Transient Receptor Potential V1 Channels Research Articles

Anticarcinogenic Effects of Odorant Substances Citral, Citrathal R and Cyclovertal on Breast Cancer in vitro.

PurposeIn 2020, breast cancer still represents the most common type of cancer in women worldwide. Depending on the specific molecular subtype, clinical breast cancer management comprises surgery, radiotherapy, chemotherapy and targeted therapy. Furthermore, there are some therapeutic approaches from the field of complementary and alternative medicine. Current research focuses on the elucidation of new therapeutic targets for treatment development. Odorant substances affect apoptosis, proliferation and cell cycle in healthy and cancerous cells. Exact signalling pathways involved are not entirely clear. The present study aims to analyse their therapeutic potential in breast cancer.MethodsThis study focuses on the effect of commonly used odorant substances (citral, citrathal R, cyclovertal, para-cymol, hexylacetat, herbavert, dihydromyrcerol and limonen) on the breast cancer cell lines MDA-MB-231, T47-D and BT474. Methodologically, this study applied cell culturing, MTT assay for detection of IC50 of the odorant substance, RNA purification followed by qRT-PCR, protein isolation and Western Blot, as well as immunocytochemistry. Further, this study investigates the role of transient receptor potential channel V1 (TRPV1), involved in the mechanisms of action for some odorant substances. Therefore, capsazepine, a TRPV1 antagonist, was used.ResultsThe odorant substances citral, citrathal R and cyclovertal have significant pro-apoptotic (p < 0.001), anti-proliferative (p < 0.001) and cell cycle-arresting effects measurable in RNA expression as well as in protein levels and immunocytochemical staining. The combination of citral and capsazepine no longer showed significant pro-apoptotic, antiproliferative, and cell cycle inhibitory effects compared to the compounds alone. This indicates that TRPV1 is necessary for the signal transduction of citral.ConclusionThis present study reveals three odorant substances with effects on cell viability, indicating their potential use in breast cancer therapy.

Electroacupuncture reduces cold stress-induced pain through microglial inactivation and transient receptor potential V1 in mice

BackgroundThe treatment, and efficacy thereof, is considered to be inadequate with specificity to alleviation of Fibromyalgia and its associated pain. Fibromyalgia patients suffer from chronic and persistent widespread pain and generalized tenderness. Transient receptor potential V1 (TRPV1), which is reported as a Ca2+ permeable ion channel that can be activated by inflammation, is reported to be involved in the development of fibromyalgia pain.MethodsThe current study explored the TRPV1 channel functions as a noxious sensory input in mice cold stress model. It remains unknown whether electroacupuncture (EA) attenuates fibromyalgia pain or affects the TRPV1 pathway.ResultsWe show that cold stress increases mechanical and thermal pain (day 7: mechanical: 1.69 ± 0.41 g; thermal: 4.68 ± 0.56 s), and that EA and Trpv1 deletion counter this increase. EA and Trpv1 deletion reduced the cold stress-induced increase in inflammatory mediators and TRPV1-related molecules in the hypothalamus, periaqueductal gray (PAG), and cerebellum of mice.ConclusionsOur results imply that EA has an analgesic effect associated with TRPV1 downregulation. We provide novel evidence that these inflammatory mediators can modulate the TRPV1 signaling pathway and suggest new potential therapeutic targets for fibromyalgia pain.

FXYD6 promotes thermal nociception by regulating TRPV1

FXYD6, an unnecessary auxiliary subunit of Na+,K+-ATPase, is expressed in the nervous system. However, its functions remain largely unclear. In the present study, we find that FXYD6 is involved in the thermal nociception. FXYD6 was mainly expressed in small-diameter DRG neurons expressing transient receptor potential channel V1 (TRPV1). In the SNS-Cre/Fxyd6F/F mice, loss of FXYD6 in these sensory neurons impaired the behavioral responses to noxious heat stimulus and intraplantar injection of capsaicin. The capsaicin-induced and TRPV1-mediated currents were decreased in the FXYD6–deficient DRG neurons. Heterologous expression of FXYD6 could increase the TRPV1 capsaicin-sensitive currents in HEK293 cells. Furthermore, we found that the negatively charged PGDEE motif in C-terminal of FXYD6 is required for the FXYD6/TRPV1 interaction and FXYD6-mediated enhancement of TRPV1. Disrupting the FXYD6/TRPV1 interaction with the TAT-PGDEE peptide could elevate the threshold of thermal nociception. Therefore, FXYD6 maintains the thermal nociception via interacting with TRPV1 channel in nociceptors.

