4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and trifluoroacetamide

Isolated beating rat hearts were perfused with trifluoroacetamide (TFM) and trifluoroacetate (TFA) and monitored by 19F-nuclear magnetic resonance (NMR). The average membrane TFA potential in spontaneously beating rat hearts, calculated according to standard principles assuming that TFA is distributed in its anionic form, was found to be -36.2 +/- 3.2 mV (n = 9) under normoxic conditions. In separate experiments, the chloride and potassium potentials were determined to be -38.5 +/- 3.6 mV (n = 7) and -85.3 +/- 3.3 mV (n = 7), respectively, from freeze-clamped heart tissue. In the presence of the anion-exchange inhibitor, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), TFA uptake into heart was significantly reduced, suggesting that TFA uptake occurs partly via the Cl(-)-HCO3- exchanger. Based on these results and the results of R. E. London and S. A. Gabel (Biochemistry 28: 2378-2382, 1989), we conclude that the distribution of TFA in hearts reflects the chloride potential (ECl) and not the membrane potential. A time-dependent change in the ECl occurs during global ischemia, and changes in ECl were also observed when the hearts were perfused with high concentrations of KCl. These results demonstrate that 19F-NMR may be utilized to monitor the ECl of perfused hearts under a variety of conditions.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetamides; Animals; Chlorides; Coronary Disease; Edetic Acid; Fluoroacetates; Heart; In Vitro Techniques; Magnetic Resonance Spectroscopy; Male; Membrane Potentials; Myocardium; Perfusion; Rats; Rats, Sprague-Dawley; Trifluoroacetic Acid

The distribution of ionic species between intra- and extracellular compartments forms one basis for the determination of cell membrane potential. It is shown that fluorine-19 NMR studies of erythrocytes in the presence of trifluoroacetate, a stable, relatively nontoxic anion with pK = -0.3, provide a sensitive probe of membrane potential. Since such measurements are based on ion concentrations, the parallel use of the neutral analogue trifluoroacetamide to provide information on intra/extracellular volume ratios was also explored. In both cases, separate 19F resonances corresponding to intra- and extracellular ions were observed, with the intracellular resonance shifted downfield by approximately 0.2 ppm and the intracellular peak typically somewhat broader than the extracellular resonance. Studies with the band 3 anion-exchange inhibitor 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) indicate that both transmembrane diffusion and flux involving the band 3 anion exchanger contribute to the observed transport of the trifluoroacetate anion. Intra/extracellular volume ratios determined on the basis of trifluoroacetamide intensity ratios were in good agreement with determinations based on measured hematocrits. On the basis of the high sensitivity of 19F NMR and the capability of monitoring volume changes simultaneously, the time resolution for these measurements can approach the lifetime of intracellular trifluoroacetate ions and hence be limited by the trifluoroacetate flux rate.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetamides; Erythrocyte Membrane; Erythrocytes; Fluorine; Fluoroacetates; Humans; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Membrane Potentials; Trifluoroacetic Acid