Uptake of [³H]ouabain and Na pump turnover rates in cells cultured in ouabain

Abstract

4. Cells incubated in ouabain* for 24 hr bind an additional amount of ouabain when exposed to 2 x 10-7 M ouabain* in K-free Krebs.5. There is a close relationship between the % of the total ouabain bound in 24 hr and the % inhibition of the Na efflux suggesting that this ouabain is bound to the Na pumps.6 LESLEY J. BOARDMAN AND OTHERS 9. These results are compatible with the hypothesis that partial blocking of Na pumps leads to the production of more pumping sites by the cell.

DOWN‐REGULATION OF THE SODIUM PUMP FOLLOWING CHRONIC EXPOSURE OF HeLa CELLS AND CHICK EMBRYO HEART CELLS TO OUABAIN

“…We think this error is negligible for two reasons. First, the 15 min incubation time used in determining the [3H]-ouabain binding is negligible compared to the 20 h half time for the release of ouabain bound to the pump site (Boardman et al, 1972); less than 1 % of the ouabain molecules would be released during the incubation period. Secondly, even if all the bound ouabain molecules were released, the maximum increase in the ouabain concentration of the incubation medium would be of the order of 10-9M, again a change of less than 1 %.…”

“…Because the half time of dissociation of ouabain from the pump site is very slow (approximately 20 h, Boardman et al, 1972) and independent of protein synthesis, the cycloheximide-sensitive component of recovery indicates the appearance of newly synthesized sodium pump sites. The time course of recovery shows that new sites first appeared within 7 h whereas cell numbers did not increase until later ( Figure 6); recovery of sodium pump site numbers was complete within 24 h. Since cycloheximide blocks this increase in [3H]-ouabain binding, we conclude that recovery of sodium pump site numbers was primarily dependent upon de novo protein synthesis.…”

“…The fraction of the 86Rb influx catalysed by the sodium pump (the ouabain sensitive 86Rb influx) was taken as the difference between the 86Rb influxes measured in the presence and absence of 10-3M ouabain. The techniques used in this study have been described in detail elsewhere Boardman, et al, 1972;Boardman, Huett, Lamb, Newton & Polson, 1974;Aiton & Lamb, 1980) and are summarised below. All experiments were carried out at 37°C.…”

“…In a previous paper we demonstrated that prolonged treatment of an established human cell line with therapeutic concentrations of ouabain caused a large decrease in sodium pump site numbers Boardman, Lamb & McCall, 1972). …”

The receptor function of the Na+, K+-activated adenosine triphosphatase system

“…The [3H]ouabain is found in a lysosomal fraction and is then released unchanged from the cells, presumably by exocytosis, with a half-time of about 70h (Pollack et al,198 la; Cook et al, 1982). The inactivated, internalized, pumps are replaced (Boardman et al, 1972) at a rate which is at least 4-fold higher than the turnover rate in the absence of ouabain (Lamb & Ogden, 1982) but slower than the internalization rate (Lamb & Ogden, 1982). As a result, the receptor are downregulated in the presence of cardioactive steroids (Aiton et al, 1981).…”