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NEUROPHARMACOLOGY

Molecular Mechanisms for Nanomolar Concentrations of Neurosteroids at NR1/NR2B Receptors

Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, Uppsala, Sweden (T.J., P.-A.F., F.N.); and Department of Neuroscience, Unit of Neurobiology, Uppsala University, Uppsala, Sweden (P.L.)

Received August 28, 2007; accepted November 14, 2007.

	Abstract

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Neurosteroids are endogenous steroids acting in the central nervous system. They participate in synaptic plasticity, memory and learning, Alzheimer's disease, and certain drug reward. Some mechanisms behind these effects are thought to be nongenomic, e.g., they modulate the function of the N-methyl-D-aspartate (NMDA) receptor complex. In this study, we used a Chinese hamster ovary cell line stably transfected with NMDA receptor constituents NR1/NR2B, to investigate the effects of nanomolar concentrations of the neurosteroids pregnenolone sulfate (PS) and pregnanolone sulfate (35βS) on binding of the radioligand [³H]ifenprodil. Neither of the steroids displaced [³H]ifenprodil, but both induced a shift in its fit from one to two binding sites. The effects of the neurosteroids were also measured as changes in intracellular calcium ([Ca²⁺]_i) after glutamate stimulation. Although the steroids did not alter the response to glutamate, they influenced the extent of ifenprodil blockade of the receptor: PS increased and 35βS decreased this effect. The coincubation of several NMDA receptor ligands in the assay indicated that PS and 35βS act via different binding sites from those for glutamate, glycine, and dithiothreitol. Combining the two steroids revealed that they do not share a common binding site. In conclusion, these results substantiate previous evidence of the allosteric modulatory effect induced by PS and 35βS on NMDA receptors at nanomolar concentrations. The neurosteroid-mediated modulation of the receptor is also reflected in an altered glutamate stimulated [Ca²⁺]_i, in response to ifenprodil.

Neurosteroids, and sulfated neurosteroids in particular, are present at relatively low concentrations in the rat brain (Higashi et al., 2003; Liu et al., 2003). Nonetheless, when PS is administered at femtomole concentrations by intracerebro-ventricular injection to rodents, processes involving the NMDA receptor are affected (Flood et al., 1995; Mathis et al., 1996; Meziane et al., 1996; Weaver et al., 1997). In contrast, in electrophysiological experiments, much higher (micromolar) concentrations of the steroids are required to achieve modulatory effects on recombinant NMDA receptors (Mukai et al., 2000; Malayev et al., 2002). In these studies, PS acted as a positive allosteric modulator, stimulating the agonist-induced response of NMDA receptor complexes composed of NR1/NR2A or NR1/NR2B subunits, whereas 35βS was inhibitory (Malayev et al., 2002). Similar effects of PS and 35βS at micromolar concentrations were found when Ca²⁺ influx was measured in CHO cells stably expressing the NMDA receptor NR1/NR2B subtype (Mukai et al., 2000).

Ifenprodil, a noncompetitive antagonist of the NMDA receptor that is selective for the NR2B subunit (Chenard and Menniti, 1999; Williams, 2001), is commonly used as a pharmacological tool. We recently published studies demonstrating that sulfated neurosteroids differentially affect the binding of ifenprodil to rat cortical membranes (Johansson and Le Greves, 2005; Johansson et al., 2005a). The altered [³H]ifenprodil binding kinetics, occurring within a narrow, nanomolar range of neurosteroid concentrations, suggests an allosteric modulation of the ifenprodil binding site. In this article, we report our investigation of the functional effects of this type of modulation by measuring Ca²⁺ influx in the CHO cell line expressing the NR1/NR2B receptor (CHO-E2) established by Uchino et al. (2001). We also characterized neurosteroid-mediated changes in [³H]ifenprodil binding to these receptors. Various binding assays, saturation/competition/dissociation studies, and fluorescence studies were used to investigate the allosteric and kinetic effects of nanomolar concentrations of PS and 35βS on [³H]ifenprodil binding to NR1/NR2B receptors.

The results show similar allosteric modulation of [³H]ifenprodil binding to that seen at cortical NMDA receptors (Johansson et al., 2005b). The neurosteroid-induced effect was also confirmed in a functional assay.

