Immunohistochemical detection of three serum protease inhibitors in mouse skeletal muscle by confocal laser scanning microscopy.

The tissue-associated counterpart of some plasmatic protease inhibitors has been studied in mouse skeletal muscle by combining immunoperoxidase confocal microscopy and Western blot analysis. To remove serum contamination all experiments were performed on C57 BL/10 adult mice perfused extensively with physiological solution under deep anesthesia. The following serum inhibitors were investigated in skeletal muscle by immunoperoxidase staining: alpha-2-macroglobulin (alpha2M), antithrombin III (ATIII) and inter-alpha-trypsin inhibitor (ITI). The resulting localization patterns were analysed by laser transmittance scanning at 488 nm using a confocal microscope. Images obtained from a series of optical sections were then digitally intensified by a computerized program, allowing detection of even negligible amounts of immunoreaction product. In all muscles examined (soleus and extensor digitorum longus mm.) an extracellular (endomysial) localization was apparent for all inhibitors. By contrast remarkable differences were observed for the intracellular component: in fact alpha2M was present in about a half of the muscle fibers; ATIII was present inside all fibers; intracellular ITI was completely absent. Western blotting analysis of muscle homogenate was performed to biochemically characterize the above immunoreactivities. In preliminary experiments alpha2M-related immunoreactivity could not be found in the soluble fraction of perfused muscle, confirming an absence of serum contamination after in vivo perfusion. By contrast experiments on detergent-solubilized extracts (0.3% Triton X-100) revealed that tissue-bound alpha2M consisted of two main bands (168-166 KDa) and a minor component (35 KDa); ATIII of a single band (50 KDA); ITI of four bands (180, 50, 45, 40 KDa). These results confirmed that the specific immunoreactivities visualized by morphological techniques corresponded to muscle-associated plasmatic inhibitors. The present data suggest that in mouse skeletal muscle i) numerous tissue-associated plasmatic inhibitors may protect the extracellular matrix from an excess of proteolysis; ii) a more restricted set of inhibitors may be also involved in the down-regulation of intracellular proteolytic processes.