scholarly journals Intracellular Ca2+ storage in acidocalcisomes of Trypanosoma cruzi

1995 ◽  
Vol 310 (3) ◽  
pp. 1005-1012 ◽  
Author(s):  
R Docampo ◽  
D A Scott ◽  
A E Vercesi ◽  
S N J Moreno
Keyword(s):  
Trypanosoma Cruzi ◽  
Acridine Orange ◽  
Bafilomycin A1 ◽  
Uptake Mechanism ◽  
Acetoxymethyl Ester ◽  
Permeabilized Cells ◽  
Fura 2 ◽  
Carbonyl Cyanide ◽  
Acidic Compartment ◽  
Proton Uptake

The use of digitonin to permeabilize the plasma membrane of Trypanosoma cruzi allowed the identification of a non-mitochondrial nigericin- or bafilomycin A1-sensitive Ca(2+)-uptake mechanism. Proton uptake, as detected by ATP-dependent Acridine Orange accumulation, was also demonstrated in these permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles. This latter process was inhibited (and reversed) by bafilomycin A1, nigericin and NH4Cl in different stages of T. cruzi. Ca2+ released Acridine Orange from permeabilized cells, suggesting that the dye and Ca2+ were being accumulated in the same acidic compartment and that Ca2+ was taken up in exchange for protons. Addition of bafilomycin A1 (5 microM), nigericin (1 microM) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 microM) to fura 2-loaded epimastigotes increased their intracellular Ca2+ concentration ([Ca2+]i). Although this effect was more noticeable in the presence of extracellular Ca2+, it was also observed in its absence. Addition of NH4Cl (10-40 mM) to different stages of T. cruzi, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry, increased both [Ca2+]i in fura 2-loaded cells, and intracellular pH (pHi) in 2′,7′-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester (BCECF)-loaded cells. Treatment of the cells with the Ca2+ ionophore ionomycin under similar conditions (nominal absence of extracellular Ca2+) resulted in an increase in [Ca2+]i and a significantly higher increase in [Ca2+]i after addition of NH4Cl, nigericin or bafilomycin A1, all agents which increase the pH of acidic compartments and make ionomycin more effective as a Ca(2+)-releasing ionophore. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that most of the Ca2+ stored in different stages of T. cruzi is present in the acidic compartment thus identified. Taken together, these results are consistent with the presence of a Ca2+/H+ exchange system in an acidic vacuole, which we have named the ‘acidocalcisome’ and which appears to be a unique organelle present in trypanosomatids.

scholarly journals Ca2+/H+ exchange in acidic vacuoles of Trypanosoma brucei

1994 ◽  
Vol 304 (1) ◽  
pp. 227-233 ◽  
Author(s):  
A E Vercesi ◽  
S N Moreno ◽  
R Docampo
Keyword(s):  
Plasma Membrane ◽  
Acridine Orange ◽  
Trypanosoma Brucei ◽  
Initial Rate ◽  
Sodium Orthovanadate ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Permeabilized Cells ◽  
Carbonyl Cyanide ◽  
Atpase System

The use of digitonin to permeabilize the plasma membrane of Trypanosoma brucei procyclic and bloodstream trypomastigotes allowed the identification of a non-mitochondrial nigericin-sensitive Ca2+ compartment. The proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was able to cause Ca2+ release from this compartment, which was also sensitive to sodium orthovanadate. Preincubation of the cells with the vacuolar H(+)-ATPase inhibitor bafilomycin A1 greatly reduced the nigericin-sensitive Ca2+ compartment. Bafilomycin A1 inhibited the initial rate of ATP-dependent non-mitochondrial Ca2+ uptake and stimulated the initial rate of nigericin-induced Ca2+ release by permeabilized procyclic trypomastigotes. ATP-dependent and bafilomycin A1- and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl)-sensitive Acridine Orange uptake was demonstrated in permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles by a process inhibited by bafilomycin A1, NBD-Cl, nigericin, and Ca2+. Vanadate or EGTA significantly increased Acridine Orange uptake, while Ca2+ released Acridine Orange from these preparations, thus suggesting that the dye and Ca2+ were being accumulated in the same acidic vacuole. Acridine Orange uptake was reversed by nigericin, bafilomycin A1 and NH4Cl. The results are consistent with the presence of a Ca2+/H(+)-ATPase system pumping Ca2+ into an acidic vacuole, that we tentatively named the acidocalcisome.

