TRPV1-dependent Manner Research Articles

Cannabidivarin and cannabigerol induce unfolded protein response and angiogenesis dysregulation in placental trophoblast HTR-8/SVneo cells

Cannabidivarin (CBDV) and cannabigerol (CBG) are minor phytocannabinoids from Cannabis sativa, whose health benefits have been reported. However, studies about the impact of these cannabinoids on fundamental cellular processes in placentation are scarce. Placental development involves physiological endoplasmic reticulum (ER) stress, however when exacerbated it can lead to altered angiogenesis and pregnancy disorders, such as intrauterine growth restriction and preeclampsia. In this work, the effects of CBDV and CBG (1–10 µM) on placental extravillous trophoblasts were studied, using the in vitro model HTR-8/SVneo cells. Both cannabinoids induced anti-proliferative effects and reactive oxygen/nitrogen species generation, which was dependent on transient receptor potential vanilloid 1 (TRPV1) activation. Moreover, CBDV and CBG significantly upregulated, in a TRPV-1 dependent manner, the gene expression of HSPA5/Glucose-regulated protein 78 (GRP78/BiP), a critical chaperone involved in ER stress and unfolded protein response (UPR) activation. Nevertheless, the UPR pathways were differentially activated. Both cannabinoids were able to recruit the IRE branch, while only CBDV enhanced the expression of downstream effectors of the PERK pathway, namely p-eIF2α, ATF4 and CHOP. It also augmented the activity of the apoptotic initiator caspases-8 and -9, though the effector caspases-3/-7 were not activated. TRB3 expression was increased by CBDV, which may hinder apoptosis termination. Moreover, both compounds upregulated the mRNA levels of the angiogenic factors VEGFA, PGF and sFLT1, and disrupted the endothelial-like behavior of HTR-8/SVneo cells, by reducing tube formation. Thus, CBDV and CBG treatment interferes with EVTs functions and may have a negative impact in placentation and in pregnancy outcome.

Piperine Attenuates Bmal1-Mediated Glucose Metabolism Disorder in a Trpv1-Dependent Manner in HepG2 Cells.

Piperine (PIP), a pungent alkaloid found in black pepper, has various pharmacological effects by activating the transient receptor potential vanilloid 1 (TRPV1) receptor. In this study, the regulating effect of PIP on glucose metabolism and the underlying mechanism were examined using an insulin-resistant cell model. Results showed that PIP alleviated glucosamine (GlcN)-induced glucose metabolism disorder (from 59.19 ± 1.90 to 88.36 ± 6.57%), restored cellular redox balance (from 148.43 ± 3.52 to 110.47 ± 3.52%), improved mitochondrial function (from 63.76 ± 4.87 to 85.98 ± 5.12%), and mitigated circadian disruption in HepG2 cells via the mediation of circadian clock gene Bmal1. After the knockdown of the Trpv1 gene, the modulating effect of PIP on Bmal1-mediated glucose metabolism was weakened, indicating that PIP alleviated Bmal1-involved insulin resistance and circadian misalignment in a Trpv1-dependent manner in HepG2 cells.

TRPV1-Mediated Microglial Autophagy Attenuates Alzheimer's Disease-Associated Pathology and Cognitive Decline.

Autophagy is a major regulator of the ageing process of the central nervous system and neurodegeneration. Autophagy dysfunction has been implicated in the pathogenesis of Alzheimer’s disease (AD). TRPV1 was reported to regulate autophagy to protect against foam cell formation and reduce the release of inflammatory factors in atherosclerosis. In this study, pharmacological activation of TRPV1 with the TRPV1 agonist capsaicin induced autophagy in a TRPV1-dependent manner in both primary microglia and BV2 cells. TRPV1-mediated autophagy regulated glycolysis and oxidative phosphorylation by controlling the expression of genes required for aerobic glycolysis and mitochondrial respiration in primary microglia. TRPV1 agonist capsaicin decreased amyloid and phosphorylated tau pathology and reversed memory deficits by promoting microglia activation, metabolism, and autophagy in 3xTg mice. These results indicate that TRPV1 was a potential therapeutic target for AD, which suggests that capsaicin should be further assessed as a possible treatment for AD.

