Turning down the body heat: A novel mechanism for TRPV1 antagonist-induced hyperthermia.

While effective analgesics, TRPV1 antagonists can dangerously alter thermoregulation. In this issue of Neuron, Huang et al.1 demonstrate that interaction with the S4-S5 linker of TRPV1 determines whether an antagonist affects core body temperature, with promising implications for analgesic development.

Protective role of TRPV2 in synaptic plasticity through the ERK1/2-CREB-BDNF pathway in chronic unpredictable mild stress rats.

PURPOSE: Chronic stress is a significant risk factor for mood disorders such as depression, where synaptic plasticity plays a central role in pathogenesis. Transient Receptor Potential Vanilloid Type-2 (TRPV2) Ion Channels are implicated in hypothalamic-pituitary-adrenal axis disorders. Previous proteomic analysis indicated a reduction in TRPV2 levels in the chronic unpredictable mild stress (CUMS) rat model, yet its role in synaptic plasticity during depression remains to be elucidated. This study aims to investigate TRPV2's role in depression and its underlying mechanisms. METHODS: In vivo and in vitro experiments were conducted using the TRPV2-specific agonist probenecid and ERK1/2 inhibitors SCH772984. In vivo, rats underwent six weeks of CUMS before probenecid administration. Depressive-like behaviors were assessed through behavioral tests. ELISA kits measured 5-HT, DA, NE levels in rat hippocampal tissues. Hippocampal morphology was examined via Nissl staining. In vitro, rat hippocampal neuron cell lines were treated with ERK1/2 inhibitors SCH772984 and probenecid. Western blot, immunofluorescence, immunohistochemical staining, and RT-qPCR assessed TRPV2 expression, neurogenesis-related proteins, synaptic markers, and ERK1/2-CREB-BDNF signaling proteins. RESULTS: Decreased hippocampal TRPV2 levels were observed in CUMS rats. Probenecid treatment mitigated depressive-like behavior and enhanced hippocampal 5-HT, NE, and DA levels in CUMS rats. TRPV2 activation countered CUMS-induced synaptic plasticity inhibition. Probenecid activated the ERK1/2-CREB-BDNF pathway, suggesting TRPV2's involvement in this pathway via ERK1/2. CONCLUSION: These findings indicate that TRPV2 activation offers protective effects against depressive-like behaviors and enhances hippocampal synaptic plasticity in CUMS rats via the ERK1/2-CREB-BDNF pathway. TRPV2 emerges as a potential therapeutic target for depression.

Discovery of first-in-class highly selective TRPV1 antagonists with dual analgesic and hypoglycemic effects.

Analgesia and blood sugar control are considered as two main unmet clinical needs for diabetes related neuropathic pain patients. Transient receptor potential vanilloid type-1 (TRPV1) channel is a highly validated target for pain perception, while no TRPV1 antagonists have been approved due to hyperthermia side effects. Herein, two series of new TRPV1 antagonists with flavonoid skeleton were designed by the structure-based drug design (SBDD) strategy. After comprehensive evaluation, compound CX-3 was identified as a promising TRPV1 antagonist. CX-3 exhibited equivalent TRPV1 antagonistic activity with classical TRPV1 antagonist BCTC in vitro, and exerted better analgesic activity in vivo than that of BCTC in the formalin induced inflammatory pain model without hyperthermia risk. Moreover, CX-3 exhibited robust glucose-lowering effects and showed high selectivity over other ion channels. Overall, these findings identified a first-in-class highly selective TRPV1 antagonist CX-3, which is a promising candidate to target the pathogenesis of diabetes related neuropathic pain.

Exploration and structure-activity relationship research of benzenesulfonamide derivatives as potent TRPV4 inhibitors for treating acute lung injury.

RN-9893, a TRPV4 antagonist identified by Renovis Inc., showcased notable inhibition of TRPV4 channels. This research involved synthesizing and evaluating three series of RN-9893 analogues for their TRPV4 inhibitory efficacy. Notably, compounds 1b and 1f displayed a 2.9 to 4.5-fold increase in inhibitory potency against TRPV4 (IC50 = 0.71 ± 0.21 µM and 0.46 ± 0.08 µM, respectively) in vitro, in comparison to RN-9893 (IC50 = 2.07 ± 0.90 µM). Both compounds also significantly outperformed RN-9893 in TRPV4 current inhibition rates (87.6 % and 83.2 % at 10 µM, against RN-9893's 49.4 %). For the first time, these RN-9893 analogues were profiled in an in vivo mouse model, where intraperitoneal injections of 1b or 1f at 10 mg/kg notably mitigated symptoms of acute lung injury induced by lipopolysaccharide (LPS). These outcomes indicate that compounds 1b and 1f are promising candidates for acute lung injury treatment.