Effects of Heating-Conduction Dry Needling Therapy on Rats with Chronic Myofascial Pain Syndrome

ObjectiveThis study aimed to investigate the effect of dry needling (DN) with 44°C heating on the pressure pain threshold (PPT) of rats with chronic myofascial pain syndrome (MPS) by regulating the transient receptor potential V1 channel. MethodsA total of 80 rats were divided into 5 groups: A, B, C, D, and E (16 in each group). The rats in Group A recovered naturally, while those in other groups received the DN treatment. The needles were heated to 40°C for the rats in Groups B and E and 44°C for the rats in Groups C and D. In addition, both Groups D and E received local capsaicin injection. PPTs of the gastrocnemius muscle and tolerance time of the plantar hot plate were measured before modeling, 1 day before the intervention, 24 hours after the intervention, and 7 days after the intervention. ResultsPPTs of the gastrocnemius muscle for rats with MPS increased 7 days after DN and heating to 40°C. For the rats receiving DN at 44°C heating, PPTs increased 24 hours and 7 days after the intervention. Further, 44°C heating and capsaicin injection improved PPT. DN and 40°C heating followed by capsaicin injection improved PPT. ConclusionDN and 44°C heating had therapeutic effects on rats with MPS at 24 hours and at 7 days after the intervention. DN and 40°C heating had therapeutic effects 7 days after the intervention. DN and 44°C heating might exert therapeutic effects by regulating the transient receptor potential V1 channel.

Effects of Honokiol on Activation of Transient Receptor Potential Channel V1 and Secretion of Thymic Stromal Lymphopoietin in HaCaT Keratinocytes

Abstract Objective: This study was performed to investigate the effects of honokiol on the activation of transient receptor potential channel V1 (TRPV1) and the secretion of thymic stromal lymphopoietin (TSLP) in a human benign epidermal keratinocyte line (HaCaT). Methods: HaCaT keratinocytes were cultivated and divided into six groups: capsaicin-induced model control group, capsazepine control group, solvent control group, and three honokiol treatment groups (7.81, 15.63, and 31.25 mg/L of honokiol). The effect of honokiol on calcium (Ca2+) influx was measured by a Ca2+ fluorescence imaging system. The fluorescence intensity (F) of cells was measured. The rate of change in F (ΔF/F 0) was calculated, and the ΔF/F 0–time curve was constructed. HaCaT keratinocytes were stimulated with polyinosinic:polycytidylic acid, recombinant human tumor necrosis factor α, and recombinant human interleukin 4. Different concentrations of honokiol (15.63, 7.81, and 3.91 mg/L) were added to the cells in the respective honokiol groups; 20 mg/L of dexamethasone or 0.5% dimethyl sulfoxide was added to the cells in the positive control group or solvent control group. The TSLP concentration in the HaCaT keratinocytes of each group was detected by enzyme-linked immunosorbent assay. Statistical analysis was performed by one-way analysis of variance and Dunnett t test. Results: The capsazepine-induced Ca2+ fluorescence intensity in HaCaT keratinocytes was significantly inhibited in the 31.25 mg/L honokiol group; ΔF/F 0 at 45 second was 0.76 in the model control group and 0 in the 31.25 mg/L honokiol group. The TSLP level in the 15.63 and 7.81 mg/L honokiol groups was lower than that in the solvent control group (t = 7.382, P = 0.003, and t = 2.766, P = 0.023, respectively), while the TSLP level in the 3.91 mg/L honokiol group was not significantly different from that in the solvent control group (t = 1.872, P = 0.124). Conclusions: Honokiol inhibited the Ca2+ influx induced by capsazepine (TRPV1 agonist) in HaCaT keratinocytes. Honokiol has an inhibitory effect on TSLP secretion in HaCaT keratinocytes.

Revisiting Tramadol: A Multi-Modal Agent for Pain Management.

