Innervation of nociceptor neurons in the spleen promotes germinal center responses and humoral immunity.

Peripheral sensory neurons widely innervate various tissues to continuously monitor and respond to environmental stimuli. Whether peripheral sensory neurons innervate the spleen and modulate splenic immune response remains poorly defined. Here, we demonstrate that nociceptive sensory nerve fibers extensively innervate the spleen along blood vessels and reach B cell zones. The spleen-innervating nociceptors predominantly originate from left T8-T13 dorsal root ganglia (DRGs), promoting the splenic germinal center (GC) response and humoral immunity. Nociceptors can be activated by antigen-induced accumulation of splenic prostaglandin E2 (PGE2) and then release calcitonin gene-related peptide (CGRP), which further promotes the splenic GC response at the early stage. Mechanistically, CGRP directly acts on B cells through its receptor CALCRL-RAMP1 via the cyclic AMP (cAMP) signaling pathway. Activating nociceptors by ingesting capsaicin enhances the splenic GC response and anti-influenza immunity. Collectively, our study establishes a specific DRG-spleen sensory neural connection that promotes humoral immunity, suggesting a promising approach for improving host defense by targeting the nociceptive nervous system.

Sensational channels.

This year's Nobel Prize in Physiology or Medicine was awarded to David Julius and Ardem Patapoutian for "explaining the molecular basis for sensing heat, cold and mechanical force." Their findings capped off a scientific quest to identify the mechanisms within the somatosensory system mediating the detection of internal and external environments.

Hypothalamic Agrp neurons drive stereotypic behaviors beyond feeding.

The nervous system evolved to coordinate flexible goal-directed behaviors by integrating interoceptive and sensory information. Hypothalamic Agrp neurons are known to be crucial for feeding behavior. Here, however, we show that these neurons also orchestrate other complex behaviors in adult mice. Activation of Agrp neurons in the absence of food triggers foraging and repetitive behaviors, which are reverted by food consumption. These stereotypic behaviors that are triggered by Agrp neurons are coupled with decreased anxiety. NPY5 receptor signaling is necessary to mediate the repetitive behaviors after Agrp neuron activation while having minor effects on feeding. Thus, we have unmasked a functional role for Agrp neurons in controlling repetitive behaviors mediated, at least in part, by neuropeptidergic signaling. The findings reveal a new set of behaviors coupled to the energy homeostasis circuit and suggest potential therapeutic avenues for diseases with stereotypic behaviors.

Aching to defend: spleen innervation drives humoral immunity.

The immune and sensory nervous systems communicate to maintain homeostasis. Wu et al. recently demonstrated that sensory neurons innervate the mouse spleen. These neurons promote calcitonin gene-related peptide (CGRP)-dependent responses in splenic B cell germinal centers (GCs) and antigen-specific antibody production. Dietary capsaicin activates these neurons to enhance humoral immunity against influenza virus infection.

Nociceptive nerves regulate haematopoietic stem cell mobilization.

Haematopoietic stem cells (HSCs) reside in specialized microenvironments in the bone marrow-often referred to as 'niches'-that represent complex regulatory milieux influenced by multiple cellular constituents, including nerves1,2. Although sympathetic nerves are known to regulate the HSC niche3-6, the contribution of nociceptive neurons in the bone marrow remains unclear. Here we show that nociceptive nerves are required for enforced HSC mobilization and that they collaborate with sympathetic nerves to maintain HSCs in the bone marrow. Nociceptor neurons drive granulocyte colony-stimulating factor (G-CSF)-induced HSC mobilization via the secretion of calcitonin gene-related peptide (CGRP). Unlike sympathetic nerves, which regulate HSCs indirectly via the niche3,4,6, CGRP acts directly on HSCs via receptor activity modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CALCRL) to promote egress by activating the Gαs/adenylyl cyclase/cAMP pathway. The ingestion of food containing capsaicin-a natural component of chili peppers that can trigger the activation of nociceptive neurons-significantly enhanced HSC mobilization in mice. Targeting the nociceptive nervous system could therefore represent a strategy to improve the yield of HSCs for stem cell-based therapeutic agents.

The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4⁺ T cells.

TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.

Sickle cell disease iPSC-derived sensory neurons exhibit increased excitability and sensitization to patient plasma.

