Transient Receptor Potential Vanilloid Receptor Type 1 Research Articles

A Novel Flp Reporter Mouse Shows That TRPA1 Expression Is Largely Limited to Sensory Neuron Subsets.

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal cation channel that is activated by electrophilic irritants, oxidative stress, cold temperature, and GPCR signaling. TRPA1 expression has been primarily identified in subsets of nociceptive sensory afferents and is considered a target for future analgesics. Nevertheless, TRPA1 has been implicated in other cell types including keratinocytes, epithelium, enterochromaffin cells, endothelium, astrocytes, and CNS neurons. Here, we developed a knock-in mouse that expresses the recombinase FlpO in TRPA1-expressing cells. We crossed the TRPA1Flp mouse with the R26ai65f mouse that expresses tdTomato in a Flp-sensitive manner. We found tdTomato expression correlated well with TRPA1 mRNA expression and sensitivity to TRPA1 agonists in subsets of TRPV1 (transient receptor potential vanilloid receptor type 1)-expressing neurons in the vagal ganglia and dorsal root ganglia (DRGs), although tdTomato expression efficiency was limited in DRG. We observed tdTomato-expressing afferent fibers centrally (in the medulla and spinal cord) and peripherally in the esophagus, gut, airways, bladder, and skin. Furthermore, chemogenetic activation of TRPA1-expressing nerves in the paw evoked flinching behavior. tdTomato expression was very limited in other cell types. We found tdTomato in subepithelial cells in the gut mucosa but not in enterochromaffin cells. tdTomato was also observed in supporting cells within the cochlea, but not in hair cells. Lastly, tdTomato was occasionally observed in neurons in the somatomotor cortex and the piriform area, but not in astrocytes or vascular endothelium. Thus, this novel mouse strain may be useful for mapping and manipulating TRPA1-expressing cells and deciphering the role of TRPA1 in physiological and pathophysiological processes.

3D Printed Lab-on-a-Chip Platform for Chemical Stimulation and Parallel Analysis of Ion Channel Function.

Functional imaging has been a widely established method for the assessment of ion channel function in vitro. Conventional infrastructure used for in vitro functional analysis of ion channels is typically proprietary, non-customizable, expensive, and requires a high level of skill to use and maintain. 3D desktop printing, which is employed in the rapid prototyping field, allows for quick engineering of alternatives to conventional imaging infrastructure that are customizable, low cost, and user friendly. Here, we describe an ultra-low-cost microfluidic lab-on-a-chip (LOC) device manufactured using acrylonitrile butadiene styrene (ABS) for in vitro functional imaging of ion channels that can quickly and easily be reconstructed using three-dimensional (3D) desktop printing. The device is light weight (<5 g), small (20 mm × 49 mm), and extremely low cost (<EUR 1). We simulate fluidics within the printed channels and assess the suitability of the engineered chamber to generate homogeneous mixtures during solution exchange. We demonstrate the usability of the 3D printed microfluidic device in a case study using Fluo-4-loaded human embryonal kidney-derived (HEK293) cells, recombinantly expressing the capsaicin receptor, transient receptor potential vanilloid receptor type 1 (TRPV1), as a model system. In the case study, we confirm its applicability to solution exchange for chemical stimulation and parallel functional time-lapse fluorescence microscopy-based calcium imaging. We assess the suitability of ABS for culturing HEK293 cells inside the microfluidic LOC, based on qualitative analysis of microscopic transmission light images of ABS-exposed HEK293 cells and confirm the previously reported biocompatibility of ABS. To highlight the versatility of the 3D printed microfluidic device, we provide an example for multiplication of the shown concept within a 3D printed multichannel microfluidic LOC to be used, for example, in a higher throughput format for parallelized functional analysis of ion channels. While this work focusses on Ca2+ imaging with TRPV1 channels, the device may also be useful for application with other ion channel types and in vitro models.

Alterations in Gastric Mucosal Expression of Calcitonin Gene-Related Peptides, Vanilloid Receptors, and Heme Oxygenase-1 Mediate Gastroprotective Action of Carbon Monoxide against Ethanol-Induced Gastric Mucosal Lesions.

