Mechanisms of Pruritogen-Induced Activation of Itch Nerves in Isolated Mouse Skin

Ru, F1., Sun, H1., Jurcakova, D.1,3, Herbstsomer, R.A1., Meixong, J2., Dong, X2, Undem, B.J. 1
Departments of Medicine1 and Neuroscience2, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA, 3Department of Pathophysiology, Biomedical Center Martin, Jessenius Medical School, Comenius University, Martin, Slovakia

Running Title: Characterization of itch causing nerves

Mechanisms of Pruritogen-Induced Activation of Itch Nerves in Isolated Mouse Skin

Key Points Summary
Chloroquine (CQ) stimulates itch nerves and causes intense scratching in mice by activating the G- Protein Coupled Receptor (GPCR) MrgprA3. It is not known how stimulation of MrgprA3 (or other GPCRs) leads to activation of the itch nerve terminals in the skin, but previous studies have found that TRPA1 gene deletion blocks CQ-induced scratching.
In the present study we used a novel dorsal skin-nerve preparation to evaluate mechanisms underlying CQ- and histamine-induced action potential discharge in itch nerve terminals.
We found that CQ activation of the nerves requires the beta3 isoform of phospholipase C, however, TRPA1 or other TRP channel are not required. Evidence is provided for a role for calcium-activated chloride channels such as TMEM16a in GPCR-activation of itch nerve terminals.
The mechanism by which TRP channels participate in pruritogen-induced scratching may involve sites of action other than the primary afferent terminals.

Tmem100 Is a Regulator of TRPA1-TRPV1 Complex and Contributes to Persistent Pain

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Hao-Jui Weng,1,2 Kush N. Patel,1 Nathaniel A. Jeske,3 Sonya M. Bierbower,3 Wangyuan Zou,4 Vinod Tiwari,5 Qin Zheng,1 Zongxiang Tang,6 Gary C.H. Mo,7 Yan Wang,1,8 Yixun Geng,1 Jin Zhang,1,7 Yun Guan,5 Armen N. Akopian,9,*
and Xinzhong Dong1,10,*
1Departments of Neuroscience and Neurosurgery, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA 2Department of Dermatology, National Taiwan University Hospital, Taipei City 100, Taiwan
3Department of Physiology, University of Texas Health Science Center, San Antonio, TX 78229, USA
4Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China
5Department of Anesthesiology and Critical Care, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA 6Nanjing University of Chinese Medicine, Nanjing 210046, China
7Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA 8West China School of Stomatology, Sichuan University, Chengdu, Sichuan 610041, China
9Department of Endodontics, University of Texas Health Science Center, San Antonio, TX 78229, USA
10Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
*Correspondence: akopian@uthscsa.edu (A.N.A.), xdong2@jhmi.edu (X.D.)
http://dx.doi.org/10.1016/j.neuron.2014.12.065

TRPA1 and TRPV1 are crucial pain mediators, but how their interaction contributes to persistent pain is unknown. Here, we identify Tmem100 as a potenti- ating modulator of TRPA1-V1 complexes. Tmem100 is coexpressed and forms a complex with TRPA1 and TRPV1 in DRG neurons. Tmem100-deficient mice show a reduction in inflammatory mechanical hyperalgesia and TRPA1- but not TRPV1-mediated pain. Single-channel recording in a heterologous system reveals that Tmem100 selectively potenti- ates TRPA1 activity in a TRPV1-dependent manner. Mechanistically, Tmem100 weakens the association of TRPA1 and TRPV1, thereby releasing the inhibi- tion of TRPA1 by TRPV1. A Tmem100 mutant, Tmem100-3Q, exerts the opposite effect; i.e., it en- hances the association of TRPA1 and TRPV1 and strongly inhibits TRPA1. Strikingly, a cell-permeable peptide (CPP) containing the C-terminal sequence of Tmem100-3Q mimics its effect and inhibits persistent pain. Our study unveils a context-depen- dent modulation of the TRPA1-V1 complex, and Tmem100-3Q CPP is a promising pain therapy.

Antioxidants Attenuate Acute and Chronic Itch: Peripheral and Central Mechanisms of Oxidative Stress in Pruritus

Feng-Ming Zhou1,3 • Ruo-Xiao Cheng1,3 • Shuai Wang1,2 • Ya Huang3 • Yong-Jing Gao4 • Yan Zhou3 • Teng-Teng Liu3 • Xue-Long Wang5 • Li-Hua Chen2 • Tong Liu1,3

Antioxidants Attenuate Acute and Chronic Itch Peripheral and Central Mechanisms of Oxidative Stress in Pruritus

Received: 1 July 2016 / Accepted: 27 August 2016
ⓒ Shanghai Institutes for Biological Sciences, CAS and Springer Science+Business Media Singapore 2016

Abstract
Itch (pruritus) is one of the most disabling syndromes in patients suffering from skin, liver, or kidney diseases. Our previous study highlighted a key role of oxidative stress in acute itch. Here, we evaluated the effects of antioxidants in mouse models of acute and chronic itch and explored the potential mechanisms. The effects of systemic administration of the antioxidants N-acetyl-L-cysteine (NAC) and N-tert-butyl-α-phenylnitrone (PBN) were determined by behavioral tests in mouse models of acute itch induced by compound 48/80 or chloroquine, and chronic itch by treatment with a mixture of acetone-diethyl-ether-water. We found that systemic administration of NAC or PBN significantly alleviated compound 48/80- and chloroquine-induced acute itch in a dose-dependent manner, attenuated dry skin-induced chronic itch, and suppressed oxidative stress in the affected skin. Antioxidants significantly decreased the accumulation of intracellular reactive oxygen species directly induced by compound 48/80 and chloroquine in the cultured dorsal root ganglia-derived cell line ND7-23. Finally, the antioxidants remarkably inhibited the compound 48/80-induced phosphorylation of extracellular signal-regulated kinase in the spinal cord. These results indicated that oxidative stress plays a critical role in acute and chronic itch in the periphery and spinal cord and antioxidant treatment may be a promising strategy for anti-itch therapy.

Keywords

Oxidative stressAntioxidantsItchTRPA1Extracellular signal-regulated kinase
Feng-Ming Zhou and Ruo-Xiao Cheng have contributed equally to this work.

Li-Hua Chen chenlihua@suda.edu.cn
Tong Liu liutong80@suda.edu.cn
1 Department of Neurology, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China
2 Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Department of Nutrition and Food Hygiene, School of Public Health, Soochow University, Suzhou 215123, China
3 Institute of Neuroscience, Soochow University, Suzhou 215123, China
4 Institute of Nautical Medicine, Nantong University,Nantong 226001, China
5 Beijing Electric Power Hospital, Beijing 100073, China

Inhibition of TRPV1 channels by a naturally occurring omega-9 fatty acid reduces pain and itch

Inhibition of TRPV1 channels by a naturally occurring omega-9 fatty acid reduces pain and itch

Sara L. Morales-La ́zaro1, Itzel Llorente1, Fe ́lix Sierra-Ram ́ırez1, Ana E. Lo ́pez-Romero1, Miguel Ort ́ız-Renter ́ıa1, Barbara Serrano-Flores1, Sidney A. Simon2, Leo ́n D. Islas3 & Tamara Rosenbaum1

The transient receptor potential vanilloid 1 (TRPV1) ion channel is mainly found in primary nociceptive afferents whose activity has been linked to pathophysiological conditions including pain, itch and inflammation. Consequently, it is important to identify naturally occurring antagonists of this channel. Here we show that a naturally occurring mono- unsaturated fatty acid, oleic acid, inhibits TRPV1 activity, and also pain and itch responses in mice by interacting with the vanilloid (capsaicin)-binding pocket and promoting the stabilization of a closed state conformation. Moreover, we report an itch-inducing molecule, cyclic phosphatidic acid, that activates TRPV1 and whose pruritic activity, as well as that of histamine, occurs through the activation of this ion channel. These findings provide insights into the molecular basis of oleic acid inhibition of TRPV1 and also into a way of reducing the pathophysiological effects resulting from its activation.

1 Instituto de Fisiolog ́ıa Celular, Universidad Nacional Auto ́noma de Me ́xico, Circuito exterior s/n, Coyoacan 04510, Mexico. 2 Department of Neurobiology, Duke University, 327C Bryan Research Building, Durham, North Carolina 27710, USA. 3 Departamento de Fisiolog ́ıa, Facultad de Medicina, Universidad Nacional Auto ́noma de Me ́xico, Circuito escolar s/n, Coyoacan 04510, Mexico. Correspondence and requests for materials should be addressed to T.R. (email: trosenba@ifc.unam.mx).

A histamine-independent itch path- way is required for allergic ocular itch

A histamine-independent itch pathway is required for allergic ocular itch

Cheng-Chiu Huang, PhDa, Yu Shin Kim, PhDb, William P. Olson, BSc, Fengxian Li, MDa, d, Changxiong Guo, BAa, Wenqin Luo, MD, PhDc, Andrew J.W. Huang, MD, MPHe, Qin Liu, PhDa, e,

From athe Department of Anesthesiology and the Center for the Study of Itch, Washing- ton University School of Medicine, St Louis, Mo; bthe Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Md; cthe Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pa; dthe Department of Anesthesiology, Zhujiang Hospital, Southern Medical University, Guangzhou, China; and ethe Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St Louis, Mo.