Tramadol-an atypical opioid analgesic-has a unique pharmacokinetic and pharmacodynamic profile, with opioidergic, noradrenergic, and serotonergic actions. Tramadol has long been used as a well-tolerated alternative to other drugs in moderate pain because of its opioidergic and monoaminergic activities. However, cumulative evidence has been gathered over the last few years that supports other likely mechanisms and uses of tramadol in pain management. Tramadol has modulatory effects on several mediators involved in pain signaling, such as voltage-gated sodium ion channels, transient receptor potential V1 channels, glutamate receptors, α2-adrenoceptors, adenosine receptors, and mechanisms involving substance P, calcitonin gene-related peptide, prostaglandin E2, and proinflammatory cytokines. Tramadol also modifies the crosstalk between neuronal and non-neuronal cells in peripheral and central sites. Through these molecular effects, tramadol could modulate peripheral and central neuronal hyperexcitability. Given the broad spectrum of molecular targets, tramadol as a unimodal analgesic relieves a broad range of pain types, such as postoperative, low back, and neuropathic pain and that associated with labor, osteoarthritis, fibromyalgia, and cancer. Moreover, tramadol has anxiolytic, antidepressant, and anti-shivering activities that could improve pain management outcomes. The aim of this review was to address these issues in the context of maladaptive physiological and psychological processes that are associated with different pain types.

The coding of cutaneous temperature in the spinal cord.

The spinal cord is the initial stage that integrates temperature information from peripheral inputs. Here we used molecular genetics and in vivo calcium imaging to investigate the coding of cutaneous temperature in the spinal cord in mice. We found that heating or cooling the skin evoked robust calcium responses in spinal neurons, and their activation threshold temperatures distributed smoothly over the entire range of stimulation temperatures. Once activated, heat-responding neurons encoded the absolute skin temperature without adaptation and received major inputs from transient receptor potential (TRP) channel V1 (TRPV1)-positive dorsal root ganglion (DRG) neurons. By contrast, cold-responding neurons rapidly adapted to ambient temperature and selectively encoded temperature changes. These neurons received TRP channel M8 (TRPM8)-positive DRG inputs as well as novel TRPV1(+) DRG inputs that were selectively activated by intense cooling. Our results provide a comprehensive examination of the temperature representation in the spinal cord and reveal fundamental differences in the coding of heat and cold.

(372) Sex differences in peripheral serotonergic pain processing in male and female rats

Many persistent pain conditions occur predominantly in women making pain a major women’s health issue. As the majority of pain research has been conducted in males, the mechanisms underlying pain in women are unclear. One theory for the prevalence of pain disorders in females is steroid hormone modulation of pain mechanisms. The monoamine neurotransmitter serotonin (5HT) has been highly implicated in various pain conditions that are more prevalent in women. Our recent studies found that peripheral 5HT (1) evokes pain via a subpopulation of trigeminal nociceptors expressing the transient receptor potential V1 channel (TRPV1) and (2) potentiates capsaicin-evoked proinflammatory peptide release from human tooth pulp during the luteal phase of the menstrual cycle. No studies have yet examined the effect of hormone modulation on 5HT-evoked pain in females. We hypothesized that peripheral 5HT evokes greater pain behaviors in female rodents during stages of the estrous cycle when steroid hormones are fluctuating. Female Sprague-Dawley rats (250-350 g) from each stage of the estrous cycle, ovariectomized rats, and intact males were acclimated to the testing apparatuses. Rats received a hindpaw injection of 5HT (2 μg / 100uL; ipl) or saline (n = 6 / group) and thermal hyperalgesia and mechanical allodynia were measured at 10, 20 and 30 minutes. Here we report that female rats in proestrus and estrus exhibited significantly greater and longer-lasting pain behaviors following 5HT injection compared to males, diestrus females, and ovariectomized females, while there was no significant change in edema. These data implicate a modulatory role of hormones on 5HT-evoked pain processing, which may underlie the greater prevalence of pain disorders in women. Our current studies are elucidating the potential enhancing effect of 17-β-estradiol treatment on serotonergic potentiation of the TRPV1 population of sensory neurons in culture. Supported by a Research Enhancement Grant from Texas Woman’s University.

A pro-nociceptive phenotype unmasked in mice lacking fatty-acid amide hydrolase.