	Materials and Methods

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Materials
[³H]Ifenprodil (67 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Ifenprodil, Folin, Ciocalten's Phenol Reagent, p-[dipropylsulfamoyl]benzoic acid (probenecid), L-glutamate, dithiothreitol (DTT), and puromycin were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). PS and 35βS were purchased from Steraloids (Newport, RI). Fura-2 acetomethoxy ester (Fura-2 AM) and glycine were purchased from Fluka (Buchs, Switzerland). G-418 and blasticidin S HCl were purchased from Invitrogen (Lidingö, Sweden). Dulbecco's modified Eagle's medium, trypsin/EDTA, and fetal bovine serum were obtained from Invitrogen (Carlsbad, CA). The CHO-E2 cell line was a generous gift from Dr. S. Uchino (Department of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan) (Uchino et al., 2001). APV was purchased from Tocris Cookson Inc. (Avonmouth, UK). Complete, Mini Protease Inhibitor Cocktail Tablets were purchased from Roche Diagnostics (Mannheim, Germany). Other chemicals were of analytical grade from commercial sources.

Cells
Cell Culturing. CHO-E2 cells were grown in a controlled environment with a humidified atmosphere containing 5% CO₂ at 37°C. The culture medium [Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1.2 mg/ml G-418 (Geneticin), 2 µg/ml blasticidin S HCl, and 10 µg/ml puromycin] was changed every sixth day. The cells were grown to approximately 80% confluence, and then they were split 1:3, generally every sixth day, using trypsin/EDTA (0.05 and 0.02%, respectively), before transferring them to fresh 100-mm culture dishes (Sarstedts, Nürnbrecht-Rommelsdorf, Germany).

Heat Induction. In the CHO-E2 cell line, the expression of functional NR1/NR2B receptors is under the control of the Drosophila 70-kDa heat shock protein promoter (Uchino et al., 2001). To induce expression, culture dishes containing 1.5 x 10⁵ cells cm^-2 were incubated for 2 h at 43°C in a controlled, humidified environment containing 5% CO₂, and then they were maintained at 37°C for 6 h in culture medium supplemented with 1 mM DL-2-amino-5-phosphonopentaoic acid (APV).

Receptor Binding
Cell Membrane Preparation for Receptor Binding. The cells were harvested after brief incubation in trypsin/EDTA at 37°C and centrifuged at 1000g for 5 min. The supernatant was decanted, and the pellet was dissolved in homogenizing buffer (50 mM Tris/HCl, pH 7.4). Centrifugation was performed three times, and the final pellet was dissolved in a small amount of the same buffer and then homogenized in a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). More homogenizing buffer was added to the homogenate, which was then centrifuged at 35,000g for 15 min. The pellet was dissolved in freezing buffer (10 mM Tris/HCl, pH 7.4, and 10% sucrose), and the well washed membranes were stored at -80°C. The protein content was measured by the method described by Lowry et al. (1951). The membrane preparation was also conducted in the presence of an enzyme inhibitor cocktail (Complete, Mini; Roche Diagnostics, Mannheim, Germany), to evaluate receptor degradation during the homogenization procedure. Because there were no significant differences in total [³H]ifenprodil specific binding, enzyme inhibitors were subsequently excluded.

[³H]Ifenprodil Binding Assay. Triplicate incubations were carried out in a total volume of 250 µl in 96-well Vee bottom microtiter plates (Sarstedts, Stockholm, Sweden) for 30 min (45 min in the saturation experiments) at 37°C (water bath), to establish equilibrium. The assays were carried out in the presence of 6.0 nM [³H]ifenprodil (except for the saturation experiments; see below) in balanced salt solution (BSS: 130 mM NaCl, 5.4 mM KCl, 2.0 mM CaCl₂, 5.5 mM glucose, and 10 mM HEPES, pH 7.3), supplemented with 50 µg of protein, 1.0 mM trifluoroperazine (to block [³H]ifenprodil binding to non-NMDA receptors), 100 µM glutamate, and 10 µM glycine. PS and 35βS were dissolved in BSS. All experiments were carried out in the absence or presence of PS or 35βS. Nonspecific binding was determined in the presence of 100 µM unlabeled ifenprodil. To make sure that no sulfatases were active, the assays were first conducted in the presence of the sulfatase inhibitor estradiol (100 µM) (Santner and Santen, 1993), but because this made no difference to the outcome, estradiol was excluded from the final assay. The assays were performed in a robot workstation (Biomek 2000; Beckman Coulter, Fullerton, CA). Bound radioligand was rapidly separated from unbound ligand by filtration (96-well harvester; Tomtec, Hamden, CT) through Filtermat B glass fiber filters (PerkinElmer Wallac, Turku, Finland), under reduced pressure. The filters were rinsed four times with 3 ml of cold homogenizing buffer. The filters were vacuum dried slightly, before being removed and left to finish drying overnight at room temperature. Solid MeltiLex B/HS (PerkinElmer Wallac) was melted onto the dried filters, and the scintillation was then measured in a beta-counter (Microbeta TriLux; PerkinElmer Wallac).

