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BEHAVIORAL PHARMACOLOGY
Department of Pharmacology and Toxicology, Virginia Commonwealth University Medical Center, Richmond, Virginia (B.H.G., F.K.K., J.K.R., W.L.D.); and Department of Pharmaceutical Sciences, Harding University College of Pharmacy, Searcy, Arkansas (F.L.S.)
Received January 17, 2007; accepted March 30, 2007.
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Abstract |
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-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors (Yoshimura and Jessell, 1990
), whereas in superficial (lamina I) and deep dorsal horn neurons, both non-NMDA and NMDA receptor (NMDAR) activation occurs in control conditions (Millan, 1999). Several genes encoding NMDAR subunits have been identified. These genes fall into three categories: NMDA-R1 subunit (NR1), A to D isoforms of NMDA-R2 subunit (NR2A-D), and NMDA-R3 subunit (Kutsuwada et al., 1992). Although NMDAR stimulation enhances synaptic transmission in the dorsal horn of the spinal cord, activation of the G-protein-coupled metabotropic glutamate receptors (mGluRs) serves mainly to modulate synaptic neurotransmission (Pin and Acher, 2002). Eight subtypes of mGluRs have been identified and divided in three groups on the basis of pharmacological and signal transduction mechanisms and amino acid homology. Group I mGluRs (mGluR1/5) are linked to phospholipase C and the activation of protein kinase C (PKC). Group II mGluRs (mGluR2/3) and group III mGluRs (mGluR4/6/7/8) are negatively coupled to adenylyl cyclase through Gi/Go to decrease cAMP accumulation (Conn and Pin, 1997).
Interestingly, group I mGluRs have been demonstrated to interact biochemically with NMDARs in vitro. For example, activating mGluR1 potentiated NMDAR currents in Xenopus oocytes, leading to an increase in intracellular Ca2+ and activation of PKC (Lan et al., 2001). Furthermore, in mice cortical slices, NMDA-induced depolarization was enhanced by the selective mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG] and blocked by the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) (Attucci et al., 2001). In contrast to in vitro studies, in vivo studies have been conducted to examine the potential coupling between group I mGluRs and NMDARs in mediating nociception in animals. The role of group I mGluRs in nociception has been well established, but less is known about the functional interaction between those receptors and the NMDARs. Activation of group I mGluRs has been demonstrated to produce nociception in animals. Intrathecal administration of (S)-3,5-DHPG was shown to induce persistent thermal hyperalgesia and mechanical allodynia (Fisher and Coderre, 1998). In addition, carrageenan-induced thermal and mechanical hyperalgesia in rats with hindpaw inflammation were blocked by i.t. of the group I mGluRs antagonist (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG] (Young et al., 1997).
There is considerable evidence that pain associated with peripheral tissue or nerve injury involves NMDAR activation (Doubell et al., 1999). Consistent with this, NMDAR antagonists have been shown to effectively alleviate pain-related behavior in animal models as well as in clinical situations (Fisher et al., 2000; Hewitt, 2000). However, NMDARs are important for normal central nervous system functions, and the use of NMDAR antagonists can often be limited by serious side effects, such as memory impairment, psychotomimetic effects, ataxia, motor incoordination, hallucinations, and agitation (Petrenko et al., 2003). Newly developed NMDAR antagonists at the glycine site, NR2B sites, and weak-binding channel blockers have demonstrated an improved side effect profile in animal models of pain (Brown and Krupp, 2006). Other recent studies indicate that modulating the activity of NMDARs with group I mGluRs drugs might be effective in treating chronic pain (Guo et al., 2004), thus limiting the incidence of NMDA-like side effects. The goal of this study was to use a model of thermal hyperalgesia to investigate the mechanisms by which group I mGluRs interact with NMDARs. The hypothesis that pharmacological stimulation of group I mGluRs results in the concomitant activation of NMDA receptors was tested.
We also evaluated, for the first time, the interaction between the mGluRs and the NR2B subunit of the NMDAR at a molecular level. Functional NR2B-containing NMDARs are formed by their coassembly with the obligatory NR1 subunits (Masuko et al., 1999). The NR1 subunits are ubiquitously expressed in the central nervous system, whereas the NR2B protein is localized predominantly in the superficial dorsal horn of the spinal cord (Boyce et al., 1999). Therefore, another aim was to investigate the role of NR2B receptor subunit in the modulation of (S)-3,5-DHPG-induced thermal hyperalgesia using a more selective experimental tool of small interfering RNA that involves a double-stranded short-hairpin RNA (shRNA) specifically silencing the mouse NR2B.
