Nitric oxide production in the mammary gland : a thesis submitted in partial fulfilment for the degree of Doctor of Philosophy at Massey University, New Zealand
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Date
2001
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Massey University
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Abstract
Although the effects of nitric oxide (NO) have been widely studied in many different cell and tissue types, very little is known of the role it plays in the mammary gland. Thus, the production of NO by mammary gland was investigated in a series of experiments. NO is a free radical gas which is produced by a wide variety of cells by the action of the enzyme nitric oxide synthase (NOS) on arginine. This results in the formation of citrulline and NO. The study first examined several methods for their suitability for the detection of NO or NOS. Evaluation of the methods revealed that the indirect measurement of NO production by the detection of nitrate and nitrite (NOx), the spontaneously produced metabolites of NO, was the most valid and reliable. The measurement of NOx was carried out in culture medium using a fluorescent-based assay, which was developed by the modification of published methods, during the course of this study. Comma-D cells (murine mammary epithelial cell line) were used to investigate the production of NOx by the inducible form of NOS (iNOS) following treatment with cytokines and cytotoxins. The cell's response was characterised and showed that mammary epithelial cells produce NOx in a dose dependent manner in response to interferon-γ (IFN-γ). Lipopolysaccharide (LPS), a component of bacterial cell walls, was employed to examine the response of the mammary epithelial cells to cytotoxins and it was found that the treated cells produced more NOx than the untreated ones, however, no dose response was apparent. The specific iNOS inhibitor, aminoguanidine (AG) and general NOS inhibitor Nω-nitro-L-arginine (L-NNA) were both used to confirm that the NOx measured in the medium was produced by NOS. The production of NOx by the mammary gland was also examined in cultured explants of mammary tissue taken from pregnant (D 12-14 of pregnancy) and lactating (D 12-14 postpartum or D 17-18 postpartum) rats. A significant difference was found in the basal production of NOx between the different developmental stages. The method of euthanasia of the rats also affected the amount of NOx produced. The inclusion of prolactin (PRL) also increased the production of NOx from both Comma-D cells and explants of mammary tissue. Xanthine oxidase (XO), an enzyme responsible for the conversion of NOx to NO under anaerobic conditions, does not interfere with the determination of NO production using the NOx assay. The measurement of NOx was carried out in the milk of cows following the intramammary infusion of Streptococcus uberis or interleukin-1β (IL-1β). By comparing the milk NOx concentration with the somatic cell count (SCC) and electrical conductivity (EC) of the milk, it was concluded that the source of the NOx in the milk could not be attributed entirely to the epithelium or the somatic cells. The experiments in this Thesis clearly show that the mammary gland is capable of the production of NO in response to a variety of situations and that the regulation of the production is very complex. The work also identifies some new areas of research, which if completed would further enhance the understanding of the role NO plays in the mammary gland.
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Nitric acid, Mammary glands, Physiology, Pathophysiology