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

Abstract

The synaptosomal transport of L-[35S]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 μM, V(max) 185±20 nmol mg protein-1 min-1), (b) high affinity sodium-independent transport (K(m) 6.90±2.1 μM, V(max) 0.485±0.060 nmol mg protein-1 min-1) and (c) low affinity sodium-independent transport (K(m) 327±29 μM, 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-[35S]cystine accumulated into synaptosomes. L-glutamate (K(i) 11.2±1.3 μM) was a non-competitive inhibitor of this transporter, and at 100 μM L-glutamate, the V(max) for L-[35S]cystine transport was reduced to 10% of control. L-cystine did not inhibit the high-affinity sodium-dependent transport of D-[3H]aspartate into synaptosomes.L-histidine and glutathione were the most potent inhibitors of the low affinity sodium-independent transport of L-[35S]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. Copyright (C) 2000 Elsevier Science Ltd.