A ROLE FOR GAMMA-GLUTAMYL-TRANSPEPTIDASE AND THE AMINO-ACID-TRANSPORTSYSTEM X(C)(-) IN CYSTINE TRANSPORT BY A HUMAN PANCREATIC DUCT CELL-LINE

1. The roles of the gamma-glutamyl cycle and the anionic amino acid tr ansport system x(c)(-) in mediating L-cystine uptake were investigated in cultured human pancreatic duct PaTu 8902 cells. This cell line exh ibits morphological features of normal pancreatic duct cells and expre sses gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2), an enzyme i nvolved in the metabolism and regulation of intracellular glutathione (GSH). 2. Uptake of L-cystine (10 mu M) was linear for up to 10 min, t emperature dependent, Na+ independent, saturable (Michaelis-Menten con stant (K-m), 86 +/- 25 mu M; maximal velocity (V-max), 109 +/- 33 nmol (mg protein)(-1) h(-1)) and reduced by 80-90% by a 50-fold excess con centration of L-glutamate and L-homocysteic acid, but not L-aspartate. These transport properties resemble those described for system x(c)(- ), 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 r ates of L-cystine and L-glutamine uptake by 41-55%. Anthglutin gamma-L -glutamyl-2-(2-carboxyphenylhyl)hydrazine), a structurally different i nhibitor of gamma-GT, also caused a concentration-dependent (0.01-1 mM ) decrease in gamma-GT activity and L-cystine uptake. 4. Neither acivi cin nor anthglutin inhibited the uptake of L-glutamate, a poor substra te for gamma-GT. 5. In the presence of a 500-fold excess concentration of glutamate, which should abolish entry of cystine via system x(c)(- ), the remaining fraction of cystine transport was inhibited by 50% by acivicin, suggesting that transport is, in part, dependent on the act ivity of gamma-GT. 6. Cystine transport was also 80-80% 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 finding s demonstrate that in human pancreatic duct PaTu 8902 cells, cystine u ptake is mediated by system x(c)(-) (50-60%) and the gamma-glutamyl cy cle. Our results provide the first evidence linking gamma-GT with cyst ine transport in human epithelial cells and are of relevance in view o f the importance of cystine as a sulphur amino acid source for GSH syn thesis in cells exposed to oxidative stress.