TRPV1 Activation Research Articles

Molecular mechanisms of two novel and selective TRPV1 channel activators

Venomous toxins hold immense value as tools in elucidating the intricate structure and underlying mechanisms of ion channels. In this article, we identified of two novel toxins, Hainantoxin-XXI (HNTX-XXI) and Hainantoxin-XXII (HNTX-XXII), derived from the venom of the Chinese spider Ornithoctonus hainana. HNTX-XXI, boasting a molecular weight of 6869.095 Da, comprises 64 amino acid residues and contains 8 cysteines. Meanwhile, HNTX-XXII, with a molecular weight of 8623.732 Da, comprises 77 amino acid residues and contains 12 cysteines. Remarkably, we discovered that both HNTX-XXI and HNTX-XXII possess the ability to activate TRPV1. They activated TRPV1 with EC50 values of 3.6 ± 0.19 μM and 862 ± 56 nM, respectively. Furthermore, the current generated by the activation of TRPV1 by these toxins can be rapidly blocked by ruthenium red. Intriguingly, our analysis revealed that the interaction between HNTX-XXI and TRPV1 is mediated by three key amino acid residues: L465, V469, and D471. Similarly, the interaction between HNTX-XXII and TRPV1 is facilitated by four key amino acid residues: A657, F659, E600, and R601. These findings provide profound insights into the molecular basis of toxin-TRPV1 interactions and pave the way for future research exploring the therapeutic potential of these toxic peptides.

Multicenter integration analysis of TRP channels revealed potential mechanisms of immunosuppressive microenvironment activation and identified a machine learning-derived signature for improving outcomes in gliomas.

This study aimed to explore the mechanisms of transient receptor potential (TRP) channels on the immune microenvironment and develop a TRP-related signature for predicting prognosis, immunotherapy response, and drug sensitivity in gliomas. Based on the unsupervised clustering algorithm, we identified novel TRP channel clusters and investigated their biological function, immune microenvironment, and genomic heterogeneity. Invitro and invivo experiments revealed the association between TRPV2 and macrophages. Subsequently, based on 96 machine learning algorithms and six independent glioma cohorts, we constructed a machine learning-based TRP channel signature (MLTS). The performance of the MLTS in predicting prognosis, immunotherapy response, and drug sensitivity was evaluated. Patients with high expression levels of TRP channel genes had worse prognoses, higher tumor mutation burden, and more activated immunosuppressive microenvironment. Meanwhile, TRPV2 was identified as the most essential regulator in TRP channels. TRPV2 activation could promote macrophages migration toward malignant cells and alleviate glioma prognosis. Furthermore, MLTS could work independently of common clinical features and present stable and superior prediction performance. This study investigated the comprehensive effect of TRP channel genes in gliomas and provided a promising tool for designing effective, precise treatment strategies.

Activation of Piezo1 or TRPV2 channels inhibits human ureteral contractions via NO release from the mucosa.

We aimed to investigate the expression and motor modulatory roles of several mechano-sensitive channels (MSCs) in human ureter. Human proximal ureters were obtained from eighty patients subjected to nephrectomy. Expression of MSCs at mRNA, protein and functional levels were examined. Contractions of longitudinal ureter strips were recorded in organ bath. A fluorescent probe Diaminofluoresceins was used to measure nitric oxide (NO). RT-PCR analyses revealed predominant expression of Piezo1 and TRPV2 mRNA in intact ureter and mucosa. Immunofluorescence assays indicate proteins of MSCs (Piezo1/Piezo2, TRPV2 and TRPV4) were mainly distributed in the urothelium. Ca2+ imaging confirmed functional expression of TRPV2, TRPV4 and Piezo1 in cultured urothelial cells. Specific agonists of Piezo1 (Yoda1, 3-300μM) and TRPV2 (cannabidiol, 3-300μM) attenuated the frequency of ureteral contractions in a dose-dependent manner while the TRPV4 agonist GSK1016790A (100nM-1μM) exerted no effect. The inhibitory effects of Piezo1 and TRPV2 agonists were significantly blocked by the selective antagonists (Dooku 1 for Piezo1, Tranilast for TRPV2), removal of the mucosa, and pretreatment with NO synthase inhibitor L-NAME (10μM). Yoda1 (30μM) and cannabidiol (50μM) increased production of NO in cultured urothelial cells. Our results suggest that activation of Piezo1 or TRPV2 evokes NO production and release from mucosa that may mediate mechanical stimulus-induced reduction of ureter contractions. Our findings support the idea that targeting Piezo1 and TRPV2 channels may be a promising pharmacological strategy for ureter stone passage or colic pain relief.

