Putting the Spotlight Back Onto the Flanker Task in Autism: Autistic Adults Show Increased Interference from Foils Compared with Non-autistic Adults.

Autistic people may have a less focused spotlight of spatial selective attention than non-autistic people, meaning that distracting stimuli are less effectively suppressed. Previous studies using the flanker task have supported this suggestion with observations of increased congruency effects in autistic participants. However, findings across studies have been mixed, mainly based on research in children and on response time measures, which may be influenced by differences in response strategy between autistic and non-autistic people rather than differences in selective attention. In this pre-registered study, 153 autistic and 147 non-autistic adults completed an online flanker task. The aims of this study were to test whether increased congruency effects replicate in autistic adults and to extend previous work by fitting a computational model of spatial selective attention on the flanker task to the data. Congruency effects were increased in the autistic group. The modelling revealed that the interference time from the foils was increased in the autistic group. This suggests that the activation of the foils was increased, meaning suppression was less effective for autistic participants. There were also differences in non-interference parameters between the groups. The estimate of response caution was increased in the autistic group and the estimate of perceptual efficiency was decreased. Together these findings suggest inefficient suppression, response strategy and perceptual processing all contribute to differences in performance on the flanker task between autistic and non-autistic people.

A single rapid heat stress episode does not result in prolonged elevations in salivary cortisol and C-reactive protein production in firefighters.

In the present experiment, we evaluated the impact of rapid heat stress (RHS) on salivary cortisol and C-reactive protein production pre-RHS, post-RHS, and 24 and 48 h post-RHS exposure among firefighters. Previous research has demonstrated that RHS increases salivary cortisol during RHS and immediately post-RHS exposure. However, no research has evaluated the duration necessary to return to baseline cortisol levels following RHS. Additionally, no studies have analyzed the impact of RHS on inflammatory biomarkers, such as C-reactive protein. This study hypothesized that salivary cortisol and C-reactive protein levels would increase following RHS and then return to pre-RHS levels within 24 h post-exposure. Twenty-four participants performed a steady-state treadmill protocol in an environmental chamber (35 °C; 45% humidity) in full firefighter personal protective equipment until reaching either a core temperature (Tc) of 39 °C or a volitional maximum. The subjects had their saliva collected via the passive drool protocol pre-RHS, post-RHS, and 24 and 48 h post-RHS. Pre-RHS of 0.23 ± 0.03 µg/dL increased post-RHS to 0.51 ± 0.06 µg/dL (p < 0.001). This finding supports previous literature demonstrating the immediate impact of RHS. There were no changes in C-reactive protein. The novel finding of this study is that salivary cortisol levels return to baseline in the 24 h post-RHS exposure. This indicates that 24 h is recommended to recover from RHS and should be applied to prevent the chronic stress response.

Stroke Alters the Function of Enteric Neurons to Impair Smooth Muscle Relaxation and Dysregulates Gut Transit.

BACKGROUND: Gut dysmotility is common after ischemic stroke, but the mechanism underlying this response is unknown. Under homeostasis, gut motility is regulated by the neurons of the enteric nervous system that control contractile/relaxation activity of muscle cells in the gut wall. More recently, studies of gut inflammation revealed interactions of macrophages with enteric neurons are also involved in modulating gut motility. However, whether poststroke gut dysmotility is mediated by direct signaling to the enteric nervous system or indirectly via inflammatory macrophages is unknown. METHODS AND RESULTS: We examined these hypotheses by using a clinically relevant permanent intraluminal midcerebral artery occlusion experimental model of stroke. At 24 hours after stroke, we performed in vivo and ex vivo gut motility assays, flow cytometry, immunofluorescence, and transcriptomic analysis. Stroke-induced gut dysmotility was associated with recruitment of muscularis macrophages into the gastrointestinal tract and redistribution of muscularis macrophages away from myenteric ganglia. The permanent intraluminal midcerebral artery occlusion model caused changes in gene expression in muscularis macrophages consistent with an altered phenotype. While the size of myenteric ganglia after stroke was not altered, myenteric neurons from post-permanent intraluminal midcerebral artery occlusion mice showed a reduction in neuronal nitric oxide synthase expression, and this response was associated with enhanced intestinal smooth muscle contraction ex vivo. Finally, chemical sympathectomy with 6-hydroxydopamine prevented the loss of myenteric neuronal nitric oxide synthase expression and stroke-induced slowed gut transit. CONCLUSIONS: Our findings demonstrate that activation of the sympathetic nervous system after stroke is associated with reduced neuronal nitric oxide synthase expression in myenteric neurons, resulting in impaired smooth muscle relaxation and dysregulation of gut transit.

