TRPV1 Receptors Research Articles

Hidden Multivalency in Phosphatase Recruitment by a Disordered AKAP Scaffold

Disordered scaffold proteins provide multivalent landing pads that, via a series of embedded Short Linear Motifs (SLiMs), bring together the components of a complex to orchestrate precise spatial and temporal regulation of cellular processes. One such protein is AKAP5 (previously AKAP79), which contains SLiMs that anchor PKA and Calcineurin, and recruit substrate (the TRPV1 receptor). Calcineurin is anchored to AKAP5 by a well-characterised PxIxIT SLiM. Here we show, using a combination of biochemical and biophysical approaches, that the Calcineurin PxIxIT-binding groove also recognises several hitherto unknown lower-affinity SLiMs in addition to the PxIxIT motif. We demonstrate that the assembly is in reality a complex system with conserved SLiMs spanning a wide affinity range. The capture is analogous to that seen for many DNA-binding proteins that have a weak non-specific affinity for DNA outside the canonical binding site, but different in that it involves (i) two proteins, and (ii) hydrophobic rather than electrostatic interactions. It is also compatible with the requirement for both stable anchoring of the enzyme and responsive downstream signalling. We conclude that the AKAP5 C-terminus is enriched in lower-affinity/mini-SLiMs that, together with the canonical SLiM, maintain a structurally disordered but tightly regulated signalosome.

Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis

Single particle cryo-EM often yields multiple protein conformations within a single dataset, but experimentally deducing the temporal relationship of these conformers within a conformational trajectory is not trivial. Here, we use thermal titration methods and cryo-EM in an attempt to obtain temporal resolution of the conformational trajectory of the vanilloid receptor TRPV1 with resiniferatoxin (RTx) bound. Based on our cryo-EM ensemble analysis, RTx binding to TRPV1 appears to induce intracellular gate opening first, followed by selectivity filter dilation, then pore loop rearrangement to reach the final open state. This apparent conformational wave likely arises from the concerted, stepwise, additive structural changes of TRPV1 over many subdomains. Greater understanding of the RTx-mediated long-range allostery of TRPV1 could help further the therapeutic potential of RTx, which is a promising drug candidate for pain relief associated with advanced cancer or knee arthritis.

Neurokinin 1 and 2 Receptors Differently Modulate PEG2‐ and Citric Acid‐Induced Cough and Ventilatory Responses

Inhalation of aerosolized citric acid (CA) or prostaglandin E2 (PGE2) provokes distinct cough patterns (Type I vs. II) and ventilatory responses (substantial hyperventilation vs. slight tachypnea) in humans and guinea pigs. Substance P (SP) and neurokinin A (NKA) are peripherally released mainly by vagal sensory fibers and capable of affecting CA‐induced cough and ventilatory response via preferentially activating neurokinin 1 and 2 receptors (NK1R and NK2R) respectively. The goal of this study was to determine the impacts of CA and PGE2 exposure on pulmonary SP and NKA levels and the roles of NK1R and NK2R in CA‐ and PGE2‐evoked cough and ventilatory responses. In unanesthetized guinea pigs, we determined: (1) pulmonary SP and NKA contents after CA or PGE2 exposure for 10 min; (2) effects of vehicle, CP‐99994 and SR‐48968 (a NK1R and a NK2R antagonist) given by intraperitoneal injection (IP) or aerosol inhalation (IH) on CA‐ and PGE2‐evoked cough and ventilation; and (3) immunoreactive expression of NK1R/NK2R in vagal sensory neurons labeled by TRPV1 (responsible for Type I cough) or EP3 receptors (responsible for Type II cough). CA and PGE2 exposure evoked Type I and II cough respectively associated with different degree of increases in pulmonary SP and NKA. CP‐99994 and SR‐48968 via both IP and IH similarly depressed the cough responses to CA with less impact on the cough response to PGE2 exposure and substantially inhibited or blocked the evoked ventilatory responses to both CA and PGE2. Moreover, NK1R and NK2R co‐expressed in vagal C‐neurons labeled by TRPV1 or EP3 receptors. These results suggest that the endogenously released SP and NKA play an important role in generating the cough and ventilatory responses to CA and maintaining the ventilatory response to PGE2, at least in part, via activation of vagal C‐neurons expressing NK1R and NK2R.

