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. 2015 May 1;10(5):e0125022.
doi: 10.1371/journal.pone.0125022. eCollection 2015.

Acidosis Mediates the Switching of Gs-PKA and Gi-PKCε Dependence in Prolonged Hyperalgesia Induced by Inflammation

Wei-Yu Huang  1 , Shih-Ping Dai  1 , Yan-Ching Chang  1 , Wei-Hsin Sun  2
Affiliations

Acidosis Mediates the Switching of Gs-PKA and Gi-PKCε Dependence in Prolonged Hyperalgesia Induced by Inflammation

Wei-Yu Huang et al. PLoS One. .

Abstract

Chronic inflammatory pain, when not effectively treated, is a costly health problem and has a harmful effect on all aspects of health-related quality of life. Previous studies suggested that in male Sprague Dawley rats, prostaglandin E2 (PGE2)-induced short-term hyperalgesia depends on protein kinase A (PKA) activity, whereas long-lasting hyperalgesia induced by PGE2 with carrageenan pre-injection, requires protein kinase Cε (PKCε). However, the mechanism underlying the kinase switch with short- to long-term hyperalgesia remains unclear. In this study, we used the inflammatory agents carrageenan or complete Freund's adjuvant (CFA) to induce long-term hyperalgesia, and examined PKA and PKCε dependence and switching time. Hyperalgesia induced by both agents depended on PKA/PKCε and Gs/Gi-proteins, and the switching time from PKA to PKCε and from Gs to Gi was about 3 to 4 h after inflammation induction. Among the single inflammatory mediators tested, PGE2 and 5-HT induced transient hyperalgesia, which depended on PKA and PKCε, respectively. Only acidic solution-induced hyperalgesia required Gs-PKA and Gi-PKCε, and the switch time for kinase dependency matched inflammatory hyperalgesia, in approximately 2 to 4 h. Thus, acidosis in inflamed tissues may be a decisive factor to regulate switching of PKA and PKCε dependence via proton-sensing G-protein-coupled receptors.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Mice show mechanical hyperalgesia with peripheral inflammation induced by complete Freund’s adjuvant (CFA) or carrageenan injection.
Wild-type male CD1/ICR mice (8–12 weeks old) received intraplantar injection in the right hind paws with 25 μl CFA (50% in saline, A), carrageenan (Carr; 20 mg/ml, B), or saline (A, B). The threshold of paw withdrawal (PWT) to mechanical stimuli was measured before (t = 0) and after injection. Data are mean±SEM of total tested mice (n = 3–6 per group). ***p
Fig 2
Fig 2. CFA- or carrageenan-induced mechanical hyperalgesia requires PKA and PKCε activity.
(A) Dosage curve for protein kinase A (PKA) and protein kinase Cε (PKCε) inhibitors. Mice received intraplantar injection with 25 μl of different doses of PKA inhibitor (PKAI, H89) before 50% CFA injection or PKCε inhibitor (PKCεI, TAT-PKCεV1-2) or control peptide (ctrl50) at 4 h after CFA injection. The PWT on ipsilateral side was measured at 90 min after the second injection. Data are mean±SEM of total tested mice (n = 6–12 per group). ##p<0.01, ###p<0.001 compared to PKAI/CFA with CFA only group and ***p<0.001 compared to PKCεI/CFA with CFA only group by one-way ANOVA with a post-hoc Bonferroni test. (B, C) Mice were injected with PKAI (50μM) or PKCεI (50μM) before (0 h) or after (3, 4, 5 h or 1 or 16 days) CFA injection. The PWT on the ipsilateral side (B) or contralateral side (C) was measured at 90 min after the second injection. Data are mean±SEM of total tested mice (n = 6 per group). ###***p<0.001 compared to CFA-injected groups by two-way ANOVA with a post-hoc Bonferroni test. (D) Mice were injected with PKAI (50μM) or PKCεI (50μM) before (0 h) or after (3, 4, 5 h or 1 or 10 days) carrageenan injection. The PWT on the ipsilateral side was measured at 90 min after the second injection. Data are mean±SEM of total tested mice (n = 6 per group). ###***p<0.001 compared to carrageenan-injected groups by two-way ANOVA with a post-hoc Bonferroni test.
Fig 3
Fig 3. Thickness of mouse paw remains unchanged after blocking PKA or PKCε.
Mice received intraplantar injection with 50μM of PKAI (H89) or PKCεI (TAT-PKCεV1-2) at 4 h or 1 day after 50% CFA injection. At 90 min after inhibitor injection, mice were killed and the thickness of injected (ipsilateral) and uninjected (contralateral) paws was measured. Data are presented as meanSEM of total tested mice (n = 3). Comparisons between CFA-injected (*), CFA/PKAI (#), or CFA/PKCεI (&) and saline-injected animals were analysed by one-way ANOVA with a post-hoc Bonferroni test. ##&&p<0.01, ***&&&p<0.001.
