Cross-talk between macrophages and gut microbiota in inflammatory bowel disease: a dynamic interplay influencing pathogenesis and therapy.

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is a group of chronic immune-mediated gastrointestinal disorders. The etiology of IBD is multifactorial, involving genetic susceptibility, environmental factors, and a complex interplay between the gut microbiota and the host's immune system. Intestinal resident macrophages play an important role in the pathogenesis and progress of IBD, as well as in maintaining intestinal homeostasis and facilitating tissue repair. This review delves into the intricate relationship between intestinal macrophages and gut microbiota, highlighting their pivotal roles in IBD pathogenesis. We discuss the impact of macrophage dysregulation and the consequent polarization of different phenotypes on intestinal inflammation. Furthermore, we explore the compositional and functional alterations in gut microbiota associated with IBD, including the emerging significance of fungal and viral components. This review also examines the effects of current therapeutic strategies, such as 5-aminosalicylic acid (5-ASA), antibiotics, steroids, immunomodulators, and biologics, on gut microbiota and macrophage function. We underscore the potential of fecal microbiota transplantation (FMT) and probiotics as innovative approaches to modulate the gut microbiome in IBD. The aim is to provide insights into the development of novel therapies targeting the gut microbiota and macrophages to improve IBD management.

Thermosensing ability of TRPC5: current knowledge and unsettled questions.

Our understanding of how the mammalian somatosensory system detects noxious cold is still limited. While the role of TRPM8 in signaling mild non-noxious coolness is reasonably understood, the molecular identity of channels transducing painful cold stimuli remains unresolved. TRPC5 was originally described to contribute to moderate cold responses of dorsal root ganglia neurons in vitro, but mice lacking TRPC5 exhibited no change in behavioral responses to cold temperature. The question of why a channel endowed with the ability to be activated by cooling contributes to the cold response only under certain conditions is currently being intensively studied. It seems increasingly likely that the physiological detection of cold temperatures involves multiple different channels and mechanisms that modulate the threshold and intensity of perception. In this review, we aim to outline how TRPC5 may contribute to these mechanisms and what molecular features are important for its role as a cold sensor.

Sex differences in the orofacial antinociceptive effect of metformin and the role of transient receptor potential channels.

Metformin is classified as a biguanide and is used in the treatment of type 2 diabetes. It is used worldwide and has been investigated in drug repositioning. The present study aims to investigate whether there is sexual dimorphism in the orofacial antinociceptive effect of metformin and the participation of TRP channels. Acute nociceptive behavior was induced by administering cinnamaldehyde or capsaicin to the upper lip. Nociceptive behavior was assessed through orofacial rubbing, and the effects of pre-treatment with metformin (125 or 250 mg/Kg) or vehicle (control) were tested on the behavior. Nociceptive behavior was also induced by formalin injected into the temporomandibular joint. The chronic pain model involved infraorbital nerve transection (IONX) was evaluated using Von Frey electronic filaments. Trpv1 gene expression was analyzed in the nerve ganglion. Docking experiments were performed. Metformin, but not the vehicle, produced antinociception (p < 0.0001) in all acute nociceptive behaviors in both sexes, and these effects were attenuated by the TRPV1 antagonist capsazepine and the TRPA1 antagonist HC-030031. In IONX with better (**p < 0.01, ****p < 0.0001 vs. control) results in females. TRPV1 gene expression was observed in the metformin treated group (*p < 0.05 vs. control). Docking experiments revealed that metformin may interact with TRPV1 and TRPA1 channels. Metformin promotes orofacial antinociception in both sexes in acute pain and is more effective in chronic pain in females than in males, through the modulation of TRPV1 and TRPA1 channels. These preclinical findings suggest a potential repositioning of metformin as an analgesic agent in acute and chronic orofacial pain states.

TRP channels in cancer: Therapeutic opportunities and research strategies.