The synthetic non pungent capsaicin analog Arvanil displays robust anti‐tumor activity in human small cell lung cancer

The nutritional compound capsaicin is the hot spicy pungent ingredient of chili peppers. Capsaicin is a pain‐relieving agent and is found in several over‐the counter pain medications. However, recent studies have shown that capsaicin has profound anti‐neoplastic effects in several types of human cancers. The applications of capsaicin as a clinically useful drug are limited by its unpleasant side effects, such as gastric irritation, stomach cramps and burning sensation. This has led to extensive research focused on the identification and rational design of second‐generation capsaicin analogs, which possess greater bioactivity than capsaicin. Our laboratory performed an SAR study using a panel of non‐pungent capsaicin analogs and identified Arvanil as the hit compound. Arvanil triggered robust apoptosis in a panel of human small cell lung cancer (SCLC). The growth‐suppressive activity of Arvanil was confirmed in chicken chorioallantoic membrane (CAM) models. Finally, the dietary administration of Arvanil potently decreased the growth rate of human SCLC tumors xenotransplanted in athymic mice. The pro‐apoptotic activity of Arvanil was mediated by the TRPV1 receptor and the calpain pathway. Non‐pungent capsaicin analogs like Arvanil may be promising drug candidates for the management and treatment of human SCLC.

Role of the Gβ5/R7‐RGS complex in the regulation of pain transmission in sensory ganglia

Neuropathic pain is a chronic disorder resulting from damage to the afferent nerve fibers or central pain pathways and is often a complication in pathological conditions such as diabetes, shingles, multiple sclerosis, and stroke. The opioid epidemic has elucidated the need for more efficacious treatments for neuropathic pain. In 2019 alone, nearly 1.6 million people were diagnosed with an opioid use disorder and 48,000 people died from a synthetic opioid overdose. Despite the addictive properties, opioids are still the most frequently prescribed pain medication, even for chronic neuropathic pain. Heterotrimeric G‐proteins consisting of the α, β, and γ subunits convey extracellular signals sensed by G‐protein coupled receptors (GPCRs) to intracellular effectors. The Gβ5 subunit is a divergent member of the G‐protein β subunit family as it does not bind to traditional γ subunits. Instead, Gβ5 complexes with the R7 subfamily of the regulators of G‐protein signaling (R7‐RGS) containing 4 members: RGS6, 7, 9 and 11. The Gβ5/R7‐RGS complex acts as a GTPase accelerating protein (GAP) for G‐protein αi/o (Gαi/o) subunits. Previous studies have established the integral role of R7‐RGS proteins in pain transmission via their interactions with Gαi/o‐coupled receptors including opioid and metabotropic gamma‐aminobutyric acid (GABA‐B) receptors. Our lab has shown the lack of Gβ5 in sensory ganglia diminishes mechanical, thermal, and chemical nociception. However, the conditional knockout of Gβ5 in Rgs7 expressing neurons reduces only mechanical nociception. This Gβ5/RGS7‐dependent mechanical nociception relies on GABA‐B receptor signaling as indicated by the rescue of mechanical nociception in Rgs7‐Cre; Gnb5 fl/fl mice after treatment with 2‐hydroxysaclofen, a GABA‐B antagonist. We also established that Rgs9 expressing neurons regulate thermal nociception via a Gβ5‐dependent pathway as assayed by the hotplate test in Rgs9‐Cre; Gnb5 fl/fl mice. The purpose of this project has been to understand the molecular role of each R7‐RGS member in the regulation of pain transmission. First, we confirmed co‐localization between the Gnb5 transcript and all four R7‐RGS mRNA transcripts in murine dorsal root ganglia (DRG) using the RNAscope HiPlex assay, a novel in situ hybridization technique. We then established the co‐localization patterns between each R7‐RGS member and various pain related receptors including Mrgprd, Trpa1, and Trpv1. Our RNAscope results support the behavioral tests since Rgs7 transcripts highly co‐express with Mrgprd, a mechanical nociceptor, while Rgs9 transcripts most frequently co‐express with Trpv1, a thermosensitive receptor. These results suggest that each R7‐RGS member might regulate unique types of nociception. We have also shown that Rgs11 transcripts co‐localize with Trpv1 and Trpa1 receptor transcripts which indicates Rgs11 might regulate the chemical nociception as tested by capsaicin and mustard oil administration in the eye‐wipe test. Next, we aim to study the possible roles of Rgs6 and Rgs11 in regulating chemical nociception using conditional Gβ5 knockout mice mediated by Rgs6‐cre and Rgs11‐cre, respectively.