Fatty-acid amide hydrolase (FAAH) is the major enzyme responsible for degradation of anandamide, an endocannabinoid. Pharmacological inhibition or genetic deletion of FAAH (FAAH KO) produces antinociception in preclinical pain models that is largely attributed to anandamide-induced activation of cannabinoid receptors. However, FAAH metabolizes a wide range of structurally related, biologically active lipid signaling molecules whose functions remain largely unknown. Some of these endogenous lipids, including anandamide itself, may exert pro-nociceptive effects under certain conditions. In our study, FAAH KO mice exhibited a characteristic analgesic phenotype in the tail flick test and in both formalin and carrageenan models of inflammatory nociception. Nonetheless, intradermal injection of the transient receptor potential channel V1 (TRPV1) agonist capsaicin increased nocifensive behavior as well as mechanical and heat hypersensitivity in FAAH KO relative to wild-type mice. This pro-nociceptive phenotype was accompanied by increases in capsaicin-evoked Fos-like immunoreactive (FLI) cells in spinal dorsal horn regions implicated in nociceptive processing and was attenuated by CB1 (AM251) and TRPV1 (AMG9810) antagonists. When central sensitization was established, FAAH KO mice displayed elevated levels of anandamide, other fatty-acid amides, and endogenous TRPV1 agonists in both paw skin and lumbar spinal cord relative to wild-type mice. Capsaicin decreased spinal cord 2-AG levels and increased arachidonic acid and prostaglandin E2 levels in both spinal cord and paw skin irrespective of genotype. Our studies identify a previously unrecognized pro-nociceptive phenotype in FAAH KO mice that was unmasked by capsaicin challenge. The heightened nociceptive response was mediated by CB1 and TRPV1 receptors and accompanied by enhanced spinal neuronal activation. Moreover, genetic deletion of FAAH has a profound impact on the peripheral and central lipidome. Thus, genetic deletion of FAAH may predispose animals to increased sensitivity to certain types of pain. More work is necessary to determine whether such changes could explain the lack of efficacy of FAAH inhibitors in clinical trials.

Evodiamine suppresses capsaicin-induced thermal hyperalgesia through activation and subsequent desensitization of the transient receptor potential V1 channels.

Evodiae fructus (EF), a fruit of Evodia rutaecarpa Bentham, has long been used as an analgesic drug in traditional Chinese and Japanese medicine. However, the underlying molecular mechanism of its pharmacological action is unclear. Here, using calcium imaging, whole-cell patch-clamp recording, and behavioral analysis, we investigated the pharmacological action of EF and its principal compound, evodiamine, on the transient receptor potential (TRP) V1 channels. Dorsal root ganglion (DRG) neurons and TRPV1- or TRPA1-transfected human embryonic kidney-derived (HEK) 293 cells were used for calcium imaging or whole-cell patch-clamp recording. Twenty male adult Sprague-Dawley rats were used for the capsaicin-induced thermal hyperalgesia behavioral analyses. We found that evodiamine induced significant increases in intracellular calcium and robust inward currents in a subpopulation of isolated rat DRG neurons, most of which were also sensitive to capsaicin. The effect of evodiamine was completely blocked by capsazepine, a competitive antagonist of TRPV1. Evodiamine induced significant inward currents in TRPV1-, but not TRPA1-transfected HEK293 cells. Pretreatment with evodiamine reduced capsaicin-induced currents significantly. Furthermore, the in vivo pre-treatment of evodiamine suppressed thermal hyperalgesia induced by intraplantar injection of capsaicin in rats. These results identify that the analgesic effect of EF and evodiamine may be due to the activation and subsequent desensitization of TRPV1 in sensory neurons.

