Inhibition Of Transient Receptor Potential Vanilloid 4 Research Articles

Protective effects of adipose-derived biogenic nanoparticles on the pulmonary microvascular endothelial barrier in mice with ventilator-induced lung injury via the TRPV4/ROCK1 signalling pathway

PurposeThe "obesity paradox" phenomenon occurs in critically ill patients who receive mechanical ventilation. Our previous studies found that the adipose-derived exosomes secreted by obese mice have a protective effect on the pulmonary microvascular endothelial barrier. However, the extraction of exosomes is cumbersome, their yield is low, and their storage is difficult. After further research, we discovered a new type of adipose-derived bioactive material called: lipoaspirate nanoparticles (Lipo-NPs). MethodsLipo-NPs were extracted and identified using a tangential flow filtration system. The Lipo-NPs were used as an intervention in ventilator-induced lung injury (VILI) models in vivo and in vitro to investigate whether they have a protective effect on lung tissue damage (haematoxylin and eosin staining), lung barrier function (lung wet/dry [W/D] weight ratio, protein concentration in bronchoalveolar lavage fluid (BALF), and Vascular endothelial (VE)-expression), as well as their related mechanisms. ResultsIn both in vivo and in vitro studies, Lipo-NPs can attenuate lung injury, reduce lung W/D ratio and protein concentration in BALF, and augment the expression of the adhesion link-protein VE-cadherin, thus playing a protective role in lung barrier function. This protective effect involves the activation of the transient receptor potential vanilloid 4 (TRPV4)/Rho-associated kinase1 (ROCK1) signalling pathway. We further verified the role of this signalling pathway via activation and inhibition of TRPV4 and ROCK1. Moreover, phosphorylation of myosin light chain 2 (MLC2) regulates F-actin and is a target of the ROCK pathway. ConclusionLipo-NPs can enhance the expression of VE-cadherin by inhibiting the TRPV4/ROCK1/pMLC2 signalling pathway in the mechanical ventilation model, thereby exerting a protective effect on the VILI pulmonary microvascular endothelial barrier.

TRPV1 Suppressed NLRP3 Through Regulating Autophagy in Microglia After Ischemia-Reperfusion Injury.

The microglia-mediated inflammatory response is one of the main causes of brain tissue damage after stroke. In recent years, it has been reported that autophagy in microglia played an important role in inflammatory response after stroke. Transient receptor potential vanilloid 1 (TRPV1) has been shown to regulate autophagy and inflammatory in microglia; however, the detailed mechanisms remain unclear. This study aimed to investigate whether autophagy regulates inflammatory is associated with TRPV1. Model of oxygen and glucose deprivation/reoxygenation (OGD/R) was established in vitro to induce cerebral ischemia-reperfusion injury (I/R). siRNA of Atg5, inhibitors, and agonists of both autophagy and TRPV1 were involved in our study. Autophagy was assayed by immunofluorescence staining LC-3 and autophagosome was observed using transmission electron microscopy (TEM). Autophagy/inflammation-related markers as Atg5, LC-3II/LC-3I, Beclin-1, NLRP3, IL-1β, and Caspase-1 were also measured in the present study. Results indicated that I/R injury-induced inflammatory injury may be impeded by inhibition of autophagy, and TRPV1 could suppress OGD/R-induced autophagy of microglia. However, the effect of TRPV1's inhibitor on inflammatory response was attenuated when the autophagy was blocked. These findings suggested that TRPV1 exhibits an anti-inflammatory effect on OGD/R-induced microglia, which was at least correlated with the anti-autophagy action of TRPV1 partially.

Transient receptor potential vanilloid 4 promotes the growth of non-small cell lung cancer by regulating Foxp3.

