A central role for R7bp in the regulation of itch sensation

Pandey, Mritunjaya; Zhang, Jian-Huaa; Mishra, Santosh K.b; Adikaram, Poorni R.a; Harris, Benjamina; Kahler, John F.a; Loshakov, Annaa; Sholevar, Roxannea; Genis, Allisona; Kittock, Clairea; Kabat, Jurajc; Ganesan, Sundarc; Neubig, Richard R.d; Hoon, Mark A.e; Simonds, William F.a,*

doi: 10.1097/j.pain.0000000000000860
Research Paper

Abstract: Itch is a protective sensation producing a desire to scratch. Pathologic itch can be a chronic symptom of illnesses such as uremia, cholestatic liver disease, neuropathies and dermatitis, however current therapeutic options are limited. Many types of cell surface receptors, including those present on cells in the skin, on sensory neurons and on neurons in the spinal cord, have been implicated in itch signaling. The role of G protein signaling in the regulation of pruriception is poorly understood. We identify here 2 G protein signaling components whose mutation impairs itch sensation. R7bp (a.k.a. Rgs7bp) is a palmitoylated membrane anchoring protein expressed in neurons that facilitates Gαi/o -directed GTPase activating protein activity mediated by the Gβ5/R7-RGS complex. Knockout of R7bp diminishes scratching responses to multiple cutaneously applied and intrathecally-administered pruritogens in mice. Knock-in to mice of a GTPase activating protein-insensitive mutant of Gαo (Gnao1 G184S/+) produces a similar pruriceptive phenotype. The pruriceptive defect in R7bp knockout mice was rescued in double knockout mice also lacking Oprk1, encoding the G protein-coupled kappa-opioid receptor whose activation is known to inhibit itch sensation. In a model of atopic dermatitis (eczema), R7bp knockout mice showed diminished scratching behavior and enhanced sensitivity to kappa opioid agonists. Taken together, our results indicate that R7bp is a key regulator of itch sensation and suggest the potential targeting of R7bp-dependent GTPase activating protein activity as a novel therapeutic strategy for pathological itch.

Knockout of R7bp significantly impairs acute and chronic itch sensation, suggesting inhibition of R7bp function could represent a novel therapeutic strategy for pathological itch.

aMetabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA. Dr. Loshakov is now with the The Commonwealth Medical College, Scranton, PA, USA and Dr. Sholevar is now with the Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, USA

bDepartment of Molecular Biomedical Sciences, North Carolina State University, College of Veterinary Medicine, Raleigh, NC, USA

cResearch Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

dDepartment of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA

eMolecular Genetics Unit, Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA

Corresponding author. Address: National Institutes of Health, Bldg 10 Room 8C-101, 10 Center Dr MSC 1752, Bethesda, MD 20892-1752, USA. Tel.: 301-496-9299; fax: 301-480-3214. E-mail address: wfs@helix.nih.gov (W. F. Simonds).

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Received October 31, 2016

Received in revised form December 16, 2016

Accepted January 18, 2017

Article Outline
Back to Top | Article Outline

1. Introduction

Itch is an irritating sensation that typically alerts us to the presence on our skin's surface of potentially harmful agents, such as fungi, parasites, insects, and certain alkaloids. The itch sensation triggers a desire to scratch. Like pain, itch is a protective sensation that promotes behavior to minimize damage to the host from harmful environmental factors. Itch detection begins in the skin, is conveyed by afferent sensory neurons that project to the dorsal horn of the spinal cord, and traverses at least 2 spinal interneurons before reaching spinal projection neurons that transmit the signal to higher brain centers resulting in conscious pruriception.4,6,9,12,28 In contrast to acute itch, pathologic itch can be a chronic and often-debilitating manifestation of systemic and organ-specific illness.46

Many types of receptors, present on cells in the skin, on sensory neurons and on neurons in the spinal cord, have been implicated in itch signaling. Such receptors include both G protein-coupled receptors (GPCR) and non-GPCR.

Regulator of G protein signaling (RGS) proteins negatively regulate GPCR signaling in many cell types.31 RGS proteins act as GTPase activating proteins (GAPs) for G protein α subunits, accelerating the intrinsic hydrolysis rate of Gα-bound GTP to rapidly terminate G protein signaling. The R7-RGS subfamily of RGS proteins (RGS6, RGS7, RGS9, or RGS11) share similar domain architecture, including a G protein-γ like (GGL) domain that enables heterodimerization with Gβ5 (Fig. 1A).3,33,38 Gβ5 and R7-RGS proteins can be found in a heterotrimeric complex with R7-binding protein (R7BP, a.k.a. RGS7BP), a palmitoylated membrane-anchoring protein (Fig. 1A) (see Refs. 3,16 for reviews). The regulatory effects of the Gβ5/R7-RGS complex on GPCR signaling and its Gαi/o-directed GAP activity are greatly enhanced by R7BP palmitoylation and membrane anchoring.7,30 A body of in vitro evidence suggests that the GAP activity of Gβ5/R7-RGS-containing complexes is specific for pertussis toxin-sensitive Gα subunits (Gαo and/or Gαi).11,30,34,35 The physiology of the endogenous Gβ5/R7-RGS/R7BP complex within signaling circuits of the nervous system in vivo however is poorly understood.

We herein identify R7bp as a previously unsuspected critical regulator of acute and chronic pruriception in vivo, whose function to promote Gαi/o-directed GAP activity may represent a novel target for the therapy of pathological itch.

Back to Top | Article Outline

2. Materials and methods

2.1. Mouse husbandry and genotyping

Mice were housed and treated in strict accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and maintained in a specific-pathogen-free facility, with 4 to 5 animals per cage in a temperature-controlled room with a 12-hour light/dark cycle and access to food and water ad libitum. Age-matched female mice from littermate cohorts, 3 to 8 months of age, were used for all the experiments. Mice with a heterozygous germline deletion of exons 1 and 2 of R7bp in a C57BL/6 background were a generous gift of Dr. Kirill A. Martemyanov, University of Minnesota (currently at the Department of Neuroscience, Scripps Florida).2 Mice heterozygous for R7bp deletion were bred to generate wild type and R7bp homozygous KO pups for testing. Kappa-opioid receptor (Oprk1) knockout mice (B6.129S2-Oprk1tm1Kff/J; Stock No. 007558) were obtained from The Jackson Laboratory (Bar Harbor, ME). The generation and characterization of mice harboring a heterozygous gain-of-function knock-in mutation in Gnao1 that prevents Gαo turnoff by regulators of G protein signaling proteins (Gnao1 G184S/+) were previously described.8,18 For this study Gnao1 G184S/+ heterozygotes in a C57BL/6 background were employed. Mouse genotyping was performed by QPCR analysis of genomic DNA extracted from mouse tails using the DirectPCR (tail) solution (Cat. No. 102-T; Viagen, Los Angeles, CA) according to manufacturer's instructions. The R7bp knock out allele was identified employing the primer pair, Fwd: 5′-CTG CAA GCC AGT AGT GCC AGT CCC-3′, and Rev: 5′-GGA ACT TCG CTA GAC TAG TAC GCG T-3′. The wild type R7bp allele was identified employing the primer pair, Fwd: 5′-TCC AAG AGT TCA ACA CGC AAG TGG-3′, and Rev: 5′-GGC CAT TTC ACA GCC TTT GGT TCT-3′. The Oprk1 knock out allele was identified employing the primer pair, Fwd: 5′-AGG GGA TTT CAA CCT GTC TG-3′, and Rev: 5′-CTC CAG ACT GCC TTG GGA AAA-3′. The wild type Oprk1 allele was identified employing the primer pair, Fwd: 5′-AGG GGA TTT CAA CCT GTC TG-3′, and Rev: 5′-CCA CAC TGC CAT TAC TGT CG-3′.

