Receptors In Primary Sensory Neurons Research Articles

TRPM8 inhibits substance P release from primary sensory neurons via PKA/GSK-3beta to protect colonic epithelium in colitis

Transient receptor potential melastatin 8 (TRPM8) is a cold sensory receptor in primary sensory neurons that regulates various neuronal functions. Substance P (SP) is a pro-inflammatory neuropeptide secreted by the neurons, and it aggravates colitis. However, the regulatory role of TRPM8 in SP release is still unclear. Our study aimed to investigate TRPM8’s role in SP release from primary sensory neurons during colitis and clarify the effect of SP on colonic epithelium. We analyzed inflammatory bowel disease patients’ data from the Gene Expression Omnibus dataset. Dextran sulfate sodium (DSS, 2.5%)-induced colitis in mice, mouse dorsal root ganglion (DRG) neurons, ND7/23 cell line, and mouse or human colonic organoids were used for this experiment. Our study found that TRPM8, TAC1 and WNT3A expression were significantly correlated with the severity of ulcerative colitis in patients and DSS-induced colitis in mice. The TRPM8 agonist (menthol) and the SP receptor antagonist (Aprepitant) can attenuate colitis in mice, but the effects were not additive. Menthol promoted calcium ion influx in mouse DRG neurons and inhibited the combination and phosphorylation of PKAca from the cAMP signaling pathway and GSK-3β from the Wnt/β-catenin signaling pathway, thereby inhibiting the effect of Wnt3a-driven β-catenin on promoting SP release in ND7/23 cells. Long-term stimulation with SP inhibited proliferation and enhanced apoptosis in both mouse and human colonic organoids. Conclusively, TRPM8 inhibits SP release from primary sensory neurons by inhibiting the interaction between PKAca and GSK-3β, thereby inhibiting the role of SP in promoting colonic epithelial apoptosis and relieving colitis.

Inflammatory Orofacial Pain Activates Peptidergic Neurons and Upregulates the Oxytocin Receptor Expression in Trigeminal Ganglion.

The majority of orofacial pain is caused by musculoskeletal and neuropathological diseases related to inflammatory processes that lead even to transcriptional alterations in the trigeminal ganglion (TG) neurons. The hypothalamic nonapeptide oxytocin has been reported to modulate nociception via binding and activating its receptor in primary sensory neurons. The purpose of this study was to analyze the gene expression of the oxytocin receptor (OTR), c-Fos, an indicator of neuronal activity, and α-calcitonin gene-related peptide (αCGRP), a characteristic neurotransmitter of the peptidergic trigeminal primary afferents in an animal model of inflammation-induced orofacial pain. Carrageenan was unilaterally injected into the vibrissal pads of male and female adult Wistar rats. RT-qPCR was performed to analyze the levels of mRNA expression in TGs 24 h after injection. The gene expression analysis revealed higher fold changes regarding the c-Fos (mean ± S.E: ♀: 3.9 ± 0.19; ♂: 3.55 ± 0.18) and αCGRP (♀: 2.84 ± 0.13; ♂: 3.39 ± 0.47) expression levels of mRNA, and a moderate rise in the expression of the OTR mRNA (♀: 1.52 ± 0.07; ♂: 1.49 ± 0.07) was observed in comparison to both vehicle(saline)-treated and untreated controls. Our results furnish evidence for inflammation-induced activation of peptidergic neurons, and it is suggested that oxytocin modulates inflammation-induced nociception by enhancing their signaling capacity due to its elevated expression in the sensory ganglion cells, thus providing new therapies for orofacial pain relief that target the OTRs.

MiR-1306-3p directly activates P2X3 receptors in primary sensory neurons to induce visceral pain in rats.

