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. 2009 May 5;160(2):501-7.
doi: 10.1016/j.neuroscience.2009.03.001. Epub 2009 Mar 9.

Activation of Gi induces mechanical hyperalgesia poststress or inflammation

O A Dina  1 , S G Khasar, R W Gear, J D Levine
Affiliations

Activation of Gi induces mechanical hyperalgesia poststress or inflammation

O A Dina et al. Neuroscience. .

Abstract

In studies of the role of primary afferent nociceptor plasticity in the transition from acute to chronic pain we recently reported that exposure to unpredictable sound stress or a prior inflammatory response induces long-term changes in the second messenger signaling pathway, in nociceptors, mediating inflammatory hyperalgesia; this change involves a switch from a G(s)-cAMP-PKA to a G(i)-PKCepsilon signaling pathway. To more directly study the role of G(i) in mechanical hyperalgesia we evaluated the nociceptive effect of the G(i) activator, mastoparan. Intradermal injection of mastoparan in the rat hind paw induces dose-dependent (0.1 ng-1 microg) mechanical hyperalgesia. The highly selective inhibitors of G(i), pertussis toxin, and of protein kinase C epsilon (PKCepsilon), PKCepsilonV(1-2), both markedly attenuate mastoparan-induced hyperalgesia in stressed rats but had no effect on mastoparan-induced hyperalgesia in unstressed rats. Similar effects were observed, at the site of nociceptive testing, after recovery from carrageenan-induced inflammation. These studies provide further confirmation for a switch to a G(i)-activated and PKCepsilon-dependent signaling pathway in primary mechanical hyperalgesia, induced by stress or inflammation.

