Uptake of cardiac glycosides by HeLa cells
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Methods for the microcarrier culture of HeLa cells were developed and optimum culture conditions established. Methods were also established for the isolation of HeLa plasma membranes on beads, based on the principle of cell lysis followed by the shrearing away of the internal cell components, leaving the plasma membrane attached to the beads. The HeLa plasma membranes isolated on beads showed 5-8 fold enrichment of plasma membrane markers, while intracellular markers were depleted to the point of non-detection (except for some slight lysosomal contamination). The preparation time for isolation of HeLa plasma membranes on beads was about 1.5 hrs. The uptake of cardiac glycosides by HeLa cells was investigated. HeLa cells were labelled with [3H]-glycoside, the non-specific binding washed off and the cells returned to normal growth medium. At various time intervals the amount of [3H]-glycoside bound to HeLa plasma membranes and HeLa cells was determined. The rate of loss of ouabain and digoxin from HeLa plasma membranes was found to be about 10%hr-1. The excretion rates of ouabain and digoxin from HeLa cells were 4%hr-1 and 10%hr-1 respectively. The nature of the uptake and excretion processes were investigated using specific inhibitors of receptor-mediated endocytic processes. No inhibitor affected the rate of loss of cardiac glycoside from HeLa plasma membranes but monensin and chloroquine slowed the excretion rate of digoxin from HeLa cells. Modulation of sodium pump numbers by various chronic stress conditions - high serum concentration, low K medium and high K medium - were investigated. High concentrations of serum had no effect on the rate of loss ouabain from HeLa plasma membrane, but low K medium reduced the rate of loss of ouabain from membranes from 10%hr-1 to 4%hr-1. The low K medium also caused a x2 increase in sodium pump numbers in the plasma membrane. Increasing the serum concentration abolished the effects due to low K medium. High K medium also caused a reduction in the rate of loss of ouabain from plasma membranes from 10%hr-1 to 4%hr-1. The results obtained for low K medium were consistent with a model whereby sodium pumps in the plasma membrane are regulated by alteration of the sodium pump turnover rate (or internalisation rate).
Thesis, PhD Doctor of Philosophy
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