Enhancement of acid-sensing ion channel activity by prostaglandin E2 in rat dorsal root ganglion neurons

Brain Research 1724 (2019) 146442

Contents lists available at ScienceDirect

Brain Research journal homepage: www.elsevier.com/locate/brainres

Research report

Enhancement of acid-sensing ion channel activity by prostaglandin E2 in rat dorsal root ganglion neurons

T

Yi-Mei Zhoua,b, Lei Wua, Shuang Weia,b, Ying Jina,b, Ting-Ting Liua,c, Chun-Yu Qiub, ⁎ Wang-Ping Hua,c, a Research Center of Basic Medical Sciences, School of Basic Medical Sciences, Hubei University of Science and Technology, 88 Xianning Road, Xianning 437100, Hubei, PR China b Department of Pharmacology, Hubei University of Science and Technology, 88 Xianning Road, Xianning 437100, Hubei, PR China c Department of Physiology, School of Basic Medical Sciences, Hubei University of Science and Technology, 88 Xianning Road, Xianning 437100, Hubei, PR China

H I GH L IG H T S

increases ASIC currents in DRG neurons and exacerbates acid-evoked pain in rats. • PGE2 of ASIC activity by PGE2 is mediated by EP1 and EP4 receptors. • Enhancement • Cross-talking reveals a novel mechanism underlying PGE2 involvement in hyperalgesia.

A R T I C LE I N FO

A B S T R A C T

Keywords: Prostaglandin E2 Acid-sensing ion channel Proton-gated current Pain Dorsal root ganglion neuron

Prostaglandin E2 (PGE2) and proton are typical inflammatory mediators. They play a major role in pain processing and hypersensitivity through activating their cognate receptors expressed in terminals of nociceptive sensory neurons. However, it remains unclear whether there is an interaction between PGE2 receptors and proton-activated acid-sensing ion channels (ASICs). Herein, we show that PGE2 enhanced the functional activity of ASICs in rat dorsal root ganglion (DRG) neurons through EP1 and EP4 receptors. In the present study, PGE2 concentration-dependently increased ASIC currents in DRG neurons. It shifted the proton concentration-response curve upwards, without change in the apparent affinity of proton for ASICs. Moreover, PGE2 enhancement of ASIC currents was partially blocked by EP1 or EP4 receptor antagonist. PGE2 failed to enhance ASIC currents when simultaneous blockade of both EP1 and EP4 receptors. PGE2 enhancement was partially suppressed after inhibition of intracellular PKC or PKA signaling, and completely disappeared after concurrent blockade of both PKC and PKA signaling. PGE2 increased significantly the expression levels of p-PKCε and p-PKA in DRG cells. PGE2 also enhanced proton-evoked action potentials in rat DRG neurons. Finally, peripherally administration of PGE2 dose-dependently exacerbated acid-induced nocifensive behaviors in rats through EP1 and EP4 receptors. Our results indicate that PGE2 enhanced the electrophysiological activity of ASICs in DRG neurons and contributed to acidosis-evoked pain, which revealed a novel peripheral mechanism underlying PGE2 involvement in hyperalgesia by sensitizing ASICs in primary sensory neurons.

1. Introduction Tissue injury and inflammation result in the release of various mediators, including ATP, prostanoids, protons, peptides and cytokines, that increase responsiveness of the peripheral terminals of nociceptor neurons and contribute to peripheral sensitization (Woolf and Ma, 2007). Among these mediators, levels of PGE2 have been shown to

increase within inflamed tissues in rodents and humans (Nantel et al., 1999; Torebjork et al., 1992; Zhang et al., 1997). Peripheral administration of PGE2 reduces nociceptor response threshold to a number of stimuli (Chen et al., 1999; Minami et al., 1999). PGE2 has been shown to sensitize peripheral terminals of primary sensory neurons and cause primary hyperalgesia (Chen et al., 2013; Minami et al., 1999; Woolf and Ma, 2007). PGE2 exerts its effects through EP receptors, termed EP1–4,

⁎ Corresponding author at: Research Center of Basic Medical Sciences, School of Basic Medical Sciences, Hubei University of Science and Technology, 88 Xianning Road, Xianning 437100, Hubei, PR China. E-mail address: [email protected] (W.-P. Hu).

https://doi.org/10.1016/j.brainres.2019.146442 Received 25 September 2018; Received in revised form 12 July 2019; Accepted 7 September 2019 Available online 09 September 2019 0006-8993/ © 2019 Elsevier B.V. All rights reserved.

Brain Research 1724 (2019) 146442

Y.-M. Zhou, et al.

currents was dependent upon the concentration of PGE2. As can be seen from Fig. 1C, the peak amplitude of IpH6.0 increased as concentration of pre-treated PGE2 increased from 3 × 10−8 M to 3 × 10−6 M in a representative DRG neuron. The enhancement of IpH6.0 was reversible after washout of PGE2. The concentration–response curve for PGE2 in Fig. 1D shows PGE2 had a maximum effect (63.7 ± 5.2%, n = 9) at a concentration of 3 × 10−6 M. And the half-maximal response (EC50) value of the concentration–response curve was 0.21 ± 0.02 μM. The results indicated that PGE2 concentration-dependently enhanced the ASIC current in rat DRG neurons. We then investigated whether the PGE2 enhancement of ASIC currents was dependent upon different pHs. ASIC currents were measured by applying a range of different pHs in the presence of PGE2 or vehicle. As can be seen from Fig. 2A, pre-application of PGE2 (1 μM) for 2 min enhanced the peak amplitude of currents evoked by three different pHs. In contrast, vehicle (0.01% ethanol) had no effect on these ASIC currents. Fig. 2B shows the concentration-response curve to protons in the presence of PGE2 (1 μM) and vehicle. First, pre-application of PGE2 shifted the concentration-response curve to protons upwards, as indicated by an increase of 60.2 ± 7.6% in the maximal current response to protons in PGE2 treated cells, while the concentration-response curve to protons did not significantly change in vehicle-treated cells. Second, the Hill coefficient or slope of curve was not significantly changed by PGE2 (pH: n = 0.92 ± 0.13; vehicle + pH: n = 0.94 ± 0.16; PGE2 + pH: n = 0.89 ± 0.14; P > 0.1, Bonferroni’s post hoc test). Third, the pH for half-maximal activation (pH0.5) values of both curves had also no statistical difference (pH: pH0.5 = 5.73 ± 0.22; vehicle + pH: 5.68 ± 0.24; PGE2 + pH: pH0.5 = 5.71 ± 0.20; P > 0.1, Bonferroni’s post hoc test). We concluded that the enhancement of ASIC current by PGE2 is not due to a change in the apparent affinity of proton for ASICs.

