Insulin enhances acid-sensing ion channel currents in rat primary sensory neurons.

Insulin has been shown to modulate neuronal processes through insulin receptors. The ion channels located on neurons may be important targets for insulin/insulin receptor signaling. Both insulin receptors and acid-sensing ion channels (ASICs) are expressed in dorsal root ganglia (DRG) neurons. However, it is still unclear whether there is an interaction between them. Therefore, the purpose of this investigation was to determine the effects of insulin on the functional activity of ASICs. A 5 min application of insulin rapidly enhanced acid-evoked ASIC currents in rat DRG neurons in a concentration-dependent manner. Insulin shifted the concentration-response plot for ASIC currents upward, with an increase of 46.2 ± 7.6% in the maximal current response. The insulin-induced increase in ASIC currents was eliminated by the insulin receptor antagonist GSK1838705, the tyrosine kinase inhibitor lavendustin A, and the phosphatidylinositol-3 kinase antagonist wortmannin. Moreover, insulin increased the number of acid-triggered action potentials by activating insulin receptors. Finally, local administration of insulin exacerbated the spontaneous nociceptive behaviors induced by intraplantar acid injection and the mechanical hyperalgesia induced by intramuscular acid injections through peripheral insulin receptors. These results suggested that insulin/insulin receptor signaling enhanced the functional activity of ASICs via tyrosine kinase and phosphatidylinositol-3 kinase pathways. Our findings revealed that ASICs were targets in primary sensory neurons for insulin receptor signaling, which may underlie insulin modulation of pain.

CXCL10 Enhances Acid-Sensing Ion Channel Currents in Rat Dorsal Root.

Both CXCL10/CXCR3 and acid-sensing ion channels (ASICs) are expressed in nociceptive sensory neurons and participate in various pain processes, but it is still unclear whether there is a link between them. Herein, we report that CXCL10 enhances the electrophysiological activity of ASICs in rat dorsal root ganglia (DRG) neurons. A brief (10 min) application of CXCL10 increased acid-evoked ASIC currents in a concentration-dependent manner. CXCL10 increased the maximum response of ASICs to acidic stimuli without changing their sensitivity. CXCL10 enhanced ASIC currents in DRG cells through CXCR3, as this enhancement was completely blocked by AMG487, a selective CXCR3 antagonist. CXCL10 also increased ASIC3 currents in CHO cells coexpressing ASIC3 and CXCR3 but not in cells expressing ASIC3 alone. The CXCL10-mediated increase in ASIC currents was prevented by the application of either the G protein inhibitor GDP-ß-S or the p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 but not by the ERK inhibitor U0126 or the JNK inhibitor SP600125. Moreover, CXCL10 increased the number of action potentials triggered by acidic stimuli via CXCR3. CXCL10 dose-dependently exacerbated acid-induced nociceptive behavior in rats through peripheral CXCR3. These results indicated that CXCL10/CXCR3 signaling enhanced ASIC-mediated electrophysiological activity in DRG neurons and nociception in rats via a p38 MAPK-dependent pathway, revealing a novel mechanism underlying pain. CXCL10/CXCR3 signaling may be an effective target in the treatment of pain associated with tissue acidification.

Lipid mediator resolvin D2 inhibits ATP currents in rat primary sensory neurons.

Resolvin D2 (RvD2), an endogenous lipid mediator derived from docosahexaenoic acid, has been demonstrated to have analgesic effects. However, little is known about the mechanism underlying RvD2 in pain relief. Herein, we demonstrate that RvD2 targeted the P2X3 receptor as an analgesic. The electrophysiological activity of P2X3 receptors was suppressed by RvD2 in rat dorsal root ganglia (DRG) neurons. RvD2 pre-application dose-dependently decreased α,ß-methylene-ATP (α,ß-meATP)-induced inward currents. RvD2 remarkably decreased the maximum response to α,ß-meATP, without influencing the affinity of P2X3 receptors. RvD2 also voltage-independently suppressed ATP currents. An antagonist of the G protein receptor 18 (GPR18), O-1918, prevented the RvD2-induced suppression of ATP currents. Additionally, intracellular dialysis of the Gαi/o -protein antagonist pertussis toxin (PTX), the PKA antagonist H89, or the cAMP analog 8-Br-cAMP also blocked the RvD2-induced suppression. Furthermore, α,ß-meATP-triggered depolarization of membrane potential along with the action potential bursts in DRG neurons were inhibited by RvD2. Lastly, RvD2 attenuated spontaneous nociceptive behaviors as well as mechanical allodynia produced by α,ß-meATP in rats via the activation of the peripheral GPR18. These findings indicated that RvD2 inhibited P2X3 receptors in rat primary sensory neurons through GPR18, PTX-sensitive Gαi/o -proteins, and intracellular cAMP/PKA signaling, revealing a novel mechanism that underlies its analgesic effects by targeting P2X3 receptors.