Back to Top | Article Outline

2.2. Behavioral testing

For all behavioral testing the evaluator was blind to the genotype of the mouse being tested. Age-matched female mice from littermate cohorts, 3 to 8 months of age, were used for all the experiments. All behavioral testing was performed during the light cycle (daylight hours). Animal experiments were conducted according to NIH guidelines using protocols approved by the Animal Care and Use Committee of the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases.

Back to Top | Article Outline

2.3. Motor coordination testing

Motor coordination testing on the accelerating rotarod and open field-testing for locomotor activity were performed as previously described.47

Back to Top | Article Outline

2.4. Testing for nociception

The hot plate test for thermal analgesia was performed as described by Terzi et al.41 Thermal nociception using the tail immersion assay was assayed as previously described by Sun et al.40 The von Frey test for mechanical pain was performed as described by Sun and Chen39 using a set of calibrated von Frey filaments. Each filament was applied 5 consecutive times and the smallest filament that evoked reflexive flinches of the paw on 3 of the 5 trials was taken as the paw withdrawal threshold. Mechanical nociception was estimated by the Randall-Selitto assay utilizing an electronic algesimeter (IITC 2500 Digital Paw Pressure Meter; IITC Life Science, Woodland Hills, CA) as previously described.37 The nocifensive response to the activation of specific nociceptors in the trigeminal sensory ganglia that innervates the cornea was assessed by the eye wipe assay, using drops (50 μL) of dilute capsaicin (100 μM, Sigma No. M2028, Sigma-Aldrich, St. Louis, MO) or mustard oil-allyl isothiocyanate (10 μM, Sigma No. W203408, Sigma-Aldrich) dissolved in PBS, as previously described.17

Back to Top | Article Outline

2.5. Behavioral responses to intradermal pruritogens

The scratch response to intradermal injection of pruritogens was assayed as previously described by Sun et al.40 Briefly, after 2 hours of cage acclimation mice were injected intradermally with a pruritogen dissolved freshly in sterile PBS (10 μL) in the nape of the neck and then returned to their cage. Hind limb scratching behavior directed towards the injection site was recorded for 30 minutes at 5-minute intervals. A scratch was defined as the lifting of the hind limb towards the injection sites and then replacing it back on the floor. This is regardless of the number of scratching strokes taking place between the 2 movements.40 The pruritogens employed were of the highest purity commercially available and included classical pruritogens (endothelin-1 [25 ng/10 μL] [Sigma, Cat. No. E7764, Sigma-Aldrich], serotonin hydrochloride [10 μg/10 μL] [Sigma, Cat. No. H9523, Sigma-Aldrich], compound 48/80 [100 μg/10 μL] [Sigma, Cat. No. C2313, Sigma-Aldrich], SLIGRL-NH2 peptide [100 μg/10 μL] [Tocris, Cat. No. 1468, Tocris-Bio-Techne, Minneapolis, MN], chloroquine diphosphate salt [200 μg/10 μL] [Sigma, Cat. No. C6628, Sigma-Aldrich], histamine dihydrochloride [500 μg/10 μL] [Sigma, Cat. No. H7520, Sigma-Aldrich], formalin [0.5%/10 μL]), Tlr3-related agents {polyinosinic:polycytidylic acid (poly [I:C], Sigma, Cat. No. P9582, Sigma-Aldrich, St. Louis, MO) and polydeoxyinosinic:polydeoxycytidylic acid (poly [dI:dC], Sigma, Cat. No. P4929, Sigma-Aldrich)}, bile salt-related pruritogens (deoxycholic acid [25 μg/50 μL] [Sigma, Cat. No. D2510, Sigma-Aldrich], and taurolithocholic acid 3-sulfate disodium salt [25 μg/50 μL] [Sigma, Cat. No. T0512, Sigma-Aldrich]), and the cytokine thymic stromal lymphopoietin (2.5 μg/10 μL) (Cat. No. 148498; eBioscience, San Diego, CA). Individual mice were used for the testing of no more than 3 cutaneous or intrathecal pruritogens in the behavioral study, with at least 15 days of rest/washout in their home cage between tests.

Back to Top | Article Outline

2.6. Behavioral responses to intrathecal pruritogens

The scratch response to the intrathecal drug injection of pruritogens was performed as described by Hylden and Wilcox13 using a sterile, disposable 30 gauge, ½ inch needle and a total injection volume of 10 μL.32 Penetration of the lumbar spinal dura mater by the needle was confirmed by the classic tail flick response.14 Scratching responses were monitored over a period of 45 minutes following injection. Peptides for intrathecal testing were of the highest purity commercially available, and included gastrin-releasing peptide (GRP) (1-5 nmol) (Bachem, Cat. No. H3120, Bachem, Torrance, CA) and B-type natriuretic peptide (BNP; Nppb) (1-5 nmol) (Sigma, Cat. No. B9901, Sigma-Aldrich).32 Individual mice were used for the testing of no more than 3 cutaneous or intrathecal pruritogens in the behavioral study, with at least 15 days of rest/washout in their home cage between tests.

Back to Top | Article Outline

2.7. Atopic dermatitis (eczema) model

A mouse model of atopic dermatitis was employed as described previously with minor modification.23,40 Mice were shaved on the nape of the neck and sensitized by painting 0.2 mL of diphenylcyclopropenone (DCP) (1% wt/vol, dissolved in acetone) once on the shaved skin. Seven days after sensitization mice were again challenged by painting 0.2 mL of DCP (0.5%) on the same location daily, for 10 consecutive days. Thirty minutes following DCP application, mice were injected with saline intraperitoneally and then beginning 30 minutes post injection scratching behavior directed toward the painted skin area was quantified for the following 30 minutes. Scratching behavior was determined on days 1 to 5 and on days 8 to 10 during the 10 consecutive days of daily topical DCP application.

On the fifth, eighth and 10th days, just after quantifying scratching behavior for 30 minutes (after the intraperitoneal saline injection), mice were given an intraperitoneal injection of the specific kappa opioid receptor agonist U50,488 at 0.1, 0.25 and 2.5 mg/kg respectively. Thirty minutes after agonist administration mice scratching behavior directed towards the DCP painted area was counted again for another 30 minutes. The relative scratching response was calculated as the ratio of bouts of scratching following U50,488 injection to that following the preceding saline injection approximately an hour earlier in the same mouse.