all of which are expressed in primary sensory neurons including DRG neurons (Oida et al., 1995; Lin et al., 2006). Proton, another inflammatory mediator, is released during issue injury and inflammation and results in tissue acidosis (Reeh and Steen, 1996). Interestingly, studies have shown that the combination of low pH and PGE2 has a synergistic role in pain. Combined application of both provokes delayed pain upon injection in muscle and skin of human volunteers (Diers et al., 2011; Reeh and Steen, 1996). Moreover, PGE2 mediates acid-induced heartburn and visceral pain hypersensitivity in humans (Kondo et al., 2013; Sarkar et al., 2003). PGE2 also sensitizes low pH-induced response in human silent nociceptors (Namer et al., 2015). In acetic acid–induced stretch model, mice lacking EP1 receptor display reduced nociceptive perception (Stock et al., 2001). Direct perfusion of acidic solutions into the skin causes pain in humans (Steen et al., 1995). The acidosis-evoked pain is mediated by proton-gated ion channels such as acid-sensing ion channels (ASICs) and TRPV1 (Woolf and Salter, 2000). Interactions between PGE2 and TRPV1 in DRG neurons are well characterized. For example, PGE2 increases capsaicininduced currents in cultured DRG neurons via activation of PKA and PKC (Moriyama et al., 2005; Schnizler et al., 2008). PGE2 also increases TRPV1 externalization in cultured DRG neurons through EP1 and EP4 receptor subtypes (Ma et al., 2017). Moreover, PGE2 enhances capsaicin-evoked pain (Sawynok et al., 2006). PGE2-induced thermal hyperalgesia is abolished in TRPV1 knockout mice (Moriyama et al., 2005). ASICs are also proton sensors and activated by extracellular acidosis. To date, at least seven ASIC subunits encoded by four genes have been identified in mammals. Most of the ASIC subunits (i.e. ASIC1a and b, ASIC2a and b, and ASIC3) are expressed in both DRG cell bodies and sensory terminals (Alvarez de la Rosa et al., 2002; Deval et al., 2008). ASICs exist as homomeric or heteromeric channels in DRG neurons and contribute to proton-evoked pain signaling. ASICs, rather than TRPV1, are shown to mainly mediate pain sensation induced by peripheral moderate (up to pH 6.0) pH (Deval et al., 2008; Lingueglia, 2007; Ugawa et al., 2002). ASICs have therefore been proposed to be involved in the perception of pain in conditions associated with tissue acidosis such as tissue injury and inflammation (Deval et al., 2011; Deval and Lingueglia, 2015). So far, it remains unclear whether there is an interaction between PGE2 and ASICs in DRG neurons. Herein, we showed that PGE2 enhanced the functional activity of ASICs in DRG neurons through EP1 and EP4 receptors, which contributes to acidosis-induced nociception in rats.

2.2. EP1 and EP4 receptor subtypes mediated the PGE2 enhancement of ASIC currents We further verify whether the enhancement of ASIC currents by PGE2 was mediated by EP receptors. We next observed the effect of SC51322, a selective EP1 receptor antagonist, on the PGE2 enhancement of ASIC currents. After SC-51322 (3 μM) was pre-applied to DRG neurons tested, PGE2 (1 μM) causes an increase of 28.6 ± 3.2% on IpH6.0, while the amplitude of IpH6.0 increased 62.9 ± 4.2% by PGE2 alone (P < 0.01, Bonferroni’s post hoc test, n = 9; Fig. 3A and B). The result suggested that PGE2-induced enhancement of ASIC currents was significantly diminished in the presence of SC-51322. As can be seen from Fig. 3A and B, the blocking effect of SC-51322 was incomplete since PGE2 still had an obvious enhancing effect on IpH6.0. L-161,982, a potent EP4 receptor antagonist, also partially blocked but not eliminated the PGE2-induced enhancement. PGE2 (1 μM) causes an increase of 34.7 ± 6.6% on IpH6.0 after pre-application of 3 μM L-161,982 (P < 0.05, compared with PGE2 alone, Bonferroni’s post hoc test, n = 9; Fig. 3A and B). Finally, we investigated the effect of the simultaneous blockade of EP1 and EP4 receptors on PGE2-induced enhancement by co-treating with SC-51322 and L-161,982. PGE2 only produced an increase of 4.4 ± 1.4% on IpH6.0 after co-treatment with 3 μM SC-51322 and 3 μM L-161,982 in nine DRG neurons tested (P < 0.01, compared with PGE2 alone, Bonferroni’s post hoc test, n = 9; Fig. 3A and B). Application of SC-51322 and L-161,982 together completely blocked the enhancing effect of PGE2, suggesting EP1 and EP4 receptors involved in PGE2 enhancement of ASIC currents.

2. Results 2.1. PGE2 enhanced ASIC currents in rat DRG neurons To purify the ASIC currents from the proton-gated currents, AMG9810 (1 μM) was added to external solution to block proton-induced TRPV1 activation (Gavva et al., 2005). As shown in Fig. 1A, a drop in extracellular pH from 7.4 to 6.0 for 5 s caused a rapid inward current (IpH6.0) in rat DRG neurons tested. The IpH6.0 could be completely blocked by 20 μM of amiloride, a broad-spectrum ASIC channel blocker. It was also blocked by 3 μM APETx2, an ASIC3 blocker. Capsaicin (30 nM) failed to evoke any membrane currents in the presence of AMG9810 (1 μM) to block TRPV1 activation. However, capsaicin (30 nM) produced a slow inward current after washout of AMG9810. Thus, these proton-induced currents were considered to be ASIC currents after AMG9810 blocked proton-induced TRPV1 activation. In most neurons sensitive to acid stimuli (74.2%, 89/120), we observed that pre-application of PGE2 for 2 min enhanced the peak amplitude of the ASIC currents (Fig. 1B–D). In contrast, vehicle (0.01% ethanol) had no effect on the ASIC currents (Fig. 1B). In addition, PGE2 (10−6 M) and vehicle itself did not evoke any membrane currents (Fig. 1B). We first investigated whether the enhancement of ASIC

2.3. Intracellular mechanisms underlying the PGE2 enhancement of ASIC currents We further explore intracellular signal transduction mechanisms underlying PGE2 enhancement of ASIC currents using an intracellular dialysis technique. Since all EP receptors belong to a subfamily of G 2

Brain Research 1724 (2019) 146442

Y.-M. Zhou, et al.