CCK-8 enhances acid-sensing ion channel currents in rat primary sensory neurons.

Cholecystokinin (CCK) is a peptide that has been implicated in pain modulation. Acid sensitive ion channels (ASICs) also play an important role in pain associated with tissue acidification. However, it is still unclear whether there is an interaction between CCK signaling and ASICs during pain process. Herein, we report that a functional link between them in rat dorsal root ganglion (DRG) neurons. Pretreatment with CCK-8 concentration-dependently increased acid-evoked ASIC currents. CCK-8 increased the maximum response of ASICs to acid, but did not changed their acid sensitivity. Enhancement of ASIC currents by CCK-8 was mediated by the stimulation of CCK2 receptor (CCK2R), rather than CCK1R. The enhancement of ASIC currents by CCK-8 was prevented by application of either G-protein inhibitor GDP-ß-S or protein kinase C (PKC) inhibitor GF109203×, but not by protein kinase A (PKA) inhibitor H-89 or JNK inhibitor SP600125. Moreover, CCK-8 increased the number of action potentials triggered by acid stimuli by activating CCK2R. Finally, CCK-8 dose-dependently exacerbated acid-induced nociceptive behavior in rats through local CCK2R. Together, these results indicated that CCK-8/CCK2R activation enhanced ASIC-mediated electrophysiological activity in DRG neurons and nociception in rats. The enhancement effect depended on G-proteins and intracellular PKC signaling rather than PKA and JNK signaling pathway. These findings provided that CCK-8/CCK2R is an important therapeutic target for ASIC-mediated pain.

Metformin inhibits spontaneous excitatory postsynaptic currents in spinal dorsal cord neurons from paclitaxel-treated rats.

Introduction: Cancer patients treated with paclitaxel often develop chemotherapy-induced peripheral neuropathy, which has not been effectively treated with drugs. The anti-diabetic drug metformin is effective in the treatment of neuropathic pain. The aim of this study was to elucidate effect of metformin on paclitaxel-induced neuropathic pain and spinal synaptic transmission. Methods: Electrophysiological experiments on rat spinal slices were performed in vitro and mechanical allodynia quantified in vitro. Results: The present data demonstrated that intraperitoneal injection of paclitaxel produced mechanical allodynia and potentiated spinal synaptic transmission. Intrathecal injection of metformin significantly reversed the established mechanical allodynia induced by paclitaxel in rats. Either spinal or systemic administration of metformin significantly inhibited the increased frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in spinal dorsal horn neurons from paclitaxel-treated rats. We found that 1 h incubation of metformin also reduced the frequency rather than the amplitude of sEPSCs in the spinal slices from paclitaxel-treated rats. Discussion: These results suggested that metformin was able to depress the potentiated spinal synaptic transmission, which may contribute to alleviating the paclitaxel-induced neuropathic pain.

Group II metabotropic glutamate receptor activation attenuates acid-sensing ion channel currents in rat primary sensory neurons.

Acid-sensing ion channels (ASICs) play an important role in pain associated with tissue acidification. Peripheral inhibitory group II metabotropic glutamate receptors (mGluRs) have analgesic effects in a variety of pain conditions. Whether there is a link between ASICs and mGluRs in pain processes is still unclear. Herein, we show that the group II mGluR agonist LY354740 inhibited acid-evoked ASIC currents and action potentials in rat dorsal root ganglia neurons. LY354740 reduced the maximum current response to protons, but it did not change the sensitivity of ASICs to protons. LY354740 inhibited ASIC currents by activating group II mGluRs. We found that the inhibitory effect of LY354740 was blocked by intracellular application of the Gi/o protein inhibitor pertussis toxin and the cAMP analogue 8-Br-cAMP and mimicked by the protein kinase A (PKA) inhibitor H-89. LY354740 also inhibited ASIC3 currents in CHO cells coexpressing mGluR2 and ASIC3 but not in cells expressing ASIC3 alone. In addition, intraplantar injection of LY354740 dose-dependently alleviated acid-induced nociceptive behavior in rats through local group II mGluRs. Together, these results suggested that activation of peripheral group II mGluRs inhibited the functional activity of ASICs through a mechanism that depended on Gi/o proteins and the intracellular cAMP/PKA signaling pathway in rat dorsal root ganglia neurons. We propose that peripheral group II mGluRs are an important therapeutic target for ASIC-mediated pain.

