journal club 2014.12.01

Antipruritic mechanisms of topical E6005, a phosphodiesterase 4 inhibitor: Inhibition of responses to proteinase-activated receptor 2 stimulation mediated by increase in intracellular cyclic AMP

Abstract

Background

Phosphodiesterase 4 (PDE4), which catalyses the conversion of cyclic adenosine 3′,5′-monophosphate (cAMP) to 5′-AMP, plays a critical role in the pathogenesis of inflammatory disorders. Pruritus is the main symptom of dermatitides, such as atopic dermatitis, and is very difficult to control. Recent studies have shown that the activation of proteinase-activated receptor 2 (PAR2) is involved in pruritus in dermatoses in humans and rodents.

Objective

To investigate the inhibitory effect of E6005, a topically effective PDE4 inhibitor, on PAR2-associated itching in mice.

Methods

Mice were given an intradermal injection of SLIGRL-NH2 (100 nmol/site), a PAR2 agonist peptide, into the rostral part of the back. E6005 and 8-bromo-cAMP were applied topically and injected intradermally, respectively, to the same site. Scratching bouts were observed as an itch-related behavior, and firing activity of the cutaneous nerve was electrophysiologically recorded. Keratinocytes were isolated from the skin of neonatal mice and cultured for in vitro experiments. The concentrations of cAMP and leukotriene B4 (LTB4) were measured by enzyme immunoassay. The distribution of PDE4 subtypes in the skin was investigated by immunostaining.

Results

Topical E6005 and intradermal 8-bromo-cAMP significantly inhibited SLIGRL-NH2-induced scratching and cutaneous nerve firing. Topical E6005 increased cutaneous cAMP content. Topical E6005 and intradermal 8-bromo-cAMP inhibited cutaneous LTB4 production induced by SLIGRL-NH2, which has been shown to elicit LTB4-mediated scratching. E6005 and 8-bromo-cAMP inhibited SLIGRL-NH2-induced LTB4 production in the cultured murine keratinocytes also. PDE4 subtypes were mainly expressed in keratinocytes and mast cells in the skin.

Conclusions

The results suggest that topical E6005 treatment inhibits PAR2-associated itching. Inhibition of LTB4 production mediated by an increase in cAMP may be partly involved in the antipruritic action of E6005.

Abbreviations

  • cAMP, cyclic adenosine 3′,5′-monophosphate;
  • EIA, enzyme immunoassay;
  • IgG, immunoglobulin G;
  • LTB4, leukotriene B4;
  • PDE, phosphodiesterase;
  • PAR2, proteinase-activated receptor 2;
  • PBS, phosphate-buffered saline

dec presentation

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Journal Club 2014.11.24

Descending Control of Itch Transmission by the Serotonergic System via 5-HT1A-Facilitated GRP-GRPR Signaling

Highlights

  • •Central 5-HT signaling facilitates itch transmission
  • •5-HT1A potentiates GRPR-mediated itch signaling
  • •5-HT1A and GRPR are present in close proximity
  • •Blockade of 5-HT1A function reduces chronic itch

Summary

Central serotonin (5-hydroxytryptophan, 5-HT) modulates somatosensory transduction, but how it achieves sensory modality-specific modulation remains unclear. Here we report that enhancing serotonergic tone via administration of 5-HT potentiates itch sensation, whereas mice lacking 5-HT or serotonergic neurons in the brainstem exhibit markedly reduced scratching behavior. Through pharmacological and behavioral screening, we identified 5-HT1A as a key receptor in facilitating gastrin-releasing peptide (GRP)-dependent scratching behavior. Coactivation of 5-HT1A and GRP receptors (GRPR) greatly potentiates subthreshold, GRP-induced Ca2+ transients, and action potential firing of GRPR+ neurons. Immunostaining, biochemical, and biophysical studies suggest that 5-HT1A and GRPR may function as receptor heteromeric complexes. Furthermore, 5-HT1A blockade significantly attenuates, whereas its activation contributes to, long-lasting itch transmission. Thus, our studies demonstrate that the descending 5-HT system facilitates GRP-GRPR signaling via 5-HT1A to augment itch-specific outputs, and a disruption of crosstalk between 5-HT1A and GRPR may be a useful antipruritic strategy.

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Journal Club 2014.11.17

Neural peptidase endothelin-converting enzyme 1 regulates endothelin 1-induced pruritus

supplementary neural peptidase

Neural peptidase endothelin-converting enzyme 1 regulates endothelin 1-induced pruritus.

Kido-Nakahara M, Buddenkotte J, Kempkes C, Ikoma A, Cevikbas F, Akiyama T, Nunes F, Seeliger S, Hasdemir B, Mess C, Buhl T, Sulk M, Müller FU, Metze D,Bunnett NW, Bhargava A, Carstens E, Furue M, Steinhoff M.

