A new method of quantifying glutathione levels in freshly isolated single superfused rat cardiomyocytes.

摘要: Introduction: Glutathione (GSH) is an important antioxidant in the heart whose content changes during cardiac insults. However, there are currently no methods for continuously monitoring free cytoplasmic GSH levels single isolated and superfused cardiomyocytes exposed to normal pathological conditions. Methods: was measured using CellTracker™ Blue CMAC (Molecular Probes), a member of new family thiol-sensitive dyes. The fluorescence 5 μM various solutions containing glutathione-S-transferase freshly inverted microscope. were by standard procedures loaded with either or monochlorobimane 15 min shaking incubation dark at room temperature followed centrifugation resuspension cells dye-free media. Cell volume calculated from 3H2O [14C]sucrose space. Results: linearly proportional 0–100 GSH, as described equation: Y=182.2 (X)+681.6 (r2=.99, P<.001). Fluorescence not affected changing level, calcium concentration, pH, neither quenched H2O2 cyanide. Exposure oxidative stress presence 0–1 mM caused progressive decrease cellular GSH. In contrast, brief exposure metabolic inhibition 2.5 NaCN evoked significant increase cardiomyocyte return control washoff. comparison monochlorobimane, gave stronger signal better retention probe. Discussion: These results suggest that will be good tool measuring because it shows expected inhibition, reversible.