ARTICLE |
Correspondence to: Nicole Mahy, Unitat de Bioquímica, Facultat de Medicina, UB, C/Casanova, 143, Barcelona E-08036, Spain. E-mail: mahy@medicina.ub.es
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Summary |
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Semicarbazide-sensitive amine oxidase (SSAO), widely distributed in highly vascularized mammalian tissues, metabolizes endogenous and xenobiotic aromatic and aliphatic monoamines. To assess whether its physiological role in humans is restricted to oxidation, we used an immunohistochemical approach to examine the cellular localization of SSAO in human peripheral tissues (adrenal gland, duodenum, heart, kidney, lung, liver, pancreas, spleen, thyroid gland, and blood vessels) and also analyzed its subcellular localization. The results are in agreement with the specific activities also determined in the same samples and are discussed with reference to the tissue distribution of monoamine oxidase A and B. Together with the oxidative deamination of monoamines, SSAO cellular localization indicates that, in most human peripheral tissues, it might participate in the regulation of physiological processes via H2O2 generation. (J Histochem Cytochem 49:209217, 2001)
Key Words: semicarbazide-sensitive amine, oxidase (SSAO), human peripheral tissues, immunohistochemistry, enzymology
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Introduction |
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AMINE OXIDASES metabolize monoamines, diamines, and polyamines of endogenous or xenobiotic origin, rendering ammonia, aldehydes, and hydrogen peroxide as final products. According to their co-factor, amine oxidases are classified into two groups. The FAD-dependent amine oxidases (E.C.1.4.3.4.) are sensitive to acetylenic type inhibitors, which include the monoamine oxidases (MAO) and polyamine oxidases. In the second group, the Cu-dependent amine oxidases (E.C.1.4.3.6), sensitive to carbonyl reagents such as semicarbazide, comprise the diamine oxidase and semicarbazide-sensitive amine oxidase (SSAO), which deaminates both aromatic and aliphatic primary amines, such as methylamine and aminoacetone.
SSAO activity is present in blood plasma and is associated with membranes in several mammalian tissues (
Despite its widespread tissue distribution, the physiological role of SSAO is far from understood. Together with its amine oxidase activity, SSAO might participate in the regulation of glucose disposal in several insulin-sensitive tissues, including cardiac and skeletal muscle and adipocytes, where it is associated with the glucose transporter GLUT4 (
Changes in plasma SSAO activity have been related to several pathological situations. Plasma SSAO activity is reduced in patients suffering from burns (
To better understand the physiological role of SSAO, the activity and cellular distribution of SSAO were determined in human tissues, including adrenal gland, duodenum, heart, kidney, lung, liver, pancreas, spleen, thyroid gland, and blood vessels, by enzymatic and immunohistochemical techniques using a specific antibody against tissue-bound SSAO (
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Materials and Methods |
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Human Tissues
Human tissues were generously provided by Dr. J. A. Bombí (Facultat de Medicina, Universitat de Barcelona, Spain) in accordance with local institutional ethical guidelines. Samples of adrenal gland, duodenum, heart, kidney, lung, liver, pancreas, spleen, thyroid gland, and vessels were obtained from nine routine autopsy cases (five men and four women). Tissues affected by the pathological state of the patient were discarded. The age range was 5084 years (mean ± SEM, 72.2 ± 3.7) and the postmortem delay ranged from 4 to 22 hr (12.6 ± 1.9).
Immediately after autopsy, tissues were divided into two groups. One group was cut into 12-cm-thick blocks for the immunohistochemical studies and the other was stored in liquid nitrogen for enzymatic activity determination.
Enzyme Assays
Samples kept in liquid nitrogen were powdered with a mechanical hammer. They were then homogenized 1:10 (w/v) in 50 mM Na+, K+ phosphate buffer, pH 7.6, and filtered through a double gauze. Aliquots were taken and stored at -80C.
SSAO and MAO-B activities were determined radiochemically (
Time-course assays were used to ensure that initial rates of the reaction were determined, and proportionality to enzyme concentration was established in each case. Protein was measured by the Bradford method (-globulin as standard. All nonradioactive products were purchased from Sigma (St Louis, MO).
Immunoblotting Analysis
Human tissues homogenates or purified porcine kidney diamine oxidase (Sigma) were fractionated by electrophoresis on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Blots were blocked for 1 hr at room temperature with 5% (w/v) non-fat dried milk in 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl and 0.05% Tween-20, then incubated with primary anti-SSAO antibody (1:500 dilution) overnight at 4C, followed by incubation for 1 hr with secondary antibody. Finally, the blots were developed with the avidinbiotinperoxidase technique, following the manufacturer's instructions (Vector Laboratories; Bretton, UK). After color development, the membrane was washed with distilled water, air-dried, and photographed. Prestained molecular weight standards used were myosin (250 kD), bovine serum albumin (98 kD), glutamate dehydrogenase (64 kD), alcohol dehydrogenase (50 kD), carbonic anhydrase (36 kD), and myoglobin (30 kD) from Novex (San Diego, CA).