Carbon monoxide (CO) has been reported to contribute to the maintenance of gastric mucosal integrity, gastroprotection, and ulcer healing. However, involvement of transient receptor potential vanilloid receptor type 1 (TRPV1) located on afferent sensory fibers endings and sensory neuropeptide calcitonin gene-related peptide (CGRP) in CO-mediated gastroprotection against ethanol-induced gastric damage has not been explored. Male Wistar rats with and without denervation of afferent sensory neurons induced by capsaicin (total dose 125 mg/kg within 3 days) were pretreated with vehicle, CO donor tricarbonyldichlororuthenium (II) dimer (CORM-2, 5 mg/kg i.g.), administered alone or with CGRP-α (10 μg/kg i.p.) or TRPV1 antagonist capsazepine (5 mg/kg i.g.), followed 30 min later by intragastric (i.g.) administration of 75% ethanol. The area of gastric damage and gastric blood flow (GBF) were assessed planimetrically and by laser flowmetry, respectively. Microscopic evaluation of ethanol-induced gastric lesions was performed after haematoxylin/eosin (H&E) or alcian blue/periodic acid-Schiff/alcian blue (AB/PAS) staining. Gastric mucosal mRNA fold change for heme oxygenase (HMOX)-1, HMOX-2, CGRP-α, CGRP-β, inducible nitric oxide synthase (iNOS), endothelial (e)NOS, neuronal (n)NOS, cyclooxygenase (COX)-1, COX-2, and protein expression for HMOX-1 and TRPV1 was determined by real-time PCR or Western blot, respectively. Pretreatment with CORM-2 combined or not with CGRP reduced ethanol-induced gastric lesions and elevated GBF. Capsaicin-denervation or co-treatment with capsazepine or CGRP and CORM-2 in capsaicin-denervated animals failed to affect these beneficial effects of CO donor. In rats with intact sensory nerves, CORM-2 increased gastric mRNA level for HMOX-1 and CGRP-α. In capsaicin-denervated rats, CORM-2 increased eNOS mRNA fold change and TRPV1 protein expression while capsaicin denervation itself decreased HMOX-1 protein expression and eNOS mRNA level. We conclude that CO prevents gastric mucosa from ethanol-induced lesions due to activation of TRPV1/CGRP-α system and accompanying increase in gastric microcirculation but independently on afferent sensory nerve activity despite the stimulation of TRPV1 protein and CGRP-α mRNA expression.

Activation of TRPV1 by 12(s)‐HpETE and 20‐HETE Releases CGRP and Protects the Heart Against the Cardiac Dysfunction Caused by LPS