Transient receptor potential vanilloid 4 (TRPV4) participates in malignant tumor. However, the role of TRPV4 in non-small cell lung cancer (NSCLC) remains unclear. In this study, we demonstrated TRPV4 was upregulated in NSCLC tissues and NSCLC cell lines. TRPV4 level in the NSCLC patients and cell lines were detected, and its function was studied both in vivo and vitro. The level of TRPV4 showed a positive correlation with tumor size of NSCLC patients. Activation TRPV4 by agonist GSK1016790A promoted cell proliferation and decreased apoptosis in A549 cells, and these effects were enhanced when the cells have overexpressed TRPV4. Moreover, GSK1016790A induced inhibitory effects on apoptosis of A549 cells was impaired when GSK1016790A used together with TRPV4 selective antagonist HC-067047, or impaired when the cells have already downregulated TRPV4 expression by TRPV4 siRNA. In vivo study, pharmacological inhibition of TRPV4 prevented A549 cells transplanted tumor growth. It was showed Foxp3 level was significantly increased in the NSCLC tissues, and showed a positive correlation with the level of TRPV4. Deactivation of TRPV4 using TRPV4 siRNA or HC-067047 significantly reduced expression of Foxp3 in GSK1016790A treated NSCLC cells. Moreover, downregulation Foxp3 by transfection of Foxp3 siRNA significantly impaired TRPV4 induced NSCLC cells proliferations in vitro. Antitumor effects caused by TRPV4 inhibition in NSCLC might be attributed to the suppression of Foxp3 which induced subsequent cell apoptosis. Thus, pharmacological inhibition of TRPV4 may be a promising option for NSCLC treatment.

Pharmacological Inhibition of Transient Receptor Potential Vanilloid 4 Reduces Vasogenic Edema after Traumatic Brain Injury in Mice.

Vasogenic edema results from blood-brain barrier (BBB) disruption after traumatic brain injury (TBI), and although it can be fatal, no promising therapeutic drugs have been developed as yet. Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable channel that is sensitive to temperature and osmotic pressure. As TRPV4 is known to be responsible for various pathological conditions following brain injury, we investigated the effects of pharmacological TRPV4 antagonists on TBI-induced vasogenic edema in this study. A TBI model was established by inflicting fluid percussion injury (FPI) in the mouse cerebrum and cultured astrocytes. Vasogenic brain edema and BBB disruption were assessed based on brain water content and Evans blue (EB) extravasation into brain tissue, respectively. After FPI, brain water content and EB extravasation increased. Repeated intracerebroventricular administration of the specific TRPV4 antagonists HC-067047 and RN-1734 dose-dependently reduced brain water content and alleviated EB extravasation in FPI mice. Additionally, real-time PCR analysis indicated that administration of HC-067047 and RN-1734 reversed the FPI-induced increase in mRNA levels of endogenous causal factors for BBB disruption, including matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor-A (VEGF-A), and endothelin-1 (ET-1). In astrocytes, TRPV4 level was observed to be higher than that in brain microvascular endothelial cells. Treatment with HC-067047 and RN-1734 inhibited the increase in mRNA levels of MMP-9, VEGF-A, and ET-1 in cultured astrocytes subjected to in vitro FPI. These results suggest that pharmacological inhibition of TRPV4 is expected to be a promising therapeutic strategy for treating TBI-induced vasogenic edema.

Inhibition of Spinal TRPV1 Reduces NMDA Receptor 2B Phosphorylation and Produces Anti-Nociceptive Effects in Mice with Inflammatory Pain.

Transient receptor potential vanilloid 1 (TRPV1) has been implicated in peripheral inflammation and is a mediator of the inflammatory response to various noxious stimuli. However, the interaction between TRPV1 and N-methyl-D-aspartate (NMDA) receptors in the regulation of inflammatory pain remains poorly understood. This study aimed to investigate the analgesic effects of intrathecal administration of capsazepine, a TRPV1 antagonist, on carrageenan-induced inflammatory pain in mice and to identify its interactions with NMDA receptors. Inflammatory pain was induced by intraplantar injection of 2% carrageenan in male ICR mice. To investigate the analgesic effects of capsazepine, pain-related behaviors were evaluated using von Frey filaments and a thermal stimulator placed on the hind paw. TRPV1 expression and NMDA receptor phosphorylation in the spinal cord and glutamate concentration in the spinal cord and serum were measured. Intrathecal treatment with capsazepine significantly attenuated carrageenan-induced mechanical allodynia and thermal hyperalgesia. Moreover, carrageenan-enhanced glutamate and phosphorylation of NMDA receptor subunit 2B in the spinal cord were suppressed by capsazepine administration. These results indicate that TRPV1 and NMDA receptors in the spinal cord are associated with inflammatory pain transmission, and inhibition of TRPV1 may reduce inflammatory pain via NMDA receptors.