Mounting evidence indicates that microRNAs (miRNAs) play critical roles in various pathophysiological conditions and diseases, but the physiological roles of extracellular miRNAs on the disease-related ion channels remain largely unknown. Here, we showed that miR-1306-3p evoked action potentials and induced inward currents of the acutely isolated rat dorsal root ganglion (DRG) neurons. The miR-1306-3p-induced effects were significantly inhibited by A317491, a potent inhibitor of the P2X3 receptor (P2X3R), or disappeared after the knockdown of P2X3Rs in DRG neurons. We further identified R180, K315, and R52 as the miR-1306-3p interaction sites on the extracellular domain of P2X3Rs, which were distinct from the orthosteric ATP-binding sites. Intrathecal injection of miR-1306-3p produced visceral pain but not somatic pain in normal control rats. Conversely, intrathecal application of a miR-1306-3p antagomir and A317491 significantly alleviated visceral pain in a rat model of chronic visceral pain. Together, our findings suggest that miR-1306-3p might function as an endogenous ligand to activate P2X3Rs, eventually leading to chronic visceral pain.

δ-Opioid receptors in primary sensory neurons tonically restrain nociceptive input in chronic pain but do not enhance morphine analgesic tolerance

δ-Opioid receptors (DORs, encoded by the Oprd1 gene) are expressed throughout the peripheral and central nervous system, and DOR stimulation reduces nociception. Previous studies suggest that DORs promote the development of analgesic tolerance of μ-opioid receptor (MOR) agonists. It is uncertain whether DORs expressed in primary sensory neurons are involved in regulating chronic pain and MOR agonist-induced tolerance. In this study, we generated Oprd1 conditional knockout (Oprd1-cKO) mice by crossing Advillin-Cre mice with Oprd1-floxed mice. DOR expression in the dorsal root ganglion was diminished in Oprd1-cKO mice. Systemic or intrathecal injection of the DOR agonist SNC-80 produced analgesia in wild-type (WT), but not Oprd1-cKO, mice. In contrast, intracerebroventricular injection of SNC-80 produced a similar analgesic effect in WT and Oprd1-cKO mice. However, morphine-induced analgesia, hyperalgesia, or analgesic tolerance did not differ between WT and Oprd1-cKO mice. Compared with WT mice, Oprd1-cKO mice showed increased mechanical and heat hypersensitivity after nerve injury or tissue inflammation. Furthermore, blocking DORs with naltrindole increased nociceptive sensitivity induced by nerve injury or tissue inflammation in WT, but not Oprd1-cKO, mice. In addition, naltrindole potentiated glutamatergic input from primary afferents to spinal dorsal horn neurons increased by nerve injury or CFA in WT mice; this effect was absent in Oprd1-cKO mice. Our findings indicate that DORs in primary sensory neurons are critically involved in the analgesic effect of DOR agonists but not morphine-induced analgesic tolerance. Presynaptic DORs at primary afferent central terminals constitutively inhibit inflammatory and neuropathic pain by restraining glutamatergic input to spinal dorsal horn neurons.

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

PurposeTumor 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.MethodsElectrophysiological recordings were carried out on rat DRG neurons, and nociceptive behavior was quantified in rats.ResultsA brief (5 min) exposure of TNF-α rapidly increased P2X3 receptor-mediated and α,β-methylene-ATP (α,β-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,β-meATP upwards, with an increase of 31.57 ± 6.81% in the maximal current response to α,β-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,β-meATP produced a synergistic effect on mechanical allodynia in rats. TNF-α-induced mechanical allodynia was also alleviated after local P2X3 receptors were blocked.ConclusionThese 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.

