TRPV1 Currents Research Articles

Crucial role for sensory nerves and Na/H exchanger inhibition in dapagliflozin- and empagliflozin-induced arterial relaxation.

Sodium/glucose transporter 2 (SGLT2 or SLC5A2) inhibitors lower blood glucose and are also approved treatments for heart failure independent of raised glucose. Various studies have showed that SGLT2 inhibitors relax arteries, but the underlying mechanisms are poorly understood and responses variable across arterial beds. We speculated that SGLT2 inhibitor-mediated arterial relaxation is dependent upon calcitonin gene-related peptide (CGRP) released from sensory nerves independent of glucose transport. The functional effects of SGLT1 and 2 inhibitors (mizagliflozin, dapagliflozin, and empagliflozin) and the sodium/hydrogen exchanger 1 (NHE1) blocker cariporide were determined on pre-contracted resistance arteries (mesenteric and cardiac septal arteries) as well as main renal conduit arteries from male Wistar rats using wire myography. SGLT2, CGRP, TRPV1, and NHE1 expression was determined by western blot and immunohistochemistry. Kv7.4/5/KCNE4 and TRPV1 currents were measured in the presence and absence of dapagliflozin and empagliflozin. All SGLT inhibitors (1-100 µM) and cariporide (30 µM) relaxed mesenteric arteries but had negligible effect on renal or septal arteries. Immunohistochemistry with TRPV1 and CGRP antibodies revealed a dense innervation of sensory nerves in mesenteric arteries that were absent in renal and septal arteries. Consistent with a greater sensory nerve component, the TRPV1 agonist capsaicin relaxed mesenteric arteries more effectively than renal or septal arteries. In mesenteric arteries, relaxations to dapagliflozin, empagliflozin, and cariporide were attenuated by the CGRP receptor antagonist BIBN-4096, depletion of sensory nerves with capsaicin, and blockade of TRPV1 or Kv7 channels. Neither dapagliflozin nor empagliflozin activated heterologously expressed TRPV1 channels or Kv7 channels directly. Sensory nerves also expressed NHE1 but not SGLT2 and cariporide pre-application as well as knockdown of NHE1 by translation stop morpholinos prevented the relaxant response to SGLT2 inhibitors. SGLT2 inhibitors relax mesenteric arteries by promoting the release of CGRP from sensory nerves in a NHE1-dependent manner.

Multi-target modulation of ion channels underlying the analgesic effects of α-mangostin in dorsal root ganglion neurons

Backgroundα-Mangostin is a xanthone isolated from the pericarps of mangosteen fruit with, and has analgesic properties. Although the effects suggest an interaction of α-mangostin with ion channels in the nociceptive neurons, electrophysiological investigation of the underlying mechanism has not been performed. HypothesisWe hypothesized that α-Mangostin exerts its analgesic effects by modulating the activity of various ion channels in dorsal root ganglion (DRG) neurons. MethodsWe performed a whole-cell patch clamp study using mouse DRG neurons, HEK293T cells overexpressing targeted ion channels, and ND7/23 cells. Molecular docking (MD) and in silico absorption, distribution, metabolism, and excretion (ADME) analyses were conducted to obtain further insights into the binding sites and pharmacokinetics, respectively. ResultsApplication of α-mangostin (1–3 µM) hyperpolarized the resting membrane potential (RMP) of small-sized DRG neurons by increasing background K+ conductance and thereby inhibited action potential generation. At micromolar levels, α-mangostin activates TREK-1, TREK-2, or TRAAK, members of the two-pore domain K+ channel (K2P) family known to be involved in RMP formation in DRG neurons. Furthermore, capsaicin-induced TRPV1 currents were potently inhibited by α-mangostin (0.43 ± 0.27 µM), and partly suppressed tetrodotoxin-sensitive voltage-gated Na+ channel (NaV) currents. MD simulation revealed that multiple oxygen atoms in α-mangostin may form stable hydrogen bonds with TREKs, TRAAK, TRPV1, and NaV channels. In silico ADME tests suggested that α-mangostin may satisfy the drug-likeness properties without penetrating the blood–brain barrier. ConclusionThe analgesic properties of α-mangostin might be mediated by the multi-target modulation of ion channels, including TREK/TRAAK activation, TRPV1 inhibition, and reduction of the tetrodotoxin-sensitive NaV current. The findings suggest that the phytochemical can be a multi-ion channel-targeting drug and an alternative drug for effective pain management.

Multiple Effects of Echinochrome A on Selected Ion Channels Implicated in Skin Physiology.

