Cytosolic free calcium in Plasmodium falciparum-infected erythrocytes and the effect of verapamil: a cytofluorimetric study

Abstract

The free Ca2+ ion concentration, measured by means of the fluorescent indicators Indo-1 and Fluo-3, has been compared in normal and parasitized erythrocytes from synchronized in vitro cultures of human blood infected with Plasmodium falciparum. The cells were loaded with the calcium probes in the form of their acetoxymethylesters. P. falciparum-infected red blood cells gradually accumulate more free Ca2+ ions than uninfected cells. The increased Ca2+ concentration is preferentially located inside a rather large central area, corresponding to the position and size of the parasite. In contrast, the Ca2+ concentration outside this area is not higher than that in normal red blood cells. This rise in calcium content becomes significant at the end of the ring stage. The concentration measured in 36-hr schizonts reaches two times that measured in uninfected erythrocytes, and it peaks to four times control values in 44-hr schizonts. The Ca2+ channel blocker verapamil (10 to 20 microM), added on the 24th hr of culture, slows down or blocks the parasite's growth at the trophozoite stage. However, the free Ca2+ concentration measured on infected red blood cells at different times after verapamil addition does not differ from that obtained in the absence of verapamil. These results demonstrate that the bulk of the free Ca2+ load of P. falciparum-infected erythrocytes is located inside the parasite or its parasitophorous vacuole. These data also indicate that the increased Ca2+ influx in P. falciparum-infected erythrocytes does not take the route of verapamil-sensitive Ca2+ channels. It also appears that the inhibitory effect of verapamil on the parasite's maturation does not depend on a change in its Ca2+ content.