Direct measurement of free Ca in organelles of gastric epithelial cells

1994 ◽  
Vol 267 (3) ◽  
pp. G442-G451 ◽  
Author(s):  
A. M. Hofer ◽  
T. E. Machen
Keyword(s):  
Cell Types ◽  
Ruthenium Red ◽  
Acetoxymethyl Ester ◽  
Gastric Glands ◽  
Free Concentration ◽  
Fura 2 ◽  
Gastric Cells ◽  
Mitochondrial Inhibitors ◽  
Carbonyl Cyanide ◽  
The Relationship

When loaded as the acetoxymethyl ester (AM) derivative, the fluorescent probe mag-fura 2 accumulates in both the cytoplasm and the subcellular compartments. The relatively high dissociation constant of this dye for Ca (53 microM) permits the measurement of changes in the free concentration of this ion in a variety of organelles where Ca concentration ([Ca]) is high. To characterize Ca stores in gastric cells, we used digitonin to permeabilize cells within isolated rabbit gastric glands loaded with mag-fura 2-AM. This allowed cytosolic dye to leak out, leaving only compartmentalized dye behind. It appears that mag-fura 2 monitors [Ca] changes in several ATP-dependent Ca-sequestering pools; an inositol 1,4,5-trisphosphate (IP3)-releasable and thapsigargin-sensitive store (which probably includes the endoplasmic reticulum), a pool that is released by the mitochondrial inhibitors oligomycin+azide, valinomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and ruthenium red (and therefore likely represents mitochondria), and a residual pool that was resistant to release by mitochondrial inhibitors and thapsigargin. Ca sequestration into all pools was sensitive to changes in [ATP], indicating that treatments that reduce cellular [ATP] will cause certain organelles to lose their Ca to the cytoplasm. Caffeine and ryanodine, which mobilize Ca from internal stores in many cell types, induced Ca sequestration into an IP3-insensitive store of gastric cells, and caffeine caused a reduction in cytoplasmic [Ca] (as measured with fura 2). We also show that the quantitation of free [Ca] in a given pool is complicated by a nonlinearity in the relationship between the mag-fura 2 ratio and [Ca]. This effect is likely a consequence of monitoring the fluorescence from multiple pools simultaneously. However, this limitation does not detract from the ability of this method to yield important qualitative information about the nature and number of Ca stores within single gastric cells.

scholarly journals Acidocalcisomes and a vacuolar H+-pyrophosphatase in malaria parasites

2000 ◽  
Vol 347 (1) ◽  
pp. 243-253 ◽  
Author(s):  
Norma MARCHESINI ◽  
Shuhong LUO ◽  
Claudia O. RODRIGUES ◽  
Silvia N. J. MORENO ◽  
Roberto DOCAMPO
Keyword(s):  
Dependent Manner ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Acetoxymethyl Ester ◽  
Malaria Parasites ◽  
Subcellular Compartment ◽  
Calcium Indicator ◽  
Thiol Reagent ◽  
Acidic Compartment ◽  
Acidic Organelles

Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by: (1) the increase in [Ca2+]i induced by bafilomycin A1, nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2+, and (2) the effect of ionomycin, which cannot take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A1, nigericin, monensin, or NH4Cl. Inorganic PPi promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PPi collapsed by addition of the K+/H+ exchanger, nigericin, and eliminated by the PPi analogue, aminomethylenediphosphonate (AMDP). Both PPi hydrolysis and proton transport were dependent upon K+, and Na+ caused partial inhibition of these activities. PPi hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H+-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca2+]i in the nominal absence of extracellular Ca2+. Ionomycin was more effective in releasing Ca2+ from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in trypanosomatids and Toxoplasma gondii, and the colocalization of the V-H+-PPase and V-H+-ATPase in these organelles.

scholarly journals Ca2+ storage in Trypanosoma brucei: the influence of cytoplasmic pH and importance of vacuolar acidity

