digitonin and 6-carboxyfluorescein

The effects of the glycoalkaloids alpha-solanine, alpha-chaconine and alpha-tomatine on different cell types were studied in order to investigate the membrane action of these compounds. Hemolysis of erythrocytes was compared to 6-carboxyfluorescein leakage from both ghosts and erythrocyte lipid vesicles, whereas leakage of enzymes from mitochondria and the apical and baso-lateral side of Caco-2 cells was determined. Furthermore, the effects of glycoalkaloids on the gap-junctional communication between Caco-2 cells was studied. From these experiments, it was found that glycoalkaloids specifically induced membrane disruptive effects of cholesterol containing membranes as was previously reported in model membrane studies. In addition, alpha-chaconine was found to selectively decrease gap-junctional intercellular communication. Furthermore, the glycoalkaloids were more potent in permeabilizing the outer membrane of mitochondria compared to digitonin at the low concentrations used.

Topics: Adenylate Kinase; Animals; Caco-2 Cells; Cell Membrane; Cell Membrane Permeability; Cholesterol; Digitonin; Erythrocyte Membrane; Erythrocytes; Fluoresceins; Fluorescent Dyes; Gap Junctions; Hemolysis; Humans; L-Lactate Dehydrogenase; Male; Mitochondria, Liver; Rats; Rats, Wistar; Solanaceous Alkaloids

Fluorescein and carboxyfluorescein have found recent application as probes of intracellular pH. The present study examines several parameters required for interpretation of the spectral information derived from fluorescein and carboxyfluorescein generated intracellularly from their permeant diacetate derivatives. Coefficients were determined for the pH dependence of the difference absorbance, of the absorbance ratios, and of the fluorescence emission intensity ratios at selected wavelength pairs for carboxyfluorescein in aqueous buffers. The effect of light scattering on the apparent pH reported by carboxyfluorescein in dilute cell suspensions was assessed. An apparent intracellular acidification associated with increasing internal dye concentration was found to result probably from interactions of the intracellular probe with itself. Working within the experimental limitations imposed by these considerations, protocols utilizing either direct measurement of absorbance or fluorescence or determination of the null spectral response observed upon release of internal carboxyfluorescein all indicate that the cytosolic space of bovine epididymal sperm is maintained at pH 6.5-6.6. The monovalent-cation-specific, carboxylic acid ionophores, nigericin and monensin, were utilized to produce transmembrane proton gradients in cells that were allowed to generate intracellular carboxyfluorescein in a preliminary incubation, then resuspended in media buffered at the same pH as the sperm cytosol but of varying cation composition. By interpolation to the null response, the initial internal Na+ and K+ concentrations in bovine sperm were estimated as 14 +/- 2 and 120 +/- 5 mM, respectively. The ability of initial transmembrane gradients of either protons or of monovalent cations to promote equivalent changes in internal pH following ionophore addition to sperm suspensions supports application of a simple model that predicts steady state cation distributions. With the assumption that the cytosolic proton buffer has a pKa near the determined internal pH, these experiments allow the additional calculation that this buffer is present at a concentration of 190 +/- 20 meq/liter in the bovine sperm.

Topics: Animals; Cations, Monovalent; Cattle; Cytosol; Digitonin; Fluorescein; Fluoresceins; Hydrogen-Ion Concentration; Light; Male; Mitochondria; Models, Biological; Monensin; Nigericin; Potassium; Scattering, Radiation; Sodium; Spectrometry, Fluorescence; Spectrophotometry; Spermatozoa