Protease inhibitors in mouse skeletal muscle: tissue-associated components of serum inhibitors and calpastatin.
The proteinase inhibitor set in skeletal muscle is poorly characterized at present. This study was aimed to investigate in mouse skeletal muscle 1) the tissue-associated counterpart, if any, of serum protease inhibitors (which may also play antiproteolytic functions in tissues) and 2) calpastatin, a tissue inhibitor of calcium-activated neutral proteases (calpains). Triton-extracts were prepared from muscle homogenates of mice, which had been perfused extensively with phosphate buffered saline (PBS) (under deep anesthesia) to remove blood inhibitors. Among various inhibitors tested, the following muscle-associated inhibitors were identified by western-blotting: alpha-2-macroglobulin (185, 165, 35 kDa), alpha-1-antitrypsin (52 kDa), inter-alpha-trypsin inhibitor (220, 180 kDa) and calpastatin (70 kDa). Combined light microscope and confocal immunohistochemical experiments revealed that, in all muscles examined (soleus, plantaris, extensor digitorum longus) the above specific immunoreactivities were localized outside the muscle fibers (in periendomysium, blood vessel wall) as well as within them. Inter-alpha-trypsin inhibitor, however, completely lacked the intracellular localization. This wide distribution of proteinase inhibitors suggests that numerous muscular structures may be normally protected from unwanted proteolysis, thus providing an essential background for further studies on pathological models with altered proteolysis (m. dystrophy, denervation atrophy, etc.).
Fumagalli
L
L
Department of Cardiovascular Sciences, University La Sapienza, Rome, Italy.
Businaro
R
R
Nori
S L
SL
Toesca
A
A
Pompili
E
E
Evangelisti
E
E
Giannetti
S
S
Ippoliti
F
F
De Renzis
G
G
D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors*; D007124:Immunoenzyme Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D011480:Protease Inhibitors; C033668:calpastatin; D002154:Calpain