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Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem.

Gazarini ML, Thomas AP, Pozzan T, Garcia CR - J. Cell Biol. (2003)

Bottom Line: This allowed selective loading of the Ca2+ probes within the PV.The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism.We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute of Biosciences, University of São Paulo, São Paulo 05508-900, Brazil.

ABSTRACT
Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

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Effects of ionomycin on the [Ca2+] of the parasite cytoplasm. P. falciparum-infected RBCs were loaded with Fluo-3/AM as described previously (Garcia et al., 1996). Where indicated, 15 μM ionomycin was added. The buffer contained 1 mM CaCl2. Identical results were obtained with P. chabaudi. (A) Phase-contrast image of the invaded RBC. (B) Initial Fluo-3 fluorescence. (C) Fluo-3 fluo-rescence 20 s after addition of 15 μM ionomycin. (D) Typical kinetics of the fluorescence changes.
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fig3: Effects of ionomycin on the [Ca2+] of the parasite cytoplasm. P. falciparum-infected RBCs were loaded with Fluo-3/AM as described previously (Garcia et al., 1996). Where indicated, 15 μM ionomycin was added. The buffer contained 1 mM CaCl2. Identical results were obtained with P. chabaudi. (A) Phase-contrast image of the invaded RBC. (B) Initial Fluo-3 fluorescence. (C) Fluo-3 fluo-rescence 20 s after addition of 15 μM ionomycin. (D) Typical kinetics of the fluorescence changes.

Mentions: Given that Fluo-3 is almost nonfluorescent in a low Ca2+ environment, the bright signal surrounding the parasite suggests that the [Ca2+] in the PV is relatively high. A simple test of this conclusion is to treat the cells with the Ca2+ ionophore ionomycin. This ionophore is known to transport Ca2+ across membranes down its electrochemical gradient. If the [Ca2+] of the PV is higher than in the RBC or parasite cytoplasm, ionomycin should transport Ca2+ from the PV into the RBC and/or parasite and thus cause a decrease of the PV [Ca2+]. Fig. 2 shows that addition of ionomycin indeed caused a slow decrease of Fluo-3 fluorescence, indicating that the ionophore was transporting Ca2+ out of the vacuolar space. Further evidence that the signal derived from the trapped Fluo-3 (free acid) reflects the PV environment and that the latter behaves differently from the parasite cytoplasm is provided by the experiment presented in Fig. 3 . In this experiment, the RBCs infected with parasites were loaded with Fluo-3/AM, which predominantly loads the parasite cytoplasm. Under these conditions, ionomycin caused a large increase in fluorescence (Fig. 3) as expected, given that this ionophore is known to penetrate into cells and release Ca2+ from intracellular, membrane bound stores. Results similar to those shown above for P. falciparum were obtained in RBCs infected with P. chabaudi (unpublished data). The important point is that Fluo-3 responds very differently to ionomycin when trapped within the parasite cytoplasm (as in Fig. 3) or within the PV (as in Fig. 2), indicating that the same dye trapped in the different compartments responds differently to the same agent.


Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem.

Gazarini ML, Thomas AP, Pozzan T, Garcia CR - J. Cell Biol. (2003)

Effects of ionomycin on the [Ca2+] of the parasite cytoplasm. P. falciparum-infected RBCs were loaded with Fluo-3/AM as described previously (Garcia et al., 1996). Where indicated, 15 μM ionomycin was added. The buffer contained 1 mM CaCl2. Identical results were obtained with P. chabaudi. (A) Phase-contrast image of the invaded RBC. (B) Initial Fluo-3 fluorescence. (C) Fluo-3 fluo-rescence 20 s after addition of 15 μM ionomycin. (D) Typical kinetics of the fluorescence changes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172890&req=5

fig3: Effects of ionomycin on the [Ca2+] of the parasite cytoplasm. P. falciparum-infected RBCs were loaded with Fluo-3/AM as described previously (Garcia et al., 1996). Where indicated, 15 μM ionomycin was added. The buffer contained 1 mM CaCl2. Identical results were obtained with P. chabaudi. (A) Phase-contrast image of the invaded RBC. (B) Initial Fluo-3 fluorescence. (C) Fluo-3 fluo-rescence 20 s after addition of 15 μM ionomycin. (D) Typical kinetics of the fluorescence changes.
Mentions: Given that Fluo-3 is almost nonfluorescent in a low Ca2+ environment, the bright signal surrounding the parasite suggests that the [Ca2+] in the PV is relatively high. A simple test of this conclusion is to treat the cells with the Ca2+ ionophore ionomycin. This ionophore is known to transport Ca2+ across membranes down its electrochemical gradient. If the [Ca2+] of the PV is higher than in the RBC or parasite cytoplasm, ionomycin should transport Ca2+ from the PV into the RBC and/or parasite and thus cause a decrease of the PV [Ca2+]. Fig. 2 shows that addition of ionomycin indeed caused a slow decrease of Fluo-3 fluorescence, indicating that the ionophore was transporting Ca2+ out of the vacuolar space. Further evidence that the signal derived from the trapped Fluo-3 (free acid) reflects the PV environment and that the latter behaves differently from the parasite cytoplasm is provided by the experiment presented in Fig. 3 . In this experiment, the RBCs infected with parasites were loaded with Fluo-3/AM, which predominantly loads the parasite cytoplasm. Under these conditions, ionomycin caused a large increase in fluorescence (Fig. 3) as expected, given that this ionophore is known to penetrate into cells and release Ca2+ from intracellular, membrane bound stores. Results similar to those shown above for P. falciparum were obtained in RBCs infected with P. chabaudi (unpublished data). The important point is that Fluo-3 responds very differently to ionomycin when trapped within the parasite cytoplasm (as in Fig. 3) or within the PV (as in Fig. 2), indicating that the same dye trapped in the different compartments responds differently to the same agent.

Bottom Line: This allowed selective loading of the Ca2+ probes within the PV.The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism.We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute of Biosciences, University of São Paulo, São Paulo 05508-900, Brazil.

ABSTRACT
Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

Show MeSH
Related in: MedlinePlus