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. 2004 Oct 13;24(41):9161-73.
doi: 10.1523/JNEUROSCI.3422-04.2004.

Group I Metabotropic Glutamate Receptor NMDA Receptor Coupling and Signaling Cascade Mediate Spinal Dorsal Horn NMDA Receptor 2B Tyrosine Phosphorylation Associated With Inflammatory Hyperalgesia

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Group I Metabotropic Glutamate Receptor NMDA Receptor Coupling and Signaling Cascade Mediate Spinal Dorsal Horn NMDA Receptor 2B Tyrosine Phosphorylation Associated With Inflammatory Hyperalgesia

Wei Guo et al. J Neurosci. .
Free PMC article

Abstract

Hindpaw inflammation induces tyrosine phosphorylation (tyr-P) of the NMDA receptor (NMDAR) 2B (NR2B) subunit in the rat spinal dorsal horn that is closely related to the initiation and development of hyperalgesia. Here, we show that in rats with Freund's adjuvant-induced inflammation, the increased dorsal horn NR2B tyr-P is blocked by group I metabotropic glutamate receptor (mGluR) antagonists [7-(hydroxyimino)cyclopropa[b] chromen-1a-carboxylate ethyl ester (CPCCOEt) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP), by the Src inhibitor CGP 77675, but not by the MAP kinase inhibitor 2'-amino-3'-methoxyflavone. Analysis of the calcium pathways shows that the in vivo NR2B tyr-P is blocked by an IP3 receptor antagonist 2-aminoethoxydiphenylborate (2APB) but not by antagonists of ionotropic glutamate receptors and voltage-dependent calcium channels, suggesting that the NR2B tyr-P is dependent on intracellular calcium release. In a dorsal horn slice preparation, the group I (dihydroxyphenylglycine), but not group II [(2R,4R)-4-aminopyrrolidine-2,3-dicarboxylate] and III [L-AP 4 (L-(+)-2-amino-4-phosphonobutyric acid)], mGluR agonists, an IP3 receptor (D-IP3) agonist, and a PKC (PMA) activator, induces NR2B tyr-P similar to that seen in vivo after inflammation. Coimmunoprecipitation indicates that Shank, a postsynaptic density protein associated with mGluRs, formed a complex involving PSD-95 (postsynaptic density-95), NR2B, and Src in the spinal dorsal horn. Double immunofluorescence studies indicated that NR1 is colocalized with mGluR5 in dorsal horn neurons. mGluR5 also coimmunoprecipitates with NR2B. Finally, intrathecal pretreatment of CPCCOEt, MPEP, and 2APB attenuates inflammatory hyperalgesia. Thus, inflammation and mGluR-induced NR2B tyr-P share similar mechanisms. The group ImGluR-NMDAR coupling cascade leads to phosphorylation of the NMDAR and appears necessary for the initiation of spinal dorsal horn sensitization and behavioral hyperalgesia after inflammation.

