TRPV1-deficient Mice Research Articles

A novel form of capsaicin-modified amygdala LTD mediated by TRPM1

Recently we have shown that capsaicin attenuates the strength of LTP in the lateral amygdala (LA) and demonstrated that this effect is mediated by the transient receptor potential (TRP) channel TRPV1. Here we further show that capsaicin, which is thought to act primarily through TRPV1, modifies long term depression (LTD) in the LA. Yet the application of various TRPV1 antagonists does not reverse this effect and it remains in TRPV1-deficient mice. In addition, voltage gated calcium channels, nitric oxide and CB1 receptors are not involved. Using pharmacology and TRPM1−/− mice, our electrophysiological data indicate that capsaicin-induced activation of TRPM1 channels contribute to the induction of LA-LTD. Whereas LA-LTD in general depends on the acitvation of NMDA receptors- and group II metabotropic glutamate receptors (mGluR), the modifying effect of capsaicin on LA-LTD via TRPM1 appears to be specifically mediated by group I mGluRs and in interaction with another member of the TRP family, TRPC5. Additionally, intact GABAergic transmission is required for the capsaicin-effect to take place. This is the first documentation that beside their function in the retina TRPM1 proteins are expressed in the brain and have a functional relevance in modifying synaptic plasticity.

Role of transient receptor potential vanilloid 1 in regulating erythropoietin-induced activation of endothelial nitric oxide synthase.

Erythropoietin (EPO), the key hormone involved in erythropoiesis, beneficially affects endothelial cells (ECs), but the detailed mechanisms are yet to be completely understood. In this study, we investigated the role of transient receptor potential vanilloid type 1 (TRPV1), a ligand-gated non-selective calcium (Ca2+ ) channel, in EPO-mediated endothelial nitric oxide synthase (eNOS) activation and angiogenesis. In ECs, EPO time dependently increased intracellular levels of calcium; this increase was abrogated by the Ca2+ chelators and pharmacological inhibitors of TRPV1 in bovine aortic ECs (BAECs) and TRPV1-transfected HEK293 cells. In addition, EPO-induced nitrite oxide (NO) production, phosphorylation of eNOS, Akt and AMP-activated protein kinase (AMPK) and the formation of TRPV1-Akt-AMPK-eNOS complex as well as tube formation were diminished by the pharmacological inhibition of TRPV1 in BAECs. Moreover, EPO time dependently induced the phosphorylation of phospholipase C-γ1 (PLC-γ1). Inhibition of PLC-γ1 activity blunted the EPO-induced Ca2+ influx, eNOS phosphorylation, TRPV1-eNOS complex formation and NO production. The phosphorylated level of eNOS increased in the aortas of EPO-treated wild-type (WT) mice or EPO-transgenic (Tg) mice but not in those of EPO-treated TRPV1-deficient (TRPV1-/- ) mice or EPO-Tg/TRPV1-/- mice. Matrigel plug assay showed that EPO-induced angiogenesis was abrogated in TRPV1 antagonist capsazepine-treated WT mice and TRPV1-/- mice. These findings indicate the EPO-induced Ca2+ influx via the activation of the PLC-γ1 signalling pathway, which leads to TRPV1 activation and consequently increases the association of the TRPV1-Akt-AMPK-eNOS complex, eNOS activation, NO production and angiogenesis.

TRPV1 sensitization mediates postinflammatory visceral pain following acute colitis.

Quiescent phases of inflammatory bowel disease (IBD) are often accompanied by chronic abdominal pain. Although the transient receptor potential vanilloid 1 (TRPV1) ion channel has been postulated as an important mediator of visceral hypersensitivity, its functional role in postinflammatory pain remains elusive. This study aimed at establishing the role of TRPV1 in the peripheral sensitization underlying chronic visceral pain in the context of colitis. Wild-type and TRPV1-deficient mice were separated into three groups (control, acute colitis, and recovery), and experimental colitis was induced by oral administration of dextran sulfate sodium (DSS). Recovery mice showed increased chemically and mechanically evoked visceral hypersensitivity 5 wk post-DSS discontinuation, at which point inflammation had completely resolved. Significant changes in nonevoked pain-related behaviors could also be observed in these animals, indicative of persistent discomfort. These behavioral changes correlated with elevated colonic levels of substance P (SP) and TRPV1 in recovery mice, thus leading to the hypothesis that SP could sensitize TRPV1 function. In vitro experiments revealed that prolonged exposure to SP could indeed sensitize capsaicin-evoked currents in both cultured neurons and TRPV1-transfected human embryonic kidney (HEK) cells, a mechanism that involved TRPV1 ubiquitination and subsequent accumulation at the plasma membrane. Importantly, although TRPV1-deficient animals experienced similar disease severity and pain as wild-type mice in the acute phase of colitis, TRPV1 deletion prevented the development of postinflammatory visceral hypersensitivity and pain-associated behaviors. Collectively, our results suggest that chronic exposure of colon-innervating primary afferents to SP could sensitize TRPV1 and thus participate in the establishment of persistent abdominal pain following acute inflammation.

