High-affinity Sodium-dependent Transport Research Articles

Characteristics of L-aspartate transport and expression of EAAC-1 in sarcolemmal vesicles and isolated cells from rat heart.

L-Aspartate is an important intermediary metabolite in the heart and has also been implicated in myocardial protection, but little is known about its transport across the cardiac sarcolemma. In this study we have tested the hypothesis that the high affinity sodium-dependent aspartate transporter, EAAC-1 is expressed in heart and have also characterised aspartate transport into the myocardium. Characteristics of L-[14C]aspartate uptake into rat heart were investigated using sarcolemmal vesicles and isolated myocytes. The expression of EAAC-1 in the two preparations was also investigated by western blotting. The K(m) and V(max) of L-aspartate uptake was 9.78+/-0.7 microM and 1.17+/-0.27 pmol/mg/s in vesicles compared to 6.53+/-1.24 microM and 13.65+/-1.0 pmol/microl/s in cells. In vesicles, L-aspartate uptake was dependent on external sodium and internal potassium, and was rheogenic. In cells, L-aspartate uptake was also dependent on external sodium. Addition of unlabelled L- and D-aspartate and L-glutamate significantly inhibited L-[14C]aspartate uptake in both preparations but D-glutamate had no effect. An antibody to the aspartate transporter, EAAC-1 recognised a protein of appropriate size in both vesicles and cells. L-aspartate uptake in heart is mediated by a high affinity sodium-dependent transporter. This is accompanied by the expression in heart of EAAC-1. The physiological significance of this transporter with respect to aspartate utilisation in the heart is discussed.

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

Abstract The synaptosomal transport of l -[ 35 S]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 -[ 35 S]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 -[ 35 S]cystine transport was reduced to 10% of control. l -cystine did not inhibit the high-affinity sodium-dependent transport of d -[ 3 H]aspartate into synaptosomes. l -histidine and glutathione were the most potent inhibitors of the low affinity sodium-independent transport of l -[ 35 S]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.