1995 ◽  
Vol 310 (3) ◽  
pp. 789-794 ◽  
Author(s):  
D A Scott ◽  
S N J Moreno ◽  
R Docampo
Keyword(s):  
Trypanosoma Brucei ◽  
Ph Effect ◽  
Calcium Ionophore ◽  
Free Calcium ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Cytosolic Ph ◽  
Permeabilized Cells ◽  
Intracellular Compartments ◽  
Acidic Compartment

The hypothesis that changes in cytosolic pH effect the release from intracellular compartments of stored calcium in Trypanosoma brucei was addressed by the use of procyclic and bloodstream trypomastigotes of T. brucei loaded with the fluorescent reagents 2′,7′-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) to measure intracellular pH (pHi), or fura 2 to measure intracellular free calcium ([Ca2+]i). Experiments were performed in EGTA-containing buffers, so increases in [Ca2+]i reflected release of stored calcium rather than Ca2+ entry. Nigericin reduced pHi and increased [Ca2+]i in loaded cells, whilst propionate reduced pHi, but did not affect [Ca2+]i, and NH4Cl increased both variables, so there appears to be no correlation between pHi and [Ca2+]i. Treatment of the cells with the calcium ionophore ionomycin under similar conditions (nominal absence of extracellular Ca2+) resulted in an increase of [Ca2+]i which was greatly increased by addition of either NH4Cl, nigericin or the vacuolar H(+)-ATPase inhibitor bafilomycin A1. Similar results were obtained when the order of additions was reversed or when digitonin-permeabilized cells were used with the Ca2+ indicator arsenazo III. The results suggest that more Ca2+ is stored in this acidic compartment in procyclic than in bloodstream forms. Taking into account the relative importance of the ionomycin-releasable and the ionomycin-plus-NH4Cl-releasable Ca2+ pools, it is apparent that a significant amount of the Ca2+ stored in T. brucei trypomastigotes is present in the acidic compartment thus identified.

A new family of fluorescent calcium indicators designed to resist leakage or for measuring calcium near membranes

1994 ◽  
Vol 52 ◽  
pp. 168-169
Author(s):  
Martin Poenie ◽  
Akwasi Minta ◽  
Charles Vorndran
Keyword(s):  
Metal Binding ◽  
Acetoxymethyl Ester ◽  
Binding Properties ◽  
Fura 2 ◽  
New Family ◽  
Anion Transporter ◽  
Calcium Indicator ◽  
Calcium Indicators ◽  
High Calcium ◽  
Intracellular Compartmentalization

The use of fura-2 as an intracellular calcium indicator is complicated by problems of rapid dye leakage and intracellular compartmentalization which is due to a probenecid sensitive anion transporter. In addition there is increasing evidence for localized microdomains of high calcium signals which may not be faithfully reported by fura-2.We have developed a new family of fura-2 analogs aimed at addressing some of these problems. These new indicators are based on a modified bapta which can be readily derivatized to produce fura-2 analogs with a variety of new properties. The modifications do not affect the chromophore and have little impact on the spectral and metal binding properties of the indicator. One of these new derivatives known as FPE3 is a zwitterionic analog of fura-2 that can be loaded into cells as an acetoxymethyl ester and whose retention in cells is much improved. The improved retention of FPE3 is important for both cuvettebased measurements of cell suspensions and for calcium imaging.

A mitochondrial uncoupler increases KCa currents but decreases KV currents in pulmonary artery myocytes

1996 ◽  
Vol 270 (1) ◽  
pp. C321-C331 ◽  
Author(s):  
X. J. Yuan ◽  
T. Sugiyama ◽  
W. F. Goldman ◽  
L. J. Rubin ◽  
M. P. Blaustein
Keyword(s):  
Pulmonary Artery ◽  
Fluorescent Imaging ◽  
K Channel ◽  
Metabolic Inhibitor ◽  
Acetoxymethyl Ester ◽  
Atp Production ◽  
Kv Channels ◽  
Kca Channels ◽  
Carbonyl Cyanide ◽  
Microscopy Techniques

