Réfulation de la (Na+ + K+)- ATPase dans une lignée cellulaire transportant le sodium : influence de l'aldostérone
(1989)
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- Authors
- Supervisors
- Crabbé, Jean
- Abstract
- This work is aimed to shed additional light on the mechanism whereby aldosterone stimulates transepithelial sodium transport. The experimental work presented here has been conducted on a cell line ‘A6) capable of sodium transport. The influence of aldosterone has been evaluated on the electrophysiological properties of these cells and on (Na+ +K+)-ATPase activity measured after homogenization as well as by means of ouabain binding onto intact cells. <BR> The A6 cells originating from the distal portion of the nephron of Xenopus leavis develop in culture as an epithelium. The basolateral surface of which comes in contact with the bottom of a Petri dish. The epithelium they form at confluence consist of a “tight” monolayer in that the permeability of the paracellular pathway is very low. Sodium is transported from the solution facing the apical membrane to the basolateral border, across which it is expelled. When grown on a permeable structure, the A6 cells thereby develop an electrical potential difference of 13-30 mV (apical side negative) with low conductance (<0.2 ms/cm²). <BR> Transepithelial sodium transport by these cells no longer occurs upon removal of sodium or after addition of amiloride both on the apical side. But the effect of this drug was observed to be practically irreversible after prolonged incubation in its presence. In fact, amiloride was found to cross the epithelium, moving probably, at least in part, through the cells. For this reason, removal of sodium in the apical compartment was given preference when attempting to assess the effect of aldosterone in the absence of sodium transport. When the preparations were incubated with the apical surface exposed to sodium free medium for 18h, sodium transporting activity upon reintroducing sodium was only one half of the control value. (Na+ +K+)-ATPase activity and sodium pump density were found to be decreased to the same extent. Interestingly, the biological half-life of the enzyme system has been reported to be 15h for this cell line. Such results are in keeping with the proposal that intracellular sodium concentration is a significant regulator of sodium pump biosynthesis. <BR> Ouabain inhibits sodium transport by A6 cells. There is a quantitative correspondence between ouabain affinity for the pump and the glycoside concentration required to inhibit sodium transport. Thus, the latter is blocked at a concentration of 5 x 10-7 M, which corresponds to saturation of ouabain binding sites onto intact cells. In addition, sodium transport (reflected by the short-circuit current) dropped by 50% when the cells are exposed to 27 mM ouabain, a value comparable to Kd (~20 nM) obtained for ouabain binding. <BR> When cells growing on a Petri dish are exposed to aldosterone ( 10-7 M) dome formation, considered as reflecting active sodium transport, appears stimulated both in terms of density and individual size. When the cells develop on a permeable support, short-circuit current was found increased 2 to 3h after addition of the hormone; there was a concomitant increase in transepithelial conductance. At that early stage, nop influence on ((Na+ +K+)-ATPase was detected. Such data are in keeping with the hormone stimulating apical sodium transport was stimulated at least 3 fold with aldosterone. At that stage, (Na+ +K+)-ATPase activity increased 50% approximately. The increase in pump density was of the same magnitude. Furthermore, from ouabain binding studies, it appeared that A6 cells are equipped with a single homogeneous population of ouabain binding sites. In addition, the binding characteristics of these sites were not modified by aldosterone. <BR> Aldosterone appeared to influence (Na+ +K+)-ATPase even in absence of sodium transport by A6 cells (after removal of sodium on the apical side). Thus, aldosterone might influence (Na+ +K+)-ATPase biosynthesis directly
- Affiliations
Citations
Leal, T. (1989). Réfulation de la (Na+ + K+)- ATPase dans une lignée cellulaire transportant le sodium : influence de l’aldostérone. https://hdl.handle.net/2078.5/111144