RationaleThe severity of cardiac dysfunction predicts mortality in patients with sepsis. Activation of transient receptor potential vanilloid receptor type 1 (TRPV1), expressed on sensory neurons innervating the heart, improves outcome in sepsis/endotoxemia. However, the identity of the endogenous activators of TRPV1 and the role of the channel in the cardiac dysfunction caused by sepsis/endotoxemia is unknown.ObjectivesWe investigated the impact of TRPV1 deletion upon the cardiac dysfunction caused by lipopolysaccharide (LPS; endotoxemia). In addition we explored the role of the arachidonic acid metabolites 12‐(S)‐hydroperoxyeicosatetraenoic acid (12(s)‐HpETE) and 20‐hydroxyeicosatetraenoic acid (20‐HETE) (potent ligands of TRPV1) and neuropeptides (downstream mediators of TRPV1 activation) in any effects seen.MethodsTRPV1−/− and wild‐type (WT, C57BL6 background) littermate mice were subjected to low (2 mg/kg, i.p.) or high dose LPS (6 mg/kg + peptidoglycan 0.1 mg/kg, i.p.). After 18 h cardiac function was determined using echocardiography. Neuronal (dorsal root ganglion) TRPV1 phosphorylation state was assessed by western blotting. Plasma levels of the neuropeptides calcitonin gene‐related peptide (CGRP), substance P (SP) and somatostatin were analyzed by ELISA. To investigate mechanisms some mice were treated with the CGRP receptor antagonist CGRP8–37 (150 mg/kg; 30 min prior to LPS and 1 and 2 h after LPS, i.v.), the 12‐lipoxygenase inhibitor cinnamyl‐3, 4‐dihydroxy‐a‐cyanocinnamate (CDC; 0.2 mg/mouse, i.p. to block 12(s)‐HpETE synthesis) or the cytochrome P450 inhibitor 17‐octadecynoic acid (17ODYA; 0.7 mg/mouse, i.p., 20‐HETE synthesis inhibitor). Data is presented as mean ± SEM and was analyzed by one‐way ANOVA followed by Bonferroni's post hoc test or unpaired Student's t‐test. P&lt;0.05 was considered to be statistically significant.ResultsWhilst low dose LPS did not alter cardiac function in WT mice [% ejection fraction (EF); vehicle: 72.4 ± 0.8, n=9 vs. LPS: 65.3 ± 4.5, n=8; p&gt;0.05], in TRPV1−/− mice a pronounced cardiac dysfunction was evident (%EF; vehicle: 69.9 ± 1.4, n=5 vs. LPS: 27.6 ± 4.5, n=7; p&lt;0.05); an effect similar to that evident in WT mice treated with high dose LPS (%EF; 34.4 ± 1.4; p&gt;0.05; n=7). Blockade of 12(s)‐HpETE or 20‐HETE synthesis augmented the cardiac dysfunction caused by low dose LPS in WT mice (%EF; vehicle: 72.3 ± 2.4, n=5; CDC: 48.5 ± 4.9, n=8; 17ODYA: 51.8 ± 4.9, n=8; CDC + 17ODYA: 40.5 ± 5.3, n=8; p&lt;0.05 for all groups compared to vehicle). LPS (2 mg/kg) caused increases in plasma CGRP, SP and somatostatin levels in WT mice; however interestingly 17ODYA or CDC/17ODYA co‐administration attenuated only the rise in CGRP; ~69% (P&lt;0.01) and 71% (p&lt;0.001) respectively. Accordingly, CGRP8–37 treatment increased cardiac dysfunction (%EF; vehicle: 67.4 ± 2.5, n=8 vs. CGRP8–37: 47.7 ± 2.5, n=7; p&lt;0.05) induced by low dose LPS, suggesting that the release of endogenous CGRP mediates the cardioprotective action of TRPV1. Western blotting confirmed activation of TRPV1 with low dose LPS treatment in WT mice with increased expression of phosphorylated TRPV1 relative to total TRPV1 (~64%, P&lt;0.05) relative to vehicle control, reflecting increased TRPV1 activation.ConclusionsActivation of TRPV1, reflected through increased phosphorylation of the channel, by 12(s)‐HpETE and 20‐HETE likely leads to the release of CGRP, which protects the heart against the cardiac dysfunction caused by LPS.Support or Funding InformationJ.C. is supported by China Scholarship Council and Queen Mary University of London. A.H. is supported by The Trustees of the Professor Derek Willoughby PhD Fund for Inflammatory Research.

2-AG promotes the expression of conditioned fear via cannabinoid receptor type 1 on GABAergic neurons.

The contribution of two major endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide (AEA), in the regulation of fear expression is still unknown. We analyzed the role of different players of the endocannabinoid system on the expression of a strong auditory-cued fear memory in male mice by pharmacological means. The cannabinoid receptor type 1 (CB1) antagonist SR141716 (3 mg/kg) caused an increase in conditioned freezing upon repeated tone presentation on three consecutive days. The cannabinoid receptor type 2 (CB2) antagonist AM630 (3 mg/kg), in contrast, had opposite effects during the first tone presentation, with no effects of the transient receptor potential vanilloid receptor type 1 (TRPV1) antagonist SB366791 (1 and 3 mg/kg). Administration of the CB2 agonist JWH133 (3 mg/kg) failed to affect the acute freezing response, whereas the CB1 agonist CP55,940 (50 μg/kg) augmented it. The endocannabinoid uptake inhibitor AM404 (3 mg/kg), but not VDM11 (3 mg/kg), reduced the acute freezing response. Its co-administration with SR141716 or SB366791 confirmed an involvement of CB1 and TRPV1. AEA degradation inhibition by URB597 (1 mg/kg) decreased, while 2-AG degradation inhibition by JZL184 (4 and 8 mg/kg) increased freezing response. As revealed in conditional CB1-deficient mutants, CB1 on cortical glutamatergic neurons alleviates whereas CB1 on GABAergic neurons slightly enhances fear expression. Moreover, 2-AG fear-promoting effects depended on CB1 signaling in GABAergic neurons, while an involvement of glutamatergic neurons remained inconclusive due to the high freezing shown by vehicle-treated Glu-CB1-KO. Our findings suggest that increased AEA levels mediate acute fear relief, whereas increased 2-AG levels promote the expression of conditioned fear primarily via CB1 on GABAergic neurons.

