scholarly journals The lysosomotrope GPN mobilises Ca2+from acidic organelles

2021 ◽  
Vol 134 (6) ◽  
pp. jcs256578 ◽  
Author(s):  
Yu Yuan ◽  
Bethan S. Kilpatrick ◽  
Susanne Gerndt ◽  
Franz Bracher ◽  
Christian Grimm ◽  
...  
Keyword(s):  
Acute Treatment ◽  
Chronic Treatment ◽  
The Novel ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Cytosolic Ph ◽  
Lysosomal Ph ◽  
Lysosomotropic Agent ◽  
Acidic Organelles ◽  
Primary Action

ABSTRACTLysosomes are acidic Ca2+stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca2+stores. Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca2+signals in many cells. However, whether these signals are the result of a primary action on lysosomes is unclear in light of recent evidence showing that GPN mediates direct ER Ca2+release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca2+signals. NH4Cl evoked comparable changes in pH but failed to affect Ca2+. The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca2+signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN action. GPN blocked Ca2+responses evoked by the novel nicotinic acid adenine dinucleotide phosphate-like agonist, TPC2-A1-N. Therefore, GPN-evoked Ca2+signals were better correlated with associated pH changes in the lysosome compared to the cytosol, and were coupled to lysosomal Ca2+release. We conclude that Ca2+signals evoked by GPN most likely derive from acidic organelles.

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 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.

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.

scholarly journals Genetically encoded ratiometric biosensor for probing lysosomal pH in mammalian cells and C. elegans

2020 ◽  
Author(s):  
Marcus Y. Chin ◽  
Anand R. Patwardhan ◽  
Kean-Hooi Ang ◽  
Austin L. Wang ◽  
Carolina Alquezar ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Multicellular Organism ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
High Content Imaging ◽  
Lysosomal Ph ◽  
C Elegans ◽  
Cellular Models

ABSTRACTLysosomes are important sites for macromolecular degradation, defined by a profoundly acidic lumenal pH of ~4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein-based pH biosensor called FIRE-pHLy (Fluorescence Indicator REporting pH in Lysosomes). This sensor was targeted to lysosomes with LAMP1 and reported lumenal pH between 3.0 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content imaging. We expressed FIRE-pHLy in several cellular models as well as the multicellular organism C. elegans and quantified its alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust and versatile lysosomal pH biosensor that has broad applications for investigating pH dynamics in aging and lysosome-related diseases, as well as in lysosome-based drug discovery.

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 Glycoprotein metabolism in normal and β-mannosidase-deficient cultured goat skin fibroblasts

1986 ◽  
Vol 234 (1) ◽  
pp. 175-183 ◽  
Author(s):  
L W Hancock ◽  
M Z Jones ◽  
G Dawson
Keyword(s):  
Culture Medium ◽  
Human Fibroblasts ◽  
Skin Fibroblasts ◽  
Medium Ph ◽  
Cytosolic Ph ◽  
Catabolic Pathways ◽  
No Conversion ◽  
Lysosomal Ph ◽  
Cultured Skin ◽  
Oligosaccharide Structures

Cultured skin fibroblasts established from goats affected with beta-mannosidosis, an inherited neurovisceral storage disorder, showed an absence of lysosomal beta-mannosidase activity and the corresponding accumulation of a trisaccharide (TS) with the structure Man beta (1→4)GlcNAc beta (1→4)GlcNAc (0.4 mumol/g) and lesser amounts (0.15 mumol/g) of a Man beta (1→4)GlcNAc disaccharide (DS). By using purified storage TS isolated from fibroblasts metabolically labelled with [3H]GlcN, no conversion of TS into DS could be demonstrated in homogenates of affected cells at either lysosomal pH (4.4) or cytosolic pH (6.1), or in the culture medium (pH 7.0) of affected cells. Both TS and DS were secreted into the culture medium by affected fibroblasts. When affected fibroblasts were treated with tunicamycin before labelling with [3H]GlcN, the accumulation of both labelled TS and DS was completely inhibited. Treatment of both affected and normal goat fibroblasts with swainsonine resulted in the inhibition of lysosomal alpha-mannosidase activity and in the accumulation of the same labelled oligosaccharides in both. The major storage pentasaccharide from both normal and affected swainsonine-treated fibroblasts was sensitive to digestion with alpha-mannosidase and endo-beta-N-acetylhexosaminidase D, suggesting a branched mannose structure and a chitobiose core. In the absence of evidence for the existence of unusual N-linked glycoprotein-associated chitotriose oligosaccharide structures in affected goat fibroblasts, it must be concluded that degradative pathways for N-linked oligosaccharides are similar in both normal and affected goat fibroblasts, and that these pathways differ from catabolic pathways in human fibroblasts.

