Gramicidin D and dithiothreitol effects on erythrocyte exovesiculation

The use of either diphenylhexatriene, trimethylamino-diphenylhexatriene, or heptadecyl-hydroxycoumarin (C17-HC) allows, simultaneously and with the same molecule, the induction of erythrocyte exovesiculation and labeling of the released vesicles with the fluorescent probe. This method was used to evaluate gramicidin D (a channel-forming peptide) and dithiothreitol (a reducing agent) effects on the human erythrocytes vesiculation process. The release of cholesterol and phospholipids in exovesicles at longer incubation times was only detectable in the presence of gramicidin or dithiothreitol. When C17-HC was used to induce the vesiculation, the presence of gramicidin or dithiothreitol lead to a drastic decrease on the [phospholipids]/[cholesterol] ratio. However, in the samples with dithiothreitol, this variation did not result in the expectable decrease of membrane fluidity. These effects can be related with the presence of lipid rafts, the transbilayer lipids reorientation induced by gramicidin or dithiothreitol, and the cholesterol-dependent gramicidin channels inactivation.