scholarly journals Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages.

1981 ◽  
Vol 90 (3) ◽  
pp. 665-669 ◽  
Author(s):  
B Poole ◽  
S Ohkuma
Keyword(s):  
Fluorescence Probe ◽  
Spectral Characteristics ◽  
Active Form ◽  
Peritoneal Macrophages ◽  
Weak Bases ◽  
Wide Range ◽  
A Minor ◽  
Energy Dependent ◽  
Mouse Peritoneal Macrophages ◽  
In Cells

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.

scholarly journals THE INTERACTION OF SOLUBLE HORSERADISH PEROXIDASE WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

1972 ◽  
Vol 55 (1) ◽  
pp. 186-204 ◽  
Author(s):  
Ralph M. Steinman ◽  
Zanvil A. Cohn
Keyword(s):  
Horseradish Peroxidase ◽  
Culture Medium ◽  
Peritoneal Macrophages ◽  
Wide Range ◽  
Mouse Macrophages ◽  
Secondary Lysosomes ◽  
Endocytic Vesicles ◽  
In Vitro Interaction ◽  
Mouse Peritoneal Macrophages

The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and cytochemical techniques. The compartmentalization of HRP in extracellular and intracellular sites has been quantitatively evaluated. A significant fraction is bound to a serum-derived layer, which coats the surface of culture vessels and may be removed by appropriate washes. Macrophages interiorize HRP as a solute in pinocytic vesicles without appreciable binding of the glycoprotein to the plasma membrane. Uptake is directly proportional to the concentration of HRP in the culture medium. 1 x 106 cells ingest 0.0025% of the administered load per hr over a wide range of concentrations. Cytochemically, all demonstrable HRP is sequestered within the endocytic vesicles and secondary lysosomes of the vacuolar apparatus. After uptake, the enzymatic activity of HRP is inactivated exponentially with a half-life of 7–9 hr, until enzyme is no longer detectable. When macrophages have pinocytosed trace-labeled HRP-125I, cell-associated isotope disappears with a t ½ of 20–30 hr and they release monoiodotyrosine-125I into the culture medium. We were unable to obtain evidence that significant amounts of HRP (>2%) can be exocytosed after uptake, can exist intact on the cell surface, or can be digested extracellularly. It is difficult to reconcile these observations with several of the postulated mechanisms whereby macrophages are thought to play a prominent role in the induction of an immune response.

scholarly journals Tubular lysosomes accompany stimulated pinocytosis in macrophages.

1987 ◽  
Vol 104 (5) ◽  
pp. 1217-1222 ◽  
Author(s):  
J Swanson ◽  
E Burke ◽  
S C Silverstein
Keyword(s):  
Phorbol Ester ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Peritoneal Macrophages ◽  
Latex Beads ◽  
Solute Accumulation ◽  
Cytoplasmic Microtubules ◽  
Macrophage Stimulation ◽  
Mouse Peritoneal Macrophages ◽  
In Cells

A network of tubular lysosomes extends through the cytoplasm of J774.2 macrophages and phorbol ester-treated mouse peritoneal macrophages. The presence of this network is dependent upon the integrity of cytoplasmic microtubules and correlates with high cellular rates of accumulation of Lucifer Yellow (LY), a marker of fluid phase pinocytosis. We tested the hypothesis that the efficiency of LY transfer between the pinosomal and lysosomal compartments is increased in the presence of tubular lysosomes by asking how conditions that deplete the tubular lysosome network affect pinocytic accumulation of LY. Tubular lysosomes were disassembled in cells treated with microtubule-depolymerizing drugs or in cells that had phagocytosed latex beads. In unstimulated peritoneal macrophages, which normally contain few tubular lysosomes and which exhibit relatively inefficient transfer of pinocytosed LY to lysosomes, such treatments had little effect on pinocytosis. However, in J774 macrophages and phorbol ester-stimulated peritoneal macrophages, these treatments markedly reduced the efficiency of pinocytic accumulation of LY. We conclude that a basal level of solute accumulation via pinocytosis proceeds independently of the tubular lysosomes, and that an extended tubular lysosomal network contributes to the elevated rates of solute accumulation that accompany macrophage stimulation. Moreover, we suggest that the transformed mouse macrophage cell line J774 exhibits this stimulated pinocytosis constitutively.

