Preventing the induction of acid saline-induced fibromyalgia pain in mice by electroacupuncture or APETx2 injection.

BACKGROUND: Fibromyalgia (FM) is a syndrome involving chronic pain, fatigue, sleep difficulties, morning stiffness and muscle cramping lasting longer than 3 months. The epidemiological prevalence is approximately 3-5% in women and increases with age. Antagonism of acid-sensing ion channel 3 (ASIC3) reportedly attenuates acid saline-induced FM pain in mice. AIMS: Whether pre-treatment with electroacupuncture (EA) or APETx2 can attenuate mechanical hyperalgesia in this murine model remains unknown. METHODS: Accordingly, we examined the analgesic effect of EA in a murine model of FM pain induced by dual injections of acid saline and investigated whether EA or APETx2 can attenuate FM pain via the ASIC3 channel. RESULTS: EA significantly reduced mechanical hyperalgesia in this model. ASIC3 antagonism, induced by injecting APETx2, also significantly reduced mechanical hyperalgesia. The expression of ASIC3 in the dorsal root ganglia, spinal cord and thalamus was increased after FM model induction. Over-expression of these nociceptive channels was attenuated by pre-treatment with EA or an ASIC3 antagonist. CONCLUSION: Our data reveal that both EA and ASIC3 blockade significantly reduce FM pain in mice via the ASIC3, Nav1.7 and Nav1.8 signalling pathways. Moreover, our findings support the potential clinical use of EA for the treatment of FM pain.

Alkaloid Lindoldhamine Inhibits Acid-Sensing Ion Channel 1a and Reveals Anti-Inflammatory Properties.

Acid-sensing ion channels (ASICs), which are present in almost all types of neurons, play an important role in physiological and pathological processes. The ASIC1a subtype is the most sensitive channel to the medium's acidification, and it plays an important role in the excitation of neurons in the central nervous system. Ligands of the ASIC1a channel are of great interest, both fundamentally and pharmaceutically. Using a two-electrode voltage-clamp electrophysiological approach, we characterized lindoldhamine (a bisbenzylisoquinoline alkaloid extracted from the leaves of Laurus nobilis L.) as a novel inhibitor of the ASIC1a channel. Lindoldhamine significantly inhibited the ASIC1a channel's response to physiologically-relevant stimuli of pH 6.5-6.85 with IC50 range 150-9 µM, but produced only partial inhibition of that response to more acidic stimuli. In mice, the intravenous administration of lindoldhamine at a dose of 1 mg/kg significantly reversed complete Freund's adjuvant-induced thermal hyperalgesia and inflammation; however, this administration did not affect the pain response to an intraperitoneal injection of acetic acid (which correlated well with the function of ASIC1a in the peripheral nervous system). Thus, we describe lindoldhamine as a novel antagonist of the ASIC1a channel that could provide new approaches to drug design and structural studies regarding the determinants of ASIC1a activation.

Analgesic Activity of Acid-Sensing Ion Channel 3 (ASIС3) Inhibitors: Sea Anemones Peptides Ugr9-1 and APETx2 versus Low Molecular Weight Compounds.

Acid-sensing ion channel 3 (ASIC3) makes an important contribution to the development and maintenance of inflammatory and acid-induced pain. We compared different ASIC3 inhibitors (peptides from sea anemones (APETx2 and Ugr9-1) and nonpeptide molecules (sevanol and diclofenac)) in anti-inflammatory action and analgesic effects. All tested compounds had distinct effects on pH-induced ASIC3 current. APETx2 inhibited only transient current, whereas Ugr9-1 and sevanol decreased transient and sustained components of the current. The effect on mice was evaluated after administering an intramuscular injection in the acetic acid writhing pain model and the complete Freund's adjuvant-induced thermal hyperalgesia/inflammation test. The bell-shaped dependence of the analgesic effect was observed for APETx2 in the acetic acid-induced writhing test, as well as for sevanol and peptide Ugr9-1 in the thermal hyperalgesia test. This dependence could be evidence of the nonspecific action of compounds in high doses. Compounds reducing both components of ASIC3 current produced more significant pain relief than APETx2, which is an effective inhibitor of a transient current only. Therefore, the comparison of the efficacy of ASIC3 inhibitors revealed the importance of ASIC3-sustained currents' inhibition for promotion of acidosis-related pain relief.

