ARTICLE |
Correspondence to: Masahiro Nishibori, Dept. of Pharmacology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: mbori@med.okayama-u.ac.jp
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Summary |
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Histamine plays important roles in gastric acid secretion, inflammation, and allergic response. Histamine N-methyltransferase (HMT; EC 2.1.1.8) is crucial to the inactivation of histamine in tissues. In this study we investigated the immunohistochemical localization of this enzyme in guinea pig tissues using a rabbit polyclonal antibody against bovine HMT. The specificity of the antibody for guinea pig HMT was confirmed by Western blotting and the lack of any staining using antiserum preabsorbed with purified HMT. There was strong HMT-like immunoreactivity (HMT-LI) in the epithelial cells in the gastrointestinal tract, especially in the gastric body, duodenum, and jejunum. The columnar epithelium in the gallbladder was also strongly positive. Almost all the myenteric plexus from the stomach to the colon was stained whereas the submucous plexus was not. Other strongly immunoreactive cells included the ciliated cells in the trachea and the transitional epithelium of the bladder. Intermediately immunoreactive cells included islets of Langerhans, epidermal cells of the skin, alveolar cells in the lung, urinary tubules in the kidney, and epithelium of semiferous tubules. HMT-LI was present in specific structures in the guinea pig tissues. The widespread distribution of HMT-LI suggests that histamine has several roles in different tissues. (J Histochem Cytochem 48:943954, 2000)
Key Words: histamine N-methyltransferase, histamine, gastrointestinal tract, epithelium, ciliated cell, gallbladder, islet of Langerhans, guinea pig
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Introduction |
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Histamine is stored in mast cells, basophils (, or concanavalin A. Histamine functions as a mediator of inflammation, an immunomodulator, a secretagogue of gastric acid secretion and a neurotransmitter in the CNS. It exerts these effects through the stimulation of three distinct subtypes of receptors, H1, H2, and H3, present on the target cells. The histamine released from storage or producing cells into the extracellular space must be removed to terminate the effect of histamine on target cells. The high-affinity uptake of monoamines, including norepinephrine and serotonin, by respective transporters has been demonstrated (
Histamine plays an important role in gastric acid secretion (
In the guinea pig trachea, the contractile response to histamine in vitro was enhanced by the addition of an HMT inhibitor to the organ bath, shifting the histamine concentrationeffect curve to the left (
In a previous study, we purified HMT from bovine brain (
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Materials and Methods |
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Animals
We followed the guidelines on animal experimentation of Okayama University Medical School. All procedures were approved by the institutional animal experimentation review committee. Male Hartley guinea pigs weighing 250 g (Shimizu Experimental Animals; Kyoto, Japan) were anesthetized with an IP injection of pentobarbital sodium at a dose of 50 mg/kg and perfused through the left ventricle with 50 ml of ice-cold saline, followed by 500 ml of 10% formalin in 0.01 M PBS. The fixed tissues were dehydrated in a series of ethanol solutions, embedded in paraffin, and cut into serial sections 57-µm thick. After the sections had been blocked with 5% normal goat serum and 2% BSA, rabbit anti-HMT antiserum (diluted 1:1500) or preimmune rabbit serum (1:1500) was applied to them for one night at 4C. The sections were rinsed three times with PBS, incubated with biotinylated anti-rabbit IgG goat serum (Vector; Burlingame, CA), washed, and soaked in streptoavidinperoxidase (Sigma; St Louis, MO). Diaminobenzidine and hematoxylin were the substrate for the enzyme reaction and counterstain, respectively. The preimmune serum did not stain any structure.
Western immunoblotting using a supernatant of the guinea pig tissues showed that anti-bovine HMT antiserum recognized a single band (Fig 1). The apparent size of the band was the same as that of purified guinea pig HMT (Fig 1). This indicates that the antibody is crossreactive for guinea pig HMT.