Discovery of N-(1,4-Benzoxazin-3-one) urea analogs as Mode-Selective TRPV1 antagonists.

A series of 1,4-benzoxazin-3-one analogs were investigated to discover mode-selective TRPV1 antagonists, since such antagonists are predicted to minimize target-based adverse effects. Using the high-affinity antagonist 2 as the lead structure, the structure activity relationship was studied by modifying the A-region through incorporation of a polar side chain on the benzoxazine and then by changing the C-region with a variety of substituted pyridine, pyrazole and thiazole moieties. The t-butyl pyrazole and thiazole C-region analogs provided high potency as well as mode-selectivity. Among them, antagonist 36 displayed potent and capsaicin-selective antagonism with IC50 = 2.31 nM for blocking capsaicin activation and only 47.5 % inhibition at 3 µM concentration toward proton activation, indicating that more than a 1000-fold higher concentration of 36 was required to inhibit proton activation than was required to inhibit capsaicin activation. The molecular modeling study of 36 with our homology model indicated that two π-π interactions with the Tyr511 and Phe591 residues by the A- and C-region and hydrogen bonding with the Thr550 residue by the B-region were critical for maintaining balanced and stable binding. Systemic optimization of antagonist 2, which has high-affinity but full antagonism for activators of all modes, led to the mode-selective antagonist 36 which represents a promising step in the development of clinical TRPV1 antagonists minimizing side effects such as hyperthermia and impaired heat sensation.

TRPV4 antagonist suppresses retinal ganglion cell apoptosis by regulating the activation of CaMKII and TNF-α expression in a chronic ocular hypertension rat model.

Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs), leading to irreversible visual function impairment. Sustained increase in intraocular pressure represents a major risk factor for glaucoma, yet the underlying mechanisms of RGC apoptosis induced by intraocular pressure remains unclear. This study aims to investigate the role of TRPV4 in RGC apoptosis in a rat model of chronic ocular hypertension (COH) and the underlying molecular mechanism. In the COH rat models, we evaluated the visual function, retinal pathological changes and RGC apoptosis. TRPV4 expression and downstream signaling molecules were also detected. We found that RGC density decreased and RGC apoptosis was induced in COH eyes compared with control eyes. TRPV4 expression increased significantly in response to elevated IOP. TRPV4 inhibition by the TRPV4 antagonist HC-067047 (HC-067) suppressed RGC apoptosis and protected visual function. HC-067 treatment upregulated the phosphorylation of CaMKII in both control and COH eyes. Finally, HC-067 treatment suppressed the production of TNF-α induced by ocular hypertension. The TRPV4 antagonist HC-067 might suppress RGC apoptosis by regulating the activation of CaMKII and inhibiting the production of TNF-α in the COH model. This indicated that TRPV4 antagonists may be a potential and novel therapeutic strategy for glaucoma.

Design, synthesis and functional validation of peptide inhibitors based on TRPV1 ion channel agonist RhTx. / é¶å‘çž¬æ—¶å—ä½“ç”µä½é¦™è‰é ¸äºšåž‹1离子通道的Nplus-RhTx多肽抑制剂理性设计及功能验证.

OBJECTIVES: To design and synthesize peptide inhibitors targeting transient receptor potential vanilloid 1 (TRPV1) ion channel, and to validate their function. METHODS: Based on previous studies on the relation of molecular structure and function of red head toxin (RhTx), a series of peptides were rationally designed and synthesized, with positive charged amino acids linked to the N terminus of RhTx. These Nplus-RhTx peptides were functionally validated by patch-clamp recordings in live cells. RESULTS: Among the 8 synthesized Nplus-RhTx peptides, four inhibited TRPV1 ion channel activated by capsaicin with IC50 of (188.3±4.7), (193.6±18.0), (282.8±11.9) and (299.5±6.4) µmol/L, respectively. CONCLUSIONS: It is feasible to develop TRPV1 peptide inhibitors by using rational design based on N terminal residues of RhTx.

