QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.062794v1 309/3/1003    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Christensen, J. K. Articles by Nielsen, E. O. Search for Related Content PubMed PubMed Citation Articles by Christensen, J. K. Articles by Nielsen, E. O.

NEUROPHARMACOLOGY

In Vitro Characterization of 5-Carboxyl-2,4-di-benzamidobenzoic Acid (NS3763), a Noncompetitive Antagonist of GLU_K5 Receptors

NeuroSearch A/S, Ballerup, Denmark

Received November 10, 2003; accepted February 24, 2004.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Accumulating preclinical data suggest that compounds that block the excitatory effect of glutamate on the kainate subtype of glutamate receptors may have utility for the treatment of pain, migraine, and epilepsy. In the present study, the in vitro pharmacological properties of the novel glutamate antagonist 5-carboxyl-2,4-di-benzamido-benzoic acid (NS3763) are described. In functional assays in human embryonic kidney (HEK)293 cells expressing homomeric GLU_K5 or GLU_K6 receptors, NS3763 is shown to display selectivity for inhibition of domoate-induced increase in intracellular calcium mediated through the GLU_K5 subtype (IC₅₀ = 1.6 µM) of kainate receptors compared with the GLU_K6 subtype (IC₅₀ > 30 µM). NS3763 inhibits the GLU_K5-mediated response in a noncompetitive manner and does not inhibit [³H]-amino-3-hydroxy-5-tertbutylisoxazole-4-propionic acid binding to GLU_K5 receptors. Furthermore, NS3763 selectively inhibits L-glutamate- and domoate-evoked currents through GLU_K5 receptors in HEK293 cells and does not significantly inhibit -amino-3-hydroxy-5-methylisoxazole-4-propionic acid- or N-methyl-D-aspartate-induced currents in cultured mouse cortical neurons at 30 µM. This is the first report on a selective and noncompetitive GLU_K5 antagonist.

Kainate receptors, as AMPA and NMDA receptors, are thought to be tetramers (for review, see Madden, 2002) formed by homo- or heteromeric association of the kainate receptor subunits GLU_K1, GLU_K2, GLU_K5, GLU_K6, and GLU_K7.

The first compound to be described as a competitive kainate antagonist was NS102, based on its ability to block low-affinity [³H]kainate binding (Johansen et al., 1993). However, functional assays yielded contradictory results because NS102 acts at GLU_K5 and GLU_K6 receptors and shows selectivity in some systems (Verdoorn et al., 1994; Wilding and Huettner, 1996) but poor selectivity in others (Paternain et al., 1996). Recently, LY382884 has been reported to bind specifically to GLU_K5 but not to GLU_K6, GLU_K7, GLU_K2, or AMPA receptor subunits (Bortolotto et al., 1999). In functional tests, LY382884 inhibits kainate-evoked currents in dorsal root ganglion neurons and is approximately 100 times less potent on AMPA- and NMDA-evoked responses in hippocampal neurons (Bleakman et al., 2002).

Kainate receptors are believed to have diverse roles under both physiological and pathological conditions, and the novel pharmacological agents have enabled insights into the involvement of GLU_K5 receptors in synaptic transmission and plasticity. In the processing of nociceptive information, kainate receptors are involved at several sites, including primary afferent fibers, superficial dorsal horn neurons, and intrinsic spinal horn neurons (Ruscheweyh and Sandkühler, 2002). Several studies have implicated kainate receptors (specifically, the GLU_K5 subtype) in pain transmission (Procter et al., 1998; Li et al., 1999), and Simmons et al. (1998) demonstrated that the selective GLU_K5 antagonist LY382884 was active in an animal model of persistent pain. More recently, GLU_K5 receptors have been linked with migraine headache, and competitive GLU_K5 antagonists have been reported to be active in animal models of acute migraine (Filla et al., 2002). Within the hippocampus, GLU_K5-containing receptors are involved in frequency facilitation and induction of long-term potentiation, and in excitatory drives of inhibitory CA1 interneurons. In addition, GLU_K5 antagonists have recently been reported to have anticonvulsant activity in animal models (Smolders et al., 2002).

Existing GLU_K5 antagonists show no overt behavioral side effect at doses where the beneficial effects are observed in animal models (Simmons et al., 1998; Smolders et al., 2002).

In summary, these data suggest that the GLU_K5 subtype of kainate receptors can be used as a target for the development of selective antagonists, which may provide a valuable approach for the future treatment of pain, migraine, and epilepsy.

In the present work, we report the in vitro pharmacology of a novel noncompetitive GLU_K5 antagonist. In contrast to the competitive antagonist NS1209, which inhibits AMPA-induced responses in cortical neurons (Nielsen et al., 1999) and kainate-evoked responses in cells expressing GLU_K5 receptors equipotently (Varming et al., 2001), NS3763 did not show significant antagonistic properties on either native AMPA or native NMDA receptors.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials and Drugs
[³H]Kainic acid (58 Ci/mmol) and [³H]ATPA (16 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA) and Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK), respectively. Fluo-4-AM (cell-permeant acetoxymethyl ester of the Ca²⁺ indicator Fluo-4) and domoic acid was purchased from Molecular Probes Europe BV (Leiden, The Netherlands) and Tocris Cookson Inc. (Bristol, UK), respectively. GYKI52466 was purchased from Sigma/RBI (Natrick, MA). NS1209 (previously known as SPD502; Nielsen et al., 1999; Varming et al., 2001) was synthesized at NeuroSearch A/S. NS3763 was identified in a compound library purchased from Chemical Diversity Labs (San Diego, CA).

Cell culture media were obtained from Invitrogen (Roskilde, Denmark). All other chemicals were purchased from regular commercial sources and were of the purest grade available.

Cell Cultures
GLU_K5- and GLU_K6-Expressing Cell Lines. HEK293 cell lines stably expressing homomeric human GLU_K5Q-1a and GLU_K6IYQ were established as described previously (Varming et al., 2001).

The cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum, in polystyrene culture flasks (175 cm²), in a humidified atmosphere of 5% CO₂, 95% air, at 37°C. Cells were cultured to 80 to 90% confluency before plating. The cells were rinsed with phosphate-buffered saline and detached from the culture flask by trypsin [0.1% (w/v)] digestion, for 5 min at 37°C. After addition of growth media, cells were resuspended by trituration and seeded at a density of 0.05 to 0.1 million cells/well in black-walled, clear-bottom 96-well plates pretreated with 0.001% (w/v) polyethyleneimine solution (75 µl/well for 30 min). Plated cells were allowed to proliferate for 24 h before loading with dye.

For experiments with the human GLU_K5Q-2b splice variant, HEK293 cells were transiently transfected using the LipofectAMINE Plus (Invitrogen) transfection kit as described by the manufacturer. Cells were used the day after transfection.

For electrophysiological studies, cells were seeded on the day of experiment. Glass coverslips (3.5 mm), precoated with poly-L-ornithine [0.005% (w/v)] and laminine [0.002% (w/v)] were placed in Petri dishes, and 2.5 ml of cell suspension (0.1 million cells/ml) was added.

Primary Cortical Neuronal Cultures. The cultures were prepared from NMRI mice (Taconic M and B, Ry, Denmark) at day 15 to 16 of gestation as described previously (Drejer et al., 1987). Briefly, dissected tissue was chopped into 0.4-mm cubes and triturated with 0.2% (w/v) trypsin and DNase (40 µg/ml), for 10 min at 37°C. The cells were suspended (1.5 million/ml) in a slightly modified DMEM (with 23 mM glucose), which contained 10% (v/v) horse serum, 7 µM p-amino benzoate, 0.5 mM L-glutamine, 100 mU/l insulin, 0.1% (w/v) penicillin, and 19.1 mM KCl. The cell suspension was subsequently inoculated into poly-D-lysine-coated [0.01% (w/v)] 35-mm Petri dishes (2 ml/dish). Glass coverslips (3.5 mm) were placed in the dishes before coating. After 24 h in culture, the medium was replaced by medium without serum but with 1% N2 supplement. Every 3 to 4 days, the culture medium was replaced with DMEM/N2 supplement. Cells were maintained in culture for 8 to 13 days before experiments were carried out.

Fluorescence Measurements
On the day of experiment, the medium was aspirated from the wells, and 50 µl of a 2 µM Fluo-4-AM loading solution was added to each well. The plates were sealed and incubated at room temperature (20-22°C) for 60 min. The loading medium was then aspirated, and the cells were washed twice with 100 µl of buffer (10 mM HEPES, 140 mM choline chloride, 5 mM KCl, 1 mM MgCl₂, and 10 mM CaCl₂; pH 7.4) to remove extracellular dye. The reason for using a relatively high CaCl₂ concentration was to enhance the fluorescent signal evoked secondary to activation of GLU_K5 and GLU_K6 receptors. Buffer (100 µl) was added to each well, and the fluorescence was measured at room temperature (excitation 488 nm, emission 510-570-nm band pass interference filter) using a fluorescent imaging plate reader (Molecular Devices, Sunnyvale, CA). Cells were preincubated for 1.5 min with test compound (50 µl) before addition of domoate (50 µl) to a final concentration of 2 µM (for GLU_K5) or 0.2 µM (for GLU_K6).

Stock solutions of test substances were made in ethanol or dimethyl sulfoxide, with final concentration of solvent never exceeding 0.5%.

Electrophysiological Studies
The electrophysiological measurements were performed in voltage clamps using conventional whole cell patch-clamp techniques (Hamill et al., 1981), and all data were obtained with an EPC-9 amplifier (HEKA Electronics, Lambrect, Germany) run by a Macintosh G3 computer. Experimental conditions and data acquisition were set and obtained using the PULSE-software accompanying the amplifier. Data were low pass filtered and sampled directly to the hard disk. Pipettes were pulled from borosilicate glass using a horizontal electrode puller (Zeitz Instrumente, Augsburg, Germany), and the final pipette resistance was approximately 2 M when filled with internal solution and submerged in the external solution used in the experiments.

Coverslips with cultured cells were transferred to a perfusion chamber mounted on the stage of an inverted microscope supplied with Nomarski optics, and cells were continuously superfused with external solution at a rate of 2.5 ml/min.

Compounds were dissolved in external solution and applied to the patched cell through double-barreled application pipettes. The application pipettes were fabricated from theta glass tubes (1.5 mm outer diameter; WPI, Sarasota, FL). The application pipettes were mounted on a piezoelectric device (PZS-100HS; Burleigh Instruments, Quebec, Canada) connected to a piezo-driver (PZ-150M; Burleigh Instruments) driven by TTL pulses from the EPC-9 amplifier. One minute after the onset of the gravity flow, a PULSE protocol was initiated and the current was recorded three times separated by 30-s waiting periods. For transfected HEK293 cells, the duration of the recording periods was 150 ms during which the application pipette was switched to the test solution for 100 ms. For the cortical neurons, the duration of the recording period was 1.5 s, and the application pipette was moved for 1 s.

GLU_K5- or GLU_K6-Expressing Cells. For recordings from GLU_K5- or GLU_K6-expressing cells, the pipette solution contained 120 mM KCl, 31 mM KOH, 10 mM EGTA, 1.8 mM MgCl₂, and 10 mM HEPES (pH 7.2). The external solution was composed of 140 mM NaCl, 5 mM KCl, 10 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES (pH 7.4). After giga-seal formation (1-2G) and establishment of the whole cell configuration, the cells were held at a holding potential of -60 mV. For each cell, a control response induced by 3 mM L-glutamate (GLU_K5) or 0.5 mM L-glutamate (GLU_K6) was recorded followed by recordings of the agonist-induced responses in the presence of increasing concentrations of NS3763. Because of reversibility of the effect of NS3763 and low, constant series resistance (<5 M), several concentrations could be tested on each cell. Series resistance was compensated by 80%.

