calpain-inhibitor-iii and Necrosis

MDL 28170 is a CNS-penetrating calpain inhibitor, and we examined the effects of MDL 28170 on hypoxic-ischemic brain injury in immature brain using the Rice-Vannucci model. Immediately after hypoxic exposure, 24 mg/kg of MDL 28170 was injected intraperitoneally as an initial dose, followed by 12 mg/kg every 4 h for a total dose of 60 mg/kg over 12 h post-HI. A vehicle control group received peanut oil injection instead. Macroscopic evaluation of brain injury revealed the neuroprotective effect of MDL 28170 after 12 h post-HI. Neuropathological quantitative analysis of cell death showed that MDL 28170 significantly decreased the number of necrotic cells in all the examined regions except for cingular cortex, and the number of apoptotic cells in caudate putamen, parietal cortex, hippocampus CA1, and laterodorsal thalamus. Western blots showed that MDL 28170 suppressed 145/150 kDa subunits of alpha-spectrin breakdown products (SBDP) in cortex, hippocampus, thalamus, and striatum, and also 120-kDa subunit of SBDP in all regions except for striatum. This suggests that MDL 28170 inhibited activation of calpain and caspase-3, respectively. Our results indicate that post-hypoxic MDL 28170 injection is neuroprotective in HI newborn rat brain by decreasing both necrosis and apoptosis. SBDP expression also suggests that MDL 28170 injection inhibits both calpain and caspase-3 activation after HI insult.

Topics: Animals; Animals, Newborn; Apoptosis; Brain; Calpain; Cell Death; Cysteine Proteinase Inhibitors; Dipeptides; Functional Laterality; Hypoxia-Ischemia, Brain; Microscopy, Electron; Necrosis; Nerve Degeneration; Rats; Spectrin; Time Factors

It has been shown that sarcolemmal rupture can occur during reenergization in cardiomyocytes in which previous ischemia has induced sarcolemmal fragility by calpain-dependent hydrolysis of structural proteins. We tested the hypothesis that attenuated calpain activation contributes to the protection against reperfusion-induced cell death afforded by ischemic preconditioning (IPC), and investigated the involvement of protein kinase A (PKA) in this effect.. Calpain activity and degradation of different structural proteins were studied along with the extent of necrosis in isolated rat hearts submitted to 60 min of ischemia and 30 min of reperfusion with or without previous IPC (two cycles of 5 min ischemia-5 min reperfusion), and the ability of different treatments to mimic or blunt the effects of IPC were analyzed.. IPC accelerated ATP depletion and rigor onset during ischemia but reduced LDH release during reperfusion by 69% (P<0.001). At the end off reperfusion, calpain activity was reduced by 66% (P<0.001) in IPC, and calpain-dependent degradation of sarcolemmal proteins was attenuated. Addition of the calpain inhibitor MDL-28170 mimicked the effects of IPC on protein degradation and reduced LDH release by 48% (P<0.001). The effects of IPC on calpain, alpha-fodrin, and LDH release were blunted by the application of the PKA inhibitor H89 or alprenolol during IPC, while transient stimulation of PKA with CPT-cAMP or isoproterenol before ischemia attenuated calpain activation, alpha-fodrin degradation, and markedly reduced LDH release (P<0.001). In hearts exposed to Na(+)-free perfusion, IPC attenuated calpain activation by 67% (P<0.001) and reduced by 56% (P<0.001) LDH release associated to massive edema occurring during Na(+) readmission without modifying its magnitude.. These results are consistent with PKA-dependent attenuation of calpain-mediated degradation of structural proteins being an end-effector mechanism of the protection afforded by IPC.

Topics: Adrenergic beta-Antagonists; Alprenolol; Animals; Calpain; Carrier Proteins; Cyclic AMP-Dependent Protein Kinases; Dipeptides; Enzyme Activation; Ischemic Preconditioning, Myocardial; Isoquinolines; L-Lactate Dehydrogenase; Male; Membrane Proteins; Microfilament Proteins; Myocardial Reperfusion Injury; Myocardium; Naloxone; Narcotic Antagonists; Necrosis; Perfusion; Rats; Rats, Sprague-Dawley; Sodium; Sulfonamides

Liver injury is known to progress even after the hepatotoxicant is long gone and the mechanisms of progressive injury are not understood. We tested the hypothesis that hydrolytic enzymes such as calpain, released from dying hepatocytes, destroy the surrounding cells causing progression of injury. Calpain inhibitor, N-CBZ-VAL-PHE-methyl ester (CBZ), administered 1 h after a toxic but nonlethal dose of CCl(4) (2 ml/kg, ip) to male Sprague Dawley rats substantially mitigated the progression of liver injury (6 to 48 h) and also led to 75% protection against CCl(4)-induced lethality following a lethal dose (LD75) of CCl(4) (3 ml/kg). Calpain leakage in plasma and in the perinecrotic areas increased until 48 h and decreased from 72 h onward paralleling progression and regression of liver injury, respectively, after CCl(4) treatment. Mitigation of progressive injury was accompanied by substantially low calpain in perinecrotic areas and in plasma after CBZ treatment. Normal hepatocytes incubated with the plasma collected from CCl(4)-treated rats (collected at 12 h when most of the CCl(4) is eliminated) resulted in extensive cell death prevented by CBZ. Cell-impermeable calpain inhibitor E64 also protected against progression of CCl(4)-induced liver injury, thereby confirming the role of released calpain in progression of liver injury. Following CCl(4) treatment, calpain-specific breakdown of alpha-fodrin increased, while it was negligible in rats receiving CBZ after CCl(4). Hepatocyte cell death in incubations containing calpain was completely prevented by CBZ. Eighty percent of Swiss Webster mice receiving a lethal dose (LD80) of acetaminophen (600 mg/kg, ip) survived if CBZ was administered 1 h after acetaminophen, suggesting that calpain-mediated progression of liver injury is neither species nor chemical specific. These findings suggest the role of calpain in progression of liver injury.

Topics: Acetaminophen; Animals; Blotting, Western; Calpain; Carbon Tetrachloride; Carrier Proteins; Chemical and Drug Induced Liver Injury; Cysteine Proteinase Inhibitors; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP2E1 Inhibitors; Dipeptides; Disease Progression; Hepatocytes; Immunohistochemistry; Liver Diseases; Male; Mice; Microfilament Proteins; Necrosis; Random Allocation; Rats; Rats, Sprague-Dawley