I need to study the effect of increase in cellular ATP on the expression of a protein. How can this be achieved in in vitro conditions? I work with cancer cell lines like HeLa.
Hi Kalkal,
I guess by mentioning "cellular ATP" you meant the soluble pool of cytoplasmic ATP and not the in situ produced ATP that is transported out of the cell. The total nucleotide levels (ATP+ADP+AMP) within cells are well preserved and increased phosphorylating of ADP to form ATP would possibly create a compromised cellular metabolism status. Theoretically increasing the pool of available ADP inside cells and preventing ATP formed to break it down to ADP+Pi (which is an important substrate cycling mechanism) or AMP+Pi+Pi would increase the soluble pool of ATP inside cell. Apyrase is an enzyme that catalyses the hydrolysis of ATP to yield AMP and inorganic phosphate. Regulated inhibition of apyrase could also be used to increase cellular ATP level. There are nucleotide transportes as well that plays significant role in terms of availability inside cell.
Since ATP is the currency that the cellular processes are constantly using (Na,K-ATPases, Ca-ATPases etc.) for survival and for health limiting ATP consumption could have adeleterious effect on cellular physiology. People already tried adding cell permeable analog of ATP (8Br-ATP) with little success to elevate cellular ATP level. Ortho vanadate is a widely used P-type ATPase inhibitor which could be used to minimize the ATP hydrolysis for a shorter duration of treatment but I will not recommend using vanadate in culture for long time.
When people want to increase the extracellular secretory pool of ATP, an Ecto-ATPase inhibitor ARL 67156 is frequently used.
There is an alder thread available addressing relevant issues avaailable in RG. For further reading this could be worth mentioning.
https://www.researchgate.net/post/Can_anyone_offer_a_protocol_or_reference_for_increasing_cellular_ATP_and_Mg2_level_pharmacologically
Hope that helps!!!
Hi Kalkal,
I guess by mentioning "cellular ATP" you meant the soluble pool of cytoplasmic ATP and not the in situ produced ATP that is transported out of the cell. The total nucleotide levels (ATP+ADP+AMP) within cells are well preserved and increased phosphorylating of ADP to form ATP would possibly create a compromised cellular metabolism status. Theoretically increasing the pool of available ADP inside cells and preventing ATP formed to break it down to ADP+Pi (which is an important substrate cycling mechanism) or AMP+Pi+Pi would increase the soluble pool of ATP inside cell. Apyrase is an enzyme that catalyses the hydrolysis of ATP to yield AMP and inorganic phosphate. Regulated inhibition of apyrase could also be used to increase cellular ATP level. There are nucleotide transportes as well that plays significant role in terms of availability inside cell.
Since ATP is the currency that the cellular processes are constantly using (Na,K-ATPases, Ca-ATPases etc.) for survival and for health limiting ATP consumption could have adeleterious effect on cellular physiology. People already tried adding cell permeable analog of ATP (8Br-ATP) with little success to elevate cellular ATP level. Ortho vanadate is a widely used P-type ATPase inhibitor which could be used to minimize the ATP hydrolysis for a shorter duration of treatment but I will not recommend using vanadate in culture for long time.
When people want to increase the extracellular secretory pool of ATP, an Ecto-ATPase inhibitor ARL 67156 is frequently used.
There is an alder thread available addressing relevant issues avaailable in RG. For further reading this could be worth mentioning.
https://www.researchgate.net/post/Can_anyone_offer_a_protocol_or_reference_for_increasing_cellular_ATP_and_Mg2_level_pharmacologically
Hope that helps!!!
We have found that you can modulate total cellular ATP levels by altering the composition of fuel sources in growth media. For example, many growth media formulations contain ~1mM pyruvate. By increasing pyruvate concentrations to 5 or 10mM, we can increase both the mitochondrial OXPHOS activity of cells as measured by oxygen consumption as well as total cellular ATP levels. We've only been able to raise ATP levels by ~20%, likely due to the homeostatic adenosine nucleotide maintenance programs mentioned by Amritlal above. Also, not all cell lines respond in this fashion, likely reflecting the intrinsically heterogeneous OXPHOS capacities of different cell types.
This is a very difficult task since ATP content is highly regulated in cells. Adenosine nucleotide pools are well preserved (AMP, ADP, and ATP) with levels of ATP in comparison to ADP and AMP being much higher. For example it is predicted that the ATP/ADP ratio in the cytosol is 1000 Increasing cellular ATP. This provides the necessary Gibbs free energy to do work in the cell. Given that cytosolic ATP levels 1000 times higher than ADP, simply adding different carbon sources may not be sufficient. This is in consideration that ATP can feedback and inhibit glycolytic enzymes (PFK and PK) and KGDH, PDH.
You may need to lower your ATP to ADP ratio which as Amritial indicated could have deleterious effects. You could possibly stimulate ATP production via activation of AMPK but this may also have some unfavorable effects.
"For example it is predicted that the ATP/ADP ratio in the cytosol is 1000"
Is there some reference for this ratio/calculations? So far I've seen values like 1 to 100-200 (free ATP/free ADP): http://www.bmb.leeds.ac.uk/teaching/icu3/lecture/19/
But it's indeed interesting - and for many people unexpected - that in fact any increase or decrease in [ATP] is essentially equivalent to an increase of the total [ANP pool] - since ATP constitutes 80-95% of adenylates under any reasonable physiological conditions.
https://www.ncbi.nlm.nih.gov/pubmed/21731132
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