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1.  Central terminal sensitization of TRPV1 by descending serotonergic facilitation modulates chronic pain 
Neuron  2014;81(4):873-887.
SUMMARY
The peripheral terminals of primary nociceptive neurons play an essential role in pain detection mediated by membrane receptors like TRPV1, a molecular sensor of heat and capsaicin. However, the contribution of central terminal TRPV1 in the dorsal horn to chronic pain has not been investigated directly. Combining primary sensory neuron-specific GCaMP3 imaging with a trigeminal neuropathic pain model, we detected robust neuronal hyperactivity in injured and uninjured nerves in the skin, soma in trigeminal ganglion, and central terminals in the spinal trigeminal nucleus. Extensive TRPV1 hyperactivity was observed in central terminals innervating all dorsal horn laminae. The central terminal TRPV1 sensitization was maintained by descending serotonergic (5-HT) input from the brainstem. Central blockade of TRPV1 or 5-HT/5-HT3A receptors attenuated central terminal sensitization, excitatory primary afferent inputs, and mechanical hyperalgesia in the territories of injured and uninjured nerves. Our results reveal new central mechanisms facilitating central terminal sensitization underlying chronic pain.
doi:10.1016/j.neuron.2013.12.011
PMCID: PMC3943838  PMID: 24462040
2.  Sensory neurons and circuits mediating itch 
Nature reviews. Neuroscience  2014;15(1):19-31.
Chemicals used experimentally to evoke itch elicit activity in diverse subpopulations of cutaneous pruriceptive neurons, all of which also respond to painful stimuli. However, itch is distinct from pain: it evokes different behaviors, such as scratching, and originates from the skin or certain mucosae but not from muscle, joints or viscera. New insights regarding the neurons that mediate the sensation of itch have been gained from experiments in which gene expression has been manipulated in different types of pruriceptive neurons as well as from comparisons between psychophysical measurements of itch and the neuronal discharges and other properties of peripheral and central pruriceptive neurons.
doi:10.1038/nrn3641
PMCID: PMC4096293  PMID: 24356071
3.  Three functionally distinct classes of C-fiber nociceptors in primate 
Nature communications  2014;5:4122.
In primate C-fiber polymodal nociceptors are broadly classified into two groups based on mechanosensitivity. Here we demonstrate that mechanically-sensitive polymodal nociceptors that respond either quickly (QC) or slowly (SC) to a heat stimulus differ in responses to a mild burn, heat sensitization, conductive properties and chemosensitivity. Superficially applied capsaicin and intradermal injection of β-alanine, a MrgprD agonist, excite vigorously all QCs. Only 40% of SCs respond to β-alanine, and their response is only half that of QCs. Mechanically-insensitive C-fibers (C-MIAs) are β-alanine insensitive but vigorously respond to capsaicin and histamine with distinct discharge patterns. Calcium imaging reveals that β-alanine and histamine activate distinct populations of capsaicin responsive neurons in primate DRG. We suggest that histamine itch and capsaicin pain are peripherally encoded in C-MIAs and that primate polymodal nociceptive afferents form three functionally distinct subpopulations with β-alanine responsive QC fibers likely corresponding to murine MrgprD- expressing, non-peptidergic nociceptive afferents.
doi:10.1038/ncomms5122
PMCID: PMC4072246  PMID: 24947823
4.  Imaging orofacial pain in mice 
Molecular Pain  2014;10(Suppl 1):O2.
doi:10.1186/1744-8069-10-S1-O2
PMCID: PMC4304368
5.  Itch Mechanisms and Circuits 
Annual review of biophysics  2014;43:331-355.