Echinochrome A (Ech A), a naphthoquinoid pigment from sea urchins, is known to have anti-inflammatory and analgesic effects that have been suggested to be mediated by antioxidant activity and intracellular signaling modulation. In addition to these mechanisms, the ion channels in keratinocytes, immune cells, and nociceptive neurons may be the target for the pharmacological effects. Here, using the patch clamp technique, we investigated the effects of Ech A on the Ca2+-permeable TRPV3, TRPV1 and Orai1 channels and the two-pore domain K+ (K2P) channels (TREK/TRAAK, TASK-1, and TRESK) overexpressed in HEK 293 cells. Ech A inhibited both the TRPV3 and Orai1 currents, with IC50 levels of 2.1 and 2.4 μM, respectively. The capsaicin-activated TRPV1 current was slightly augmented by Ech A. Ech A alone did not change the amplitude of the TREK-2 current (ITREK2), but pretreatments with Ech A markedly facilitated ITREK2 activation by 2-APB, arachidonic acid (AA), and acidic extracellular pH (pHe). Similar facilitation effects of Ech A on TREK-1 and TRAAK were observed when they were stimulated with 2-APB and AA, respectively. On the contrary, Ech A did not affect the TRESK and TASK-1 currents. Interestingly, the ITREK2 maximally activated by the combined application of 2-APB and Ech A was not inhibited by norfluoxetine but was still completely inhibited by ruthenium red. The selective loss of sensitivity to norfluoxetine suggested an altered molecular conformation of TREK-2 by Ech A. We conclude that the Ech A-induced inhibition of the Ca2+-permeable cation channels and the facilitation of the TREK/TRAAK K2P channels may underlie the analgesic and anti-inflammatory effects of Ech A.

Scorpion Venom peptide, AGAP inhibits TRPV1 and potentiates the analgesic effect of lidocaine

The current study was designed to test the hypothesis that BmK AGAP (AGAP) potentiates the analgesic effect of lidocaine. The chronic constrictive injury was performed on 72 rats to induce a rapid onset and long-lasting pain. The rats were randomly assigned to one of six groups; Group A (n = 12) received an intrathecal administration of saline, Group B (n = 12) received an intrathecal injection of lidocaine, Group C (n = 12) received an intrathecal administration of AGAP, Group D, E, and F (n = 12 each) received an intrathecal administration of lidocaine 0.005 mg/ml + AGAP 25, 50, 100 μg/kg respectively. The von Frey filaments were used to assess mechanical allodynia. Nav1.7 and TRPV1 currents were recorded by the whole-cell aspiration patch-clamp technique, and KCNQ2/3 currents were recorded by the whole-cell drilling patch-clamp technique. The whole-cell aspiration patch-clamp technique showed that AGAP inhibited TRPV1and KCNQ2/3 currents and increased the analgesic effect of lidocaine. AGAP may have a synergistic effect with lidocaine which demonstrates a potential therapeutic approach for optimizing post-operative analgesia.

The mechanism of Annexin A1 to modulate TRPV1 and nociception in dorsal root ganglion neurons

BackgroundAnnexin A1 (ANXA1) exerts anti-nociceptive effect through ANXA1 receptor formyl peptide receptor 2 (FPR2/ALX (receptor for lipoxin A4), FPR2) at the dorsal root ganglia (DRG) level. However, the mechanisms remain elucidated. By using radiant heat, hot/cold plate, tail flick, von Frey, and Randall-Selitto tests to detect nociception in intact and chemical (capsaicin, menthol, mustard oil, formalin or CFA) injected AnxA1 conditional knockout (AnxA1−/−) mice, applying calcium imaging and patch clamp recordings in cultured DRG neurons to measure neuronal excitability, conducting immunofluorescence and western blotting to detect the protein levels of TRPV1, FPR2 and its downstream molecules, and performing double immunofluorescence and co-immunoprecipitation to investigate the interaction between Calmodulin (CaM) and TRPV1; we aim to uncover the molecular and cellular mechanisms of ANXA1’s role in antinociception.ResultsAnxA1−/− mice exhibited significant sensitivity to noxious heat (mean ± SD, 6.2 ± 1.0 s vs. 9.9 ± 1.6 s in Hargreaves test; 13.6 ± 1.5 s vs. 19.0 ± 1.9 s in hot plate test; n = 8; P < 0.001), capsaicin (101.0 ± 15.3 vs. 76.2 ± 10.9; n = 8; P < 0.01), formalin (early phase: 169.5 ± 32.8 s vs. 76.0 ± 21.9 s; n = 8; P < 0.05; late phase: 444.6 ± 40.1 s vs. 320.4 ± 33.6 s; n = 8; P < 0.01) and CFA (3.5 ± 0.8 s vs. 5.9 ± 1.4 s; n = 8; P < 0.01). In addition, we found significantly increased capsaicin induced Ca2+ response, TRPV1 currents and neuronal firing in AnxA1 deficient DRG neurons. Furthermore, ANXA1 mimic peptide Ac2-26 robustly increased intracellular Ca2+, inhibited TRPV1 current, activated PLCβ and promoted CaM-TRPV1 interaction. And these effects of Ac2-26 could be attenuated by FPR2 antagonist Boc2.ConclusionsSelective deletion of AnxA1 in DRG neurons enhances TRPV1 sensitivity and deteriorates noxious heat or capsaicin induced nociception, while ANXA1 mimic peptide Ac2-26 desensitizes TRPV1 via FPR2 and the downstream PLCβ-Ca2+-CaM signal. This study may provide possible target for developing new analgesic drugs in inflammatory pain.