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Materials and Methods |
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Drugs and Chemicals. The following agents were purchased from Tocris Cookson Bioscience (Ellisville, MO): (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG; mGluR1/5 agonist]; -amino-5-carboxy-3-methyl-2-thiopheneacetic acid (3-MATIDA; mGluR1 antagonist; Moroni et al., 2002); MPEP hydrochloride (mGluR5 antagonist); cis-4-[phosphomethyl]-piperidine-2-carboxylic acid (CGS 19755; selective competitive NMDAR antagonist); and [(1S)-1-[[(7-bromo-1,2,3,4-tetrahydro-2,3-dioxo-5-quinoxalinyl)methyl]amino]-ethyl]phosphonic acid (CGP 78608; selective NMDAR antagonist that acts through the glycine site). The selective NR2B subunit antagonists -(4-hydroxyphenyl)-
-methyl-4-benzyl-1-piperidineethanol tartrate salt (ifenprodil) and [R-(R*,S*)]--(4-hydroxyphenyl)--methyl-4-(phenylmethyl)-1-piperidinepropanol hydrochloride (Ro 25-6981) were purchased from Sigma (St. Louis, MO). The following agents were purchased from Calbiochem (San Diego, CA). 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (bisindolylmaleimide I HCl; selective inhibitor of PKC, I, II, 
, 
, and 
isoforms), 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Go-7874; selective inhibitor of PKC, I, II, and 
isoforms), and myristoylated PKI-(14–22)-amide [Myr-N-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-NH2; selective PKA inhibitor] as well as H-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Leu-Arg-Lys-Lys-Lys-Lys-Lys-His-OH (DT-3; selective PKG inhibitor). [(R)-[(S)-1-(4-bromophenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077; selective NR2A subunit antagonist) was a generous gift from Novartis Pharma (Basel, Switzerland). The drugs were dissolved in saline or mixture of 10% dimethyl sulfoxide (Sigma, St. Louis, MO), 20% Cremophor EL (Sigma), and 70% saline. The appropriate drug vehicle was used as a control.
NR2B shRNA. The vector directing the transcription of a shRNA specifically targeting the NR2B (grin2b) subunit of the mouse NMDAR, as well as a shRNA nonsilencing (NS) control vector, were purchased from Open Biosystems (Huntsville, AL) as glycerol stocks of transformed Escherichia coli. The base vector for both plasmids is a retroviral pSM2. The pSM2-grin2b is designed for high-level expression of shRNA for the mouse NR2B subunit under the control of the mouse U6 promoter. The pSM2-NS represents a negative control shRNA vector. E. coli were grown according to the manufacturer's recommended protocols. The plasmids were gradient-purified using cesium chloride (CsCl). The sequence of the shRNA insert in pSM2-grin2b was confirmed using a primer developed for the U6 promoter region (5'-TGT GGA AAG GAC GAA ACA CC-3'). Sequence analysis was performed using an Applied Biosystems 377 Prism XL automated DNA sequencer (Applied Biosystems, Foster City, CA) by the Massey Cancer Center Nucleic Acid Synthesis and Analysis Core Resource (Virginia Commonwealth University, Richmond, VA).
Arrest-In, a polymeric transfection reagent (Open Biosystems, Huntsville, AL), was used for the highly efficient delivery of both pSM2-grin2b and pSM2-NS plasmids into neurons. Once in the cells, Arrest-In promotes the entry of the shRNA-containing plasmid into the nucleus where it is transcribed into a hairpin, enters the cytoplasm, and is processed by the endogenous RNA interference machinery into functional small interfering RNAs.
Intrathecal Injections. Intrathecal injections were made through the intervertebral space in unanaesthetized mice between the L5 and L6 of the spinal cord, as described by Hylden and Wilcox (1980). In brief, a volume of 5 µl was administered i.t. with a 28-gauge 1/2-inch stainless steel needle connected to a 50-µl Hamilton microsyringe, the animal being lightly restrained to maintain the position of the needle. Puncture of the dura was indicated behaviorally by a slight flick of the tail.
