monensin and methylamine

The presence of various genomic sequences in the pool of extracellular DNA was studied by cloning and sequencing the DNA eluted from the surface of HeLa cells. Sequences of 19 genes, 10 pseudogenes, and 41 repeated elements were found in 103 clones. Sequences of LINE repeats were found in 17% of the clones; consequently real-time PCR assay for quantification of LINE repeats was chosen to study the influence of protein transport inhibitors on the concentration of free and cell-surface-bound DNA in the cultures of HUVEC and HeLa cells. Treatment of both cell lines with the inhibitors did not interfere with the concentration of extracellular DNA in the growth medium, except for chloroquine, which doubled the concentration of extracellular DNA in HUVEC culture. The treatment of HUVECs with monensin, glyburide, or methylamine decreases the cell-surface-bound DNA concentration by 30, 35, and 19%, respectively. The incubation of HeLa cells with monensin reduces the concentration of cell-surface-bound DNA by 15%; however, the treatment with glyburide increases cell-surface-bound DNA concentration by 50%. The data obtained demonstrate the involvement of vesicular transport in generation of extracellular DNA.

Topics: Antirheumatic Agents; Base Sequence; Cell Line; Chloroquine; Cytochalasin B; DNA; Enzyme Inhibitors; Glyburide; Humans; Hypoglycemic Agents; Ionophores; Methylamines; Molecular Sequence Data; Monensin; Ouabain; Protein Transport; Sequence Analysis, DNA

There are an increasing number of studies reporting the presence of Hsps in human serum. We have investigated the release of Hsp70 into blood and culture medium from peripheral blood mononuclear cells (PBMCs), and whether this release is due to cell damage or active secretion from the cells. Intact Hsp70 was released from cells within whole blood and from purified PBMCs under normal culture conditions. Hsp70 release was rapid (0.1 ng/10(6) cells/h) over the first 2 h of culture and continued at a reduced rate up to 24 h (<0.025 ng/10(6) cells/h). Using viable cell counts and lactate dehydrogenase release we were able to confirm that the release of Hsp70 was not due to cellular damage. Hsp70 release was inhibited by monensin, methyl-beta-cyclodextrin, and methylamine, but not by brefeldin A. These data suggest that Hsp70 is released from cells via a non-classical pathway, possibly involving lysosomal lipid rafts.

Topics: B-Lymphocytes; beta-Cyclodextrins; Blotting, Western; Brefeldin A; Cell Survival; Culture Media; HSP70 Heat-Shock Proteins; Humans; L-Lactate Dehydrogenase; Leukocytes, Mononuclear; Lysosomes; Membrane Microdomains; Methylamines; Monensin; Protein Synthesis Inhibitors; RNA, Messenger; T-Lymphocytes; Temperature; Time Factors

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

Calpain was generally believed to exist and function only in the cytoplasm. However, m-calpain has now been detected in the extracellular spaces of some kinds of tissue. In this study, we demonstrated the existence of m-calpain in the medium surrounding MC3T3-E1 cultures, and its activity by zymography. At the same time, the amount of lactate dehydrogenase in medium of MC3T3-E1 culture was extremely low compared with other cell cultures, suggesting that m-calpain found in the culture medium of MC3T3-E1 cells originated mainly from active secretion. Moreover, the secretion of m-calpain was not blocked by brefeldin A, implying that m-calpain may be secreted by a nonclassical pathway. Recently, MC3T3-E1 has been reported to produce matrix vesicles and media vesicles, and we demonstrated m-calpain in these vesicles produced by MC3T3-E1 cultures. We therefore concluded that these vesicles are partly responsible for the secretion of m-calpain into the culture medium of MC3T3-E1 cells.

Topics: Animals; Brefeldin A; Calcium Channel Blockers; Calpain; Cell Division; Cell Line; Culture Media, Conditioned; Enzyme Precursors; Extracellular Matrix; Humans; Immunoblotting; Ionophores; Isoenzymes; L-Lactate Dehydrogenase; Methylamines; Mice; Monensin; Osteoblasts; Protein Synthesis Inhibitors; Secretory Vesicles; Verapamil

To clarify the function of the multidrug transporter P-glycoprotein in mast cells we used the green fluorescent compound Bodipy-FL-verapamil, which is a substrate of P-glycoprotein. This compound is also transported by Multidrug Resistance-related Protein (MRP), another membrane transport protein expressed in many tumour resistant cells as well as in normal cells. When rat peritoneal mast cells were incubated with Bodipy-verapamil, a rapid uptake of this compound was observed. Pretreatment with modulators of P-glycoprotein activity, such as verapamil and vinblastine, increased Bodipy-verapamil intracellular concentrations. In addition, Bodipy-verapamil efflux from these cells was rapid and also inhibited by verapamil and vinblastine. In contrast, no effect was observed when cells were treated with agents, such as probenecid and indomethacin, that are known inhibitors of MRP. Methylamine and monensin, substances that modify the pH values in the granules, were able to lower the concentrations of Bodipy-verapamil. Microscopical observations, conducted in both rat and beige mouse mast cells, demonstrated that the fluorochrome accumulated in the cytoplasmic secretory granules. RT-PCR performed on rat peritoneal mast cells revealed the presence of MDR1a and MDR1b mRNAs; on the contrary, MRP mRNA was not expressed. Mast cells were further treated with the fluorescent probe LysoSensor Blue, a weak base that becomes fluorescent when inside acidic organelles. This substance accumulated in mast cell granular structures and its fluorescence was reduced either by treatment with P-glycoprotein modulators or with agents that disrupt pH gradients. In conclusion, these data further confirm the presence of an active P-glycoprotein, but not of MRP, in rat peritoneal mast cells. These findings, coupled with previous ultrastructural data, lend further support to the assumption that this protein is located on the mast cell perigranular membrane. The functional role of P-glycoprotein in these cells is at present unclear, but a possible involvement in the transport of molecules from the granules to the cytosol can be hypothesized. Alternatively, this protein might be indirectly implicated in changes of pH values inside secretory granules.

Topics: Animals; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Calcium Channel Blockers; Drug Resistance, Multiple; Fluorescent Dyes; Indicators and Reagents; Ionophores; Mast Cells; Methylamines; Mice; Mice, Inbred Strains; Microscopy, Fluorescence; Monensin; Multidrug Resistance-Associated Proteins; Peritoneum; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Secretory Vesicles; Spectrometry, Fluorescence; Verapamil; Vinblastine

Conditions for the use of both [14C]methylamine and 5, 5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin bloodstream forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99-7.03, the pH of the mitochondrial matrix was 7.71-7.73, and the algebraic average pH of the various endosomal compartments was 5.19-5.50. Similar values were found when cells were respiring aerobically on glycerol. However, bloodstream forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0-7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Deltapsip, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Deltapsip. Oligomycin abolished the mitochondrial pH gradient (DeltapHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (DeltapHe). The pH gradient across the plasma membrane (DeltapHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (DeltaPp) of -52 mV and an inwardly directed sodium-motive force (DeltaNp) of -149 mV, whereas the mitochondrial inner membrane had only an inwardly directed DeltaPm of -195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed DeltaPl within the range + 89 to +110 mV, depending on the measurement method.

Topics: Animals; Antimalarials; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Chloroquine; Dimethadione; Dose-Response Relationship, Drug; Endosomes; Glycerol; Hydrogen-Ion Concentration; Ionophores; Kinetics; Membrane Potentials; Methylamines; Mitochondria; Monensin; Nystatin; Oligomycins; Potassium Chloride; Proton-Motive Force; Protons; Pyruvates; Sodium; Time Factors; Trypanosoma brucei brucei; Uncoupling Agents; Valinomycin; Water

The aggregability of rabbit platelets was studied under various cytoplasmic pHs (pHi). Nigericin, a K+/H+ ionophore, which can induce a decrease in pHi, at 2-10 microM in 2 min incubation reduced both platelet aggregation and an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) stimulated with thrombin or U46619. The reduced aggregability recovered 10 min after incubation with nigericin in parallel with an increase in pHi. In contrast, when pHi was increased by simultaneous addition of NH4Cl, methylamine or monensin, aggregation in response to a low concentration of thrombin, U46619, arachidonic acid or A23187 was enhanced significantly. The enhancing effect of NH4Cl was lowered by prolonged incubation with NH4Cl, by which the increased pHi was improved concomitantly. Indomethacin, an inhibitor of cyclooxygenase, failed to inhibit the enhancement of aggregation by NH4Cl under stimulation with U46619. In addition, treatment with NH4Cl enhanced an increase in [Ca2+]i in response to U46619 in a concentration-dependent manner, although the treatment by NH4Cl alone did not affect [Ca2+]i. When pHi was artificially altered during the ranges of 6.6-7.4 by treatment with nigericin in K+-rich medium, aggregation by low concentrations of thrombin was dependent on the pHi. These data indicate that pHi is an important factor for platelet activation including intracellular Ca2+ mobilization and aggregation.

Topics: Ammonium Chloride; Animals; Biological Transport; Blood Platelets; Calcium; Cytoplasm; Hydrogen-Ion Concentration; In Vitro Techniques; Ionophores; Methylamines; Monensin; Nigericin; Platelet Activation; Platelet Aggregation; Rabbits

The mechanism and the kinetics of rubella virus (RV) penetration into Vero cells were studied. By using pronase or acid treatment to inactivate virus which had adsorbed to cell membrane but had not been internalized, it was found that a period of 7 h was required in order for all of the adsorbed virus to enter the host cells. Lysosomotropic agents (monensin, methylamine, ammonium chloride and chloroquine) were used to study the mechanism by which RV penetrates host cells. Virus replication was inhibited if treatment of cells with these compounds was performed for at least 9 h after infection. However, if extracellular adsorbed virions were eliminated by acid treatment following removal of the lysosomotropic compounds, RV replication was completely inhibited by treatment with these drugs for any time period after adsorption. This indicated that the prolonged period of treatment with these compounds necessary to inhibit virus replication is due to the slow rate of RV internalization. None of the compounds had any effect on infection initiated by transfection of RV RNA, confirming that these drugs were exerting their inhibitory activity at penetration. The inhibition of RV replication by lysosomotropic compounds indicates that RV penetrates host cells by the endosomal pathway.

Topics: Ammonium Chloride; Animals; Chlorocebus aethiops; Chloroquine; Hydrogen-Ion Concentration; Methylamines; Monensin; Rubella virus; Transfection; Vero Cells

Surfactant protein C (SP-C) is a hydrophobic protein synthesized and secreted exclusively by alveolar type II cells through proteolysis of a 21-kDa propeptide (SP-C21) to produce the 3.7-kDa surface active form. Previous studies from this laboratory have demonstrated that early processing of proSP-C involves extensive intracellular proteolysis of the COOH terminus of proSP-C21 in subcellular compartments, which include the acidic type II cell-specific subcellular organelle, the lamellar body. (Beers, M. F., Kim, C. Y., Dodia, C., and Fisher, A. B.(1994) J. Biol. Chem. 269, 20318-20328). The role of intracellular pH gradients in SP-C processing was studied in freshly isolated rat type II cells. Using vital fluorescence microscopy, the pH indicator acridine orange (AO) identified intense fluorescence staining of acidic cytoplasmic vesicles within fresh type II cells. The AO vesicular staining pattern was similar in cells labeled with the lamellar body marker phosphine 3R and the phospholipid dye nile red. AO fluorescence was quenched by the addition of a membrane-permeable weak base, methylamine. Immunoprecipitation of cell lysates with anti-proSP-C antisera following pulse-chase labeling (0-2 h) with 35S-Translabel demonstrated rapid synthesis of 35S-proSP-C21 with a time-dependent appearance of 16- and 6-kDa intermediates (SP-C16 and SP-C6). Tricine polyacrylamide gel electrophoresis analysis of organic extracts of cell lysates showed time-dependent appearance of mature SP-C3.7. The addition of 5 mM methylamine significantly blocked the post-translational processing of proSP-C resulting in disruption of normal precursor-product relationships and inhibition of SP-C3.7 formation. Methylamine-treated cells exhibited slow accumulation of SP-C16 and SP-C6, a persistence of SP-C21, and an absence of SP-C3.7 for the duration of the chase period. The lysosomotropic agent chloroquine, the proton ionophore monensin, and bafilomycin A1, a specific vacuolar H+-ATPase inhibitor, each caused inhibition of proSP-C processing in a similar manner. These results demonstrate that normal post-translational proteolysis of proSP-C occurs in acidic intracellular compartments, which include the lamellar body, and that complete processing to SP-C3.7 is dependent upon maintenance of transmembrane pH gradients by a vacuolar H+-ATPase.

Topics: Animals; Anti-Bacterial Agents; Antibodies; Cells, Cultured; Chloroquine; Enzyme Inhibitors; Epitopes; Hydrogen-Ion Concentration; Ionophores; Kinetics; Lung; Macrolides; Methylamines; Models, Biological; Monensin; Protein Precursors; Protein Processing, Post-Translational; Proteolipids; Pulmonary Surfactants; Rats; Rats, Sprague-Dawley; Subcellular Fractions; Sulfur Radioisotopes; Time Factors; Vacuoles

Lung lamellar bodies, the storage organelles for lung surfactant phosphatidylcholine (PC), maintain an acidic pH that can be increased with weak bases. This study investigates the effect of a weak base, methylamine, on the pH in lamellar bodies and on the trafficking and packaging of newly synthesized PC in lamellar bodies. Methylamine increased the pH of isolated lung lamellar bodies and of lamellar bodies in intact cells. Metabolic labelling of isolated type II cells with [methyl-3H]choline showed that although methylamine (2.5-10 mM) did not alter the labelling of cellular or microsomal PC and disaturated PC, it decreased the labelling of the PC and disaturated PC in lamellar bodies. The packaging of PC in lamellar bodies (the specific activities ratio between the PC in lamellar bodies and the microsomal PC) also decreased in a time- and concentration-dependent manner. The cellular synthesis of PC or its packaging into lamellar bodies was unaltered by brefeldin A, suggesting that the Golgi was not involved in PC packaging. Although methylamine also increased surfactant secretion, the inhibition of PC packaging in lamellar bodies seems unrelated to the secretagogue effect, (1) on the basis of metabolic consequences of increased secretion and (2) because ATP, another secretagogue, did not inhibit PC packaging. Methylamine seems to inhibit PC packaging by inhibiting trafficking of PC to lipid-rich light subcellular fractions. Together our results suggest that the trafficking of surfactant PC into lamellar bodies might be sensitive to changes in the pH of lamellar bodies.

Topics: Acridine Orange; Adenosine Triphosphate; Ammonium Chloride; Animals; Brefeldin A; Cells, Cultured; Centrifugation, Density Gradient; Choline; Cyclopentanes; Hydrogen-Ion Concentration; Ionophores; Methylamines; Microscopy, Fluorescence; Monensin; Organelles; Phosphatidylcholines; Phospholipids; Pulmonary Alveoli; Pulmonary Surfactants; Quinacrine; Rats

The hepatocanalicular transport of a large number of organic anions, such as bilirubin glucuronides and glutathione conjugates in the rat, is mediated by an adenosine triphosphate (ATP)-dependent transport system, which is termed canalicular multispecific organic anion transporter (cMOAT). This system is mainly defined by its deficiency in mutant TR rats. We have previously reported that in cultured hepatocytes the fluorescent organic anion glutathione-bimane (GS-B) accumulates in intracellular vesicles and that this transport is mediated by cMOAT. We now show that this intracellular accumulation of fluorescent organic anion is largely absent in freshly isolated hepatocytes but appears when cells are incubated in suspension at 37 degrees C or cultured for periods of 1 to 24 hours. The appearance of intracellular cMOAT activity coincides with the disappearance of 70% of cMOAT activity from the plasma membrane as measured by the transport activity of the cells for the organic anion dinitrophenyl-glutathione (GS-DNP). Both the appearance of intracellular cMOAT and the disappearance of transport activity from the plasma membrane were completely inhibited at temperatures below 20 degrees C. Residual cMOAT activity in 24-hour cultured hepatocytes could be further diminished by incubation of the cells with 1 mumol/L monensin or 10 mmol/L methylamine. We conclude that after disruption of the cell polarity by collagenase isolation of the hepatocytes, remnants of apical membrane containing cMOAT are rapidly endocytosed when the cells are kept at 37 degrees C. Evidence suggests that at least part of the transporters may recycle back to the plasma membrane after endocytosis. These observations may be relevant for the understanding of regulation of canalicular transport.