Cytokine activin C ameliorates chronic neuropathic pain in peripheral nerve injury rodents by modulating the TRPV1 channel.

The cytokine activin C is mainly expressed in small-diameter dorsal root ganglion (DRG) neurons and suppresses inflammatory pain. However, the effects of activin C in neuropathic pain remain elusive. Male rats and wild-type and TRPV1 knockout mice with peripheral nerve injury - sciatic nerve axotomy and spinal nerve ligation in rats; chronic constriction injury (CCI) in mice - provided models of chronic neuropathic pain. Ipsilateral lumbar (L)4-5 DRGs were assayed for activin C expression. Chronic neuropathic pain animals were treated with intrathecal or locally pre-administered activin C or the vehicle. Nociceptive behaviours and pain-related markers in L4-5 DRGs and spinal cord were evaluated. TRPV1 channel modulation by activin C was measured. Following peripheral nerve injury, expression of activin βC subunit mRNA and activin C protein was markedly up-regulated in L4-5 DRGs of animals with axotomy, SNL or CCI. [Correction added on 26 November 2020, after first online publication: The preceding sentence has been corrected in this current version.] Intrathecal activin C dose-dependently inhibited neuropathic pain in spinal nerve ligated rats. Local pre-administration of activin C decreased neuropathic pain, macrophage infiltration into ipsilateral L4-5 DRGs and microglial reaction in L4-5 spinal cords of mice with CCI. In rat DRG neurons, activin C enhanced capsaicin-induced TRPV1 currents. Pre-treatment with activin C reduced capsaicin-evoked acute hyperalgesia and normalized capsaicin-evoked persistent hypothermia in mice. Finally, the analgesic effect of activin C was abolished in TRPV1 knockout mice with CCI. Activin C inhibits neuropathic pain by modulating TRPV1 channels, revealing potential analgesic applications in chronic neuropathic pain therapy.

Intrathecal Injection of Scorpion Venom Peptide, BmK AGAP Inhibits KCNQ2/3, TRPV1 and Decreases TNF-α and IL-1β Expression to Attenuates Pain in Rat

The current study was designed to test the hypothesis that Recombinant Buthus martensii Kasch Analgesic-Antitumor Peptide (rBmK AGAP) potentiates lidocaine induced analgesia. The chronic constrictive injury was performed on 72 rats to induce a rapid onset and long-lasting pain. The rats were randomly assigned to one of six groups; Group A (n=12) received an intrathecal injection of saline, Group B (n=12) received an intrathecal injection of lidocaine alone, Group C (n=12) received an intrathecal injection of rBmK AGAP alone, Group D, E, and F (n=12 each) received an intrathecal injection of lidocaine + rBmK AGAP (0.5, 1, or 2 mg.kg-1). The von Frey filaments were used to assessed mechanical allodynia. Nav1.7 and TRPV1 currents were recorded by whole-cell aspiration patch-clamp technique, and KCNQ2/3 currents were recorded by whole-cell drilling patch-clamp technique. We performed qPCR, ELISA and western blot to determine the expression of TNF-α, IL-1β, IL-6, IL-10, p65/NF-κB, p-p65/NF-κB, IKKα, IκBα in rats following rBmK AGAP treatment. The rats from the lidocaine + BmK AGAP group exhibited significantly increased Paw Withdrawal Threshold (PWT) after 30 minutes of drug administration which gradually decreased to baseline PWT at 360 minutes. The whole-cell aspiration patch-clamp technique showed that rBmK AGAP significantly inhibited KCNQ2/3 and TRPV1 currents to potentiate lidocaine induced analgesia. We observed significantly decreased expression of TNF-α and IL-1β following the combined treatment compared with lidocaine or rBmK AGAP alone. Intrathecal injection of rBmK AGAP inhibits KCNQ2/3, TRPV1 and decreases pro-inflammatory cytokines, TNF-α, and IL-1β expression and potentiates lidocaine induced analgesia. Funding Statement: This study was supported by the Key Laboratory of Liaoning Provincial Education Department (Grant NO: LZ2016002) and Liaoning Natural Science Foundation (Grant NO: 20170540290). Declaration of Interests: All authors declared no conflict of interests. Ethics Approval Statement: The animal ethics committee of the Dalian Medical University approved all the study protocols and were by the declaration of the national institutes of health guide for the care and use of laboratory animals.

