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 asse ssed by a fluorescent probe as an alternative to direct methods. We used th e fluorescent cationic dye rhodamine 6G, a lipophilic probe with high perme ability in cell membranes. A linear relationship was obtained between fluor escence intensity (F.I.) and membrane potential (Em) as a function of the e xtracellular 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 membr ane potential. The resting plasma membrane potential obtained was -65 +/- 7 mV. Nigericin (27 mu M), ouabain (1 mM), and bradykinin (20 nM.) induced a decrease in F.I. (depolarization), while ATP (25-100 mu M) induced an incr ease in F.I. (hyperpolarization). Mitochondrial membrane potential inhibito rs myxothiazol (3 mu M) and oligomycin (4 mu M) did not influence F.I. meas ured in the cultured bovine aortic endothelial cells. The results indicate that rhodamine 6G; can be used as a sensitive and specific dye in studies o f substances that affect the membrane potential of endothelial cells. (C) 1 999 Academic Press.