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.

Plumbagin, Juglone, and Boropinal as Novel TRPA1 Agonists
Kerstin Hill,*,† Serena Fiorito,‡ Vito Alessandro Taddeo,‡ Anja Schulze,§ Marion Leonhardt,† Francesco Epifano,*,‡ and Salvatore Genovese‡
†Rudolf-Boehm-InstitutfürPharmakologieundToxikologie,UniversitaẗLeipzig,Har̈telstr.16-18,04107Leipzig,Germany ‡Department of Pharmacy, University “G. D’Annunzio” of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti Scalo (CH), Italy §Fraunhofer-Institut für Zelltherapie und Immunologie IZI, Biozentrum, Weinbergweg 22, 06120 Halle, Germany

Plumbagin, Juglone, and Boropinal as Novel TRPA1 Agonists

ABSTRACT: A series of seven oxyprenylated phenylpropanoids and naphthoquinones were tested regarding their ability to activate transient receptor potential ankyrin subtype 1 channel (TRPA1). Three of the assayed compounds, namely, boropinal (3), juglone (5), and plumbagin (7), acted as strong modulators of TRPA1 channels with EC50 values of 9.8, 1.7,and 0.5 μM, respectively, as assessed by Ca2+ assays. Moreover, the compounds elicited TRPA1 currents in electrophysiological whole cell recordings. We additionally provide evidence that plumbagin activated TRPA1-positive neurons isolated from mouse dorsal root ganglion neurons but did not affect sensory neurons from TRPA1-deficient mice. The high potencies of plumbagin and juglone to activate TRPA1 channels may explain the molecular basis of the mucosal irritant properties of these compounds as well as of related naphthoquinones and phytopreparations, as widely reported in the literature.

PPARγ ameliorated LPS induced inflammation of HEK cell line expressing both human Toll-like receptor 4 (TLR4) and MD2.

Voltage-Gated Potassium Channels Involved in Regulation of Physiological Function in MrgprA3-Specific Itch Neurons.

TRPV1 and PLC Participate in Histamine H4 Receptor-Induced Itch

¹College of Basic Medicine, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, China
²College of Life Science, Nanjing Normal University, Nanjing 210046, China
³College of Basic Medicine, Guangxi University of Chinese Medicine, 13 Wuhe Road, Nanning 530200, China

TRPV1 and PLC Participate in Histamine H4 Receptor-Induced Itch

GROWTH FACTORS, CYTOKINES, AND CELL CYCLE MOLECULES
Histamine Released from Epidermal Keratinocytes Plays a Role in a-MelanocyteeStimulating Hormone-Induced Itching in Mice

Histamine Released from Epidermal Keratinocytes Plays a Role in a-MelanocyteeStimulating Hormone-Induced Itching in Mice

Kyoko Shimizu,* Tsugunobu Andoh,y Yoko Yoshihisa,* and Tadamichi Shimizu*
From the Departments of Dermatology* and Applied Pharmacology,y Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan

Sunburn, wound repair, and chronic renal failure with hemodialysis are usually accompanied by both pigmentation and itching. Proopiomelanocortin-derived a-melanocyteestimulating hormone (a-MSH) is produced in response to external stimuli, such as UV irradiation, and is involved in cutaneous pigmen- tation. However, it is unclear whether a-MSH is also involved in the itching. We therefore investigated whether a-MSH elicited itch-related responses in mice. We found that an intradermal injection of a-MSH induced hind-paw scratching, an itch-related response, in mice. The a-MSHeinduced scratching was inhibited by the m-opioid receptor antagonist naltrexone and the H1 histamine receptor antagonist terfenadine. In mast cell-deficient mice, a-MSH also elicited scratching, which was inhibited by terfe- nadine. The immunoreactivity for L-histidine decarboxylase, a key enzyme required for the production of histamine, histamine, and the melanocortin 1 and 5 receptors were shown in not only mast cells but also keratinocytes in murine skin. In addition to the expression of L-histidine decarboxylase and melanocortin 1 and 5 receptors, the mouse keratinocyte cell lines (Pam212) also showed immunoreactivity for L-histidine decarboxylase, histamine, and melanocortin 1 and 5 receptors. The application of a-MSH induced the release of histamine from Pam212 cells. These findings indicate that a-MSH may play an important role in the itching associated with pigmented cutaneous lesions and that the histamine released from keratinocytes is involved in this a-MSHeinduced itching. (Am J Pathol 2015, 185: 3003e3010; http://dx.doi.org/10.1016/j.ajpath.2015.07.015)

