Life and Medical Sciences
Cell & Developmental Biology
Wiley InterScience Backfile Collection 1832-2000
Na+ transport properties of neuroblastoma (clone Neuro-2A) cells have been characterized both in exponentially growing cells and during the cell cycle. In exponentially growing cells, Na+ influx followed the kinetics for a one-compartment system with an influx rate of 9.54 ± 0.96 nmoles/minute/106 cells, equilibrium being reached within 2 minutes. The initial rate of influx in the presence of ouabain (5 mM), a concentration completely inhibiting the (Na+-K+)ATPase and thus backflux of tracer, was 11.33 ± 1.50 nmoles/minute/106 cells and, within error, the same as in the absence of ouabain, provided determinations were made within 3 minutes of ouabain addition.Na+ influx rate was determined at intervals during the cell cycle of synchronized Neuro-2A cells using a 3-minute pulse of 22Na+ in the presence of ouabain. On passing from mitosis to G1-phase Na+ influx decreases from 12.13 ± 1.93 to 7.40 ± 0.90 nmoles/minute/106 cells but increases rapidly and transiently approximately twofold upon entry into S-phase. This transient increase coincides with a transient stimulation of the (Na+-K+) pump activity. It then returns to a steady level of ˜ 12 nmoles/minute/106 cells for most of the remainder S-phase.Intracellular Na+ concentration during the cell cycle was determined from the equilibrium content of 24Na+ and data on the intracellular H2O volume, published previously (Boonstra et al., 1981b). Na+ concentration is maximal in mitosis at 56.96 ± 6.05 mM and decreases rapidly tourfold as cells enter G1-phase. With progression through S-phase a steady increase from 13.80 ± 1.25 mM to 37.35 ± 2.91 mM is observed. Combining the Na+ concentration with the K+ concentration obtained previously (Boonstra et al., 1981b), the K+:Na+ ratio was obtained during the cell cycle. The ratio had a value of between 3 and 5 during most of the cell cycle, but was significantly higher in G1-phase, where the loss of Na+ is considerably greater than the loss of K+. The values for Na+ concentration were combined with membrane potential measurements reported previously (Boonstra et al., 1980b) to obtain the Na+ electrochemical gradient across the cell membrane in the cell cycle. This had a value of 63.2 ± 2.6 mV in mitosis and increased rapidly to reach a maximum value of 84.0 ± 5.5 mV during G1-phase, thereafter maintaining a value of between 73 and 80 mV. The transient increase in Na+ influx and in (Na+-K+)ATPase-mediated K+ influx at the G1/S-phase transition are specifically inhibitable by the diuretic amiloride (0.2 mM) but amiloride has little effect on Na+ or K+ influx in other phases of the cell cycle. This indicated activation of an amiloride-sensitive transport system specically at the G1/S-phase transition and a causal relationship between increased Na+ entry and transient stimulation of the pump at this point in the cell cycle. Further, amiloride (0.1, 0.2 mM) addition was shown to give rise to a significant lengthening of the cell cycle and to cause a partial blockage of the cells specifically in G1-phase, suggesting an important role for the transient ion flux changes in controlling entry into and progression through S-phase.
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