Inhibition of nonlysosomal calcium-dependent proteolysis by glycine during anoxic injury of rat hepatocytes.
OBJECTIVE
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 mu-calpain protease activity.


CONCLUSIONS
Glycine may exert its cytoprotective activity during lethal anoxic hepatocyte injury, in part by inhibiting Ca(2+)-dependent degradative, nonlysosomal proteases, including calpains.
Nichols
J C
JC
Department of Internal Medicine, Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota.
Bronk
S F
SF
Mellgren
R L
RL
Gores
G J
GJ
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D005998:Glycine / Q000494:pharmacology*; D000860:Hypoxia / Q000473:pathology*; D008099:Liver / Q000187:drug effects / Q000473:pathology*; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D011480:Protease Inhibitors; D010447:Peptide Hydrolases; D002154:Calpain; D002118:Calcium; D005998:Glycine