ABSTRACT: Individuals living with sickle cell disease (SCD) experience severe recurrent acute and chronic pain. Challenges to gaining mechanistic insight into pathogenic SCD pain processes include differential gene expression and function of sensory neurons between humans and mice with SCD, and extremely limited availability of neuronal tissues from patients with SCD. Here, we used induced pluripotent stem cells (iPSCs), derived from patients with SCD, differentiated into sensory neurons (SCD iSNs) to begin to overcome these challenges. We characterize key gene expression and function of SCD iSNs to establish a model to investigate intrinsic and extrinsic factors that may contribute to SCD pain. Despite similarities in receptor gene expression, SCD iSNs show pronounced excitability using patch clamp electrophysiology. Furthermore, we find that plasma taken from patients with SCD during acute pain associated with a vaso-occlusive event increases the calcium responses to the nociceptive stimulus capsaicin in SCD iSNs compared with those treated with paired plasma from patients with SCD at steady state baseline or healthy control plasma samples. We identified high levels of the polyamine spermine in baseline and acute pain states of plasma from patients with SCD, which sensitizes SCD iSNs to subthreshold concentrations of capsaicin. Together, these data identify potential intrinsic mechanisms within SCD iSNs that may extend beyond a blood-based pathology.

Antithetic effects of agonists and antagonists on the structural fluctuations of TRPV1 channel.

Transient receptor potential vanilloid member 1 (TRPV1) is a heat and capsaicin receptor that allows cations to permeate and cause pain. As the molecular basis for temperature sensing, the heat capacity (ΔCp) model [D. E. Clapham, C. Miller, Proc. Natl. Acad. Sci. U.S.A. 108, 19492-19497 (2011).] has been proposed and experimentally supported. Theoretically, heat capacity is proportional to a variance in enthalpy, presumably related to structural fluctuation; however, the fluctuation of TRPV1 has not been directly visualized. In this study, we directly visualized single-molecule structural fluctuations of the TRPV1 channels in a lipid bilayer with the ligands resiniferatoxin (agonist, 1,000 times hotter than capsaicin) and capsazepine (antagonist) by high-speed atomic force microscopy. We observed the structural fluctuations of TRPV1 in an apo state and found that RTX binding enhances structural fluctuations, while CPZ binding suppresses fluctuations. These ligand-dependent differences in structural fluctuation would play a key role in the gating of TRPV1.

TRPV1 activation in human Langerhans cells and T cells inhibits mucosal HIV-1 infection via CGRP-dependent and independent mechanisms.

Upon its mucosal transmission, HIV type 1 (HIV-1) rapidly targets genital antigen-presenting Langerhans cells (LCs), which subsequently transfer infectious virus to CD4+ T cells. We previously described an inhibitory neuroimmune cross talk, whereby calcitonin gene-related peptide (CGRP), a neuropeptide secreted by peripheral pain-sensing nociceptor neurons innervating all mucosal epithelia and associating with LCs, strongly inhibits HIV-1 transfer. As nociceptors secret CGRP following the activation of their Ca2+ ion channel transient receptor potential vanilloid 1 (TRPV1), and as we reported that LCs secret low levels of CGRP, we investigated whether LCs express functional TRPV1. We found that human LCs expressed mRNA and protein of TRPV1, which was functional and induced Ca2+ influx following activation with TRPV1 agonists, including capsaicin (CP). The treatment of LCs with TRPV1 agonists also increased CGRP secretion, reaching its anti-HIV-1 inhibitory concentrations. Accordingly, CP pretreatment significantly inhibited LCs-mediated HIV-1 transfer to CD4+ T cells, which was abrogated by both TRPV1 and CGRP receptor antagonists. Like CGRP, CP-induced inhibition of HIV-1 transfer was mediated via increased CCL3 secretion and HIV-1 degradation. CP also inhibited direct CD4+ T cells HIV-1 infection, but in CGRP-independent manners. Finally, pretreatment of inner foreskin tissue explants with CP markedly increased CGRP and CCL3 secretion, and upon subsequent polarized exposure to HIV-1, inhibited an increase in LC-T cell conjugate formation and consequently T cell infection. Our results reveal that TRPV1 activation in human LCs and CD4+ T cells inhibits mucosal HIV-1 infection, via CGRP-dependent/independent mechanisms. Formulations containing TRPV1 agonists, already approved for pain relief, could hence be useful against HIV-1.

