RAPID STIMULATION OF EAAC1-MEDIATED NA-DEPENDENT L-GLUTAMATE TRANSPORT ACTIVITY IN C6 GLIOMA-CELLS BY PHORBOL ESTER()

C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na+-dependent L-[H-3]glutamate transport. Although a 3 0-min preincubation with forskolin had no effect on transport activity , preincubation with phorbol 12-myristate 13-acetate (PMA) increased t ransport activity two- to threefold, PMA caused a time-dependent and c oncentration-dependent increase in EAAC1-mediated L-[H-3]glutamate tra nsport activity. A 2-min preincubation with PMA was sufficient to caus e 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 EC(50) value of PMA for stimulation of transport activity was 80 n M. Kinetic analyses demonstrated that the increase in transport activi ty was due to a 2.5-fold increase in V-max with no change in K-m. PMA also increased the transport of the non metabolizable analogue, D-[H-3 ] aspartate to the same extent. In parallel assays, PMA did not, howev er, increase Na+-dependent glycine transport activity in C6 glioma. Th e inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate, did not st imulate L-[H-3]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 effe ct on the stimulation of transport activity caused by PMA. The Ca2+ io nophore A23187 did not act synergistically to increase PMA stimulation . In previous studies, PMA caused a rapid increase in amiloride-sensit ive Na+/H+ transport activity in C6 glioma, In the present study, pre- and coincubation with amiloride had no effect on the stimulation of t ransport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transpo rt activity. This rapid increase in Na+-dependent L-[H-3]glutamate tra nsport activity may provide a novel mechanism for protection against a cute insults to the CNS.