The itch-scratch reflex serves as a protective mechanism in everyday life. However, chronic persistent itching can be devastating. Despite the clinical importance of the itch sensation, its mechanism remains elusive. In the past decade, substantial progress has been made to uncover the mystery of itching. Here, we review the molecules, cells, and circuits known to mediate the itch sensation, which, coupled with advances in understanding the pathophysiology of chronic itching conditions, will hopefully contribute to the development of new anti-itch therapies.
doi:10.1146/annurev-biophys-051013-022826
PMCID: PMC4081479  PMID: 24819620
itch; pruritus; neurons; neural circuits; sensitization; pain
6.  Tolerance develops to the antiallodynic effects of the peripherally acting opioid loperamide hydrochloride in nerve-injured rats 
Pain  2013;154(11):10.1016/j.pain.2013.07.023.
Peripherally acting opioids are potentially attractive drugs for the clinical management of certain chronic pain states due to the lack of centrally mediated adverse effects. However, it remains unclear whether tolerance develops to peripheral opioid analgesic effects under neuropathic pain conditions. We subjected rats to L5 spinal nerve ligation (SNL) and examined the analgesic effects of repetitive systemic and local administration of loperamide hydrochloride, a peripherally acting opioid agonist. We found that the inhibition of mechanical hypersensitivity, an important manifestation of neuropathic pain, by systemic loperamide (1.5 mg/kg subcutaneously) decreased after repetitive drug treatment (tolerance-inducing dose: 0.75 to 6.0 mg/kg subcutaneously). Similarly, repeated intraplantar injection of loperamide (150 µg/50 µL intraplantarly) and D-Ala2-MePhe4-Glyol5 enkephalin (300 µg/50 µL), a highly selective mu-opioid receptor (MOR) agonist, also resulted in decreased inhibition of mechanical hypersensitivity. Pretreatment with naltrexone hydrochloride (5 mg/kg intraperitoneally) and MK-801 (0.2 mg/kg intraperitoneally) attenuated systemic loperamide tolerance. Western blot analysis showed that repetitive systemic administration of morphine (3 mg/kg subcutaneously), but not loperamide (3 mg/kg subcutaneously) or saline, significantly increased MOR phosphorylation in the spinal cord of SNL rats. In cultured rat dorsal root ganglion neurons, loperamide dose-dependently inhibited KCl-induced increases in [Ca2+]i. However, this drug effect significantly decreased in cells pretreated with loperamide (3 µM, 72 hours). Intriguingly, in loperamide-tolerant cells, the delta-opioid receptor antagonist naltrindole restored loperamide’s inhibition of KCl-elicited [Ca2+]i increase. Our findings indicate that animals with neuropathic pain may develop acute tolerance to the antiallodynic effects of peripherally acting opioids after repetitive systemic and local drug administration.
doi:10.1016/j.pain.2013.07.023
PMCID: PMC3863910  PMID: 23880055
Nerve injury; Neuropathic pain; Peripheral opioid receptor; Rats; Tolerance
7.  Immunohistological demonstration of CaV3.2 T-type voltage-gated calcium channel expression in soma of dorsal root ganglion neurons and peripheral axons of rat and mouse 
Neuroscience  2013;250:263-274.
Previous behavioural studies have revealed that CaV3.2 T-type calcium channels support peripheral nociceptive transmission and electrophysiological studies have established the presence of T-currents in putative nociceptive sensory neurons of dorsal root ganglion (DRG). To date, however, the localization pattern of this key nociceptive channel in the soma and peripheral axons of these cells has not been demonstrated due to lack of isoform-selective anti-CaV3.2 antibodies. In the present study a new polyclonal CaV3.2 antibody is used to localize CaV3.2 expression in rodent DRG neurons using different staining techniques including confocal and electron microscopy. Confocal microscopy of both acutely dissociated cells and short-term cultures demonstrated strong immunofluorescence of anti-CaV3.2 antibody that was largely confined to smaller diameter DRG neurons where it co-localized with established immuno-markers of unmyelinated nociceptors, such as, CGRP, IB4 and peripherin. In contrast, a smaller proportion of these CaV3.2-labeled DRG cells also co-expressed NF-200, a marker of myelinated sensory neurons. In the rat sciatic nerve preparation, confocal microscopy demonstrated anti-CaV3.2 immunofluorescence which was co-localized with both peripherin and NF-200. Further, electron microscopy revealed immuno-gold labelling of CaV3.2 preferentially in association with un-myelinated sensory fibres from mouse sciatic nerve. Finally, we demonstrated the expression of CaV3.2 channels in peripheral nerve endings of mouse hindpaw skin as shown by co-localisation with Mrgpd-GFP-positive fibres. The CaV3.2 expression within the soma and peripheral axons of nociceptive sensory neurons further demonstrates the importance of this channel in peripheral pain transmission.
