Evaluation of the Presence of a Thapsigargin-Sensitive Calcium Store in Trypanosomatids Using Trypanosoma evansi as a Model

Abstract

Ca2+ plays an important role in the regulation of several important activities in different trypanosomatids. These parasites possess a Ca2+ transport system in the endoplasmic reticulum (ER) involved in Ca2+ homeostasis, which has been reported to be insensitive to thapsigargin, a classical inhibitor of the sarcoplasmic-ER Ca2+ adenosine triphosphatase (ATPase) (SERCA) in most eukaryotic cells. However, currently there is a controversy regarding the existence of a thapsigargin-sensitive ER Ca2+ store in these parasites. Therefore, we decided to explore the effect of this inhibitor using different methodological approaches. First, we selected Trypanosoma evansi as a parasite model to warrant the homogeneity of the population because this parasite has only a single life cycle, i.e., bloodstream-form trypomastigotes. Second, we compared the thapsigargin effect on Ca2+ homeostasis by spectrophotometrical Ca2+ measurements using 3 different approaches: whole-cell populations, cells that have been permeabilized by treatment with digitonin, and intact single cells. Our results demonstrate that a low concentration of thapsigargin induces Ca2+ release from intracellular Ca2+ stores in this parasite, which can be observed independently of the method used. Furthermore, the addition of thapsigargin before or after nigericin did not abolish its effect, showing that thapsigargin acts specifically on the ER. In conclusion, our results indicate the presence of a nonmitochondrial thapsigargin-sensitive Ca2+ store in T. evansi.

Promastigote to amastigote differentiation of Leishmania is markedly delayed in the absence of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation

“…ER stress can be achieved in vitro by exposing cells to pharmacological agents, such as thapsigargin that inhibits the sarcoplasmic ER Ca 2+ ATPase and causes the accumulation of cytoplasmic Ca 2+ in most eukaryotic cells (Pozzan et al ., 1994). In T. brucei and Trypanosoma evansi , thapsigargin has been shown to inhibit the activity of an ER Ca 2+ ATPase and to specifically act on the ER by modulating cellular Ca 2+ stores (Nolan et al ., 1994; Mendoza et al ., 2004). Here we report that exposure of L. infantum promastigotes to 1 µM thapsigargin for 6 h resulted in a dramatic decrease in general translation as exemplified by polysome profiling analysis (Fig.…”

Unfolded Protein Response Pathways in Bloodstream-Form Trypanosoma brucei?

“…In our hands both of the DTT concentrations used by prior investigators, 4 mM (4) and 1 mM (12), had ϳ50% kill rates for BSF trypanosomes at 2 h of incubation and were essentially 100% lethal at 4 h. DTT at 0.5 mM was less toxic, with ϳ80% and ϳ30% survival at 2 and 4 h, respectively, but the morphology of viable cells at the later time points was severely impacted (data not shown). Thapsigargin at a concentration (5 M) known to inhibit the ER Ca ϩϩ transporter in trypanosomes (27)(28)(29)(30) completely blocked growth over 24 h, but lower concentrations (1 and 2.5 M) had only a modest impact (Fig. 4A, center).…”

Anti-VSG antibodies induce an increase inTrypanosoma evansiintracellular Ca2+concentration

“…8d) (Moreno and Docampo, 2003; Mendoza et al . 2002, 2004 b ), and (2) an influx of this ion through channels located on its plasma membrane (Fig. 8e).…”