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.

Effects of bafilomycin A1 on cytosolic pH of sheep alveolar and peritoneal macrophages: evaluation of the pH-regulatory role of plasma membrane V-ATPases.

1995 ◽  
Vol 198 (8) ◽  
pp. 1711-1715 ◽  
Author(s):  
T A Heming ◽  
D L Traber ◽  
F Hinder ◽  
A Bidani
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Initial Rate ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Cytosolic Ph ◽  
Phi Regulation

The role of plasma membrane V-ATPase activity in the regulation of cytosolic pH (pHi) was determined for resident alveolar and peritoneal macrophages (m theta) from sheep. Cytosolic pH was measured using 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The baseline pHi of both cell types was sensitive to the specific V-ATPase inhibitor bafilomycin A1. Bafilomycin A1 caused a significant (approximately 0.2 pH units) and rapid (within seconds) decline in baseline pHi. Further, bafilomycin A1 slowed the initial rate of pHi recovery (dpHi/dt) from intracellular acid loads. Amiloride had no effects on baseline pHi, but reduced dpHi/dt (acid-loaded pHi nadir < 6.8) by approximately 35%. Recovery of pHi was abolished by co-treatment of m theta with bafilomycin A1 and amiloride. These data indicate that plasma membrane V-ATPase activity is a major determinant of pHi regulation in resident alveolar and peritoneal m theta from sheep. Sheep m theta also appear to possess a Na+/H+ exchanger. However, Na+/H+ exchange either is inactive or can be effectively masked by V-ATPase-mediated H+ extrusion at physiological pHi values.

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.

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 Active transport of l-proline in the protozoan parasite Trypanosoma brucei brucei

1993 ◽  
Vol 291 (1) ◽  
pp. 297-301 ◽  
Author(s):  
C L'Hostis ◽  
M Geindre ◽  
J Deshusses
Keyword(s):  
Trypanosoma Brucei ◽  
Membrane Vesicles ◽  
Protozoan Parasite ◽  
Protonmotive Force ◽  
Atpase Inhibitor ◽  
Plasma Membrane Vesicles ◽  
Proline Transport ◽  
Carbonyl Cyanide ◽  
Uptake Activity ◽  
Filtration Technique

The characteristics of L-proline transport in the procyclic form of Trypanosoma brucei were studied by using L-[14C]proline and a quick separation technique by centrifugation through an oil mixture. L-Proline uptake displayed typical Michaelis-Menten kinetics, with a Km of 19 microM and a maximum transport velocity of 17 nmol/min per 10(8) cells at 27 degrees C. The maximum concentration gradient factor obtained after 1 min of incubation was 270-fold in 0.02 mM proline. Cells permeabilized with 80 microM digitonin were still able to accumulate 14C label, but to a lower extent. The temperature-dependence of proline uptake gave an apparent activation energy of 74.9 kJ.mol-1. In competition studies with a 10-fold excess of structural analogues, L-alanine, L-cysteine and L-azetidine-2-carboxylate were found to inhibit L-proline uptake. Variation of pH or addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (‘CCCP’) did not affect proline transport, showing that it is not driven by a protonmotive force. The absence of Na+, with or without monensin, did not affect proline transport. The absence of K+ and the addition of the Na+,K(+)-ATPase inhibitor ouabain had no significant effect on proline uptake activity. The thiol-modifying reagent iodoacetate (10 mM) decreased proline uptake by half. KCN (1 mM) inhibited proline uptake to a lesser extent, and the degree of inhibition was proportional to the intracellular ATP concentration. Preliminary experiments on proline transport in plasma-membrane vesicles of the cells, using a filtration technique, showed an uptake of proline (0.67 nmol/mg of protein) by the vesicles, but only in the presence of intravesicular ATP. The results thus obtained suggest that the proline carrier system in T. brucei is ATP-driven and independent of Na+, K+ or H+ co-transport.

Recycling of AQP2 occurs through a temperature- and bafilomycin-sensitive trans-Golgi-associated compartment

2000 ◽  
Vol 278 (2) ◽  
pp. F317-F326 ◽  
Author(s):  
Corinne E. Gustafson ◽  
Toshiya Katsura ◽  
Mary McKee ◽  
Richard Bouley ◽  
James E. Casanova ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Hormonal Stimulation ◽  
Intracellular Location ◽  
Membrane Location ◽  
Recycling Pathway ◽  
Intracellular Vesicles

