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Research Report
Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons Qi Caia,b,1, Chun-Yu Qiua,1, Fang Qiua, Ting-Ting Liua, Zu-Wei Qua, Yu-Min Liub,nn, Wang-Ping Hua,n a Department of Pharmacology, Hubei University of Science and Technology, 88 Xianning Road, Xianning 437100, Hubei, PR China b Neurology Department, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, PR China
art i cle i nfo
ab st rac t
Article history:
Extracellular acidosis is a common feature in pain-generating pathological conditions.
Accepted 24 January 2014
Acid-sensing ion channels (ASICs), pH sensors, are distributed in peripheral sensory neurons and participate in nociception. Morphine exerts potent analgesic effects through the activation of opioid receptors for various pain conditions. A cross-talk between ASICs
Keywords: Acid-sensing ion channel Proton-gated current
and opioid receptors in peripheral sensory neurons has not been shown so far. Here, we have found that morphine inhibits the activity of native ASICs in rat dorsal root ganglion (DRG) neurons. Morphine dose-dependently inhibited proton-gated currents mediated by
Morphine
ASICs in the presence of the TRPV1 inhibitor capsazepine. Morphine shifted the proton
Opioid receptor Dorsal root ganglion neuron Electrophysiology
concentration–response curve downwards, with a decrease of 51.473.8% in the maximum current response but with no significant change in the pH0.5 value. Another μ-opioid receptor agonist DAMGO induced a similar decrease in ASIC currents compared with
Pain
morphine. The morphine inhibition of ASIC currents was blocked by naloxone, a specific opioid receptor antagonist. Pretreatment of forskolin, an adenylyl cyclase activator, or the addition of cAMP reversed the inhibitory effect of morphine. Moreover, morphine altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. Finally, peripheral applied morphine relieved pain evoked by intraplantar of acetic acid in rats. Our results indicate that morphine can inhibit the activity of ASICs via μ-opioid receptor and cAMP dependent signal pathway. These observations demonstrate a cross-talk between ASICs and opioid receptors in peripheral sensory neurons, which was a novel analgesic mechanism of morphine. & 2014 Published by Elsevier B.V.
Abbreviations: ASICs,
acid-sensing ion channels; DRG,
potential vanilloid type 1; PKA,
protein kinase A; AC,
dorsal root ganglion; IpH,
adenylyl cyclase; 8-Br-cAMP,
proton-gated current; TRPV1, 8-bromo-cAMP; DAMGO,
transient receptor
[D-Ala2,N-Me-Phe4,
Gly5-ol]-enkephalin; ANOVA, analysis of variance. n Corresponding author. Fax: þ86 715 82256221. nn Corresponding author. E-mail addresses: [email protected] (Y.-M. Liu), [email protected] (W.-P. Hu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.brainres.2014.01.042 0006-8993 & 2014 Published by Elsevier B.V.
Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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1.
Introduction
Tissue acidosis is a prominent feature of multiple pathological conditions such as inflammation, ischemic stroke, infections, and cancer. Protons are produced or released by the injured tissues, resulting in tissue acidosis (Deval et al., 2010). It is well known that the local extracellular pH levels drop to 5.4 in acute inflammation and to 4.7 in fracture-related hematomas (Staruschenko et al., 2007; Steen et al., 1992). Severe ischemia also induces the reduction of pH to 6.3 or even lower (Smith et al., 1986; Zha, 2013). It has been demonstrated that accumulations of protons depolarize the terminals of nociceptive primary sensory neurons to cause pain sensation, and that the depolarization is caused by a direct activation of proton gated ionic channels (Deval et al., 2003; Steen et al., 1995). Direct application of an acidic solution into the skin induces non-adapting pain (Jones et al., 2004; Steen et al., 1995). Although both acid-sensing ion channels (ASICs) and transient receptor potential vanilloid receptor type 1 (TRPV1) could be involved, studies have suggested that ASICs, rather than TRPV1, mediate pain sensation induced by acid injection (Deval et al., 2008; Wemmie et al., 2006). Acidosis-induced pain is suppressed by APETx2, a specific blocker of ASIC3, or knockdown of ASIC3 with siRNA (Deval et al., 2008). ASICs inhibitors have been shown to relieve pain in a variety of pain syndromes (Deval
Decrease of IpH5.5 (%)
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et al., 2010; Dube et al., 2009). Thus, ASICs emerge as a potential therapeutic target for pain therapy (Kweon and Suh, 2013; Wemmie et al., 2013). ASICs are pH sensors for detecting a wide range of pH fluctuations during pathological conditions. To date, seven subunits of ASICs (1a, 1b1, 1b2, 2a, 2b, 3, and 4) encoded by four genes have been identified (Deval et al., 2008). All other ASICs, except ASIC4, are present in primary sensory neurons of the trigeminal, vagal, and dorsal root ganglia (Alvarez de la Rosa et al., 2002; Benson et al., 2002). In peripheral sensory neurons, ASICs have been found on cell bodies and sensory terminals, where they have been suggested to be important for nociception and mechanosensation (Price et al., 2000, 2001). Among the ASIC subunits, ASIC3, which is the most essential pH sensor for pain, is specifically localized in nociceptive fibers innervating the skeletal and cardiac muscles, joints, and bone (Ikeuchi et al., 2008; Noel et al., 2010). It has been shown that inflammation and tissue injury increase the expression levels of ASICs mRNA in dorsal root ganglion (DRG) neurons (Voilley et al., 2001). Moreover, the activity of ASICs is upregulated by pro-inflammatory mediators such as serotonin, bradykinin, arachidonic acid and nitric oxide (Cadiou et al., 2007; Qiu et al., 2012; Smith et al., 2007; Yan et al., 2013). ASICs are also the subject of various pharmacological actions. For example, ASICs are inhibited by cannabinoids and nonsteroid anti-inflammatory drugs (Liu et al., 2012; Voilley et al., 2001).
60 40 20 0 10-7
10-6
10-5
10-4
Concentration of Morphine [M] Fig. 1 – Concentration-dependent inhibition of the proton-gated current by morphine in rat DRG neurons. The proton-gated currents were recorded in DRG neurons. (A) Example traces show that the current was inhibited by pre-application of 10 5 M morphine for 60 s. Also, this proton-induced current could be completely blocked by 10 4 M amiloride (Amil), a broadspectrum ASIC channel blocker. (B) Sequential current traces illustrate the inhibition of proton-induced currents by different concentrations of morphine on a DRG neuron with membrane potential clamped at 60 mV. (C) Morphine decreased protongated currents (IpH 5.5) in a concentration-dependent manner (3 10 7–3 10 5 M). Each point represents the mean7SEM of 6–9 neurons. Proton-induced currents were evoked by extracellular application of a pH 5.5 solution for 5 s in the presence of the TRPV1 inhibitor capsazepine (10 μM). Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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we measured proton-gated currents in the presence of the TRPV1 inhibitor, capsazepine (10 μM), in the whole-cell patchclamp configuration (Poirot et al., 2006). In most native DRG neurons (70.3%, 97/138), an inward current (IpH 5.5) was evoked by application of a pH 5.5 solution for 5 s (Fig.1A). IpH 5.5 was repeated stably within 30 min and the change in amplitude was within 7.0%, when a pH 5.5 solution was applied regularly for 5 s durations with 3 min intervals. ASICs may be the only channels known to mediate the acid currents in the presence of the TRPV1 inhibitor, since all observed acid currents could be completely blocked by 100 mM amiloride, a broad-spectrum ASIC channel blocker (Fig. 1A). Therefore, we considered them as ASIC currents. In the majority of the neurons sensitive to acid stimuli (72.2%, 70/97), we observed that the proton-evoked currents were inhibited by the pre-application of morphine for 60 s (Fig. 1). The morphine evoked inhibitory effect was reversible in washout experiments. Moreover, the inhibition of protongated currents was dependent on the concentrations of morphine. Fig. 1B shows that the amplitudes of IpH 5.5 further decreased when the concentration of morphine increased from 10 6 M to 10 5 M. Fig. 1C shows the dose–response curve for morphine in the inhibition of proton-gated currents. Morphine caused the maximum effect (55.778.2%, n¼ 7) at a concentration of 30 μM. The half-maximal response (IC50) value of the dose–response curve for morphine was 2.3270.21 μM (Fig. 1C). The results indicated that morphine inhibited the proton-gated current in a dose-dependent manner.
