Selection of an ASIC1a-blocking combinatorial antibody that protects cells from ischemic death.

Acid-sensing ion channels (ASICs) have emerged as important, albeit challenging therapeutic targets for pain, stroke, etc. One approach to developing therapeutic agents could involve the generation of functional antibodies against these channels. To select such antibodies, we used channels assembled in nanodiscs, such that the target ASIC1a has a configuration as close as possible to its natural state in the plasma membrane. This methodology allowed selection of functional antibodies that inhibit acid-induced opening of the channel in a dose-dependent way. In addition to regulation of pH, these antibodies block the transport of cations, including calcium, thereby preventing acid-induced cell death in vitro and in vivo. As proof of concept for the use of these antibodies to modulate ion channels in vivo, we showed that they potently protect brain cells from death after an ischemic stroke. Thus, the methodology described here should be general, thereby allowing selection of antibodies to other important ASICs, such as those involved in pain, neurodegeneration, and other conditions.

Ion Channel Pharmacology for Pain Modulation.

A large series of different ion channels have been identified and investigated as potential targets for new medicines for the treatment of a variety of human diseases, including pain. Among these channels, the voltage gated calcium channels (VGCC) are inhibited by drugs for the treatment of migraine, neuropathic pain or intractable pain. Transient receptor potential (TRP) channels are emerging as important pain transducers as they sense low pH media or oxidative stress and other mediators and are abundantly found at sites of inflammation or tissue injury. Low pH may also activate acid sensing ion channels (ASIC) and mechanical forces stimulate the PIEZO channels. While potent agonists of TRP channels due to their desensitizing action on pain transmission are used as topical applications, the potential of TRP antagonists as pain therapeutics remains an exciting field of investigation. The study of ASIC or PIEZO channels in pain signaling is in an early stage, whereas antagonism of the purinergic P2X3 channels has been reported to provide beneficial effects in chronic intractable cough. The present chapter covers these intriguing channels in great detail, highlighting their diverse mechanisms and broad potential for therapeutic utility.

Black mamba venom peptides target acid-sensing ion channels to abolish pain.

Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway, including in humans. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia.

Emerging Targets for the Management of Osteoarthritis Pain.

Worldwide, osteoarthritis (OA) is one of the leading causes of chronic pain, for which adequate relief is not available. Ongoing peripheral input from the affected joint is a major factor in OA-associated pain. Therefore, this review focuses predominantly on peripheral targets emerging in the preclinical and clinical arena. Nerve growth factor is the most advanced of these targets, and its blockade has shown tremendous promise in clinical trials in knee OA. A number of different types of ion channels, including voltage-gated sodium channels and calcium channels, transient receptor potential channels, and acid-sensing ion channels, are important for neuronal excitability and play a role in pain genesis. Few channel blockers have been tested in preclinical models of OA, with varying results. Finally, we discuss some examples of G-protein coupled receptors, which may offer attractive therapeutic strategies for OA pain, including receptors for bradykinin, calcitonin gene-related peptide, and chemokines. Since many of the pathways described above can be selectively and potently targeted, they offer an exciting opportunity for pain management in OA, either systemically or locally.

Randomized clinical trial: efficacy and safety of PPC-5650 on experimental esophageal pain and hyperalgesia in healthy volunteers.

