium concentration was maintained close to control values of 138 ± 10 mM in HN2-treated cells because of an approx. 35% fall in cell volume. The results suggest that the Na⁺/K⁺/Cl⁻ cotransporter is a selectively inhibitable target for HN2, and the lesion is discussed with reference to the cytotoxic effects of this agent.