Opiates such as morphine, a major class of analgesics widely used in the clinical treatment of pain condition, exert their effects through the activation of opioid receptors (Costantino et al., 2012). All three types of opioid receptors are synthesized and expressed in the cell bodies of DRG neurons. These opioid receptors are intra-axonally transported into the neuronal processes and are detectable on peripheral C- and A-nerve terminals. Peripheral opioid agonists are particularly effective in inflammatory hyperalgesia (Stein et al., 2003). It has been demonstrated that opioids suppression channels such as calcium, sodium channels and TRPV1, which are mainly expressed in nociceptors (EndresBecker et al., 2007; Gold and Levine, 1996; Ingram and Williams, 1994; Schroeder and McCleskey, 1993). However, a cross-talk between ASICs and opioid receptors in DRG neurons has not been shown so far. In this study, we show that morphine can inhibit the activity of ASICs in rat DRG neurons via μ-opioid receptors in a cAMP-dependent manner.
2.
Results
2.1. Inhibition of the proton-gated current by morphine in rat DRG neurons All current measurements in this study were done in small and medium diameter (15–35 μm) acutely isolated DRG neurons of adult rats. To functionally characterize ASIC currents,
Normlized peak current
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1.0
3
pH pH + Morphine
0.8 0.6 0.4 0.2 0.0
7.0 6.5 6.0 5.5 5.0 4.5 pH
Fig. 2 – Concentration–response relationship for proton with or without the pre-application of morphine. (A) Sequential currents evoked by different pHs in the absence and presence of 10 5 M morphine. (B) The concentration–response curves for proton with or without 10 5 M morphine pre-application for 60 s. The concentration–response curve for proton with morphine pretreatment shifted downwards. Each point represents the mean7SEM of 7–10 neurons. All current values were normalized to the current response induced by pH 4.5 applied alone (marked with asterisk). Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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2.2. Concentration–response relationship for proton-gated currents with and without pretreatment of morphine We then investigated whether the inhibition of morphine was dependent on the concentrations of proton. Fig. 2 shows that currents evoked by different pHs were inhibited by morphine (10 5 M). First, pretreatment of morphine shifted the concentration–response curve to protons downwards, as indicated by a decrease of 51.473.8% in the maximal current response to protons when morphine was pre-applied. However, the slopes or Hill coefficients of those two curves were essentially similar (n¼ 1.29 in the absence of morphine versus n¼ 1.31 in the presence of morphine; P40.1, Bonferroni's post hoc test). Second, the pH0.5 values of both curves had no statistical difference (pH0.5 of 5.8870.18 with morphine pretreatment versus pH0.5 of 6.0170.13 without morphine pretreatment; P40.1, Bonferroni's post hoc test). Third, the threshold pH value was essentially the same.
2.3. Receptor and intracellular signal transduction mechanisms underlying suppression of proton-gated currents by morphine To verify whether the suppression of proton-gated currents by morphine was mediated by the opioid receptor, we examined the effect of naloxone, a specific opioid receptor antagonist, on the morphine-induced inhibition of protongated currents. As shown in Fig. 3A and B, the suppression of IpH 5.5 by pretreatment with morphine (10 5 M) was reversed
100
60
*
75
50
25
0
by the addition of 10 5 M naloxone (n ¼10, Po0.01, one way analysis of variance followed by post hoc Bonferroni's test). Another μ-opioid receptor agonist (DAMGO; 10 5 M) induced a similar decrease (51.776.3%, n¼ 10) in ASIC currents compared with 10 5 M morphine (55.075.9%, n ¼10, P40.1, unpaired t-test). Opioid receptor belongs to Gi/o protein-coupled receptor family, and the activation of the receptor leads to a cascade of events that inhibit adenylyl cyclase (AC) and therefore decreases the intracellular cAMP level (Law et al., 2000). We determined if Gi/o inhibition with pertussis toxin (PTX) preexposure altered the effect by morphine on ASIC currents. Fig. 3C shows that the pre-treatment with PTX (500 ng/mL) significantly blocked morphine-induced suppression of ASIC currents. To further explore intracellular signal transduction mechanisms underlying suppression of proton-gated currents by morphine, an experiment using forskolin (an AC activator) and 8-Br-cAMP was carried out. As shown in Fig. 3C, the inhibition of morphine on proton-gated current was blocked by application of forskolin (10 5 M) or 8-Br-cAMP (10 3 M). Suppression of IpH 5.5 induced by morphine was 55.075.9% (n¼ 10) in the control experiment. In contrast, the suppression of IpH 5.5 induced by morphine was 12.773.9% after treatment of forskolin (n¼ 10, Po0.01, post hoc Bonferroni's test), and 8.173.6% after treatment of 8-Br-cAMP (n ¼10, Po0.01, post hoc Bonferroni's test). In addition, PTX treatment in the absence of morphine did not alter ASIC currents significantly, the relative amplitude of ASIC currents was 98.974.9% of the control condition (n¼ 10, P40.1, paired
Inhibition ofIpH5.5 (%)
Relative amplitude of IpH5.5
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Control
Morphine
Morphine + Naloxone
50 40 30 20
**
10 0
** Control
PTX
Forskolin
** 8-Br-cAMP
** H89
Fig. 3 – The receptor and intracellular signal transduction mechanisms underlying inhibition of proton-gated currents by morphine. The current traces in (A) and the bar graph in (B) show that the morphine inhibition of IpH 5.5 was abolished by the co-application of naloxone (10 5 M), a specific opioid receptor antagonist nPo0.05, one way analysis of variance followed by post hoc Bonferroni's test, n ¼ 10. The bar graph in (C) shows the percentage decreases in the IpH 5.5 induced by 10 5 M morphine in control and pre-treatment of pertussis toxin (PTX, 500 ng/mL), forskolin (10 5 M), 8-Br-cAMP (10 3 M) and H89 (10 6 M) conditions. nnPo0.01, one way analysis of variance followed by post hoc Bonferroni's test, compared with control, n ¼10/each column. Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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t-test). Forskolin or 8-Br-cAMP treatment alone showed an increased tendency on ASIC currents, the relative amplitudes of ASIC currents were 111.576.7% and 114.979.0% of the control condition, respectively (n ¼10, P¼ 0.067 and 0.068, paired t-test). We then studied the effect of a membranepermeant PKA inhibitor, H89, on the inhibitory effect of morphine. We first perfused H89 (10 6 M) onto the recorded cell and then observed the change of ASIC currents. Indeed, H89 mimicked the action of morphine, the ASIC currents were reduced to 43.377.2% of control (n ¼10) after 10 min pretreatment with H89. In the presence of H89 (10 6 M), morphine no longer could inhibit ASIC currents (n¼ 10, Po0.01, post hoc Bonferroni's test; Fig. 3C), further suggesting that PKA is involved in the inhibitory effect of morphine. Taken together, these results indicated that morphineinduced inhibition of proton-gated currents is mediated through the μ-opioid receptor and cAMP signaling pathway.
