Small Changes of Cytosolic Sodium in Rat Ventricular Myocytes Measured with SBFI in Emission Ratio Mode

The spectral properties of SBFI (sodium-binding benzofurzan isophthalate) were re-examined to arrive at a more specific and sensitive method to measure small changes of intracellular sodium ([Na+]i) particularly at low concentration. Relative to spectra of SBFI in protein- and cell-free solution, binding of SBFI to intracellular proteins caused a shift of excitation and emission spectra, and increased quantum efficiency. Excitation of SBFI at 340 nm caused an exclusively sodium-dependent fluorescence from 400–420 nm, and hardly any change of fluorescence above 530 nm upon replacing sodium by potassium. Due to these spectral and quantum efficiency changes, SBFI excitated at 340 nm can be used in a dual emission ratio mode to measure [Na+]i. In dual emission ratio mode (410 and 590 nm, respectively), the fluorescence ratio increased by a factor of 13 upon replacing sodium for potassium. The apparent equilibrium constant measured in single isolated rat ventricular myocytes was 22.5±0.3 mmol/l. Control [Na+]iwas 9.6±0.4 mmol/l. After abrupt reduction of extracellular sodium from 156 to 29 or 11 mmol/l, [Na+]idecreased mono-exponentially to 2.5±0.3 and 1.9±0.3 mmol/l, respectively, with a rate constant of about 0.02/s. We conclude that SBFI used in dual emission mode provides a more sensitive and more specific method to measure small changes of [Na+]iin single myocytes down to cytosolic sodium concentration as low as about 1 mmol/l.