Itch is the cardinal symptom of allergic conjunctivitis and afflicts 15% to 20% of the population worldwide. Histamine produced by conjunctival mast cells has been implicated as the principal itch mediator that activates histamine receptors on primary sensory fibers to induce allergic ocular itch.1 However, antihistamines cannot completely relieve ocular itch in many cases, suggesting the involvement of a histamine-independent itch pathway. Herein, we sought to identify the histamine- independent neural pathway involved in allergic conjunctivitis and to develop new therapeutic strategies for allergic ocular itch.

Enhanced itch elicited by capsaicin in a chronic itch model

Enhanced itch elicited by capsaicin in a chronic itch model

Guang Yu, PhD1,2, Niuniu Yang, MD1, Fengxian Li, MD2,3, Meijuan Chen, PhD1, Changxiong J Guo, BS2, Changming Wang, PhD1, Danyou Hu, BS1, Yan Yang, BS1, Chan Zhu, BS1,
Zhongli Wang, PhD1, Hao Shi, MD1, Tana Gegen, MD1, Ming Tang, MS1, Qian He, MS1, Qin Liu, PhD2 and Zongxiang Tang, PhD1

Abstract
Chronic itch (pruritus) is an important clinical problem. However, the underlying molecular basis has yet to be understood. The Transient Receptor Potential Vanilloid 1 channel is a heat-sensitive cation channel expressed in primary sensory neurons and involved in both thermosensation and pain, but its role in chronic itch remains elusive. Here, we for the first time revealed an increased innervation density of Transient Receptor Potential Vanilloid 1-expressing sensory fibers in the skin afflicted with chronic itch. Further analysis indicated that this phenomenon is due to an expansion of Transient Receptor Potential Vanilloid 1-expressing sensory neurons under chronic itch conditions. As a functional correlates of this neuronal expansion, we observed an enhanced neuronal responsiveness to capsaicin under the dry skin conditions. Importantly, the neuronal hypersensitivity to capsaicin results in itch, rather than pain sensation, suggesting that the up-regulated Transient Receptor Potential Vanilloid 1 underlies the pain-to-itch switch under chronic itchy conditions. The study shows that there are different mechanisms of chronic pain and itching, and Transient Receptor Potential Vanilloid 1 plays an important role in chronic itch.

Keywords
Chronic itch, pain, Transient Receptor Potential Vanilloid 1, calcium imaging Date received: 5 January 2016; revised: 20 March 2016; accepted: 20 March 2016

Anti-pruritic effect of baicalin and its metabolites, baicalein and oroxylin A, in mice

Anti-pruritic effect of baicalin and its metabolites, baicalein and oroxylin A, in mice

Hien-trung TRINH1, Eun-ha JOH1, Ho-young KWAK2, Nam-in BAEK2, Dong-hyun KIM1, *
1Department of Pharmaceutical Science and Department of Life and Pharmaceutical Sciences, Kyung Hee University, 1, Hoegi, Dongdaemun-ku, Seoul 130–701, Korea; 2Graduate School of Biotechnology and PMRC, Kyung Hee University, 1, Seochunri, Suwon, Kyunggi-Do 449–701, Korea
Aim: To explore whether intestinal micro ora plays a role in anti-pruritic activity of baicalin, a main constituent of the rhizome of Scutellaria baicalensis (SB).
Methods: Baicalin was anaerobically incubated with human fecal micro ora, and its metabolites, baicalein and oroxylin A, were isolated. The inhibitory effect of baicalin and its metabolites was accessed in histamine- or compound 48/80-induced scratching behavior in mice.
Results: Baicalin was metabolized to baicalein and oroxylin A, with metabolic activities of 40.2±26.2 and 1.2±1.1 nmol·h-1·mg-1 wet weight of human fecal micro ora, respectively. Baicalin (20, 50 mg/kg) showed more potent inhibitory effect on histamine-induced scratching behavior when orally administered than intraperitoneally. In contrast, baicalein and oroxylin A had more potent inhibitory effect when the intraperitoneally administered. The anti-scratching behavior activity of oral baicalin and its metabolites was in proportion to their inhibition on histamine-induced increase of vascular permeability with oroxylin A more potent than baicalein and baicalin. In Magnus test using guinea pig ileum, oroxylin A is more potent than baicalein and baicalin in inhibition of histamine-induced contraction. The anti-scratching behavioral effect of oral baicalin was signi cantly reduced when oral antibiotics were simultaneously administered, whereas the effect of baicalein and oroxylin A were not affected.
Conclusion: Oral baicalin may be metabolized by intestinal micro ora into baicalein and oroxylin A, which ameliorate pruritic reactions through anti-histamine action.
Keywords: Scutellaria baicalensis; baicalin; baicalein; oroxylin A; scratching behavior; metabolism; histamine Acta Pharmacologica Sinica (2010) 31: 718–724; doi: 10.1038/aps.2010.42; published online 10 May 2010