ETA Receptor Blockade and Vascular Function in Patients with Sickle Cell Disease

Endothelin-1 (ET1) is a potent vasoconstrictor, mitogen, proinflammatory mediator, and a mediator of nociception, whose synthesis is increased by hypoxia, ischemia, shear stress, oxidative stress and reduced nitric oxide (NO) bioavailability, all of which are well documented mechanisms in the pathophysiology of sickle cell disease (SCD). We earlier reported on our studies on the role of ET-1 in SCD, and in transgenic mouse models showed that ETA receptor blockade with ambrisentan provides renal protection by preventing the development of glomerular hyperfiltration and proteinuria (Kasztan et al, 2017, Taylor et al, 2019, Kasztan and Pollock, 2019). ETA receptor blockade was also shown to decrease pulmonary inflammation in response to hypoxia/reoxygenation and LPS (Meiler et al.). Additionally, Lutz et al (2018) showed that pharmacologic inhibition or neuron specific knockdown of ETA receptor in primary sensory neurons of dorsal root ganglia in Berk mice alleviated basal and post-hypoxia evoked pain sensitivity. More recently (Kutlar et al, Blood 2019, 617,130036) we reported the preliminary results of a placebo controlled, double-blind phase I trial of ambrisentan in 26 subjects with SCD (SS and S-ß0 thalassemia) and microalbuminuria, and showed that the drug was well tolerated without any dose limiting toxicities and patients on ambrisentan had a reduction in microalbuminuria, especially in the subgroup who had been on concomitant ACEi/ARB therapy at a stable dose for at least six months (n=6, 286.1 mg/g Cr at baseline to 197.7 mg/g on day 85, p=0.06). FMD measurements showed increased arterial diameter, and improved microvascular function. We analyzed data collected on secondary end points during the phase I study, including Tricuspid regurgitant jet velocity (TRV), Transcranial Doppler (TCD) velocities in distal internal carotid (dICA) and middle cerebral (MCA) arteries, and eGFR (calculated with CKD-Epi formula). Fig. 1a shows eGFRs in ambrisentan and placebo groups; Fig. 1b and Fig. 1c show a breakdown of ambrisentan and placebo groups based on concomitant ACEi/ARB usage. There is a reduction in glomerular hyperfiltration in the ambrisentan group compared to placebo, more pronounced in the subgroup who have not been on ACEi/ARBs. Fig. 1d depicts the change in flow velocity in MCA (TAMMV, Time Averaged Mean Maximal Velocity, cm/sec) between the ambrisentan and placebo groups; similarly, Fig. 1e and 1f show the breakdown of baseline and Day 85 TAMMVs in MCA according to ACEi/ARB usage. A similar trend is also observed in dICA flow (fig. 1g-1i), and suggests a synergistic effect of ETA receptor blockade with ACEi/ARBs in preventing an increase in blood flow velocities. TRV was available on 7 subjects, 6 of which were in the ambrisentan group. Fig. 1j shows the change in TRV in the ambrisentan group, and again is suggestive of a synergistic effect of ambrisentan and ACEi/ARBs in decreasing TRV. These data are clearly very preliminary, and are obtained on a small number of subjects, and as such, do not warrant any conclusions and or speculations. Nevertheless, an interesting observation is the apparent interaction of ETA receptor blockade and ACEi/ARBs in altering vascular flow/function in SCD patients. Decrease in microalbuminuria has been reported with ACEi and ARBs in SCD (Yee et al, 2018), without any effect on GFR. A reduction in hyperfiltration would likely have a significant renoprotective effect, at an earlier stage in the development of sickle nephropathy. ETA receptor antagonists are approved for the treatment of pulmonary arterial hypertension; thus, a decrease in TRV would have a beneficial effect. Increase in blood flow velocity in major intracranial vessels is a well established risk factor for ischemic stroke in children with SCD; however, much less is known in adults. In summary, the effect of ETA receptor blockade with or without ACEi/ARB, may have a significant effect on vascular function/blood flow in different organ systems, and should be explored in a large, multi-center phase II trial, with and without concomitant and or serial ACEi/ARBs, for a longer period of time, with a dose escalation, to further clarify the pleiotropic effects on multiple aspects of SCD pathology. Figure 1 Disclosures Kutlar: NIH/NHLBI (SCDIC): Research Funding; Global Blood Therapeutics: Research Funding, Speakers Bureau; Micelle Biopharma: Consultancy; Novartis Pharmaceuticals: Consultancy, Research Funding; Novo Nordisk: Research Funding; Forma Therapeutics: Research Funding; REACH: Other: DSMB Member; NOHARM: Other: DSMB Member; Bluebird Bio: Other: DSMB Member.

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,β-methylene-ATP (α,β-meATP)-evoked inward currents, which were mediated by P2X3 receptors. ET-1 shifted the α,β-meATP concentration–response curve upwards, with an increase of 34.38 ± 4.72% in the maximal current response to α,β-meATP in the presence of ET-1. ET-1 potentiation of α,β-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,β-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.

Ru-Rong Ji, Ph.D., Recipient of the 2020 Excellence in Research Award.