Saturation Studies. Saturation isotherms were created from three independent series of [³H]ifenprodil saturation binding experiments (using eight concentrations between 0.01 and 40 nM). The results were analyzed using nonlinear curve fitting and compared for the best fit (one or two binding sites), using the equations for one binding site:

(1)

and for two binding sites:

(2)

where B_max is the maximum density of NR1/NR2B receptors, and K_d is the dissociation constant of [³H]ifenprodil at the NR1/NR2B receptor.

Competition Studies. The displacement studies of [³H]ifenprodil were performed with different concentrations of unlabeled ifenprodil (10 pM–1 µM). The results were analyzed using nonlinear curve fitting and compared for the best fit (one or two binding sites), using the equations for one binding site:

(3)

and for two binding sites:

(4)

where B_nsb is nonspecifically bound [³H]ifenprodil, B_sb is specifically bound [³H]ifenprodil, IC₅₀ is the concentration of unlabeled ifenprodil that causes 50% inhibition at the binding site, and f is the fraction of binding sites (first and second sites refer to binding sites with high and low affinity for [³H]ifenprodil). K_i values were calculated using the Cheng-Prusoff equation (Cheng and Prusoff, 1973):

(5)

Dissociation Studies. The dissociation of [³H]ifenprodil from the NMDA receptor was studied at a fixed concentration of 6.0 nM [³H]ifenprodil. The experiment began at equilibrium, reached after 30 min, and dissociation was initiated by the addition of excess unlabeled ifenprodil (1.0 mM, final concentration). The results were analyzed using nonlinear curve fitting and compared for the best fit (one or two binding sites), using the equations for one binding site:

(6)

and for two binding sites:

(7)

where B_t is the specific binding at time t, B_eq is the specific binding at equilibrium, B_{t 2nd site} is the plateau between the first and second binding sites, and K_{off 1} and K_{off 2} are the dissociation rate constants.

Interaction Studies. To determine whether the two neurosteroids share the same site, two interaction experiments were conducted. First, we tested the effects of PS (0.1–10 nM) on total specific [³H]ifenprodil binding in the presence of 1 or 10 nM 35βS. The reversed situation was also tested: 35βS (1–100 nM) in the presence of 1 or 10 nM PS. To test whether the modulatory sites for the two neurosteroids at nanomolar concentrations are shared with other NMDA receptor ligands, experiments on total specific [³H]ifenprodil (6.0 nM) binding in the presence of PS or 35βS and one on glutamate (100 µM), the glutamate antagonist APV (1 mM), glycine (10 µM), or the reducing (redox site ligand) DTT (4 nM) were carried out.

Calcium Measurements
Cell Preparation for [Ca²⁺]_i Measurements. The cells were harvested after brief incubation in trypsin/EDTA at 37°C, and then they were centrifuged at 300g for 5 min. The supernatant was decanted, and the pellet was dissolved in BSS. Centrifugation was performed three times, and the pellet was then dissolved in a small amount of BSS.

Loading of Fluorescence. Heat-activated cells were gently centrifuged (300g; 3 min); resuspended in 100 µl of BSS supplemented with 5 µM Fura-2 AM, 0.001% cremophore EL, and 1 mM probenecid; and incubated for 45 min. Extracellular Fura-2 AM was removed by dilution, gentle centrifugation, and decantation, and the cells were resuspended in fresh BSS (50,000 cells in 100 µl), now supplemented with 100 µM trifluoroperazine, and then distributed in 96-well black, round-bottomed plates (NUNC A/S, Copenhagen, Denmark) for immediate [Ca²⁺]_i measurement.

[Ca²⁺]_i Measurement. [Ca²⁺]_i measurements were run in a fluorescence plate reader (POLARstar; BMG, Offenburg, Germany) at 37°C. The cells were illuminated alternately at 340 and 380 nm. The emitted signals were recorded at 510 nm. The quotient of the emission generated from 380 nm divided by the emission generated from 340 nm was calculated. Baseline measurements were established, and a mixture of glutamate and glycine (final concentrations of 100 and 10 µM, respectively, dissolved in BSS) was added to the well by a pump (100 ml/min^-1). The cells were preincubated, initially with the steroids and then with other substances, before measurement. The baseline was established, and the glutamate and glycine solution was added. The peak signal was monitored for 45 s per well. The peak counts were compared with the maximum stimulation level, and they were then translated into calcium ion concentrations. Each experiment was performed four times in quadruplicates.