Activation of TRPV1 channel by dietary capsaicin improves visceral fat remodeling through connexin43-mediated Ca2+ influx.

BackgroundThe prevalence of obesity has dramatically increased worldwide and has attracted rising attention, but the mechanism is still unclear. Previous studies revealed that transient receptor potential vanilloid 1 (TRPV1) channels take part in weight loss by enhancing intracellular Ca2+ levels. However, the potential mechanism of the effect of dietary capsaicin on obesity is not completely understood. Ca2+ transfer induced by connexin43 (Cx43) molecules between coupled cells takes part in adipocyte differentiation. Whether TRPV1-evoked alterations in Cx43-mediated adipocyte-to-adipocyte communication play a role in obesity is unknown.Materials and methodsWe investigated whether Cx43 participated in TRPV1-mediated adipocyte lipolysis in cultured 3T3-L1 preadipocytes and visceral adipose tissues from humans and wild-type (WT) and TRPV1-deficient (TRPV1-/-) mice.ResultsTRPV1 and Cx43 co-expressed in mesenteric adipose tissue. TRPV1 activation by capsaicin increased the influx of Ca2+ in 3T3-L1 preadipocytes and promoted cell lipolysis, as shown by Oil-red O staining. These effects were deficient when capsazepine, a TRPV1 antagonist, and 18 alpha-glycyrrhetinic acid (18α-GA), a gap-junction inhibitor, were administered. Long-term chronic dietary capsaicin reduced the weights of perirenal, mesenteric and testicular adipose tissues in WT mice fed a high-fat diet. Capsaicin increased the expression levels of p-CaM, Cx43, CaMKII, PPARδ and HSL in mesenteric adipose tissues from WT mice fed a high-fat diet, db/db mice, as well as obese humans, but these effects of capsaicin were absent in TRPV1-/- mice. Long-term chronic dietary capsaicin decreased the body weights and serum lipids of WT mice, but not TRPV1-/- mice, fed a high-fat diet.ConclusionThis study demonstrated that capsaicin activation of TRPV1-evoked increased Ca2+ influx in Cx43-mediated adipocyte-to-adipocyte communication promotes lipolysis in both vitro and vivo. TRPV1 activation by dietary capsaicin improves visceral fat remodeling through the up-regulation of Cx43.

PD9-04 RECEPTOR CASCADE MEDIATING POST-UTI CHRONIC PAIN

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Interstitial Cystitis1 Apr 2014PD9-04 RECEPTOR CASCADE MEDIATING POST-UTI CHRONIC PAIN John Rosen and David Klumpp John RosenJohn Rosen More articles by this author and David KlumppDavid Klumpp More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.786AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES E. coli evoke a spectrum of pain responses when instilled into the bladder, ranging from no pelvic pain to post-UTI chronic pain that persists after transient infection. Previous studies indicate that E. coli-induced pain is mediated by TLR4 independent of inflammation. Here, we evaluated the hypothesis that TRPV1 and/or CCR2 mediators of post-UTI chronic pain downstream of TLR4. METHODS Post-UTI chronic pain was evaluated in mice by tactile allodynia in response to von Frey filaments applied to the pelvic region. Viscero-motor reflex (VMR) of abdominal muscles in response to bladder distension with saline was used as an independent measure of visceral pain. Anxiety-like/depression-like behavior was quantified as a central manifestation of chronic pain using novelty-suppressed feeding (NSF). RESULTS Mice infected with E. coli strain S[phi]874 acquired chronic pelvic allodynia that persisted long after bacterial clearance. Treatment with the TRPV1 antagonist capsazepine at the time of infection signficantly inhibited the development of chronic allodynia. In contrast, capsazepine administered after the onset of chronic allodynia had no effect, and these allodynia findings were corroborated at the levels of VMR activity and NSF. Similarly, allodynia and VMR activity did not develop in TRPV1-deficient mice infected with S[phi]874. Induction of MCP-1 and CCR2 was noted in sacral DRGs, and administration of a CCR2 antagonist significantly reduced allodynia. CONCLUSIONS These data suggest that TRPV1 mediates the establishment of chronic pelvic pain in response to a transient E. coli infection but is not required for pain maintenance. Conversely, CCR2 is required for maintenance of post-UTI chronic pain. Together with previous studies, our data suggest that E. coli may induce chronic pelvic pain through a cascade of TLR4-TRPV1-CCR2 in the periphery. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e212-e213 Advertisement Copyright & Permissions© 2014MetricsAuthor Information John Rosen More articles by this author David Klumpp More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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.