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Figures

Figure 1
Figure 1
Effect of Pertussis toxin (PTx) on prostaglandin E2 (PGE2)-induced mechanical hyperalgesia at a site on the dorsum of the hind paw that had experienced a prior inflammatory response. Five days after injection of carrageenan, when nociceptive threshold had returned to baseline, PGE2 (100 ng/μl) injected at the same site produced mechanical hyperalgesia that was still maximal 4 hrs later (Aley et al., 2000). At this time injection of PTx (100 ng/μl) completely reversed PGE2 hyperalgesia. In this and subsequent figures, data are plotted as mean ± standard error of the mean (S.E.M.). Left three bars: Two-way repeated measures ANOVA with one within-subjects factor (treatments with two levels, post-PGE2 and post-PTx or saline) and one between-subjects factor (treatment group with two levels, PTx or saline) demonstrated a significant treatment × treatment group interaction (F1,14=90.834; p<0.001) as well as a significant main effect of treatment group (F1,14=97.000; p<0.001), indicating that PTx reverses PGE2 hyperalgesia. The effect of PTx alone was studied in a control group of carrageenan-treated rats. Right two bars: PTx administered alone and measured at 0.5 or 4 h, had no effect (p=NS) on mechanical nociceptive threshold in rats not previously treated with PGE2.
Figure 2
Figure 2
Dose-response relationship for the mechanical hyperalgesia induced by intradermal injection of mastoparan, in the rat hind paw. Different groups of naïve rats (each, n = 6 paws) were used to determine nociceptive thresholds to each dose of mastoparan (1 ng – 1 μg) at 0.5 h and 24 h. Two- way ANOVA showed a significant time by dose interaction (F3,20=29.751; p<0.001) and a significant main effect of group (F3,20=23.977; p<0.001). Because the interaction was significant, separate one-way ANOVAs (dose with four levels) were performed for each time point. The effect of dose at 0.5 h was significant (F3,20=81.476; p<0.001); Scheffé post hoc analyses showed that both the 1 ng and the 10 ng doses were significantly different from all other doses, but the two highest doses (100 and 1000 ng) were not significantly different (p=NS) from each other. The of at 24 h was not significant (F 3,20=0.202; p=NS), indicating that the effects observed at 0.5 h were dissipated by 24 hours.
Figure 3
Figure 3
Mastoparan-induced hyperalgesia is enhanced by sound stress. (A) Dose-response curves for the effect of mastoparan in naïve (non-stressed) and sound-stressed rats show a significant enhancement of mastoparan-induced hyperalgesia in sound stressed rats. The two-way ANOVA with one within-subjects factor (dose with 4 levels) and one between-subjects factor (stress treatment with two levels) showed a significant stress treatment × dose interaction (F3,30=6.364; p=0.004) as well as a significant main effect of stress (F1,10=19.420; p=0.001). Based on the significant interaction, a one-way between-subjects ANOVA (stress treatment with two levels) was performed for each dose to identify the doses showing significant differences. Because this method of analysis required four separate comparisons, the alpha level was set at p<0.0125. These analyses showed that groups that received sound stress were significantly different from the groups that did not, for each of the three lowest doses (p=0.011, p<0.001, p<0.01, respectively); however, the groups receiving the highest dose were not significantly different (p=0.738). These results suggest a leftward shift in the dose response curve such that the maximal effect of mastoparan was achieved with a dose an order of magnitude smaller in stressed animals. (B) Peak hyperalgesia to doses of mastoparan (1 ng – 1 μg), determined after the 1 μg dose, was not significantly different when comparing naïve and stressed rats (A and B). In both groups, hyperalgesia was no longer evident 24 h after the initial administration of mastoparan.
Figure 4
Figure 4
Mastoparan-induced hyperalgesia is profoundly sensitive to PTx or PKCHε inhibitor post sound stress. (A) The selective Gi inhibitor PTx (100 ng/μl) markedly attenuated mastoparan-induced hyperalgesia 14 days post stress but not in non-stressed naïve rats. The three-way ANOVA with two between-subjects factors (stress treatment with two levels and PTx treatment with two levels) and one within-subjects factor (dose with 5 levels) demonstrated a significant 3-way interaction (F4,80=15.882; p<0.001) and a significant main effect of PTx (F1,20=32.880; p<0.001). Based on the significant interaction, separate two-way repeated measures ANOVAs were performed for the two stress treatment groups. The between-subjects factor was PTx treatment with two levels. These analyses revealed that, for the two groups exposed to sound stress, the group receiving PTx differed significantly from the group receiving vehicle. There was a significant dose by PTx treatment interaction (F4,40=10.116; p<0.001) and a significant main effect of PTx treatment (F1,10=65.408; p< 0.001). There were no significant main effect or interaction between the two PTx treatment groups not exposed to sound stress, indicating that the effect of PTx depends on prior exposure to stress. (B) Similarly, in stressed, but not in unstressed rats, mastoparan-induced hyperalgesia was markedly attenuated by a PKCε inhibitor, PKCεV1–2. Three-way ANOVA with two between-subjects factors (stress treatment with two levels and PKCε inhibitor (PKCε-I) treatment with two levels, active and scrambled peptide sequences) and one within-subjects factor (dose with 5 levels) demonstrated a significant 3-way interaction (F4,80=5.858; p<0.001) and a significant main effect of PKCε-I treatment (F1,20=95.435; p<0.001). Based on the significant interaction, separate two-way repeated measures ANOVAs were performed for the two stress treatment groups. The between-subjects factor was PKCε-I treatment with two levels. These analyses revealed that, for the two groups exposed to sound stress, the group receiving active PKCε-I differed significantly from the group receiving scrambled PKCε-I. There was a significant dose by PKCε-I treatment interaction (F4,40=9.356; p<0.001) and a significant main effect of PKCε-I treatment (F1,10=220.826; p< 0.001). There were no significant main effects or interactions between the two PKCε treatment groups not exposed to sound stress, indicating that the effect of PKCε depends on prior exposure to stress.
Figure 5
Figure 5
Mastoparan-induced hyperalgesia is profoundly sensitive to PTx or PKCε inhibition following recovery from carrageenan-induced inflammation. (A) Mastoparan hyperalgesia was inhibited by PTx (100 ng/μl; p<0.01). Two-way ANOVA with one between-subjects factor (PTx treatment with two levels) and one within-subjects factor (dose with 5 levels) demonstrated a significant dose × PTx treatment interaction (F4,40=14.585; p<0.001) and a significant main effect of PTx treatment (F1,10=24.250; p<0.001). (B). Mastoparan hyperalgesia was similarly inhibited by the selective PKCε inhibitor peptide (1μg; p<0.001). The two-way ANOVA with one between-subjects factor (PKCε treatment with two levels) and one within-subjects factor (dose with 5 levels) demonstrated a significant main effect of PKCε treatment (F1,10=96.610; p<0.001); the dose × PKCε treatment interaction was not significant (F4,40=2.641; p<0.073). Both inhibitors were applied at the site of nociceptive testing that had previously experienced a localized inflammatory response induced by carrageenan.
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