Characterization of L-glutamic acid transport by glioma cells in culture: evidence for sodium-independent, chloride-dependent high affinity influx

Abstract

The transport of radiolabeled L-glutamic acid by LRM55 glioma cells in culture was examined. Time course studies indicated that L-[3H]glutamic acid is rapidly accumulated, and then 3H is lost from the cell, presumably in the form of glutamate metabolites. Kinetic analysis of L-glutamate uptake provided evidence for two components of transport. A low affinity component was found to persist at 0 to 4 degrees C and was not saturable, influx being proportional to the substrate concentration. A high affinity component, resolved by subtraction of the influx at 0 to 4 degrees C, followed Michaelis-Menten kinetics having a Km of 123 microM and a Vmax of 2.99 nmol/min/mg of protein. The transport system was highly substrate-specific: At least 27-fold larger concentrations of the most potent analogues--cysteic acid, cysteine sulfinic acid, and L-aspartic acid--were required to compete effectively with glutamate. Second, the system was not severely affected by exposure to inhibitors of oxidative phosphorylation or gamma-glutamyltranspeptidase. Third, only 65% of the high affinity uptake was dependent upon the presence of sodium, the other 35% being dependent upon chloride. These observations were supported by the findings that uptake was only partially inhibited by ouabain and quite effectively reduced by several inhibitors of chloride transport. The results of this study provide information on the properties of low affinity glutamate transport, as well as the first description of sodium-independent, chloride-dependent high affinity glial transport. The high affinity component of influx is stimulated by elevated potassium and inhibited by several pharmacological agents. The sodium independence of a significant proportion of high affinity glutamate transport suggests that glutamate binding studies done in sodium-free medium with intact cells may be confounded by a considerable amount of intracellular uptake.