Characterization of multiple cysteine and cystine transporters in rat alveolar type II cells

Abstract

Cysteine availability is rate limiting for the synthesis of glutathione, an important antioxidant in the lung. We used rat alveolar epithelial type II cells to study the mechanism of cysteine and cystine uptake. Consistent with carrier-mediated transport, each uptake process was saturable with Michaelis-Menten kinetics and was inhibited at 4°C and by micromolar levels of amino acids or analogs known to be substrates for a specific transporter. A unique system XAG was found that transports cysteine and cystine (as well as glutamate and aspartate, the only substrates previously described for system XAG). We also identified a second Na+-dependent cysteine transporter system, system ASC, and two Na+-independent transporter systems, system xc for cystine and system L for cysteine. In the presence of glutathione at levels measured in rat plasma and alveolar lining fluid, cystine was reduced to cysteine and was transported on systems ASC and XAG, doubling the transport rate. Cysteinylglycine, released from glutathione at the cell surface by γ-glutamyl transpeptidase, also stimulated uptake after reduction of cystine. These findings suggest that, under physiological conditions, cysteine and cystine transport is influenced by the extracellular redox state.

The Phagocytosis-Associated Respiratory Burst in Human Monocytes Is Associated with Increased Uptake of Glutathione

“…(In this paper we refer to the choline buffer as Na ϩ -free HEPES buffer.) Uptake was initiated by replacing the above solution with 1.0 ml of HEPES buffer containing 0.9 Ci of L- [ 35 S]cystine (0.8 nM) with 1 M cold cystine, 0.9 Ci L- [ 35 S]cysteine (0.8 nM) with 1 M cold cysteine in the presence of 1 mM GSH as a reducing agent, or 0.9 Ci L- [ 35 S]GSH (2.1 nM) in the presence of 1 M cold GSH, as we previously described (14). The radioactivity of the mixture of radioactive and cold thiols was measured by liquid scintillation counting, and the specific activity of the thiol was calculated as counts per minute per picomole.…”

“…Characterization of cysteine and cystine uptake was performed using inhibitors selective for each transporter system. ASC, L, X AG , and x c were inhibited using 1 mM serine, 1 mM 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, 400 M L-aspartic acid-␤-hydroxamate, or 200 M quisqualate, respectively (14). All chemicals were obtained from Sigma.…”

“…The K i , which is the concentration of inhibitor that doubles the apparent K m (or decreases the affinity) of the substrate, was determined using the Dixon plot (14).…”

“…Transporters for cysteine, cystine, or both can be differentiated by their substrate and inhibitor specificity and whether Na ϩ is necessary for transport activity. We explored cystine and cysteine uptake (5 min, 37°C) in monocytes under basal culture conditions by determining transport in the presence of these selective inhibitors (14). Cystine transport was predominantly (72%) Na ϩ independent (Figs.…”

“…The rate of transport and the intracellular availability of cysteine/ cystine appear to control the rate of GSH synthesis (9 -12). Transport of these amino acids is accomplished by well-characterized amino acid transporters (13,14). More recently, distinct transport systems for GSH have been described on the canalicular and sinusoidal membranes of rat and human liver, and analysis of cell lines and tissues suggests that a transport mechanism also exists on certain other cell types (15)(16)(17)(18).…”