journal club 2015.01.23.

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Protein kinase Cdelta mediates histamine-evoked itch and responses in pruriceptors

Molecular Pain 2015, 11:1 doi:10.1186/1744-8069-11-1

Manouela V Valtcheva (valtchevam@wusm.wustl.edu) Steve Davidson (sdavidson@wustl.edu) Chengshui Zhao (zhaoc@anest.wustl.edu) Michael Leitges (michael.leitges@biotek.uio.no) Robert W Gereau IV (gereaur@wustl.edu)

1744-8069-11-1

Abstract Background

Itch-producing compounds stimulate receptors expressed on small diameter fibers that innervate the skin. Many of the currently known pruritogen receptors are Gq-Protein Coupled Receptors (GqPCR), which activate Protein Kinase C (PKC). Specific isoforms of PKC have been previously shown to perform selective functions; however, the roles of PKC isoforms in regulating itch remain unclear. In this study, we investigated the novel PKC isoform PKCδ as an intracellular modulator of itch signaling in response to histamine and the non- histaminergic pruritogens chloroquine and β-alanine.

Results

Behavioral experiments indicate that PKCδ knock-out (KO) mice have a 40% reduction in histamine-induced scratching when compared to their wild type littermates. On the other hand, there were no differences between the two groups in scratching induced by the MRGPR agonists chloroquine or β-alanine. PKCδ was present in small diameter dorsal root ganglion (DRG) neurons. Of PKCδ-expressing neurons, 55% also stained for the non-peptidergic marker IB4, while a smaller percentage (15%) expressed the peptidergic marker CGRP. Twenty-nine percent of PKCδ-expressing neurons also expressed TRPV1. Calcium imaging studies of acutely dissociated DRG neurons from PKCδ-KO mice show a 40% reduction in the total number of neurons responsive to histamine. In contrast, there was no difference in the number of capsaicin-responsive neurons between KO and WT animals. Acute pharmacological inhibition of PKCδ with an isoform-specific peptide inhibitor (δV1-1) also significantly reduced the number of histamine-responsive sensory neurons.

Conclusions

Our findings indicate that PKCδ plays a role in mediating histamine-induced itch, but may be dispensable for chloroquine- and β-alanine-induced itch.

journal club – 2014.12.15.

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A natural dye, Niram improves atopic dermatitis through down-regulation of TSLP

Na-Ra Hana, Jin-Young Parkb, Jae-Bum Jangb, Hyun-Ja Jeongc,∗, Hyung-Min Kima,∗∗

a Department of Pharmacology, College of Korean Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
b Regional Innovation Center and Inflammatory Disease Research Center, Hoseo University, 165, Sechul-ri, Baebang-myun, Asan, Chungnam 336-795, Republic of Korea

c Department of Food Technology, Biochip Research Center, and Inflammatory Disease Research Center, Hoseo University, 165, Sechul-ri, Baebang-myun, Asan, Chungnam 336-795,Republic of Korea

1-s2.0-S1382668914002476-main

abstract

Naju Jjok (Polygonum tinctorium Lour.) has been known to treat skin diseases in traditional Korean medicine. A natural textile dye, Niram made from Naju Jjok has traditionally been used to dye clothes. Thymic stromal lymphopoietin (TSLP) plays an important role in the development of atopic dermatitis (AD). Thus, we investigated that Niram might amelio- rate AD through regulation of TSLP. Niram significantly inhibited the levels of TSLP through blockade of caspase-1/receptor-interacting protein 2 pathway in stimulated mast cells. Fur- ther, Niram ameliorated clinical symptoms in AD mouse. Niram significantly inhibited the infiltration of inflammatory cells in lesional skin. The levels of TSLP, caspase-1, IL-4, and IL-6 were inhibited in lesional skin applied topically with Niram. Niram significantly inhibited the serum levels of IgE and histamine in AD mouse. Finally, Niram significantly inhibited the levels of TSLP in polyriboinosinic polyribocytidylic acid-stimulated human keratinocyte HaCaT cells. These results establish Niram as a functional dye embracing the aspects of not only a traditional use but also a pharmacological effect.

