Hi,
I am studying ROS accumulation in the mitochondria of Leishmania parasites, that are defective in sterol biosynthesis. Staining with MitoSox shows that the mutants accumulate very high level of ROS in the mitochondria, as compared to that of the wild type (WT) parasites. However, when I stained these cells with DHE or H2DCFDA, I did not see much accumulation of ROS into the cytoplasm. Infact staining with DCFDA showed much less signal into the cytoplasm indicating that the peroxide level is probably much less than the WT.
Additionally I measured the mitochondrial membrane potential and the mutants showed higher potential than WT. All together the data look like this:
mutants: higher mitochondrial membrane potential (measured by Rh123, JC-1), swelled mitochondria (by Mitoracker CMXROs using epifluorescene micoscopy and by transmission electron microscopy), higher accumulation of mitochondrial accumulation of ROS (by MitoSox Red), lower accumulation of cellular hydrogen peroxide (by H2DCFDA) and finally higher SOD acitivity from whole cell lysate. All are compared to the WT. My questions are:
1. Could higher membrane potential block export of ROS from mitochondria to cytoplasm?
2. How could higher membrane potential lead to more ROS generation in the mitochondria?
Thank you very much for your suggestions!
Sumit
Dear, Dr. Sumit,
Starting from the second question. Please find one possible mechanism of increased mitochondrial ROS production by increased mitochondrial potential in the paper: https://www.researchgate.net/publication/13861125_KorshunovS_S_SkulachevV_P_Starkov_A_A_High_protonic_potential_actuates_a_mechanism_of_production_of_reactive_oxygen_species_in_mitochondria_FEBS_Lett_416_15-18
But note, that this phenomenon was obtained in islotaed mitochondria. The situation in cell could me much more complicated due to existence of cytoplasmic antioxidant enzymes and ROS-generating enzymes. The cross-talk between these systems should be taken in account. There is positive-feedback between mitochondrial and cytoplasmic ROS-generating systems. But also the increased activity of ROS-generating enzymes increases the expression of antioxidant enzymes. I recomend you to read two review dedicated to the problem (https://www.researchgate.net/publication/263709362_Mitochondrial_reactive_oxygen_species_ROS_and_ROS-induced_ROS_release?ev=prf_pub
https://www.researchgate.net/publication/51510171_Crosstalk_between_mitochondria_and_NADPH_oxidases).
First question. H2O2 can easily penetrate through membarenes. It's generaly considered that primary ROS produced on respiratory chain (compl. I, III IV) is superoxide. But then it is rapidly converted to hydrogen peroxide by Mn-SOD. So it is impossible to trap ROS inside mitochondria.
One more thing. You should be very cautious with your methods beacause it is very strange that you have simultaneously increased mitochondrial potential and mitochondrial swelling. In most cases the processes that results in swelling also results in dissipation of mitochodnrial potential.
Good luck!
Article Korshunov,S. S. , Skulachev,V. P. & Starkov, A. A. High prot...
Article Crosstalk between mitochondria and NADPH oxidases
Article Mitochondrial reactive oxygen species (ROS) and ROS-induced ...
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