Topics: Alkaloids; Animals; Anion Transport Proteins; Bile Canaliculi; Biological Transport; Carrier Proteins; Cell Membrane; Cells, Cultured; Glutathione; Kinetics; Liver; Male; Methylamines; Mice; Mice, Mutant Strains; Monensin; Protein Kinase C; Rats; Rats, Wistar; Staurosporine; Temperature; Tetradecanoylphorbol Acetate

The cellular mechanisms underlying the generation of beta A4 in Alzheimer's disease and its relationship to the normal metabolism of the amyloid protein precursor (APP) are unknown. In this report, we show that expression of the C-terminal 100 residues of APP, with (SPA4CT) or without (A4CT) a signal sequence in the N-terminal position, in human neuroblastoma cells results in secretion of a 4 kDa beta A4-like peptide. In A4CT and SPA4CT expressing SY5Y cells, beta A4 generation could not be inhibited by the lysosomotropic amines chloroquine and ammonium chloride but was inhibited by brefeldin A, monensin and methylamine. The last also selectively inhibits APP secretion in neuroblastoma cells [1]. The finding that chloroquine and ammonium chloride inhibit beta A4 generation from full length APP but not from A4CT and SPA4CT are consistent with the assumption that the two cleavages necessary to generate beta A4 operate in two different compartments. Our data suggest the cleavage which generates the C-terminus of beta A4 takes place in the same compartment (late Golgi or endosomal vesicles) in which the APP-secretase operates.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Brefeldin A; Culture Media, Conditioned; Cyclopentanes; Gene Expression; Humans; Kinetics; Methylamines; Monensin; Neuroblastoma; Peptide Fragments; Plasmids; Transfection; Tumor Cells, Cultured

In the classical secretory pathway proteins containing a signal peptide are translocated from the cytoplasm of the cell into the lumen of the endoplasmic reticulum (ER). From the ER they are transported to the Golgi apparatus and finally to the plasma membrane (PM) where they are released into the extracellular compartment. However, some proteins are synthesized without a signal peptide and maintain a predominantly cytosolic distribution until they are released from the cell. As a marker for this nonclassical secretory pathway we have chosen L-29, a soluble lectin of M(r) about 29,000, that has affinity for lactose and other beta-galactoside containing glycoconjugates. We were interested in determining if cultured epithelial cells secrete L-29 and if they do so in a polarized fashion. Madin-Darby canine kidney (MDCK)-II cells were found to express large quantities of L-29 (about 1% of the detergent soluble protein). The lectin was diffusely distributed in the cytosol, with little or none in vesicular compartments. The polarity of L-29 secretion, when analyzed in pulse-chase experiments, was selectively into the apical compartment of filter-grown MDCK cells. This secretion was not inhibited by brefeldin A or monensin, drugs that are known to inhibit protein transport through the ER-Golgi-PM pathway. Secretion of L-29 was augmented 3-5-fold by the calcium ionophore A23187 and by increasing the temperature to 42 degrees C, whereas lowering the temperature to 20 degrees C or addition of nocodazole prevented secretion. These results demonstrate the polarized secretion of a cytosolic protein by a nonclassical secretory pathway.

Topics: Animals; Brefeldin A; Calcimycin; Cell Line; Chromatography, Affinity; Cyclopentanes; Cysteine; Cytosol; Dogs; Electrophoresis, Polyacrylamide Gel; Immunohistochemistry; Kidney; Kinetics; L-Lactate Dehydrogenase; Lectins; Methionine; Methylamines; Molecular Weight; Monensin; Nocodazole; Sulfur Radioisotopes; Temperature; Verapamil

Anthracycline accumulation was evaluated by flow cytometry or radiolabeled drug assays in cells and cytoplasts (enucleated cells) prepared from parental and multidrug-resistant human K562 leukemia cells. Treatment with energy inhibitors, such as dinitrophenol (DNP) or sodium azide/deoxyglucose, led to a marked decrease in daunorubicin accumulation in parental cells and cytoplasts. Another ionophore, monensin, also caused a significant decrease in daunorubicin accumulation; however, ATPase inhibitors ouabain, vanadate, and N-ethylamaleimide had little or no effect. The lysosomatropic agents chloroquine and methylamine caused a moderate decrease in anthracycline accumulation. Fluorescence microscopy showed that the DNP-sensitive daunorubicin uptake occurred in a nonnuclear subcellular compartment. Studies using increasing daunorubicin concentrations demonstrated fluorescence quenching that occurred in the nonnuclear, DNP-sensitive compartment. The effect of inhibitors on the accumulation of rhodamine 123 and acridine orange strongly implicated lysosomes as the principal compartment of this inhibitable daunorubicin accumulation. Cytoplasts from P-glycoprotein containing multidrug-resistant K562 cells demonstrated a verapamil-reversible, decreased daunorubicin accumulation that was observed in resistant whole cells. Verapamil pretreatment of cytoplasts from resistant cells revealed the subcellular DNP-sensitive uptake present in parental cytoplasts. These studies demonstrate that cytoplasts are an effective means to study drug transport in mammalian cells without nuclear drug binding. Parental K562 cells and cytoplasts exhibit an energy-dependent accumulation of daunorubicin into cytoplasmic organelles that is also present in resistant cells and cytoplasts when P-glycoprotein mediated efflux is inhibited.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Cell Compartmentation; Chloroquine; Daunorubicin; Deoxyglucose; Dinitrophenols; Drug Interactions; Drug Resistance; Energy Metabolism; Ethylmaleimide; Humans; Leukemia; Membrane Glycoproteins; Methylamines; Microscopy, Fluorescence; Monensin; Ouabain; Sodium Azide; Tumor Cells, Cultured; Vanadates; Verapamil

Astroviruses are intestinal pathogens associated with gastroenteritis in man and animals. The mechanism of internalization into host cells has not been reported previously. The cell entry pathway of serotype 1 human astrovirus into 293 cell line was studied biochemically and morphologically. Viral infection was monitored by indirect immunofluorescence. Infected cells were treated with the lysosomotropic agents ammonium chloride, methylamine, and dansylcadaverine or the ionophore monensin to raise the intraendosomal and intralysosomal pH. All drugs tested inhibited the early stages of infection whereas they did not interfere with the viral binding to the plasma membrane. The presence of astrovirus particles was detected by electron microscopy in coated pits and later in coated vesicles. The data indicate adsorptive endocytosis as the most probable mechanism by which astroviruses enter susceptible cells.