The Tail Immersion Test. The warm-water tail immersion test was performed according to Coderre and Rollman (1983). The mouse was gently wrapped in a towel, held at a 45° angle to a thermostatically controlled water bath set at 50 ± 1°C. The latency between submersion of the tail and its removal from the water by the animal was recorded, with a maximal cut-off time of 10 s to avoid tissue damage. Pretreatment latencies were determined in mice, three times with an interval of 24 h starting 3 days before drug administration, to obtain a stable predrug response (basal latency). Mice with a latency value between 5.0 and 7.0 s were used. The average basal latency for the experiments was 5.7 ± 0.01 s. The test latency after drug administration was assessed at the appropriate time. The tail immersion response was expressed as percentage of basal latency.
Immunoblotting. To examine changes in expression of NR2B protein in the spinal cord, seven groups of mice (n = 6) were used for immunoblot analysis: naive, three pSM2-NS-treated groups (0.5, 1.0, and 1.5 µg/mouse), and three pSM2-grin2b-treated groups (0.5, 1.0, and 1.5 µg/mouse). Animals were euthanized by decapitation under anesthesia (120 mg/kg i.p. pentobarbital). Spinal cords were extracted by injecting normal saline under pressure through a 16-gauge needle into the upper sacral spine, thus forcing the spinal cord out rostrally. The L4–L6 dorsal spinal cord segments were rapidly dissected and homogenized with a hand-held pellet pestle in 250 µl of lysis buffer [20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS, and Complete protease inhibitor cocktail from Roche (Indianapolis, IN; 1 tablet/50 ml of lysis buffer)] and centrifuged at 13,000g for 3 min at 25°C. The supernatant was decanted and used for the subsequent Western blot analyses. The protein concentration of the supernatant was measured using a Pierce bicinchoninic acid protein assay kit (Pierce, Norcross, GA). NR2B protein levels were determined using an immunoblotting procedure essentially as described previously (Ritter et al., 1999; Guillemette et al., 2000). Proteins were separated on a 7.5% SDS-polyacrylamide gel (Bio-Rad; Hercules, CA) and transferred to a nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were blocked with SuperBlock T-20 (Tris-buffered saline) blocking buffer (Pierce) and incubated overnight at 4°C with rabbit polyclonal antibody to NR2B (1:500 dilution; Novus Biologicals, Littleton, CO). Proteins were visualized by incubation with ECL Plus Western Blotting detection reagents (GE Healthcare, Piscataway, NJ) and scanned at 200-µm resolution using the Molecular Dynamics STORM system (GE Healthcare). Images were analyzed by using ImageQuant software 5.2 (GE Healthcare), and data were expressed as volume of pixilation. Because of the molecular weight disparity between NR2B and -actin, duplicate blots were prepared at the same time and probed with a purified mouse monoclonal anti--actin, peroxidase (1:35,000 dilution; Sigma), and visualized as described above and used as a loading control.
Pharmacological Treatments. In the first experiment, a dose-response curve for (S)-3,5-DHPG (1.5–15 nmol/mouse) was performed at 10-min intervals until 80 min after the drug administration. The following experiments then examined whether the hyperalgesic effects of (S)-3,5-DHPG could be inhibited by pretreatment with various antagonists. MPEP (12.5–50 nmol/mouse), 3-MA-TIDA (0.5–5 nmol/mouse), CGS 19755 (0.01–5 pmol/mouse), CGP 78608 (1–100 pmol/mouse), ifenprodil (0.5–2 nmol/mouse), Ro 25-6981 (0.02–2 nmol/mouse), NVP-AAM077 (0.005–10 nmol/mouse), bisindolylmaleimide I (1–100 pmol/mouse), Go-7874 (0.25–2.5 nmol/mouse), PKI-(14–22)-amide (0.025–2.5 nmol/mouse), or DT-3 (0.01–10 nmol/mouse) was administered 5 min before a 15 nmol/mouse i.t. injection of (S)-3,5-DHPG (the dose that produced maximal hyperalgesia) and then reassessed for the tail immersion test 30 min later (time point that showed maximal hyperalgesia).
