Abstract
We recently found evidence for anatomic and physical linkages between the astroglial Na+-dependent glutamate transporters (GLT-1/EAAT2 and GLAST/EAAT1) and mitochondria. In these same studies, we found that the glutamate dehydrogenase (GDH) inhibitor, epigallocatechin-monogallate (EGCG), inhibits both glutamate oxidation and Na+-dependent glutamate uptake in astrocytes. In the present study, we extend this finding by exploring the effects of EGCG on Na+-dependent l-[3H]-glutamate (Glu) uptake in crude membranes (P2) prepared from rat brain cortex. In this preparation, uptake is almost exclusively mediated by GLT-1. EGCG inhibited l-[3H]-Glu uptake in cortical membranes with an IC50 value of 230 μM. We also studied the effects of two additional inhibitors of GDH, hexachlorophene (HCP) and bithionol (BTH). Both of these compounds also caused concentration-dependent inhibition of glutamate uptake in cortical membranes. Pre-incubating with HCP for up to 15 min had no greater effect than that observed with no pre-incubation, showing that the effects occur rapidly. HCP decreased the Vmax for glutamate uptake without changing the Km, consistent with a non-competitive mechanism of action. EGCG, HCP, and BTH also inhibited Na+-dependent transport of d-[3H]-aspartate (Asp), a non-metabolizable transporter substrate, and [3H]-γ-aminobutyric acid (GABA). In contrast to the forebrain, glutamate uptake in crude cerebellar membranes (P2) is likely mediated by GLAST (EAAT1). Therefore, the effects of these compounds were examined in cerebellar membranes. In this region, none of these compounds had any effect on uptake of either l-[3H]-Glu or d-[3H]-Asp, but they all inhibited [3H]-GABA uptake. Together these studies suggest that GDH is preferentially required for glutamate uptake in forebrain as compared to cerebellum, and GDH may be required for GABA uptake as well. They also provide further evidence for a functional linkage between glutamate transport and mitochondria.
Highlights
Glutamate is the predominant excitatory neurotransmitter in the mammalian CNS and mediates the vast majority of cell-to-cell communication in the brain [for review, see [1]]
We examined the effect of an inhibitor of glutamate dehydrogenase (GDH), a mitochondrial enzyme that could contribute to glutamate oxidation, on glutamate uptake and found that it inhibited uptake in astrocytes [19]
The first compound examined, hexachlorophene (HCP), caused a concentration-dependent inhibition of Na+-dependent glutamate uptake (Figure 2B). When these data were fit to a single site, the IC50 value was 3.9 μM, RESULTS We recently found that EGCG, a compound extracted from green tea that inhibits GDH [21], blocks Na+-dependent glutamate uptake in astrocytes
Summary
Glutamate is the predominant excitatory neurotransmitter in the mammalian CNS and mediates the vast majority of cell-to-cell communication in the brain [for review, see [1]]. Results from several different types of studies strongly suggest that GLT-1 and GLAST mediate the bulk of glutamate uptake in the mammalian brain [(9); for review, see [10]]. This clearance into astroglia differentiates glutamate from most of the other classical neurotransmitters that are directly recycled back into the presynaptic nerve terminal [for reviews, see [11, 12]]. These transporters co-transport three Na+ ions and one H+ with glutamate in the inward direction; the cycle is completed with the counter-transport one K+ ion [13]. These transporters are capable of generating up to a one million-fold concentration gradient of glutamate across the plasma membrane [for review, see [14]]
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