Wei-Hsin Sun

A role for ASIC3 in the modulation of high-intensity pain stimuli

Acid-sensing ion channel 3 (ASIC3), a proton-gated ion channel of the degenerins/epithelial sodium channel (DEG/ENaC) receptor family is expressed predominantly in sensory neurons including nociceptive neurons responding to protons. To study the role of ASIC3 in pain signaling, we generated ASIC3 knockout mice. Mutant animals were healthy and responded normally to most sensory stimuli. However, in behavioral assays for pain responses, ASIC3 null mutant mice displayed a reduced latency to the onset of pain responses, or more pain-related behaviors, when stimuli of moderate to high intensity were used. This unexpected effect seemed independent of the modality of the stimulus and was observed in the acetic acid-induced writhing test (0.6 vs. 0.1–0.5%), in the hot-plate test (52.5 and 55 vs. 50°C), and in tests for mechanically induced pain (tail-pinch vs. von Frey filaments). We postulate that ASIC3 is involved in modulating moderate- to high-intensity pain sensation.

Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M–G1 transition in mammals

To investigate the events leading to initiation of DNA replication in mammalian chromosomes, the time when hamster origin recognition complexes (ORCs) became functional was related to the time when Orc1, Orc2 and Mcm3 proteins became stably bound to hamster chromatin. Functional ORCs, defined as those able to initiate DNA replication, were absent during mitosis and early G1 phase, and reappeared as cells progressed through G1 phase. Immunoblotting analysis revealed that hamster Orc1 and Orc2 proteins were present in nuclei at equivalent concentrations throughout the cell cycle, but only Orc2 was stably bound to chromatin. Orc1 and Mcm3 were easily eluted from chromatin during mitosis and early G1 phase, but became stably bound during mid‐G1 phase, concomitant with the appearance of a functional pre‐replication complex at a hamster replication origin. Since hamster Orc proteins are closely related to their human and mouse homologs, the unexpected behavior of hamster Orc1 provides a novel mechanism in mammals for delaying assembly of pre‐replication complexes until mitosis is complete and a nuclear structure has formed.

Nociceptors of dorsal root ganglion express proton-sensing G-protein-coupled receptors.

One major goal in pain research is to identify novel pain targets. Tissue injury, inflammation, and ischemia are usually accompanied by local tissue acidosis, the degree of associated pain or discomfort well correlated with the magnitude of acidification. Proton-sensing ion channels, transient receptor potential/vanilloid receptor subtype 1, and acid-sensing ion channel 3 are involved in acidosis-linked pain. However, whether recently identified proton-sensing G-protein-coupled receptors (GPCRs) also have some contributions is unclear. Proton-sensing GPCRs, including OGR1, GPR4, G2A, and TDAG8, are fully activated at pH 6.4-6.8 in vitro. To understand whether the proton-sensing GPCRs are expressed in nociceptors, we cloned the four mouse genes and examined their tissue distribution and localization in pain-relevant loci, the dorsal root ganglion (DRG). The OGR1 family members were widely expressed in neuronal and non-neuronal tissues. Their transcripts were expressed in the DRG, and most (75-82%) were present in small-diameter neurons responsible for nociception. Approximately 31-40% of total DRG neurons expressed at least two proton-sensing GPCRs. We have also demonstrated that gene expression of proton-sensing GPCRs is changed in ASIC3 knockout mice. Our finding suggests that proton-sensing GPCRs could have some roles in nociception or in compensation of loss of ASIC3 gene.

Serotonin Receptor 5-HT2B Mediates Serotonin-Induced Mechanical Hyperalgesia

Serotonin [5-hydroxytryptamine (5-HT)] released from mast cells or platelets in peripheral tissues is one of the important inflammatory mediators in pain and hyperalgesia. The involvement of 5-HT in pain is complex because it could inhibit or facilitate nociceptive transmission, reflecting the presence of multiple 5-HT subtype receptors on peripheral and central nociceptors. The present study aimed to investigate the involvement of 5-HT2B in 5-HT-induced pain and whether the subtype exists in dorsal root ganglion (DRG) neurons. Injecting the 5-HT or 5-HT2 agonist in hindpaws of mice induced significant hyperalgesia to mechanical stimuli, which was inhibited by the 5-HT2B/2C antagonist but not by 5-HT1A, 5-HT2A, or 5-HT3A antagonists. Therefore, 5-HT2B or 5-HT2C may be involved in 5-HT-induced mechanical hyperalgesia. The 5-HT2B/2C antagonist also blocked 5-HT-induced transient [Ca2+] signaling in DRG neurons. All subtypes of 5-HT receptors except 5-HT2C and 5-HT6 are present in DRGs. In situ hybridization also demonstrated 5-HT2B mainly expressed in small- to medium-diameter DRG neurons that respond to pain. Likely, 5-HT2B mediates 5-HT-induced mechanical hyperalgesia in mice.

