BRIEF REPORT |
Correspondence to: Durval R. Borges, Rua Jabuticabeiras 807, 05674-011 São Paulo, Brazil. E-mail: drborges@epm.br
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Summary |
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The liver is important for the kallikreinkinin system modulation. This system plays a role in the inflammatory cascade with anticoagulant, profibrinolytic, and anti-adhesive attributes. The metalloendopeptidase EP24.15 is a major hepatic kininase. We studied the tissue distribution and subcellular localization of this enzyme in rat liver by cell fractionation and immunohistochemistry. Our results showed that EP24.15 is predominant in the soluble fraction of the liver homogenate and is present in the cytoplasm of hepatocytes, particularly in the perivenous zone (Z3). This localization is relevant because most hepatotoxin-induced necrosis, as well as ischemic hepatocellular injury, is predominant in Z3. (J Histochem Cytochem 51:125127, 2003)
Key Words: bradykinin, kininase, endopeptidase-24.15, liver
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Introduction |
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THE LIVER plays a role in the systemic inflammatory response and it is relevant in modulation of the kinin system. This system, in turn, plays a role in the inflammatory cascade featuring anticoagulant, profibrinolytic and anti-adhesive attributes (
Adult male Wistar rats, raised and provided by the animal house of the Universidade Federal de São Paulo, were manipulated in accordance with the "International Guiding Principles for Biomedical Research Involving Animals" (Council For International Organization of Medical Sciences 1985, Geneva).
Liver exsanguination was performed at 37C as previously described (
Cell fractionation was performed according to EM = 420 nm and
EX = 320 nm). The amount of protein varied among the fractions but after 10 min of incubation less than 10% of the substrate was consumed, even for the cytoplasmic fraction, which contained the highest activity, indicating first-order kinetics. The assay was performed in both the absence (total kininase activity) or presence (5 µM) of the EP24.15 specific inhibitor (EP24.15 activity), the N-[1(RS)-carboxy-3-phenyl-propyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), a generous gift from Dr. M.J. Glucksman (Mount Sinai School of Medicine, New York, NY). The enzymatic activity was expressed in µmoles of substrate hydrolyzed/min and the total fraction activity obtained by multiplying the activity by the fraction volume. The results were analyzed by the GraphPad Prism software (version 1.03).
For immunohistochemistry, the exsanguinated livers were removed and fragments of 2 cm were fixed by immersion for 8 hr with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Liver samples were then paraffin-embedded and 5-µm-thick sections were made and placed on poly-L-lysine-coated glass slides. The rabbit antiserum to detect EP24.15 was a gift of Dr. E.S. Ferro (Biomedical Sciences Institute, USP, Brazil). Detection of EP24.15 was performed as follows. The liver sections were re-hydrated and after several washes in PBS the unspecific sites were blocked with Superblock blocking buffer (Pierce; Rockford, IL) containing 5% normal goat serum (NGS) and 5% bovine serum albumin (BSA) for 30 min at room temperature (RT) after incubation with the EP24.15 antiserum diluted 1:500 in PBS containing 3% NGS, 5% BSA, and 0.3% Triton X-100 in PBS for 18 hr in a humid chamber at 4C. After washing steps sections were incubated with Cy-3 labeled anti-rabbit IgG (Sigma; St Louis, MO) diluted 1:200 in PBS for 2 hr at RT. The sections were washed and finally coverslipped using Vectashield with DAPI (4',6'diamino-2-phenylindole) (Vector Laboratories; Burlingame, CA) as mounting medium. The analysis was made in a Nikon Eclipse-E800 equipped with epifluorescence. The images were acquired using a CCD camera and processed for better contrast using Adobe's Photoshop 5.0.
The mitochondrial, lysosomal, microsomal and cytoplasmic fractions were characterized by the presence of their specific markers. Considering as 100% the EP24.15 activity measured in all fractions (N, Mt, L, M, and C), 90% of its total activity was found within the cytoplasmic fraction (Fig 1). Its specific activity (U/g protein) in this fraction was 4, 7, and 16 times greater compared to the Mt, L, and M fractions, respectively. These results are in agreement with the typical cytosolic activity of the EP24.15 found in mammalian tissues (
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Immunohistochemistry demonstrated EP24.15 in the cytoplasm of hepatocytes (Fig 2A), mainly in those surrounding the centrolobular vein (zone 3). Other cell types, such as Kupffer cells and stellate cells, were not labeled. Although cells of this region receive fewer nutrients and less oxygenated blood supply compared to hepatocytes of zone 1, they are enriched with endoplasmatic reticulum and therefore have great metabolic activity. Observation with a UV filter (Fig 2B) to visualize DAPI nuclear staining ruled out the existence of EP24.15 immunoreactivity in the nucleus of the cells. We conclude that EC 3.4.24.15 is a cytoplasmic enzyme present mainly in hepatocytes of perivenous regions. This localization is relevant because most hepatotoxins induce necrosis which, in addition to ischemic hepatocellular injury, is predominant in zone 3.
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Acknowledgments |
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Supported by grants from FAPESP 99/12435-2 and PRONEX (41.96.0873.00) and by FADA/UNIFESP.
We thank Dr E.S. Ferro (Biomedical Sciences Institute, USP, Brazil) who provided the rabbit antiserum against recombinant EP24.15; Dr L. Juliano (Biophysics Department, Unifesp, Brazil) for the fluorogenic substrate; and Dr M.J. Glucksman (Mount Sinai School of Medicine, NY) for the inhibitor of EP24.15; and Erika Suzuki for image editing.
Received for publication May 13, 2002; accepted August 7, 2002.
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