Immunohistochemical Assays
After autopsy, 12-cm tissue blocks were immediately fixed by immersion in paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4C. Different fixation times (2, 4, 7, or 24 hr) and fixative concentrations [0, 0.1, 1, 2, 4 % (w/v)] were tested. After fixation, tissues were immersed for 72 hr in 15% (w/v) sucrose in 0.1 M phosphate buffer, pH 7.4, for cryoprotection, frozen with dry ice, and stored at -30C. Sections of fresh frozen tissue were also postfixed with acetone or paraformaldehyde 4% (w/v) for 2 or 5 min.
Cryostat sections (12 µm) were cut from the fixed tissue blocks and mounted on gelatinized slides. The immunohistochemical avidinbiotin method was used. Endogenous peroxidase activity was inhibited by a 30-min preincubation in 30% (v/v) H2O2 in 10 mM Tris-buffered saline (TBS), pH 7.6, followed by a 15-min wash in TBS. Then sections were incubated in 20% (v/v) normal goat serum (NGS) in TBS for 20 min. A rabbit polyclonal anti-bovine SSAO antibody (-gliadin, which has been typed as subclass IgG1) was diluted 1:400 and used to define the nonspecific staining. After washing, sections were incubated for 30 min at room temperature in biotinylated goat anti-rabbit IgG (1:300 in TBS1% NGS), rinsed, and washed in TBS, and then incubated for 2 hr at room temperature in ExtraAvidinhorseradish peroxidase (1:250 in TBS1% NGS). After washing, sections were developed for 15 min in a 50 mM Tris-HCl solution, pH 7.6, containing 0.03% (w/v) diaminobenzidine and 0.006% (v/v) H2O2. Some sections were counterstained with Mayer's hematoxylin for morphology. Secondary antibodies and reagents were purchased from Sigma.
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Results |
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Specific Activities
Specific activities of MAO-A, MAO-B, and SSAO were detected in most of the tissues assayed, with marked differences (see Table 1). The highest specific activity of MAO-A was observed in liver, duodenum, and kidney (10031158 pmol·min-1·mg prot-1). Heart, lung, thyroid and adrenal glands presented about half the value found in the former tissues, and aorta, spleen, muscle, and vena cava showed the lowest activity. Low activity was detected in pancreas. For MAO-B, the highest activity was found in heart, liver, duodenum, and kidney (209322 pmol·min-1·mg prot-1). Half this amount was found in adrenal gland, and negligible activity was observed in pancreas and spleen.
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SSAO specific activity was found in all the tissues assayed. Aorta showed the highest activity (338 pmol· min-1·mg prot-1); lung, duodenum, and venous cava showed lower activity (86133 pmol·min-1·mg prot-1) and the remaining tissues much lower (<54 pmol·min-1·mg prot-1). Comparison of MAO-B and SSAO contribution to benzylamine oxidative deamination indicates that SSAO is the main oxidase in aorta, pancreas, lung, and vena cava, whereas MAO-B is the main oxidase in heart, liver, duodenum, kidney, and adrenal gland.
Distribution and Cellular Localization of SSAO Immunoreactivity
To test the specificity of the polyclonal antibody anti-SSAO used in this study, immunoblotting analysis were performed using different human tissue homogenates. Fig 1 (Lanes 1 and 2) shows that human heart and adrenal gland homogenates rendered a single immunoreactive band of 100 kD, similar to the apparent molecular weight of SSAO protein reported in bovine (
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Widespread SSAO distribution was observed in all tissues except the thyroid gland and endocrine pancreas, which were devoid of specific SSAO immunoreactivity (IR) (Fig 2 and Fig 3; Table 2 and Table 3). In most tissues, SSAO IR appeared to be bound to the cytoplasmic membrane of the labeled cells. Only a weak and diffuse stain was detected in the presence of the 9H7 antibody. The optimal condition for fixation was found to be 7 hr of immersion in 2% paraformaldehyde. Longer fixation times and higher paraformaldehyde concentrations reduced the intensity of staining. Postfixation with acetone was discarded because of poor tissue preservation. A primary antibody dilution of 1:400 was considered an optimal compromise because higher dilution reduced the signal in several tissues.
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In the adrenal gland, strong SSAO IR was observed in the cytoplasmic membrane, and also intracellularly in the capsule cells. Intracellular positive staining was also seen in both secretory and interstitial cells of the cortex (Fig 2a and Fig 2b). A weak SSAO stain was seen in the medulla, where most cells were negative, and the only SSAO IR was observed in the wall of the central vein and in some scattered cells (Fig 2c and Fig 2d).
