Cell death in response to oxidative stress plays a role in a variety of neurodegenerative diseases and can be studied in detail in the neuronal cell line HT22, where extracellular glutamate causes glutathione depletion by inhibition of the glutamate/cystine antiporter system xc(-), elevation of reactive oxygen species and eventually programmed cell death caused by cytotoxic calcium influx. Using this paradigm, we screened 54 putative extracellular peptide or small molecule ligands for effects on cell death and identified extracellular cyclic guanosine monophosphate (cGMP) as a protective substance. Extracellular cGMP was protective, whereas the cell-permeable cGMP analog 8-pCPT-cGMP or the inhibition of cGMP degradation by phosphodiesterases was toxic. Interestingly, metabolites GMP and guanosine were even more protective than cGMP and the inhibition of the conversion of GMP to guanosine attenuated its effect, suggesting that GMP offers protection through its conversion to guanosine. Guanosine increased system xc(-) activity and cellular glutathione levels in the presence of glutamate, which can be explained by transcriptional upregulation of xCT, the functional subunit of system xc(-). However, guanosine also provided protection when added late in the cell death cascade and significantly reduced the number of calcium peaking cells, which was most likely not mediated by transcriptional mechanisms. We observed no changes in the classical protective pathways such as phosphorylation of Akt, ERK1/2 or induction of Nrf2 or ATF4. We conclude that extracellular guanosine protects against endogenous oxidative stress by two probably independent mechanisms involving system xc(-) induction and inhibition of cytotoxic calcium influx.
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