The exo- and endocytotic pathway in which aquaporin-2 (AQP2) travels between the plasma membrane and intracellular vesicles is only partially characterized. It is known that the antidiuretic hormone vasopressin induces a translocation of AQP2 from an intracellular to a plasma membrane location, both in kidney collecting duct principal cells and in transfected epithelial cells. Here we provide evidence suggesting that while AQP2 shifts from an intracellular location to the cell surface in response to vasopressin, AQP2 also constitutively recycles through a similar pathway in transfected LLC-PK1 cells even in the absence of hormonal stimulation. Incubating cells at 20°C blocks AQP2 recycling in a perinuclear compartment, regardless of whether vasopressin is present. The H+-ATPase inhibitor bafilomycin A1 also blocks the recycling pathway of AQP2 in a perinuclear compartment adjacent to the Golgi in the presence and absence of vasopressin stimulation, indicating a role of vesicle acidification in both the constitutive and regulated recycling of AQP2. Colocalization of AQP2 with clathrin, but not with giantin, after both bafilomycin treatment and a 20°C block suggests that the compartment in which recycling AQP2 is blocked may be the trans-Golgi, and not cis- and medial-Golgi cisternae.

Evidence for active H+ secretion by rat alveolar epithelial cells

1989 ◽  
Vol 257 (6) ◽  
pp. L438-L445 ◽  
Author(s):  
R. L. Lubman ◽  
S. I. Danto ◽  
E. D. Crandall
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Epithelial Cells ◽  
Initial Rate ◽  
Alveolar Epithelial Cells ◽  
Metabolic Inhibitor ◽  
Free Medium ◽  
Atpase Inhibitor ◽  
Alveolar Epithelial ◽  
Ethanesulfonic Acid

A plasma membrane proton-translocating adenosinetriphosphatase (ATPase) has been identified in rat alveolar pneumocytes in primary culture using the pH-sensitive fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Intracellular pH (pHi) was acutely lowered by NH3 prepulse in HCO3(-)-free medium buffered with 6 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and its recovery was measured thereafter under control conditions, in the presence of amiloride to inhibit Na(+)-H+ antiport, and in the presence of N-ethylmaleimide (NEM), a plasma membrane H(+)-ATPase inhibitor. Initial rate of pHi recovery was reduced by 67% in the presence of amiloride, 52% in the presence of NEM, and 96% in the presence of both. Recovery was decreased but not abolished in Na(+)-free buffer, was essentially abolished when NEM was present in the absence of Na+, and was also abolished by addition of the metabolic inhibitor KCN in glucose- and Na(+)-free medium. These data suggest that alveolar epithelial cells possess a plasma membrane H(+)-ATPase. In Na(+)-containing buffer at pH 7.4, steady-state pHi was 7.50. This value was unaffected by amiloride but decreased to 7.01 in the presence of NEM, suggesting active H(+)-ATPase and inactive Na(+)-H+ antiport at steady-state pHi. We conclude that this plasma membrane proton-translocating ATPase in alveolar pneumocytes may be an important mechanism contributing to regulation of steady-state pHi, recovery from acute intracellular acidification, and modulation of extracellular alveolar fluid pH.

scholarly journals Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

1998 ◽  
Vol 330 (2) ◽  
pp. 853-860 ◽  
Author(s):  
N. J. Silvia MORENO ◽  
Li ZHONG ◽  
Hong-Gang LU ◽  
Wanderley DE SOUZA ◽  
Marlene BENCHIMOL
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Laser Scanning ◽  
Membrane Surface ◽  
Surface Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Cytoplasmic Ph ◽  
Bafilomycin A1

Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

scholarly journals Retrograde Transport from the Pre-Golgi Intermediate Compartment and the Golgi Complex Is Affected by the Vacuolar H+-ATPase Inhibitor Bafilomycin A1

1998 ◽  
Vol 9 (12) ◽  
pp. 3561-3578 ◽  
Author(s):  
Harri Palokangas ◽  
Ming Ying ◽  
Kalervo Väänänen ◽  
Jaakko Saraste
Keyword(s):  
Brefeldin A ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Marker Proteins ◽  
Golgi Region ◽  
Intermediate Compartment ◽  
Acidic Compartments

The effect of the vacuolar H+-ATPase inhibitor bafilomycin A1 (Baf A1) on the localization of pre-Golgi intermediate compartment (IC) and Golgi marker proteins was used to study the role of acidification in the function of early secretory compartments. Baf A1 inhibited both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER), whereas anterograde ER-to-Golgi transport remained largely unaffected. Furthermore, p58/ERGIC-53, which normally cycles between the ER, IC, and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive ∼80-nm Golgi (coatomer protein I) vesicles was reduced, suggesting that the drug inhibits the retrieval of the protein from post-ER compartments. In parallel, redistribution of β-coatomer protein from the Golgi to peripheral pre-Golgi structures took place. The small GTPase rab1p was detected in short pre-Golgi tubules in control cells and was efficiently recruited to the tubules accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of newly synthesized, anterogradely transported proteins, indicating that they participate in retrograde transport. These results suggest that the pre-Golgi structures contain an active H+-ATPase that regulates retrograde transport at the ER–Golgi boundary. Interestingly, although Baf A1 had distinct effects on peripheral pre-Golgi structures, only more central, p58-containing elements accumulated detectable amounts of 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine (DAMP), a marker for acidic compartments, raising the possibility that the lumenal pH of the pre-Golgi structures gradually changes in parallel with their translocation to the Golgi region.