OBJECTIVE: Gastroesophageal reflux disease (GERD) is a common condition associated with symptoms as heart burn, regurgitation, chest pain, and gastrointestinal discomfort. PPC-5650 is a new pharmacological agent that can modulate acid-sensing ion channel activity, potentially leading to reduction in the pain signal. In healthy volunteers the esophagus was sensitized with acid to mimic GERD with the aims: 1) to assess the efficacy of a single bolus of PPC-5650 locally applied to the esophagus using multimodal pain stimulations, and 2) to assess the safety profile of PPC-5650. MATERIALS AND METHODS: The study was a randomized, double-blinded, placebo-controlled, crossover trial in healthy males. Esophageal electrical, thermal, mechanical, and chemical stimulations were performed, pain perception was rated, and referred pain areas were drawn. Sensitization was induced by intraluminal esophageal acid perfusions. Adverse events were registered. RESULTS: Twenty-five healthy males completed the study (mean age 23.4 ± 2.0 years). About 90 min after drug administration, PPC-5650 increased the volume tolerated at moderate pain during mechanical stimulation compared to placebo (difference 13.5, 95% CI: 0.58-26.47, p = 0.04), but there was no effects on thermal-, electrical-, and chemical-induced pain (all p > 0.05). PPC-5650 did not affect referred pain areas to any stimulation (all p > 0.05). Ten participants reported adverse events during the placebo treatment period, and nine participants reported adverse events during the PPC-5650 treatment period (p = 0.8). CONCLUSION: Sensitization to mechanical stimulation of the esophagus was reduced by PPC-5650 compared to placebo. The overall safety and tolerability of PPC-5650 was acceptable. Thus, PPC-5650 may play a role in the future treatment of patients with GERD.

[Hypertension in the course of primary aldosteronism during pregnancy]. / Nadcisnienie tetnicze w przebiegu pierwotnego hiperaldosteronizmu u kobiet w ciazy

Hypertension is one of the most common cardiovascular diseases during pregnancy. Primary hyperaldosteronism (PHA) is the most frequent endocrinological, secondary cause of hypertension, rarely diagnosed in pregnant women. In the available literature about 50 cases of PHA in pregnant women have been described. PHA is often a cause of resistant hypertension. PHA can cause life-threatening complications both for the pregnant woman and the fetus. Diagnosis of PHA in pregnancy is difficult due to the antagonistic effect of progesterone on aldosterone, physiological increase of aldosterone release during gestation and frequent normokalaemic clinical course. Typical pharmacological treatment of PHA is limited due to the anti­androgenic effect of spironolactone, lack of data concerning the safety of eplerenone and limited access to amiloride in Poland. Surgical treatment is a therapeutic option only in early pregnancy. This paper presents the current state of knowledge on diagnostic methods and treatment of PHA in pregnant women and a systematic review of cases described in the literature.

Inhibition of ASICs reduces rat hepatic stellate cells activity and liver fibrosis: an in vitro and in vivo study.

Hepatic fibrosis is a chronic inflammation-associated disease, which is involved in the infiltration of inflammatory cells and releasing of proinflammatory cytokines. In the pathological process, protons are released by damaged cells and acidosis is considered to play a critical role in cell injury. Although the underlying mechanism (s) remain ill-defined, ASICs (acid-sensing ion channels) are assumed to be involved in this process. The diuretic, amiloride, is neuroprotective in models of cerebral ischemia, a property attributable to the inhibition of central ASICs by the drug. However, the effect of inhibition of ASICs by amiloride in the liver fibrotic process remains unclear. We found that amiloride (25, 50, or 100 µM) could restrain acid-induced HSCs at pH6 in vitro. In vivo experiments showed that amiloride could significantly alleviate liver injury, decreasing levels of profibrogenic cytokines, collagen deposition, and reducing pathological tissue damage. In summary, amiloride inhibits hepatic fibrosis in vivo and in vitro, which is probably associated with the downregulation of ASICs.

Targeting ASIC1 in primary progressive multiple sclerosis: evidence of neuroprotection with amiloride.

