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Comparative Study
. 2010 Jan 6;30(1):38-46.
doi: 10.1523/JNEUROSCI.4346-09.2010.

Ca2+/calmodulin-dependent protein kinase II alpha is required for the initiation and maintenance of opioid-induced hyperalgesia

Yan Chen  1 , Cheng Yang, Zaijie Jim Wang
Affiliations
Comparative Study

Ca2+/calmodulin-dependent protein kinase II alpha is required for the initiation and maintenance of opioid-induced hyperalgesia

Yan Chen et al. J Neurosci. .

Abstract

Repeated administration of opioids not only leads to tolerance and dependence, but also results in nociceptive enhancement called opioid-induced hyperalgesia (OIH). Nociceptive mediators involved in OIH generation remain poorly understood. In the present study, we tested the hypothesis that Ca(2+)/calmodulin-depent protein kinase II (CaMKIIalpha) is critical for OIH. Opioid-induced hyperalgesia was produced by repeated morphine administration or pellet implantation in mice. Correlating with the development of tactile allodynia and thermal hyperalgesia, spinal CaMKIIalpha activity was significantly increased in OIH. KN93, a CaMKII inhibitor, dose- and time-dependently reversed OIH and CaMKII activation without impairing locomotor coordination. To elucidate the specific CaMKII isoform involved, we targeted CaMKIIalpha by using small interfering RNA and demonstrated that knockdown of spinal CaMKIIalpha attenuated OIH. Furthermore, morphine failed to induce OIH in CaMKIIalpha(T286A) point mutant mice, although wild-type littermate mice developed robust OIH after repeated treatments with morphine. These data implicate, for the first time, an essential role of CaMKIIalpha as a cellular mechanism leading to and maintaining opioid-induced hyperalgesia.