Native AMPA and NMDA Receptors in Cortical Neurons. For recordings in native AMPA and NMDA receptors in cortical neurons, the pipette solution contained 135 mM CsCl, 11 mM EGTA, 1.2 mM MgCl₂, 0.5 mM CaCl₂, 4 mM ATP, and 10 mM HEPES (pH 7.3). The external solution was composed of 140 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 25 mM D-glucose, 0.5 µM tetrodotoxin, and 10 mM HEPES (pH 7.4). After giga-seal formation (1-3 G) and establishment of the whole cell configuration, the cells were held at a holding potential of -60 mV. For the measurement of AMPA responses, 4 mM MgCl₂ was added to the external solution, whereas 10 µM glycine was added for the measurement of NMDA responses. Agonists (100 µM AMPA or 100 µM NMDA) were dissolved in the extracellular solution, and for each cell a control agonist response was recorded followed by recordings of the agonist-induced response in the presence of 30 µM NS3763.

For all electrophysiological measurements, the current amplitudes were measured at the peak of the responses, and the effect of NS3763 was calculated as the amplitude during compound application divided by the amplitude of the agonist-induced current evoked before the application of compound. All experiments were performed at room temperature (20-22°C).

Receptor Binding
GLU_K5- and GLU_K6-expressing cells were harvested and washed once with 50 mM Tris-HCl (pH 7.1) and stored at -80°C until the day of experiment. The thawed membrane pellets were resuspended in >100 volumes of ice-cold Tris-HCl buffer and centrifuged for 10 min (27,000g). The final pellets were resuspended in Tris-HCl buffer and used for binding experiments. All procedures were performed at 0 to 4°C.

Binding conditions for GLU_K5 and GLU_K6 were as described previously (Varming et al., 2001). Briefly, binding to GLU_K5 receptors was performed using 3 nM [³H]ATPA at 46 to 84 µg of protein/assay, and GLU_K6 receptors were labeled with 5 nM [³H]kainate at 22 to 27 µg of protein/assay. The samples were incubated in a final volume of 550 µl for 60 min at 2°C. Nonspecific binding was determined in the presence of 0.6 mM L-glutamate, and binding was terminated by rapid filtration. Radioactivity was determined by conventional liquid scintillation counting.

Data Analysis
In binding and functional studies, compounds were tested over a wide range of concentrations, and IC₅₀ values and Hill coefficients were determined based on the equation Y = Bottom + (Top - Bottom)/(1 + (X/IC₅₀)ⁿ), where Y is binding/calcium increase/current in percentage of total binding/calcium increase/current; X the concentration of test compound; and n is the Hill coefficient. The EC₅₀ values for domoate in stimulation of intracellular calcium in HEK293 cells were determined by using the equation Y = 100· Xⁿ/(EC₅₀ⁿ + Xⁿ).

Estimates of IC₅₀ and EC₅₀ values were calculated with the nonlinear curve-fitting program GraphPad Prism (version 2.0; GraphPad Software Inc., San Diego, CA). K_i values were calculated from IC₅₀ values using the Cheng and Prusoff equation: K_i = IC₅₀/(1 + (L/K_d)). The K_d values were as follows: 2.9 nM for [³H]ATPA at GLU_K5 receptors and 5.7 nM for [³H]kainate at GLU_K6 receptors, respectively. All results are given as means ± S.E.M.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Calcium Measurements. Domoate concentration dependently increased intracellular calcium in GLU_K5- and GLU_K6-expressing cells with EC₅₀ values of 1.5 ± 0.2 and 0.13 ± 0.03 µM, respectively (data not shown). Based on these potencies, the following inhibition studies were conducted at 2 µM domoate for GLU_K5 and 0.2 µM for GLU_K6 cells. NS3763 (Fig. 1) inhibited GLU_K5-mediated responses with an IC₅₀ value of 1.6 ± 0.2 µM, whereas no inhibition of GLU_K6-mediated response was seen at concentrations up to 30 µM (n = 4; Table 1; Fig. 2). ATPA did not induce agonist responses at concentrations ranging from 0.01 to 3 µM, but the compound potently and selectively inhibited domoate-induced increase in intracellular calcium in GLU_K5 cells with an IC₅₀ value of 0.21 ± 0.03 µM. No inhibition of GLU_K6-mediated responses was seen at concentrations up to 300 µM (n = 3; Table 1).

View larger version (12K): [in this window] [in a new window]   Fig. 1. Chemical structure of NS3763.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Inhibition of domoate-induced increase in intracellular calcium in HEK293 cells expressing homomeric GLUK5 or GLUK6 receptors by NS3763 (, ) and NS1209 (, ). Closed symbols represent GLUK5 and open symbols represent GLUK6. Each point represents the mean ± S.E.M. of three separate experiments. Curve fitting was performed as described in "Data Analysis". The IC50 values are given in Table 1.

The competitive AMPA/kainate antagonist NS1209 potently inhibited domoate-induced calcium increase mediated by GLU_K5 receptors with an IC₅₀ value of 0.63 ± 0.09 µM (n = 4). GLU_K6-mediated responses were inhibited with an IC₅₀ value of 65 ± 4 µM by NS1209 (n = 4; Table 1; Fig. 2).

Electrophysiology on GLU_K5 and GLU_K6 Receptors. Both L-glutamate and domoate evoked concentration-dependent inward currents in cells stably expressing GLU_K5 and GLU_K6 receptors. The estimated EC₅₀ values for L-glutamate were 3.6 and 1.0 mM for GLU_K5 and GLU_K6 (data not shown), respectively, and for the inhibition studies 3 and 0.5 mM L-glutamate were chosen as agonist concentration for GLU_K5 and GLU_K6, respectively. The peak inward current responses to 3 mM L-glutamate in GLU_K5-expressing cells ranged from 122 to 1147 pA (n = 31). The L-glutamate-evoked currents were considerably larger in GLU_K6-expressing cells ranging from 1.1 to 11.8 nA (n = 23) at 0.5 mM L-glutamate.

In the presence of 10 µM NS3763, the amplitude of the L-glutamate-evoked response in GLU_K5 cells was inhibited by 44 ± 10% (n = 4), whereas no effect was seen on GLU_K6-mediated responses up to 30 µM (n = 3, Fig. 3, A and B).

