Introduction

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Ifenprodil was the first neuroprotective compound found to be selective for NMDA receptors containing NR2B subunits (Carter et al., 1989; Williams et al., 1993; Williams, 1993). NMDA receptors are ligand gated ion channels, which are activated by glutamate in the required presence of glycine (Ascher and Johnson, 1994). They are thought to play a role in physiological functions, neurodegenerative disease, neurotrauma (Monaghan et al., 1989; Dingledine et al., 1999), psychiatric disorders (Thornberg and Saklad, 1996), and pain (Woolf and Thompson, 1991). Rat NMDA receptors consist of heteromeric combinations of two major subunits designated NR1 and NR2 (Yamakura and Shimoji, 1999). There are eight splice variants of the NR1 subunit, and four major types of the NR2 subunit designated NR2A, NR2B, NR2C, and NR2D. An additional not fully characterized subunit designated as NR3A also has been cloned (Sucher et al., 1995). Specific combinations of heteromeric subunits differ as to their sensitivity to agonists, polyamines, and allosteric regulation. Receptors containing NR2B subunits are widely expressed in cerebral cortex and spinal cord. Ifenprodil interacts with high affinity at a distinct, voltage-independent, polyamine-regulated site on NMDA receptors containing NR2B subunits (Carter et al., 1989; Reynolds and Miller, 1989; Williams, 1993; Gallagher et al., 1996).

Ifenprodil and its halogenated analog eliprodil are effective neuroprotective agents in vitro and in vivo but are devoid of many of the side effects that have limited the therapeutic usefulness of other types of NMDA antagonists (Scatton et al., 1994; Carter et al., 1997). They have a potential use in Parkinson's disease (Zeevalk et al., 1994) as well as in stroke and head trauma (Gotti et al., 1988; Toulmond et al., 1993) but appear to lack deleterious properties, which include locomotor stimulation, abuse potential, learning impairment, and neuronal pathology (Carter et al., 1997). This improved therapeutic profile is thought to be associated with selectivity for NR2B subunit containing NMDA receptors and has triggered extensive efforts to find other novel compounds with improved potency, efficacy, and NR2B selectivity (Chenard and Menniti, 1999).

[³H]Ifenprodil is a commercially available tool for the discovery of novel NR2B subtype-selective agents. Its binding is fully inhibited by a number of polyamines and interacts with the cations zinc and magnesium (Schoemaker et al., 1990). Antagonists of the NMDA glutamate and glycine recognition sites also regulate it in a reversible manner (Carter et al., 1997). However, promiscuous binding to other sites limits its usefulness. Ifenprodil has affinity for calcium channels, adrenergic, histamine, and serotonin receptors. It also binds with nanomolar affinity to  binding sites (Contreras et al., 1990; Hashimoto and London, 1993; Hashimoto and London, 1995) and can be inhibited by imidazoline ligands (Moebius et al., 1998). At higher concentrations it binds to a voltage-dependent site within the NMDA ion channel (Williams, 1993; Marvizon and Baudry, 1994). This affinity for multiple binding sites complicates the isolation of its binding to the voltage-independent high affinity site on native NMDA receptors containing NR2B subunits.

[³H]Ifenprodil receptor binding studies have focused on separating the polyamine-sensitive, NMDA-associated sites from high affinity  binding sites (Schoemaker et al., 1990; Dana et al., 1991; Hashimoto and London, 1993). At 4°C inclusion of the  site blocker GBR-12909 isolates polyamine-sensitive, ifenprodil binding sites (Hashimoto et al., 1994). Even in the presence of GBR-12909, both high and low affinity [³H]ifenprodil sites remain (Dana et al., 1991; Nicolas and Carter, 1994; Coughenour and Cordon, 1997). Nicolas and Carter (1994) found that high affinity, polyamine-sensitive, binding sites in rat brain slices match the distribution of NR2B mRNA. Several calmodulin antagonists selectively inhibit the low affinity sites, which are ubiquitously distributed. Among the agents used, trifluoroperazine appears to be particularly useful because of its negligible affinity for the high affinity ifenprodil sites, its piperazine structure, and its ability to block other receptors that bind ifenprodil.