The influence of gut microbiota on transient receptor potential (TRP) channels has been identified as an important element in the development of gastrointestinal conditions, yet its involvement in cancer progression is not as thoroughly understood. This review explores the multifaceted roles of TRP channels in oncogenesis and emphasizes their significance in cancer progression and therapeutic outcomes. Critical focus was placed on the influence of traditional medicines, such as traditional Chinese medicine (TCM) related aromatic medicines, on TRP channel functions. Moreover, we explored the interplay between the gut microbiota and TRP channels in cancer signaling, highlighting the therapeutic potential of targeting this axis in cancer treatment. The impact of current therapies on TRP channel function was examined, demonstrating the need for a comprehensive understanding of how different modalities affect TRP channels in cancer. Technological advancements, including artificial intelligence (AI) tools and computer-aided drug development (CADD), have been discussed in the context of leveraging TRP channels for innovative cancer therapies. Future directions emphasize the potential applications of TRP channel research in advancing cancer treatment and enhancing patients' well-being.

Origami of KR-12 Designed Antimicrobial Peptides and Their Potential Applications.

This review describes the discovery, structure, activity, engineered constructs, and applications of KR-12, the smallest antibacterial peptide of human cathelicidin LL-37, the production of which can be induced under sunlight or by vitamin D. It is a moonlighting peptide that shows both antimicrobial and immune-regulatory effects. Compared to LL-37, KR-12 is extremely appealing due to its small size, lack of toxicity, and narrow-spectrum antimicrobial activity. Consequently, various KR-12 peptides have been engineered to tune peptide activity and stability via amino acid substitution, end capping, hybridization, conjugation, sidechain stapling, and backbone macrocyclization. We also mention recently discovered peptides KR-8 and RIK-10 that are shorter than KR-12. Nano-formulation provides an avenue to targeted delivery, controlled release, and increased bioavailability. In addition, KR-12 has been covalently immobilized on biomaterials/medical implants to prevent biofilm formation. These constructs with enhanced potency and stability are demonstrated to eradicate drug-resistant pathogens, disrupt preformed biofilms, neutralize endotoxins, and regulate host immune responses. Also highlighted are the safety and efficacy of these peptides in various topical and systemic animal models. Finaly, we summarize the achievements and discuss future developments of KR-12 peptides as cosmetic preservatives, novel antibiotics, anti-inflammatory peptides, and microbiota-restoring agents.

Inhibition of transient receptor potential vanilloid 3 channels by antimalarial hydroxychloroquine alleviates TRPV3-dependent dermatitis.

Transient receptor potential vanilloid 3 channel (TRPV3) is closely associated with skin inflammation, but there is a lack of effective and specific inhibitors for clinical use. In this study, we identified antimalarial hydroxychloroquine (HCQ) as a selective TRPV3 inhibitor following the prediction by network pharmacology data analysis. In whole-cell patch-clamp recordings, HCQ inhibited the current of the TRPV3 channel, with an IC50 of 51.69 ± 4.78 µM. At the single-channel level, HCQ reduced the open probability of TRPV3 and decreased single-channel conductance. Molecular docking and site-directed mutagenesis confirmed that residues in the pore domain were critical for the activity of HCQ. In vivo, HCQ effectively reduced carvacrol-induced epidermal thickening, erythema, and desquamation. Additionally, the serum immunoglobulin E and inflammatory factors such as tumor necrosis factor-α and interleukin-6 were markedly decreased in the dorsal skin tissues in the HCQ treatment group, as compared to the model group. Our results suggested the antimalarial HCQ may represent a potential alleviator for treating skin inflammation by inhibiting TRPV3 channels.

Flufenamic acid abolishes epileptiform activity in the entorhinal cortex slices by reducing the temporal summation of glutamatergic responses.

Flufenamic acid (FFA) is an anti-inflammatory drug that affects multiple targets and is a widely used research tool in ion channel studies. This pharmacological compound has a low level of selectivity for the transient receptor potential (TRP) channel superfamily, blocking calcium-activated nonselective cation current (ICAN) as well as afterdepolarizations (ADP) induced by it. A number of studies have demonstrated that FFA exerts an anti-epileptic effect in vitro, although the precise mechanism of this effect is not yet identified. The present study used whole-cell patch-clamp recordings and demonstrated that FFA (25 µM) can abolish the generation of seizure-like events (SLE) in entorhinal cortex slices perfused with a 4-aminopyridine-containing solution, depending on the time of application. FFA decreased the temporal summation of synaptic potentials at the onset of SLEs. However, as the epileptiform activity evolved and the SLE onset phase became more abrupt, the blocking effect of FFA diminished. FFA effectively abolished TRP channel-mediated slow ADPs, exerted a weak blockade and slowed the kinetics of GABAa receptor-mediated currents, and did not affect NMDA receptor-mediated evoked currents induced by extracellular stimulation. Although FFA did not directly inhibit NMDA receptor-mediated evoked currents, it decreased the summation of NMDA receptor-mediated potentials in a manner comparable to its effect on the initiation phase of SLE. This suggests that ICAN blockade may be responsible for this effect. Furthermore, our results showed that the selective blocker of melastatin TRP channels (TRPM4) 9-phenanthrol effectively abolished epileptiform activity in a manner analogous to FFA. In contrast, ML-204, the blocker of canonical TRP channels (TRPC), had no discernible effect on this phenomenon. In conclusion, the study demonstrate that FFA abolishes epileptiform activity in the entorhinal cortex by blocking TRPM4 channels and, consequently, decreasing the effectiveness of temporal summation of glutamatergic potentials.