Renal Sympathetic Nerve Activity Responses to Hypoxia and Hypercapnia in Rats Exposed to Chronic Intermittent Hypoxia

Increased responsiveness of the carotid bodies to acute hypoxia was shown in animals exposed to chronic intermittent hypoxia (CIH). The kidney is sensitive to hypoxia and decreased cortical partial pressure of oxygen (PO2) levels was revealed in rats exposed to moderate CIH. We hypothesized that the response of the renal sympathetic nerve activity (RSNA) to acute hypoxia and hypercapnia is enhanced in rats exposed to CIH. Wistar rats were exposed to 96 cycles of CIH (6% O2, 8 hr/day, n=6) or to normoxia (sham, 21% O2, n=9) for 14 days. On day 15, rats were anesthetized (induction: urethane, α‐chloralose and sodium pentobarbitone mixture i.p. (416, 27 and 33mg/kg), maintenance: urethane and α‐chloralose mixture i.v. (62 and 4mg/kg)) to record cardiovascular parameters and RSNA. Rats were provided with supplementary oxygen to maintain PO2 levels > 84 mmHg. During hypoxia, rats breathed 10% O2for two minutes. This was followed with a hypercapnic challenge during which rats breathed 10% CO2 for 5 minutes. Following a 90‐minute recovery period, capsaizepine (CPZ, 5µg/ml/hr), a TRPV1 blocker, was infused into the right kidney. Then, hypoxic and hypercapnic challenges were repeated following the intra‐renal infusion of CPZ. Data are expressed as mean±SD and were analyzed using t‐test or ANOVA, where relevant. CIH rats were hypertensive (130±9 vs. 118±9mmHg, p=0.039) with comparable basal HR (425±38 vs. 407±34bpm), and RSNA (% of max, 33±12 vs. 25±12) compared with sham rats. In response to acute hypoxia, RSNA was increased to an equivalent extent in CIH‐exposed and sham rats (Maximum change in RSNA of baseline, 128±23 vs. 125±16%; RSNA (% of max), 36±7 vs. 44±22%). Additionally, the increase in HR in response to acute hypoxia was similar between CIH‐exposed and sham rats (∆HR, +42±29 vs. +30±27bpm). Mean arterial blood pressure (MAP) was similarly decreased in CIH‐exposed and sham rats during acute hypoxia (Maximum change in MAP, ‐33±24 vs. ‐27±17mmHg). The decrease in PaO2 levels during hypoxia was comparable between exposure groups (∆PaO2, ‐48±15 vs. ‐47±10mmHg). Hypercapnia induced a comparable increase in the RSNA in CIH‐exposed and sham rats (Maximum change in RSNA of baseline, +121±15 vs. +113±8%). The analysis of absolute RSNA when normalized to maximum RSNA showed that CIH‐exposed rats had increased RSNA compared with sham rats before and after exposure to hypercapnia (RSNA (% of max), 37±8 vs. 26±10%, p=0.037). The increase in PaCO2 levels during hypercapnia was similar between CIH and sham groups (∆PaCO2, +24±8 vs. +19±6mmHg). Intra‐renal CPZ did not alter the increase in RSNA and HR in response to hypoxia and hypercapnia in CIH‐exposed and sham rats. Protein expression of TRPV1 receptors in kidney tissue was equivalent in both exposure groups (1.88±0.23 vs. 1.68±0.26ng/mg). Our results indicate that the responsiveness of RSNA to acute hypoxia and hypercapnia is not altered following exposure to moderate CIH sufficient to increase RSNA and blood pressure.

A Novel Curcumin Isomer, cis‐transCurcumin, Activates Adenosine Receptor Subtypes A1 And A3: Potential for Pain Therapy

A wide variety of plants produce compounds that contain the 4‐hydroxy‐3‐methoxybenzyl or “vanillyl” group. These vanilloid compounds include such well‐known food molecules as vanillin, capsaicin, gingerol, and curcumin. Vanilloid compounds have been widely studied for their medicinal properties, and one property that most of them have in common is the ability to induce analgesia. Studies have indicated multiple potential cellular mechanisms for this analgesic activity, including TRPV1 receptors, opioid receptors, and adenosine receptors (ARs). There are only a few studies on the interaction of vanilloid compounds with ARs, and to our knowledge no previous study has measured the binding affinity of any vanilloid compound with either of the two AR subtypes that are linked to the inhibitory Gαsubunit (subtypes A1and A3). Our study objective was to assess the binding affinity and receptor activation of a novel curcumin isomer, cis‐transcurcumin (CTCUR), at AR subtypes A1and A3. Since agonism of these subtypes is associated with analgesia, we hypothesized that CTCUR would be an agonist at AR subtypes A1 and A3. Two lines of Chinese hamster ovary cells were used, each transfected to overexpress one of the AR subtypes. Cell survival assays were performed to assess toxicity. Competitive binding assays and confocal microscopy were performed to measure binding affinity. Computer docking was also performed, to assess location of binding. Assays of intracellular cAMP levels were performed to measure receptor activation. Survival results indicate that CTCUR is not toxic to CHO cells at any of the concentrations tested (the highest being 100 μM). Competition assay results indicate that CTCUR binds to subtype A1 with a Ki of 306 nM and to subtype A3 with a Ki of 400 nM. Microscopy results confirm sub‐micromolar affinity for A1, but not for A3. Docking results confirm sub‐micromolar affinity for both subtypes, and furthermore indicate that CTCUR binds to the “toggle switch” domain of α helix 6 that is important for receptor activation. Intracellular cAMP results indicate that CTCUR acts as an agonist of ARs. Thus, we fail to reject our hypothesis. These results are of interest because they provide the first in vitro confirmation of the hypothesis that vanilloid compounds can activate ARs, in accordance with the results of previous studies in vivo. Vanilloid compounds that activate ARs are therefore worthy of further study, as a potential class of analgesic therapeutics.