Data Analysis. The data were assembled and analyzed using nonlinear regression in Microsoft Excel (Microsoft, Redmond, WA) and Prism 4.0 (GraphPad Software Inc., San Diego, CA). Where appropriate, one-way analysis of variance (ANOVA) or partial F-test was used for data analysis, considering P < 0.05 as significant level. ANOVA was followed by Dunnett's multiple comparisons post-hoc test.

	Results

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[³H]Ifenprodil Binding and Effects of Ifenprodil on [Ca²⁺]_i in CHO-E2 Cells. To be able to compare receptor binding data for the CHO-E2 cell line to those obtained from the functional Ca²⁺ influx assay, it was necessary to optimize the receptor binding assay at 37°C in the presence of glutamate, because ifenprodil displays temperature-(Hashimoto and London, 1993) and glutamate-dependent receptor binding kinetics to the NMDA receptor (Kew et al., 1996). The results showed that the double amount of protein and a 10-fold concentration of radioligand were required to achieve a total binding similar to that seen at 25°C. In accordance, the concentration of trifluoroperazine [used to block other [³H]ifenprodil targets, including ₁ receptors, the piperazine acceptor site (Coughenour and Barr, 2001), and ₁ receptors (U'Prichard et al., 1977)] was also used at 10 times higher concentration compared with assays performed at 25°C. The addition of 1 mM trifluoroperazine neither affected the [³H]ifenprodil specific binding to the NR2B site, nor its affinity (Fig. 1A). The binding to nonheat-activated cells was low (Fig. 1B). Binding was concentration-dependent and saturable (Fig. 2A; Table 1); the data fitted a monophasic curve. The displacement of [³H]ifenprodil by unlabeled ifenprodil fitted a one-site model best (Fig. 2B; Table 2). The dissociation of [³H]ifenprodil from the membranes was significantly better described by a single exponential decay function for [³H]ifenprodil than by a double exponential decay function (P < 0.01) (Fig. 2C; Table 3). Neither of the steroids displaced [³H]ifenprodil from the membranes (data not shown). Glutamate-induced elevation of [Ca²⁺]_i was dose-dependently inhibited by the presence of ifenprodil (Fig. 2D; Table 4), yielding a one-site inhibition curve.

View larger version (11K): [in this window] [in a new window]   Fig. 1. [3H]Ifenprodil binding to membranes of CHO-E2 cells extracted from saturation experiments. A, Bmax and Kd in the presence (white) or absence (black) of trifluoroperazine on heat-activated cells. B, Bmax in heat-activated cells (white) and nonheat-activated cells (gray). The binding of [3H]ifenprodil was performed as described under Materials and Methods.

View larger version (13K): [in this window] [in a new window]   Fig. 2. A, saturation isotherms of specific [3H]ifenprodil binding. B, displacement of [3H]ifenprodil by unlabeled ifenprodil. C, dissociation of [3H]ifenprodil induced by unlabeled ifenprodil. D, inhibition of glutamate-stimulated [Ca2+]i elevation by ifenprodil. The data comprise means ± S.E.M. (n = 3 or 4) for the fit to data from the individual experiments. The binding of [3H]ifenprodil to membranes of and [Ca2+]i measurements on heat-activated CHO-E2 cells were performed as described under Materials and Methods.

Effects of PS on [³H]Ifenprodil Binding and [Ca²⁺]_i. The influence of PS was significantly greater between 0.1 and 100 nM than at other concentrations. The activity curve was bell-shaped and seemed to peak at 1 nM (Fig. 3). The presence of 0.1 to 10 nM PS exposed another binding site for [³H]ifenprodil with different characteristics. This effect was seen in saturation (Fig. 4A; Table 1), competition (Fig. 4B; Table 2), and dissociation (Fig. 4C; Table 3) experiments. Contributory evidence was also provided by the ability of PS to modulate the low-affinity site in this concentration range, by increasing the affinity for [³H]ifenprodil, as shown in saturation and dissociation experiments. These results are in line with our previous experiments conducted on rat frontal cortex membranes (Johansson et al., 2005a).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Total specific binding of 6.0 nM [3H]ifenprodil (displayed as 100%; ) to membranes of heat-activated CHO-E2 cells and the impact of different concentrations of PS () or 35βS (). The data are means ± S.E.M. (n = 4) to data from the individual experiments. The significance of the deviation from 100% was calculated for each mean value; *, P < 0.05; **, P < 0.01 (t test). The binding of [3H]ifenprodil was performed as described under Materials and Methods.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Influence of 10 pM PS (), 0.1 nM PS (x), 1 n MPS (*), 10 nM PS (+), and 100 nM PS (). Control (), absence of PS. A, saturation isotherms of specific [3H]ifenprodil binding in the absence or presence of PS, clearly displaying the effect of 0.1 to 10 nM PS. B, displacement of [3H]ifenprodil by unlabeled ifenprodil in the absence or presence of PS. C, dissociation of [3H]ifenprodil induced by unlabeled ifenprodil in the absence or presence of PS. D, inhibition of glutamate-stimulated [Ca2+]i elevation by ifenprodil in the absence or presence of PS. The data are means ± S.E.M. (n = 3 or 4) for the fit to data from the individual experiments. The binding of [3H]ifenprodil to membranes of and [Ca2+]i measurements on heat-activated CHO-E2 cells were performed as described under Materials and Methods.

