Receptor-mediated phagocytosis of zymosan is unaffected by some conditions which reduce macrophage lysosomal enzyme secretion

1983 ◽  
Vol 3 (11) ◽  
pp. 1053-1061 ◽  
Author(s):  
Judith L. Bodmer ◽  
Roger T. Dean
Keyword(s):  
Cell Line ◽  
Lysosomal Enzyme ◽  
Secretory Pathway ◽  
Mannose Receptor ◽  
Enzyme Secretion ◽  
Surface Binding ◽  
Previous Suggestion ◽  
Mannose 6 Phosphate

We have previously shown that several agents which interfere with binding of Iigands to the mannose-glycoprotein receptor on macrophages can inhibit zymosan-induced lysosomal enzyme secretion. Here we show that mannose only reduces the association of zymosan with macrophages during the first hour of exposure; after longer periods of uptake no effect is detectable. We have previously shown that mannose reduces surface binding of zymosan, probably by interfering selectively with binding to the mannose receptor. The present inhibition of association of zymosan with macrophages during short exposures can be entirely explained by this reduction of binding. Macrophages must therefore internalize zymosan at sites in addition to the mannose receptor. In contrast to macrophages the routine macrophage-like cell line P388D1 is lacking the mannose-glycoprotein receptor. Accordingly we find that binding of zymosan to P388D1 is much slighter than to macrophages and is unaffected by mannose or mannose-6-phosphate. The spontaneous lysosomal enzyme secretion of P388D1 is also unaffected by mannose. The data on macrophages confirm our previous suggestion that agents interfering with the mannose receptor inhibit the induction of lysosomal enzyme secretion by acting directly on the receptor. The data on P388D1 ceils support this assertion by excluding effects at later steps in the secretory pathway.

scholarly journals Spontaneous lysosomal enzyme secretion by a murine macrophage-like cell line

1980 ◽  
Vol 190 (3) ◽  
pp. 847-850 ◽  
Author(s):  
W Jessup ◽  
R T Dean
Keyword(s):  
Cell Line ◽  
Lysosomal Enzyme ◽  
Peritoneal Macrophages ◽  
Enzyme Secretion ◽  
Murine Macrophage ◽  
Lysosomal Hydrolase

Lysosomal enzyme secretion by the murine macrophage-like cell line, P388D1, was compared with that of normal peritoneal macrophages. Unlike macrophages, lysosomal hydrolase secretion by P388D1 cells occurred spontaneously in vitro and was not further stimulated by the presentation of inflammatory agents such as zymosan and asbestos.

scholarly journals Toxicity Induced by Cytokines, Glucose, and Lipids Increase Apoptosis and Hamper Insulin Secretion in the 1.1E7 Beta Cell-Line

2021 ◽  
Vol 22 (5) ◽  
pp. 2559
Author(s):  
Antonia Diaz-Ganete ◽  
Aranzazu Quiroga-de-Castro ◽  
Rosa M. Mateos ◽  
Francisco Medina ◽  
Carmen Segundo ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
High Glucose ◽  
Secretory Pathway ◽  
Early Stage ◽  
Hybrid Cell ◽  
Basic Research ◽  
Stress Markers ◽  
Beta Cell Line ◽  
Starting Point

Basic research on types 1 and 2 diabetes mellitus require early stage studies using beta cells or cell lines, ideally of human origin and with preserved insulin secretion in response to glucose. The 1.1E7 cells are a hybrid cell line resulting from the electrofusion of dispersed human islets and PANC-1 cells, capable of secreting insulin in response to glucose, but their survival and function under toxic conditions remains untested. This characterization is the purpose of the present study. We treated these cells with a cytokine mix, high glucose, palmitate, and the latter two combined. Under these conditions, we measured cell viability and apoptosis (MTT, Caspase Glo and TUNEL assays, as well as caspase-8 and -9 levels by Western blotting), endoplasmic reticulum stress markers (EIF2AK3, HSPA4, EIF2a, and HSPA5) by real-time PCR, and insulin secretion with a glucose challenge. All of these stimuli (i) induce apoptosis and ER stress markers expression, (ii) reduce mRNA amounts of 2–5 components of genes involved in the insulin secretory pathway, and (iii) abrogate the insulin release capability of 1.1E7 cells in response to glucose. The most pronounced effects were observed with cytokines and with palmitate and high glucose combined. This characterization may well serve as the starting point for those choosing this cell line for future basic research on certain aspects of diabetes.