Topics: Ammonium Chloride; Cadaverine; Cell Line; Dose-Response Relationship, Drug; Humans; Immunosuppressive Agents; Mamastrovirus; Methylamines; Microscopy, Electron; Monensin; Virus Replication

We have identified nerve growth factor receptor (NGFR) on the cell surface and a truncated nerve growth factor receptor (NGFRt) in the conditioned medium of NGFR-negative cells that have been transfected with either the gene or the cDNA for the full-length receptor. By using cell surface iodination or metabolic labeling of A875 human melanoma cells, coupled with immunoprecipitation, we have determined the half-life of the cell-associated receptor to be approximately 7 h. Concomitant with receptor degradation is the accumulation of NGFRt in the extracellular medium. Approximately one-fifth of the labeled receptor can be recovered as the truncated species. These data support the hypothesis that NGFRt is generated by proteolysis of previously intact receptor. Furthermore, although no specific protease inhibitor assayed could affect this processing, NGFR degradation and truncation were retarded by treatment with: 1) the weak base amines, ammonium chloride or methylamine; 2) the carboxylic ionophore, monensin; or 3) the vacuolar ATPase inhibitor, bafilomycin A1, all agents that dissipate endosomal/lysosomal proton gradients via alternate mechanisms. Incubation of cells at 4 degrees C precluded NGFR degradation and truncation. The presence of ligand did not alter the time course of receptor truncation.

Topics: Ammonium Chloride; Anti-Bacterial Agents; Blotting, Western; DNA; Electrophoresis, Polyacrylamide Gel; Endocytosis; Endopeptidases; Humans; Hydrolysis; Macrolides; Melanoma; Methylamines; Monensin; Precipitin Tests; Protease Inhibitors; Receptors, Cell Surface; Receptors, Nerve Growth Factor; Transfection; Tumor Cells, Cultured

Rat Nb2 node lymphoma cells proliferate in response to lactogens, but the signal transduction mechanism involved remains unclear. Specific binding, internalization, and degradation of ovine PRL (oPRL) were examined under a variety of experimental conditions to characterize the metabolism of receptor-bound hormone by these cells. Stationary-phase cells were incubated with [125I]oPRL in Fischer's medium containing horse serum. Cell suspensions were centrifuged, and the cell pellets were assayed to determine specific cell-associated radioactivity. Internalized ligand was measured by exposing the cells to an acidic buffer before centrifugation to dissociate hormone from plasma membrane receptors, and cell-surface ligand was calculated by subtracting internalized hormone from the total [125I]oPRL bound by the cells. Hormone degradation was assessed by measuring the radioactivity in an acid-soluble fraction prepared from the incubation medium. Endocytosis of [125I]oPRL was observed within 30 min at 37 C, and the internalized component accounted for approximately 50% of the bound hormone under steady-state conditions. Hormone degradation was detectable within 1 h at 37 C and continued at a relatively linear rate thereafter; by 4 h, 8% of the added [125I]oPRL was acid soluble. Chloroquine (0.2 mM), methylamine (20 mM) and monensin (20 microM) prevented [125I]oPRL degradation and elevated both cell-surface and intracellular hormone 2-fold during a 4-h incubation. Leupeptin (0.2 mM) decreased degradation by only 15% under the same conditions. Phorbol 12-myristate 13-acetate (PMA; 20 nM), a comitogen for lactogen-stimulated Nb2 cells, increased cell-surface hormone by 20% and decreased intracellular hormone by a corresponding amount 1 h after administration. Calcium ionophore A23187 (1 microM) produced similar changes, and a synergistic effect was noted when cells were exposed to both agents for 4 h. Amiloride (125 microM), an inhibitor of Nb2 cell mitogenesis, decreased [125I]oPRL degradation by 25% during a 4-h incubation. This response was abolished when the cells were exposed simultaneously to PMA. These experiments demonstrate that receptor-bound oPRL is rapidly internalized and extensively degraded via the endosome-lysosome pathway when Nb2 cells are maintained at 37 C. The inhibitory effect of PMA on oPRL internalization may help to explain the comitogenic action of this phorbol on Nb2 cells. Since amiloride also produced major changes in oPRL metabolism, pos

Topics: Amiloride; Animals; Calcimycin; Cell Division; Cell Membrane; Chloroquine; Endocytosis; Kinetics; Leupeptins; Lymphoma; Methylamines; Mitosis; Monensin; Prolactin; Rats; Receptors, Prolactin; Sheep; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

A murine B lymphoid cell line, 70Z/3, has proven to be a useful model to study the developmental transition from membrane immunoglobulin (mIgM)-negative to mIgM-positive B cells. 70Z/3 cells have normal intracellular levels of mu heavy chain but kappa mRNA is transcribed at a very low rate. Because both kappa and mu chains are required for IgM to localize on the external membrane the cells remain essentially mIgM negative. A number of biologic agents can stimulate kappa mRNA transcription leading to increased surface IgM expression. Pharmacologic agents which cause cytoplasmic alkalinization have also been shown to increase the level of surface IgM expression when assessed by FITC-labeled anti-IgM antibodies. This increase has been used to argue that cytoplasmic alkalinization is sufficient to stimulate 70Z/3 differentiation but previous studies did not measure kappa mRNA levels or rate of synthesis. In this study we set out to determine whether the increased FITC staining that follows cytoplasmic alkalinization results from increased surface IgM expression and whether that increase results from activation of kappa mRNA transcription. When cells were treated with methylamine, NH4Cl, or monensin there was transient cytoplasmic alkalinization. The levels of surface IgM expression were measured by flow cytometry of cells stained with FITC-labeled anti-mouse kappa and mu. Using this criterion, a 24-hr treatment with either weak bases or monensin increased the level of surface IgM. The increase in fluorescence was not the result of nonspecific binding or uptake by the cell because there was no increase in fluorescence when methylamine-treated cells were stained with FITC-labeled antibody directed against an antigen not found on these cells. Iodination of surface proteins confirmed that the increase in fluorescence was the result of increased levels of IgM protein on the cell surface. However, exposure of the cells to weak bases or monensin caused no increase in either the steady-state level of kappa light chain mRNA, or in the level of kappa protein. We conclude that the transient alkalinization is not sufficient to induce differentiation of the 70Z/3 cells.

Topics: Ammonium Chloride; Animals; B-Lymphocytes; Blotting, Western; Cell Compartmentation; Cell Line; Cell Membrane; Gene Expression; Genes, Immunoglobulin; Humans; Hydrogen-Ion Concentration; Immunoglobulin kappa-Chains; Immunoglobulin M; In Vitro Techniques; Methylamines; Mice; Monensin; Receptors, Antigen, B-Cell; RNA, Messenger; Sodium

These studies were undertaken to characterize the physiological fate of the insulin-like growth factor-I (IGF-I)-type I receptor complex in MG-63, an IGF-responsive human osteosarcoma cell line. To investigate this, a photoreactive iodinated derivative of IGF-I [5-azido-2-nitrobenzoyl-125I-IGF-I (ANBz-125I-IGF-I)] was synthesized. This derivative retained biological activity and photolabeled a cell surface component on MG-63 cells with the size and binding specificity characteristic of the type I IGF receptor. To assess the stability of the photoaffinity-labeled IGF-I receptor complex, quiescent monolayers of MG-63 cells were incubated with ANBz-125I-IGF-I at 2 C, photolyzed, and then warmed to 37 C. At various times the monolayers were solubilized and the receptor complex was identified by quantitative autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under these conditions the photoaffinity-labeled IGF-I receptor complex was relatively stable, with a half-life of 11 +/- 2 h in five experiments. To determine if the complex undergoes internalization, its susceptibility to hydrolysis by trypsin at 2 C was measured. When cells that were labeled at 2 C were warmed to 37 C, a trypsin-insensitive band appeared within 20 min, reached a maximum by 1 h, and declined thereafter; however, an average of only 7% (5-8% in four experiments) of the total labeled receptor pool was present intracellularly at 1 h, and this declined to less than 1% by 4 h. A similar distribution of receptors was observed in cells photolabeled after incubation with ANBz-125I-IGF-I at 37 C, indicating that this distribution was not the result of altered metabolism of the photolabeled receptor complex. Lysosomotropic agents inhibited degradation of the complex, but did not alter its distribution between the cell surface and interior. These results indicated that the IGF-I-type I receptor complex is relatively stable and does not undergo rapid ligand-induced down-regulation and degradation. These observations suggest a model in which the IGF-receptor complex functions while present at or near the cell surface.