Fig. 1. Enhancement of proton-gated currents by PGE2 in DRG neurons in a concentration-dependent manner A. Representative traces show currents evoked by a pH 6.0 acidic solution for 5 s in a DRG neuron in the presence of AMG9810 (1 μM) to block proton-induced TRPV1 activation. The proton-gated current could be blocked by 20 μM amiloride, a broad-spectrum ASIC channel blocker. It was also blocked by 3 μM APETx2, an ASIC3 blocker. Capsaicin (30 nM) failed to evoke any membrane currents in the presence of AMG9810 (1 μM) to block TRPV1 activation. However, capsaicin (30 nM) produced a slow inward current after washout of AMG9810. Membrane potential was clamped at −60 mV. B. The current traces illustrate that currents induced by pH 6.0 was enhanced by pre-application of PGE2 (10−6 M) for 2 min, but not by vehicle (0.01% ethanol). C. The sequential current traces illustrate enhancement of proton-induced currents by different concentration of PGE2 in a DRG neuron tested. PGE2 was pre-applied to external solution for 2 min. D. The graph shows PGE2 concentration-dependently enhanced the peak amplitude of proton-gated currents with an EC50 of 0.21 ± 0.02 μM. Each point represents the mean ± SEM of 9–11 cells.

Fig. 2. Effect of PGE2 on concentration-response curve for proton A. Sequential currents evoked by different pHs in the absence and presence of PGE2 (1 μM) or vehicle (0.01% ethanol). B. The concentration-response curves for proton. PGE2 (1 μM) pretreatment shifted the concentration-response curve for proton upwards. Each point represents the mean ± S.E.M. of 8–11 neurons. All peak current values were normalized to the current response induced by pH 5.0 applied alone in control solution (marked with asterisk).

normal internal solution, n = 9; Fig. 3C). The results suggested that the enhancing effect of PGE2 on ASIC current was partially blocked but not eliminated by dialysis of GF109203X or H89.When a mixture consisting of GF109203X (2 μM) and H89 (2 μM) was applied internally to DRG neurons through recording patch pipettes, PGE2 only produced an increase of 8.7 ± 2.4% on IpH6.0 (P < 0.01, post hoc Bonferroni’s test, compared with 62.9 ± 4.2% in normal internal solution, n = 9; Fig. 3C). Simultaneous blockade of PKC and PKA almost completely blocked PGE2-induced enhancement, suggesting the enhancement of ASIC currents was dependent upon PKC and PKA signal pathways.

protein-coupled receptors (GPCRs), GDP-β-S, a non-hydrolyzable GDP analog, was applied internally to DRG neurons through recording patch pipettes. As shown in Fig. 3C, PGE2 only produced an increase of 7.1 ± 1.2% on IpH6.0 when GDP-β-S (500 μM) was included in the pipette solution (P < 0.01, post hoc Bonferroni’s test, compared with 62.9 ± 4.2% in normal internal solution, n = 9; Fig. 3C). After PKC inhibitor GF109203X (2 μM) or PKA inhibitor H89 (2 μM) was applied internally to DRG neurons through recording pipettes, PGE2 produced increases of 25.6 ± 2.3% and 30.8 ± 4.5% on IpH6.0, separately (P < 0.01, post hoc Bonferroni’s test, compared with 62.9 ± 4.2% in 3

Brain Research 1724 (2019) 146442

Y.-M. Zhou, et al.

Fig. 3. The receptor and intracellular signal transduction mechanisms underlying enhancement of proton-gated currents by PGE2 The current traces in (A) and the bar graph in (B) show that the enhancement of IpH6.0 by PGE2 (1 μM) pre-applied alone was partially abolished by selective EP1 antagonist SC-51322 (3 μM) or potent EP4 receptor antagonist L-161,982 (3 μM). Both SC-51322 (3 μM) and L-161,982 (3 μM) applied together almost completely blocked PGE2 enhancement of IpH6.0. *P < 0.05, **P < 0.01, one way analysis of variance followed by post hoc Bonferroni’s test. n = 9 in each column. The bar graph in (C) shows the percentage increases in the IpH6.0 induced by PGE2 (1 μM) with recording pipettes filled with the normal internal solution, non-hydrolyzable GDP analog GDP-β-S (500 μM), PKC inhibitor GF109203X (2 μM), PKA inhibitor H89 (2 μM), or the combination of GF109203X (2 μM) and H89 (2 μM) containing internal solution. Intracellular dialysis of GDP-β-S or the combination of GF109203X and H89 almost completely abolished enhancement of IpH6.0 by PGE2. And dialysis of GF109203X or H89 alone blocked PGE2 effect partially. **P < 0.01, post hoc Bonferroni’s test, compared with normal internal solution. n = 9 in each column.

2.5. PGE2 exacerbated acid-induced nocifensive behaviors in rats

We then verify whether EP1 and EP4 expressed in DRG neurons. Fig. 4A shows existence of EP1 and EP4 in isolated DRG cells using western blotting. In addition, western blotting also used to detect the expression levels of p-PKCε and p-PKA in the absence or presence of PGE2 pretreatment (10−6 M, 2 min). As shown in Fig. 4B and C, PGE2 increased significantly the expression levels of p-PKCε and p-PKA by 1.5-fold and 1.7-fold, separately, suggesting the activation of PKCε and PKA in DRG neurons by PGE2.

We finally determined whether the PGE2 enhancing effect on ASICmediated responses in vitro could be observed behaviorally. Intraplantar injection of acetic acid (0.6%, 50 μl) elicits an intense flinch/shaking response in rats, and the flinch response is mediated by ASICs and mainly occurred during 0–5 min after injection of acetic acid. Acetic acid-evoked nociceptive response in rats was potently blocked by treatment with amiloride (100 μM, 50 μl), a broad-spectrum ASIC channel blocker (Fig. 6A), demonstrating the involvement of ASICs in the acidosis-induced nociception. We found that intraplantar pretreatment with PGE2 dose-dependently exacerbated the acidosis-induced nocifensive behaviors. The mean number of flinches induced by acetic acid significantly increased in rats pretreated with > 10 ng PGE2, compared with that observed in rats pretreated with vehicle (Bonferroni’s post hoc test, p < 0.05 and p < 0.01, n = 10; Fig. 6A). However, intraplantar pretreatment with 3 ng PGE2 had no effect on the acidosis-induced nocifensive behaviors (Bonferroni’s post hoc test, p > 0.1, n = 10; Fig. 6A). As a contrast, after 5 min rats received intraplantar injection with 100 ng PGE2, normal saline (50 μl) was subcutaneously administered into the hind paw. We failed to observe spontaneous flinch behaviors (Fig. 6A). When rats were intraplantarly co-treated with PGE2 (100 ng) and SC-51322 (1 μg), acetic acid induced a weaker nocifensive behaviors than that observed in rats treated with 100 ng PGE2 alone (Bonferroni’s post hoc test, P < 0.05, compared with 100 ng PGE2 alone, n = 10; Fig. 6B). However, SC-51322 only partially blocked the exacerbating

