Inhibition of the high-affinity uptake of D-[3H]aspartate in rate by L-alpha-aminoadipate and arachidonic acid.

ABSTRACT

The mechanism of inhibition of the high-affinity sodium-dependent transport of D-[3H]aspartate by the gliotoxin, L-alpha-aminoadipate, and also by the endogenous fatty acid, arachidonic acid (cis-5,8,11,14 eicosatetraenoic acid), into rat brain synaptosomes has been investigated. L-alpha-Aminoadipate competitively inhibited the transport of D-[3H]aspartate with a K1 value of 192 microM. Superfusion of coronal slices of rat brain for 40 min with 1 mM L-alpha-aminoadipate reduced the glutathione concentration of the tissue by 20%. Neither glutamate nor kainate depleted the glutathione level of the slices. Pre-incubation of synaptosomes with arachidonic acid (10 microM) for 10-60 min produced a marked potentiation of the inhibition of D-[3H]aspartate transport, compared to experiments in which the acid was added concurrently with the D-[3H]aspartate ('co-incubation' experiments). Inhibition of D-[3H]aspartate transport by arachidonic acid was not blocked by addition of nordihydroguaretic acid to the pre-incubation medium. Staurosporine (50 nM) reduced the inhibition of transport occurring during pre-incubation with 10 microM arachidonic acid, and there was no longer any significant difference from the level of inhibition obtained in co-incubation experiments. Phorbol, 12-myristate, 13-acetate (1 microM) reduced the transport of D-[3H]aspartate to 73% of control after 20 min pre-incubation of the synaptosomes. This study highlights the fact that inhibition of glutamate transport may affect brain function in a number of different ways. Competitive inhibition by a structural analogue of glutamate, such as L-alpha-aminoadipate, leads to a reduction in the glutathione level, which may be an important factor in L-alpha-aminoadipate-mediated toxicity. On the other hand, the more long-term effects of non-competitive inhibition of glutamate transport by arachidonic acid, in a mechanism involving protein kinase C, may represent a physiological means for regulation of transporter activity in the brain.