doi:10.1016/j.neuroscience.2013.07.005
PMCID: PMC3796369  PMID: 23867767
nociceptors; low-voltage-activated; T-currents; DRG; Ca2+
8.  Conventional and Kilohertz-frequency Spinal Cord Stimulation Produces Intensity- and Frequency-dependent Inhibition of Mechanical Hypersensitivity in a Rat Model of Neuropathic Pain 
Anesthesiology  2013;119(2):422-432.
Background
Spinal cord stimulation (SCS) is a useful neuromodulatory technique for treatment of certain neuropathic pain conditions. However, the optimal stimulation parameters remain unclear.
Methods
In rats after L5 spinal nerve ligation, we compared the inhibitory effects on mechanical hypersensitivity from bipolar SCS of different intensities (20%, 40%, 80% motor threshold) and frequencies (50-Hz, 1-kHz, and 10-kHz). We then compared the effects of 1-kHz and 50-Hz dorsal column stimulation at high and low stimulus intensities on conduction properties of afferent Aα/β-fibers and spinal wide-dynamic-range neuronal excitability.
Results
Three consecutive daily SCS at different frequencies progressively inhibited mechanical hypersensitivity in an intensity-dependent manner. At 80% motor threshold, the ipsilateral paw withdrawal threshold (%preinjury) increased significantly from pre-SCS measures, beginning with the first day of SCS at the frequencies of 1-kHz (50.2 ± 5.7% from 23.9 ± 2.6%, n = 19, mean ± SEM) and 10-kHz (50.8 ± 4.4 % from 27.9 ± 2.3%, n = 17), while it was significantly increased beginning on the second day in the 50-Hz group (38.9 ± 4.6% from 23.8 ± 2.1%, n = 17). At high intensity, both 1-kHz and 50-Hz dorsal column stimulation reduced Aα/β-compound action potential size recorded at the sciatic nerve, but only 1-kHz stimulation was partially effective at the lower intensity. The number of actions potentials in C-fiber component of wide-dynamic-range neuronal response to windup-inducing stimulation was significantly decreased after 50-Hz (147.4 ± 23.6 from 228.1 ± 39.0, n = 13), but not 1-kHz (n = 15), dorsal column stimulation.
Conclusions
Kilohertz SCS attenuated mechanical hypersensitivity in a time course and amplitude that differed from conventional 50-Hz SCS, and may involve different peripheral and spinal segmental mechanisms.
doi:10.1097/ALN.0b013e31829bd9e2
PMCID: PMC3763697  PMID: 23880991
9.  Development and Evaluation of Small Peptidomimetic Ligands to Protease-Activated Receptor-2 (PAR2) through the Use of Lipid Tethering 
PLoS ONE  2014;9(6):e99140.
Protease-activated receptor-2 (PAR2) is a G-Protein Coupled Receptor (GPCR) activated by proteolytic cleavage to expose an attached, tethered ligand (SLIGRL). We evaluated the ability for lipid-tethered-peptidomimetics to activate PAR2 with in vitro physiological and Ca2+ signaling assays to determine minimal components necessary for potent, specific and full PAR2 activation. A known PAR2 activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG3-Hdc) provided a potent agonist starting point (physiological EC50 = 1.4 nM; 95% CI: 1.2–2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG3-Hdc retained potency (EC50 = 2.1 nM; 1.3–3.4 nM) with improved selectivity for PAR2 over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR2 peptide agonist, SLIGRL-NH2. 2at-LIG-PEG3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20–100 nM). 2at-LI-PEG3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260–360 nM) but displayed partial PAR2 activation in both physiological and Ca2+ signaling assays. Further truncation (2at-L-PEG3-Hdc and 2at-PEG3-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR2 in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG3-Hdc lacked efficacy. Minimum peptidomimetic PAR2 agonists were developed with known heterocycle substitutes for Ser1 (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu2. Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR2 specificity, however, only the heterocycle-tetrapeptides displayed full PAR2 agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR2 that allows for selective probing of PAR2 function across a broad range of physiological systems.
