The Protective Effect Of Glycine In ATP Depleted Injury

Both ischemia and hypoxia are important in human pathophysiology because they occur in a wide variety of important clinical diseases, including myocardial infarction, acute renal failure, and stroke. As a result of the decreased blood flow, cells in ischemic tissues are depleted of oxygen and nutrients, and lose their capacity for ATP production and become energy deprived, which culminate in cell irreversible injury and death. A decade ago, glycine was found to inhibit the ATP depletion-induced cell injury and to improve cell survival during recovery. However, the mechanism of the cytoprotection by glycine was not clear yet. We investigated the effect of glycine on the cell damage, proliferation and metabolism during the ATP depletion and recovery, and explore the role of glycine receptor on the cytoprotection of glycine.Firstly, MDCK cells were exposed to ATP depletion with respiratory chain inhibitors and glucose-free medium. During ATP depletion and recovery, the effects of glycine on the damage, proliferation and metabolism of MDCK cells were assessed by light microscope, Trypan Blue (TB) exclusion test, the lactate dehydrogenase (LDH) release assay, the uptake of ~3H-TdR and MTT assay. After 2h ATP depletion, thepercentage of TB stained cells in Gly-treated reduced 50%, compared with glycine untreated ATP-depleted group. The LDH releases decreased greatly. And the uptake of ~3H-TdR was 2 times higher than that in ATP-depleted group. During recovery, the cell damage seen with light microscope attenuated greatly in Gly-treated group. The percentage of TB stained cells and LDH release in Gly-treated group were significantly lower than that in ATP-depleted group. The uptake of ~3H-TdR and MTT were higher. Then, we transfected the human neuronal GlyR a 1 cDNA-encoded plasmid into HEK293 cells, which lack native GlyR. Then the transfected and the wild type HEK293 and MDCK cells were kept to ATP-free condition at the same time. Glycine was found to protect the transfected HEK293 and MDCK cells, but not the wild type HEK293 cells. And the native GlyR was found to express in MDCK cells by RT-PCR.Our experiments demonstrated that glycine protected MDCK cells from ATP depletion injury and increased the cell survival during recovery, by preventing membrane permeabilization, and improving the proliferation and metabolism activities of injured cells. And these results suggested that the GlyR might play an important role in glycine-mediated cytoprotection.