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Oxidative stress induces release of 2'-AMP from microglia
Travis C Jackson1, Shawn E Kotermanski2, Patrick M Kochanek1
1Department of Critical Care Medicine, Children's Hospital of Pittsburgh, Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Background:
Microglia metabolize exogenous 2'-AMP and 3'-AMP (non-canonical nucleotides) to adenosine and exogenous 2'-AMP and 3'-AMP (via conversion to adenosine) inhibit the production of inflammatory cytokines by microglia. This suggests that if microglia release endogenous 2'-AMP and/or 3'-AMP in response to injurious stimuli, this would complete an autocrine/paracrine mechanism that attenuates the over-activation of microglia during brain injury. Here we investigated in microglia (and for comparison astrocytes and neurons) the effects of injurious stimuli on extracellular and intracellular levels of 2',3'-cAMP (2'-AMP and 3'-AMP precursor), 2'-AMP, and 3'-AMP.
Methods:
Experiments were conducted in primary cultures of rat microglia, astrocytes, and neurons. Cells were exposed to oxygen/glucose deprivation, iodoacetate plus 2,4-dinitrophenol (metabolic inhibitors), glutamate, or H2O2 for one hour, and extracellular and intracellular 2',3'-cAMP, 2'-AMP, and 3'-AMP were measured by UPLC-MS/MS.
Key Results:
In microglia, H2O2 increased extracellular levels of 2'-AMP, but not 3'-AMP, by ∼16-fold (from 0.17 ± 0.11 to 2.78 ± 0.27 ng/106 cells; n = 13; mean ± SEM; P < 0.000005). H2O2 also induced oxidative changes in cellular proteins as detected by an increased number of carbonyl groups in protein side chains. In contrast, oxygen/glucose deprivation, metabolic inhibitors, or glutamate had no effect on either extracellular 2'-AMP or 3'-AMP levels. In astrocytes and neurons, none of the injurious stimuli increased extracellular 2'-AMP or 3'-AMP.
Conclusions:
Oxidative stress (but not oxygen/glucose deprivation, energy deprivation, or excitotoxicity) induces microglia (but not astrocytes or neurons) to release 2'-AMP, but not 3'-AMP. The 2',3'-cAMP/2'-AMP/adenosine pathway mechanism may serve to prevent over-activation of microglia in response to oxidative stress.
Insights
Oxidative stress triggers microglia to release 2'-AMP, a nucleotide that helps prevent over-activation. This finding reveals a protective mechanism against brain injury involving microglia and adenosine signaling.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Microglia metabolize non-canonical nucleotides (2 extprime extprime-AMP and 3 extprime extprime-AMP) to adenosine.
- Exogenous 2 extprime extprime-AMP and 3 extprime extprime-AMP inhibit microglial inflammatory cytokine production.
- Endogenous release of these nucleotides could mediate a protective autocrine/paracrine mechanism against microglial over-activation during brain injury.
Purpose of the Study:
- To investigate the effects of various injurious stimuli on extracellular and intracellular levels of 2 extprime extprime,3 extprime extprime-cAMP, 2 extprime extprime-AMP, and 3 extprime extprime-AMP in microglia.
- To compare these effects in microglia with those in astrocytes and neurons.
Main Methods:
- Primary cultures of rat microglia, astrocytes, and neurons were utilized.
- Cells were exposed to injurious stimuli including oxygen/glucose deprivation, metabolic inhibitors (iodoacetate plus 2,4-dinitrophenol), glutamate, or H extsubscript{2} extsubscript{O} extsubscript{2} for one hour.
- Extracellular and intracellular levels of 2 extprime extprime,3 extprime extprime-cAMP, 2 extprime extprime-AMP, and 3 extprime extprime-AMP were quantified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).
Main Results:
- Hydrogen peroxide (H extsubscript{2} extsubscript{O} extsubscript{2}) significantly increased extracellular 2 extprime extprime-AMP levels in microglia by approximately 16-fold, while 3 extprime extprime-AMP levels remained unchanged.
- H extsubscript{2} extsubscript{O} extsubscript{2} exposure also led to oxidative damage in microglial proteins.
- Oxygen/glucose deprivation, metabolic inhibitors, or glutamate did not alter extracellular 2 extprime extprime-AMP or 3 extprime extprime-AMP levels in microglia, astrocytes, or neurons.
Conclusions:
- Oxidative stress, specifically, induces microglia but not astrocytes or neurons to release 2 extprime extprime-AMP.
- This release of 2 extprime extprime-AMP is not accompanied by 3 extprime extprime-AMP release.
- The 2 extprime extprime,3 extprime extprime-cAMP/2 extprime extprime-AMP/adenosine pathway may function as a protective mechanism to attenuate microglial over-activation in response to oxidative stress.