© 2014 Elsevier B.V. All rights reserved.

Keywords : Niram, TSLP, Caspase-1, Mast cell, Atopic dermatitis, NC/Nga mice

journal club – 14.10.27.

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Propofol-induced pain sensation involves multiple mechanisms in sensory neurons

Rei Nishimoto & Makiko Kashio & Makoto Tominaga

Propofol

Received: 18 June 2014 / Revised: 22 September 2014 / Accepted: 25 September 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Propofol, a commonly used intravenous anesthetic agent, is known to at times cause pain sensation upon injection in humans. However, the molecular mechanisms underlying this effect are not fully understood. Although propofol was reported to activate human transient receptor potential ankyrin 1 (TRPA1) in this regard, its action on human TRP vanilloid 1 (TRPV1), another nociceptive receptor, is unknown. Further- more, whether propofol activates TRPV1 in rodents is con- troversial. Here, we show that propofol activates human and mouse TRPA1. In contrast, we did not observe propofol- evoked human TRPV1 activation, while the ability of propofol to activate mouse TRPV1 was very small. We also found that propofol caused increases in intracellular Ca2+ concentrations in a considerable portion of dorsal root gangli- on (DRG) cells from mice lacking both TRPV1 and TRPA1, indicating the existence of TRPV1- and TRPA1-independent mechanisms for propofol action. In addition, propofol pro- duced action potential generation in a type A γ-amino butyric acid (GABAA) receptor-dependent manner. Finally, we found that both T-type and L-type Ca2+ channels are activated downstream of GABAA receptor activation by propofol. Thus, we conclude that propofol may cause pain sensation through multiple mechanisms involving not only TRPV1 and TRPA1 but also voltage-gated channels downstream of GABAA receptor activation.

Keywords Propofol . TRPV1 . TRPA1 . Voltage-gated Ca2+ channel . GABAA receptor

journal club 20-10-2014

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Extracellular MicroRNAs Activate Nociceptor Neurons to Elicit Pain via TLR7 and TRPA1

Chul-Kyu Park,1,2 Zhen-Zhong Xu,1,2 Temugin Berta,1,2 Qingjian Han,1 Gang Chen,1 Xing-Jun Liu,1 and Ru-Rong Ji1,* 1Pain Signaling and Plasticity Laboratory, Departments of Anesthesiology and Neurobiology, Duke University Medical Center, Durham, NC 27710, USA
2Co-first authors

*Correspondence: ru-rong.ji@duke.edu http://dx.doi.org/10.1016/j.neuron.2014.02.011 Extracellular MicroRNAs Activate Nociceptor neurins to elicit pain via TLR7 and TRPV1

Intracellular microRNAs (miRNAs) are key regulators of gene expression. The role of extracellular miRNAs in neuronal activation and sensory behaviors are unknown. Here we report an unconventional role of extracellular miRNAs for rapid excitation of nocicep- tor neurons via toll-like receptor-7 (TLR7) and its coupling to TRPA1 ion channel. miRNA-let-7b induces rapid inward currents and action potentials in dorsal root ganglion (DRG) neurons. These re- sponses require the GUUGUGU motif, only occur in neurons coexpressing TLR7 and TRPA1, and are abolished in mice lacking Tlr7 or Trpa1. Furthermore, let-7b induces TLR7/TRPA1-dependent single-chan- nel activities in DRG neurons and HEK293 cells over- expressing TLR7/TRPA1. Intraplantar injection of let-7b elicits rapid spontaneous pain via TLR7 and TRPA1. Finally, let-7b can be released from DRG neurons by neuronal activation, and let-7b inhibitor reduces formalin-induced TRPA1 currents and spon- taneous pain. Thus, secreted extracellular miRNAs may serve as novel pain mediators via activating TLR7/TRPA1 in nociceptor neurons.

Journal Club 2014. 09. 29.