Lysosomal TFEB-TRPML1 Axis in Astrocytes Modulates Depressive-like Behaviors.

Lysosomes are important cellular structures for human health as centers for recycling, signaling, metabolism and stress adaptation. However, the potential role of lysosomes in stress-related emotions has long been overlooked. Here, it is found that lysosomal morphology in astrocytes is altered in the medial prefrontal cortex (mPFC) of susceptible mice after chronic social defeat stress. A screen of lysosome-related genes revealed that the expression of the mucolipin 1 gene (Mcoln1; protein: mucolipin TRP channel 1) is decreased in susceptible mice and depressed patients. Astrocyte-specific knockout of mucolipin TRP channel 1 (TRPML1) induced depressive-like behaviors by inhibiting lysosomal exocytosis-mediated adenosine 5'-triphosphate (ATP) release. Furthermore, this stress response of astrocytic lysosomes is mediated by the transcription factor EB (TFEB), and overexpression of TRPML1 rescued depressive-like behaviors induced by astrocyte-specific knockout of TFEB. Collectively, these findings reveal a lysosomal stress-sensing signaling pathway contributing to the development of depression and identify the lysosome as a potential target organelle for antidepressants.

Evaluation of SOX-10 immunohistochemical expression in canine melanoma and non-melanocytic tumors by tissue microarray.

Melanoma is the most common malignant oral tumor in dogs. It frequently presents a diagnostic challenge as many melanomas lack or contain scant melanin and may have a variable microscopic phenotype. Previous studies evaluating immunohistochemical markers for diagnosing melanoma have shown limited sensitivity and/or specificity for S-100, PNL2, melan A, TRP-1, TRP-2, and HMB-45. Sry-related HMG-box gene 10 (SOX-10) is a transcription factor associated with melanocytic, peripheral neural crest, and peripheral nervous system development. In humans, SOX-10 expression has been demonstrated in melanoma, breast carcinoma, glioma, and schwannoma, but has only recently been explored in veterinary species. In this study, 198 tumors comprised of 147 melanocytic neoplasms and 51 non-melanocytic neoplasms were evaluated by immunohistochemistry using a tissue microarray for SOX-10, PNL2, melan A, TRP-1, and TRP-2 expressions. The SOX-10 had the highest diagnostic sensitivity (96.7%) in melanomas. In addition, SOX-10 had the highest percentage (91.5%; 130/142) of melanomas label at least 75% of neoplastic cells. Of the 51 selected non-melanocytic tumors examined, SOX-10 labeling was observed in mammary carcinomas (6/6), gliomas (4/4), and oral soft tissue sarcomas (4/18). Of the 41 non-melanocytic oral neoplasms evaluated, SOX-10 had a specificity of 92.7%. Therefore, SOX-10 represents a useful immunohistochemical screening marker for the diagnosis of canine melanoma given its extremely high sensitivity and robust labeling intensity. The SOX-10 may have utility in diagnosing some non-melanocytic neoplasms in the dog, although this requires further investigation.

TRPM3, TRPM4, and TRPM5 as thermo-sensitive channels.

Temperature detection is essential for the survival and perpetuation of any species. Thermoreceptors in the skin sense body temperature as well as the temperatures of ambient air and objects. Since Dr. David Julius and his colleagues discovered that TRPV1 is expressed in small-diameter primary sensory neurons, and activated by temperatures above 42 °C, 11 of thermo-sensitive TRP channels have been identified. TRPM3 expressed in sensory neurons acts as a sensor for noxious heat. TRPM4 and TRPM5 are Ca2⁺-activated monovalent cation channels, and their activity is drastically potentiated by temperature increase. This review aims to summarize the expression patterns, electrophysiological properties, and physiological roles of TRPM3, TRPM4, and TRPM5 associated with thermosensation.

