Acta Histochem. Cytochem. 35 (5): 417–422, 2002

Monoamine Oxidase Type B is Localized to Mitochondrial Outer Membranes in Mast Cells, Schwann Cells, Endothelial Cells and Fibroblasts of the Rat Tongue

Yang Xu1, Kazusada Yoshitake2, Akio Ito3 and Ryohachi Arai1

1Department of Anatomy and 2Department of Oral and Maxillofacial Surgery, Shiga University of Medical Science, Otsu, Shiga 520–2192 and 3Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Fukuoka 812–8581

Received September 5, 2002; accepted October 11, 2002

We have used immunohistochemistry to outer membranes of mitochondria in examine the subcellular localization of mast cells, Schwann cells, capillary endo- type B (MAOB) in thelial cells and fibroblasts. The present the rat tongue. Light microscopy showed study is the first to immunohistochemi- that MAOB-immunoreactive structures cally reveal the intracellular localization were present in connective tissues. Elec- of MAOB in tissues outside of the central tron microscopy revealed that MAOB nervous system. Possible functions of immunoreactivity was localized to the MAOB in these cells are discussed.

Key words: MAOB, Tele-methylhistamine, Immunohistochemistry, Electron microscopy, Subcellular localization

I. Introduction chondria in catecholaminergic neurons of the brain and spinal cord of rat and monkey [33]; and that MAOB immu- Monoamine oxidases are enzymes that degrade biogen- noreactivity is found on the mitochondrial outer membrane ic monoamines [1, 30]. There are two types of monoamine in serotonergic neurons and astrocytes of the rat brain [2]. In oxidase enzymes. Monoamine oxidase type A (MAOA) has peripheral organs, MAOB activity has been shown to be far a higher affinity for the substrates, and noradrena- more abundant in the outer membrane fraction of rat liver line, and the inhibitor clorgyline, whereas monoamine oxi- mitochondria than in the inner membrane fraction [28]. Us- dase type B (MAOB) has a higher affinity for the substrate, ing an enzyme histochemical method employing a substrate -phenylethylamine, and the inhibitor deprenyl [17, 18]. common to both MAOA and MAOB, subcellular localiza- is a common substrate of both MAOA and tion of MAO activity has been examined in the heart, liver, MAOB [8]. MAOA and MAOB are comprised of distinct adrenal cortex and medulla, and kidney of rats [9, 24]. How- amino acid sequences in humans [3] and rats [16, 19], and ever, to our knowledge, no previous study has examined, by their genes are located at adjacent sites on the X-chromo- immunohistochemistry, the individual subcellular localiza- some in both humans [20, 21] and mice [7]. MAOA knock- tion of MAOA or MAOB enzyme in peripheral tissues. out mice have elevated brain levels of serotonin, noradrena- Recently, we raised and characterized a rabbit antiserum line and dopamine; and show aggressive behavior [4]. In against bovine MAOB, which crossreacts with rat MAOB contrast, MAOB knock-out mice do not exhibit aggression, [2, 27]. Using this antiserum, we have now performed and only levels of -phenylethylamine in the brain are in- immunohistochemistry at the electron microscopic level, to creased [12]. Both MAOA- and MAOB-deficient mice examine the subcellular localization of MAOB in the rat demonstrate an increased response to stress [4, 12]. tongue. This organ of humans is known to contain MAOB The subcellular localization of MAOA and MAOB has activity [22]. been examined in the central nervous system. A biochemical study demonstrated that MAOA and MAOB activities are II. Materials and Methods found predominantly in the mitochondrial fraction of rat brain homogenates [31]. Immunohistochemical studies us- Animals ing electron microscopy have shown that MAOA immuno- Male Sprague-Dawley rats (n3, 180–200 g) were ob- reactivity is associated with the outer membrane of mito- tained from Japan SLC (Hamamatsu, Japan). All experi- ments were carried out according to the Guidelines for Correspondence to: Dr. Ryohachi Arai, Department of Anatomy, Animal Experimentation at Shiga University of Medical Shiga University of Medical Science, Otsu, Shiga 520–2192, Japan. Science. All efforts were made to minimize both the number

