PROTEASE INHIBITORS IN MOUSE SKELETAL-MUSCLE - TISSUE-ASSOCIATED COMPONENTS OF SERUM INHIBITORS AND CALPASTATIN

The proteinase inhibitor set in skeletal muscle is poorly characterize d at present. This study was aimed to investigate in mouse skeletal mu scle 1) the tissue-associated counterpart, if any, of serum protease i nhibitors (which may also play antiproteolytic functions in tissues) a nd 2) calpastatin, a tissue inhibitor of calcium-activated neutral pro teases (calpains). Triton-extracts were prepared from muscle homogenat es of mice, which had been perfused extensively with phosphate buffere d saline (PBS) (under deep anesthesia) to remove blood inhibitors. Amo ng various inhibitors tested, the following muscle-associated inhibito rs were identified by western-blotting: alpha-2-macroglobulin (185, 16 5, 35 kDa), alpha-1-antitrypsin (52 kDa), inter-alpha-trypsin inhibito r (220, 180 kDa) and calpastatin (70 kDa). Combined light microscope a nd confocal immunohistochemical experiments revealed that, in all musc les examined (soleus, plantaris, extensor digitorum longus) the above specific immunoreactivities were localized outside the muscle fibers ( in peri-endomysium, blood vessel wall) as well as within them. Inter-a lpha-trypsin inhibitor, however, completely lacked the intracellular l ocalization. This wide distribution of proteinase inhibitors suggests that numerous muscular structures may be normally protected from unwan ted proteolysis, thus providing an essential background for further st udies on pathological models with altered proteolysis (m. dystrophy, d enervation atrophy, etc.).