sq-23377 and bis(1-3-dibutylbarbiturate)trimethine-oxonol

Quantitative determinations of the cell membrane potential of lymphocytes (Wilson et al., J Cell Physiol 1985;125:72-81) and thymocytes (Krasznai et al., J Photochem Photobiol B 1995;28:93-99) using the anionic dye DiBAC4 (3) proved that dye depletion in the extracellular medium as a result of cellular uptake can be negligible over a wide range of cell densities. In contrast, most flow cytometric studies have not verified this condition but rather assumed it from the start. Consequently, the initially prepared extracellular dye concentration has usually been used for the calculation of the Nernst potential of the dye. In this study, however, external dye depletion could be observed in both large IGR-1 and small LCL-HO cells under experimental conditions, which have often been applied routinely in spectrofluorimetry and flow cytometry. The maximum cell density at which dye depletion could be virtually avoided was dependent on cell size and membrane potential and definitely needed to be taken into account to ensure reliable results. In addition, accepted calibration procedures based on the partition of sodium and potassium (Goldman-Hodgkin-Katz equation) or potassium alone (Nernst equation) were performed by flow cytometry on cell suspensions with an appropriately low cell density. The observed extensive lack of concordance between the correspondingly calculated membrane potential and the equilibrium potential of DiBAC4 (3) revealed that these methods require the additional measurement of cation parameters (membrane permeability and/or intracellular concentration). In contrast, due to the linear relation between fluorescence and low DiBAC4 (3) concentrations, the Nernst potential of the dye for totally depolarized cells can be reliably used for calibration with an essentially lower effort and expense.

Topics: Algorithms; Barbiturates; Calcium Ionophores; Calibration; Cell Line; Cell Membrane Permeability; Flow Cytometry; Fluorescent Dyes; Gramicidin; Humans; Ionomycin; Isoxazoles; Membrane Potentials; Models, Biological; Staining and Labeling

Guanosine 5' triphosphate (GTP), acting synergistically with the nerve growth factor (NGF), enhances the proportion of neurite-bearing cells in cultures of PC12 rat pheochromocytoma cells. We studied the transduction mechanisms activated by GTP in PC12 cells and found that addition of GTP (100 microM) increased intracellular calcium concentration ([Ca(2+)](i)) in cells that were between 60 and 70% confluent. Addition of GTP also enhanced activation of NGF-induced extracellular regulated kinases (ERKs) and induced Ca(2+) mobilization. This mobilization, due to the activation of voltage-sensitive and ryanodine-sensitive calcium channels, as well as pertussis toxin-sensitive purinoceptors, modulates Ca(2+)-activated K(+) channels not involved in activation of ERKs. The results presented here indicate that GTP-triggered [Ca(2+)](i) increase may be a key event in GTP signal transduction, which can modulate activity of ERKs. The physiological importance of the GTP effect lies in its capacity to interact with the NGF-activated pathway to enhance neurite outgrowth from PC12 cells.

Topics: Animals; Barbiturates; Blotting, Western; Calcium; Calcium Channel Blockers; Cell Count; Cell Differentiation; Chelating Agents; Clotrimazole; Diagnostic Imaging; Dose-Response Relationship, Drug; Drug Synergism; Egtazic Acid; Enzyme Inhibitors; Extracellular Space; Fluorescent Antibody Technique; Fluorescent Dyes; Gallic Acid; Growth Inhibitors; Guanosine Triphosphate; Ionomycin; Ionophores; Isoxazoles; Membrane Potentials; Microscopy, Confocal; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nerve Growth Factor; Neurites; Nifedipine; PC12 Cells; Pertussis Toxin; Pyridoxal Phosphate; Rats; Signal Transduction; Suramin; Time Factors; Triazines

CD3 mAb induced calcium movements are unaffected by hyperpolarization of the membrane in Jurkat T cells treated with valinomycin. By contrast, the CD3 induced Ca2+ influx was impaired by depolarization of the membrane with either gramicidin or by equimolar substitution of KCl for NaCl in the medium. In depolarized cells, the synthesis of phosphatidylserine was strongly diminished as a result of impaired transport of the [3H]serine substrate. In depolarized cells, the CD3-induced release of Ca2+ from intracellular stores (endoplasmic reticulum) was unaffected. Emptying of the Ca2+ stores by CD3 was shown by the lack of effect of additional treatment of the cells with the Ca2+ ionophore, ionomycin. The empty status of the calcium stores was also confirmed by measurements of phosphatyidylserine synthesis through the Ca2+ -dependent base exchange enzyme system that was found to be significantly decreased despite the low amount synthesized in the presence of a defective [3H]serine transport in depolarized cells.

Topics: Antibodies, Monoclonal; Barbiturates; Calcium; CD3 Complex; Fluorescent Dyes; Gramicidin; Humans; Indoles; Ionomycin; Ionophores; Isoxazoles; Jurkat Cells; Membrane Potentials; Phosphatidylserines; T-Lymphocytes; Valinomycin