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Production of Antiserum
Bovine brain HMT was purified as described previously (
Purification of HMT from Guinea Pig Brain and Intestine
The brain and intestine from 10 male Hartley guinea pigs were homogenized with four volumes of 20 mM Tris-HCl buffer, pH 7.9, containing 1 mM DTT and 1% polyethylene glycol (PEG) (average MW 300). The homogenate was centrifuged at 36,000 x g for 60 min at 4C. To the resultant supernatant, ammonium sulfate was added slowly with gentle stirring to attain 40% saturation, and 20 min later the suspension was centrifuged at 6000 x g for 30 min. The precipitate was discarded and ammonium sulfate was added to the supernatant fraction to attain 75% saturation. The suspension was centrifuged as described above. The precipitate was dissolved in 20 mM Tris-HCl buffer, pH 7.9, containing 1 mM DTT and 1% PEG and dialyzed overnight against the same buffer with three changes of the buffer. The dialyzed sample was applied to a DEAE-cellulose column (2.5 x 12 cm) equilibrated with the buffer and the HMT activity was eluted with 15 mM histamine in the same buffer. The active fractions were pooled and concentrated by an ultrafiltration cell. The concentrate was dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.5, containing 0.15 M NaCl, 1 mM DTT, and 1% PEG, with two changes of the buffer. The dialyzed sample was applied to an AmodiaquineSepharose 6B column (5 ml bed volume) equilibrated with 10 mM sodium phosphate buffer, pH 7.5, containing 0.15 M NaCl, 1 mM DTT, and 1% PEG. After extensive washing with 300 ml of the equilibration buffer, HMT activity was eluted with 50 mM histamine dihydrochloride containing 1 mM DTT and 1% PEG. The eluate both from guinea pig brain and from intestine showed a single band on SDS-PAGE with Coomassie Blue or silver staining.
Determination of HMT Activity
The tissues were homogenized with 4 volumes of 50 mM sodium phosphate buffer, pH 7.4, containing 1 mM dithiothreitol and 1% PEG (average MW 300). Centrifugation was conducted at 36,000 x g for 1 hr at 4C, and the resultant supernatants were used to determine the HMT activity in each tissue as previously described (
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Results |
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Specificity of the Antibody Used
As shown in Fig 1, anti-bovine HMT antiserum recognized a single band in the samples from different guinea pig tissues, which was the same size as that of the HMT purified from guinea pig intestine and brain. This indicated that the antiserum crossreacted with guinea pig HMT and that the antibody was specific for HMT. The specificity of the antibody was also confirmed in immunohistochemical studies by the lack of staining with preimmune serum (not shown) or immune serum preabsorbed with purified HMT from guinea pig intestine (Fig 2A and Fig 2E). There was no staining with preimmune serum or immune serum preabsorbed with purified HMT in the tissue sections examined in the present study.
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Immunohistochemical Staining of HMT-LI in the Alimentary Tract of Guinea Pig
Immunohistochemical studies revealed that HMT-LI was distributed throughout the peripheral guinea pig tissues. In the gastrointestinal tract, the epithelial cell layer from stomach to colon showed HMT-LI (Fig 2 Fig 3 Fig 4). In the stomach, the intensity of the staining was higher in the surface mucous cells in the body (Fig 3D). The immunoreactivity in the gastric glands decreased at the glandular base (Fig 3C and Fig 3D). In the cardia and pylorus, the immunoreactive staining in the epithelial cell layer was weaker than that in body glands (Fig 3B and Fig 3E). There were HMT-LI neurons in the myenteric plexus of the stomach (Fig 3F and Fig 3G), duodenum (Fig 2C), jejunum (Fig 2G), ileum (Fig 4B), and colon (Fig 4F). A strong immunoreactive plexus was observed in the intestinal region. Immunoreactive fibers originating from the plexus were also seen in the ileum (Fig 4B, arrowheads). In contrast to the myenteric plexus, the submucous plexus showed no immunoreactivity from stomach to colon. In the liver, the hepatocytes surrounding the central veins were moderately immunoreactive and exhibited a lobular structure. A few lobules were devoid of staining (Fig 5A). In the pancreas, the islets of Langerhans were intermediately immunoreactive and the intensity of the staining differed depending on the type of cell (Fig 5C). The centroacinar cells were moderately stained, whereas the acinar cells were weakly positive (Fig 5C). In the gallbladder, the columnar epithelial layer was strongly immunoreactive (Fig 5B). However, the staining in the submucosa and muscular layer was almost negative.
