| Glutamic acid is generally accepted as one of the major excitatory neurotransmitters in the central nervous system. To study the glutamate receptor and its regulation under carefully controlled conditions, the specific binding of {lcub}('3)H{rcub}glutamic acid was characterized in washed membranes isolated from N18-RE-105, a neuroblastoma-retinal hybrid cell line.; {lcub}('3)H{rcub}Glutamic acid bound in a saturable and reversible fashion with a K(,D) of 654 nM and a B(,max) of 15.5 pmole/mg protein. Pharmacologic characterization of the site indicates that it closely resembles the Na('+)-independent binding site for glutamate found on brain membranes, and thought to be an excitatory amino acid receptor. Thus, while kainate, N-methyl-D,L-aspartate and nonamino acid ligands did not displace {lcub}('3)H{rcub}glutamate, quisqualate and ibotenate were potent inhibitors of specific binding. Furthermore, this binding site has regulatory properties which resemble those described in the hippocampus (Baudry and Lynch, Nature 282:748, 1979). As in the hippocampus, preincubation of the membranes in the presence of Ca('++) for 10 min caused a dose dependent increase in specific binding that was the result of an increased number of binding sites with no change in their affinity. Maximal stimulation required Ca('++) concentrations in excess of 1 mM and resulted in greater than 100% stimulation of specific binding. In contrast, preincubation with monovalent cations resulted in a decrease in the number of sites. La('+++) was the only other cation tested which enhanced the specific binding of {lcub}('3)H{rcub}glutamic acid.; N18-RE-105 cells grown in the presence of 10 mM glutamate for 72 hours exhibited a two-fold increase in the number of binding sites for {lcub}('3)H{rcub}glutamic acid with no alteration in K(,D); binding site density was further increased by preincubation of the membranes with Ca('++). This regulation of the binding site after exposure to glutamate, and the further increase seen after preincubation with Ca('++), may model long-term potentiation in the hippocampus, a cellular correlate to learning and memory. In summary, N18-RE-105 cells possess a pharmacologically relevant binding site for {lcub}('3)H{rcub}glutamate, which exhibits regulatory properties resembling those previously described in hippocampal membranes. |