Transient Receptor Potential Vanilloid 4 Mediates Hypersensitivity in Chronic Sickle Cell Disease Pain

Sickle cell disease (SCD) pain is debilitating and severely diminishes patients' quality of life. A significant cause of SCD pain is vaso-oclusion, however, pain in SCD can also arise from chronic tissue inflammation and neuropathy. Our lab is interested in identifying novel peripheral targets for treating chronic SCD pain. Transient receptor potential (TRP) channels are among these targets, as they have known somatosensory functions. One of these, TRPV4, is a nonselective cation channel involved in naïve somatosensation, as well as inflammatory and neuropathic pain. TRPV4 is expressed in dorsal root ganglia (DRG) neurons, the primary detectors and transducers of somatosensory information, and in keratinocytes, which compose the majority of the epidermis and are located proximal to afferent terminals in the skin. Further, inflammatory factors that interact with TRPV4, including neutrophil elastase and endothelin-1, are prevalent in SCD. Thus, TRPV4 is a likely contributor to SCD pain. In this study we investigated the role of TRPV4 in persistent SCD somatic hypersensitivity. First, we demonstrated that punctate mechanical allodynia observed in SCD mice was completely and dose-dependently blocked by a TRPV4 antagonist. Functional analysis of small-diameter DRG neurons (many of which are nociceptors in rodents) from SCD mice revealed a moderate TRPV4-mediated increase of calcium flux, and blocking TRPV4 receptors reduced the proportion of mechanically-sensitive neurons from SCD mice. In contrast to these modest DRG data, we found keratinocytes from SCD mice display robust TRPV4 agonist-induced calcium flux compared to WT controls. Given keratinocytes are profuse throughout the skin and are positioned alongside sensory nerve terminals, this evidence suggests keratinocytes play a pivotal role in TRPV4-mediated SCD hypersensitivity. Together, these data suggest TRPV4 plays an integral role in SCD evoked behavioral hypersensitivity, an effect that is likely due to sensitized TRPV4-mediated keratinocyte, and to a lesser extent DRG, activity in these mice. Grant support from R01 NS070711 NIH/NINDS.

Participation of Potential Transient Receptors in the Antinociceptive Effect of Pharmacopuncture.

Despite the widespread clinical use of acupuncture in painful situations, the use of this treatment should be further clarified. Nociception is mediated by the activation of nociceptors, such as transient receptor potentials (TRPs). The family of TRPs includes TRPV1, TRPM8, and TRPA1, which can be stimulated by substances such as capsaicin, menthol, and methyl salicylate, respectively. This study aimed to investigate the role of TRPs in antinociception via the administration of agonists of these receptors in the Zusanli acupoint (ST36) in models of inflammatory, acute, and neuropathic pain. Male Wistar rats were used for this experiment. All rats received a subcutaneous injection of TRP agonists (capsaicin, menthol, or methyl salicylate) in ST36; saline was injected as control. Nociception was evaluated using the electronic mechanical threshold test and tail-flick test before the administration of complete Freund's adjunct or chronic constriction injury of the sciatic nerve and after the administration of TRP agonists. Results: Nociception was found to be attenuated after treatment with TRP agonists. The administration of different doses (0.03, 0.3, and 3.0 μg/20 μL) of capsaicin, menthol, and methyl salicylate in the different pain models (neuropathic, inflammatory, and nociceptive) induced antinociception in most of the evaluated time points. Based on the findings, we suggest that the activation of TRPV1, TRPM8, and TRPA1 receptors results in the antinociceptive effect of the stimulation of the ST36 acupoint. Thus, TRP receptors may present a new therapeutic opportunity for the control of inflammatory and neuropathic pain.