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Immunohistochemical Staining of HMT-LI in the Urinary and Genital Tract of Guinea Pig
In the kidney, all urinary tubule systems, including proximal, Henle, and distal tubules as well as collecting ducts, were intermediately immunoreactive (Fig 5D). On the other hand, renal corpuscles were negative (Fig 5D, inset). In the bladder, the transitional epithelium was strongly immunoreactive (Fig 5E). The smooth muscle cell layer was weakly positive.
Immunohistochemical Staining of HMT-LI in the Respiratory Tract of Guinea Pig
In the respiratory tract, ciliated cells were strongly immunoreactive. The cilia as well as cell bodies were stained homogeneously (Fig 6A). In contrast, goblet cells were devoid of staining. Cartilage cells in the trachea were immunoreactive to some extent (Fig 6C). Tracheal smooth muscle in the posterior portion lacking the cartilage structure was immunoreactive compared with the remaining portions of the ring structure (Fig 6C). In the bronchus, the epithelium, smooth muscle cell layer, and cartilage cells were moderately immunoreactive (Fig 6D). The epithelium and smooth muscle cells in the bronchioles were also moderately stained (Fig 6E). In the lung, irregularly shaped immunoreactive cells were distributed diffusely throughout the alveolar walls (Fig 6F).
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Immunohistochemical Staining of HMT-LI in Other Tissues of Guinea Pig
In the skin of nose, ear, and foot pad, the epidermis exhibited intermediate HMT-LI, which distributed with an increasing gradient to the granular layer (Fig 7A, Fig 7C, and Fig 7D). In the hair follicle, the internal root sheath was intermediately immunoreactive and there were a few strongly immunoreactive cells in the external root sheath (Fig 7B). In the tongue, the epithelium was moderately stained (Fig 7E) and the bundles of nerve fibers among the glossal muscle were weakly stained (Fig 7F). The myocardium of the heart was stained moderately in both the atrium and the ventricle (not shown). The tissue distribution of HMTlike immunoreactivity and HMT activities is summarized in Table 1 and Table 2.
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Discussion |
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We concluded that the anti-bovine HMT polyclonal antibody recognized the guinea pig HMT specifically because the antibody recognized a single band on Western blotting, using different tissue extracts, which was the same size as purified HMT from guinea pig intestine as well as from brain. Immunohistochemical staining also supported the specificity of the antibody because the antiserum preabsorbed with purified guinea pig as well as preimmune serum did not stain any structure in the tissues examined.
The gastrointestinal tract has the highest HMT activity. Consistent with this finding, the epithelial cell layer of the gastrointestinal tract was strongly immunoreactive for HMT. In general, the HMT-LI in the mucosa distributed along a gradient from the luminal side to the deeper mucosa propria. Specific cells in the mucosal epithelium were not stained. This distribution suggested a role for epithelial HMT in the inactivation of histamine from food.
Endogenous histamine is stored in enterochromaffin-like cells in the gastric mucosa and in enterochromaffin cells in the intestinal mucosa (
The smooth muscle cell layer of the gastrointestinal tract was intermediately immunoreactive for HMT. The immunoreactivity was relatively high in the ileum, where histamine-induced contraction through the stimulation of H1 receptors has been used for biological assay of histamine. Interestingly, almost all the neurons in the myenteric plexus were HMT-immunoreactive from the stomach to colon. The neurons in the duodenum and jejunum were strongly positive compared with those in other gastrointestinal regions.
The other strongly immunoreactive cells for HMT were ciliated cells in the trachea. It was reported that the contractile response of guinea pig trachea to exogenous histamine was enhanced by inhibition of HMT and by removal of the tracheal epithelial layer (
The epithelial cells in the gallbladder and the bladder were strongly immunoreactive for HMT. The sensitivity of gallbladder contraction to histamine correlated well with the severity of cystitis (
In this study we showed the localization of HMT in specific cells in the peripheral tissues. Concerning the alternative pathway of histamine catabolism, involving diamine oxidase, few immunohistochemical data are available. Although
Received for publication December 16, 1999; accepted February 16, 2000.
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