Neurodegeneration is the main cause for permanent disability in multiple sclerosis. The effect of current immunomodulatory treatments on neurodegeneration is insufficient. Therefore, direct neuroprotection and myeloprotection remain an important therapeutic goal. Targeting acid-sensing ion channel 1 (encoded by the ASIC1 gene), which contributes to the excessive intracellular accumulation of injurious Na(+) and Ca(2+) and is over-expressed in acute multiple sclerosis lesions, appears to be a viable strategy to limit cellular injury that is the substrate of neurodegeneration. While blockade of ASIC1 through amiloride, a potassium sparing diuretic that is currently licensed for hypertension and congestive cardiac failure, showed neuroprotective and myeloprotective effects in experimental models of multiple sclerosis, this strategy remains untested in patients with multiple sclerosis. In this translational study, we tested the neuroprotective effects of amiloride in patients with primary progressive multiple sclerosis. First, we assessed ASIC1 expression in chronic brain lesions from post-mortem of patients with progressive multiple sclerosis to identify the target process for neuroprotection. Second, we tested the neuroprotective effect of amiloride in a cohort of 14 patients with primary progressive multiple sclerosis using magnetic resonance imaging markers of neurodegeneration as outcome measures of neuroprotection. Patients with primary progressive multiple sclerosis underwent serial magnetic resonance imaging scans before (pretreatment phase) and during (treatment phase) amiloride treatment for a period of 3 years. Whole-brain volume and tissue integrity were measured with high-resolution T(1)-weighted and diffusion tensor imaging. In chronic brain lesions of patients with progressive multiple sclerosis, we demonstrate an increased expression of ASIC1 in axons and an association with injury markers within chronic inactive lesions. In patients with primary progressive multiple sclerosis, we observed a significant reduction in normalized annual rate of whole-brain volume during the treatment phase, compared with the pretreatment phase (P = 0.018, corrected). Consistent with this reduction, we showed that changes in diffusion indices of tissue damage within major clinically relevant white matter (corpus callosum and corticospinal tract) and deep grey matter (thalamus) structures were significantly reduced during the treatment phase (P = 0.02, corrected). Our results extend evidence of the contribution of ASIC1 to neurodegeneration in multiple sclerosis and suggest that amiloride may exert neuroprotective effects in patients with progressive multiple sclerosis. This pilot study is the first translational study on neuroprotection targeting ASIC1 and supports future randomized controlled trials measuring neuroprotection with amiloride in patients with multiple sclerosis.

Effect of amiloride on experimental acid-induced heartburn in non-erosive reflux disease.

BACKGROUND: Acid-sensing ion channels (ASICs) are esophageal nociceptors that are candidates to mediate heartburn in non-erosive reflux disease (NERD). Amiloride, a diuretic, is known to inhibit ASICs. For this reason, we sought a role for ASICs in mediating heartburn by determining whether amiloride could block heartburn in NERD induced by esophageal acid perfusion. METHODS: In a randomized double-blind crossover study, we perfused the esophagus with amiloride or (saline) placebo prior to eliciting acid-induced heartburn in patients with a history of proton pump inhibitor-responsive NERD. Those with NERD and positive modified Bernstein test were randomized to perfusion with amiloride, 1 mmol/l, or placebo for 5 min, followed by repeat acid-perfusion. Heartburn severity and time to onset was measured and the process repeated following crossover to the alternative agent. RESULTS: 14 subjects completed the study. Amiloride did not reduce the frequency (100 vs. 100 %) or severity of acid-induced heartburn (Mean 2.50 ± SEM 0.33 vs. 2.64 ± 0.45), respectively. There was a trend towards longer time to onset of heartburn for amiloride versus placebo (Mean 2.93 ± SEM 0.3 vs. 2.36 ± 0.29 min, respectively), though these differences did not reach statistical significance (p > 0.05). CONCLUSIONS: Amiloride had no significant effect on acid-induced heartburn frequency or severity in NERD, although there was a trend towards prolonged time to onset of symptoms.

u-PA inhibitor amiloride suppresses peritoneal metastasis in gastric cancer.

BACKGROUND: Peritoneal metastasis in gastric cancer represents a ubiquitous human health problem but effective therapies with limited side effects are still lacking. Although previous research suggested that u-PA was involved in some tumor metastasis such as lung-specific metastasis, the role of u-PA for peritoneal metastasis in gastric cancer is still unclear. The aim of this study was to explore whether selective pharmacological blockade of u-PA is able to affect the peritoneal metastasis of gastric cancer both in vivo and in vitro. METHODS: In the present study, we evaluated the effects and explored the anti-tumor mechanisms of amiloride, a selective u-PA inhibitor, on a panel of gastric cancer cell lines and in a murine model of human gastric cancer MKN45. RESULTS: The study showed that amiloride significantly inhibited the tumor growth and prolonged the survival of the tumor-bearing mice. In vitro, compared with controls, amiloride could not only significantly down-regulate the mRNA expression and protein level of u-PA from MKN45 cells with dose dependence but also inhibit the adhesion of HMrSV5 cells, migration and invasion of MKN45 cells. CONCLUSIONS: The findings in our current report provide evidence that selective u-PA inhibitor amiloride has potent effects against peritoneal metastasis in gastric cancer, suggesting its possible therapeutic value for the treatment of gastric cancer.

