The calcium content and transport processes of Plasmodium chabaudi-infected rat erythrocytes were analyzed by atomic absorption spectroscopy and 45Ca2+ flux measurements. Infected erythrocytes, after fractionation on metrizamide gradients according to stage of parasite development, exhibited progressively increasing levels of Ca2+ with schizont and gametocytes containing 10- to 20-fold greater calcium levels than normal cells (0.54 +/- 0.25 nmol/10(8) cells). 45Ca2+ flux experiments showed both increased influx and decreased efflux in infected erythrocytes. Tris/NH4Cl lysis of normal erythrocytes preloaded with 45Ca2+ with the Ca2+ ionophore A23187 released less than 90% of cell calcium after incubation in ethyleneglycol bis(aminoethylether) N,N'-tetraacetic acid containing buffer, whereas lysis of the infected erythrocyte membrane resulted in release of 10-20% cell Ca2+, with the remaining portion associated with the isolated parasite fraction. This information together with the effects of various metabolic inhibitors indicates the presence of a parasite Ca2+ compartment in P. chabaudi-infected erythrocytes. Dicyclohexylcarbodiimide (DCCD) an inhibitor of proton ATPases of chloroplasts, bacteria, yeast, and mitochondria, and the proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), inhibited Ca2+ influx and stimulated efflux from infected cells. These results combined with evidence for a DCCD- and CCCP-sensitive membrane potential in P. chabaudi-infected cells (Mikkelsen et al., accompanying manuscript) suggest that Ca2+ transport of intraerythrocytic parasites is coupled to a proton-motive force across the Plasmodia plasma membrane.
The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP). The SCN- and TPMP-measured Em of normal erythrocytes are -6.5 +/- 3 mV and -10 +/- 4 mV, respectively. The TPMP-measured Em of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Em = -35 mV "early stages (ring and copurifying noninfected) by a low Em (-16 mV). The SCN-determined Em of infected cells was -7 mV regardless of parasite stage. Studies with different metabolic inhibitors including antimycin A, a proton ionophore (carbonylcyanide m-chlorophenylhydrazone [CCCP] ), and a H+ -ATPase inhibitor (N,N'-dicyclohexylcarbodiimide, [DCCD] ) indicate that SCN monitors the Em across the erythrocyte membrane of infected and normal cells whereas TPMP accumulation reflects the Em across the plasma membranes of both erythrocyte and parasite. These inhibitor studies also implicated proton fluxes in Em-generation of parasitized cells. Experiments with weak acids and bases to measure intracellular pH further support this proposal. Methylamine distribution and direct pH measurement after saponin lysis of erythrocyte membranes demonstrated an acidic pH for the erythrocyte matrix of infected cells. The transmembrane distributions of weak acids (acetate and 5,5-dimethyloxazolidine-2,4-dione) indicated a DCCD-sensitive alkaline compartment. The combined results suggest that the intraerythrocyte parasite Em and delta pH are in part the consequence of an electrogenic proton pump localized to the parasite plasma membrane.