Voltage-dependent modulation of TRPA1 currents by diphenhydramine

Diphenhydramine (DPH) has been broadly used to treat allergy. When used as a topical medicine, DPH temporarily relieves itching and pain. Although transient receptor potential type A1 (TRPA1) channel is known to play roles in both acute and chronic itch and pain, whether DPH affects the activities of TRPA1 remains unclear. Using whole-cell patch clamp recordings, we demonstrated that DPH modulates the voltage-dependence of TRPA1. When co-applied with a TRPA1 agonist, DPH significantly enhanced the inward currents while suppressing the outward currents of TRPA1, converting the channel from outwardly rectifying to inwardly rectifying. This effect of DPH occurred no matter TRPA1 was activated by an electrophilic or non-electrophilic agonist and for both mouse and human TRPA1. The modulation of TRPA1 by DPH was maintained in the L906C mutant, which by itself also causes inward rectification of TRPA1, indicating that additional acting sites are present for the modulation of TRPA1 currents by DPH. Our recordings also revealed that DPH partially blocked capsaicin evoked TRPV1 currents. These data suggest that DPH may exert its therapeutic effects on itch and pain, through modulation of TRPA1 in a voltage-dependent fashion.

Liquiritin, a novel inhibitor of TRPV1 and TRPA1, protects against LPS-induced acute lung injury

TRPV1 and TRPA1 are cation channels that play key roles in inflammatory signaling pathways. They are co-expressed on airway C-fibers, where they exert synergistic effects on causing inflammation and cough. Licorice, the root of Glycyrrhiza uralensis, has been widely used in China as an anti-inflammatory and anti-coughing herb. To learn if TRPV1 and TRPA1 might be key targets of the anti-inflammatory and antitussive effects of licorice, we examined liquiritin, the main flavonoid compound and active ingredient of licorice, on agonist-evoked TRPV1 and TRPA1 activation. Liquiritin inhibited capsaicin- and allyl isothiocyanate-evoked TRPV1 and TRPA1 whole-cell currents, respectively, with a similar potency and maximal inhibition. In a mouse acute lung injury (ALI) model induced by the bacterial endotoxin lipopolysaccharide, which involves both TRPV1 and TRPA1, an oral gavage of liquiritin prevented tissue damage and suppressed inflammation and the activation of NF-κB signaling pathway in the lung tissue. Liquiritin also suppressed LPS-induced increase in TRPV1 and TRPA1 protein expression in the lung tissue, as well as TRPV1 and TRPA1 mRNA levels in cells contained in mouse bronchoalveolar lavage fluid. In cultured THP-1 monocytes, liguiritin, or TRPV1 and TRPA1 antagonists capsazepine and HC030031, respectively, diminished not only cytokine-induced upregulation of NF-κB function but also TRPV1 and TRPA1 expression at both protein and mRNA levels. We conclude that the anti-inflammatory and antitussive effects of liquiritin are mediated by the dual inhibition of TRPV1 and TRPA1 channels, which are upregulated in nonneuronal cells through the NF-κB pathway during airway inflammation via a positive feedback mechanism.

Zoledronic Acid Activates TRPV1 Channels: Possible Role in Cell Proliferation and Pain

