valinomycin and Leukemia--Erythroblastic--Acute

Dielectrophoresis measurements obtained using an image processing technique are reported over the frequency range 1 Hz to 100 kHz for the Friend murine erythroleukemia cell lines DS19 and R1 before and after treatment with hexamethylene bisacetamide and dimethylsulfoxide, agents that induce terminal differentiation in DS19 but not in R1 cells. Data are analyzed according to the single shell dielectric model of the cell. The membrane capacitance was found to fall by 30% and membrane conductivity by a factor of at least 5 when DS19 cells were induced to differentiate. R1 cells showed no such response. While the theoretical model was found to be useful for comparing differences in data for the different cell lines, several significant discrepancies between its predictions and the experimental data were observed, including positive dielectrophoretic collection at frequencies below 20 Hz and a smaller than predicted response to the membrane permeabilizing agents saponin and valinomycin. Factors that may have accounted for these discrepancies include surface charge effecgs, conduction parallel to the plasma membrane surface, and intracellular compartments.

Topics: Cell Differentiation; Cell Line; Cell Membrane; Electric Conductivity; Electrophoresis; Leukemia, Erythroblastic, Acute; Membrane Potentials; Saponins; Valinomycin

This study deals with the modulation of the plasma membrane potential (delta psi p) of murine erythroleukemia (MEL) cells by cell-substratum or cell-cell contact. delta psi p was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane; it appeared strongly, and inversely, influenced by the two types of cell contacts. Contact with the culture surface produced a delta psi p hyperpolarization directly proportional to average distance among the ideal centers of the cells on this surface (d) within the range 10-80 microns. A detailed mathematical analysis of the function delta psi p = f(d) is presented, as well as experiments involving the use of ionophores (valinomycin and A23187) and the conditioning of the culture surface. We concluded that the d-dependent hyperpolarization (dDH) was the result of a complex interplay between the activating properties of substratum on Ca2+-dependent K+ channels (KCa) and some substratum-adherent factors that are shed by MEL cells and antagonize KCa activation (substratum-attached cellular factors = SACF). By contrast, contact of the cells with each other, obtained by incubating MEL cells at d smaller than the average cell diameter (phi = 10 microns), produced a marked delta psi p depolarization. This intercellular contact-dependent depolarization (ICDD) was unaffected by valinomycin; it was abolished by substituting Na+ in the external medium with a nondiffusible cation (choline), which shows that ICDD was sustained by Na+ influxes, probably mediated by stretch-activated (s.a.) cation channels.

Topics: Animals; Biomechanical Phenomena; Calcimycin; Calcium; Cations; Cell Communication; Cell Membrane; Cells, Cultured; Electrophysiology; Extracellular Space; Ion Channels; Ions; Leukemia, Erythroblastic, Acute; Mice; Potassium Channels; Sodium; Valinomycin

The role of the plasma membrane potential (delta psi p) in the commitment to differentiation of murine erythroleukemia (MEL) cells has been studied by analyzing the ionic basis and the time course of this potential in the absence or the presence of different types of inducers. delta psi p was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane and displayed a 22-hour depolarization phase (from -28 to +5 mV) triggered by factors contained in foetal calf serum (FCS) and followed by a nearly symmetrical repolarization phase. After measuring the electrochemical equilibrium potential of Na+, K+, and Cl-, the relative contribution of these ions to delta psi p was evaluated by means of ion substitution experiments and by the addition of ion flux inhibitors (tetrodotoxin [TTX], 4-acetoamide-4'-isothiocyanostilbene-2,2'-disulfonate [SITS]) and ionophores (Valinomycin, A23187). The Na+ contribution to delta psi p appeared negligible, the potential being essentially generated by K+ and Cl- fluxes. When evaluated by a new mathematical approach, the effects of Valinomycin and A23187 at different times of incubation provided evidence that both the depolarization and the repolarization phase were due to variations of the K+ permeability across the plasma membrane (PK) mediated by Ca2+-activated K+ channels. All the inducers tested (dimethylsulfoxide [DMSO], hexamethylen-bis-acetamide [HMBA], diazepam), although they did not modify the ionic basis of delta psi p, strongly attenuated the depolarization rate of this potential. This attenuation was not brought about when the inducers were added to noninducible MEL cell clonal sublines. Cell commitment occurred only during the depolarization phase and increased proportionally to the attenuation of this phase up to a threshold beyond which the further increase of the attenuation was associated with the inhibition of commitment. The major role of the inducers apparently consisted of the stabilization of the Ca2+-activated K+ channels, suggesting that a properly modulated delta psi p depolarization through these channels is primarily involved in the signal generation for MEL cell commitment to differentiation.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetamides; Animals; Calcimycin; Calcium; Cell Differentiation; Cell Membrane Permeability; Chlorides; Diazepam; Dimethyl Sulfoxide; Electrochemistry; Ion Channels; Leukemia, Erythroblastic, Acute; Mathematics; Membrane Potentials; Mice; Potassium; Sodium; Tetraethylammonium Compounds; Tetrodotoxin; Time Factors; Valinomycin

The plasmamembrane potential (delta psi p) of murine erythroleukemia (MEL) cells has been determined by measuring the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) across the plasmamembrane. TPP+ accumulation within the cells (experimental accumulation ratio, AR exp) was measured by adding 2 microM TPP+ directly to the culture flasks, avoiding any other perturbation of the experimental system. The mitochondrial contribution to AR exp, evaluated by adding carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or 2,4-dinitrophenol (DNP), was apparently negligible in standard cultures, AR exp being substantially the same in either the absence or presence of these uncouplers. However, the addition of oligomycin produced a strong AR exp enhancement, which was abolished by FCCP, suggesting that MEL cell mitochondria are in state 3. The aspecific TPP+ binding was estimated by a new mathematical approach worked out to fit AR exp values measured in the presence of valinomycin at various extracellular K+ concentrations and plotted against the ratio of intracellular to extracellular K+ concentration ([K+]i/[K+]e). This binding was found to be close to zero in MEL cells. By applying the Nernst equation directly to AR exp values, delta psi p of these cells was then measured; this potential varying from -65 mV to -16 mV (inside negative) is inversely related to the cell density on the culture surface on which the cells sediment (cells/cm2; CD). The dependence of delta psi p on CD is practically eliminated by valinomycin and appears to be related to a cell interaction with the culture surface of the flasks, suggesting that in the immediate environment of MEL cells one or more factors are produced that modulate the K+ plasma membrane permeability (PK).

Topics: Animals; Cell Count; Cell Line; Cell Membrane Permeability; Culture Media; Leukemia, Erythroblastic, Acute; Mathematics; Membrane Potentials; Mice; Mitochondria; Onium Compounds; Organophosphorus Compounds; Potassium; Valinomycin