6]-shogaol induces Ca²⁺ signals by activating the TRPV1 channels in the rat insulinoma INS-1E cells.

[6]-shogaol is a vanilloid compound present in steamed ginger (Zingiber officinale), a commonly used spice. Pancreatic beta-cells respond to nutrients like glucose, amino acids and fatty acids, by an increase in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺](i)), which mediates diverse cellular processes in these cells. Some vanilloid compounds activate the transient receptor potential vanilloid receptor type 1 (TRPV1) channel. We investigated whether [6]-shogaol could trigger Ca²⁺ signals in the beta-cell. [Ca²⁺](i) was measured from single INS-1E cells by microscope-based fluorometry using fura-2 as the Ca²⁺ indicator. In fura-2 loaded single rat insulinoma INS-1E cells, a widely used model of beta-cell, [6]-shogaol increased [Ca²⁺](i) in a concentration-dependent manner. [Ca²⁺](i) increase by [6]-shogaol was completely blocked when Ca²⁺ was omitted from the extracellular medium. Capsazepine, an inhibitor of the TRPV1 ion channel completely inhibited the [6]-shogaol-induced [Ca²⁺](i) increase. [Ca²⁺](i) increase obtained by 1 µM [6]-shogaol was greater than that obtained by 10 mM glucose. Moreover, a sub-stimulatory concentration of [6]-shogaol (300 nM), significantly enhanced the glucose-induced [Ca²⁺](i) increase in these cells. We conclude that [6]-shogaol induces Ca²⁺ signals in the beta-cell by activating the TRPV1 channels, and it sensitizes the beta-cells to stimulation by glucose.

The essential role of transient receptor potential vanilloid 1 in simvastatin-induced activation of endothelial nitric oxide synthase and angiogenesis.

We investigated the role of transient receptor potential vanilloid receptor type 1 (TRPV1) in simvastatin-mediated activation of endothelial nitric oxide synthase (eNOS) and angiogenesis. Fluo-8 NW assay was for Ca(2+) detection; Griess's assay was for NO bioavailability; Western blotting and immunoprecipitation were for protein phosphorylation and interaction; tube formation and Matrigel plug assay were for angiogenesis. In endothelial cells (ECs), treatment with simvastatin time-dependently increased intracellular level of Ca(2+). Pharmacological inhibition or genetic disruption of TRPV1 abrogated simvastatin-mediated elevation of intracellular Ca(2+) in ECs or TRPV1-transfected HEK293 cells. Loss of TRPV1 function abolished simvastatin-induced NO production and phosphorylation of eNOS and calmodulin protein kinase II (CaMKII) in ECs and in aortas of mice. Inhibition of TRPV1 activation prevented the simvastatin-elicited increase in the formation of TRPV1-Akt-CaMKII-AMPK-eNOS complex. In mice, Matrigel plug assay showed that simvastatin-evoked angiogenesis was abolished by TRPV1 antagonist and genetic ablation of TRPV1. Additionally, our results demonstrated that TRP ankyrin 1 (TRPA1) is the downstream effector in the simvastatin-activated TRPV1-Ca(2+) signalling and in the consequent NO production and angiogenesis as evidence by that re-expression of TRPA1 further augmented simvastatin-elicited Ca(2+) influx in TRPV1-expressed HEK293 cells and ablation of TRPA1 function profoundly inhibited the simvastatin-induced increase in the phosphorylation of eNOS and CaMKII, formation of TRPV1-Akt-CaMKII-AMPK-eNOS complex, NO bioavailability, tube formation and angiogenesis in ECs or mice. Simvastatin-induced Ca(2+) influx may through the activation of TRPV1-TRPA1 signalling, which leads to phosphorylation of CaMKII, increases in the formation of TRPV1-CaMKII-AMPK-eNOS complex, eNOS activation, NO production and, ultimately, angiogenesis in ECs.

