Travis C Jackson1, Shawn E Kotermanski2, Patrick M Kochanek3, Edwin K Jackson4. 1. Department of Critical Care Medicine, Children's Hospital of Pittsburgh, Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: tcj10@pitt.edu. 2. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: sek128@pitt.edu. 3. Department of Critical Care Medicine, Children's Hospital of Pittsburgh, Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: kochanekpm@ccm.upmc.edu. 4. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: edj@pitt.edu.
Abstract
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.
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.
Authors: Jonathan D Verrier; Travis C Jackson; Rashmi Bansal; Patrick M Kochanek; Ava M Puccio; David O Okonkwo; Edwin K Jackson Journal: J Neurochem Date: 2012-03-20 Impact factor: 5.372
Authors: Travis C Jackson; Jonathan D Verrier; Susan Semple-Rowland; Ashok Kumar; Thomas C Foster Journal: J Neurochem Date: 2010-09-28 Impact factor: 5.372
Authors: M Lee Haselkorn; David K Shellington; Edwin K Jackson; Vincent A Vagni; Keri Janesko-Feldman; Raghvendra K Dubey; Delbert G Gillespie; Dongmei Cheng; Michael J Bell; Larry W Jenkins; Gregg E Homanics; Jurgen Schnermann; Patrick M Kochanek Journal: J Neurotrauma Date: 2010-05 Impact factor: 5.269
Authors: Nils Ludwig; Juliana H Azambuja; Aparna Rao; Delbert G Gillespie; Edwin K Jackson; Theresa L Whiteside Journal: Purinergic Signal Date: 2020-05-22 Impact factor: 3.765