Selective inhibition of overactive warmth-sensitive Ca2+-permeable TRPV3 channels by antispasmodic agent flopropione for alleviation of skin inflammation.

The temperature-sensitive Ca2+-permeable TRPV3 ion channel is robustly expressed in the skin keratinocytes, and its gain-of-function mutations are involved in the pathology of skin lesions. Here, we report the identification of an antispasmodic agent flopropione that alleviates skin inflammation by selective inhibition of TRPV3. In whole-cell patch clamp recordings, flopropione selectively inhibits macroscopic TRPV3 currents in a concentration-dependent manner with an IC50 value of 17.8 ± 3.5 µM. At the single-channel level, flopropione inhibits TRPV3 channel open probability without alteration of its unitary conductance. In an in vivo mouse model of skin inflammation induced by the skin sensitizer DNFB, flopropione also alleviates dorsal skin lesions and ear skin swelling. Further molecular docking combined with site-directed mutagenesis reveals that two residues E501 and I505 in the channel S2-helix are critical for flopropione-mediated inhibition of TRPV3. Taken together, our findings demonstrate that the spasmolytic drug flopropione as a selective inhibitor of TRPV3 channel not only provides a valuable tool molecule for understanding of TRPV3 channel pharmacology but also holds repurposing potential for therapy of skin disorders, such as dermatitis and pruritus.

Structural mechanism of TRPV5 inhibition by econazole.

The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in calcium homeostasis. Recently, cryoelectron microscopy (cryo-EM) provided molecular details of TRPV5 modulation by exogenous and endogenous molecules. However, the details of TRPV5 inhibition by the antifungal agent econazole (ECN) remain elusive due to the low resolution of the currently available structure. In this study, we employ cryo-EM to comprehensively examine how the ECN inhibits TRPV5. By combining our structural findings with site-directed mutagenesis, calcium measurements, electrophysiology, and molecular dynamics simulations, we determined that residues F472 and L475 on the S4 helix, along with residue W495 on the S5 helix, collectively constitute the ECN-binding site. Additionally, the structure of TRPV5 in the presence of ECN and PI(4,5)P2, which does not show the bound activator, reveals a potential inhibition mechanism in which ECN competes with PI(4,5)P2, preventing the latter from binding, and ultimately pore closure.

Higenamine inhibits acute and chronic inflammatory pain through modulation of TRPV4 channels.

Pain is the cardinal symptom of many debilitating diseases and results in heavy health and economic burdens worldwide. Asarum (Asarum sieboldii Miq.) is a commonly used analgesic in Chinese medicine. However, the analgesic components and mechanisms of asarum in acute and chronic pain mice model remain unknown. In this study, we first generated asarum water extract and confirmed strong analgesic properties in mice in both the acute thermal and mechanical pain models, as well as in the complete Freund's adjuvant (CFA) induced chronic inflammatory pain model. Second, we identified higenamine as a major component of asarum and found that higenamine significantly inhibited thermal and mechanical induced acute pain and CFA induced chronic inflammatory pain. Then, using Trpv4-/- mice, we found that TRPV4 is necessary for CFA induced thermal and mechanical allodynia, and demonstrated that higenamine analgesia in the CFA model is partly through TRPV4 channel inhibition. Finally, we found that GSK1016790A, a TRPV4 agonist, induced calcium response was significantly inhibited by higenamine in both cultured DRG neurons and TRPV4 transfected HEK293 cells. Consistent with calcium imaging results, higenamine pretreatment also dose-dependently inhibited GSK1016790A induced acute pain. Taken together, our behavior and calcium imaging results demonstrate that the asarum component higenamine inhibits acute and chronic inflammatory pain by modulation of TRPV4 channels.

Simulated microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts can be prevented by protection of primary cilia.