Citral: A monoterpene with prophylactic and therapeutic anti-nociceptive effects in experimental models of acute and chronic pain

Citral (3,7-dimethyl-2,6-octadienal) is an open-chain monoterpenoid present in the essential oils of several medicinal plants. The aim of this work was to evaluate the effects of orally administered citral in experimental models of acute and chronic nociception, inflammation, and gastric ulcers caused by non-steroidal anti-inflammatory drugs (NSAIDs). Oral treatment with citral significantly inhibited the neurogenic and inflammatory pain responses induced by intra-plantar injection of formalin. Citral also had prophylactic and therapeutic anti-nociceptive effects against mechanical hyperalgesia in plantar incision surgery, chronic regional pain syndrome, and partial ligation of sciatic nerve models, without producing any significant motor dysfunction. In addition, citral markedly attenuated the pain response induced by intra-plantar injection of glutamate and phorbol 12-myristate 13-acetate (PMA, a protein kinase C activator), as well as by intrathecal (i.t.) injection of ionotropic and metabotropic glutamate receptor agonists (N-methyl-d-aspartic acid [NMDA] and 1-amino-1,3-dicarboxycyclopentane [trans-ACPD], respectively), substance P, and cytokine tumour necrosis factor-α. However, citral potentiated behaviours indicative of pain caused by i.t., but not intra-plantar, injection of a transient receptor potential vanilloid receptor type 1 (TRPV1) agonist. Finally, the anti-nociceptive action of citral was found to involve significant activation of the 5-HT2A serotonin receptor. The effect of citral was accompanied by a gastro-protective effect against NSAID-induced ulcers. Together, these results show the potential of citral as a new drug for the treatment of pain.

Implication of transient receptor potential vanilloid type 1 in 14,15-epoxyeicosatrienoic acid-induced angiogenesis.

14,15-epoxyeicosatrienoic acid (14,15-EET) is implicated in regulating physiological functions of endothelial cells (ECs), yet the potential molecular mechanisms underlying the beneficial effects in ECs are not fully understood. In this study, we investigated whether transient receptor potential vanilloid receptor type 1 (TRPV1) is involved in 14,15-EET-mediated Ca2+ influx, nitric oxide (NO) production and angiogenesis. In human microvascular endothelial cells (HMECs), 14,15-EET time-dependently increased the intracellular level of Ca2+. Removal of extracellular Ca2+, pharmacological inhibition or genetic disruption of TRPV1 abrogated 14,15-EET-mediated increase of intracellular Ca2+ level in HMECs or TRPV1-transfected HEK293 cells. Furthermore, removal of extracellular Ca2+ or pharmacological inhibition of TRPV1 decreased 14,15-EET-induced NO production. 14,15-EET-mediated tube formation was abolished by TRPV1 pharmacological inhibition. In an animal experiment, 14,15-EET-induced angiogenesis was diminished by inhibition of TRPV1 and in TRPV1-deficient mice. TRPV1 may play a crucial role in 14,15-EET-induced Ca2+ influx, NO production and angiogenesis.

Control of synaptic function by endocannabinoid-mediated retrograde signaling.