Intracellular free Ca2+ concentration ([Ca2+]i) and ATP play important roles in the regulation of K- channels in pulmonary artery (PA) myocytes. Previous studies have demonstrated that hypoxia and the metabolic inhibitor, 2-deoxy-D-glucose, decrease voltage-gated K+ (KV) currents [IK(V)] and thereby depolarize PA myocytes; these effects lead to a rise in [Ca2+]i. Here, we used carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a protonophore that uncouples mitochondrial respiration from ATP production, to test whether the inhibition of oxidative phosphorylation affects K+ channel activities in rat PA myocytes. Patch-clamp and fluorescent-imaging microscopy techniques were used to measure K+ currents (IK) and [Ca2+]i, respectively. FCCP (3-5 microM) reversibly raised [Ca2-]i in the presence and absence of external Ca2+. This effect was prevented by pretreating the cells with the membrane-permeable Ca2+ chelator, 1,2-bis(2-amino-phenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). This suggests that much of the FCCP-evoked rise in [Ca2-]i was due to Ca2+ release from intracellular stores. Brief exposure to FCCP (approximately 2 min) reversibly enhanced Ik. This augmentation was not influenced by glibenclamide, an ATP-sensitive K channel blocker, but was eliminated by pretreatment with BAPTA-AM. This implies that the FCCP-evoked rise in [Ca2+]i activated Ca(2+)-activated K- (Kca) channels. Furthermore, in BAPTA-treated cells, longer application (> or = 6 min) of FCCP reversibly decreased IK(V) in PA cells bathed in Ca(2+)-free solution. These results demonstrate that FCCP affects KCa and Kv channels by different mechanisms. FCCP increases IK[Ca] by raising [Ca2+]i primarily as a result of Ca2+ release, but decreases IK(V) by a Ca(2+)-independent mechanism, presumably the inhibition of oxidative ATP production.

Properties of neuroprotective cell-permeant Ca2+ chelators: effects on [Ca2+]i and glutamate neurotoxicity in vitro

1994 ◽  
Vol 72 (4) ◽  
pp. 1973-1992 ◽  
Author(s):  
M. Tymianski ◽  
M. P. Charlton ◽  
P. L. Carlen ◽  
C. H. Tator
Keyword(s):  
Time Course ◽  
Dependent Manner ◽  
Protective Effects ◽  
Tetraacetic Acid ◽  
Acetoxymethyl Ester ◽  
Neuroprotective Effects ◽  
Concentration Dependent Manner ◽  
Fura 2 ◽  
Glutamate Neurotoxicity

1. Cell-permeant Ca2+ chelators such as 1,2-bis-(2-amino-phenoxy)ethane- N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) protect neurons against excitotoxic and ischemic neuronal injury in vitro and in vivo. Here we provide the first steps toward characterizing the mechanisms by which these agents produce their neuroprotective effects. 2. Cultured mouse spinal neurons were simultaneously loaded with the Ca2+ indicator fura-2 and with one of three permeant chelators derived from the fast Ca2+ buffer BAPTA, or with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM). Adding these chelators did not interfere with the fluorescence spectrum of fura-2 and had no effect on baseline [Ca2+]i. 3. The neurons were challenged with 250 microM L-glutamate for 50 min, producing a marked transient [Ca2+]i increase followed by a decay of [Ca2+]i to a lower “plateau.” About 80% of control neurons succumbed to this excitotoxic insult. Neurons that survived adjusted their plateau [Ca2+]i to lower levels than those that succumbed. 4. Neurons that were pretreated with permeant Ca2+ chelators became more resistant to these neurotoxic challenges. 5. We examined whether this reduction in glutamate neurotoxicity could be related to the given buffer's known Ca2+ affinity (Kd), its Ca2+ binding kinetics, and its ability to attenuate glutamate-induced [Ca2+]i increases. 6. Pretreatment of neurons with BAPTA analogues having Kds ranging from 100 to 3,600 microM 1) attenuated the amplitude and 2) lengthened the time constant describing the rise and decay of the glutamate-evoked [Ca2+]i transient. The magnitude of these effects paralleled the affinity of the chelator for Ca2+. 7. BAPTA-AM and its analogues dramatically attenuated the early neurotoxicity of glutamate, reducing cell deaths by up to 80%. However, in contrast with the graded effects of chelators having different Ca2+ affinities on Ca2+ transients, all BAPTA analogues were equally protective. These protective effects did not relate to the chelators' Ca2+ affinity within a Kd range of 100 nM (for BAPTA) to 3,600 nM (for 5,5'-dibromo BAPTA). 8. BAPTA-AM protected neurons in a concentration-dependent manner with 50% protection obtained with 10 microM, a concentration having no effect on the [Ca2+]i transient amplitude. 9. EGTA, a slow Ca2+ buffer with a similar Ca2+ affinity to BAPTA produced the same effects as BAPTA on [Ca2+]i transient kinetics. However, it was far less protective than BAPTA. 10. The time course of early glutamate neurotoxicity was altered by the BAPTA analogues, but not EGTA. BAPTA analogues caused a small increase in cell deaths in the first minutes of each experiment, followed by relative sparing from further neurodegeneration. 11. The ability of low Ca2+ affinity chelators such as 5,5'-dibromo BAPTA to protect neurons without markedly attenuating measured [Ca2+]i increases conflicts with the hypothesis that global elevations in [Ca2+]i are responsible for triggering neurotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular hypercalcemia is an early event in deoxycholate injury of rabbit gastric mucosal cells