Group II metabotropic glutamate receptor activation suppresses ATP currents in rat dorsal root ganglion neurons.

P2X3 receptors and group II metabotropic glutamate receptors (mGluRs) have been found to be expressed in primary sensory neurons. P2X3 receptors participate in a variety of pain processes, while the activation of mGluRs has an analgesic effect. However, it's still unclear whether there is a link between them in pain. Herein, we reported that the group II mGluR activation inhibited the electrophysiological activity of P2X3 receptors in rat dorsal root ganglia (DRG) neurons. Group II mGluR agonist LY354740 concentration-dependently decreased P2X3 receptor-mediated and α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents in DRG neurons. LY354740 significantly suppressed the maximum response of P2X3 receptor to α,ß-meATP, but did not change their affinity. Inhibition of ATP currents by LY354740 was blocked by the group II mGluR antagonist LY341495, also prevented by the intracellular dialysis of either the Gi/o protein inhibitor pertussis toxin, the cAMP analog 8-Br-cAMP, or the protein kinase A (PKA) inhibitor H-89. Moreover, LY354740 decreased α,ß-meATP-induced membrane potential depolarization and action potential bursts in DRG neurons. Finally, intraplantar injection of LY354740 also relieved α,ß-meATP-induced spontaneous nociceptive behaviors and mechanical allodynia in rats by activating peripheral group Ⅱ mGluRs. These results indicated that peripheral group II mGluR activation inhibited the functional activity of P2X3 receptors via a Gi/o protein and cAMP/PKA signaling pathway in rat DRG neurons, which revealed a novel mechanism underlying analgesic effects of peripheral group II mGluRs. This article is part of the Special Issue on "Purinergic Signaling: 50 years".

A1 Adenosine Receptor Activation Inhibits P2X3 Receptor-Mediated ATP Currents in Rat Dorsal Root Ganglion Neurons.

Purinergic signaling is involved in multiple pain processes. P2X3 receptor is a key target in pain therapeutics, while A1 adenosine receptor signaling plays a role in analgesia. However, it remains unclear whether there is a link between them in pain. The present results showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) concentration dependently suppressed P2X3 receptor-mediated and α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents in rat dorsal root ganglion (DRG) neurons. CPA significantly decreased the maximal current response to α,ß-meATP, as shown a downward shift of the concentration-response curve for α,ß-meATP. CPA suppressed ATP currents in a voltage-independent manner. Inhibition of ATP currents by CPA was completely prevented by the A1 adenosine receptor antagonist KW-3902, and disappeared after the intracellular dialysis of either the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, or the cAMP analog 8-Br-cAMP. Moreover, CPA suppressed the membrane potential depolarization and action potential bursts, which were induced by α,ß-meATP in DRG neurons. Finally, CPA relieved α,ß-meATP-induced nociceptive behaviors in rats by activating peripheral A1 adenosine receptors. These results indicated that CPA inhibited the activity of P2X3 receptors in rat primary sensory neurons by activating A1 adenosine receptors and its downstream cAMP signaling pathway, revealing a novel peripheral mechanism underlying its analgesic effect.

Potentiation of ASIC currents by lysophosphatidic acid in rat dorsal root ganglion neurons.

Lysophosphatidic acid (LPA) is a phospholipid which has been implicated in pain. Acid-sensing ion channels (ASICs) are important players in pain associated with tissue acidification. However, it is still unclear whether there is a link between LPA signaling and ASICs in pain processes. Herein, we show that a functional interaction between them in rat dorsal root ganglia (DRG) neurons. Pre-application of LPA enhanced ASIC-mediated and acid-evoked inward currents in a concentration-dependent manner. LPA shifted the concentration-response curve for protons upwards, with an increase of 41.79 ± 4.71% in the maximal current response of ASICs to protons in the presence of LPA. Potentiation of ASIC currents by LPA was blocked by the LPA1 receptor antagonist Ki16198, but not by the LPA2 receptor antagonist H2L5185303. The LPA-induced potentiation was also prevented by intracellular application of either G protein inhibitor or protein kinase C (PKC) inhibitor, but not by Rho inhibitor. LPA also enhanced ASIC3 currents in CHO cells co-expressing ASIC3 and LPA1 receptors, but not in cells expressing ASIC3 alone. Moreover, LPA increased the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, LPA exacerbated acid-induced nociceptive behaviors in rats. These results suggested that LPA enhanced ASIC-mediated electrophysiological activity and nociception via a LPA1 receptor and its downstream PKC rather than Rho signaling pathway, which provided a novel peripheral mechanism underlying the sensitization of pain.