In another series of experiments, mice were administered Arrest-In vehicle pSM2-NS (0.5, 1.0, and 1.5 µg/mouse) or pSM2-grin2b (0.5, 1.0, and 1.5 µg/mouse), 7 days before a 15 nmol/mouse i.t. injection of (S)-3,5-DHPG, and then reassessed for the tail immersion test 30 min later. Spinal cords were then extracted for Western blot analyses. Groups of 6 to 12 mice per dose per treatment were tested for each compound following baseline determination (discussed in the section entitled "The Tail Immersion Test").
Statistical Analysis. Data are expressed as mean values ± S.E.M. Time course studies were analyzed using two-factor (drug treatment x time) repeated measures analysis of variance. A significant F-value led to post hoc analysis of data using the Student Newman-Keuls test with Instat 3.0 software (GraphPad Software, San Diego, CA). The influence of increasing doses of antagonists and inhibitors to block (S)-3,5-DHPG-induced hyperalgesia was compared with mice treated with vehicle + (S)-3,5-DHPG using analysis of variance. Dose-dependent antagonism of (S)-3,5-DHPG-induced hyperalgesia led to calculation of ID50 values, using least-squares linear regression analysis followed by calculation of 95% confidence limits (95% CL) by the method of Bliss (1967).
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50% decrease in the tail immersion reaction time) (P < 0.001). All hyperalgesic effects disappeared between 70 and 80 min from the injection time of (S)-3,5-DHPG. mGluR1/5 Antagonists Inhibit (S)-3,5-DHPG-Mediated Hyperalgesia. The mGluR5 antagonist MPEP, injected i.t. 5 min before (S)-3,5-DHPG, blocked in a dose-dependent manner the (S)-3,5-DHPG-mediated hyperalgesia [F(3,20) = 23.8; P < 0.001] (Table 1; Fig. 2). Post hoc analyses indicated that the 12.5, 25, and 50 nmol/mouse MPEP producing a 45, 61, and 93% inhibition of (S)-3,5-DHPG-induced hyperalgesia, respectively, were all significant (P < 0.001). Likewise, the mGluR1 antagonist 3-MATIDA, administered i.t. 5 min before (S)-3,5-DHPG, resulted in a dose-dependent inhibition of the (S)-3,5-DHPG-induced hyperalgesia [F(3,20) = 27.7; P < 0.001] (Table 1; Fig. 2). Post hoc analyses indicated that the 0.5, 1, and 5 nmol/mouse 3-MATIDA producing a 51, 67, and 98% inhibition of (S)-3,5-DHPG-induced hyperalgesia, respectively, were all significant (P < 0.001). The two antagonists did not change the tail immersion response in control mice within the range of doses used (data not shown).
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NMDAR Antagonists Inhibit (S)-3,5-DHPG-Mediated Hyperalgesia. Experiments were conducted to test the hypothesis that activation of group I mGluRs leads to stimulation of NMDARs. The (S)-3,5-DHPG-induced hyperalgesia was blocked in a dose-dependent fashion when mice were pretreated with the competitive NMDA receptor antagonist CGS 19755 [F(3,20) = 63.4; P < 0.001] (Table 1; Fig. 3). Post hoc analyses indicated that the 0.01, 1, and 5 pmol/mouse doses of CGS 19755 producing a 32, 53, and 91% inhibition of (S)-3,5-DHPG-induced hyperalgesia, respectively, were all significant (P < 0.001). In addition, the NMDA glycine site antagonist CGP 78608, administered i.t. 5 min before (S)-3,5-DHPG, induced a dose-dependent inhibition of the (S)-3,5-DHPG-induced hyperalgesia [F(3,49) = 65.5; P < 0.001] (Table 1; Fig. 3). Post hoc analyses indicated that the 1, 10, and 100 pmol/mouse doses of CGP 78608 producing a 19, 57, and 94% inhibition of (S)-3,5-DHPG-induced hyperalgesia, respectively, were all significant (P < 0.001).
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All of the doses of the drugs that blocked (S)-3,5-DHPG-induced hyperalgesia were inactive when administered alone to mice (data not shown). Very high doses of NVP-AAM077 (5–10 nmol/mouse) impaired motor activity in control mice and produced a maximal response in the tail-immersion test (data not shown). The highest inactive dose of NVP-AAM077 (0.5 nmol/mouse) that did not elicit toxic effects in control mice was tested, and it had no effect on the (S)-3,5-DHPG-induced hyperalgesia (Fig. 3).