View larger version (10K): [in this window] [in a new window]   Fig. 3. NS3763 selectively blocks L-glutamate-evoked currents in HEK293 cells expressing homomeric GLUK5 receptors. A, inhibition of L-glutamate-evoked current in a GLUK5-expressing cell by 10 µM NS3763. The cell was voltage clamped at -60 mV in the whole cell configuration and exposed to a 100-ms pulse of 3 mM L-glutamate. After attainment of responses of a repeatable amplitude (only one response shown), 10 µM NS3763 was included in the solution as indicated by the black horizontal bar. B, effect of 30 µM NS3763 on L-glutamate-evoked current in a GLUK6-expressing cell. The experiment was performed as described above but using 0.5 mM L-glutamate. The interval between stimulations was 30 s. The recordings are representative of findings in three to four separate experiments.

It was examined whether the effect of NS3763 was due to changes in the desensitization kinetics of GLU_K5 receptors. For selected pairs of current responses evoked by 3 mM L-glutamate in the presence and absence of 30 µM NS3763, the decay phase of the peak-current was fitted to a double-exponential function and compared by paired t test. The decay time constant of the fast component, _fast, in the presence of NS3763 (1.75 ± 0.08 ms; n = 7) was not significantly different from the control value of 1.62 ± 0.10 ms (n = 7). Similarly, the slow component, _slow, was not different in the presence (8.87 ± 1.81 ms; n = 7) and absence (7.20 ± 0.69 ms; n = 7) of NS3763. The ratio of the two components was also unaffected by the application of the compound, the fast component accounting for 0.78 ± 0.03 (control) and 0.81 ± 0.04 (30 µM NS3763).

Domoate was a more potent agonist than L-glutamate in evoking currents in GLU_K5- and GLU_K6-expressing cells, and the estimated EC₅₀ values were 7.5 and 0.4 µM, respectively (data not shown). The peak inward current responses obtained at the estimated EC₅₀ value for domoate in GLU_K5 cells ranged from 41 to 1474 pA (n = 9), and the amplitude of the response evoked by 8 µM domoate was inhibited by 63 ± 10% in the presence of 10 µM NS3763 (n = 5; Fig. 4A). NS3763 (0.01-30 µM) caused a concentration-dependent inhibition of GLU_K5 responses to 8 µM domoate with an IC₅₀ value of 1.3 µM, and a maximal inhibitory effect of approximately 60% (n = 5; Fig. 4B). Currents evoked by 30 µM domoate were inhibited 58 ± 5% (n = 6) in the presence of 10 µM NS3763.

View larger version (12K): [in this window] [in a new window]   Fig. 4. NS3763 blocks domoate-evoked currents in HEK293 cells expressing homomeric GLUK5 receptors. A, inhibition of domoate-evoked current by 10 µM NS3763. The cell was voltage clamped at -60 mV in the whole cell configuration and exposed to a 100-ms pulse of 8 µM domoate. After attainment of responses of a repeatable amplitude (only one response shown), 10 µM NS3763 was included in the solution as indicated by the black horizontal bar. B, concentration-dependent inhibition of domoate-induced currents by NS3763. The experiment was performed as described above. The current amplitudes were expressed as percentage of the current induced by 8 µM domoate. Each data point represents the mean ± S.E.M. of five separate experiments. Curve fitting was performed as described in "Data Analysis".

All data reported above were obtained using the GLU_K5-1a isoform, which contains 15 extra amino acids in the NH₂-terminal domain and has a shorter COOH-terminal domain than the GLU_K5-2b isoform reported for rat (Sommer et al., 1992) and for human by Korczak et al. (1995). To investigate whether NS3763 had different modulatory effects at GLU_K5-1a and GLU_K5-2b receptors, HEK293 cells transiently expressing the GLU_K5-2b isoform were used. In these cells an EC₅₀ value of 1.2 mM was obtained for L-glutamate (data not shown).

As illustrated in Fig. 5, NS3763 caused a concentration-dependent inhibition of L-glutamate responses in both isoforms; however, the drug was approximately 10-fold more potent at the GLU_K5-2b (IC₅₀ = 0.38 µM; n = 3) than the GLU_K5-1a isoform (IC₅₀ = 5.7 µM; n = 4).

View larger version (12K): [in this window] [in a new window]   Fig. 5. Effect of NS3763 on L-glutamate-induced currents in HEK293 cells expressing the homomeric GLUK5 receptor isoform GLUK5-1a () and GLUK5-2b (). The cells were voltage clamped at -60 mV. The current amplitudes were expressed as percentage of the current induced by 3 mM L-glutamate. Each data point represents the mean ± S.E.M. of three to four separate experiments. Curve fitting was performed as described in "Data Analysis".

In electrophysiological studies, as well as in assays measuring intracellular calcium (Fig. 2), only a partial inhibition of the responses could be obtained.

Mechanism of Action of NS3763 on GLU_K5 Receptors. To investigate the mechanism of inhibition by NS3763, its effect on the concentration-response relationship for domoate was characterized. As illustrated in Fig. 6A, the maximal increase in intracellular calcium occurred at 30 µM domoate, and 3 µM NS3763 caused a reduction of the maximal response. However, the EC₅₀ value for domoate in the presence of NS3763 (1.7 ± 0.2 µM; n = 3) was similar to the control value of 1.4 ± 0.1 µM (n = 3). In contrast, 3 µM NS1209 caused a concentration-dependent rightward shift in the concentration-response curve with no change in the maximal response (Fig. 6A); the EC₅₀ value for domoate in the presence of 3 µM NS1209 was 7.1 ± 1.3 µM (n = 3). Thus, unlike NS1209, which interacts competitively at the domoate binding site, NS3763 inhibits domoate responses by a noncompetitive mechanism.