Bisphosphonates (BPs) are the most used bone specific anti‐resorptive drugs. Lately there is an increasing interest in BPs capability to prolong osteoblasts lifespan and mineralize; however, the mechanism of action which underpin these effects are nowadays poorly understood. In addition, an increasing concern about BPs pain‐relieving effects is now emerging. Empirical evidence showed BPs may exert anti‐nociceptive, anti‐hyperalgesia and anti‐allodynic properties against multiple stimuli; targets involved in these analgesic effects remains elusive. FDA alert showed that the chronic use of BPs is linked with an increased risk of musculoskeletal pain; mechanisms underlying this sensation are not known. TRPV1 channel is actually recognized to play a key role in bone formation process; as well, it appeared to be involved in bone pain sensation. Capsaicin, a selective agonist of TRPV1 channel, is used for treating different bone pain conditions. We have recently demonstrated that the BP Zoledronic acid (ZOL) can activate TRPV1 currents on osteoblasts. In the mineralization assay, the co‐application of ZOL with capsazepine, an antagonist of TRPV1 ion channel, reduced ZOL‐induced mineralization (Scala et al., Cancer 2019). We tested ZOL effects on TRPV1 ion channel in differentiated neuronal SH‐SY5Y cells and in HEK293 cells transfected with cDNA codifying for WT and PI(4,5)P2‐binding site mutant TRPV1 ion channels. In whole‐cell patch clamp experiments on neuronal SH‐SY5Y cells, ZOL (100μM) activated strong outward currents both at negative and positive voltages. At positive membrane voltages ZOL (100μM)‐ evoked currents were closed after the application of capsazepine (1μM). On WT TRPV1 channel expressed on HEK293 cells, ZOL (100 μM) activates currents by 37% at −60 mV, by +81% at +60 mV and +80% at +180 mV (Vm). As well, on K694A mutant, ZOL was able to activate currents by +10%, +63% and +76% at −60 mV, +60 mV and +180 mV (Vm), respectively. On the contrary, no activation of currents was observed on the R579A mutant.The ZOL‐induced activation of TRPV1 channel contributes to the mineralization of osteoblasts and to the pain‐relieving effect of ZOL in neurones. Experiments on mutant TRPV1 ion channel suggest that the arginine in the S4–S5 linker of the channel is essential for the drug interaction.Support or Funding InformationThis research was funded by Reg. Puglia (Italy) project “Cluster in Bioimaging” code QZYCUM0 D.T and A.S., and Fondecyt grants 1150273, 1190293 R.L.. R.S. was supported by GLOBAL THESIS Study (2016–2017) by Univ. degli Studi di Bari “Aldo Moro” at CINV “Centro Interdisciplinario De Neurociencia De Valparaíso”.

Myrcene and terpene regulation of TRPV1

ABSTRACTNociceptive Transient Receptor Potential channels such as TRPV1 are targets for treating pain. Both antagonism and agonism of TRP channels can promote analgesia, through inactivation and chronic desensitization. Since plant-derived mixtures of cannabinoids and the Cannabis component myrcene have been suggested as pain therapeutics, we screened terpenes found in Cannabis for activity at TRPV1. We used inducible expression of TRPV1 to examine TRPV1-dependency of terpene-induced calcium flux responses. Terpenes contribute differentially to calcium fluxes via TRPV1 induced by Cannabis-mimetic cannabinoid/terpenoid mixtures. Myrcene dominates the TRPV1-mediated calcium responses seen with terpenoid mixtures. Myrcene-induced calcium influx is inhibited by the TRPV1 inhibitor capsazepine and Myrcene elicits TRPV1 currents in the whole-cell patch-clamp configuration. TRPV1 currents are highly sensitive to internal calcium. When Myrcene currents are evoked, they are distinct from capsaicin responses on the basis of Imax and their lack of shift to a pore-dilated state. Myrcene pre-application and residency at TRPV1 appears to negatively impact subsequent responses to TRPV1 ligands such as Cannabidiol, indicating allosteric modulation and possible competition by Myrcene. Molecular docking studies suggest a non-covalent interaction site for Myrcene in TRPV1 and identifies key residues that form partially overlapping Myrcene and Cannabidiol binding sites. We identify several non-Cannabis plant-derived sources of Myrcene and other compounds targeting nociceptive TRPs using a data mining approach focused on analgesics suggested by non-Western Traditional Medical Systems. These data establish TRPV1 as a target of Myrcene and suggest the therapeutic potential of analgesic formulations containing Myrcene.

Inhibitive effect of loureirin B plus capsaicin on tetrodotoxin-resistant sodium channel

