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. 2012 Oct 11:222:392-403.
doi: 10.1016/j.neuroscience.2012.07.004. Epub 2012 Jul 13.

Transient decrease in nociceptor GRK2 expression produces long-term enhancement in inflammatory pain

L F Ferrari  1 , O Bogen, N Alessandri-Haber, E Levine, R W Gear, J D Levine
Affiliations

Transient decrease in nociceptor GRK2 expression produces long-term enhancement in inflammatory pain

L F Ferrari et al. Neuroscience. .

Abstract

In heterozygous mice, attenuation of G-protein-coupled receptor kinase 2 (GRK2) level in nociceptors is associated with enhanced and prolonged inflammatory hyperalgesia. To further elucidate the role of GRK2 in nociceptor function we reversibly decreased GRK2 expression using intrathecal antisense oligodeoxynucleotide (AS-ODN). GRK2 AS-ODN administration led to an enhanced and prolonged hyperalgesia induced by prostaglandin E(2), epinephrine and carrageenan. Moreover, this effect persisted unattenuated 2weeks after the last dose of antisense, well after GRK2 protein recovered, suggesting that transient attenuation of GRK2 produced neuroplastic changes in nociceptor function. Unlike hyperalgesic priming induced by transient activation of protein kinase C epsilon (PKCε), (Aley et al., 2000; Parada et al., 2003b), the enhanced and prolonged hyperalgesia following attenuation of GRK2 is PKCε- and cytoplasmic polyadenylation element binding protein (CPEB)-independent and is protein kinase A (PKA)- and Src tyrosine kinase (Src)-dependent. Finally, rats treated with GRK2 AS-ODN exhibited enhanced and prolonged hyperalgesia induced by direct activation of second messengers, adenyl cyclase, Epac or PKA, suggesting changes downstream of G-protein-coupled receptors. Because inflammation can produce a decrease in GRK2, such a mechanism could help explain a predilection to develop chronic pain, after resolution of acute inflammation.