Since the first reports in 2001, great advances have been made towards the understanding of endocannabinoid-mediated synaptic modulation. Electrophysiological studies have revealed that one of the two major endocannabinoids, 2-arachidonoylglycerol (2-AG), is produced from membrane lipids upon postsynaptic Ca2+ elevation and/or activation of Gq/11-coupled receptors, and released from postsynaptic neurons. The released 2-AG then acts retrogradely onto presynaptic cannabinoid CB1 receptors and induces suppression of neurotransmitter release either transiently or persistently. These forms of 2-AG-mediated retrograde synaptic modulation are functional throughout the brain. The other major endocannabinoid, anandamide, mediates a certain form of endocannabinoid-mediated long-term depression (LTD). Anandamide also functions as an agonist for transient receptor potential vanilloid receptor type 1 (TRPV1) and mediates endocannabinoid-independent and TRPV1-dependent forms of LTD. It has also been demonstrated that the endocannabinoid system itself is plastic, which can be either up- or down-regulated by experimental or environmental conditions. In this review, I will make an overview of the mechanisms underlying endocannabinoid-mediated synaptic modulation.

Calcium‐sensing receptor in rat vagal bronchopulmonary sensory neurons regulates the function of the capsaicin receptor TRPV1

Extracellular calcium-sensing receptor (CaSR) has been known to play a critical role in the maintainance of systemic Ca(2+) homeostasis. Recent studies have shown that CaSR is also expressed in many tissues that are not directly related to plasma Ca(2+) regulation, such as the central and peripheral nervous system, where the function of this receptor remains to be defined. In this study, we aimed to investigate the expression of CaSR and its potential interaction with transient receptor potential vanilloid receptor type 1 (TRPV1) in rat vagal bronchopulmonary sensory neurons. Our immunohistochemical experiments demonstrated the expression of CaSR in these sensory neurons as well as in trachea and lung parenchyma. Results from our whole-cell patch-clamp recordings in isolated neurons showed that strong activation of CaSR with high concentrations of its agonists, including spermine, NPS R-568 and Ca(2+), inhibited the capsaicin-evoked whole-cell inward current. Blockade of CaSR with its antagonists NPS 2390 and NPS 2143 significantly enhanced the capsaicin-evoked TRPV1 current. These data suggest that CaSR is likely to be involved in the integration of primary bronchopulmonary sensory inputs in physiological and/or pathophysiological conditions.

Mo2101 Japanese ‘Kampo’ Formula Daikenchuto, in Comparison With Its Similar Formula Ninjinto, Inhibits Gastric Acid Secretion Through Activation of Transient Receptor Potential Vanilloid Receptor Type 1 (TRPV1) Channels in Conscious Mice

Mo2101 Japanese ‘Kampo’ Formula Daikenchuto, in Comparison With Its Similar Formula Ninjinto, Inhibits Gastric Acid Secretion Through Activation of Transient Receptor Potential Vanilloid Receptor Type 1 (TRPV1) Channels in Conscious Mice

Development of ELISA to measure TRPV1 protein in rat tissues

The transient receptor potential vanilloid receptor type 1 (TRPV1) is a non-selective cation channel expressed in both the peripheral and the central nervous systems. To quantitatively determine TRPV1 protein levels in native rat tissues, novel monoclonal antibodies were raised against full-length recombinant human TRPV1 protein and utilized to develop a sandwich ELISA assay. Monoclonal antibody 10E3-1A2 specifically recognized TRPV1 protein and the recognition epitope was determined to reside in amino acids 45–58 of human and rat TRPV1. Using the TRPV1 polyclonal antibody ABRK4 as the capturing antibody and the monoclonal antibody 10E3-1A2 as the detection antibody, a sandwich ELISA that detected both human and rat TRPV1 protein was established. Recombinant human TRPV1 heterologously expressed in mammalian HEK293-F cells, which showed high ligand-binding affinity, was purified by TRPV1 monoclonal antibody affinity chromatography and used as protein standard to quantify TRPV1 protein levels. This ELISA detected TRPV1 protein as low as 1.5 ng/ml (15 pM), and was able to determine TRPV1 protein levels in native rat tissues such as DRG and spinal cord. This is the first TRPV1 sandwich ELISA that determines the abundance of TRPV1 protein in different tissues. It provides a powerful tool to quantify changes of TRPV1 protein levels in pathological states.

