Modulation of functional and optimal (Na+-K+)ATPase activity during the cell cycle of neuroblastoma cells

Abstract

Functional and optimal activities of the (Na⁺‐K⁺)ATPase, as determined by ouabain‐sensitive K⁺ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro‐2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain‐sensitive K⁺ influx decreased more than fourfold from 1.62 ± 0.11 nmoles/min/10⁶ cells to 0.36 ± 0.25 nmoles/min/10⁶ cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 ± 0.30 nmoles/min/10⁶ cells was observed, followed by a rapid decline, after which the active K⁺ influx rose again steadily from 1.03 ± 0.25 nmole/min/10⁶ cells in early S phase to 2.10 ± 0.92 nmoles/min/10⁶ cells just prior to the next mitosis. The ouabain‐insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K⁺ influx values with efflux data obtained previously showed that net loss of K⁺ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid‐S phase net K⁺ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na⁺‐K⁺)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na⁺‐K⁺)ATPase mediated K⁺ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; k_m = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na⁺‐K⁺)ATPase mediated K⁺ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K⁺ influx is necessary for normal rate of progression through the rest of the cell cycle.