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Figures

Figure 1
Figure 1. Inflammatory hyperalgesia during GRK2 knockdown
A Western blot analysis. Analysis by Western blotting demonstrates a significant decrease in GRK2 protein levels in the saphenous nerve of rats treated for 3 consecutive days with antisense (AS)-ODN for GRK2 mRNA (39±9% when compared to the MM-treated rats; p=0.012, N=6 per group); B Potentiation of mechanical PGE2or epinephrine (EPI)induced hyperalgesia during GRK2 knockdown. Groups of rats were treated with antisense (AS)- or mismatch (MM)-ODN for GRK2 mRNA, for 3 consecutive days. On the 4th day, PGE2 (20 ng or 100 ng) or EPI (20 ng or 100 ng) was injected into the hind paw, in separate groups of rats. Mechanical nociceptive thresholds were evaluated 30 min, 4 h and 24 h later, using the Randall-Selitto paw-withdrawal test (N=6 for all groups). Average paw withdrawal threshold immediately before injection of PGE2 or EPI was 110.6 ± 1.0 g for the AS- and 110.8 ± 0.9 g for the MM-ODN groups. Paired Student’s t-test showed no difference in the mechanical threshold before and after the ODN treatments: p=0.5360 for the AS and p=0.3138 for the MM groups (N=24, data not shown). Left panels, low dose (20 ng) of PGE2 or EPI: Two-way repeated measures ANOVA showed a significant group time interaction (PGE2: F3.30=30.743; p<0.001; EPI: F3.30=17.254; p<0.001) and a significant main effect of group (PGE2: F1,10=300.167; p<0.001; EPI: F1,10=193.704; p<0.001). Based on the significant interaction, one-way repeated measures ANOVA with simple contrasts were performed for the AS and MM groups, showing that, for both groups, there was a significant main effect of time (PGE2 MM: F3,15=14.226; p=0.001; PGE2 AS: F3,15=124.234; p<0.001; EPI MM: F3,15=21.104; p=0.001; EPI AS: F3,15=43.756; p<0.001). However, while in the MM groups only the 30 min time point differed significantly from baseline (PGE2: p<0.001; EPI: p=0.002), in the AS groups the 30 min, 4 h, and 24 h time points were all significantly different from baseline (PGE2: *p<0.001 in each case; EPI: **p≤0.001 in each case); Right panels, high dose (100 ng) of PGE2 or EPI: Two-way repeated measures ANOVA showed a significant group time interaction (PGE2: F3,30=60.135; p<0.001; EPI: F3.30=35.221; p<0.001) and a significant main effect of group (PGE2: F1,10=68.594; p<0.001; EPI: F1,10=209.275; p<0.001). Based on the significant interaction, one-way repeated measures ANOVA with simple contrasts were performed for the AS and MM groups, showing that, for both groups, there was a significant main effect of time (PGE2 MM: F3,15=94.239; p<0.001; PGE2 AS: F3,15=49.454; p<0.001; EPI MM: F3,15=94.239; p<0.001; EPI AS: F3,15=49.454; p<0.001). However, in the MM groups, only the 30 min time point differed significantly from baseline (p<0.001 in both cases), while, in the AS groups, the 30 min, 4 h, and 24 h time points were all significantly different from baseline (#p≤0.004 in both cases).
Figure 2
Figure 2
A Carrageenan hyperalgesia during GRK2 knockdown. Rats were treated with antisense (AS)- or mismatch (MM)-ODN to GRK2 mRNA for 3 consecutive days. On the 4th day carrageenan (0.1%) was injected into the hind paw. Mechanical thresholds in the AS- and MM-treated groups right before carrageenan injection were 112.3 ± 1.5 g and 107.3 ± 2.2 g, respectively. No significant differences were observed on the mechanical threshold in both groups before and after ODN treatments: AS-treated group: p=0.2666; MM-treated group: p= 0.4818, paired Student’s t-test, N=6 per group, data not shown). Two-way repeated measures ANOVA showed a significant group time interaction (F3,30=36.847; p<0.001) and a significant main effect of group (F1,10=17.174; p=0.002). Based on the significant interaction, one-way repeated measures ANOVA with simple contrasts were performed for each of the two groups, showing that, for the MM group, there was a significant main effect of time (F3,15=65.668; p=0.001) but only the 3 h and 1 d time points differed significantly from baseline (p<0.001 in both cases). However, for the AS group, although there was also a significant main effect of time (F3,15=94.568; p<0.001), the 3 d time point also was significantly different from baseline (*p<0.001); B - Prolonged PGE2-induced hyperalgesia in primed rats during GRK2 knockdown. Rats received intradermal injection of carrageenan (1%) in the hind paw. One week later (when the mechanical threshold returned to the baseline), GRK2 AS- or MM-ODN was injected for 3 consecutive days. Test with PGE2 was performed on the 4th day and the AS/MM treatment continued until the MM group thresholds returned to baseline (N=6 per group). Mechanical thresholds on the 3rd day of the AS-/MM-ODN treatment (right before PGE2 injection) were 106.0 ± 1.8 g in the AS- and 108.0 ± 2.8 g in the MM-treated group. ODN treatments did not induce significant changes in the mechanical threshold (AS-treated group: p= 0.1464; MMtreated group: p= 0.9067, paired Student’s t-test, data not shown). Two-way repeated measures ANOVA showed a significant group time interaction (F4,40=82.270; p<0.001) and a significant main effect of group (F1,10=102.059; p<0.001). Based on the significant interaction, one-way repeated measures ANOVA with simple contrasts were performed for each of the two groups, showing that, for the MM group, there was a significant main effect of time (F4,20=55.933; p<0.001) with the 30 min and 4 h time points significantly different from the baseline (p<0.001); for the AS group there was also a significant main effect of time (F4,20=47.785; p<0.001). However, all time points were significantly different from baseline (**p≤0.001).