417 418 Xu et al. of animals used and their suffering. III. Results Primary antiserum Light microscopy showed that MAOB immunoreactiv- MAOB purified from bovine liver mitochondria was ity was present in connective tissues, but not in the epitheli- used to generate a rabbit anti-MAOB antiserum [27]. This um or muscle fibers (Fig. 1). Electron microscopy showed anti-MAOB antiserum has been shown to immunoprecipi- that MAOB immunoreactivity was present in mast cells tate MAOB from rat liver lysate [27]. Immunohistochemical (Fig. 2A), Schwann cells (Fig. 2B), capillary endothelial analysis has shown that this antiserum stains MAOB-con- cells (Fig. 3A) and fibloblasts (Fig. 3B). In these cells, mito- taining cells but not MAOA-containing cells in the rat chondria showed MAOB immunohistochemical reaction brain, indicating that the antiserum specifically recognizes products on and around their outer membranes (Figs. 2, 3). rat MAOB [2]. Some mitochondria in capillary endothelial cells were very weakly stained for MAOB (Fig. 3A). Electron microscopic immunohistochemistry Three rats were anesthetized with sodium pentobarbital IV. Discussion (60 mg/kg body weight, i.p.) and perfused through the ascending aorta with 50 ml of 0.01 M phosphate-buffered In summary, we have shown that MAOB is localized to saline (pH 7.4, room temperature), followed by 300 ml of a the mitochondrial outer membranes in mast cells, capillary fixative containing 4% paraformaldehyde, 0.3% glutaralde- endothelial cells, Schwann cells and fibroblasts of the rat hyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.4, 4LC). The tongues were dissected and placed in another fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer for 24 hr at 4LC. Frontal, 50 m thick sections were cut using a microslicer (Dosaka, Kyoto, Japan) and collected in 0.1 M phosphate-buffered saline (PBS). For MAOB staining, we employed an immunoperoxi- dase technique using an avidin-biotin-peroxidase complex [15]. Sections were incubated in the following solutions: (1) 0.001% trypsin (type III, Sigma, St. Louis, MO, USA) in PBS for 5 min at room temperature; (2) 5% normal goat serum in PBS for 1 hr at room temperature; (3) rabbit anti- MAOB antiserum (1: 60,000 in PBS) with 1% normal goat serum for 48 hr at 4LC; (4) biotinylated goat anti-rabbit immunoglobulin G (BA-1000, Vector, Burlingame, USA, 1:1,000 in PBS) for 2 hr at room temperature; (5) avidin- biotin-peroxidase complex (PK-4000, Vector, 1:1,000 in PBS) for 2 hr at room temperature; and (6) 0.025% 3,3'- diaminobenzidine (Dojindo, Kumamoto, Japan), 0.6% nickel ammonium sulfate (Nacalai Tesque, Kyoto, Japan), and 0.006% hydrogen peroxide in 0.05 M Tris-HCl buffer (pH 7.6) for 10 min at room temperature. The stained sec- tions were fixed with 1% osmium tetroxide (Nacalai Tesque) in 0.1 M phosphate buffer for 1 hr at 4LC, dehydrated and flat-embedded in epoxy resin (Luveak-812, Nacalai Tesque). The embedded sections were examined under a light micro- scope (BX51, Olympus, Tokyo, Japan). Small areas contain- ing the epithelium, connective tissue and muscle fibers were trimmed from the embedded section and cut on an ultrami- crotome. Ultrathin sections were collected on grids, stained with uranyl acetate (Nacalai Tesque) and lead stain solution (Sigma-Aldrich Japan, Tokyo, Japan), and examined in a transmission electron microscope (5000, Hitachi, Tokyo, Japan). As a control for the immunohistochemical staining, Fig. 1. Presence of MAOB in connective tissues (thin arrows), but sections were processed as described above, omitting the not in the epithelium (thick arrows) or muscle fibers (asterisks). primary antiserum. No immunoreactivity was found in the Frontal section of the rat tongue was stained for MAOB with the control sections. immunoperoxidase method, fixed with osmium, and embedded in Epon. Bar30 m. Monoamine Oxidase Type B in Tongue 419

Fig. 2 420 Xu et al.