2.4. Effect of morphine on proton-evoked action potentials of rat DRG neurons Activation of ASICs by protons induces sodium influx, resulting in membrane depolarization and neuronal excitation. In the presence of the TRPV1 inhibitor, capsazepine (10 μM), a steep pH drop from 7.4 to 5.5 could trigger bursts of action potentials under current-clamp conditions in a neuron tested, while the whole-cell current was also induced by pH 5.5 in the same cell with voltage-clamp recording (Fig. 4A). Consistent with the inhibition of acid currents by morphine, the acid-induced action potentials were also inhibited by the
pre-application of morphine (10 5 M, 60 s). The mean number of action potentials was 4.6370.43 during exposure to pH 5.5 for 5 s in seven neurons tested. In contrast, the mean number of action potentials decreased to 2.3870.34 after the pretreatment of morphine (n ¼7, Po0.05, paired t-test) (Fig. 4B). After 20 min washout of morphine, the mean number of action potentials evoked by acid were 3.9770.46, which was not a significant difference with control condition (4.6370.43, n¼ 7, P40.1, paired t-test) (Fig. 4B). These results indicated that morphine reversibly inhibited proton-induced excitability of rat DRG neurons.
2.5. Effect of morphine on nociceptive responses to intraplantar injection of acetic acid in rats Intraplantar injection of acetic acid elicited an intense flinch/ shaking response in rats (Deval et al., 2008; Omori et al., 2008). The flinch response mainly occurred during 0–5 min after injection of acetic acid. In the present study, intraplantar injection of 20 μL acetic acid solution (0.6%) in the presence of the TRPV1 inhibitor capsazepine (100 μM) caused an intense flinch/shaking response (Fig. 5). And the acid-evoked pain was potently blocked by treatment of 200 μM amiloride, a broadspectrum ASIC channel blocker. The number of flinches decreased from 9.9771.12 of control conditions to 1.9170.54 with 200 μM amiloride pretreatment (n¼10, Po0.01, unpaired t-test), suggesting the involvement of ASICs (Fig. 5). The pretreatment of morphine decreased flinching behavior induced by acetic acid in a dose-dependent manner (10 7–10 5 M) in 10 rats/each group (Fig. 5). The number of flinches decreased
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Mean number of APsA
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Morphine
Wash
Fig. 4 – Effect of morphine on proton-evoked action potentials of rat DRG neurons. (A) Original current and action potentials recordings from the same DRG neuron. Left panel, pH 5.5 induced an inward current with voltage–clamp recording. Right panel, pH 5.5 produced action potentials with current–clamp recording in the same neuron. The pretreatment of 10 5 M morphine decreased the acid-induced number of action potentials. (B) Bar graph shows the effect of 10 5 M morphine on the number of action potentials produced by pH 5.5 in acid perfusions. After 20 min washout of morphine, acid-evoked action potentials recovered to control condition nPo0.05, paired t-test, compared with control, n¼ 7/each column. Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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Mean number of flinches
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&
10 8
*
6 4
** ##
2 0
Control Amiloride
Naloxone 10-7
10-6
10-5
10-5
Morphine Fig. 5 – Effect of morphine on nociceptive responses to injection of acetic acid in rats. Intraplantar injection acetic acid (0.6%, 20 μl) evoked a flinch/shaking response. The bar graph shows acid-evoked pain was blocked by pretreatment of 200 μM amiloride. The pretreatment of morphine decreased flinching behavior induced by acetic acid in a dose-dependent manner (10 7–10 5 M). The inhibitory effect of morphine was blocked by naloxone (10 5 M), an opioid receptor antagonist. ##Po0.01, unpaired t-test, compared with control column; nPo0.05, nnPo0.01, one way analysis of variance followed by post hoc Bonferroni's test, compared with control column; and Po0.05, unpaired t-test, compared with 10 5 M morphine column. n¼ 10/each column. Flinching shaking of paw was recorded as the number of flinches per observation period (5 min).
from 9.9771.12 of control conditions to 4.7670.46 with 10 5 M morphine pretreatment (n¼ 10, Po0.01, post hoc Bonferroni's test). The inhibiting effect of morphine on acetic acid-induced pain behavior failed to exert when naloxone was co-injected together with morphine. The number of acid-evoked flinches was 8.8671.15 with co-treatment of both 10 5 M naloxone and 10 5 M morphine, and there was a significant difference compared with treatment of 10 5 M morphine alone (4.7670.46, n¼ 10, Po0.05, unpaired t-test) (Fig. 5). These results indicated that locally applied morphine relieved acidosis-evoked pain.
3.
Discussion
The present study demonstrated that morphine inhibited the activity of ASICs in dissociated rat DRG neurons. Morphine decreased the amplitude of ASIC currents, the number of action potentials induced by acid stimuli. Locally applied morphine inhibited nociceptive responses to intraplantar injection of acetic acid in vivo. We further showed that the μ-opioid receptor and cAMP-dependent signal pathway likely underlie intracellular mechanism in inhibiting ASIC activity by morphine. A rapid drop in the extracellular pH from 7.4 to 5.5 for 5 s was found to evoke an inward current in most native DRG neurons. The acid-evoked currents may be involved in the activation of ASIC and TRPV1 channels (Bevan and Geppetti, 1994). The contribution of TRPV1 was ruled out here since all observed acid currents could be completely blocked by amiloride, a broad-spectrum ASIC channel blocker. Our previous work showed that the acid currents were partly inhibited by homomeric ASIC1a channel antagonist PcTx1, not by the TRPV1 blocker AMG 9810 (Liu et al., 2012).