Acute Ongoing Nociception Delays Recovery of Consciousness from Sevoflurane Anesthesia via a Midbrain Circuit.

Although anesthesia provides favorable conditions for surgical procedures, recent studies have revealed that the brain remains active in processing noxious signals even during anesthesia. However, whether and how these responses affect the anesthesia effect remains unclear. The ventrolateral periaqueductal gray (vlPAG), a crucial hub for pain regulation, also plays an essential role in controlling general anesthesia. Hence, it was hypothesized that the vlPAG may be involved in the regulation of general anesthesia by noxious stimuli. Here, we found that acute noxious stimuli, including capsaicin-induced inflammatory pain, acetic acid-induced visceral pain, and incision-induced surgical pain, significantly delayed recovery from sevoflurane anesthesia in male mice, whereas this effect was absent in the spared nerve injury-induced chronic pain. Pretreatment with peripheral analgesics could prevent the delayed recovery induced by acute nociception. Furthermore, we found that acute noxious stimuli, induced by the injection of capsaicin under sevoflurane anesthesia, increased c-Fos expression and activity in the GABAergic neurons of the ventrolateral periaqueductal gray. Specific reactivation of capsaicin-activated vlPAGGABA neurons mimicked the effect of capsaicin and its chemogenetic inhibition prevented the delayed recovery from anesthesia induced by capsaicin. Finally, we revealed that the vlPAGGABA neurons regulated the recovery from anesthesia through the inhibition of ventral tegmental area dopaminergic neuronal activity, thus decreasing dopamine (DA) release and activation of DA D1-like receptors in the brain. These findings reveal a novel, cell- and circuit-based mechanism for regulating anesthesia recovery by nociception, and it is important to provide new insights for guiding the management of the anesthesia recovery period.

An evolutionary view of vanillylamine synthase pAMT, a key enzyme of capsaicinoid biosynthesis pathway in chili pepper.

Pungent capsaicinoid is synthesized only in chili pepper (Capsicum spp.). The production of vanillylamine from vanillin is a unique reaction in the capsaicinoid biosynthesis pathway. Although putative aminotransferase (pAMT) has been isolated as the vanillylamine synthase gene, it is unclear how Capsicum acquired pAMT. Here, we present a phylogenetic overview of pAMT and its homologs. The Capsicum genome contained 5 homologs, including pAMT, CaGABA-T1, CaGABA-T3, and two pseudogenes. Phylogenetic analysis indicated that pAMT is a member of the Solanaceae cytoplasmic GABA-Ts. Comparative genome analysis found that multiple copies of GABA-T exist in a specific Solanaceae genomic region, and the cytoplasmic GABA-Ts other than pAMT are located in the region. The cytoplasmic GABA-T was phylogenetically close to pseudo-GABA-T harboring a plastid transit peptide (pseudo-GABA-T3). This suggested that Solanaceae cytoplasmic GABA-Ts occurred via duplication of a chloroplastic GABA-T ancestor and subsequent loss of the plastid transit signal. The cytoplasmic GABA-T may have been translocated from the specific Solanaceae genomic region during Capsicum divergence, resulting in the current pAMT locus. A recombinant protein assay demonstrated that pAMT had higher vanillylamine synthase activity than those of other plant GABA-Ts. pAMT was expressed exclusively in the placental septum of mature green fruit, whereas tomato orthologs SlGABA-T2/4 exhibit a ubiquitous expression pattern in plants. These findings suggested that both the increased catalytic efficiency and transcriptional changes in pAMT may have contributed to establish vanillylamine synthesis in the capsaicinoid biosynthesis pathway. This study provides insights into the establishment of pungency in the evolution of chili peppers.

How side effects can improve treatment efficacy: a randomized trial.

