The effect of
glycine on
hypoxia- and
ionomycin-induced increases in
calpain activity in rat proximal tubules was determined.
Calpain activity was determined both in vitro and in the intact cell using the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl
coumarin (N-succinyl-
Leu-Leu-
Val-Tyr-AMC) and Western blotting for
calpain-specific
spectrin breakdown products (BDP), respectively. At 7.5 minutes of
hypoxia (prelethal injury model) there was a significant (10-fold) increase in in vitro
calpain activity that was not inhibited by
glycine. At 15 minutes of
hypoxia (postlethal injury model) there was a further increase in
calpain activity that was inhibited by
glycine. Normoxic tubules incubated with the
calcium ionophore ionomycin (5 microM) for two minutes and 10 minutes had a significant increase in
calpain activity that was not inhibited by
glycine. After 15 minutes of
hypoxia in the presence of
glycine, there was an increase in
calpain-specific
spectrin breakdown products (BDP) in both
Triton X-100 soluble and cytosolic extracts from proximal tubules.
Glycine in concentrations up to 10 mM had no direct effect on the in vitro
calpain activity of purified calpains. The present study demonstrates that: (1) prelethal increases in
calpain activity stimulated by
hypoxia and
ionomycin treatment are not affected by
glycine; (2)
calpain-mediated
spectrin breakdown during
hypoxia occurs in the presence of
glycine; (3)
glycine does prevent the additional postlethal increase in
calpain activity probably by maintaining membrane integrity to
calcium influx.