monensin and pepstatin

Previously we showed that the redox active Cu(2+) was much more effective than Cd(2+) at inducing reactive oxygen species ("ROS") formation in hepatocytes and furthermore "ROS" scavengers prevented Cu(2+)-induced hepatocyte cytotoxicity (Pourahmad and O'Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu(2+), but not Cd(2+), was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, "ROS" formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu(2+), but not Cd(2+), as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu(2+)-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu(2+)-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu(2+). Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu(2+)-induced hepatocyte cytotoxicity. It is proposed that Cu(2+)-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by "ROS" generated by lysosomal Cu(2+) redox cycling.

Topics: Acridine Orange; Adenine; Animals; Aurothioglucose; Cadmium; Cell Death; Chloroquine; Copper; Endopeptidases; Enzyme Activation; Gentamicins; Leupeptins; Lipid Peroxidation; Liver; Lysosomes; Male; Methylamines; Monensin; Oxidation-Reduction; Pepstatins; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

CPAE endothelial cells were cultured in the presence of pepstatin, NH4Cl, or chloroquine in order to assess their effects on the secretion of endothelin-1 (ET-1). The first of these is an inhibitor of aspartyl proteases and the last two are known to neutralize acidic intracellular compartments. The pepstatin was encapsulated into liposomes to aid in its uptake, and uptake was confirmed by measuring the residual aspartyl protease activity in washed, lysed cells. Pepstatin had no effect (less than 5%) on the secretion of ET-1, 25 mM NH4Cl decreased secretion by 30-47%, and 25 microM chloroquine increased secretion by 37-79%. In contrast, each of these reagents is known to inhibit lysosomal degradation of intracellular proteins by 75-90%. Additionally, big ET was shown to be a very poor substrate, in terms of kcat/Km values, for aspartyl proteases. The rate constants were less than 10(4) M-1 s-1, which is approximately 1% of the value for the best substrates. The data, therefore, do not support a role for aspartyl proteases in the formation of ET-1. Similar to chloroquine, 0.5 microM monensin increased the secretion of ET-1 by 40-60%. Both of these reagents have previously been shown to increase the rate of constitutive secretion of peptides by affecting their partitioning between packaging into storage granules and constitutive secretion. The results would therefore provide supportive evidence for the existence of a storage form of ET-1 in endothelial cells.

Topics: Ammonium Chloride; Aspartic Acid; Aspartic Acid Endopeptidases; Cells, Cultured; Chloroquine; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Endothelium; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Monensin; Pepstatins; Protein Precursors

We examined the role of proteolytic ligand modification in endosomal targeting using vitellogenin (VTG) uptake by Xenopus oocytes as a model system. Non-cleavable VTG is internalized, but does not appear in yolk platelets. We identified two inhibitors of VTG processing into the yolk proteins: the ionophore monensin and pepstatin A, a specific inhibitor of cathepsin D. Pepstatin neither affected ligand binding and internalization, nor inhibited the degradation of nonspecifically incorporated proteins, whereas monensin inhibited all of these processes. Inhibiting VTG processing prevented its deposition into yolk platelets by strongly interfering with endosome-yolk platelet fusion. Monensin treatment resulted in morphologically abnormal endosomes, while pepstatin only inhibited VTG cleavage and the subsequent fusion of endosomes with yolk platelets. Since VTG cleavage is initiated prior to its deposition in platelets, we postulate that ligand proteolysis could be necessary for normal endosomal targeting.