2.4. PGE2 enhanced proton-evoked action potentials in rat DRG neurons In the presence of 1 μM AMG9810 to block proton-induced TRPV1 activation, a stimulus of pH drop from 7.4 to 6.0 could induced a wholecell inward current with voltage-clamp recording in a DRG neuron, meanwhile the pH drop could also trigger bursts of action potentials under current-clamp conditions in the same cell tested (Fig. 5A). Similar to that observed under voltage-clamp conditions, PGE2 also increased proton-evoked action potentials in DRG neurons (Fig. 5A). After PGE2 (1 μM) was applied to DRG neurons tested for 2 min, the mean number of action potentials evoked by stimuli of pH 6.0 increased from 5.58 ± 0.70 to 8.52 ± 1.12 (n = 9, P < 0.01, paired t-test; Fig. 5B). After washout of PGE2, the mean number of action potentials evoked by acid recovered to control condition. These results indicated that PGE2 reversibly exerted an enhancing effect on proton-evoked action potentials in rat DRG neurons.

4

Brain Research 1724 (2019) 146442

Y.-M. Zhou, et al.

Fig. 5. Enhancement of proton-evoked action potentials by PGE2 in rat DRG neurons A. Original current and spikes recordings from the same DRG neuron in the presence of AMG9810 (1 μM). Left panel: a pH 6.0 acid stimulus induced an inward current with voltage-clamp recording. Right panel: the pH 6.0 acid stimulus produced cell spikes with current-clamp recording. The pretreatment of PGE2 (1 μM) increased the acid-induced the number of action potentials. B. The bar graph shows PGE2 increased significantly the number of action potentials produced by pH 6.0 acid perfusions. After 20 min washout of PGE2, acid-evoked action potentials recovered to control condition. ** P < 0.01, paired t-test, compared with control, n = 9 in each column.

Fig. 4. Increased expression p-PKCε and p-PKA by PGE2 in isolated DRG cells A. Expression of EP1 and EP4 in DRG neurons. B. Detection using western blotting of p-PKCε and p-PKA in isolated DRG cells in the absence (vehicle) or presence of PGE2 pretreatment (10−6 M, 2 min). C. The bar graph shows PGE2 increased significantly the expression levels of p-PKCε and p-PKA.

Since the 1980s, it has been known that extracellular protons generate currents in a subset of primary afferents (Krishtal and Pidoplichko, 1980). The acidosis-evoked currents are mediated by proton-gated ion channels such as ASICs and TRPV1 (Woolf and Salter, 2000). In the presence of AMG9810 to block the activation of TRPV1 channels, the present proton-gated currents were mediated by ASICs, since they could be completely blocked by ASIC channel blockers. We thus considered them to be ASIC currents, although precise ASIC subunits need to be identified. Besides ASIC4 subunit, other six ASIC subunits are present in DRG neurons (Alvarez de la Rosa et al., 2002). And ASIC3 have emerged as critical pH sensors predominantly expressed in nociceptors (Deval et al., 2008). ASIC subunits can form a variety of homomeric or heteromeric channels in DRG neurons and contribute to proton-evoked pain signaling. The current study showed that PGE2 can enhance ASIC currents in rat DRG neurons in a dose-dependent manner. PGE2 sensitized ASICs by shifting the proton concentration–response curve upward without changing the pH0.5. Thus, the enhancement of ASIC currents is not due to a change in the apparent affinity of proton for ASICs. Activation of ASICs by extracellular protons mainly induces sodium influx, resulting in membrane depolarization and bursts of action potentials (Kellenberger and Schild, 2015). The current clamp experiments showed that PGE2 also increased the number of action potentials evoked by extracellular acid stimuli in DRG neurons, which was consistent with the results that PGE2 enhanced ASIC currents in voltage clamp experiments. Further, behavioral studies showed that PGE2 dosedependently exacerbated nocifensive responses to acetic acid in rats. Obviously, the electrophysiological data corroborated the behavioral

effect of PGE2 on acidosis-induced nocifensive behaviors. Co-treatment with PGE2 (100 ng) and SC-51322 (1 μg) still exacerbated the acidosisinduced nocifensive behaviors compared with control rats (15.43 ± 1.09 vs 10.64 ± 1.47, P < 0.05, n = 10). Similar results were also observed in rats co-treated with 100 ng PGE2 and 1 μg L161,982 (Fig. 6B). Further, intraplantar application of SC-51322 (1 μg) and L-161,982 (1 μg) together completely blocked the exacerbating effect of PGE2 on acidosis-induced nocifensive behaviors (Fig. 6B). These results indicated that PGE2 contributed to acid-induced nocifensive behaviors at the periphery through EP1 and EP4 receptor subtypes in rats. We further observed the action of PGE2 on other flinching behavior in physiological pH models without acid. As shown in Fig. 6C, intraplantar administration of αβ-methylene ATP (αβ-me-ATP; 500 nmol, 50 µl) or capsaicin (0.05%, 50 µl) also caused flinching behavior in rats. We found that intraplantar pretreatment with 100 ng PGE2 significantly increased the flinching behavior by induced capsaicin, but not by induced αβ-me-ATP. 3. Discussion We demonstrated here that PGE2 enhanced the functional activity of ASICs in rat primary sensory neurons. PGE2 not only increased ASICmedicated currents and action potentials in dissociated DRG neurons but also exacerbated acid-induced nocifensive behaviors in rats. The enhancement of PGE2 was mediated by EP1 and EP4 via intracellular PKC and PKA signaling pathways. 5

Brain Research 1724 (2019) 146442

Y.-M. Zhou, et al.

Fig. 6. Effect of PGE2 on nociceptive responses to intraplantar injection of acetic acid in rats A. The nociceptive responses evoked by intraplantar injection of acetic acid (0.6%, 50 μl) in the presence of the TRPV1 inhibitor AMG9810 (10 μM). The acidosis-evoked nociception was blocked by pretreatment with 100 μM amiloride. The pretreatment of PGE2, but not vehicle, increased the flinching behavior induced by acetic acid in dose-dependent manner (3–100 ng). In contrast, normal saline (NS, 50 μl) was subcutaneously administered into the hind paw did not produce spontaneous flinches after the pretreatment of 100 ng PGE2. Each bar represents the number of flinches that animals spent licking/lifting the injected paw during first 5-min observation period. *P < 0.05, **P < 0.01, Bonferroni’s post hoc test, compared with vehicle (0.01% ethanol) column. Each group represents the mean ± S.E.M. of 10 rats. B. The effect of PGE2 (100 ng) on the flinching behavior was partially blocked by co-treatment of SC-51322 (1 μg) or L-161,982 (1 μg). Both SC-51322 (1 μg) and L-161,982 (1 μg) applied together almost completely blocked PGE2 enhancement of the flinching behavior. *P < 0.05, **P < 0.01, Bonferroni’s post hoc test, n = 10 in each column. C. Flinching behavior induced by intraplantar administration of αβ-me-ATP (500 nmol, 50 µl) or capsaicin (0.05%, 50 µl). The pretreatment of 100 ng PGE2 increased the flinching behavior induced by capsaicin, but not by induced αβ-me-ATP. **P < 0.01, unpaired t-test, n = 8 in each column.