Thus, ASICs may be the only channels known to mediate the acid currents, particularly in the presence of the TRPV1 inhibitor capsazepine. The present study shows that morphine exerted an inhibitory effect on ASIC currents in a dosedependent manner. Inhibition of morphine cannot be due to the decrease in the affinity of ASICs to proton since this effect was observed at the saturating concentration of proton. Morphine shifted the proton concentration–response curve downwards, with a decrease in the maximum current response but with no significant change in the pH0.5 and threshold values. The present study disclosed a cross-talk between ASICs and μ-opioid receptors in rat DRG neurons. Morphine inhibition of ASICs was opioid receptor-mediated, because it was blocked by naloxone, a specific opioid receptor antagonist. Furthermore, another μ-opioid receptor agonist DAMGO had a similar inhibitory effect on ASIC currents. μ-opioid receptors are coupled to Gi/o proteins and their activation leads to inhibition of AC activity. As a result, formation of cAMP is inhibited, and protein kinase A (PKA) cannot be activated (Law et al., 2000). The morphine-induced inhibition of ASIC currents may involve intracellular signal transduction. Our observations that both forskolin and 8-Br-cAMP can reverse morphine inhibition of ASIC currents indicated that this effect may be mediated mainly via a cAMP/PKA-dependent pathway. Consistent with our previous study, cannabinoids attenuation of ASICs is mediated by the inhibition of cAMP/ PKA-dependent pathway through CB1 receptors (Liu et al., 2012). In addition, μ-opioid activation can also inhibit the activity of TRPV1 via Gi/o proteins and the cAMP pathway (Endres-Becker et al., 2007). A-kinase anchoring protein 150 (AKAP150) has been reported to be co-localized with ASIC1a and ASIC2a in rat cortical neurons, and regulates the activity of these channels. It has also been reported that the
Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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inhibition of protein kinase A (PKA) binding to AKAP150 reduces the amplitude of ASIC currents, suggesting that AKAP150 mediates the PKA-dependent phosphorylation of ASICs (Chai et al., 2007). Therefore, the reduction of PKA activity may be involved in the morphine inhibition of ASIC currents in primary sensory neurons. ASICs are extracellular pH sensors and permeable to cations. In peripheral sensory neurons, ASICs have been found on cell bodies and sensory terminals. Activation of ASICs, particularly ASIC3 and ASIC1a channels, by acidic pH and the resultant depolarization of nociceptive primary sensory neurons, participate in nociception (Leng et al., 2013). ASICs are found to mediate cutaneous acid-induced pain (Jones et al., 2004; Ugawa et al., 2002). Intraplantar injection of acetic acid elicited an intense flinch/shaking response in rats (Deval et al., 2008; Omori et al., 2008). In the present study, the acid-evoked pain was significantly blocked by amiloride, a broad-spectrum ASIC channel blocker, indicating the involvement of ASICs. Behavioral experiments demonstrated that locally applied morphine relieved the acidosisevoked pain in a dose-dependent manner. The analgesic effects of peripheral morphine were mediated by opioid receptors, since it was blocked by naloxone, a specific opioid receptor antagonist. The results were also consistent with morphine inhibitory effects on acid-evoked action potentials in current clamp experiments. A number of pain-causing stimuli, such as inflammation, lower extracellular pH (Wemmie et al., 2013). ASICs are a family of cation channels and present both in the cell body and at terminals of peripheral nociceptive neurons. Thus, activation of ASICs contributes to pain. ASICs have emerged as a potential therapeutic target for new pain medication (Kweon and Suh, 2013; Wemmie et al., 2013). The ASIC inhibitor amiloride is approved for use in humans, and a few small translational experiments have demonstrated its potential for reducing cutaneous pain and migraine (Holland et al., 2012; Jones et al., 2004; Ugawa et al., 2002). It has been shown that analgesic effects of peripheral opioids are particularly prominent during inflammatory pain (Stein et al., 2003). In the present study, our results strongly indicated morphine can exert their analgesic action by interaction with ASICs in the primary afferent neurons. In conclusion, this study identified a cross-talk between ASICs and μ-opioid receptors in rat primary sensory neurons. We showed that the activity of ASICs can be inhibited by morphine in DRG neurons. Morphine decreased ASIC currents, action potentials and pain induced by acid stimuli. The morphine inhibition was blocked by naloxone, a specific opioid receptor antagonist. Our results reveal a novel peripheral analgesic mechanism of morphine by modulating native ASICs.
guidelines on the ethical use of animals, and every effort was made to minimize the number of animals used and their suffering. Two- to three-week old Sprague–Dawley male rats were anesthetized with ethyl ether and then decapitated. The DRGs were taken out and transferred immediately into DMEM (Sigma-Aldrich, St. Louis, MO, US) 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 1C 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 another 60 min before electrophysiological recordings. The neurons selected for electrophysiological experiment were 15–35 μm in diameter.
4.2.
Experimental procedures
4.1.
Isolation of the dorsal root ganglia neurons
The experimental protocol was approved by the Animal Research Ethics Committee of Hubei University of Science and Technology. All procedures conformed to international
Electrophysiological recordings
Whole-cell patch clamp and voltage-clamp recordings were carried out at room temperature (22–25 1C) using a MultiClamp-700B amplifier and Digidata-1440A A/D converter (Axon Instruments, Foster City, CA, USA). Recording pipettes were pulled using a Sutter P-97 puller (Sutter Instruments, Novato, CA, USA). The micropipettes were filled with internal solution containing KCl 140 mM, MgCl2 2.5 mM, HEPES 10 mM, EGTA 11 mM and ATP 5 mM; 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 NaCl 150 mM, KCl 5 mM, CaCl2 2.5 mM, MgCl2 2 mM, HEPES 10 mM and D-glucose 10 mM; its osmolarity was adjusted to 330 mOsm/L with sucrose and 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 gigaseal and then negative pressure was applied to rupture it, thus establishing a whole-cell configuration. The adjustment of capacitance compensation and series resistance compensation was 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 10–50 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).
4.3.
4.
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Drug application
Drugs used in the experiments were purchased from Sigma Chemical Co. and include hydrochloric acid, morphine (catalog # M-005), naloxone, amiloride, forskolin, 8-Br-cAMP, DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) and capsazepine. Stocks of drugs were made up in dimethyl sulfoxide and diluted daily in the external solution at a minimum of 1:1000 to a final working concentration. The final concentration of dimethyl
Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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sulfoxide used was 0.1%, which has no effect on ASICs. Except for hydrochloric acid, the pH for all working solutions was adjusted daily to 7.470.1 with HCl or NaOH. Next, they were held in a linear array of fused silica tubes (o.d./i.d.¼ 500 μm/ 200 μm) connected to a series of independent reservoirs. The application pipette tips were positioned 30 μm away from the recorded neurons. 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 with a PC-controlled micromanipulator. Cells were constantly bathed in normal external solution flowing from one tube connected to a larger reservoir between drug applications. To functionally characterize ASIC activity, we used capsazepine (10 μM) to block TRPV1 in this study (Poirot et al., 2006).
4.4.
Nociceptive behavior induced by acetic acid in rats
Sprague–Dawley male rats (3–4 weeks old) were kept with a 12 h light/dark cycle and with ad libitum access to food and water. Animals were placed in a 30 30 30 cm3 Plexiglas chamber and allowed to habituate for at least 30 min before nociceptive behavior experiments. After pretreatment with 10 μL capsazepine (100 μM), a double-blind experiment was carried out. Twenty microliters of acetic acid solution (0.6%) together with 20 μL external solution, amiloride, morphine and/or naloxone were coded, and the other experimenters subcutaneously administered them into the dorsal face of the hind paw using a 30 gauge needle connected to a 100 μL Hamilton syringe. 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).
4.5.
Data analysis
Data were statistically compared using 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 mean7standard error of the mean (SEM).
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81171039) to WPH, Program for New Century Excellent Talents in University (NCET-11-0967) to WPH, Team of Outstanding Young Scientific and Technological Innovation of the Higher Education Institutions of Hubei Province (T201113) to WPH, Key Project of Chinese Ministry of Education (210142) to WPH, and Scientific Research Staring Foundation for the Returned Overseas Chinese Scholars, Ministry of Education of China to WPH.
