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. 2015 Nov;156(11):2364-2372.
doi: 10.1097/j.pain.0000000000000295.

A-kinase anchoring protein 79/150 coordinates metabotropic glutamate receptor sensitization of peripheral sensory neurons

Kalina Szteyn  1 , Matthew P Rowan, Ruben Gomez, Junhui Du, Susan M Carlton, Nathaniel A Jeske
Affiliations

A-kinase anchoring protein 79/150 coordinates metabotropic glutamate receptor sensitization of peripheral sensory neurons

Kalina Szteyn et al. Pain. 2015 Nov.

Abstract

Glutamate serves as the primary excitatory neurotransmitter in the nervous system. Previous studies have identified a role for glutamate and group I metabotropic receptors as targets for study in peripheral inflammatory pain. However, the coordination of signaling events that transpire from receptor activation to afferent neuronal sensitization has not been explored. Herein, we identify that scaffolding protein A-kinase anchoring protein 79/150 (AKAP150) coordinates increased peripheral thermal sensitivity after group I metabotropic receptor (mGluR5) activation. In both acute and persistent models of thermal somatosensory behavior, we report that mGluR5 sensitization requires AKAP150 expression. Furthermore, electrophysiological approaches designed to record afferent neuronal activity reveal that mGluR5 sensitization also requires functional AKAP150 expression. In dissociated primary afferent neurons, mGluR5 activation increases TRPV1 responses in an AKAP-dependent manner through a mechanism that induces AKAP association with TRPV1. Experimental results presented herein identify a mechanism of receptor-driven scaffolding association with ion channel targets. Importantly, this mechanism could prove significant in the search for therapeutic targets that repress episodes of acute pain from becoming chronic in nature.