Memory Consolidation Depends on Endogenous Hippocampal Levels of Anandamide: CB1 and M4, but Possibly not TRPV1 Receptors Mediate AM404 effects

The endocannabinoid system is involved in the fine-tuning of local synaptic plasticity in the hippocampus during the initial steps of memory formation/transformation. In spite of extensive studies, endocannabinoid modulation of these processes is still poorly understood. Here we studied the effects of intra-CA1 infused AM404, an anandamide (AEA) transport/metabolism inhibitor, upon an aversive memory consolidation with or without prior systemic administration of metyrapone, as well the concomitant intra-CA1 administration of AM404 plus AM251 (CB1 receptor inverse-agonist), capsazepine (TRPV1 receptor antagonist) or tropicamide (M4 receptor antagonist). We also investigated the effect of AM404 on memory retrieval and Long-Term Potentiation induction. Adult male Wistar rats were trained in the Contextual Fear Conditioning task and tested 48 h later. AM404 disrupted both memory consolidation and retrieval, and abolished LTP induction. The post-training effect, however, was reverted by metyrapone – which was amnestic by itself – corroborating the known co-dependency between glucocorticoids and endocannabinoids, and suggesting that some level of aversiveness is necessary for an adequate consolidation. In the coadministration experiments, while AM251 and tropicamide were able to revert the AM404 amnestic effect, capsazepine had no effect. This confirms that CB1 actually mediate the amnestic effect caused by the augmented AEA pool, but TRPV1 does not. The tropicamide result suggests an interesting comodulatory interaction between the endocannabinoid and the cholinergic systems. We propose a steady-state model centered in the idea of an optimal, stable extracellular concentration of anandamide as a necessary condition to ensure the consolidation of a stable memory trace in the CA1 area.

Anti-Inflammatory and Antiviral Effects of Cannabinoids in Inhibiting and Preventing SARS-CoV-2 Infection.

The COVID-19 pandemic caused by the SARS-CoV-2 virus made it necessary to search for new options for both causal treatment and mitigation of its symptoms. Scientists and researchers around the world are constantly looking for the best therapeutic options. These difficult circumstances have also spurred the re-examination of the potential of natural substances contained in Cannabis sativa L. Cannabinoids, apart from CB1 and CB2 receptors, may act multifacetedly through a number of other receptors, such as the GPR55, TRPV1, PPARs, 5-HT1A, adenosine and glycine receptors. The complex anti-inflammatory and antiviral effects of cannabinoids have been confirmed by interactions with various signaling pathways. Considering the fact that the SARS-CoV-2 virus causes excessive immune response and triggers an inflammatory cascade, and that cannabinoids have the ability to regulate these processes, it can be assumed that they have potential to be used in the treatment of COVID-19. During the pandemic, there were many publications on the subject of COVID-19, which indicate the potential impact of cannabinoids not only on the course of the disease, but also their role in prevention. It is worth noting that the anti-inflammatory and antiviral potential are shown not only by well-known cannabinoids, such as cannabidiol (CBD), but also secondary cannabinoids, such as cannabigerolic acid (CBGA) and terpenes, emphasizing the role of all of the plant’s compounds and the entourage effect. This article presents a narrative review of the current knowledge in this area available in the PubMed, Scopus and Web of Science medical databases.

Synthesis, molecular docking, ADMET study and in vitro pharmacological research of 7-(2-chlorophenyl)-4-(4-methylthiazol-5-yl)-4,6,7,8-tetrahydroquinoline-2,5(1H,3H)-dione as a promising non-opioid analgesic drug

Introduction: The discovery of novel drugs that can block the transmission of pain signals for treating the pain of various etiologies is an urgent topic in pharmaceutics. The aim of this paper is to synthesize and to investigate in vitro and in silico characteristics of a promising novel compound: 7-(2-chlorophenyl)-4-(4-methylthiazol-5-yl)-4,6,7,8-tetrahydroquinoline-2,5(1H,3H)-dione (HSV-DKH-0450).
 Materials and methods: The specific activity and the inhibitory mechanism of HSV-DKH-0450 were studied using the HEK293 culture cells expressing the IPTG-induced TRPA1 ion channels. Cardiotoxicity was determined by estimating the binding of HSV-DKH-0450 to the hERG channel. Inhibition of human liver cytochromes was determined by the effect on the activity of cytochromes 1A2, 2C9, 2D6, 2C8, and 3A4. Cellular toxicity was assessed by the effect on the viability of human hepatocytes. ADMET properties were evaluated using admetSAR and SwissADME web-based tools. Molecular docking was carried out using AutoDock Vina tools to predict the binding affinity of all HSV-DKH-0450 stereoisomers toward the TRPA1 and TRPV1 receptors.
 Results and discussion: In silico predictions of ADMET properties of HSV-DKH-0450 showed that it has optimal pharmaceutical profiles. A series of in vitro pharmacological studies revealed that HSV-DKH-0450 is a promising antagonist of the TRPA1 ion channel with the IC50 of 91.3 nM. The molecular docking of HSV-DKH-0450 stereoisomers against the TRPA1 and TRPV1 receptors demonstrates that they all are characterized by an approximately similar high binding affinity.
 Conclusion: The obtained data for substance HSV-DKH-0450 look promising for its further development as a potential therapeutic agent for pain relief.

