A role for gamma-glutamyl transpeptidase and the amino acid transport system xc- in cystine transport by a human pancreatic duct cell line.
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.
Sweiry
J H
JH
Biomedical Sciences Division, King's College, London, UK.
Sastre
J
J
Viña
J
J
Elsässer
H P
HP
Mann
G E
GE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism*; D002460:Cell Line; D003553:Cystine / Q000378:metabolism*; D005971:Glutamates / Q000494:pharmacology; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D007700:Kinetics; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D010183:Pancreatic Ducts / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D011761:Pyrrolidonecarboxylic Acid / Q000494:pharmacology; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors / Q000502:physiology*
D000596:Amino Acids; D005971:Glutamates; D007555:Isoxazoles; D003553:Cystine; D000255:Adenosine Triphosphate; D005723:gamma-Glutamyltransferase; C018830:anthglutin; C006303:acivicin; D011761:Pyrrolidonecarboxylic Acid