Effects of 35βS on [³H]Ifenprodil Binding and [Ca²⁺]_i. The influence of 35βS was significantly greater at concentrations of 1 to 100 nM than at other concentrations. The activity curve of 35βS was U-shaped, reaching its nadir at 10 nM (Fig. 3). Between 1 and 100 nM, 35βS induced changes to [³H]ifenprodil binding characteristics (Fig. 5B; Table 2). In the [Ca²⁺]_i experiments (Fig. 5D; Table 4), 35βS significantly decreased ifenprodil inhibition at these same concentrations. Like PS, 35βS exposed another binding site for [³H]ifenprodil, but, in contrast to PS, it decreased the affinity of [³H]ifenprodil for the low-affinity site, as shown in saturation (Fig. 5A; Table 1) and dissociation (Fig. 5C; Table 3) experiments. Moreover, the affinity of [³H]ifenprodil for the high-affinity binding site was lower with 35βS than with PS. 35βS also changed the one-site inhibition slope into a two-site slope at 10 nM concentration (Table 5).

View larger version (20K): [in this window] [in a new window]   Fig. 5. Influence of 0.1 nM 35βS (), 1 nM 35βS (), 10 nM 35βS (), 100 nM 35βS (), and 1 µM 35βS (). Control (), absence of 35βS. A, saturation isotherms of specific [3H]ifenprodil binding to membranes of heat-activated CHO-E2 cells in the absence or presence of 35βS, clearly displaying the effect of 1 to 100 nM 35βS. B, displacement of [3H]ifenprodil by unlabeled ifenprodil from membranes of heat-activated CHO-E2 cells in the absence or presence of 35βS. C, dissociation of [3H]ifenprodil induced by unlabeled ifenprodil from membranes of heat-activated CHO-E2 cells in the absence or presence of 35βS. D, inhibition of glutamate-stimulated [Ca2+]i elevation by ifenprodil in the absence or presence of 35βS. The data are means ± S.E.M. (n = 3 or 4) for the fit to data from the individual experiments. The binding of [3H]ifenprodil to membranes of and [Ca2+]i measurements on heat-activated CHO-E2 cells were performed as described under Materials and Methods.

Influence of Other NMDA Receptor Ligands on the Effects of PS and 35βS on [³H]Ifenprodil Binding and [Ca²⁺]_i.

Interaction Experiments between PS and 35βS on the Effect on [³H]Ifenprodil Binding. PS (Fig. 6A) and 35βS (Fig. 6B) show great structural similarities. If the two neurosteroids share a common binding site, competition between the steroids would have resulted in a parallel shift in dose-effect curves on [³H]ifenprodil total binding. Because this was not found, it was concluded that PS and 35βS have separate binding targets on the NMDA receptor (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 6. Structures of the neurosteroids PS (A) and 35βS (B).

	Discussion

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To characterize the [³H]ifenprodil binding specificity to the NR1/NR2B receptors on the CHO-E2 cell line, we studied the binding on heat- and nonheat-activated cells. [³H]Ifenprodil displayed no or very low binding to nonheat-activated cells, indicating that its affinity to activated cells almost entirely was to NR1/NR2B receptors (Fig. 1B). Further strengthening the absence of [³H]ifenprodil binding to non-NMDA receptors in heat-activated cells was the low binding at 37°C, a temperature known to favor ₁ receptor binding (Hashimoto and London, 1993) and the finding that trifluoroperazine did not lower B_max for radio ligand (Fig. 1A). However, we included trifluoroperazine in the assay to emulate the conditions as in the studies on cortical membranes (Johansson and Le Greves, 2005; Johansson et al., 2005a). Other workers have also suggested that [³H]ifenprodil binds to two different (low- and high-affinity) sites on the NMDA receptor (Nicolas and Carter, 1994). The low-affinity binding site on the NMDA receptor was discriminated against by the use of low concentrations of [³H]ifenprodil in the competition and dissociation measurements.