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Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons Qi Caia,b,1, Chun-Yu Qiua,1, Fang Qiua, Ting-Ting Liua, Zu-Wei Qua, Yu-Min Liub,nn, Wang-Ping Hua,n a Department of Pharmacology, Hubei University of Science and Technology, 88 Xianning Road, Xianning 437100, Hubei, PR China b Neurology Department, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, PR China
art i cle i nfo
ab st rac t
Article history:
Extracellular acidosis is a common feature in pain-generating pathological conditions.
Accepted 24 January 2014
Acid-sensing ion channels (ASICs), pH sensors, are distributed in peripheral sensory neurons and participate in nociception. Morphine exerts potent analgesic effects through the activation of opioid receptors for various pain conditions. A cross-talk between ASICs
Keywords: Acid-sensing ion channel Proton-gated current
and opioid receptors in peripheral sensory neurons has not been shown so far. Here, we have found that morphine inhibits the activity of native ASICs in rat dorsal root ganglion (DRG) neurons. Morphine dose-dependently inhibited proton-gated currents mediated by
Morphine
ASICs in the presence of the TRPV1 inhibitor capsazepine. Morphine shifted the proton
Opioid receptor Dorsal root ganglion neuron Electrophysiology
concentration–response curve downwards, with a decrease of 51.473.8% in the maximum current response but with no significant change in the pH0.5 value. Another μ-opioid receptor agonist DAMGO induced a similar decrease in ASIC currents compared with
Pain
morphine. The morphine inhibition of ASIC currents was blocked by naloxone, a specific opioid receptor antagonist. Pretreatment of forskolin, an adenylyl cyclase activator, or the addition of cAMP reversed the inhibitory effect of morphine. Moreover, morphine altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. Finally, peripheral applied morphine relieved pain evoked by intraplantar of acetic acid in rats. Our results indicate that morphine can inhibit the activity of ASICs via μ-opioid receptor and cAMP dependent signal pathway. These observations demonstrate a cross-talk between ASICs and opioid receptors in peripheral sensory neurons, which was a novel analgesic mechanism of morphine. & 2014 Published by Elsevier B.V.
Abbreviations: ASICs,
acid-sensing ion channels; DRG,
potential vanilloid type 1; PKA,
protein kinase A; AC,
dorsal root ganglion; IpH,
adenylyl cyclase; 8-Br-cAMP,
proton-gated current; TRPV1, 8-bromo-cAMP; DAMGO,
transient receptor
[D-Ala2,N-Me-Phe4,
Gly5-ol]-enkephalin; ANOVA, analysis of variance. n Corresponding author. Fax: þ86 715 82256221. nn Corresponding author. E-mail addresses: [email protected] (Y.-M. Liu), [email protected] (W.-P. Hu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.brainres.2014.01.042 0006-8993 & 2014 Published by Elsevier B.V.
Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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1.
Introduction
Tissue acidosis is a prominent feature of multiple pathological conditions such as inflammation, ischemic stroke, infections, and cancer. Protons are produced or released by the injured tissues, resulting in tissue acidosis (Deval et al., 2010). It is well known that the local extracellular pH levels drop to 5.4 in acute inflammation and to 4.7 in fracture-related hematomas (Staruschenko et al., 2007; Steen et al., 1992). Severe ischemia also induces the reduction of pH to 6.3 or even lower (Smith et al., 1986; Zha, 2013). It has been demonstrated that accumulations of protons depolarize the terminals of nociceptive primary sensory neurons to cause pain sensation, and that the depolarization is caused by a direct activation of proton gated ionic channels (Deval et al., 2003; Steen et al., 1995). Direct application of an acidic solution into the skin induces non-adapting pain (Jones et al., 2004; Steen et al., 1995). Although both acid-sensing ion channels (ASICs) and transient receptor potential vanilloid receptor type 1 (TRPV1) could be involved, studies have suggested that ASICs, rather than TRPV1, mediate pain sensation induced by acid injection (Deval et al., 2008; Wemmie et al., 2006). Acidosis-induced pain is suppressed by APETx2, a specific blocker of ASIC3, or knockdown of ASIC3 with siRNA (Deval et al., 2008). ASICs inhibitors have been shown to relieve pain in a variety of pain syndromes (Deval
Decrease of IpH5.5 (%)
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et al., 2010; Dube et al., 2009). Thus, ASICs emerge as a potential therapeutic target for pain therapy (Kweon and Suh, 2013; Wemmie et al., 2013). ASICs are pH sensors for detecting a wide range of pH fluctuations during pathological conditions. To date, seven subunits of ASICs (1a, 1b1, 1b2, 2a, 2b, 3, and 4) encoded by four genes have been identified (Deval et al., 2008). All other ASICs, except ASIC4, are present in primary sensory neurons of the trigeminal, vagal, and dorsal root ganglia (Alvarez de la Rosa et al., 2002; Benson et al., 2002). In peripheral sensory neurons, ASICs have been found on cell bodies and sensory terminals, where they have been suggested to be important for nociception and mechanosensation (Price et al., 2000, 2001). Among the ASIC subunits, ASIC3, which is the most essential pH sensor for pain, is specifically localized in nociceptive fibers innervating the skeletal and cardiac muscles, joints, and bone (Ikeuchi et al., 2008; Noel et al., 2010). It has been shown that inflammation and tissue injury increase the expression levels of ASICs mRNA in dorsal root ganglion (DRG) neurons (Voilley et al., 2001). Moreover, the activity of ASICs is upregulated by pro-inflammatory mediators such as serotonin, bradykinin, arachidonic acid and nitric oxide (Cadiou et al., 2007; Qiu et al., 2012; Smith et al., 2007; Yan et al., 2013). ASICs are also the subject of various pharmacological actions. For example, ASICs are inhibited by cannabinoids and nonsteroid anti-inflammatory drugs (Liu et al., 2012; Voilley et al., 2001).
60 40 20 0 10-7
10-6
10-5
10-4
Concentration of Morphine [M] Fig. 1 – Concentration-dependent inhibition of the proton-gated current by morphine in rat DRG neurons. The proton-gated currents were recorded in DRG neurons. (A) Example traces show that the current was inhibited by pre-application of 10 5 M morphine for 60 s. Also, this proton-induced current could be completely blocked by 10 4 M amiloride (Amil), a broadspectrum ASIC channel blocker. (B) Sequential current traces illustrate the inhibition of proton-induced currents by different concentrations of morphine on a DRG neuron with membrane potential clamped at 60 mV. (C) Morphine decreased protongated currents (IpH 5.5) in a concentration-dependent manner (3 10 7–3 10 5 M). Each point represents the mean7SEM of 6–9 neurons. Proton-induced currents were evoked by extracellular application of a pH 5.5 solution for 5 s in the presence of the TRPV1 inhibitor capsazepine (10 μM). Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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we measured proton-gated currents in the presence of the TRPV1 inhibitor, capsazepine (10 μM), in the whole-cell patchclamp configuration (Poirot et al., 2006). In most native DRG neurons (70.3%, 97/138), an inward current (IpH 5.5) was evoked by application of a pH 5.5 solution for 5 s (Fig.1A). IpH 5.5 was repeated stably within 30 min and the change in amplitude was within 7.0%, when a pH 5.5 solution was applied regularly for 5 s durations with 3 min intervals. ASICs may be the only channels known to mediate the acid currents in the presence of the TRPV1 inhibitor, since all observed acid currents could be completely blocked by 100 mM amiloride, a broad-spectrum ASIC channel blocker (Fig. 1A). Therefore, we considered them as ASIC currents. In the majority of the neurons sensitive to acid stimuli (72.2%, 70/97), we observed that the proton-evoked currents were inhibited by the pre-application of morphine for 60 s (Fig. 1). The morphine evoked inhibitory effect was reversible in washout experiments. Moreover, the inhibition of protongated currents was dependent on the concentrations of morphine. Fig. 1B shows that the amplitudes of IpH 5.5 further decreased when the concentration of morphine increased from 10 6 M to 10 5 M. Fig. 1C shows the dose–response curve for morphine in the inhibition of proton-gated currents. Morphine caused the maximum effect (55.778.2%, n¼ 7) at a concentration of 30 μM. The half-maximal response (IC50) value of the dose–response curve for morphine was 2.3270.21 μM (Fig. 1C). The results indicated that morphine inhibited the proton-gated current in a dose-dependent manner.