TRPV1‐mediated Ca2+ Influx and Middle Meningeal Artery Constriction

The causes of migraine and cluster headaches are not clear, but may involve dilation of the meningeal vasculature. Recent evidence indicates that transient receptor potential channel V1 (TRPV1) agonists may benefit patients with migraine or cluster headaches. Here, we test the hypothesis that capsaicin, a TRPV1 agonist, constricts middle meningeal artery (MMA) via activation of smooth muscle (SM) TRPV1 channels. Consistent with this hypothesis, capsaicin induced constriction of isolated pressurized rat MMA (EC50 ∼ 100 nM) that was blocked by the TRPV1‐selective antagonist, capsazepine (1 µM). Interestingly, capsaicin (10 µM) did not constrict cerebral arteries. Capsaicin‐induced MMA constriction was reduced ∼ 34% by the voltage‐dependent calcium channel (VDCC) blocker diltiazem (100 µM) and abolished by removal of extracellular Ca2+. To assess the direct impact of capsaicin on Ca2+ influx, MMA myocytes were freshly isolated from transgenic (acta2‐GCaMP5‐mCherry) mice with SM‐specific expression of Ca2+‐sensitive GCaMP5 and Ca2+‐independent mCherry proteins. In these cells, 1 µM capsaicin increased the fluorescence ratio of GCaMP5/mCherry by 4‐fold. Further, optical imaging of unitary Ca2+ events (sparklets) in intact MMA using Fluo‐4 revealed elementary capsaicin‐induced signals with a quantal fractional fluorescence change consistent with TRPV1 channels. In sum, these findings suggest capsaicin‐induced MMA constriction results from activation of TRPV1 channels on vascular myocytes. Capsaicin‐induced Ca2+ influx in MMA SM likely reflects both direct Ca2+ entry via TRPV1 channels and VDCC activation via TRPV1‐mediated Na+ influx. Supported by: Totman Trust, Peter Martin Fund, AHA and NIH.

Auto-oxidation Products of Epigallocatechin Gallate Activate TRPA1 and TRPV1 in Sensory Neurons

The sensation of astringency is elicited by catechins and their polymers in wine and tea. It has been considered that catechins in green tea are unstable and auto-oxidized to induce more astringent taste. Here, we examined how mammalian transient receptor potential V1 (TRPV1) and TRPA1, which are nociceptive sensors, are activated by green tea catechins during the auto-oxidation process. Neither TRPV1 nor TRPA1 could be activated by any of the freshly prepared catechin. When one of the major catechin, epigallocatechin gallate (EGCG), was preincubated for 3h in Hank's balanced salt solution, it significantly activated both TRP channels expressed in HEK293 cells. Even after incubation, other catechins showed much less effects. Results suggest that only oxidative products of EGCG activate both TRPV1 and TRPA1. Dorsal root ganglion (DRG) sensory neurons were also activated by the incubated EGCG through TRPV1 and TRPA1 channels. Liquid chromatography-mass spectrometry revealed that theasinensins A and D are formed during incubation of EGCG. We found that purified theasinensin A activates both TRPV1 and TRPA1, and that it stimulates DRG neurons through TRPV1 and TRPA1 channels. Results suggested a possibility that TRPV1 and TRPA1 channels are involved in the sense of astringent taste of green tea.

A human capsaicin model to quantitatively assess salivary CGRP secretion.

Capsaicin induces the release of calcitonin gene-related peptide (CGRP) via the transient receptor potential channel V1 (TRPV1). The CGRP response after capsaicin application on the tongue might reflect the "activation state" of the trigeminal nerve, since trigeminal CGRP-containing vesicles are depleted on capsaicin application. We tested (i) the quantitative CGRP response after oral capsaicin application; (ii) the optimal concentration of red chili homogenate; and (iii) the day-to-day variability in this response. Saliva was collected for two consecutive days after oral application of eight capsaicin dilutions (red chili homogenates) of increasing concentrations in 13 healthy individuals. Effects of homogenate concentration were assessed. Consecutively, saliva was sampled after application of vehicle and undiluted homogenates. CGRP secretion (pg/ml) increased dose-dependently with homogenate concentration (p < 0.001). CGRP levels were highest after application of nondiluted homogenate (vs. baseline: 13.3 (5.0) vs. 9.7 (2.9); p = 0.003, as was total CGRP secretion in five minutes (pg) with undiluted (vs. baseline): 89.2 (44.1) vs. 14.1 (2.8); p < 0.001. The dose-dependent response in CGRP was not affected by day (p = 0.14) or day*concentration (p = 0.60). Increase in CGRP (undiluted - baseline; pg/ml) did not differ between measurements on dose-finding (p = 0.67) and follow-up days (p = 0.46). Oral application of red chili homogenate is well tolerated and causes a dose-dependent CGRP release in saliva, without day-to-day effects in this response. This model could be used to noninvasively study the activation state of the trigeminal nerve innervating salivary glands.