Activation of transient receptor potential vanilloid 4 exacerbates myocardial ischemia-reperfusion injury via JNK-CaMKII phosphorylation pathway in isolated mice hearts

Previous studies, including our own, have demonstrated that transient receptor potential vanilloid 4 (TRPV4) is involved in myocardial ischemia-reperfusion (IR) injury, yet its underlying molecular mechanism remains unclear. In this study, we isolated mice hearts for a Langendorff perfusion test and used HL-1 myocytes for in vitro assessments. We first confirmed that TRPV4 agonist (GSK101) enhanced myocardial IR injury, as demonstrated by the reduced recovery of cardiac function, larger myocardial infarct size, and more apoptotic cells. We also found that GSK101 could further increase the phosphorylation of JNK and CaMKII in isolated hearts during IR. Notably, GSK101 dose-dependently evoked the phosphorylation of JNK and CaMKII in isolated normal hearts. All above GSK101-induced effects could be significantly blocked by the pharmacological inhibition or genetic ablation of TRPV4. More importantly, JNK inhibition (with SP600125) or CaMKII inhibition (with KN93 or in transgenic AC3-I mice) could prevent GSK101-induced myocardial injury during IR. In HL-1 myocytes, GSK101 triggered Ca2+ influx and evoked the phosphorylation of JNK and CaMKII but these effects were abolished by removing extracellular Ca2+ or in the presence of a TRPV4 antagonist. Finally, we showed that in HL-1 myocytes and isolated hearts during IR, JNK inhibition significantly inhibited the phosphorylation of CaMKII induced by GSK101 but CaMKII inhibition had no effect on JNK activation induced by GSK101. Our data suggest that TRPV4 activation exacerbates myocardial IR injury via the JNK-CaMKII phosphorylation pathway.

Suppression of joint pain in transient receptor potential vanilloid 4 knockout rats with monoiodoacetate-induced osteoarthritis.

Transient receptor potential vanilloid 4 (TRPV4) modulates osteoarthritic (OA) pain in animal models. However, the pathophysiological function of TRPV4 in regulating OA pain remains poorly understood. We developed TRPV4-knockout (TRPV4-KO) rats and assessed the effects of Trpv4 gene deficiency in a monoiodoacetate (MIA)-induced OA pain model (MIA rats) by examining pain-related behavior, pathological changes, and electrophysiological changes in dorsal root ganglion (DRG) neurons. The changes detected in TRPV4-KO rats were confirmed in wild-type rats using a TRPV4 antagonist. Transient receptor potential vanilloid 4-KO rats showed the same pain threshold as wild-type rats for thermal or pressure stimuli under normal conditions. Trpv4 gene deletion did not suppress the development of osteoarthritis pathologically in MIA rats. However, the OA-related mechanical pain behaviors observed in MIA rats, including decreased grip strength, increased mechanical allodynia, and reduced weight-bearing on the ipsilateral side, were completely suppressed in TRPV4-KO rats. The DRG neurons in wild-type but not TRPV4-KO MIA rats were depolarized with increased action potentials. Transient receptor potential vanilloid 4 antagonist treatments recapitulated the effects of genetic Trpv4 deletion. Transient receptor potential vanilloid 4 was sensitized in the DRG neurons of MIA rats and played a critical role in the development of OA pain. These results suggest that the inhibition of TRPV4 might be a novel potent analgesic strategy for treating OA pain.

De Novo Design of Peptidic Positive Allosteric Modulators Targeting TRPV1 with Analgesic Effects.

Transient receptor potential vanilloid 1 (TRPV1) ion channel is a nociceptor critically involved in pain sensation. Direct blockade of TRPV1 exhibits significant analgesic effects but also incurs severe side effects such as hyperthermia, causing failures of TRPV1 inhibitors in clinical trials. In order to selectively target TRPV1 channels that are actively involved in pain‐sensing, peptidic positive allosteric modulators (PAMs) based on the high‐resolution structure of the TRPV1 intracellular ankyrin‐repeat like domain are de novo designed. The hotspot centric approach is optimized for protein design; its usage in Rosetta increases the success rate in protein binder design. It is demonstrated experimentally, with a combination of fluorescence resonance energy transfer (FRET) imaging, surface plasmon resonance, and patch‐clamp recording, that the designed PAMs bind to TRPV1 with nanomolar affinity and allosterically enhance its response to ligand activation as it is designed. It is further demonstrated that the designed PAM exhibits long‐lasting in vivo analgesic effects in rats without changing their body temperature, suggesting that they have potentials for developing into novel analgesics.