Back to Top | Article Outline

2.8. Preparation of probes for in situ hybridization

Digoxigenin-labeled RNA probes were made by in vitro transcription using DIG RNA labeling Kit (SP6/T7) (Cat. No. 11175025910; Roche Life Science, Indianapolis, IN). For RNA probe generation from the SP6/T7 dual promoter vector, DNA templates in the range of 500 to 700 bp derived from cDNA were used. After preparation, probes were analyzed for RNA integrity and stored in aliquots at −70°C. PCR primers used to generate murine cDNA-derived DNA template inserts for the SP6/T7 dual promoter vector were: R7bp, Fwd: 5′-CTG TAC CGA GAG TTG GTC ATT T-3′, Rev: 5′-GAA CCT TCT CTT CCG TCT TCT G-3′; Rgs7, Fwd: 5′-CAT GGC TAC TTC TTT CCC ATC T-3′, Rev: 5′-CCC TCT GTT GAC TTG GTT CTT-3′; Gnb5, Fwd: 5′-ATC TGC CCT CAG GTC ATA GA-3′, Rev: 5′-GCT TGT GGT GGT CTG GSA TAA TA-3′; Rgs9, Fwd: 5′-GAC ACA GAC TAC GCC ATC TAT C-3′, Rev: 5′-CAT CTC TCC ACT CGC ATC TT-3′; Oprk1, Fwd: 5′-AAG TCA GGG AAG ATG TGG ATG T-3′, Rev: 5′-ACT GCA ACT ACT ACC AGC ACC A-3′. Primers used to generate other ISH probes were as previously described.32

Back to Top | Article Outline

2.9. In situ hybridization

Mice were anesthetized with avertin and their spinal cord and dorsal root ganglia were rapidly dissected out. Tissues were frozen with OCT embedding medium (Electron Microscopy Sciences [EMS], Hatfield, PA) and 12 micron-thick sections were cut on a cryostat at −20°C. Pairs of wild type and R7bp knockout sections, from either lumbar spinal cord or pooled dorsal root ganglia, were carefully collected onto the same slides and allowed to dry for 10 minutes at room temperature. Sections were fixed in 4% (wt/vol) paraformaldehyde (EMS) dissolved in RNase-free PBS for 10 minutes. Sections were washed in RNase-free PBS 3 times and then permeabilized in 10% TritonX-100 (Sigma Cat. No. T8787, Sigma-Aldrich) (vol/vol) for 5 minutes. After permeabilization, sections were acetylated in acetylation buffer for 10 minutes at room temperature. (Acetylation buffer was prepared by combining 675 μL triethanolamine [Cat. No. 90279; Sigma-Aldrich] with 125 μL of acetic anhydride [Cat. No. 320102; Sigma-Aldrich] in 50 mL of DEPC-treated water; acetylation buffer was prepared fresh and used within 10 minutes of preparation.) After acetylation, sections were washed in PBS 3 times. Sections were then incubated with digoxigenin-labeled RNA probes overnight at 68°C. Sections were then washed in ×0.2 SSC buffer at 70°C for 30 minutes. Sections were twice washed in a buffer containing 0.1 M maleic acid (Cat. No. M0375; Sigma-Aldrich) in 0.15 M NaCl, brought to pH 7.5 with solid NaOH, and then blocked with 1% (wt/vol) BSA for 30 minutes. Next, sections were incubated with alkaline phosphatase-labeled anti-digoxigenin antibodies (Roche) for 6 to 7 hours at room temperature. After incubation the sections were washed extensively and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) (Roche) developing agent was added. Sections were observed at different time intervals during color development. Pictures were taken using an inverted Leica microscope.

For double fluorescent in situ hybridization (FISH), digoxigenin-labeled RNA probes were made for R7bp and fluorescein-labeled RNA probes were made for Oprk1 by in vitro transcription as described above. Horseradish peroxidase-conjugated anti-digoxigenin antibody (Cat. No. 11207733910; Roche) and alkaline phosphatase-conjugated anti-fluorescein antibody (Cat. No. 11426346910; Roche) were used for detecting the RNA probes. The HNPP Fluorescent Detection Set (Cat. No. 11758888001; Roche) was used for detecting alkaline phosphatase-conjugated antibody and TSA Plus Fluorescence Kit (Cat. No. NEL741001KT; Perkin Elmer, Waltham, MA) was used for detecting horseradish peroxidase-conjugated antibody. Sections were washed at the end of reaction in PBS 5 to 6 times and mounted with mounting medium containing DAPI. Sections were immediately analyzed using a Leica inverted fluorescent microscope with capture of digital images.

Back to Top | Article Outline

2.10. Quantification of dorsal root ganglion cells expressing pruriceptive markers

Microscopic sections of dorsal root ganglion (DRG) cells expressing MrgprA3 or Nppb by in situ hybridization (ISH) were analyzed using the count tool of the Adobe Photoshop CC 2015 software. Images of ISH-stained sections were opened in TIFF format and each stained cell of every DRG section was counted by manually with computer mouse clicks. Six different DRG sections were counted for each genotype (wild type and R7BP knockout mice), with the evaluator blind to the genotype of the mouse from which the section originated.

Back to Top | Article Outline

2.11. Induction and quantification of spinal neuron c-Fos expression in response to injected pruritogen

Hundred microgram of compound 48/80 or 200 μg of chloroquine dissolved in PBS in a volume of 50 μL were injected subcutaneously into the nape of the neck by an investigator blind to genotype of the mouse under study. Four to 5 pairs of wild-type and R7bp knockout mouse littermates were analyzed per pruritogen. As a control, 50 μL of PBS vehicle only was injected in wild-type mice to estimate the effect of the skin prick sensation as a background signal. Two hours after injection, mice were anesthetized using Avertin and perfused with 20 mL of cold PBS followed by 25 mL of cold 4% paraformaldehyde dissolved in PBS. Whole spinal cords were dissected and tissues were post-fixed for 1 hour at 4°C, washed extensively with PBS, cryopreserved in 20% sucrose in PBS overnight, embedded in OTC (Electron Microscopy Sciences, Hatfield, PA), and frozen. Serial sections of 20 μm thickness were cut from cervical spinal cord on a cryostat and placed on slides. Tissue sections from wild-type and R7bp knockout mice were processed on the same slide to minimize differences in handling. For immunostaining, sections were blocked in 10% BSA and 0.25% Triton X-100 in PBS for 1 hour at RT. Sections were incubated with primary antibodies (c-Fos, sc-52; Santa Cruz, Dallas, TX; Anti-NeuN Antibody, clone A60, MAB377; EMD Millipore, Billerica, MA) in blocking solution overnight at 4°C. Slides were washed 4 times 5 minutes in PBS containing 0.1% Triton X-100. Detection was carried out using Alexa-Fluor-labeled secondary antibodies diluted 1: 500 in blocking solution. Sections were mounted using glycerol containing the nuclear dye DAPI for 1 minute at room temperature. Sections were visualized using a BZ-9000 fluorescence microscope (Keyence) at ×10 magnification and digital images were captured for counting. Four to 11 sections per animal were used for quantification, sampled across ∼1200 to 1500 μm axial distance. Quantification was performed by manual counting of the number of fluorescent c-Fos positive cells in the lateral dorsal horn by a second investigator blind to the genotype. The number of c-Fos cells from each lateral dorsal horn (R and L) in the sections was separately counted. The data presented represents the number of c-Fos positive cells per dorsal horn (spinal sections viewed at ×10 magnification) for different mouse genotypes and test substances (compound 48/80, chloroquine, or PBS vehicle).

