ARTICLE |
Correspondence to: Ramaswamy K. Iyer, Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles Young Drive (South), Los Angeles, CA 90095-1732. E-mail: riyer@mednet.ucla.edu
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Summary |
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Arginase I (AI), the fifth and final enzyme of the urea cycle, detoxifies ammonia as part of the urea cycle. In previous studies from others, AI was not found in extrahepatic tissues except in primate blood cells, and its roles outside the urea cycle have not been well recognized. In this study we undertook an extensive analysis of arginase expression in postnatal mouse tissues by in situ hybridization (ISH) and RT-PCR. We also compared arginase expression patterns with those of ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT). We found that, outside of liver, AI was expressed in many tissues and cells such as the salivary gland, esophagus, stomach, pancreas, thymus, leukocytes, skin, preputial gland, uterus and sympathetic ganglia. The expression was much wider than that of arginase II, which was highly expressed only in the intestine and kidney. Several co-localization patterns of AI, ODC, and OAT have been found: (a) AI was co-localized with ODC alone in some tissues; (b) AI was co-localized with both OAT and ODC in a few tissues; (c) AI was not co-localized with OAT alone in any of the tissues examined; and (d) AI was not co-localized with either ODC or OAT in some tissues. In contrast, AII was not co-localized with either ODC or OAT alone in any of the tissues studied, and co-localization of AII with ODC and OAT was found only in the small intestine. The co-localization patterns of arginase, ODC, and OAT suggested that AI plays different roles in different tissues. The main roles of AI are regulation of arginine concentration by degrading arginine and production of ornithine for polyamine biosynthesis, but AI may not be the principal enzyme for regulating glutamate biosynthesis in tissues and cells. (J Histochem Cytochem 51:11511160, 2003)
Key Words: arginase, ornithine decarboxylase, ornithine aminotransferase, in situ hybridization, expression
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Introduction |
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ARGINASE is an enzyme that catalyzes the hydrolysis of arginine to ornithine and urea. In mammals, two isoforms of arginase have been identified. They have different tissue distribution, immunological reactivity, physiological function, and subcellular location. Arginase I (AI, hepatic arginase) is expressed highly in the liver cytosol, and its function in ureagenesis has been very well characterized. In contrast to the other urea cycle enzymes, arginase has another isoform, arginase II (AII, extrahepatic arginase). Arginase activity ascribed to AII was first detected two decades ago by a study of patients with AI deficiency (
We recently reported the patterns of arginase isozyme expression in mouse brain and embryo (
AI was found only in human and mouse liver by Northern blotting analysis (
Compared with other urea cycle enzymes, arginase has a much wider distribution in tissues, which suggests that arginase has important physiological roles aside from the urea cycle. These include potential roles as a regulator of the synthesis of polyamines, glutamate, proline, and NO (-ketoglutarate to glutamic-
-semialdehyde and glutamate (Fig 1). Depending on the physiological circumstances, OAT functions in arginine metabolism, leading to glutamate or proline biosynthesis, or by the reverse reaction to de novo ornithine synthesis (
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Materials and Methods |
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Animal and Tissue Preparation
C57BL/6 mice were maintained in the UCLA vivarium facilities and were free of infection with known pathogens. They were fed normal lab chow, were given access to water ad lib, and were maintained on 12-hr lightdark cycles. The studies were approved by the UCLA Animal Welfare Committee.
C57BL/6 mice at ages 1, 2, 3, 6, and 12 weeks were used (except skin for RT-PCR). For ISH study, the animals were perfused with 510 ml of PBS to flush the blood from the circulatory system through the left ventricle, and then perfused with 25 ml of 4% paraformaldehyde in PBS. The tissues were postfixed with 4% paraformaldehyde in PBS overnight, cryoprotected in 30% sucrose in PBS for 24 hr at 4C, embedded with OCT on dry ice, and stored at -80C until sectioning on a cryostat. Sections were cut at 20-µm thickness, thaw-mounted on Superfrost slides (Fisher Scientific; Pittsburgh, PA), and kept at -80C until use. The tissues for RT-PCR analysis were flash-frozen in liquid nitrogen and kept at -80C until use.
