Proceedings of The Physiological Society

Newcastle University (2009) Proc Physiol Soc 16, PC14

Poster Communications

Inhibition of the glycine transporter GLYT1 blocks glycine protection of intestinal cells.

S. Javed1, I. Tahir1, A. Howard1, B. H. Hirst1

1. Epithelial Research Group, ICAMB, Faculty of Medical Sciences, Newcastle University, Newcastle-upon-Tyne, United Kingdom.

Glycine is cytoprotective against a range of toxic challenges, particularly, in intestine, against ischemia-reperfusion injury and oxidative stress (Schaefer et al, 2008; Kallakuri et al, 2003; Lee et al, 2002). To investigate the mechanism of cytoprotection in intestine we used the model cell lines Caco-2 and HCT-8, representative of enterocytes and colonocytes respectively, where GLYT1 activity accounts for 30-40% of basolateral glycine uptake (Christie et al, 2001; Tahir et al, 2005). Cells grown on 96-well plates were exposed to the oxidising agent tert-butylhydroperoxide (t-BOOH) which induced formation of reactive oxygen species (ROS) and a reduction in cell viability (viability following 150µmol L-1 t-BOOH, 28 ± 2 (mean ± S.E.M.)% of control cells; n = 12, p< 0.05 (ANOVA with Dunnett’s post-test)) and intracellular glutathione concentration (concentration following 100µmol L-1 t-BOOH, 77.5 ± 1.8 % of control, n = 12, p <0.05). Pre-treatment with glycine (1 or 5 mmol L-1 ) was protective against t-BOOH challenge and increased cell viability (63 ± 7 and 71.5 ± 3.3% of control respectively, p<0.05 compared to t-BOOH treated cells), reduced induced ROS concentration (75.9 ± 2.17 and 77.02 ± 4.34 % of t-BOOH treated respectively, p<0.05) and increased glutathione levels (concentration in 5mmol L-1 glycine pretreated cells: 99.6 ± 3.2% of control, p<0.01 compared to t-BOOH treated). Glycine given concurrent with or after oxidative challenge had no protective effect. Inhibition of GLYT1 activity with the specific inhibitor ALX-5407 (N [3-(4’-fluorophenyl)-3-(4’-phenylphenoxy)-propyl] sarcosine (NFPS)) reduced Na+ -dependent, alanine insensitive glycine uptake by ~75%, whereas the GLYT2 inhibitor ALX-1393 (O-[(2-Benzyloxyphenyl-3-flurophenyl)methyl]-L-serine) had no effect, consistent with data demonstrating lack of GLYT2 expression in these cell lines. When cells were incubated with ALX-5407 prior to glycine treatment and subsequent oxidative challenge, the protective effects of glycine were blocked: cell viability (t-BOOH treated: 53.3 ± 5.2 % of control; 5mmol L-1 glycine pre-treated: 48.3 ± 1.7 % of control, p>0.05 compared to t-BOOH treated) and glutathione concentration (72.2 ± 4.42 % of control, p< 0.05) were reduced compared to controls and ROS concentration was comparable to that in cells treated with t-BOOH alone (105.9 ± 3.83 and 102.1 ± 4.65% for 1 and 5mmol L-1 glycine pre-treated cells respectively, p>0.05). We conclude that glycine acquired across the basolateral membrane of intestinal cells by GLYT1 is used in cell defence and in maintaining intracellular antioxidant potential.

Where applicable, experiments conform with Society ethical requirements