The lysosomal protease cathepsin D is efficiently sorted to and secreted from regulated secretory compartments in the rat basophilic/mast cell line RBL

2000 ◽  
Vol 113 (18) ◽  
pp. 3289-3298 ◽  
Author(s):  
A. Dragonetti ◽  
M. Baldassarre ◽  
R. Castino ◽  
M. Demoz ◽  
A. Luini ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Cell Line ◽  
Cathepsin D ◽  
Secretory Granules ◽  
Bioactive Molecules ◽  
Blue Staining ◽  
Lysosomal Protease ◽  
Mannose 6 Phosphate ◽  
Mast Cell Line

Basophils and mast cells contain a peculiar class of inflammatory granules that discharge their content upon antigen-mediated crosslinking of IgE-membrane receptors. The pathways for granule biogenesis and exocytosis in these cells are still largely obscure. In this study we employed the rat basophilic leukemia (RBL)/mast cell line to verify the hypothesis that inflammatory granules share common bioactive molecules and functional properties with lysosomes. We demonstrate that inflammatory granules, as identified by the monoclonal 5G10 antibody (which recognises an integral membrane protein) or by Toluidine Blue staining, have an intralumenal acidic pH, possess lysosomal enzymes and are accessible by fluid-phase and membrane endocytosis markers. In addition, we studied the targeting, subcellular localisation and regulated secretion of the lysosomal aspartic protease cathepsin D (CD) as affected by IgE receptor stimulation in order to obtain information on the pathways for granule biogenesis and exocytosis. Stimulation with DNP-BSA of specific IgE-primed RBL cells led to a prompt release of processed forms of CD, along with other mature lysosomal hydrolases. This release could be prevented by addition of EGTA, indicating that it was dependent on extracellular calcium influx. Antigen stimulation also induced exocytosis of immature CD forms accumulated by ammonium chloride, suggesting the existence of an intermediate station in the pathway for granule biogenesis still sensitive to regulated exocytosis. The targeting of molecules to secretory granules may occur via either a mannose-6-phosphate-dependent or mannose-6-phosphate-independent pathway. We conclude that endosomes and lysosomes in basophils/mast cells can act as regulated secretory granules or actually identify with them.

Abstract P611: Immortalization of Sheep Proximal Tubule Cells Retain the Ability to Internalize Angiotensinogen that Traffics to the Mitochondria and Nucleus

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Nildris Cruz-Diaz ◽  
Yixin Su ◽  
James C Rose ◽  
Bryan A Wilson ◽  
Mark C Chappell
Keyword(s):  
Cell Line ◽  
Time Course ◽  
Secretory Pathway ◽  
Protein A ◽  
Renin Angiotensin System ◽  
Subcellular Fractionation ◽  
Cell System ◽  
Ang Ii ◽  
Mitochondrial Fractions ◽  
Intracellular Expression

Although there is compelling evidence for an intracellular renin-angiotensin system (RAS) that includes localization of AT1, AT2 and AT7/Mas receptors (R) on the nucleus and mitochondria of various cell types, the mechanism for the intracellular expression of angiotensins remains equivocal as the precursor protein angiotensinogen (Aogen) enters the secretory pathway upon synthesis. Proximal tubules (PTs) of the kidney present a unique cell system since the PTs internalize Aogen and transgenic mice lacking either the PT protein transporter megalin or liver Aogen exhibit reduced renal content of both Aogen and Ang II. We reported that isolated sheep PTs readily internalize Aogen, and subcellular fractionation revealed that Aogen was evident in the nuclear and mitochondrial fractions. The present study sought to establish a permanent cell line derived from the sheep PT to facilitate the characterization of Aogen internalization and processing. Sheep PT cells were isolated by protease digestion and Percoll density gradient separation, maintained in culture to promote epithelial cell growth and immortalized by SV-40 transfection. A clone (SPT-1) was obtained that expressed the SGLT-2 protein, a selective PT marker. SPT-1 cells were incubated with recombinant 125 I-Aogen at 37°C in DMEM/F12 media. A time course [0.5 to 6 hrs] revealed linear uptake of Aogen [r = 0.995] that did not saturate by 6 hrs. Pre-treatment of the SPT-1 cells with renin/ACE/neprilysin/chymase inhibitors [INHIB] or AT1R/AT2R/AT7/MasR antagonists [ANTAG] failed to attenuate Aogen internalization [Control: 209 ± 22; INHIB: 200 ± 21; ANTAG: 217 ± 15 fmol/hr/mg, n=3] while Ang II or Ang-(1-7) [10 μM, each] also did not inhibit, but tended to increase Aogen uptake [238 ± 24 and 244 ± 15 fmol/hr/mg, respectively, n=3]. Subcellular fractionation studies revealed that 12.0 ± 0.2% [n=3] of the total internalized Aogen was localized to the mitochondrial fraction with a higher content in the nucleus following an 18 hr uptake. We conclude that the established SPT-1 cell line which retains the capacity to internalize Aogen and expresses a similar pattern of protein trafficking to isolated PTs, may constitute a relevant model to elucidate the pathway for intracellular expression of angiotensins.