The role of mast cells, interleukin‐13 and transient receptor potential channels in a mouse model of chemical‐induced airway hyperresponsiveness

Method On days 1 and 8, wild type C57Bl/6 mice, IL-13, TRP (Transient Receptor Potential) A1, TRPV1 and mast cell deficient mice were dermally sensitized with 1% TDI (toluene-2.4-diisocyanate) or vehicle (acetone/olive oil) on both ears. On day 15, the mice received a single intranasal challenge with 0.1% TDI or vehicle. In a second experiment TDI or vehicle sensitized wild type C57Bl/6 mice received an intraperitoneal injection of the NK1R antagonist RP67580 (1μg/μl) prior to the challenge. Airway reactivity to methacholine, lung inflammation, lymphocyte subpopulations in the draining auricular lymph nodes and total serum IgE were assessed 24h after the challenge.

TRPV1, but not TRPA1, in Primary Sensory Neurons Contributes to Cutaneous Incision-Mediated Hypersensitivity

BackgroundMechanisms underlying postoperative pain remain poorly understood. In rodents, skin-only incisions induce mechanical and heat hypersensitivity similar to levels observed with skin plus deep incisions. Therefore, cutaneous injury might drive the majority of postoperative pain. TRPA1 and TRPV1 channels are known to mediate inflammatory and nerve injury pain, making them key targets for pain therapeutics. These channels are also expressed extensively in cutaneous nerve fibers. Therefore, we investigated whether TRPA1 and TRPV1 contribute to mechanical and heat hypersensitivity following skin-only surgical incision.ResultsBehavioral responses to mechanical and heat stimulation were compared between skin-incised and uninjured, sham control groups. Elevated mechanical responsiveness occurred 1 day post skin-incision regardless of genetic ablation or pharmacological inhibition of TRPA1. To determine whether functional changes in TRPA1 occur at the level of sensory neuron somata, we evaluated cytoplasmic calcium changes in sensory neurons isolated from ipsilateral lumbar 3–5 DRGs of skin-only incised and sham wild type (WT) mice during stimulation with the TRPA1 agonist cinnamaldehyde. There were no changes in the percentage of neurons responding to cinnamaldehyde or in their response amplitudes. Likewise, the subpopulation of DRG somata retrogradely labeled specifically from the incised region of the plantar hind paw showed no functional up-regulation of TRPA1 after skin-only incision. Next, we conducted behavior tests for heat sensitivity and found that heat hypersensitivity peaked at day 1 post skin-only incision. Skin incision-induced heat hypersensitivity was significantly decreased in TRPV1-deficient mice. In addition, we conducted calcium imaging with the TRPV1 agonist capsaicin. DRG neurons from WT mice exhibited sensitization to TRPV1 activation, as more neurons (66%) from skin-incised mice responded to capsaicin compared to controls (46%), and the sensitization occurred specifically in isolectin B4 (IB4)-positive neurons where 80% of incised neurons responded to capsaicin compared to just 44% of controls.ConclusionsOur data suggest that enhanced TRPA1 function does not mediate the mechanical hypersensitivity that follows skin-only surgical incision. However, the heat hypersensitivity is dependent on TRPV1, and functional up-regulation of TRPV1 in IB4-binding DRG neurons may mediate the heat hypersensitivity after skin incision injury.