Figure 3
Figure 3
A Hyperalgesia after recovery of GRK2 levels. Rats were treated with antisense (AS)- or mismatch (MM)-ODN for GRK2 mRNA for 3 consecutive days. 7 days after the last dose of the AS or MM, the saphenous nerves were harvested and Western blot analysis was performed. No differences in the GRK2 protein levels were observed between these two groups (p=0.3309, unpaired Student’s t-test, N=6 per group); B - Potentiation of PGE2- or epinephrine (EPI)induced hyperalgesia. Rats were treated with GRK2 antisense (AS)- or mismatch (MM)-ODN for 3 consecutive days. One week after the last dose of AS- or MM-ODN, when levels of GRK2 had recovered to pretreatment levels, PGE2 (20 ng or 100 ng) or EPI (20 ng or 100 ng) was injected into the hind paw. Mechanical thresholds were evaluated 30 min later and at further time points, until nociceptive threshold had returned to the baseline values (N=6 per group). Average paw withdrawal threshold right before injection of PGE2 or EPI was 110.6 ± 1.0 g in the AS group and 110.8 ± 0.9 g in the MM group. Comparison of thresholds before and after ODN treatments showed no significant changes after 3 injections of AS or MM: p=0.5360 for the AS and p=0.3138 for the MM group (paired Student’s t-test, data not shown). Left panels, low dose (20 ng) of PGE2 or EPI: Two-way repeated measures ANOVA showed a significant group time interaction (PGE2: F8,80=22.499; EPI: F8,80=21.874; p<0.001 for both cases) and a significant main effect of group (PGE2: F1,10=65.755; EPI: F1,10=25.784; p<0.001 for both cases). Based on the significant interaction, one-way repeated measures ANOVA with simple contrasts were performed for each of the two groups (AS and MM), showing that, in the MM groups, there was a significant main effect of time (PGE2: F8,40=39.788; EPI: F8,40=47.896; p<0.001 for both cases), with significant difference from baseline (p<0.001 in all cases) only in the 30 min and 1 h time points. For the AS groups there was also a significant main effect of time (PGE2: F8,40=54.597; EPI: F8,40=44.886; p< 0.001 for both cases), with significant difference from baseline in the first five time points (*p≤0.003); Right panels, high dose (100 ng) of PGE2 or EPI: The two-way repeated measures ANOVA showed a significant group time interaction (PGE2: F8,80=28.320; EPI: F8,80=23.774; p<0.001 for both cases) and a significant main effect of group (PGE2: F1,10=54.643; EPI: F1,10=46.212; p<0.001 for both cases). Based on the significant interaction, one-way repeated measures ANOVA with simple contrasts were performed for each of the two groups (AS and MM). For the MM groups, a significant main effect of time (PGE2: F8,40=120.213; EPI: F8,40=65.319; p<0.001 for both cases) was observed, and simple contrasts showed that, for PGE2, the first three time points, 30 min, 60 min and 3 h, and for EPI, the first two time points, 30 min and 60 min, differed significantly from baseline (p<0.001 in each case). For the AS groups there was also a significant main effect of time (PGE2: F8,40=95.097; EPI: F8,40=105.009; p<0.001), and simple contrasts showed that the first six time points were significantly different from baseline (PGE2: **p≤0.004, EPI: #p≤0.001 in all cases).
Figure 4
Figure 4. Role of PKCε in prolongation of PGE2 hyperalgesia induced by knockdown of GRK2
Rats were treated with antisense (AS)- or mismatch (MM)-ODN for PKC? for 6 (upper panel) or 13 (lower panel) days; GRK2 AS-ODN was administered from day 4 to 6 and did not induce significant changes in the mechanical threshold (upper panel: before and after AS: p=0.8771; lower panel: before and after AS: p=0.8771, paired Student’s t-test, data not shown). PGE2 (100 ng, i.d.) was injected into the hind paw on the 7th or 14th day. Mechanical thresholds were evaluated before PGE2 injections and 30 min, 4 h and 24 h later – (upper panel shows the readings until the 5th day) (N=6 per group). Average baseline mechanical thresholds in each group immediately before PGE2 injection were: upper panel - GRK2 AS/PKCε AS: 110.6 ± 2.2 g; GRK2 AS/ PKCε MM: 108.0 ± 2.0 g; lower panel - GRK2 AS/PKCε AS: 111.3 ± 0.9 g; GRK2 AS/ PKCε MM: 101.3 ± 0.6 g. Although the two-way repeated measures ANOVA showed a significant hyperalgesia for both groups (significant main effect of time: upper panel: F5,35=104.906; lower panel: F3,21=273.564; p<0.001 in both cases), there was no effect of the PKCε AS-ODN on the prolongation of PGE2 hyperalgesia, neither during GRK2 knockdown (group time interaction: F5,35=0.220; p=0.857; main effect of group: F1,7=0.006; p=0.941) nor after GRK2 recovery (group time interaction: F3,21=0.737; p=0.495; main effect of group: F1,7=0.061; p=0.812).
