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Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy
Paul Facer, Maria A Casula, Graham D Smith, Christopher D Benham, Iain P Chessell, Chas Bountra, Marco Sinisi, Rolfe Birch, Praveen Anand
BMC Neurology , 2007, DOI: 10.1186/1471-2377-7-11
Abstract: We have studied TRPV1, TRPV3, TRPV4, and TRPM8 in nerves (n = 14) and skin from patients with nerve injury, avulsed dorsal root ganglia (DRG) (n = 11), injured spinal nerve roots (n = 9), diabetic neuropathy skin (n = 8), non-diabetic neuropathic nerve biopsies (n = 6), their respective control tissues, and human post mortem spinal cord, using immunohistological methods.TRPV1 and TRPV3 were significantly increased in injured brachial plexus nerves, and TRPV1 in hypersensitive skin after nerve repair, whilst TRPV4 was unchanged. TRPM8 was detected in a few medium diameter DRG neurons, and was unchanged in DRG after avulsion injury, but was reduced in axons and myelin in injured nerves. In diabetic neuropathy skin, TRPV1 expressing sub- and intra-epidermal fibres were decreased, as was expression in surviving fibres. TRPV1 was also decreased in non-diabetic neuropathic nerves. Immunoreactivity for TRPV3 was detected in basal keratinocytes, with a significant decrease of TRPV3 in diabetic skin. TRPV1-immunoreactive nerves were present in injured dorsal spinal roots and dorsal horn of control spinal cord, but not in ventral roots, while TRPV3 and TRPV4 were detected in spinal cord motor neurons.The accumulation of TRPV1 and TRPV3 in peripheral nerves after injury, in spared axons, matches our previously reported changes in avulsed DRG. Reduction of TRPV1 levels in nerve fibres in diabetic neuropathy skin may result from the known decrease of nerve growth factor (NGF) levels. The role of TRPs in keratinocytes is unknown, but a relationship to changes in NGF levels, which is produced by keratinocytes, deserves investigation. TRPV1 represents a more selective therapeutic target than other TRPs for pain and hypersensitivity, particularly in post-traumatic neuropathy.The cloning of the vanilloid receptor-1 (TRPV1) [1,2] has led to greater understanding of the mechanisms of thermosensation and the effects of capsaicin, the noxious component from chilli peppers. TRPV1 is a non
TRPV3 and TRPV4 ion channels are not major contributors to mouse heat sensation
Susan M Huang, Xiaoxin Li, YinYin Yu, Juan Wang, Michael J Caterina
Molecular Pain , 2011, DOI: 10.1186/1744-8069-7-37
Abstract: TRPV3 knockout mice on the C57BL6 background exhibited no obvious alterations in thermal preference behavior. On the 129S6 background, absence of TRPV3 resulted in a more restrictive range of occupancy centered around cooler floor temperatures. TRPV3 knockout mice showed no deficits in acute heat nociception on either background. Mice deficient in both TRPV3 and TRPV4 on a C57BL6 background showed thermal preference behavior similar to wild-type controls on the thermal gradient, and little or no change in acute heat nociception or inflammatory heat hyperalgesia. Masking of TRPV1 by the TRPV1 antagonist JNJ-17203212 did not reveal differences between C57BL6 animals deficient in TRPV3 and TRPV4, compared to their wild-type counterparts.Our results support the notion that TRPV3 and TRPV4 likely make limited and strain-dependent contributions to innocuous warm temperature perception or noxious heat sensation, even when TRPV1 is masked. These findings imply the existence of other significant mechanisms for heat perception.TRPV1 is a non-selective cation channel that can be activated by heat (at > ~42°C) or a wide range of chemical agonists such as capsaicin and acid [1]. TRPV1 is highly expressed in small diameter primary sensory neurons. Mice