Analysis of T cells in hapten-induced contact dermatitis on TRPV1-deficient mice

Analysis of T cells in hapten-induced contact dermatitis on TRPV1-deficient mice

Effects of single swim stress on changes in TRPV1-mediated plasticity in the amygdala

By examining the involvement of transient receptor potential vanilloid type 1 (TRPV1) in the stress modulation of learning and memory processes in mice, we evaluated the effects of endovanilloid N-oleoyldopamine (OLDA) on the long-term potentiation (LTP) of the lateral nucleus of the amygdala (LA). After high-frequency stimulation of external capsule fibers we found that LA-LTP is reduced in OLDA-treated slices derived from adult C57BL/6 control mice. The specificity of the TRPV1 receptor activation by OLDA was confirmed by blocking the OLDA-induced inhibitory effect on LA-LTP with the specific TRPV1 receptor antagonist AMG 9810. The specificity of OLDA was further supported by using TRPV1 deficient mice, where the effect of OLDA on LA-LTP was missing. Following exposure to a forced swim test (FST) OLDA enhanced LA-LTP in control but not TRPV1-deficient mice. The results also show that a short period of acute stress significantly impairs LA-LTP. Since we have recently shown the involvement of cannabinoid CB1 receptors in the mediation of capsaicin-induced inhibitory effects on LA-LTP ([23] Zschenderlein et al., 2011), it is reasonable to assume that the OLDA-induced enhancement of LA-LTP after the forced swim test can be attributed to the up-regulation of TRPV1 and the action of ligands such as anandamide on TRPV1. As a result, stimulation of TRPV1 receptors rescues LTP in slices derived from swim-stressed mice.

Severe ulceration with impaired induction of growth factors and cytokines in keratinocytes after trichloroacetic acid application on TRPV1-deficient mice

Transient receptor potential vanilloid 1 (TRPV1) is a highly polymodal TRP channel activated by various stimuli, including capsaicin, heat and acids. TRPV1 expression can be detected widely but is highest in sensory neurons and its activation alerts the body to noxious signals via neurogenic pain. Although TRPV1 is reportedly localized in the epidermis, it remains unclear how TRPV1 is involved in the chemical peeling processes with cytotoxic acids. Therefore, in this study, the role of TRPV1 on the effects of trichloroacetic acid (TCA) peeling was assessed using TRPV1-deficient mice. Following the confirmation of TRPV1 expression in murine keratinocytes with reverse transcription-polymerase chain reaction and immunohistochemistry, the effects of TCA on TRPV1-deficient mouse skin were compared with those on wild-type mouse skin. Our results indicated that TRPV1 expression was not required for TCA-induced DNA damage, as shown by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, but was indispensable for the TCA-induced production of distinct growth factors and cytokines by keratinocytes. Ulceration after TCA peeling was actually more severe in the absence of TRPV1, suggesting that the TRPV1-mediated epidermal production of growth factors and cytokines affected the damaging and healing processes of TCA-peeled skin to induce rejuvenation.

Tu1983 Activation of TRPA1, but Not of TRPV1, Improves Gut Motility in a Murine Model of Postoperative Ileus

Background and Aim: Agonists of transient receptor potential vanilloid (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) have been reported to induce contraction of intestinal smooth muscle In Vitro, but it is unclear whether activation of TRPV1 or TRPA1 affects gastrointestinal (GI) transit In Vivo. In this study, we investigated the effects of capsaicin (TRPV1 agonist), allyl isothiocyanate (AITC, TRPA1 agonist), cinnamaldehyde (CA, TRPA1 agonist), daikenchuto (TU-100, TRPA1/TRPV1 agonist), and 6-shogaol (6SG, TRPA1/TRPV1 agonist), the main constituent of TU-100, on murine postoperative ileus (POI) induced by surgical manipulation of mouse intestines. TU-100, a traditional Japanese medicine that has been prescribed to improve paralytic POI in Japan, is currently undergoing clinical trials on various intestinal diseases such as postoperative ileus, functional constipation, and Crohn's disease in the USA (http://clinicaltrials.gov/ct2/results?term=TU-100). Methods: Male C57Bl/ 6 mice underwent laparotomy and large bowel manipulation. Fluorescein isothiocyanatedextran (FD70) was administered orally at 23.5 h postoperatively, and the mice were killed at 30 min post gavage. GI transit was estimated by calculating the geometric center (GC) value. Serotonin release from RIN-14B cell, an enterochromaffin-like cell line, was measured by EIA. Results: Colonic manipulation decreased small intestinal transit to 70.2±2.7% of control. TRPA1 agonists (AITC, CA, TU-100, and 6SG) significantly improved the delayed GI transit induced by intestinal manipulation by 68.3±12.9%, 83.6±9.8%, 65.7±13.8%, and 26.3±6.7%, respectively. On the other hand, the TRPV1 agonist capsaicin had no effect. Freshly isolated intestinal epithelial cells and RIN-14B cells expressed abundant TRPA1 mRNA but no TRPV1 mRNA. POI was induced in TRPV1-deficient mice to a similar degree as in wild-type mice, and TU-100 was effective in POI of TRPV1-deficient mice. AITC, CA, TU-100, and 6SG stimulated RIN-14B cell to release serotonin. Conclusions: These results suggest that TRPA1 is essential to induction and alleviation of POI while TRPV1 stimulation may play little role. Stimulation of TRPA1 may be a novel therapeutic approach for the treatment of POI.