TRPV1 Antagonist Prevents Neonatal Sevoflurane-Induced Synaptic Abnormality and Cognitive Impairment in Mice Through Regulating the Src/Cofilin Signaling Pathway.

Long-term neurodevelopmental disorders following neonatal anesthesia have been reported both in young animals and in children. The activation of transient receptor potential vanilloid 1 (TRPV1) channels in hippocampus adversely affects neurodevelopment. The current study explored the underlying mechanism of TRPV1 channels on long-lasting cognitive dysfunction induced by anesthetic exposure to the developing brain. we demonstrated that TRPV1 expression was increased after sevoflurane exposure both in vitro and in vivo. Sevoflurane exposure to hippocampal neurons decreased the synaptic density and the surface GluA1 expression, as well as increased co-localization of internalized AMPAR in early and recycling endosomes. Sevoflurane exposure to newborn mice impaired learning and memory in adulthood, and reduced AMPAR subunit GluA1, 2 and 3 expressions in the crude synaptosomal fractions from mouse hippocampus. The inhibition of TRPV1 reversed the phenotypic changes induced by sevoflurane. Moreover, sevoflurane exposure increased Src phosphorylation at tyrosine 416 site thereby reducing cofilin phosphorylation. TRPV1 blockade reversed these suppressive effects of sevoflurane. Our data suggested that TRPV1 antagonist may protect against synaptic damage and cognitive dysfunction induced by sevoflurane exposure during the brain developing stage.

TRPV1 Hyperfunction Contributes to Renal Inflammation in Oxalate Nephropathy.

Inflammation worsens oxalate nephropathy by exacerbating tubular damage. The transient receptor potential vanilloid 1 (TRPV1) channel is present in kidney and has a polymodal sensing ability. Here, we tested whether TRPV1 plays a role in hyperoxaluria-induced renal inflammation. In TRPV1-expressed proximal tubular cells LLC-PK1, oxalate could induce cell damage in a time- and dose-dependent manner; this was associated with increased arachidonate 12-lipoxygenase (ALOX12) expression and synthesis of endovanilloid 12(S)-hydroxyeicosatetraenoic acid for TRPV1 activation. Inhibition of ALOX12 or TRPV1 attenuated oxalate-mediated cell damage. We further showed that increases in intracellular Ca2+ and protein kinase C α activation are downstream of TRPV1 for NADPH oxidase 4 upregulation and reactive oxygen species formation. These trigger tubular cell inflammation via increased NLR family pyrin domain-containing 3 expression, caspase-1 activation, and interleukin (IL)-1β release, and were alleviated by TRPV1 inhibition. Male hyperoxaluric rats demonstrated urinary supersaturation, tubular damage, and oxidative stress in a time-dependent manner. Chronic TRPV1 inhibition did not affect hyperoxaluria and urinary supersaturation, but markedly reduced tubular damage and calcium oxalate crystal deposition by lowering oxidative stress and inflammatory signaling. Taking all these results together, we conclude that TRPV1 hyperfunction contributes to oxalate-induced renal inflammation. Blunting TRPV1 function attenuates hyperoxaluric nephropathy.

Targeting TRPV1-mediated autophagy attenuates nitrogen mustard-induced dermal toxicity

