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.
Keywords: 2’,3’-cAMP; 2’-AMP; 3’-AMP; Astrocytes; CNPase; Microglia; Neurons; Oxidative stress.
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