In this study we explored the use of trifluoroperazine to isolate high affinity [³H]ifenprodil binding to NMDA receptors in rat brain membranes. We examined the interaction of [³H]ifenprodil binding sites remaining in the presence of trifluoroperazine with selective NR2B antagonists, polyamines, Mg²⁺, Zn²⁺, protons, and NMDA agonists and antagonists to determine whether their pharmacology matched that of NMDA receptors of the NR2B subtype.

	Experimental Procedures

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Membrane Preparation

Extensively washed, rat brain (Zivic-Miller Laboratories, Inc., Zelienople, PA), buffy coat membranes were prepared as previously described and stored at 70°C (Coughenour and Cordon, 1997). On the day of the assay, each pellet was thawed and disrupted with a Brinkmann/KINEMATICA POLYTRON PT 10/35 (Brinkmann Instruments, Inc., Westbury, NY) homogenizer in 35 ml of 20 mM HEPES buffer, pH 7.4. Following incubation at 37°C for 30 min in a shaking water bath, the homogenates were centrifuged at 40,000g for 10 min at 4°C and the supernatant decanted. This wash step without the incubation was repeated three more times. For use in the assays each pellet was resuspended using the homogenizer in 40 ml of the assay buffer and pooled. These were termed well washed membranes and used for [³H]ifenprodil and [³H]TCP binding experiments.

For [³H]DTG binding experiments crude rat brain membranes were prepared as previously described (Weber et al., 1986). Whole rat brains minus the brainstem and cerebellum were homogenized in 0.32 M sucrose and centrifuged at 40,000g for 10 min. Pellets were resuspended in 50 mM Tris-HCl buffer, pH 8.0, and incubated at 37°C for 30 min. After centrifugation as above, pellets were stored at 70°C. On the day of the assay, pellets were thawed and resuspended by homogenization in 50 mM Tris-HCl buffer, pH 8.0.

Binding Studies

[³H]Ifenprodil Binding. Triplicate incubations were carried out in a volume of 0.5 ml in 1.2-ml polyethylene tubes (Marsh Biomedical Products Inc., Rochester, NY) for 2 h at room temperature. Incubations contained test agents, membranes (200-300 µg of protein) and 4 nM [³H]ifenprodil in 20 mM HEPES-KOH buffer, pH 7.4. In some experiments the assay buffer contained 100 µM trifluoroperazine. For experiments at pH 6.8 and 8.0, membranes were prepared in buffer at the respective pH. Assays were started by addition of the membranes. Bound radioligand was separated by filtration under reduced pressure using a 96-well cell harvester (Mach II, Tomtec Inc., Orange, CO). Filtration was through Whatman GF/B glass fiber filters (Whatman Ltd., Maidstone, England) that had been soaked for at least 15 min in 0.3% polyethyleneimine and allowed to air dry. The filters were rinsed with 3 ml of ice-cold assay buffer within 6 s, and air was allowed to pass through the filters for an additional 10 s to remove residual moisture. The filter mat was supported on a cold (20°C) Teflon support, and filters from individual wells were separated and placed in Mini Poly-Q vials (Beckman Instruments Inc., Fullerton, CA) and filled with 4 ml of scintillation cocktail (Beckman Ready Protein⁺). Radioactivity retained on the filter was determined by liquid scintillation spectrophotometry. Nonspecific binding was defined as the binding in the presence of 1 mM ifenprodil. Specific binding was usually 80%.

[³H]TCP Binding. Binding was carried out as described above but at nonequilibrium conditions using an incubation time of 10 min. Incubations contained test agents, membranes (200-300 µg of protein), 10 µM glutamate, 10 µM glycine, 10 µM spermidine, and 2 nM [³H]TCP in 20 mM HEPES-KOH buffer, pH 7.4. Nonspecific binding was defined as the binding in the presence of 100 µM (+)MK-801. In the presence of 10 µM glutamate, glycine and spermidine specific binding was 90%.

[³H]DTG Binding. [³H]DTG binding was carried out as described above using crude rat membranes for 2 h at room temperature. Incubations contained test agents, membranes (300-400 µg of protein), and 4 nM [³H]DTG in 0.5 ml of 50 mM Tris-HCl buffer, pH 8.0. Nonspecific binding was defined as the binding in the presence of 100 µM haloperidol. Specific binding was >80%.