Osthole inhibits histamine-dependent itch via modulating TRPV1 activity

srep25657

Osthole, an active coumarin isolated from Cnidium monnieri (L.) Cusson, has long been used in China as an antipruritic herbal medicine; however, the antipruitic mechanism of osthole is unknown. We studied the molecular mechanism of osthole in histamine-dependent itch by behavioral test, Ca2+ imaging,
and electrophysiological experiments. First, osthole clearly remitted the scratching behaviors of mice induced with histamine, HTMT, and VUF8430. Second, in cultured dorsal root ganglion (DRG) neurons, osthole showed a dose-dependent inhibitory e ect to histamine. On the same neurons, osthole also decreased the response to capsaicin and histamine. In further tests, the capsaicin-induced inward currents were inhibited by osthole. These results revealed that osthole inhibited histamine-dependent itch by modulating TRPV1 activity. This study will be helpful in understanding how osthole exerts anti- pruritus e ects and suggests that osthole may be a useful treatment medicine for histamine-dependent itch.

Long-Term Activation of Group I Metabotropic Glutamate Receptors Increases Functional TRPV1- Expressing Neurons in Mouse Dorsal Root Ganglia

Takayoshi Masuoka1*, Makiko Kudo1, Junko Yoshida1, Takaharu Ishibashi1,2, Ikunobu Muramatsu1, Nobuo Kato3, Noriko Imaizumi1 and Matomo Nishio1
1 Department of Pharmacology, School of Medicine, Kanazawa Medical University, Uchinada, Japan, 2 Department
of Pharmacology, School of Nursing, Kanazawa Medical University, Uchinada, Japan, 3 Department of Physiology I, School of Medicine, Kanazawa Medical University, Uchinada, Japan

fncel-10-00079

Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1) in dorsal root ganglion (DRG) neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h) treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG) increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC), a transient receptor potential ankyrin type 1 (TRPA1) agonist. Increase in the proportion was suppressed by phospholipase C (PLC), protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for 4 h post injection. This chronic hyperalgesia was attenuated by treatment with either mGluR1 or mGluR5 antagonists. These results suggest that long-term activation of mGluR1/5 by peripherally released glutamate may increase the number of neurons expressing functional TRPV1 in DRG, which may be strongly associated with chronic hyperalgesia.
Keywords: metabotropic glutamate receptors, TRPV1, TRPA1, dorsal root ganglion, heat hyperalgesia

A Cell-Based Functional Assay Using a Green Fluorescent Protein-Based Calcium Indicator dCys-GCaMP
Bin Cai,1 Xia Chen,1 Fang Liu,1 Jun Li,1 Lijuan Gu,2 Jason R. Liu,3 and Jay Liu1
1Rugen Therapeutics Ltd., Suzhou Industrial Park, China. 2Biotech Development Co., Ltd., Suzhou Industrial Park, China. 3Summer Intern from Penn State University, University Park, Pennsylvania.

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Measurement of the changes in intracellular Ca2+ levels is an important assay for drug discovery. In this report, we describe a novel Ca2+ indicator, dCys-GCaMP, based on the green fluorescent protein and the development of a rapid and simple cell-based functional assay using this new Ca2+ indicator. We demonstrated the sensitivity and reliability of the assay by measuring the cellular responses to the agonists, antagonists, channel blockers, and modulators of the ionotropic N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. HEK293 cells coexpressing the NMDA receptor and dCys-GCaMP displayed a strong increase in fluorescence intensity when stimulated with the agonist glutamate. This increase in the fluorescence signal was agonist concentration dependent and could be blocked by NMDAR antagonists and channel blockers. The pharmacological parameters measured with the dCys-GCaMP assay are in close agreement with those derived from conventional assays with synthetic dye fluo-4 and literature values. In addition, we showed that this assay could be used on G protein-coupled receptors as well, as exemplified by studies on the a1A adrenergic receptor. A limited scale evaluation of the assay performance in a 96-well compound screening format suggests that the dCys-GCaMP assay could be easily adapted to a high-throughput screening environ- ment. The most important advantage of this new assay over the con- ventional fluo-4 and aequorin assays is the elimination of the dye-loading or substrate-loading process.