Design and experimental validation of a new radiolabeled analog of N-(3-hydroxy-4-methoxy-phenyl-methyl) ferrocene-carboxamide (VFC) targeting the TRPV1 receptor

The TRPV1 receptor has been recognized to play a role in cancer development, being overexpressed in prostate, breast, lung, and colon cancers. Since TRPV1 activation promotes cancer cell proliferation, invasion, and migration, TRPV1 antagonists may show potential as anti-cancer agents. Capsaicin and capsaicinoids are phytochemicals derived from homovanillic acid found in abundance in chili peppers and responsible for its pungent properties. Capsaicin acts as a potent agonist of TRPV1 with recognized antineoplastic properties. Here, we employ computational approaches including molecular modeling and docking to refine the 3D structure of human TRPV1 and assess its interaction with the newly synthesized N-(3-hydroxy-4-methoxyphenylmethyl)ferrocenecarboxamide (VFC) and its cyclopentadienyl tricarbonyl rhenium and technetium analogs. Radiolabeling of VFC with 99mTc was achieved by double ligand exchange to afford 99mTc-VFC in high radiochemical purity and yield. Biodistribution studies in mice demonstrated preferential accumulation of 99mTc-VFC in the bladder, liver, kidney, and lung. These findings may contribute to developing efficient TRPV1-targeted radiotracers for molecular imaging of tumors by SPECT.

TRPV4 mediates IL-1-induced Ca2+ signaling, ERK activation and MMP expression.

Ca2+ permeation through TRPV4 in fibroblasts is associated with pathological matrix degradation. In human gingival fibroblasts, IL-1β binding to its signaling receptor (IL-1R1) induces activation of extracellular regulated kinase (ERK) and MMP1 expression, processes that require Ca2+ flux across the plasma membrane. It is not known how IL-1R1, which does not conduct Ca2+, generates Ca2+ signals in response to IL-1. We examined whether TRPV4 mediates the Ca2+ fluxes required for ERK signaling in IL-1 stimulated gingival fibroblasts. TRPV4 was immunostained in fibroblasts of human gingival connective tissue and in focal adhesions of cultured mouse gingival fibroblasts. Human gingival fibroblasts treated with IL-1β showed no change of TRPV4 expression but there was increased MMP1 expression. In mouse, gingival fibroblasts expressing TRPV4, IL-1 strongly increased [Ca2+]i. Pre-incubation of cells with IL-1 Receptor Antagonist blocked Ca2+ entry induced by IL-1 or the TRPV4 agonist GSK101. Knockout of TRPV4 or expression of a non-Ca2+-conducting TRPV4 pore-mutant or pre-incubation with the TRPV4 inhibitor RN1734, blocked IL-1-induced Ca2+ transients and expression of the mouse interstitial collagenase, MMP13. Treatment of mouse gingival fibroblasts with GSK101 phenocopied Ca2+ and ERK responses induced by IL-1; these responses were absent in TRPV4-null cells or cells expressing a non-conducting TRPV4 pore-mutant. Immunostained IL-1R1 localized with TRPV4 in adhesions within cell extensions. While TRPV4 immunoprecipitates analyzed by mass spectrometry showed no association with IL-1R1, TRPV4 associated with Src-related proteins and Src co-immunoprecipitated with TRPV4. Src inhibition reduced IL-1-induced Ca2+ responses. The functional linkage of TRPV4 with IL-1R1 expands its repertoire of innate immune signaling processes by mediating IL-1-driven Ca2+ responses that drive matrix remodeling in fibroblasts. Thus, inhibiting TRPV4 activity may provide a new pharmacological approach for blunting matrix degradation in inflammatory diseases.

Graphdiyne as a Highly Efficient and Neuron-Targeted Photothermal Transducer for in Vivo Neuromodulation.