Synthesis and Characterization of Novel Mono- and Bis-Guanyl Hydrazones as Potent and Selective ASIC1 Inhibitors Able to Reduce Brain Ischemic Insult.

Acid-sensitive ion channels (ASICs) are sodium channels partially permeable to Ca2+ ions, listed among putative targets in central nervous system (CNS) diseases in which a pH modification occurs. We targeted novel compounds able to modulate ASIC1 and to reduce the progression of ischemic brain injury. We rationally designed and synthesized several diminazene-inspired diaryl mono- and bis-guanyl hydrazones. A correlation between their predicted docking affinities for the acidic pocket (AcP site) in chicken ASIC1 and their inhibition of homo- and heteromeric hASIC1 channels in HEK-293 cells was found. Their activity on murine ASIC1a currents and their selectivity vs mASIC2a were assessed in engineered CHO-K1 cells, highlighting a limited isoform selectivity. Neuroprotective effects were confirmed in vitro, on primary rat cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation, and in vivo, in ischemic mice. Early lead 3b, showing a good selectivity for hASIC1 in human neurons, was neuroprotective against focal ischemia induced in mice.

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.

Acid-Sensing Ion Channels Structural Aspects, Pathophysiological Importance and Experimental Mutational Data Available Across Various Species to Target Human ASIC1.

The H+-gated (proton) currents are widely present in brain sensory neuronal system and various studies identified the structural units and deciphered the physiological and pathological function of ion channels. The normal neuron requires an optimal pH to carry out its functions. In acidosis, the ASICs (Acid-sensing Ion Channels) are activated in both the CNS (central nervous system) and PNS (peripheral nervous system). ASICs are related to degenerin channels (DEGs), epithelial sodium cation channels (ENaCs), and FMRF-amide (Phe-Met-Arg-Phe-NH2)-gated channels (FaNaC). Its activation leads physiologically to pain perception, synaptic plasticity, learning and memory, fear, ischemic neuronal injury, seizure termination, neuronal degeneration, and mechanosensation. It detects the level of acid fluctuation in the extracellular environment and responds to acidic pH by increasing the rate of membrane depolarization. It conducts cations like Na+ (Sodium) and Ca2+ (Calcium) ions across the membrane upon protonation. The ASICs subtypes are characterized by differing biophysical properties and pH sensitivities. The subtype ASIC1 is involved in various CNS diseases and therefore focusing on its specific functional properties will guide in drug design methods. The review highlights the cASIC1 (Chicken ASIC1) crystal structures, involvement in physiological environment and limitations of currently available inhibitors. In addition, it details the mutational data available to design an inhibitor against hASIC1 (Human ASIC1).

Blockade of Acid-Sensing Ion Channels Attenuates Recurrent Hypoglycemia-Induced Potentiation of Ischemic Brain Damage in Treated Diabetic Rats.