Role of trpv1 (transient receptor potential v1 channel) in piperine induced hyperalgesia: a preliminary study

Background: The inflammatory disease is associated with chronic pain and hypersensitivity to noxious stimuli in form of hyperalgesia. Transient receptor potential vanilloid type-1 (TRPV1) activated by various inflammatory mediators; plays a key role in occurrence of hyperalgesia. Piperine is the primary pungent alkaloid of Piper nigrum. Piperine has common binding site with capsaicin (TRPV1 agonist) on TRPV1 and it has more efficacy for activation of TRPV1 than capsaicin. Objective: The purpose of the study was to investigate the role of TRPV1 in piperine induced hyperalgesia in diabetic mice. Methods: Diabetes mellitus was induced in mice using streptozotocin (STZ). Nociception was measured by three types of thermal nociceptive tests (Hot plate method, radient heat tail-flick method and tail immersion test). Pilot study (using 2.5, 5, 10 and 50 mg kg-1 piperine, i.p.) was carried out and 5 mg kg-1 piperine is used as optimum dose for further study. Capsaicin, capsazepine and piperine were administered to their respected groups and nociceptive threshold was noted on day 4, 7 and 10 in all groups. Results: Piperine showed a significant decrease in nociceptive threshold as compared to diabetic control group, while there was a significant increase in reaction time, tail flick latency and tail withdrawal latency, in the animals pre-treated with capsazepine before piperine administration as compare to only piperine treated diabetic group. Conclusion: Activation of TRPV1 might be one of the possible mechanisms of Piperine induced hyperalgesia. Keywords: Capsaicin, Capsazepine, Hyperalgesia, Nociceptive Threshold, Piperine, TRPV1.

Role of TRPV1 and ASIC3 in formalin-induced secondary allodynia and hyperalgesia

BackgroundIn the present study we determined the role of transient receptor potential V1 channel (TRPV1) and acid-sensing ion channel 3 (ASIC3) in chronic nociception. Methods1% formalin was used to produce long-lasting secondary allodynia and hyperalgesia in rats. Western blot was used to determine TRPV1 and ASIC3 expression in dorsal root ganglia. ResultsPeripheral ipsilateral, but not contralateral, pre-treatment (−10min) with the TRPV1 receptor antagonists capsazepine (0.03–0.3μM/paw) and A-784168 (0.01–1μM/paw) prevented 1% formalin-induced secondary mechanical allodynia and hyperalgesia in the ipsilateral and contralateral paws. Likewise, peripheral ipsilateral, but not contralateral, pre-treatment with the non-selective and selective ASIC3 blocker benzamil (0.1–10μM/paw) and APETx2 (0.02–2μM/paw), respectively, prevented 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. Peripheral ipsilateral post-treatment (day 6 after formalin injection) with capsazepine (0.03–0.3μM/paw) and A-784168 (0.01–1μM/paw) reversed 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. In addition, peripheral ipsilateral post-treatment with benzamil (0.1–10μM/paw) and APETx2 (0.02–2μM/paw), respectively, reversed 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. TRPV1 and ASIC3 proteins were expressed in dorsal root ganglion in normal conditions, and 1% formalin injection increased expression of both proteins in this location at 1 and 6 days compared to naive rats. ConclusionsData suggest that TRPV1 and ASIC3 participate in the development and maintenance of long-lasting secondary allodynia and hyperalgesia induced by formalin in rats. The use of TRPV1 and ASIC3 antagonists by peripheral administration could prove useful to treat chronic pain.