Ru-Rong Ji, Ph.D., Recipient of the 2020 Excellence in Research Award.

Histone methyltransferase G9a diminishes expression of cannabinoid CB1 receptors in primary sensory neurons in neuropathic pain

Type 1 cannabinoid receptors (CB1Rs) are expressed in the dorsal root ganglion (DRG) and contribute to the analgesic effect of cannabinoids. However, the epigenetic mechanism regulating the expression of CB1Rs in neuropathic pain is unknown. G9a (encoded by the Ehmt2 gene), a histone 3 at lysine 9 methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined G9a's role in regulating CB1R expression in the DRG and in CB1R-mediated analgesic effects in an animal model of neuropathic pain. We show that nerve injury profoundly reduced mRNA levels of CB1Rs but increased the expression of CB2 receptors in the rat DRG. ChIP results indicated increased enrichment of histone 3 at lysine 9 dimethylation, a G9a-catalyzed repressive histone mark, at the promoter regions of the CB1R genes. G9a inhibition in nerve-injured rats not only up-regulated the CB1R expression level in the DRG but also potentiated the analgesic effect of a CB1R agonist on nerve injury-induced pain hypersensitivity. Furthermore, in mice lacking Ehmt2 in DRG neurons, nerve injury failed to reduce CB1R expression in the DRG and to decrease the analgesic effect of the CB1R agonist. Moreover, nerve injury diminished the inhibitory effect of the CB1R agonist on synaptic glutamate release from primary afferent nerves to spinal cord dorsal horn neurons in WT mice but not in mice lacking Ehmt2 in DRG neurons. Our findings reveal that nerve injury diminishes the analgesic effect of CB1R agonists through G9a-mediated CB1R down-regulation in primary sensory neurons.

μ-Opioid receptors in primary sensory neurons are involved in supraspinal opioid analgesia

Both inhibiting ascending nociceptive transmission and activating descending inhibition are involved in the opioid analgesic effect. The spinal dorsal horn is a critical site for modulating nociceptive transmission by descending pathways elicited by opioids in the brain. μ-Opioid receptors (MORs, encoded by Oprm1) are highly expressed in primary sensory neurons and their central terminals in the spinal cord. In the present study, we tested the hypothesis that MORs expressed in primary sensory neurons contribute to the descending inhibition and supraspinal analgesic effect induced by centrally administered opioids. We generated Oprm1 conditional knockout (Oprm1-cKO) mice by crossing AdvillinCre/+ mice with Oprm1flox/flox mice. Immunocytochemical labeling in Oprm1-cKO mice showed that MORs are completely ablated from primary sensory neurons and are profoundly reduced in the superficial spinal dorsal horn. Intracerebroventricular injection of morphine or fentanyl produced a potent analgesic effect in wild-type mice, but such an effect was significantly attenuated in Oprm1-cKO mice. Furthermore, the analgesic effect produced by morphine or fentanyl microinjected into the periaqueductal gray was significantly greater in wild-type mice than in Oprm1-cKO mice. Blocking MORs at the spinal cord level diminished the analgesic effect of morphine and fentanyl microinjected into the periaqueductal gray in both groups of mice. Our findings indicate that MORs expressed at primary afferent terminals in the spinal cord contribute to the supraspinal opioid analgesic effect. These presynaptic MORs in the spinal cord may serve as an interface between ascending inhibition and descending modulation that are involved in opioid analgesia.