Back to Top | Article Outline

2.12. Culture of dorsal root ganglion cells and intracellular calcium mobilization assay

Preparation and dissociation of mouse dorsal root ganglion cells was performed following the procedure of Malin et al.,29 with minor modification as noted. Wild-type and R7bp knockout cage-matched littermate mice were anesthetized using avertin and the dorsal root ganglia were rapidly dissected (without prior animal perfusion). The sensory ganglia were washed once in cold Hanks' Balanced Salt solution without calcium or magnesium (GIBCO, Gaithersburg, MD) and incubated with papain (Cat. No. NM100200; NeuroPapain, Genlantis, San Diego, CA) for 10 minutes at 37° followed by collagenase, type 2 (Cat. No. LS004174; Worthington Biochemical Corp, Lakewood, NJ) and dispase II (Cat. No. 17-105-041, GIBCO) at 37°. The ganglia were suspended in culture Ham's F12 medium containing 10% fetal calf serum, penicillin and streptomycin and triturated with fire polished Pasteur pipettes to isolate single neurons. The neurons with medium were plated onto Poly-D-lysine/laminin coated cover slips (Cat. No. 354087; Corning, Inc., Corning, NY) and grown in culture at 37° with 5% CO2.

A fluorescence-based assay for detecting intracellular calcium mobilization in the cultured dorsal root ganglia cells employed the Rhod-4 No Wash Calcium Assay Kit (Cat. No. ab112157; Abcam, Cambridge, MA), following the manufacturer's instructions for intracellular calcium assay. In short, culture medium was removed and Rhod-4 dye containing buffer was gently added to cells, followed by incubation at 37° for 30 minutes followed by 20 minutes at room temperature. For imaging and quantification of ligand-induced intracellular calcium mobilization, confocal images were acquired using a Leica DMI 6000 confocal microscope (Leica Microsystems, Exton, PA) enabled with ×20 multi-immersion objective NA 1.25. Images were acquired using high sensitive hybrid detectors to achieve a maximum signal to noise ratio and the time lapse images of the confocal scan were acquired every second over a 3 minute period. Sensory neuronal cultures were treated with either chloroquine or histamine (50 μM, final concentration), added after the first 10 seconds to establish a baseline and observe the steady-state changes in calcium mobilization in response to the ligand treatment. Acquired confocal images were post-processed using Imaris image processing software (Bitplane USA, Concord, MA). Fluorescent intensity changes were quantified using fixed parameters across the entire set of captured images.

Back to Top | Article Outline

2.13. Statistical methods

No sample size calculation was used to predetermine sample sizes. Sample size was chosen as a balance between establishing confidence in reproducibility (on one hand) and practical considerations, such as the time required to breed animals of a particular age, gender, and genotype (on the other). Sample size is reported in the legend to each figure. Data distribution was assumed to be normal, but this was not formally tested. For comparison of 2 data sets, the 2-tailed unpaired Student's t test or 2-way ANOVA test was employed, as appropriate. No data points were excluded. Significance level was set at P < 0.05 and all data are reported as mean ± SEM. Prism computer software, Version 5.0f (GraphPad Software, Inc, La Jolla, CA) was employed for the statistical analysis.

Back to Top | Article Outline

3. Results

3.1. Nociceptive somatosensation is largely preserved in mice lacking R7bp

R7bp is thought to function as a component of a heterotrimeric complex with Gβ5 and R7-RGS proteins (Fig. 1A), and R7bp and Rgs7 proteins are expressed in mouse dorsal root ganglia (DRG).24 The expression of R7bp and its potential functional partners in the heterotrimeric Gβ5/R7-RGS/R7BP complex was verified by in situ hybridization (ISH) in wild-type and R7bp KO mice. R7bp, Gβ5, Rgs7, and Rgs9 transcripts were readily identified in wild-type DRG (Fig. 1B upper panel), and knockout of R7bp did not appear to significantly affect the expression of the latter transcripts (Fig. 1B lower panel). We therefore wondered if the R7bp present in sensory neurons might regulate one or more modality of somatosensation.

Vertebrates utilize the capsaicin–sensitive Trpv1 channel expressed in peripheral sensory neurons for thermal nociception. DRG harvested from wild-type and R7bp KO mice showed no apparent difference in the expression of Trpv1 (Fig. 1C). Acute thermal nociception, as determined by the eye wipe test with the Trpv1 agonist capsaicin and the acute tail immersion assay, respectively, were unchanged in R7bp KO mice (Fig. 1D, E). Thermal nociception using the hot plate test was diminished in the R7bp KO mice as evidenced by enhanced basal latency of paw withdrawal (Fig. 1F), as previously reported.48

In adult mice, sensory neurons expressing the G protein-coupled receptor Mrgprd are required for the behavioral response to noxious mechanical stimuli.5 There was no obvious difference in the expression of Mrgprd in DRG from R7bp KO mice vs control (Fig. 1G). Furthermore mechanical nociception was preserved in R7bp KO mice (Fig. 1H, I).

Acute chemical nociception in response to mustard oil (acting through Trpa1 receptors) also appeared unchanged in R7bp KO mice (Fig. 1J). Since nociception was largely preserved in R7bp KO mice we investigated if the sense of itch was altered.

Back to Top | Article Outline

3.2. R7bp-knockout mice exhibit impaired pruriception

The scratching response to the intradermal application of a variety of classical histamine-dependent and histamine-independent pruritogens acting directly and indirectly through GPCRs was markedly deficient in R7bp KO mice compared to that of their wild-type littermates (Fig. 2A–E). To exclude motor difficulties that might impact their ability to scratch, we confirmed that mice lacking R7bp exhibited normal motor coordination and locomotor activity, consistent with previous reports (data not shown).1 We proceeded to study a more diverse set of cutaneous pruritogens in order to better demarcate the pruriceptive defect in these mutant mice.

In wild-type mice we confirmed that the toll-like receptor TLR3 agonist polyinosinic:polycytidylic acid (poly [I:C]), a structural analog of double-stranded (ds) RNA such as that found in dsRNA viruses, but not the TLR3-inactive synthetic double-stranded DNA analog polydeoxyinosinic:polydeoxycytidylic acid (dI:dC), was a weak pruritogen (Fig. 2F) as was previously reported.27 Mice lacking R7bp failed to respond to the pruritogenic effects of poly (I:C) (Fig. 2G).

The R7bp KO mice also failed to respond to the cytokine thymic stromal lymphopoietin (TSLP), implicated in the pathogenesis of atopic dermatitis in humans15 and shown to evoke scratching behavior in mice44 (Fig. 2H), endothelin-1, a paracrine factor released from endothelial cells (Fig. 2I), and the bile salts deoxycholate (DCA) and taurolithocholate (TLCA) implicated in the pruritus characteristic of cholestatic liver disease20 (Fig. 2J, K).