simultaneous blockade of EP1 and EP4 receptors by co-treating with SC-51322 and L-161,982 almost completely blocked PGE2-induced enhancement. Four EP receptors (EP1-4) that are encoded by different genes have been identified, all of which are expressed in primary sensory neurons including DRG neurons (Oida et al., 1995). PGE2 sensitizes peripheral nociceptors through activation of EP receptors present on the peripheral terminals. EP1 receptor subtype has been shown to play a major, but not exclusive, role in mediating the contribution of PGE2 to peripheral sensitization (Ma et al., 2017; Moriyama et al., 2005; Sarkar et al., 2003; Stock et al., 2001). Subcutaneous or systemic administration of EP1 receptor antagonists attenuate peripheral hypersensitivity and hyperalgesia induced by PGE2 (Johansson et al., 2011; Omote et al., 2001). Mice lacking EP1 receptor display a significant reduction in local PGE2-induced thermal hyperalgesia (Moriyama et al., 2005). Expressional levels of EP4 receptor subtype are increased in DRG neurons after adjuvant injection and mainly contribute to peripheral inflammatory pain hypersensitivity (Lin et al., 2006).). Blockade of the EP4 receptor shows analgesic effects in inflammation models (Clark et al., 2008). EP4-deficient mice display a reduction in the development of joint inflammation in a model of rheumatoid arthritis (McCoy et al., 2002). It has been shown that PGE2 or EP4 agonists stimulated EP4 externalization in cultured DRG neurons, which contributes to nociceptor sensitization by PGE2 (Ma and StJacques, 2018; St-Jacques and Ma, 2013). Both EP1 and EP4 receptors have been shown to mediate PGE2 potentiation of TRPV1 activity in

studies and vice versa. Many studies have shown that PGE2 sensitizes nociceptors and induces hyperalgesia. For example, intra-plantar PGE2 sensitizes the rodent hind paw to mechanical stimuli in physiological pH (Villarreal et al., 2009). So we cannot rule out the exacerbated nociceptive response evoked by acetic acid was due to sensitization of nociceptors by PGE2. But we found that intra-plantar PGE2 failed to increase the flinching behavior induced by intraplantar administration of αβ-me-ATP in physiological pH. PGE2 does not increase all flinching behavior although it sensitizes nociceptors. Thus, the action of PGE2 on flinching behavior may be related to channels which mediated the behavior. Consistent with the result that PGE2 potentiation of TRPV1 current in DRG neurons (Lin et al., 2006; Moriyama et al., 2005), intraplantar PGE2 increased capsaicin-induced flinching behavior. The present study revealed an interaction between PGE2/EPs and ASICs. PGE2 can enhance ASIC- mediated currents and action potentials in rat DRG neurons. So it’s also possible that PGE2 exacerbated acid-induced nocifensive behaviors by sensitizing ASICs in nociceptors. PGE2 enhanced the currents and action potentials medicated by ASICs in primary sensory neurons, resulting in amplification of proton-induced pain. The present study provided electrophysiological and behavioral evidences that PGE2 can enhance the functional activity of ASICs. We found that PGE2 enhanced ASIC currents and acid-induced nocifensive behaviors through EP1 and EP4 receptor subtypes, since the PGE2 enhancement was partially blocked by selective EP1 receptor antagonist SC-51322 or potent EP4 receptor antagonist. Further, 6

Brain Research 1724 (2019) 146442

Y.-M. Zhou, et al.

5. Methods and materials

cultured DRG neurons (Lin et al., 2006; Moriyama et al., 2005). Detection using western blotting exposed EP1 and EP4 expressed in the present isolated DRG cells. The current study demonstrated a functional interaction between ASICs and EP1 and EP4 receptors. Both EP1 and EP4 receptors together enhanced the functional activity of ASICs in primary sensory neurons. Since ASICs play an important role in pain processing, PGE2 enhancement of ASICs would provide a way for sensory neurons to specifically respond to tissue injury and inflammation. The current study showed that enhancement of ASIC currents by PGE2 was blocked by intracellular dialysis of GDP-β-S, indicating an involvement of GPCRs. We found that PGE2 enhancement was partially suppressed by intracellular dialysis of PKC inhibitor GF109203X or PKA inhibitor H89. Further, PGE2 failed to enhance ASIC currents when simultaneous blockade of PKC and PKA. The results of western blotting showed that PGE2 pretreatment also increased significantly the expression levels of p-PKCε and p-PKA in DRG neurons. These results suggested that activation of PKC and PKA played a major role in PGE2 enhancement of ASIC currents. Both EP1 and EP4 receptors belong to a subfamily of GPCRs. EP1 receptor Gq/11 to activate PLC, which in turn produces IP3 and DAG, followed by cytosolic calcium mobilization and activation of PKC (Kawabata, 2011). Whereas EP4 receptor couples Gs to activate AC that generates cAMP and results in activation of PKA (Kawabata, 2011; Yokoyama et al., 2013). It has been reported that PGE2 increases intracellular cAMP levels in DRG neurons and sensitizes TRPV1 (Moriyama et al, 2005). It has been shown that both PKA and PKC participate in PGE2-mediated hypernociception (Sachs et al., 2009). Both PKC and PKA dependent signaling pathways are also involved in PGE2 potentiation of TRPV1 currents and tetrodotoxin resistant sodium channels in DRG neurons (Gold et al., 1998; Moriyama et al., 2005; Schnizler et al., 2008). Recently, Ma et al reported that PGE2 increases TRPV1 externalization in cultured DRG neurons via EP1/PKC and EP4/PKA signaling pathways (Ma et al., 2017). Our previous studies showed that ASICs are modulated by PKC and PKA signaling (Liu et al., 2012; Wu et al., 2017). Thus, PGE2 enhancement of ASIC currents may be also dependent upon EP1/PKC and EP4/PKA signaling pathways, although physiological relevance of the two different pathways remains to be elucidated. . During issue injury and inflammation, protons are released from damaged cells and the de-granulation of mast cells. These released protons result in a drop of the extracellular pH locally, which is enough to activate ASICs (Reeh and Steen, 1996). PGE2 is synthesized and released from damaged or inflamed tissue and its levels have been shown to increase within inflamed tissues in rodents and humans (Nantel et al., 1999; Torebjork et al., 1992; Zhang et al., 1997). Once both protons and PGE2 are locally released together at the site inflammation, they could initiate and/or sensitize nociceptive process through activating their cognate receptors expressed in surrounding terminals of nociceptive sensory neurons. Herein, we showed that the ASICs are targets of PGE2. The released PGE2 could sensitize ASICs through EP1 and EP4 receptors, which further exacerbated proton-evoked pain.