Oxidative stress has been considered to be closely related to spaceflight-induced bone loss; however, mechanism is elusive and there are no effective countermeasures. Using cultured rat calvarial osteoblasts exposed to microgravity simulated by a random positioning machine, this study addressed the hypotheses that microgravity-induced shortening of primary cilia leads to oxidative stress and that primary cilium protection prevents oxidative stress and osteogenesis loss. Microgravity was found to induce oxidative stress (as represented by increased levels of reactive oxygen species (ROS) and malondialdehyde production, and decreased activities of antioxidant enzymes), which was perfectly replicated in osteoblasts growing in NG with abrogated primary cilia (created by transfection of an interfering RNA), suggesting the possibility that shortening of primary cilia leads to oxidative stress. Oxidative stress was accompanied by mitochondrial dysfunction (represented by increased mitochondrial ROS and decreased mitochondrial membrane potential) and intracellular Ca2+ overload, and the latter was found to be caused by increased activity of Ca2+ channel transient receptor potential vanilloid 4 (TRPV4), as also evidenced by TRPV4 agonist GSK1016790A-elicited Ca2+ influx. Supplementation of HC-067047, a specific antagonist of TRPV4, attenuated microgravity-induced mitochondrial dysfunction, oxidative stress, and osteogenesis loss. Although TRPV4 was found localized in primary cilia and expressed at low levels in NG, microgravity-induced shortening of primary cilia led to increased TRPV4 levels and Ca2+ influx. When primary cilia were protected by miR-129-3p overexpression or supplementation with a natural flavonoid moslosooflavone, microgravity-induced increased TRPV4 expression, mitochondrial dysfunction, oxidative stress, and osteogenesis loss were all prevented. Our data revealed a new mechanism that primary cilia function as a controller for TRPV4 expression. Microgravity-induced injury on primary cilia leads to increased expression and overactive channel of TRPV4, causing intracellular Ca2+ overload and oxidative stress, and primary cilium protection could be an effective countermeasure against microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts.

Ocular Pharmacology and Toxicology of TRPV1 Antagonist SAF312 (Libvatrep).

Purpose: To evaluate the pharmacology and toxicology of SAF312, a transient receptor potential vanilloid 1 (TRPV1) antagonist. Methods: TRPV1 expression in human ocular tissues was evaluated with immunohistochemistry. Inhibition of calcium influx in Chinese hamster ovary (CHO) cells expressing human TRPV1 (hTRPV1) and selectivity of SAF312 were assessed by a fluorescent imaging plate reader assay. Ocular tissue and plasma pharmacokinetics (PK) were assessed following a single topical ocular dose of SAF312 (0.5%, 1.0%, 1.5%, 2.5%) in rabbits. Safety and tolerability of SAF312 were evaluated in rabbits and dogs. Effects of SAF312 on corneal wound healing after photorefractive keratectomy (PRK) surgery were assessed in rabbits. Results: TRPV1 expression was noted in human cornea and conjunctiva. SAF312 inhibited calcium influx in CHO-hTRPV1 cells induced by pH 5.5 (2-[N-morpholino] ethanesulfonic acid), N-arachidonoylethanolamine, capsaicin, and N-arachidonoyl dopamine, with IC50 values of 5, 10, 12, and 27 nM, respectively, and inhibition appeared noncompetitive. SAF312 demonstrated high selectivity for TRPV1 (>149-fold) over other TRP channels. PK analysis showed highest concentrations of SAF312 in cornea and conjunctiva. SAF312 was found to be safe and well tolerated in rabbits and dogs up to the highest feasible concentration of 2.5%. No delay in wound healing after PRK was observed. Conclusions: SAF312 is a potent, selective, and noncompetitive antagonist of hTRPV1 with an acceptable preclinical safety profile for use in future clinical trials. Translational Relevance: SAF312, which was safe and well tolerated without causing delay in wound healing after PRK in rabbits, may be a potential therapeutic agent for ocular surface pain.

TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway.

Cisplatin resistance along with chemotherapy-induced neuropathic pain is an important cause of treatment failure for many cancer types and represents an unmet clinical need. Therefore, future studies should provide evidence regarding the mechanisms of potential targets that can overcome the resistance as well as alleviate pain. Here, we show that the emergence of cisplatin resistance is highly associated with EGFR hyperactivation, and that EGFR hyperactivation is arisen by a transcriptional increase in the pain-generating channel, TRPV1, via NANOG. Furthermore, TRPV1 promotes autophagy-mediated EGF secretion via Ca2+ influx, which activates the EGFR-AKT signaling and, consequentially, the acquisition of cisplatin resistance. Importantly, TRPV1 inhibition renders tumors susceptible to cisplatin. Thus, our findings indicate a link among cisplatin resistance, EGFR hyperactivation, and TRPV1-mediated autophagic secretion, and implicate that TRPV1 could be a crucial drug target that could not only overcome cisplatin resistance but also alleviate pain in NANOG+ cisplatin-resistant cancer.

