Gq rather than G11 preferentially mediates nociceptor sensitization
© Wirotanseng et al.; licensee BioMed Central Ltd. 2013
Received: 14 June 2013
Accepted: 20 October 2013
Published: 25 October 2013
The Gq/11-protein signaling mechanism is essential throughout the nervous system, but little is known about the contribution of the individual G-protein GPCR signaling branches towards nociceptor activation and their specific role on nociceptor sensitization. We aimed to unravel the contribution of the Gq/11-signaling pathway towards nociceptor activation via a variety of classical inflammatory mediators signalling via different G-protein GPCRs and investigated the specific contribution of the individual Gq and G11 G-Proteins in nociceptors.
Using different transgenic mouse lines, lacking Gαq, Gα11 or both α-subunit of the G-proteins in primary nociceptive neurons, we analyzed the mechanical- and heat-sensitivity upon application of different GPCR-agonists that are known to play an important role under inflammatory conditions (e.g. ATP, Glutamate, Serotonin etc.). We found that the Gq/11-GPCR signaling branch constitutes a primary role in the manifestation of mechanical allodynia and a minor role in the development of thermal hyperalgesia. Moreover, with respect to the mediators used here, the Gq-protein is the principle G-protein among the Gq/11-protein family in nociceptive neurons leading to nociceptor sensitization.
Our results demonstrate that the Gq/11 signaling branch plays a primary role in nociceptor sensitization upon stimulation with classical GPCR ligands, contributing primarily towards the development of mechanically allodynia. Moreover, the deletion of the individual G-proteins led to the finding that the Gq-protein dominates the signalling machinery of the Gq/11 family of G-proteins in nociceptive neurons.
G-protein coupled receptors (GPCRs) represent the largest family of seven transmembrane receptors and downstream signaling constitutes one of the most important signaling pathways to regulate physiological processes. GPCR family members represent a major primary target for drug development [1, 2] and their signaling is a predominant focus in the development of novel analgesic therapeutics .
Peripheral sensitization is accompanied by an inflammatory milieu, acting on receptors and channels on the peripheral nerve terminals (reviewed in ). Most of these sensitizers are known to bind to GPCRs of the Gq/11 family, the Gi/o, Gs and G12/13 family of heterotrimeric G-proteins.
We have recently elucidated the specific significance of the Gq/11 pathway in modulating properties of nociceptors in vivo in the context of physiological pain and pathological states . We found that Gq/11 is involved in sensitization mechanisms in pathological states and tonically modulates basal nociception and acute pain .
There are four members of the Gq/11-protein family, namely Gq, G11, G14 and G15/16, which activate Phospholipase C beta isoforms to regulate intracellular calcium. G15/16 overall show very low levels of expression whereas G14 has been shown to be expressed at high levels selectively in some tissues (e.g. kidney, lung and spleen; reviewed by ), and for the first time Han et al. showed that G14 is expressed in a subset of DRG neurons  but does not compensate for a loss of Gq/11.
The aim of this study was to investigate the individual role of the Gq or G11 signaling branch towards acute nociceptive behavior induced by different GPCR ligands specifically activating Gq/11-coupled GPCRs or GPCRs that are capable to couple different G-protein classes. This is the first study addressing the distinct roles of Gq and G11 towards nociceptor sensitization.
All animal use procedures were in accordance with ethical guidelines imposed by the local governing body (Regierungspräsidium Karlsruhe, Germany). All behavioral measurements were done in awake, unrestrained, age-matched mice that were more than 3 months old, by individuals who were blinded to the genotype of the mice being analyzed. Genotypes were identified by genomic tail DNA PCR (as described earlier ). Animals were kept on a 12-hour light–dark cycle with constant room temperature and behavioral tests were performed in an appropriate quiet room between 11 am and 4 pm.
We used the following mice, which have been described in detail (except SNS-Gαq-/- mice) before ([5, 8]): Homozygous mice deficient for Gα11 (Gα11-/-) carrying the floxed allele of the mouse Gαq (Gαqfl/fl) gene (SNS-Cre-;Gαqfl/fl;Gα11-/-; referred to as Gα11-/- in this manuscript), sensory neuron-specific conditional double deficient mice for Gαq and Gα11 (SNS-Cre+;Gαqfl/fl;Gα11-/- mice; referred to as SNS-Gαq/11-/- in this manuscript), sensory neuron-specific conditional single deficient mice for Gαq (SNS-Cre+;Gαqfl/fl;Gα11+/+; referred to as SNS-Gαq-/- in this manuscript) and mice carrying the floxed allele of Gαq (Gαqfl/fl; referred to as control in this manuscript).