1995 ◽  
Vol 269 (2) ◽  
pp. G287-G296 ◽  
Author(s):  
A. J. Dziki ◽  
S. Batzri ◽  
J. W. Harmon ◽  
M. Molloy
Keyword(s):  
Cell Death ◽  
Bile Salt ◽  
Cell Injury ◽  
Early Event ◽  
Acetoxymethyl Ester ◽  
Gastric Mucosal Cells ◽  
Maximal Increase ◽  
Fura 2 ◽  
Gastric Cells ◽  
Mucosal Cells

Ca2+ entry into the cell may be an early event in the pathophysiology of bile salt-induced gastric mucosal injury. The aim of this study was to characterize the rise in cytosolic free Ca2+ associated with bile salt injury and its association with cell injury and death. Rabbit gastric mucosal cells were preloaded with the Ca2+ indicator fura 2-acetoxymethyl ester (fura 2-AM) for 20 min at 37 degrees C and then exposed to graded concentrations of the bile salt deoxycholate (DC). Cytosolic free Ca2+ concentration ([Ca2+]i) was estimated by spectrofluorometry. The resting [Ca2+]i in gastric cells was 177 +/- 15 nM (n = 6). When cells were subjected to 0.5 mM DC, there was a time-dependent rise in [Ca2+]i. An increase in [Ca2+]i was observed within 2 min, at which time [Ca2+]i rose from 177 +/- 15 to 480 +/- 30 nM. The maximal increase in [Ca2+]i was observed after 20 min of exposure to 0.5 mM DC (639 +/- 49 nM), and [Ca2+]i remained unchanged for at least 2 h. The increase in [Ca2+]i depended on the concentration of DC. The minimum effective dose of DC was 0.2 mM, with which [Ca2+]i was increased by 1.6-fold (from 177 to 285 nM). At 0.5 mM DC also caused a rise in 45Ca2+ influx into the cells and reduced the viability of gastric cells from 96% to 58% at 2 h. The DC-induced rise in cytosolic free Ca2+ depended on the presence of extracellular Ca2+. In the absence of extracellular Ca2+ there was no rise in cytosolic Ca2+ and gastric cells were protected from cell death caused by DC. The DC-induced cell death was reduced from 26% to 10% and from 37% to 16% at 60 and 90 min, respectively, by removal of extracellular Ca2+. The association of DC with gastric cells was not altered by removing extracellular Ca2+. This suggests decreased DC-induced injury in the absence of extracellular Ca2+ is due to the protection from cellular hypercalcemia rather than some other mechanism related to reduced binding and/or association of DC to gastric cells. These experiments show that rising [Ca2+]i appears to be an early pathophysiological event in bile salt-induced cellular injury and that extracellular Ca2+ is critical to produce this effect.