Tu1984 Hydroxy a-Sanshool, a Major Ingredient of TU-100 (Daikenchuto), Augments Migrating Motor Complex of Proximal Colon Triggered by Other Prokinetic Stimuli

Background and Aim: Agonists of transient receptor potential vanilloid (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) have been reported to induce contraction of intestinal smooth muscle In Vitro, but it is unclear whether activation of TRPV1 or TRPA1 affects gastrointestinal (GI) transit In Vivo. In this study, we investigated the effects of capsaicin (TRPV1 agonist), allyl isothiocyanate (AITC, TRPA1 agonist), cinnamaldehyde (CA, TRPA1 agonist), daikenchuto (TU-100, TRPA1/TRPV1 agonist), and 6-shogaol (6SG, TRPA1/TRPV1 agonist), the main constituent of TU-100, on murine postoperative ileus (POI) induced by surgical manipulation of mouse intestines. TU-100, a traditional Japanese medicine that has been prescribed to improve paralytic POI in Japan, is currently undergoing clinical trials on various intestinal diseases such as postoperative ileus, functional constipation, and Crohn's disease in the USA (http://clinicaltrials.gov/ct2/results?term=TU-100). Methods: Male C57Bl/ 6 mice underwent laparotomy and large bowel manipulation. Fluorescein isothiocyanatedextran (FD70) was administered orally at 23.5 h postoperatively, and the mice were killed at 30 min post gavage. GI transit was estimated by calculating the geometric center (GC) value. Serotonin release from RIN-14B cell, an enterochromaffin-like cell line, was measured by EIA. Results: Colonic manipulation decreased small intestinal transit to 70.2±2.7% of control. TRPA1 agonists (AITC, CA, TU-100, and 6SG) significantly improved the delayed GI transit induced by intestinal manipulation by 68.3±12.9%, 83.6±9.8%, 65.7±13.8%, and 26.3±6.7%, respectively. On the other hand, the TRPV1 agonist capsaicin had no effect. Freshly isolated intestinal epithelial cells and RIN-14B cells expressed abundant TRPA1 mRNA but no TRPV1 mRNA. POI was induced in TRPV1-deficient mice to a similar degree as in wild-type mice, and TU-100 was effective in POI of TRPV1-deficient mice. AITC, CA, TU-100, and 6SG stimulated RIN-14B cell to release serotonin. Conclusions: These results suggest that TRPA1 is essential to induction and alleviation of POI while TRPV1 stimulation may play little role. Stimulation of TRPA1 may be a novel therapeutic approach for the treatment of POI.

