monensin and 4-4-difluoro-4-bora-3a-4a-diaza-s-indacene

The spatial distribution of acid membrane organelles and their relationships with normal and vacuolated transverse tubules has been studied in living frog skeletal muscle fibres using confocal microscopy. Acridine orange (AO) was used to evaluate acid compartments, while a lipophilic styryl dye, RH 414, was employed to stain the membranes of the T-system. AO accumulated in numerous spherical granules located near the poles of nuclei and between myofibrils where they were arranged in short parallel rows, triplets or pairs. AO granules could be divided into three groups: green (monomeric AO), red (aggregated AO), and mixed green/red. As demonstrated by lambda-scanning, most granules were mixed. Double staining of muscle fibres with AO and RH 414 revealed almost all AO granules located near the transverse tubules. Vacuolation of the T-system was induced by glycerol loading and subsequent removal. The close juxtaposition of AO granules and the T-system was preserved in vacuolated fibres. The lumens of vacuoles did not accumulate AO. It is concluded that AO granules represent an accumulation of AO in lysosome-related organelles and fragmented Golgi apparatus and a possible functional role of the spatial distribution of such acidic compartments is discussed.

Topics: Acridine Orange; Amines; Animals; Anura; Boron Compounds; Ceramides; Cytoplasmic Granules; Monensin; Muscle Fibers, Skeletal; Muscle, Skeletal; Organelles; Spectrometry, Fluorescence; Staining and Labeling; Vacuoles

Mammalian polyamine carriers have not yet been molecularly identified. The fluoroprobe Spd-C2-BODIPY faithfully reports polyamine transport and accumulates almost exclusively in polyamine-sequestering vesicles (PSVs). Polyamines might thus be imported first by a plasma membrane carrier and then sequestered into pre-existing PSVs (model A), or be directly captured by polyamine receptors undergoing endocytosis (model B). Spd-C2-BODIPY uptake was unaffected in receptor-mediated endocytosis-deficient Chinese hamster ovary cell mutants. PSVs strongly colocalized with acidic vesicles of the late endocytic compartment and the trans Golgi. Virtually perfect colocalization between PSVs and acidic vesicles was found in Chinese hamster ovary cell mutants that are blocked either in the late endosome/lysosome fusion process or in the maturation of multivesicular bodies. Prior inhibition of the V-ATPase dramatically decreased total Spd-C2-BODIPY accumulation while increasing cytosolic fluorescence. Conversely, cells pre-loaded with the probe slowly released it from PSVs upon V-ATPase inhibition. The present data thus support model A, and indicate that polyamine accumulation is primarily driven by the activity of a vesicular H+:polyamine carrier.

Topics: Animals; Biological Transport; Boron Compounds; CHO Cells; Cricetinae; Endocytosis; Endosomes; Fluorescent Dyes; Golgi Apparatus; Image Processing, Computer-Assisted; Lysosomes; Macrolides; Microscopy, Confocal; Microscopy, Fluorescence; Models, Biological; Models, Chemical; Monensin; Mutation; Polyamines; Protons; Spermidine; Time Factors; Vacuolar Proton-Translocating ATPases

A number of cell types express inducible nitric-oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide or proinflammatory cytokines. Although it has been known for some time that the N-terminal end of NOS2 suffers a post-translational modification, its exact identification has remained elusive. Using radioactive fatty acids, we show herein that NOS2 becomes thioacylated at Cys-3 with palmitic acid. Site-directed mutagenesis of this single residue results in the absence of the radiolabel incorporation. Acylation of NOS2 is completely indispensable for intracellular sorting and .NO synthesis. In fact, a C3S mutant of NOS2 is completely inactive and accumulates to intracellular membranes that almost totally co-localize with the Golgi marker beta-cop. Likewise, low concentrations of the palmitoylation blocking agents 2-Br-palmitate or 8-Br-palmitate severely affected the .NO synthesis of both NOS2 induced in muscular myotubes and transfected NOS2. However, unlike endothelial NOS, palmitoylation of inducible NOS is not involved in its targeting to caveolae. We have created 16 NOS2-GFP chimeras to inspect the effect of the neighboring residues of Cys-3 on the degree of palmitoylation. In this regard, the hydrophobic residue Pro-4 and the basic residue Lys-6 seem to be indispensable for palmitoylation. In addition, agents that block the endoplasmic reticulum to Golgi transit such as brefeldin A and monensin drastically reduced NOS2 activity leading to its accumulation in perinuclear areas. In summary, palmitoylation of NOS2 at Cys-3 is required for both its activity and proper intracellular localization.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antifungal Agents; Biological Transport; Boron Compounds; Brefeldin A; Cell Line; Cells, Cultured; Cloning, Molecular; COS Cells; Cysteine; Escherichia coli; Fluorescent Dyes; Golgi Apparatus; Green Fluorescent Proteins; Hydroxylamine; Lysine; Mice; Molecular Sequence Data; Monensin; Mutagenesis, Site-Directed; Mutation; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Palmitic Acid; Proline; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Recombinant Proteins; Serine; Time Factors; Transfection