deficient for TRPV1 show blunted noxious heat perception in tests of acute heat nociception and inflammatory heat hyperalgesia [2]. Although these responses are impaired, mice devoid of TRPV1 are still able to respond to heat. For example, although TRPV1 knockout mice exhibit a 4-fold longer tail withdrawal latency at 50°C, they still withdraw their tails in response to hot water. In one TRPV1 knockout line, behavioral deficits were reported in thermal hyperalgesia but not in acute heat nociception [3]. Moreover, thermal selection behavior on a thermal gradient is normal in the absence of TRPV1 [4]. Significant residual responses to heat have also been observed in skin-nerve explants derived from TRPV1 knockout mice [2,5,6]. Thus,
Tumor necrosis factor-alpha (TNF-α) enhances functional thermal and chemical responses of TRP cation channels in human synoviocytes
Mikhail Y Kochukov, Terry A McNearney, Huaizhi Yin, Liping Zhang, Fei Ma, Larissa Ponomareva, Sarah Abshire, Karin N Westlund
Molecular Pain , 2009, DOI: 10.1186/1744-8069-5-49
Abstract: Fluorescent imaging of Fura-2 loaded human SW982 synoviocytes reveals immediate and delayed cytosolic calcium oscillations elicited by (1) TRPV1 agonists capsaicin and resiniferatoxin (20 – 40% of cells), (2) moderate and noxious temperature change, and (3) osmotic stress TRPV4 activation (11.5% of cells). TNF-alpha pre-treatment (1 ng/ml, 8 – 16 hr) significantly increases (doubles) capsaicin responsive cell numbers and [Ca2+]i spike frequency, as well as enhances average amplitude of temperature induced [Ca2+]i responses. With TNF-alpha pre-treatment for 8, 12, and 16 hr, activation with 36 or 45 degree bath solution induces bimodal [Ca2+]i increase (temperature controlled chamber). Initial temperature induced rapid transient spikes and subsequent slower rise reflect TRPV1 and TRPV4 channel activation, respectively. Only after prolonged TNF-alpha exposure (12 and 16 hr) is recruitment of synoviocytes observed with sensitized TRPV4 responses to hypoosmolarity (3–4 fold increase). TNF-alpha increases TRPV1 (8 hr peak) and TRPV4 (12 hr peak) immunostaining, mRNA and protein expression, with a TRPV1 shift to membrane fractions.TNF-α provides differentially enhanced synoviocyte TRPV1 and TRPV4 expression and [Ca2+]i response dependent on the TRP stimulus and time after exposure. Augmented relevance of TRPV1 and TRPV4 as inflammatory conditions persist would provide calcium mediated cell signaling required for pathophysiological responses of synoviocytes in inflammatory pain states.Temperature sensitive transient receptor potential (TRP) channels belonging to the V- (or vanilloid related) subfamily are widely expressed in mammalian cells. Four members of this subfamily, TRPV1–4 conduct mono- and di-valent cations when activated by temperatures ranging from > 23°C (TRPV3 and TRPV4) to > 43°C (TRPV1) or > 53°C (TRPV2). In addition, TRPV1–4 function as important membrane sensors for extracellular chemical, osmotic, or mechanical stimuli. TRPV1 channels are activated by low
Quantitative Real-Time PCR detection of TRPV1–4 gene expression in human leukocytes from healthy and hyposensitive subjects
Giacomo Spinsanti, Raffaella Zannolli, Cristina Panti, Ilaria Ceccarelli, Letizia Marsili, Valeria Bachiocco, Francesco Frati, Anna Aloisi
Molecular Pain , 2008, DOI: 10.1186/1744-8069-4-51