5.1. Isolation of DRG neurons The experimental protocol was approved by the animal research ethics committee of Hubei University of Science and Technology. All procedures were made to minimize the number of animals used and their sufferings. Five- to six-week old Sprague-Dawley male rats were sacrificed after anesthetized. The DRGs were taken out and transferred immediately into Dulbecco’s modified Eagle’s medium (DMEM, Sigma) at pH 7.4. After the removal of the surrounding connective tissues, the DRGs were minced with fine spring scissors and the ganglion fragments were placed in a flask containing 5 ml of DMEM in which trypsin (type II-S, Sigma) 0.5 mg/ml, collagenase (type I-A, Sigma) 1.0 mg/ml and DNase (type IV, Sigma) 0.1 mg/ml had been dissolved, and incubated at 35 °C in a shaking water bath for 25–30 min. Soybean trypsin inhibitor (type II-S, Sigma) 1.25 mg/ml was then added to stop trypsin digestion. Dissociated neurons were placed into a 35-mm Petri dish and kept for at least 1 h in normal external solution before the start of electrophysiological experiments. The neurons selected for electrophysiological experiment were 15–35 μm in diameter, which are thought to be nociceptive neurons. 5.2. Electrophysiological recordings Whole-cell patch clamp and voltage-clamp recordings were carried out at room temperature (22–25 °C) using a MultiClamp-700B amplifier and Digidata-1440A A/D converter (Axon Instruments, CA, USA). Recording pipettes were pulled using a Sutter P-97 puller (Sutter Instruments, CA, USA). The micropipettes were filled with internal solution containing (mM): KCl 140, MgCl2 2.5, HEPES 10, EGTA 11 and ATP 5; its pH was adjusted to 7.2 with KOH and osmolarity was adjusted to 310 mOsm/L with sucrose. Cells were bathed in an external solution containing (mM): NaCl 150, KCl 5, CaCl2 2.5, MgCl2 2, HEPES 10, d-glucose 10; its osmolarity was adjusted to 330 mOsm/L with sucrose and its pH to 7.4. The resistance of the recording pipette was in the range of 3–6 MΩ. A small patch of membrane underneath the tip of the pipette was aspirated to form a giga seal and then a negative pressure was applied to rupture it, thus establishing a whole-cell configuration. The series resistance was compensated for by 70–80%. The adjustment of capacitance compensation was also done before recording the membrane currents. The membrane voltage was maintained at −60 mV in all voltage-clamp experiments unless otherwise specified. Current-clamp recordings were obtained by switching to current-clamp mode after a stable whole-cell configuration was formed in voltage-clamp mode. Only cells with a stable resting membrane potential (more negative than −50 mV) were used in the study. Signals were sampled at 10 to 50 kHz and filtered at 2–10 kHz, and the data were stored in compatible PC computer for off-online analysis using the pCLAMP 10 acquisition software (Axon Instruments, CA, USA). 5.3. Drug application

4. Conclusion

Drugs were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and used in the experiments include: hydrochloric acid, PGE2, SC51322, L-161,982, amiloride, APETx2, capsaicin, AMG 9810, and αβmethylene ATP (αβ-me-ATP). Different pH values were configured with hydrochloric acid and external solution. PGE2 was dissolved in ethanol and freshly prepared in normal external solution just before use and held in a linear array of fused silica tubes (o.d./i.d. = 500 μm/200 μm) connected to a series of independent reservoirs. The application pipette tips were positioned ~30 μm away from the recorded neurons. Cells were constantly bathed in normal external solution. The application of each drug was driven by gravity and controlled by the corresponding valve, and rapid solution exchange could be achieved within about 100 ms by shifting the tubes horizontally. In some experiments where

In summary, our results indicated that PGE2 enhanced the electrophysiological activity of ASICs in DRG neurons and contributed to acidosis-evoked pain, which revealed a novel peripheral mechanism underlying PGE2 involvement in hyperalgesia by sensitizing ASICs in primary sensory neurons. ASICs represent downstream targets of PGE2 and therapy targeting ASICs is likely useful for treating inflammatory and neuropathic pain.

7

Brain Research 1724 (2019) 146442

Y.-M. Zhou, et al.

manuscript.

GDP-β-S (Sigma), GF109203X (RBI) and H89 (Sigma) were applied for intracellular dialysis through recording patch pipettes, they were dissolved in the internal solution before use. To ensure that the cell interior was perfused with the dialysis drug, there was at least a 30 min interval between establishment of whole-cell access and the current measurement. To functionally characterize ASIC activity, we used AMG9810 (1 μM) to block TRPV1 in the extracellular solution (Gavva et al., 2005).

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements

5.4. Western blots This work was supported by the National Natural Science Foundation of China (No. 81671101, No. 31471062), and Natural Science Foundation of Hubei Province of China (No. 2015CFA145).

Dissociated DRG neurons were placed into a 35-mm Petri dish and kept for at least 1 h before western blot analysis. After the DRG neurons were pre-treated by PGE2 or vehicle, total proteins were prepared by homogenizing neurons in a lysis buffer (50 mM Tris–HCl at pH 6.8, 2% SDS and 10% glycerol) with 1 × NaF, 1 × NaVO4, and 1 × Protein inhibitor cocktail. The supernatant was obtained by centrifugation, and the protein concentration was determined using the PierceTM BCA protein Assay kit (Thermo Fisher, Waltham mass, USA). Proteins were resolved by SDS–PAGE, transferred onto a polyvinylidene fluoride membrane and blocked in 5% skim milk/Tris-buffered saline that contained 0.1% Tween 20 at room temperature for 1 h. The membranes were incubated with the primary antibodies at 4 °C overnight, and then were incubated with second antibody at room temperature for 1 h. After washing, the bands were visualized with Enhanced chemiluminescence Western blotting detection reagents. The band density was quantified using Image J software. The antibodies were listed as following: antiEP1 (1:1000; 101740, Cayman Chemical Co., USA), anti-EP4 (1:1000, 101770, Cayman Chemical Co., USA), anti-p-PKCε (1:1000; ab63387, Abcam, UK), anti-p-PKA (1:1000; ab32390, Abcam, UK), anti-β-actin (1:1000; A2228, Sigma, USA).