Signal Transduction between Type I mGluRs and NMDAR Activation. The hyperalgesia observed in (S)-3,5-DHPG-treated mice was blocked by pretreatment with the selective inhibitor of PKC, I, II, , , and , bisindolylmaleimide I, the selective inhibitor of PKC, I, II, and , Go-7874, or the selective PKA inhibitor PKI-(14–22)-amide (Table 1; Fig. 4). Bisindolylmaleimide I, injected i.t. 5 min before (S)-3,5-DHPG, blocked in a dose-dependent manner the (S)-3,5-DHPG-induced hyperalgesia [F(3,44) = 61.4; P < 0.001]. Post hoc analyses indicated that the 0.1, 10, and 100 pmol/mouse of bisindolylmaleimide I producing a 27, 56, and 89% inhibition of (S)-3,5-DHPG-induced hyperalgesia, respectively, were all significant (P < 0.001). In a similar fashion, Go-7874 dose-dependently inhibited the (S)-3,5-DHPG-mediated hyperalgesia [F(3,47) = 78.7; P < 0.001] (Table 1; Fig. 4). Post hoc analyses indicated that the 0.5, 1, and 2.5 nmol/mouse Go-7874 producing a 20, 60, and 100% inhibition of (S)-3,5-DHPG-induced hyperalgesia, respectively, were all significant (P < 0.001).
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On the other hand, the selective PKA inhibitor PKI-(14–22)-amide was also shown to block the (S)-3,5-DHPG-mediated hyperalgesia in a dose-dependent manner [F(3,38) = 70.4; P < 0.001] (Table 1; Fig. 4). Post hoc analyses indicated that the 0.25 and 2.5 nmol/mouse PKI-(14–22)-amide producing a 58 and 91% inhibition of (S)-3,5-DHPG-induced hyperalgesia, respectively, were significant (P < 0.001). In contrast, the dose of 0.025 nmol/mouse PKI-(14–22)-amide did not produce any significant effect in inhibiting the (S)-3,5-DHPG-induced hyperalgesia. It is worth mentioning that the selective PKG inhibitor DT-3 had no effect on hyperalgesia (Fig. 4).
All of the doses of the PKC and PKA inhibitors that blocked (S)-3,5-DHPG-induced hyperalgesia were inactive when administered alone to mice (data not shown). High doses of DT-3 (0.01–10 nmol/mouse) induced motor deficits in control mice and produced a maximal response in the tail-immersion test. However, the highest inactive dose of DT-3 (2 nmol/mouse) failed to block (S)-3,5-DHPG-induced hyperalgesia (Fig. 4).
pSM2-grin2b Injection Silences the NR2B Gene and Blocks the (S)-3,5-DHPG-Mediated Hyperalgesia. Mice were injected i.t. with 1.5 µg of either pSM2-grin2b, an NR2B shRNA expression plasmid, or pSM2-NS, a nonsilencing shRNA. As seen in Fig. 5, the (S)-3,5-DHPG-induced hyperalgesia, 7 days later, was blocked by the pSM2-grin2b expression plasmid but not by the pSM2-NS control vector treatment [F(3,20) = 29.3; P < 0.001]. The blockade of hyperalgesia was correlated with diminished protein levels of the NR2B subunit of the NMDAR shown in Western blotting. Mice injected with pSM2-grin2b showed a dose-dependent decrease in NR2B subunit protein levels [F(3,20) = 30.4; P < 0.001] (Fig. 6). Post hoc analyses indicated that the 0.5, 1, and 1.5 µg/mouse pSM2-grin2b producing a 30, 41, and 53% decreases in the relative NR2B protein levels as percentage of control, respectively, were all significant (P < 0.001). The (S)-3,5-DHPG-induced hyperalgesia was unaffected by the transfecting agent Arrest-In. It is worth mentioning that the nonsilencing shRNA had no effect on the tail immersion reaction time in tested mice.