Expression and function of proton-sensing G-protein-coupled receptors in inflammatory pain

BackgroundChronic inflammatory pain, when not effectively treated, is a costly health problem and has a harmful effect on all aspects of health-related quality of life. Despite the availability of pharmacologic treatments, chronic inflammatory pain remains inadequately treated. Understanding the nociceptive signaling pathways of such pain is therefore important in developing long-acting treatments with limited side effects. High local proton concentrations (tissue acidosis) causing direct excitation or modulation of nociceptive sensory neurons by proton-sensing receptors are responsible for pain in some inflammatory pain conditions. We previously found that all four proton-sensing G-protein-coupled receptors (GPCRs) are expressed in pain-relevant loci (dorsal root ganglia, DRG), which suggests their possible involvement in nociception, but their functions in pain remain unclear.ResultsIn this study, we first demonstrated differential change in expression of proton-sensing GPCRs in peripheral inflammation induced by the inflammatory agents capsaicin, carrageenan, and complete Freunds adjuvant (CFA). In particular, the expression of TDAG8, one proton-sensing GPCR, was increased 24 hours after CFA injection because of increased number of DRG neurons expressing TDAG8. The number of DRG neurons expressing both TDAG8 and transient receptor potential vanilloid 1 (TRPV1) was increased as well. Further studies revealed that TDAG8 activation sensitized the TRPV1 response to capsaicin, suggesting that TDAG8 could be involved in CFA-induced chronic inflammatory pain through regulation of TRPV1 function.ConclusionEach subtype of the OGR1 family was expressed differently, which may reflect differences between models in duration and magnitude of hyperalgesia. Given that TDAG8 and TRPV1 expression increased after CFA-induced inflammation and that TDAG8 activation can lead to TRPV1 sensitization, it suggests that high concentrations of protons after inflammation may not only directly activate proton-sensing ion channels (such as TRPV1) to cause pain but also act on proton-sensing GPCRs to regulate the development of hyperalgesia.

Cell cycle-dependent regulation of the association between origin recognition proteins and somatic cell chromatin

Previous studies have suggested that cell cycle‐dependent changes in the affinity of the origin recognition complex (ORC) for chromatin are involved in regulating initiation of DNA replication. To test this hypothesis, chromatin lacking functional ORCs was isolated from metaphase hamster cells and incubated in Xenopus egg extracts to initiate DNA replication. Intriguingly, Xenopus ORC rapidly bound to hamster somatic chromatin in a Cdc6‐dependent manner and was then released, concomitant with initiation of DNA replication. Once pre‐replication complexes (pre‐RCs) were assembled either in vitro or in vivo, further binding of XlORC was inhibited. Neither binding nor release of XlORC was affected by inhibitors of either cyclin‐dependent protein kinase activity or DNA synthesis. In contrast, inhibition of pre‐RC assembly, either by addition of Xenopus geminin or by depletion of XlMcm proteins, augmented ORC binding by inhibiting ORC release. These results demonstrate a programmed release of XlORC from somatic cell chromatin as it enters S phase, consistent with the proposed role for ORC in preventing re‐initiation of DNA replication during S phase.

Targeting ASIC3 for pain, anxiety, and insulin resistance

The acid-sensing ion channel 3 (ASIC3) is a pH sensor that responds to mild extracellular acidification and is predominantly expressed in nociceptors. There is much interest in targeting ASIC3 to relieve pain associated with tissue acidosis, and selective drugs targeting ASIC3 have been used to relieve acid-evoked pain in animal models and human studies. There is accumulating evidence that ASIC3 is widely expressed in many neuronal and non-neuronal cells, such as neurons in the brain and adipose cells, albeit to a lesser extent than in nociceptors. Asic3-knockout mice have reduced anxiety levels and enhanced insulin sensitivity, suggesting that antagonizing ASIC3 has additional benefits. This view is tempered by recent studies suggesting that Asic3-knockout mice may experience cardiovascular disturbances. Due to the development of ASIC3 antagonists as analgesics, we review here the additional benefits, safety, risks, and strategy associated with antagonizing ASIC3 function.

