gramicidin-a and bis(1-3-diethylthiobarbiturate)trimethineoxonol

The short-term (20-minute) action of beta[1-40]-amyloid on the resting transmembrane potential was investigated by means of flow-cytofluorimetric studies in M26-1F cells, an immortalized rat striatal cell line, using the potential-sensitive fluorescent probe bis-oxonol. The distribution of the individual cell-associated probe fluorescence was found to be shifted to lower levels in cells treated with beta-amyloid[1-40] for 20 minutes as compared with that of their untreated counterparts. A change in the same direction was caused by valinomycin, a hyperpolarizing ionophore, whereas gramicidin, a depolarizing ionophore, induced a shift to higher fluorescence intensities. These findings, together with the reported behaviour of this particular fluorescent probe at different transmembrane potential levels, indicate that beta-amyloid[1-40] is capable of inducing early hyperpolarization in M26-1F cells. This is one of the earliest cell physiological effect of beta-amyloid peptides that has been reported so far. Moreover, our findings indicate an ionophore-like action of amyloid peptides.

Topics: Alleles; Amyloid beta-Peptides; Animals; Anti-Bacterial Agents; Antigens, Polyomavirus Transforming; Cell Line, Transformed; Cell Polarity; Corpus Striatum; Diffusion; Flow Cytometry; Fluorescent Dyes; Gramicidin; Ionophores; Membrane Potentials; Peptide Fragments; Rats; Temperature; Thiobarbiturates; Valinomycin

We tested the hypothesis that membrane depolarization may initiate oxidant generation in the endothelial cell. Depolarization was produced in bovine pulmonary arterial endothelial cells (BPAEC) in monolayer culture with varying external K+, or with glyburide (10 microM), tetraethylammonium (TEA, 10 mM), gramicidin (1 microM), or nigericin (2 microM). Evaluation of bisoxonol fluorescence of BPAEC indicated concentration-dependent depolarization by high K+ (2% change in fluorescence/mV change in membrane potential in the 5.9-48 mM range of K+) and essentially complete depolarization with glyburide. Generation of oxidants was assessed with o-phenylenediamine dihydrochloride (o-PD) oxidation in the presence of horseradish peroxidase (HRP). There was a time-dependent increase in o-PD oxidation with 24 mM K+, nigericin, and gramicidin over 2 hours compared with control. In 1 hour o-PD oxidation increased 2.8-fold for 24 mM and 3.7-fold for 48 mM K+ compared with control. Catalase reduced 24 mM K(+)-induced o-PD oxidation by 50%, while Cu/Zn-superoxide dismutase (SOD) abolished the increase. Oxidation of o-PD was reduced by 57% in the absence of HRP in the system. With K+ channel blockade, o-PD oxidation increased 3.8-fold with glyburide and 4.6-fold with TEA compared with control. These data indicate formation of H2O2 and possibly other oxidants with depolarization and suggest involvement of K(+)-channels in this process.

Topics: Animals; Cattle; Cell Membrane; Cells, Cultured; Electrophysiology; Endothelium, Vascular; Fluorescent Dyes; Gramicidin; Nigericin; Oxidants; Phenylenediamines; Potassium; Potassium Channel Blockers; Thiobarbiturates

We studied, in different ionic conditions, the effect of various agents on the membrane potential of rat peritoneal mast cells using the fluorescent probe bisoxonol. Ouabain and ionophore A23187 lead to a fast depolarization of the plasma membrane of mast cells, while compound 48/80 and thapsigargin induced membrane hyperpolarization, which was more pronounced in the case of compound 48/80. When using compound 48/80, the amount of gramicidin necessary to depolarize the cells was twice the amount required in resting cells, which indicates that compound 48/80 increases considerably the activity of the Na+/K+ pump. On the other hand, the ionophore A23187 elicited a clear depolarization which was oblated in the absence of intracellular calcium. The increase in the osmolarity of the medium causes a depolarization in the plasma membrane of mast cells. Hypertonicity-stimulated depolarization is inhibited by removing sodium and potassium.

Topics: Animals; Calcimycin; Calcium-Transporting ATPases; Fluorescent Dyes; Gramicidin; Ions; Mast Cells; Membrane Potentials; p-Methoxy-N-methylphenethylamine; Rats; Rats, Sprague-Dawley; Terpenes; Thapsigargin; Thiobarbiturates