Diabetes is a chronic metabolic disease and cerebral ischemia is a serious complication of diabetes. Anti-diabetic therapy mitigates this complication but increases the risk of exposure to recurrent hypoglycemia (RH). We showed previously that RH exposure increases ischemic brain damage in insulin-treated diabetic (ITD) rats. The present study evaluated the hypothesis that increased intra-ischemic acidosis in RH-exposed ITD rats leads to pronounced post-ischemic hypoperfusion via activation of acid-sensing (proton-gated) ion channels (ASICs). Streptozotocin-diabetic rats treated with insulin were considered ITD rats. ITD rats were exposed to RH for 5 days and were randomized into Psalmotoxin1 (PcTx1, ASIC1a inhibitor), APETx2 (ASIC3 inhibitor), or vehicle groups. Transient global cerebral ischemia was induced overnight after RH. Cerebral blood flow was measured using laser Doppler flowmetry. Ischemic brain injury in hippocampus was evaluated using histopathology. Post-ischemic hypoperfusion in RH-exposed rats was of greater extent than that in control rats. Inhibition of ASICs prevented RH-induced increase in the extent of post-ischemic hypoperfusion and ischemic brain injury. Since ASIC activation-induced store-operated calcium entry (SOCE) plays a role in vascular tone, next we tested if acidosis activates SOCE via activating ASICs in vascular smooth muscle cells (VSMCs). We observed that SOCE in VSMCs at lower pH is ASIC3 dependent. The results show the role of ASIC in post-ischemic hypoperfusion and increased ischemic damage in RH-exposed ITD rats. Understanding the pathways mediating exacerbated ischemic brain injury in RH-exposed ITD rats may help lower diabetic aggravation of ischemic brain damage.

Targeted Acid-Sensing Ion Channel Therapies for Migraine.

Acid-sensing ion channels (ASICs) are a family of ion channels, consisting of four members; ASIC1 to 4. These channels are sensitive to changes in pH and are expressed throughout the central and peripheral nervous systems-including brain, spinal cord, and sensory ganglia. They have been implicated in a number of neurological conditions such as stroke and cerebral ischemia, traumatic brain injury, and epilepsy, and more recently in migraine. Their expression within areas of interest in the brain in migraine, such as the hypothalamus and PAG, their demonstrated involvement in preclinical models of meningeal afferent signaling, and their role in cortical spreading depression (the electrophysiological correlate of migraine aura), has enhanced research interest into these channels as potential therapeutic targets in migraine. Migraine is a disorder with a paucity of both acute and preventive therapies available, in which at best 50% of patients respond to available medications, and these medications often have intolerable side effects. There is therefore a great need for therapeutic development for this disabling condition. This review will summarize the understanding of the structure and CNS expression of ASICs, the mechanisms for their potential role in nociception, recent work in migraine, and areas for future research and drug development.

Acid Sensing Ion Channel 1a (ASIC1a) Mediates Activity-induced Pain by Modulation of Heteromeric ASIC Channel Kinetics.

Chronic muscle pain is acutely worsened by exercise. Acid sensing ion channels (ASIC) are heteromeric channels expressed in muscle sensory neurons that detect decreases in pH. We have previously shown ASIC3 is important in activity-induced hyperalgesia. However, ASICs form heteromers with ASIC1a being a key component in sensory neurons. Therefore, we studied the role of ASIC1a in mice using behavioral pharmacology and genetic deletion in a model of activity-induced hyperalgesia. We found ASIC1a-/- mice developed mechanical hyperalgesia similar to wild-type mice, but antagonism of ASIC1a, with psalmotoxin, prevented development of mechanical hyperalgesia in wild-type mice, but not in ASIC1a-/- mice. To explain this discrepancy, we then performed electrophysiology studies of ASICs and examined the effects of psalmotoxin on ASIC heteromers. We expressed ASIC1a, 2 and 3 heteromers or ASIC1 and 3 heteromers in CHO cells, and examined the effects of psalmotoxin on pH sensitivity. Psalmotoxin significantly altered the properties of ASIC hetomeric channels. Specifically, in ASIC1a/2/3 heteromers, psalmotoxin slowed the kinetics of desensitization, slowed the recovery from desensitization, and inhibited pH-dependent steady-state desensitization, but had no effect on pH-evoked current amplitudes. We found a different pattern in ASIC1a/3 heteromers. There was a significant leftward shift in the pH dose response of steady-state desensitization and decrease in pH-evoked current amplitudes. These results suggest that blockade of ASIC1a modulates the kinetics of heteromeric ASICs to prevent development of activity-induced hyperalgesia. These data suggest ASIC1a is a key subunit in heteromeric ASICs and may be a pharmacological target for treatment of musculoskeletal pain.

TRPA1 and TRPV1 Antagonists Do Not Inhibit Human Acidosis-Induced Pain.