The neurosteroids themselves did not displace [³H]ifenprodil, nor did they alter glutamate stimulated increase in [Ca²⁺]_i. The kinetics for the radioligand was only affected by their continual presence, indicating an allosteric modulation of the binding site. An allosteric interaction is best studied and evaluated by examining the pattern of radioligand dissociation from the receptor (Kostenis and Mohr, 1996; Limbird, 2004). Because the neurosteroids changed the dissociation pattern of [³H]ifenprodil without displacing it, an allosteric interaction with the NMDA receptor can be assumed. This is further supported by our results for the Hills coefficient (n_H). An n_H value different from -1 suggests the presence of another binding site, and, in our competitive experiments, PS (0.1–10 nM) and 35βS (1–100 nM) both affected the competition curves to present n_H values significantly different from -1. The saturation experiments in the continual presence of the neurosteroids also suggested the presence of second binding sites. The results from the experiments on total specific binding, performed at a nonsaturated concentrations of [³H]ifenprodil (below K_d), clearly illustrate the differential modulation generated by PS and 35βS (Fig. 3). Taken together, the results from the binding experiments on NR1/NR2B-expressing CHO cells show great similarities with those obtained on rat cortical membranes (Johansson et al., 2005b), indicating that the NMDA receptor studied in the two investigations are of equal subunit composition.

In rat cortical membranes, we previously demonstrated that the positive allosteric effect of PS on [³H]ifenprodil binding was decreased in the presence of glutamate (Johansson et al., 2005b). This effect, observed at 30 nM PS, was proposed to originate from an interaction between the PS site and the site of action for glutamate. In CHO-E2 cells, decreased modulatory effect in the presence of glutamate was seen at all three PS concentrations inducing a two-site fit binding curve. The negative modulatory effect of 35βS on [³H]ifenprodil binding was, as in rat cortical membranes, not sensitive to glutamate, supporting the notion that PS and 35βS act at separate sites on the NR1/NR2B receptor. This is further strengthened by the fact that the two neurosteroids did not interact competitively on one another's modulatory effect curves. The addition of ligands for some other known targets on the NMDA receptor (redox, APV, and glycine sites) did not interfere with the effects of the neurosteroids.

In addition, in the functional calcium-influx assay, data suggest an allosteric modulation induced by the steroids. PS changed the dose-response curve for ifenprodil inhibition of glutamate-stimulated [Ca²⁺]_i from an one- to a two-site fit at all three concentrations altering the binding characteristics for [³H]ifenprodil. For 35βS, this effect was seen only at 10 nM. Moreover, and in line with the receptor binding results, PS potentiated whereas 35βS decreased ifenprodil inhibition of glutamate-activated calcium influx. It seems likely that the effects of the steroids seen in the functional assay reflect those observed in the receptor binding studies.

Ifenprodil acts via the proton sensor site on the NMDA receptor, enhancing the negative modulation of receptor activity by protons (Mott et al., 1998), an effect that can be counteracted by spermine (Traynelis et al., 1995). Based on studies of chimeric NMDA receptor subunits, Jang et al. (2004) identified a steroid modulatory domain on the NR2B subunit. The 78-amino acid segment was shown to be important for the action of PS, spermine, and protons but not for ifenprodil. The interaction between PS (at nanomolar concentrations) and ifenprodil as seen in our experiments may thus involve other site(s) of action for PS compared with those studied at much higher concentrations. These targets can represent high- and low-affinity sites for some neurosteroids where the former sites may mediate in vivo effects seen after administration of very low amounts of these compounds (Flood et al., 1995; Mathis et al., 1996; Meziane et al., 1996; Weaver et al., 1997). We propose that PS and 35βS at physiological concentration can change ifenprodil binding by converting the receptor site from a one-site state into a two-site state. This will subsequently affect proton sensitivity and the ion channel opening.

Taken together, these findings support our previous results that neurosteroids allosterically induce new [³H]ifenprodil binding characteristics, including altered affinity for the NR1/NR2B NMDA receptor. We have shown that neurosteroids at nanomolar concentrations act via binding sites separate from several known targets on the NMDA receptor, suggesting the existence of unique targets for the steroids. The results are also supported by the altered ifenprodil inhibitory effect on glutamate stimulated [Ca²⁺]_i. In the continuation, the PS-induced positive allosteric modulation of [³H]ifenprodil binding to the NMDA receptor may favor the neuroprotective properties of ifenprodil. By using PS as an adjunct to ifenprodil, the dose may be lowered to levels where side effects are absent or clinically tolerated but still effective at the NMDA receptor.