Nitrogen mustard (NM) causes severe vesicating skin injury, which lacks effective targeted therapies. The major limitation is that the specific mechanism of NM-induced skin injury is not well understood. Recently, autophagy has been found to play important roles in physical and chemical exposure-caused cutaneous injuries. However, whether autophagy contributes to NM-induced dermal toxicity is unclear. Herein, we initially confirmed that NM dose-dependently caused cell death and induced autophagy in keratinocytes. Suppression of autophagy by 3-methyladenine, chloroquine, and bafilomycin A1 or ATG5 siRNA attenuated NM-induced keratinocyte cell death. Furthermore, NM increased transient receptor potential vanilloid 1 (TRPV1) expression, intracellular Ca2+ content, and the activities of Ca2+/calmodulin-dependent kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), unc-51-like kinase 1 (ULK1), and mammalian target of rapamycin (mTOR). NM-induced autophagy in keratinocytes was abolished by treatment with inhibitors of TRPV1 (capsazepine), CaMKKβ (STO-609), AMPK (compound C), and ULK1 (SBI-0206965) as well as TRPV1, CaMKKβ, and AMPK siRNA transfection. In addition, an mTOR inhibitor (rapamycin) had no significant effect on NM-stimulated autophagy or cell death of keratinocytes. Finally, the results of the in vivo experiment in NM-treated skin tissues were consistent with the findings of the in vitro experiment. In conclusion, NM-caused dermal toxicity by overactivating autophagy partially through the activation of TRPV1-Ca2+-CaMKKβ-AMPK-ULK1 signaling pathway. These results suggest that blocking TRPV1-dependent autophagy could be a potential treatment strategy for NM-caused cutaneous injury.

The interaction between P2X3 and TRPV1 in the dorsal root ganglia of adult rats with different pathological pains

Peripheral inflammatory and neuropathic pain are closely related to the activation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3) and transient receptor potential vanilloid 1 (TRPV1), but the interaction between P2X3 and TRPV1 in different types of pathological pain has rarely been reported. In this study, complete Freund’s adjuvant (CFA)-induced inflammatory pain and spared nerve injury (SNI)-induced neuropathic pain models were established in adult rats. The interactions between P2X3 and TRPV1 in the dorsal root ganglion were observed by pharmacological, co-immunoprecipitation, immunofluorescence and whole-cell patch-clamp recording assays. TRPV1 was shown to promote the induction of spontaneous pain caused by P2X3 in the SNI model, but the induction of spontaneous pain behaviour by TRPV1 was not completely dependent on P2X3 in vivo. In both the CFA and SNI models, the activation of peripheral P2X3 enhanced the effect of TRPV1 on spontaneous pain, while the inhibition of peripheral TRPV1 reduced the induction of spontaneous pain by P2X3 in the CFA model. TRPV1 and P2X3 had inhibitory effects on each other in the inflammatory pain model. During neuropathic pain, P2X3 facilitated the function of TRPV1, while TRPV1 had an inhibitory effect on P2X3. These results suggest that the mutual effects of P2X3 and TRPV1 differ in cases of inflammatory and neuropathic pain in rats.

Inhibition of transient receptor potential vanilloid 1 (TRPV1) channel regulates chikungunya virus infection in macrophages.

Chikungunya virus (CHIKV), a virus that induces pathogenic inflammatory host immune responses, is re-emerging worldwide, and there are currently no established antiviral control measures. Transient receptor potential vanilloid 1 (TRPV1), a non-selective Ca2+-permeable ion channel, has been found to regulate various host inflammatory responses including several viral infections. Immune responses to CHIKV infection in host macrophages have been reported recently. However, the possible involvement of TRPV1 during CHIKV infection in host macrophages has not been studied. Here, we investigated the possible role of TRPV1 in CHIKV infection of the macrophage cell line RAW 264.7. It was found that CHIKV infection upregulates TRPV1 expression in macrophages. To confirm this observation, the TRPV1-specific modulators 5'-iodoresiniferatoxin (5'-IRTX, a TRPV1 antagonist) and resiniferatoxin (RTX, a TRPV1 agonist) were used. Our results indicated that TRPV1 inhibition leads to a reduction in CHIKV infection, whereas TRPV1 activation significantly enhances CHIKV infection. Using a plaque assay and a time-of-addition assay, it was observed that functional modulation of TRPV1 affects the early stages of the viral lifecycle in RAW 264.7 cells. Moreover, CHIKV infection was found to induce of pNF-κB (p65) expression and nuclear localization. However, both activation and inhibition of TRPV1 were found to enhance the expression and nuclear localization of pNF-κB (p65) and production of pro-inflammatory TNF and IL-6 during CHIKV infection. In addition, it was demonstrated by Ca2+ imaging that TRPV1 regulates Ca2+ influx during CHIKV infection. Hence, the current findings highlight a potentially important regulatory role of TRPV1 during CHIKV infection in macrophages. This study might also have broad implications in the context of other viral infections as well.