Topics: Affinity Labels; Cell Line; Chloroquine; Humans; Insulin-Like Growth Factor I; Light; Methylamines; Monensin; Photolysis; Receptors, Cell Surface; Receptors, Somatomedin; Somatomedins; Trypsin

Ticlopidine is a potent inhibitor of ADP-induced aggregation of rabbit platelets ex vivo. In vivo, however, multiple agonists play a role in platelet activation. In this study, we examined the effect of epinephrine on the antiplatelet action of ticlopidine in rabbit platelets. Epinephrine reversed the inhibitory effect of drug on ADP-induced platelet aggregation. The potentiating effect of epinephrine was mediated through alpha 2-adrenergic receptors, was reversed by pretreatment with the Na+/H+ exchange inhibitor dimethylamiloride, and was mimicked by agents that increased intracellular sodium or pH. Ticlopidine had no effect on resting intracellular pH, an indication that the effect of epinephrine was not compensating for a drug-induced intracellular acidification. While this potentiation was also found to be inhibited by aspirin, it did not involve enhanced release of thromboxane A2. Our results demonstrate that epinephrine can overcome the inhibitory effect of ticlopidine on ADP-induced aggregation through a mechanism involving activation of Na+/H+ exchange and through an as yet unidentified mechanism sensitive to aspirin.

Topics: Adenosine Diphosphate; Amiloride; Animals; Aspirin; Drug Interactions; Epinephrine; Hydrogen-Ion Concentration; Male; Methylamines; Monensin; Platelet Aggregation; Platelet Aggregation Inhibitors; Rabbits; Receptors, Adrenergic, alpha; Sodium; Thromboxane A2; Ticlopidine

Lysis of HeLa cells infected with poliovirus revealed intact virus; 135S particles, devoid of VP4 but containing the viral RNA; and 80S empty capsids. During infection the kinetics of poliovirus uncoating showed a continuous decrease of intact virus, while the number of 135S particles and empty shells increased. After 1.5 h of infection conformational transition to altered particles resulted in complete disappearance of intact virions. To investigate the mechanism of poliovirus uncoating, which has been suggested to depend on low pH in endosomal compartments of cells, we used lysosomotropic amines to raise the pH in these vesicles. In the presence of ammonium chloride, however, the kinetics of uncoating were similar to those for untreated cells, whereas in cells treated with methylamine, monensin, or chloroquine, uncoating was merely delayed by about 30 min. This effect could be attributed to a delay of virus entry into cells after treatment with methylamine and monensin, whereas chloroquine stabilized the viral capsid itself. Thus, elevation of endosomal pH did not affect virus uncoating. We therefore propose a mechanism of poliovirus uncoating which is independent of low pH.

Topics: Ammonium Chloride; Cell Transformation, Viral; Chloroquine; Endocytosis; Glutathione; HeLa Cells; Hot Temperature; Humans; Hydrogen-Ion Concentration; Kinetics; Methylamines; Monensin; Poliovirus; Tritium; Virus Replication

Rapid and parallel secretion of lysosomal beta-N-acetylglucosaminidase and preloaded fluorescein-labelled dextran was initiated in macrophages by agents affecting intracellular pH (methylamine, chlorpromazine, and the ionophores monensin and nigericin). In order to evaluate the relative role of changes in lysosomal and cytosolic pH, these parameters were monitored by using pH-sensitive fluorescent probes [fluorescein-labelled dextran or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein]. All agents except chlorpromazine caused large increases in lysosomal pH under conditions where they induced secretion. By varying extracellular pH and ion composition, the changes in lysosomal and cytosolic pH could be dissociated. Secretion was then found to be significantly modulated by changes in cytosolic pH, being enhanced by alkalinization and severely inhibited by cytosolic acidification. However, changes in cytosolic pH in the absence of stimulus were unable to initiate secretion. Dissociation of the effects on lysosomal and cytosolic pH was also achieved by combining stimuli with either nigericin or acetate. Further support for a role of intracellular pH in the control of lysosomal enzyme secretion was provided by experiments where bicarbonate was included in the medium. The present study demonstrates that an increase in lysosomal pH is sufficient to initiate lysosomal enzyme secretion in macrophages and provides evidence for a significant regulatory role of cytosolic pH.

Topics: Acetates; Acetic Acid; Acetylglucosaminidase; Animals; Cells, Cultured; Chlorpromazine; Cytosol; Female; Hydrogen-Ion Concentration; Kinetics; Lysosomes; Macrophages; Methylamines; Mice; Mice, Inbred Strains; Monensin; Nigericin

The effect in vitro of some cytoplasmic structure and function inhibitors on the different stages of rabies virus infection was investigated. Treatment of fibroblasts (CER) and human neuroblastoma cells (IMR-32) with substances acting on low pH intracellular compartments (methylamine and monensin) prevented rabies virus genome delivery in the cytosol. An early inhibition of viral infection was also obtained in the presence of B and D cytochalasins and trifluoperazine which interact with microfilament structures. Treatment with colchicine and vinblastine did not affect rabies multiplication, suggesting that microtubules are not involved in this process. However, the multiplication of prebound virions did not take place in the presence of inhibitors of oxidative phosphorylation (sodium azide and CCCP) and of glycolysis (2-deoxy-D-glucose) indicating that rabies virus replication is largely energy-dependent in both host cells examined.

Topics: Animals; Azides; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Colchicine; Cytochalasin B; Cytochalasin D; Deoxyglucose; Dose-Response Relationship, Drug; Fibroblasts; Humans; Methylamines; Monensin; Neuroblastoma; Rabies virus; Sodium Azide; Trifluoperazine; Tumor Cells, Cultured; Vinblastine; Virus Replication

Interleukin 1 (IL-1) is a major soluble mediator of inflammation. Two human IL-1 genes, alpha and beta, have been isolated, which encode polypeptides with only 20-30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that IL-1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum--Golgi route. Drugs which block the intracellular transport of IL-6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of IL-1 beta. Secretion of IL-1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. IL-1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of IL-1 beta are made but none is secreted. In these cells IL-1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain IL-1 beta are part of the protein secretory pathway. We conclude that IL-1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions.

Topics: Brefeldin A; Cyclopentanes; Hot Temperature; Humans; Interleukin-1; Kinetics; Lipopolysaccharides; Methylamines; Monensin; Monocytes; Protein Processing, Post-Translational; Subcellular Fractions

Liposome-encapsulated (LSOD) or free (FSOD), human recombinant Cu-Zn superoxide dismutase prevented the killing of cultured rat hepatocytes by tert-butyl hydroperoxide (TBHP). A dose of 32 U/ml of LSOD reduced the cell killing by 50%. By contrast, it required 288 U/ml of FSOD to similarly reduce the toxicity of TBHP by 50%. Both LSOD and FSOD increased the cell-associated superoxide dismutase activity of the cultured hepatocytes. Whereas 64 U/ml of LSOD increased cell-associated superoxide dismutase activity fourfold, it required 500 U/ml of FSOD to achieve a similar increase. Furthermore, methylamine, benzyl alcohol, cytochalasin B, oligomycin, and monensin, all inhibitors of endocytosis, prevented the increase in cell-associated superoxide dismutase produced by 500 U/ml of FSOD. These same inhibitors had no effect on the increase in cell-associated superoxide dismutase activity produced by a much lower concentration of LSOD. Thus, liposome-encapsulated superoxide dismutase prevented the cell killing by TBHP more efficiently than free superoxide dismutase because it more efficiently entered the hepatocytes by a mechanism that was independent of the endocytosis responsible for the uptake of FSOD. These data further define the conditions of the toxicity of TBHP. The target hepatocyte must contribute superoxide anions, in addition to the previously shown ferric iron. It is hypothesized that superoxide anions reduce ferric to ferrous iron; the latter then reacts with the hydroperoxide to form tert-butyl alkoxyl radicals. Such radicals are potent oxidizing agents that can initiate the peroxidation of cellular lipids previously shown to lethally injure the hepatocytes.