Figure 5
Figure 5. Prolonged hyperalgesia induced by previous knockdown of GRK2 is not dependent on CPEB
Rats were treated with mismatch (MM, clear bars)- or antisense (AS, black bars)-ODN for CPEB, for 6 or 14 days. In both cases GRK2 AS-ODN was administered from days 4 to 6. No significant changes in the mechanical threshold were observed after GRK2 AS-ODN treatment (7th day: before and after AS: p=0.7227; 14th day: before and after AS: p= 0.6639, paired Student’s t-test, data not shown). PGE2 (100 ng, i.d.) was injected into the hind paw on the 7th or 14th day. Mechanical threshold was evaluated before PGE2 injections and 30 min and 4 h later (N=6 per group). Average mechanical thresholds immediately before PGE2 injection were 111.3 ± 1.9 g (GRK2 AS/CPEB MM, 7th day), 114.0 ± 1.9 g (GRK2 AS/CPEB AS, 7th day), 110.0 ± 1.5 g (GRK2 AS/CPEB MM, 14th day) and 113.0 ± 2.3 4g (GRK2 AS/CPEB AS, 14th day). The two-way repeated measures ANOVA showed a significant main effect of time (F4,40=207.197; p<0.001), indicating an overall significant mechanical hyperalgesia in the two groups, but neither the group time interaction (F4,40=0.636; p=0.566) nor the main effect of group (F1,10=0.562; p=0.471) were significant, indicating no difference between the groups treated with CPEB AS or MM.
Figure 6
Figure 6. Second messengers involved in the prolongation of PGE2 hyperalgesia induced by previous knockdown of GRK2
Rats were treated with antisense (AS) for GRK2 for 3 consecutive days. At least one week after AS treatment, PGE2 (100 ng, i.d.) was injected into the hind paw. A. Rats previously (1 week before) treated with GRK2 AS received AS- or MM-ODN for PLC-β3 for 3 consecutive days. Test with PGE2 was performed on the 4th day. Average baseline mechanical thresholds immediately before PGE2 injection were 107.0 ± 2.4 g (GRK2 AS/PLC-β3 MM) and 110.3 ± 2.3 g (GRK2 AS/ PLC-β3 AS). Importantly, treatment with ODNs did not induce significant changes on the baseline thresholds (data not shown, see Section 2.6). Although paired Student’s t-test showed no difference in the PGE2-induced hyperalgesia 30 min and 4 h after injection in the PLC-β3 MM group (p=0.6392, N=6 per group), in the AS group there was significant decrease in the magnitude of mechanical hyperalgesia on the 4th h after the injection (*p=0.0046, N=6 per group); B. Inhibitors of signaling pathways were injected, in different groups of rats, 3.5 h after PGE2, when the mechanical threshold had decreased, in average, 31.6 ± 0.6 % (comparison of the mechanical thresholds before (110.8 ± 0.6 g) and 3.5 h after PGE2 injection (75.7 ± 0.7 g, immediately before inhibitors injection) showed significant hyperalgesia: p<0.0001, paired Student’s t-test, N=48, data not shown). Readings were taken before and after the inhibitors (roscovitine: Cdk5 inhibitor / wortmannin: PI-3K inhibitor / U73122: PLCγ inhibitor / UO126: ERK/MEK inhibitor / BIM: non-selective PKC inhibitor / SU6656: Src inhibitor / WIPTIDE: PKA inhibitor) (N=6 per group; each group of bars represents a different group of rats/inhibitor test). Paired Student’s t-test for each inhibitor showed lack of effect of roscovitine (p=0.8945), wortmannin (p=0.6109) and U73122 (p=0.1852). However, significant inhibition of prolonged PGE2 hyperalgesia was observed after injection of U0126 (*p=0.0074), BIM (**p=0.0026), SU6656 (***p=0.0002), WIPTIDE (#p=0.0010), and SU6656+WIPTIDE (***p=0.0002).
Figure 7
Figure 7. Prolongation of hyperalgesia induced by direct activation of intracellular second messengers by previous knockdown of GRK2
Rats were treated with antisense (AS, black bars) or mismatch (MM, clear bars) for GRK2 for 3 consecutive days. Two weeks after ODN treatments, activators of adenyl cyclase (forskolin, 1 μg) or Epac (CPToMe, 10 ng), or the cAMP analog 8-bromo-cAMP (1 μg) were injected into the hind paw of different groups of rats. Mechanical thresholds (not significantly affected by AS- and MM-ODN treatment, data not shown), just before the injection of the second messenger activators were: forskolin/GRK2 AS: 112.3 ± 2.0 g; forskolin/GRK2 MM: 115.0 ± 2.2 g; CPToMe/GRK2 AS: 113.3 ± 3.3 g; CPToMe/GRK2 MM: 103.3 ± 2.1 g; 8-bromo-cAMP/GRK2 AS: 115.0 ± 2.2 g; 8-bromo-cAMP/GRK2 MM: 112.0 ±1.6 g. Measurements of nociceptive thresholds were performed 30 min and 4 h after the injections. Comparison between GRK2 AS- and MMODN groups at the 4th h post injections showed prolongation of hyperalgesia in all cases (GRK2 AS/forskolin at 4th h X GRK2 MM/forskolin at 4th h: *p< 0.0001; GRK2 AS/8-bromocAMP at 4th h X GRK2 MM/8-bromo-cAMP at 4th h: *p< 0.0001; GRK2 AS/CPToMe at 4th h X GRK2 MM/ CPToMe at 4th h: *p< 0.0001, unpaired Student’s t-test, N=6 per group). In addition, contrary to forskolin (p=0.9523) and 8-bromo-cAMP (p=0.8912), for CPToMe, unpaired Student’s t-test showed a significant difference, between GRK2 AS and MM groups, in the hyperalgesia 30 min after injection of lower dose of CPToMe (**p=0.0137, N=6 per group), suggesting potentiation of the effect in the GRK2 AS-treated group.
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