The role of extracellular pH in the uptake of chloroquine and its effect on mucopolysaccharide metabolism in normal and Hurler's-disease fibroblasts

Abstract

The preceding paper (Danpure & Patel, 1983) demonstrated the different effects of the lysosomotropic drug chloroquine on mucopolysaccharide metabolism in normal human fibroblasts and those from patients suffering from the mucopolysaccharidosis Hurler’s disease (a-L-iduronidase deficiency). It was noted that there was considerable quantitative inter-experiment variability with respect to chloroquine uptake and its effect on 35S-labelled mucopolysaccharide metabolism, especially in normal fibroblasts. Chloroquine is a weak base and concentrates in lysosomes by proton entrapment (de Duve et al., 1974; Wibo & Poole, 1974; Reijngoud & Tager, 1976), and therefore its uptake is highly dependent on the difference in pH between the lysosomes and the extracellular medium. The pH of tissueculture medium is notoriously difficult to control for any length of time in the presence of actively metabolizing cells (Eagle, 1971), and the present study concerns an investigation into the effect of extracellular pH on the uptake of chloroquine and its effect on 3sS-labelled mucopolysaccharide metabolism in normal and Hurler’s-disease fibroblasts. Monolayers of human skin fibroblasts from normal volunteers and patients suffering from Hurler’s disease were grown to confluence in Ham’s F 10 medium (pH 7.2) supplemented with 15% foetal-calf serum. Before the experiments the medium was replaced with Eagle’s minimal essential medium containing 15% foetal-calf serum. The pH was adjusted by adding appropriate amounts of NaHCO, (Taylor, 1962) and gassed with air/CO, (19 : 1). The cells were labelled with Na25S04 (10pCi/ml, 1 mM) for 3 days (the medium being changed every 24 h), again in the presence or absence of chloroquine. For the chloroquine-uptake experiments [ 14Clchloroquine (Amersham International; 13 nCi/ml, 2 0 , ~ ~ ) was used. At the end of the experiments the cells were washed with phosphate-buffered saline (Dulbecco formula Ca2+and Mg2+free) and harvested by scraping with a ‘rubber policeman’ in this medium, sonicated (IOs, 1 O p ~ amplitude) and assayed for protein (Lowry et al., 1951), and for radioactivity by liquidscintillation counting. The effect of chloroquine on the [35Slsulphate labelling of cellular mucopolysaccharides was found to be markedly dependent on the pH of the medium (Fig. 1). As shown previously (Lie et al., 1972, 1973), in the absence of chloroquine normal cells progressively labelled more cellular mucopolysaccharide as the pH was increased from 6.8 to 7.6. In the presence of chloroquine the optimum pH for the increased labelling in normal cells was 7.2. At pH 7.6 chloroquine had no effect on [35Slsulphate uptake. The effect of pH on Hurler cells was quite different. In the absence of chloroquine there was no clear effect of pH. However, chloroquine slightly increased labelling at pH6.8-7.0, but decreased it rapidly at more alkaline pH values, to reach only 30% of the control value at pH 7.6. The changes in I 35Slsulphate uptake with pH were to some extent paralleled by total cellular protein, so that the plots of [35Slsulphate/mg of protein against pH were almost horizontal. The uptake of [ 14Clchloroquine was also highly dependent on the pH of the medium, especially in normal cells (Fig. 2). In normal cells the uptake of chloroquine increased gradually as the pH of the medium increased from 6.8 to 7.4 (see Polet, 1970), but still remained lower than the equivalent Hurler-cell uptake, which showed little change with pH of the medium. However, at more alkaline pH (7.6), the uptake of chloroquine by normal cells increased dramatically, to extents similar to those in Hurler cells (the 5 h uptake point being greater). The pH optimum for the effect of chloroquine on [35S]sulphate metabolism in normal cells was not directly related to chloroquine uptake. The pH-dependence of chloroquine uptake by normal fibroblasts in the present study is at variance with the observations of Ohkuma & Poole (1981), who showed a pH optimum of 7.0. However, they used macrophages and 100pM-chloroquine, which we have found to be toxic in our