ARTICLE |
Correspondence to: Koki Hirasawa, First Department of Medicine, Hamamatsu U. School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka, 431-3192, Japan. E-mail: k_shimo@bea.hi-ho.ne.jp
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Summary |
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Angiotensin II (Ang II) regulates water and sodium transport in renal tubules and gastrointestinal tract. Two types of Ang II receptors have been cloned, but their distributions have not been determined in human colon. In addition, tissue reninangiotensin systems (RAS) are believed to exist and to regulate local actions in human colon. We studied by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) the presence and localization of Ang II receptors Type 1 (AT1), Type 2 (AT2), and RAS components [angiotensinogen, renin, and angiotensin-converting enzyme (ACE)] in normal human colon. AT1 receptors were localized in vessel walls, myofibroblasts, macrophages, and surface epithelium. AT2 receptors were found in mesenchymal cells and weakly in parts of surface epithelium. Renin and ACE were distributed in vessel walls, mesenchymal cells, and in parts of surface epithelium. Angiotensinogen was also detected by RT-PCR. These findings demonstrated that Ang II receptors and RAS components were present in human colon, suggesting the possibility of its local regulation. (J Histochem Cytochem 50:275282, 2002)
Key Words: reninangiotensin system, angiotensin II receptor, human colon
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Introduction |
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Angiotensin ii (Ang II) has many different actions, most of which are related, either directly or indirectly, to the regulation of blood pressure and of fluid and electrolyte homeostasis. Ang II modulates the transfer of sodium and water across epithelial tissues of the proximal renal tubules and gastrointestinal tract (
In addition to the classical pathway of Ang II synthesis, tissue reninangiotensin systems (RAS) have been identified in a number of organs, suggesting that various tissues have the ability to synthesize Ang II independently of the circulating RAS (
To the best of our knowledge, no report is available concerning the localization of AT1 and AT2 receptors and the distribution of RAS components in human colon. We therefore investigate the presence and localization at the mRNA and protein levels of Ang II receptors and RAS components in human colon mucosa.
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Materials and Methods |
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Samples
Two pieces of biopsy specimens were obtained of normal-appearing colon mucosa from patients positive for fecal occult blood. The subjects were 40- to 60-year-old men without hypertension, heart failure, renal disease, sarcoidosis, diabetes mellitus, or inflammatory bowel disease. The research protocol was approved by the Research and Development Committee of the Hamamatsu University School of Medicine.
RT-PCR
Total RNA was purified from biopsy samples by the acid guanidinephenolchloroform method (
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Immunohistochemical Procedures
Other biopsy samples were immediately washed in cold distilled water and fixed for 24 hr by immersion in 4% paraformaldehyde in PBS 0.1 M (pH 7.4) at 4C, and subsequently replaced with 0.1 M PBS (pH 7.4) for 24 hr. Paraformaldehyde-fixed biopsy specimens were embedded in paraffin and cut at 5 µm thickness. Rabbit anti-AT1 receptor polyclonal antibody (sc-1173) and goat anti-AT2 receptor polyclonal antibody (sc-7420) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-ACE monoclonal antibody (CD-143) was purchased from Chemicon International (Temecula, CA). The selectivity of these antibodies has been confirmed in Western blotting analysis as previously described (
Double Immunofluorescence Studies
To confirm types of mesenchymal cells that showed immunoreactivity for AT1 receptors, we used a double-immunofluorescence method. Because, in kidney and heart, myofibroblasts and macrophages are known to express AT1 receptors on their cell surfaces (
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Results |
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RT-PCR for AT1 and AT2
AT1 receptor mRNA was found in the samples of sigmoid colon and ascending colon (Fig 1A). AT2 receptor mRNA transcripts were also detected in sigmoid and ascending colon (Fig 1B).
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RT-PCR for the Components of RAS
All RAS component (angiotensinogen, renin, and ACE) mRNA transcripts were found in both sigmoid colon and ascending colon (Fig 1C). Without RT (for negative control), no mRNA was found. The nucleotide sequences of the above PCR products were identical to the known human genes.