doi:10.1371/journal.pone.0099140
PMCID: PMC4057235  PMID: 24927179
10.  A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons 
Nature neuroscience  2013;16(8):1024-1031.
Neuropathic pain is a refractory disease characterized by maladaptive changes in gene transcription and translation within the sensory pathway. Long noncoding RNAs (lncRNAs) are emerging as new players in gene regulation, but how lncRNAs operate in the development of neuropathic pain is unclear. Here we identify a conserved lncRNA for Kcna2 (named Kcna2 antisense RNA) in first-order sensory neurons of rat dorsal root ganglion (DRG). Peripheral nerve injury increases Kcna2 antisense RNA expression in injured DRG through activation of myeloid zinc finger protein 1, a transcription factor that binds to Kcna2 antisense RNA gene promoter. Mimicking this increase downregulates Kcna2, reduces total Kv current, increases excitability in DRG neurons, and produces neuropathic pain symptoms. Blocking this increase reverses nerve injury-induced downregulation of DRG Kcna2 and attenuates development and maintenance of neuropathic pain. These findings suggest native Kcna2 antisense RNA as a new therapeutic target for the treatment of neuropathic pain.
doi:10.1038/nn.3438
PMCID: PMC3742386  PMID: 23792947
11.  Pirt Functions as an Endogenous Regulator of TRPM8 
Nature communications  2013;4:2179.
Pirt is a membrane protein that is specifically expressed in the peripheral nervous system, where it has been shown to increase the sensitivity of the transient receptor potential vanilloid 1 (TRPV1) channel and modulate its role in heat pain. The broad expression of Pirt among dorsal root ganglion neurons suggests it may modulate other TRPs, such as the menthol and cooling sensor TRPM8. The discrepancies in the channel properties of TRPM8 in native neurons versus heterologous cells indicate the existence of endogenous modulators of the channel. Here we show that Pirt regulates the function of TRPM8 and its role in detecting cold. Pirt−/− mice exhibit decreased behavioral responses to cold and cool temperatures, and Pirt increases the sensitivity of TRPM8 to menthol and cool temperature. Our data suggest Pirt is an endogenous regulator of TRPM8.
doi:10.1038/ncomms3179
PMCID: PMC3748931  PMID: 23863968
12.  A subpopulation of nociceptors specifically linked to itch 
Nature neuroscience  2012;16(2):174-182.
Itch-specific neurons have been sought for decades. The existence of such neurons is in doubt recently due to the observation that itch-mediating neurons also respond to painful stimuli. Here, we genetically labeled and manipulated MrgprA3+ neurons in dorsal root ganglion (DRG) and found that they exclusively innervate the epidermis of the skin and respond to multiple pruritogens. Ablation of MrgprA3+ neurons led to significant reductions in scratching evoked by multiple pruritogens and occurring spontaneously under chronic itch conditions whereas pain sensitivity remained intact. Importantly, mice with TRPV1 exclusively expressed in MrgprA3+ neurons exhibited only itch- and not pain behavior in response to capsaicin. Although MrgprA3+ neurons are sensitive to noxious heat, activation of TRPV1 in these neurons by noxious heat did not alter pain behavior. These data suggest that MrgprA3 defines a specific subpopulation of DRG neurons mediating itch. Our study opens new avenues for studying itch and developing anti-pruritic therapies.
doi:10.1038/nn.3289
PMCID: PMC3557753  PMID: 23263443
13.  Analgesic properties of loperamide differ following systemic and local administration to rats after spinal nerve injury 
Background
The analgesic properties and mechanisms of loperamide hydrochloride, a peripherally acting opioid receptor agonist, in neuropathic pain warrant further investigation.
