Application of resonance raman spectroscopy on manganese porphyrin detection and redox state in endothelial cells: implications on cytoprotection towards peroxynitrite

Manganese(III) ortho N-substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can reach mitochondria offering protection against reactive species in different animal models of disease. In this scenario, we applied Resonance Raman (RR) spectroscopy as a direct method to detect both the uptake and the redox state of MnP in living cells. Using RR we were able to analyze the intracellular redox state changes of two potent MnP (MnTnBuOE-2-PyP 5+ and MnTnHex-2-PyP 5+ ) within endothelial cells exposed to SIN-1. RR experiments with cells and isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. We also evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems.