calpain and methylamine

Calpain was generally believed to exist and function only in the cytoplasm. However, m-calpain has now been detected in the extracellular spaces of some kinds of tissue. In this study, we demonstrated the existence of m-calpain in the medium surrounding MC3T3-E1 cultures, and its activity by zymography. At the same time, the amount of lactate dehydrogenase in medium of MC3T3-E1 culture was extremely low compared with other cell cultures, suggesting that m-calpain found in the culture medium of MC3T3-E1 cells originated mainly from active secretion. Moreover, the secretion of m-calpain was not blocked by brefeldin A, implying that m-calpain may be secreted by a nonclassical pathway. Recently, MC3T3-E1 has been reported to produce matrix vesicles and media vesicles, and we demonstrated m-calpain in these vesicles produced by MC3T3-E1 cultures. We therefore concluded that these vesicles are partly responsible for the secretion of m-calpain into the culture medium of MC3T3-E1 cells.

Topics: Animals; Brefeldin A; Calcium Channel Blockers; Calpain; Cell Division; Cell Line; Culture Media, Conditioned; Enzyme Precursors; Extracellular Matrix; Humans; Immunoblotting; Ionophores; Isoenzymes; L-Lactate Dehydrogenase; Methylamines; Mice; Monensin; Osteoblasts; Protein Synthesis Inhibitors; Secretory Vesicles; Verapamil

The effect of dexamethasone on protein degradation and the involvement of different proteolytic pathways were examined in cultured L6 myotubes. Treatment of the cells with dexamethasone resulted in an approximately 20% increase in protein degradation at a hormone concentration of 10(-7) to 10(-6) M. By using various proteolytic blockers, evidence was found that the dexamethasone-induced increase in protein breakdown mainly reflected energy-proteasome-dependent proteolysis and to a lesser extent calcium-dependent protein breakdown. In contrast, the hormone treatment did not increase lysosomal proteolysis. mRNA levels for cathepsin B, ubiquitin, and the proteasome subunit C3 were increased by dexamethasone. The results suggest that glucocorticoids stimulate calcium and energy-proteasome-dependent muscle proteolysis and that changes in mRNA levels for proteolytic enzymes do not necessarily reflect the involvement of different proteolytic pathways.

Topics: Adenosine Triphosphate; Animals; Calcium; Calpain; Cathepsin B; Cathepsin D; Cells, Cultured; Chloroquine; Cysteine Endopeptidases; Deoxyglucose; Dexamethasone; Leucine; Leupeptins; Methylamines; Mifepristone; Multienzyme Complexes; Muscle, Skeletal; Proteasome Endopeptidase Complex; Proteins; Rats; Tyrosine; Ubiquitins