One explanation for the above findings could be a loss of pruriceptive sensory neurons in mice lacking R7bp. Using DRG sections labeled by ISH, we found that specific pruriceptive sensory neuronal markers, such as natriuretic peptide B (Nppb)32 and mas-related GPCR MrgprA3,25 appeared to be expressed normally in DRG from R7bp KO mice (Fig. 2L). Cell counting experiments showed that knockout of R7bp caused no reduction in the number of DRG cells expressing either pruriceptive marker (Fig. 2M, N). If anything in fact, the R7bp KO mice tended to have more DRG cells expressing the pruriceptive markers than controls, a difference that did not however achieve statistical significance.

Another explanation for the loss of scratching behavior observed in the R7bp KO mice could be a loss of sensitivity to pruritogens in primary pruriceptive neurons. To test this hypothesis, DRG were harvested from WT and R7bp KO mice, and primary sensory neurons were isolated by gentle protease treatment and cultured in vitro for testing, using ligand-induced mobilization of intracellular calcium as a functional assay. Treatment of the isolated sensory neurons with the prototypical pruritogens histamine and chloroquine triggered intracellular calcium release in a subset of sensory cells from both WT and R7bp KO mice over the 3 minute study period (Fig. 2O, P), with no significant difference between genotypes detected (P = 0.73, histamine; P = 0.24, chloroquine, by 2-way ANOVA).

Given that loss of R7bp diminished the scratching behavior in response to multiple pruritogens acting through disparate classes of receptors, including non-GPCRs (Table 1), we questioned our underlying hypothesis linking loss of R7bp's canonical function, namely facilitation of Gαi/o-directed GAP activity, to the pruriceptive defects. We therefore sought independent confirmation of a linkage between loss of Gαi/o-directed GAP activity and loss of pruriception.

Back to Top | Article Outline

3.3. Mice heterozygous for an RGS protein-insensitive mutant of Gαo exhibit a pruriceptive defect similar to that of R7bp knockout mice

Within R7bp- and Gβ5/R7-RGS complex-expressing neurons, the amplitude of the intracellular signal transduced from agonist-activated Gi/o-coupled GPCRs represents a balance between receptor-driven Gi/o activation and Gi/o de-activation (Fig. 3A). Signaling in neurons from mice lacking R7bp (Fig. 3B) or harboring an RGS protein-insensitive mutant form of Gαi oρ Gαo (Fig. 3C) might be similarly enhanced.

Given the similarity of the predicted signaling phenotypes, we hypothesized that the pruriceptive phenotype of mice expressing a Gαi/o subunit insensitive to the GAP activity of RGS proteins might be similar to that of R7bp KO mice if, in fact, the R7bp KO phenotype was due to loss of its canonical role in facilitating the Gαi/o-directed GAP activity of Gβ5/R7-RGS complexes.

We therefore tested pruriception in mice heterozygous for the G184S mutant of Gαo (Gnao1 G184S/+).8 This approach afforded the opportunity to test the hypothesis without any knowledge regarding the upstream Gi/o-coupled GPCR(s) that might be involved in R7bp-dependent pruriception. Because of the single amino acid mutation, the G184S mutant form of Gαo fails to bind RGS proteins with high affinity and is insensitive to RGS protein GAP activity.22 Mice homozygous for this mutation are not viable.8

We found the scratching response to the intradermal application of both compound 48/80 and chloroquine was markedly deficient in Gnao1 G184S heterozygous mice (Fig. 3D, E respectively). The similarity of the pruriceptive phenotype of Gnao1 G184S/+ mice to that of R7bp KO mice strongly suggested that it was loss of R7bp's canonical role as facilitator of Gαi/o-directed GAP activity that was critical to the phenotype of the latter mice.

To help explain why the scratching response to such a diverse array of cutaneously applied pruritogens was affected in R7bp KO mice (Table 1), and to account for the apparent lack of effect of R7bp KO on the number or sensitivity of primary pruriceptors (Fig. 2L–P), we decided to consider possible effects of R7bp loss in the central nervous system. To evaluate this possibility, we quantified behavioral responses to intrathecally-administered pruritogens.

Back to Top | Article Outline

3.4. Mice lacking R7bp show reduced scratching behavior in response to intrathecally-administered pruritogens

The itch sensation is conveyed by primary sensory neurons that release Nppb onto secondary pruriceptors in the spinal dorsal horn (Fig. 4A).32 Accordingly the intrathecal administration of Nppb induces scratching behavior in mice.32 Secondary pruriceptors release gastrin-releasing peptide (GRP) onto tertiary pruriceptors in the superficial dorsal horn that express gastrin-releasing peptide receptors (Grpr) (Fig. 4A).32,39,40 Scratching behavior resulting from the intrathecal administration of Nppb is blocked by pretreatment with a Grpr antagonist.19 Wild-type and Nppb −/− mice, but not Grpr −/− mice, exhibit scratching behavior in response to the intrathecal administration of GRP.32,39

We studied the scratching behavior in response to intrathecal Nppb and GRP in R7bp KO mice. The intrathecal administration of either Nppb (Fig. 4B) or GRP (Fig. 4C) stimulated scratching behavior that was greatly diminished in mice lacking R7bp (Fig. 4B, C) (Table 1). We observed that, as in R7bp KO mice, Gnao1 G184S/+ mice also exhibited reduced scratching behavior in response to intrathecal GRP (Fig. 4D).

The diminished responsiveness to intrathecal pruritogens observed suggested that the pruriceptive defect in R7bp KO mice involves R7bp actions at the spinal level. If this model were true, R7bp KO mice should also exhibit reduced activation of spinal neurons in response to cutaneously applied pruritogens. We tested this using an immunohistochemical assay for the activation of spinal neurons.

Back to Top | Article Outline

3.5. Mice lacking R7bp show impaired activation of spinal cord dorsal horn neurons in response to cutaneous pruritogens

We first confirmed the expression of R7bp and other components of the heterotrimeric Gβ5/R7-RGS/R7BP complex in the spinal cord by ISH (Fig. 5A). Transcript for R7bp was expressed widely in cells throughout the dorsal horn and intermediate zone of grey matter in wild-type mice (Fig. 5A upper panel, left). Knockout of R7bp did not affect the level or pattern of expression of transcripts for Gβ5, Rgs7, and Rgs9 (Fig. 5A, lower panel).

Cutaneous pruritogens can activate neurons in the superficial dorsal horn as evidenced by induction of the immediate early gene c-Fos in neuronal nuclei.10,45 We therefore reasoned that if the pruriceptive defect in R7bp KO mice resulted from diminished inter-neuronal signaling at the spinal level, there might be a corresponding reduction in the pruritogen-induced activation of c-Fos expression in neurons present in the superficial dorsal spinal cord. Intradermal administration of compound 48/80 induced c-Fos immunoreactivity in lateral spinal dorsal horn neurons of wild-type mice (Fig. 5B upper panel; 5C). Significantly fewer neurons demonstrated c-Fos induction in 48/80-treated R7bp KO mice or in PBS-treated wild-type mice (Fig. 5B lower panel; Fig. 5C). Spinal lateral dorsal horn neuronal c-Fos induction in response to intradermal chloroquine administration was also reduced in R7bp KO mice (Fig. 5D).