1. Transport-P is an uptake process for amines in peptidergic neurones of the hypothalamus. It differs from other uptake processes by its anatomical location in post-synaptic neurones, its functional properties and by the structure of its ligands. Transport-P accumulates amines in intracellular vesicles, derives its energy from the electrochemical proton gradient and is linked to vacuolar-type ATPase (V-ATPase). Transport-P is blocked by antidepressants. We have now studied the release of amines following uptake by Transport-P in a cell line of hypothalamic peptidergic neurones. 2. Release of prazosin was not inhibited by the antidepressant desipramine; as Transport-P is blocked by desipramine, this indicated that amines are released by a mechanism which is independent of Transport-P. 3. Release of prazosin was sensitive to temperature and conformed to the Arrhenius equation. Release was minimal in the range 0-25 degrees C but accelerated exponentially at higher temperatures up to 33 degrees C. The activation energy for the release of prazosin is 83.1 kJ x mol(-1), corresponding to a temperature quotient (Q10) value of 3. 4. Release was accelerated by the organic base chloroquine, the ionophore monensin, bafilomycinA1 which inhibits V-ATPase and by increasing extracellular pH. Thus, retention of prazosin requires an intracellular proton gradient which is generated by V-ATPase. 5. Fluorescence microscopy demonstrated that release of BODIPY FL prazosin was temperature dependent and was accelerated by chloroquine and monensin. 6. Thus, following uptake by Transport-P, amines are accumulated in acidified intracellular stores. Their retention in peptidergic neurones requires intracellular acidity. The amines are released by a temperature-dependent process which is resistant to antidepressants.

Topics: Antidepressive Agents; Biological Transport; Boron Compounds; Cells, Cultured; Chloroquine; Desipramine; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Hypothalamus; Monensin; Prazosin; Temperature

Tamoxifen has been reported to have numerous physiological effects that are independent of the estrogen receptor, including sensitization of resistant tumor cells to many chemotherapeutic agents. Drug-resistant cells sequester weak base chemotherapeutics in acidic organelles away from their sites of action in the cytosol and nucleus. This work reports that tamoxifen causes redistribution of weak base chemotherapeutics from acidic organelles to the nucleus in drug-resistant cells. Agents that disrupt organelle acidification (e.g., monensin, bafilomycin A1) cause a similar redistribution. Measurement of cellular pH in several cell lines reveals that tamoxifen inhibits acidification of endosomes and lysosomes without affecting cytoplasmic pH. Similar to monensin, tamoxifen decreased the rate of vesicular transport though the recycling and secretory pathways. Organellar acidification is required for many cellular functions, and its disruption could account for many of the side effects of tamoxifen.

Topics: Anti-Bacterial Agents; Biological Transport; Boron Compounds; Breast Neoplasms; Cytoplasm; Doxorubicin; Drug Resistance, Neoplasm; Endosomes; Female; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Lysosomes; Macrolides; Monensin; Neuroblastoma; Receptors, Estrogen; Tamoxifen; Transferrin; Tumor Cells, Cultured

Fluorescent lipid analogues of the lipids ceramide and sphingomyelin, namely BODIPY FL C5-ceramide ((N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-pentanoyl) sphingosine) and BODIPY C5-sphingomyelin ((N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingosyl phosphocholine), respectively, were used to investigate the presence of a sphingomyelin cycle in Schistosoma mansoni adult males. The parasites were able to convert BODIPY FL C5-ceramide into a fluorescent sphingomyelin analogue, and traffic it to the outer monolayer where it was lost to the medium. The vesicular trafficking inhibitors Brefeldin A and monensin were found, however, to have no effect on either the rate of sphingomyelin synthesis or its trafficking to the surface. Parasites were also shown to break BODIPY FL sphingomyelin down, forming a fluorescent ceramide analogue. Inhibitors of lysosomal function, NH4Cl, desipramine and perhexiline, did not inhibit this breakdown, suggesting that endocytosis and trafficking to lysosomes was not involved. In addition, assays carried out on parasite homogenates for sphingomyelinase activity were unable to detect sphingomyelin breakdown at acidic pH, but did detect activity at pH 7.4. This activity was stimulated by arachidonic acid and MgCl2. The results are discussed with respect to tegument synthesis and turnover, and cellular signalling.

Topics: Ammonium Chloride; Animals; Boron Compounds; Brefeldin A; Ceramides; Cyclopentanes; Desipramine; Fluorescent Dyes; Lysosomes; Male; Monensin; Perhexiline; Schistosoma mansoni; Sphingomyelins