Characteristics of an amiloride‐sensitive sodium entry pathway in cultured rodent glial and neuroblastoma cells

Abstract

We have studied the induction of an amiloride‐sensitive sodium influx into C6 glioma, NIE, and NB2A neuroblastoma cell lines. In late log phase, cells grown continuously in the presence of 10% fetal calf serum showed Na⁺ influxes of approximately 25–30 nmol/mg protein min; < 5% of this flux was inhibited by amiloride. Removal of serum for 24 h caused a decrease in the total Na⁺ influx to 15–20 nmol/mg protein/min. Upon readdition of serum to the incubation medium, there was an increase in total Na⁺ influx, depending on the cell type, of 20–400% within 2 min. This increment in Na⁺ influx represented an increase in amiloride‐sensitive Na⁺ transport with an apparent K′, of 0.4 mM. By adding serum back at various times after serum deprivation, it was determined that 4 h was required to observe a detectable increase in the amiloride‐sensitive Na⁺ flux. Thus, serum removal results in the induction of the amiloride transport system which, however, remains latent until the reintroduction of serum to the medium. Addition of 5 μg/ml of cycloheximide blocked the increase in Na⁺ transport, indicating that de novo protein synthesis mediated this serum deprivation–induced increase in Na⁺ transport. Moreover, inhibition of de novo lipid synthesis by 0.1 mM fenfluramine also blocked the induction of this transport activity, suggesting that a coordinated synthesis of lipid and protein is required for the expression of this sodium transport site. We have also found that this serum stimulated Na⁺ influx did not saturate with Na⁺ concentration, up to 140 mM. Also, among commonly used inhibitors of passive Na⁺ entry into epithelial tissues, only amiloride was capable of inhibiting this transport system in these neural cell lines.