Methods
We examined the effects of systemic or local administration of loperamide on heat and mechanical hyperalgesia in rats after an L5 spinal nerve ligation (SNL).
Results
1) Systemic loperamide (0.3–10 mg/kg, subcutaneous in the back) dose-dependently reversed heat hyperalgesia in SNL rats, but did not produce thermal analgesia. Systemic loperamide (3 mg/kg) did not induce thermal antinociception in naïve rats; 2) Systemic loperamide-induced anti-heat hyperalgesia was blocked by pretreatment with intraperitoneal naloxone methiodide (5 mg/kg), but not by intraperitoneal naltrindole (5 mg/kg) or intrathecal naltrexone (20 μg/10 μL); 3) Local administration of loperamide (150 μg), but not vehicle, into plantar or dorsal hind paw tissue induced thermal analgesia in SNL rats and thermal antinociception in naïve rats; 4) The analgesic effect of intraplantar loperamide (150 μg/15 μL) in SNL rats at 45 min, but not 10 min, post-injection was blocked by pretreatment with an intraplantar injection of naltrexone (75 μg/10 μL); 5) Systemic (3.0 mg/kg) and local (150 μg) loperamide reduced the exaggerated duration of hind paw elevation to noxious pinprick stimuli in SNL rats. Intraplantar injection of loperamide also decreased the frequency of pinprickevoked response in naïve rats.
Conclusions
These findings suggest that both systemic and local administration of loperamide induce an opioid receptor-dependent inhibition of heat and mechanical hyperalgesia in nerve-injured rats, but that local paw administration of loperamide also induces thermal and mechanical antinociception.
doi:10.1002/j.1532-2149.2012.00148.x
PMCID: PMC3389202  PMID: 22508374
14.  Preso1 dynamically regulates group I metabotropic glutamate receptors 
Nature neuroscience  2012;15(6):836-844.
Group I metabotropic glutamate receptors (mGluRs), including mGluR1 and mGluR5, are G protein–coupled receptors (GPCRs) that are expressed at excitatory synapses in brain and spinal cord. GPCRs are often negatively regulated by specific G protein–coupled receptor kinases and subsequent binding of arrestin-like molecules. Here we demonstrate an alternative mechanism in which group I mGluRs are negatively regulated by proline-directed kinases that phosphorylate the binding site for the adaptor protein Homer, and thereby enhance mGluR–Homer binding to reduce signaling. This mechanism is dependent on a multidomain scaffolding protein, Preso1, that binds mGluR, Homer and proline-directed kinases and that is required for their phosphorylation of mGluR at the Homer binding site. Genetic ablation of Preso1 prevents dynamic phosphorylation of mGluR5, and Preso1−/− mice exhibit sustained, mGluR5-dependent inflammatory pain that is linked to enhanced mGluR signaling. Preso1 creates a microdomain for proline-directed kinases with broad substrate specificity to phosphorylate mGluR and to mediate negative regulation.
doi:10.1038/nn.3103
PMCID: PMC3434267  PMID: 22561452
15.  Mechanisms of itch evoked by β-alanine 
β-alanine, a popular supplement for muscle building, induces itch and tingling after consumption, but the underlying molecular and neural mechanisms are obscure. Here we show that, in mice, β-alanine elicited itch-associated behavior that requires MrgprD, a G protein-coupled receptor expressed by a subpopulation of primary sensory neurons. These neurons exclusively innervate the skin, respond to β-alanine, heat and mechanical noxious stimuli but do not respond to histamine. In humans, intradermally injected β-alanine induced itch but neither wheal nor flare suggesting that the itch was not mediated by histamine. Thus, the primary sensory neurons responsive to β-alanine are likely part of a histamine-independent itch neural circuit and a target for treating clinical itch that is unrelieved by anti-histamines.
doi:10.1523/JNEUROSCI.3509-12.2012
PMCID: PMC3491570  PMID: 23077038
16.  TLR3 deficiency impairs spinal cord synaptic transmission, central sensitization, and pruritus in mice 
The Journal of Clinical Investigation  2012;122(6):2195-2207.
