The effect of the newly developed, nonpeptide,
calpain inhibitor,
PD 150606, on
hypoxia and
ionomycin-induced increases in
calpain activity in rat proximal tubules (PT) was determined.
PD150606 inhibited both
hypoxia and
ionomycin-induced
calpain activity as determined by the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl
coumarin (N-succinyl-
Leu-Leu-
Val-Tyr-AMC). This decrease in
calpain activity was accompanied by dose-dependent cytoprotection against
hypoxia and
ionomycin-induced cell membrane damage.
PD150606 had no effect on
cathepsin B and L activity in PT as measured by the fluorescent substrate, benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methyl
coumarin (
Z-Phe-Arg-AMC). The effects of low intracellular pH (pHi) or low free cytosolic
calcium [Ca2+]i on this
hypoxia-induced
calpain activity were also determined. Both low pHi and low [Ca2+]i attenuated the
hypoxia-induced increase in
calpain activity. This attenuation of
calpain activity was observed early before
hypoxia-induced membrane damage and was associated with marked reduction in the typical pattern of
hypoxia-induced cell membrane damage observed in this model. To identify the
isoform of
calpain activated in rat proximal tubules, normoxic, hypoxic and
ionomycin treated tubules were fractionated by
MONO-Q anion exchange chromatography and the fractions were assayed for
calpain activity. A single peak of
calpain activity characteristic of
mu-calpain was found. The
calcium dependency of the
calpain activity was in the nanomolar range, further confirming that the activity was the low Ca(2+)-sensitive
mu-calpain. The present study suggests that in rat proximal tubules: (1)
PD 150606 is a specific inhibitor of
calpain and not
cathepsins B and L; (2) the cytoprotective effects of low pHi and low [Ca2+]i are mediated, at least in part, by inhibition of
calpain activity; and (3) the predominant active form of
calpain is the
isoenzyme mu-calpain.