TRPV4 is expressed by enteric glia and muscularis macrophages of the colon but does not play a prominent role in colonic motility.

Background: Mechanosensation is an important trigger of physiological processes in the gastrointestinal tract. Aberrant responses to mechanical input are associated with digestive disorders, including visceral hypersensitivity. Transient Receptor Potential Vanilloid 4 (TRPV4) is a mechanosensory ion channel with proposed roles in visceral afferent signaling, intestinal inflammation, and gut motility. While TRPV4 is a potential therapeutic target for digestive disease, current mechanistic understanding of how TRPV4 may influence gut function is limited by inconsistent reports of TRPV4 expression and distribution. Methods: In this study we profiled functional expression of TRPV4 using Ca2+ imaging of wholemount preparations of the mouse, monkey, and human intestine in combination with immunofluorescent labeling for established cellular markers. The involvement of TRPV4 in colonic motility was assessed in vitro using videomapping and contraction assays. Results: The TRPV4 agonist GSK1016790A evoked Ca2+ signaling in muscularis macrophages, enteric glia, and endothelial cells. TRPV4 specificity was confirmed using TRPV4 KO mouse tissue or antagonist pre-treatment. Calcium responses were not detected in other cell types required for neuromuscular signaling including enteric neurons, interstitial cells of Cajal, PDGFRα+ cells, and intestinal smooth muscle. TRPV4 activation led to rapid Ca2+ responses by a subpopulation of glial cells, followed by sustained Ca2+ signaling throughout the enteric glial network. Propagation of these waves was suppressed by inhibition of gap junctions or Ca2+ release from intracellular stores. Coordinated glial signaling in response to GSK1016790A was also disrupted in acute TNBS colitis. The involvement of TRPV4 in the initiation and propagation of colonic motility patterns was examined in vitro. Conclusions: We reveal a previously unappreciated role for TRPV4 in the initiation of distension-evoked colonic motility. These observations provide new insights into the functional role of TRPV4 activation in the gut, with important implications for how TRPV4 may influence critical processes including inflammatory signaling and motility.

Understanding the post-diagnostic support priorities of autistic adults in the United Kingdom: A co-produced modified Delphi study.

LAY ABSTRACT: Autistic adults in the United Kingdom report that support for themselves and their peers is not suitable for their needs. There has been an increase in adults receiving an autism diagnosis, which many have reported as having a positive impact on their lives. However, the lack of support and understanding after diagnosis, combined with long wait times for an assessment to obtain a diagnosis and to access follow-on support, is having a negative impact on people's lives. This study took place to find out what support autistic people need and want after receiving their diagnosis. It was co-designed with a group of 10 autistic adults which means that the researchers and group members collaboratively designed the research. For the study, 43 autistic adults, diagnosed aged 18 or older, completed three questionnaires. A fourth questionnaire followed that was completed by 139 autistic people who received their diagnosis in adulthood. These questionnaires aimed to help people identify their own priorities when it came to the support they would have liked to receive after being given their autism diagnosis. Participants ranked access to support where they live, training of professionals, support to process the impact of a late diagnosis, use of their preferred mode of contact and a personalised support plan as their top priorities. This demonstrates that local support is highly valued by autistic adults, as are well-trained professionals who offer a range of contact options, support to process a late-in-life autism diagnosis and help to develop and implement support plans.

When 2 become 1: Autistic simultaneity judgements about asynchronous audiovisual speech.

It has been proposed that autistic people experience a temporal distortion whereby the temporal binding window of multisensory integration is extended. Research to date has focused on autistic children so whether these differences persist into adulthood remains unknown. In addition, the possibility that the previous observations have arisen from between-group differences in response bias, rather than perceptual differences, has not been addressed. Participants completed simultaneity judgements of audiovisual speech stimuli across a range of stimulus-onset asynchronies. Response times and accuracy data were fitted to a drift-diffusion model so that the drift rate (a measure of processing efficiency) and starting point (response bias) could be estimated. In Experiment 1, we tested a sample of non-autistic adults who completed the Autism Quotient questionnaire. Autism Quotient score was not correlated with either drift rate or response bias, nor were there between-group differences when splitting based on the first and third quantiles of scores. In Experiment 2, we compared the performance of autistic with a group of non-autistic adults. There were no between-group differences in either drift rate or starting point. The results of this study do not support the previous suggestion that autistic people have an extended temporal binding window for audiovisual speech. In addition, exploratory analysis revealed that operationalising the temporal binding window in different ways influenced whether a group difference was observed, which is an important consideration for future work.

