Spinal cord NR1 serine phosphorylation and NR2B subunit suppression following peripheral inflammation

Abstract

Background: Spinal cord N-methyl-D-aspartate (NMDA) receptors are intimately involved in the development and maintenance of central sensitization. However, the mechanisms mediating the altered function of the NMDA receptors are not well understood. In this study the role of phosphorylation of NR1 splice variants and NR2 subunits was examined following hind paw inflammation in rats. We further examined the level of expression of these proteins following the injury.

Results: Lumbar spinal cord NR1 subunits were found to be phosphorylated on serine residues within two hours of the induction of hind paw inflammation with carrageenan. The enhanced NR1 serine phosphorylation reversed within six hours. No phosphorylation on NR1 threonine or tyrosine residues was observed. Likewise, no NR2 subunit phosphorylation was observed on serine, threonine or tyrosine residues. An analysis of NR1 and NR2 protein expression demonstrated no change in the levels of NR1 splice variants or NR2A following the inflammation. However, spinal cord NR2B expression was depressed by the hind paw inflammation. The expression of NR2B remained depressed for more than one week following initiation of the inflammation.

Conclusion: These data suggest that NR1 serine phosphorylation leads to an initial increase in NMDA receptor activity in the spinal cord following peripheral injury. The suppression of NR2B expression suggests compensation for the enhanced nociceptive activity. These data indicate that spinal cord NMDA receptors are highly dynamic in the development, maintenance and recovery from central sensitization following an injury. Thus, chronic pain therapies targeted to NMDA receptors should be designed for the exact configuration of NMDA receptor subunits and post-translational modifications present during specific stages of the disease.

Figure 1

Selectivity of rabbit polyclonal antibodies raised against splice variants of the NR1 subunit of the NMDA receptor. Antibodies were raised against synthetic peptides and used for western blots. The peptide sequences used to raise the antibodies were SKKRNYENLDQLSYDNKRGPC, DRKSGRAEPDPKKKATFRAC, PRRAIEREEGQLQLC, and QYHPTDITGPLNLSDPS for exons 5 (N1), 21 (C1), 22 (C2) and 22 minus (C2') respectively. The antibodies were purified with affinity columns and tested against cloned splice variants of the NR1 subunit transfected into COS-7 cells. The three numbers at the top of the columns identify the splice variant clones. The first number indicates the presence (1) or absence (0) of N1, the second C1 and the third C2. The protein was compared to protein taken from non-transfected cells and rat brain homogenates.

Figure 2

Time course of carrageenan induced inflammation on heat hyperalgesia and mechanical allodynia. A. Paw withdrawal latencies from a thermal stimulus before and following injection of 6 mg carrageenan into the plantar surface of the left hind paw. Data are means ± SEM (N = 8 rats). Asterisks indicate p < 0.05 ANOVA followed by Dunnett's test. B. Thresholds for paw withdrawal from a mechanical stimulus before and following the injection of 6 mg carrageenan into the plantar surface of the left hind paw. Data are means ± SEM (N = 8 rats). Asterisks indicate p < 0.05 ANOVA followed by Dunnett's test.

Figure 3

Serine phosphorylation of spinal cord NR1 subunits following carrageenan induced hind paw inflammation. A. Time course of serine phosphorylation on NR1 subunits immunoprecipitated by antibodies that recognized all NR1 subunits, N1 containing splice variants or C1 containing splice variants. The western blots at the top are representative experiments where the immunoprecipitated protein was probed with anti-phosphoserine antibodies. The bands migrated at approximately 118 kd. The graph summarizes the band density data from the western blots. Data are means ± SEM of the normalized band density data (NR1: N = 6 rats per time point, N1: N = 5 rats per time point, C1: N = 6 rats per time point). Asterisks indicate p < 0.05 ANOVA followed by Dunnett's test on the raw data. B. Comparison of serine phosphorylation on NR1 subunits from the dorsal and ventral spinal cord four hours following the injection of either saline or carrageenan (6 mg) into the left hind paws. At the top is a representative western blot demonstrating the immunoprecipitated NR1 after it was probed with anti-phosphoserine antibodies. The graph is a summary of the normalized band density data for control animals (N = 5), saline injected animals (N = 5) and carrageenan injected animals (N = 5). Data are means ± SEM. Asterisks indicate p < 0.05 ANOVA followed by Dunnett's test.

