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ASIC3-dependent metabolomics profiling of serum and urine in a mouse model of fibromyalgia


Senior Member

ASIC3-dependent metabolomics profiling of serum and urine in a mouse model of fibromyalgia.
Hsu WH1, Lee CH2, Chao YM1, Kuo CH3, Ku WC4, Chen CC5,6, Lin YL7.
august 2019

Fibromyalgia (FM) is characterized by chronic widespread pain.
The pathogenesis of FM remains unclear. No specific biomarkers are available. Animal models of FM may provide an opportunity to explore potential biomarkers in a relative homogenous disease condition. Here, we probed the metabolomics profiles of serum and urine in a mouse model of FM induced by intermittent cold stress (ICS).

We focused on the role of acid-sensing ion channel 3 (ASIC3) in the metabolomics profiling because ICS treatment induced chronic widespread muscle pain lasting for 1 month in wild-type (Asic3+/+) but not Asic3-knockout (Asic3-/-) mice.

Serum and urine samples were collected from both genotypes at different ICS stages, including before ICS (basal level) and post-ICS at days 10 (middle phase, P10) and 40 (recovery phase, P40).
Control naïve mice and ICS-induced FM mice differed in 1H-NMR- and LC-MS-based metabolomics profiling.
On pathway analysis, the leading regulated pathways in Asic3+/+ mice were taurine and hypotaurine, cysteine and methionine, glycerophospholipid, and ascorbate and aldarate metabolisms, and the major pathways in Asic3-/- mice involved amino acid-related metabolism.

Finally, we developed an algorithm for the impactful metabolites in the FM model including cis-aconitate, kynurenate, taurine, pyroglutamic acid, pyrrolidonecarboxylic acid, and 4-methoxyphenylacetic acid in urine as well as carnitine, deoxycholic acid, lysoPC(16:0), lysoPC(20:3), oleoyl-L-carnitine, and trimethylamine N-oxide in serum.

Asic3-/- mice were impaired in only muscle allodynia development but not other pain symptoms in the ICS model, so the ASIC3-dependent metabolomics changes could be useful for developing diagnostic biomarkers specific to chronic widespread muscle pain, the core symptom of FM. Further pharmacological validations are needed to validate these metabolomics changes as potential biomarkers for FM diagnosis and/or treatment responses.


Senior Member
Involvement of Acid-Sensing Ion Channel 1b in the Development of Acid-Induced Chronic Muscle Pain.
Chang CT1, Fong SW1,2, Lee CH1, Chuang YC1, Lin SH1,3, Chen CC1,2.
Nov 2019

Acid-sensing ion channels (ASICs) are important acid sensors involved in neural modulation in the central nervous system and pain-associated tissue acidosis in the peripheral system.

Among ASIC subtypes, ASIC1b is the most selectively expressed in peripheral sensory neurons.

However, the role of ASIC1b is still elusive in terms of its functions and expression profile.

In this study, we probed the role of ASIC1b in acid-induced muscle pain in Asic1b-knockout (Asic1b -/-) and Asic1b-Cre transgenic (Asic1b Cre ) mice.

We tested the effect of ASIC1b knockout in a mouse model of fibromyalgia induced by dual intramuscular acid injections. In this model, a unilateral acid injection to the gastrocnemius muscle induced transient bilateral hyperalgesia in wild-type (Asic1b + / +) but not Asic1b -/- mice; a second acid injection, spaced 1 or 5 days apart, to the same muscle induced chronic hyperalgesia lasting for 4 weeks in Asic1b + / + mice, but the duration of hyperalgesia was significantly shortened in Asic1b -/- mice. Mambalgin-1, an ASIC1b-containing channel inhibitor that was mixed with acid saline at the first injection, dose-dependently blocked the acid-induced transient and chronic hyperalgesia in Asic1b + / + mice. In contrast, psalmotoxin 1 (PcTx1), an ASIC1a-selective antagonist, had no effect on acid-induced transient or chronic hyperalgesia. We used whole-cell patch clamp recording to study the properties of acid-induced currents in ASIC1b-expressing dorsal root ganglia (DRG) neurons from Asic1b Cre -TdTomato reporter mice. Medium- to large-sized ASIC1b-expressing DRG neurons mainly exhibited an amiloride-sensitive ASIC-like biphasic current (I ASIC) in response to acid stimulation, whereas small- to medium-sized ASIC1b-expressing DRG neurons predominantly exhibited an amiloride-insensitive sustained current. Specifically, mambalgin-1 selectively inhibited the I ASIC in most ASIC1b-expressing DRG neurons. However, PcTx1 or APETx2 (an ASIC3-selective antagonist) had only a mild inhibitory effect on I ASIC in about half of the ASIC1b-expressing DRG neurons.