Activation of TRPV1 channel antagonizes diabetic nephropathy through inhibiting endoplasmic reticulum-mitochondria contact in podocytes

The impairment of podocyte protein filtration function caused by excessive mitochondrial calcium intake is a critical feature of diabetic nephropathy (DN). Ca2+ channel transient receptor potential cation channel subfamily V member 1 (TRPV1) has been reported to protect against ischemia-reperfusion induced acute renal injury, but there is no report about its role in DN. Here, we report that dietary capsaicin potently inhibits and reverses chronic renal structural and functional damages in db/db or streptozotocin (STZ)-induced diabetic mice in a TRPV1-dependent manner. Activation of TRPV1 by capsaicin alleviated hyperglycemia-induced mitochondrial dysfunction in podocytes, accompanied by reduced mitochondria-associated membranes (MAMs) formation and fewer Ca2+ transport from endoplasmic reticulum (ER) to mitochondria. Mechanistically, TRPV1-mediated transient Ca2+ influx activated 5′ AMP-activated protein kinase (AMPK) that reduced the transcription of Fundc1, a key molecule participating in MAMs formation. Inhibition of AMPK or overexpression of Fundc1 obviously blocked the inhibitory effect of capsaicin on MAMs formation and functional decline in podocytes. These findings emphasize the critical role of mitochondrial Ca2+ homeostasis in the maintenance of normal renal function and suggest an effective intervention method to counteract DN.

Acute inflammation sensitizes knee-innervating sensory neurons and decreases mouse digging behavior in a TRPV1-dependent manner

Ongoing, spontaneous pain is characteristic of inflammatory joint pain and reduces an individual's quality of life. To understand the neural basis of inflammatory joint pain, we made a unilateral knee injection of complete Freund's adjuvant (CFA) in mice, which reduced their natural digging behavior. We hypothesized that sensitization of knee-innervating dorsal root ganglion (DRG) neurons underlies this altered behavior. To test this hypothesis, we performed electrophysiological recordings on retrograde labeled knee-innervating primary DRG neuron cultures and measured their responses to a number of electrical and chemical stimuli. We found that 24-h after CFA-induced knee inflammation, knee neurons show a decreased action potential generation threshold, as well as increased GABA and capsaicin sensitivity, but have unaltered acid sensitivity. The inflammation-induced sensitization of knee neurons persisted for 24-h in culture, but was not observed after 48-h in culture. Through immunohistochemistry, we showed that the increased knee neuron capsaicin sensitivity correlated with enhanced expression of the capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) in knee-innervating neurons of the CFA-injected side. We also observed an increase in the co-expression of TRPV1 with tropomyosin receptor kinase A (TrkA), which is the receptor for nerve growth factor (NGF), suggesting that NGF partially induces the increased TRPV1 expression. Lastly, we found that systemic administration of the TRPV1 antagonist, A-425619, reversed the decrease in digging behavior induced by CFA injection, further confirming the role of TRPV1, expressed by knee neurons, in acute inflammatory joint pain.

Zinc Inhibits TRPV1 to Alleviate Chemotherapy-Induced Neuropathic Pain.

Zinc is a transition metal that has a long history of use as an anti-inflammatory agent. It also soothes pain sensations in a number of animal models. However, the effects and mechanisms of zinc on chemotherapy-induced peripheral neuropathy remain unknown. Here we show that locally injected zinc markedly reduces neuropathic pain in male and female mice induced by paclitaxel, a chemotherapy drug, in a TRPV1-dependent manner. Extracellularly applied zinc also inhibits the function of TRPV1 expressed in HEK293 cells and mouse DRG neurons, which requires the presence of zinc-permeable TRPA1 to mediate entry of zinc into the cytoplasm. Moreover, TRPA1 is required for zinc-induced inhibition of TRPV1-mediated acute nociception. Unexpectedly, zinc transporters, but not TRPA1, are required for zinc-induced inhibition of TRPV1-dependent chronic neuropathic pain produced by paclitaxel. Together, our study demonstrates a novel mechanism underlying the analgesic effect of zinc on paclitaxel-induced neuropathic pain that relies on the function of TRPV1.SIGNIFICANCE STATEMENT The chemotherapy-induced peripheral neuropathy is a major limiting factor affecting the chemotherapy patients. There is no effective treatment available currently. We demonstrate that zinc prevents paclitaxel-induced mechanical hypersensitivity via inhibiting the TRPV1 channel, which is involved in the sensitization of peripheral nociceptors in chemotherapy. Zinc transporters in DRG neurons are required for the entry of zinc into the intracellular side, where it inhibits TRPV1. Our study provides insight into the mechanism underlying the pain-soothing effect of zinc and suggests that zinc could be developed to therapeutics for the treatment of chemotherapy-induced peripheral neuropathy.