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Figures

Figure 1.
Figure 1.
Repeated intermittent morphine administration-induced mechanical allodynia (A) and thermal hyperalgesia (B). Mice received saline or morphine sulfate (day 1–3: 20 mg/kg; day 4: 40 mg/kg; twice daily, s.c.). The paw withdrawal threshold to von Frey filament probing and withdrawal latency to radiant heat were determined. Data are expressed as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001, compared with the saline-treated group; n = 5 for each group.
Figure 2.
Figure 2.
Morphine pellet implantation-induced mechanical allodynia (A) and thermal hyperalgesia (B). Mice were implanted subcutaneously with morphine or placebo pellets. The paw withdrawal threshold to von Frey filament probing and withdrawal latency to radiant heat were measured daily. Morphine initially produced antinociception followed by a steep decrease in mechanical threshold (A) and thermal withdrawal latency (B), indicative of the development of OIH. Data are expressed as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001, compared with the placebo pellet group; n = 5 for each group.
Figure 3.
Figure 3.
Reversal of morphine-induced mechanical allodynia (A) and thermal hyperalgesia (B) by KN93. OIH was induced by intermittent morphine injections. On day 5, mice received KN93 (15–45 nmol, i.t.), KN92 (45 nmol, i.t.), or saline (intrathecally) at time 0. Mechanical allodynia and thermal hyperalgesia were tested at the different time points as indicated. KN93, but not KN92, reversed the established morphine-induced mechanical allodynia and thermal hyperalgesia in a dose- and time-dependent manner. Data are expressed as mean ± SEM *p < 0.05, ***p < 0.001, compared with the saline-treated group; †p < 0.05, †††p < 0.001, compared with the morphine-treated group; n = 8 for each group.
Figure 4.
Figure 4.
Effect of KN93 on locomotor activity. Mice were trained to remain on a fixed speed (4 rpm) rotarod for 60 s. On the following day, each mouse was retrained to ensure that it could stay on the rotarod for at least 60 s. Baseline was tested by placing the mice on an accelerating rotarod (4–40 rpm over 300 s). The latency to fall from the rotarod was recorded. Mice were then administered with either KN93 (45 nmol), KN92 (45 nmol) or saline intrathecally and retested 0.5, 1, 2, 4 and 8 h later. Neither KN93 nor KN92 significantly impaired locomotor activity. Data are expressed as mean ± SEM p > 0.05, compared with the saline-treated group; n = 5 for each group.
Figure 5.
Figure 5.
Suppression of morphine-induced CaMKIIα activation by KN93. Morphine or saline-treated mice were administered intrathecally with KN93 (15–45 nmol), KN92 (45 nmol), or saline on day 5. One hour later, mice were killed and the lumbar spinal cords were taken for the analysis of CaMKIIα activation using the immunoblotting method, by determining the degree of CaMKIIα autophosphorylation (pCaMKIIα). Morphine enhanced pCaMKIIα (A), without altering CaMKIIα expression (B). KN93, but not its inactive analog, KN92, reversed morphine enhanced CaMKIIα activation. Data are expressed as mean ± SEM, *p < 0.05, compared with the saline-treated group; †p < 0.05, compared with the morphine-treated group; n = 4 for each group.
Figure 6.
Figure 6.
Immunohistochemical staining of pCaMKIIα expression after the treatment with KN93. I, Morphine- or saline-treated mice were administered (intrathecally) with KN93 (15–45 nmol), KN92 (45 nmol), or saline on day 5. Mice were killed 1 h after the treatment with saline or KN93 (45 nmol, i.t.). The lumbar spinal section was dissected out and immunostained with pCaMKIIα antibody. II, Quantitative analysis of pCaMKIIα immunoreactivity was performed by counting the number of positively stained cells using the MetaMorph Imaging Software. No pCaMKIIα immunoreactivity was detected if the first antibody was omitted (D) or if the first antibody was incubated in the presence of pCaMKIIαT286 blocking peptide (Santa Cruz Biotechnology). Data are expressed as mean ± SEM, †††p < 0.001, compared with the morphine-treated group; n = 3 for each group. Scale bars are 200 μm (A-1, B-1, C-1, D, E), 100 μm (A-2, B-2, C-2), or 20 μm (A-3, B-3, C-3).
Figure 7.
Figure 7.
Reversal of OIH by siRNA-mediated CaMKIIα knockdown. In phase 1, mice received repeated saline or morphine administration. Mechanical and thermal sensitivities were measured on day 5. In phase 2, after OIH had fully developed, mice were treated with CaMKIIα siRNA or scrambled siRNA (2 μg, twice per day for 3 d). Mechanical and thermal sensitivities were tested daily. Established OIH was reversed by CaMKIIα siRNA. Data are expressed as mean ± SEM ***p < 0.001, compared with the saline-treated group; †††p < 0.001, compared with the morphine-treated group; n = 5 for each group. Arrows indicated saline, CaMKIIα, or scrambled siRNA administration.
Figure 8.
Figure 8.
Suppression of morphine-induced CaMKIIα activation by CaMKIIα siRNA. I, Naive mice (A) or mice with OIH were treated with saline (B), CaMKIIα siRNA (2 μg, twice daily for 3 d) (C), or scrambled siRNA (2 μg, twice daily for 3 d). One day after the last injection of siRNA, the spinal lumbar region was immunostained with pCaMKIIα antibody. II, Quantitative analysis of pCaMKIIα immunoactivity was performed by counting the number of positively stained cells using MetaMorph Imaging Software. Data are expressed as mean ± SEM, †††p < 0.001, compared with the morphine-treated group; n = 3 for each group. Scale bars are 200 μm (A-1, B-1, C-1, D-1), 100 μm (A-2, B-2, C-2, D-2), or 20 μm (A-3, B-3, C-3, D-3).
Figure 9.
Figure 9.
Morphine failed to induce hyperalgesia in CaMKIIαT286A mutant mice. Male CaMKIIαT286A mutant and littermate wild-type mice were subcutaneously administered saline or morphine sulfate (day 1–3: 20 mg/kg/d; day 4: 40 mg/kg, twice daily). Baseline pain thresholds were not significantly different between wild-type and mutant mice. Mechanical and thermal sensitivities were tested on day 5. Morphine treatment successfully induced hyperalgesia in CaMKIIαT286A+/+ mice (**p < 0.01, compared with the baseline). However, CaMKIIαT286A−/− mice failed to develop OIH (p > 0.05, compared with the baseline). Data are expressed as mean ± SEM; n = 6 for each group.
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References

    1. Angst MS, Clark JD. Opioid-induced hyperalgesia: a qualitative systematic review. Anesthesiology. 2006;104:570–587. - PubMed
    1. Baraldi PG, del Carmen Nuñez M, Morelli A, Falzoni S, Di Virgilio F, Romagnoli R. Synthesis and biological activity of N-arylpiperazine-modified analogues of KN-62, a potent antagonist of the purinergic P2X7 receptor. J Med Chem. 2003;46:1318–1329. - PubMed
    1. Bridge AJ, Pebernard S, Ducraux A, Nicoulaz AL, Iggo R. Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet. 2003;34:263–264. - PubMed
    1. Brüggemann I, Schulz S, Wiborny D, Höllt V. Colocalization of the mu-opioid receptor and calcium/calmodulin-dependent kinase II in distinct pain-processing brain regions. Brain Res Mol Brain Res. 2000;85:239–250. - PubMed
    1. Carlton SM. Localization of CaMKIIalpha in rat primary sensory neurons: increase in inflammation. Brain Res. 2002;947:252–259. - PubMed

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