Enhancement of P2X3 Receptor-Mediated Currents by Lysophosphatidic Acid in Rat Primary Sensory Neurons.

Lysophosphatidic acid (LPA), a lipid metabolite, plays a role in both neuropathic and inflammatory pain through LPA1 receptors. P2X3 receptor has also been shown to participate in these pathological processes. However, it is still unclear whether there is a link between LPA signaling and P2X3 receptors in pain. Herein, we show that a functional interaction between them in rat dorsal root ganglia (DRG) neurons. Pretreatment of LPA concentration-dependently enhanced α,ß-methylene-ATP (α,ß-meATP)-induced inward currents mediated by P2X3 receptors. LPA significantly increased the maximal current response of α,ß-meATP, showing an upward shift of the concentration-response curve for α,ß-meATP. The LPA enhancement was independent on the clamping-voltage. Enhancement of P2X3 receptor-mediated currents by LPA was prevented by the LPA1 receptor antagonist Ki16198, but not by the LPA2 receptor antagonist H2L5185303. The LPA-induced potentiation was also attenuated by intracellular dialysis of either G-protein inhibitor or protein kinase C (PKC) inhibitor, but not by Rho inhibitor. Moreover, LPA significantly changed the membrane potential depolarization and action potential burst induced by α,ß-meATP in DRG neurons. Finally, LPA exacerbated α,ß-meATP- induced nociceptive behaviors in rats. These results suggested that LPA potentiated the functional activity of P2X3 receptors in rat primary sensory neurons through activation of the LPA1 receptor and its downstream PKC rather than Rho signaling pathway, indicating a novel peripheral mechanism underlying the sensitization of pain.

Resveratrol inhibits the activity of acid-sensing ion channels in male rat dorsal root ganglion neurons.

Resveratrol can relieve pain under various pain conditions. One of the mechanisms of resveratrol analgesia is the regulation of ion channels. Acid-sensing ion channels (ASICs) are expressed predominantly in nociceptive sensory neurons to detect changes in extracellular pH. ASICs are important players in pain associated with tissue acidification. However, it is still unclear whether ASICs are resveratrol targets. Electrophysiological recordings showed that resveratrol decreased acid-induced and ASIC-mediated currents in male rat dorsal root ganglion (DRG) neurons in a concentration-dependent manner. Resveratrol downwardly shifted the concentration-response curve for protons, suggesting that it inhibited ASICs not by changing the pH0.5 , but by suppressing the proton-induced maximum response. It also suppressed acid-triggered action potentials in the rat DRG neurons. Finally, intraplantar pretreatment with resveratrol relieved acid-induced nociceptive responses in male rats in a dose-dependent manner. These results indicated that resveratrol inhibited ASIC-mediated electrophysiological activity and nociception, suggesting a novel peripheral mechanism underlying its analgesic effect.

Suppression of ASIC activity by the activation of A1 adenosine receptors in rat primary sensory neurons.

Peripheral A1 adenosine receptor signaling has been shown to have analgesic effects in a variety of pain conditions. However, it is not yet fully elucidated for the precise molecular mechanisms. Acid sensing ion channels (ASICs) are expressed predominantly in nociceptive sensory neurons responding to protons. Given that both A1 adenosine receptors and ASICs are present in dorsal root ganglia (DRG) neurons, we therefore investigated whether there was a cross-talk between the two types of receptors. Herein, electrophysiological recordings showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) suppressed acid-induced currents and action potentials, which were mediated by ASICs, in rat DRG neurons. CPA inhibited the maximum response to protons, as shown a downward shift of concentration-response curve for protons. The CPA-induced suppression of ASIC currents was blocked by the A1 adenosine receptor antagonist KW-3902 and also prevented by intracellular application of the Gi/o-protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8-Br-cAMP. Finally, intraplantar pretreatment of CPA dose-dependently relieved acid-induced nociceptive responses in rats through peripheral A1 adenosine receptors. These results suggested that CPA suppressed ASICs via A1 adenosine receptors and intracellular Gi/o-proteins and cAMP signaling cascades in rat DRG neurons, which was a novel potential mechanism underlying analgesia of peripheral A1 adenosine receptors.