Photosensitization of TRPA1 and TRPV1 by 7-dehydrocholesterol: implications for the Smith-Lemli-Opitz syndrome.

Loss-of-function mutations in the enzyme 7-dehydrocholesterol reductase are responsible for the Smith-Lemli-Opitz syndrome, in which 7-dehydrocholesterol (7-DHC) levels are markedly increased in the plasma and tissues of patients. This increase in 7-DHC is probably associated with the painful and itchy photosensitivity reported by the majority of patients with Smith-Lemli-Opitz syndrome. To identify the molecular targets involved in the activation and photosensitization of primary afferents by 7-DHC, we focused on TRPA1 and TRPV1, two ion channels expressed in nociceptive nerve endings and previously shown to respond to ultraviolet and visible light under pathophysiological circumstances. Recombinant human TRPA1 is activated and photosensitized in the presence of 7-DHC. Prolonged preexposure to 7-DHC causes more pronounced photosensitization, and while TRPV1 contributes less to the acute effect, it too becomes highly photosensitive upon preincubation with 7-DHC for 1 to 15 hours. Dorsal root ganglion neurons in primary culture display acute sensitivity to 7-DHC in the dark and also light-evoked responses in the presence of 7-DHC, which are exclusively dependent on TRPA1 and TRPV1. Similarly, prolonged exposure of mouse dorsal root ganglion neurons to 7-DHC renders these cells photosensitive in a largely TRPA1- and TRPV1-dependent manner. Single-fiber recordings in mouse skin-nerve preparations demonstrate violet light-evoked activation and a sensitization to 7-DHC exposure. Vice versa, 7-DHC pretreatment of the isolated trachea leads to a TRPA1- and TRPV1-dependent increase of the light-induced calcitonin gene-related peptide release. Taken together, our results implicate TRPA1 and TRPV1 channels as potential pharmacological targets to address the 7-DHC-induced hypersensitivity to light in patients.

The multiplicity of spinal AA-5-HT anti-nociceptive action in a rat model of neuropathic pain

There is considerable evidence to support the role of anandamide (AEA), an endogenous ligand of cannabinoid receptors, in neuropathic pain modulation. AEA also produces effects mediated by other biological targets, of which the transient receptor potential vanilloid type 1 (TRPV1) has been the most investigated. Both, inhibition of AEA breakdown by fatty acid amide hydrolase (FAAH) and blockage of TRPV1 have been shown to produce anti-nociceptive effects. Recent research suggests the usefulness of dual-action compounds, which may afford greater anti-allodynic efficacy. Therefore, in the present study, we examined the effect of N-arachidonoyl-serotonin (AA-5-HT), a blocker of FAAH and TRPV1, in a rat model of neuropathic pain after intrathecal administration. We found that treatment with AA-5-HT increased the pain threshold to mechanical and thermal stimuli, with highest effect at the dose of 500nM, which was most strongly attenuated by AM-630, CB2 antagonist, administration. The single action blockers PF-3845 (1000nM, for FAAH) and I-RTX (1nM, for TRPV1) showed lower efficacy than AA-5-HT. Moreover AA-5-HT (500nM) elevated AEA and palmitoylethanolamide (PEA) levels. Among the possible targets of these mediators, only the mRNA levels of CB2, GPR18 and GPR55, which are believed to be novel cannabinoid receptors, were upregulated in the spinal cord and/or DRG of CCI rats. It was previously reported that AA-5-HT acts in CB1 and TRPV1-dependent manner after systemic administration, but here for the first time we show that AA-5-HT action at the spinal level involves CB2, with potential contributions from GRP18 and/or GPR55 receptors.

Eact, a small molecule activator of TMEM16A, activates TRPV1 and elicits pain- and itch-related behaviours.

