Biochemical SocietyTransactions ( 1 992) 20

neuroblastoma cells possess sensitive glutamate binding sites.

C1300

quisqualate

James B. Uney, Sian Thomas, Brian H. Anderton and lain C. Campbell. Department of Neuroscience, Institute of Psychiatry, DeCrespigny Park. London SE5 8AF. UK. Glutamate is accepted as being the major excitatory neurotransmitter in the central nervous system (CNS) [ I ] and its effects are mediated by at least three receptor sub-types (N-methylD-aspartate (NMDA), kainate, and quisqualate) which have been identified using selective agonists [2]. A fourth binding site, which is reported to account for 5040% of specific binding in standard assays [3,4,5], has been shown to have a high-affinity for quisqualate and L-glutamate and is blocked by the antagonist L-2-amino-4-phmphonobutryic acid (L-APB). This binding site is also characterized by a strong dependency on chloride, and controversy exists as to whether they are receptors involved in synaptic transmission [6] or are involved in the chloride-dependent sequestration of glutamate [7]. Most experimental preparations of central nervous system neurones express multiple EAA receptor types and it has therefore proved to be difficult to study individual populations. A clonal cell line expressing only one EAA receptor sub-type would greatly facilitate the elucidation of the biochemical and physiological characteristics of EAA receptors.We have used radioligand binding assays to pharmacologically characterise 3Hglutamate-binding sites in mouse-derived C1300 N 2 A neuroblastoma cells: quisqualate-sensitive glutamate binding sites were present on the neuroblastoma cells and fluorometric experiments to measure [CaZ+li were carried out to determine whether this binding site is a receptor or is a glutamate sequestration site. Figure l a shows 3H-glutamate binding to differentiated mouse C1300 neuroblastoma cells and demonstrates that there is a high ratio of specific to non-specific binding and that the KD+_SEM was

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610+58 nM and the BmaxLSEM was 16.8+1.8pmol/mg o f protein. To determine the nature of this glutamate binding site, displacement studies were performed (Figure lb). Quisqualate and glutamic acid were equally potent competitive blockers (Ki=0.6 and 0.8pM respectively) of 3H-glutamate (400nM) binding to the washed C1300 N2A sonicated membranes. Kainic acid and NMDA had virtually no effect on binding of 3H-glutamate to the C1300 N2A neuroblastoma membranes (Kb100pM for both kainate and NMDA). [CaZ+]imeasured in f u n - 2 loaded C1300 N2A cells was calculated to be 219+4.5nM (median value+SEM). Incubation of the cells (from 1-20 minutes) with quisqualate, kainate, glutamic acid and NMDA (10pM-1.5mM) did not elevate the intracellular calcium concentration in the cells. However, incubation of the C1300 neuroblastoma cells with potassium (8mM) produced a 75% increase in [Caz+Ii (383+6.3nM, mean value+SEM). Glutamate binding sites have not previously been reported to exist on C1300 neuroblastoma cells although Malouf el al, [8] have reported the presence of a quisqualate sensitive glutamic acid binding site (Kd 650nM and Bmax 16pmollmg of protein) on N18-RE-105 neuroblastoma hybrid cells (N18TG-2 x Fisher rat 18-day embryonic neural retina) and suggested that these site probably represented a neurotransmitter binding site. Fluorometric measurements made in this study showed that there was n o agonist induced rise in intracellular calcium levels in C1300 neuroblastoma cells although high levels of potassium depolarised the cells and caused an increase in the concentration of [Caz+Ii. Therefore, the quisqualate sensitive glutamate binding site present in C1300 N2A neuroblastoma cells probably is a glutamate uptake site, similar to that previously described in NI8-RE- 105 cells. This is the first demonstration of glutamate uptake binding sites in neuroblastoma cells (and represents an easily manipulable model for the further study of this system). We are grateful to the Parkinson Disease Society of Great Britain and Action Research for financial support and would like to thank Dr David Ward for h s help with the cell cultures. 1. Fonnum, F. (1984). J.Neurochem. 42, 1-11. 2. Foster, A. C. & Fagg, G.E. (1984) Brain Res.Rev. 7, 103164. 3. Fagg, G.E., Foster, A.C., Mena, E.E & Cottman, C. (1982). J. Neurosci. 3, 958-965. 4. Mena, E.E., Pagnozzi, M.J. & Gullak, M.F. ( 1986) J.Neurochem. 47, 1052- 1060. 5. Kessler, M.,Peterson, G.,Vu, H. M.,Baudry, M & Lynch, G. L- (1987) J. Neurochem. 48, 1191-1200. 6. Baudry, M. & Lynch, G. (1981) Mol. Cell. Biochem. 36, 518.

7. Pin,J-P., Bockaer1.J. & Recasens, M. (1984). FEBS Lett. 175, 31-36. 8.Malouf, A.T.,Schnaar,R. L.& Coyle, J.T.(1984) J.Biol.Chem. 259, 12756-12763.

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3H-glutamate (nM)

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Figure I . Association isotherm (A) for 3Wglutamate h i d i n g and Competition curves (B) for the displacement of 3II-glulamate by excitatory amino acids ~n C1300 N3A neurohlasloma cells. Each data point is the mean of triplicate determinations f r o m three separate experiments.