Structural determinants of TRPV4 inhibition and identification of new antagonists with antiviral activity.

Background and PurposeThe transient receptor potential vanilloid 4 (TRPV4) cation channel participates in multiple physiological processes and is also at the core of different diseases, making this channel an interesting pharmacological target with therapeutic potential. However, little is known about the structural elements governing its inhibition.Experimental ApproachWe have now combined in silico drug discovery and molecular dynamics simulation based on Xenopus tropicalis xTRPV4 structure with functional studies measuring cell Ca2+ influx mediated by human TRPV4 channel to characterize the binding site of known TRPV4 inhibitors and to identify novel small molecule channel modulators.Key ResultsWe have found that the inhibitor HC067047 binds to a pocket conformed by residues from S2–S3 linker (xTRPV4‐D542), S4 (xTRPV4‐M583 and Y587 and S5 (xTRPV4‐D609 and F613). This pocket was also used for structure‐based virtual screening in the search of novel channel modulators. Forty potential hits were selected based on the lower docking scores (from ~250,000 compounds) and their effect upon TRPV4 functionally tested. Three were further analysed for stability using molecular dynamics simulation and functionally tested on TRPV4 channels carrying mutations in the binding pocket. Compound NSC151066, shown to require residue xTRPV4‐M583 for its inhibitory effect, presented an IC50 of 145 nM and demonstrated to be an effective antiviral against Zika virus with a potency similar to HC067047.Conclusion and ImplicationsTogether, we propose structural insights into the inhibition of TRPV4 and how this information can be used for the design of novel channel modulators.LINKED ARTICLESThis article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc

Inhibition of TRPV1 by SHP-1 in nociceptive primary sensory neurons is critical in PD-L1 analgesia.

Recently programmed death-ligand 1 (PD-L1) receptor PD-1 was found in dorsal root ganglion (DRG) neurons, and PD-L1 activates PD-1 to inhibit inflammatory and neuropathic pain by modulating neuronal excitability. However, the downstream signaling of PD-1 in sensory neurons remains unclear. Here, we show that PD-L1 activated Src homology 2 domain-containing tyrosine phosphatase-1 (SHP-1) to downregulate transient receptor potential vanilloid 1 (TRPV1) in DRG neurons and inhibit bone cancer pain in mice. Local injection of PD-L1 produced analgesia. PD-1 in DRG neurons colocalized with TRPV1 and SHP-1. PD-L1 induced the phosphorylation of SHP-1 in DRG TRPV1 neurons and inhibited TRPV1 currents. Loss of TRPV1 in mice abolished bone cancer–induced thermal hyperalgesia and PD-L1 analgesia. Conditioned deletion of SHP-1 in NaV1.8+ neurons aggravated bone cancer pain and diminished the inhibition of PD-L1 on TRPV1 currents and pain. Together, our findings suggest that PD-L1/PD-1 signaling suppresses bone cancer pain via inhibition of TRPV1 activity. Our results also suggest that SHP-1 in sensory neurons is an endogenous pain inhibitor and delays the development of bone cancer pain via suppressing TRPV1 function.

Thermal Hyperalgesia and Mechanical Allodynia Elicited by Histamine and Non-histaminergic Itch Mediators: Respective Involvement of TRPV1 and TRPA1

