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. 2011 Jul;152(7):1548-1554.
doi: 10.1016/j.pain.2011.02.044. Epub 2011 Apr 1.

Metallopeptidase inhibition potentiates bradykinin-induced hyperalgesia

Ruben Gomez  1 , Elaine D Por, Kelly A Berg, William P Clarke, Marc J Glucksman, Nathaniel A Jeske
Affiliations

Metallopeptidase inhibition potentiates bradykinin-induced hyperalgesia

Ruben Gomez et al. Pain. 2011 Jul.

Abstract

The neuropeptide bradykinin (BK) sensitizes nociceptor activation following its release in response to inflammatory injury. Thereafter, the bioactivity of bradykinin is controlled by the enzymatic activities of circulating peptidases. One such enzyme, the metalloendopeptidase EC3.4.24.15 (EP24.15), is co-expressed with bradykinin receptors in primary afferent neurons. In this study, using approaches encompassing pharmacology, biochemistry, cell biology, and behavioral animal models, we identified a crucial role for EP24.15 and the closely related EP24.16 in modulating bradykinin-mediated hyperalgesia. Pharmacological analyses indicated that EP24.15 and EP24.16 inhibition significantly enhances bradykinin type-2 receptor activation by bradykinin in primary trigeminal ganglia cultures. In addition, bradykinin-induced sensitization of TRPV1 activation was increased in the presence of the EP24.15/16 inhibitor JA-2. Furthermore, behavioral analyses illustrated a significant dose-response relationship between JA-2 and bradykinin-mediated thermal hyperalgesia. These results indicate an important physiological role for the metallopeptidases EP24.15 and EP24.16 in regulating bradykinin-mediated sensitization of primary afferent nociceptors.

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Figures

Figure 1
Figure 1
EP24.15/16 inhibition increases bradykinin efficacy in trigeminal neurons. Cultured TG neurons were treated in the presence of the EP24.16-specific inhibitor, Pro-Ile (200 μM, n=4), the EP24.15/16 inhibitor JA-2 (5 μM, n=6), or the vehicle (0.005% DMSO/H2O, n=10) prior to treatment with increasing concentrations of bradykinin. Total IP accumulation was measured and expressed as percent above basal IP accumulation in non-treated neurons.
Figure 2
Figure 2
Functional expression of EP24.15 on the extracellular surface of the plasma membrane in trigeminal neurons. Cultured TG neurons were treated with 0.025% Trypsin-EDTA in serum-free media conditions. Cultures were harvested, and fractionated to separate plasma membrane from cytoplasm. A. 35 μg samples were resolved by SDS-PAGE, and probed for EP24.15, EP24.16 and caveolin-1. Results are representative of three independent trials. B. 10 μg samples were combined with QFS reaction materials to measure EP24.15 activity. n=6, ** p<0.01, two-way ANOVA.
Figure 3
Figure 3
EP24.15/16 inhibition potentiates bradykinin-sensitization of TRPV1 in trigeminal neurons. Cultured TG neurons were pre-treated with vehicle (0.005% DMSO/H2O) or JA-2 (5 μM, 3 min), then treated with vehicle (H2O) or BK (50 nM, 30 sec), and then stimulated with capsaicin (CAP, 50 nM, 30 sec). A. Representative traces of calcium accumulation following the illustrated experimental paradigm, treatment conditions indicated in legend. B. Quantified cumulative results of calcium accumulation, n for each treatment paradigm indicated along the X-axis. Colored bars correspond to colors in A. ** p<0.01, *** p < 0.005, as determined by one-way ANOVA, with Bonferroni correction ad-hoc. C. Quantified sensitization values for each treatment paradigm shown in A and B, normalized to veh/veh treatment, expressed as percentage. *** p < 0.005, as determined by one-way ANOVA, with Bonferroni correction ad-hoc.
Figure 4
Figure 4
EP24.15/16 inhibition potentiates bradykinin-mediated re-sensitization of desensitized TRPV1. Cultured TG neurons were pre-treated with vehicle (0.005% DMSO/H2O) or JA-2 (5 μM, 3 min), stimulated with CAP (50 nM, 30 sec), then treated with vehicle (H2O) or BK (50 nM, 30 sec), and again with CAP (50 nM, 30 sec). A. Representative traces of calcium accumulation following the illustrated experimental paradigm, treatment conditions indicated in legend. B. Quantified cumulative results of calcium accumulation, n=62-98 for each treatment paradigm indicated along the X-axis. Colored bars correspond to colors in A. * p<0.05, as determined by one-way ANOVA, with Bonferroni correction ad-hoc. C. Quantified desensitization percentages for each treatment paradigm shown in A and B, normalized to the initial CAP response. ** p<0.01, *** p < 0.005, as determined by one-way ANOVA, with Bonferroni correction ad-hoc.
Figure 5
Figure 5
JA-2 pre-treatment increases bradykinin-induced thermal hyperalgesia. Male, adult rats were injected i.pl. with 25 μl of either vehicle (0.005% DMSO/0.9% Saline) or JA-2 (5 μM), and tested for paw withdrawal latency to a thermal stimulus following same-paw administration of BK (1μM or 0.1μM, 25μl). A. Experimental timeline that was followed for each animal injected with BK. B. Paw withdrawal latencies for animals treated with 1 μM BK, n = 6-8 per treatment group. C. Paw withdrawal latencies for animals treated with 0.1 μM BK, n = 6-10 per treatment group. * p < 0.05, ** p<0.01, *** p < 0.005 (as shown between indicated groups), as determined by one-way ANOVA, with Bonferroni correction ad-hoc.
Figure 6
Figure 6
JA-2 pre-treatment does not significantly affect CFA-induced thermal hyperalgesia. A. Experimental timeline that was followed for each animal injected with complete Freund’s adjuvant (CFA). B. Male, adult rats were injected i.pl. with 25 μl of either vehicle (0.005% DMSO/0.9% Saline) or JA-2 (5 μM), and tested for paw withdrawal latency to a thermal stimulus 24 h after same-paw administration of CFA (0.5 mg/ml), n = 6 per treatment group. NS= no significance (as shown between indicated groups), ** p<0.01, *** p < 0.005 (compared to respective baseline readings), as determined by one-way ANOVA, with Bonferroni correction ad-hoc.
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