Taken together, the results above were most consistent with a model in which the impaired scratching behavior in response to the cutaneous application of pruritogens in R7bp KO mice resulted (1) primarily from disruption of the itch sensory circuitry at the spinal level, and (2) from the loss of the canonical function of R7bp to facilitate GAP activity directed against Gai/o. We therefore considered candidate Gi/o-coupled GPCR-regulated pathways in the spinal cord that were normally inhibitory to pruriception. Loss of R7bp-facilitated GAP activity in such a pathway could lead to an exaggeration of the normal anti-pruriceptive inhibitory signal, resulting in a loss-of-pruriception phenotype.

Back to Top | Article Outline

3.6. Knockout of the kappa-opioid receptor rescues the pruriceptive defects of R7bp knockout mice

Recently, a population of inhibitory interneurons in the dorsal horn was identified that express the transcription factor Bhlhb5 and are abolished by knockout of Bhlhb5 during development.36 These spinal interneurons, termed B5-I neurons, are involved in the tonic inhibition of itch.17,36 B5-I neurons, thought to be GABAergic or glycinergic, also utilize dynorphin as an inhibitory neuromodulator.17,36 Dynorphin is the endogenous ligand for the kappa-opioid receptor, a Gi/o-coupled GPCR encoded by Oprk1. Loss of R7bp from neurons expressing kappa-opioid receptors and positioned downstream from B5-I neurons in the pruriceptive signaling pathway could lead to an exaggeration of normal inhibitory neuromodulation and a loss-of-pruriception phenotype. We therefore hypothesized that, if this were the case, knockout of the kappa-opioid receptor might reverse the loss-of-pruriception phenotype in R7bp KO mice.

To explore this possibility, pruriception was tested in mice singly deficient for Oprk1 or for R7bp, in Oprk1/R7bp double KO mice, and in their wild-type littermates. As previously shown, the scratching response to the intradermal application of both compound 48/80 and chloroquine was deficient in R7bp KO mice compared to their wild-type littermates (Fig. 6A, B). While the knockout of Oprk1 alone had no effect, the double knockout of Oprk1 and R7bp completely reversed the deficiency of scratching behavior in response to intradermal 48/80 and chloroquine observed in mice deficient for R7bp alone (Fig. 6A, B).

In response to intrathecal GRP, R7bp KO mice exhibited diminished scratching behavior, as previously shown (Fig. 6C). Mice deficient for Oprk1 also showed a diminished response to intrathecal GRP, however mice doubly knocked out for Oprk1 and R7bp showed a normal scratching response to intrathecal GRP, indicating mutual rescue of the diminished scratching responses seen with either single gene deficiency (Fig. 6C). That single knockout of Oprk1 resulted in diminished scratching in response to intrathecal GRP was an incidental and unexpected finding, not readily explained by current models.

Back to Top | Article Outline

3.7. Mice lacking R7bp exhibit diminished scratching behavior and enhanced sensitivity to kappa opioid agonists in a model of atopic dermatitis

We showed above that R7bp KO mice exhibit diminished scratching in response to cutaneous application of the cytokine TSLP (Fig. 2I). Since TSLP signaling has been implicated in the pathogenesis of atopic dermatitis,15 we studied the scratching behavior R7bp KO mice and their wild-type littermates in a mouse model of atopic dermatitis induced by the chronic topical application of diphenylcyclopropenone (DCP).23 Following the sensitization period, mice lacking R7bp exhibited significantly diminished scratching behavior over the 10 days of the trial compared to control (P < 0.0001, 2-way ANOVA) (Fig. 6D).

Since we previously showed the effects of R7bp KO on acute pruritogen-induced scratching behavior depended on the presence of kappa opioid receptors, we tested the effect of U50,488, a specific kappa receptor agonist, on chronic itch during the latter portion of DCP trial. Since during this time the control and R7bp KO mice had different levels of scratching behavior, we compared the relative scratching behavior following U50,488 injection to that following a preceding saline injection an hour earlier in the same mouse. Control mice exhibited no difference in relative scratching behavior in response to the 0.1 and 0.25 mg/kg doses of U50,488, whereas R7bp KO mice showed a significant reduction (Fig. 6E). A heightened sensitivity to U50,488 treatment in the R7bp KO mice was particularly evident with the 0.25 mg/kg dose following which the mutant mice showed a nearly 90% reduction in relative scratching behavior compared to control (Fig. 6E).

Back to Top | Article Outline

4. Discussion

We demonstrate here that R7bp critically regulates the acute and pathologic sensation of itch in vivo. Our results indicate that the loss-of-pruriception phenotype seen in R7bp KO mice results primarily from regulatory actions by R7bp on itch signaling at the level of the spinal cord, or more centrally. This is because the loss of R7bp fails to reduce the number or sensitivity of primary pruriceptive sensory neurons yet results in inhibition of the scratching response to intrathecally-applied Nppb and GRP. Regulation by R7bp occurring at a central node in the pruriceptive pathway can explain why R7bp loss affects responses to a diverse set of peripherally administered pruritogens (Table 1). Our findings do not exclude however the possibility that R7bp expressed in primary sensory neurons, at their cutaneous or spinal nerve terminals, may play a secondary role in pruriception.

A loss-of-pruriception phenotype very similar to that seen in R7bp KO mice was observed in Gnao1 G184S heterozygotes, expressing an RGS protein-insensitive mutant of Gαo. This indicates that the central effect of R7bp loss likely results from loss of R7bp's canonical function as facilitator of Gαi/o-directed GAP activity.

If this is true, and since our results show that R7bp effects on pruriception depend on Oprk1 expression, the simplest model would be that R7bp normally enhances pruriception in the CNS by inhibiting Gi/o protein signaling directly coupled to kappa-opioid receptors (Fig. 7B). Our observation that R7bp KO potentiates the antipruritic effects of exogenous kappa opioid agonists would be consistent with this model. Our preliminary expression analysis by double fluorescent ISH in the spinal cord identified a subset of R7bp+ cells in the dorsal horn that were also Oprk1+, a finding also consistent with our proposed model (Fig. 7A).

