valinomycin and 3-3--dihexyl-2-2--oxacarbocyanine

Topics: Carbocyanines; Cell Membrane; Cell Survival; Flow Cytometry; Fluorescent Dyes; Gramicidin; Humans; In Vitro Techniques; Indicators and Reagents; Leukocytes, Mononuclear; Membrane Potentials; Models, Biological; Valinomycin

Mitochondrial membrane potential, in situ, is an important indicator of mitochondrial function and dysfunction. Because of recent interest in the role of mitochondria in signaling, cell injury and cell death, there is a need for a convenient, sensitive and accurate method for the measurement of the mitochondrial membrane potential, Deltapsim, in situ, in a heterogeneous cell population. We have adapted a flow cytometry method for the quantitative measurement of DeltaPsim which utilizes the lipophilic, cationic, fluorescent probe 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)). We developed a new protocol in which cells are equilibrated with very low dye concentrations (<1 nM). Only under these condition, the cell fluorescence appears to be correlated with the magnitude of DeltaPsim, as evident from the sensitivity of the fluorescence to low concentrations of uncouplers, ionophores and inhibitors of the mitochondrial proton pumps. The magnitude of the plasma membrane potential, DeltaPsip, also affects cell fluorescence, and a procedure that corrects for this effect is outlined. This method offers a distinct advantage over existing methods for estimation of Deltapsim by flow cytometry.

Topics: Animals; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Count; Cell Respiration; Flow Cytometry; Fluorescent Dyes; Intracellular Membranes; Lymphocytes; Membrane Potentials; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mitochondria; Oligomycins; Potassium Chloride; Proton Pump Inhibitors; Rotenone; Spleen; Valinomycin

Cyclosporin A (CsA) produced dose-dependent membrane depolarization of human peripheral blood lymphocytes. The phenomenon was investigated applying the membrane potential probe dihexyloxacarbocyanine iodide in a flow cytometer in combination with ionophores, hormones and monoclonal antibodies binding to different subclasses of lymphocytes and the anti-interleukin 2 receptor antibody. Human interferon-gamma abolished the depolarizing effect of cyclosporin on lymphocytes. Interleukin 2 caused depolarization and also enhanced the effect of CsA. OKT4 and OKT8 monoclonal antibodies slightly hindered depolarization by CsA while OKT3, OKT11 and OKIa1 antibodies had no such effect. Valinomycin decreased CsA's effect on the membrane potential while the ionophore A-23187 and ionomycin caused depolarizations that were additive with CsA's. CsA treatment released the isotope from 42K-loaded human lymphocytes in a dose-dependent fashion. CsA addition increased intracellular calcium content. CsA decreased the motional freedom of a spin probe in the membrane, but did not hinder the binding of fluoresceinated antibodies to the cell surface. These results suggest immediate alteration in membrane structure upon CsA treatment, causing potassium leakage and calcium ion uptake. These are the earliest detected effects of CsA on cells so far.

Topics: Antibodies, Monoclonal; Calcium; Carbocyanines; Cell Membrane; Cyclosporins; Cytoplasm; Dimethyl Sulfoxide; Electron Spin Resonance Spectroscopy; Flow Cytometry; Humans; Interferon-gamma; Interleukin-2; Intracellular Membranes; Ion Channels; Lymphocytes; Membrane Fluidity; Membrane Potentials; Potassium Radioisotopes; Spectrometry, Fluorescence; Valinomycin

Potential-dependent accumulation of the lipophilic cationic dye 3,3' dihexyloxacarbocyanine (DiOC6(3)) in macrophages has been investigated. Resulting fluorescence of cells was measured by flow cytometry. Alterations of membrane potential of macrophages were induced by ionophore treatment (valinomycin and gramicidin) in a dose-dependent (10(-5) M-10(-7) M) and time-dependent (0 min-45 min) manner. Resulting changes in relative fluorescence intensity were compared with changes of transmembrane potential measured by intracellular recordings obtained by applying glass microelectrodes. The comparative studies offer the possibility to calibrate the flow cytometric estimate of membrane potential of suspended cells. Equilibration of dye partition between cells and surrounding medium is strictly potential-dependent at dye concentrations between 5 X 10(-8) M and 10(-7) M and within an incubation interval from 10 min up to 30 min after addition of dye. Conclusions are drawn concerning the field of application of the optical method. Dynamics of electrical processes following ionophore treatment are discussed in terms of molecular mechanisms of altered ionic transport.

Topics: Animals; Carbocyanines; Flow Cytometry; Gramicidin; Guinea Pigs; Kinetics; Macrophages; Membrane Potentials; Microelectrodes; Valinomycin

Some quaternary ammonium derivatives of ellipticine are active antitumor drugs on both experimental and human tumors. Because of their positive charge, the cellular uptake of these molecules is expected to be influenced by the electric membrane potential. Experimental variations of the potential were produced by changing the external potassium concentration and the potassium permeability by the addition of valinomycin. Using the fluorescent lipophilic cationic dye 3,3-dihexyloxacarbocyanine iodide, the L1210 cell membrane potential was estimated at -35 mV by flow cytometric analysis, and the same technique was then used to study the effects of the membrane potential variations on 2-N-methyl-ellipticinium (NME) cellular uptake. Our results show that indeed NME uptake depends on the cell membrane potential, which might then influence its pharmacological properties.

Topics: Alkaloids; Animals; Carbocyanines; Cell Line; Coloring Agents; Ellipticines; Flow Cytometry; Leukemia L1210; Membrane Potentials; Mice; Potassium; Valinomycin