Mo1860 Identification of Kappa Opioid Receptors on Colonic Sensory Nerves and Their Up-Regulation in a Mouse Model of Chronic Visceral Hypersensitivity

Background: Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract. This relapsing condition is often associated with debilitating abdominal pain, which in some cases can persist throughout quiescent stages of the disease. The regulatory mechanisms involved in central sensitization and peripheral neuroplasticity in the context of IBD-related visceral pain remains elusive. A growing body of evidence suggests a role for the transient receptor potential vanilloid 1 (TRPV1) in visceral hypersensitivity. TRPV1 is expressed in sensory dorsal root ganglion (DRG) neurons and is directly involved in nociception. Previous studies have demonstrated that TRPV1-deficient mice experience reduced thermal hyperalgesia during inflammation, thus suggesting a role for TRPV1 in inflammatory pain. The objective of the present study was therefore to investigate the role of TRPV1 in visceral pain in the dextran sulfate sodium (DSS) murine model of colitis. Methods: Mice were given DSS in drinking water for 7 days, after which spontaneous visceral pain was assessed using the non-invasive, animal behavior recognition system LABORAS. Control and DSS-treated mice were then sacrificed and colon-innervating DRGs collected for quantitative PCR, immunostaining, and neuronal dissociation. In Vitro studies were performed with human neuroblastoma NG108 cells. Results: DSS-treated mice showed reduced mobility and frequency of grooming and climbing, which likely reflect spontaneous visceral pain. Remarkably, while controls cells showed very few processes, dissociated DRG neurons isolated from DSS-treated mice displayed extensive neurite outgrowth after 24 h in culture. Immunocytochemistry later revealed TRPV1 expression along the entire length of these axons. These results are supported by In Vitro data demonstrating that exposure to a mixture of proinflammatory cytokines (bradykinin, histamine, and serotonin) induces neurite outgrowth and TRPV1 membrane trafficking in TRPV1-transfected neuronal NG108 cells. Finally, PCR analysis demonstrated that the neuronal morphological changes observed in DSS-treated animals were associated with a significant decrease in the expression of the small Rho GTPase RhoA and Rho kinase, both of which are known to modulate neurite outgrowth. Conclusion: These results demonstrate that DSS-colitis induces changes in neuronal morphology and modulation of TRPV1 trafficking, which may play a role in visceral hypersensitivity in the context of gastrointestinal inflammatory disorders. Current experiments using pharmacological inhibitors of the RhoA/Rho kinase signaling axis and TRPV1deficient animals will provide important insights into the exact role of TRPV1 in IBD-related visceral hypersensitivity and perhaps identify new targets for the treatment of chronic pain in these patients.

Mo1859 Visceral Pain Correlates With Neuronal Outgrowth and Modulation of TRPV1 Trafficking in Dorsal Root Ganglion Neurons in a Murine Model of Colitis

Background: Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract. This relapsing condition is often associated with debilitating abdominal pain, which in some cases can persist throughout quiescent stages of the disease. The regulatory mechanisms involved in central sensitization and peripheral neuroplasticity in the context of IBD-related visceral pain remains elusive. A growing body of evidence suggests a role for the transient receptor potential vanilloid 1 (TRPV1) in visceral hypersensitivity. TRPV1 is expressed in sensory dorsal root ganglion (DRG) neurons and is directly involved in nociception. Previous studies have demonstrated that TRPV1-deficient mice experience reduced thermal hyperalgesia during inflammation, thus suggesting a role for TRPV1 in inflammatory pain. The objective of the present study was therefore to investigate the role of TRPV1 in visceral pain in the dextran sulfate sodium (DSS) murine model of colitis. Methods: Mice were given DSS in drinking water for 7 days, after which spontaneous visceral pain was assessed using the non-invasive, animal behavior recognition system LABORAS. Control and DSS-treated mice were then sacrificed and colon-innervating DRGs collected for quantitative PCR, immunostaining, and neuronal dissociation. In Vitro studies were performed with human neuroblastoma NG108 cells. Results: DSS-treated mice showed reduced mobility and frequency of grooming and climbing, which likely reflect spontaneous visceral pain. Remarkably, while controls cells showed very few processes, dissociated DRG neurons isolated from DSS-treated mice displayed extensive neurite outgrowth after 24 h in culture. Immunocytochemistry later revealed TRPV1 expression along the entire length of these axons. These results are supported by In Vitro data demonstrating that exposure to a mixture of proinflammatory cytokines (bradykinin, histamine, and serotonin) induces neurite outgrowth and TRPV1 membrane trafficking in TRPV1-transfected neuronal NG108 cells. Finally, PCR analysis demonstrated that the neuronal morphological changes observed in DSS-treated animals were associated with a significant decrease in the expression of the small Rho GTPase RhoA and Rho kinase, both of which are known to modulate neurite outgrowth. Conclusion: These results demonstrate that DSS-colitis induces changes in neuronal morphology and modulation of TRPV1 trafficking, which may play a role in visceral hypersensitivity in the context of gastrointestinal inflammatory disorders. Current experiments using pharmacological inhibitors of the RhoA/Rho kinase signaling axis and TRPV1deficient animals will provide important insights into the exact role of TRPV1 in IBD-related visceral hypersensitivity and perhaps identify new targets for the treatment of chronic pain in these patients.