Opiates such as morphine, a major class of analgesics widely used in the clinical treatment of pain condition, exert their effects through the activation of opioid receptors (Costantino et al., 2012). All three types of opioid receptors are synthesized and expressed in the cell bodies of DRG neurons. These opioid receptors are intra-axonally transported into the neuronal processes and are detectable on peripheral C- and A-nerve terminals. Peripheral opioid agonists are particularly effective in inflammatory hyperalgesia (Stein et al., 2003). It has been demonstrated that opioids suppression channels such as calcium, sodium channels and TRPV1, which are mainly expressed in nociceptors (EndresBecker et al., 2007; Gold and Levine, 1996; Ingram and Williams, 1994; Schroeder and McCleskey, 1993). However, a cross-talk between ASICs and opioid receptors in DRG neurons has not been shown so far. In this study, we show that morphine can inhibit the activity of ASICs in rat DRG neurons via μ-opioid receptors in a cAMP-dependent manner.
2.
Results
2.1. Inhibition of the proton-gated current by morphine in rat DRG neurons All current measurements in this study were done in small and medium diameter (15–35 μm) acutely isolated DRG neurons of adult rats. To functionally characterize ASIC currents,
Normlized peak current
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1.0
3
pH pH + Morphine
0.8 0.6 0.4 0.2 0.0
7.0 6.5 6.0 5.5 5.0 4.5 pH
Fig. 2 – Concentration–response relationship for proton with or without the pre-application of morphine. (A) Sequential currents evoked by different pHs in the absence and presence of 10 5 M morphine. (B) The concentration–response curves for proton with or without 10 5 M morphine pre-application for 60 s. The concentration–response curve for proton with morphine pretreatment shifted downwards. Each point represents the mean7SEM of 7–10 neurons. All current values were normalized to the current response induced by pH 4.5 applied alone (marked with asterisk). Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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2.2. Concentration–response relationship for proton-gated currents with and without pretreatment of morphine We then investigated whether the inhibition of morphine was dependent on the concentrations of proton. Fig. 2 shows that currents evoked by different pHs were inhibited by morphine (10 5 M). First, pretreatment of morphine shifted the concentration–response curve to protons downwards, as indicated by a decrease of 51.473.8% in the maximal current response to protons when morphine was pre-applied. However, the slopes or Hill coefficients of those two curves were essentially similar (n¼ 1.29 in the absence of morphine versus n¼ 1.31 in the presence of morphine; P40.1, Bonferroni's post hoc test). Second, the pH0.5 values of both curves had no statistical difference (pH0.5 of 5.8870.18 with morphine pretreatment versus pH0.5 of 6.0170.13 without morphine pretreatment; P40.1, Bonferroni's post hoc test). Third, the threshold pH value was essentially the same.
2.3. Receptor and intracellular signal transduction mechanisms underlying suppression of proton-gated currents by morphine To verify whether the suppression of proton-gated currents by morphine was mediated by the opioid receptor, we examined the effect of naloxone, a specific opioid receptor antagonist, on the morphine-induced inhibition of protongated currents. As shown in Fig. 3A and B, the suppression of IpH 5.5 by pretreatment with morphine (10 5 M) was reversed
100
60
*
75
50
25
0
by the addition of 10 5 M naloxone (n ¼10, Po0.01, one way analysis of variance followed by post hoc Bonferroni's test). Another μ-opioid receptor agonist (DAMGO; 10 5 M) induced a similar decrease (51.776.3%, n¼ 10) in ASIC currents compared with 10 5 M morphine (55.075.9%, n ¼10, P40.1, unpaired t-test). Opioid receptor belongs to Gi/o protein-coupled receptor family, and the activation of the receptor leads to a cascade of events that inhibit adenylyl cyclase (AC) and therefore decreases the intracellular cAMP level (Law et al., 2000). We determined if Gi/o inhibition with pertussis toxin (PTX) preexposure altered the effect by morphine on ASIC currents. Fig. 3C shows that the pre-treatment with PTX (500 ng/mL) significantly blocked morphine-induced suppression of ASIC currents. To further explore intracellular signal transduction mechanisms underlying suppression of proton-gated currents by morphine, an experiment using forskolin (an AC activator) and 8-Br-cAMP was carried out. As shown in Fig. 3C, the inhibition of morphine on proton-gated current was blocked by application of forskolin (10 5 M) or 8-Br-cAMP (10 3 M). Suppression of IpH 5.5 induced by morphine was 55.075.9% (n¼ 10) in the control experiment. In contrast, the suppression of IpH 5.5 induced by morphine was 12.773.9% after treatment of forskolin (n¼ 10, Po0.01, post hoc Bonferroni's test), and 8.173.6% after treatment of 8-Br-cAMP (n ¼10, Po0.01, post hoc Bonferroni's test). In addition, PTX treatment in the absence of morphine did not alter ASIC currents significantly, the relative amplitude of ASIC currents was 98.974.9% of the control condition (n¼ 10, P40.1, paired
Inhibition ofIpH5.5 (%)
Relative amplitude of IpH5.5
241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300
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Control
Morphine
Morphine + Naloxone
50 40 30 20
**
10 0
** Control
PTX
Forskolin
** 8-Br-cAMP
** H89
Fig. 3 – The receptor and intracellular signal transduction mechanisms underlying inhibition of proton-gated currents by morphine. The current traces in (A) and the bar graph in (B) show that the morphine inhibition of IpH 5.5 was abolished by the co-application of naloxone (10 5 M), a specific opioid receptor antagonist nPo0.05, one way analysis of variance followed by post hoc Bonferroni's test, n ¼ 10. The bar graph in (C) shows the percentage decreases in the IpH 5.5 induced by 10 5 M morphine in control and pre-treatment of pertussis toxin (PTX, 500 ng/mL), forskolin (10 5 M), 8-Br-cAMP (10 3 M) and H89 (10 6 M) conditions. nnPo0.01, one way analysis of variance followed by post hoc Bonferroni's test, compared with control, n ¼10/each column. Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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t-test). Forskolin or 8-Br-cAMP treatment alone showed an increased tendency on ASIC currents, the relative amplitudes of ASIC currents were 111.576.7% and 114.979.0% of the control condition, respectively (n ¼10, P¼ 0.067 and 0.068, paired t-test). We then studied the effect of a membranepermeant PKA inhibitor, H89, on the inhibitory effect of morphine. We first perfused H89 (10 6 M) onto the recorded cell and then observed the change of ASIC currents. Indeed, H89 mimicked the action of morphine, the ASIC currents were reduced to 43.377.2% of control (n ¼10) after 10 min pretreatment with H89. In the presence of H89 (10 6 M), morphine no longer could inhibit ASIC currents (n¼ 10, Po0.01, post hoc Bonferroni's test; Fig. 3C), further suggesting that PKA is involved in the inhibitory effect of morphine. Taken together, these results indicated that morphineinduced inhibition of proton-gated currents is mediated through the μ-opioid receptor and cAMP signaling pathway.
