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+/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.

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.

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 pH-dependent Cargo Sorting from the Golgi

2011 ◽  
Vol 286 (12) ◽  
pp. 10058-10065 ◽  
Author(s):  
Chunjuan Huang ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Atpase Activity ◽  
Secretory Pathway ◽  
Ph Dependence ◽  
Glucose Deprivation ◽  
Ph Homeostasis ◽  
Plasma Membrane Proteins ◽  
Cytosolic Ph ◽  
Cargo Sorting

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.

scholarly journals Lysosomal Fusion Is Essential for the Retention of Trypanosoma cruzi Inside Host Cells

2004 ◽  
Vol 200 (9) ◽  
pp. 1135-1143 ◽  
Author(s):  
Luciana O. Andrade ◽  
Norma W. Andrews
Keyword(s):  
Plasma Membrane ◽  
Life Cycle ◽  
Trypanosoma Cruzi ◽  
Parasitophorous Vacuole ◽  
Cell Types ◽  
Significant Fraction ◽  
Host Cells ◽  
Productive Infection ◽  
Kinase Inhibition

Trypomastigotes, the highly motile infective forms of Trypanosoma cruzi, are capable of infecting several cell types. Invasion occurs either by direct recruitment and fusion of lysosomes at the plasma membrane, or through invagination of the plasma membrane followed by intracellular fusion with lysosomes. The lysosome-like parasitophorous vacuole is subsequently disrupted, releasing the parasites for replication in the cytosol. The role of this early residence within lysosomes in the intracellular cycle of T. cruzi has remained unclear. For several other cytosolic pathogens, survival inside host cells depends on an early escape from phagosomes before lysosomal fusion. Here, we show that when lysosome-mediated T. cruzi invasion is blocked through phosophoinositide 3-kinase inhibition, a significant fraction of the internalized parasites are not subsequently retained inside host cells for a productive infection. A direct correlation was observed between the lysosomal fusion rates after invasion and the intracellular retention of trypomastigotes. Thus, formation of a parasitophorous vacuole with lysosomal properties is essential for preventing these highly motile parasites from exiting host cells and for allowing completion of the intracellular life cycle.

scholarly journals Detachment of cultured cells from the substratum induced by the neutrophil-derived oxidant NH2Cl: synergistic role of phosphotyrosine and intracellular Ca2+ concentration.

1995 ◽  
Vol 131 (2) ◽  
pp. 509-524 ◽  
Author(s):  
T Y Nakamura ◽  
I Yamamoto ◽  
H Nishitani ◽  
T Matozaki ◽  
T Suzuki ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Cultured Cells ◽  
Tyrosine Phosphatase ◽  
Cell Types ◽  
Human Neutrophils ◽  
Atpase Inhibitor ◽  
Culture Dish ◽  
Protein Tyrosine Phosphatase Inhibitor ◽  
Peeling Off

The neutrophil-derived, membrane-permeating oxidant, NH2Cl, (but not the non-membrane-permeating chloramine, taurine-NHCl) induced detachment of fetal mouse cardiac myocytes and other cell types (fibroblasts, epithelial cells, and endothelial cells) from the culture dish, concomitant with cell shrinkage ("peeling off"). Stimulated human neutrophils also induced peeling off of cultured mouse cardiac myocytes when the latter were pretreated with inhibitors of .OH and elastase. Immunofluorescence microscopy revealed that the NH2Cl-induced peeling off of WI-38 fibroblasts is accompanied by disorganization of integrin alpha 5 beta 1, vinculin, stress fibers, and phosphotyrosine (p-Tyr)-containing proteins. Decrease in the content of the p-Tyr-containing proteins of the NH2Cl-treated cells was analyzed by immunoblotting techniques. Coating of fibronectin on the culture dish prevented both NH2Cl-induced peeling off and a decrease in p-Tyr content. Preincubation with a protein-tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4), also prevented NH2Cl-induced peeling off, suggesting that dephosphorylation of p-Tyr is necessary for peeling off. NH2Cl-induced peeling off was accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiac myocytes and WI-38 fibroblasts. The absence of extracellular Ca2+ prevented both NH2Cl-induced peeling off and increased [Ca2+]i, both of which did occur on subsequent incubation of the cells in Ca2+-containing medium. These observations suggest that an increase in [Ca2+]i is also necessary for peeling off. Depletion of microsomal and cytosolic Ca2+ by incubation with the microsomal Ca2+-ATPase inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ) plus EGTA prevented both NH2Cl-induced increases in [Ca2+]i and peeling off. Direct inhibition of microsomal Ca2+ pump activity by NH2Cl may participate in the NH2Cl-induced [Ca2+]i increment. A combination of p-Tyr dephosphorylation by genistein (an inhibitor of tyrosine kinase) and an increase in [Ca2+]i by BHQ could also induce peeling off. All these observations suggest a synergism between p-Tyr dephosphorylation and increased [Ca2+]i in NH2Cl-induced peeling off.

scholarly journals Studies on the mechanism of phagocytosis. I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane.