View larger version (14K): [in this window] [in a new window]   Fig. 6. NS3763 interacts noncompetitively with homomeric GLUK5 receptors. A, comparison of the effects of NS3763 and NS1209 on domoate concentration-response curves in HEK293 cells expressing homomeric GLUK5 receptors. Concentration-response relationships for domoate (0.1-50 µM) under control conditions () and in the presence 3 µM NS3763 () or 3 µM NS1209 () were obtained by measurements of intracellular calcium. Values were normalized to maximum calcium increase (30 µM domoate; 100%) and represent the mean ± S.E.M. of three separate experiments. B, effect of NS3763 on L-glutamate-induced currents in HEK293 cells expressing homomeric GLUK5 receptors. Concentration-response relationships for L-glutamate (0.1-30 mM) under control conditions () and in the presence 10 µM NS3763 () were obtained in electrophysiological studies. The cell was voltage clamped at -60 mV in the whole cell configuration and exposed to a 100-ms pulse of L-glutamate. For each cell, the data were normalized to the current evoked by 30 mM L-glutamate. Points indicate the mean ± S.E.M. of the peak current amplitude values for three to nine cells separate experiments. Curve fitting was performed as described in "Data Analysis".

The noncompetitive action of NS3763 was supported by radioligand binding studies, because NS3763 did not inhibit [³H]ATPA binding to GLU_K5 receptors (Table 1). In contrast, NS1209 inhibited ligand binding to GLU_K5 and GLU_K6 (Varming et al., 2001), whereas ATPA only displaced binding to GLU_K5.

The noncompetitive mechanism of action for NS3763 was confirmed in electrophysiological studies (Fig. 6B); the EC₅₀ value for L-glutamate in the presence of 10 µM NS3763 (3.28 mM; n = 5-9) was similar to the control value of 3.35 mM (n = 3-7; p > 0.05; paired t test).

Native AMPA and NMDA Receptors. The possible antagonistic activity of NS3763 on native AMPA- and NMDA receptors was investigated in cultured mouse cortical neurons. The peak inward current responses to 100 µM AMPA or NMDA ranged from 104 to 244 pA (n = 4) and from 525 to 1237 pA (n = 4), respectively. In the presence of 30 µM NS3763, the amplitude of the responses to 100 µM AMPA was inhibited by -0.6 ± 5.4% (n = 4; Fig. 7A). In contrast, the AMPA-induced current was blocked by 89 ± 2% in the presence of 30 µM GYKI52466, a noncompetitive AMPA antagonist (n = 4; Fig. 7A). The response to 100 µM NMDA was blocked by 10 ± 4% in the presence of 30 µM NS3763 and by 79 ± 11% by 50 µM APV (n = 4; Fig. 7B). The inhibition of AMPA- and NMDA-induced currents by NS3763 was not significant (p > 0.05; paired t test).

View larger version (14K): [in this window] [in a new window]   Fig. 7. NS3763 has no effect on AMPA- and NMDA-induced responses in cultured mouse cortical neurons. A, effect of 30 µM NS3763 on AMPA-induced current in a cortical neuron. The cell was voltage clamped at -60 mV in the whole cell configuration and exposed to 1-s pulses of 100 µM AMPA. After attainment of responses of a repeatable amplitude, 30 µM NS3763 was included in the solution as indicated by the black horizontal bar. Finally, the AMPA-induced responses were inhibited by 30 µM GYKI52466 ("GYKI'). The interval between stimulations was 30 s. B, effect of 30 µM NS3763 on NMDA-induced current in a cortical neuron. The cell was voltage clamped at -60 mV in the whole cell configuration and exposed to 1-s pulses of 100 µM NMDA. The experiments were performed using Mg2+-free solutions containing 10 µM glycine. After attainment of responses of a repeatable amplitude, 30 µM NS3763 was included in the solution as indicated by the black horizontal bar. The NMDA-induced current was blocked by 50 µM APV (APV). The interval between stimulations was 30 s. The recordings are representative of findings in three separate experiments.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The present study shows that NS3763 selectively and noncompetitively inhibits homomeric GLU_K5 receptor-mediated responses. This is the first demonstration of a noncompetitive kainate antagonist.

In studies measuring intracellular calcium, NS3763 was shown to inhibit domoate-induced responses in GLU_K5 receptor-expressing HEK293 cells (IC₅₀ = 1.6 µM). ATPA and the mixed AMPA/kainate receptor antagonist NS1209 (Nielsen et al., 1999; Varming et al., 2001) also inhibited domoate-induced calcium responses in GLU_K5-expressing cells with IC₅₀ values of 0.21 and 0.63 µM, respectively. The lack of agonist response observed for ATPA at concentrations up to 3 µM and its inhibitory action on domoate-induced responses is most likely due to rapid desensitization of homomeric GLU_K5 receptors (Lerma et al., 1993, 2001). Similarly, no agonist response could be obtained for L-glutamate (data not shown).

The effects of the two antagonists NS3763 and NS1209 on domoate responses, however, were different. NS1209 shifted domoate concentration-response curves to the right in a parallel manner indicative of competitive antagonism, whereas the effect of NS3763 was noncompetitive. The noncompetitive mechanism of action of NS3763 was further supported by the fact that NS3763, in contrast to ATPA and NS1209, did not inhibit [³H]ATPA binding to GLU_K5 receptors.

In electrophysiological studies, NS3763 inhibited domoate-evoked currents in GLU_K5-expressing cells (IC₅₀ = 1.3 µM), with a potency similar to that determined using intracellular calcium measurements. The inhibition of agonist-evoked currents by NS3763 was, as expected from its noncompetitive mechanism of action, independent of the concentration of agonist used. Currents evoked by 30 µM domoate were inhibited by 10 µM NS3763 to the same extend (58 ± 5%) as seen at 8 µM of the agonist (Fig. 4B). Despite the different functional endpoints (current versus cytosolic calcium) in electrophysiological and imaging studies, the potency and maximal inhibitory effect (60-70%) of NS3763 against domoate-induced responses were very similar.

Currents evoked by the endogenous ligand L-glutamate were also inhibited in the low micromolar range by NS3763 with a maximal inhibitory effect varying from 30 to 50% (Figs. 5 and 6B), and the noncompetitive mechanism of action of the compound was confirmed.