Phase-I Study of ETA Receptor Antagonist Ambrisentan in Sickle Cell Disease

Endothelin-1 (ET1) is a potent vasoconstrictor, mitogen, proinflammatory mediator and a mediator of nociception. Its synthesis is increased by hypoxia, ischemia, shear stress, oxidative stress and reduced nitric oxide (NO) bioavailability, all of which are well documented mechanisms in the pathophysiology of sickle cell disease. Two distinct receptors, ETA and ETB mediate opposing actions of ET1; while ETA mediates vasoconstriction, inflammation, cellular adhesion and vascular remodeling, ETB stimulation leads to increased NO production, vasodilatation, anticoagulation and anti-adhesion. We studied the role of ET1 in SCD in transgenic mouse models, and showed that selective ETA receptor blockade with ambrisentan provides renal protection by preventing the development of glomerular hyperfiltration, and preventing proteinuria (Kasztan et al, 2017; Taylor et al, 2019; Kasztan & Pollock, 2019).Additionaly, ETA receptor blockade was shown to decrease pulmonary inflammation in response to different stimuli, including hypoxia/reoxygenation and LPS (Meiler et al). Lutz et al (2018) showed that pharmacologic inhibition, or neuron specific knockdown of ETA receptor in primary sensory neurons of dorsal root ganglia in Berk mice alleviated basal and post-hypoxia evoked pain hypersensitivity. We then conducted a Phase 1, double blind, placebo controlled study to elucidate the role of ET-1 in 26 patients with SCD (SS or Sb0thal); patients were randomized to either ambrisentan 5 mg/day (N=13) or placebo (N=13) for 12 weeks. The primary end point was safety and tolerability. We also measured microalbuminuria, pressure pain threshold, transcranial doppler (TCD), echocardiograms (TR jet velocity), vascular endothelial function by flow mediated dilation (FMD), and inflammatory cytokines. Urine specimens were collected for measurement of markers of glomerular and tubular injury (NGAL, KIM-1, netrin and nephrin) at baseline and at the end of study (Day 85). Ambrisentan was well tolerated without any adverse events or fluid retention. No change in systolic and diastolic blood pressures were observed in either group (p=0.88, and p=049, respectively). No significant change in weight or serum creatinine was observed in the ambrisentan group (69.1→68.6 Kg, p=0.99, and 0.74→0.69 mg/dl, p=0.43). In the ambrisentan group, there was a tendency toward reduced microalbuminuria, (-36.6 mg/g Cr, vs +91.6), especially in the subgroup of patients who had been on a stable dose of ACE or ARB for 6 months (n=6, 286.1 mg/g Cr at baseline to 197.7 mg/g on day 85, p=0.06). FMD measurements showed increased arterial diameter, and improved microvascular function. Data collected for secondary end points including, TCD, TRV, inflammatory cytokines, markers of glomerular and tubular injury, quantitative sensory testing (QST) with pressure pain measurements, and von Frey monofilament readings, as well as quality of life measures (ASCQMe) are in the process of being analyzed. These data suggest that ETA receptor blockade is safe, well tolerated and has the potential to impact various aspects of disease pathophysiology in SCD. Disclosures Kutlar: Novartis: Consultancy; Global Blood Therapeutics, Inc. (GBT): Research Funding; Bluebird Bio: Other: DSMB Member; Novo Nordisk: Research Funding; Micelle Biopharma: Other: DSMB Chair. OffLabel Disclosure: Ambrisentan is an approved FDA drug used for treatment of another condition but it is expected that it will also help improve and/or prevent kidney damage that can be caused by sickle cell disease.

μ-Opioid receptors in primary sensory neurons are essential for opioid analgesic effect on acute and inflammatory pain and opioid-induced hyperalgesia.