Fig 4
Fig 4. Blocking of G-protein signalling reduces CFA and carrageenan-induced mechanical hyperalgesia.
(A) Mice were injected with 25 μl of AC inhibitor (SQ22536) before CFA injection or with Gi inhibitor (pertussis toxin [PTX]), PLCβ inhibitor (U73122), or Gβγ inhibitor (gallein) 1 day after CFA injection. Data are mean±SEM of total tested mice (n = 6–12 per group). #$*p<0.05, &&P<0.01, $$$###&&&***p<0.001 compared to no inhibitor-injected groups by one-way ANOVA with a post-hoc Bonferroni test. (B-C) Mice were injected with AC inhibitor (SQ22536, 1 mM), Gi inhibitor (PTX, 100 ng) or PLCβ inhibitor (U73122, 500 μM), before (0 h) or 4 h or 1 day after CFA (B) or carrageenan (C) injection. The threshold of paw withdrawal was measured at 90 min after the second injection. Data are mean±SEM of total tested mice (n = 6–8 per group). $$$###&&&***p<0.001 compared to CFA-injected or carrageenan-injected groups by two-way ANOVA with a post-hoc Bonferroni test.
Fig 5
Fig 5. Blocking PKA or PKCε reduces PGE2 or 5-HT-induced mechanical hyperalgesia, respectively.
(A) Mice were intraplantarly injected with 25 μl PGE2, followed by mechanical test at 90 min after injection. ***p<0.001 compared with saline-injected by one-way ANOVA with a post-hoc Bonferroni test. (B) Mice were injected with PGE2 (100 ng). The threshold of paw withdrawal was measured before (t = 0) and after injection. ***p<0.001 compared to contralateral side by two-way ANOVA with a post-hoc Bonferroni test. (C) Mice were injected with PKAI (50μM, H89) before PGE2 (100 ng) injection or with PKCεI (50μM, TAT-PKCεV1-2) 2 h after PGE2 injection. The threshold of paw withdrawal was measured at 90 min after the PGE2 injection. ***p<0.01, compared to PGE2-injected group by one-way ANOVA with a post-hoc Bonferroni test. (D) Mice were pre-injected with PKAI (50μM) or PKCεI (50μM) before 5-HT (10μM) injection, followed by measurement of the threshold of paw withdrawal at 30 min after 5-HT injection. ***p<0.001 compared with 5-HT-injected by one-way ANOVA with a post-hoc Bonferroni test. Data are mean±SEM of total tested mice (n = 6–10 per group).
Fig 6
Fig 6. Acid-induced mechanical hyperalgesia requires PKA and PKCε activity.
(A) Mice received intraplantar injection with 25 μl of acid solution (pH 5.0). The threshold of paw withdrawal was measured before (t = 0) and after injection. ***p
Fig 7
Fig 7. Acid induces PKA and PKCε translocation.
Primary dorsal root ganglia (DRG) from CD1/ICR mice were cultured for 12~16 h, then stimulated with acid solution, pH 7.6, 6.4 or 5.5 for 5 or 30 min or 15 s, respectively. After immunostaining with anti-PKA or anti-PKCε antibodies, cell images with red fluorescence for PKA (A) or PKCε (D) were observed by confocal microscopy. Bar = 10μm. (B, C, E, F) Fluoresence intensity (F0) in a line bisecting the neuronal soma (see dashed line in A, D) was measured and the profile of intensity (F0/Favg) is shown in B, E. Favg = the mean intensity of the dashed line. Lines were positioned to avoid nuclei. Fluorescence intensity within the peripheral (0–10% and 90–100% of cell distance) region was the membrane fraction and within the central region (10–90% of cell distance) the cytosolic fraction. Shows the mean intensity of membrane or cytosolic fractions (n = 3) (C, F). ***P<0.001 compared to cytosolic fraction (10–90%) by one-way ANOVA with a post-hoc Bonferroni test.
Fig 8
Fig 8. Gene expression patterns of OGR1 family after inhibition of PKA or PKCε activity.
Mice received intraplantar injection with 25 μl CFA (A), with pre-injection with PKAI (B) or post-injection with PKCεI 1 day after CFA injection (C). At 90 min and 4 and 24 h after CFA injection or at 90 min after the second injection, mice were killed. RNA was obtained from lumbar 4–6 DRG ipsilateral and contralateral to injected paws for RT-PCR. The expression of each gene on the ipsilateral DRG was normalized to mGAPDH expression, then represented as relative expression to contralateral controls (fold change in expression). Data are mean±SEM of triplicate measurements (n = 3 mice). **p
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