Photothermal modulation of neural activity offers a promising approach for understanding brain circuits and developing therapies for neurological disorders. However, the low neuron selectivity and inefficient light-to-heat conversion of existing photothermal nanomaterials significantly limit their potential for neuromodulation. Here, we report that graphdiyne (GDY) can be developed into an efficient neuron-targeted photothermal transducer for in vivo modulation of neuronal activity through rational surface functionalization. We functionalize GDY with polyethylene glycol (PEG) through noncovalent hydrophobic interactions, followed by antibody conjugation to specifically target the temperature-sensitive transient receptor potential cation channel subfamily V member 1 (TRPV1) on the surface of neural cells. The nanotransducer not only exhibits high photothermal conversion efficiency in the near-infrared region but also shows great TRPV1-targeting capability. This enables photothermal activation of TRPV1, leading to neurotransmitter release in cells and modulation of neural firing in living mice. With its precision and selectivity, the GDY-based transducer provides an innovative avenue for understanding brain function and developing therapeutic strategies for neurodegenerative diseases.

A neural-mast cell axis regulates skin microcirculation in diabetes.

Changes in microcirculation lead to the progression of organ pathology in diabetes. Although neuroimmune interactions contribute to a variety of conditions, it is still unclear whether abnormal neural activities affect microcirculation related to diabetes. Using laser speckle contrast imaging, we examined the skin of patients with type 2 diabetes and found that their microvascular perfusion was significantly compromised. This phenomenon was recapitulated in a high-fat-diet-driven murine model of type 2 diabetes-like disease. In this setting, although both macrophages and mast cells were enriched in the skin, only mast cells and associated degranulation were critically required for the microvascular impairment. Sensory neurons exhibited enhanced TRPV1 activities, which triggered mast cells to degranulate and compromise skin microcirculation. Chemical and genetic ablation of TRPV1+ nociceptors robustly improve skin microcirculation status. Substance P (SP) is a neuropeptide and was elevated in the skin and sensory neurons in the context of type 2 diabetes. Exogenous administration of SP resulted in impaired skin microcirculation, whereas neuronal knockdown of SP dramatically prevented mast cell degranulation and consequently improved skin microcirculation. Overall, our findings indicate a neural-mast cell axis underlying skin microcirculation disturbance in diabetes and shed light on neuroimmune therapeutics for diabetes-related complications.

Dermal TRPV1 innervations engage a macrophage- and fibroblast-containing pathway to activate hair growth in mice.

Pain, detected by nociceptors, is an integral part of injury, yet whether and how it can impact tissue physiology and recovery remain understudied. Here, we applied chemogenetics in mice to locally activate dermal TRPV1 innervations in naive skin and found that it triggered new regenerative cycling by dormant hair follicles (HFs). This was preceded by rapid apoptosis of dermal macrophages, mediated by the neuropeptide calcitonin gene-related peptide (CGRP). TRPV1 activation also triggered a macrophage-dependent induction of osteopontin (Spp1)-expressing dermal fibroblasts. The neuropeptide CGRP and the extracellular matrix protein Spp1 were required for the nociceptor-triggered hair growth. Finally, we showed that epidermal abrasion injury induced Spp1-expressing dermal fibroblasts and hair growth via a TRPV1 neuron and CGRP-dependent mechanism. Collectively, these data demonstrated a role for TRPV1 nociceptors in orchestrating a macrophage and fibroblast-supported mechanism to promote hair growth and enabling the efficient restoration of this mechano- and thermo-protective barrier after wounding.

Far-infrared irradiation increases the uptake of LDL cholesterol by downregulating PCSK9 through the activation of TRPV4 calcium channels in HepG2 cells

This study investigated the effects of far-infrared (FIR) irradiation on low-density lipoprotein cholesterol (LDL-C) uptake by human hepatocellular carcinoma G2 (HepG2) cells via the regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9). FIR irradiation for 30 min significantly decreased PCSK9 expression (p < 0.01) in HepG2 cells. FIR irradiation substantially increased the low-density lipoprotein receptor (p < 0.0001) and LDL-C uptake (p < 0.01). Activation of transient receptor potential vanilloid (TRPV) channels mimicked the effects of FIR irradiation, significantly decreasing the protein expression of PCSK9 (p < 0.05). Conversely, inhibition of TRP channels using ruthenium red reversed the reduction in PCSK9 protein expression following FIR irradiation (p < 0.01). The specific activation of TRPV4 using 4α-PDD mimicked the effect of FIR irradiation (p < 0.01), whereas PCSK9 reduction by FIR irradiation was significantly reversed by the inhibition of TRPV4 using RN1734 (p < 0.05). These findings implied that FIR irradiation emitted from a ceramic lamp specifically increased TRPV4 activity. These findings provide insights into a novel therapeutic approach using FIR irradiation for LDL-C regulation and its implications for cardiovascular health.