Suppression of P2X3 receptor-mediated currents by the activation of α2A -adrenergic receptors in rat dorsal root ganglion neurons.

AIMS: The α2 -adrenergic receptor (α2 -AR) agonists have been shown to be effective in the treatment of various pain. For example, dexmedetomidine (DEX), a selective α2A -AR agonist, can be used for peripheral analgesia. However, it is not yet fully elucidated for the precise molecular mechanisms. P2X3 receptor is a major receptor processing nociceptive information in primary sensory neurons. Herein, we show that a functional interaction of α2A -ARs and P2X3 receptors in dorsal root ganglia (DRG) neurons could contribute to peripheral analgesia of DEX. METHODS: Electrophysiological recordings were carried out on rat DRG neurons, and nociceptive behavior was quantified in rats. RESULTS: The activation of α2A -ARs by DEX suppressed P2X3 receptor-mediated and α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents in a concentration-dependent and voltage-independent manner. Pre-application of DEX shifted the α,ß-meATP concentration-response curve downwards, with a decrease of 50.43 ± 4.75% in the maximal current response of P2X3 receptors to α,ß-meATP in the presence of DEX. Suppression of α,ß-meATP-evoked currents by DEX was blocked by the α2A -AR antagonist BRL44408 and prevented by intracellular application of the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8-Br-cAMP. DEX also suppressed α,ß-meATP-evoked action potentials through α2A -ARs in rat DRG neurons. Finally, the activation of peripheral α2A -ARs by DEX had an analgesic effect on the α,ß-meATP-induced nociception. CONCLUSIONS: These results suggested that activation of α2A -ARs by DEX suppressed P2X3 receptor-mediated electrophysiological and behavioral activity via a Gi/o proteins and cAMP signaling pathway, which was a novel potential mechanism underlying analgesia of peripheral α2A -AR agonists.

Acute P38-Mediated Enhancement of P2X3 Receptor Currents by TNF-α in Rat Dorsal Root Ganglion Neurons.

PURPOSE: Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine and involves in a variety of pain conditions. Some findings suggest that TNF-α may act directly on primary afferent neurons to induce acute pain hypersensitivity through non-transcriptional regulation. This study investigated whether TNF-α had an effect on functional activity of P2X3 receptors in primary sensory neurons. Herein, we report that a brief (5 min) application of TNF-α rapidly enhanced the electrophysiological activity of P2X3 receptors in rat dorsal root ganglia (DRG) neurons. METHODS: Electrophysiological recordings were carried out on rat DRG neurons, and nociceptive behavior was quantified in rats. RESULTS: A brief (5 min) exposure of TNF-α rapidly increased P2X3 receptor-mediated and α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents in a dose-dependent manner. The potentiation of P2X3 receptor-mediated ATP currents by TNF-α was voltage-independent. TNF-α shifted the concentration-response curve for α,ß-meATP upwards, with an increase of 31.57 ± 6.81% in the maximal current response to α,ß-meATP. This acute potentiation of ATP currents by TNF-α was blocked by p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190, but not by non-selective cyclooxygenase inhibitor indomethacin, suggesting involvement of p38 MAPK, but not cyclooxygenase. Moreover, intraplantar injection of TNF-α and α,ß-meATP produced a synergistic effect on mechanical allodynia in rats. TNF-α-induced mechanical allodynia was also alleviated after local P2X3 receptors were blocked. CONCLUSION: These results suggested that TNF-α rapidly sensitized P2X3 receptors in primary sensory neurons via a p38 MAPK dependent pathway, which revealed a novel peripheral mechanism underlying acute mechanical hypersensitivity by peripheral administration of TNF-α.

Dexmedetomidine Inhibits ASIC Activity via Activation of α2A Adrenergic Receptors in Rat Dorsal Root Ganglion Neurons.