Thermosensation and TRP Channels.

Somatosensory neurons can sense external temperature by converting sensation of temperature information to neural activity via afferent input to the central nervous system. Various populations of somatosensory neurons have specialized gene expression, including expression of thermosensitive transient receptor potential (TRP) ion channels. Thermosensitive TRP channels are responsible for thermal transduction at the peripheral ends of somatosensory neurons and can sense a wide range of temperatures. Here we focus on several thermosensitive TRP channels including TRPV1, TRPV4, TRPM2, TRPM3, TRPM8, TRPC5, and TRPA1 in sensory neurons. TRPV3, TRPV4, and TRPC5 are also involved in somatosensation in nonneuronal cells and tissues. In particular, we discuss whether skin senses ambient temperatures through TRPV3 and TRPV4 activation in skin keratinocytes and the involvement of TRPM2 expressed by hypothalamic neurons in thermosensation in the brain.

Temperature-Dependent Activation of Thermosensitive Transient Receptor Potential Channels.

Temperature detection is essential for the survival and perpetuation of any species. Thermoreceptors in the skin sense the body temperature and also the temperatures of the ambient air and the objects. In 1997, Dr. David Julius and his colleagues found that a receptor expressed in small-diameter primary sensory neurons was activated by capsaicin (the pungent chemical in hot pepper). This receptor was also activated by temperature above 42 °C. That was the first time that a thermal receptor in primary sensory neurons has been identified. This receptor is named transient receptor potential vanilloid 1 (TRPV1). Now, 11 thermosensitive TRP channels are known. In this chapter, we summarize the reports and analyze thermosensitive TRP channels in a variety of ways to clarify the activation mechanisms by which temperature changes are sensed.

TRPV1 corneal neuralgia mutation: Enhanced pH response, bradykinin sensitization, and capsaicin desensitization.

The factors that contribute to pain after nerve injury remain incompletely understood. Laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) are common surgical techniques to correct refractive errors. After LASIK or PRK, a subset of patients suffers intense and persistent pain, of unknown origin, described by patients as feeling like shards of glass in their eye. Here, we evaluated a TRPV1 variant, p.V527M, found in a 49-y-old woman who developed corneal pain after LASIK and subsequent PRK enhancement, reporting an Ocular Surface Disease Index score of 100. Using patch-clamp and Ca2+ imaging, we found that the V527M mutation enhances the response to acidic pH. Increasing proton concentration induced a stronger leftward shift in the activation curve of V527M compared to WT, resulting in channel activity of the mutant in acidic pH at more physiological membrane potentials. Finally, comparing the responses to consecutive applications of different agonists, we found in V527M channels a reduced capsaicin-induced desensitization and increased sensitization by the arachidonic acid metabolite 12-hydroxyeicosatetraenoic acid (12-HETE). We hypothesize that the increased response in V527M channels to protons and enhanced sensitization by 12-HETE, two inflammatory mediators released in the cornea after tissue damage, may contribute to the pathogenesis of corneal neuralgia after refractive surgery.

TRPA1-Related Diseases and Applications of Nanotherapy.

Transient receptor potential (TRP) channels, first identified in Drosophila in 1969, are multifunctional ion channels expressed in various cell types. Structurally, TRP channels consist of six membrane segments and are classified into seven subfamilies. Transient receptor potential ankyrin 1 (TRPA1), the first member of the TRPA family, is a calcium ion affinity non-selective cation channel involved in sensory transduction and responds to odors, tastes, and chemicals. It also regulates temperature and responses to stimuli. Recent studies have linked TRPA1 to several disorders, including chronic pain, inflammatory diseases, allergies, and respiratory problems, owing to its activation by environmental toxins. Mutations in TRPA1 can affect the sensory nerves and microvasculature, potentially causing nerve pain and vascular problems. Understanding the function of TRPA1 is important for the development of treatments for these diseases. Recent developments in nanomedicines that target various ion channels, including TRPA1, have had a significant impact on disease treatment, providing innovative alternatives to traditional disease treatments by overcoming various adverse effects.

A proteomics-based study of the mechanism of oxymatrine to ameliorate hepatic fibrosis in mice.