Heat acclimation and heat stress have different effects on cholinergic-induced calcium mobilization

2001 ◽  
Vol 280 (6) ◽  
pp. R1688-R1696 ◽  
Author(s):  
Pavel Kaspler ◽  
Michal Horowitz
Keyword(s):  
Heat Stress ◽  
Submaxillary Gland ◽  
Heat Acclimation ◽  
Cytosolic Calcium ◽  
Calcium Signal ◽  
Permeabilized Cells ◽  
Fura 2 ◽  
Submaxillary Glands ◽  
Intracellular Ca ◽  
Glandular Cells

There is evidence that the signal transduction array responsible for the secretion of water in evaporative cooling by the submaxillary gland of the rat is subject to heat acclimatory responses. The objectives of the present study were 1) to examine whether heat acclimation affects intracellular Ca2+ mobilization and, in turn, submaxillary glandular responsiveness; 2) to assess whether the acclimatory responses differ from those evoked on heat stress (HS). Experiments were conducted on submaxillary glands of rats acclimated at 34°C for 0, 2 [short-term heat acclimation (STHA)], and 30 [long-term heat acclimation (LTHA)] days. The resting cytosolic calcium concentration ([Ca2+]c) and the carbamylcholine-evoked calcium signal ([Ca2+]s) of dispersed glandular cells were measured using the fluorescent dye fura 2 AM. Inositol-1,4,5-trisphosphate (IP3)-sensitive endoplasmic reticulum Ca2+ stores were determined in permeabilized cells using fura 2 potassium salt. STHA resulted in a drop in both [Ca2+]s and IP3-sensitive Ca2+ stores. On LTHA, the [Ca2+]samplitude reverted to the preacclimation value, whereas the IP3-sensitive Ca2+ stores remained low. The drop in [Ca2+]s on STHA is in accord with the decreased glandular output (measured by 86Rb efflux) observed during this acclimation phase. However, after LTHA the enhanced glandular output despite reduced [Ca2+]s levels suggests an increased efficiency of cellular secretory mechanisms in that group. Collectively, the alterations in [Ca2+]ssupport our biphasic acclimation model (Horowitz M, Kaspler P, Marmari Y, and Oron Y. J Appl Physiol 80: 77–85, 1996.). In nonacclimated glands, HS caused an elevation in [Ca2+]s coincidentally with a decrease in the IP3 Ca2+ stores. In contrast, [Ca2+]s in both STHA and LTHA glands was not affected by HS, despite a marked increase in the IP3-sensitive Ca2+ stores in the LTHA glands. The opposing responses to HS and heat acclimation in calcium signaling and stores confirm the specificity of each process.

scholarly journals Acidocalcisomes in Toxoplasma gondii tachyzoites

1996 ◽  
Vol 313 (2) ◽  
pp. 655-659 ◽  
Author(s):  
Silvia N. J. MORENO ◽  
Li ZHONG
Keyword(s):  
Endoplasmic Reticulum ◽  
Toxoplasma Gondii ◽  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Mitochondrial Atpase ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Extracellular Medium ◽  
Acidic Compartment ◽  
Acidic Organelles

Toxoplasma gondii tachyzoites were loaded with the fluorescent indicator fura 2 to investigate the transport mechanisms involved in maintaining their intracellular Ca2+ homoeostasis. The mitochondrial ATPase inhibitor oligomycin and the endoplasmic-reticulum Ca2+-ATPase inhibitor thapsigargin increased the intracellular Ca2+ concentration ([Ca2+]i), thus indicating the requirement for ATP and the involvement of the endoplasmic reticulum in maintaining intracellular Ca2+ homoeostasis. The effect of thapsigargin was more accentuated in the presence of extracellular Ca2+, clearly showing that, as occurs with other eukaryotic cells, depletion of intracellular Ca2+ pools led to an increase in the uptake of Ca2+ from the extracellular medium. In addition to these results, we found evidence that, in contrast with what occurs in mammalian cells, T. gondii tachyzoites possess a significant amount of Ca2+ stored in an acidic compartment, termed the acidocalcisome, as indicated by: (1) the increase in [Ca2+]i induced by bafilomycin A1 (a specific inhibitor of H+-ATPases), nigericin (a K+/H+ exchanger) or the weak base NH4Cl, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry; and (2) the effect of ionomycin, a Ca2+-releasing ionophore that cannot take Ca2+ out of acidic organelles and that was more effective after alkalinization of these compartments by addition of bafilomycin A1, nigericin or NH4Cl. Considering the relative importance of the ionomycin-releasable and the ionomycin+NH4Cl-releasable Ca2+ pools, it is apparent that T. gondii tachyzoites contain a significant amount of Ca2+ stored in acidocalcisomes.

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