Dexmedetomidine (DEX), a selective α2 adrenergic receptor (α2-AR) agonist, has been shown to have peripheral analgesic effects in a variety of pain conditions. However, the precise molecular mechanisms have not yet been fully elucidated. Acid sensing ion channels (ASICs) are the major player in pain associated with tissue acidosis. Given that both α2-ARs and ASICs exist in dorsal root ganglia (DRG) neurons, we therefore investigated the effects of DEX on the functional activity of ASICs. Herein, whole-cell patch-clamp recordings demonstrated that DEX suppressed ASIC-mediated and acid-evoked currents and action potentials in dissociated rat DRG neurons. DEX shifted downwards concentration-response curve to protons, with a decrease of 35.83 ± 3.91% in the maximal current response to pH 4.5. DEX-induced inhibition of ASIC currents was blocked by the α2A-AR antagonist BRL44408 in DRG neurons. DEX also inhibited ASIC3 currents in CHO cells co-expressing ASIC3 and α2A-ARs, but not in ASIC3 transfected CHO cells without α2A-ARs expression. DEX-induced inhibition of ASIC currents was mimicked by the protein kinase A inhibitor H-89, and blocked by intracellular application of the Gi/o protein inhibitor pertussis toxin and the cAMP analog 8-Br-cAMP. In addition, peripherally administration of DEX dose-dependently relieved nociceptive responses to intraplantar injection of acetic acid in rats through local α2A-ARs. Our results indicated that DEX inhibited the functional activity of ASICs via α2A-ARs and intracellular Gi/o proteins and cAMP/protein kinase A signaling pathway in rat DRG neurons, which was a novel potential mechanism that probably mediated peripheral analgesia of DEX.

TNF-α acutely enhances acid-sensing ion channel currents in rat dorsal root ganglion neurons via a p38 MAPK pathway.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine involved in pain processing and hypersensitivity. It regulates not only the expression of a variety of inflammatory mediators but also the functional activity of some ion channels. Acid-sensing ion channels (ASICs), as key sensors for extracellular protons, are expressed in nociceptive sensory neurons and contribute to pain signaling caused by tissue acidosis. It is still unclear whether TNF-α has an effect on functional activity of ASICs. Herein, we reported that a brief exposure of TNF-α acutely sensitized ASICs in rat dorsal root ganglion (DRG) neurons. METHODS: Electrophysiological experiments on rat DRG neurons were performed in vitro and acetic acid induced nociceptive behavior quantified in vitro. RESULTS: A brief (5min) application of TNF-α rapidly enhanced ASIC-mediated currents in rat DRG neurons. TNF-α (0.1-10 ng/ml) dose-dependently increased the proton-evoked ASIC currents with an EC50 value of 0.12 ± 0.01 nM. TNF-α shifted the concentration-response curve of proton upwards with a maximal current response increase of 42.34 ± 7.89%. In current-clamp recording, an acute application of TNF-α also significantly increased acid-evoked firing in rat DRG neurons. The rapid enhancement of ASIC-mediated electrophysiological activity by TNF-α was prevented by p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190, but not by non-selective cyclooxygenase inhibitor indomethacin, suggesting that p38 MAPK is necessary for this enhancement. Behaviorally, TNF-α exacerbated acid-induced nociceptive behaviors in rats via activation of local p38 MAPK pathway. CONCLUSIONS: These results suggest that TNF-α rapidly enhanced ASIC-mediated functional activity via a p38 MAPK pathway, which revealed a novel peripheral mechanism underlying TNF-α involvement in rapid hyperalgesia by sensitizing ASICs in primary sensory neurons.

Inhibition of ASIC-Mediated Currents by Activation of Somatostatin 2 Receptors in Rat Dorsal Root Ganglion Neurons.

Somatostatin (SST) and its analogues like octreotide (OCT) have analgesic effect on a variety of pain through peripheral SST receptors (SSTRs). However, the precise molecular mechanisms have not yet been fully elucidated. This research aimed to identify possible antinociceptive mechanisms, showing functional links of the SSTR2 and acid-sensing ion channels (ASICs). Herein, we reported that OCT inhibited the electrophysiological activity of ASICs in rat dorsal root ganglia (DRG) neurons. OCT concentration-dependently decreased the peak amplitude of acid-evoked inward currents, which were mediated by ASICs. OCT shifted concentration-response curve to protons downwards, with a decrease of 36.53 ± 5.28% in the maximal current response to pH 4.5 in the presence of OCT. OCT inhibited ASIC-mediated currents through SSTR2, since the inhibition was blocked by Cyn 154806, a specific SSTR2 antagonist. The OCT inhibition of ASIC-mediated currents was mimicked by H-89, a membrane-permeable inhibitor of PKA, and reversed by internal treatment of an adenylyl cyclase activator forskolin or 8-Br-cAMP. OCT also decreased the number of action potentials induced by acid stimuli through SSTR2. Finally, peripheral administration of 20 µM OCT, but not 2 µM OCT, significantly relieved nociceptive responses to intraplantar injection of acetic acid in rats. This occurred through local activation of SSTR2 in the injected hindpaw and was reversed following co-application of Cyn 154806. Our results indicate that activation SSTR2 by OCT can inhibit the activity of ASICs via an intracellular cAMP and PKA signaling pathway in rat DRG neurons. These observations demonstrate a cross-talk between ASICs and SSTR2 in peripheral sensory neurons, which was a novel peripheral analgesic mechanism of SST and its analogues.