TRPV3 facilitates lipolysis and attenuates diet-induced obesity via activation of the NRF2/FSP1 signaling axis

Transient receptor potential vanilloid (TRPV) ion channels play a crucial role in various cellular functions by regulating intracellular Ca2+ levels and have been extensively studied in the context of several metabolic diseases. However, the regulatory effects of TRPV3 in obesity and lipolysis are not well understood. In this study, utilizing a TRPV3 gain-of-function mouse model (TRPV3G568V/G568V), we assessed the metabolic phenotype of both TRPV3G568V/G568V mice and their control littermates, which were randomly assigned to either a 12-week high-fat diet or a control diet. We investigated the potential mechanisms underlying the role of TRPV3 in restraining obesity and promoting lipolysis both in vivo and in vitro. Our findings indicate that a high-fat diet led to significant obesity, characterized by increased epididymal and inguinal white adipose tissue weight and higher fat mass. However, the gain-of-function mutation in TRPV3 appeared to counteract these adverse effects by enhancing lipolysis in visceral fat through the upregulation of the major lipolytic enzyme, adipocyte triglyceride lipase (ATGL). In vitro experiments using carvacrol, a TRPV3 agonist, demonstrated the promotion of lipolysis and antioxidation in 3T3-L1 adipocytes after TRPV3 activation. Notably, carvacrol failed to stimulate Ca2+ influx, lipolysis, and antioxidation in 3T3-L1 adipocytes treated with BAPTA-AM, a cell-permeable calcium chelator. Our results revealed that TRPV3 activation induced the action of transcriptional factor nuclear factor erythroid 2-related factor 2 (NRF2), resulting in increased expression of ferroptosis suppressor protein 1 (FSP1) and superoxide dismutase2 (SOD2). Moreover, the inhibition of NRF2 impeded carvacrol-induced lipolysis and antioxidation in 3T3-L1 adipocytes, with downregulation of ATGL, FSP1, and SOD2. In summary, our study suggests that TRPV3 promotes visceral fat lipolysis and inhibits diet-induced obesity through the activation of the NRF2/FSP1 signaling axis. We propose that TRPV3 may be a potential therapeutic target in the treatment of obesity.

Unlocking the therapeutic potential of TRPV3: Insights into thermosensation, channel modulation, and skin homeostasis involving TRPV3.

Recent insights reveal the significant role of TRPV3 in warmth sensation. A novel finding elucidated how thermosensation is affected by TRPV3 membrane abundance that is modulated by the transmembrane protein TMEM79. TRPV3 is a warmth-sensitive ion channel predominantly expressed in epithelial cells, particularly skin keratinocytes. Multiple studies investigated the roles of TRPV3 in cutaneous physiology and pathophysiology. TRPV3 activation by innocuous warm temperatures in keratinocytes highlights its significance in temperature sensation, but whether TRPV3 directly contributes to warmth sensations in vivo remains controversial. This review explores the electrophysiological and structural properties of TRPV3 and how modulators affect its intricate regulatory mechanisms. Moreover, we discuss the multifaceted involvement of TRPV3 in skin physiology and pathology, including barrier formation, hair growth, inflammation, and itching. Finally, we examine the potential of TRPV3 as a therapeutic target for skin diseases and highlight its diverse role in maintaining skin homeostasis.

TRPV3 activation by different agonists accompanied by lipid dissociation from the vanilloid site.