Summary of behavioral results showing main impact of G q -mediating sensitization processes
Thermal hyperalgesia – paw withdrawal latency
Mechanical allodynia – paw withdrawal response frequency
(Mean % change over pre-injection)
(Mean Δ increase over pre-injection) 0.4 g von Frey Filament
-51.5 ± 3.3 (n=8)
-37.1 ± 2.5 (n=7)
-7.4 ± 11.5* (n=7)
-8 ± 2.7* (n=7)
54.1 ± 5.5
27.8 ± 8*
14.4 ± 3.6*
6.7 ± 5.9*
-44.2 ± 10.4 (n=8)
-23.8 ± 7.2 (n=8)
-9.7 ± 7.5* (n=7)
-9.8 ± 5.1* (n=8)
32.8 ± 3.2
20 ± 8.9
4.8 ± 3.5*
6.7 ± 6.0*
-41 ± 5.7 (n=8)
-38.3 ± 5 (n=8)
-17.7 ± 3.3* (n=8)
-14.3 ± 8.1* (n=6)
-3.3 ± 3.1
4.2 ± 1.8
0.8 ± 0.8
-31,3 ± 6.9 (n=7)
-29.2 ± 2.3 (n=8)
21.1 ± 8.7
6.7 ± 2.7
-48.4 ± 4.6 (n=8)
-42.2 ± 6.1 (n=7)
-16.7 ± 5.5* (n=8)
-12.3 ± 4.3* (n=8)
27.5 ± 2
17.1 ± 6.5
4.8 ± 3.8*
5 ± 2.4*
Gq/11, Gs, G12/13
-39.5 ± 4 (n=8)
-42.3 ± 6.2 (n=7)
-21.7 ± 3.2* (n=8)
-25.5 ± 3.9* (n=7)
26.7 ± 8.5
26.7 ± 5.7
-0.8 ± 4.4*
4.8 ± 5*
Gq/11, Gs, Gi/o
-26.8 ± 5.6 (n=8)
-22.2 ± 8.8 (n=8)
-6.6 ± 8 (n=8)
-8 ± 5.7 (n=7)
15.6 ± 5.8
7.5 ± 4.8
-0.8 ± 2.9*
4.8 ± 5*
Gq/11, Gs, Gi/o
-41.9 ± 3.9 (n=8)
-34.4 ± 4.8 (n=8)
-31.4 ± 3.1 (n=8)
-24 ± 3.2* (n=7)
29.2 ± 5.9
14.2 ± 5.1*
7.5 ± 2.3*
2.9 ± 2*
-45.1 ± 11.8 (n=9)
-8.2 ± 6.5 * (n=8)
-11.2 ± 4.5* (n=9)
-12.5 ± 6.7* (n=9)
19.3 ± 9.7
30.4 ± 8.7
3.3 ± 6.3
6 ± 4.7
-49 ± 1.8 (n=7)
-38.7 ± 4.8 (n=8)
-30.7 ± 5 (n=7)
-26.7 ± 8.3 (n=8)
30.5 ± 5.8
36.7 ± 7.4
-1 ± 3.1*
5.8 ± 5.6*
Gq/11, Gi/o, G12/13
-27.5 ± 6.2 (n=8)
-30.1 ± 5 (n=8)
20 ± 3.3
16.7 ± 8.2
Gq/11, Gi/o, G12/13
-51.6 ± 5.7 (n=8)
-50.8 ± 5 (n=8)
-32.3 ± 4.5* (n=8)
-34.2 ± 7.3* (n=8)
23.3 ± 5
20 ± 7.1
4.2 ± 2.5*
0.8 ± 4.1*
All data are presented as mean ± standard error of the mean (S.E.M.). For multiple comparisons, Analysis of Variance (ANOVA) for random measures was performed followed by post-hoc Bonferroni’s test.
We analyzed the algogen-induced behavior in Gq/11 double deficient mice and found a complete loss of thermal hyperalgesia triggered by PGE2, Bradykinin, Glutamate, UTP and ATP, as well as mechanical hyperalgesia elicit by PGE2, Trypsin, Glutamate, UTP, Serotonin, ET1, S1P, PAF and ATP (Examples in Figure 1A, 1B, 1D-F, Table 1). There were minor changes with respect to thermal hyperalgesia upon ET1 and S1P application (Example in Figure 1B, Table 1), whereas thermal hyperalgesia towards Thrombin, CGRP and Serotonin and mechanical hyperalgesia towards Thrombin and CGRP was fully preserved in Gq/11 double deficient mice (Example in Figure 1C, Table 1). Interestingly, the deletion of Gq/11 in nociceptors had a stronger impact on mechanical allodynia than on thermal hyperalgesia.