Functional validation of co-culture model of human keratinocytes and neuronal cell line for sensitive skin by using transient receptor potential channel vanilloid subfamily member 1 antagonist.

BACKGROUND: Sensitive skin is a subjective cutaneous hyper-reactivity that occurs in response to various innocuous stimuli. Keratinocytes have recently been shown to participate in sensory transduction by releasing many neuroactive molecules that bind to intra-epidermal free nerve endings and modulate nociception. In the literature, the characterization of these interactions has been based on the co-culture of keratinocyte and mammalian-origin neuronal cell lines. In this study, we established an in vitro model based on a co-culture of primary human keratinocytes and differentiated SH-SY5Y cells, a human neuronal cell line. METHODS: Human epidermal keratinocytes and SH-SY5Y cells were monocultured and co-cultured. Changes in calcium influx, substance P, inflammatory cytokines, and neuropeptides between the monoculture and co-culture groups treated with capsaicin only and capsaicin with transient receptor potential channel vanilloid subfamily member 1 (TRPV1) antagonist, trans-4-tert-butylcyclohexanol (TTBC), together. In addition, the difference in stinging sensation was evaluated by applying it to the volunteers. RESULTS: When SH-SY5Y cells were co-cultured with keratinocytes, they had no significant effect on axonal development. Substance P was also released after capsaicin treatment and reduced by TTBC under co-culture conditions. Moreover, the expression of inflammatory cytokines and neuropeptides was significantly increased in co-cultured keratinocytes compared to that under monoculture conditions. In addition, the stinging sensation was significantly induced after the application of capsaicin in vivo and was relieved after the application of the TRPV1 antagonist. CONCLUSION: We demonstrated that the novel co-culture model is functionally valid through capsaicin and TRPV1 antagonist. We also confirmed that TTBC could be used for the treatment of sensitive skin through a co-culture model and in vivo tests. This co-culture model of keratinocytes and SH-SY5Y cells may be useful in vitro alternatives for studying the close communication between keratinocytes and neuronal cells and for screening therapeutic drugs for sensitive skin.

Diosgenin Exerts Analgesic Effects by Antagonizing the Selective Inhibition of Transient Receptor Potential Vanilloid 1 in a Mouse Model of Neuropathic Pain.

Diosgenin is a botanical steroidal saponin with immunomodulatory, anti-inflammatory, anti-oxidative, anti-thrombotic, anti-apoptotic, anti-depressant, and anti-nociceptive effects. However, the effects of diosgenin on anti-nociception are unclear. Transient receptor potential vanilloid 1 (TRPV1) plays an important role in nociception. Therefore, we investigated whether TRPV1 antagonism mediates the anti-nociceptive effects of diosgenin. In vivo mouse experiments were performed to examine nociception-related behavior, while in vitro experiments were performed to examine calcium currents in dorsal root ganglion (DRG) and Chinese hamster ovary (CHO) cells. The duration of capsaicin-induced licking (pain behavior) was significantly reduced following oral and intraplantar administration of diosgenin, approaching levels observed in mice treated with the TRPV1 antagonist N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl) tetrahydropyrazine-1(2H)-carbox-amide. Additionally, oral administration of diosgenin blocked capsaicin-induced thermal hyperalgesia. Further, diosgenin reduced capsaicin-induced Ca2+ currents in a dose-dependent manner in both DRG and CHO cells. Oral administration of diosgenin also improved thermal and mechanical hyperalgesia in the sciatic nerve constriction injury-induced chronic pain model by reducing the expression of TRPV1 and inflammatory cytokines in DRG cells. Collectively, our results suggest that diosgenin exerts analgesic effects via antagonism of TRPV1 and suppression of inflammation in the DRG in a mouse model of neuropathic pain.

Loratadine, an antihistaminic drug, suppresses the proliferation of endometrial stromal cells by inhibition of TRPV2.