Fig. 3 Monoamine Oxidase Type B in Tongue 421 tongue connective tissue. The present study is the first to humans) or experimental methods (electron and light show the presence of MAOB in mast cells and capillary microscopy). Our finding of MAOB in capillary endothelial endothelial cells. We are also the first to immunohisto- cells of the rat supports an idea that the enzyme activity in chemically reveal the intracellular localization of MAOB microvessels plays a protective role for the brain by degra- at electron microscopic levels in peripheral tissues. dation of extraneous monoamines, such as a neurotoxin By electron microscopy, we found MAOB immuno- 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, in the blood histochemical reaction products on and around the mito- [25]. Similar roles are possibly played by MAOB in capil- chondrial outer membranes. Our question is now concerned lary endothelial cells of peripheral organs. with whether MAOB itself is localized not only on but also In the present study, MAOB immunohistochemical re- around the mitochondrial outer membranes. The immuno- action products were found on and around the mitochondrial histochemical signal in our assay derives from 3,3'-diamino- outer membranes of Shwann cells. A previous study using benzidine oxidation products formed by peroxidase activity enzyme histochemistry showed that MAOB reaction prod- in the avidin-biotin-peroxidase complex [15]. In immuno- ucts were localized throughout the whole extent of the cyto- histochemistry using peroxidase for electron microscopy, plasm of Shwann cells in cerebral arteries of the rat [29]. 3,3'-diaminobenzidine oxidation products can spread out However, taking into consideration the possible diffusion of from the site of the antigen [6, 23]. It therefore seems likely the reaction products, it is still likely that the primary site of that the primary site of MAOB localization is on the mito- MAOB localization is on the mitochondrial outer membrane chondrial outer membrane. of Shwann cells. We have also shown that MAOB is local- Tele-methylhistamine is the most likely substrate of ized to the mitochondrial outer membranes in fibroblasts of MAOB in mast cells. Mast cells synthesize , which the rat tongue. This is consistent with findings of a previous is stored in granules that are discharged upon antigenic chal- immunohistochemical study at light microscopic levels that lenge or tissue damage [5]. Histamine released from mast showed fibroblasts of an artery of the human kidney contain cells acts mainly through H1-receptors [13]. Histamine MAOB [26]. Exact roles of MAOB in Scwann cells and occurs by two distinct pathways: oxidative fibroblasts are not known, but the enzyme may be involved deamination by diamine oxidase, and methylation by hista- in the metabolism of biogenic and extraneous monoamines. mine methyltransferase (HMT) [11]. The histamine metabo- tele lite, -methylhistamine, is further processed for oxidative V. Acknowledgments deamination by MAOB [14]. Tele-methylhistamine is de- tected in histamine-containing granules but not in the cyto- This work was supported by Grant-in-Aid for Scientific plasm of mast cells of the rat peritoneum [10]. Although Research (KAKENHI 13680819) of Japan Society for the mast cells of the rat peritoneum have no detectable HMT Promotion of Science. activity [10], the epithelium of the guinea pig tongue was immunohistochemically stained for HMT [32]. Therefore, it is likely that histamine released from mast cells of the VI. 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Fig. 2. A: Subcellular localization of MAOB in mast cells. Immunohistochemical reaction products (arrows) are observed on and around mitochondrial outer membranes. G, granule; Nu, nucleus. Bar1 m. B: Subcellular localization of MAOB in Schwann cells. Immunohisto- chemical reaction products (arrows) are observed on and around mitochondrial outer membranes. Ax, Axon; My, myelin sheath; Nu, nucleus. Bar0.5 m. Fig. 3. A: Subcellular localization of MAOB in capillary endothelial cells. Immunohistochemical reaction products (arrows) are observed on and around mitochondrial outer membranes. Some mitochondria are very weakly stained for MAOB (arrowhead). Nu, nucleus. Bar1 m. B: Subcellular localization of MAOB in fibroblasts. Immunohistochemical reaction products (arrows) are observed on and around mitochondrial outer membranes. Col, collagen fibers; Nu, nucleus. Bar1 m. 422 Xu et al.

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