Characterization of the Intracellular Ca2+ Pools Involved in the Calcium Homeostasis in Herpetomonas sp. Promastigotes

Sodré, Cátia Lacerda; Moreira, B. L. M.; Nobrega, F.B; Gadelha, Fernanda Ramos; Meyer-Fernandes, J.R.; Dutra, Patrícia Maria Lourenço; Vercesi, Anı́bal E.; Lopes, Angela H.; Scofano, Helena M.; Barrabin, Héctor

doi:10.1006/abbi.2000.1899

Cited by 19 publications

(22 citation statements)

References 38 publications

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“…Our data are consistent with observations showing that ecto-nucleotidases are widely distributed in nature, as they have been described in plants (Traverso-Cori et al, 1965), several mammalian cell types (Plesner, 1995), and in insects (Sarkis et al, 1986;Valenzuela et al, 1998Valenzuela et al, , 2001MeyerFernandes et al, 2000). The great physiological importance of extracellular ATP in different organisms is characterized on the one hand by its direct involvement in the control of diverse cellular functions, and on the other hand by the regulatory role of biologically active compounds derived from subsequent nucleotide breakdown (Schlosser et al, 1996).…”

Section: January 2006supporting

confidence: 93%

“…Table 1 shows that both nucleotidase activities were insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase (Meyer-Fernandes et al, 1997); bafilomycin A 1 , a V-ATPase inhibitor (Browman et al, 1988); ouabain, a Na + K + -ATPase inhibitor (Caruso-Neves et al, 1998), and vanadate, a potent inhibitor of PATPases and acid phosphatases (Sodre et al, 2000;Dutra et al, 2001;De Jesus et al, 2002). Molybdate and sodium fluoride, two potent inhibitors of acid phosphatase activity (Meyer-Fernandes et al, 1999), did not inhibit either ecto-nucleotidase activity (Table 1).…”

Section: Resultsmentioning

confidence: 99%

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Ecto-nucleotidase activities in the fat body ofRhodnius prolixus

Entringer

Gondim

Meyer‐Fernandes

2005

Arch. Insect Biochem. Physiol.

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In this study, we describe the ability of intact fat body of an insect, Rhodnius prolixus, to hydrolyze extracellular ATP. In these fat bodies, the ATP hydrolysis was low in the absence of any divalent metal, and was stimulated by MgCl 2 . Both activities (in the absence or presence of MgCl 2 ) were linear with time for at least 30 min. In order to confirm the observed nucleotidase activities as ecto-nucleotidases, we used an impermeant inhibitor, DIDS (4, 4′-diisothiocyanostylbene 2′-2′-disulfonic acid). This reagent inhibited both nucleotidase activities and its inhibitory effect was suppressed by ATP. Both ecto-nucleotidase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin, ouabain, vanadate, molybdate, sodium fluoride, levamizole, tartrate, p-NPP, sodium phosphate, and suramin. Concanavalin A, activator of some ecto-ATPases, was able to stimulate the Mg 2+ -independent nucleotidase activity, but not the Mg 2+ -dependent one. The Mg 2+ -independent nucleotidase activity was enhanced with increases in the pH in the range between 6.4-8.0, but the Mg 2+ -dependent nucleotidase activity was not affected. Besides MgCl 2 , the ecto-ATPase activity was also stimulated by CaCl 2 , MnCl 2 , and SrCl 2 , but not by ZnCl 2 . ATP, ADP, and AMP were the best substrates for the Mg 2+ -dependent ecto-nucleotidase activity, and CTP, GTP, and UTP produced very low reaction rates. However, the Mg 2+ -independent nucleotidase activity recognized all these nucleotides producing similar reaction rates, but GTP was a less efficient substrate. The possible role of the two ecto-nucleotidase activities present on the cell surface of fat body of Rhodnius prolixus, which are distinguished by their substrate specificity and their response to Mg 2+ , is discussed. Arch. Insect Biochem. Physiol. 61:1-9, 2006.

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Section: January 2006supporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Ecto-nucleotidase activities in the fat body ofRhodnius prolixus

Entringer

Gondim

Meyer‐Fernandes

2005

Arch. Insect Biochem. Physiol.

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“…In a previous work, we have identified and characterized two Ca 2+ -transport systems in Herpetomonas sp. permeabilized with digitonin [29]. One, sensitive to antimycin A, is driven by an electrical potential in mitochondria, and a second, vanadate sensitive, is promoted by a SERCA-type (sarcoplasmic-endoplasmic reticulum).…”

Section: Introductionmentioning

confidence: 99%

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

Medeiros

Moreira

Miranda

et al. 2005

Molecular and Biochemical Parasitology

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Acidocalcisomes are acidic calcium storage organelles found in several microorganisms. They are characterized by their acidic nature, high electron density, high content of polyphosphates and several cations. Electron microscopy contrast tuned images of Herpetomonas sp. showed the presence of several electron dense organelles ranging from 100 to 300 nm in size. In addition, X-ray element mapping associated with energy-filtering transmission electron microscopy showed that most of the cations, namely Na, Mg, P, K, Fe and Zn, are located in their matrix. Using acridine orange as an indicator dye, a pyrophosphate-driven H + uptake was measured in cells permeabilized by digitonin. This uptake has an optimal pH of 6.5-6.7 and was inhibited by sodium fluoride (NaF) and imidodiphosphate (IDP), two H + -pyrophosphatase inhibitors. H + uptake was not promoted by ATP. Addition of 50 M Ca 2+ induced the release of H + , suggesting the presence of a Ca 2+ /H + countertransport system in the membranes of the acidic compartments. Na + was unable to release protons from the organelles. The pyrophosphate-dependent H + uptake was dependent of ion K + and inhibited by Na + Herpetomonas sp. immunolabeled with monoclonal antibodies raised against a Trypanosoma cruzi V-H + -pyrophosphatase shows intense fluorescence in cytoplasmatic organelles of size and distribution similar to the electron-dense vacuoles.Together, these results suggest that the electron dense organelles found in Herpetomonas sp. are homologous to the acidocalcisomes described in other trypanosomatids. They possess a vacuolar H + -pyrophosphatase and a Ca 2+ /H + antiport. However, in contrast to the other trypanosomatids so far studied, we were not able to measure any ATP promoted H + transport in the acidocalcisomes of this parasite.

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“…These results contrast with those reported in other trypanosomatids, where thapsigargin (1-8 M) did not have any effect on intracellular Ca 2ϩ release in digitonin-treated cells of T. cruzi, T. brucei (Moreno, Vercesi et al, 1992;Vercesi et al, 1993), or Herpetomonas sp. (Sodré et al, 2000). Figure 1Ab shows that the addition of the Ca 2ϩ ionophore A23187 produces a large Ca 2ϩ release, probably from other compartments, such as acidocalcisomes, in which Ca 2ϩ uptake is also an ATP-dependent process (Docampo and Moreno, 2001).…”

Section: Ingestion Of Cryptosporidium Oocysts By Caenorhabditis Elegansmentioning

confidence: 96%

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

Mendoza

Mijares

Rojas

et al. 2004

Journal of Parasitology

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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.

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Characterization of the Intracellular Ca2+ Pools Involved in the Calcium Homeostasis in Herpetomonas sp. Promastigotes

Cited by 19 publications

References 38 publications

Ecto-nucleotidase activities in the fat body ofRhodnius prolixus

Ecto-nucleotidase activities in the fat body ofRhodnius prolixus

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

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

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