ARTICLE |
Correspondence to: Makoto Onoda, First Research Group, Nat. Inst. of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi, Chiba, 263-8555, Japan..
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Summary |
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We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female WistarMS rats primed by implantation with pellets of 17ß-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 µM) in the presence or absence of LPS (0.5 µg/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 µM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 ± 2 kD) and eNOS (152 ± 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands. (J Histochem Cytochem 46:12691278, 1998)
Key Words: nitric oxide, nitric oxide synthase, mammary gland, organ culture, immunohistochemistry, rat
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Introduction |
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Nitric oxide (NO) is a unique biological messenger molecule that is synthesized from L-arginine by nitric oxide synthase (NOS) (
NOS has at least three distinct isoforms, including the neuronal (nNOS), brain (bNOS or NOS1), inducible (iNOS or NOS2), and endothelial (eNOS or NOS3) types (
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Materials and Methods |
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Materials and Animals
Reagents and chemicals were purchased from Sigma Chemical (St Louis, MO) unless stated otherwise. All animals used in the present study were treated and handled according to the "Recommendations for the Handling of Laboratory Animals for Biomedical Research" complied by the Committee of the Safety and Handling Regulations for Laboratory Animal Experiments in our Institute.
Organ Culture of Rat Mammary Glands
Female WistarMS rats (8-week-old) bred at Nippon SLC (Hamamatsu, Japan) were primed by implantation with pellets of 17ß-estradiol (0.5 mg/3-week-release type; Innovative Research of America, Toledo, OH) and progesterone (35 mg/3-week-release type, Innovative Research). After 3 weeks of priming the rats were sacrificed by CO2 asphyxiation and the inguinal mammary glands were excised aseptically for organ culture. The isolated mammary glands were diced into approximately 3-mm cubes, and three of these cubes were cultured in a well of 24-multiwell plate containing 2 ml of 10% fetal calf serum (FCS)/Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) and an antimycotic (0.25 µg/ml amphotericin B) in a mixture of 5% CO2/95% air at 37C for 2 days. The medium was then replaced with 10% FCS/DMEM plus 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO, 100 µM; Dojindo Laboratories, Kumamoto, Japan) in the presence or absence of bacterial lipopolysaccharide (LPS, 0.5 µg/ml), and the culture was maintained for another 2 days. At the end of the culture, the conditioned media were collected for determination of the nitrite concentration as described below, and the cultured mammary glands were further processed for the preparation of mammary gland homogenates and frozen thin sections as described below.
Determination of Nitrite
NO produced and secreted by mammary glands into the culture medium was estimated by measuring the nitrite (NO2-) converted from NO with a Griess reagent mixture. The nitrite concentrations in conditioned media were determined immediately after the termination of the culture by a modification of a previously described colorimetric method (
Preparation of Mammary Gland Homogenates and Immunoblot Analysis of Nitric Oxide Synthase Isoforms
The mammary glands cultured with or without LPS were minced and homogenized in ice-chilled 5 mM Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose, 5 mM EGTA, and inhibitors (1 M phenylmethylsulfonyl fluoride, 2 mM sodium vanadate, 10 µg/ml aprotinin, 5 µg/ml leupeptin) (
Immunohistochemistry of Nitric Oxide Synthase Isoforms
The mammary glands incubated as mentioned above were cut into small cubes, fixed in 10% neutral buffered formalin at 4C, usually for about 20 hr, immersed in ascending grades of sucrosePBS, and brought finally to 20% sucrose5% glycerolPBS. The fixed tissues then were embedded in Tissue-Tek OCT compound (Sakura Finetechnical; Tokyo, Japan) and frozen in hexane at -60C. Frozen sections (12 µm thick) were prepared in a Jung MC1900 cryostat (Leica; Nussloch, Germany), placed on gelatinchromium (III)potassium sulfate-coated slide glasses, and allowed to cool air-dry for a half hour. The sections were rehydrated in PBS before immunostaining. Streptavidinbiotin peroxidase immunostaining was carried out using Histofine SAB-PO kits (Nichirei; Tokyo, Japan) as described previously (
NADPH-diaphorase Staining
The NADPH-diaphorase staining was performed according to the method described by
Statistical Analysis
Data are expressed as mean ± SE, and statistical comparisons among control and other groups were performed using ANOVA with StatView-J4.5 software (Abacus Concepts; Berkeley, CA) for Fisher's PLSD of multiple comparisons test. The level of significance was defined as p<0.05.
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Results |
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NO Production by Cultured Mammary Glands
The amount of NO produced spontaneously by mammary glands in culture was relatively minute for the 2-day culture period, whereas the NO concentration in the conditioned media was significantly increased (by almost fivefold of the control) by the addition of LPS (0.5 µg/ml) to the culture system (Table 1). The secretion of NO from mammary glands cultured with LPS increased in a dose-dependent manner up to a concentration of 0.5 µg/ml, and was slightly inhibited at higher concentrations of LPS up to 2 µg/ml (Figure 1). This enhancement of NO secretion by the mammary glands in culture with LPS was completely eliminated by addition of 3 µM hydrocortisone to the culture (Table 1). Hydrocortisone and corticosterone inhibited NO production/secretion into the culture medium from mammary glands with LPS stimulation in a dose-dependent manner, and the median inhibition doses (ID50) of hydrocortisone and corticosterone were approximately 40 nM and 150 nM, respectively (Figure 2). These findings suggest that the cultured rat mammary gland contains at least a certain cell population that responds to LPS stimulation and that this cell population produces NO.