Preparation of cDNA Templates
The AI and AII cDNAs fragments were 956 bp (bases 2371192;
Synthesis of DIG RNA Probes and ISH
Digoxigenin (DIG)-labeled RNA probes were transcribed from cDNA fragments inserted into plasmids. On the basis of the cDNA sequence, the plasmid DNAs were linearized by BamHI and XbaI or EcoRV and SpeI for antisense and sense preparation. The linearized plasmid DNAs were purified using the QIAGEN quick PCR purification kit (QIAGEN; Valencia, CA), then checked by 1% agarose gel. Linearized plasmid DNA (>1 µg) template was incubated with T7 or SP6 RNA polymerase, 5 x transcription buffer (Promega; Madison, WI), and DIGRNA labeling mixture (Boehringer Mannheim, Indianapolis, IN) for 2 hr at 37C. It was then incubated with 1 µl deoxyribonuclease I (20 U/µl) for 15 min at 37C to digest template DNA. After ethanol precipitation, the pellet was rinsed with 70% ethanol, dried under vacuum, and reconstituted in RNase-free TE buffer.
Prepared sections were dried for 1 hr at 37C after removal from a -80C freezer. After treatment with 0.25% acetic anhydride in 0.1 M triethanolamine, the sections were incubated with a pre-hybridization solution (Boehringer Mannheim) for 2 hr at 65C and then hybridized overnight at the same temperature with DIG-labeled RNA probe and hybridization solution (Boehringer Mannheim). After washing with 5 x SSC at 65C for 5 min, the slides were washed with 0.2 x SCC at 65C twice for 30 min. Then the sections were incubated with anti-DIG-AP FAb fragments (Boehringer Mannheim) for 2 hr and developed in development buffer (Boehringer Mannheim) overnight at room temperature. Sections were observed and photographed with a CCD camera attached to a Nikon microscope. A brown signal, which was only present in the antisense slides, was judged as positive. No brown signal was observed in sense probe slides (for photos of sense control or AI antisense negative in AI knockout mouse tissues, please refer to
RT-PCR
Total RNA was isolated from 50100 mg of tissue using a total RNA purification system (Sigma). The quality and quantity of total RNA were detected by 230/260/320-nm UV light. To avoid contamination of hair, skin tissue was taken from 2-day-old neonates. RT-PCR was performed using the ThermoScript RT-PCR System (Invitrogen). cDNA was transcribed from 1 µg of total RNA for each one reaction; 2 µl of the cDNA reaction products was used as template in 50 µl of PCR reaction. Amplification was done using 30 cycles consisting of 30 sec at 94C, 30 sec at 55C, and 30 sec at 72C. Reaction mixture without template cDNA was used as a negative control. The PCR reaction (9 µl) was run on a 1.5% agarose gel to visualize the products. All primers for PCR were the same as for ISH.
Histochemistry and Cytochemistry
PAS (periodic acidSchiff) staining was performed for identification of mucopolysaccharides of the salivary gland. Slides were stained in periodic acid solution for 5 min, then in Schiff's reagent solution for 15 min (Sigma). The nuclei were stained with hematoxylin. The confirmation of histology for other tissues was completed using hematoxylin and eosin staining from the contiguous section.
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Results |
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RT-PCR
The expression of AI, AII, ODC, and OAT genes in mouse tissues by RT-PCR is shown in Fig 2. AI expression was seen in most of the tissues studied except kidney. Strong expression was observed in the salivary gland, esophagus, stomach, liver, skin, and preputial gland. Strong AII expression was seen only in the intestine and kidney. ODC was expressed in all the tissues, with strongest expression in the kidney. OAT was present in every tissue studied.
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In Situ Hybridization
AI Expression
The widespread expression of AI, as detected by ISH, is shown in Fig 3. AI was expressed in the salivary gland, esophagus, stomach, intestine, pancreas, liver, thymus, lung, lymph node, skin, preputial gland, uterus, and sympathetic ganglia. In the salivary gland, esophagus, stomach, and preputial gland, AI was located in the epithelium. In the lung, thymus, and lymph node, AI was expressed in the leukocytes. Compared to adults (12 weeks), stronger expression was seen in fetal esophagus, stomach, skin, and lung.
