Induction of cystine transport via system x c− and maintenance of intracellular glutathione levels in pancreatic acinar and islet cell lines

Abstract

The relationship between L-cystine transport and intracellular glutathione (GSH) levels was investigated in cultured pancreatic AR42J acinar and βTC3 islet cells exposed to diethylmaleate, an electrophilic agent known to activate cellular antioxidant responses. Cystine transport was mediated predominantly by the Na +-independent anionic amino acid transport system x c −, with influx inhibited potently by glutamate and homocysteate but unaffected by cationic or neutral amino acids. Saturable cystine transport was 10-fold higher in AR42J (531 pmol (mg protein) −1 min −1) than in βTC3 (49 pmol (mg protein) −1 min −1) cells, and GSH levels were higher in AR42J cells. Treatment with 2-mercaptoethanol increased GSH levels in βTC3 cells from 7.5 to 36 nmol (mg protein) −, whilst the GSH content in AR42J cells (64 nmol (mg protein) −1) was not altered significantly. Incubation of AR42J or βTC3 cells with homocysteate (2.5 mM, 0–48 h), a competitive inhibitor of cystine transport via system x c −, reduced intracellular GSH levels and resulted in a time-dependent (6–24 h) induction of system x c − transport activity. Treatment of AR42J cells with diethylmaleate (100 μM 0–48 h) resulted in a time- (5–10 h) and protein synthesis-dependent induction of cystine transport, with intracellular GSH levels initially decreasing and then increasing 2-fold above control levels after 24 h. Diethylmaleate also depressed GSH levels in βTC3 cells, but cystine transport was not elevated significantly. In both AR42J and βTC3 cells, inhibition of γ-glutamyl cysteine synthetase by buthionine sulphoximine (100 μM, 24 h) reduced GSH levels but had no effect on cystine transport. The present findings establish that induction of system x c − leads to changes in GSH levels in pancreatic AR42J acinar and βTC3 islet cells, with changes in the intracellular redox state stimulating transporter expression. Induction of activity of system x c −, together with adaptive increases in GSH synthesis in response to oxidative stress, may contribute to cellular antioxidant defences in pancreatic disease.