Topics: Animals; Cell Compartmentation; Egg Proteins; Endocytosis; Female; Membrane Fusion; Monensin; Oocytes; Organoids; Pepstatins; Peptide Hydrolases; Vitellogenins; Xenopus laevis

Rabbit alveolar macrophages rapidly internalize and degrade mannosylated bovine serum albumin (125I-mannose-BSA). Trichloroacetic acid-soluble degradation products appear in the cells as early as 6 min after uptake at 37 degrees C, and in the extracellular medium after 10 min. Incubation of endocytic vesicles containing this ligand in isotonic buffers at pH 7.4 + ATP resulted in intravesicular proteolysis, which was inhibited by monensin, nigericin, or ammonium chloride. At pH 5.0, degradation proceeded rapidly and was abolished by lysis of the vesicles with 0.1% Triton X-100. Readdition of lysosomes to the incubation mixture did not increase the rate of prelysosomal degradation. Proteolysis of 125I-mannose-BSA was optimal at pH 4.5, and inhibited by low concentrations of the cathepsin D inhibitor pepstatin A. After subcellular fractionation of the macrophages on Percoll gradients, 125I-mannose-BSA sedimented with prelysosomal vesicles and was not transported to secondary lysosomes. Addition of pepstatin A to extracellular medium during internalization of prebound 125I-mannose-BSA partially inhibited degradation of ligand, and resulted in transfer of undegraded 125I-mannose-BSA to lysosomes after 20 min. Using 125I-bovine serum albumin as a substrate for the protease in the presence of 0.1% Triton X-100, we have shown that as much as 36% of the total pepstatin A-sensitive activity sediments with nonlysosomal membranes. After intraendosomal iodination using lactoperoxidase, a labeled protease was isolated by affinity chromatography on pepstatin-agarose. The labeled protease, which had a subunit size of 46 kDa, was detected in endocytic vesicles after 5 min of internalization. These results suggest that a cathepsin D-like protease is responsible for the degradation of 125I-mannose-BSA in macrophages, and that this ligand is degraded in a prelysosomal vesicle.

Topics: Ammonium Chloride; Animals; Cathepsin D; Chromatography, Affinity; Endocytosis; Hydrogen-Ion Concentration; Ligands; Lysosomes; Macrophages; Mannose; Monensin; Nigericin; Pepstatins; Peptide Hydrolases; Protease Inhibitors; Pulmonary Alveoli; Rabbits; Serum Albumin; Serum Albumin, Bovine; Time Factors

The kinetics of cytotoxicity induced by ricin and a series of immunotoxins consisting of ricin A-chain coupled to antibodies against cell-surface antigens has been studied. The inhibition of protein synthesis in cells treated with immunotoxins or ricin occurs after a lag period. The rate of protein synthesis decreases according to a mono-exponential function, indicating a first-order process. With increasing concentration of immunotoxin, a maximal rate of inhibition is reached. The inactivation rate induced by immunotoxins was much slower than that achieved with ricin, even when products were compared on a basis of an identical number of molecules bound per cell, demonstrating the real higher efficacy of ricin. The time required to reduce protein synthesis by 90%, denoted T10, was 1.4-1.6 h with ricin, 60 h with anti-T65 immunotoxin on CEM human T leukemia cells (T65 positive), 65 h with anti-p97 immunotoxin on SK-MEL 28 human melanoma cells (p97 positive), and 20 h with an IgM anti-Thy 1.2 immunotoxin on WEHI-7 mouse T leukemia cells (Thy 1.2 positive). In this latter case, when the IgM antibody was replaced by an IgG anti-Thy 1.2, a 5-fold increase in the inactivation rate was obtained, demonstrating the importance of the binding moiety for the immunotoxins. Lysosomotropic amines such as ammonium chloride, chloroquine, and methylamine and carboxylic ionophores such as monensin, which are known to interfere with the uptake of certain macromolecules, strongly increased the rate of protein synthesis inhibition by all immunotoxins tested and increased 4-50,000-fold the sensitivity of cells to the immunotoxin. Enhancement in the inactivation rate was as much as 7-10-fold when either of these compounds was added, generating T10 values comparable to those of ricin.

Topics: Amines; Ammonium Chloride; Animals; Antigens, Surface; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Humans; Ionophores; Isoantibodies; Kinetics; Leukemia, Experimental; Leupeptins; Mice; Monensin; Nigericin; Pepstatins; Protein Biosynthesis; Ricin