Data Analysis. Date analysis was performed using GraphPad Prism version 3.0 software (GraphPad Software Inc., San Diego, CA). When binding inhibition curves were statistically analyzed for a best one- or two-site competition fit, the normalized data was fit by nonweighted nonlinear regression to either

(1)

or

(2)

Where noted, data were analyzed using the four-parameter logistic equation (GraphPad Prism).

(3)

Control data was entered as 100%, and unless noted no parameters were constrained. Inhibition curves were compared by ANOVA with post-test comparisons of the log of the concentration inhibiting 50% of the specific binding (IC₅₀) (GraphPad InStat version 3.0 software).

Materials. TCP ([piperidyl-3,4-³H(N)], specific activity, 41.8-57.6 Ci/mmol), ifenprodil ([phenyl-³H], specific activity, 40-55.3 Ci/mmol), and DTG ([5-³H(N)](1,3-di-o-tolylguanidine, [p-ring-³H]), specific activity, 31 Ci/mmol) were purchased from PerkinElmer (Boston, MA). The following were purchased from Sigma-RBI (St. Louis, MO): agmatine sulfate, arcaine, cirazoline hydrochloride, clonidine hydrochloride, DEAP, l-glutamic acid, glycine, haloperidol, HEPES, ifenprodil tartrate, MDL 105,519, (+)MK-801, magnesium chloride, nylidrin, putrescine dihydrochloride, trifluperidol hydrochloride, trifluoroperazine dihydrochloride, and zinc chloride. Spermine tetrahydrochloride and spermidine trihydrochloride were purchased from United States Biochemical Corp. (Cleveland, OH). PPBP and (R,S)-CPP were purchased from Tocris Cookson (St. Louis, MO). Clozapine was obtained from Sandoz Chemicals Corp. (Charlotte, NC). Eliprodil was synthesized by Christopher Bigge (Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, MI). DTG was synthesized by Stephen Bergmeier (Pfizer Global Research and Development). CP-101606 was synthesized by Mark Lovdahl and Tracey Gregory (Pfizer Global Research and Development). Co 101244, Co 101314, and Co 101313 were obtained from CoCensys Inc. (Irvine, CA).

	Results

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Trifluoroperazine inhibited the binding of [³H]ifenprodil to rat brain membranes with two affinity states (Fig. 1). Thirty-five percent of the binding was inhibited with high affinity (IC₅₀ = 1.9 µM) and the remainder with lower affinity (IC₅₀ = 79 µM). In the presence of 1 mM spermidine the specific binding was reduced by 58%. Only the high affinity inhibition by trifluoroperazine remained (IC₅₀ = 2.8 µM).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Inhibition of [3H]ifenprodil binding by trifluoroperazine in the absence () and presence () of 1 mM spermidine. Binding was carried out as described under Experimental Procedures. Data are the mean ± S.E.M. from three independent experiments. Specific binding in the absence of spermidine was 17,752 ± 1,699 dpm and 7,447 ± 987 dpm in its presence.

The trifluoroperazine-insensitive portion of the binding was examined for its selectivity for NMDA receptors containing the NR2B subunit by inhibiting the binding in the absence or presence of 100 µM trifluoroperazine with ifenprodil (Fig. 2A) and the highly selective NR1a/NR2B antagonist CP-101,606 (Fig. 2B). The inhibition curves of both ifenprodil and CP-101,606 were best fit with two-site nonlinear regression curves in the absence of trifluoroperazine. Ifenprodil inhibited 81% of the binding with high affinity (IC₅₀ = 0.018 µM) and the remaining portion with an IC₅₀ equal to 5.4 µM. CP-101,606 inhibited the high affinity fraction of the binding (55%) with an IC₅₀ of 0.010 µM and the low affinity portion with an IC₅₀ equal to 0.724 µM. Thirteen percent of the binding was not inhibited at concentrations of 100 µM CP-101,606. In the presence of trifluoroperazine both ifenprodil and CP-101,606 completely inhibited the binding in a monophasic manner with IC₅₀ values of 0.054 µM for ifenprodil and 0.006 µM for CP-101,606. In the presence of 3 µM GBR-12909, the inhibition curve of CP-101,606 also was monophasic with an IC₅₀ of 0.003 µM, but 24% of the binding was not inhibited at 100 µM.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   A, inhibition of [3H]ifenprodil binding by ifenprodil in the absence () and presence () of 100 µM trifluoroperazine; B, CP-101,606 in the absence () and presence () of 100 µM trifluoroperazine or () 3 µM GBR-12909. Experiments were carried out as described under Experimental Procedures. Data are the mean ± S.E.M. from six ifenprodil and five CP-101,606 independent experiments in the absence of trifluoroperazine, nine ifenprodil and five CP-101606 experiments in the presence of trifluoroperazine, and three CP-101,606 experiments in the presence of GBR-12909. Specific binding (mean ± S.E.M.) for the eleven experiments in the absence of trifluoroperazine was 15,176 ± 879 dpm, 5,893 ± 224 dpm for the fourteen experiments in the presence of trifluperazine and 7,518 ± 1,919 dpm for the three experiments in the presence of GBR-12909.