Potentiation of P2X3 receptor mediated currents by endothelin-1 in rat dorsal root ganglion neurons.

Endothelin-1 (ET-1), an endogenous vasoconstrictor, has been known as a pro-nociceptive agent involved in multitude of pain. ET-1 acts on endothelin receptors on vascular endothelial cells, sensitizes release of ATP, which then acts on P2X3 receptors on nociceptors and results in mechanical hyperalgesia. Both endothelin receptors and P2X3 receptors are present in primary sensory neuron, where it remains unclear whether there is an interaction between them. Herein, we reported that ET-1 potentiated the electrophysiological activity of P2X3 receptors in rat dorsal root ganglia (DRG) neurons. ET-1 concentration-dependently increased α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents, which were mediated by P2X3 receptors. ET-1 shifted the α,ß-meATP concentration-response curve upwards, with an increase of 34.38 ± 4.72% in the maximal current response to α,ß-meATP in the presence of ET-1. ET-1 potentiation of α,ß-meATP-evoked currents was voltage-independent. ET-1 potentiated P2X3 receptor-mediated currents through endothelin-A receptors (ETAR), but not endothelin-B receptors (ETBR). ET-1 potentiation was supressed by blockade of intracellular G-protein or protein kinase C (PKC) signaling. Moreover, there is a synergistic effect on mechanical allodynia induced by intraplantar injection of ET-1 and α,ß-meATP in rats. Pharmacological blockade of P2X3 receptors also alleviated ET-1-induced mechanical allodynia. These results suggested that ET-1 sensitized P2X3 receptors in primary sensory neurons via an ETAR and PKC signaling pathway. Our data provide evidence that cutaneous ET-1 induced mechanical allodynia not only by increasing the release of ATP from vascular endothelial cells, but also by sensitizing P2X3 receptors on nociceptive DRG neurons.

Endothelin-1 enhances acid-sensing ion channel currents in rat primary sensory neurons.

Endothelin-1 (ET-1), an endogenous vasoactive peptide, has been found to play an important role in peripheral pain signaling. Acid-sensing ion channels (ASICs) are key sensors for extracellular protons and contribute to pain caused by tissue acidosis. It remains unclear whether an interaction exists between ET-1 and ASICs in primary sensory neurons. In this study, we reported that ET-1 enhanced the activity of ASICs in rat dorsal root ganglia (DRG) neurons. In whole-cell voltage-clamp recording, ASIC currents were evoked by brief local application of pH 6.0 external solution in the presence of TRPV1 channel blocker AMG9810. Pre-application with ET-1 (1-100 nM) dose-dependently increased the proton-evoked ASIC currents with an EC50 value of 7.42 ± 0.21 nM. Pre-application with ET-1 (30 nM) shifted the concentration-response curve of proton upwards with a maximal current response increase of 61.11% ± 4.33%. We showed that ET-1 enhanced ASIC currents through endothelin-A receptor (ETAR), but not endothelin-B receptor (ETBR) in both DRG neurons and CHO cells co-expressing ASIC3 and ETAR. ET-1 enhancement was inhibited by blockade of G-protein or protein kinase C signaling. In current-clamp recording, pre-application with ET-1 (30 nM) significantly increased acid-evoked firing in rat DRG neurons. Finally, we showed that pharmacological blockade of ASICs by amiloride or APETx2 significantly alleviated ET-1-induced flinching and mechanical hyperalgesia in rats. These results suggest that ET-1 sensitizes ASICs in primary sensory neurons via ETAR and PKC signaling pathway, which may contribute to peripheral ET-1-induced nociceptive behavior in rats.

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

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.