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Determination of NOS Isoforms in Mammary Gland Extracts by Immunoblot Analysis
The NOS isoforms that immunoreacted with anti-iNOS and anti-eNOS antisera were detected, respectively, in the mammary gland extracts obtained at the terminus of the organ culture of mammary glands (Figure 3 and Figure 4). These immunoreactive bands of iNOS and eNOS had apparent molecular weights of 122 ± 2 kD and 152 ± 3 kD, respectively. In addition to this eNOS immunoreactive band (152 kD), in some cases a few other minor immunoreactive bands with molecular weights of approximately 97 kD, 78 kD, and 46 kD were detected by the anti-eNOS antibody (data not shown). These molecular species may be immunoreactive degradation products of eNOS, which were produced during the preparation of mammary gland extract owing to the presence of certain proteases (
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Immunohistochemical Observation of NOS Isoforms in Mammary Gland
Because the presence of NOS isoforms in the rat mammary gland was demonstrated by the immunoblot analyses, we carried out immunohistochemical staining of NOS isoforms in the rat mammary glands obtained after the organ culture with or without LPS stimulation to identify the particular location of NOS isoforms with specific antisera.
The immunoreactive signal of iNOS was clearly localized to the basal layers of alveoli and lactiferous ducts of the mammary glands obtained from the organ culture with LPS treatment (Figure 5b and Figure 5b'). These component cells are probably myoepithelial cells, because the staining pattern of mammary epithelial cells with anti-transferrin and anti-casein antisera was evidently different from the iNOS staining pattern in the alveoli and lactiferous ducts (Figure 6). In addition to the mammary parenchymal cells, the endothelial cell layers of mammary blood vessels were also intensively stained with iNOS antiserum (Figure 5b). Similar immunoreactivity with anti-iNOS antiserum was present in the mammary glands obtained from organ cultures without LPS treatment, although less intense staining with anti-iNOS antiserum was noted (data not shown). The histochemical staining of NADPH-diaphorase activity, which is identical to NOS (
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Discussion |
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Nitric oxide is increasingly appreciated as a major regulator in the nervous, immune, and cardiovascular systems. Apart from being an autocrine and paracrine mediator of homeostasis, NO has been found to inflict damage on important biomolecules, and it was suggested to contribute to the cytocidal action of macrophages and to many pathological events. Recent reports also implicate the overproduction of NO in inflammation, arthritis, myositis, and other diseases, and a role of NO in the carcinogenic process and tumor progression has been described (
The tentative presence of NOS activity in the mammary gland was first reported by
Interestingly, iNOS was present not only in LPS-treated mammary glands but also in the mammary glands that were not stimulated with LPS, although the amount of iNOS in the normal unstimulated mammary glands was relatively minute in comparison with the LPS-stimulated mammary glands. This was also reflected as spontaneous NO production in the NO concentration of culture medium in the mammary gland organ cultures without LPS stimulation. This is suggestive of a basal expression of iNOS and of other constitutive NOS isoforms in the unstimulated rat mammary glands. Because there are precedents for the expression of iNOS isoform in normal rat kidney (
Another surprising finding of our study is that the expression of the eNOS isoform in the mammary gland declined after the exposure of the mammary glands to LPS in the culture, although the iNOS-derived NO production masked a reduced NO production by eNOS in the cultures with LPS treatment. The downregulation of constitutive NOS expression by iNOS was recently reported not only for eNOS in cultured bovine coronary venular endothelial cells (
It is known that the growth and differentiation of the mammary gland are essentially controlled by multiple interactions of several peptide and steroid hormones from endocrine organs, such as the pituitary, ovary, and adrenal gland. Subsequently, development occurs in several phases, characterized by distinct morphological features (
Involution of the mammary gland is a tissue remodeling process, of which an integral component is the apoptosis of mammary epithelial cells (
In any event, our present data demonstrate that the presence of three NOS isoforms in the rat mammary gland is certain and that these NOS isoforms may correlate with mammary gland development and regulatory functions. Additional studies are needed to elucidate the precise roles of NO in the physiology and pathophysiology of the mammary gland.
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Acknowledgments |
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Supported by grants for Special Project Research for Experimental Studies on Radiation Health, Detriment and Its Modifying Factors, and for Research Programme on Bioregulatory Mechanisms of the National Institute of Radiological Sciences.
We are grateful to Dr Yuji Ishikawa, Senior Researcher in the Division of Biology and Oncology, for helpful advice on the preparation of the frozen sections from cultured mammary glands.
Received for publication June 29, 1998; accepted June 30, 1998.
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