To further define the selectivity of ifenprodil's trifluoroperazine-insensitive binding sites, eight additional agents with potencies ranging from nanomolar to the high micromolar range as antagonists of NR1a/NR2B NMDA receptors were examined (Fig. 3). All of the agents completely inhibited the binding with a single affinity state in the presence of trifluoroperazine (Table. 1). Linear regression analysis demonstrated a high correlation between the inhibition of [³H]ifenprodil binding and the inhibition of NR1a/NR2B receptors expressed in Xenopus oocytes (Fig. 4A). The same high correlation was found between their inhibition of [³H]ifenprodil and [³H]TCP binding to NMDA receptors containing NR2B subunits in rat brain membranes (Fig. 4B).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Structures of NR1a/NR2B-selective agents.

View larger version (9K): [in this window] [in a new window]   Fig. 4.   Correlation of [3H]ifenprodil binding with (A) inhibition of NR1a/2B receptors expressed in Xenopus oocytes or (B) inhibition of [3H]TCP binding to NR2B subunit containing NMDA receptors in rat brain membranes or (C) inhibition of [3H]DTG binding to  sites in rat brain membranes. See Table 1 for experimental data.

Because several of these compounds are known to have high affinity for  sites, we determined the potencies of these same compounds to inhibit the binding of [³H]DTG to rat brain membranes (Table 1). There was no correlation between their affinity for the [³H]ifenprodil trifluoroperazine-insensitive sites and [³H]DTG sites (Fig. 4C).

View this table: [in this window] [in a new window]   TABLE 1 Inhibition of NR2B-selective compounds at NMDA receptors containing NR2B subunits labeled with [3H]ifenprodil or [3H]TCP and at  binding sites labeled with [3H]DTG in rat brain membranes The binding experiments were carried out as described under Experimental Procedures. Data are the geometric mean and 95% CI from N independent experiments. Inhibition curves were compared for one- or two-site competition using Prism software (GraphPad). For [3H]ifenprodil binding in the presence of 100 µM trifluoroperazine binding, all curves were best fit with a one-site competition model. For [3H]DTG binding, all inhibition curves were best fit with a two-site competition model except for Co 101314, ifenprodil, and nylidrin, which were best fit with a two-site competition model with 90% of the binding in the high affinity fraction. For [3H]TCP binding, curves were best fit with two-site competition model except for Co 101313, which only inhibited 37% of the binding at 100 µM. Inhibition data at NR1a/NR2B receptors expressed in Xenopus oocytes are from the sources indicated.

The ability of agents from other pharmacological classes to inhibit trifluoroperazine-insensitive [³H]ifenprodil sites was examined. The ₂-adrenergic agonist and imidazoline antagonist, clonidine, and the atypical antipsychotic, clozapine, which has affinity for dopamine and serotonin receptors, were weak inhibitors with IC₅₀ values about 100 µM or greater (Table 2). Both agents were less potent inhibitors in the presence of trifluoroperazine than in its absence (Fig. 5, A and B). Another ₂-adrenergic agonist and imidazoline antagonist, cirazoline, was equally potent in the absence (IC₅₀ = 12 µM) as in the presence (IC₅₀ = 15 µM) of trifluoroperazine (Fig. 5C). The ₁-adrenergic antagonist prazosin, the NMDA channel blocker (+)MK-801, the histamine antagonist mepyramine, the voltage-dependent calcium channel blocker flunarizine, and the  site ligand DTG inhibited <20% of the binding at 10 µM in the presence of trifluoroperazine (Table 2).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Inhibition of [3H]ifenprodil binding by clonidine (A), clozapine (B), and ciralozine (C) in the absence () and presence () of 100 µM trifluoroperazine. See Table 2 for experimental data.

