calpain-inhibitor-iii and leupeptin

The K-Cl cotransporter KCC2 plays a crucial role in neuronal chloride regulation. In mature central neurons, KCC2 is responsible for the low intracellular Cl(-) concentration ([Cl(-)](i)) that forms the basis for hyperpolarizing GABA(A) receptor-mediated responses. Fast changes in KCC2 function and expression have been observed under various physiological and pathophysiological conditions. Here, we show that the application of protein synthesis inhibitors cycloheximide and emetine to acute rat hippocampal slices have no effect on total KCC2 protein level and K-Cl cotransporter function. Furthermore, blocking constitutive lysosomal degradation with leupeptin did not induce significant changes in KCC2 protein levels. These findings indicate a low basal turnover rate of the total KCC2 protein pool. In the presence of the glutamate receptor agonist NMDA, the total KCC2 protein level decreased to about 30% within 4 h, and this effect was blocked by calpeptin and MDL-28170, inhibitors of the calcium-activated protease calpain. Interictal-like activity induced by incubation of hippocampal slices in an Mg(2+)-free solution led to a fast reduction in KCC2-mediated Cl(-) transport efficacy in CA1 pyramidal neurons, which was paralleled by a decrease in both total and plasmalemmal KCC2 protein. These effects were blocked by the calpain inhibitor MDL-28170. Taken together, these findings show that calpain activation leads to cleavage of KCC2, thereby modulating GABAergic signaling.

Topics: Action Potentials; Analysis of Variance; Animals; Animals, Newborn; Calcium; Calcium Ionophores; Calpain; Cycloheximide; Cysteine Proteinase Inhibitors; Dipeptides; Dizocilpine Maleate; Dose-Response Relationship, Drug; Emetine; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Hippocampus; In Vitro Techniques; Ionomycin; K Cl- Cotransporters; Leupeptins; Magnesium; Male; Membrane Potentials; N-Methylaspartate; Patch-Clamp Techniques; Protein Synthesis Inhibitors; Pyramidal Cells; Rats; Rats, Wistar; Statistics, Nonparametric; Symporters; Valine

E64, an inhibitor of calpain (EC 3.4.22.17) and other cysteine proteases, slows the rate of formation of cataract in cultured rat lenses. The purpose of this study was to determine (1) why E64, a charged compound with little cell permeability, was effective in reducing cataract in cultured lens and (2) whether uncharged more permeable protease inhibitors are more effective than E64 in preventing cataract. Results showed that E64 entered the lens, but only after the lens was treated with the calcium ionophore, A23187, or sodium selenite, both of which cause cataracts. Therefore, the uptake and subsequent effectiveness of E64 may be related to a generalized increase in membrane permeability during induction of cataract in culture. Three protease inhibitors, reported to have improved cell permeability, were compared with E64 for their ability to prevent cataracts in cultured lenses. cBz-ValPheH, calpain inhibitors I and II, are uncharged-aldehyde inhibitors of calpain. Calpain inhibitors I and II even at high concentrations were not effective at reducing lens opacity caused by calcium ionophore and were toxic to the lens. cBz-ValPheH, which is slightly toxic to the lens, was able to significantly reduce lens opacity induced by calcium ionophore. The presented data suggest that while E64 decreases cataract formation in cultured lens, the more cell permeable inhibitor, cBz-ValPheH, may have greater efficacy as an anticataract drug in vivo.

Topics: Amino Acid Sequence; Animals; Calcimycin; Calpain; Cataract; Cell Membrane Permeability; Chromatography, High Pressure Liquid; Culture Techniques; Cysteine Proteinase Inhibitors; Dipeptides; Drug Interactions; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Lens, Crystalline; Leucine; Leupeptins; Molecular Sequence Data; Rats; Rats, Sprague-Dawley

A synthetic inhibitor of calpain protects rat erythrocyte membrane-associated cytoskeletal proteins from proteolytic degradation (IC50 = 1 microM) which occurs when the cells are rendered permeable to Ca++. Leupeptin, a naturally occurring inhibitor of the enzyme, does not afford any protection at concentrations up to 100 microM.

Topics: Animals; Blotting, Western; Calcimycin; Calcium; Calpain; Cell Membrane Permeability; Cytoskeletal Proteins; Dipeptides; Erythrocyte Membrane; Hydrolysis; Leupeptins; Membrane Proteins; Molecular Weight; Peptide Hydrolases; Protease Inhibitors; Rats; Spectrin