Lipid-Mediated Interaction of Double-Knot Toxin with TRPV1 Channels

The Transient Receptor Potential V1 (TRPV1) channel is a cation channel that opens in response to diverse chemical and physical stimuli. These stimuli include capsaicin, temperature, pH and bioactive lipids. Recently, a novel peptide named Double cystine Knot Toxin (DkTx) was purified from tarantula venom and shown to activate the channel from the extracellar site of the membrane and proposed to bind the pore region (Bohlen CJ., et al, 2010). In order to characterize DkTx-TRPV1 interactions, we expressed and purified recombinant His-tagged DkTx in E. coli and 1D4-tagged TRPV1 in Saccharomyces cerevisiae. Expressed TRPV1 binds the specific TRPV1 agonist, 3H- resiniferatoxin (RTx), with high affinity (Kd∼500 pM) and in the presence of DkTx the affinity of RTx increases by ∼4-fold, indicating that both RTx and DkTx interact with the TRPV1 channel in membranes, but at distinct sites that interact allosterically. Pull-down experiments with full-length and truncated TRPV1 (containing the pore-forming S5-S6 segments and the C-terminus suggest that DkTx interacts with both detergent purified channel constructs, supporting an interaction of the toxin with the external pore region. DkTx and TRPV1 constructs do not, however, co-elute as a complex in size-exclusion or affinity chromatography, suggesting that in detergent solution the interactions are weak. To investigate the strength of TRPV1 interactions with DkTx in detergent micelles we utilized surface plasmon resonance with TRPV1 deposited on a gold chip using 1D4 tag. DkTx interacts with TRPV1 in detergent micelles, but with much lower apparent affinity than observed in membranes. The isolated K2 lobe of DkTx exhibited higher affinity than K1, in agreement with the previous electrophysiology studies. These results support the notion that DkTx interacts with the external pore of TRPV1 and suggest that the membrane lipids play a critical role in toxin-channel interaction.

TRPV1 channels make major contributions to behavioral hypersensitivity and spontaneous activity in nociceptors after spinal cord injury

Chronic neuropathic pain is often a severe and inadequately treated consequence of spinal cord injury (SCI). Recent findings suggest that SCI pain is promoted by spontaneous activity (SA) generated chronically in cell bodies of primary nociceptors in dorsal root ganglia (DRG). Many nociceptors express transient receptor potential V1 (TRPV1) channels, and in a preceding study most dissociated DRG neurons exhibiting SA were excited by the TRPV1 activator, capsaicin. The present study investigated roles of TRPV1 channels in behavioral hypersensitivity and nociceptor SA after SCI. Contusive SCI at thoracic segment T10 increased expression of TRPV1 protein in lumbar DRG 1month after injury and enhanced capsaicin-evoked ion currents and Ca2+ responses in dissociated small DRG neurons. A major role for TRPV1 channels in pain-related behavior was indicated by the ability of a specific TRPV1 antagonist, AMG9810, to reverse SCI-induced hypersensitivity of hind limb withdrawal responses to mechanical and thermal stimuli at a dose that did not block detection of noxious heat. Similar reversal of behavioral hypersensitivity was induced by intrathecal oligodeoxynucleotides antisense to TRPV1, which knocked down TRPV1 protein and reduced capsaicin-evoked currents. TRPV1 knockdown also decreased the incidence of SA in dissociated nociceptors after SCI. Prolonged application of very low concentrations of capsaicin produced nondesensitizing firing similar to SA, and this effect was enhanced by prior SCI. These results show that TRPV1 makes important contributions to pain-related hypersensitivity long after SCI, and suggest a role for TRPV1-dependent enhancement of nociceptor SA that offers a promising target for treating chronic pain after SCI.

Sex differences in serotonin enhancement of capsaicin-evoked calcitonin gene-related peptide release from human dental pulp

Serotonin (5HT) is a pronociceptive mediator in the periphery, and evidence implicates involvement in trigeminal pain processing. However, the mechanism(s) by which 5HT modulates trigeminal nociceptors remains unclear. Trigeminal pain can be evoked by the transient receptor potential V1 channel (TRPV1), which is expressed by nociceptive trigeminal neurons and induces release of proinflammatory calcitonin gene-related peptide (CGRP). In our preclinical models, 5HT evoked thermal hyperalgesia and enhanced calcium influx and CGRP release from the TRPV1 population of trigeminal nociceptors. Whether this occurs in humans is unknown. As dental pulp is densely innervated by trigeminal nociceptors, routine tooth extractions offer a unique opportunity to examine whether 5HT enhances CGRP release from human nociceptors. Pulpal tissue was collected from 140 extracted molar teeth from men and women, and basal release samples were collected before treatment with saline or 5HT 100μmol/L. CGRP release was then stimulated with the TRPV1 agonist capsaicin 1μmol/L and quantitated by enzyme immunoassay. Additional samples were collected for Western blots to examine 5HT receptor expression. We report that 5HT induced a significant increase in capsaicin-evoked CGRP release, and that this enhancement was observed only in female dental pulp, with no effect of 5HT on male dental pulp. The greatest amount of CGRP release occurred in dental pulp from women in the luteal phase of the menstrual cycle. These results indicate that 5HT enhances capsaicin-evoked CGRP release from human trigeminal nociceptors in a sexually dimorphic manner providing a mechanistic basis for prevalence of trigeminal pain disorders in women.

