Characterization of Na⁺ transport in normal human fibroblasts and neoplastic H.Ep.2 cells and the role of inhibitin

Summary

Na⁺ transport was characterized in normal human fibroblasts and neoplastic H.Ep. 2 cells in order to investigate the role of the endogenous peptidic factor ‘inhibitin’ that is secreted by a variety of neoplastic cells (including H.Ep. 2) and inhibits Na⁺/Na⁺ exchange in human erythrocytes. Although active (Na⁺, K⁺-ATPase mediated) Na⁺ fluxes were similar in the two cell types, H.Ep. 2 cells maintained higher intracellular Na[su+] concentration (26mm) compared to fibroblasts (12mm). An analysis of passive Na⁺ fluxes showed a difference in the handling of Na⁺ via ouabain and bumetanide-insensitive transport between the two cell types: H.Ep. 2 cells achieved net Na⁺ influx via an amiloride-sensitive pathway that was only demonstrated in fibroblasts when 10% fetal calf serum (FCS) was present. Kinetic studies were undertaken to investigate the interaction between Na⁺ flux via Na⁺/H⁺ and Na⁺/Na⁺ exchanges. for this purpose, an outwardly directed Na⁺ gradient was created by loading the cells with Na⁺ (Na _i >100mm) to activate the reverse functioning of Na⁺/H⁺ exchange (i.e., Na ⁺_out H ⁺_in ). The rates of ouabain-and bumetanide-insensitive Na⁺ efflux were measured over a range of extracellular Na⁺ concentrations (Na ⁺ _o 14–140mm). In the presence of 10% FCS, the two cell types showed different responses: in fibroblasts the Na⁺ efflux rate showed an inverse correlation with extracellular Na⁺ concentration, while H.Ep. 2 cells significantly increased their rate of Na⁺ efflux as extracellular Na⁺ concentration increased. So although the thermodynamic force would direct net Na⁺ efflux when Na ⁺ _i >Na ⁺ _o , H.Ep.2 cells were under kinetic control to perform Na⁺/Na⁺ exchange.

When exogenous inhibitin was tested on fibroblasts, the steady-state intracellular Na⁺ concentration increased from 14 to 19mm (p<0.01). In Na⁺-loaded fibroblasts, serum-stimulated Na⁺ efflux was partially inhibitin sensitive and the maximal inhibitory effect was seen when extracellular Na⁺ concentration was 14mm and presumably the Na⁺/H⁺ exchanger operating in the reverse mode. This study demonstrated that, in contrast to fibroblasts, H.Ep.2 cells have a modified Na⁺/H⁺ exchange system whereby it acts in the Na ⁺_in H ⁺_out mode without exogenous growth factor activation and resists functioning in the reversed mode. It is proposed that inhibitin, is the endogenous modifier of this transport system in H.Ep.2 cells with the result that H.Ep.2 cells maintain a higher concentration of intracellular Na⁺ compared to fibroblasts.