Abstract

In humans, pruritus (itch) is a common but poorly understood symptom in numerous skin and systemic diseases. Endothelin 1 (ET-1) evokes histamine-independent pruritus in mammals through activation of its cognate G protein-coupled receptor endothelin A receptor (ETAR). Here, we have identified neural endothelin-converting enzyme 1 (ECE-1) as a key regulator of ET-1-induced pruritus and neural signaling of itch. We show here that ETAR, ET-1, and ECE-1 are expressed and colocalize in murine dorsal root ganglia (DRG) neurons and human skin nerves. In murine DRG neurons, ET-1 induced internalization of ETAR within ECE-1-containing endosomes. ECE-1 inhibition slowed ETAR recycling yet prolonged ET-1-induced activation of ERK1/2, but not p38. In a murine itch model, ET-1-induced scratching behavior was substantially augmented by pharmacological ECE-1 inhibition and abrogated by treatment with an ERK1/2 inhibitor. Using iontophoresis, we demonstrated that ET-1 is a potent, partially histamine-independent pruritogen in humans. Immunohistochemical evaluation of skin from prurigo nodularis patients confirmed an upregulation of the ET-1/ETAR/ECE-1/ERK1/2 axis in patients with chronic itch. Together, our data identify the neural peptidase ECE-1 as a negative regulator of itch on sensory nerves by directly regulating ET-1-induced pruritus in humans and mice. Furthermore, these results implicate the ET-1/ECE-1/ERK1/2 pathway as a therapeutic target to treat pruritus in humans.

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journal club 2014-11-10

The Gardenia jasminoides extract and its constituent, geniposide, elicit anti-allergic effects on atopic dermatitis by inhibiting histamine in vitro and in vivo

Abstract

Ethnopharmacological relevance

Gardenia jasminoides Ellis has been used in traditional medicine for treatment of inflammation, edema, and dermaitis. The aim of this study was to investigate the mechanism by which Gardenia jasminoides extract (GJE) elicits anti-allergic effects in mast cells and in mice with atopic dermatitis (AD).

Materials and methods

We investigated the effects of GJE and its fractions on compound 48/80-induced histamine release from MC/9 cells and Dermatophagoides farinae-exposed NC/Nga mice. The effects of its constituents on histamine release from MC/9 cells were also investigated.

Results

GJE and its ethyl acetate fraction (GJE-EA) inhibited compound 48/80-induced histamine release from MC/9 mast cells. The topical application of GJE or GJE-EA to Dermatophagoides farinae-exposed NC/Nga mice reduced the symptoms of AD, inhibited the infiltration of inflammatory cells, and lowered the serum levels of immunoglobulin E and histamine. Both GJE and GJE-EA reduced the expression of cytokines (interleukin [IL]-4, IL-6, and tumor necrosis factor-alpha) and adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) in ear lesions. In addition, the quantitative analysis of GJE and GJE-EA by high-performance liquid chromatography revealed the presence of crocin and geniposide. Geniposide, but not crocin, inhibited the release of histamine from mast cells, which may contribute to the anti-allergic effect of GJE and GJE-EA.

Conclusions

These results suggest that GJE and GJE-EA can suppress mast cell degranulation-induced histamine release, and geniposide may be potential therapeutic candidates for AD

 

gardenia paper

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Journal Club 2014.11.3

 

TLR4 enhances histamine-mediated pruritus by potentiating TRPV1 activity (1)
Filename : tlr4-enhances-histamine-mediated-pruritus-by-potentiating-trpv1-activity-1.pdf (2 MB)
Caption :

TLR4 enhances histamine-mediated pruritus by potentiating TRPV1 activity.

Min H, Lee H, Lim H, Jang YH, Chung SJ, Lee CJ, Lee SJ1.

Author information

Abstract

BACKGROUND:

Recent studies have indicated that Toll-like receptor 4 (TLR4), a pathogen-recognition receptor that triggers inflammatory signals in innate immune cells, is also expressed on sensory neurons, implicating its putative role in sensory signal transmission. However, the possible function of sensory neuron TLR4 has not yet been formally addressed. In this regard, we investigated the role of TLR4 in itch signal transmission.

RESULTS:

TLR4 was expressed on a subpopulation of dorsal root ganglia (DRG) sensory neurons that express TRPV1. In TLR4-knockout mice, histamine-induced itch responses were compromised while TLR4 activation by LPS did not directly elicit an itch response. Histamine-induced intracellular calcium signals and inward currents were comparably reduced in TLR4-deficient sensory neurons. Reduced histamine sensitivity in theTLR4-deficient neurons was accompanied by a decrease in TRPV1 activity. Heterologous expression experiments in HEK293T cells indicated thatTLR4 expression enhanced capsaicin-induced intracellular calcium signals and inward currents.