1975 ◽  
Vol 142 (5) ◽  
pp. 1263-1282 ◽  
Author(s):  
F M Griffin ◽  
J A Griffin ◽  
J E Leider ◽  
S C Silverstein
Keyword(s):  
Plasma Membrane ◽  
Sheep Erythrocyte ◽  
Peritoneal Macrophages ◽  
Membrane Receptors ◽  
Macrophage Receptors ◽  
Specific Receptors ◽  
Particle Ingestion ◽  
Plasma Membrane Receptors ◽  
Mouse Peritoneal Macrophages

These experiments were designed to evaluate the role of macrophage plasma membrane receptors for the third component of complement (C) and for the Fc portion of IgG in the ingestion phase of phagocytosis. Sheep erythrocyte (E) were coated with anti-E IgG [E(IgG)]; these E(IgG) were then attached to cultivated monolayers of mouse peritoneal macrophages under conditions which reversibly inhibit ingestion of E(IgG). The E(IgG)-macrophage complexes were further incubated under similar conditions with an antimacrophage IgG fraction which blocks Fc receptor-mediated ingestion but has no effect upon ingestion mediated by other phagocytic receptors. When these cultures were subsequently incubated under conditions optimal for particle ingestion, phagocytosis of the IgG-coated erythrocytes did not occur; the erythrocytes remained bound to the Fc receptors of the macrophage plasma membrane. To determine whether ligands must cover the entire surface of an attached particle to permit ingestion of that particle, C-coated E [E(IgM)C] were bound to the C receptors of thioglycollate-induced (activated) macrophages at 4 degrees C. E(IgM)C-macrophage complexes were then trypsinized at 4 degrees C, a procedure which resulted in cleavage of erythrocyte-bound C3b molecules to a form of C3 not recognized by the macrophage receptors for C3b. Under the conditions used, trypsin did not affect the attachment of E(IgM)C to the macrophage surface or the macrophage receptors for C3b. When these trypsin treated E(IgM)C-macrophage complexes were incubated at 37 degrees C, the bound E(IgM)C were not ingested; the erythrocytes remained attached to the macrophage plasma membrane via the macrophage's C receptors. These results indicate that attachment of a particle to specific receptors on the macrophage plasma membrane is not sufficient to trigger ingestion of that particle. Rather, ingestion requires the sequential, circumferential interaction of particle-bound ligands with specific plasma membrane receptors not involved in the initial attachment process.

scholarly journals Down-regulation of mannosyl receptor-mediated endocytosis and antigen F4/80 in bacillus calmette-guerin-activated mouse macrophages. Role of T lymphocytes and lymphokines

1982 ◽  
Vol 155 (6) ◽  
pp. 1623-1637 ◽  
Author(s):  
AB Ezekowitz ◽  
S Gordon
Keyword(s):  
Plasma Membrane ◽  
Single Cell Analysis ◽  
Target Population ◽  
Peritoneal Macrophages ◽  
Membrane Receptors ◽  
Lymphocyte Function ◽  
Bacillus Calmette Guerin ◽  
Down Regulation ◽  
Receptor Mediated Endocytosis

Bacillus Calmette-Guerin (BCG) infection alters the surface and endocytic properties of mouse peritoneal macrophages (PM) compared with thioglycollate- elicited (TPM) or resident PM (RPM). Expression of Ia antigen (Ag) is enhanced up to fourfold, but plasma membrane receptors that mediate binding and uptake of mannosyl/fucosyl-terminated glycoconjugates (MFR), Fc receptors, and the macrophage (mφ)-specific Ag F4/80 are reduced by 50-80 percent. Levels of Mac-1 remain relatively stable. These changes are accompanied by enhanced secretion of O(2)(-), after further stimulation with phorbyl myristate acetate, and of plasminogen activator. Both these products are released by TPM, but not RPM. The characteristic surface phenotype of BCG-PM can also be induced by injection of C. parvum, another mφ- activating agent, but not by thioglycollate broth, lipopolysaccharide, or proteose peptone. Purified protein derivative (PPD) and N-acetylmuramyl-L- alanyl-D-isoglutamine. 2H(2)0 are soluble agents with partial activity. Alteration of mφ markers by BCG infection depends on T lymphocyte function, although studies with nude mice indicate that other pathways may also serve to modify the surface of the mφ. Mφ from uninfected animals displayed all markers of activation after adoptive transfer of specifically-sensitised lymphocytes with PPD, intraperitoneally, or after co- cultivation. Treatment of primed lymphocytes with anti-Thy-1 antibody and complement ablated this effect. Lymphokines obtaned by Ag or mitogen stimulation induced similar changes in TPM and RPM. Mannose-specific endocytosis decayed rapidly, time 1/2 approximately equal to 16 h and stabilized at approximately 25 percent of control values. Single-cell analysis showed that residual MFR activity was uniform in the target population. Loss of Ag F4/80 after activation by lymphocyte and PPD was less marked than after infection (35 percent vs 80 percent), unlike MFR activity, which declined to a similar extent. Induction of mφ Ia by lymphokine reached a peak after 2-3 d and was lost within 2 d of its removal. Recovery of MFR and F4/80 was incomplete under these conditions. These studies establish that activated mφ known to display enhanced antimicrobial/anticellular activity express markedly different surface properties distinct from elicited or resident cells. The role of antigen- stimulated T cell products in regulating mφ function is confirmed, and down-regulation of mannosyl-receptor-mediated endocytosis provides a sensitive, quantitative, and cell-specific new marker to study their properties and mechanism of action. Extensive, but selective remodeling of mφ plasma membrane structure could play an important role in controlling recognition and effector mechanisms of the activated mφ.

Sign in / Sign up

Export Citation Format

Share Document