Inhibition of nonlysosomal calcium-dependent proteolysis by glycine during anoxic injury of rat hepatocytes

Background/Aims: The mechanism by which glycine protects against hepatocyte death during anoxia remains unclear. Nonlysosomal proteolysis, including calpain proteolysis, has been implicated as a mechanism of lethal cell injury. However, the effect of glycine on nonlysosomal proteolysis is unknown. The aim of this study was to ascertain if glycine cytoprotection is associated with inhibition of nonlysosomal proteolysis. Methods: Rat hepatocyte suspensions were rendered anoxic using an anaerobic chamber. Cell viability was measured by propidium iodide fluorometry. Nonlysosomal protease activity was quantitated by the release of trichloroacetic acid-soluble free amines or tyrosine. Calpain protease activity was measured using a fluorogenic substrate. Results: Glycine and alanine (but not valine) markedly improved cell viability during anoxia in a concentration-dependent manner. During anoxia, the majority of nonlysosomal proteolysis (60%) was dependent on extracellular Ca²⁺. Glycine only inhibited that portion of nonlysosomal proteolysis that was dependent on extracellular Ca²⁺. Amino acids inhibited the anoxia-stimulated increase in calpain protease activity with the same specificity and concentration-dependence observed for cytoprotection. Glycine was more potent in directly inhibiting purified m-calpain as compared with μ-calpain protease activity. Conclusions: Glycine may exert its cytoprotective activity during lethal anoxic hepatocyte injury, in part by inhibiting Ca²⁺-dependent degradative, nonlysosomal proteases, including calpains.