Root bark of Discaria americana attenuates pain: A pharmacological evidence of interaction with opioidergic system and TRP/ASIC channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Discaria americana (Rhamnaceae) root bark infusion have been used in traditional medicine as antipyretic, tonic, ameliorative of stomach and skin diseases and diabetes. This study was designed to investigate whether the methanolic extract of the root bark of Discaria americana (MEDa) exhibits antinociceptive effects in mice. Furthermore, it was investigated the involvement of the opioidergic system in MEDa mechanism of action as well the interactions with TRP/ASIC channels in its effect. MATERIALS AND METHODS: The antinociceptive effect of intra-gastric gavage (i.g.) of MEDa (0.3-300 mg/kg) was evaluated in mice subjected to acute chemical (acetic-acid, formalin, glutamate, capsaicin, cinnamaldehyde, and acidified saline) or thermal (hot plate) tests of pain. The involvement of opioid system was evaluated in the formalin test. A nonspecific effect of MEDa was observed by measuring locomotor activity and exploratory behavior in open field test. RESULTS: MEDa significantly reduced the number of writhing induced by acetic acid and inhibited the nociception in the two phases of formalin. These effects were inhibited by pretreatment with naloxone. The nociception induced by hot plate and intraplantar injection of glutamate, capsaicin, cinnamaldehyde and acidified saline were significantly inhibited by MEDa. Only the dose of 300 mg/kg altered the locomotor activity. CONCLUSIONS: Our results demonstrated, for the first time, that the methanolic extract of the root bark of Discaria americana presents antinociceptive effect in chemical and thermal stimuli and its analgesic properties can be due activation of the opioidergic system. These results support the use of Discaria americana in traditional medicine and demonstrate that this plant presents a therapeutic potential for the development of phytomedicines with antinociceptive profile.

Cerebrospinal Fluid-Contacting Neurons Sense pH Changes and Motion in the Hypothalamus.

CSF-contacting (CSF-c) cells are present in the walls of the brain ventricles and the central canal of the spinal cord and found throughout the vertebrate phylum. We recently identified ciliated somatostatin-/GABA-expressing CSF-c neurons in the lamprey spinal cord that act as pH sensors as well as mechanoreceptors. In the same neuron, acidic and alkaline responses are mediated through ASIC3-like and PKD2L1 channels, respectively. Here, we investigate the functional properties of the ciliated somatostatin-/GABA-positive CSF-c neurons in the hypothalamus by performing whole-cell recordings in hypothalamic slices. Depolarizing current pulses readily evoked action potentials, but hypothalamic CSF-c neurons had no or a very low level of spontaneous activity at pH 7.4. They responded, however, with membrane potential depolarization and trains of action potentials to small deviations in pH in both the acidic and alkaline direction. Like in spinal CSF-c neurons, the acidic response in hypothalamic cells is mediated via ASIC3-like channels. In contrast, the alkaline response appears to depend on connexin hemichannels, not on PKD2L1 channels. We also show that hypothalamic CSF-c neurons respond to mechanical stimulation induced by fluid movements along the wall of the third ventricle, a response mediated via ASIC3-like channels. The hypothalamic CSF-c neurons extend their processes dorsally, ventrally, and laterally, but as yet, the effects exerted on hypothalamic circuits are unknown. With similar neurons being present in rodents, the pH- and mechanosensing ability of hypothalamic CSF-c neurons is most likely conserved throughout vertebrate phylogeny.SIGNIFICANCE STATEMENT CSF-contacting neurons are present in all vertebrates and are located mainly in the hypothalamic area and the spinal cord. Here, we report that the somatostatin-/GABA-expressing CSF-c neurons in the lamprey hypothalamus sense bidirectional deviations in the extracellular pH and do so via different molecular mechanisms. They also serve as mechanoreceptors. The hypothalamic CSF-c neurons have extensive axonal ramifications and may decrease the level of motor activity via release of somatostatin. In conclusion, hypothalamic somatostatin-/GABA-expressing CSF-c neurons, as well as their spinal counterpart, represent a novel homeostatic mechanism designed to sense any deviation from physiological pH and thus constitute a feedback regulatory system intrinsic to the CNS, possibly serving a protective role from damage caused by changes in pH.