Therapeutic potential of modulation of the ion channel activity of vanilloid receptors TRPV1 in oncological practice

Background. Type 1 vanilloid receptors (TRPV1 ) play an important role in tumoral genesis and cancer development, because the expression levels of TRPV1 change in a lot of types of cancer cells. At present, the regulation of functional activity and sensitivity of TRPV1 is an object of intensive research. Purpose – to characterize the modern concept of therapeutic potential of modulation of the ion channel activity of vanilloid receptors TRPV1 in oncological practice according to the data from open literature sources.
 Materials and methods. The publications were selected from the following databases: PubMed, EBSCO, Clinical Key, etc. In these publications the data on the ion channels of the transistor receptor potential were elucidated, particularly on type 1 vanilloid receptors, their role in tumoral genesis and the therapeutic potential of the modulation of their activity. Results. Binding of exogenous agonists to the TRPV1 receptor is accompanied by the influx of Ca2+ ions from the cytosol to the cell. It is known that Ca2+ ions are one of the main secondary messengers, since they play an important role in lots of fundamental physiological processes, including cell excitability, vitality, apoptosis and transcription. The disbalance of intracellular flow of Ca2+ is associated with characteristics of different types of cancer. The latest studies have shown that Ca2+ also contributes to certain malignant appearances, such as proliferation, invasion, migration and metastasis. Moreover, TRPV1 activation modulates the apoptosis-proliferation balance through the mechanisms beyond Ca2+ signaling, and in some works TRPV1 role in metastasis of cancer cells is mentioned. Conclusions. Selective TRPV1 activation or the increase in its expression has therapeutic potential, conditioned by pleiotropic influence on the apoptosis-proliferation balance in cancer cells. TRPV1 blockage or reduction of its expression can mitigate hyperalgesia caused by the tumor. In addition, TRPV1 act as biomarkers of a range of cancers (invasive breast carcinoma, epithelial ovarian and cervical cancer).

Anti-Inflammatory Effect of Beta-Caryophyllene Mediated by the Involvement of TRPV1, BDNF and trkB in the Rat Cerebral Cortex after Hypoperfusion/Reperfusion.

We have previously shown that bilateral common carotid artery occlusion followed by reperfusion (BCCAO/R) is a model to study early hypoperfusion/reperfusion-induced changes in biomarkers of the tissue physiological response to oxidative stress and inflammation. Thus in this study, we investigate with immunochemical assays if a single dose of beta-caryophyllene (BCP), administered before the BCCAO/R, can modulate the TRPV1, BDNF, and trkB receptor in the brain cortex; the glial markers GFAP and Iba1 were also examined. Frontal and temporal-occipital cortical regions were analyzed in two groups of male rats, sham-operated and submitted to BCCAO/R. Six hours before surgery, one group was gavage fed a dose of BCP (40 mg/per rat in 300 μL of sunflower oil), the other was pre-treated with the vehicle alone. Western blot analysis showed that, in the frontal cortex of vehicle-treated rats, the BCCAO/R caused a TRPV1 decrease, an increment of trkB and GFAP, no change in BDNF and Iba1. The BCP treatment caused a decrease of BDNF and an increase of trkB levels in both sham and BCCAO/R conditions while inducing opposite changes in the case of TRPV1, whose levels became higher in BCCAO/R and lower in sham conditions. Present results highlight the role of BCP in modulating early events of the cerebral inflammation triggered by the BCCAO/R through the regulation of TRPV1 and the BDNF-trkB system.

Capsaicin, a Phytochemical From Chili Pepper, Alleviates the Ultraviolet Irradiation-Induced Decline of Collagen in Dermal Fibroblast via Blocking the Generation of Reactive Oxygen Species.

Capsaicin, a major ingredient in chili pepper, has broad pharmaceutical applications, including relieving pain, anti-inflammation, and treating psoriasis. In dermatological biology, capsaicin has been shown to prevent the ultraviolet (UV)-induced melanogenesis via TRPV1 receptor. To strengthen the roles of capsaicin in skin function, the damaged skin, triggered by exposure to UV, was reversed by capsaicin in both in vitro and in vivo models. In cultured dermal fibroblasts, the exposure to UV induced a decrease of collagen synthesis and increases expression of matrix metalloproteinases (MMPs), generation of reactive oxygen species (ROS), and phosphorylation of Erk and c-Jun, and these events subsequently led to skin damage. However, the UV-mediated damages could be reversed by pre-treatment with capsaicin in a dose-dependent manner. The effect of capsaicin in blocking the UV-mediated collagen synthesis was mediated by reducing generation of ROS in dermal fibroblasts, instead of the receptor for capsaicin. Hence, capsaicin has high potential value in applying as an agent for anti-skin aging in dermatology.