TMEM16A, also known as anoctamin 1 channel, is a member of the Ca(2+)-activated chloride channels family and serves as a heat sensor in the primary nociceptors. Eact is a recently discovered small molecule activator of the TMEM16A channel. Here, we asked if Eact produces pain- and itch-related responses in vivo and investigated the cellular and molecular basis of Eact-elicited responses in dorsal root ganglia (DRG) neurons. We employed behavioural testing combined with pharmacological inhibition and genetic ablation approaches to identify transient receptor potential vanilloid 1 (TRPV1) as the prominent mediator for Eact-evoked itch- or pain-related responses. We investigated the effects of Eact on TRPV1 and TMEM16A channels expressed in HEK293T cells and in DRG neurons isolated from wild type and Trpv1(-/-) mice using Ca(2+) imaging and patch-clamp recordings. We also used site-directed mutagenesis to determine the molecular basis of Eact activation of TRPV1. Administration of Eact elicited both itch- and pain-related behaviours. Unexpectedly, the Eact-elicited behavioural responses were dependent on the function of TRPV1, as shown by pharmacological inhibition and genetic ablation studies. Eact activated membrane currents and increased intracellular free Ca(2+) in both TRPV1-expressing HEK293T cells and isolated DRG neurons in a TRPV1-dependent manner. Eact activation of the TRPV1 channel was severely attenuated by mutations disrupting the capsaicin-binding sites. Our results suggest that Eact activates primary sensory nociceptors and produces both pain and itch responses mainly through direct activation of TRPV1 channels.

The anti-inflammatory mediator palmitoylethanolamide enhances the levels of 2-arachidonoyl-glycerol and potentiates its actions at TRPV1 cation channels.

Palmitoylethanolamide (PEA) is an endogenous congener of anandamide and potentiates its actions at cannabinoid CB1 and CB2 receptors, and at transient receptor potential vanilloid type-1 (TRPV1) channels. The other endocannabinoid, 2-arachidonoylglycerol (2-AG), was recently suggested to act as a TRPV1 channel agonist. We investigated if PEA enhanced levels of 2-AG in vitro or in vivo and 2-AG activity at TRPV1 channels. Endogenous lipid levels were measured by LC-MS in (i) human keratinocytes incubated with PEA (10-20 μM, 40 min, 6 and 24 h, 37°C); (ii) the blood of spontaneously Ascaris suum hypersensitive beagle dogs given a single oral dose of ultramicronized PEA (30 mg·kg(-1), 1, 2, 4 and 8 h from administration); (iii) the blood of healthy volunteers given a single oral dose of micronized PEA (300 mg, 2, 4 and 6 h from administration). Effects of 2-AG at TRPV1 channels were assessed by measuring intracellular Ca(2+) in HEK-293 cells over-expressing human TRPV1 channels. PEA elevated 2-AG levels in keratinocytes (∼3-fold) and in human and canine plasma (∼2 and ∼20-fold respectively). 2-AG dose-dependently raised intracellular Ca(2+) in HEK-293-TRPV1 cells in a TRPV1-dependent manner and desensitized the cells to capsaicin. PEA only slightly enhanced 2-AG activation of TRPV1 channels, but significantly increased 2-AG-induced TRPV1 desensitization to capsaicin (IC50 from 0.75 ± 0.04 to 0.45 ± 0.02 μM, with PEA 2 μM). These observations may explain why several effects of PEA are attenuated by cannabinoid receptor or TRPV1 channel antagonists. This article is part of a themed section on Endocannabinoids. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.7/issuetoc.

The role of TRPV1 channels in carrageenan-induced mechanical hyperalgesia in mice

Peripheral inflammation leads to ipsilateral and contralateral mechanical hyperalgesia. The transient receptor potential channel vanilloid type 1 (TRPV1), a nonselective cation channel expressed in mammalian primary sensory neurons and the spinal cord, may be involved in peripheral inflammation, but there is no consensus on the role of this channel in inflammation-induced mechanical hyperalgesia. Here, we examined the role of TRPV1 channels in carrageenan-induced mechanical hyperalgesia using wild-type and TRPV1-knockout (KO) mice and compared the results with those obtained in mice peripherally administered capsazepine, a TRPV1 antagonist, or capsaicin, a TRPV1 agonist. In the TRPV1-KO mice, ipsilateral mechanical hyperalgesia was significantly reduced during the acute phase (10-60 min), and the contralateral mechanical hyperalgesia nearly disappeared during both the acute and subacute phases. Blocking peripheral TRPV1 using capsazepine before carrageenan administration resulted in similar effects as those observed in the TRPV1-KO mice, except that it was less effective against contralateral mechanical hyperalgesia during the subacute phase. In contrast, capsaicin remarkably decreased ipsilateral and contralateral mechanical hyperalgesia throughout both phases, but this analgesic effect was not observed in the TRPV1-KO mice. Thus, TRPV1 channels could be involved in the development of both ipsilateral and contralateral mechanical hyperalgesia after inflammation. Peripheral TRPV1 could participate in acute hyperalgesia, whereas central TRPV1 may participate in subacute secondary hyperalgesia. Capsaicin potentially acts on both primary and secondary hyperalgesia in a TRPV1-dependent manner.