P.3.d.002 Modulation of neurotrophic mechanisms after chronic treatment with the novel antipsychotic lurasidone in rats

2011 ◽  
Vol 21 ◽  
pp. S507
Author(s):  
A. Luoni ◽  
F. Fumagalli ◽  
F. Calabrese ◽  
M. Ogasa ◽  
G. Racagni ◽  
...  
Keyword(s):  
Chronic Treatment ◽  
The Novel ◽  
Novel Antipsychotic

Effect of tianeptine on neuroendocrine, enzyme and behavioral responses to restraint stress in male rats

1993 ◽  
Vol 8 (S2) ◽  
pp. 67s-73s ◽  
Author(s):  
R Fontanges ◽  
J Mimouni ◽  
X de Grieve ◽  
J Picard ◽  
M Pugeat ◽  
...  
Keyword(s):  
Open Field ◽  
Restraint Stress ◽  
Wistar Rats ◽  
Chronic Treatment ◽  
Single Injection ◽  
Behavioral Responses ◽  
Chronic Administration ◽  
Exploratory Activity ◽  
Male Rats ◽  
The Novel

SummaryThe effects of the novel antidepressant tianeptine, after acute or chronic administration, were compared in normal and restraint-stressed (30 min or 2 h) Wistar rats. Tianeptine, at the dose of 10 mg/kg, did not exert any effect in non-stressed rats. However, in animals restrained for 30 min, tianeptine reduced the increase of circulating ACTH and β-endorphin levels without modification of corticosterone. Moreover, it antagonized the deficit of vertical exploratory activity in an open field. In rats restrained for 2 hours, a single injection of tianeptine suppressed the stress-induced increase of TAT hepatic activity and moderately attenuated the deficit of activity in the open field. This effect was less marked and not statistically significant after chronic treatment.

Mesenteric vascular responses to vasopressin during development of DOCA-salt hypertension in male and female rats

1995 ◽  
Vol 268 (1) ◽  
pp. R40-R49 ◽  
Author(s):  
J. N. Stallone
Keyword(s):  
Sex Differences ◽  
Acute Treatment ◽  
Vascular Reactivity ◽  
Chronic Treatment ◽  
Female Rats ◽  
Salt Treatment ◽  
Male And Female Rats ◽  
Salt Hypertension ◽  
Vascular Responsiveness

Deoxycorticosterone acetate (DOCA)-salt hypertension develops to a greater extent in male (M) than in female (F) rats. To determine the role of the vasculature, reactivity to arginine vasopressin (AVP) and prostanoid output were examined in the isolated perfused mesenteric vasculature of hypertensive (HT) and normotensive-control (NTC) M and F rats after acute (1-wk) and chronic (4-wk) DOCA-salt treatment. Systolic blood pressure was significantly higher in M than in F HT rats (187 +/- 3 vs. 151 +/- 3 mmHg after 4 wk; P < 0.02). After acute treatment, vascular reactivity to AVP (maximal perfusion pressure) in HT was elevated in M (181 +/- 18 mmHg; P < 0.02) but not in F (135 +/- 6 mmHg) compared with NTC (90 +/- 6 mmHg, M vs. 119 +/- 5 mmHg, F). After chronic treatment, vascular reactivity to AVP in HT was elevated in both sexes (P < 0.02), although more in F (175 +/- 13 mmHg) than in M (141 +/- 11 mmHg). In contrast, vascular responsiveness to phenylephrine did not differ significantly between M and F NTC or HT preparations after either acute or chronic treatment. Sex differences in basal and AVP-induced 6-ketoprostaglandin (6-keto-PG) F1 alpha and PGE2 output by HT and NTC vasculature were reciprocal to sex differences in the vasoconstriction responses to AVP. After acute treatment, AVP-stimulated 6-keto-PGF1 alpha output by HT was elevated slightly in F (33.6 +/- 1.7 ng/3 min; P < or = 0.02) but not in M (49.9 +/- 4.3 ng/3 min) compared with NTC (23.5 +/- 2.6 ng/3 min, F vs. 34.7 +/- 4.9 ng/3 min, M). After chronic treatment, output by HT was enhanced in both sexes (P < or = to 0.02), although more in M (109 +/- 15.4 ng/3 min) than in F (68 +/- 6.6 ng/3 min)> These findings suggest that sex differences in the relative balance between AVP-induced vasoconstriction and vasodilatory prostanoid release may contribute to male-female differences in mesenteric vascular reactivity to AVP in NT and that disturbances in this balance may be responsible, at least in part, for the sex- and time-dependent changes in reactivity to AVP observed during the development of DOCA-salt hypertension.

Sign in / Sign up

Export Citation Format

Share Document