Acute itch is elicited by histamine, as well as non-histaminergic itch mediators including chloroquine, BAM8-22 and Ser-Leu-Ile-Gly-Arg-Leu (SLIGRL). When injected intradermally, histamine binds to histamine H1 and H4 receptors that activate transient receptor potential vanilloid 1 (TRPV1) to depolarize pruriceptors. Chloroquine, BAM8-22, and SLIGRL, respectively, bind to Mas-related G-protein-coupled receptors MrgprA3, MrgprC11, and MrgprC11/PAR2 that in turn activate transient receptor potential ankyrin 1 (TRPA1). In this study we tested if histamine, chloroquine, BAM8-22 and SLIGRL elicit thermal hyperalgesia and mechanical allodynia in adult male mice. We measured the latency of hindpaw withdrawal from a noxious heat stimulus, and the threshold for hindpaw withdrawal from a von Frey mechanical stimulus. Intraplantar injection of histamine resulted in significant thermal hyperalgesia (p < 0.001) and mechanical allodynia (p < 0.001) ipsilaterally that persisted for 1 h. Pretreatment with the TRPV1 antagonist AMG-517 (10 or 20 μg), but not the TRPA1 antagonist HC-030031 (50 or 100 μg), significantly attenuated the magnitude and time course of thermal hyperalgesia and mechanical allodynia elicited by histamine (p < 0.001 for both), indicating that these effects are mediated by TRPV1. In contrast, pretreatment with the TRPA1 antagonist significantly reduced thermal hyperalgesia and mechanical allodynia elicited by chloroquine (p < 0.001 for both ), BAM-822 (p < 0.01, p < 0.001, respectively) and SLGRL (p < 0.05, p < 0.001, respectively), indicating that effects elicited by these non-histaminergic itch mediators require TRPA1. TRPV1 and TRPA1 channel inhibitors thus may have potential use in reducing hyperalgesia and allodynia associated with histaminergic and non-histaminergic itch, respectively.

Sustained laryngeal transient receptor potential vanilloid 1 activation inhibits mechanically induced swallowing in anesthetized rats.

A major component of gastric acid is hydrochloric acid (HCl), which can activate transient receptor potential vanilloid 1 (TRPV1). In the present study, we investigated how sustained laryngeal TRPV1 activation affects the frequency of the swallowing reflex. Experiments were carried out on 85 male Sprague-Dawley rats. The effects of short and sustained application of chemicals (3 µl of 0.1 N HCl or capsaicin) on the frequency of swallowing and on time-dependent changes in the occurrence of swallowing evoked by supralaryngeal nerve stimulation were determined. To evaluate vascular permeability of the larynx, Evans blue dye was intravenously injected after 5 or 60 min of sustained TRPV1 activation. SB366791 (a TRPV1 inhibitor) and Cap/QX-314 (a TRPV1-expressed neuronal inhibitor) significantly inhibited HCl/capsaicin-induced swallowing, but air flow-induced swallowing was not affected. Although the number of air flow-induced swallows followed by capsaicin stimulation was not affected within 5 min, it was significantly reduced by 60-min capsaicin or HCl application. The swallowing threshold associated with supralaryngeal nerve stimulation did not significantly change throughout the recording period. Evans blue dye concentrations in the larynx were significantly higher at 60 min in the 10-5 M capsaicin group than in the control group. Our results suggest that sustained TPRV1 activation not only desensitizes TRPV1 but also inactivates mechanoreceptors, which may be attributed to increases in vascular permeability and edema, as part of an inflammatory process.NEW & NOTEWORTHY Although a transient receptor potential vanilloid 1 (TRPV1) inhibitor or TRPV1-expressed neuronal inhibitor significantly inhibited HCl/capsaicin-evoked swallowing, air flow-induced swallowing was not affected. The number of air flow-induced swallows was significantly reduced within 60 min of TRPV1 activation. Evans blue dye concentration in the larynx increased 60 min after capsaicin application. TPRV1 activation not only desensitizes TRPV1 but also inactivates mechanoreceptors caused by increases in vascular permeability and edema.

Hydrogen Protons Modulate Perivascular Axo-axonal Interactions in the Middle Cerebral Artery of Rats.

