The relation between dicarbocyanine dye fluorescence and the membrane potential of human red blood cells set at varying Donnan equilibria.

Abstract

The fluorescence, F, of two dicarbocyanine dyes, diS-Ca(5) and diI-Ca(5), depends both on the membrane potential, E, and on the intracellular pH, pile, of human red blood cells. Compositions of isotonic media have been devised in which the equilibrium Donnan potential, E, varies at constant pHc and in which pHc varies at constant E. Dye fluorescence measurements in these suspensions yield calibrations of + 1.7 %6~F/mV for diS-Ca(5) and + 0.6 %~tF/ mV for diI-Ca (5). While pHo does not affect F of either dye, changes in pHc of 0. I unit at constant E cause changes of F equivalent to those induced by 2-3 mV. Based on these results, a method is given for estimating changes in E from dye fluorescence in experiments in which E and pile co-vary. The relation of F to E also depends in a complex way on the type and concentration of cells and dye, and the wavelengths employed. The equilibrium calibration of dye fluorescence, when applied to diffusion potentials induced by 1 /tM valinomycin, yields a value for the permeability ratio, PK.VAL/Po, of 20 + 5, in agreement with previous estimates by other methods. The calibration of F is identical both for diffusion potentials and for equilibrium potentials, implying that diS-Ca(5) responds to changes in voltage independently of ionic fluxes across the red cell membrane. Changes in the absorption spectra of dye in the presence of red cells in response to changes in E show that formation of non fluorescent dimers contributes to fluorescence quenching of diS-Ca(5). In contrast, only a hydrophobic interaction of dye monomers need be considered for diI-Ca(5), indicating the occurrence of a simpler mechanism of fluorescence quenching.

The Escherichia coli Enterotoxin STb Permeabilizes Piglet Jejunal Brush Border Membrane Vesicles

“…However, in the N-methyl-D-glucamine hydrochloride solution, both in the presence and in the absence of STb, fluorescence levels decreased gradually as pH was increased. This result is consistent with earlier studies indicating that, although the fluorescence of diS-C 3 (5) is independent of pH in solution, it is strongly pH dependent in the presence of cells or cell membranes (13,21,23,41). The pattern illustrated in Fig.…”

“…This result deviates from the linear relationship predicted by the Nernst equation, although a straight line can be fitted reasonably well to the data for ratios of 1 to approximately 10. At higher concentration ratios, however, the slope of the curve increases sharply before starting to level off gradually as predicted by the Goldman-Hodgkin-Katz equation which, in contrast with the Nernst equation, takes into account the permeability of the (8,13,22). Interestingly, linear relationships were reported for experiments performed with rabbit renal brush border membrane vesicles but, in these studies, the potassium concentration ratio was only varied from 1 to 10 (6, 43).…”

Volume, pH, and ion-content regulation in human red cells: Analysis of transient behavior with an integrated model

“…It can be seen that when the diffusional anion permeability is about a hundred times that reported for CI-, the anion permeability is no longer limiting to the rate of K loss. The diffusional anion permeability determines not only the rates of K and A loss, but also the pattern of change in membrane potential [28,63]. With normal anion permeability, hyperpolarization is maximal at the onset of the K permeability increase, as a result of the increased Goldman potential determined by the new ratio of the increased diffusional permeability of K to that of A.…”

The activity of powdery-mildew haustoria after feeding the host cells with different sugars, as measured with a potentiometric cyanine dye

“…At high concentrations, generally in a range of 10 -4 M, further uptake of dye into the membranes results in a quenching of fluorescence. This reduction of fluorescence can be explained by the formation of nonfluorescent di-or oligomers of the dye or by hydrophobic interaction between monomers of the dyes (Freedman and Hoffman 1979;Guillet and Kimmich 1981 ;Emaus et al 1986). Therefore, the interpretation of an increase or decrease of fluorescence should be made with care.…”