Afferent drive elicits ongoing pain in a model of advanced osteoarthritis

Osteoarthritis (OA) is a chronic condition characterized by pain during joint movement. Additionally, patients with advanced disease experience pain at rest (ie, ongoing pain) that is generally resistant to nonsteroidal antiinflammatory drugs. Injection of monosodium iodoacetate (MIA) into the intraarticular space of the rodent knee is a well-established model of OA that elicits weight-bearing asymmetry and referred tactile and thermal hypersensitivity. Whether ongoing pain is present in this model is unknown. Additionally, the possible relationship of ongoing pain to MIA dose is not known. MIA produced weight asymmetry, joint osteolysis, and cartilage erosion across a range of doses (1, 3, and 4.8mg). However, only rats treated with the highest dose of MIA showed conditioned place preference to a context paired with intraarticular lidocaine, indicating relief from ongoing pain. Diclofenac blocked the MIA-induced weight asymmetry but failed to block MIA-induced ongoing pain. Systemic AMG9810, a transient receptor potential V1 channel (TRPV1) antagonist, effectively blocked thermal hypersensitivity, but failed to block high-dose MIA-induced weight asymmetry or ongoing pain. Additionally, systemic or intraarticular HC030031, a TRPA1 antagonist, failed to block high-dose MIA-induced weight asymmetry or ongoing pain. Our studies suggest that a high dose of intraarticular MIA induces ongoing pain originating from the site of injury that is dependent on afferent fiber activity but apparently independent of TRPV1 or TRPA1 activation. Identification of mechanisms driving ongoing pain may enable development of improved treatments for patients with severe OA pain and diminish the need for joint replacement surgery.

The endocannabinoid N-arachidonoyldopamine (NADA) exerts neuroprotective effects after excitotoxic neuronal damage via cannabinoid receptor 1 (CB1)

Endocannabinoids exert numerous effects in the CNS under physiological and pathological conditions. The aim of the present study was to examine whether the endocannabinoid N-arachidonoyldopamine (NADA) may protect neurons in excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). OHSC were excitotoxically lesioned by application of N–methyl-d-aspartate (NMDA, 50 μM) for 4 h and subsequently treated with different NADA concentrations (0.1 pM–50 μM) alone or in combination with cannabinoid receptor antagonists. NADA protected dentate gyrus granule cells and caused a slight reduction in the number of microglial cells. The number of degenerated neurons significantly decreased between 100 pM and 10 μM NADA (p < 0.05). To identify the responsive receptor type of NADA mediated neuroprotection, we applied the cannabinoid (CB) receptor 1 (CB1) inverse agonist/antagonist AM251, CB2 inverse agonist/antagonist AM630, abnormal-cannabidiol (abn-CBD)-sensitive receptor antagonist O-1918, transient receptor potential channel V1 (TRPV1) antagonist 6-iodonordihydrocapsaicin and A1 (TRPA1) antagonist HC-030031. Neuroprotective properties of low (1 nM) but not high (10 μM) NADA concentrations were solely blocked by AM251 and were absent in CB1−/− mice. AM630, O-1918, 6-iodonordihydrocapsaicin and HC-030031 showed no effects at all NADA concentrations applied. Our findings demonstrate that NADA protects dentate gyrus granule cells by acting via CB1. NADA reduced the number of microglial cells at distinct concentrations. TRPV1 and TRPA1 were not involved in NADA mediated neuroprotection. Thus, our data implicate that NADA mediated activation of neuronal CB1 may serve as a novel pharmacological target to mitigate symptoms of neuronal damage.