Topics: Animals; Benzyl Alcohol; Benzyl Alcohols; Cytochalasin B; Endocytosis; In Vitro Techniques; Liposomes; Liver; Methylamines; Monensin; Oligomycins; Peroxides; Rats; Rats, Inbred Strains; Solubility; Superoxide Dismutase; tert-Butylhydroperoxide

T cells recognize foreign proteins as peptides bound to self molecules encoded by the major histocompatibility complex (MHC). The kinetics of interaction between purified class II MHC molecules and peptides is unusual, in that the rate of association is very slow, but once formed, the complexes are extremely stable. This raises the question of how the antigen-presenting cell provides a sufficient number of free MHC binding sites to ensure T cell immunity. We present results suggesting that an exchange of peptide in MHC binding sites may take place under physiological conditions.

Topics: Ammonium Chloride; Animals; Antigen-Presenting Cells; Cell Line; Chloroquine; Fixatives; Glutaral; Histocompatibility Antigens Class II; Hydrogen-Ion Concentration; Interleukin-2; Kinetics; Methylamines; Mice; Monensin; Muramidase; Peptides; Protein Binding; T-Lymphocytes; Temperature

The precursor of mitochondrial aspartate aminotransferase accumulates in the cytosol of cultured chicken embryo fibroblasts if its import into mitochondria is inhibited by an uncoupling agent. However, its accumulation is limited by degradation with a half-life of only approximately 5 min (Jaussi, R., Sonderegger, P., Flückiger, J., and Christen, P. (1982) J. Biol. Chem. 257, 13334-13340). The aim of the present study was the characterization of the proteolytic system(s) responsible for this very rapid intracellular degradation. On depleting chicken embryo fibroblasts of ATP, the rate of degradation of the precursor was lowered by approximately 70%. Chicken embryo fibroblasts depleted of divalent metal ions showed a degradative activity of 10% of the initial value. Reconstitution of these cells with Mg2+ and Ca2+ increased the degradative activity from 10 to 107 and 24%, respectively. Thiol reagents almost completely prevented the degradation, whereas specific peptide inhibitors of cysteine proteases or inhibitors of intralysosomal proteolysis decreased the rate of degradation by only approximately 30%. Inhibitors of serine proteases had little effect. No rapid degradation of the precursor was observed in crude extracts of chicken embryo fibroblasts. The data indicate that the bulk of the precursor accumulated under conditions of import block is degraded by one or several cytosolic proteases dependent on ATP, Mg2+, and thiol groups of unknown localization, conceivably by proteolytic enzymes identical with or similar to one of the high molecular weight cytosolic proteases (Waxman, L., Fagan, J.M., Tanaka, K., and Goldberg, A. L. (1985) J. Biol. Chem. 260, 11994-12000). The rest of the precursor appears to be degraded by lysosomes.

Topics: Adenosine Triphosphate; Animals; Antipain; Aspartate Aminotransferases; Chick Embryo; Cytosol; Enzyme Precursors; Fibroblasts; Half-Life; Leupeptins; Methylamines; Mitochondria; Monensin

Lysosomotropic amines can raise the acidic internal pH of the neutrophil lysosome and inhibit neutrophil function. Because pH and calcium regulation are intimately connected in various types of excitable cells, we studied the effects of several lysosomotropic weak bases on neutrophil calcium homeostasis. Base-treated cells had normal to minimally elevated resting cytosol free calcium, but weak bases produced significant release of calcium from organelles when this release was directly measured in permeabilized cells, even after depletion of inositol-triphosphate-sensitive stores. Collapse of transmembrane pH gradients with monensin similarly released organelle calcium. The initial cytosol calcium response to f-met-leu-phe was enhanced by some of the lysosomotropic amines but the calcium rise was more transient in base-treated cells than in control samples. These findings suggest that existence of an acidic intracellular compartment, such as the lysosome, is important to normal calcium homeostasis in the neutrophil and that pH sensitivity and inositol triphosphate sensitivity may define two pools of releasable organelle calcium. The effect of pH perturbation on calcium homeostasis may partially account for the inhibition of neutrophil function by lysosomotropic amines.

Topics: Adult; Aminoquinolines; Ammonium Chloride; Calcium; Clindamycin; Cytosol; Fluorescent Dyes; Homeostasis; Humans; Kinetics; Lysosomes; Methylamines; Monensin; Neutrophils; Propranolol; Sodium Chloride

It has been demonstrated recently by acridine orange fluorescence that pancreatic zymogen granules are acidic in situ, with respect to the cytoplasm. To evaluate the relationship between the acidic intragranular pH and hormone-stimulated secretion, mouse pancreatic acini were treated with lysosomotropic agents to collapse the zymogen granule pH gradient. Methylamine, monensin, and chloroquine collapsed the granule pH gradient as evidenced by a disappearance of acridine orange fluorescence. Cholecystokinin octapeptide (CCK-8)-stimulated acinar amylase secretion was unaffected in the presence of up to 30 mM methylamine and slightly enhanced in the presence of 0.3-10 microM monensin or 3-300 microM chloroquine. Acini were also preincubated for 15 min before addition of either CCK-8 or bombesin with concentrations of the lysosomotropic agents that dissipated the granule acridine orange fluorescence within this time. With preincubation, basal amylase release was unaffected, while stimulated secretion was slightly enhanced by all three lysosomotropic agents. Monensin and methylamine caused vacuolization of Golgi and lysosomal membranes and inhibition of intracellular transport of newly synthesized proteins. Chloroquine affected lysosomes similarly but had little effect on Golgi membranes or on intracellular protein transport. We also demonstrate that parotid secretory granules are acidic in situ by the acridine orange technique. Thus acidified secretory granules may be a general feature of exocrine secretory granules, but the acid pH is not requisite for the final steps in protein secretion from isolated pancreatic acini.

Topics: Amylases; Animals; Chloroquine; Cytoplasmic Granules; Enzyme Precursors; Exocytosis; Hydrogen-Ion Concentration; Male; Methylamines; Mice; Microscopy, Electron; Monensin; Pancreas; Secretory Rate; Sincalide

Binding of porcine interleukin 1, radiolabeled with Bolton-Hunter reagent (125I IL 1), to monolayers of porcine synovial fibroblasts (PSF) was found to be a temperature-dependent process. The rate of uptake and the amount of cell-associated ligand was higher at 37 degrees C than at 4 degrees C or 19 degrees C, and exceeded the apparent equilibrium binding capacity. The amount of bound 125I IL 1 that was removed by brief treatment with acidic buffers decreased from 80% at 4 degrees C to 35% for PSF incubated at 37 degrees C; this procedure was used to distinguish surface-bound from internalized ligand. In untreated PSF, surface binding was maximal at 1 hr and was maintained for at least 5 hr during which time the internal pool continued to increase. The lysosomotropic agent methylamine (20 mM) decreased surface binding by 50%; monensin (20 microM) decreased the rate and extent of internalization. Cycloheximide (10 micrograms/ml) did not affect ligand uptake, hence, continual expression of surface receptors could not be ascribed to their de novo synthesis. 40% of the radioactivity taken up by PSF during incubation at 37 degrees C subsequently appeared in the culture medium upon prolonged postincubation (5 hr) in the absence of added 125I IL 1: 60% of this fraction was trichloroacetic acid-soluble in untreated cultures, but the extent of degradation was halved by treatment with methylamine or monensin. Direct measurement of the rate of internalization of prebound 125I IL 1 was obtained by monitoring the formation of covalently cross-linked ligand-receptor complexes after warming PSF monolayers to 37 degrees C. By using gel electrophoresis we observed a decrease (t1/2 = 9 to 11 min) in labeling of the major cross-linkable species.