CONCLUSIONS:

Our data show that TLR4 on sensory neurons enhances histamine-induced itch signal transduction by potentiating TRPV1 activity. The results suggest that TLR4 could be a novel target for the treatment of enhanced itch sensation.

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journal club – 14.10.27.

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 – 14.10.27. Read More »

journal club 20-10-2014

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.

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journal club 13-10-2014

B-type natriuretic peptide is neither itch-specific nor functions upstream of the GRP-GRPR signaling pathway

Xian-Yu Liu12, Li Wan12, Fu-Quan Huo12, Devin M Barry12, Hui Li12, Zhong-Qiu Zhao12and Zhou-Feng Chen1234

Abstract

Background

A recent study by Mishra and Hoon identified B-type natriuretic peptide (BNP) as an important peptide for itch transmission and proposed that BNP activates spinal natriuretic peptide receptor-A (NPRA) expressing neurons, which release gastrin releasing peptide (GRP) to activate GRP receptor (GRPR) expressing neurons to relay itch information from the periphery to the brain (Science340:968–971, 2013). A central premise for the validity of this novel pathway is the absence of GRP in the dorsal root ganglion (DRG) neurons. To this end, they showed that Grp mRNA in DRG neurons is either absent or barely detectable and claimed that BNP but not GRP is a major neurotransmitter for itch in pruriceptors. They showed that NPRA immunostaining is perfectly co-localized with Grp-eGFP in the spinal cord, and a few acute pain behaviors in Nppb-/- mice were tested. They claimed that BNP is an itch-selective peptide that acts as the first station of a dedicated neuronal pathway comprising a GRP-GRPR cascade for itch. However, our studies, along with the others, do not support their claims.

Findings

We were unable to reproduce the immunostaining of BNP and NPRA as shown by Mishra and Hoon. By contrast, we were able to detect Grp mRNA in DRGs using in situ hybridization and real time RT-PCR. We show that the expression pattern of Grp mRNA is comparable to that of GRP protein in DRGs. Pharmacological and genetic blockade of GRP-GRPR signaling does not significantly affect intrathecal BNP-induced scratching behavior. We show that BNP inhibits inflammatory pain and morphine analgesia.

Conclusions

Accumulating evidence demonstrates that GRP is a key neurotransmitter in pruriceptors for mediating histamine-independent itch. BNP-NPRA signaling is involved in both itch and pain and does not function upstream of the GRP-GRPR dedicated neuronal pathway. The site of BNP action in itch and pain and its relationship with GRP remain to be clarified.

Keywords:

BNP; NPRA; GRP; GRPR; Itch; Pain; Spinal cord; DRG

B type natriuretic peptide is neither itch specific nor

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Journal Club 2014-10-06

1-s2.0-S156757691300297X-main
Filename : 1-s2-0-s156757691300297x-main.pdf (953 KB)
Caption :

Int Immunopharmacol. 2013 Nov;17(3):502-7. doi: 10.1016/j.intimp.2013.07.012. Epub 2013 Aug 9.

Nobiletin and tangeretin ameliorate scratching behavior in mice by inhibiting the action of histamine and the activation of NF-κB, AP-1 and p38.

Jang SE1, Ryu KR, Park SH, Chung S, Teruya Y, Han MJ, Woo JT, Kim DH.

Abstract

Nobiletin and tangeretin are polymethoxy flavonoids that are abundantly present in the pericarp of Citrus unshiu (family Rutaceae) and the fruit of Citrus depressa (family Rutaceae). They exhibit various biological activities, including anti-inflammatory and anti-asthmatic effects. To evaluate the anti-allergic effects of nobiletin and tangeretin, we measured their inhibitory effects in histamine- or compound 48/80-induced scratching behavioral mice. Nobiletin and tangeretin potently inhibited scratching behavior, as well as histamine-induced vascular permeability. Furthermore, they inhibited the expression of the allergic cytokines, IL-4 and TNF-α as well as the activation of their transcription factors NF-κB, AP-1 and p38 in histamine-stimulated skin tissues. They also inhibited the expression of IL-4 and TNF-α and the activation of NF-κB and c-jun in PMA-stimulated RBL-2H3 cells. Furthermore, nobiletin and tangeretin inhibited protein kinase C (PKC) activity and the IgE-induced degranulation of RBL-2H3 cells. These agents showed potent anti-histamine effect through the Magnus test when guinea pig ileum was used. Based on these results, nobiletin and tangeretin may ameliorate scratching behavioral reactions by inhibiting the action of histamine as well as the activation of the transcription factors NF-κB and AP-1 via PKC.

© 2013.

KEYWORDS:

1-s2.0-S156757691300297X-mainPMID:

 

23938254

 

[PubMed – indexed for MEDLINE]

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Journal Club 2014. 09. 29.

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. 09. 29. Read More »

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