Tmem100 Is a Regulator of TRPA1-TRPV1 Complex and Contributes to Persistent Pain

TRPA1 and TRPV1 are crucial pain mediators, but how their interaction contributes to persistent pain is unknown. Here, we identify Tmem100 as a potentiating modulator of TRPA1-V1 complexes. Tmem100 is coexpressed and forms a complex with TRPA1 and TRPV1 in DRG neurons. Tmem100-deficient mice show a reduction in inflammatory mechanical hyperalgesia and TRPA1- but not TRPV1-mediated pain. Single-channel recording in a heterologous system reveals that Tmem100 selectively potentiates TRPA1 activity in a TRPV1-dependent manner. Mechanistically, Tmem100 weakens the association of TRPA1 and TRPV1, thereby releasing the inhibition of TRPA1 by TRPV1. A Tmem100 mutant, Tmem100-3Q, exerts the opposite effect; i.e., it enhances the association of TRPA1 and TRPV1 and strongly inhibits TRPA1. Strikingly, a cell-permeable peptide (CPP) containing the C-terminal sequence of Tmem100-3Q mimics its effect and inhibits persistent pain. Our study unveils a context-dependent modulation of the TRPA1-V1 complex, and Tmem100-3Q CPP is a promising pain therapy.

Activation of TRPV1 attenuates high salt-induced cardiac hypertrophy through improvement of mitochondrial function.

High-salt diet induces cardiac remodelling and leads to heart failure, which is closely related to cardiac mitochondrial dysfunction. Transient receptor potential (TRP) channels are implicated in the pathogenesis of cardiac dysfunction. We investigated whether activation of TRP vanilloid (subtype 1) (TRPV1) channels by dietary capsaicin can, by ameliorating cardiac mitochondrial dysfunction, prevent high-salt diet-induced cardiac hypertrophy. Male wild-type (WT) and TRPV1(-/-) mice were fed a normal or high-salt diet with or without capsaicin for 6 months. Their cardiac parameters and endurance capacity were assessed. Mitochondrial respiration and oxygen consumption were measured using high-resolution respirometry. The expression levels of TRPV1, sirtuin 3 and NDUFA9 were detected in cardiac cells and tissues. Chronic high-salt diet caused cardiac hypertrophy and reduced physical activity in mice; both effects were ameliorated by capsaicin intake in WT but not in TRPV1(-/-) mice. TRPV1 knockout or high-salt diet significantly jeopardized the proficiency of mitochondrial Complex I oxidative phosphorylation (OXPHOS) and reduced Complex I enzyme activity. Chronic dietary capsaicin increased cardiac mitochondrial sirtuin 3 expression, the proficiency of Complex I OXPHOS, ATP production and Complex I enzyme activity in a TRPV1-dependent manner. TRPV1 activation by dietary capsaicin can antagonize high-salt diet-mediated cardiac lesions by ameliorating its deleterious effect on the proficiency of Complex I OXPHOS. TRPV1-mediated amendment of mitochondrial dysfunction may represent a novel target for management of early cardiac dysfunction.

Abstract LB-33: TRPV1 suppresses intestinal tumorigenesis by negatively regulating EGFR signaling.