Itch, also known as pruritus, is a common, intractable symptom of several skin diseases, such as atopic dermatitis and xerosis. TLRs mediate innate immunity and regulate neuropathic pain, but their roles in pruritus are elusive. Here, we report that scratching behaviors induced by histamine-dependent and -independent pruritogens are markedly reduced in mice lacking the Tlr3 gene. TLR3 is expressed mainly by small-sized primary sensory neurons in dorsal root ganglions (DRGs) that coexpress the itch signaling pathway components transient receptor potential subtype V1 and gastrin-releasing peptide. Notably, we found that treatment with a TLR3 agonist induces inward currents and action potentials in DRG neurons and elicited scratching in WT mice but not Tlr3–/– mice. Furthermore, excitatory synaptic transmission in spinal cord slices and long-term potentiation in the intact spinal cord were impaired in Tlr3–/– mice but not Tlr7–/– mice. Consequently, central sensitization–driven pain hypersensitivity, but not acute pain, was impaired in Tlr3–/– mice. In addition, TLR3 knockdown in DRGs also attenuated pruritus in WT mice. Finally, chronic itch in a dry skin condition was substantially reduced in Tlr3–/– mice. Our findings demonstrate a critical role of TLR3 in regulating sensory neuronal excitability, spinal cord synaptic transmission, and central sensitization. TLR3 may serve as a new target for developing anti-itch treatment.
doi:10.1172/JCI45414
PMCID: PMC3366391  PMID: 22565312
17.  Itch: Cells, Molecules, and Circuits 
ACS chemical neuroscience  2011;2(1):17-25.
The itch field has made great advances in recent years, building upon earlier work to form a clearer picture of the biology behind this important sensory modality. Models for how itch is encoded have emerged that fit with physiological, molecular, and behavioral data. The molecular mechanisms of itch, both peripherally and centrally, are being revealed with the aid of newer animal models. Future work must address shortcomings in our current understanding of itch including limitations of current experimental methods. Here we review what is known about the cells, molecules, and circuits involved in itch and highlight key questions that remain to be answered.
doi:10.1021/cn100085g
PMCID: PMC3123905  PMID: 21720568
Itch; pain; DRG neurons; skin; spinal cord; Mrgpr; GRPR; histamine; TRP
18.  Itch: Cells, Molecules, and Circuits 
ACS Chemical Neuroscience  2010;2(1):17-25.
The itch field has made great advances in recent years, building upon earlier work to form a clearer picture of the biology behind this important sensory modality. Models for how itch is encoded have emerged that fit with physiological, molecular, and behavioral data. The molecular mechanisms of itch, both peripherally and centrally, are being revealed with the aid of newer animal models. Future work must address shortcomings in our current understanding of itch including limitations of current experimental methods. Here we review what is known about the cells, molecules, and circuits involved in itch and highlight key questions that remain to be answered.
doi:10.1021/cn100085g
PMCID: PMC3123905  PMID: 21720568
Itch; pain; DRG neurons; skin; spinal cord; Mrgpr; GRPR; histamine; and TRP
19.  The Distinct Roles of Two GPCRs, MrgprC11 and PAR2, in Itch and Hyperalgesia 
Science signaling  2011;4(181):ra45.
