Protection by glycine against hypoxic injury of rat hepatocytes: inhibition of ion fluxes through nonspecific leaks

Background/Aims: Glycine has long been shown to exert strong protective effects against hypoxic injury of hepatocytes. Recently, it was suggested that glycine exerts this protection via inhibition of ligand-gated chloride channels, thereby secondarily inhibiting sodium influx. The purpose of this study was to examine this suggestion. Methods: Cultured rat hepatocytes were incubated under normoxic and hypoxic conditions. Loss of viability was determined by release of lactate dehydrogenase. Cytosolic ion concentrations were measured using digital fluorescence microscopy. Results: Glycine prevented the hypoxic increase in cytosolic sodium and strongly protected against hypoxic injury. The amino acid was not only protective in Krebs-Henseleit buffer but also in a chloride-free modification thereof and offered additional protection in a sodium-free medium (which already yielded substantial protection in its own right). Glycine also prevented the hypoxic release of the anionic fluorescent dye Newport Green and appeared to prevent the hypoxic entrance of the “nonphysiological” cations cobalt and nickel. Conclusion: The results strongly argue against inhibition of ligand-gated chloride channels as being responsible for the potent protective effect of glycine against hypoxic injury of hepatocytes. Instead, they suggest that glycine prevents the formation of nonspecific leaks for small ions including sodium, thereby providing protection.