μ-Opioid receptors (MORs) are expressed peripherally and centrally, but the loci of MORs responsible for clinically relevant opioid analgesia are uncertain. Crossing Oprm1flox/flox and AdvillinCre/+ mice completely ablates MORs in dorsal root ganglion neurons and reduces the MOR expression level in the spinal cord. Presynaptic MORs expressed at primary afferent central terminals are essential for synaptic inhibition and potentiation of sensory input by opioids. MOR ablation in primary sensory neurons diminishes analgesic effects produced by systemic and intrathecal opioid agonists and abolishes chronic opioid treatment-induced hyperalgesia. These findings demonstrate a critical role of MORs expressed in primary sensory neurons in opioid analgesia and suggest new strategies to increase the efficacy and reduce adverse effects of opioids. The pain and analgesic systems are complex, and the actions of systemically administered opioids may be mediated by simultaneous activation of μ-opioid receptors (MORs, encoded by the Oprm1 gene) at multiple, interacting sites. The loci of MORs and circuits responsible for systemic opioid-induced analgesia and hyperalgesia remain unclear. Previous studies using mice in which MORs are removed from Nav1.8- or TRPV1-expressing neurons provided only an incomplete and erroneous view about the role of peripheral MORs in opioid actions in vivo. In the present study, we determined the specific role of MORs expressed in primary sensory neurons in the analgesic and hyperalgesic effects produced by systemic opioid administration. We generated Oprm1 conditional knockout (Oprm1-cKO) mice in which MOR expression is completely deleted from dorsal root ganglion neurons and substantially reduced in the spinal cord, which was confirmed by immunoblotting and immunocytochemical labelling. Both opioid-induced inhibition and potentiation of primary sensory input were abrogated in Oprm1-cKO mice. Remarkably, systemically administered morphine potently inhibited acute thermal and mechanical nociception and persistent inflammatory pain in control mice but had little effect in Oprm1-cKO mice. The analgesic effect of intrathecally administered morphine was also profoundly reduced in Oprm1-cKO mice. Additionally, chronic morphine treatment-induced hyperalgesia was absent in Oprm1-cKO mice. Our findings directly challenge the notion that clinically relevant opioid analgesia is mediated mostly by centrally expressed MORs. MORs in primary sensory neurons, particularly those expressed presynaptically at the first sensory synapse in the spinal cord, are crucial for both opioid analgesia and opioid-induced hyperalgesia.

Endothelin type A receptors mediate pain in a mouse model of sickle cell disease.

Sickle cell disease is associated with acute painful episodes and chronic intractable pain. Endothelin-1, a known pain inducer, is elevated in the blood plasma of both sickle cell patients and mouse models of sickle cell disease. We show here that the levels of endothelin-1 and its endothelin type A receptor are increased in the dorsal root ganglia of a mouse model of sickle cell disease. Pharmacologic inhibition or neuron-specific knockdown of endothelin type A receptors in primary sensory neurons of dorsal root ganglia alleviated basal and post-hypoxia evoked pain hypersensitivities in sickle cell mice. Mechanistically, endothelin type A receptors contribute to sickle cell disease-associated pain likely through the activation of NF-κB-induced Nav1.8 channel upregulation in primary sensory neurons of sickle cell mice. Our findings suggest that endothelin type A receptor is a potential target for the management of sickle cell disease-associated pain, although this expectation needs to be further verified in clinical settings.

A mathematical model of histamine-mediated neural activation during acupuncture.

Acupoints (Xuewei) are the focus of acupuncture on the body in traditional Chinese medicine treatment. Mast and nerve cells share a perivascular location and are abundantly found at these acupoints. Both environmental factors and medical treatments (chemical and physical stimuli) can stimulate local mast cells (MCs) to degranulate and thus release histamine which then activates the nearby nerves and therefore contributes to a signal transmission from the peripheral to the central nervoussystem. In this paper, a mathematical model is constructed to describe the signaling pathways that originate from the cells located at an acupoint. We show that: (1) peripheral stimuli induce the release of histamine from MCs; (2) histamine excites receptors in primary sensory neurons and leads to membrane action potentials; (3) histamine released from MCs at an acupoint plays a key role in response to acupuncture. These results are consistent with experimental observations. Furthermore, this study may facilitate our understanding of the signal transmission process that occurs in response to peripheral stimuli as well as provide a useful modeling tool for quantitatively analyzing immune and acupuncture mechanisms that involve MCs.

The role of endogenous opioid peptides in the antinociceptive effect of 15-deoxyΔ12,14-prostaglandin J2 in the temporomandibular joint

We have previously demonstrated that peripheral administration of 15d-PGJ2 in the Temporomandibular joint (TMJ) of rats can prevent nociceptor sensitization, mediated by peroxisome proliferator activated receptor-γ (PPAR-γ), and κ- and δ- opioid receptors. However, the mechanism that underlies the signaling of PPAR-γ (upon activation by 15d-PGJ2) to induce antinociception, and how the opioid receptors are activated via 15d-PGJ2 are not fully understood. This study demonstrates that peripheral antinociceptive effect of 15d-PGJ2 is mediated by PPAR-γ expressed in the inflammatory cells of TMJ tissues. Once activated by 15d-PGJ2, PPAR-γ induces the release of β-endorphin and dynorphin, which activates κ- and δ-opioid receptors in primary sensory neurons to induce the antinociceptive effect.

