Kinetic and pharmacological analysis of L-[35S]cystine transport into rat brain synaptosomes

The synaptosomal transport of L-[S-35]cystine occurs by three mechanisms that are distinguishable on the basis of their ionic dependence, kinetics of transport and the specificity of inhibitors. They are (a) low affinity sodium-dependent transport (K-m 463 +/- 86 mu M, V-max 185 +/- 20 nmol mg protein(-1) min(-1)), (b) high affinity sodium-independent transport (K-m 6.90 +/- 2.1 mu M, V-max 0.485 +/- 0.060 mmol mg protein(-1) min(-1)) and (c) low affinity sodium-independent transport (K-m 327 +/- 29 mu M, V-max 4.18 +/- 0.25 nmol mg protein(-1) min(-1)). The sodium-dependent transport of L-cystine was mediated by the X-AG- family of glutamate transporters, and accounted for almost 90% of the total quantity of L-[S-35]cystine accumulated into synaptosomes. L-glutamate (K-i 11.2 +/- 1.3 mu M) was a noncompetitive inhibitor of this transporter, and at 100 mu M L-glutamate, the V-max for L-[S-35]cystine transport was reduced to 10% of control. L-cystine did not inhibit the high-affinity sodium-dependent transport of D-[H-3]aspartate into synaptosomes. L-histidine and glutathione were the most potent inhibitors of the low affinity sodium-independent transport of L-[S-35]cystine. L-homocysteate, L-cysteine sulphinate and L-homocysteine sulphinate were also effective inhibitors. 1 mM L-glutamate reduced the sodium-independent transport of L-cystine to 63% of control. These results suggest that the vast majority of the L-cystine transported into synaptosomes occurs by the high-affinity glutamate transporters, but that L-cystine may bind to a site that is distinct from that to which L-glutamate binds. The uptake of L-cystine by this mechanism is sensitive to inhibition by increased extracellular concentrations of L-glutamate. The importance of these results for understanding the mechanism of glutamate-mediated neurotoxicity is discussed. (C) 2000 Elsevier Science Ltd. All rights reserved.