sq-23377 and 3-3--dihexyl-2-2--oxacarbocyanine

The aim of this paper was to establish whether actin polymerization modulated cytosolic Ca2+ storage in human neutrophils. Over the concentration ranges which inhibit actin polymerization, cytochalasins A, B, and D liberated Ca2+ from membrane-bound stores within neutrophils. Two Ca2+ storage sites were identified in neutrophils by the accumulation of the Ca2+ binding probe, chlortetracycline: one at the center of the cell and the other at the cell periphery. Confocal imaging demonstrated that cytochalasins released Ca2+ from the neutrophil periphery, but not from the central Ca2+ store. Ca2+ store release was coupled to Ca2+ influx, suggesting that the peripheral site may be a physiological store containing a Ca2+ influx factor. 3,3'-Dihexyloxacarbocyanine iodide staining organelles, which correlate with Ca2+ release sites, coalesced in neutrophils after treatment with cytochalasins. We propose that peripheral Ca2+ storage sites are restricted from coalescence by cortical polymerized actin and that Ca2+ store coalescence and Ca2+ release are coupled events.

Topics: Actins; Animals; Botulinum Toxins; Calcium; Carbocyanines; Chelating Agents; Chlortetracycline; Cytochalasins; Cytosol; Fluorescent Dyes; Humans; Intracellular Membranes; Ionomycin; Ionophores; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Poly(ADP-ribose) Polymerases; Polymers; Rats

Biological characteristics of the globular substance, which is considered to be a precursor of otoconia, were investigated by means of confocal laser scanning microscopy. The shape of the globular substance was a complete sphere, 3-10 microns in diameter. Its surface stained positively with both rhodamine 123 and DiOC6(3), implying similarity to intracellular organelles, whereas no fluorescence was seen when stained with chlortetracycline, a membrane-associated Ca2+ dye. Meanwhile, this substance showed very little affinity for six kinds of lectins, indicating the lack of a surface structure of carbohydrates. The fluorescence of fluo-3 in the globular substance increased markedly after the application of ionomycin. But this was completely inhibited by the depletion of external Ca2+. This reaction suggests that the surface of the globular substance exhibits characteristics of a biological membrane and that the influx of external Ca2+ occurs through membrane-combined ionomycin. Internal free Ca2+ concentration varied from 1.1 x 10(-9) to 1.6 x 10(-4) M, the geometric mean being 3.3 x 10(-7) M, which is higher than normal resting level of intracellular Ca2+ concentration but lower than the calcium content of the globular substance estimated by X-ray microanalysis in previous studies.

Topics: Animals; Binding Sites; Calcium; Carbocyanines; Electron Probe Microanalysis; Fluorescent Dyes; Guinea Pigs; Ionomycin; Lectins; Microscopy, Confocal; Otolithic Membrane; Reproducibility of Results; Rhodamine 123; Rhodamines; Staining and Labeling

Cytoplasmic membrane potential of mouse lymphocytes was determined with flow cytometry and fluorescence spectroscopy using 3,3'-dihexylcarbocyanine iodide (DiOC6(3)). The amount of this lipophilic cation incorporated into the cytoplasmic membrane is dependent upon the transmembrane potential, so the dye is suitable for continuous monitoring of this parameter, under controlled conditions. Membrane potential of the cells was decreased in the presence of cyclosporin A and cyclosporin G in a dose-dependent manner. However, the depolarization caused by Ca2+ ionophores, ionomycin and A23187, was reduced in the presence of cyclosporin A. Electron spin resonance spectroscopy with 5-doxylstearic acid as a probe indicated that cyclosporin A decreased the apparent motional freedom of membrane lipids. These data suggest incorporation of cyclosporin A into the cytoplasmic membrane, causing changes in ion fluxes. The membrane potential change induced by cyclosporin A may have selective biological consequences in certain subpopulations of lymphocytes.

Topics: Animals; Calcimycin; Carbocyanines; Cell Membrane; Cyclosporine; Cyclosporins; Dose-Response Relationship, Drug; Drug Interactions; Electron Spin Resonance Spectroscopy; Ethers; Humans; Insulin; Ionomycin; Lymphocyte Activation; Lymphocytes; Membrane Lipids; Membrane Potentials; Mice; Mice, Inbred Strains; Quinolines