LOCALIZATION OF NITRIC-OXIDE SYNTHASES AND NITRIC-OXIDE PRODUCTION INTHE RAT MAMMARY-GLAND

We investigated nitric oxide (NO) production and the presence of nitri c oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the in guinal parts of female Wistar-MS rats primed by implantation with pell ets of 17 beta-estradiol and progesterone and were diced into approxim ately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 mu M) in the presence or absence of LPS (0.5 mu g/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 signif icantly enhanced by the presence of LPS. This enhancement of NO produc tion was completely eliminated by addition of hydrocortisone (3 mu M), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immun oblot analyses with specific antisera against NOS isoforms such as iNO S, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepa red from the mammary glands in the culture without LPS. The immunoreac tive band of iNOS was highly intense after the treatment of mammary gl ands 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 (compose d of myoepithelial cells of alveoli and lactiferous ducts) of the mamm ary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining fo r NADPH-diaphorase activity, which is identical to NOS, showed localiz ation similar to that of iNOS in the mammary glands. Similar observati ons were noted in the immunohistochemistry of eNOS. in contrast, the i mmunoreactive signal with the bNOS antiserum was barely detected in th e epithelial parts of alveoli and lactiferous ducts of the mammary gla nds. These observations demonstrate that three isoforms of NOS are pre sent not only in the endothelium of blood vessels but also in the pare nchymal cells (the glandular epithelium) of the rat mammary gland, suc h as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.