Glycine transporter GLYT1 is essential for glycine-mediated protection of human intestinal epithelial cells against oxidative damage

Abstract

Glycine protects mammalian intestine against oxidative damage caused by ischaemiareperfusion (IR) injury and prevents or reverses experimentally-induced colitis. However the mechanism of protection remains largely unknown. The objectives of the current study were to demonstrate directly glycine-mediated protection of human intestinal epithelial cells and to determine the requirement for glycine uptake by the specific transporter GLYT1. Exogenous glycine protected human intestinal Caco-2 and HCT-8 cells against the oxidative agent tert-butylhydroperoxide and reduced the intracellular concentration of reactive oxygen species, when applied prior to but not concomitant with the oxidative challenge. Glycine given prior to oxidative challenge preserved intracellular glutathione concentration but had no effect on the rate of glycine uptake. Protection was dependent on GLYT1 activity, being blocked by a specific GLYT1 inhibitor, 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. However expression of the genes encoding GLYT1 and the glutathione synthesising enzymes glutamate-cysteine ligase, both catalytic and modifier subunits, and glutathione synthetase was not altered by glycine or tert-butylhydroperoxide, suggesting transcriptional regulation is not involved. This work has demonstrated a novel role of GLYT1 in intestine and shown that intestinal epithelial cells respond directly to oxidative challenge without reliance on extra-epithelial tissues or functions such as neurone, blood-flow or immune responses for antioxidant defence. The protective actions of glycine and maintenance of epithelial antioxidant defences suggest it may be beneficial in treatment of inflammatory bowel disease.

P2Y Purinergic Regulation of the Glycine Neurotransmitter Transporters

“…Alternatively, the combined effects of PKG-I and PKA on GLYT1 activity may result in activation instead of inhibition of the transport. The rise in GLYT1 transport by NO may explain the finding that GLYT1 activity has been reported as essential for glycine-mediated protection of human intestinal epithelial cells against oxidative damage (74). This issue deserves future research.…”

The Glycine Transporter GLYT1 in Human Intestine: Expression and Function

“…Measurements made using a GLYT2 specific inhibitor, ALX-1393, confirmed molecular data indicating the absence of this transporter in human intestinal cells. 15) 2.2. Function That GLYT1 activity predominates at the basolateral membrane leads to questions about its function.…”

“…In both enterocytes and colonocytes, GLYT1 is restricted to the plasma membrane and located at both apical and basolateral surfaces. 14,15) While it has not been possible to determine by functional or immunological methods the GLYT1 isoform/s present in intestine, as suitable antibodies and selective inhibitors 12) remain elusive, studies at the molecular level have shown that the predominant transcript variant is that which encodes GLYT1A. 16) Although several other GLYT1 transcript variants were detectable in human intestine by polymerase chain reaction (PCR), including those for GLYT1B and GLYT1C and a number which were previously undescribed, these were all found at very low concentration and detection was inconsistent, suggesting again that some if not all arose as unstable intermediates or errors of the splicing process.…”

“…42) In contrast, oxidative stress caused by excess reactive oxygen species (ROS) stimulates basolateral uptake of glycine by GLYT1 in a mechanism as yet undetermined but apparently independent of transcriptional activation. 15) Oxidative stress-enhanced influx of glycine increases intracellular glutathione concentration, reduces ROS concentration and improves cell viability. dual role, mediating both glycine export and uptake given appropriate electrochemical gradients has been demonstrated in several experimental models including mouse cerebellar slices, 22) HEK293 23) and Chinese hamster ovary (CHO) 24) cells, and Xenopus laevis oocytes.…”

“…15) Oxidative injury was chosen for investigation as it is a significant component of intestinal inflammatory diseases such as Crohn's disease and ulcerative colitis. The oxidising agent tert-butylhydroperoxide (t-BOOH) stimulated production of reactive oxygen species in intestinal cells, reduced intracellular glutathione concentration and led to reduced cell viability.…”