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Digestive System AI was expressed in the salivary gland, esophagus, and stomach in the surface epithelium, duct epithelium, and secretory epithelium. In the salivary glands, AI was seen in both the duct epithelium (Fig 3B and Fig 3C) and the secretory epithelium (Fig 3D and Fig 3E). In the serous gland and mixed gland, only serous gland epithelium expressed AI. No AI expression was seen in the mucous gland epithelium of the mucous gland and mixed gland (Fig 3A1, 3C, and 3D). In the esophagus, the expression was located in the surface stratified squamous epithelium; the expression in neonates (Fig 3F) was much stronger than in adults. In the stomach, strong expression was seen only in the surface epithelium of the cardia and pylorus (Fig 3J, Fig 3K, and Fig 6D). AI expression in the intestine was very weak and was seen only in the 1- and 2-week-old neonates.
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In the liver, AI was expressed in the periportal hepatocytes. No expression was seen in the pericentral hepatocytes (Fig 5A).
Pancreas In the pancreas, very weak expression of AI was seen in the gland cells of the exocrine portion but moderate AI expression was present in the peripheral cells of the islet of Langerhans (endocrine portion) (Fig 3M).
Leukocytes In the lung, scattered AI-positive macrophages/monocytes were present in the alveolar wall and in the alveolar cavity. Concentrated AI positive macrophages/monocytes were also observed in the fetal lung (Fig 3F, Fig 3H, and Fig 4A). AI-positive macrophages were also present in the pericardial and pleural cavity (Fig 3F and Fig 3G). In the thymus, the expression was located in the thymocytes (lymphocytes) in the cortex (Fig 3L and Fig 4C). In the lymph node, the expression was located in the lymphocytes (Fig 3I and Fig 4B).
Sympathetic Ganglia AI was expressed in the ganglion neurons in the sympathetic ganglia. Expression was seen during postnatal development from 1 week to 12 weeks (Fig 3N and 3O).
Other Tissues AI was expressed in the skin and in some reproductive tissues such as preputial gland and uterus. In the skin, AI was expressed in the hair follicle; expression was detected during postnatal day 1 to 2 weeks (Fig 3Q and Fig 6G). In the preputial gland, AI was expressed in the duct epithelium but not glandular epithelium (Fig 3P and Fig 6A). In the uterus, expression was seen in the endometrium (Fig 4D).
AII Expression
By ISH, strongest AII expression was seen in the small intestine (data not shown). Expression in the kidney was weaker than that in the small intestine. In the kidney, AII was present in the inner zone of proximal tubules (Fig 6J).
Expression and Localization Patterns of AI, AII, ODC, and OAT Genes
A comparison of AI, AII, ODC, and OAT expression by ISH is shown in Table 1. Several patterns can be deciphered. (a) AI is the principal form of arginase to be expressed in various tissues and cells, except intestine and kidney. (b) AI co-localized with ODC alone in many tissues, such as esophagus, uterus, macrophages in lung, and lymphocytes in thymus and lymph node (Fig 4). (c) AI co-localized with both OAT and ODC in some tissues, such as the salivary gland epithelium, but AI did not co-localize with OAT alone in any tissue examined. (d) AI was expressed strongly but was not co-localized with either ODC or OAT in a few tissues, such as in preputial gland, stomach, and skin (Fig 6). (e) ODC was highly expressed in the spleen and the bone marrow, in which arginase expression was not seen, and only very low expression of OAT was present (Fig 5). (f) OAT was co-localized with ODC alone in some tissues, such as surface epithelium of respiratory tract and hair bulb of skin (Fig 6). (g) AII was highly expressed in the intestine and kidney and AII was co-localized with ODC and OAT in the small intestine (Table 1). (h) AI expression was not detected in the kidney, whereas no AII expression was found in the liver. AI, OAT, and ODC were not co-localized in the same region of the liver (Fig 5), and AII, ODC, and OAT were not co-localized in the same region of the kidney (Fig 6).
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Histochemistry and Cytochemistry
PAS staining identified subtypes of the salivary glands. The mucous gland epithelium was stained reddish-purple, whereas the serous salivary gland was stained blue-purple only by hematoxylin (Fig 3A2). H&E staining was used for confirmation of leukocytes and other tissues, as demonstrated in our previous study (
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Discussion |
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The present study has shown, for the first time, that AI is not only widely expressed in mouse tissues but that it is also the principal form of arginase expressed in most mouse tissues and cells, except small intestine and kidney (Fig 2 Fig 3 Fig 4 Fig 5 Fig 6). AI is co-localized with ODC in many tissues and with both ODC and OAT in some tissues, whereas co-localization of AII with ODC and OAT was seen only in the small intestine.