Nerve Injury Diminishes Opioid Analgesia through Lysine Methyltransferase-mediated Transcriptional Repression of μ-Opioid Receptors in Primary Sensory Neurons

The μ-opioid receptor (MOR, encoded by Oprm1) agonists are the mainstay analgesics for treating moderate to severe pain. Nerve injury causes down-regulation of MORs in the dorsal root ganglion (DRG) and diminishes the opioid effect on neuropathic pain. However, the epigenetic mechanisms underlying the diminished MOR expression caused by nerve injury are not clear. G9a (encoded by Ehmt2), a histone 3 at lysine 9 methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined the role of G9a in diminished MOR expression and opioid analgesic effects in animal models of neuropathic pain. We found that nerve injury in rats induced a long-lasting reduction in the expression level of MORs in the DRG but not in the spinal cord. Nerve injury consistently increased the enrichment of the G9a product histone 3 at lysine 9 dimethylation in the promoter of Oprm1 in the DRG. G9a inhibition or siRNA knockdown fully reversed MOR expression in the injured DRG and potentiated the morphine effect on pain hypersensitivity induced by nerve injury. In mice lacking Ehmt2 in DRG neurons, nerve injury failed to reduce the expression level of MORs and the morphine effect. In addition, G9a inhibition or Ehmt2 knockout in DRG neurons normalized nerve injury-induced reduction in the inhibitory effect of the opioid on synaptic glutamate release from primary afferent nerves. Our findings indicate that G9a contributes critically to transcriptional repression of MORs in primary sensory neurons in neuropathic pain. G9a inhibitors may be used to enhance the opioid analgesic effect in the treatment of chronic neuropathic pain.

G9a inhibits CREB-triggered expression of mu opioid receptor in primary sensory neurons following peripheral nerve injury.

Neuropathic pain, a distressing and debilitating disorder, is still poorly managed in clinic. Opioids, like morphine, remain the mainstay of prescribed medications in the treatment of this disorder, but their analgesic effects are highly unsatisfactory in part due to nerve injury-induced reduction of opioid receptors in the first-order sensory neurons of dorsal root ganglia. G9a is a repressor of gene expression. We found that nerve injury-induced increases in G9a and its catalyzed repressive marker H3K9m2 are responsible for epigenetic silencing of Oprm1, Oprk1, and Oprd1 genes in the injured dorsal root ganglia. Blocking these increases rescued dorsal root ganglia Oprm1, Oprk1, and Oprd1 gene expression and morphine or loperamide analgesia and prevented the development of morphine or loperamide-induced analgesic tolerance under neuropathic pain conditions. Conversely, mimicking these increases reduced the expression of three opioid receptors and promoted the mu opioid receptor-gated release of primary afferent neurotransmitters. Mechanistically, nerve injury-induced increases in the binding activity of G9a and H3K9me2 to the Oprm1 gene were associated with the reduced binding of cyclic AMP response element binding protein to the Oprm1 gene. These findings suggest that G9a participates in the nerve injury-induced reduction of the Oprm1 gene likely through G9a-triggered blockage in the access of cyclic AMP response element binding protein to this gene.

Wnt/Ryk signaling contributes to neuropathic pain by regulating sensory neuron excitability and spinal synaptic plasticity in rats.