To examine the interaction of the trifluoroperazine-insensitive sites with polyamines, we determined the inhibition of [³H]ifenprodil binding by the endogenous polyamines, spermine, spermidine, putrescine, and agmatine, and the constrained polyamines, DEAP and arcaine, in the presence and absence of trifluoroperazine. All the polyamines except for spermidine inhibited the binding more potently in the presence of trifluoroperazine than in its absence (Table 2). Spermine and spermidine were the most potent inhibitors and were the most selective. They only inhibited about 60% of the binding in the absence of trifluoroperazine at concentrations up to 1 mM. DEAP and arcaine fully inhibited the trifluoroperazine-insensitive binding but also had substantial affinity for trifluoroperazine sites. Putrescine and agmatine were weak inhibitors, partially inhibiting the binding at concentrations up to 1 mM.

The inhibition of both sites by the divalent cations, Mg²⁺ and Zn²⁺, was determined. MgCl₂ partially inhibited the binding at concentrations up to 1 mM. The inhibition curve of magnesium was left-shifted 3.7-fold in the presence of trifluoroperazine (Table 2). ZnCl₂ was an equipotent inhibitor of trifluoroperazine-sensitive and trifluoroperazine-insensitive sites. The interactions of spermidine and MgCl₂ were examined in more detail by determining the inhibition curves of ifenprodil at trifluoroperazine-insensitive sites in the presence of increasing concentrations of spermidine and MgCl₂ alone or in combination (Table 3). Spermidine at 10 and 100 µM shifted the inhibition curve of ifenprodil to the right in a parallel fashion 2.3- and 11.7-fold, respectively. Only 20% of the binding remained in the presence of 1 mM spermidine. A similar 5-fold right shift of the ifenprodil curve was found in the presence of 1000 µM MgCl₂. Over 80% of the binding was inhibited in the presence of 10,000 µM MgCl₂. At a concentration of 100 µM, MgCl₂ did not significantly affect the ifenprodil curve. When the inhibition of ifenprodil to trifluoroperazine-insensitive sites was tested in the presence of both 10 µM spermidine and 1000 µM MgCl₂, the shift was not different from that found in the presence of 1000 µM MgCl₂ alone.

Regulation of the trifluoroperazine-insensitive sites by NMDA agonists and antagonists was examined. Glutamate (100 µM) increased the binding of 4 nM [³H]ifenprodil by 23%, and glycine (100 µM) decreased the binding by 17% (Table 4). The glycine antagonist MDL 105,519 increased the binding in a concentration-dependent manner with an EC₅₀ of approximately 500 nM (data not shown). The binding was maximally increased to between 23% and 30% at concentrations of 1, 10, and 100 µM. MDL 105,519, at concentrations of 10 and 100 µM, reversed the decrease with glycine to at or above control levels. The increase in binding observed with MDL 105,519 appeared to be additive to that of glutamate. The competitive NMDA receptor antagonist CPP at concentrations of 1, 10, and 100 µM decreased the binding by 32% to 52%. These decreases were reversed by glutamate to above control levels. The decreases observed with CPP appeared additive with those of glycine.

To determine the effect of proton concentration on the affinity of the ifenprodil binding site, the inhibition of [³H]ifenprodil binding also was examined in buffers of pH 6.8 and 8.0 (Table 5). The potency of trifluoroperazine and spermidine to inhibit [³H]ifenprodil binding was not affected by changes in buffer pH. The potencies of ifenprodil, Co 101244, haloperidol, and nylidrin to inhibit [³H]ifenprodil binding in the presence of trifluoroperazine also were unaffected by changes in proton concentration in this range.