Because both AI and ODC exist in the cytoplasm and the reaction catalyzed by the ODC reaction is irreversible, co-localization of AI and ODC could shift the metabolic fate of arginine to the direction of arginineornithine
putrescine. The co-localization pattern of AI and ODC presented here supports the hypothesis that arginase contributes to polyamine biosynthesis (
In tissues where AI co-localizes with ODC and OAT, it is difficult to determine whether the role of OAT is to produce glutamic--semialdehyde/glutamate by the forward reaction (arginine
ornithine
glutamate) or to supply ornithine for polyamine biosynthesis by the reverse reaction. However, if AI co-localized with OAT alone, it would increase the possibility that the metabolic pathway proceeds in the forward direction. The present study shows that AI does not co-localize with OAT alone in any of the tissues examined but does co-localize with both OAT and ODC in a few tissues. Therefore, though ornithine produced by AI could serve as substrate for both glutamate and polyamine biosynthesis pathways in these tissues, the importance of AI in these metabolic pathways is unknown, because a reversible OAT reaction could also supply ornithine for this purpose. Contrary to our expectations, in some tissues, such as the respiratory tract surface epithelium and hair bulb, no arginase expression was seen but strong OAT expression along with ODC expression was observed (Fig 6). Endogenous ornithine is needed for growth and regeneration in these tissues with a relative low blood circulation. The co-localization pattern of OAT and ODC increases the possibility that endogenous ornithine can be supplied by the reverse OAT reaction. The results presented here suggest that AI may not be the principal enzyme producing ornithine for glutamate or proline biosynthesis in most tissues. Instead, OAT may be the principal enzyme that provides endogenous ornithine in some tissues.
Another possible role for AI is regulation of arginine concentration by degradation of arginine in extrahepatic tissues. This is suggested by the AI expression pattern in the gland duct epithelium and the surface epithelium of the upper GI tract. AI was strongly expressed in the duct epithelium of the salivary gland and preputial gland, without co-localization with ODC or OAT (Fig 6). We also examined NO synthase mRNAs (iNOS and eNOS) expression in these tissues but did not find co-expression with AI (data not shown). The duct epithelium AI may regulate the secretory fluid's arginine concentration to influence its calcium concentration and pH for the physiological needs (
In the present study we found that AI was strongly expressed in the macrophages/monocytes in the lung, in the pericardial and pleural cavities, and in the lymphocytes under physiological conditions. AI not only is induced by pathological conditions (
Arginase II was highly expressed in the small intestine, and was co-localized with OAT and ODC in the small intestine (Table 1). Similar results have also been reported by others (
During embryonic development, we found strong AI expression in the sensory ganglion neurons in the dorsal root ganglia and retina (
In the present study, the expression of ISH is concordant with the RT-PCR data in most of the tissues studied. For example, strong AI expression in the salivary gland, esophagus, stomach, liver, and preputial gland was detected by RT-PCR, and strong AI signal was also found in those tissues by ISH. However, in some tissues small amounts of mRNA can be detected only by RT-PCR but not by ISH.
In summary, a central and well-known role of AI is catalyzation of the hydrolysis of arginine to ornithine and urea as the final step of the urea cycle. Furthermore, the role of AI outside the urea cycle has not yet been well recognized. The present study is the first to show that AI is widely expressed in mouse tissues. AI was found to be the principal form expressed in most tissues and cells, except in the intestine and kidney. AI co-localized alone with ODC, but not OAT, in some tissues, whereas AII was not co-localized with either ODC or OAT alone in the tissues examined. AI was also expressed alone in some tissues without co-localization with both ODC and OAT. The arginase, ODC, and OAT expression patterns suggest a dichotomous role of AI, which is production of ornithine for polyamine biosynthesis and regulation of arginine concentration by degrading arginine.
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Acknowledgments |
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Supported by USPHS grants HD-06576 and HD-04612, and in part by the Mental Retardation Research Program at UCLA.
Received for publication November 22, 2002; accepted March 26, 2003.
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