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The Bile Acid Receptor TGR5 Activates the TRPA1 Channel to Induce Itch in Mice Short Title: Bile Acid Evoked Itch

The Bile Acid Receptor TGR5 Activates the TRPA1 Channel to Induce Itch in Mice

TinaMarie Lieu, Gihan Jayaweera, Peishen Zhao, Daniel P. Poole, Dane Jensen, Megan Grace, Peter McIntyre, Romke Bron, Yvette M. Wilson, Matteus Krappitz, Silke Haerteis, Christoph Korbmacher, Martin S. Steinhoff, Romina Nassini, Serena Materazzi, Pierangelo Geppetti, Carlos U. Corvera, Nigel W. Bunnett

Abstract
Background & Aims: Patients with cholestatic disease have increased systemic concentrations of bile acids (BAs) and profound pruritus. The G protein-coupled BA receptor 1 TGR5 (encoded by GPBAR1) is expressed by primary sensory neurons; its activation induces neuronal hyperexcitability and scratching, by unknown mechanisms. We investigated whether the transient receptor potential ankyrin 1 (TRPA1) is involved in BA-evoked, TGR5-dependent pruritus in mice.

Methods: Co-expression of TGR5 and TRPA1 in cutaneous afferent neurons isolated from mice

was analyzed by immunofluorescence, in situ hybridization, and single-cell PCR. TGR5-induced

activation of TRPA1 was studied in in HEK293 cells, Xenopus laevis oocytes, and primary 2+

sensory neurons by measuring Ca signals. The contribution of TRPA1 to TGR5-induced release of pruritogenic neuropeptides, activation of spinal neurons, and scratching behavior were studied using TRPA1 antagonists or Trpa1–/– mice.

Results: TGR5 and TRPA1 protein and mRNA were expressed by cutaneous afferent neurons. In

HEK cells, oocytes, and neurons co-expressing TGR5 and TRPA1, BAs caused TGR5-dependent

activation and sensitization of TRPA1 by mechanisms that required Gβγ, protein kinase C and 2+

Ca . Antagonists or deletion of TRPA1 prevented BA-stimulated release of the pruritogenic neuropeptides gastrin-releasing peptide and atrial natriuretic peptide B in the spinal cord. Disruption of Trpa1 in mice blocked BA-induced expression of Fos in spinal neurons and prevented BA-stimulated scratching. Spontaneous scratching was exacerbated in transgenic mice that overexpressed TRG5. Administration of a TRPA1 antagonist or the BA sequestrant colestipol, which lowered circulating levels of BAs, prevented exacerbated spontaneous scratching in TGR5 overexpressing mice.

Conclusions: BAs induce pruritus in mice by co-activation of TGR5 and TRPA1. Antagonists of TGR5 and TRPA1, or inhibitors of the signaling mechanism by which TGR5 activates TRPA1, might be developed for treatment of cholestatic pruritus.

KEYWORDS: liver, mouse model, itching, signal transduction

Journal club 2014.07.25.

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Activation of TRPV1 mediates thymic stromal lymphopoietin release via the Ca2+/NFAT pathway in airway epithelial cells

Xinying Jia a, Hong Zhang a, Xu Cao b,⇑, Yuxin Yin a, Bo Zhang a,⇑ a Department of Pathology, Peking University Health Science Center, 100191 Beijing, China

b Department of Neurology, Peking University Health Science Center, 100191 Beijing, China

1-s2.0-S0014579314004773-main-2 S0014579314004773-fx1.jpg

1-s2.0-S0014579314004773-main-2

Abstract

The airway epithelium is exposed to a range of irritants in the environment that are known to trig- ger inflammatory response such as asthma. Transient receptor potential vanilloid 1 (TRPV1) is a Ca2+-permeable cation channel critical for detecting noxious stimuli by sensory neurons. Recently increasing evidence suggests TRPV1 is also crucially involved in the pathophysiology of asthma on airway epithelium in human. Here we report that in airway epithelial cells TRPV1 activation potently induces allergic cytokine thymic stromal lymphopoietin (TSLP) release. TSLP induction by protease-activated receptor (PAR)-2 activation is also partially mediated by TRPV1 channels.

Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Journal club 2014-06-27

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Ligand determinants of fatty acid activation of the pronociceptive ion channel TRPA1

peerj-248

William John Redmond1, Liuqiong Gu2, Maxime Camo1, Peter McIntyre2,3 and Mark Connor1

  1. 1  Australian School of Advanced Medicine, Macquarie University, NSW, Australia
  2. 2  Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia
  3. 3  Health Innovations Research Institute and School of Medical Sciences, RMIT University, Melbourne,

    Victoria, Australia

ABSTRACT

Background and purpose. Arachidonic acid (AA) and its derivatives are important modulators of cellular signalling. The transient receptor potential cation channel subfamily A, member 1 (TRPA1) is a cation channel with important functions in mediating cellular responses to noxious stimuli and inflammation. There is limited information about the interactions between AA itself and TRPA1, so we investigated the effects of AA and key ethanolamide and amino acid/neurotransmitter deriva- tives of AA on hTRPA1.

Experimental approach. HEK 293 cells expressing hTRPA1 were studied by mea- suring changes in intracellular calcium ([Ca]i) with a fluorescent dye and by stan- dard whole cell patch clamp recordings.
Key results. AA (30 μM) increased fluorescence in hTRPA1 expressing cells by 370% (notional EC50 13 μM). The covalent TRPA1 agonist cinnamaldehyde

(300 μM) increased fluorescence by 430% (EC50, 11 μM). Anandamide (230%) and N -arachidonoyl tyrosine (170%) substantially activated hTRPA1 at 30 μM, how- ever, N -arachidonoyl conjugates of glycine and taurine were less effective while
N -acyl conjugates of 5-HT did not affect hTRPA1. Changing the acyl chain length or the number and position of double bonds reduced fatty acid efficacy at hTRPA1. Mutant hTRPA1 (Cys621, Cys641 and Cys665 changed to Ser) could be activated by AA (100 μM, 40% of wild type) but not by cinnamaldehyde (300 μM). Conclusions and implications. AA is a more potent activator of TRPA1 than its ethanolamide or amino acid/neurotransmitter derivatives and acts via a mechanism distinct from that of cinnamaldehyde, further underscoring the likelyhood of multi- ple pharmacologically exploitable sites on hTRPA1.

Journal Club 2014-04-11

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BEHAVIORAL MODEL OF ITCH, ALLOKNESIS, PAIN AND ALLODYNIA IN THE LOWER HINDLIMB AND CORRELATIVE RESPONSES
OF LUMBAR DORSAL HORN NEURONS IN THE MOUSE

T. AKIYAMA, M. NAGAMINE, M. I. CARSTENS AND E. CARSTENS *

University of California, Davis, Department of Neurobiology, Physiology & Behavior, 1 Shields Avenue, Davis, CA 95616, USA

1-s2.0-S0306452214001018-main

Abstract—We have further developed a behavioral model of itch and pain in the lower hindlimb (calf) originally reported by LaMotte et al. (2011) that allows comparisons with responses of lumbar dorsal horn neurons to pruritic and noxious stimuli. Intradermal (id) microinjection of the prurit- ogens histamine, SLIGRL-NH2 (agonist of PAR-2 and MrgprC11) and chloroquine (agonist of MrgprA3) into the calf of the lower limb elicited significant biting and a small amount of licking directed to the injection site, over a 30- min time course. Following id injection of histamine, low-threshold mechanical stimuli reliably elicited discrete episodes of biting (alloknesis) over a longer time course; significantly less alloknesis was observed following id injec- tion of SLIGRL-NH2. Capsaicin injections elicited licking but little biting. Following id injection of capsaicin, low-thresh- old mechanical stimuli elicited discrete hindlimb flinches (allodynia) over a prolonged (>2 h) time course. In single- unit recordings from superficial lumbar dorsal horn neurons, low-threshold mechanically evoked responses were significantly enhanced, accompanied by receptive field expansion, following id injection of histamine in histamine- responsive neurons. This was not observed in histamine- insensitive neurons, or following id injection of saline or SLIGRL-NH2, regardless of whether the latter activated the neuron or not. These results suggest that itch-responsive neurons are selectively sensitized by histamine but not SLI- GRL-NH2 to account for alloknesis. The presently described ‘‘calf’’ model appears to distinguish between itch- and pain- related behavioral responses, and provides a basis to inves- tigate lumbar spinal neural mechanisms underlying itch, alloknesis, pain and allodynia. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO.