Figures

Figure 1.
Figure 1.
Selective increase in NR2B tyr-P after hindpaw inflammation. Proteins were extracted from L4,5 spinal cord of noninflamed naive (N) rats and rats at 24 hr after complete Freund's adjuvant or saline injection. The spinal dorsal horn was divided at the midline into the ipsilateral (ipsi) and contralateral (Con) halves. Cervical spinal cord (Cer) was used as a control. The top blot shows the immunoreactive bands against anti-phosphotyrosine 4G-10 (PY-NR2B) after immunoprecipitation of extracted proteins with anti-NR2B antibodies. The bottom blot shows immunobands against NR2B antibodies after stripping and reprobing the same membrane previously probed with 4G-10 antibodies. The levels of tyr-P were normalized to the respective NR2B immunoreactive bands. The relative phosphotyrosine protein levels (mean ± SEM) are expressed as a percentage of the naive controls for the purpose of illustration. Raw data were used for statistical comparisons. The asterisks indicate significant differences (p < 0.05) from the naive controls. n = 5 per time point. The dashed line indicates the control levels in noninflamed rats.
Figure 2.
Figure 2.
Inflammation-induced in vivo NR2B tyr-P was blocked by group I mGluR antagonists and a Src inhibitor. The drugs were injected intrathecally 10 min before CFA injection. DMSO (0.01 ml) was a vehicle control. Dorsal spinal cord tissues were collected at 30 min after inflammation of both hindpaws. In each panel, representative immunoblots against anti-4G-10 (PY-NR2B) and anti NR2B (bottom) antibodies are shown at the top. The relative levels of tyrosine-phosphorylated NR2B proteins from four individual experiments are shown below as a percentage of the naive controls (mean ± SEM). The dashed line indicates the control level. Statistical comparisons were made among all groups using raw data. *p < 0.05 versus naive rats. A, The effect of CPCCOEt (2 μmol; n = 6; left), an mGluR1 antagonist, and MPEP (170 nmol; n = 4; right), a mGluR5 antagonist. B, The inflammation-induced NR2B tyr-P requires Src activity. The NR2B tyr-P was blocked by intrathecal pretreatment of CGP 77675 (100 pmol; n = 5), an Src family protein tyrosine kinase inhibitor (left). PD98059 (4 nmol; n = 6; i.t.), a MAPKK inhibitor, did not prevent NR2Btyr-P after inflammation (right). C, The basal levels of NR2Btyr-P in naive rats were not affected by the same dose of CGP 77675, CPCCOEt, MPEP, and 2APB compared with vehicle (VEH)-treated rats (n = 2).
Figure 3.
Figure 3.
Contribution of calcium-releasing pathways to NR2B tyr-P. Drugs were injected intrathecally 10 min before CFA injection into the two hindpaws. DMSO (0.01 ml) and saline (0.01 ml) were vehicle controls. Dorsal horn tissues were collected at 30 min after inflammation. A, IP3 receptor inhibitor 2APB (1.0 nmol; n = 6; right) blocked inflammation-induced NR2B tyr-P. B, Ionotropic glutamate receptor antagonists MK-801 (60 nmol; n = 4; left) and NBQX (13 nmol; n = 3; right) did not block NR2Btyr-P. C, VDCC blockers ω-conotoxin GVIA (1.3 nmol; n = 3; left), NIMO (120 nmol; n = 3; left), andω-conotoxin MVIIC (100 nmol; n = 4; right) did not prevent inflammation-induced NR2B tyr-P. *p < 0.05 versus naive rats.
Figure 4.
Figure 4.
The mGluR agonists induced NR2B tyr-P in vitro. The transverse spinal cord slice was obtained from adult 8- to 10-week-old rats. Spinal slices were incubated with drugs for 15 min before protein extraction. In all panels, representative immunoblots against anti-4G-10 (PY-NR2B) and anti-NR2B antibodies are shown at the top, and mean relative levels of tyrosine-phosphorylated NR2B proteins are shown in the bar graphs. The dashed line in the bar graphs indicates the percentage of naive level. *p < 0.05 versus naive untreated slices. A, Selective group I (DHPG; 100 μm; n = 10) but not group II (APDC; 100 μm; n = 6) and group III (l-AP-4; 100 μm; n = 6) mGluR agonists induced NR2Btyr-P in spinal slice. DHPG-induced NR2Btyr-P was blocked by CPCCOEt (100 μm; n = 6) and MPEP (10 μm; n = 6) (B), an Src inhibitor, CGP 77675 (CGP; 10 μm; n = 6) (C, left), a PKC inhibitor, chelerythrine (Che; 10 μm; n = 4) (C, middle), and an IP3 receptor inhibitor 2APB (72 μm; n = 6) (C, right). D, The basal levels of NR2B tyr-P in naive rats were not affected by the same dose of Che, CGP 77675, CPCCOEt, MPEP, and 2APB compared with vehicle (VEH)-treated rats (n = 2).
Figure 5.
Figure 5.
IP3 and PMA induced NR2B tyr-P in vitro. A, d-IP3 (100 μm; n = 4), an IP3 receptor activator, induced a significant increase in NR2B tyr-P compared with untreated (naive) slices. The increased NR2B tyr-P was blocked by pretreatment with CGP 77675 (10 μm; n = 4). l-IP3 (100 μm; n = 4), an inactive analog of d-IP3, did not produce a significant effect. The slices were permeabilized by exposure to a brief (10 sec) application of saponin (0.001%) to allow penetration of IP3 through cell membrane. Saponin itself [saponin plus vehicle (Veh)] did not affect the level of NR2B tyr-P. B, Direct application of PMA to spinal slices to activate PKC, a downstream event to IP3-mediated intracellular release, also mimicked CFA-induced NR2B tyr-P. PMA (1 μm; n = 5) produced a significant increase in NR2B tyr-P in the spinal slice. The inactive analog 4αPDD (10 μm; n = 5) did not produce a significant effect. The PMA-induced NR2B tyr-P was blocked by inhibitors of Src (CGP 77675, 10 μm) and PKC [chelerythrine (Che)]. *p < 0.05 versus naive untreated slices.
