Localization of collagenase in normal rat liver is studied by the electron-histochemicaI method. Enzyme activity is detected in Kupffer and endothelial cells and on hepatocyte microvilli.
Key Words: collagenase; fiver, electron histochemistryCollagenase is a key enzyme in collagen metabolism. Tissue collagenases are zinc-dependent metalloenzymes that cleave various types of interstitial collagens across the three chains into 1/4 and 3/4 length fragments [6]. The investigation of collagenases has been hampered by a number of difficulties, one of them being that the enzymes are not accumulated in the cell but are synthesized as necessary. Therefore, only small amounts of enzyme have been detected in cells and tissues, and the majority of collagenase studies have been carried out in vitro. Morphological identification of the enzyme is very difficult. In the liver, two forms of collagenase are present: active and latent [7]. Proenzyme can be activated by trypsin, plasmin, kallikrein [1,11], and cathepsins B [2] and G [14]. Since collagenase normally occurs in the latent state, it can be visualized only by immunomorphological methods. Collagenase activity in the liver was first described by Japanese scientists [10] and then confirmed by other researchers [4,12]. It is generally accepted that Kupffer cells are the major producers of collagenase [3,5]. They contain the enzyme in the active form. In addition, two types of collagenase have been demonstrated in the liver; one type is produced by hepatocytes and the other by sinusoidal cells [9]. However, none of these biochemical investigations provide information regarding the intracellular localization of the enzyme in the liver. The only morphological study of collagenase at the light microscopy level with the use of immuno- logical methods has shown the enzyme to be localized extracellularly, being associated with collagen fibrils and basal membranes [8].This study was designed to identify the cells producing collagenase and to reveal the structural localization of active collagenase in normal rat liver.
MATERIALS AND METHODSLivers of male albino rats weighing 180-200 g were studied. Electron-histochemical identification of collagenase was performed using the techniques developed in our laboratory, based on the reaction of successive nitrogen coupling [3]. Tissue pieces (0.5 mm 3) were fixed for 3 h in 1.5% glutaraldehyde on 0.05 M cacodylate buffer (pH 7.4) containing 2% sucrose. The material was then washed with the same buffer containing 7% sucrose. The incubation medium contained 10 ml 0.05 M Tris-HCI buffer (pH 7.5), 10 mM CaCI 2 [14], and 10 mg Z-Pro-Ala-Gly-Pro-4-MBNA substrate (Serva) dissolved in dimethylformamide (DMF, 10 ~1/10 ml final volume). After 1-h incubation at 37~ the material was washed with cacodylate buffer (0.1 M, pH 6.5) and incubated in the same buffer containing hexazotated pararosaniline (50 ~l/mt) at 37~ for 30 min. After washing with cacodylate buffer (0.05 M, pH 7.4) containing 7% sucrose, the material was treated with 2% OsO 4 for 90 rain...
The location of cathepsin L in rat liver was studied by electron histochemical methods. Enzyme activity was detected in lysosomes of hepatocytes, Kupffer cells, and endotheliocytes and extracellularly on hepatocyte microvilli.
The location of elastase in rat uterus during its postpartum involution was studied by electron histochemical method. Intense extracellular activity of the enzyme was detected: the reaction product was found on the cytolemma of smooth-muscle cells and, to a lesser extent, on the cytolemma of macrophages and fibroblasts and on adjacent collagen fibrils. The reaction product was also found in vacuoles with collagen in macrophages and fibroblasts.
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