OBJECTIVE: This study investigated the protective effect of oxymatrine (OMT) on carbon tetrachloride (CCl4)-induced hepatic fibrosis in mice and explored its possible targets and signaling pathways. METHODS: Male BALB/c mice were randomly divided into blank control, model, positive drug (silymarin), and OMT administration groups, respectively, with 10 mice in each group. Hepatic fibrosis was induced in mice using CCl4 and the corresponding drug intervention was given. After the final administration, ultrasonography tests, blood tests, and analysis of liver differential proteins using tandem mass tag labeling and liquid chromatography-mass spectrometry were performed. RESULTS: OMT intervention ameliorated CCl4-induced hepatic fibrosis in mice, significantly reduced serum alanine aminotransferase and aspartate aminotransferase levels, down-regulated the expression of fibrosis factors, such as type IV collagen IV, laminin, type III procollagen III, and alpha-smooth muscle actin, and improved liver function. The results of the proteomic analysis showed that the intervention of OMT significantly down-regulated 130 out of 440 up-regulated proteins and up-regulated 70 out of 294 down-regulated proteins, primarily involving the transient receptor potential (TRP) signaling pathway, the peroxisome proliferator-activated receptors (PPAR) signaling pathway, and the metabolic pathway of arachidonic acid. The main differential proteins involved were Cyp2c37, SCP-2, and Tbxas1. In addition, OMT intervention significantly reversed the expression of sterol carrier protein-2 (SCP2) and upregulated the expression of peroxisome proliferator-activated receptor gamma, Cyp2c37, and transient receptor potential cation channel subfamily V member 1 proteins. CONCLUSION: OMT inhibited the proliferative capacity of hepatic stellate cells, induced apoptotic properties, and suppressed the development of fibrosis by elevating Cyp2c37/TRP signaling axis activity and upregulating PPAR pathway activity by inhibiting SCP2.

Dementia and neurodegenerative diseases: What is known and what is promising at the cellular and molecular level.

Millions of people worldwide are affected by neurodegenerative diseases and cognitive impairment, which includes dementia, while there are only symptomatic treatments available for this syndrome at present. However, several important prospective drug targets have been identified in recent years that can potentially arrest or even reverse the progression of neurodegenerative diseases. Their natural or synthetic ligands are currently in the experimental stage of drug development. In vitro and preclinical (e.g. using animal models) studies confirm their therapeutic potential, but clinical trials often fail or produce conflicting results. Here, we first review the complexity and typology of dementia, followed by the discussion of currently available treatments, and, finally, some novel molecular and cellular approaches to this problem.

Cathepsin B-Activatable Bioactive Peptide Nanocarrier for High-Efficiency Immunotherapy of Asthma.

Introduction: Asthma, a chronic respiratory disease closely associated with inflammation, presents ongoing treatment challenges. IALLIPF (le-Ala-Leu-Leu-Ile-Pro-Phe) is one of millet prolamins peptides (MPP) which shows anti-oxidant bioactivity by reducing the production of reactive oxygen species (ROS). Tryptophan (Trp, W) is an amino acid that has been demonstrated to possess anti-inflammatory effects. We introduce a novel cathepsin B-activatable bioactive peptides nanocarrier, PEG-IALLIPF-GFLG-W (MPP-Trp), designed for immunotherapy of asthma. Methods: MPP-Trp is synthesized, purified, and its characteristics are investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The yield of nitric oxide (NO) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß) are examined to evaluate anti-inflammatory effects of IALLIPF, Trp and MPP-Trp. The immunomodulatory effects of IALLIPF, Trp and MPP-Trp on Th1/Th2 cell populations and cytokines are investigated by flow cytometry, qRT-PCR and ELISA assays. We explore the therapeutic effect of MPP-Trp in the mouse model of asthma by the analysis of lung histology and ELISA. It is necessary to study the biocompatibility of MPP-Trp by CCK8 assay and histopathologic analysis using hematoxylin and eosin (HE) staining. Results: In asthmatic peripheral blood mononuclear cells (PBMCs), IALLIPF, Trp and MPP-Trp are able to significantly alleviate inflammation by inhibiting the yield of nitric oxide (NO) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß), especially MPP-Trp. MPP-Trp significantly upregulates Th1 cell levels while notably reducing Th2 cell levels. Furthermore, MPP-Trp effectively elevates the expression and production of interferon-gamma (IFN-γ), an essential cytokine from Th1 cells. Additionally, MPP-Trp markedly diminishes the mRNA expression and levels of key asthma pathogenesis cytokines, such as interleukin-4 (IL-4), interleukin-13 (IL-13), and interleukin-5 (IL-5), in asthma PBMCs. MPP-Trp ameliorates pulmonary pathological alterations and significantly inhibits OVA-induced inflammation in mice with asthma. It has little influence on the cell viability in Asthma-PBMCs treated with various concentrations or durations of MPP-Trp. No pathological changes, including in the heart, liver, spleen, lung, and kidney tissues, are observed in non-sensitized and non-challenged mice treated with MPP-Trp (20 mg/kg). Discussion: Our research demonstrates that MPP-Trp has immunomodulatory effects on Th1/Th2 cell populations, essential in managing asthma. It considerably alleviates OVA-induced asthma by shifting the immune response towards a Th1-dominant profile, thereby reducing Th2-driven inflammation. Therefore, this novel bioactive peptide nanocarrier, MPP-Trp, holds promise as a candidate for asthma immunotherapy.