While treatment side effects may adversely impact patients, they could also potentially function as indicators for effective treatment. In this study, we investigated whether and how side effects can trigger positive treatment expectations and enhance treatment outcomes. In this pre-registered trial (DRKS00026648), 77 healthy participants were made to believe that they will receive fentanyl nasal sprays before receiving thermal pain in a controlled experimental setting. However, nasal sprays did not contain fentanyl, rather they either contained capsaicin to induce a side effect (mild burning sensation) or saline (inert). After the first session, participants were randomized to two groups and underwent functional MRI. One group continued to believe that the nasal sprays could contain fentanyl while the other group was explicitly informed that no fentanyl was included. This allowed for the independent manipulation of the side effects and the expectation of pain relief. Our results revealed that nasal sprays with a side effect lead to lower pain than inert nasal sprays without side effects. The influence of side effects on pain was dependent on individual beliefs about how side effects are related to treatment outcome, as well as on expectations about received treatment. Functional MRI data indicated an involvement of the descending pain modulatory system including the anterior cingulate cortex and the periaqueductal gray during pain after experiencing a nasal spray with side effects. In summary, our data show that mild side effects can serve as a signal for effective treatment thereby influencing treatment expectations and outcomes, which is mediated by the descending pain modulatory system. Using these mechanisms in clinical practice could provide an efficient way to optimize treatment outcome. In addition, our results indicate an important confound in clinical trials, where a treatment (with potential side effects) is compared to placebo.

Genetically-encoded BRET probes shed light on ligand bias-induced variable ion selectivity in TRPV1 and P2X5/7.

Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.

How much does TRPV1 deviate from an ideal MWC-type protein?

Many ion channels are known to behave as an allosteric protein, coupling environmental stimuli captured by specialized sensing domains to the opening of a central pore. The classic Monod-Wyman-Changeux (MWC) model, originally proposed to describe binding of gas molecules to hemoglobin, has been widely used as a framework for analyzing ion channel gating. Here, we address the issue of how accurately the MWC model predicts activation of the capsaicin receptor TRPV1 by vanilloids. Taking advantage of a concatemeric design that makes it possible to lock TRPV1 in states with zero to four bound vanilloid molecules, we showed quantitatively that the overall gating behavior is satisfactorily predicted by the MWC model. There is, however, a small yet detectable subunit position effect: ligand binding to two kitty-corner subunits is 0.3-0.4 kcal/mol more effective in inducing opening than binding to two neighbor subunits. This difference-less than 10% of the overall energetic contribution from ligand binding-might be due to the restriction on subunit arrangement imposed by the planar membrane; if this is the case, then the position effect is not expected in hemoglobin, in which each subunit is related equivalently to all the other subunits.

Capsaicin enhances cisplatin-induced anti-metastasis of nasopharyngeal carcinoma by inhibiting EMT and ERK signaling via serpin family B member 2.

Cisplatin (DDP)-based combined chemotherapy or concurrent chemoradiotherapy is the mainstay treatment for advanced-stage nasopharyngeal carcinoma (NPC), but needs improvement due to its severe side effects. Capsaicin (CAP) can enhance the anti-tumor activity of cytotoxic drugs. The aim of this study was to investigate the anti-metastasis activity of CAP in combination with DDP in NPC. Herein, CAP and DDP showed synergistic cytotoxic effects on NPC cells. CAP alone and DDP alone inhibited NPC migration and invasion in vitro and in vivo, and the combination of CAP and DDP had the greatest effect. Moreover, CAP upregulated the mRNA and protein expressions of serpin family B member 2 (SERPINB2). Further results showed that both SERPINB2 mRNA and protein expressions were downregulated in NPC cell lines and tissues and SERPINB2 overexpression inhibited NPC migration and invasion in vitro and in vivo, while silencing SERPINB2 acted oppositely. In addition, SERPINB2 was abnormally expressed in head and neck squamous cell carcinoma and other multiple cancers, and downregulation of SERPINB2 predicted poor prognosis in head and neck squamous cell carcinoma according to the Cancer Genome Atlas database. We further found that SERPINB2 overexpression inhibited epithelial-mesenchymal transition (EMT) and the phosphorylated extracellular signal-regulated kinase (p-ERK), and the inhibitory effect was enhanced by CAP and DDP. Altogether, our results suggest that the combined inhibition of CAP and DDP on NPC metastasis may be related to the inhibition of epithelial-mesenchymal transition and ERK signals mediated by SERPINB2, and CAP may help to improve the efficacy of DDP in the treatment of NPC and develop new therapeutic approaches.

Capsiate ameliorates secondary hyperparathyroidism by improving insulin sensitivity and inhibiting angiogenesis.

