Expression of carbonic anhydrase isozymes (CA-I, CA-II, CA-III) during postnatal development of the rat gastrointestinal tract

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Expression of carbonic anhydrase isozymes (CA-I, CA-II, CA-III) during postnatal development of the rat gastrointestinal tract Shin-ichi Igarashi, Kohshi Miura*, Nobutune Ichihara*, Yutaka Kano*, Toshiho Nishita**, Masao Asari*, and H. Amasaki*** Toxicology Laboratory, Chugai Pharmaceutical Co., Ltd. 14016 Minowa, Nagano 399-46, *Department of Anatomy 1, **Department of Physiology 1, School of Veterinary Medicine, Azabu University, Fuchinobe 1-17-71, Sagamihara, Kanagawa 229, and ***Department of Veterinary Anatomy, Nippon Veterinary and Animal Science University, Musashino city, Tokyo 180, Japan

Introduction Carbonic anhydrase (CA) catalyzes hydration of CO 2 , and the dehydration processes of H 2C0 3 (C0 2 + H 20 <::! H 2C0 3 <::! HC0 3 - + H+) occur quite rapidly. CA is a very important enzyme and is essential to most living things such as bacteria (Veitch and Blankenship 1963), plants (Wayggod 1955), and animals (Wayggod 1955; Hennigar et al. 1983; Kumpulainen 1979; Menghi et al. 1983; Spicer et al. 1982). The precise histolocalization of CA isozymes needs to be understood for the elucidation of the action of the isozymes in organs and tissue. The immunohistochemical localization of CA-I, CA-Il and CA-III in the stomach and large intestine of the adult rat have been previously reported (Igarashi et al. 1992). In this study immunohistochemical examination was made of the expression and distribution of CA-I, CA-II and CA-III in the rat gastrointestinal tract during postnatal development.

Materials and methods Animals: postnatal rats ranging from birth to 35 days from Sprague Dawley (Sic: SD) strains were used. After a lethal overdose of anesthetic ether, the samples of gastrointestinal tract were excised from the animals and examined immunohistochemically. For histochemistry, the samples were taken from the non-glandular and glandular (cardiac, fundic, and pyloric glands) stomach, duodenum, jejunum, ileum, cecum, proximal and distal colon. and rectum.

Correspondence to: M. Asari

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Ann Anat (1996) 178: 369-373 Gustav Fischer Verlag ] ena

Antibodies: CA-I and CA-II were purified from equine erythrocytes (Deutsch et al. 1972), and CA-III from equine striated muscle (Nishita and Deutsch 1981). The cross reactivity and specificity of rabbit anti-equine CA-I, CA-II and CA-III has been previously examined (Igarashi et al. 1992). Immunohistochemistry: tissue pieces were fixed in Bouin's solution overnight, dehydrated in a graded series of ethanol and embedded in Paraplast (Monject, U. S. A.). Serial sections were made at 4 ~m, deparaffinized and rehydrated as usual, pretreated with 3070 H 20 2 in 99.51110 methanol for 5 min at room temperature and incubated with 2070 normal goat serum in 0.01 M PBS, pH 7.4, for 20 min at 37°C. This was followed by incubation with antiequine CA-I, CA-II and CA-III IgG, or normal rabbit IgG (for the negative controls) for 1 hr at 37°C and then treated with the avidinbiotin-peroxidase complex (Vectastain Elite ABC-POD Reagent Kit, Vector, U. S. A.). Each section was counter stained with hematoxylin and observed under a light microscope.

Results The results are summarized in Fig. 1. In the stomach, parietal cells of the glandular region showed immunoreactivity only to CA-II in a 7-day-old rat (Fig. 2a), and likewise surface epithelial cells in a 9-day-old rat. Staining intensity clearly increased during maturation. The non-glandular region and other gastric gland cells showed negative reactions at all stages. In the large intestine, the early expression of CA-II in a 4-day-old rat was apparent in the lower portion of fetal villi in the cecum (Fig. 2 b, c) and in the proximal colon. The expression of CA-l was detected in a 12-day-old rat (Fig. 2d), and that of CA-III in a 21-day-old rat. In the latter, each CA isozyme was stained in a similar way to that in the adult rat

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Fig. 1 a-e. Expression of carbonic anhydrase isozyme-positive cells during postnatal development in the gastrointestinal tract. In the stomach, CA-positive epithelial cells appeared ill a 7-day-old rat, and staining intensity clearly increased during maturation l ). In the large intestine, CA-II-positive epithelial cells were apparent in the cecum and proximal colon in a 4-day-old rat. The expression of CAl-positive epithelial cells detected in the proximal (olon in a 12-day-old rat, and that of CAIII-positive epithelial cells in the same region of a 21-day-old rat. In the latter, staining intensit y clearly increased during maturation 2). Intensity of staining was graded subjectively on a scale of 0 representing nonreactivity to 3 representing very intense dark brown to black staining. I) The CA-positive cells in the stomach include a parietal cell (from 7 days after birth) and a surface mucous cell (from 9 days after birth).