OBJECTIVETo investigate whether the effect of loureirin B plus capsaicin on tetrodotoxin-resistant (TTX-R) sodium channel. METHODSBy using whole-cell patch-clamp recordings, in acutely isolated dorsal root ganglion (DRG) neurons, the combined effects of loureirin B and capsaicin on TTX-R sodium channel were observed. Based on the data, the interaction between loureirin B and capsaicin in their modulation on TTX-R sodium channel was assessed. RESULTSLoureirin B could not induce transient inward TRPV1 current. Capsazepine, a transient receptor potential vanilloid l (TRPV1) antagonist, could not attenuate the block of 0.64 mmol/L loureirin B on TTX-R sodium channel. There was no significant difference (P > 0.05) between IC50 of loureirin B (0.37 mmol/L) on TTX-R sodium channel in capsaicin-sensitive DRG neurons and that (0.38 mmol/L) in capsaicin-insensitive DRG neurons. However, there was a significant difference (P < 0.05) between the IC50 of capsaicin (0.28 μmol/L) on TTX-R sodium channel in capsaicin-sensitive DRG neurons and that (52.24 μmol/L) in capsaicin-insensitive DRG neurons. Four combinations composed of various concentrations of loureirin B and capsaicin could all inhibit TTX-R sodium currents but have different interactions between loureirin B and capsaicin. CONCLUSIONLoureirin B plus capsaicin could produce double blockage on TRPV1 and modulation on TTX-R sodium channel. The action of loureirin B on TTX-R sodium channel was independent of TRPV1 but similar with that of capsaicin on TTX-R sodium channel in capsaicin-insensitive DRG neurons.

Calorie restriction protects against apoptosis, mitochondrial oxidative stress and increased calcium signaling through inhibition of TRPV1 channel in the hippocampus and dorsal root ganglion of rats.

The TRPV1 channel is activated in neurons by capsaicin, oxidative stress, acidic pH and heat factors, and these factors are attenuated by the antioxidant role of calorie restriction (CR). Hence, we investigated the hypothesis that the antioxidant roles of CR and food frequency (FF) may modulate TRPV1 activity and apoptosis through inhibition of mitochondrial oxidative stress in hippocampal (HIPPON) and dorsal root ganglion neurons (DRGN). We investigated the contribution of FF and CR to neuronal injury and apoptosis through inhibition of TRPV1 in rats. We assigned rats to control, FF and FF + CR groups. A fixed amount of food ad libitum was supplemented to the control and FF groups for 20weeks, respectively. FF + CR group were fed the same amount of food as the control group but with 20% less calories during the same period. In major results, TRPV1 currents, intracellular Ca2+ levels, apoptosis, reactive oxygen species, mitochondrial depolarization, PARP-1 expression, caspase 3 and 9 activity and expression values were found to be increased in the HIPPON and DRGN following FF treatment, and these effects were decreased following FF + CR treatment. The FF-induced decrease in cell viability of HIPPO and DRGN, and vitamin E concentration of brain, glutathione peroxidase, vitamin A, and β-carotene values of the HIPPO, DRGN, plasma, liver and kidney were increased by FF + DR treatment, although lipid peroxidation levels in the same samples were decreased. In conclusion, CR reduces FF-induced increase of oxidative stress, apoptosis and Ca2+ entry through TRPV1 in the HIPPON and DRGN. Our findings may be relevant to the etiology and treatment of obesity following CR treatment.

917 - Butyrate-Producing Synbiotics Reduce Visceral Hyperalgesia and Anxiety-Like Behaviors in a Mouse Model of Irritable Bowel Syndrome, Associated with Inhibition of TRPV1 Currents in Sensory Neurons

917 - Butyrate-Producing Synbiotics Reduce Visceral Hyperalgesia and Anxiety-Like Behaviors in a Mouse Model of Irritable Bowel Syndrome, Associated with Inhibition of TRPV1 Currents in Sensory Neurons

Long term exposure to cell phone frequencies (900 and 1800MHz) induces apoptosis, mitochondrial oxidative stress and TRPV1 channel activation in the hippocampus and dorsal root ganglion of rats.

Mobile phone providers use electromagnetic radiation (EMR) with frequencies ranging from 900 to 1800MHz. The increasing use of mobile phones has been accompanied by several potentially pathological consequences, such as neurological diseases related to hippocampal (HIPPON) and dorsal root ganglion neuron (DRGN). The TRPV1 channel is activated different stimuli, including CapN, high temperature and oxidative stress. We investigated the contribution TRPV1 to mitochondrial oxidative stress and apoptosis in HIPPON and DRGN following long term exposure to 900 and 1800MHz in a rat model. Twenty-four adult rats were equally divided into the following groups: (1) control, (2) 900MHz, and (3) 1800MHz exposure. Each experimental group was exposed to EMR for 60min/ 5days of the week during the one year. The 900 and 1800MHz EMR exposure induced increases in TRPV1 currents, intracellular free calcium influx (Ca2+), reactive oxygen species (ROS) production, mitochondrial membrane depolarization (JC-1), apoptosis, and caspase 3 and 9 activities in the HIPPON and DRGN. These deleterious processes were further increased in the 1800MHz experimental group compared to the 900MHz exposure group. In conclusion, mitochondrial oxidative stress, programmed cell death and Ca2+ entry pathway through TRPV1 activation in the HIPPON and DRGN of rats were increased in the rat model following exposure to 900 and 1800MHz cell frequencies. Our results suggest that exposure to 900 and 1800MHz EMR may induce a dose-associated, TRPV1-mediated stress response.