Roles of Proton-Sensing Receptors in the Transition from Acute to Chronic Pain.

Chronic pain, when not effectively treated, is a leading health and socioeconomic problem and has a harmful effect on all aspects of health-related quality of life. Therefore, understanding the molecular mechanism of how pain transitions from the acute to chronic phase is essential for developing effective novel analgesics. Accumulated evidence has shown that the transition from acute to chronic pain is determined by a cellular signaling switch called hyperalgesic priming, which occurs in primary nociceptive afferents. The hyperalgesic priming is triggered by inflammatory mediators and is involved in a signal switch from protein kinase A (PKA) to protein kinase Cε (PKCε) located in both isolectin B4 (IB4)–positive (nonpeptidergic) and IB4-negative (peptidergic) nociceptors. Acidosis may be the decisive factor regulating the PKA-to-PKCε signal switch in a proton-sensing G-protein-coupled receptor-dependent manner. Protons can also induce the hyperalgesic priming in IB4-negative muscle nociceptors in a PKCε-independent manner. Acid-sensing ion channel 3 (ASIC3) and transient receptor potential/vanilloid receptor subtype 1 (TRPV1) are 2 major acid sensors involved in the proton-induced hyperalgesic priming. The proton-induced hyperalgesic priming in muscle afferents can be prevented by a substance P–mediated signaling pathway. In this review, we summarize the factors that modulate hyperalgesic priming in both IB4-positive and IB4-negative nociceptors and discuss the role of acid signaling in inflammatory and noninflammatory pain as well as orofacial muscle pain.

Acidosis Mediates the Switching of Gs-PKA and Gi-PKCε Dependence in Prolonged Hyperalgesia Induced by Inflammation

Chronic inflammatory pain, when not effectively treated, is a costly health problem and has a harmful effect on all aspects of health-related quality of life. Previous studies suggested that in male Sprague Dawley rats, prostaglandin E2 (PGE2)-induced short-term hyperalgesia depends on protein kinase A (PKA) activity, whereas long-lasting hyperalgesia induced by PGE2 with carrageenan pre-injection, requires protein kinase Cε (PKCε). However, the mechanism underlying the kinase switch with short- to long-term hyperalgesia remains unclear. In this study, we used the inflammatory agents carrageenan or complete Freund’s adjuvant (CFA) to induce long-term hyperalgesia, and examined PKA and PKCε dependence and switching time. Hyperalgesia induced by both agents depended on PKA/PKCε and Gs/Gi-proteins, and the switching time from PKA to PKCε and from Gs to Gi was about 3 to 4 h after inflammation induction. Among the single inflammatory mediators tested, PGE2 and 5-HT induced transient hyperalgesia, which depended on PKA and PKCε, respectively. Only acidic solution-induced hyperalgesia required Gs-PKA and Gi-PKCε, and the switch time for kinase dependency matched inflammatory hyperalgesia, in approximately 2 to 4 h. Thus, acidosis in inflamed tissues may be a decisive factor to regulate switching of PKA and PKCε dependence via proton-sensing G-protein–coupled receptors.

RINGdb: An integrated database for G protein-coupled receptors and regulators of G protein signaling

BackgroundMany marketed therapeutic agents have been developed to modulate the function of G protein-coupled receptors (GPCRs). The regulators of G-protein signaling (RGS proteins) are also being examined as potential drug targets. To facilitate clinical and pharmacological research, we have developed a novel integrated biological database called RINGdb to provide comprehensive and organized RGS protein and GPCR information.ResultsRINGdb contains information on mutations, tissue distributions, protein-protein interactions, diseases/disorders and other features, which has been automatically collected from the Internet and manually extracted from the literature. In addition, RINGdb offers various user-friendly query functions to answer different questions about RGS proteins and GPCRs such as their possible contribution to disease processes, the putative direct or indirect relationship between RGS proteins and GPCRs. RINGdb also integrates organized database cross-references to allow users direct access to detailed information. The database is now available at http://ringdb.csie.ncu.edu.tw/ringdb/.ConclusionRINGdb is the only integrated database on the Internet to provide comprehensive RGS protein and GPCR information. This knowledgebase will be useful for clinical research, drug discovery and GPCR signaling pathway research.