STRUCTURAL SELECTIVITY AND MOLECULAR NATURE OF L-GLUTAMATE TRANSPORT IN CULTURED HUMAN FIBROBLASTS

Uptake of L-[H-3]glutamate by monolayers of fibroblasts cultured from human embryonic skin has been studied in the presence of several nonra dioactive structural analogs of glutamate and aspartate. Results have suggested that the structural specificities of glutamate transporters in cultured human fibroblasts are similar to those of glutamate transp orters in the mammalian brain Only subtle differences have been detect ed: in the mammalian cerebral cortex, enantiomers of threo-3-hydroxyas partate are almost equipotent as inhibitors of L-[H-3]glutamate uptake while, in human fibroblasts, the D-isomer has been found to be an ord er of magnitude less potent than the corresponding L-isomer. Kinetic a nalysis of a model in which substrates are recognized by the glutamate transporter binding site(s) as both alpha and beta-amino acids indica ted that such a mechanism cannot explain the apparent negative coopera tivity characterizing the effects of D- and L-aspartate. Molecular mod eling has been used to estimate the optimum conformation of L-glutamat e as it interacts with the transporter(s). Flow cytometry has indicate d that all fibroblasts in culture express at least moderate levels of four glutamate transporters cloned from human brain. Small subpopulati ons (<3%) of cells, however, mere strongly labeled with antibodies aga inst EAAT1 (GLAST) and EAAT2 (GLT-1) transporters. We conclude that th ese two transporters-known to be strongly expressed in brain tissue-ca n be principally responsible for the ''high affinity'' transport of gl utamate also in nonneural cells. (C) 1998 Academic Press.