Effect of an Acid-sensing Ion Channels Inhibitor on Pain-related Behavior by Nucleus Pulposus Applied on the Nerve Root in Rats.

STUDY DESIGN: Controlled, interventional animal study. OBJECTIVE: To examine the effect of an inhibitor of acid-sensing ion channel 3 (ASIC3) on pain-related behavior induced by application of the nucleus pulposus (NP) onto the dorsal root ganglion (DRG) in rats. SUMMARY OF BACKGROUND DATA: ASIC3 is associated with acidosis pain in inflamed or ischemic tissues and is expressed in sensory neurons and NP cells. The ASIC3 inhibitor, APETx2, increases the mechanical threshold of pain in models of knee osteoarthritis or postoperative pain. However, the efficacy of APETx2 for pain relief in the NP application model remains unknown. METHODS: Autologous NP was applied to the left L5 nerve root of 183 adult female Sprague-Dawley rats. The DRGs were treated with NP plus one of the following four treatments: saline solution (SM), low (0.01 µg: LD), medium (0.1 µg: MD), or high dose (1.0 µg: HD) of APETx2. Behavioral testing was performed to investigate the mechanical withdrawal threshold using von Frey hairs. Expression of nerve growth factor, hypoxia-inducible factor-1α (HIF1α), activating transcription factor-3, and ionized calcium-binding adaptor molecule-1 was evaluated using immunohistochemistry. Statistical differences among multiple groups were assessed using the Steel test, the Tukey-Kramer test, and the Dunnett test. P < 0.05 were considered significant. RESULTS: The thresholds in the HD group were higher than those in the SM group at Days 14 and 21 (P < 0.05). In the MD group, the threshold was higher than in the SM group at Day 14 (P < 0.05). High doses of APETx2 reduced the expression of HIF1α after Day 14 compared with the SM group (P < 0.05). CONCLUSION: APETx2 significantly improved pain-related behavior in a dose-dependent manner. APETx2 may inhibit ASIC3 and partly inhibit Nav1.8 channels. This ASIC3 channel inhibitor may be a potential therapeutic agent in early-stage lumbar disc herniation. LEVEL OF EVIDENCE: N/A.

Gastrodin protects against chronic inflammatory pain by inhibiting spinal synaptic potentiation.

Tissue injury is known to produce inflammation and pain. Synaptic potentiation between peripheral nociceptors and spinal lamina I neurons has been proposed to serve as a trigger for chronic inflammatory pain. Gastrodin is a main bioactive constituent of the traditional Chinese herbal medicine Gastrodia elata Blume, which has been widely used as an analgesic since ancient times. However, its underlying cellular mechanisms have remained elusive. The present study demonstrated for the first time that gastrodin exhibits an analgesic effect at the spinal level on spontaneous pain, mechanical and thermal pain hypersensitivity induced by peripheral inflammation, which is not dependent on opioid receptors and without tolerance. This analgesia by gastrodin is at least in part mediated by depressing spinal synaptic potentiation via blockade of acid-sensing ion channels. Further studies with miniature EPSCs and paired-pulse ratio analysis revealed the presynaptic origin of the action of gastrodin, which involves a decrease in transmitter release probability. In contrast, neither basal nociception nor basal synaptic transmission was altered. This study revealed a dramatic analgesic action of gastrodin on inflammatory pain and uncovered a novel spinal mechanism that could underlie the analgesia by gastrodin, pointing the way to a new analgesic for treating chronic inflammatory pain.

Acidic pH promotes intervertebral disc degeneration: Acid-sensing ion channel -3 as a potential therapeutic target.