Figure 6.
Figure 6.
Coimmunoprecipitation (IP) of Shank, NR2B, PSD-95, and Src in the spinal dorsal horn. Spinal dorsal horn tissues were taken from naive rats and inflamed rats at 30 min after injection of CFA into the hindpaw. Proteins were immunoprecipitated with Src (middle) or Shank (right) antibodies and probed with antibodies against Shank, NR2B, GluR1, PSD-95, and Src. Note that boiling of the samples before IP eliminated associated proteins, leaving only Src (middle, bottom) of Shank (left, top) that was directly pulled down by respective antibodies. GluR1 protein was not coimmunoprecipitated with Src or Shank antibodies.
Figure 7.
Figure 7.
Immunohistochemical localization of Shank and Src in the spinal cord of the rat. Shank-like (A, C) and Src-like (B, D) immunoreactivity is distributed in spinal neurons. Low-power views of the lumbar spinal cord are shown in A and B. The horizontal lines in A and B separate the spinal cord into the dorsal (top) and ventral (bottom) halves. The dorsal half of the spinal cord was used for phosphorylation analysis. C and D show enlarged fields of dorsal horn from A and B, respectively. C1-2 and D1-2 illustrate images enlarged from the respective superficial (C1, D1) and deep (C2, D2) dorsal horn regions in C and D, indicated by small rectangles. Scale bars: A, B, 0.4 mm; C, D, 0.2 mm; C1-2, D1-2, 0.025 mm.
Figure 8.
Figure 8.
Colocalization of NMDAR and mGluR5 in the spinal dorsal horn. A, NMDAR NR1 subunit-like immunoreactivity is visualized with Cy3-tyramide (red). B, mGluR5-like immunoreactivity is shown as green fluorescence after reacting with goat anti-rabbit IgG conjugated to Alexa Fluor 488. C, Overlap of panels A and B illustrating dorsal horn neurons that exhibit double fluorescence (yellow-orange; arrows), suggesting colocalization of NMDAR/mGluR5. The area shown includes laminas IV-VI of the lateral spinal dorsal horn medial to the lateral funiculus (lfu). A1, B1, and C1 are enlarged from rectangles in A, B, and C, respectively. The NR1 staining showed numerous punctate profiles in the cell membrane, dendrite, and cytoplasm (A1), whereas mGluR5-like immunoreactivity appeared uniformly in the cell membrane and cytoplasm (B1). C1 shows the same neuron in A1 and B1 that is double labeled with anti-NR1 and mGluR5 antibodies. Scale bars: A-C, 0.04 mm; A1-C1, 0.01 mm. D, Coimmunoprecipitation of NR2B and mGluR5 in the spinal dorsal horn. Spinal dorsal horn tissues were taken from naive rats and inflamed rats at 30 min after injection of CFA into the hindpaw. Proteins were immunoprecipitated with NR2B antibodies and probed with antibodies against NR2B and mGluR5. Note that boiling of the samples before IP eliminated mGluR5, leaving only NR2B that was directly pulled down by NR2B antibodies.
Figure 9.
Figure 9.
Effects of selective group I mGluR antagonists on inflammatory hyperalgesia-allodynia. A, Stimulus-response frequency curves illustrating examples of the intensity-dependent paw withdrawal responses to mechanical stimuli. Each curve was established with a series of subthreshold to suprathreshold range of von Frey filament forces, and the response frequency is plotted against the stimulus intensity. Inflammation was induced in the left hindpaw. The stimulus-response curves for vehicle, CPCCOEt (mGluR1 antagonist), and MPEP (mGluR5 antagonist) overlapped before inflammation (pre-CFA), indicating consistent responses. There was a leftward shift of the curve in vehicle-treated rats at 30 min post-CFA, compared with the pre-CFA curve, suggesting the development of mechanical hyperalgesia and allodynia. At 30 min post-CFA in CPCCOEt- and MPEP-treated rats, the stimulus-response curves were shifted to the right compared with vehicle-treated rats, suggesting an attenuation of hyperalgesia-allodynia. At 2 hr post-CFA, the curves shifted back to the left in CPCCOEt- and MPEP-treated rats as hyperalgesia was recovering from the drug effect. B, EF50 values, defined as the von Frey filament force (g) that produces a 50% response frequency, were derived from the stimulus-response function curve and used as a measure of mechanical sensitivity. Pretreatment (10 min before CFA) of rats with CPCCOEt (2 μmol; n = 5), MPEP (170 nmol; n = 5), or 2APB (1 nmol; n = 6) prevented the early development of hyperalgesia-allodynia on the inflamed paw as shown by significantly higher EF50 values when compared with vehicle-treated rats (DMSO; 0.01 ml; n = 4). The responses of the contralateral noninflamed paw were not affected by the drug treatment. C, CPCCOEt (2 μmol; n = 4), MPEP (170 nmol; n = 5), or 2APB (1 nmol; n = 6) were administered intrathecally at 1 d post-CFA when mechanical hyperalgesia-allodynia had developed. There was a slight increase in EF50 values that lasted for ∼5 min after CPCCOEt and MPEP treatment. The 95% confidence limit of the EF50 values is shown as a vertical line in each bar in B and C.

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