Abstract: To study the gene expression of TRPV1–4 genes in human leukocytes, a quantitative Real-Time PCR (qRT-PCR) method, based on the calculation of their relative expression, has been developed and validated. The four commonly used house-keeping genes (HKGs), β-Actin (Act-B), glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxanthine ribosyltransferase (HPRT1), and cyclophilin B (hCyPB), were tested for the stability of their expression in several human leukocyte samples, and used in the normalization procedure to determine the mRNA levels of the TRPV 1–4 genes in 30 healthy subjects. cDNAs belonging to all the TRPV1–4 genes were detected in leukocytes but the genes appear to be expressed at different levels. Our analysis did not show significant sex differences in TRPV1–4 cDNA levels in the 30 healthy subjects. The same qRT-PCR assay was used to compare TRPV1–4 expression between healthy controls and patients hyposensitive to capsaicin, pain and thermal stimuli: an almost doubled up-regulation of the TRPV1 gene was found in the pathological subjects.The qRT-PCR assay developed and tested in this study allowed us to determine the relative expression of TRPV1–4 genes in human leukocytes: TRPV3 is the least expressed gene of this pool, followed by TRPV4, TRPV1 and TRPV2. The comparison of TRPV1–4 gene expression between two groups of healthy and hyposensitive subjects highlighted the evident up-regulation of TRPV1, which was almost doubly expressed (1.9× normalized fold induction) in the latter group. All the four house-keeping genes tested in this work (Act-B, GAPDH, hCyPB, HPRT1) were classified as optimal controls and showed a constant expression in human leukocytes samples. We recommend the use of these genes in similar qRT-PCR studies on human blood cells.Transient receptor potential (TRP) channels have been identified as cellular sensors that respond to diverse external and internal stimuli and play a fundamental role in the cytosolic free Ca2+ concentration, either by act
Acute Heat-Evoked Temperature Sensation Is Impaired but Not Abolished in Mice Lacking TRPV1 and TRPV3 Channels  [PDF]
Irène Marics, Pascale Malapert, Ana Reynders, Stéphane Gaillard, Aziz Moqrich
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0099828
Abstract: The discovery of heat-sensitive Transient Receptor Potential Vanilloid ion channels (ThermoTRPVs) greatly advanced our molecular understanding of acute and injury-evoked heat temperature sensation. ThermoTRPV channels are activated by partially overlapping temperatures ranging from warm to supra-threshold noxious heat. TRPV1 is activated by noxious heat temperature whereas TRPV3 can be activated by warm as well as noxious heat temperatures. Loss-of-function studies in single TRPV1 and TRPV3 knock-out mice have shown that heat temperature sensation is not completely abolished suggesting functional redundancies among these two channels and highlighting the need of a detailed analysis of TRPV1::TRPV3 double knock-out mice (V1V3dKO) which is hampered by the close proximity of the loci expressing the two channels. Here we describe the generation of a novel mouse model in which trpv1 and trpv3 genes have been inactivated using bacterial artificial chromosome (BAC)-based homologous recombination in embryonic stem cells. In these mice, using classical thermosensory tests such hot plate, tail flick and the thermotaxis gradient paradigms, we confirm that TRPV1 is the master channel for sensing noxious heat temperatures and identify a cooperative role of TRPV1 and TRPV3 for sensing a well-defined window of acute moderate heat temperature. Using the dynamic hot plate assay, we unravel an intriguing and unexpected pronounced escape behavior in TRPV1 knock-out mice that was attenuated in the V1V3dKO. Together, and in agreement with the temperature activation overlap between TRPV1 and TRPV3 channels, our data provide in vivo evidence of a cooperative role between skin-derived TRPV3 and primary sensory neurons-enriched TRPV1 in modulation of moderate and noxious heat temperature sensation and suggest that other mechanisms are required for heat temperature sensation.