Abstract The intestinal epithelium is the most rapidly self-renewing tissue. This dynamic process is mainly driven by Wnt, Notch and EGFR signaling in the crypts of Lieberkühn. These signaling pathways are negatively regulated by multiple control mechanisms to restrain intestinal epithelial cell (IEC) turnover and thereby reduce the risk of oncogenic transformation. Here we report a novel homeostatic feedback loop, intrinsic to IEC, between the EGFR and a member of the transient receptor potential (TRP) ion channel family. We identified that TRP vanilloid 1 (TRPV1), a polymodal pain receptor, is expressed by IEC and that this channel negatively regulates EGFR-induced cell proliferation in vivo. Intestinal crypt cultures (organoids) from Trpv1-/- mice showed hyperproliferation, crypt elongation and a reduced requirement for exogenous EGF compared to WT controls. Importantly, genetic deletion of Trpv1 increased the intestinal tumor load in multiple intestinal neoplasia (Apc+/min) mice, which was reversed by EGFR kinase inhibitor treatment. Continuous oral administration of a dietary TRPV1 agonist, capsaicin, suppressed intestinal tumorigenesis in Apc+/min mice in a TRPV1-dependent manner. In addition we found that capsaicin worked synergistically with a COX2 inhibitor, celecoxib, in the prevention of intestinal neoplasia development in these mice. Mechanistically, we show that TRPV1 is activated by EGFR signaling through phospholipase C (PLC). TRPV1 signaling subsequently restrains EGFR signaling through Ca2+-dependent triggering of protein tyrosine phosphatase (PTP) activity. Together, these findings suggest an intrinsic mechanism that exerts negative feedback on EGFR signaling through Ca2+ channel TRPV1 that thereby acts as a tumor suppressor in the intestinal epithelium. Citation Format: Petrus R. de Jong, Naoki Takahashi, Alexandra R. Harris, Jihyung Lee, Samuel Bertin, Eyal Raz. TRPV1 suppresses intestinal tumorigenesis by negatively regulating EGFR signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-33. doi:10.1158/1538-7445.AM2013-LB-33

Transient receptor potential vanilloid 1 activation induces autophagy in thymocytes through ROS-regulated AMPK and Atg4C pathways

Autophagy is a highly conserved process involved in lymphocyte development and differentiation. Herein, we demonstrated for the first time that triggering of TRPV1 by the specific agonist CPS induces autophagy in mouse thymocytes. TRPV1-dependent autophagy required [Ca(2+)](i) and ROS generation, resulting in AMPK activation. CPS specifically increased Atg4C mRNA expression and induced oxidation of Atg4C protein by ROS generation. TRPV1-triggered autophagy was Atg6/Beclin-1-dependent, as demonstrated by the use of Beclin-1(+/-) transgenic mice, and involved ROS- and AMPK-mediated up-regulation of Beclin-1 expression. Autophagy is activated as a prosurvival process, as its inhibition triggered apoptosis of thymocytes: this effect was accompanied by down-regulation of Atg4C, Bcl-X(L), and Irgm1 mRNA expression, decreased Bcl-X(L) and Beclin-1 protein levels, and caspase-3 activation, suggesting the existence of a molecular interplay between autophagic and apoptotic programs. TRPV1 activation by CPS altered the expression of CD4 and CD8α antigens, inducing the development of DP(dull). Interestingly, we found that CPS induces autophagy of DP(dull) cells, and inhibition of CPS-induced autophagy by the 3-MA autophagic inhibitor induces apoptosis of DP(dull) cells, suggesting the presence of an interplay between autophagic survival and apoptotic cell death. Overall, our findings suggest that DP(dull) cells constitute a distinct thymocyte subpopulation involved in the homeostatic control of cellularity and in the responses to chemical stress signals during thymocyte maturation, via regulating autophagy and apoptosis in a TRPV1-dependent manner.

Transient receptor potential vanilloid-1 participates in the inhibitory effect of ginsenoside Rg1 on capsaicin-induced interleukin-8 and prostaglandin E2 production in HaCaT cells

Ginsenoside Rg1 (GRg1), one of the major active constituents of Panax notoginseng, has shown anti-inflammatory and antinocioceptic activity, but its role in keratinocytes needs further study. We have examined the inhibitory effect of GRg1 on transient receptor potential vanilloid-1 (TRPV1) activation in keratinocyte HaCaT cells and explored its involved mechanism. HEK 293T cells over-expressing exogenous TRPV1 were constructed and named HEK 293T-TRPV1 cells. The effects of GRg1 on production of interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2) ), calcium influx, the expression of cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB) transcriptional activity in HEK 293T-TRPV1 and HaCaT cells were examined by ELISA, Fluo 3-AM fluorescence probe, Western blot and Dual-Luciferase Reporter Assay, respectively. The results showed that GRg1 blocked intracellular calcium by both capsaicin and proton activation in a TRPV1-dependent manner. Furthermore, GRg1 inhibited the expression of COX-2 and NF-κB transcriptional activity induced by capsaicin in keratinocytes. The inhibitory effect of GRg1 was similar to capsazepine, an antagonist of TRPV1. More importantly, GRg1 dose-dependently inhibited capsaicin-induced PGE(2) and IL-8 secretion in HaCaT cells and HEK 293T-TRPV1 cells. These data showed that GRg1 could inhibit TRPV1 mediated responses in HaCaT cells, indicating that GRg1 acted as a TRPV1 antagonist.