TRPV3 represents both temperature- and ligand-activated transient receptor potential (TRP) channel. Physiologically relevant opening of TRPV3 channels by heat has been captured structurally, while opening by agonists has only been observed in structures of mutant channels. Here, we present cryo-EM structures that illuminate opening and inactivation of wild-type human TRPV3 in response to binding of two types of agonists: either the natural cannabinoid tetrahydrocannabivarin (THCV) or synthetic agonist 2-aminoethoxydiphenylborane (2-APB). We found that THCV binds to the vanilloid site, while 2-APB binds to the S1-S4 base and ARD-TMD linker sites. Despite binding to distally located sites, both agonists induce similar pore opening and cause dissociation of a lipid that occupies the vanilloid site in their absence. Our results uncover different but converging allosteric pathways through which small-molecule agonists activate TRPV3 and provide a framework for drug design and understanding the role of lipids in ion channel function.

Mechanosensor channels of the lens and ciliary body: patch clamp studies

Epithelia from the lens and the ciliary body possess mechanosensors, including Piezo1 and transient receptor potential (TRP) channels, that may partake in the regulation of lens and aqueous humor volume. Although these mechanosensor channels commonly allow calcium entry, the outcomes of elevated intracellular calcium vary with the type of channel activated. For instance, separate activation of TRPV4 and TRPV1 increases Na-K-ATPase and Na/K/2Cl cotransporter activities, respectively. To further understand the role of mechanosensitivity in lens and ciliary body, we are currently using patch clamp techniques to characterize the response of primary cultured cells to chemical agonists and antagonists, as well as direct mechanical stimulation. Previously, we found that in mouse lens epithelial cells (mLEC), TRPV4 activation by agonist GSK1016790A (GSK) is followed by disappearance of a fast, transient K+-like outward current (Ipeak), decrease of a steady state outward current (Iss), fast depolarization of resting membrane potential (RMP) values from near -50 mV to near 0 mV, increase of Cx43 hemichannel-like events, and increase of overall membrane conductance ( gm) at holding potential (HP) = -50mV. More recently, we found that in mLEC, shear stress (streaming of external solution from a pipette tip over the cell membrane) reduced Ipeak but did not cause large RMP changes. Porcine non-pigmented epithelial cells (pNPE) in normal conditions display no Ipeak, small Iss, small RMP near -20 mV and spontaneous Cx43 hemichannel-like event activity. Nevertheless, GSK caused further depolarization of pNPE cells, and while no gm changes were seen when GSK was applied while holding cells at -50 mV, gm did increase when GSK exposure occurred during repeated ±80 mV pulses. Preliminary experiments show that activation of TRPM3 by its agonist pregnenolone causes a minimal hyperpolarization in mLEC. In comparison, in a line of human LEC (HLE B3) expressing TRPM3 channels, and which appear generally depolarized, pregnenolone caused a proportionally larger shift toward more negative RMP values; more intriguing, GSK has no measurable effect on the gm of HLE B3 cells. Taken together, and apart from considerations of cell- and species-specific differences, the data suggest that mechanosensor channels have different and/or opposite effects on the regulation of membrane ion channel activity, and thus on cell function. R01EY029171 and R01EY009532. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TRPV3-Activated PARP1/AIFM1/MIF Axis through Oxidative Stress Contributes to Atopic Dermatitis

TRPV3 is a temperature-sensitive calcium-permeable channel. In previous studies, we noticed prominent TUNEL-positive keratinocytes in patients with Olmsted syndrome and Trpv3+/G568V mice, both of which carry gain-of-function variants in the TRPV3 gene. However, it remains unclear how the keratinocytes die and whether this process contributes to more skin disorders. In this study, we showed that gain-of-function variant or pharmacological activation of TRPV3 resulted in poly(ADP-ribose) polymerase 1 (PARP1)/AIFM1/macrophage migration inhibitory factor axis-mediated parthanatos, which is an underestimated form of cell death in skin diseases. Chelating calcium, scavenging ROS, or inhibiting nitric oxide synthase effectively rescued the parthanatos, indicating that TRPV3 regulates parthanatos through calcium-mediated oxidative stress. Furthermore, inhibiting PARP1 downregulated TSLP and IL33 induced by TRPV3 activation in HaCaT cells, reduced immune cell infiltration, and ameliorated epidermal thickening in Trpv3+/G568V mice. Marked parthanatos was also detected in the skin of MC903-treated mice and patients with atopic dermatitis, whereas inhibiting PARP1 largely alleviated the MC903-induced dermatitis. In addition, stimulating parthanatos in mouse skin with methylnitronitrosoguanidine recapitulated many features of atopic dermatitis. These data demonstrate that the TRPV3-regulated parthanatos-associated PARP1/AIFM1/macrophage migration inhibitory factor axis is a critical contributor to the pathogenesis of Olmsted syndrome and atopic dermatitis, suggesting that modulating the PARP1/AIFM1/macrophage migration inhibitory factor axis is a promising therapy for these conditions.