Ion Channel Expression and Characterization in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Background Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are providing new possibilities for the biological study, cell therapies, and drug discovery. However, the ion channel expression and functions as well as regulations in hiPSC-CMs still need to be fully characterized. Methods Cardiomyocytes were derived from hiPS cells that were generated from two healthy donors. qPCR and patch clamp techniques were used for the study. Results In addition to the reported ion channels, INa, ICa-L, ICa-T, If, INCX, IK1, Ito, IKr, IKs IKATP, IK-pH, ISK1–3, and ISK4, we detected both the expression and currents of ACh-activated (KACh) and Na+-activated (KNa) K+, volume-regulated and calcium-activated (Cl-Ca) Cl−, and TRPV channels. All the detected ion currents except IK1, IKACh, ISK, IKNa, and TRPV1 currents contribute to AP duration. Isoprenaline increased ICa-L, If, and IKs but reduced INa and INCX, without an effect on Ito, IK1, ISK1–3, IKATP, IKr, ISK4, IKNa, ICl-Ca, and ITRPV1. Carbachol alone showed no effect on the tested ion channel currents. Conclusion Our data demonstrate that most ion channels, which are present in healthy or diseased cardiomyocytes, exist in hiPSC-CMs. Some of them contribute to action potential performance and are regulated by adrenergic stimulation.

Involvement of TRPM2 and TRPV1 channels on hyperalgesia, apoptosis and oxidative stress in rat fibromyalgia model: Protective role of selenium

Fibromyalgia (FM) results in pain characterized by low selenium (Se) levels, excessive Ca2+ influx, reactive oxygen species (ROS) production, and acidic pH. TRPM2 and TRPV1 are activated by ROS and acid; nevertheless, their roles have not been elucidated in FM. Therefore, we investigated the contribution of TRPM2 and TRPV1 to pain, oxidative stress, and apoptosis in a rat model of FM and the therapeutic potential of Se. Thirty-six rats were divided into four groups: control, Se, FM, and FM + Se. The Se treatment reduced the FM-induced increase in TRPM2 and TRPV1 currents, pain intensity, intracellular free Ca2+, ROS, and mitochondrial membrane depolarization in the sciatic (SciN) and dorsal root ganglion (DRGN) neurons. Furthermore, Se treatment attenuated the FM-induced decrease in cell viability in the DRGN and SciN, glutathione peroxidase, and reduced glutathione and α-tocopherol values in the DRGN, SciN, brain, muscle, and plasma; however, lipid peroxidation levels were decreased. Se also attenuated PARP1, caspase 3, and 9 expressions in the SciN, DRGN, and muscle. In conclusion, Se treatment decreased the FM-induced increase in hyperalgesia, ROS, apoptosis, and Ca2+ entry through TRPM2 and TRPV1 in the SciN and DRGN. Our findings may be relevant to the elucidation and treatment of FM.

Scalaradial Is a Potent Inhibitor of Transient Receptor Potential Melastatin 2 (TRPM2) Ion Channels.

TRPM2 is a Ca2+-permeable, nonselective cation channel that plays a role in oxidant-induced cell death, insulin secretion, and cytokine release. Few TRPM2 inhibitors have been reported, which hampers the validation of TRPM2 as a drug target. While screening our in-house marine-derived chemical library, we identified scalaradial and 12-deacetylscalaradial as the active components within an extract of an undescribed species of Cacospongia (class Demospongiae, family Thorectidae) that strongly inhibited TRPM2-mediated Ca2+ influx in TRPM2-overexpressing HEK293 cells. In whole-cell patch-clamp experiments, scalaradial (and similarly 12-deacetylscalaradial) inhibited TRPM2-mediated currents in a concentration- and time-dependent manner (∼20 min to full onset; IC50 210 nM). Scalaradial inhibited TRPM7 with less potency (IC50 760 nM) but failed to inhibit CRAC, TRPM4, and TRPV1 currents in whole-cell patch clamp experiments. Scalaradial's effect on TRPM2 channels was shown to be independent of its well-known ability to inhibit secreted phospholipase A2 (sPLA2) and its reported effects on extracellular signal-regulated kinases (ERK) and Akt pathways. In addition, scalaradial was shown to inhibit endogenous TRPM2 currents in a rat insulinoma cell line (IC50 330 nM). Based on its potency and emerging specificity profile, scalaradial is an important addition to the small number of known TRPM2 inhibitors.