Kynurenic acid protects against ischemia/reperfusion injury by modulating apoptosis in cardiomyocytes.

Acute myocardial infarction, often associated with ischemia/reperfusion injury (I/R), is a leading cause of death worldwide. Although the endogenous tryptophan metabolite kynurenic acid (KYNA) has been shown to exert protection against I/R injury, its mechanism of action at the cellular and molecular level is not well understood yet. Therefore, we examined the potential involvement of antiapoptotic mechanisms, as well as N-methyl-D-aspartate (NMDA) receptor modulation in the protective effect of KYNA in cardiac cells exposed to simulated I/R (SI/R). KYNA was shown to attenuate cell death induced by SI/R dose-dependently in H9c2 cells or primary rat cardiomyocytes. Analysis of morphological and molecular markers of apoptosis (i.e., membrane blebbing, apoptotic nuclear morphology, DNA double-strand breaks, activation of caspases) revealed considerably increased apoptotic activity in cardiac cells undergoing SI/R. The investigated apoptotic markers were substantially improved by treatment with the cytoprotective dose of KYNA. Although cardiac cells were shown to express NMDA receptors, another NMDA antagonist structurally different from KYNA was unable to protect against SI/R-induced cell death. Our findings provide evidence that the protective effect of KYNA against SI/R-induced cardiac cell injury involves antiapoptotic mechanisms, that seem to evoke independently of NMDA receptor signaling.

Dynamic remodeling of TRPC5 channel-caveolin-1-eNOS protein assembly potentiates the positive feedback interaction between Ca2+ and NO signals.

The cell signaling molecules nitric oxide (NO) and Ca2+ regulate diverse biological processes through their closely coordinated activities directed by signaling protein complexes. However, it remains unclear how dynamically the multicomponent protein assemblies behave within the signaling complexes upon the interplay between NO and Ca2+ signals. Here we demonstrate that TRPC5 channels activated by the stimulation of G-protein-coupled ATP receptors mediate Ca2+ influx, that triggers NO production from endothelial NO synthase (eNOS), inducing secondary activation of TRPC5 via cysteine S-nitrosylation and eNOS in vascular endothelial cells. Mutations in the caveolin-1-binding domains of TRPC5 disrupt its association with caveolin-1 and impair Ca2+ influx and NO production, suggesting that caveolin-1 serves primarily as the scaffold for TRPC5 and eNOS to assemble into the signal complex. Interestingly, during ATP receptor activation, eNOS is dissociated from caveolin-1 and in turn directly associates with TRPC5, which accumulates at the plasma membrane dependently on Ca2+ influx and calmodulin. This protein reassembly likely results in a relief of eNOS from the inhibitory action of caveolin-1 and an enhanced TRPC5 S-nitrosylation by eNOS localized in the proximity, thereby facilitating the secondary activation of Ca2+ influx and NO production. In isolated rat aorta, vasodilation induced by acetylcholine was significantly suppressed by the TRPC5 inhibitor AC1903. Thus, our study provides evidence that dynamic remodeling of the protein assemblies among TRPC5, eNOS, caveolin-1, and calmodulin determines the ensemble of Ca2+ mobilization and NO production in vascular endothelial cells.