TRPV1+ Sensory nerves modulate antigen specific immune responses

Abstract Sensory neurons recognize harmful or noxious stimuli and initiate a protective response by eliciting pain and defensive behavior. Nociceptors also interact with immune cells to regulate immune responses and play a key role in modulating antibody class switching to IgE. However, it is still unknown whether nociceptors participate in regulating primary IgG antibody responses to novel antigens. Here, we show that TRPV1 expressing nociceptors are required to develop an antigen specific immune response and that direct activation of TRPV1+ signals by selective optogenetic stimulation enhances primary IgG antibody responses to novel antigen. Specific ablation of TRPV1+ neurons, which include nociceptors, significantly blunts NP-specific IgG antibody responses in TRPV1-DTA mice compared to controls (anti-NP2 IgG AU, Day 28 control, 18,401 ± 3393 n=18 versus TRPV1-DTA, 9,028 ± 1,498, n=18, p&amp;lt; 0.001). Selective optogenetic stimulation of TRPV1+ nociceptors on the dorsum of the paw with blue light (473nm, 3Hz, 20%DC, 15min) immediately prior to a local immunization in TRPV1-ChR2 mice significantly increases KLH-specific IgG antibody responses compared to sham stimulation controls (anti-KLH IgG ng/mL Day 28, TRPV1-ChR2: 2,581 ± 476, n= 6 versus Control, 1,224 ± 332, n=8; * p&amp;lt;0.05). Together these studies with genetic and selective functional evidence indicates that nociceptors are required during immunization to produce primary antigen-specific IgG antibody responses to novel antigens.

Trpv4 Mechanosensing Mediates Valvular Interstitial Cell Differentiation to Myofibroblast

Aortic valve stenosis (AVS) is a heart disease that gradually worsens, marked by the fibrosis, inflammation, and stiffening of the aortic valve leaflets, which results in obstructed blood flow and increased pressure on the left ventricle. This condition is associated with a significantly high risk of death. As AVS progresses, valvular interstitial cells (VICs) transform into myofibroblasts due to various soluble and physical cues, leading to detrimental changes in the surrounding cellular matrix. The transformation of VICs into myofibroblasts and the subsequent stiffening of the aortic valve are key factors in the advancement of AVS. Published work by our group and others show that Trpv4 (Transient Receptor Potential subfamily V member 4), a mechanosensitive, calcium-permeant channel/receptor, is activated by a range of soluble and physical stimuli. In mice, Trpv4 deficiency is associated with fibrosis and inflammation. In this study, we tested the hypothesis that Trpv4 regulates the transformation of VICs into myofibroblasts. Our research yielded several key findings: 1) Trpv4 is active in mouse VICs; 2) blocking Trpv4 activity in VICs prevents their transformation into myofibroblasts when exposed to TGF or matrix stiffness; 3) Trpv4 is needed for traction force generation in VICs as determined by traction force microscopy; and 4) Trpv4 residues 100 to 130 are required for myofibroblast differentiation and traction force generation. Altogether, our results highlight the crucial role of Trpv4 mechanosensing in regulating the transformation of VICs to myofibroblasts, a central process in AVS. We believe these findings could lead to novel Trpv4-targeted treatments to mitigate the effects of AVS. NIH (R01 AI172086) grant to Shaik O. Rahaman. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Low-Intensity Extracorporeal Shock Wave Therapy Ameliorates Detrusor Hyperactivity with Impaired Contractility via Transient Potential Vanilloid Channels: A Rat Model for Ovarian Hormone Deficiency.

