INTERNALIZATION OF OUABAIN AND REPLACEMENT OF SODIUM PUMPS IN THE PLASMA MEMBRANES OF HeLa CELLS FOLLOWING BLOCK WITH CARDIAC GLYCOSIDES

Abstract

SUMMARYThe cardiac glycosides, ouabain or digoxin, when applied to HeLa cells at a concentration of 1 /SM, block all Na/K pumping. The recovery of the cells from such a block involves a process of internalization of the glycoside, with its subsequent excretion and the appearance of a working Na pump at the plasma membrane. The rate of recovery of pump function probably does not depend on the direct dissociation of the glycoside from the sodium pumps on the plasma membrane. Low temperature and vinblastine slow the rate of glycoside loss from the cells (the t1 is normally about 20 h). No other agent examined altered the excretion rate. The recovery ofpump function (ti of 6 h) was reduced by all factors examined, i.e. low temperature, vinblastine, low serum concentration, cycloheximide and chloropromazine. We think this means that glycoside excretion and pump recovery are not directly related, though recovery may ultimately depend on glycoside excretion. The recovery in pump function which does occur in the absence of protein synthesis is thought to be due to a store of preformed sodium pumps held in the cell. Fresh HeLa cells 'turnover' their sodium pumps at a slow rate, i.e. they internalize their pumps and replace them with new ones at a rate of about 1 % per hour. When pumps on the plasma membrane are blocked with cardiac glycosides, the rate of replacement can be increased by a factor of at least 4. This, therefore, constitutes a repair process. It is shown that HeLa cells provide a good model for the detoxification mechanism present in the human heart treated with therapeutic concentrations of glycosides.

Fast degradation of the auxiliary subunit of Na+/K+-ATPase in the plasma membrane of HeLa cells

“…When cells were chased in the presence of 1 μM ouabain, the α1-subunit was swiftly internalized within 1 hour and remained inside the cell for the next few hours without degradation (data not shown). These data match well those of previous studies in which ouabain treatment induces the internalization of the enzyme (Cook et al, 1982;Griffiths et al, 1983;Lamb and Ogden, 1982;Nunez-Duran et al, 1996;NunezDuran et al, 1988).…”

The effects of chloroquine and other weak bases on the accumulation and efflux of digoxin and ouabain in HeLa cells

“…For example, recovery from a complete block ought to be partly due to pump renewal, as well as dissociation of cardiac glycosides from the pumps. Both Cook & Brake (1978) and Lamb & Ogden (1977, 1982a have presented evidence for this in HeLa cells. When pumps are internalised it would be expected that ©) The Macmillan Press Ltd 1983 878 N. GRIFFITHS etal. cardiac glycosides bound to them would also be internalised.…”

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).…”