Treating neuropathic pain continues to be a major clinical challenge and underlying mechanisms of neuropathic pain remain elusive. We have recently demonstrated that Wnt signaling, which is important in developmental processes of the nervous systems, plays critical roles in the development of neuropathic pain through the β-catenin-dependent pathway in the spinal cord and the β-catenin-independent pathway in primary sensory neurons after nerve injury. Here, we report that Wnt signaling may contribute to neuropathic pain through the atypical Wnt/Ryk signaling pathway in rats. Sciatic nerve injury causes a rapid-onset and long-lasting expression of Wnt3a, Wnt5b, and Ryk receptors in primary sensory neurons, and dorsal horn neurons and astrocytes. Spinal blocking of the Wnt/Ryk receptor signaling inhibits the induction and persistence of neuropathic pain without affecting normal pain sensitivity and locomotor activity. Blocking activation of the Ryk receptor with anti-Ryk antibody, in vivo or in vitro, greatly suppresses nerve injury-induced increased intracellular Ca and hyperexcitability of the sensory neurons, and also the enhanced plasticity of synapses between afferent C-fibers and the dorsal horn neurons, and activation of the NR2B receptor and the subsequent Ca-dependent signals CaMKII, Src, ERK, PKCγ, and CREB in sensory neurons and the spinal cord. These findings indicate a critical mechanism underlying the pathogenesis of neuropathic pain and suggest that targeting the Wnt/Ryk signaling may be an effective approach for treating neuropathic pain.

Enhancement of acid-sensing ion channel activity by metabotropic P2Y UTP receptors in primary sensory neurons.

Peripheral purinergic signaling plays an important role in nociception. Increasing evidence suggests that metabotropic P2Y receptors are also involved, but little is known about the underlying mechanism. Herein, we report that selective P2Y receptor agonist uridine 5'-triphosphate (UTP) can exert an enhancing effect on the functional activity of acid-sensing ion channels (ASICs), key sensors for extracellular protons, in rat dorsal root ganglia (DRG) neurons. First, UTP dose-dependently increased the amplitude of ASIC currents. UTP also shifted the concentration-response curve for proton upwards, with a 56.6 ± 6.4% increase of the maximal current response to proton. Second, UTP potentiation of proton-gated currents can be mimicked by adenosine 5'-triphosphate (ATP), but not by P2Y1 receptor agonist ADP. Potentiation of UTP was blocked by P2Y receptor antagonist suramin and by inhibition of intracellular G protein, phospholipase C (PLC), protein kinase C (PKC), or protein interacting with C-kinase 1 (PICK1) signaling. Third, UTP altered acidosis-evoked membrane excitability of DRG neurons and caused a significant increase in the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, UTP dose-dependently exacerbated nociceptive responses to injection of acetic acid in rats. These results suggest that UTP enhanced ASIC-mediated currents and nociceptive responses, which reveal a novel peripheral mechanism underlying UTP-sensitive P2Y2 receptor involvement in hyperalgesia by sensitizing ASICs in primary sensory neurons.

Adrenergic signaling mediates mechanical hyperalgesia through activation of P2X3 receptors in primary sensory neurons of rats with chronic pancreatitis

The mechanism of pain in chronic pancreatitis (CP) is poorly understood. The aim of this study was designed to investigate roles of norepinephrine (NE) and P2X receptor (P2XR) signaling pathway in the pathogenesis of hyperalgesia in a rat model of CP. CP was induced in male adult rats by intraductal injection of trinitrobenzene sulfonic acid (TNBS). Mechanical hyperalgesia was assessed by referred somatic behaviors to mechanical stimulation of rat abdomen. P2XR-mediated responses of pancreatic dorsal root ganglion (DRG) neurons were measured utilizing calcium imaging and whole cell patch-clamp-recording techniques. Western blot analysis and immunofluorescence were performed to examine protein expression. TNBS injection produced a significant upregulation of P2X3R expression and an increase in ATP-evoked responses of pancreatic DRG neurons. The sensitization of P2X3Rs was reversed by administration of β-adrenergic receptor antagonist propranolol. Incubation of DRG neurons with NE significantly enhanced ATP-induced intracellular calcium signals, which were abolished by propranolol, and partially blocked by protein kinase A inhibitor H-89. Interestingly, TNBS injection led to a significant elevation of NE concentration in DRGs and the pancreas, an upregulation of β2-adrenergic receptor expression in DRGs, and amplification of the NE-induced potentiation of ATP responses. Importantly, pancreatic hyperalgesia was markedly attenuated by administration of purinergic receptor antagonist suramin or A317491 or β2-adrenergic receptor antagonist butoxamine. Sensitization of P2X3Rs, which was likely mediated by adrenergic signaling in primary sensory neurons, contributes to pancreatic pain, thus identifying a potential target for treating pancreatic pain caused by inflammation.