This study explores low-intensity extracorporeal shock wave therapy (LiESWT)'s efficacy in alleviating detrusor hyperactivity with impaired contractility (DHIC) induced by ovarian hormone deficiency (OHD) in ovariectomized rats. The rats were categorized into the following four groups: sham group; OVX group, subjected to bilateral ovariectomy (OVX) for 12 months to induce OHD; OVX + SW4 group, underwent OHD for 12 months followed by 4 weeks of weekly LiESWT; and OVX + SW8 group, underwent OHD for 12 months followed by 8 weeks of weekly LiESWT. Cystometrogram studies and voiding behavior tracing were used to identify the symptoms of DHIC. Muscle strip contractility was evaluated through electrical-field, carbachol, ATP, and KCl stimulations. Western blot and immunofluorescence analyses were performed to assess the expressions of various markers related to bladder dysfunction. The OVX rats exhibited significant bladder deterioration and overactivity, alleviated by LiESWT. LiESWT modified transient receptor potential vanilloid (TRPV) channel expression, regulating calcium concentration and enhancing bladder capacity. It also elevated endoplasmic reticulum (ER) stress proteins, influencing ER-related Ca2+ channels and receptors to modulate detrusor muscle contractility. OHD after 12 months led to neuronal degeneration and reduced TRPV1 and TRPV4 channel activation. LiESWT demonstrated potential in enhancing angiogenic remodeling, neurogenesis, and receptor response, ameliorating DHIC via TRPV channels and cellular signaling in the OHD-induced DHIC rat model.

Functional and structural insights into activation of TRPV2 by weak acids

Transient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO2 activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.

A phytosphingosine derivative mYG-II-6 inhibits histamine-mediated TRPV1 activation and MRGPRX2-dependent mast cell degranulation

BackgroundPhytosphingosine and its derivative are known for their skin-protective properties. While mYG-II-6, a phytosphingosine derivative, has shown anti-inflammatory and antipsoriatic effects, its potential antipruritic qualities have yet to be explored. This study aimed to investigate mYG-II-6′s antipruritic properties. MethodsThe calcium imaging technique was employed to investigate the activity of ion channels and receptors. Mast cell degranulation was confirmed through the β-hexosaminidase assay. Additionally, in silico molecular docking and an in vivo mouse scratching behavior test were utilized. ResultsUsing HEK293T cells transfected with H1R and TRPV1, we examined the impact of mYG-II-6 on histamine-induced intracellular calcium rise, a key signal in itch-mediating sensory neurons. Pretreatment with mYG-II-6 significantly reduced histamine-induced calcium levels and inhibited TRPV1 activity, suggesting its role in blocking the calcium influx channel. Additionally, mYG-II-6 suppressed histamine-induced calcium increase in primary cultures of mouse dorsal root ganglia, indicating its potential antipruritic effect mediated by histamine. Interestingly, mYG-II-6 exhibited inhibitory effects on human MRGPRX2, a G protein-coupled receptor involved in IgE-independent mast cell degranulation. However, it did not inhibit mouse MrgprB2, the ortholog of human MRGPRX2. Molecular docking analysis revealed that mYG-II-6 selectively interacts with the binding pocket of MRGPRX2. Importantly, mYG-II-6 suppressed histamine-induced scratching behaviors in mice. ConclusionsOur findings show that mYG-II-6 can alleviate histamine-induced itch sensation through dual mechanisms. This underscores its potential as a versatile treatment for various pruritic conditions.

Sens-ocular model: Cell-based assay to evaluate eye stinging potential of chemicals and baby cosmetic formulations.

The TRPV1 receptor, which is known to contribute significantly to pain perception, has recently been identified as a useful tool for predicting eye stinging potential in cosmetics. In this study, HEK-293 cells with high TRPV1 expression were utilized to evaluate calcium influx related to receptor activation triggered by chemicals and cosmetic formulations. The cells were exposed to increasing concentrations of substances to cause or not some aggression to the eye, and TRPV1 activity was assessed by measuring intracellular FURA-2 AM fluorescence signal. To confirm TRPV1 channel activation, capsazepine, a capsaicin antagonist, was employed in addition to using capsaicin as a positive control. The study's results indicate that this novel model can identify compounds known to cause some aggression to the eye, such as stinging, considering a cut-off value of 60% of Ca2+ influx exposed to the lowest evaluated concentration (0.00032%). When applied to the cosmetic baby formulation, although the presented model exhibited higher sensitivity by classifying as stinging formulations that had previously undergone clinical testing and were deemed non-stinging, the assay could serve as a valuable in vitro tool for predicting human eye stinging sensation and can be used as a tier 1 in an integrated testing strategy.