Surprisingly, the single deletion of Gq caused the same behavioral phenotype as the double deletion of Gq and G11, (examples in Figure 1, Table 1) indicating a predominant role for Gq- over G11- proteins in nociceptive neurons.
We found that a particular G-protein pathway can contribute differentially to the action of diverse algogens and that a particular algogen can employ different G-protein pathways to elicit thermal hyperalgesia and mechanical allodynia. The Gq/11 G-protein signaling pathway plays an important role for nociceptor sensitization and the transduction of GPCR signaling towards the development of mechanical allodynia and thermal hyperalgesia with respect to the mediators tested in this manuscript.
To our surprise Gq has a major impact over G11 mediated nociceptor sensitization. Although Gq and G11 are nearly ubiquitously expressed in overlapping patterns , including the dorsal root ganglia and spinal cord , it cannot be ruled out that specific, highly localized differences may exist between the expression pattern and subcellular distribution of Gq and G11 in central circuits mediating hyperalgesia. Previous studies showing no difference in receptor-coupling with respect to Gq or G11 are performed in vitro[10–13] and thereby might not reflect the in vivo situation. It is more likely that different expression levels as shown for different brain regions [14–17] or membrane compartmentalization might account for the observed phenotypes. With respect to the DRGs it seems that there is a signaling succession for members of the Gq/11 family. G15/16 are not expressed, G14, G11 and Gq are expressed, while G14 has no specific role, G11 plays only a minor role for nociceptor sensitization and Gq is the most prominent G-proteins of this important signaling family. The classical deletion of Gq is known to be lethal , indicating essential requirement for this particular G-protein and no possible compensation of other G-proteins from different G-protein classes. Within the Gq/11 G-protein class, a preferential signaling role of Gq over G11 signaling has been demonstrated in various systems [14–16, 18–20] and the Gq-protein mediated signaling pathway in DRGs seems to have the major role over all other possible G-protein pathways which are involved in signal transduction upon receptor activation after application of ligands. We used the Cre-lox P system for conditional deletion such that the gene deletion only commences prenatally, thereby excluding early developmental deficits in SNS-Gq-/- mice, but we cannot rule out compensatory mechanisms of Gq in G11-/- mice as it has been suggested earlier .
Moreover, we were surprised to see the predominant contribution of the Gq/11 signaling pathway over Gs or Gi/o signaling with respect to those substances that are known to activate GPCRs which can bind different classes of G-protein, e.g. ATP, ET1, Glutamate, PAF, PGE2, Serotonin, S1P or Thrombin. Whereas the inhibitory Gi/o proteins contribute to anti-nociceptive signaling pathway, Gs and Gq/11 protein signaling mediates pro-nociceptive signaling (reviewed in ). For example PGE2, a crucial mediator for inflammatory pain couples to Gq/11-, Gi/o- and Gs-GPCRs but does not elicit thermal hyperalgesia or mechanical allodynia in mice lacking Gq, indicating a major contribution of the Gq-GPCR signaling pathway. Similarly, ATP or Serotonin, which can bind Gq/11- and Gs-GPCRs, do not lead to mechanical allodynia in Gq-deficient mice indicating a dominant role of Gq over the other G-proteins which are known to couple to the same receptors. On the contrary, thermal hyperalgesia and mechanical allodynia induced by CGRP (which can activate Gq/11- and Gs- coupled GPCRs) or Thrombin (which can bind Gq/11- Gi/o- and G12/13-GPCRs) are fully preserved, indicating that compensatory mechanisms via other G-proteins are functional.
Interestingly, with respect to thermal hyperalgesia only ATP-mediated heat hyperalgesia is abrogated in Gq-deficient mice whereas Serotonin-induced heat hyperalgesia is preserved in these animals. This predominant role of the Gq-protein in mediating mechanical allodynia over thermal hyperalgesia was also found for Endothelin and to some extend for S1P. It seems that the Gq/11 signaling pathway contributes significantly to mechanical allodynia elicited via a broad range of inflammatory mediators herein tested and that GPCR agonist-induced heat hyperalgesia is mediated via distinct G-protein GPCRs or other receptors.
Our results constitute a valuable tool to work out in vivo conditions of established nociceptive sensitizers. Moreover, this tool can be used for studying the mechanisms of action of new mediators in pain sensitization.
The authors thank Stefan Offermanns and Nina Wettschureck for providing Gαq/11 (Gαqfl/fl;Gα11-/-) mice. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to A.TT.
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