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Antagonism of Type I mGluRs and NMDARs Blocks the (S)-3,5-DHPG-Induced Hyperalgesia. It is known that activation of group I mGluRs produces nocifensive behaviors in rats, although the mechanisms of these effects were unclear. A number of studies have indicated a role for group I mGluRs in central sensitization and hyperalgesia (Fisher and Coderre, 1996; Neugebauer et al., 1999; Karim et al., 2001). In contrast, i.t. application of group II and group III agonists did not produce nociceptive effects (Fisher and Coderre, 1996); moreover, in a rat model of neuropathic pain, they were shown to decrease mechanical and cold allodynia (Fisher et al., 2002). Evidence thus far has suggested that group I mGluRs tend to be excitatory, whereas groups II and III mGluRs have an inhibitory role in the spinal cord dorsal horn. In the present study, i.t. administration of the mGluR1/5 agonist (S)-3,5-DHPG to mice resulted in significant hyperalgesia as assessed by the tail immersion test. The pretreatment of mice with mGluR1 or mGluR5 antagonists dose-dependently blocked the development of thermal hyperalgesia in mice. These antagonists given i.t. did not produce an effect in control mice, suggesting a selective effect on (S)-3,5-DHPG-induced hyperalgesia. Importantly, competitive antagonists of the NMDAR or its glycine-binding site significantly blocked group I mGluR-mediated hyperalgesia, suggesting that the coupling between mGluR-NMDAR facilitates ionotropic synaptic transmission and spinal desensitization.
NR2B Gene Knockdown Blocks the (S)-3,5-DHPG-Induced Hyperalgesia. The present study addresses the coupling of the group I mGluRs to the NMDARs in mediating thermal hyperalgesia in mice. Competitive antagonism of the NMDAR or its glycine-binding site significantly blocked group I mGluR-mediated hyperalgesia We also demonstrated for the first time that gene knockdown of the NR2B subunit of the NMDAR or pretreatment with a selective NR2B subunit antagonist blocked (S)-3,5-DHPG-induced hyperalgesia. These results demonstrate that NR2B subunits in the spinal cord play an important role in maintaining functional NMDARs. In contrast, recent evidence indicates that nociceptive stimuli were more selective at increasing NR2A subunit phosphorylation in the brain rather than in the spinal cord. In a model of inflammation, tyrosine-phosphorylation of NR2A was up-regulated in the rostral ventromedial medulla (Turnbach et al., 2003), whereas it was unchanged at the spinal level (Guo et al., 2002). This could account for why the NR2A subunit antagonist NVP-AAM077 failed to significantly block (S)-3,5-DHPG-induced hyperalgesia (Fig. 3). These findings suggest that the activation of mGluRs is key to initiating enhanced NMDA receptor responses.
Signal Transductions Mechanisms Linking the Type I mGluRs and the NMDARs. The (S)-3,5-DHPG-induced hyperalgesia was blocked by PKC and PKA, but not PKG inhibitors, indicating that activation of mGluR receptors leads to increases in the activity of both PKC and PKA. mGluR1/5 receptors stimulate phospholipase C through Gq/11 proteins to convert phosphatidylinositol bisphosphate into diacylglycerol and IP3. Diacylglycerol activates Ca2+-dependent (conventional) and independent (novel) PKC isoforms, which can phosphorylate serine/threonine sites on the NMDAR (Sánchez-Pérez and Felipo, 2005). Stimulation of mGluR receptors in rats with neuropathic pain leads to NMDAR-induced sensitization and increases in PKC activity. This effect was prevented by transient knockdown of mGluR1 receptors with antisense (Fundytus et al., 2001). In addition, mGluR stimulation could increase the activity of PKA through homer protein-linked stimulation of IP3 receptors (Sala et al., 2005). The Ca2+ that is released from IP3-sensitive Ca2+ pools is known to stimulate the Ca2+-dependent isoforms of adenylyl cyclase. The finding that a selective PKG inhibitor could not block the (S)-3,5-DHPG-induced hyperalgesia confirms the lack of linkage between mGluRs stimulation and activation of the guanylyl cyclase pathway. Taken together, it could be suggested that the ionotropic function of the NMDARs in mediating thermal hyperalgesia in vivo is subject to regulation that is initiated by mGluRs/G-protein-linked mechanisms.
