No abstract

06 glioma cells were used as a model system to study the regulation of EAAC1 -mediated Na~-dependent L-[ 3H]glutamate transport. Although a 30-mm preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two-to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated L-[3H]glutamate transport activity. A 2-mm preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The E0 50 value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in Vmax with no change in Km. PMA also increased the transport of the nonmetabolizable analogue, D-[ 3H]aspartate to the same extent. In parallel assays, PMA did not, however, increase Nat-dependent glycine transport activity in 06 glioma. The inactive phorbol ester 4a-phorbol 12,13-didecanoate, did not stimulate L-[3H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca2~ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na~/Htransport activity in 06 glioma. In the present study, pre-and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Nat-dependent L-[3H]glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS. Key Words: Glutamate-Transport-Uptake-EAAC 1-06 glioma-Phorbol ester.

Some data suggest that the sodium-dependent, high-affinity L-glutamate (Glu) transport sites in forebrain are different from those in cerebellum. In the present study, sodium-dependent transport of L-[3H]Glu was characterized in cerebellum and cortex. In both cerebellar and cortical tissue, activity was enriched in synaptosomes. Approximately 100 excitatory amino acid analogues were tested as potential inhibitors of transport activity. Many of the compounds tested inhibited transport activity by < 65% at 1 mM and were not studied further. One group of compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were < 10-fold selective as inhibitors of transport activity. These included three of the putative endogenous substrates for transport: L-Glu, L-aspartate, and L-cysteate. Four of the compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were > 10-fold selective as inhibitors. These included beta-N-oxalyl-L-alpha,beta-diaminopropionate, alpha-methyl-DL-glutamate, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, and (2S,1'S,2'S,3'S)-2-(2-carboxy-3-methoxymethylcyclopropyl)glycine. Data obtained with a few of the inhibitors were consistent with two sites in one or both of the brain regions. (2S,1'R,2'R)-2-(Carboxycyclopropyl)glycine (L-CCG-II) was identified as the most potent (IC50 = 5.5 microM) and selective (60-100-fold) inhibitor of transport activity in cerebellum. One of the potential endogenous substrates, L-homocysteate, was also a selective inhibitor of cerebellar transport activity. The data for inhibition of transport activity in cortex by both L-CCG-II and L-homocysteate were best fit to two sites. Kainate was equipotent as an inhibitor of transport activity, and in both brain regions the data for inhibition were best fit to two sites. The possibility that there are four subtypes of excitatory amino acid transport is discussed. Altering sodium and potassium levels affects cerebellar and cortical transport activity differently, suggesting that the differences extend to other recognition sites on these transporters.