Key words: itch, alloknesis, pain, allodynia, scratching, dor- sal horn neuron.

Journal Club 2014-03-28

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TRPV3 is a calcium-permeable temperature-sensitive cation channel

12077604-Nature-2002-TRPV3 is a calcium-permeable temperature-sensitive

Haoxing Xu*†, I. Scott Ramsey*†, Suhas A. Kotecha*†,
Magdalene M. Moran‡, Jayhong A. Chong*, Deborah Lawson§, Pei Ge§, Jeremiah Lilly§, Inmaculada Silos-Santiago§, Yu Xie§,
Peter S. DiStefano§k, Rory Curtis§k & David E. Clapham*

* Howard Hughes Medical Institute, Children’s Hospital, Harvard Medical School, Enders 1309, 320 Longwood Avenue, Boston, Massachusetts 02115, USA ‡ Program in Neuroscience, Harvard Medical School, Goldenson Building B2-228, 220 Longwood Avenue, Boston, Massachusetts 02115, USA
§ Millennium Pharmaceuticals Inc., 75 Sidney Street, Cambridge, Massachusetts 02139, USA
† These authors contributed equally to this work

Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin (>43 8C; TRPV1 (ref. 1)), noxious heat (>52 8C; TRPV2 (ref. 2)), and cooling (< 22 8C; TRPM8 (refs 3, 4)) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between ,22 8C and 40 8C. Here we have identified a member of the vanilloid channel family, human TRPV3 (hTRPV3) that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 228C to 408C in mammalian cells transfected with hTRPV3 elevated intracellular calcium by activating a nonselective cat- ionic conductance. As in published recordings from sensory neurons, the current was steeply dependent on temperature, sensitized with repeated heating, and displayed a marked hyster- esis on heating and cooling5–10. On the basis of these properties, we propose that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1.

Journal Club 2014-03-22

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Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1

nature02282

Sven-Eric Jordt1, Diana M. Bautista1, Huai-hu Chuang1,
David D. McKemy1, Peter M. Zygmunt3, Edward D. Ho ̈ gesta ̈ tt3, Ian D. Meng2* & David Julius1

1Department of Cellular and Molecular Pharmacology and 2Department of Neurology, University of California, San Francisco, California 94143-2140, USA 3Department of Clinical Pharmacology, Institute of Laboratory Medicine,
Lund University Hospital, SE-221 85 Lund, Sweden

* Present address: Department of Physiology, College of Osteopathic Medicine, University of New England, 11 Hills Beach Road, Biddeford, Maine 04005, USA ………………………………………………………………………………………………………………………………………………………..

Wasabi, horseradish and mustard owe their pungency to iso- thiocyanate compounds. Topical application of mustard oil (allyl isothiocyanate) to the skin activates underlying sensory nerve endings, thereby producing pain, inflammation and robust

1,2 hypersensitivity to thermal and mechanical stimuli . Despite

their widespread use in both the kitchen and the laboratory, the molecular mechanism through which isothiocyanates mediate their effects remains unknown. Here we show that mustard oil depolarizes a subpopulation of primary sensory neurons that are also activated by capsaicin, the pungent ingredient in chilli peppers, and by D9-tetrahydrocannabinol (THC), the psycho- active component of marijuana. Both allyl isothiocyanate and THC mediate their excitatory effects by activating ANKTM1, a member of the transient receptor potential (TRP) ion channel family recently implicated in the detection of noxious cold3,4. These findings identify a cellular and molecular target for the pungent action of mustard oils and support an emerging role for TRP channels as ionotropic cannabinoid receptors5–8.

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