The aetiology of intervertebral disc (IVD) degeneration remains poorly understood. Painful IVD degeneration is associated with an acidic intradiscal pH but the response of NP cells to this aberrant microenvironmental factor remains to be fully characterised. The aim here was to address the hypothesis that acidic pH, similar to that found in degenerate IVDs, leads to the altered cell/functional phenotype observed during IVD degeneration, and to investigate the involvement of acid-sensing ion channel (ASIC) -3 in the response. Human NP cells were treated with a range of pH, from that of a non-degenerate (pH 7.4 and 7.1) through to mildly degenerate (pH 6.8) and severely degenerate IVD (pH 6.5 and 6.2). Increasing acidity of pH caused a decrease in cell proliferation and viability, a shift towards matrix catabolism and increased expression of proinflammatory cytokines and pain-related factors. Acidic pH resulted in an increase in ASIC-3 expression. Importantly, inhibition of ASIC-3 prevented the acidic pH induced proinflammatory and pain-related phenotype in NP cells. Acidic pH causes a catabolic and degenerate phenotype in NP cells which is inhibited by blocking ASIC-3 activity, suggesting that this may be a useful therapeutic target for treatment of IVD degeneration.

Inhibition of acid-sensing ion channels by levo-tetrahydropalmatine in rat dorsal root ganglion neurons.

Levo-tetrahydropalmatine (l-THP), a main bioactive Chinese herbal constituent from the genera Stephania and Corydalis, has been in use in clinical practice for years in China as a traditional analgesic agent. However, the mechanism underlying the analgesic action of l-THP is poorly understood. This study shows that l-THP can exert an inhibitory effect on the functional activity of native acid-sensing ion channels (ASICs), which are believed to mediate pain caused by extracellular acidification. l-THP dose dependently decreased the amplitude of proton-gated currents mediated by ASICs in rat dorsal root ganglion (DRG) neurons. l-THP shifted the proton concentration-response curve downward, with a decrease of 40.93% ± 8.45% in the maximum current response to protons, with no significant change in the pH0.5 value. Moreover, l-THP can alter the membrane excitability of rat DRG neurons to acid stimuli. It significantly decreased the number of action potentials and the amplitude of the depolarization induced by an extracellular pH drop. Finally, peripherally administered l-THP inhibited the nociceptive response to intraplantar injection of acetic acid in rats. These results indicate that l-THP can inhibit the functional activity of ASICs in dissociated primary sensory neurons and relieve acidosis-evoked pain in vivo, which for the first time provides a novel peripheral mechanism underlying the analgesic action of l-THP.

Acid sensing ion channel 1 in lateral hypothalamus contributes to breathing control.

Acid-sensing ion channels (ASICs) are present in neurons and may contribute to chemoreception. Among six subunits of ASICs, ASIC1 is mainly expressed in the central nervous system. Recently, multiple sites in the brain including the lateral hypothalamus (LH) have been found to be sensitive to extracellular acidification. Since LH contains orexin neurons and innervates the medulla respiratory center, we hypothesize that ASIC1 is expressed on the orexin neuron and contributes to acid-induced increase in respiratory drive. To test this hypothesis, we used double immunofluorescence to determine whether ASIC1 is expressed on orexin neurons in the LH, and assessed integrated phrenic nerve discharge (iPND) in intact rats in response to acidification of the LH. We found that ASIC1 was co-localized with orexinA in the LH. Microinjection of acidified artificial cerebrospinal fluid increased the amplitude of iPND by 70% (pH 7.4 v.s. pH 6.5:1.05±0.12 v.s. 1.70±0.10, n = 6, P<0.001) and increased the respiratory drive (peak amplitude of iPND/inspiratory time, PA/Ti) by 40% (1.10±0.23 v.s. 1.50±0.38, P<0.05). This stimulatory effect was abolished by blocking ASIC1 with a nonselective inhibitor (amiloride 10 mM), a selective inhibitor (PcTX1, 10 nM) or by damaging orexin neurons in the LH. Current results support our hypothesis that the orexin neuron in the LH can exert an excitation on respiration via ASIC1 during local acidosis. Since central acidification is involved in breathing dysfunction in a variety of pulmonary diseases, understanding its underlying mechanism may improve patient management.