Secondary hyperparathyroidism has a significant impact on the overall well-being of the body. Capsiates, known for their antioxidant and metabolic properties, have emerged as a promising alternative treatment for secondary hyperparathyroidism. This study aims to evaluate the effects and mechanisms of capsiates in the treatment of secondary hyperparathyroidism. To achieve our research objectives, we conducted a study on patients' serum and examined changes in metabolic markers using serum metabolomics. We induced secondary hyperparathyroidism in rat through dietary intervention and divided them into four groups. The first group, referred to as the Parathyroid Hormone (PTH) group, received a low-calcium and high-phosphate diet (0.2% calcium, 1.2% phosphorus). The second group served as the control group, receiving a standard phosphate and calcium diet (0.6% calcium, 0.6% phosphorus). The third group, called the capsiates group, consisted of rat from the control group treated with capsiates (intraperitoneal injection of 2 mg/kg capsiates for 2 weeks after 2 weeks of dietary intervention). The fourth group was the capsiates-treated PTH group. Subsequently, we conducted ribose nucleic acid (RNA) sequencing on parathyroid gland cells and evaluated serum thyroxine levels, oxidative stress, expression of proteins associated with vascular neogenesis, measurement of SOD, GSH and 3-nitrotyrosine, micro-CT and histological staining. The serum metabolomic data revealed a significant decrease in capsiate levels in the secondary hyperparathyroidism group. Administration of capsiates to PTH rat resulted in increased calcium levels compared to the PTH group. Additionally, the PTH + Capsiates group showed significantly lower levels of PTH and phosphate compared to the PTH group. The PTH group exhibited a notable increase in the quantity and size of mitochondria compared to the control group. Following capsiates administration to the PTH group, there was a significant reduction in the number of mitochondria and length of microvilli, but an increase in the size of mitochondria compared to the PTH group. Sequencing analysis revealed that vascular endothelial growth factor (VEGF) and Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) play crucial roles in this process. Vascular-related variables and downstream signalling were significantly elevated in hyperthyroidism and were alleviated with capsaicin treatment. Finally, combining capsiates with the PTH group improved bone mineral density, Tb.N, BV.TV, Cs.Th, Tt.Ar, OPG, Ob.TV and Oc.TV, as well as the mineral apposition rate, but significantly decreased Tb.Sp and Receptor Activator for Nuclear Factor-κ B Ligand (RANKL) compared to the PTH group. The findings suggest that capsiates can improve secondary hyperparathyroidism and ameliorated osteoporosis outcomes by inhibiting angiogenesis and reducing oxidative stress.

Contribution of transient receptor potential vanilloid 1 (TRPV1) channel to cholinergic contraction of rat bladder smooth muscle.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel that is gated by the pungent constituent of red chili pepper, capsaicin, and by related chemicals from the group of vanilloids, in addition to noxious heat. It is expressed mostly in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Although TRPV1 is also found outside the sensory nervous system, its expression and function in the bladder detrusor smooth muscle (DSM) remain controversial. Here, by using Ca2+ imaging and patch clamp on isolated rat DSM cells, in addition to tensiometry on multicellular DSM strips, we show that TRPV1 is expressed functionally in only a fraction of DSM cells, in which it acts as an endoplasmic reticulum Ca2+-release channel responsible for the capsaicin-activated [Ca2+]i rise. Carbachol-stimulated contractions of multicellular DSM strips contain a TRPV1-dependent component, which is negligible in the circular DSM but reaches ≤50% in the longitudinal DSM. Activation of TRPV1 in rat DSM during muscarinic cholinergic stimulation is ensured by phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists. Immunofluorescence detection of TRPV1 protein in bladder sections and isolated DSM cells confirmed both its preferential expression in the longitudinal DSM sublayer and its targeting to the endoplasmic reticulum. We conclude that TRPV1 is an essential contributor to the cholinergic contraction of bladder longitudinal DSM, which might be important for producing spatial and/or temporal anisotropy of bladder wall deformation in different regions during parasympathetic stimulation. KEY POINTS: The transient receptor potential vanilloid 1 (TRPV1) heat/capsaicin receptor/channel is localized in the endoplasmic reticulum membrane of detrusor smooth muscle (DSM) cells of the rat bladder, operating as a calcium-release channel. Isolated DSM cells are separated into two nearly equal groups, within which the cells either show or do not show TRPV1-dependent [Ca2+]i rise. Carbachol-stimulated, muscarinic ACh receptor-mediated contractions of multicellular DSM strips contain a TRPV1-dependent component. This component is negligible in the circular DSM but reaches ≤50% in longitudinal DSM. Activation of TRPV1 in rat DSM during cholinergic stimulation involves phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists.

