The Fluorescent Cationic Dye Rhodamine 6G as a Probe for Membrane Potential in Bovine Aortic Endothelial Cells

The membrane potential of cultured bovine aortic endothelial cells was assessed by a fluorescent probe as an alternative to direct methods. We used the fluorescent cationic dye rhodamine 6G, a lipophilic probe with high permeability in cell membranes. A linear relationship was obtained between fluorescence intensity (F.I.) and membrane potential (Em) as a function of the extracellular Na+ concentration in the presence of the ionophore gramicidin. From the equation derived from the linear relationship F.I. = −0.004 Em + 0.03 (P < 0.001), the fluorescence measurements could be converted to membrane potential. The resting plasma membrane potential obtained was −65 ± 7 mV. Nigericin (27 μM), ouabain (1 mM), and bradykinin (20 nM) induced a decrease in F.I. (depolarization), while ATP (25–100 μM) induced an increase in F.I. (hyperpolarization). Mitochondrial membrane potential inhibitors myxothiazol (3 μM) and oligomycin (4 μM) did not influence F.I. measured in the cultured bovine aortic endothelial cells. The results indicate that rhodamine 6G can be used as a sensitive and specific dye in studies of substances that affect the membrane potential of endothelial cells.