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Comparative Study
. 2003 Jan 7;100(1):271-6.
doi: 10.1073/pnas.0136822100. Epub 2002 Dec 20.

Contribution of GIRK2-mediated postsynaptic signaling to opiate and alpha 2-adrenergic analgesia and analgesic sex differences

Affiliations
Comparative Study

Contribution of GIRK2-mediated postsynaptic signaling to opiate and alpha 2-adrenergic analgesia and analgesic sex differences

Igor Mitrovic et al. Proc Natl Acad Sci U S A. .

Abstract

The analgesia produced by inhibitory G protein-coupled receptor agonists involves coordinated postsynaptic inhibition via G protein-coupled inwardly rectifying potassium channels (GIRKs) and presynaptic inhibition of neurotransmitter release through regulation of voltage-gated Ca(2+) channels. Here, we used mice lacking the GIRK2 channel subunit to assess the relative contribution of these two effector systems to nociceptive processing in male and female mice. Compared with female WT mice, male WT mice exhibited higher pain thresholds and enhanced opioid (morphine) and alpha(2)-adrenergic (clonidine) receptor-induced antinociception in a spinal reflex test. The GIRK2-null mutation reduced the "pain" threshold in male but not in female mice, effectively eliminating the sex differences in pain threshold. In addition, deletion of GIRK2 channels in mutant mice largely eliminated clonidine antinociception and significantly decreased morphine antinociception. Furthermore, the more pronounced morphine and clonidine-induced antinociception in male mice disappeared in the GIRK2 mutants. Based on the almost complete loss of clonidine-induced antinociception in the mutant mice, we conclude that it is primarily mediated by postsynaptic alpha(2)-adrenergic receptors. In contrast, the significant residual morphine effect in the mutant mice points to the presynaptic mu opioid receptor as a major contributor to its analgesic action. Finally, our results suggest that the reduced pain responsiveness of male compared with female mice results in part from GIRK2-coupled postsynaptic receptors that are activated by endogenous antinociceptive systems.

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Figures

Figure 1
Figure 1
Baseline threshold tests. (a) Tail-flick test responses in male and female WT and GIRK2-null mutant (ko) mice. Between 14 and 19 mice per group were tested. One-way ANOVA (F3,63 = 5.17) followed by Bonferroni post test indicated that male WT mice exhibited a higher withdrawal threshold than the other three groups of mice (*, P < 0.05). (b) Hot plate responses in the same mice (one mouse was excluded because of seizures). ANOVA (F3,62 = 2.7) followed by Bonferroni post test indicated significant differences between male WT and mutant mice (*, P < 0.05). (c) Pain behaviors elicited by 5% formalin injection into the animals' hind paws. Between four and five mice were tested in each group. One-way ANOVA (F3,16 = 7.131) analysis of behavior in the normally quiescent interphase (5–15 min after the formalin injection) followed by Bonferroni post test indicated that male and female mutant mice (unlike their WT counterparts) do not exhibit a cessation of pain-related behaviors. (There was no difference in formalin-elicited behaviors between any two groups of mice in either the first or second phase of the response. *, P < 0.05 between the WT mice and their respective mutant counterparts.)
Figure 2
Figure 2
Effects of the GIRK2-null mutation on morphine antinociception in male and female mice. Between 14 and 19 mice per group were tested. All mice received all of the doses in a randomized order, with a 1-week interval between treatments. (a) Morphine dose–response curve generated with tail-flick test (legend is the same as in b). (b) Comparison of areas under the morphine dose-response curves for WT males, WT females, mutant males, and mutant females. One-way ANOVA (F3,64 = 31.55) followed by Bonferroni post test indicated a significant difference between male and female WT animals in response to morphine (*, P < 0.05). The GIRK2-null mutation decreased antinociceptive effectiveness of morphine in both male and female mice (#, P < 0.001, compared with either male or female WT mice). At the same time, the sex difference present in WT mice was eliminated by the null mutation of the GIRK2 gene. (c) Morphine dose–response curves generated in the hot plate test. (d) Comparison of areas under the dose–response curves for WT males, WT females, mutant males, and mutant females. One-way ANOVA (F3,62 = 35.99) followed by Bonferroni post test indicated the same morphine response profile as the one in the tail-flick test: (i) sex difference in WT animals (*, P < 0.001) disappeared in mutant mice; (ii) morphine antinociceptive effectiveness in male and female mutant mice was decreased relative to either male or female WT animals (#, P < 0.001). (e) Effects of morphine on pain-related behaviors elicited by injection of 5% formalin (between five and nine animals per group; because there were no sex differences in the baseline test, male and female animals in the baseline group were combined). Both 3.0 and 10 mg/kg morphine inhibited formalin-elicited first-phase (0–5 min) behaviors (*, P < 0.001, compared with the baseline). The 10 mg/kg dose had a greater antinociceptive effect in WT mice ($, P < 0.001, compared with the effects observed in mutant mice). Both doses of morphine eliminated the effects of the GIRK2-null mutation in the interphase (5–15 min; *, P < 0.001). The 3.0 mg/kg dose of morphine reduced and the 10 mg/kg dose eliminated the second phase (15–40 min) of responses (#, P < 0.05 and *, P < 0.001, compared with the baseline).
Figure 3
Figure 3
Effects of the GIRK2-null mutation on clonidine-mediated antinociception. Between 13 and 19 mice per group were tested. (All mice were treated with all of the doses in a randomized order with a 1-week interval between treatments.) (a) Clonidine dose–response curve generated by using the tail-flick test. (b) Comparison of areas under the dose–response curves for WT males, WT females, mutant males, and mutant females. One-way ANOVA (F3,56 = 24.12) followed by Bonferroni post test revealed sex differences in clonidine antinociception in WT mice (*, P < 0.001 compared with male WT mice). The GIRK2-null mutation decreased clonidine-mediated antinociception (#, P < 0.05 compared with WT mice) and eliminated the sex differences. (c) Clonidine dose–response curve generated with the hot plate test. (d) Comparison of effects of the GIRK2-null mutation on morphine vs. clonidine antinociception. Data are represented as areas under the morphine and clonidine dose–response curves (AUCs) normalized to the MPE observed in WT male animals. Decreases in the antinociceptive effectiveness of each of the two analgesics caused by the GIRK2-null mutation were compared. Two-tailed t test indicates significantly greater effect of the mutation on clonidine antinociception (*, P < 0.001).
Figure 4
Figure 4
Transverse section of the spinal cord (from the L5 segment) immunostained with the GIRK2 antiserum (1:2,500), with a nickel-enhanced diaminobenzidine glucose-oxidase reaction. (a) Immunostaining in a WT animal. Dark bands of staining (arrowheads) represent GIRK2 immunoreactivity in lamina II. (b) Immunostaining for GIRK2 is absent in a mutant animal. (Bar = 0.2 mm.)

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