Businaro R, Nori S L, Toesca A, De Renzis G, Ortolani F, De Santis E, Fumagalli L
Department of Cardiovascular Sciences, University La Sapienza, Rome, Italy.
Ital J Anat Embryol. 1995;100 Suppl 1:123-30.
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.
通过结合免疫过氧化物酶共聚焦显微镜和蛋白质印迹分析，在小鼠骨骼肌中研究了一些血浆蛋白酶抑制剂的组织相关对应物。为了去除血清污染，所有实验均在深度麻醉下用生理溶液大量灌注的C57 BL/10成年小鼠身上进行。通过免疫过氧化物酶染色在骨骼肌中研究了以下血清抑制剂：α-2-巨球蛋白（α2M）、抗凝血酶III（ATIII）和α-胰蛋白酶抑制剂（ITI）。使用共聚焦显微镜在488 nm处通过激光透射扫描分析所得的定位模式。然后通过计算机程序对从一系列光学切片获得的图像进行数字增强，从而能够检测到即使是极少量的免疫反应产物。在所有检查的肌肉（比目鱼肌和趾长伸肌）中，所有抑制剂均表现出细胞外（肌内膜）定位。相比之下，在细胞内成分方面观察到了显著差异：事实上，α2M存在于约一半的肌纤维中；ATIII存在于所有纤维内部；细胞内ITI完全不存在。对肌肉匀浆进行蛋白质印迹分析以对上述免疫反应进行生化表征。在初步实验中，在灌注肌肉的可溶部分中未发现与α2M相关的免疫反应性，证实体内灌注后不存在血清污染。相比之下，对去污剂溶解提取物（0.3% Triton X-100）的实验表明，与组织结合的α2M由两条主要条带（168 - 166 kDa）和一个次要成分（35 kDa）组成；ATIII为单一条带（50 kDa）；ITI为四条条带（180、50、45、40 kDa）。这些结果证实，通过形态学技术观察到的特异性免疫反应性对应于与肌肉相关的血浆抑制剂。目前的数据表明，在小鼠骨骼肌中，i）许多与组织相关的血浆抑制剂可能保护细胞外基质免受过度的蛋白水解；ii）一组更有限的抑制剂也可能参与细胞内蛋白水解过程的下调。