Furthermore, the data indicates that NS3763 is selective for homomeric GLU_K5 over homomeric GLU_K6 receptors at concentrations up to 30 µM. NS3763 is somewhat less potent than NS1209 (IC₅₀ = 0.075 µM; Varming et al., 2001) in inhibiting GLU_K5 receptor-mediated currents, but whereas NS1209 displays equipotent inhibition of AMPA-induced responses in cortical neurons (Nielsen et al., 1999) and kainate evoked responses in cells expressing GLU_K5 receptors (Varming et al., 2001), NS3763 did not show significant antagonistic properties on either native AMPA or native NMDA receptors at concentrations up to 30 µM. NS3763 thus shows selectivity for GLU_K5 receptors and displays a selectivity profile different from NS1209. These data are the first describing a selective noncompetitive antagonist of GLU_K5 receptors.

Several compounds interacting noncompetitively with AMPA receptors are known, and it is well described that AMPA receptor-mediated responses can be either diminished or enhanced by drugs acting at allosteric sites. The 2,3-benzodiazepines such as GYKI52466 and GYKI53655 are well characterized as negative allosteric modulators (Bleakman et al., 1996). The known positive allosteric modulators are benzothiadiazines (e.g., cyclothiazide) and benzoylpiperidines (e.g., aniracetam and CX516). The precise mechanism by which 2,3-benzodiazepines act as negative modulators is not known; they do not affect deactivation or desensitization and are not open channel blockers (Donevan and Rogawski, 1993; Rammes et al., 1998).

Rapid desensitization is one of the most characteristic features of kainate receptors, and modification of this feature is one of the means by which kainate receptor responses can be altered by pharmacological agents. The lectin concanavalin A has long been known to markedly reduce kainate receptor desensitization by an allosteric mechanism (Huettner, 1990), and thereby potentiates current responses of native and recombinant receptors (Huettner, 1990; Partin et al., 1993). The inhibition of L-glutamate-evoked currents by NS3763 is apparently not due to an increase in kainate receptor desensitization because the drug had no effect on the rate of L-glutamate current desensitization.

The binding site for NS3763 at homomeric GLU_K5 receptors is not known. However, the compound seemed to distinguish between two isoforms of GLU_K5, being 10-fold more potent in inhibiting L-glutamate-evoked currents in the GLU_K5-2b than in the GLU_K5-1a isoform, which has 15 additional amino acids present in the NH₂ terminal (Sommer et al., 1992). The reason for these potency differences is currently being studied.

The potencies of NS3763 in inhibiting agonist-induced responses in assays measuring intracellular calcium and in electrophysiological studies were very similar, but it seems that only a partial inhibition of the responses can be obtained. The reason for this is not known, but it should be noted that the compound has limited water solubility [<0.05 mg/ml (<125 µM) at pH 7.4]. However, it cannot be excluded that NS3763 is unable to inhibit the functional responses completely due to its noncompetitive interaction with the GLU_K5 receptor.

ATPA has been shown to be not completely selective for homomeric GLU_K5 receptors because it also activates heteromeric GLU_K5 receptors containing GLU_K2 or GLU_K6 subunits and heteromers consisting of GLU_K6/K2 receptor subunits (Paternain et al., 2000). LY382884 has recently been reported to also be effective against heteromeric assemblies of GLU_K5 and GLU_K6 subunits (Smolders et al., 2002), so there is still a need for new pharmacological tools. The activity of NS3763 on heteromeric kainate receptors and the potential analgesic effect of the drug in animal models of pain are currently being studied.

	Acknowledgements

 
We thank Anne B. Fisher, Kirsten V. Haugegaard, Kristina Christensen, and Aino Munch (Departments of Biochemical Screening and Receptor Biochemistry) for skillful technical assistance.

	Footnotes

 
J.K.C. was supported by the Danish Academy of Technical Sciences.

DOI: 10.1124/jpet.103.062794.

ABBREVIATIONS: NMDA, N-methyl-D-aspartate; AMPA, -amino-3-hydroxy-5-methylisoxazole-4-propionic acid; APV, D-2-amino-5-phosphovaleric acid; NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline; ATPA, -amino-3-hydroxy-5-tertbutylisoxazole-4-propionic acid; GYKI52466, 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine; NS1209, 8-methyl-5-(4-(N,N-dimethylsulfamoyl)phenyl)-6,7,8,9,-tetrahydro-1H-pyrrolo[3,2-h]-isoquinoline-2,3-dione-3-O-(4-hydroxybutyric acid-2-yl)oxime; NS3763, 5-carboxyl-2,4-di-benzamidobenzoic acid; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; GYI53655, 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine; LY382884, 3S,4aR,6S,8aR-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-deca-hydroisoquinoline-3-carboxylic acid; NS102, 5-nitro-6,7,8,9-tetrahydrobenzo[g]indole-2,3-dione-3-oxime.

Address correspondence to: Dr. Elsebet Ø. Nielsen, Department of Receptor Biochemistry, NeuroSearch A/S, 93 Pederstrupvej, DK-2750 Ballerup, Denmark. E-mail: eon{at}neurosearch.dk

	References

Top Abstract Materials and Methods Results Discussion References

 

Bleakman D, Ballyk BA, Schoepp DD, Palmer AJ, Bath CP, Sharpe EF, Woolley ML, Bufton HR, Kamboj RK, Tarnawa I, and Lodge D (1996) Activity of 2,3-benzodiazepines at native rat and recombinant human glutamate receptors in vitro: stereospecificity and selectivity profiles. Neuropharmacology 35: 1689-1702.[CrossRef][Medline]

Bleakman D, Gates MR, Ogden AM, and Mackowiak M (2002) Kainate receptor agonists, antagonists and allosteric modulators. Curr Pharm Des 8: 873-885.[CrossRef][Medline]

Bortolotto ZA, Clarke VR, Delany CM, Parry MC, Smolders I, Vignes M, Ho KH, Miu P, Brinton BT, Fantaske R, et al. (1999) Kainate receptors are involved in synaptic plasticity. Nature (Lond) 402: 297-301.[CrossRef][Medline]

