Digestive vacuolar pH of intact intraerythrocytic P-falciparum either sensitive or resistant to chloroquine

We present the first single cell-level analysis of digestive vacuolar pH fo r representative chloroquine resistant (strain Dd2) versus sensitive (strai n HB3) malarial parasites. Human red blood cells harboring intact intraeryt hrocytic parasites were attached to glass substrate, continuously perfused with appropriate buffer, and pH was analyzed via single cell imaging and ph otometry techniques. We find that digestive vacuolar pH (pH(vac)) is near 5 .6 for HB3 parasites. Surprisingly, we also find that pH(vac) of Dd2 is mor e acidic relative to HB3. Notably, in vitro pH titration of hematin confirm s a very steep transition between soluble heme (capable of binding chloroqu ine) and insoluble heme (not capable of binding chloroquine, but still capa ble of polymerization to hemozoin) with a distinct midpoint at pH 5.6. We s uggest the similarity between the hematin pH titration midpoint and the mea sured value of HB3 pH(vac) is not coincidental, and that decreased pH(vac) for Dd2 titrates limited initial drug target (i.e. soluble heme) to lower c oncentration. That is, changes in pH(vac) or drug resistant Dd2 relative to drug sensitive HB3 are consistent with lowering drug target levels, but no t directly lowering vacuolar concentrations of drug via the predictions of weak base partitioning theory. Regardless, lowering either would of course decrease the efficiency of drug/target interaction and hence the net cellul ar accumulation of drug over time, as is typically observed for resistant p arasites. These observations contrast sharply with the common expectation t hat decreased chloroquine accumulation in drug resistant malarial parasites is likely linked to elevated pH(vac), but nonetheless illustrate important differences in vacuolar ion transport for drug resistant, malarial parasit es. In the accompanying paper (Ursos, L. et al., following paper this issue ) we describe how pH(vac) is affected by exposure to chloroquine and verapa mil for HB3 versus Dd2. (C) 2000 Elsevier Science B.V. All rights reserved.