Implication of AMP-Activated Protein Kinase in Transient Receptor Potential Vanilloid Type 1-Mediated Activation of Endothelial Nitric Oxide Synthase

We investigated whether AMP-activated protein kinase (AMPK), a multifunctional regulator of energy homeostasis, is involved in transient receptor potential vanilloid type 1 (TRPV1)-mediated activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) and mice. In ECs, treatment with evodiamine, the activator of TRPV1, increased the phosphorylation of AMPK, acetyl-CoA carboxylase (ACC) and eNOS, as revealed by Western blot analysis. Inhibition of AMPK activation by compound C or dominant-negative AMPK mutant abrogated the evodiamine-induced increase in phosphorylation of AMPK and eNOS and NO bioavailability, as well as tube formation in ECs. Immunoprecipitation and two-hybrid analysis demonstrated that AMPK mediated the evodiamine-induced increase in the formation of a TRPVl-eNOS complex. Additionally, TRPV1 activation by evodiamine increased the phosphorylation of AMPK and eNOS in aortas of wild-type mice but did not activate eNOS in aortas of TRPV1-deficient mice. In mice, inhibition of AMPK activation by compound C markedly decreased evodiamine-evoked angiogenesis in matrigel plugs and in a hind-limb ischemia model. Moreover, evodiamine-induced phosphorylation of AMPK and eNOS in aortas of apolipoprotein E-deficient (ApoE−/−) mice was abrogated in TRPVl-deficient ApoE−/− mice. In conclusion, TRPV1 activation may trigger AMPK-dependent signaling, which leads to enhanced activation of AMPK and eNOS and retarded development of atherosclerosis.

Aging reverses the role of the transient receptor potential vanilloid-1 channel in systemic inflammation from anti-inflammatory to proinflammatory

Studies in young rodents have shown that the transient receptor potential vanilloid-1 (TRPV1) channel plays a suppressive role in the systemic inflammatory response syndrome (SIRS) by inhibiting production of tumor necrosis factor (TNF)α and possibly by other mechanisms. We asked whether the anti-inflammatory role of TRPV1 changes with age. First, we studied the effect of AMG517, a selective and potent TRPV1 antagonist, on aseptic, lipopolysaccharide (LPS)-induced SIRS in young (12 wk) mice. In agreement with previous studies, AMG517 increased LPS-induced mortality in the young. We then studied the effects of TRPV1 antagonism (AMG517 or genetic deletion of TRPV1) on SIRS in middle-aged (43–44 wk) mice. Both types of TRPV1 antagonism delayed and decreased LPS-induced mortality, indicating a reversal of the anti-inflammatory role of TRPV1 with aging. In addition, deletion of TRPV1 decreased the serum TNFα response to LPS, suggesting that the suppressive control of TRPV1 on TNFα production is also reversed with aging. In contrast to aseptic SIRS, polymicrobial sepsis (induced by cecal ligation and puncture) caused accelerated mortality in aged TRPV1-deficient mice as compared with wild-type littermates. The recovery of TRPV1-deficient mice from hypothermia associated with the cecal ligation and puncture procedure was delayed. Hence, the reversal of the anti-inflammatory role of TRPV1 found in the aged and their decreased systemic inflammatory response are coupled with suppressed defense against microbial infection. These results caution that TRPV1 antagonists, widely viewed as new-generation painkillers, may decrease the resistance of older patients to infection and sepsis.

