EFFECT OF GLYCINE ON PRELETHAL AND POSTLETHAL INCREASES IN CALPAIN ACTIVITY IN RAT RENAL PROXIMAL TUBULES

The effect of glycine on hypoxia- and ionomycin-induced increases in c alpain activity in rat proximal tubules was determined. Calpain activi ty was determined both in vitro and in the intact cell using the fluor escent substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl coumarin (N-succinyl-Leu-Leu-Val-Tyr-AMC) and Western blotting for calpain-spec ific spectrin breakdown products (BDP), respectively. At 7.5 minutes o f hypoxia (prelethal injury model) there was a significant (10-fold) i ncrease in in vitro calpain activity that was not inhibited by glycine . At 15 minutes of hypoxia (postlethal injury model) there was a furth er increase in calpain activity that was inhibited by glycine. Normoxi c tubules incubated with the calcium ionophore ionomycin (5 mu M) for two minutes and 10 minutes had a significant increase in calpain activ ity that was not inhibited by glycine. After 15 minutes of hypoxia in the presence of glycine, there was an increase in calpain-specific spe ctrin breakdown products (BDP) in both Triton X-100 soluble and cytoso lic 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 duri ng hypoxia occurs in the presence of glycine; (3) glycine does prevent the additional postlethal increase in calpain activity probably by ma intaining membrane integrity to calcium influx.