Hi,
I study lipid metabolism in Leishmania. Recently, I had some interesting observations and I would like to have your comments on these. Compared to the wild type parasites, the lipid mutants showed higher mitochondrial membrane potential as was determined by JC-1 staining. I also, looked at the ATP production. For this I treated the parasites with Na-pyruvate (substrate for mitochondrial respiration) and deoxy-D-glucose (hexokinase inhibitor) in an isotonic salt solution. I found that the mitochondria generated ATP production was much higher in the lipid mutants compared to the wild type.
Finally I analyzed the cellular ROS production by staining the parasites with H2DCFDA. Here my observation was that lipid mutants are generating less ROS than wild type. Overall, to summarize, the lipid mutants showed Higher mitochondrial membrane potential and higher mitochondrial ATP production but lower cellular ROS production as compared to the wild type parasites.
My question is how mitochondrial oxidation is related to the ROS production? I could be wrong, but my understanding was higher mitochondrial activity would generate more ROS. However, my observation was just the opposite. Is there any alternative explanation?
I really appreciate your comments!
Thanks
Sumit
I think most people are under the same impression as you. Generally, higher MMP results in greater ATP production and greater ROS production. Did you measure any endogenous antioxidant enzyme activities? Did you measure JC-1 on whole cells? JC-1 can give you a lot of non-specific fluorescence unless used with isolated mitochondria. Maybe confirm the MMP measurements with TMRM?
from https://www.ncbi.nlm.nih.gov/pubmed/12765765: "An exponential increase of ROS production has been measured in mitochondria at membrane potentials of ΔΨm>140 mV [96], [97] and [98]." (Although this was not in Leishmania of course)
[96] S.S. Liu Generating, partitioning, targeting and functioning of superoxide in mitochondria Biosci. Rep., 17 (1997), pp. 259–272
[97] S.S. Liu Cooperation of a “reactive oxygen cycle” with the Q cycle and the proton cycle in the respiratory chain—superoxide generating and cycling mechanism in mitochondria J. Bioenerg. Biomembr., 31 (1999), pp. 367–376
[98] S.S. Korshunov, V.P. Skulachev, A.A. Starkov High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria FEBS Lett., 416 (1997), pp. 15–18
Just giving you a model. There could be other ways too.
ROS to JNK and JNK to p53
http://www.nature.com/cdd/journal/v21/n4/full/cdd2013186a.html
then
p53 to MOMP
https://www.ncbi.nlm.nih.gov/pubmed/18504456
So you may check JNK, p53 and p47phox differences between WT and lipid mutants (Leishmania).
Mitochondria that have a high membrane potential are more prone to ROS generation. Active mitochondria that run 'smoothly' tend to have a normal membrane potential and produce less ROS. I can recommend the works of David G. Nicholls who studied the functional relationships between ROS production and the mitochondrial membrane potential in detail. Have a look at one of his articles in the attachment. It serves as a good starter for delving into this fascinating topic.
With best regards, Christian
Article Mitochondrial membrane potential and aging
More classical references on the subject:
- T.V. Votyakova, I.J. Reynolds, Delta psi(m)-dependent and -independent production of reactive oxygen species by rat brain mitochondria, J. Neurochem., 79 (2001), pp. 266–277
- A.A. Starkov, G. Fiskum, Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state, J. Neurochem., 86 (2003), pp. 1101–1107
Using different assays can confirm results of mitochondrial membrane potential, and also ROS (oxidative markers?).
Alternative explanations?
- changes in (enzymatic/non-enzymatic) antioxidant systems
- other sources of ROS (independent of mitochondrial membrane potential).
H2DCF is a low-specificity probe, reacting with different ROS (H2O2, lipid hydroperoxide, peroxynitrite), also Fe, by mechanisms depending on several conditions.
Hi Sumit,
You have mentioned that you have checked for ROS using DCFDA dye. In my understanding you will get to know about CYTOSOLIC ROS using this particular probe.
You may use MitoSOX red dye for mitochondrial ROS. May be your mutant parasite produces a higher amount of antioxidants to cope up for the excess ROS as an adaptive mechanism, although you may need to find out all these as indicated by the others in the reply series.
With regards
shubhankar
What is the level of catalase? i.e, peroxisomes...and aren't these mitochondria functioning out of glycolytic pathway like in tumour cells?using fumarate or lactate?
After all these are parasites adapted and maybe the mitochondria are also adapted.
For example, see Pierre Sonveaux published papers.
'Healthy mitochondria' that have normal membrane potential may produce less ROS due to low 'e' leakage. In addition, you may check the status of antioxidant enzymes as its a mutant. Nicholls' article may be helpful to you. Good luck!
Dear Dr. Sumit Mukherjee, my greetings.
In order to answer your question I would like to have a little more information about your model and experimental conditions. I also would warn you not to rely heavily on JC1. because it is a very unreliable indicator prone to a many errors. With your interesting data it would be useful to use some alternative method, TPP electrode, for example.
My questions to the methodology:
1. How you receive your parasitic cells? Do you culture them, do you use streptomycin?
2. Do you use whole cells or isolate mitochondria?
3.How you incubate cells and what concentrations of substrate, do you measure oxygen consumption (with Oroboros for example)?
and 4. What is the essence of the mutation? Do the cells consume more fatty acids?
PS. Thank you Dr. Christian Q. Scheckhuber for the PDFs. I retired and not all papers are accessible to me.
Sincerely,
Alexander Panov
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology