Quantification of intracellular free sodium ions by using a new fluorescent indicator, sodium-binding benzofuran isophthalate in guinea pig myocytes

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Abstract

Isolated guinea pig myocytes were loaded with the Na+-sensitive fluorescent probe, sodium-binding benzofuran isophthalate (SBFI). The 340380nm fluorescence ratios were measured with fluorescence microscopy. The distribution of intracellular Na+ concentration ([Na+]i) was homogenous, and the mean resting [Na+]i was 8.4 ± 0.5 mM. There was a significant relationship (r=0.66, p<0.001) between elevation of [Na+]i and shortening of longitudinal length of the cells, during the perfusion of 100 μM strophanthidin. It is concluded that this method is suitable for measuring [Na+]i in isolated myocytes.

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    The mean fluorescence signals from the 10 SBFI-unloaded wells at 340 nm and 380 nm were subtracted from the individual signals of the SBFI-loaded wells at each wavelength [22]. The in vivo calibration of SBFI was accomplished, similar to that described in previous reports, by exposing the cardiomyocytes to various concentrations of extracellular [Na+] (0–20 mM) in the presence of 1 mg/l Gramicidin D (MP Biomedicals), 100 μM of strophanthidin (Sigma-Aldrich) and 2 mM EGTA, and the pH was adjusted to 7.1 with Tris base [22–25]. The solutions were prepared by mixing two solutions of equal ionic strength.

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