Role of glycine and GLYT-1 transporter in intestinal cell protection

Glycine has been shown to protect a broad range of tissues and cell types against oxidative stress. In the intestine, these effects have been observed against Ischaemia-Reperfusion injury, hypothermic ischaemia and chemically induced colitis. The process by which glycine exerts its protective effects remain uncertain. In this thesis, the ability of glycine to protect human intestinal cells against tert-butyl hydroperoxide (t-BuOOH) induced cell death, and the requirement for GLYT-1 in the protective mechanism were explored. The human i1eo-caecal adenocarcinoma cell line, HCT-8, was chosen as an in vitro mfldel of human intestinal epithlium. RT-PCR with mRNA from HCT-8 cells and the human enterocytic Caco-2 cells was used to determine exwession of a range of amino acid transporters. Immunohistochemistry techniques using a GLYT-1 specific antibody were employed to determine the localisation of GLYT-1 in this cell line and human large intestine. Uptake of glycine in HCT-8 cell monolayers was measured using radio-labelled uptake experiments. For cytoprotection studies, the MIT assay and the ApoGSH assay were used to determine cell viability and glutathione concentration respectively, following t-BuOOH treatment. Expression of GLYT-1 and other specific amino acid transporters known to be expressed in the small and large intestine was determined, confirming the suitability of this cell line as a model for the study of amino acid absorption. GLYT-1 protein was shown to be localised along the apical and basolateral membranes of both human colon and HCT-8 cells. A proportion of Na+/Cr -,.dependent glycine uptake in HCT-8 cells was inhibited by sarcosine and the GLYT-1 inhibitor .ALX-5407, at both apical and basolateral membranes, and was attributed to GLYT-1. Exogenous glycine supplementation, prior to t·BuOOH treatment, protected HCT-8 cells against cell death and preserved intracellular glutathione concentration. Protection was specific to glycine and dependent on GLYT-1 activity. Glycine cytoprotection requires GLYT-1 activity, supporting a requirement for intracellular glycine accumulation. Maintained intracellular glutathione content is indicated as a mechanism through which the protective effect may in part be mediated.EThOS - Electronic Theses Online ServiceGBUnited Kingdo