Microtubule modulation of biliary excretion of endogenous and exogenous hepatic lysosomal constituents

1984 ◽  
Vol 246 (1) ◽  
pp. G8-G15 ◽  
Author(s):  
R. B. Sewell ◽  
S. S. Barham ◽  
A. R. Zinsmeister ◽  
N. F. LaRusso
Keyword(s):  
Lysosomal Enzyme ◽  
Biliary Excretion ◽  
Lysosomal Enzymes ◽  
Male Rats ◽  
Enzyme Secretion ◽  
Initial Increase ◽  
Acute Administration ◽  
Subsequent Decrease ◽  
Before And After

We tested the hypothesis that hepatocyte microtubules modulate the biliary excretion of endogenous and exogenous constituents of hepatocyte lysosomes. We collected bile via bile fistulas from male rats before and after acute administration of colchicine and vinblastine, agents known to bind to hepatocyte microtubules; rats were then killed and livers were homogenized for biochemical analyses or processed for electron microscopy. Colchicine caused biphasic, parallel alterations in the biliary excretion of three lysosomal enzymes compared with control rats given saline or lumicolchicine; a peak rise in enzyme outputs of approximately 175% at 45-60 min after colchicine administration was followed by a sustained fall to approximately 25% of control values, which persisted for 2-4 h. When hepatocyte lysosomes were prelabeled in vivo by administration of [3H]Triton WR-1339, a nonionic detergent that is sequestered in hepatic lysosomes, the biliary excretion of radiolabel in response to colchicine paralleled the biliary excretion of the three lysosomal enzymes. Vinblastine also induced a biphasic response in biliary lysosomal enzyme output that was similar to that produced by colchicine administration. Morphometric analysis of electron micrographs of rat livers demonstrated changes in the number of lysosomelike vesicles in the vicinity of bile canaliculi after colchicine and vinblastine administration; the initial increase in lysosomal enzyme secretion was associated with a significant decrease in the number of pericanalicular lysosomes after both agents, while the subsequent decrease in enzyme secretion coincided with an increase in the number of pericanalicular lysosomes after vinblastine.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Impaired secretion of mutant alpha 2-plasmin inhibitor (alpha 2 PI- Nara) from COS-7 and HepG2 cells: molecular and cellular basis for hereditary deficiency of alpha 2-plasmin inhibitor

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1092-1096 ◽  
Author(s):  
O Miura ◽  
N Aoki
Keyword(s):  
Cell Line ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Hepatoma Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Secretory Process ◽  
Hepatoma Cell Line ◽  
Pi Deficiency ◽  
Plasmin Inhibitor

Abstract The elongated mutant of alpha 2-plasmin inhibitor (alpha 2 PI) designated as alpha 2 PI-Nara is caused by a frameshift mutation found near the 3′ end of the coding region of the alpha 2 PI gene. To elucidate the mechanism by which this molecular abnormality leads to alpha 2 PI deficiency in plasma, we transfected an expression plasmid for alpha 2 PI-Nara into a monkey kidney cell line COS-7 or human hepatoma cell line HepG2 synthesizing alpha 2 PI, and analyzed the secretory process of the expressed alpha 2 PI-Nara by radioimmunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. The results obtained showed that the recombinant alpha 2 PI-Nara was retained within the cells for prolonged periods as an endoglycosidase H- sensitive precursor form, and only a small portion of the recombinant protein was secreted into the medium as a neuraminidase-sensitive mature form. These results suggest that instead of being secreted from the cells, most of the alpha 2 PI-Nara undergoes degradation within the cells while its transport is retarded in the intracellular secretory pathway; thus, alpha 2 PI-Nara should lead to the alpha 2 PI deficiency primarily by causing a block in the intracellular transport from the endoplasmic reticulum to the Golgi complex.

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