Glutamate Receptor (gamma-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionate and Kainate Subtype) Activity Enhanced by Dizocilpine (MK801) in Rat Hippocampus. Rapid Chemical Kinetic Measurements of Sodium Flux and Receptor Desensitization with Native Membranes

Abstract

l-Glutamate-mediated transmembrane influx of 22Na+ into a native membrane vesicle preparation from rat hippocampus was resolved into components due to glutamate receptor and Na+-glutamate symport, by kinetic analysis and pharmacological specificity. Measurements made with a quench-flow technique showed that receptor-mediated cation exchange proceeded in two phases, the faster phase progressively attenuated by a desensitization process with a half time of approximately 90 ms. The influx of 22Na+ continued in the second phase with a half time of 6 s. Receptor-mediated 22Na+ influx was replicated with the glutamate mimetics, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate and kainate but not with N-methyl-d-aspartate and was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione but not by 2-amino-5-phosphonovaleric acid. Receptor-mediated 22Na+ influx did not require glycine and was pH dependent; influx was inhibited at pH values less than pH 5.0. Thus, the receptor-mediated activity was of the γ-amino-3-hydroxy-5-methyl-4-isoxazole propionate-kainate subtype. The N-methyl-d-aspartate receptor inhibitor 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine or MK801), increased receptor-mediated transmembrane 22Na+ flux in both phases, but did not accelerate receptor desensitization. Receptor-mediated radiotracer exchange was the major contribution to 22Na+ influx in times less than 5 s and the receptor-mediated responses could be measured with a small correction for sodium-glutamate symport or in the presence of a glutamate-uptake inhibitor. A third phase of 22Na+ influx accessed a different internal volume and proceeded with the same first-order rate constant as [3H]glutamate uptake when measured simultaneously. This was shown to be mediated by the glutamate uptake mechanism, with a half time of 20–40 s varying with the preparation. The Na+ -glutamate symport became the largest cause of 22Na+ influx after at least 5 s, the final magnitude being, on average, twofold larger than the receptor-mediated 22Na+ influx.