scholarly journals Metalloproteinases secretion from cultured cells: Differentiation by detergents

2018 ◽  
Vol 72 ◽  
pp. 1132-1137
Author(s):  
Rafał Seredyński ◽  
Katarzyna Hotowy ◽  
Elżbieta Czapińska ◽  
Nicole Nowak ◽  
Ewa Terlecka ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cultured Cells ◽  
Intracellular Localization ◽  
Active Form ◽  
Dermal Fibroblasts ◽  
Fluorimetric Assay ◽  
Enzyme Distribution ◽  
A Minor ◽  
Triton X ◽  
In Cells

Aim: Matrix metalloproteinases, particularly MMP-2 and MMP-9, are the active factors influencing the structure and properties of the extracellular matrix. This effect is enhanced in metastasis. Hence, it is necessary to enrich the knowledge of the relation between the accumulation and distribution of the enzymes in cells and the intensity of their secretion. Material/Methods: The study was conducted on three cell lines of dermal origin: normal line of human dermal fibroblasts (NHDF) and two melanoma lines (BM and B16F10). The results were obtained with substrate zymography, immunofluorescence microscopy and detergent-complemented fluorimetric assay. Results: All the studied cell lines revealed relatively rich content of MMP-2 (latent and active) with random intracellular localization. Nevertheless, the enzyme secretion was differentiated in various types of cells. The most intensive proMMP-2 secretion was obtained for NHDF, relatively lower for BM, and the lowest for B16F10 line. Zymographic detection of the active form of MMP-2 was restricted to NHDF and BM cell lines. On the other hand, differentiating usage of detergents Brij 35P and Triton X-100 evidenced an active fraction of MMP-2 present in cells of all studied lines. Triton X-100-generated lysis of cells of high MMP-2 secretion (NHDF, BM) revealed the presence of intracellular inhibitors. Conclusions: From the obtained results it can be concluded that the active form of MMP-2 is localized pericellularly in studied cells, being a minor fraction of a total amount of cellular MMP-2. The rest of the enzyme content is located deeper in cell cytoplasm in a latent form. The activity of the pericellular fraction of the enzyme can be measured with detergent-complemented fluorimetric assay, constituting a potentially useful tool for evaluating the enzyme distribution.

scholarly journals Cytoplasmic vacuolation of mouse peritoneal macrophages and the uptake into lysosomes of weakly basic substances.

1981 ◽  
Vol 90 (3) ◽  
pp. 656-664 ◽  
Author(s):  
S Ohkuma ◽  
B Poole
Keyword(s):  
Peritoneal Macrophages ◽  
Weak Base ◽  
Neutral Form ◽  
Osmotic Swelling ◽  
Cytoplasmic Vacuolation ◽  
Weak Bases ◽  
Protonated Forms ◽  
Basic Compounds ◽  
Mouse Peritoneal Macrophages ◽  
Basic Substances

With few exceptions, weakly basic compounds that are sufficiently lipophilic in their neutral forms and sufficiently hydrophilic in their protonated forms accumulate in lysosomes. When the concentration within the lysosomes becomes sufficiently high, osmotic swelling occurs. The cells than take on a vacuolated appearance. The concentrations at which different weak bases cause lysosomal vacuolation vary over almost three orders of magnitude. For any particular weak base, it is the concentration of the neutral form that determines the extent of uptake and the degree of vacuolation. Chloroquine is anomalous in that concentrations greater than approximately 30 microM cause less uptake and less vacuolation than do lower concentrations.

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