The transient receptor potential (TRP) channel TRPV2 is widely expressed in a variety of different cell types and tissues. However, elucidating the exact biological functions of TRPV2 is significantly hampered by the lack of selective pharmacological tools to modulate channel activity in vitro and in vivo. This study aimed to identify new compounds that modify TRPV2 activity via the use of a plate-based calcium imaging approach to screen a drug repurposing library. Three antihistaminic drugs, loratadine, astemizole and clemizole were identified to reduce calcium-influx evoked by the TRPV2 agonist tetrahydrocannabivarin in HEK293 cells expressing murine TRPV2. Using single-cell calcium-microfluorimetry and whole-cell patch clamp recordings, we further confirmed that all three compounds induced a concentration-dependent block of TRPV2-mediated Ca2+ influx and whole-cell currents, with loratadine being the most potent antagonist of TRPV2. Moreover, this study demonstrated that loratadine was able to block both the human and mouse TRPV2 orthologs, without inhibiting the activity of other closely related members of the TRPV superfamily. Finally, loratadine inhibited TRPV2-dependent responses in a primary culture of mouse endometrial stromal cells and attenuated cell proliferation and migration in in vitro cell proliferation and wound healing assays. Taken together, our study revealed that the antihistaminic drugs loratadine, astemizole and clemizole target TRPV2 in a concentration-dependent manner. The identification of these antihistaminic drugs as blockers of TRPV2 may form a new starting point for the synthesis of more potent and selective TRPV2 antagonists, which could further lead to the unravelling of the physiological role of the channel.

TRPV6 Regulation by Cis-22a and Cholesterol.

The highly calcium-selective transient receptor potential vanilloid-type channel TRPV6 is important for epithelial Ca2+ transport. Proper regulation of the inherently constitutively active TRPV6 channels is intricate in preserving Ca2+ homeostasis, whereby structural and functional data suggest that lipids hold an essential role. Altered expression levels or specific TRPV6 mutations may lead to diseases, hence, TRPV6 represents an interesting target for pharmacological modulation. Recent cryo-EM data identified that the specific TRPV6 blocker cis-22a binds, apart from the pore, to a site within the tetrameric channel that largely matches a lipid binding pocket, LBS-2. Therein, cis-22a may replace a lipid such as cholesterol that is bound in the open state. Based on site-directed mutagenesis and functional recordings, we identified and characterized a series of residues within LBS-2 that are essential for TRPV6 inhibition by cis-22a. Additionally, we investigated the modulatory potential of diverse cholesterol depletion efforts on TRPV6 activity. While LBS-2 mutants exhibited altered maximum currents, slow Ca2+-dependent inactivation (SCDI) as well as less inhibition by cis-22a, TRPV6 activity was resistant to cholesterol depletion. Hence, lipids other than cholesterol may predominate TRPV6 regulation when the channel is expressed in HEK293 cells.

Capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption by blocking TRPV4 channels in healthy and colitic mice.

Although capsaicin has been studied extensively as an activator of the transient receptor potential vanilloid cation channel subtype 1 (TRPV1) channels in sensory neurons, little is known about its TRPV1-independent actions in gastrointestinal health and disease. Here, we aimed to investigate the pharmacological actions of capsaicin as a food additive and medication on intestinal ion transporters in mouse models of ulcerative colitis (UC). The short-circuit current (Isc) of the intestine from WT, TRPV1-, and TRPV4-KO mice were measured in Ussing chambers, and Ca2+ imaging was performed on small intestinal epithelial cells. We also performed Western blots, immunohistochemistry, and immunofluorescence on intestinal epithelial cells and on intestinal tissues following UC induction with dextran sodium sulfate. We found that capsaicin did not affect basal intestinal Isc but significantly inhibited carbachol- and caffeine-induced intestinal Isc in WT mice. Capsaicin similarly inhibited the intestinal Isc in TRPV1 KO mice, but this inhibition was absent in TRPV4 KO mice. We also determined that Ca2+ influx via TRPV4 was required for cholinergic signaling-mediated intestinal anion secretion, which was inhibited by capsaicin. Moreover, the glucose-induced jejunal Iscvia Na+/glucose cotransporter was suppressed by TRPV4 activation, which could be relieved by capsaicin. Capsaicin also stimulated ouabain- and amiloride-sensitive colonic Isc. Finally, we found that dietary capsaicin ameliorated the UC phenotype, suppressed hyperaction of TRPV4 channels, and rescued the reduced ouabain- and amiloride-sensitive Isc. We therefore conclude that capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption predominantly by blocking TRPV4 channels to exert its beneficial anti-colitic action.

A novel antagonist of TRPM2 and TRPV4 channels: Carvacrol.