References Alvarez de la Rosa, D., Zhang, P., Shao, D., White, F., Canessa, C.M., 2002. Functional implications of the localization and activity of acid-sensitive channels in rat peripheral nervous system. Proc. Natl. Acad. Sci. U.S.A. 99, 2326–2331. Chen, L., Yang, G., Grosser, T., 2013. Prostanoids and inflammatory pain. Prostaglandins Other Lipid Mediat. 104–105, 58–66. Chen, X., Tanner, K., Levine, J.D., 1999. Mechanical sensitization of cutaneous C-fiber nociceptors by prostaglandin E2 in the rat. Neurosci. Lett. 267, 105–108. Clark, P., Rowland, S.E., Denis, D., Mathieu, M.C., Stocco, R., Poirier, H., Burch, J., Han, Y., Audoly, L., Therien, A.G., Xu, D., 2008. MF498 [N-{[4-(5,9-Diethoxy-6-oxo-6,8dihydro-7H-pyrrolo[3,4-g]quinolin-7-yl)-3-methylbe nzyl]sulfonyl}-2-(2-methoxyphenyl)acetamide], a selective E prostanoid receptor 4 antagonist, relieves joint inflammation and pain in rodent models of rheumatoid and osteoarthritis. J. Pharmacol. Exp. Ther. 325, 425–434. Deval, E., Noel, J., Lay, N., Alloui, A., Diochot, S., Friend, V., Jodar, M., Lazdunski, M., Lingueglia, E., 2008. ASIC3, a sensor of acidic and primary inflammatory pain. EMBO J. 27, 3047–3055. Deval, E., Noel, J., Gasull, X., Delaunay, A., Alloui, A., Friend, V., Eschalier, A., Lazdunski, M., Lingueglia, E., 2011. Acid-sensing ion channels in postoperative pain. J. Neurosci. 31, 6059–6066. Deval, E., Lingueglia, E., 2015. Acid-Sensing Ion Channels and nociception in the peripheral and central nervous systems. Neuropharmacology 94, 49–57. Diers, M., Schley, M.T., Rance, M., Yilmaz, P., Lauer, L., Rukwied, R., Schmelz, M., Flor, H., 2011. Differential central pain processing following repetitive intramuscular proton/prostaglandin E(2) injections in female fibromyalgia patients and healthy controls. Eur. J. Pain 15, 716–723. Gavva, N.R., Tamir, R., Qu, Y., Klionsky, L., Zhang, T.J., Immke, D., Wang, J., Zhu, D., Vanderah, T.W., Porreca, F., Doherty, E.M., Norman, M.H., Wild, K.D., Bannon, A.W., Louis, J.C., Treanor, J.J., 2005. AMG 9810 [(E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide], a novel vanilloid receptor 1 (TRPV1) antagonist with antihyperalgesic properties. J. Pharmacol. Exp. Ther. 313, 474–484. Gold, M.S., Levine, J.D., Correa, A.M., 1998. Modulation of TTX-R INa by PKC and PKA and their role in PGE2-induced sensitization of rat sensory neurons in vitro. J. Neurosci. 18, 10345–10355. Johansson, T., Narumiya, S., Zeilhofer, H.U., 2011. Contribution of peripheral versus central EP1 prostaglandin receptors to inflammatory pain. Neurosci. Lett. 495, 98–101. Kawabata, A., 2011. Prostaglandin E2 and pain–an update. Biol. Pharm. Bull. 34, 1170–1173. Kellenberger, S., Schild, L., 2015. International union of basic and clinical pharmacology. XCI. Structure, function, and pharmacology of acid-sensing ion channels and the epithelial Na+ channel. Pharmacol. Rev. 67, 1–35. Kondo, T., Oshima, T., Tomita, T., Fukui, H., Watari, J., Okada, H., Kikuchi, S., Sasako, M., Matsumoto, T., Knowles, C.H., Miwa, H., 2013. Prostaglandin E(2) mediates acidinduced heartburn in healthy volunteers. Am. J. Physiol. Gastrointest. Liver Physiol. 304, G568–G573. Krishtal, O.A., Pidoplichko, V.I., 1980. A receptor for protons in the nerve cell membrane. Neuroscience 5, 2325–2327. Lin, C.R., Amaya, F., Barrett, L., Wang, H., Takada, J., Samad, T.A., Woolf, C.J., 2006. Prostaglandin E2 receptor EP4 contributes to inflammatory pain hypersensitivity. J. Pharmacol. Exp. Ther. 319, 1096–1103. Lingueglia, E., 2007. Acid-sensing ion channels in sensory perception. J. Biol. Chem. 282, 17325–17329. Liu, Y.Q., Qiu, F., Qiu, C.Y., Cai, Q., Zou, P., Wu, H., Hu, W.P., 2012. Cannabinoids inhibit acid-sensing ion channel currents in rat dorsal root ganglion neurons. PLoS ONE 7, e45531. Ma, W., St-Jacques, B., 2018. Signalling transduction events involved in agonist-induced PGE2/EP4 receptor externalization in cultured rat dorsal root ganglion neurons. Eur. J. Pain 22, 845–861. Ma, W., St-Jacques, B., Rudakou, U., Kim, Y.N., 2017. Stimulating TRPV1 externalization and synthesis in dorsal root ganglion neurons contributes to PGE2 potentiation of TRPV1 activity and nociceptor sensitization. Eur. J. Pain 21, 575–593. McCoy, J.M., Wicks, J.R., Audoly, L.P., 2002. The role of prostaglandin E2 receptors in the pathogenesis of rheumatoid arthritis. J. Clin. Invest. 110, 651–658. Minami, T., Okuda-Ashitaka, E., Hori, Y., Sakuma, S., Sugimoto, T., Sakimura, K., Mishina, M., Ito, S., 1999. Involvement of primary afferent C-fibres in touch-evoked