Capsaicin-induced vasodilatation in human nasal vasculature is mediated by modulation of cyclooxygenase-2 activity and abrogated by sulprostone

Extensively based on evidence gained from experimental animal models, the transient receptor potential vanilloid receptor type 1 (TRPV1)-activator capsaicin is regarded as a valuable tool in the research on neurogenic inflammation. Although capsaicin-related drugs gained renewed interest as a therapeutic tool, there is also controversy as whether neurogenic inflammation actually takes place in humans. In this study, we verified the involvement of capsaicin in vascular responses that are regarded to be implicated in the cascade of neurogenic inflammatory mechanisms. By means of ex vivo functional experiments on human nasal mucosal vascular beds, the effect and mechanism of action of capsaicin was assessed in the absence and presence of various agents that interfere with potentially related transduction pathways. Ten micromolars of capsaicin induced vasodilatations that were reduced by the selective EP(1) prostanoid receptor antagonist SC19220 (10 μM) and almost abolished by the selective COX-2 inhibitor NS398 (1 μM) and the EP(1/3) receptor agonist sulprostone (0.1-10 nM), but not affected by the TRPV1-antagonists capsazepine (5 μM), the neurokinin NK(1) receptor antagonist GR20517A (1 μM), and the calcitonin-gene-related peptide (CGRP) receptor antagonist CGRP8-37 (100 nM). Spontaneously released PGE(2) and PGD(2) levels were significantly reduced in the presence of capsaicin. In conclusion, capsaicin-at concentrations clinically applied or under investigation for diverse disease backgrounds-induces a vasodilatory response in human nasal mucosa via a mechanism involving TRPV1-independent reduction of PGE(2) production by modulation of COX-2 enzymatic activity. These vasodilatations can be suppressed by the EP(1/3) receptor agonist sulprostone at subnanomolar concentrations.

Epithelial expression of vanilloid and cannabinoid receptors: a potential role in burning mouth syndrome pathogenesis

Burning mouth syndrome is an intraoral burning sensation in which the oral mucosa has a normal appearance and no medical or dental causes can be found. It remains an unknown disease for which long-term treatment is still lacking. The aim of this study is to assess in epithelial human tongue the expression of three receptors involved in pain transmission, such as a transient receptor potential vanilloid receptor type 1 (TRPV1) which mediates the sensation produced by chilli peppers, cannabinoid receptors type 1 (CB1) and type 2 (CB2), which are pathway-related to TRPV1. Epithelial cells express TRPV1, CB1 and CB2 receptors suggesting a role for these cells in sensory transduction. The study was performed on 8 healthy and 9 BMS patients. All patients underwent a 3-mm punch biopsy at the anterolateral aspect of the tongue close to the tip. Specimens were included in paraffin and serially cut to obtain 6um thick sections. The sections were processed for TRPV1, CB1 and CB2 immunohistochemistry. The analysis showed an altered expression of the studied receptors. In particular we observed an increase of TRPV1, a decrease of CB1 and an increase of CB2 expression in epithelial cells of the tongue with a change in morphological localization. So, these receptors seem to be correlated with BMS. These data could be useful for future characterization of BMS markers and specific therapies.

Characterization of Anandamide-Stimulated Cannabinoid Receptor Signaling in Human ULTR Myometrial Smooth Muscle Cells