The capsaicin binding affinity of wildtype and mutant TRPV1 ion channels.

Vanilloids such as capsaicin and resiniferatoxin are highly selective and potent activators for transient receptor potential vanilloid subfamily, member 1, a nociceptor for heat and pain perception. However, the intrinsic vanilloid binding affinity, key for understanding transient receptor potential vanilloid subfamily, member 1 function, remains unknown despite intensive investigations by electrophysiological, structural, and computational methods. In this study, we determined capsaicin binding affinity under physiological conditions by isolating individual binding steps to each subunit with concatemers. We estimated the capsaicin association constant of a wildtype subunit to be in the order of 106 M-1 and that of the Y511A mutant subunit to be a hundred times lower, in the order of 104 M-1. The Y511A mutation, located at the entrance of the vanilloid binding pocket, reduces binding affinity without a noticeable effect on activation gating. We further affirmed that there is little cooperativity between vanilloid binding steps. Models based on independent binding and equally cooperative subunit gating can accurately describe capsaicin activation.

Opening of capsaicin receptor TRPV1 is stabilized equally by its four subunits.

Capsaicin receptor TRPV1 is a nociceptor for vanilloid molecules, such as capsaicin and resiniferatoxin (RTX). Even though cryo-EM structures of TRPV1 in complex with these molecules are available, how their binding energetically favors the open conformation is not known. Here, we report an approach to control the number of bound RTX molecules (0-4) in functional rat TRPV1. The approach allowed direct measurements of each of the intermediate open states under equilibrium conditions at both macroscopic and single-molecule levels. We found that RTX binding to each of the four subunits contributes virtually the same activation energy, which we estimated to be 1.70 to 1.86 kcal/mol and found to arise predominately from destabilizing the closed conformation. We further showed that sequential bindings of RTX increase open probability without altering single-channel conductance, confirming that there is likely a single open-pore conformation for TRPV1 activated by RTX.

TRPV1 and mast cell involvement in repeated variate stress-induced urinary bladder dysfunction in adult female mice.

The etiology of interstitial cystitis/bladder pain syndrome (IC/BPS) is unknown but likely multifactorial. IC/BPS symptoms can be exacerbated by psychological stress, but underlying mechanisms remain to be defined. Transient receptor potential vanilloid 1 (TRPV1) channels, expressed on nerve fibers, have been implicated in bladder dysfunction and colonic hypersensitivity with stress in rodents. Histamine/H1R activation of TRPV1+ nerves increases bladder afferent fiber sensitivity to distension. TRPV1 channels are also expressed on mast cells, previously implicated in contributing to IC/BPS etiology and symptoms. We have examined the contribution of TRPV1 and mast cells to bladder dysfunction after repeated variate stress (RVS). RVS increased (P ≤ 0.05) serum and fecal corticosterone expression and induced anxiety-like behavior in wild-type (WT) mice. Intravesical instillation of the selective TRPV1 antagonist capsazepine (CPZ) rescued RVS-induced bladder dysfunction in WT mice. Trpv1 knockout (KO) mice did not increase voiding frequency with RVS and did not exhibit increased serum corticosterone expression despite exhibiting anxiety-like behavior. Mast cell-deficient mice (B6.Cg-Kitw-sh) failed to demonstrate RVS-induced increased voiding frequency or serum corticosterone expression, whereas control (no stress) mast cell-deficient mice had similar functional bladder capacity to WT mice. TRPV1 protein expression was significantly increased in the rostral lumbar (L1-L2) spinal cord and dorsal root ganglia (DRG) in WT mice exposed to RVS, but no changes were observed in lumbosacral (L6-S1) spinal segments or DRG. These studies demonstrated TRPV1 and mast cell involvement in RVS-induced increased voiding frequency and suggest that TRPV1 and mast cells may be useful targets to mitigate stress-induced urinary bladder dysfunction.NEW & NOTEWORTHY Using pharmacological tools and transgenic mice in a repeated variate stress (RVS) model in female mice, we demonstrate that transient receptor potential vanilloid 1 (TRPV1) and mast cells contribute to the increased voiding frequency observed following RVS. TRPV1 and mast cells should continue to be considered as targets to improve bladder function in stress-induced bladder dysfunction.