Figure 4

Phosphorylation of threonine and tyrosine residues on spinal cord NR1 subunits. No phosphorylation of NR1 threonine or tyrosine residues was found in response to peripheral inflammation. A. Lumbar spinal cord NR1 subunits were immunoprecipitated with an antibody that recognizes all NR1 splice variants or an antibody that recognizes splice variants with the C1 cassette before and after left hind paw inflammation with carrageenan. The protein was run on western blots and probed with an anti-phosphothreonine antibody. The western blots at the top are representative experiments and the graph at the bottom presents the mean ± SEM band density of all experiments (Total NR1; N = 8 rats per time point, C1 cassette; N = 9 rats per time point). ANOVA p > 0.05. The molecular weight of the bands on the western blots was ~118 kd. B. NR1 subunit protein was immunoprecipitated with an antibody that recognizes all NR1 splice variants or an antibody that recognizes splice variants with the C1 cassette, run on western blots and probed with an anti-phosphotyrosine antibody. The western blots at the top are representative experiments and the graph at the bottom presents the mean ± SEM band density of all experiments (Total NR1; N = 4 rats per time point, C1 cassette; N = 4 rats per time point). ANOVA p > 0.05.

Figure 5

Phosphorylation of NR2A and NR2B subunits. NR2A and NR2B were immunoprecipitated from rat lumbar spinal before and after carrageenan induced inflammation of the left hind paw. The protein was run on western blots and probed with anti-phosphoserine, anti-phosphothreonine or anti-phosphotyrosine antibodies. Single bands were identified on the western blots that migrated at ~180 kd. A. Phosphoserine on NR2A and NR2B subunits. The western blots at the top demonstrate representative experiments and the graph below presents the mean ± SEM band density for all experiments (NR2A N = 4 rats per time point, NR2B N = 4 rats per time point). ANOVA p > 0.05. B. Phosphothreonine on NR2A and NR2B subunits. The western blots at the top demonstrate representative experiments and the graph below presents the mean ± SEM band density for all experiments (NR2A N = 4 rats per time point, NR2B N = 4 rats per time point). ANOVA p > 0.05. C. Phosphotyrosine on NR2A and NR2B subunits. The western blots at the top demonstrate representative experiments and the graph below presents the mean ± SEM band density for all experiments (NR2A N = 4 rats per time point, NR2B N = 4 rats per time point). ANOVA p > 0.05.

Figure 6

Peripheral inflammation does not influence NR1 protein expression in the lumbar spinal cord. Spinal cords were harvested before and after carrageenan injection into the left hind paw of rats, homogenized and run on western blots. The western blots were probed with antibodies that recognized all NR1 splice variants (N = 6 rats per time point), splice variants containing the N1 cassette (N = 8 rats per time point), splice variants with the C1 cassette (N = 8 rats per time point), splice variants with the C2 cassette (N = 4 rats per time point) and splice variants with the C2' cassette (N = 4 rats per time point). The western blots at the top are representative experiments and the graph a summary of all experiments. Bars are the mean ± SEM. ANOVA p > 0.05.

Figure 7

Expression of spinal cord NR2A and NR2B protein following peripheral inflammation. Rat lumbar spinal cords were harvested before and after carrageenan induced hind paw inflammation. The tissue was homogenized and run on western blots. The membranes were probed with antibodies that were selective for either NR2A (N = 4 rats per time point) or NR2B (N = 4 rats per time point). Single bands were identified at ~180 kd. The western blots are representative for NR2B and actin. Bars on the graph are means ± SEM of band density data normalized to the control bands. Asterisks indicate p < 0.05 ANOVA followed by Dunnett's test on the raw band densities.

Figure 8

Correlation between NR2B protein and recovery of mechanical allodynia following carrageenan induced hind paw inflammation. Data from 6 hours to 120 hours from the NR2B western blots (figure 7) and the mechanical allodynia data (figure 2) were plotted against each other. Deming linear regression indicates r² = -0.93, p = 0.0363.