In duodenum, SSAO IR was present mostly in the membrane of the enterocytes of villi and Lieberkühn crypts. However, the cytoplasm of the muscularis mucosa and both outer and inner muscular layers showed strong SSAO IR (Fig 2e2g). No signal was found in the Brunner glands.
In heart, a strong intracellular SSAO IR was found in the cardiomyocytes but not in the surrounding connective tissue (Fig 2h2j). In kidney, intracellular SSAO IR was observed in the epithelial cells of renal tubuli, whereas stronger staining was seen in the collecting ducts. Glomeruli were devoid of SSAO IR because neither Bowman's capsule nor podocytes showed specific staining (Fig 2k2m). Interstitial tissue was also devoid of SSAO staining.
In liver, strong SSAO IR was observed intracellularly and in the membranes of hepatocytes (Fig 2n2p). A similar pattern was observed in the bile duct and central vein. No portal-to-central vein IR gradient was detected.
In lung, strong staining was detected in bronchiolar cells, both ciliated and Clara cells, and in all pneumocytes of alveoli (Fig 3a and Fig 3b). In this tissue, SSAO IR was stronger in the membrane than in the cytoplasm. In spleen, SSAO staining was observed in the membrane of the capsule and trabecular cells, and in some cells in the white pulp (Fig 3c and Fig 3d). In contrast, no signal was detected in the red pulp, and spleen lymphocytes were negative for SSAO. In the thyroid gland, all the follicular and parafollicular cells were SSAO IR-negative (not shown).
In the exocrine pancreas, intracellular SSAO IR was seen in all acinar cells (Fig 3e3g). Centroacinar cells, intercalated ducts, and epithelial cells of pancreatic ducts showed specific IR in the cell membrane (Fig 3h and Fig 3i). The walls of pancreatic ducts and the connective tissue showed no SSAO-specific staining. The endocrine pancreas was devoid of SSAO IR.
In all the organs studied, endothelial cells of arteries and veins were devoid of anti-SSAO staining. However, fibroblasts surrounding the internal elastic lamina of arteries observed at a higher magnification showed strong membrane and intracellular SSAO IR (Fig 3j and Fig 3k). A moderate level of IR was seen in the membrane of vascular muscle-layer cells. In contrast to blood vessels, endothelial cells of lymphatic vessels gave a very strong SSAO IR (Fig 3l). Because these cells are very narrow, we were unable to localize more precisely their SSAO label.
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Discussion |
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We have studied the human tissue and cellular distribution of SSAO using a polyclonal SSAO antibody (
Our results confirm the presence of MAO-A, MAO-B, and SSAO in most human peripheral tissues, with a higher MAO-A specific activity. In agreement with previous reports (
SSAO specific activities found in this study are mainly in accordance with their tissue distribution revealed by immunohistochemistry. The high specific activity and strong immunostaining found in highly vascularized tissues such as duodenum, liver, lung, and blood vessels, but not heart, could reflect the metabolic deamination of the circulating amines of endogenous or exogenous origin.
The presence of SSAO in adrenal gland, kidney, exocrine pancreas, and lymphatic vessels in cells lacking MAO-B (
In the production of pancreatic juice, there is a contribution from two cell types from the exocrine pancreas: acinar cells, producing digestive enzymes and co-factors, and centroacinar/duct cells, producing bicarbonate and water. We found SSAO in both cell populations where MAO-A is also present, whereas MAO-B appears only in the centroacinar/duct cells (
In addition, SSAO IR was observed only in the membrane of the smooth muscle of the blood vessels, probably related to the sympathetic regulation of blood pressure (
In cardiomyocytes and acinar cells, SSAO localization was mainly intracellular, but it was mainly membranous in adrenal gland, duodenum, liver, lung, and exocrine pancreas. Both liver and lung contribute to the degradation of physiological amines. The membrane and intracellular SSAO IR localization observed in these tissues suggests that it might also modulate hormonal action. Thus, as shown in adipocytes (
We found only two tissues devoid of SSAO IR: the thyroid gland, rich in MAO-A, and the endocrine pancreas, with MAO-A localized mainly in ß-cells and MAO-B restricted to -cells (
In conclusion, these results are the first attempt to quantify and map SSAO localization in human peripheral tissues. As suggested by its cell localization, its role may not be restricted to amine oxidation. Further experiments are necessary to provide such evidence and understand the physiological importance of SSAO in humans.
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Acknowledgments |
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Supported by SGR 99-224 from the Generalitat de Catalunya and the DGES grant SAF99-0093 from the Ministerio de Educación y Cultura.
We wish to thank Dr J. A. Bombí (Departament de Biologia Cel·lular i Anatomia Patològica, Facultat de Medicina, Universitat de Barcelona, Spain) for the gift of the human tissue samples, and Dr Ellen E. Billett (Department of Life Sciences, Faculty of Science and Mathematics, The Nottingham Trent University, UK) for the gift of the 9H7 antibody.
Received for publication May 18, 2000; accepted October 4, 2000.
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