Topics: Animals; Cell Compartmentation; Cycloheximide; Fibroblasts; Hydrogen-Ion Concentration; Interleukin-1; Intracellular Membranes; Kinetics; Lysosomes; Membrane Proteins; Methylamines; Molecular Weight; Monensin; Receptors, Immunologic; Receptors, Interleukin-1; Swine; Synovial Membrane; Temperature

Carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), a protonophore, and methylamine, a weak base, agents that dissipate hydrogen gradients across cellular membranes, were used to probe the coupling of hydrogen gradients to the processing and secretion of the glycoprotein hormone hCG by human choriocarcinoma cells (JAR) in culture. Both drugs disrupted the processing of asparagine-linked oligosaccharides such that the secreted hCG forms contained mostly high mannose rather than complex oligosaccharide chains. As the concentrations of FCCP were increased above 1 microgram/ml and those of methylamine above 12.5 mg/ml, the secretion of the labeled hCG dimer and free alpha-subunit was progressively inhibited. Both FCCP and methylamine also inhibited the incorporation of [35S] methionine and [3H]mannose into hCG subunits. Nevertheless, the inhibition of secretion was clearly apparent as an intracellular accumulation of the hCG subunit precursors in spite of the diminished incorporation of radioactive substrates. The intracellular hCG precursors that accumulated in the drug-treated cells contained predominantly Man8-9GlcNAc2 units, structures characteristic of glycoproteins localized in the endoplasmic reticulum. Both FCCP and methylamine inhibited hCG secretion at concentrations that did not lower the cellular content of ATP. We postulate on the basis of these results that a hydrogen gradient across the membrane either of the rough endoplasmic reticulum or the transitional vesicle is coupled to the rough endoplasmic reticulum to Golgi translocation step such that dissipation of the proton gradient blocks the secretion of hCG.

Topics: Adenosine Triphosphate; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Female; Humans; Methylamines; Monensin; Nitriles; Oligosaccharides; Oxygen Consumption; Pregnancy; Protein Processing, Post-Translational

When normal human fibroblasts are brought to a steady state with 125I-labeled epidermal growth factor (125I-EGF), greater than 90% of the radioactivity is intracellular. We investigated this material to determine whether the 125I-EGF is intact or degraded. Our results show that 125I-EGF is rapidly processed after internalization and can be resolved into four peaks by native gel electrophoresis. These different forms were isolated and tested for their ability to bind to cell-surface EGF receptors. The first processed form was fully capable of binding to EGF receptors, but the second processed form could not. The third form was a collection of small degradation products. We calculated that at steady state about 60% of internalized "125I-EGF" was in a form still able to bind to EGF receptors. We then investigated the ability of different reported inhibitors of EGF "degradation" to block the processing of EGF. Although inhibitors of cathepsin B (leupeptin, antipain, N alpha-p-tosyl-L-lysine chloromethyl ketone, and chymostatin) were able to inhibit the release of monoiodotyrosine from treated cells in a time- and concentration-dependent manner, they had little effect on the processing step that apparently inactivates 125I-EGF. In contrast, agents that raised intravesicular pH, such as methylamine and monensin, inhibited the initial steps in EGF processing as well as the later steps. Low temperatures inhibited the transfer of 125I-EGF to the lysosomes and inhibited the conversion of EGF to a nonbindable form, but had little effect on the initial processing. We conclude that the intracellular processing of EGF is a multistep process that is initiated prior to lysosomal fusion, involves cathepsin B activity, and requires an acidic pH. In addition, many of the protease inhibitors that have been utilized to investigate the role of EGF degradation in mitogenesis do not block the conversion of EGF to a form that is apparently unable to interact with its receptor.

Topics: Animals; Cathepsin B; Cathepsins; Cell Line; Cold Temperature; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Humans; Hydrogen-Ion Concentration; Leupeptins; Methylamines; Mice; Models, Biological; Monensin; Receptors, Cell Surface

Topics: Amiloride; Animals; Biological Transport, Active; Carrier Proteins; Chickens; Chlorides; Electrochemistry; Hydrogen-Ion Concentration; Hydroxides; Hydroxyl Radical; Intestinal Mucosa; Kinetics; Membrane Potentials; Methylamines; Monensin; Protons; Sodium; Sodium-Hydrogen Exchangers

Cultured fibroblasts were treated with the carboxylic ionophore monensin to study the effect on 125I-epidermal growth factor binding, internalization, and mitogenic response. Monensin enhanced the accumulation of the ligand by both preventing its degradation in lysosomes and causing a redistribution of receptors from the plasma membrane to an intracellular compartment. Monensin also prevented the mitogenic activity of EGF. This ionophore, like alkylamines, raises the pH of endosomes and lysosomes. These data are consistent with the hypothesis that exposure of EGF-receptor complexes to an acid environment is part of the pathway that leads to a mitogenic response.

Topics: Cell Division; Cell Line; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Furans; Humans; Iodine Radioisotopes; Kinetics; Methylamines; Mitogens; Monensin; Receptors, Cell Surface

A conjugate of Pseudomonas exotoxin and epidermal growth factor (PE-EGF) inhibits proteins synthesis in KB cells, and this inhibition is increased by adenovirus. Protein synthesis inhibition is dependent on the amount of adenovirus and PE-EGF used and the time of incubation of cells with these agents. With 1 microgram of adenovirus and 0.5 micrograms of PE-EGF per ml, protein synthesis is inhibited about 80% in a 60-min experiment. Under these conditions neither adenovirus nor PE-EGF alone has any effect. In the presence of several weak bases or monensin, the enhancement of toxicity was substantially inhibited; half-maximal inhibition was achieved with 40 microM chloroquine, 10 mM ammonium chloride, 5 mM methylamine, 0.1 mM N-hexylamine and 1 microM monensin. At the concentrations employed, none of the inhibitors affected the amount of virus taken up or bound to the cell surface, and chloroquine had no effect on the amount of EGF taken up in 60 min. Chloroquine did not prevent the toxicity of the PE-EGF (5 micrograms/ml) alone. Because these compounds are known to elevate the pH in receptosomes, it seems likely that the acidification of the receptosome either enhances the lysis of the membrane by adenovirus or enhances some other step in the release of PE-EGF.

Topics: Adenoviruses, Human; Amines; Ammonium Chloride; Carcinoma; Cell Line; Chloroquine; Drug Synergism; Epidermal Growth Factor; Exotoxins; Humans; Hydrogen-Ion Concentration; Kinetics; Methylamines; Monensin; Mouth Neoplasms; Protein Biosynthesis; Pseudomonas; Receptors, Virus

Methylamine and monensin are two agents known to interfere with the recycling of membrane receptors. In the present investigation, we studied their effects on the binding, intracellular distribution, and action of insulin in isolated pancreatic acini prepared from diabetic mice. These drugs had several similar effects on these cells. In a dose-dependent fashion, both increased the amount of [125I]insulin associated with acini. Methylamine approximately doubled and monensin tripled the amount of insulin associated with cells. Employing electron microscope autoradiographs, we observed the accumulation of hormone in large vacuoles in the Golgi-lysosomal area after treatment with methylamine and in smaller swollen Golgi vesicles after treatment with monensin. The influences of both agents on the biological effects of insulin in acini were then investigated. Both agents blocked insulin stimulation of [3H]2-deoxy-D-glucose uptake in acini. The effect of methylamine was also studied on [3H]leucine incorporation into protein and was found to only partially block the effect of insulin. Since insulin and its receptor are internalized, it is likely that the accumulation of insulin in acini induced by these two agents was due to their inhibition of the cellular processing of insulin and its receptor. Moreover, the effects of these two agents to inhibit insulin-stimulated glucose transport may have resulted from either the inhibition of the recruitment of glucose carriers from the Golgi to the plasma membrane or an abnormal interaction of internalized insulin with the Golgi.