Previous studies have demonstrated that nicotine can induce relaxation of the middle cerebral artery (MCA). However, whether this relaxation is associated with the activity of sensory calcitonin gene-related peptide (CGRP) nerves and whether this is modulated by hydrogen protons (H), facilitating the release of CGRP from sensory CGRPergic nerve terminals in the MCA, remains unclear. In this study, we examined the role of H in the modulation of neurogenic vasomotor responses in the rat-isolated endothelium-denuded MCA. Wire myography was used to measure vasoreactivity and indicated that nicotine-induced relaxation was sensitive to tetrodotoxin and lidocaine and drastically reduced levels of guanethidine (an adrenergic neuronal blocker), N-nitro-L-arginine (L-NNA), CGRP8-37, vasoactive intestinal polypeptide (VIP)6-28, capsaicin, capsazepine (a transient receptor potential vanilloid-1 inhibitor), and tetraethylammonium. However, this nicotine-induced relaxation was not sensitive to propranolol. Lowering the pH of the buffer solution with HCl caused pH-dependent vasorelaxation and deceased intracellular pH in the MCA rings, which was sensitive to L-NNA, CGRP8-37, VIP6-28, capsazepine, 4-aminopyridine (a voltage-gated potassium channel antagonist), and paxilline (a large conductance Ca-activated K channel antagonist). However, HCl-induced relaxation was not inhibited by glibenclamide (an ATP-sensitive K channel blocker). These results suggested that electrical and chemical activation of cerebral perivascular adrenergic nerves led to the release of H, which then facilitated the release of NO, VIP, and CGRP, resulting in vasorelaxation. Lowering the pH of the buffer solution caused potassium channels of vascular smooth muscle cells and perivascular nerves to open. In conclusion, our results demonstrated that H may act as a modulator on MCA perivascular nerves and/or smooth muscles.

The nonselective cation channel TRPV4 inhibits angiotensin II receptors

G-protein-coupled receptors (GPCRs) are a ubiquitously expressed family of receptor proteins that regulate many physiological functions and other proteins. They act through two dissociable signaling pathways: the exchange of GDP to GTP by linked G-proteins and the recruitment of β-arrestins. GPCRs modulate several members of the transient receptor potential (TRP) channel family of nonselective cation channels. How TRP channels reciprocally regulate GPCR signaling is less well-explored. Here, using an array of biochemical approaches, including immunoprecipitation and fluorescence, calcium imaging, phosphate radiolabeling, and a β-arrestin-dependent luciferase assay, we characterize a GPCR-TRP channel pair, angiotensin II receptor type 1 (AT1R), and transient receptor potential vanilloid 4 (TRPV4), in primary murine choroid plexus epithelial cells and immortalized cell lines. We found that AT1R and TRPV4 are binding partners and that activation of AT1R by angiotensin II (ANGII) elicits β-arrestin-dependent inhibition and internalization of TRPV4. Activating TRPV4 with endogenous and synthetic agonists inhibited angiotensin II-mediated G-protein-associated second messenger accumulation, AT1R receptor phosphorylation, and β-arrestin recruitment. We also noted that TRPV4 inhibits AT1R phosphorylation by activating the calcium-activated phosphatase calcineurin in a Ca2+/calmodulin-dependent manner, preventing β-arrestin recruitment and receptor internalization. These findings suggest that when TRP channels and GPCRs are co-expressed in the same tissues, many of these channels can inhibit GPCR desensitization.

Pregabalin reduces oxaliplatin-induced oxidative neurotoxicity through modulation of TRPV1 channels in DBTRG neuronal cell line.

As a member of the platinum drug group, oxaliplatin (OXAL) is used to treat brain tumors, although its use is limited through excessive calcium ion (Ca) influx and reactive oxygen species (ROS) production in neurons. The Ca permeable transient receptor potential vanilloid 1 (TRPV1) channel is activated by ROS, and its activity might be reduced by the antioxidant property of pregabalin (PREGAB). This study aimed to investigate the protective action of PREGAB against OXAL-induced oxidative neurotoxicity in human glioblastoma (DBTRG) cells. The DBTRG cells were divided into four treatment groups: control, PREGAB (500 µM for 1 h), OXAL (25 µM for 24 h), and PREGAB + OXAL. In the laser confocal microscope and plate reader analyses, apoptosis, mitochondrial membrane depolarization (JC-1), cell death (propidium iodide/Hoechst rate), and ROS-level production increased by activating TRPV1 in the cells using the OXAL treatment, although the cell viability values decreased. However, these values were recovered in the PREGAB + OXAL group using PREGAB and TRPV1 inhibitor (capsazepine) treatments. In the patch-clamp analyses, OXAL-induced TRPV1 channel activation in the OXAL group also decreased in the PREGAB + OXAL group using the PREGAB and capsazepine treatments. In conclusion, the apoptosis and oxidant actions of OXAL were increased by activation of the TRPV1 channel, but this effect was diminished by the PREGAB treatment. PREGAB treatment has the potential to be an effective strategy in the treatment of OXAL-induced oxidative neurotoxicity.