Is the Mitochondrial Membrane Potential ( increment ψ) Correctly Assessed? Intracellular and Intramitochondrial Modifications of the increment ψ Probe, Rhodamine 123

The mitochondrial membrane potential ( increment psi) is the driving force providing the electrical component of the total transmembrane potential of hydrogen ions generated by proton pumps, which is utilized by the ATP synthase. The role of increment psi is not limited to its role in bioenergetics since it takes part in other important intracellular processes, which leads to the mandatory requirement of the homeostasis of increment psi. Conventionally, increment psi in living cells is estimated by the fluorescence of probes such as rhodamine 123, tetramethylrodamine, etc. However, when assessing the fluorescence, the possibility of the intracellular/intramitochondrial modification of the rhodamine molecule is not taken into account. Such changes were revealed in this work, in which a comparison of normal (astrocytic) and tumor (glioma) cells was conducted. Fluorescent microscopy, flow cytometry, and mass spectrometry revealed significant modifications of rhodamine molecules developing over time, which were prevented by amiodarone apparently due to blocking the release of xenobiotics from the cell and their transformation with the participation of cytochrome P450. Obviously, an important role in these processes is played by the increased retention of rhodamines in tumor cells. Our data require careful evaluation of mitochondrial increment psi potential based on the assessment of the fluorescence of the mitochondrial probe.

Automated summary

The mitochondrial membrane potential plays a crucial role in bioenergetics and other intracellular processes. It is traditionally estimated by the fluorescence of probes, but the possible modifications of fluorescent molecules in cells are often overlooked. This study reveals significant changes in rhodamine molecules and suggests the importance of evaluating mitochondrial membrane potential with caution.