	Acknowledgements

 
We thank Dr. Shigeo Uchino for generous support in providing the CHO-E2 cell line.

	Footnotes

 
This study was funded by the Swedish Medical Research Council Grant 9459 and Gunvor och Josef Anérs stiftelse.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.130518.

ABBREVIATIONS: NMDA, N-methyl-D-aspartate; NR, N-methyl-D-aspartate receptor; PS, pregnenolone sulfate (5-pregnen-3β-ol-20-one sulfate); 35βS, pregnanolone sulfate (5β-pregnan-3-ol-20-one sulfate); CHO, Chinese hamster ovary; APV, DL-2-amino-5-phosphonopentaoic acid; BSS, balanced salt solution; DTT, dithiothreitol; Fura-2 AM, Fura-2 acetomethoxy ester; ANOVA, analysis of variance.

Address correspondence to: Dr. Tobias Johansson, Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, P.O. Box 591, S-751 24, Uppsala, Sweden. E-mail: tobias.johansson{at}farmbio.uu.se

	References

Top Abstract Materials and Methods Results Discussion References

 

Chenard BL and Menniti FS (1999) Antagonists selective for NMDA receptors containing the NR2B subunit. Curr Pharm Des 5: 381-404.[Medline]

Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22: 3099-3108.[CrossRef][Medline]

Compagnone NA and Mellon SH (2000) Neurosteroids: biosynthesis and function of these novel neuromodulators. Front Neuroendocrinol 21: 1-56.[CrossRef][Medline]

Coughenour LL and Barr BM (2001) Use of trifluoroperazine isolates a [(3)H]Ifenprodil binding site in rat brain membranes with the pharmacology of the voltage-independent ifenprodil site on N-methyl-D-aspartate receptors containing NR2B subunits. J Pharmacol Exp Ther 296: 150-159.[Abstract/Free Full Text]

Flood JF, Morley JE, and Roberts E (1995) Pregnenolone sulfate enhances posttraining memory processes when injected in very low doses into limbic system structures: the amygdala is by far the most sensitive. Proc Natl Acad Sci U S A 92: 10806-10810.[Abstract/Free Full Text]

Hashimoto K and London ED (1993) Further characterization of [3H]ifenprodil binding to sigma receptors in rat brain. Eur J Pharmacol 236: 159-163.[CrossRef][Medline]

Higashi T, Sugitani H, Yagi T, and Shimada K (2003) Studies on neurosteroids XVI. Levels of pregnenolone sulfate in rat brains determined by enzyme-linked immunosorbent assay not requiring solvolysis. Biol Pharm Bull 26: 709-711.[CrossRef][Medline]

Horak M, Vlcek K, Petrovic M, Chodounska H, and Vyklicky L Jr (2004) Molecular mechanism of pregnenolone sulfate action at NR1/NR2B receptors. J Neurosci 24: 10318-10325.[Abstract/Free Full Text]

Jang MK, Mierke DF, Russek SJ, and Farb DH (2004) A steroid modulatory domain on NR2B controls N-methyl-D-aspartate receptor proton sensitivity. Proc Natl Acad Sci U S A 101: 8198-8203.[Abstract/Free Full Text]

Johansson T and Le Greves P (2005) The effect of dehydroepiandrosterone sulfate and allopregnanolone sulfate on the binding of [(3)H]ifenprodil to the N-methyl-D-aspartate receptor in rat frontal cortex membrane. J Steroid Biochem Mol Biol 94: 263-266.[CrossRef][Medline]

Johansson T, Frandberg PA, Nyberg F, and Le Greves P (2005a) Low concentrations of neuroactive steroids alter kinetics of [3H]ifenprodil binding to the NMDA receptor in rat frontal cortex. Br J Pharmacol 146: 894-902.[CrossRef][Medline]

Johansson T, Frandberg PA, Nyberg F, and Le Greves P (2005b) Low concentrations of neuroactive steroids alter kinetics of [(3)H]ifenprodil binding to the NMDA receptor in rat frontal cortex. Br J Pharmacol 146: 894-902.[CrossRef][Medline]

Kew JN, Trube G, and Kemp JA (1996) A novel mechanism of activity-dependent NMDA receptor antagonism describes the effect of ifenprodil in rat cultured cortical neurones. J Physiol 497: 761-772.[Abstract/Free Full Text]

Klangkalya B (2005) Modulation of brain synaptic plasticity by steroid hormones. J Med Assoc Thai 88 (Suppl 3): S354-S362.[Medline]

Kostenis E and Mohr K (1996) Two-point kinetic experiments to quantify allosteric effects on radioligand dissociation. Trends Pharmacol Sci 17: 280-283.[CrossRef][Medline]

Limbird L (2004) Cell Surface Receptors a Short Course on Theory and Methods, Springer-Verlag New York Inc., New York.

