European Journal of Pharmacology - Molecular Pharmacology Section, 244 (1993) 103-104 103 ¢3 1993 Elsevier Science Publishers B.V. All rights reserved 0922-4106/93/$06.00 EJPMOL 0123R Rapid communication NMDA receptor activation by spermine requires glutamate but not glycine Juan-Carlos Marviz6n and Michel Baudry Neurobiology Program, UniL~ersity of Southern California, Los Angeles, CA, USA Received 12 November 1992, accepted 18 November 1992 Stimulation by spermine of [3H]MK-801 binding to N-methyl-D-aspartate (NMDA) receptors was additive with the effect of glutamate and glycine, but was completely abolished by the glutamate antagonist 3-(carboxypiperazin-4-yl)propyl-l-phosphonate (CPP, 10/xM) or the glycine antagonist 7-chlorokynurenate (10/zM). Blockade by 7-chlorokynurenate could be overcome in the presence of glutamate, whereas blockade by CPP was unaffected by glycine. Therefore, NMDA receptors can be activated by glutamate and spermine but not by glycine and spermine. MK-801; NMDA (N-methyl-D-aspartate); Polyamines N-Methyl-D-aspartate (NMDA) receptors are posi- tively modulated by agonists of three different sites: the glutamate or neurotransmitter site, the glycine site and the polyamine site (Monaghan et al., 1989; Williams et al., 1991). Although it is not clear yet how these sites interact to modulate receptor function, electrophysio- logical studies (Kleckner and Dingledine, 1988) indi- cate that glycine is absolutely required for the activa- tion of the receptor by glutamate. The channel blocker MK-801 (dizocilpine) binds selectively to the open state of the NMDA receptor channel (Huettner and Bean, 1988), and the binding of its radiolabeled form has been widely used to measure receptor activation (Wil- liams et al., 1991). We show here that NMDA receptor activation, measured by the stimulation of [3H]MK-801 binding to extensively washed membranes, does not require glycine when both glutamate and the polyamine spermine are present. Binding experiments were performed as described (Marviz6n and Skolnick, 1991). Forebrains from male Sprague-Dawley rats (150-250 g) were homogenized with a Tissumizer (Tekmar Corp.) in 10 ml/g of 0.32 M sucrose, 1 mM HEPES-K +, pH 7.4, and centrifuged 10 min at 1000 x g in 50 ml/g of the same solution. The supernatant was centrifuged 20 min at 23,000 x g and the resulting pellet was washed 8 times. Each washing Correspondence to: Juan-Carlos Marviz6n, University Park Campus, HNB-311, University of Southern California, Los Angeles, CA 90089-2520, USA. cycle consisted of resuspension in 50 ml/g of buffer A (5 mM HEPES-K +, pH 7.4) followed by centrifugation for 20 min at 23,000 x g. Membranes were stored at -80°C in 5 ml/g of buffer A, and washed one more time immediately before binding assays. [3H]MK-801 binding was measured by incubating membranes (50- 100/zg protein/assay) at 25°C for 120 min in buffer A with 4.5 nM [3H]MK-801 (22.3 Ci/mmol, DuPont- NEN) and other compounds. Incubations were termi- nated by filtration using Whatman GF/C filters and a Brandel MB-24 manifold, followed by one wash with cold buffer A (5 ml/assay). Non-specific binding was determined with 10 /zM MK-801. Protein concentra- tions were determined by the method of Bradford (1976) using bovine serum albumin as a standard. Spermine-induced stimulation of [3H]MK-801 bind- ing was assessed in the presence of saturating concen- trations of agonists and antagonists of the glutamate or the glycine sites of NMDA receptors (fig. 1). In the presence of glutamate and glycine, [3H]MK-801 bind- ing was further increased by spermine, indicating that spermine acts at a binding site different from the glutamate and the glycine sites, a result in agreement with previous studies (Williams et al., 1991). Spermine also enhanced [3H]MK-801 binding in the absence of added glutamate and glycine, but not when the gluta- mate site was blocked with the antagonists 3-(carboxy- piperazin-4-yl)propyl-l-phosphonate (CPP, 10 /xM) or 2-amino-5-phosphonopentanoate (AP-5, 30/zM), or in the presence of the glycine antagonists 7-chloro- kynurenate (10 p.M) or 6,7-dinitroquinoxaline-2,3-di-