2.4. Effect of morphine on proton-evoked action potentials of rat DRG neurons Activation of ASICs by protons induces sodium influx, resulting in membrane depolarization and neuronal excitation. In the presence of the TRPV1 inhibitor, capsazepine (10 μM), a steep pH drop from 7.4 to 5.5 could trigger bursts of action potentials under current-clamp conditions in a neuron tested, while the whole-cell current was also induced by pH 5.5 in the same cell with voltage-clamp recording (Fig. 4A). Consistent with the inhibition of acid currents by morphine, the acid-induced action potentials were also inhibited by the
pre-application of morphine (10 5 M, 60 s). The mean number of action potentials was 4.6370.43 during exposure to pH 5.5 for 5 s in seven neurons tested. In contrast, the mean number of action potentials decreased to 2.3870.34 after the pretreatment of morphine (n ¼7, Po0.05, paired t-test) (Fig. 4B). After 20 min washout of morphine, the mean number of action potentials evoked by acid were 3.9770.46, which was not a significant difference with control condition (4.6370.43, n¼ 7, P40.1, paired t-test) (Fig. 4B). These results indicated that morphine reversibly inhibited proton-induced excitability of rat DRG neurons.
2.5. Effect of morphine on nociceptive responses to intraplantar injection of acetic acid in rats Intraplantar injection of acetic acid elicited an intense flinch/ shaking response in rats (Deval et al., 2008; Omori et al., 2008). The flinch response mainly occurred during 0–5 min after injection of acetic acid. In the present study, intraplantar injection of 20 μL acetic acid solution (0.6%) in the presence of the TRPV1 inhibitor capsazepine (100 μM) caused an intense flinch/shaking response (Fig. 5). And the acid-evoked pain was potently blocked by treatment of 200 μM amiloride, a broadspectrum ASIC channel blocker. The number of flinches decreased from 9.9771.12 of control conditions to 1.9170.54 with 200 μM amiloride pretreatment (n¼10, Po0.01, unpaired t-test), suggesting the involvement of ASICs (Fig. 5). The pretreatment of morphine decreased flinching behavior induced by acetic acid in a dose-dependent manner (10 7–10 5 M) in 10 rats/each group (Fig. 5). The number of flinches decreased
6
Mean number of APsA
301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360
5
4
* 2
0
Control
Morphine
Wash
Fig. 4 – Effect of morphine on proton-evoked action potentials of rat DRG neurons. (A) Original current and action potentials recordings from the same DRG neuron. Left panel, pH 5.5 induced an inward current with voltage–clamp recording. Right panel, pH 5.5 produced action potentials with current–clamp recording in the same neuron. The pretreatment of 10 5 M morphine decreased the acid-induced number of action potentials. (B) Bar graph shows the effect of 10 5 M morphine on the number of action potentials produced by pH 5.5 in acid perfusions. After 20 min washout of morphine, acid-evoked action potentials recovered to control condition nPo0.05, paired t-test, compared with control, n¼ 7/each column. Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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Mean number of flinches
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&
10 8
*
6 4
** ##
2 0
Control Amiloride
Naloxone 10-7
10-6
10-5
10-5
Morphine Fig. 5 – Effect of morphine on nociceptive responses to injection of acetic acid in rats. Intraplantar injection acetic acid (0.6%, 20 μl) evoked a flinch/shaking response. The bar graph shows acid-evoked pain was blocked by pretreatment of 200 μM amiloride. The pretreatment of morphine decreased flinching behavior induced by acetic acid in a dose-dependent manner (10 7–10 5 M). The inhibitory effect of morphine was blocked by naloxone (10 5 M), an opioid receptor antagonist. ##Po0.01, unpaired t-test, compared with control column; nPo0.05, nnPo0.01, one way analysis of variance followed by post hoc Bonferroni's test, compared with control column; and Po0.05, unpaired t-test, compared with 10 5 M morphine column. n¼ 10/each column. Flinching shaking of paw was recorded as the number of flinches per observation period (5 min).
from 9.9771.12 of control conditions to 4.7670.46 with 10 5 M morphine pretreatment (n¼ 10, Po0.01, post hoc Bonferroni's test). The inhibiting effect of morphine on acetic acid-induced pain behavior failed to exert when naloxone was co-injected together with morphine. The number of acid-evoked flinches was 8.8671.15 with co-treatment of both 10 5 M naloxone and 10 5 M morphine, and there was a significant difference compared with treatment of 10 5 M morphine alone (4.7670.46, n¼ 10, Po0.05, unpaired t-test) (Fig. 5). These results indicated that locally applied morphine relieved acidosis-evoked pain.
3.
Discussion
The present study demonstrated that morphine inhibited the activity of ASICs in dissociated rat DRG neurons. Morphine decreased the amplitude of ASIC currents, the number of action potentials induced by acid stimuli. Locally applied morphine inhibited nociceptive responses to intraplantar injection of acetic acid in vivo. We further showed that the μ-opioid receptor and cAMP-dependent signal pathway likely underlie intracellular mechanism in inhibiting ASIC activity by morphine. A rapid drop in the extracellular pH from 7.4 to 5.5 for 5 s was found to evoke an inward current in most native DRG neurons. The acid-evoked currents may be involved in the activation of ASIC and TRPV1 channels (Bevan and Geppetti, 1994). The contribution of TRPV1 was ruled out here since all observed acid currents could be completely blocked by amiloride, a broad-spectrum ASIC channel blocker. Our previous work showed that the acid currents were partly inhibited by homomeric ASIC1a channel antagonist PcTx1, not by the TRPV1 blocker AMG 9810 (Liu et al., 2012).