	Discussion

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In this study we explored the use of trifluoroperazine to block the binding of [³H]ifenprodil in rat brain membranes to sites other than NMDA receptors and determined if this would isolate its binding to the voltage-independent regulatory site on NMDA receptors containing NR2B subunits. Previous ifenprodil binding studies have used the  site blocker GBR-12909 to isolate polyamine-sensitive sites associated with the NMDA receptor (Schoemaker et al., 1990; Hashimoto et al., 1994). Other studies have found additional, low affinity, polyamine-sensitive, ifenprodil binding sites remaining in the presence of GBR-12909 (Dana et al., 1991; Nicolas and Carter, 1994; Coughenour and Cordon, 1997). In rat brain slices several calmodulin antagonists, including trifluoroperazine, block these low affinity sites (Nicolas and Carter, 1994). The inclusion of 100 µM trifluoroperazine in the present study reduced the binding of 4 nM [³H]ifenprodil to rat brain homogenates by 61% and allowed it to bind to a single high affinity state in rat brain membranes with a K_D of 50 nM.

We did not use GBR-12909, because trifluoroperazine also occupies  sites (Weber et al., 1986). In addition, it occupied sites not inhibited by GBR-12909. The selective NR2B agent CP-101,606 revealed three [³H]ifenprodil affinity states in the absence of trifluoroperazine (see Fig. 2). Trifluoroperazine but not GBR-12909 was able to block all but the high affinity inhibition of the binding by CP-101,606.

These remaining, high affinity, trifluoroperazine-insensitive sites were inhibited by nine additional NR2B agents with a single affinity and a wide range of potency. The structure-activity relationship of these agents highly correlated with published results using cloned NR1a/NR2B receptors expressed in Xenopus oocytes and to the fraction of [³H]TCP binding in rat brain membranes that is bound to NRs containing NR2B subunits (Williams et al., 1993; Coughenour and Cordon, 1997). The lack of correlation between [³H]ifenprodil binding to these sites and [³H]DTG binding to rat brain  sites provided further evidence that trifluoroperazine effectively masked  sites in this assay. These findings also were consistent with those of Nicolas and Carter (1994), which demonstrated that the distribution of the high affinity trifluoroperazine-insensitive [³H]ifenprodil binding sites matched that of NR2B mRNA.

The high affinity trifluoroperazine-insensitive sites had little or no affinity for agents selective for dopamine, serotonin, -adrenergic, and histamine receptors or for the calcium channel blocker flunarizine or the NMDA channel blocker (+)MK-801. Cirazoline, an ₂-adrenergic agonist and imidazoline antagonist, inhibited both the trifluoroperazine-sensitive and -insensitive components of the binding equally with micromolar potency. It is possible that cirazoline has moderate affinity for or allosterically regulates the ifenprodil binding site. It seems less likely that its inhibition reflects the affinity of [³H]ifenprodil for -adrenergic or imidazoline receptors.

The nature of the low affinity, trifluoroperazine-sensitive, ifenprodil sites was difficult to define. These sites had only low to moderate sensitivity to polyamines. DEAP, arcaine, and cirazoline inhibited them at µM concentrations. Both clozapine and clonidine exhibited only high µM affinity. These sites may correspond to the low affinity polyamine sites described by Nicolas and Carter (1994) or include what previous [³H]ifenprodil homogenate studies have described as low affinity piperazine sites (Schoemaker et al., 1990).

The second objective of the study was to characterize the high affinity trifluoroperazine-insensitive [³H]ifenprodil sites with regard to the allosteric pharmacology that is known for NMDA receptors containing NR2B subunits. There is evidence from many studies demonstrating that ifenprodil is inhibited by polyamines, although this inhibition is no longer thought to be competitive as was initially proposed (Scatton et al., 1994). Recent electrophysiological and molecular studies suggest that the binding sites are allosterically linked or overlap (Williams, 1993; Kashiwagi et al., 1996; Gallagher et al., 1998). The rank order of potency among the polyamines we examined was spermine > spermidine > DEAP > arcaine > agmatine > putrescine. This is in agreement with the findings of previous receptor binding studies (Hashimoto et al., 1994; Carter et al., 1997).

Spermine and spermidine were not only the most potent inhibitors within this class but were highly selective for the trifluoroperazine-insensitive sites. Spermidine, 10 and 100 µM, shifted the ifenprodil inhibition curve to the right in a parallel manner. The interactions of the polyamines with trifluoroperazine-insensitive, ifenprodil binding sites confirm previous findings that ifenprodil binding is regulated by polyamines (Schoemaker et al., 1994).