Immunohistochemistry for Ang II Receptor
On IHC examination of human sigmoid colon, immunoreactivity for AT1 receptors appeared to be localized in vessel walls, surface epithelium, crypt bases, and in mesenchymal cells in the lamina propria (Fig 2B and Fig 2C). To confirm the types of mesenchymal cells, we performed double immunofluorescence staining. As with IHC staining, AT1 receptor-positive cells were seen in surface epithelium, crypt bases, and in mesenchymal cells in the lamina propria (Fig 3D and Fig 3G). Most of the mesenchymal cells below the surface epithelium were macrophages (Fig 3E), some of which showed immunoreactivity for AT1 receptors (Fig 3F). -Smooth muscle actin was seen in crypt bases and weakly in some mesenchymal cells (Fig 3H). Double immunofluorescence staining with AT1 receptors and
-smooth muscle actin antibodies showed that some of myofibroblasts also expressed AT1 receptors (Fig 3I). Immunoreactivity for AT2 receptors was observed in parts of mesenchymal cells and weakly in parts of surface epithelium in sigmoid colon (Fig 2E and Fig 2F). A small number of crypt cells were also immunoreactive for AT2 receptors. The ascending colon exhibited the same pattern of immunoreactivity for AT1 and AT2 receptors (data not shown). No immunostaining was found in control sections incubated with preabsorbed sera instead of primary antibodies (Fig 2A and Fig 2D).
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Immunohistochemistry for Components of RAS
On IHC examination of human colon, immunoreactivity for renin appeared to be localized in the surface epithelium, vessel walls, muscularis mucosae, and in mesenchymal cells in the lamina propria (Fig 4B and Fig 4C). Immunoreactivity for ACE appeared to be shown in vessel walls and mesenchymal cells in lamina propria and submucosa and weakly in parts of the surface epithelium in ascending colon (Fig 4D and Fig 4E). Heterogeneity in immunoreactivity between sigmoid colon and ascending colon could not be found in renin and ACE (data not shown). No immunostaining was found in control sections incubated with preimmune sera instead of primary antibodies (Fig 4A).
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Discussion |
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To the best of our knowledge, no report is available concerning localization of AT1 and AT2 receptors in human colon.
Ang II has many physiological functions in vivo. Most of the functions of Ang II, including, among others, vascular constriction, cell proliferation, and release of aldosterone, are known to mediate via AT1 receptors. AT1 receptors in vessel walls in human colon mucosa may regulate vascular constriction (
Intestinal subepithelial myofibroblasts (ISEMFs) are located in the lamina propria under the epithelial cells. They exist as a syncytium and have important roles in releasing chemical mediators and extracellular matrix proteins (
Classically, angiotensin has been considered as a plasma hormone. The discovery of components of the RAS in many tissues has led to the hypothesis that, in addition to being a plasma hormone, Ang II may be formed locally and may therefore possess an additional paracrine and/or autocrine action. There are currently inconsistent issues concerning the origin of tissue RAS components in the cardiovascular system. It has not been clearly shown whether or not local RAS components are derived from plasma or local production in tissues, or from both (
In conclusion, we obtained the following findings. (a) AT1 receptors were localized in vessel walls, macrophages, and myofibroblasts in the lamina propria and in the surface epithelium of human colon. (b) AT2 receptors were localized in parts of mesenchymal cells and weakly in the surface epithelium. (c) Renin and ACE were distributed in parts of the surface epithelium, vessel walls, and mesenchymal cells. These findings suggest the possibility that RAS exists in human colon mucosa. However, the definition of existence of local RAS is required to demonstrate generation of Ang II in colon, in addition to the existence of its components. Further studies will be needed.
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Acknowledgments |
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We thank Dr Akiyoshi Fukamizu for his kind gift of anti-human renin antibody. The help of Ms Satoko Takebayashi, Ms Yoko Takei, Ms Hiroko Murakami, Ms Kazuko Tsukioka, and Ms Kyoko Nishitani, the nurses in the Division of Endoscopy, is greatly appreciated.
Received for publication June 15, 2001; accepted October 1, 2001.
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