5.5. Nociceptive behaviour induced by acetic acid in rats Rats were placed in a 30 × 30 × 30 cm Plexiglas chamber and allowed to habituate for at least 30 min before nociceptive behavior experiments. A double-blind experiment was carried out. Separate groups of rats were coded and pretreated with 50 μl AMG 9810 (10 μM) together with vehicle (0.01% ethanol), different dosages of PGE2, SC51322, L-161,982, or amiloride in ipsilateral hindpaw before injection of acetic acid. After 5 min, the other experimenters who did not know the above experimental condition subcutaneously administered acetic acid solution (0.6%, 50 μl) into the hind paw using a 30 gauge needle connected to a 100 μl Hamilton syringe. And nociceptive behavior (that is, number of flinches) was counted over a 5 min period starting immediately after the injection (Deval et al., 2008; Omori et al., 2008). Other flinching behaviors induced by intraplantar administration of αβ-me-ATP (500 nmol, 50 µl) or capsaicin (0.05%, 50 µl). Flinching was observed for 30 min following αβ-me-ATP injection and 5 min following capsaicin injection. A 1% capsaicin stock solution was diluted with the capsaicin vehicle (7% Tween-80 in saline) in order to produce a working solution of 0.05% capsaicin. 5.6. Data analysis Data were statistically compared using the Student’s t-test or analysis of variance (ANOVA), followed by Bonferroni’s post hoc test. Statistical analysis of concentration-response data was performed using nonlinear curve-fitting program ALLFIT. Data are expressed as mean ± S.E.M. Contributors WPH and YMZ designed this research; YMZ, LW, SW, YJ, TTL and CYQ performed the experiments and YMZ participated in data analysis. All authors contributed substantially to this research and reviewed this 8

Brain Research 1724 (2019) 146442

Y.-M. Zhou, et al.

Schnizler, K., Shutov, L.P., Van Kanegan, M.J., Merrill, M.A., Nichols, B., McKnight, G.S., Strack, S., Hell, J.W., Usachev, Y.M., 2008. Protein kinase A anchoring via AKAP150 is essential for TRPV1 modulation by forskolin and prostaglandin E2 in mouse sensory neurons. J. Neurosci. 28, 4904–4917. St-Jacques, B., Ma, W., 2013. Prostaglandin E2/EP4 signalling facilitates EP4 receptor externalization in primary sensory neurons in vitro and in vivo. Pain 154, 313–323. Steen, K.H., Issberner, U., Reeh, P.W., 1995. Pain due to experimental acidosis in human skin: evidence for non-adapting nociceptor excitation. Neurosci. Lett. 199, 29–32. Stock, J.L., Shinjo, K., Burkhardt, J., Roach, M., Taniguchi, K., Ishikawa, T., Kim, H.S., Flannery, P.J., Coffman, T.M., McNeish, J.D., Audoly, L.P., 2001. The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure. J. Clin. Invest. 107, 325–331. Torebjork, H.E., Lundberg, L.E., LaMotte, R.H., 1992. Central changes in processing of mechanoreceptive input in capsaicin-induced secondary hyperalgesia in humans. J. Physiol. 448, 765–780. Ugawa, S., Ueda, T., Ishida, Y., Nishigaki, M., Shibata, Y., Shimada, S., 2002. Amilorideblockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors. J. Clin. Invest. 110, 1185–1190. Villarreal, C.F., Funez, M.I., Figueiredo, F., Cunha, F.Q., Parada, C.A., Ferreira, S.H., 2009. Acute and persistent nociceptive paw sensitisation in mice: the involvement of distinct signalling pathways. Life Sci. 85, 822–829. Woolf, C.J., Salter, M.W., 2000. Neuronal plasticity: increasing the gain in pain. Science 288, 1765–1769. Woolf, C.J., Ma, Q., 2007. Nociceptors–noxious stimulus detectors. Neuron 55, 353–364. Wu, J., Liu, T.T., Zhou, Y.M., Qiu, C.Y., Ren, P., Jiao, M., Hu, W.P., 2017. Sensitization of ASIC3 by proteinase-activated receptor 2 signaling contributes to acidosis-induced nociception. J. Neuroinflammation 14, 150. Yokoyama, U., Iwatsubo, K., Umemura, M., Fujita, T., Ishikawa, Y., 2013. The prostanoid EP4 receptor and its signaling pathway. Pharmacol. Rev. 65, 1010–1052. Zhang, Y., Shaffer, A., Portanova, J., Seibert, K., Isakson, P.C., 1997. Inhibition of cyclooxygenase-2 rapidly reverses inflammatory hyperalgesia and prostaglandin E2 production. J. Pharmacol. Exp. Ther. 283, 1069–1075.

pain (allodynia) induced by prostaglandin E2. Eur. J. Neurosci. 11, 1849–1856. Moriyama, T., Higashi, T., Togashi, K., Iida, T., Segi, E., Sugimoto, Y., Tominaga, T., Narumiya, S., Tominaga, M., 2005. Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol. Pain 1, 3. Namer, B., Schick, M., Kleggetveit, I.P., Orstavik, K., Schmidt, R., Jorum, E., Torebjork, E., Handwerker, H., Schmelz, M., 2015. Differential sensitization of silent nociceptors to low pH stimulation by prostaglandin E2 in human volunteers. Eur. J. Pain 19, 159–166. Nantel, F., Denis, D., Gordon, R., Northey, A., Cirino, M., Metters, K.M., Chan, C.C., 1999. Distribution and regulation of cyclooxygenase-2 in carrageenan-induced inflammation. Br. J. Pharmacol. 128, 853–859. Oida, H., Namba, T., Sugimoto, Y., Ushikubi, F., Ohishi, H., Ichikawa, A., Narumiya, S., 1995. In situ hybridization studies of prostacyclin receptor mRNA expression in various mouse organs. Br. J. Pharmacol. 116, 2828–2837. Omori, M., Yokoyama, M., Matsuoka, Y., Kobayashi, H., Mizobuchi, S., Itano, Y., Morita, K., Ichikawa, H., 2008. Effects of selective spinal nerve ligation on acetic acid-induced nociceptive responses and ASIC3 immunoreactivity in the rat dorsal root ganglion. Brain Res. 1219, 26–31. Omote, K., Kawamata, T., Nakayama, Y., Kawamata, M., Hazama, K., Namiki, A., 2001. The effects of peripheral administration of a novel selective antagonist for prostaglandin E receptor subtype EP(1), ONO-8711, in a rat model of postoperative pain. Anesth. Analg. 92, 233–238. Reeh, P.W., Steen, K.H., 1996. Tissue acidosis in nociception and pain. Prog. Brain Res. 113, 143–151. Sachs, D., Villarreal, C., Cunha, F., Parada, C., Ferreira, S., 2009. The role of PKA and PKCepsilon pathways in prostaglandin E2-mediated hypernociception. Br. J. Pharmacol. 156, 826–834. Sarkar, S., Hobson, A.R., Hughes, A., Growcott, J., Woolf, C.J., Thompson, D.G., Aziz, Q., 2003. The prostaglandin E2 receptor-1 (EP-1) mediates acid-induced visceral pain hypersensitivity in humans. Gastroenterology 124, 18–25. Sawynok, J., Reid, A., Meisner, J., 2006. Pain behaviors produced by capsaicin: influence of inflammatory mediators and nerve injury. J. Pain 7, 134–141.

9