Contributions of TRPV1, endovanilloids, and endoplasmic reticulum stress in lung cell death in vitro and lung injury.

Endogenous agonists of transient receptor potential vanilloid-1 (TRPV1) (endovanilloids) are implicated as mediators of lung injury during inflammation. This study tested the hypothesis that endovanilloids produced following lipopolysaccharide (LPS) treatment activate TRPV1 and cause endoplasmic reticulum stress/GADD153 expression in lung cells, representing a mechanistic component of lung injury. The TRPV1 agonist nonivamide induced GADD153 expression and caused cytotoxicity in immortalized and primary human bronchial, bronchiolar/alveolar, and microvascular endothelial cells, proportional to TRPV1 mRNA expression. In CF-1 mice, Trpv1 mRNA was most abundant in the alveoli, and intratracheal nonivamide treatment promoted Gadd153 expression in the alveolar region. Treatment of CF-1 mice with LPS increased Gadd153 in the lung, lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid, and lung wet-to-dry weight ratio. Cotreating mice with LPS and the TRPV1 antagonist LJO-328 reduced Gadd153 induction and LDH in BAL but did not inhibit increases in lung wet-to-dry ratio. In Trpv1(-/-) mice treated with LPS, Gadd153 induction and LDH in BAL were reduced relative to wild-type mice, and the wet-to-dry weight ratios of lungs from both wild-type and Trpv1(-/-) mice decreased. Organic extracts of blood collected from LPS-treated mice were more cytotoxic to TRPV1-overexpressing cells compared with BEAS-2B cells and extracts from control mice, however, most pure endovanilloids did not produce cytotoxicity in a characteristic TRPV1-dependent manner. Collectively, these data indicate a role for TRPV1, and endogenous TRPV1 agonists, in ER stress and cytotoxicity in lung cells but demonstrate that ER stress and cytotoxicity are not essential for pulmonary edema.

Hydrogen Sulfide and Neurogenic Inflammation in Polymicrobial Sepsis: Involvement of Substance P and ERK-NF-κB Signaling

Hydrogen sulfide (H2S) has been shown to induce transient receptor potential vanilloid 1 (TRPV1)-mediated neurogenic inflammation in polymicrobial sepsis. However, endogenous neural factors that modulate this event and the molecular mechanism by which this occurs remain unclear. Therefore, this study tested the hypothesis that whether substance P (SP) is one important neural element that implicates in H2S-induced neurogenic inflammation in sepsis in a TRPV1-dependent manner, and if so, whether H2S regulates this response through activation of the extracellular signal-regulated kinase-nuclear factor-κB (ERK-NF-κB) pathway. Male Swiss mice were subjected to cecal ligation and puncture (CLP)-induced sepsis and treated with TRPV1 antagonist capsazepine 30 minutes before CLP. DL-propargylglycine (PAG), an inhibitor of H2S formation, was administrated 1 hour before or 1 hour after sepsis, whereas sodium hydrosulfide (NaHS), an H2S donor, was given at the same time as CLP. Capsazepine significantly attenuated H2S-induced SP production, inflammatory cytokines, chemokines, and adhesion molecules levels, and protected against lung and liver dysfunction in sepsis. In the absence of H2S, capsazepine caused no significant changes to the PAG-mediated attenuation of lung and plasma SP levels, sepsis-associated systemic inflammatory response and multiple organ dysfunction. In addition, capsazepine greatly inhibited phosphorylation of ERK1/2 and inhibitory κBα, concurrent with suppression of NF-κB activation even in the presence of NaHS. Furthermore, capsazepine had no effect on PAG-mediated abrogation of these levels in sepsis. Taken together, the present findings show that H2S regulates TRPV1-mediated neurogenic inflammation in polymicrobial sepsis through enhancement of SP production and activation of the ERK-NF-κB pathway.