Accumulating evidence highlights the importance of the endocannabinoid anandamide (AEA) as a key mediator in reproductive physiology. Current data suggest potential roles for AEA in gametogenesis, fertilization, and parturition. AEA exerts its actions through two G protein-coupled receptors, termed cannabinoid receptor 1 (CB1), and 2 (CB2), and the ligand-gated transient receptor potential vanilloid receptor type 1 (TRPV1) ion channel. At present, the cellular mechanism(s) and consequences of AEA signaling in reproductive tissues, especially the myometrium, are poorly understood. Here, we examine the expression of CB1, CB2, and TRPV1 in the human myometrial smooth muscle cell-line (ULTR) and characterize intracellular signaling after stimulation with AEA. Radioligand binding analysis revealed a total CB receptor expression of 76 +/- 24 fmol/mg protein, with both quantitative PCR and competition binding studies indicating a negligible CB2 component. AEA caused Galpha(i/o)-dependent inhibition of adenylate cyclase to reduce intracellular cAMP levels. In addition, AEA caused a 2.5- to 3.5-fold increase in ERK activation, which was ablated by inhibition of Galpha(i/o), phosphoinositide-3-kinase and Src-kinase activities, but not by inhibition of Ca(2+)/calmodulin-dependent protein kinase or protein kinase C activities. TRPV1 channel activation with capsaicin failed to activate ERK. Consistent with these findings, the selective agonists, arachidonyl-2-chloroethylamide (CB1) and L759656 (CB2), and selective antagonists AM251 (CB1) and JTE907 (CB2), provided pharmacological evidence that the ERK signaling pathway is activated through endogenously expressed CB1. These findings provide an insight into myometrial AEA signaling, highlighting a potential role for endocannabinoids in the regulation of gene expression in myometrial smooth muscle cells.

Involvement of afferent neurons in the pathogenesis of endotoxin-induced ileus in mice: Role of CGRP and TRPV1 receptors

Activation of neuronal reflex pathways by inflammatory mediators is postulated as an important pathogenic mechanism in postoperative ileus. In this study, we investigated the involvement of afferent neurons and more specifically the role of the transient receptor potential vanilloid receptor type 1 (TRPV1) and calcitonin gene-related peptide (CGRP) in endotoxin-induced motility disturbances in mice. Mice were injected with either lipopolysaccharides (LPS) or saline (control) and pre-treated with hexamethonium (blocker of neuronal transmission), capsaicin (neurotoxin), CGRP 8-37 (CGRP antagonist) or BCTC (TRPV1 receptor antagonist). We measured gastric emptying and intestinal transit of Evans blue next to rectal temperature and a global sickness behaviour scale. In vehicle-treated mice, LPS significantly delayed gastric emptying, small intestinal transit and rectal temperature while the sickness behaviour scale was increased. Hexamethonium, capsaicin, CGRP8-37 and BCTC all reversed the endotoxin-induced delay in gastric emptying and significantly reduced the delay in intestinal transit without effect on the endotoxin-induced decrease in rectal temperature and increase in sickness behaviour scale. Our findings provide evidence for the involvement of afferent nerves in the pathogenesis of endotoxin-induced motility disturbances in mice mediated via CGRP and TRPV1 receptors. Blockade of CGRP and TRPV1 receptors may offer a novel strategy for the treatment of endotoxin-induced ileus.

Capsaicin-induced, capsazepine-insensitive relaxation of the guinea-pig ileum

The mechanisms underlying transient receptor potential vanilloid receptor type 1 (TRPV1)-independent relaxation elicited by capsaicin were studied by measuring isometric force and phosphorylation of 20-kDa regulatory light chain subunit of myosin (MLC 20) in ileum longitudinal smooth muscles of guinea-pigs. In acetylcholine-stimulated tissues, capsaicin (1–100 μM) and resiniferatoxin (10 nM–1 μM) produced a concentration-dependent relaxation. The relaxant response was attenuated by 4-aminopyridine and high-KCl solution, but not by capsazepine, tetraethylammonium, Ba 2+, glibenclamide, charybdotoxin plus apamin nor antagonists of cannabinoid receptor type 1 and calcitonin-gene related peptide. A RhoA kinase inhibitor reduced the relaxant effect of capsaicin at 30 μM. Capsaicin and resiniferatoxin reduced acetylcholine- and caffeine-induced transient contractions in a Ca 2+-free, EGTA solution. Capsaicin at 30 μM for 20 min did not alter basal levels of MLC 20 phosphorylation, but abolished an increase by acetylcholine in MLC 20 phosphorylation. It is suggested that the relaxant effect of capsaicin at concentrations used is not mediated by TRPV1, but by 4-aminopyridine-sensitive K + channels, and that capsaicin inhibits contractile mechanisms involving Ca 2+ release from intracellular storage sites. The relaxation could be explained by a decrease in phosphorylation of MLC 20.