TRPV1 activation improves exercise endurance and energy metabolism through PGC-1α upregulation in mice

Impaired aerobic exercise capacity and skeletal muscle dysfunction are associated with cardiometabolic diseases. Acute administration of capsaicin enhances exercise endurance in rodents, but the long-term effect of dietary capsaicin is unknown. The capsaicin receptor, the transient receptor potential vanilloid 1 (TRPV1) cation channel has been detected in skeletal muscle, the role of which remains unclear. Here we report the function of TRPV1 in cultured C2C12 myocytes and the effect of TRPV1 activation by dietary capsaicin on energy metabolism and exercise endurance of skeletal muscles in mice. In vitro, capsaicin increased cytosolic free calcium and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression in C2C12 myotubes through activating TRPV1. In vivo, PGC-1α in skeletal muscle was upregulated by capsaicin-induced TRPV1 activation or genetic overexpression of TRPV1 in mice. TRPV1 activation increased the expression of genes involved in fatty acid oxidation and mitochondrial respiration, promoted mitochondrial biogenesis, increased oxidative fibers, enhanced exercise endurance and prevented high-fat diet-induced metabolic disorders. Importantly, these effects of capsaicin were absent in TRPV1-deficient mice. We conclude that TRPV1 activation by dietary capsaicin improves energy metabolism and exercise endurance by upregulating PGC-1α in skeletal muscles. The present results indicate a novel therapeutic strategy for managing metabolic diseases and improving exercise endurance.

Therapeutic Targeting of TRPV1 by Resiniferatoxin, from Preclinical Studies to Clinical Trials

In primary sensory neurons, the capsaicin receptor TRPV1 functions as a molecular integrator for a broad range of seemingly unrelated chemical and physical noxious stimuli, including heat and altered pH. Indeed, TRPV1 is thought to be a major transducer of the thermal hyperalgesia that follows inflammation and tissue injury as this response is impaired in TRPV1-deficient mice. Following the molecular cloning of TRPV1 in 1997, over a dozen companies embarked on efforts to find clinically useful TRPV1 antagonists, but side-effects and limited efficacy have thus far prevented any compounds from progressing beyond phase II. This has rekindled interest in desensitization of nociceptive neurons to TRPV1 agonists (e.g. capsaicin and its ultrapotent analog resiniferatoxin) as an alternative pharmacological approach to block pain in the periphery where it is generated. The clinical value of capsaicin is, however, limited by its unfavorable irritancy to desensitization ratio. In animal experiments, resiniferatoxin treatment is a powerful approach to achieve long-lasting analgesia. In patients with overactive bladder, intravesical resiniferatoxin improves bladder function (or even restores continence) without significant irritancy and/or toxicity. In this review, we argue that resiniferatoxin is an attractive alternative to capsaicin in that it achieves lasting desensitization without the side effects that complicate capsaicin therapy.

Molecular mechanisms of activation of endothelial nitric oxide synthase mediated by transient receptor potential vanilloid type 1

We investigated the molecular mechanism underlying the role of transient receptor potential vanilloid type 1 (TRPV1), a Ca(2+)-permeable non-selective cation channel, in the activation of endothelial nitric oxide (NO) synthase (eNOS) in endothelial cells (ECs) and mice. In ECs, TRPV1 ligands (evodiamine or capsaicin) promoted NO production, eNOS phosphorylation, and the formation of a TRPV1-eNOS complex, which were all abrogated by the TRPV1 antagonist capsazepine. TRPV1 ligands promoted the phosphorylation of Akt, calmodulin-dependent protein kinase II (CaMKII) and TRPV1, and increased the formation of a TRPV1-Akt-CaMKII complex. Removal of extracellular Ca(2+) abolished the ligand-induced increase in the phosphorylation of Akt and CaMKII, formation of a TRPV1-eNOS complex, and eNOS activation. Inhibition of PI3K and CaMKII suppressed the ligand-induced increase in TRPV1 phosphorylation, formation of a TRPV1-eNOS complex, and eNOS activation. TRPV1 activation increased the phosphorylation of Akt, CaMKII, and eNOS in the aortas of wild-type mice but failed to activate eNOS in TRPV1-deficient aortas. Additionally, TRPV1 ligand-induced angiogenesis was diminished in eNOS- or TRPV1-deficient mice. When compared with apolipoprotein E (ApoE)-deficient mice, ApoE/TRPV1-double-knockout mice displayed reduced phosphorylation of eNOS, Akt, and CaMKII in aortas but worsened atherosclerotic lesions. TRPV1 activation in ECs may trigger Ca(2+)-dependent PI3K/Akt/CaMKII signalling, which leads to enhanced phosphorylation of TRPV1, increased TRPV1-eNOS complex formation, eNOS activation and, ultimately, NO production.