Therapeutic antagonism of the neurokinin 1 receptor in endosomes provides sustained pain relief.

The hypothesis that sustained G protein-coupled receptor (GPCR) signaling from endosomes mediates pain is based on studies with endocytosis inhibitors and lipid-conjugated or nanoparticle-encapsulated antagonists targeted to endosomes. GPCR antagonists that reverse sustained endosomal signaling and nociception are needed. However, the criteria for rational design of such compounds are ill-defined. Moreover, the role of natural GPCR variants, which exhibit aberrant signaling and endosomal trafficking, in maintaining pain is unknown. Herein, substance P (SP) was found to evoke clathrin-mediated assembly of endosomal signaling complexes comprising neurokinin 1 receptor (NK1R), Gαq/i, and ßarrestin-2. Whereas the FDA-approved NK1R antagonist aprepitant induced a transient disruption of endosomal signals, analogs of netupitant designed to penetrate membranes and persist in acidic endosomes through altered lipophilicity and pKa caused sustained inhibition of endosomal signals. When injected intrathecally to target spinal NK1R+ve neurons in knockin mice expressing human NK1R, aprepitant transiently inhibited nociceptive responses to intraplantar injection of capsaicin. Conversely, netupitant analogs had more potent, efficacious, and sustained antinociceptive effects. Mice expressing C-terminally truncated human NK1R, corresponding to a natural variant with aberrant signaling and trafficking, displayed attenuated SP-evoked excitation of spinal neurons and blunted nociceptive responses to SP. Thus, sustained antagonism of the NK1R in endosomes correlates with long-lasting antinociception, and domains within the C-terminus of the NK1R are necessary for the full pronociceptive actions of SP. The results support the hypothesis that endosomal signaling of GPCRs mediates nociception and provides insight into strategies for antagonizing GPCRs in intracellular locations for the treatment of diverse diseases.

Therapeutic potential of allosteric modulators for the treatment of gastrointestinal motility disorders.

Gastrointestinal motility is tightly regulated by the enteric nervous system (ENS). Disruption of coordinated enteric nervous system activity can result in dysmotility. Pharmacological treatment options for dysmotility include targeting of G protein-coupled receptors (GPCRs) expressed by neurons of the enteric nervous system. Current GPCR-targeting drugs for motility disorders bind to the highly conserved endogenous ligand-binding site and promote indiscriminate activation or inhibition of the target receptor throughout the body. This can be associated with significant side-effect liability and a loss of physiological tone. Allosteric modulators of GPCRs bind to a distinct site from the endogenous ligand, which is typically less conserved across multiple receptor subtypes and can modulate endogenous ligand signalling. Allosteric modulation of GPCRs that are important for enteric nervous system function may provide effective relief from motility disorders while limiting side-effects. This review will focus on how allosteric modulators of GPCRs may influence gastrointestinal motility, using 5-hydroxytryptamine (5-HT), acetylcholine (ACh) and opioid receptors as examples.

Sustained endosomal release of a neurokinin-1 receptor antagonist from nanostars provides long-lasting relief of chronic pain.

Soft polymer nanoparticles designed to disassemble and release an antagonist of the neurokinin 1 receptor (NK1R) in endosomes provide efficacious yet transient relief from chronic pain. These micellar nanoparticles are unstable and rapidly release cargo, which may limit the duration of analgesia. We examined the efficacy of stable star polymer nanostars containing the NK1R antagonist aprepitant-amine for the treatment of chronic pain in mice. Nanostars continually released cargo for 24 h, trafficked through the endosomal system, and disrupted NK1R endosomal signaling. After intrathecal injection, nanostars accumulated in endosomes of spinal neurons. Nanostar-aprepitant reversed mechanical, thermal and cold allodynia and normalized nociceptive behavior more efficaciously than free aprepitant in preclinical models of neuropathic and inflammatory pain. Analgesia was maintained for >10 h. The sustained endosomal delivery of antagonists from slow-release nanostars provides effective and long-lasting reversal of chronic pain.

Time perception in autistic adults: Interval and event timing judgments do not differ from nonautistics.

