THE USE OF THE POTENTIAL-SENSITIVE FLUORESCENT-PROBE BISOXONOL IN MAST-CELLS

The regulation of the plasma membrane potential of rat peritoneal mast cells at the resting state and during activation was investigated using bisoxonol as a potential-sensitive fluorescent dye. Fluorescence microphotography showed that this negatively charged probe was not only present in the plasma membrane, but was also distributed in the cytoplasm. The intracellular localization of bisoxonol was confirmed by conducting experiments which showed that bisoxonol fluorescence was not enhanced in ATP-permeabilized mast cells. Rotenone (10(-7) M) and oligomycin (10(-6) M) did not change the fluorescence of bisoxonol showing, therefore, that mitochondrial depolarization was not recorded with bisoxonol and suggesting that bisoxonol may represent a useful probe to study plasma membrane potential changes in the absence of exocytosis. We showed that, in non-stimulated mast cells, the blockade of the sodium pump enhanced the fluorescence of bisoxonol as did gramicidin a non selective ionophore used to fully depolarize the cells. High concentration of potassium (30 mM) as well as different ionic channel blockers did not significantly change the fluorescence intensity of bisoxonol, suggesting that ionic channel permeabilities were not involved in maintaining the resting plasma membrane potential of mast cells. Mast cells stimulated by compound 48/80 completely lost the fluorescence, shown by fluorescence microphotography, suggesting that exocytotic phenomena might induce a dye redistribution which is not only due to changes in the plasma membrane potential. In mast cells pretreated with pertussis toxin, which blocks mast cell-exocytosis, compound 48/80 induced a delayed (2 min) decrease of bisoxonol fluorescence which was shown to be dependent on the activity of the sodium pump. Considering that bisoxonol is a useful potential-sensitive probe in exocytosis-deprived mast cells, our results suggest that the sodium pump is mainly involved in the changes of plasma membrane potential of mast cells.