A52 GUT-INNERVATING TRPV1+ NEURONS DRIVE CHRONIC VISCERAL PAIN VIA MICROGLIAL P2Y12 RECEPTOR

BackgroundLong-lasting changes in neural pain circuits precipitate the transition from acute to chronic pain in patients living with inflammatory bowel diseases (IBDs). While significant improvement in IBD therapy has been made to reduce inflammation, a large subset of patients continues to suffer throughout quiescent phases of the disease. Peripheral and central mechanisms contribute to the transition from acute to chronic pain during active disease and clinical remission. Lower mechanical threshold and hyperexcitability of visceral afferents induce gliosis in central pain circuits, leading to persistent visceral hypersensitivity (VHS). In the spinal cord, microglia, the immune sentinels of the central nervous system, undergo activation in multiple models of VHS. Using the Dextran Sodium Sulfate (DSS) model of colitis, we found that microglial G-CSF was able to sensitize colonic nociceptors that express the pain receptor TRPV1. While TRPV1+ nociceptors have been implicated in peripheral sensitization, their contribution to central sensitization via microglia remains unknown.AimsHere we investigated the mechanisms of microglia activation to identify centrally acting analgesics for chronic IBD pain.MethodsUsing Designer Receptors Exclusively Activated by Designer Drugs (DREADD) expressed in TRPV1-expressing visceral neurons that sense colonic inflammation, we tested whether neuronal activity was indispensable to control microglia activation and VHS. We then investigated the neuron-microglia signaling system involved in visceral pain chronification.ResultsWe found that chemogenetic inhibition of TRPV1+ visceral afferents prevents microglial activation in the spinal cord and subsequent VHS in colitis mice. In contrast, chemogenetic activation, in the absence of colitis, enhanced microglial activation associated with VHS. We identified a purinergic signaling mechanism mediated by neuronal ATP and microglial P2RY12 receptor, triggering VHS in colitis. Inhibition of P2RY12 prevented microglial reactivity and chronic VHS post-colitis.ConclusionsOverall, these data provide novel insights into the central mechanisms of chronic visceral pain and suggest that targeting microglial P2RY12 signaling could be harnessed to relieve pain in IBD patients who are in remission.Funding AgenciesCCC

Botulinum toxin promotes orofacial antinociception by modulating TRPV1 and NMDA receptors in adult zebrafish

The aim of the study was to evaluate the possible involvement of transient receptor potential (TRP) channels, Acid-sensing ion channels (ASIC) and N-Methyl-D-aspartate receptor (NMDAR) in the orofacial antinociceptive behaviour effect of botulinum toxin type A (BoNT/A) in adult zebrafish. Initially, the open field test was performed to evaluate the effect of BoNT/A on the locomotor activity of zebrafish. Subsequently, the animals were pretreated with BoNT/A (0.05U, 0.1U or 0.5U/masseter) and acute orofacial nociception was induced by cinnamaldehyde, capsaicin, menthol, acid saline or glutamate applied to the lip or masseter muscle. In another group of experiments, animals were pre-treated with capsazepine (TRPV1 antagonist) or ketamine (NMDAR antagonist) to investigate the mechanism of antinociception. The possible involvement of central C-fibre afferents was also investigated using capsaicin desensitized animals. A molecular docking study was performed to observe the in silico interaction of BoNT/A with TRPV1 and NMDA channels. Pretreatment with BoNT/A reduced the nociceptive behaviour induced by capsaicin and glutamate. Antinociception was effectively inhibited by capsazepine and ketamine, as well as by capsaicin-induced desensitization. Consistent with these in vivo findings, the molecular docking study indicated that BoNT/A can interact with TRPV1 and NMDAR. The results indicate the involvement of TRP and NMDAR mechanisms in the orofacial antinociceptive behaviour effect of BoNT/A. The results also confirm the pharmacological relevance of BoNT/A as an inhibitor of orofacial nociception behaviour.

The Effect of Anti-Chemokine Oral Drug XC8 on Cough Triggered by The Agonists of TRPA1 But Not TRPV1 Channels in Guinea Pigs.