Protein Structure Linking mGluRs to NMDARs. The spinal dorsal horn is the major site for initial nociceptive processing. The results of our study confirm previous coimmunoprecipitation experiments, which suggested in the dorsal horn, that the mGluR-Homer-Shank complex and NMDAR are biochemically linked through related postsynaptic density proteins such as PSD-95 and Shank (Guo et al., 2004). The cytoskeletal elements and adaptor proteins that make up the postsynaptic complex linking the mGluR to the NMDAR also include protein kinases (Tezuka et al., 1999; Lei et al., 2002; Guo et al., 2004) that are important modulators of NMDAR channels. Accordingly, we suggested that the NR2B subunit is phosphorylated by kinases intrinsic to this protein complex. The present study show that the effects of group I mGluR agonists are coupled to NR2B phosphorylation and that this coupling requires PKC and PKA, but not PKG activation. The coupling of the mGluRs to the NMDARs in dorsal horn neurons is supported by the fact that selective mGluR antagonists block inflammation-induced NR2B tyrosine-phosphorylation and selective mGluR agonist-induced NR2B tyrosine-phosphorylation in spinal slices (Guo et al., 2004). Previous studies have shown that activation of group I mGluRs enhances NMDA-induced currents (Bond and Lodge, 1995) and the NMDA-mediated increase in intracellular calcium concentration in dorsal horn neurons (Bleakman et al., 1992). A role of the mGluRs in central sensitization and persistent pain has been suggested previously (Morisset and Nagy, 1996; Neugebauer et al., 1999; Karim et al., 2001). In addition, it has been found that a coactivation of hippocampal mGluRs and NMDARs is required for the induction of long-term potentiation (LTP) (Fujii et al., 2004). Moreover, in mice lacking mGluR5, LTP was significantly reduced in NMDAR-dependent pathways, such as the CA1 region and dentate gyrus of the hippocampus. However, LTP was not altered in the CA3 region, an NMDAR-independent pathway (Lu et al., 1997).
NMDAR-NR2B Subunit Provides a New Target for Pain Management. In summary, our results demonstrated a coupling between the mGluR and the NR2B subunit of the NMDAR in the spinal cord, and this coupling involved the activation of PKC and PKA mechanisms. Ongoing projects in our laboratory are targeting the structural proteins that link mGluRs and NMDARs and their role in pain transmission in acute and chronic animal pain models. These biochemical events are correlated with the development of thermal hyperalgesia and may underlie the mechanisms of spinal sensitization. An important implication from the present study is that mGluRs facilitate ionotropic synaptic transmission as a mechanism of modulation in the spinal cord. It is known that phosphorylation of the NMDAR results in an increase in NMDAR channel current and Ca2+ influx through the channel (Wang and Salter, 1994). Thus, one functional consequence of mGluR activation under painful stimuli is to prime NMDAR for further enhanced hyperexcitability. This mechanism may be a critical initiator for central nociceptive sensitization.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NMDA, N-methyl-D-aspartic acid; NMDAR, N-methyl-D-aspartic acid receptor; NR1, NMDA-R1 subunit; NR2, NMDA-R2 subunit; mGluRs, metabotropic glutamate receptors; PKC, protein kinase C; PKA, protein kinase A; PKG, protein kinase G; (S)-3,5-DHPG, (S)-3,5-dihydroxyphenylglycine; (S)-4C3HPG, (S)-4-carboxy-3-hydroxyphenylglycine; MPEP, 2-methyl-6-(phenylethynyl)-pyridine; 3-MATIDA, -amino-5-carboxy-3-methyl-2-thiopheneacetic acid; CGS 19755, cis-4-[phosphomethyl]-piperidine-2-carboxylic acid; CGP 78608, [(1S)-1-[[(7-bromo-1,2,3,4-tetrahydro-2,3-dioxo-5-quinoxalinyl)methyl]amino]ethyl]phosphonic acid; Ro 25-6981, [R-(R*,S*)]--(4-hydroxyphenyl)--methyl-4-(phenylmethyl)-1-piperidinepropanol hydrochloride; Go-7874, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; PKI-(14–22)-amide, Myr-N-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-NH2; DT-3, H-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Leu-Arg-Lys-Lys-Lys-Lys-Lys-His-OH; NVP-AAM077, [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid; shRNA, short-hairpin RNA; pSM2, pShag Magic version 2; NS, nonsilencing; IP3, inositol triphosphate; LTP, long-term potentiation.
Address correspondence to: Dr. William L. Dewey, Department of Pharmacology and Toxicology, Virginia Commonwealth University Medical Center, P. O. Box 980613, Richmond, VA 23298-0613. E-mail: wdewey{at}vcu.edu
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