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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 extracellular Ca2+ on the Ca2+ content of the parasite intracellular stores. P. chabaudi were loaded with Fluo-3/AM and analyzed at the confocal level. The loaded cells were incubated for 90 min in media containing different concentrations of CaCl2. (A and B) The medium was supplemented with 1 mM CaCl2; in C and D, no CaCl2 was added and 100 μM EGTA was included ([Ca2+] about 100 nM); in E and F, the medium was supplemented with 100 μM CaCl2. For the experiments in C–F, the cells were loaded with Fluo-3 before treating with 10 μM digitonin. The detergent was washed away after 5 min. The arrows indicate the additions of 10 μM THG and 100 μM of melatonin (MLT). The kinetics of the fluorescence of three typical single cells are presented in each panel.
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fig5: Effects of extracellular Ca2+ on the Ca2+ content of the parasite intracellular stores. P. chabaudi were loaded with Fluo-3/AM and analyzed at the confocal level. The loaded cells were incubated for 90 min in media containing different concentrations of CaCl2. (A and B) The medium was supplemented with 1 mM CaCl2; in C and D, no CaCl2 was added and 100 μM EGTA was included ([Ca2+] about 100 nM); in E and F, the medium was supplemented with 100 μM CaCl2. For the experiments in C–F, the cells were loaded with Fluo-3 before treating with 10 μM digitonin. The detergent was washed away after 5 min. The arrows indicate the additions of 10 μM THG and 100 μM of melatonin (MLT). The kinetics of the fluorescence of three typical single cells are presented in each panel.

Mentions: As a final test to prove that parasites within intact RBCs are indeed exposed to a relatively high Ca2+ environment, the experiments presented in Fig. 5 were carried out. RBCs infected with P. chabaudi were first loaded with Fluo-3/AM, then after 90 min incubation in 1 mM CaCl2, the Ca2+ content of the parasite intracellular Ca2+ stores was verified by the addition of THG (Fig. 5 A). In a parallel experiment, after loading with Fluo-3/AM, the RBC plasma membrane was permeabilized with digitonin and then, after washing out the digitonin, the cells were incubated in a low [Ca2+] medium (∼100 nM) for 90 min (Fig. 5 C). The rationale of the experiment is as follows: digitonin completely permeabilizes the RBC plasma membrane and the PVM. On the other hand, the plasma membrane of the parasites is not affected by the detergent at these concentrations, as revealed by the maintenance of Fluo-3 fluorescent signal within the Plasmodium. However, under these conditions, the parasite is exposed not to the [Ca2+] of the PV, but to that of the extracellular medium. Addition of THG under these conditions resulted in no [Ca2+] increase, revealing that exposure of the parasite to a low [Ca2+] medium for 90 min results in almost complete emptying of its intracellular Ca2+ stores (Fig. 5 C). On the contrary, if the cells were permeabilized with digitonin (see previous paragragh), but incubated in a medium containing 100 μM CaCl2 (i.e., a concentration close to that calculated to be in the PV), the intracellular release caused by THG was similar to that observed in controls, i.e., without digitonin permeabilization (compare Fig. 5 E with Fig. 5 A). Results similar to those shown above for P. chabaudi were obtained in RBCs infected with P. falciparum (unpublished data).


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 extracellular Ca2+ on the Ca2+ content of the parasite intracellular stores. P. chabaudi were loaded with Fluo-3/AM and analyzed at the confocal level. The loaded cells were incubated for 90 min in media containing different concentrations of CaCl2. (A and B) The medium was supplemented with 1 mM CaCl2; in C and D, no CaCl2 was added and 100 μM EGTA was included ([Ca2+] about 100 nM); in E and F, the medium was supplemented with 100 μM CaCl2. For the experiments in C–F, the cells were loaded with Fluo-3 before treating with 10 μM digitonin. The detergent was washed away after 5 min. The arrows indicate the additions of 10 μM THG and 100 μM of melatonin (MLT). The kinetics of the fluorescence of three typical single cells are presented in each panel.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Effects of extracellular Ca2+ on the Ca2+ content of the parasite intracellular stores. P. chabaudi were loaded with Fluo-3/AM and analyzed at the confocal level. The loaded cells were incubated for 90 min in media containing different concentrations of CaCl2. (A and B) The medium was supplemented with 1 mM CaCl2; in C and D, no CaCl2 was added and 100 μM EGTA was included ([Ca2+] about 100 nM); in E and F, the medium was supplemented with 100 μM CaCl2. For the experiments in C–F, the cells were loaded with Fluo-3 before treating with 10 μM digitonin. The detergent was washed away after 5 min. The arrows indicate the additions of 10 μM THG and 100 μM of melatonin (MLT). The kinetics of the fluorescence of three typical single cells are presented in each panel.
Mentions: As a final test to prove that parasites within intact RBCs are indeed exposed to a relatively high Ca2+ environment, the experiments presented in Fig. 5 were carried out. RBCs infected with P. chabaudi were first loaded with Fluo-3/AM, then after 90 min incubation in 1 mM CaCl2, the Ca2+ content of the parasite intracellular Ca2+ stores was verified by the addition of THG (Fig. 5 A). In a parallel experiment, after loading with Fluo-3/AM, the RBC plasma membrane was permeabilized with digitonin and then, after washing out the digitonin, the cells were incubated in a low [Ca2+] medium (∼100 nM) for 90 min (Fig. 5 C). The rationale of the experiment is as follows: digitonin completely permeabilizes the RBC plasma membrane and the PVM. On the other hand, the plasma membrane of the parasites is not affected by the detergent at these concentrations, as revealed by the maintenance of Fluo-3 fluorescent signal within the Plasmodium. However, under these conditions, the parasite is exposed not to the [Ca2+] of the PV, but to that of the extracellular medium. Addition of THG under these conditions resulted in no [Ca2+] increase, revealing that exposure of the parasite to a low [Ca2+] medium for 90 min results in almost complete emptying of its intracellular Ca2+ stores (Fig. 5 C). On the contrary, if the cells were permeabilized with digitonin (see previous paragragh), but incubated in a medium containing 100 μM CaCl2 (i.e., a concentration close to that calculated to be in the PV), the intracellular release caused by THG was similar to that observed in controls, i.e., without digitonin permeabilization (compare Fig. 5 E with Fig. 5 A). Results similar to those shown above for P. chabaudi were obtained in RBCs infected with P. falciparum (unpublished data).

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