Prenatal Nicotinic Exposure (PNE) Prolongs Superior Laryngeal C‐Fibers (SLCFs)‐mediated Apnea through Upregulating TRPV1 Receptor

Maternal cigarette smoke is a critical factor in the pathogenesis of sudden infant death syndrome (SIDS). Simulation of SLCFs evokes an immediate apnea and cardiovascular instability, which are potentially life‐threatening and implicated in pathogenesis of SIDS. To date, the impact of PNE on SLCFs has not been explored. We tested hypothesis whether PNE could prolong the SLCF‐mediated apnea, and if so, whether this prolongation is associated with an upregulated expression of TRPV1 on nodose/jugular (N/J) ganglionic neurons retrogradely traced from the superior laryngeal nerve (SLN). To this end, we compared following outcomes between Ctrl and PNE rat pups (postnatal days 11–14): 1) the apneic response to intralaryngeal application of capsaicin (10 μg/kg, 50 μl) to stimulate local TRPV1 in anesthetized preparation; 2) immunoreactivity of TRPV1 in laryngeal sensory neurons of N/J ganglia retrogradely traced by DiI; and 3) TRPV1 currents recorded by whole‐cell patch clamp on cultured sensory laryngeal C‐neurons from N/J ganglia retrogradely traced by DiI. Our results showed that PNE: 1) markedly prolonged the SLCF‐mediated apnea; 2) significantly increased the expression TRPV1 receptors in N/J ganglionic C‐neurons; and 3) augmented the amplitudes of TRPV1 currents on these neurons. The results suggest that PNE is able to prolong the SLCF‐mediated apnea likely though upregulating TRPV1 receptor expression, which may contribute to SIDS.Support or Funding InformationSupported by HL‐107462 and HL‐119683

Capsaicin‐sensitive TRPV1 Currents Are Enhanced in Pulmonary Artery Smooth Muscle Cells from Patients with Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a fatal and progressive disease. Sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling are two major causes for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with PAH. Ca2+ signaling in pulmonary artery smooth muscle cells (PASMC) plays a central role in the development and progression of PAH as it is involved with both vasoconstriction, by triggering PASMC contraction, and vascular remodeling, via stimulation of PASMC proliferation. Enhanced Ca2+ signaling has been observed in PASMCs from patients with PAH and is due in part to increased Ca2+ influx through upregulated plasmalemmal Ca2+‐permeable channels. Several members of the transient receptor potential (TRP) channel family are known to facilitate Ca2+ entry. TRP channels are relatively nonselective cation channels consisting of six subfamilies, including the vanilloid (TRPV) ion channels. TRPV1 channels respond to physical stimulus such as noxious temperatures as well as ligand stimuli like capsaicin. TRPV1 is expressed in PASMCs and has been implicated in vascular remodeling and possibly vasoconstriction of the pulmonary artery. However, TRPV1 currents have not been fully characterized in PASMCs. To study this, we used the patch clamp technique to measure capsaicin‐induced single‐channel currents in PASMCs in cell‐attached mode and to investigate whether channel activity is altered in PASMC from idiopathic PAH (IPAH) patients. Under conditions where membrane potential was set to ~0 mV (using high K+ bath solution), channel openings were detected with reversal potential of −3.49 mV and −3.57 mV for normal PASMCs and IPAH‐PASMCs, respectively, in the presence of capsaicin (1 μM). The TRPV1 antagonist capsazepine (10 μM) decreased the steady‐state open probability (Popen) of the single‐channel currents without altering the amplitude of the currents. The slope conductance for normal and IPAH PASMCs was 131 pS and 135 pS, respectively. To rule out any potential influence from other channels, we used an inhibitor cocktail consisting of paxilline and apamin (Ca2+‐activated K+ channel blockers), tetraethylammonium (voltage‐gated K+ channel blocker), and niflumic acid (Cl− channel inhibitor) in the pipette solution. Under these conditions, the open probability of capsaicin‐activated single‐channel currents was markedly enhanced, compared to the vehicle groups for IPAH‐PASMCs. In addition, Western blot studies indicated that the protein expression level of TRPV1 in IPAH‐PASMCs was greater than in normal PASMCs.. Taken together, these data suggest that upregulated expression of TRPV1 channels is associated with the increased Popen of capsaicin‐activated single channel currents in IPAH‐PASMC compare to normal PASMC. This could explain, in part, the enhancement of Ca2+ influx observed in PASMCs from IPAH patients.Support or Funding InformationResearch supported by NIH Grants (HL115014, HL066012 and HL098053).