scholarly journals Resynthesis of phosphatidylinositol in permeabilized neutrophils following phospholipase Cβ activation: transport of the intermediate, phosphatidic acid, from the plasma membrane to the endoplasmic reticulum for phosphatidylinositol resynthesis is not dependent on soluble lipid carriers or vesicular transport

1999 ◽  
Vol 341 (2) ◽  
pp. 435-444 ◽  
Author(s):  
Jacqueline WHATMORE ◽  
Claudia WIEDEMANN ◽  
Pennti SOMERHARJU ◽  
Philip SWIGART ◽  
Shamshad COCKCROFT
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Vesicular Transport ◽  
Human Neutrophils ◽  
Permeabilized Cells ◽  
Transfer Protein ◽  
Membrane Contact ◽  
Phosphatidylinositol Transfer ◽  
Pi Hydrolysis ◽  
Hydrolysis Of

Receptor-mediated phospholipase C (PLC) hydrolysis of phosphoinositides is accompanied by the resynthesis of phosphatidylinositol (PI). Hydrolysis of phosphoinositides occurs at the plasma membrane, and the resulting diacylglycerol (DG) is converted into phosphatidate (PA). Two enzymes located at the endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be followed in permeabilized cells. In the presence of [γ-32P]ATP, DG generated by PLC activation accumulates label when converted into PA. The 32P-labelled PA is subsequently converted into labelled PI. The formation of labelled PI reports the arrival of labelled PA from the plasma membrane to the ER. Cytosol-depleted, permeabilized human neutrophils are capable of PI resynthesis following stimulation of PLCβ (in the presence of phosphatidylinositol-transfer protein), provided that CTP and inositol are also present. We also found that wortmannin, an inhibitor of endocytosis, or cooling the cells to 15 °C did not stop PI resynthesis. We conclude that PI resynthesis is dependent neither on vesicular transport mechanisms nor on freely diffusible, soluble transport proteins. Phosphatidylcholine-derived PA generated by the ADP-ribosylation-factor-stimulated phospholipase D pathway was found to accumulate label, reflecting the rapid cycling of PA to DG, and back. This labelled PA was not converted into PI. We conclude that PA derived from the PLC pathway is selected for PI resynthesis, and its transfer to the ER could be membrane-protein-mediated at sites of close membrane contact.

scholarly journals Amplification of the thapsigargin-evoked increase in the cytosolic free Ca2+ concentration by acetylcholine in acutely isolated mouse submandibular acinar cells

1996 ◽  
Vol 317 (3) ◽  
pp. 779-783 ◽  
Author(s):  
Peter. M. SMITH ◽  
Helen. E. REED
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Acinar Cells ◽  
Fold Increase ◽  
Extrusion Process ◽  
Significant Alteration ◽  
Atpase Inhibitor ◽  
Fura 2

The intracellular Ca2+ concentration was measured in single, acutely isolated, mouse submandibular acinar cells loaded with fura-2 AM. All experiments were performed in the absence of extracellular Ca2+ in order to eliminate Ca2+ influx. The microsomal ATPase inhibitor, thapsigargin, was used to release Ca2+ from intracellular stores and simultaneously prevent re-uptake into the stores. Sequential application of thapsigargin (2 μM) and the Ca2+ ionophore ionomycin (500 nM) indicated that thapsigargin was able to mobilize practically all intracellular Ca2+. Furthermore, in comparison with results obtained following inhibition of the plasma membrane Ca2+-ATPase by La3+ (2 mM), it may be shown that slowly unloading the intracellular Ca2+ stores using thapsigargin does not normally cause a massive, cytotoxic, increase in the cytosolic Ca2+ concentration, because Ca2+ is rapidly extruded from the cell across the plasma membrane. Application of a submaximal dose of acetylcholine (500 nM) during the rising phase of the response to thapsigargin caused a 3–4-fold increase in the amplitude of the rise in the cytosolic Ca2+ concentration without any significant alteration of the time course of the response. As thapsigargin alone is capable of mobilizing all releasable Ca2+, this increase in amplitude is most likely the result of inhibition of the Ca2+ extrusion process by acetylcholine.

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