Clarke VR, Ballyk BA, Hoo KH, Mandelzys A, Pellizzari A, Bath CP, Thomas J, Sharpe EF, Davies CH, Ornstein PL, et al. (1997) A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission. Nature (Lond) 389: 599-603.[CrossRef][Medline]

Collingridge GL and Lester RA (1989) Excitatory amino acid receptors in the vertebrate central nervous system. Pharmacol Rev 41: 143-210.[Medline]

Davies J, Francis AA, Jones AW, and Watkins JC (1981) 2-Amino-5-phosphonovalerate (2APV), a potent and selective antagonist of amino acid-induced and synaptic excitation. Neurosci Lett 21: 77-81.[CrossRef][Medline]

Donevan SD and Rogawski MA (1993) GYKI 52466, a 2,3-benzodiazepine, is highly selective, noncompetitive antagonist of AMPA/kainate receptor responses. Neuron 10: 51-59.[CrossRef][Medline]

Drejer J, Honoré T, and Schousboe A (1987) Excitatory amino acid-induced release of [³H]GABA from cultured mouse cerebral cortex interneurons. J Neurosci 7: 2910-2916.[Abstract]

Filla SA, Winter MA, Johnson KW, Bleakman D, Bell MG, Bleisch TJ, Castaño AM, Clemens-Smith A, del Prado M, Dieckman DK, et al. (2002) Ethyl (3S,4aR,6S,8aR)-6-(4-ethoxycarbonylimidazol-1-ylmethyl)decahydroisoquinoline-3-carboxylic ester: a prodrug of a GluR5 kainate receptor antagonist active in two animal models of acute migraine. J Med Chem 45: 4383-4386.[CrossRef][Medline]

Fletcher EJ, Martin D, Aram JA, Lodge D, and Honoré T (1988) Quinoxalinediones selectively block quisqualate and kainate receptors and synaptic events in rat neocortex and hippocampus and frog spinal cord in vitro. Br J Pharmacol 95: 585-597.[Medline]

Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recordings from cells and cell-free membrane patches. Pflueg Arch 391: 85-100.[CrossRef][Medline]

Honoré T, Davies SN, Drejer J, Fletcher EJ, Jacobsen P, Lodge D, and Nielsen FE (1988) Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists. Science (Wash DC) 241: 701-703.[Abstract/Free Full Text]

Huettner JE (1990) Glutamate receptor channels in rat DRG neurons: activation by kainate and quisqualate and blockade of desensitization by Con A. Neuron 5: 255-266.[CrossRef][Medline]

Johansen TH, Drejer J, Wätjen F, and Nielsen EØ (1993) A novel non-NMDA receptor antagonist shows selective displacement of low-affinity [³H]kainate binding. Eur J Pharmacol 246: 195-204.[CrossRef][Medline]

Korczak B, Nutt SL, Fletcher EJ, Hoo KH, Elliott CE, Rampersad V, McWhinnie EA, and Kamboj RK (1995) cDNA cloning and functional properties of human glutamate receptor EAA3 (GluR5) in homomeric and heteromeric configuration. Receptors Channels 3: 41-49.[Medline]

Lauridsen J, Honoré T, and Krogsgaard-Larsen P (1985) Ibotenic acid analogues. Synthesis, molecular flexibility and in vitro activity of agonists and antagonists at central glutamic acid receptors. J Med Chem 28: 668-672.[CrossRef][Medline]

Lerma J, Paternain AV, Naranjo JR, and Mellström B (1993) Functional kainate-selective glutamate receptors in cultured hippocampal neurons. Proc Natl Acad Sci USA 90: 11688-11692.[Abstract/Free Full Text]

Lerma J, Paternain AV, Rodriquez-Moreno A, and Lopez-Garcia JC (2001) Molecular physiology of kainate receptors. Physiol Rev 81: 971-998.[Abstract/Free Full Text]

Li P, Wilding TJ, Kim SJ, Calejesan AA, Huettner JE, and Zhuo M (1999) Kainate-receptor-mediated sensory synaptic transmission in mammalian spinal cord. Nature (Lond) 397: 161-164.[CrossRef][Medline]

Lodge D, Jones MG, and Palmer AJ (1991) Excitatory amino acids: new tools for old stories or pharmacological subtypes of glutamate receptors: electrophysiological studies. Can J Physiol Pharmacol 69: 1123-1128.[Medline]

Madden DR (2002) The structure and function of glutamate receptor ion channels. Nat Rev Neurosci 3: 91-101.[Medline]

Mulle C, Sailer A, Swanson GT, Brana C, O'Gorman S, Bettler B, and Heinemann SF (2000) Subunit composition of kainate receptors in hippocampal interneurons. Neuron 28: 475-484.[CrossRef][Medline]

Nielsen EØ, Varming T, Mathiesen C, Jensen LH, Møller A, Gouliaev AH, Wätjen F, and Drejer J (1999) SPD 502: a water-soluble and in vivo long-lasting AMPA antagonist with neuroprotective activity. J Pharmacol Exp Ther 289: 1492-1501.[Abstract/Free Full Text]

Partin KM, Patneau DK, Winters CA, Mayer ML, and Buonanno A (1993) Selective modulation of desensitization at AMPA versus kainate receptors by cyclothiazide and concanavalin A. Neuron 11: 1069-1082.[CrossRef][Medline]

Paternain AV, Herrera MT, Nieto MA, and Lerma J (2000) GluR5 and GluR6 kainate receptor subunits coexist in hippocampal neurons and coassemble to form functional receptors. J Neurosci 20: 196-205.[Abstract/Free Full Text]

Paternain AV, Morales M, and Lerma J (1995) Selective antagonism of AMPA receptors unmasks kainate receptor-mediated responses in hippocampal neurons. Neuron 14: 185-189.[CrossRef][Medline]

Paternain AV, Vicente MA, Nielsen EØ and Lerma J (1996) Comparative antagonism of kainate-activated AMPA and kainate receptors in hippocampal neurons. Eur J Neurosci 8: 2129-2136.[CrossRef][Medline]