Thus, ASICs may be the only channels known to mediate the acid currents, particularly in the presence of the TRPV1 inhibitor capsazepine. The present study shows that morphine exerted an inhibitory effect on ASIC currents in a dosedependent manner. Inhibition of morphine cannot be due to the decrease in the affinity of ASICs to proton since this effect was observed at the saturating concentration of proton. Morphine shifted the proton concentration–response curve downwards, with a decrease in the maximum current response but with no significant change in the pH0.5 and threshold values. The present study disclosed a cross-talk between ASICs and μ-opioid receptors in rat DRG neurons. Morphine inhibition of ASICs was opioid receptor-mediated, because it was blocked by naloxone, a specific opioid receptor antagonist. Furthermore, another μ-opioid receptor agonist DAMGO had a similar inhibitory effect on ASIC currents. μ-opioid receptors are coupled to Gi/o proteins and their activation leads to inhibition of AC activity. As a result, formation of cAMP is inhibited, and protein kinase A (PKA) cannot be activated (Law et al., 2000). The morphine-induced inhibition of ASIC currents may involve intracellular signal transduction. Our observations that both forskolin and 8-Br-cAMP can reverse morphine inhibition of ASIC currents indicated that this effect may be mediated mainly via a cAMP/PKA-dependent pathway. Consistent with our previous study, cannabinoids attenuation of ASICs is mediated by the inhibition of cAMP/ PKA-dependent pathway through CB1 receptors (Liu et al., 2012). In addition, μ-opioid activation can also inhibit the activity of TRPV1 via Gi/o proteins and the cAMP pathway (Endres-Becker et al., 2007). A-kinase anchoring protein 150 (AKAP150) has been reported to be co-localized with ASIC1a and ASIC2a in rat cortical neurons, and regulates the activity of these channels. It has also been reported that the
Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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inhibition of protein kinase A (PKA) binding to AKAP150 reduces the amplitude of ASIC currents, suggesting that AKAP150 mediates the PKA-dependent phosphorylation of ASICs (Chai et al., 2007). Therefore, the reduction of PKA activity may be involved in the morphine inhibition of ASIC currents in primary sensory neurons. ASICs are extracellular pH sensors and permeable to cations. In peripheral sensory neurons, ASICs have been found on cell bodies and sensory terminals. Activation of ASICs, particularly ASIC3 and ASIC1a channels, by acidic pH and the resultant depolarization of nociceptive primary sensory neurons, participate in nociception (Leng et al., 2013). ASICs are found to mediate cutaneous acid-induced pain (Jones et al., 2004; Ugawa et al., 2002). Intraplantar injection of acetic acid elicited an intense flinch/shaking response in rats (Deval et al., 2008; Omori et al., 2008). In the present study, the acid-evoked pain was significantly blocked by amiloride, a broad-spectrum ASIC channel blocker, indicating the involvement of ASICs. Behavioral experiments demonstrated that locally applied morphine relieved the acidosisevoked pain in a dose-dependent manner. The analgesic effects of peripheral morphine were mediated by opioid receptors, since it was blocked by naloxone, a specific opioid receptor antagonist. The results were also consistent with morphine inhibitory effects on acid-evoked action potentials in current clamp experiments. A number of pain-causing stimuli, such as inflammation, lower extracellular pH (Wemmie et al., 2013). ASICs are a family of cation channels and present both in the cell body and at terminals of peripheral nociceptive neurons. Thus, activation of ASICs contributes to pain. ASICs have emerged as a potential therapeutic target for new pain medication (Kweon and Suh, 2013; Wemmie et al., 2013). The ASIC inhibitor amiloride is approved for use in humans, and a few small translational experiments have demonstrated its potential for reducing cutaneous pain and migraine (Holland et al., 2012; Jones et al., 2004; Ugawa et al., 2002). It has been shown that analgesic effects of peripheral opioids are particularly prominent during inflammatory pain (Stein et al., 2003). In the present study, our results strongly indicated morphine can exert their analgesic action by interaction with ASICs in the primary afferent neurons. In conclusion, this study identified a cross-talk between ASICs and μ-opioid receptors in rat primary sensory neurons. We showed that the activity of ASICs can be inhibited by morphine in DRG neurons. Morphine decreased ASIC currents, action potentials and pain induced by acid stimuli. The morphine inhibition was blocked by naloxone, a specific opioid receptor antagonist. Our results reveal a novel peripheral analgesic mechanism of morphine by modulating native ASICs.
guidelines on the ethical use of animals, and every effort was made to minimize the number of animals used and their suffering. Two- to three-week old Sprague–Dawley male rats were anesthetized with ethyl ether and then decapitated. The DRGs were taken out and transferred immediately into DMEM (Sigma-Aldrich, St. Louis, MO, US) 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 1C 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 another 60 min before electrophysiological recordings. The neurons selected for electrophysiological experiment were 15–35 μm in diameter.
4.2.
Experimental procedures
4.1.
Isolation of the dorsal root ganglia neurons
The experimental protocol was approved by the Animal Research Ethics Committee of Hubei University of Science and Technology. All procedures conformed to international
Electrophysiological recordings
Whole-cell patch clamp and voltage-clamp recordings were carried out at room temperature (22–25 1C) using a MultiClamp-700B amplifier and Digidata-1440A A/D converter (Axon Instruments, Foster City, CA, USA). Recording pipettes were pulled using a Sutter P-97 puller (Sutter Instruments, Novato, CA, USA). The micropipettes were filled with internal solution containing KCl 140 mM, MgCl2 2.5 mM, HEPES 10 mM, EGTA 11 mM and ATP 5 mM; 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 NaCl 150 mM, KCl 5 mM, CaCl2 2.5 mM, MgCl2 2 mM, HEPES 10 mM and D-glucose 10 mM; its osmolarity was adjusted to 330 mOsm/L with sucrose and 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 gigaseal and then negative pressure was applied to rupture it, thus establishing a whole-cell configuration. The adjustment of capacitance compensation and series resistance compensation was 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 10–50 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).
4.3.
4.
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Drug application
Drugs used in the experiments were purchased from Sigma Chemical Co. and include hydrochloric acid, morphine (catalog # M-005), naloxone, amiloride, forskolin, 8-Br-cAMP, DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) and capsazepine. Stocks of drugs were made up in dimethyl sulfoxide and diluted daily in the external solution at a minimum of 1:1000 to a final working concentration. The final concentration of dimethyl
Please cite this article as: Cai, Q., et al., Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.01.042
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sulfoxide used was 0.1%, which has no effect on ASICs. Except for hydrochloric acid, the pH for all working solutions was adjusted daily to 7.470.1 with HCl or NaOH. Next, they were held in a linear array of fused silica tubes (o.d./i.d.¼ 500 μm/ 200 μm) connected to a series of independent reservoirs. The application pipette tips were positioned 30 μm away from the recorded neurons. 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 with a PC-controlled micromanipulator. Cells were constantly bathed in normal external solution flowing from one tube connected to a larger reservoir between drug applications. To functionally characterize ASIC activity, we used capsazepine (10 μM) to block TRPV1 in this study (Poirot et al., 2006).
4.4.
Nociceptive behavior induced by acetic acid in rats
Sprague–Dawley male rats (3–4 weeks old) were kept with a 12 h light/dark cycle and with ad libitum access to food and water. Animals were placed in a 30 30 30 cm3 Plexiglas chamber and allowed to habituate for at least 30 min before nociceptive behavior experiments. After pretreatment with 10 μL capsazepine (100 μM), a double-blind experiment was carried out. Twenty microliters of acetic acid solution (0.6%) together with 20 μL external solution, amiloride, morphine and/or naloxone were coded, and the other experimenters subcutaneously administered them into the dorsal face of the hind paw using a 30 gauge needle connected to a 100 μL Hamilton syringe. 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).
4.5.
Data analysis
Data were statistically compared using 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 mean7standard error of the mean (SEM).
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81171039) to WPH, Program for New Century Excellent Talents in University (NCET-11-0967) to WPH, Team of Outstanding Young Scientific and Technological Innovation of the Higher Education Institutions of Hubei Province (T201113) to WPH, Key Project of Chinese Ministry of Education (210142) to WPH, and Scientific Research Staring Foundation for the Returned Overseas Chinese Scholars, Ministry of Education of China to WPH.
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