Acidification of rat TRPV1 alters the kinetics of capsaicin responses
Torben R Neelands, Michael F Jarvis, Ping Han, Connie R Faltynek, Carol S Surowy
Molecular Pain , 2005, DOI: 10.1186/1744-8069-1-28
Abstract: The vanilloid receptor 1 (TRPV1) is a member of the transient receptor potential family (TRP) of non-selective cation channels [1]. These receptors are activated by a variety of lipids, acidic conditions and temperatures above 42°C. TRPV1 channels are tetramers composed of subunits with six transmembrane spanning domains, a pore loop between TM5 and TM6, and large N- and C-terminal intracellular domains [2]. An intracellular domain just C-terminal to TM6 has been characterized as being important in the tetramerization of the channel and is coincident, in part, with the TRP box that is common among this family of ion channels [3].The structural features of TRPV1 suggest that the primary ligand interaction site(s) and important regulatory mechanisms for the channel are intracellular. Indeed, multiple mutagenesis studies have shown that distinct intracellular regions are necessary for the binding of the exogenous TRPV1 agonist, capsaicin [4-6] although an extracellular site may also contribute to capsaicin binding [7]. In contrast, extracellular site acidic residues have been implicated in proton activation (at pH < 6) and sensitization of TRPV1 [8]. Further evidence that TRPV1 activation mechanisms are different for capsaicin and protons is provided by site-directed mutagenesis studies that disrupt capsaicin activation of the channel but leave proton actions intact [9]. Despite these differences, there is evidence of some commonality in the gating of the channel in response to capsaicin or acidic pH activation [10].Under pathological conditions multiple agents may simultaneously influence the activity of TRPV1 receptors. For instance, inflammation, ischemia, and infections result in elevated proton concentrations that can reduce the pH below 6 in the surrounding tissues [11]. Acidic pH has been shown to stimulate a subpopulation of sensory nerves that are also activated by capsaicin [12]. In addition, disruption of the TRPV1 gene attenuates proton-induced excitation o
Attenuation of TRPV1 and TRPV4 Expression and Function in Mouse Inflammatory Pain Models Using Electroacupuncture
Wei-Hsin Chen,Jason T. C. Tzen,Ching Liang Hsieh,Yung Hsiang Chen,Tzu-Jou Lin,Shih-Yin Chen,Yi-Wen Lin
Evidence-Based Complementary and Alternative Medicine , 2012, DOI: 10.1155/2012/636848
Abstract: Although pain is a major human affliction, our understanding of pain mechanisms is limited. TRPV1 (transient receptor potential vanilloid subtype 1) and TRPV4 are two crucial receptors involved in inflammatory pain, but their roles in EA- (electroacupuncture-) mediated analgesia are unknown. We injected mice with carrageenan (carra) or a complete Freund’s adjuvant (CFA) to model inflammatory pain and investigated the analgesic effect of EA using animal behavior tests, immunostaining, Western blotting, and a whole-cell recording technique. The inflammatory pain model mice developed both mechanical and thermal hyperalgesia. Notably, EA at the ST36 acupoint reversed these phenomena, indicating its curative effect in inflammatory pain. The protein levels of TRPV1 and TRPV4 in DRG (dorsal root ganglion) neurons were both increased at day 4 after the initiation of inflammatory pain and were attenuated by EA, as demonstrated by immunostaining and Western blot analysis. We verified DRG electrophysiological properties to confirm that EA ameliorated peripheral nerve hyperexcitation. Our results indicated that the AP (action potential) threshold, rise time, and fall time, and the percentage and amplitude of TRPV1 and TRPV4 were altered by EA, indicating that EA has an antinociceptive role in inflammatory pain. Our results demonstrate a novel role for EA in regulating TRPV1 and TRPV4 protein expression and nerve excitation in mouse inflammatory pain models.