IntroductionChronic cough heavily affects patients’ quality of life, and there are no effective licensed therapies available. Cough is a complication of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection, asthma, and other diseases. Patients with various diseases have a different profile of tussive responses to diverse cough triggers, thereby suggesting sundry mechanisms of neuronal dysfunctions. Previously, we demonstrated that the small molecule drug XC8 shows a clinical anti-asthmatic effect. The objective of the present study was to investigate the effect of XC8 on cough.MethodsWe studied the antitussive effect of XC8 on cough induced by agonists activating human transient receptor potential (TRP) cation channels TRPA1 or TRPV1 in guinea pigs. We checked the agonistic/antagonistic activity of XC8 on the human cation channels TRPA1, TRPV1, TRPM8, P2X purinoceptor 2 (P2X2), and human acid sensing ion channel 3 (hASIC3) in Fluorescent Imaging Plate Reader (FLIPR) assay.ResultsXC8 demonstrated clear antitussive activity and dose-dependently inhibited cough in guinea pigs induced by citric acid alone (up to 67.1%) or in combination with IFN-γ (up to 76.4%). XC8 suppressed cough reflexes induced by the repeated inhalation of citric acid (up to 80%) or by cinnamaldehyde (up to 60%). No activity of XC8 against cough evoked by capsaicin was revealed. No direct agonistic/antagonistic activity of XC8 on human TRPA1, TRPV1, TRPM8, P2X2, or hASIC3 was detected.ConclusionsXC8 acts against cough evoked by the activation of TRPA1 (citric acid/cinnamaldehyde) but not TRPV1 (capsaicin) channels. XC8 inhibits the cough reflex and suppresses the cough potentiation by IFN-γ. XC8 might be of significant therapeutic value for patients suffering from chronic cough associated with inflammation.

Controlled Activation of TRPV1 Channels on Microglia to Boost Their Autophagy for Clearance of Alpha-Synuclein and Enhance Therapy of Parkinson's Disease.

Parkinson's disease (PD) is characterized with accumulation of Lewy bodies with a major component of fibrillar alpha-synuclein (α-syn). Herein, boosting PD therapeutic efficacy by enhancing the autophagy of microglia to phagocytose and degrade α-syn via controlled opening of their surface TRPV1 channels with rationally designed photothermal nanoagent is reported. The Cu2- x Se-anti-TRPV1 nanoparticles (CS-AT NPs) are fabricated to target the microglia and open their surface TRPV1 channels under the second near infrared (NIR-II) laser irradiation to cause influx of Ca2+ to activate ATG5 and Ca2+ /CaMKK2/AMPK/mTOR signaling pathway, which promote phagocytosis and degradation of α-syn. The CS-AT NPs are efficiently delivered by focused ultrasound into striatum of PD mice with high expression of TRPV1 receptors. The athletic ability of PD mice treated by CS-AT NPs and NIR-II irradiation is significantly improved due to the phagocytotic clearance of α-syn by microglia with enhanced autophagy. The enzyme tyrosine hydroxylase, ionized calcium binding adapter protein 1, glial fibrillary acidic protein, and pSer129-α-syn (p-α-syn) of treated PD mice are almost recovered to the normal levels of healthy mice. This study provides insights into the activation of microglial autophagy by targeting surface ion channels to improve the treatment of PD and other neurodegenerative diseases.

The Role of the Possible Receptors and Intracellular Pathways in Protective Effect of Exogenous Anandamide in Kindling Model of Epilepsy.

In this research, the involvement of CB1 and TRPV1 receptors in the possible protective effects of anandamide were investigated in the kindling model of epilepsy. The basolateral amygdala of the rat brain was chosen to put stimulating electrodes. Semi-rapid kindling was induced by a repetitive sub-threshold stimulation for 5-9 consecutive days. There were seven groups, six of which were kindled and used for drug testing by intracerebroventricular (i.c.v.) microinjection. (i) Sham, (ii) control group received vehicles, (iii) anandamide (AEA; 100ng/rat), (iv) capsazepine (TRPV1 antagonist; 100ng/rat), (v) AM251 (CB1 antagonist; 100ng/rat), (vi) AM251 + anandamide, and (vii) capsazepine + anandamide. The after-discharge duration, seizure duration, and stage five duration were measured in rats. Moreover, the expressions of the extracellular signal-regulated kinase (ERK) and the cAMP responsive element binding (CREB) proteins in the hippocampus were also studied. The anandamide-treated group showed a significant decrease in seizure scores, while no change was shown in seizure scores in the capsazepine- and AM251-treated groups compared with the control group. Co-administrations of either capsazepine + AEA or AM251 + AEA attenuated the protective effect of AEA against seizure. Furthermore, the group received AEA showed a decrease in the expressions of CREB and p-CREB possibly through the activation of the CB1 and TRPV1 receptors. Activation of CB1 and TRPV1 receptors might be involved in AEA anticonvulsant effect in kindling model of epilepsy. This effect could be due to suppression of CREB phosphorylation in hippocampal neurons.