Acidosis occurs in a variety of pathophysiological and painful conditions where it is thought to excite or contribute to excitation of nociceptive neurons. Despite potential clinical relevance the principal receptor for sensing acidosis is unclear, but several receptors have been proposed. We investigated the contribution of the acid-sensing ion channels, transient receptor potential vanilloid type 1 (TRPV1) and transient receptor potential ankyrin type 1 (TRPA1) to peripheral pain signaling. We first established a human pain model using intraepidermal injection of the TRPA1 agonist carvacrol. This resulted in concentration-dependent pain sensations, which were reduced by experimental TRPA1 antagonist A-967079. Capsaicin-induced pain was reduced by the TRPV1 inhibitor BCTC. Amiloride was used to block acid-sensing ion channels. Testing these antagonists in a double-blind and randomized experiment, we probed the contribution of the respective channels to experimental acidosis-induced pain in 15 healthy human subjects. A continuous intraepidermal injection of pH 4.3 was used to counter the buffering capacity of tissue and generate a prolonged painful stimulation. In this model, addition of A-967079, BCTC or amiloride did not reduce the reported pain. In conclusion, target-validated antagonists, applied locally in human skin, have excluded the main hypothesized targets and the mechanism of the human acidosis-induced pain remains unclear. PERSPECTIVE: An acidic milieu is a trigger of pain in many clinical conditions. The aim of this study was to identify the contribution of the currently hypothesized sensors of acid-induced pain in humans. Surprisingly, inhibition of these receptors did not alter acidosis-induced pain.

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.

Pharmacological evaluation of NSAID-induced gastropathy as a "Translatable" model of referred visceral hypersensitivity.

AIM: To evaluate whether non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastropathy is a clinically predictive model of referred visceral hypersensitivity. METHODS: Gastric ulcer pain was induced by the oral administration of indomethacin to male, CD1 mice (n = 10/group) and then assessed by measuring referred abdominal hypersensitivity to tactile application. A diverse range of pharmacological mechanisms contributing to the pain were subsequently investigated. These mechanisms included: transient receptor potential (TRP), sodium and acid-sensing ion channels (ASICs) as well as opioid receptors and guanylate cyclase C (GC-C). RESULTS: Results showed that two opioids and a GC-C agonist, morphine, asimadoline and linaclotide, respectively, the TRP antagonists, AMG9810 and HC-030031 and the sodium channel blocker, carbamazepine, elicited a dose- and/or time-dependent attenuation of referred visceral hypersensitivity, while the ASIC blocker, amiloride, was ineffective at all doses tested. CONCLUSION: Together, these findings implicate opioid receptors, GC-C, and sodium and TRP channel activation as possible mechanisms associated with visceral hypersensitivity. More importantly, these findings also validate NSAID-induced gastropathy as a sensitive and clinically predictive mouse model suitable for assessing novel molecules with potential pain-attenuating properties.

Analgesic effects of mambalgin peptide inhibitors of acid-sensing ion channels in inflammatory and neuropathic pain.

Mambalgins are 57-amino acid peptides isolated from snake venom that evoke naloxone-resistant analgesia after local (intraplantar) and central (intrathecal) injections through inhibition of particular subtypes of acid-sensing ion channels (ASICs). We now show that mambalgins also have an opioid-independent effect on both thermal and mechanical inflammatory pain after systemic intravenous (i.v.) administration and are effective against neuropathic pain. By combining the use of knockdown and knockout animals, we show the critical involvement of peripheral ASIC1b-containing channels, along with a contribution of ASIC1a-containing channels, in the i.v. effects of these peptides against inflammatory pain. The potent analgesic effect on neuropathic pain involves 2 different mechanisms depending on the route of administration, a naloxone-insensitive and ASIC1a-independent effect associated with i.v. injection and an ASIC1a-dependent and partially naloxone-sensitive effect associated with intrathecal injection. These data further support the role of peripheral and central ASIC1-containing channels in pain, demonstrate their participation in neuropathic pain, and highlight differences in the repertoire of channels involved in different pain conditions. They also strengthen the therapeutic potential of mambalgin peptides that are active in a broader range of experimental pain models and through i.v. systemic delivery.