Itch has been defined as an unpleasant skin sensation that triggers the urge to scratch. Primary sensory dorsal root ganglia neurons detect itch stimuli through peripheral axons in the skin, playing an important role in generating itch. Itch is broadly categorized as histaminergic (sensitive to antihistamines) or nonhistaminergic. The peptide Ser-Leu-Ile-Gly-Arg-Leu (SLIGRL) is an itch-inducing agent widely used to study histamine-independent itch. Here, we show that Mrgprs (Mas-related G protein–coupled receptors), particularly MrgprC11, rather than PAR2 (protease-activated receptor 2) as previously thought, mediate this type of itch. A shorter peptide, SLIGR, which specifically activates PAR2 but not MrgprC11, induced thermal pain hypersensitivity in mice but not a scratch response. Therefore, although both Mrgpr and PAR2 are SLIGRL-responsive G protein–coupled receptors present in dorsal root ganglia, each plays a specific role in mediating itch and pain.
doi:10.1126/scisignal.2001925
PMCID: PMC3144551  PMID: 21775281
20.  BAM8–22 peptide produces itch and nociceptive sensations in humans independent of histamine release 
Chronic itch accompanying many dermatological, neurological and systemic diseases is unresponsive to antihistamines. Our knowledge of endogenous chemicals that evoke histamine-independent itch and their molecular targets is very limited. Recently it was demonstrated in behavioral and cellular experiments that bovine adrenal medulla 8–22 peptide (BAM8–22), a proteolytically cleaved product of proenkephalin A, is a potent activator of Mas-related G protein-coupled receptors (Mrgprs), MrgprC11 and hMrgprX1, and induces scratching in mice in a Mrgpr-dependent manner. To study the sensory qualities that BAM8–22 evokes in humans we tested the volar forearm of 15 healthy volunteers with heat-inactivated cowhage spicules previously soaked in the peptide. BAM8–22 produced itch in each subject, usually accompanied by sensations of pricking/stinging and burning. The sensations were occasionally accompanied by one or more mechanically evoked dysesthesias, namely alloknesis, hyperknesis, and hyperalgesia, but no wheal or neurogenic flare in the skin surrounding the application site. The inactive truncated peptide BAM8–18 produced weak or no sensations. Pretreatment of the tested skin with an antihistamine cream (doxepin) inhibited the histamine-induced sensations, dysesthesias and skin reactions but not the sensations and dysesthesias evoked by BAM8–22. We show that BAM8–22 produces itch and nociceptive sensations in humans in a histamine-independent manner. Thus, BAM8–22 may be an endogenous itch mediator that activates, in humans, MrgprX1, a novel target for potential anti-itch treatments.
doi:10.1523/JNEUROSCI.1192-11.2011
PMCID: PMC3111068  PMID: 21593341
21.  TRPA1 is required for histamine-independent, Mas-related G protein-coupled receptor-mediated itch 
Nature neuroscience  2011;14(5):595-602.
SUMMARY
Itch, the unpleasant sensation that evokes a desire to scratch, accompanies numerous skin and nervous system disorders. In many cases, pathological itch is insensitive to antihistamine treatment. Recent studies have identified members of the Mas-related GPCR (Mrgpr) family that are activated by mast cell mediators and promote histamine-independent itch. MrgprA3 and MrgprC11 act as receptors for the pruritogens chloroquine and BAM8–22, respectively. However, the signaling pathways and transduction channels activated downstream of these pruritogens are largely unknown. We found that TRPA1 is the downstream target of both MrgprA3 and MrgprC11, in cultured sensory neurons and heterologous cells. TRPA1 is required for Mrgpr-mediated signaling, as sensory neurons from TRPA1-deficient mice exhibited profoundly diminished responses to chloroquine and BAM8–22. Likewise, TRPA1-deficient mice displayed little to no scratching in response to these pruritogens. Our findings demonstrate that TRPA1 is an essential component of the signaling pathways that promote histamine-independent itch.
doi:10.1038/nn.2789
PMCID: PMC3181150  PMID: 21460831
22.  An Itch To Be Scratched 
Neuron  2010;68(3):334-339.