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Figures

Figure 1
Figure 1. DHPG sensitization of thermal sensitivity in AKAP WT and KO mice
AKAP WT and KO mice were monitored for baseline paw withdrawal latency responses to a thermal stimulus prior to experimentation. AKAP WT and KO mice were injected intraplantarly (ipl) with DHPG (50nmol in 10μl PBS) or vehicle (Veh) as indicated, and tested 10min later for paw withdrawal latency to a thermal stimulus. n=6 mice/treatment paradigm, **p<0.01, *p<0.05, NS = not significant, two-way ANOVA with Bonferroni correction.
Figure 2
Figure 2. Carageenan priming of PGE2-induced thermal allodynia requires mGluR5 and AKAP
A. Rats were injected intraplantarly (ipl) with vehicle (Veh) or carrageenan (Cg, 5μl of 1% solution in 50μl PBS final) on day 1 (see Materials and Methods for full protocol). Rats were then injected with Veh or MTEP (400μg in 50 μl PBS) on days 2, 3, and 4. Rats were tested for baseline paw withdrawal latency day 5, then injected with PGE2 (indicated by arrow, 100ng in 50μl PBS, ipl) and tested again for paw withdrawal latency to a thermal stimulus at 15 min, 30 min, 2 h, and 4 h. Paw withdrawal latency displayed as change from baseline, n=6 rats/treatment paradigm, **p<0.01, *p<0.05, two-way ANOVA with Bonferroni correction. B. Mice were tested for baseline paw withdrawal latency to a thermal stimulus before intraplantar (ipl) injections (Pre-Cg). Mice were then injected with Cg (5μl of 1% solution in 10μl PBS final, ipl) on day 1. Mice were tested for baseline PWL on the Day 5 (Post-Cg), then injected with PGE2 (indicated by arrow, 50ng in 10μl, ipl) and tested for PWL at 15 min, 30 min, 2 h, and 4 h. Paw withdrawal latency displayed as change from baseline illustrated, n=6 mice/group, ***p<0.005, two-way ANOVA with Bonferroni correction.
Figure 3
Figure 3. mGluR Sensitization of Heat Response in Nociceptors from AKAP WT and KO Mice
A. Baseline activity in nociceptors was measured for 2 min prior to any manipulation. Mean discharge rate is higher in KO compared to WT, however, the increase is not significant. B. A 10 sec heat ramp, ascending from 34 to 51°C, demonstrates that the mean threshold to heat activation is significantly lower in KO compared to WT mice. *p < 0.05, Mann Whitney U test. C. Traces of unfiltered raw data show unit responses a few seconds before and after a 10 sec heat stimulus is applied to the receptive field of a nociceptor. In the KO mice there is no change in heat-induced activity following a 2 min treatment with 1 or 3 mM DHPG. D. In contrast, a unit from WT shows an enhanced discharge rate to heat following 3mM DHPG. The population data are summarized in E, F showing that a 2 min application of 1 or 3 mM DHPG to nociceptors in KO has no effect on heat-induced discharge rate (E) or threshold (F) but both are modified in WT. *p < 0.05, Kruskal-Wallis followed by Dunn's post hoc test.
Figure 4
Figure 4. mGluR Sensitization of Capsaicin Response in Nociceptors from AKAP WT and KO Mice
A, B Traces of unfiltered raw data demonstrate that repeated 2-min exposures of receptive fields to 10 μM DHPG has no effect on discharge rate in WT and KO. C. The response to CAP alone is similar in KO vs WT mice. However, 10μM DHPG greatly enhances 1 μM CAP-induced activity in WT (A,C) compared to KO (B,C) *p < 0.05, Kruskal-Wallis followed by Dunn's posthoc test. The percent of CAP responders is similar in KO compared to WT, however, deletion of AKAP significantly reduces the percent of nociceptors enhanced by DHPG-CAP (D) *p < 0.05, Fischer exact test.
Figure 5
Figure 5. AKAP plays a role in DHPG enhanced Ca2+ responses following sequential capsaicin treatment
Dorsal root ganglia neurons were isolated from WT and AKAP KO mice. Changes in intracellular Ca2+ levels were assessed by measurements with Fura2 calcium indicator. A. Representative tracings of single cell measurements. Starting with establishment of baseline, cells were treated with CAP (100 nM) for 30 s, washed with constant perfusion of SES buffer for 5 min, followed by administration of the second CAP treatment. In DHPG groups, cells were washed with SES buffer for 3 min and DHPG (100 μM) was administered for 2 min prior to second CAP treatment. B. The net change of intracellular Ca2+ concentration was calculated by subtraction of established basal concentration from peak concentration following CAP administration, n=28-76 neurons per group. In case of the second peak, concentration at the end of wash was taken as base value. C. The percentage of desensitization was determined by comparison of responses after first and second CAP application. The first Ca2+ increase was normalized to 100 %, and the second response was calculated as its percentage and the difference between the two illustrated the rate of desensitization for each group. ***p<0.001, NS = no significance, as determined by one-way ANOVA, with Bonferroni correction.
Figure 6
Figure 6. DHPG reversal of TRPV1 pharmacological desensitization is reversed by inhibition of PLC
Dorsal root ganglia neurons were isolated from WT mice. Changes in intracellular Ca2+ levels were assessed by measurements with Fura2 calcium indicator. A. Representative tracings of single cell measurements. Starting with establishment of baseline, cells were treated with CAP (100 nM) for 30 s, washed with constant perfusion of SES buffer for 5 min, followed by administration of the second CAP treatment. In DHPG treatment groups, cells were washed with SES buffer for 3 min and vehicle, DHPG (100 μM) and vehicle, DHPG and U73122 (1μM), or DHPG and U73343 (1μM) was administered for 2 min prior to second CAP treatment. B. The net change of intracellular Ca2+ concentration was calculated by subtraction of established basal concentration from peak concentration following CAP administration, n=77-107 neurons per group. In case of the second peak, concentration at the end of wash was taken as base value. C. The percentage of desensitization was determined by comparison of responses after first and second CAP application. The first Ca2+ increase was normalized to 100 %, and the second response was calculated as its percentage and the difference between the two illustrated the rate of desensitization for each group. ***p<0.001, NS = no significance, as determined by two-way ANOVA, with Bonferroni correction.
Figure 7
Figure 7. Figure 1. mGluR1/5 activation stimulates AKAP association with TRP Receptors
A. Cultured rat TG neurons were treated with DHPG (100μM, 5 min) and AKAP:TRPV1 association was determined by co-immunoprecipitation (Co-IP). Increased Co-IP is inhibited by pre-treatment with the mGluR5 negative allosteric modulator, fenobam (1μM, 5 min prior to DHPG). B. Quantified Co-IP results from A, normalized to total AKAP IP. Results representative of 3 independent trials, *p<0.05, **p<0.01, two-way ANOVA with Bonferroni correction. C. Cultured rat TG neurons were pre-treated with PLC inhibitor U73122 (1μM, 5 min) or negative control U73433 (1μM, 5 min) prior to DHPG (100μM, 5 min). AKAP:TRPV1 association was determined by Co-IP. D. Quantified Co-IP results from C, normalized to total AKAP IP. Results representative of 3 independent trials, *p<0.05, **p<0.01, two-way ANOVA with Bonferroni correction. For A and C, molecular weights of immunoreactive proteins in kilo Daltons (kDa) are indicated to the left of representative Western blots.
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