Mg²⁺ selectively inhibits the polyamine-sensitive NMDA-associated fraction of [³H]ifenprodil binding, whereas zinc inhibits [³H]ifenprodil binding to NMDA sites and  sites equally (Hashimoto et al., 1994). In agreement with these studies we found that ZnCl₂ was equipotent regardless of the presence of trifluoroperazine, but MgCl₂ was 3.7-fold more potent if trifluoroperazine was present. In the earlier [³H]ifenprodil studies the Hill slope of Mg²⁺ inhibition was somewhat less than unity, but the Hill slope of Zn²⁺ was 1.8 (Schoemaker et al., 1994). We also found that the Hill slope for Mg²⁺ was <1 regardless of the presence of trifluoroperazine. However, the presence of trifluoroperazine reduced the Hill slope of Zn²⁺ from about 2 to unity. The difference in these results may be due to the block of low affinity binding sites in addition to  sites by trifluoroperazine.

Mg²⁺ not only plays a major role in maintaining a physiological blockade of the NMDA receptor channel, but at lower concentrations it enhances NMDA receptor function (Dingledine et al., 1999). It has been suggested that Mg²⁺ may be the endogenous agonist at the polyamine site on the NMDA receptor. In support of this, electrophysiological studies have shown that the enhancing effects of polyamines and Mg²⁺ are not additive (Kew and Kemp, 1998). In our hands Mg²⁺ (1 mM) shifted the inhibition curve of ifenprodil 5-fold to the right in a similar manner to that of spermidine (10 µM), which shifted the curve 2.3-fold. The addition of 10 µM spermidine to 1 mM MgCl₂ did not cause a further shift of the ifenprodil inhibition curve. This is consistent with the electrophysiological studies and suggested that Mg²⁺ and spermidine are not additive and may be interacting at the same site to decrease the affinity of [³H]ifenprodil.

Allosteric interactions of ifenprodil with the glutamate and glycine recognition sites have been demonstrated (Scatton et al., 1994). Glutamate antagonists decrease and glycine antagonists increase [³H]ifenprodil binding (Carter et al., 1997). No effect of glutamate or glycine was observed at concentrations up to 1 mM, although the agonists reversed the effect of their respective antagonists. In agreement with these studies we found that the glycine antagonist MDL 105,519 increased [³H]ifenprodil binding to trifluoroperazine-insensitive sites and that the glutamate antagonist CPP decreased the binding. These effects were reversed in a concentration-dependent manner by glycine and glutamate, respectively. In addition, we observed a decrease in the binding in the presence of 100 µM glycine and an increase in the binding in the presence of 100 µM glutamate. The decrease caused by CPP and that by glycine appeared additive, as did the increase caused by MDL 105,519 and glutamate, suggesting that they were separate interactions at their respective agonist recognition sites. The effect with glutamate and glycine could be detected in the absence of trifluoroperazine (L. L. Coughenour, personal observation). Possibly, the extensive washing of the membranes allowed the detection of these small but significant and reproducible changes by the agonists as well as the previously reported more robust changes with the antagonists.

Another potentially important regulator of NMDA receptors is proton concentration (Dingledine et al., 1999). Recently, it was reported that the potency of CP-101,606, ifenprodil, and nylidrin to block the NR1a/NR2B receptor is increased at acidic pH (Whittemore et al., 1997; Mott et al., 1998). These data suggest that ifenprodil and related agents may act in some manner to enhance the tonic inhibition of the proton sensor on the NMDA receptor. We found no effect of pH within the range of 6.8 to 8.0 on the potency of ifenprodil, haloperidol, Co 101244, and nylidrin to inhibit [³H]ifenprodil binding. Within this range the buffer pH did not affect the potency of either spermidine or trifluoroperazine to inhibit the binding. These results indicated that the effect of protons to enhance the potency of NR2B subtype-selective agents is not a direct effect of proton concentration on the [³H]ifenprodil binding site.

The results of this study suggested that, in the presence of trifluoroperazine, [³H]ifenprodil binds to a site corresponding to ifenprodil's high affinity voltage-independent site present on NMDA receptors containing NR2B subunits and not to  sites or other receptors and ion channels. These binding sites exhibited several of the allosteric interactions that have been shown for NR2B subunit containing NMDA receptors. This assay should be a simple and useful method for determining the potency of agents at NR2B subunit containing NMDA receptors in rat brain.