In situ hybridization revealed that ASIC1b-positive DRG neurons co-expressed highly with ASIC1a and ASIC2a mRNA and partially with ASIC3 and ASIC2b. Thus, ASIC1b might form a wide variety of heteromeric channels. ASIC1b-containing heteromeric channels might be promising targets for the therapeutic treatment of acid-induced chronic muscle pain.


Senior Member

2020 paper​


One class of commonly taken over the counter, and also widely prescribed, analgesics are non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit cyclooxygenase (COX) enzymes to reduce prostaglandin production and thus relieve pain by preventing the sensitizing effects of prostaglandins upon sensory neurones.

However, it has also been shown that COX-independent mechanisms exist that contribute to the analgesic effect of NSAIDs [Citation170].

The Lazdunski lab observed that inflammation of rat hindpaws significantly upregulated the mRNA levels of some ASIC subunits (ASIC1a, ASIC2b, and ASIC3) in rat DRG neurones and that this could largely be suppressed by corticosteroids, as well as by some NSAIDS [Citation129].

They further investigated if there were any direct effects of NSAIDs on ASIC subunits when expressed in CHO cells and observed that certain NSAIDs, at high micromolar concentrations, could inhibit ASIC currents in a somewhat isoform-selective manner.

Interestingly, salicylic acid (IC50 of 260 ± 21 μM, Figure 5a), aspirin (IC50 not specified, Figure 5b), and diclofenac (IC50 of 92 ± 19 μM, Figure 5c) inhibited the sustained current component of ASIC3 but not the transient component, whereas the ASIC1a current was inhibited by ibuprofen (IC50 not specified) and flurbiprofen (IC50 of 349 ± 40 μM) only.

A range of other NSAIDs, namely piroxicam, etodolac, nimesulide, naproxen indomethacin and acetaminophen had no effect on either ASIC1a or ASIC3.

However, subsequent work from Pless lab established that ibuprofen and some of its analogues could inhibit the transient currents of ASIC3 albeit with very weak potency, i.e. ibuprofen had an IC50 of 51.3 mM (Figure 5d) at pH 6.4 [Citation171]. Using site-directed mutagenesis, they identified a region comprising the upper of transmembrane helices and extracellular domain β9-α4 loop of rASIC1a to be the main site of ibuprofen binding.

Most of the key residues identified for ibuprofen recognition are largely conserved across rASIC1a, rASIC1b, and rASIC2a, but rASIC3 has different residues in two key positions namely Thr in place of Lys76 (K76T) and has Ala in place of a critical Lys (K422A).

It is intriguing to note that despite these amino acid differences, ibuprofen’s potency for inhibiting the ASIC3 peak current was comparable to that for inhibiting ASIC1a (IC50 of 29.5 mM), which suggests that other binding regions are likely involved.

The structural determinants for the inhibition of ASIC3’s sustained current by salicylic acid, aspirin, and diclofenac remain unknown. In outside-out patches, extracellular application of aspirin or salicylic acid inhibits ASIC3, thus making a direct interaction with an extracellular region of ASIC3 highly likely. In addition, the reversibility of aspirin’s inhibition, as well as inhibition by salicylic acid, suggest that such ASIC3 inhibition by these specific NSAIDS does not involve acetylation [Citation129]. However, employing salicylic acid or high dose salicylates to block ASIC3 and reduce pain is not a clinical option as high doses and/or chronic exposure causes tinnitus and sepsis-like disease [Citation172].