tosylphenylalanyl-chloromethyl-ketone and Brain-Injuries

N-Tosyl-L-phenylalanyl-chloromethyl ketone (TPCK) is neuroprotective in rat pups. We measured bcl-2, Bax and caspase-3 to determine the mechanisms. Seven-day-old rats had the right carotid artery ligated and were subjected to 2.5 h of 8% oxygen. Ten mg/kg of PTCK or vehicle was given intraperitoneally 15 min prior to hypoxia. At 24 h after hypoxia the brains were removed. Bcl-2 in the hippocampus increased from 0.149 +/- (SE) 0.023 in the shams to 0.289 +/- 0.037 with injury and vehicle (p < 0.05 vs. shams), which was reduced to 0.177 +/- 0.030 by TPCK ( p < 0.05 vs. vehicle). Bcl-2 in the cortex increased from 0.180 +/- 0.037 in the shams to 0.655 +/- 0.078 with injury and vehicle (p < 0.01 vs. shams), which was reduced to 0.354 +/- 0.035 by TPCK (p < 0.01 vs. vehicle). Bax, measured only in the mitochondrial enriched fraction of the cortex, was unchanged. Caspase-3 activity increased with injury to 245 +/- 38% of baseline in the hippocampus (p < 0.01) and to 261 +/- 69% in the cortex (p < 0.01). Treatment with TPCK reduced this to 132 +/- 16% in the hippocampus (p < 0.01 vs. vehicle) and 140 +/- 14% in the cortex (p < 0.05 vs. vehicle). In this experiment TPCK reduces bcl-2 and caspase-3 concentration in animals who have been shown in our previous studies to be protected by TPCK from hypoxic ischemic brain injury. This is consistent with the hypothesis that TPCK produces neuroprotection by blocking the apoptotic cascade between bcl-2 and caspase-3.

Topics: Animals; Animals, Newborn; Brain Injuries; Caspase 3; Caspase Inhibitors; Caspases; Female; Hypoxia-Ischemia, Brain; Male; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Tosylphenylalanyl Chloromethyl Ketone

N-Tosyl-l-phenylalanyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin-like serine protease (CSP), prevents DNA fragmentation and apoptotic cell death in certain blood cell lines and was reported to reduce hippocampal neuronal damage caused by cerebral ischemia. We examined the role of CSP on recovery after lateral fluid percussion-induced traumatic brain injury (TBI) in rats, as well as on cell survival in various in vitro models of neuronal cell death. TBI caused significant time-dependent upregulation of CSP activity, but not trypsin-like serine protease activity in injured cortex. Intracerebroventricular administration of TPCK to rats after TBI did not significantly affect deficits of spatial learning but exacerbated motor dysfunction after injury. Moreover, TPCK did not prevent apoptotic neuronal cell death caused by serum/K(+) deprivation or by application of staurosporine or etoposide in cultured rat cerebellar granule cells, rat cortical neurons, or in the human neuroblastoma SH-SY5Y cell line. Instead, at doses from 10 to 100 microM, TPCK was cytotoxic in all cultures tested. Similar results were obtained in cultures treated with another CSP inhibitor, 3,4-dichloroisocoumarin. Cell death caused by CSP inhibitors was neither caspase-dependent nor associated with oligonucleosomal DNA fragmentation. Taken together, these data do not support a neuroprotective role for CSP inhibitors. Rather, they suggest that CSPs may serve an endogenous neuroprotective role, possibly by modulating necrotic cell death.

Topics: Animals; Apoptosis; Behavior, Animal; Brain Injuries; Caspase 3; Caspases; Cell Survival; Cells, Cultured; Coumarins; Culture Media, Serum-Free; Disease Models, Animal; Dose-Response Relationship, Drug; Injections, Intraventricular; Isocoumarins; Male; Motor Activity; Neurons; Nucleic Acid Synthesis Inhibitors; Potassium; Rats; Rats, Sprague-Dawley; Serine Endopeptidases; Serine Proteinase Inhibitors; Spatial Behavior; Staurosporine; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone; Wounds, Nonpenetrating