Liu S, Sjovall J, and Griffiths WJ (2003) Neurosteroids in rat brain: extraction, isolation, and analysis by nanoscale liquid chromatography-electrospray mass spectrometry. Anal Chem 75: 5835-5846.[Medline]

Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275.[Free Full Text]

Malayev A, Gibbs TT, and Farb DH (2002) Inhibition of the NMDA response by pregnenolone sulphate reveals subtype selective modulation of NMDA receptors by sulphated steroids. Br J Pharmacol 135: 901-909.[CrossRef][Medline]

Mathis C, Vogel E, Cagniard B, Criscuolo F, and Ungerer A (1996) The neurosteroid pregnenolone sulfate blocks deficits induced by a competitive NMDA antagonist in active avoidance and lever-press learning tasks in mice. Neuropharmacology 35: 1057-1064.[CrossRef][Medline]

Meziane H, Mathis C, Paul SM, and Ungerer A (1996) The neurosteroid pregnenolone sulfate reduces learning deficits induced by scopolamine and has promnestic effects in mice performing an appetitive learning task. Psychopharmacology (Berl) 126: 323-330.[CrossRef][Medline]

Mott DD, Doherty JJ, Zhang S, Washburn MS, Fendley MJ, Lyuboslavsky P, Traynelis SF, and Dingledine R (1998) Phenylethanolamines inhibit NMDA receptors by enhancing proton inhibition. Nat Neurosci 1: 659-667.[CrossRef][Medline]

Mukai H, Uchino S, and Kawato S (2000) Effects of neurosteroids on Ca²⁺ signaling mediated by recombinant N-methyl-D-aspartate receptor expressed in Chinese hamster ovary cells. Neurosci Lett 282: 93-96.[CrossRef][Medline]

Nicolas C and Carter C (1994) Autoradiographic distribution and characteristics of high- and low-affinity polyamine-sensitive [3H]ifenprodil sites in the rat brain: possible relationship to NMDAR2B receptors and calmodulin. J Neurochem 63: 2248-2258.[Medline]

Park-Chung M, Wu FS, Purdy RH, Malayev AA, Gibbs TT, and Farb DH (1997) Distinct sites for inverse modulation of N-methyl-D-aspartate receptors by sulfated steroids. Mol Pharmacol 52: 1113-1123.[Abstract/Free Full Text]

Santner SJ and Santen RJ (1993) Inhibition of estrone sulfatase and 17 beta-hydroxysteroid dehydrogenase by antiestrogens. J Steroid Biochem Mol Biol 45: 383-390.[CrossRef][Medline]

Shimizu E, Tang YP, Rampon C, and Tsien JZ (2000) NMDA receptor-dependent synaptic reinforcement as a crucial process for memory consolidation. Science 290: 1170-1174.[Abstract/Free Full Text]

Traynelis SF, Hartley M, and Heinemann SF (1995) Control of proton sensitivity of the NMDA receptor by RNA splicing and polyamines. Science 268: 873-876.[Abstract/Free Full Text]

U'Prichard DC, Greenberg DA, and Snyder SH (1977) Binding characteristics of a radiolabeled agonist and antagonist at central nervous system alpha noradrenergic receptors. Mol Pharmacol 13: 454-473.[Abstract/Free Full Text]

Uchino S, Watanabe W, Nakamura T, Shuto S, Kazuta Y, Matsuda A, Nakajima-Iijima S, Kudo Y, Kohsaka S, and Mishina M (2001) Establishment of CHO cell lines expressing four N-methyl-D-aspartate receptor subtypes and characterization of a novel antagonist PPDC. FEBS Lett 506: 117-122.[CrossRef][Medline]

Weaver CE Jr, Marek P, Park-Chung M, Tam SW, and Farb DH (1997) Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-D-aspartate receptor. Proc Natl Acad Sci U S A 94: 10450-10454.[Abstract/Free Full Text]

Weill-Engerer S, David JP, Sazdovitch V, Liere P, Eychenne B, Pianos A, Schumacher M, Delacourte A, Baulieu EE, and Akwa Y (2002) Neurosteroid quantification in human brain regions: comparison between Alzheimer's and nondemented patients. J Clin Endocrinol Metab 87: 5138-5143.[Abstract/Free Full Text]

Williams K (2001) Ifenprodil, a novel NMDA receptor antagonist: site and mechanism of action. Curr Drug Targets 2: 285-298.[CrossRef][Medline]

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