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Regulation of PKC mediated signaling by calcium during visceral leishmaniasis.

Roy N, Chakraborty S, Paul Chowdhury B, Banerjee S, Halder K, Majumder S, Majumdar S, Sen PC - PLoS ONE (2014)

Bottom Line: Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+.Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden.These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, West Bengal, India.

ABSTRACT
Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca2+ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca2+ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca2+ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca2+ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis.

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Effect of inhibitors on plasma membrane Ca2+-ATPases during Leishmania infection.Macrophages were treated with thapsigargin (TG, 1 µM) and trifluoperazine (TFP, 10 µM) followed by infection with L.donovani in 1∶10 ratio. Unbound parasites were washed off after 4 hr and further incubated for 20 hr. Cells were collected, plasma membrane was isolated and Ca2+-ATPase activity was measured. (A). In a similar experiment, inhibitor treated and infected macrophages were collected in Trizol for RNA extraction and semi-quantitative RT-PCR were performed (B). Macrophages were seeded on 8 well chamber slides (10,000 cells/well) and treated with inhibitors followed by infection with L.donovani in a 1∶10 ratio. Cells were incubated for 24 hr, fixed with chilled methanol, and stained with Giemsa. Amastigotes were then microscopically counted. ***P<.001, *P<.05 and ns = non-significant for the comparison with infected one (C). Data represented means ± SD from three different experiments (A and C). Result shown in B is a representative one from three different experiments.
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pone-0110843-g005: Effect of inhibitors on plasma membrane Ca2+-ATPases during Leishmania infection.Macrophages were treated with thapsigargin (TG, 1 µM) and trifluoperazine (TFP, 10 µM) followed by infection with L.donovani in 1∶10 ratio. Unbound parasites were washed off after 4 hr and further incubated for 20 hr. Cells were collected, plasma membrane was isolated and Ca2+-ATPase activity was measured. (A). In a similar experiment, inhibitor treated and infected macrophages were collected in Trizol for RNA extraction and semi-quantitative RT-PCR were performed (B). Macrophages were seeded on 8 well chamber slides (10,000 cells/well) and treated with inhibitors followed by infection with L.donovani in a 1∶10 ratio. Cells were incubated for 24 hr, fixed with chilled methanol, and stained with Giemsa. Amastigotes were then microscopically counted. ***P<.001, *P<.05 and ns = non-significant for the comparison with infected one (C). Data represented means ± SD from three different experiments (A and C). Result shown in B is a representative one from three different experiments.

Mentions: Calcium appears to be vital in Leishmania apoptosis. It has been also established that more parasites die in the presence of Ca2+ than in its absence. Therefore, we focused on the parasitic load of the infected macrophages. In these experiments, macrophages were infected as described in Materials and Methods. Figure 5C shows that parasitic load was significantly increased in presence of thapsigargin; whereas a sharp decline was observed in presence trifluoperazine in comparison to the control. When both inhibitors were used parasitic load was found to be lower compared to the control.


Regulation of PKC mediated signaling by calcium during visceral leishmaniasis.

Roy N, Chakraborty S, Paul Chowdhury B, Banerjee S, Halder K, Majumder S, Majumdar S, Sen PC - PLoS ONE (2014)

Effect of inhibitors on plasma membrane Ca2+-ATPases during Leishmania infection.Macrophages were treated with thapsigargin (TG, 1 µM) and trifluoperazine (TFP, 10 µM) followed by infection with L.donovani in 1∶10 ratio. Unbound parasites were washed off after 4 hr and further incubated for 20 hr. Cells were collected, plasma membrane was isolated and Ca2+-ATPase activity was measured. (A). In a similar experiment, inhibitor treated and infected macrophages were collected in Trizol for RNA extraction and semi-quantitative RT-PCR were performed (B). Macrophages were seeded on 8 well chamber slides (10,000 cells/well) and treated with inhibitors followed by infection with L.donovani in a 1∶10 ratio. Cells were incubated for 24 hr, fixed with chilled methanol, and stained with Giemsa. Amastigotes were then microscopically counted. ***P<.001, *P<.05 and ns = non-significant for the comparison with infected one (C). Data represented means ± SD from three different experiments (A and C). Result shown in B is a representative one from three different experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4201563&req=5

pone-0110843-g005: Effect of inhibitors on plasma membrane Ca2+-ATPases during Leishmania infection.Macrophages were treated with thapsigargin (TG, 1 µM) and trifluoperazine (TFP, 10 µM) followed by infection with L.donovani in 1∶10 ratio. Unbound parasites were washed off after 4 hr and further incubated for 20 hr. Cells were collected, plasma membrane was isolated and Ca2+-ATPase activity was measured. (A). In a similar experiment, inhibitor treated and infected macrophages were collected in Trizol for RNA extraction and semi-quantitative RT-PCR were performed (B). Macrophages were seeded on 8 well chamber slides (10,000 cells/well) and treated with inhibitors followed by infection with L.donovani in a 1∶10 ratio. Cells were incubated for 24 hr, fixed with chilled methanol, and stained with Giemsa. Amastigotes were then microscopically counted. ***P<.001, *P<.05 and ns = non-significant for the comparison with infected one (C). Data represented means ± SD from three different experiments (A and C). Result shown in B is a representative one from three different experiments.
Mentions: Calcium appears to be vital in Leishmania apoptosis. It has been also established that more parasites die in the presence of Ca2+ than in its absence. Therefore, we focused on the parasitic load of the infected macrophages. In these experiments, macrophages were infected as described in Materials and Methods. Figure 5C shows that parasitic load was significantly increased in presence of thapsigargin; whereas a sharp decline was observed in presence trifluoperazine in comparison to the control. When both inhibitors were used parasitic load was found to be lower compared to the control.

Bottom Line: Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+.Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden.These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, West Bengal, India.

ABSTRACT
Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca2+ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca2+ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca2+ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca2+ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis.

Show MeSH
Related in: MedlinePlus