The overload cytosolic free Ca2+ (cCa2+) influx-mediated excessive generation of oxidative stress in the pathophysiological conditions induces neuronal and cellular injury via the activation of cation channels. TRPM2 and TRPV4 channels are activated by oxidative stress, and their specific antagonists have not been discovered yet. The antioxidant and anti-Covid-19 properties of carvacrol (CARV) were recently reported. Hence, I suspected possible antagonist properties of CARV against oxidative stress (OS)/ADP-ribose (ADPR)-induced TRPM2 and GSK1016790A (GSK)-mediated TRPV4 activations in neuronal and kidney cells. I investigated the antagonist role of CARV on the activations of TRPM2 and TRPV4 in SH-SY5Y neuronal, BV-2 microglial, and HEK293 cells. The OS/ADPR and GSK in the cells caused to increase of TRPM2/TRPV4 current densities and overload cytosolic free Ca2+ (cCa2+) influx with an increase of mitochondrial membrane potential, cytosolic (cROS), and mitochondrial (mROS) ROS. The changes were not observed in the absence of TRPM2 and TRPV4 or the presence of Ca2+ free extracellular buffer and PARP-1 inhibitors (PJ34 and DPQ). When OS-induced TRPM2 and GSK-induced TRPV4 activations were inhibited by the treatment of CARV, the increase of cROS, mROS, lipid peroxidation, apoptosis, cell death, cCa2+ concentration, caspase -3, and caspase -9 levels were restored via upregulation of glutathione and glutathione peroxidase. In conclusion, the treatment of CARV modulated the TRPM2 and TRPV4-mediated overload Ca2+ influx and may provide an avenue for protecting TRPM2 and TRPV4-mediated neurodegenerative diseases associated with the increase of mROS and cCa2+. The possible TRPM2 and TRPV4 blocker action of carvacrol (CARV) via the modulation oxidative stress and apoptosis in the SH-SY5Y neuronal cells. TRPM2 is activated by DNA damage-induced (via PARP-1 activation) ADP-ribose (ADPR) and reactive oxygen species (ROS) (H2O2), although it is inhibited by nonspecific inhibitors (ACA and 2-APB). TRPV4 is activated by the treatments of GSK1016790A (GSK), although it is inhibited by a nonspecific inhibitor (ruthenium red, RuRe). The treatment of GSK induces excessive generation of ROS. The accumulation of free cytosolic Ca2+ (cCa2+) via the activations of TRPM2 and TRPV4 in the mitochondria causes the increase of mitochondrial membrane depolarization (ΔΨm). In turn, the increase of ΔΨm causes the excessive generation of ROS. The TRPM2 and TRPV4-induced the excessive generations of ROS result in the increase of apoptosis and cell death via the activations of caspase -3 (Casp-3) and caspase -9 (Casp-9) in the neuronal cells, although their oxidant actions decrease the glutathione (GSH) and glutathione peroxidase (GSHPx) levels. The oxidant and apoptotic adverse actions of TRPM2 and TRPV4 are modulated by the treatment of CARV.

Inhibition of transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 by mosquito and mouse saliva.

ABSTRACT: Arthropods are the largest group of living organisms, and among them, mosquitoes spread parasites and viruses causing deadly diseases. They can easily spread these pathogens because of their painless skin piercing. Although the lack of pain is mainly due to the thinness of their fascicle, it is possible that mosquito saliva, which is discharged during their piercing, might also contribute to it. If mosquito saliva contains antinociceptive substances, it should act on the sensory neurons innervating the epidermis where there are several ion channels that can detect noxious stimuli, such as the transient receptor potential (TRP) channels. We found that mosquito head homogenates and mouse saliva inhibit TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) channels, either heterologously expressed in HEK293T cells or endogenously expressed in native mouse sensory neurons. Among the different substances contained in mosquito head homogenates or mouse saliva, we have also identified sialorphin as a candidate antinociceptive peptide because it showed similar inhibition effects on TRPV1 and TRPA1. Finally, we confirmed the antinociceptive effects of mosquito head homogenates, mouse saliva, and sialorphin in vivo by observing decreased pain-related behaviors in mice coinjected with these substances. Similar inhibitory effects of mosquito head homogenates and mouse saliva on TRPV1 and TRPA1 suggest that the antinociceptive effects of saliva are universal, which could explain why many animals including humans often lick their wounds. These findings would lead to the development of novel and safe antinociceptive agents.