Capsaicin-Induced Changes in LTP in the Lateral Amygdala Are Mediated by TRPV1  [PDF]
Carsten Zschenderlein,Christine Gebhardt,Oliver von Bohlen und Halbach,Christoph Kulisch,Doris Albrecht
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0016116
Abstract: The transient receptor potential vanilloid type 1 (TRPV1) channel is a well recognized polymodal signal detector that is activated by painful stimuli such as capsaicin. Here, we show that TRPV1 is expressed in the lateral nucleus of the amygdala (LA). Despite the fact that the central amygdala displays the highest neuronal density, the highest density of TRPV1 labeled neurons was found within the nuclei of the basolateral complex of the amygdala. Capsaicin specifically changed the magnitude of long-term potentiation (LTP) in the LA in brain slices of mice depending on the anesthetic (ether, isoflurane) used before euthanasia. After ether anesthesia, capsaicin had a suppressive effect on LA-LTP both in patch clamp and in extracellular recordings. The capsaicin-induced reduction of LTP was completely blocked by the nitric oxide synthase (NOS) inhibitor L-NAME and was absent in neuronal NOS as well as in TRPV1 deficient mice. The specific antagonist of cannabinoid receptor type 1 (CB1), AM 251, was also able to reduce the inhibitory effect of capsaicin on LA-LTP, suggesting that stimulation of TRPV1 provokes the generation of anandamide in the brain which seems to inhibit NO synthesis. After isoflurane anesthesia before euthanasia capsaicin caused a TRPV1-mediated increase in the magnitude of LA-LTP. Therefore, our results also indicate that the appropriate choice of the anesthetics used is an important consideration when brain plasticity and the action of endovanilloids will be evaluated. In summary, our results demonstrate that TRPV1 may be involved in the amygdala control of learning mechanisms.
Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1) by Diacylglycerol (DAG)
Dong Woo, Sung Jung, Mei Zhu, Chul-Kyu Park, Yong Kim, Seog Oh, C Justin Lee
Molecular Pain , 2008, DOI: 10.1186/1744-8069-4-42
Abstract: The capsaicin receptor, TRPV1 (transient receptor potential channel vanilloid subtype 1), is a molecular sensor that detects a wide range of painful stimuli such as capsaicin, heat, and acid in nociceptive sensory neurons [1-4]. Since TRPV1 plays a pivotal role in thermal nociception and inflammatory hyperalgesia [2,3] and is also widely found in the central nervous system [5], considerable effort has been made to identify endogenous activators for TRPV1. The products of lipoxygenases, anandamide, and other endocannabinoids [6-11] and even phosphorylation by protein kinase C (PKC) [12] in the absence of any other agonists have been shown to directly activate TRPV1. However, their roles under physiological condition are still debatable.Multiple chemical mediators such as bioactive peptides or plasma proteins are generated in inflammatory sites, and many of these mediators heightens the sensitivity of nociceptive sensory neurons after binding to their respective G-protein coupled receptors (GPCR) [13]. Indeed, many Gαq coupled receptors such as bradykinin receptor 2, prostaglandin receptor, protease activated receptor 2, histamine receptor 1, and metabotropic glutamate receptors (mGluR1 and mGluR5), are implicated in sensitization of sensory neurons via TRPV1 modulation during inflammation-induced thermal hyperalgesia [8,14-18]. Diacylglycerol (DAG) is at the core of GPCR signaling pathway and has been shown to directly activate subfamilies of TRP channels. Mammalian homologues of TRP family (TRPC3, C6 and C7) are activated by DAG [19-21], raising the possibility that DAG directly activates TRPV1. Thus, in the present work, we set out to evaluate the possibility of TRPV1 activation by DAG.Dorsal root ganglia (DRG) were prepared as previously described [22]. Briefly, Sprague-Dawley rat (OrientBio, Korea) was decapitated, and DRG were rapidly removed under aseptic conditions, placed in HBSS (Gibco). DRG were digested in 0.1% collagenase and 1% collagenase/dispase (Boehr
Human Keratinocytes Are Vanilloid Resistant  [PDF]
László Pecze, Kornélia Szabó, Márta Széll, Katalin Jósvay, Krisztián Kaszás, Erzsébet Kúsz, Tamás Letoha, János Prorok, István Koncz, András Tóth, Lajos Kemény, Csaba Vizler, Zoltán Oláh
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003419
Abstract: Background Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. Methods To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. Results Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 μM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. Conclusion TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials.
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