calpain-inhibitor-iii and acetylleucyl-leucyl-norleucinal

α-latrotoxin and snake presynaptic phospholipases A2 neurotoxins target the presynaptic membrane of axon terminals of the neuromuscular junction causing paralysis. These neurotoxins display different biochemical activities, but similarly alter the presynaptic membrane permeability causing Ca(2+) overload within the nerve terminals, which in turn induces nerve degeneration. Using different methods, here we show that the calcium-activated proteases calpains are involved in the cytoskeletal rearrangements that we have previously documented in neurons exposed to α-latrotoxin or to snake presynaptic phospholipases A2 neurotoxins. These results indicate that calpains, activated by the massive calcium influx from the extracellular medium, target fundamental components of neuronal cytoskeleton such as spectrin and neurofilaments, whose cleavage is functional to the ensuing nerve terminal fragmentation.

Topics: Acrylates; Animals; Animals, Newborn; Calcium Signaling; Calpain; Cell Membrane Permeability; Cells, Cultured; Cytoskeleton; Dipeptides; Leupeptins; Nerve Degeneration; Neurofilament Proteins; Neurons; Neurotoxins; Phospholipases A2; Presynaptic Terminals; Rats; Rats, Wistar; Snake Venoms; Spectrin; Spider Venoms

Matrix metalloproteinase (MMP)-2 is a zinc-dependent endopeptidase which, alongside its known extracellular actions, plays fundamental roles in oxidative stress-induced injury to the heart. Intracellular cleavage targets of MMP-2 selectively mediating this injury include the sarcomeric proteins troponin I, myosin light chain-1 and titin; some of these are also targeted by calpains. In myocardial ischemia and reperfusion injury, inhibitors of MMP-2 and some calpain inhibitors were shown to improve the recovery of contractile function. We hypothesized that the protective effects of calpain inhibitors may be due in part to their ability to inhibit MMP-2. Four calpain inhibitors (calpain inhibitor III, ALLM, ALLN, and PD-150606) were tested for their ability to inhibit MMP-2 in comparison to the selective MMP inhibitor ONO-4817. At 100 μM, all calpain inhibitors, except ALLM, showed significant inhibition of MMP-2 gelatinolytic activity. When assessed by the troponin I proteolysis assay, both ALLN and PD-150606, but neither ALLM nor calpain inhibitor III (at 20 μM), significantly inhibited MMP-2 activity. Using a fluorogenic MMP substrate peptide OmniMMP in a kinetic assay the rank order of IC(50) values against MMP-2 were: PD-150606

Topics: Acrylates; Calpain; Catalysis; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; Gelatin; Humans; Inhibitory Concentration 50; Leupeptins; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Oligopeptides; Phenyl Ethers; Troponin I

The role of neuronal N-methyl-D-aspartate (NMDA) receptor-mediated intracellular signaling has been elucidated in both physiological and pathological conditions. However, the details of relative vulnerability for excitotoxicity remain unknown. Retinal excitotoxicity is involved in various diseases leading to irreversible blindness. Here, we used the visual system and explored the mechanistic details of the NMDA-elicited intracellular events, especially in the amacrine cells, which are the most vulnerable type of neuron in the retina. G-substrate, a specific substrate of cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase, is colocalized with amacrine cells and acts as an endogenous inhibitor of protein phosphatase. To elucidate how G-substrate was involved in NMDA-induced amacrine cell death, the immunohistochemical analysis with G-substrate antibody was performed following NMDA injury. In vivo, NMDA immediately decreased G-substrate immunoreactivity, and the suppression of calpain activation using ALLN or calpain III, an inhibitor of calpain, blocked this decrease. In vitro, degraded fragments of G-substrate were detected within 10 min after coincubation of G-substrate and calpain. Moreover, G-substrate knockout (G-substrate(-/-)) mice were more susceptible to NMDA injury than wild-type mice. ALLN did not have a neuroprotective effect in G-substrate(-/-) mice. These data strongly suggest that calpain-mediated loss of G-substrate represents an important mechanism contributing to NMDA-induced amacrine cell death.

Topics: Amacrine Cells; Analysis of Variance; Animals; Blotting, Western; Calpain; Cell Death; Cysteine Proteinase Inhibitors; Dipeptides; Immunohistochemistry; In Situ Nick-End Labeling; Leupeptins; Mice; Mice, Inbred C57BL; Mice, Knockout; N-Methylaspartate; Nerve Tissue Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Retina

p120-catenin contributes to the cadherin-mediated adhesion and aggregation of cells. mu-Calpain was activated and p120-catenin was degraded after 36 h of ischemia in differentiated SH-SY5Y cells. Calpain inhibitors Cbz-Val-Phe-H (MDL28170, 20 microM) and N-acetyl-leucyl-leucyl-norleucinal (ALLN, 20 microM) increased the levels of dephosphorylated p120-catenin, aggregation, and cell survival as detected by reduced LDH release in ischemic cells. However, a proteasome inhibitor lactacystin had no such effects. This is the first report of the calpain-mediated degradation of p120-catenin and an association between the level of dephosphorylated p120-catenin and cell aggregation in ischemic neuronal cells.

Topics: Acetylcysteine; Calpain; Catenins; Cell Adhesion Molecules; Cell Aggregation; Cell Death; Cell Line, Tumor; Delta Catenin; Dipeptides; Humans; Ischemia; Leupeptins; Neuroblastoma; Phosphoproteins