Dynorphin is the endogenous ligand of the kappa-opioid receptor whose knockout reverses the dominant loss-of-pruriception phenotype observed in R7bp KO mice. Ross et al. showed that dynorphin is synthesized and utilized as a neuromodulator by B5-I spinal interneurons that are inhibitory to itch sensation.17 Our model would further be consistent with the possibility that the functionally relevant kappa-opioid receptors, whose knockout reverses the dominant loss-of-pruriception phenotype in R7bp KO mice as demonstrated here, are normally responsive to the dynorphin released from these B5-I spinal interneurons (Fig. 7B). If that is true, it implies that dynorphin is released tonically from B5-I cells under basal conditions. Basal activation of kappa-opioid receptors by dynorphin released from B5-I spinal interneurons could explain how KO of R7bp produces a loss-of-pruriception phenotype under un-stimulated conditions. Confirmation and rigorous testing of this model will require at least 2 parallel sets of experiments utilizing R7bp conditional KO mice. One set of experiments would target and limit the KO of R7bp to anatomically and spatially defined subpopulations of neurons within the peripheral and central nervous system. The other line of experiments would restrict the KO of R7bp to neurons functionally defined by their expression of specific signaling molecules, such as G o-coupled GPCRs (particularly including Oprk1), specific R7-RGS subfamily RGS proteins, or other signaling molecules and their regulators.

Current therapeutic options for pathologic itch are suboptimal.46 The pruritus associated with diseases such as cholestatic liver disease, uremia, neuropathies, and eczematous, psoriasiform, and other types of dermatitis frequently does not respond to antihistamines or general anti-inflammatory agents. Centrally acting antipruritic therapies are being explored and clinical studies suggest that the kappa-opioid receptor agonists hold promise for the treatment of uremia-associated pruritus.21,42 Pharmacologic agents acting at the kappa-opioid receptor likely mimic the antipruritic action of the endogenous itch-inhibitory dynorphinergic B5-I neurons identified by Ross et al.17

Our findings suggest therefore that an endogenous kappa opioid pathway signals tonically to inhibit itch, and that manipulation of that tone can have striking effects on pruriception and the response to exogenous kappa opioids. Since our studies indicate that loss of R7bp diminishes pruriception to a wide range of pruritogens and has pronounced effects on both acute and chronic itch, inhibitors of R7bp-dependent GAP activity could represent a novel and more specific therapeutic strategy for pathological itch.

Back to Top | Article Outline

Conflict of interest statement

The authors have no conflicts of interest to declare.

M. Pandey and J.-H. Zhang contributed equally to this work.

Back to Top | Article Outline

Acknowledgements

The authors thank Dr. Kirill A. Martemyanov, Department of Neuroscience, Scripps Florida, for his generous gift of the R7bp knockout mice. The authors grateful to Dr. Ahmed Kablan for instruction in the tail-flick assay, to Mr. Jeffrey R. Leipprandt for help with the Gnao1 mutant mice and genotyping advice, Dr. Rashad Riazuddin for help with mouse phenotyping, and Dr. Owen M. Schwartz for help with the calcium imaging. The Intramural Research Programs of the National Institute of Diabetes and Digestive and Kidney Diseases (DK043304-23) and the National Institute of Dental and Craniofacial Research (DE000721-10) supported this research.