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'/ ') Fig. 2. Paraffin sections of the gastrointestinal tract in rat neonates, showing immunohistochemical staining with anti-equine CA-I, antiequine CA-ll and anti-equine CA-IJI IgG as the primary antibody. Stomach (a) of a 7-day-old rat reacted for CA-JI. The parietal cells of the glandular region showed immunoreactivity (arrow head) . In the cecum (b), the early expression of CA-JI in a 4-day-old rat was apparent in the lower portion of the fetal villi. The proximal colon (c) of an 8-day-old rat reacted for CA-IJ in the same region. The first expression of CA-I detected in a 12-dayold rat in the epithelial line of the cecum (d). In photographs (c) and (d), some reactive cells, possibly lymphoid or mast cells, are strongly positive in the lamina propria of the immunohistochemical controls on the basis of nonspecific affinity for the antibody. Hematoxylin counterstain. x 620 (a, c, d) and x 340 (b)

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in the upper portion of the intestinal glands. Staining intensity clearly increased during maturation. The upper portions of the intestinal glands in the cecum and proximal colon were intensely positive to CA-I and CA-II and moderately to CA-III. Activity gradually decreased from the distal colon to the rectum. Absorptive columnar cells in the lower portion of the intestinal glands and goblet cells showed no immunoreactivity to CA-I, CA-II and CA-Ill. CA isozymes (CA-I, CA-II and CA-III) were not detected in the small intestine. Immunohistochemical findings were essentially the same for both males and females.

Discussion The immunohistolocalization of CA isozymes in the mature rat gastrointestinal (01) tract has been described in detail by Igarashi et a1. (1992). In the rat GI tract, surface epithelial and parietal cells of the glandular region in the stomach showed a positive reaction to CA-II. In the large intestine, immunoreactivity to CA-I, CA-II and CA-III was localized in the upper portion of the intestinal glands, but, absent throughout the small intestine. Generally, cytosolic isozymes of carbonic anhydrase, CAl, CA-II and CA-III are present in various mammalian tissues. The enzyme activity of CA-III is much less than that of CA-I, which has a lower activity than CA-II (Deutsch 1987). CA- II, possessing the greatest activity. is thus the main isozyme and is present in many different types of cells. CA activity may possibly serve different purposes, depending on the biological activity of the cell types. In the stomach, CA-II is involved in the production and secretion of hydrogen ions by the gastric parietal cells and the maintenance of intracellular pH. CA may be regulated by the requirement for the maintenance of intracellular pH during cellular proliferation and by exposure of the gastric surface epithelium to the highly acidic luminal environment of a mature stomach. Here, parietal cells of the stomach showed more immunoreactivity to CA-II in a 7-day-old rat, than did surface epithelial cells in a 9-day-old rat. Marino et al. (1990) have demonstrated that the fundic tissue of developing rat pups, abundantly possesses CA-II-mRNA, even in the mucosa of 1-week-old pups. by in situ hybridization. We observed a correlation between CA-II protein content and mRNA within the gastric mucosa. It is difficult to explain why gastric epithelial cells producing the CA-II isozyme appear when they do during postnatal development. The delay in differentiation of parietal and chief cells may possibly be related ill sume way to the mechanism of the transfer of immunoglobulins (passive immunity) from the mother to offspring. The early differentiation of parietal and chief cells will surely have an adverse effect on the transfer if it occurs via mil k; Ihis has been confirmed in many species including the rat (Rodenwald 1973). The period of absorption from the gut varies from species to species. Absorption may cease (closure) dunng the first week after birth in cattle (Asari et a1. 1987). bur llot until

about 18 days after birth in rats (Halliday 1955; Clark and Hardy 1969). CA is essential to the regulation of luminal and intracellular pH in the large intestine. That is, enteric bacteria flora (EBF) usually develop in the large intestine and produce volatile fatty acids (VFAs), the end products of microbial carbohydrate digestion. The acids are produced faster than they can be absorbed, and consequently it is necessary to neutralize them by HC0 3 buffer systems to keep the luminal pH constant. At this site, CA is closely related to the secretion of HC0 3 in the lumen of the large gut. VFAs produced by EBF are absorbed with H ions into the intestinal epithelial cells. Hydrogen ions that have entered the cytoplasm are neutralized by a buffer system in the cell. A tissue buffer system (HC0 3) is also made available from the hydration of CO 2 by CA (Agenzio 1984). Fetuses are bacteria-free, and intestinal flora colonize the cecum and colon of animals within the first few weeks after birth. The colonization of flora causes increase in VFAs and a reduction in the luminal and intracellular pH in the large intestine. CA would thus appear essential for maintaining the pH at these site early in the postnatal period. In the intestinal surroundings in developing rats, CA-II is expressed first and it is the most active cytosolic CA isozyme in a 4-day-old rat.

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