| Abstrakt: |
1. The roles of the gamma‐glutamyl cycle and the anionic amino acid transport system xc‐ in mediating L‐cystine uptake were investigated in cultured human pancreatic duct PaTu 8902 cells. This cell line exhibits morphological features of normal pancreatic duct cells and expresses gamma‐glutamyl transpeptidase (gamma‐GT, EC 2.3.2.2), an enzyme involved in the metabolism and regulation of intracellular glutathione (GSH). 2. Uptake of L‐cystine (10 microM) was linear for up to 10 min, temperature dependent, Na+ independent, saturable (Michaelis‐Menten constant (Km), 86 +/‐ 25 microM; maximal velocity (Vmax), 109 +/‐ 33 nmol (mg protein)‐1 h‐1) and reduced by 80‐90% by a 50‐fold excess concentration of L‐glutamate and L‐homocysteic acid, but not L‐aspartate. These transport properties resemble those described for system xc‐, which exchanges cystine for intracellular glutamate. 3. Acivicin, a known inhibitor of gamma‐GT, decreased gamma‐GT activity from 2.58 +/‐ 0.96 to 0.97 +/‐ 0.11 mU (mg protein)‐1 and decreased the initial rates of L‐cystine and L‐glutamine uptake by 41‐55%. Anthglutin (1‐gamma‐L‐glutamyl‐2‐(2‐carboxyphenylhyl)hydrazine), a structurally different inhibitor of gamma‐GT, also caused a concentration‐dependent (0.01‐1 mM) decrease in gamma‐GT activity and L‐cystine uptake. 4. Neither acivicin nor anthglutin inhibited the uptake of L‐glutamate, a poor substrate for gamma‐GT. 5. In the presence of a 500‐fold excess concentration of glutamate, which should abolish entry of cystine via system xc‐, the remaining fraction of cystine transport was inhibited by 50% by acivicin, suggesting that transport is, in part, dependent on the activity of gamma‐GT. 6. Cystine transport was also 60‐80% inhibited by a series of gamma‐glutamyl amino acids (5 mM) including gamma‐glutamyl‐glutamate, gamma‐glutamyl‐glutamine and gamma‐glutamyl‐glycine. alpha‐Dipeptides inhibited cystine transport by only 6‐22%. 7. These findings demonstrate that in human pancreatic duct PaTu 8902 cells, cystine uptake is mediated by system xc‐ (50‐60%) and the gamma‐glutamyl cycle. Our results provide the first evidence linking gamma‐GT with cystine transport in human epithelial cells and are of relevance in view of the importance of cystine as a sulphur amino acid source for GSH synthesis in cells exposed to oxidative stress. |
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