Procter MJ, Houghton AK, Faber ES, Chizh BA, Ornstein PL, Lodge D, and Headley PM (1998) Actions of kainate and AMPA selective glutamate receptor ligands on nociceptive processing in the spinal cord. Neuropharmacology 37: 1287-1297.[CrossRef][Medline]

Rammes G. Swandulla D, Spielmanns P, and Parsons CG (1998) Interactions of GYKI 52466 and NBQX with cyclothiazide at AMPA receptors: experiments with outside-out patches and EPSCs in hippocampal neurones. Neuropharmacology 37: 1299-1320.[CrossRef][Medline]

Ruscheweyh R and Sandkühler J (2002) Role of kainate receptors in nociception. Brain Res Rev 40: 215-222.[CrossRef][Medline]

Schoepp DD, Jane DE, and Monn JA (1999) Pharmacological agents acting at subtypes of metabotropic glutamate receptors. Neuropharmacology 38: 1431-1476.[CrossRef][Medline]

Sheardown MJ, Nielsen EØ, Hansen AJ, Jacobsen P and Honoré T (1990) 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline: a neuroprotectant for cerebral ischemia. Science (Wash DC) 247: 571-574.[Abstract/Free Full Text]

Simmons RM, Li DL, Hoo KH, Deverill M, Ornstein PL, and Iyengar S (1998) Kainate GluR5 receptor subtype mediates the nociceptive response to formalin in the rat. Neuropharmacology 37: 25-36.[CrossRef][Medline]

Smolders I, Bortolotto ZA, Clarke VR, Warre R, Khan GM, O'Neill MJ, Ornstein PL, Bleakman D, Ogden A, Weiss B, et al. (2002) Antagonists of GLU_K5-containing kainate receptors prevent pilocarpine-induced limbic seizures. Nat Neurosci 5: 796-804.[Medline]

Sommer B, Burnashev N, Verdoorn TA, Keinanen K, Sakmann B, and Seeburg PH (1992) A glutamate receptor channel with high affinity for domoate and kainate. EMBO J 11: 1651-1656.[Medline]

Varming T, Ahring PK, Sager TN, Mathiesen C, Johansen TH, Wätjen F, and Drejer J (2001) Optimization of isatine oximes as neuroprotective AMPA receptor antagonists: in vitro and in vivo evaluation of SPD 502, in Excitatory Amino Acids: 10 Years Later (Turski L, Schoepp DD, and Cavalheiro EA eds) pp 193-205, IOS Press, Amsterdam.

Verdoorn TA, Johansen TH, Drejer J and Nielsen EØ (1994) Selective block of recombinant glur6 receptors by NS-102, a novel non-NMDA receptor antagonist. Eur J Pharmacol 269: 43-49.[CrossRef][Medline]

Wilding TJ and Huettner JE (1996) Antagonist pharmacology of kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-preferring receptors. Mol Pharmacol 49: 540-546.[Abstract]

Wilding TJ and Huettner JE (2001) Functional diversity and developmental changes in rat neuronal kainate receptors. J Physiol (Lond) 532: 411-421.[Abstract/Free Full Text]

Wong EH, Kemp JA, Priestly T, Knight AR, Woodruff GN, and Iversen LL (1986) The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc Natl Acad Sci USA 83: 7104-7108.[Abstract/Free Full Text]

This article has been cited by other articles:

				H. Y. Sun, A. F. Bartley, and L. E. Dobrunz Calcium-Permeable Presynaptic Kainate Receptors Involved in Excitatory Short-Term Facilitation Onto Somatostatin Interneurons During Natural Stimulus Patterns J Neurophysiol, February 1, 2009; 101(2): 1043 - 1055. [Abstract] [Full Text] [PDF]

				J.-C. Platel, T. Heintz, S. Young, V. Gordon, and A. Bordey Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in whole-mounts of the subventricular zone J. Physiol., August 15, 2008; 586(16): 3783 - 3793. [Abstract] [Full Text] [PDF]

				L. L. Lash, J. M. Sanders, N. Akiyama, M. Shoji, P. Postila, O. T. Pentikainen, M. Sasaki, R. Sakai, and G. T. Swanson Novel Analogs and Stereoisomers of the Marine Toxin Neodysiherbaine with Specificity for Kainate Receptors J. Pharmacol. Exp. Ther., February 1, 2008; 324(2): 484 - 496. [Abstract] [Full Text] [PDF]

				B. Weiss, A. Alt, A. M. Ogden, M. Gates, D. K. Dieckman, A. Clemens-Smith, K. H. Ho, K. Jarvie, G. Rizkalla, R. A. Wright, et al. Pharmacological Characterization of the Competitive GLUK5 Receptor Antagonist Decahydroisoquinoline LY466195 in Vitro and in Vivo J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 772 - 781. [Abstract] [Full Text] [PDF]

				J. M. Sanders, O. T. Pentikainen, L. Settimo, U. Pentikainen, M. Shoji, M. Sasaki, R. Sakai, M. S. Johnson, and G. T. Swanson Determination of Binding Site Residues Responsible for the Subunit Selectivity of Novel Marine-Derived Compounds on Kainate Receptors Mol. Pharmacol., June 1, 2006; 69(6): 1849 - 1860. [Abstract] [Full Text] [PDF]

				J. M. Sanders, K. Ito, L. Settimo, O. T. Pentikainen, M. Shoji, M. Sasaki, M. S. Johnson, R. Sakai, and G. T. Swanson Divergent Pharmacological Activity of Novel Marine-Derived Excitatory Amino Acids on Glutamate Receptors J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1068 - 1078. [Abstract] [Full Text] [PDF]

				J. K. Christensen, A. V. Paternain, S. Selak, P. K. Ahring, and J. Lerma A Mosaic of Functional Kainate Receptors in Hippocampal Interneurons J. Neurosci., October 13, 2004; 24(41): 8986 - 8993. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.062794v1 309/3/1003    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Christensen, J. K. Articles by Nielsen, E. O. Search for Related Content PubMed PubMed Citation Articles by Christensen, J. K. Articles by Nielsen, E. O.