Divergent calcium signaling in RBCs from Tropidurus torquatus (Squamata – Tropiduridae) strengthen classification in lizard evolution

Abstract\ud \ud \ud \ud Background\ud \ud We have previously reported that a Teiid lizard red blood cells (RBCs) such as Ameiva ameiva and Tupinambis merianae controls intracellular calcium levels by displaying multiple mechanisms. In these cells, calcium stores could be discharged not only by: thapsigargin, but also by the Na+/H+ ionophore monensin, K+/H+ ionophore nigericin and the H+ pump inhibitor bafilomycin as well as ionomycin. Moreover, these lizards possess a P2Y-type purinoceptors that mobilize Ca2+ from intracellular stores upon ATP addition.\ud \ud \ud \ud Results\ud \ud Here we report, that RBCs from the tropidurid lizard Tropidurus torquatus store Ca2+ in endoplasmic reticulum (ER) pool but unlike in the referred Teiidae, these cells do not store calcium in monensin-nigericin sensitive pools. Moreover, mitochondria from T. torquatus RBCs accumulate Ca2+. Addition of ATP to a calcium-free medium does not increase the [Ca2+]c levels, however in a calcium medium we observe an increase in cytosolic calcium. This is an indication that purinergic receptors in these cells are P2X-like.\ud \ud \ud \ud Conclusion\ud \ud T. torquatus RBCs present different mechanisms from Teiid lizard red blood cells (RBCs), for controlling its intracellular calcium levels. At T. torquatus the ion is only stored at endoplasmic reticulum and mitochondria. Moreover activation of purinergic receptor, P2X type, was able to induce an influx of calcium from extracelullar medium. These studies contribute to the understanding of the evolution of calcium homeostasis and signaling in nucleated RBCs.We thank Fundação de Amparo à Pesquisa de São Paulo (FAPESP) for funding CRSG. FHB received fellowship from FAPESP. We thank Robson Sartorello and Miguel T. Rodrigues for collecting the lizards.We thank Fundação de Amparo à Pesquisa de São Paulo (FAPESP) for funding CRSG. FHB received fellowship from FAPESP. We thank Robson Sartorello and Miguel T. Rodrigues for collecting the lizards

Antimalarials and the fight against malaria in Brazil

Malaria, known as the “fevers,” has been treated for over three thousand years in China with extracts of plants of the genus Artemisia (including Artemisia annua, A. opiacea, and A. lancea) from which the active compound is artemisin, a sesquiterpene that is highly effective in the treatment of the disease, especially against young forms of the parasite. South American Indians in the seventeenth century already used an extract of the bark of chinchona tree, commonly named “Jesuits’ powder.” Its active compound was isolated in 1820 and its use spread all over the world being used as a prophylactic drug during the construction of the Madeira–Mamoré railroad in the beginning of the twentieth century. During the 1920s to the 1940s, new antimalarial drugs were synthesized to increase the arsenal against this parasite. However, the parasite has presented systematic resistence to conventional antimalarial drugs, driving researchers to find new strategies to treat the disease. In the present review we discuss how Brazil treats Plasmodium-infected patients

Extracellular ATP triggers proteolysis and cytosolic Ca²⁺ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites.

BACKGROUND: Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. METHODS: Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca²⁺ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. RESULTS: The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 μM), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 μM) to isolated parasites previously loaded with Fluo4/AM in a Ca²⁺-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms (82% ± 4, n = 11) and a decrease in trophozoite forms (18% ± 4, n = 11). CONCLUSIONS: The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway

Camobilization in Fluo-3-labelled RBCs of the lizard : A) RBCs incubated in calcium medium (1 mM CaCl) were treated where indicated with the SERCA inhibitor, thapsigargin (5 μM)

<p><b>Copyright information:</b></p><p>Taken from "Divergent calcium signaling in RBCs from (Squamata – Tropiduridae) strengthen classification in lizard evolution"</p><p>http://www.biomedcentral.com/1472-6793/7/7</p><p>BMC Physiology 2007;7():7-7.</p><p>Published online 23 Aug 2007</p><p>PMCID:PMC2018699.</p><p></p> as in A, but CaClwas omitted and the medium was supplemented with 15 mM EGTA THG (5 μM) was add before addition of Caionophore ionomycin (10 μM). C) Effect of THG (5 μM) in RBC pre incubated with ionomycin (10 μM). D) Monensin (25 μM) was not able to induce an intracellular Caincrease

Increase of mitochondrial Capromotes an increase of NADPH in RBCs loaded with mitochondrial CaRhod-2

<p><b>Copyright information:</b></p><p>Taken from "Divergent calcium signaling in RBCs from (Squamata – Tropiduridae) strengthen classification in lizard evolution"</p><p>http://www.biomedcentral.com/1472-6793/7/7</p><p>BMC Physiology 2007;7():7-7.</p><p>Published online 23 Aug 2007</p><p>PMCID:PMC2018699.</p><p></p> A) Increase of mitochondrial Ca[Ca]and fluorescence of NADPH induced by ATP (50 μM) in RBCs loaded with Rhod-2 AM, in the Camedium. B) similar experiments in a Cafree medium C) Ionomycin (10 μM) promotes a increase of [Ca]with concomitant increase of NADPH fluorescence

Antimalarial drugs disrupt ion homeostasis in malarial parasites

Plasmodium chabaudi malaria parasite organelles are major elements for ion homeostasis and cellular signaling and also target for antimalarial drugs. By using confocal imaging of intraerythrocytic parasites we demonstrated that the dye acridine orange (AO) is accumulated into P. chabaudi subcellular compartments. The AO could be released from the parasite organelles by collapsing the pH gradient with the K+/H+ ionophore nigericin (20 μM), or by inhibiting the H+-pump with bafilomycin (4 μM). Similarly, in isolated parasites loaded with calcium indicator Fluo 3-AM, bafilomycin caused calcium mobilization of the acidic calcium pool that could also be release with nigericin. Interestingly after complete release of the acidic compartments, addition of thapsigargin at 10 μM was still effective in releasing parasite intracellular calcium stores in parasites at trophozoite stage. The addition of antimalarial drugs chloroquine and artemisinin resulted in AO release from acidic compartments and also affected maintenance of calcium in ER store by using different drug concentrations