Back to Top | Article Outline

References

[1]. Anderson GR, Cao Y, Davidson S, Truong HV, Pravetoni M, Thomas MJ, Wickman K, Giesler GJ Jr, Martemyanov KA. R7BP complexes with RGS9-2 and RGS7 in the striatum differentially control motor learning and locomotor responses to cocaine. Neuropsychopharmacol 2010;35:1040–50.
[2]. Anderson GR, Lujan R, Semenov A, Pravetoni M, Posokhova EN, Song JH, Uversky V, Chen CK, Wickman K, Martemyanov KA. Expression and localization of RGS9-2/G 5/R7BP complex in vivo is set by dynamic control of its constitutive degradation by cellular cysteine proteases. J Neurosci 2007;27:14117–27.
[3]. Anderson GR, Posokhova E, Martemyanov KA. The R7 RGS protein family: multi-subunit regulators of neuronal G protein signaling. Cell Biochem Biophys 2009;54:33–46.
[4]. Bautista DM, Wilson SR, Hoon MA. Why we scratch an itch: the molecules, cells and circuits of itch. Nat Neurosci 2014;17:175–82.
[5]. Cavanaugh DJ, Lee H, Lo L, Shields SD, Zylka MJ, Basbaum AI, Anderson DJ. Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli. Proc Natl Acad Sci U S A 2009;106:9075–80.
[6]. Dhand A, Aminoff MJ. The neurology of itch. Brain 2014;137(pt 2):313–22.
[7]. Drenan RM, Doupnik CA, Jayaraman M, Buchwalter AL, Kaltenbronn KM, Huettner JE, Linder ME, Blumer KJ. R7BP augments the function of RGS7/Gβ5 complexes by a plasma membrane-targeting mechanism. J Biol Chem 2006;281:28222–31.
[8]. Goldenstein BL, Nelson BW, Xu K, Luger EJ, Pribula JA, Wald JM, O'Shea LA, Weinshenker D, Charbeneau RA, Huang X, Neubig RR, Doze VA. Regulator of G protein signaling protein suppression of Galphao protein-mediated alpha2A adrenergic receptor inhibition of mouse hippocampal CA3 epileptiform activity. Mol Pharmacol 2009;75:1222–30.
[9]. Han L, Dong X. Itch mechanisms and circuits. Annu Rev Biophys 2014;43:331–55.
[10]. Han L, Ma C, Liu Q, Weng HJ, Cui Y, Tang Z, Kim Y, Nie H, Qu L, Patel KN, Li Z, McNeil B, He S, Guan Y, Xiao B, Lamotte RH, Dong X. A subpopulation of nociceptors specifically linked to itch. Nat Neurosci 2013;16:174–82.
[11]. Hooks SB, Waldo GL, Corbitt J, Bodor ET, Krumins AM, Harden TK. RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity. J Biol Chem 2003;278:10087–93.
[12]. Hoon MA. Molecular dissection of itch. Curr Opin Neurobiol 2015;34C:61–6.
[13]. Hylden JL, Wilcox GL. Intrathecal morphine in mice: a new technique. EurJPharmacol 1980;67:313–16.
[14]. Hylden JL, Wilcox GL. Intrathecal opioids block a spinal action of substance P in mice: functional importance of both mu- and delta-receptors. EurJPharmacol 1982;86:95–8.
[15]. Indra AK. Epidermal TSLP: a trigger factor for pathogenesis of atopic dermatitis. Expert Rev Proteomics 2013;10:309–11.
[16]. Jayaraman M, Zhou H, Jia L, Cain MD, Blumer KJ. R9AP and R7BP: traffic cops for the RGS7 family in phototransduction and neuronal GPCR signaling. Trends Pharmacol Sci 2009;30:17–24.
[17]. Kardon AP, Polgar E, Hachisuka J, Snyder LM, Cameron D, Savage S, Cai X, Karnup S, Fan CR, Hemenway GM, Bernard CS, Schwartz ES, Nagase H, Schwarzer C, Watanabe M, Furuta T, Kaneko T, Koerber HR, Todd AJ, Ross SE. Dynorphin acts as a neuromodulator to inhibit itch in the dorsal horn of the spinal cord. Neuron 2014;82:573–86.
[18]. Kehrl JM, Sahaya K, Dalton HM, Charbeneau RA, Kohut KT, Gilbert K, Pelz MC, Parent J, Neubig RR. Gain-of-function mutation in Gnao1: a murine model of epileptiform encephalopathy (EIEE17)? Mamm Genome 2014;25:202–10.
[19]. Kiguchi N, Sukhtankar DD, Ding H, Tanaka K, Kishioka S, Peters CM, Ko MC. Spinal functions of B-Type natriuretic peptide, gastrin-releasing peptide, and their cognate receptors for regulating itch in mice. J Pharmacol Exp Ther 2016;356:596–603.
[20]. Kremer AE, Bolier R, van Dijk R, Oude Elferink RP, Beuers U. Advances in pathogenesis and management of pruritus in cholestasis. Dig Dis 2014;32:637–45.
[21]. Kumagai H, Ebata T, Takamori K, Miyasato K, Muramatsu T, Nakamoto H, Kurihara M, Yanagita T, Suzuki H. Efficacy and safety of a novel k-agonist for managing intractable pruritus in dialysis patients. Am J Nephrol 2012;36:175–83.
[22]. Lan KL, Sarvazyan NA, Taussig R, Mackenzie RG, DiBello PR, Dohlman HG, Neubig RR. A point mutation in Galphao and Galphai1 blocks interaction with regulator of G protein signaling proteins. J Biol Chem 1998;273:12794–7.
[23]. Li LF, Fiedler VC, Kumar R. The potential role of skin protein kinase C isoforms alpha and delta in mouse hair growth induced by diphencyprone-allergic contact dermatitis. J Dermatol 1999;26:98–105.
[24]. Liapis E, Sandiford S, Wang Q, Gaidosh G, Motti D, Levay K, Slepak VZ. Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells. J Neurochem 2012;122:568–81.
[25]. Liu Q, Tang Z, Surdenikova L, Kim S, Patel KN, Kim A, Ru F, Guan Y, Weng HJ, Geng Y, Undem BJ, Kollarik M, Chen ZF, Anderson DJ, Dong X. Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus. Cell 2009;139:1353–65.
[26]. Liu Q, Weng HJ, Patel KN, Tang Z, Bai H, Steinhoff M, Dong X. The distinct roles of two GPCRs, MrgprC11 and PAR2, in itch and hyperalgesia. Sci Signal 2011;4:ra45.
[27]. Liu T, Berta T, Xu ZZ, Park CK, Zhang L, Lu N, Liu Q, Liu Y, Gao YJ, Liu YC, Ma Q, Dong X, Ji RR. TLR3 deficiency impairs spinal cord synaptic transmission, central sensitization, and pruritus in mice. J Clin Invest 2012;122:2195–207.
[28]. Liu T, Ji RR. New insights into the mechanisms of itch: are pain and itch controlled by distinct mechanisms? Pflugers Arch 2013;465:1671–85.
[29]. Malin SA, Davis BM, Molliver DC. Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity. Nat Protoc 2007;2:152–60.
[30]. Masuho I, Xie K, Martemyanov KA. Macromolecular composition dictates receptor and G protein selectivity of regulator of G protein signaling (RGS) 7 and 9-2 protein complexes in living cells. J Biol Chem 2013;288:25129–42.
[31]. McCoy KL, Hepler JR. Regulators of G protein signaling proteins as central components of G protein-coupled receptor signaling complexes. Prog Mol Biol Transl Sci 2009;86:49–74.
[32]. Mishra SK, Hoon MA. The cells and circuitry for itch responses in mice. Science 2013;340:968–71.
[33]. Morhardt DR, Guido W, Chen CK. The role of Gbeta5 in vision. Prog Mol Biol Transl Sci 2009;86:229–48.
[34]. Posner BA, Gilman AG, Harris BA. Regulators of G protein signaling 6 and 7- Purification of complexes with Gβ5 and assessment of their effects on G protein-mediated signaling pathways. J Biol Chem 1999;274:31087–93.
[35]. Rose JJ, Taylor JB, Shi J, Cockett MI, Jones PG, Hepler JR. RGS7 is palmitoylated and exists as biochemically distinct forms. J Neurochem 2000;75:2103–12.
[36]. Ross SE, Mardinly AR, McCord AE, Zurawski J, Cohen S, Jung C, Hu L, Mok SI, Shah A, Savner EM, Tolias C, Corfas R, Chen S, Inquimbert P, Xu Y, McInnes RR, Rice FL, Corfas G, Ma Q, Woolf CJ, Greenberg ME. Loss of inhibitory interneurons in the dorsal spinal cord and elevated itch in Bhlhb5 mutant mice. Neuron 2010;65:886–98.
[37]. Santos-Nogueira E, Redondo Castro E, Mancuso R, Navarro X. Randall-Selitto test: a new approach for the detection of neuropathic pain after spinal cord injury. J neurotrauma 2012;29:898–904.
[38]. Slepak VZ. Structure, function, and localization of Gbeta5-RGS complexes. Prog Mol Biol Transl Sci 2009;86:157–203.
[39]. Sun YG, Chen ZF. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord. Nature 2007;448:700–3.
[40]. Sun YG, Zhao ZQ, Meng XL, Yin J, Liu XY, Chen ZF. Cellular basis of itch sensation. Science 2009;325:1531–4.
[41]. Terzi D, Cao Y, Agrimaki I, Martemyanov KA, Zachariou V. R7BP modulates opiate analgesia and tolerance but not withdrawal. Neuropsychopharmacol 2012;37:1005–12.
[42]. Wikstrom B, Gellert R, Ladefoged SD, Danda Y, Akai M, Ide K, Ogasawara M, Kawashima Y, Ueno K, Mori A, Ueno Y. Kappa-opioid system in uremic pruritus: multicenter, randomized, double-blind, placebo-controlled clinical studies. J Am Soc Nephrol 2005;16:3742–7.
[43]. Wilson SR, Gerhold KA, Bifolck-Fisher A, Liu Q, Patel KN, Dong X, Bautista DM. TRPA1 is required for histamine-independent, Mas-related G protein-coupled receptor-mediated itch. Nat Neurosci 2011;14:595–602.
[44]. Wilson SR, The L, Batia LM, Beattie K, Katibah GE, McClain SP, Pellegrino M, Estandian DM, Bautista DM. The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell 2013;155:285–95.
[45]. Yao GL, Tohyama M, Senba E. Histamine-caused itch induces Fos-like immunoreactivity in dorsal horn neurons: effect of morphine pretreatment. Brain Res 1992;599:333–7.
[46]. Yosipovitch G, Bernhard JD. Clinical practice. Chronic pruritus. N Engl J Med 2013;368:1625–34.
[47]. Zhang JH, Pandey M, Seigneur EM, Panicker LM, Koo L, Schwartz OM, Chen W, Chen CK, Simonds WF. Knockout of G protein beta5 impairs brain development and causes multiple neurologic abnormalities in mice. J Neurochem 2011;119:544–54.
[48]. Zhou H, Chisari M, Raehal KM, Kaltenbronn KM, Bohn LM, Mennerick SJ, Blumer KJ. GIRK channel modulation by assembly with allosterically regulated RGS proteins. Proc Natl Acad Sci USA 2012;109:19977–82.
© 2017 International Association for the Study of Pain