The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons

3-iodothyroacetic acid, a metabolite of thyroid hormone, induces itch and reduces threshold to noxious and to painful heat stimuli in mice.

Sensory Neuron-Specific GPCR Mrgprs Are Itch Receptors Mediating Chloroquine-Induced Pruritus

Qin Liu,1 Zongxiang Tang,1 Lenka Surdenikova,2,4 Seungil Kim,5 Kush N. Patel,1 Andrew Kim,1 Fei Ru,2 Yun Guan,3
Hao-Jui Weng,1 Yixun Geng,1 Bradley J. Undem,2 Marian Kollarik,2 Zhou-Feng Chen,5 David J. Anderson,6,7
and Xinzhong Dong1,7,*

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 antimalarial 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 coexpress gastrin-releasing
peptide, a peptide involved in itch sensation,
and MrgprC11. Activation of these neurons with the
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.

2.Sensory Neuron-Specific GPCR Mrgprs

Toll-like receptor 4 contributes to chronic itch, alloknesis and spinal astrocyte activation in male mice
Tong Liu , Qingjian Han , Gang Chen , Ya Huang , Lin-Xia Zhao , Temugin Berta ,Yong-Jing Gao , and Ru-Rong Ji

Running title: TLR4 and glial signaling in acute and chronic itch

00006396-900000000-99651

Abstract
Increasing evidence suggests that Toll-like receptor 4 (TLR4) contributes importantly to spinal cord glial activation and chronic pain sensitization; however, its unique role in acute and chronic itch is unclear. In this study, we investigated the involvement of TLR4 in acute and chronic itch models in male mice using both transgenic and pharmacological approaches. Tlr4−/− mice exhibited normal acute itch induced by compound 48/80 and chloroquine, but these mice showed substantial reductions in scratching in chronic itch models of dry skin, induced by acetone and diethyether followed by water (AEW), contact dermatitis, and allergic contact dermatitis on the neck. Intrathecal (spinal) inhibition of TLR4 with lipopolysaccharide Rhodobacter sphaeroides (LPS-RS) did not affect acute itch but suppressed AEW-induced chronic itch. Compound 48/80 and AEW also produced robust alloknesis, a touch-elicited itch in wild-type mice, which was suppressed by intrathecal LPS-RS and Tlr4−/− deletion. AEW induced persistent upregulation of Tlr4 mRNA and increased TLR4 expression in GFAP-expressing astrocytes in spinal cord dorsal horn. AEW also induced TLR4-dependent astrogliosis (GFAP upregulation) in spinal cord. Intrathecal injection of astroglial inhibitor L-α- aminoadipate reduced AEW-induced chronic itch and alloknesis without affecting acute itch. Spinal TLR4 was also necessary for AEW-induced chronic itch in the cheek model. Interestingly, scratching plays an essential role in spinal astrogliosis, since AEW-induced astrogliosis was abrogated by putting Elizabethan Collars on the neck to prevent scratching the itchy skin. Our findings suggest that spinal TLR4 signaling is important for spinal astrocyte activation and astrogliosis that may underlie alloknesis and chronic itch.
Key words: Alloknesis (touch-evoked itch), astrogliosis, dry skin, innate immunity, lipopolysaccharide (LPS), Toll-like receptor 4 (TLR4)