The description of itch (formally known as pruritus) as an “unpleasant sensation that elicits the desire or reflex to scratch” (Ikoma et al., 2006) is immediately familiar. Research in the field of pruritoception has added to our understanding of this area of sensory neurobiology as it pertains to both normal and pathological conditions. In particular, much progress has been made on the mechanisms and circuits of itch, which we review here.
doi:10.1016/j.neuron.2010.10.018
PMCID: PMC3111050  PMID: 21040839
23.  Pirt, a TRPV1 Modulator, Is Required for Histamine-Dependent and -Independent Itch 
PLoS ONE  2011;6(5):e20559.
Itch, or pruritus, is an important clinical problem whose molecular basis has yet to be understood. Recent work has begun to identify genes that contribute to detecting itch at the molecular level. Here we show that Pirt, known to play a vital part in sensing pain through modulation of the transient receptor potential vanilloid 1 (TRPV1) channel, is also necessary for proper itch sensation. Pirt−/− mice exhibit deficits in cellular and behavioral responses to various itch-inducing compounds, or pruritogens. Pirt contributes to both histaminergic and nonhistaminergic itch and, crucially, is involved in forms of itch that are both TRPV1-dependent and -independent. Our findings demonstrate that the function of Pirt extends beyond nociception via TRPV1 regulation to its role as a critical component in several itch signaling pathways.
doi:10.1371/journal.pone.0020559
PMCID: PMC3105090  PMID: 21655234
24.  Sensory neuron-specific GPCRs Mrgprs are itch receptors mediating chloroquine-induced pruritus 
Cell  2009;139(7):1353-1365.
SUMMARY
The cellular and molecular mechanisms mediating histamine-independent itch in primary sensory neurons are largely unknown. Itch induced by chloroquine (CQ) is a common side-effect of this widely used anti-malarial drug. Here we show that Mrgprs, a family of G protein-coupled receptors expressed exclusively in peripheral sensory neurons, function as itch receptors. Mice lacking a cluster of Mrgpr genes display significant deficits in itch induced by CQ but not histamine. CQ directly excites sensory neurons in an Mrgpr-dependent manner. CQ specifically activates mouse MrgprA3 and human MrgprX1. Loss- and gain-of-function studies demonstrate that MrgprA3 is required for CQ responsiveness in mice. Furthermore, MrgprA3-expressing neurons respond to histamine and co-express Gastrin-Releasing Peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with MrgprC11-specific agonist BAM8-22 induces itch in wild-type but not mutant mice. Therefore, Mrgprs may provide molecular access to itch-selective neurons and constitute novel targets for itch therapeutics.
doi:10.1016/j.cell.2009.11.034
PMCID: PMC2989405  PMID: 20004959
25.  Pirt, a Phosphoinositide-Binding Protein, Functions as a Regulatory Subunit of TRPV1 
Cell  2008;133(3):475-485.
SUMMARY
Transient receptor potential vanilloid 1 (TRPV1) is a molecular sensor of noxious heat and capsaicin. Its channel activity can be modulated by several mechanisms. Here we identify a membrane protein, Pirt, as a regulator of TRPV1. Pirt is expressed in most nociceptive neurons in the dorsal root ganglia (DRG) including TRPV1-positive cells. Pirt null mice show impaired responsiveness to noxious heat and capsaicin. Noxious heat- and capsaicin-sensitive currents in Pirt-deficient DRG neurons are significantly attenuated. Heterologous expression of Pirt strongly enhances TRPV1-mediated currents. Furthermore, the C terminus of Pirt binds to TRPV1 and several phosphoinositides, including phosphatidylinositol-4,5-bisphosphate (PIP2), and can potentiate TRPV1. The PIP2 binding is dependent on the cluster of basic residues in the Pirt C terminus and is crucial for Pirt regulation of TRPV1. Importantly, the enhancement of TRPV1 by PIP2 requires Pirt. Therefore, Pirt is a key component of the TRPV1 complex and positively regulates TRPV1 activity.
doi:10.1016/j.cell.2008.02.053
PMCID: PMC2605970  PMID: 18455988

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