Topics: Animals; Cell Membrane; Deoxyglucose; Diabetes Mellitus, Experimental; Furans; Golgi Apparatus; Insulin; Leucine; Male; Methylamines; Mice; Microscopy, Electron; Monensin; Pancreas; Vacuoles

Chloroquine (CQ), the most widely used antimalarial drug, is an acidotropic agent (De Duve, 1983) which accumulates to high levels in malaria-infected erythrocytes. A possible site of accumulation of the drug, the parasite's food vacuole, has been implicated in the mode of action of CQ. We have defined the various compartments of Plasmodium falciparum-parasitized human erythrocytes in terms of their pH and capacity to accumulate bases. The host cell and the parasite cytosols were differentially labeled in situ with pH-sensitive fluorescein, and the parasite food vacuole was revealed by targeting fluoresceinated dextran via endocytosis. The pH of the various compartments obtained from fluorescence excitation spectra were 6.9 for the cytosol of normal and infected erythrocytes and 5.2 for the parasite food vacuole. Determination of CQ and methylamine accumulation in infected erythrocytes, in conjunction with morphometric determination of the relative sizes of the various cellular compartments, provided an independent assessment of the vacuolar pH, yielding a value of 5.0-5.2. Perturbation of the proton gradient, either by lowering extracellular pH or by alkalinization of the food vacuole with NH4Cl or monensin, resulted in a concomitant and reversible decrease in accumulation of the probe. We conclude that drug accumulation in malaria-infected erythrocytes can be fully accounted for by the steady-state proton gradients across the barriers delineating the various cellular compartments and the acidotropic properties of the drug.

Topics: Ammonium Chloride; Animals; Chloroquine; Endocytosis; Erythrocytes; Humans; Hydrogen-Ion Concentration; Kinetics; Malaria; Methylamines; Monensin; Plasmodium falciparum

L cells and mouse 3T3 cells, which are very sensitive to Pseudomonas aeruginosa exotoxin A (PEA), were protected with weak bases and low concentrations of monensin. BHK cells and a number of other cell lines which are much less sensitive to PEA were much less protected under these conditions. Trifluoperazine, dansylcadaverine, and several other calmodulin antagonists strongly sensitized BHK cells to the toxin whereas they did not affect the sensitivity of the mouse 3T3 and L cells. The sensitization of the BHK cells was counteracted by treatment with weak bases or low concentrations of monensin. Calmodulin antagonists also sensitized cells to toxin which had become inaccessible to antitoxin, indicating that the effect of the calmodulin antagonists is exerted on a process taking place after the toxin is endocytosed.

Topics: ADP Ribose Transferases; Ammonium Chloride; Animals; Bacterial Toxins; Cadaverine; Calmodulin; Cell Line; Cricetinae; Drug Synergism; Endocytosis; Exotoxins; Humans; Methylamines; Mice; Monensin; Pseudomonas aeruginosa Exotoxin A; Trifluoperazine; Virulence Factors

Agents that affect intracellular cation and pH gradients and inhibit energy production have been tested for their ability to modulate the processing and secretion of the free alpha subunit and the alpha beta dimer of human chorionic gonadotropin (hCG) by cultured human trophoblastic cells (JAR). Incubation of JAR cells with monensin or nigericin, monovalent cation ionophores that produce equilibration of Na+ and K+ across cellular membranes, dicyclohexylcarbodiimide, an agent that inhibits intracellular membrane ATPases, and methylamine, which neutralizes intracellular pH gradients, produced similar effects on hCG processing and secretion. All these agents inhibited the processing of the asparagine-linked oligosaccharide chains of free alpha subunit and the alpha and beta subunits contained in the hCG dimer. Moreover, after treatment of JAR cells with these agents, there was an intracellular accumulation of precursor forms and an inhibition of secretion of "mature" forms of hCG. Monensin affected the processing and secretion of hCG subunits differently at different concentrations. At 5 X 10(-7) M, monensin inhibited the processing of the asparagine-linked oligosaccharides of hCG without altering the rate-limiting step in the secretory pathway or blocking hCG secretion. The intracellular hCG subunit precursors in both control and monensin-treated cells contained a similar array of high mannose oligosaccharides, predominantly of the Man8GlcNAc2 and Man9GlcNAc2 types. However, monensin-treated cells secreted hCG subunits that contained endo H-sensitive oligosaccharides of the high mannose (mostly Man5GlcNAc2) and hybrid types rather than the endo H-resistant complex chains synthesized by control cells. Nevertheless, a full complement of serine-linked oligosaccharides was added to the hCG-beta subunit in monensin-treated cells. These results indicate that the intracellular movement of hCG from the rough endoplasmic reticulum to the cell surface was not inhibited by monensin at a concentration that impaired Golgi-localized steps in the processing of asparagine-linked oligosaccharides. At 5 X 10(-6) M, monensin significantly inhibited secretion of hCG and created a new rate-limiting step in the processing pathway. hCG subunits bearing Man5GlcNAc2 units accumulated intracellularly, suggesting that the equilibration of intracellular Na+/K+ pools blocked oligosaccharide processing at an intra-Golgi point, perhaps by inhibiting movement of the glycoprotein hormone

Topics: Calcimycin; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Chorionic Gonadotropin; Dicyclohexylcarbodiimide; Female; Glycoside Hydrolases; Humans; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Methylamines; Monensin; Oligomycins; Trophoblasts

Three different carboxylic ionophores (monensin, nigericin and lasalocid) were each found capable of causing a relatively complete block of the lysosomal (i.e., methylamine-sensitive) protein degradation in isolated rat hepatocytes. Monensin was found to be the most specific in action, as it had no effect on non-lysosomal degradation and did not bring about any substantial inhibition of protein synthesis. Morphometric examination of electron micrographs revealed that monensin causes an accumulation of early forms of autophagic vacuoles and blocks the swelling of lysosomes seen in the presence of methylamine. The results indicate that monensin inhibits lysosomal protein degradation by affecting lysosomal pH.

Topics: Animals; Autophagy; Dose-Response Relationship, Drug; Golgi Apparatus; Ionophores; Lasalocid; Liver; Lysosomes; Male; Methylamines; Monensin; Nigericin; Proteins; Rats; Rats, Inbred Strains; Vacuoles

It has been shown that endocytic vesicles in BALB/c 3T3 cells have a pH of 5.0 (Tycko and Maxfield, Cell, 28:643-651). In this paper, a method for measuring the effect of various agents, including weak bases and ionophores, on the pH of endocytic vesicles is presented. The method is based on the increase in fluorescein fluorescence with 490-nm excitation as the pH is raised above 5.0. Intensities of cells were measured using a microscope spectrofluorometer after internalization of fluorescein-labeled alpha 2-macroglobulin by receptor-mediated endocytosis. The increase in endocytic vesicle pH was determined from the increase in fluorescence after addition of various concentrations of the test agents. The following agents increased endocytic vesicle pH above 6.0 at the indicated concentrations: monensin (6 microM), FCCP (10 microM), chloroquine (140 microM), ammonia (5 mM), methylamine (10 mM). The ability of many of these agents to raise endocytic vesicle pH may account for many of their effects on receptor-mediated endocytosis. Dansylcadaverine caused no effect on vesicle pH at 1 mM. The observed increases in vesicle pH were rapid (1-2 min) and could be reversed by removal of the perturbant. This reversibility indicates that the vesicles themselves contain a mechanism for acidification. The increase in vesicle pH due to these treatments can be observed visually using an SIT video camera. Using this method, it is shown that endocytic vesicles become acidic at very early times (i.e., within 5-7 min of continuous uptake at 37 degrees C).

Topics: alpha-Macroglobulins; Ammonia; Animals; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Line; Chloroquine; Endocytosis; Epidermal Growth Factor; Fibroblasts; Hydrogen-Ion Concentration; Ionophores; Methylamines; Mice; Monensin; Organoids