It has previously been proposed that autistic people have problems with timing which underlie the behavioral and cognitive differences in the condition. However, the nature of this postulated timing issue has not been well specified and the existing experimental literature has generated mixed findings. In the current study, we attempted a systematic investigation of timing processes in autistic adults using scalar expectancy theory as a theoretical framework. Autistic (n = 58) and nonautistic (n = 91) adults matched for age, sex, and full-scale IQ completed a battery of auditory and visual timing tasks measuring basic subsecond duration perception (temporal discrimination thresholds), clock processes (verbal estimation), clock and memory processes (temporal generalization), and event timing (temporal order judgments). Participants also completed suprasecond retrospective duration estimates where the participant was not warned in advanced that they would be required to make a timing judgment, and questionnaires measuring self-reported timing behaviors in daily life. The groups reported differences on questionnaires, but measures of timing performance were comparable overall. In an exploratory analysis, we performed principal components analysis to investigate the relationship between timing judgments and participants' self-reported social-communicative, sensory, and motor traits. Measures of timing performance were not well correlated with these questionnaire scores. The current study, the largest conducted on time and autism to date, shows no clear evidence for reduced timing performance in autistic adults. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Mice expressing fluorescent PAR2 reveal that endocytosis mediates colonic inflammation and pain.

G protein-coupled receptors (GPCRs) regulate many pathophysiological processes and are major therapeutic targets. The impact of disease on the subcellular distribution and function of GPCRs is poorly understood. We investigated trafficking and signaling of protease-activated receptor 2 (PAR2) in colitis. To localize PAR2 and assess redistribution during disease, we generated knockin mice expressing PAR2 fused to monomeric ultrastable green fluorescent protein (muGFP). PAR2-muGFP signaled and trafficked normally. PAR2 messenger RNA was detected at similar levels in Par2-mugfp and wild-type mice. Immunostaining with a GFP antibody and RNAScope in situ hybridization using F2rl1 (PAR2) and Gfp probes revealed that PAR2-muGFP was expressed in epithelial cells of the small and large intestine and in subsets of enteric and dorsal root ganglia neurons. In healthy mice, PAR2-muGFP was prominently localized to the basolateral membrane of colonocytes. In mice with colitis, PAR2-muGFP was depleted from the plasma membrane of colonocytes and redistributed to early endosomes, consistent with generation of proinflammatory proteases that activate PAR2 PAR2 agonists stimulated endocytosis of PAR2 and recruitment of Gαq, Gαi, and ß-arrestin to early endosomes of T84 colon carcinoma cells. PAR2 agonists increased paracellular permeability of colonic epithelial cells, induced colonic inflammation and hyperalgesia in mice, and stimulated proinflammatory cytokine release from segments of human colon. Knockdown of dynamin-2 (Dnm2), the major colonocyte isoform, and Dnm inhibition attenuated PAR2 endocytosis, signaling complex assembly and colonic inflammation and hyperalgesia. Thus, PAR2 endocytosis sustains protease-evoked inflammation and nociception and PAR2 in endosomes is a potential therapeutic target for colitis.

Mini-review: Dissecting receptor-mediated stimulation of TRPV4 in nociceptive and inflammatory pathways.

Transient Receptor Potential Vanilloid 4 (TRPV4) is a polymodal, non-selective cation channel that detects thermal, mechanical, and environmental cues and contributes to a range of diverse physiological processes. The effects of chronic TRPV4 stimulation and gain-of-function genetic mutations suggest that TRPV4 may also be a valuable therapeutic target for pathophysiological events including neurogenic inflammation, peripheral neuropathies, and impaired wound healing. There has been significant interest in defining how and where TRPV4 may promote inflammation and pain. Endogenous stimuli such as osmotic stress and lipid binding are established TRPV4 activators. The TRP channel family is also well-known to be controlled by 'receptor-operated' pathways. For example, G protein-coupled receptors (GPCRs) expressed by primary afferent neurons or other cells in inflammatory pathways utilize TRPV4 as an effector protein to amplify nociceptive and inflammatory signaling. Contributing to disorders including arthritis, neuropathies, and pulmonary edema, GPCRs such as the protease-activated receptor PAR2 mediate activation of kinase signaling cascades to increase TRPV4 phosphorylation, resulting in sensitization and enhanced neuronal excitability. Phospholipase activity also leads to production of polyunsaturated fatty acid lipid mediators that directly activate TRPV4. Consistent with the contribution of TRPV4 to disease, pharmacological inhibition or genetic ablation of TRPV4 can diminish receptor-mediated inflammatory events. This review outlines how receptor-mediated signaling is a major endogenous driver of TRPV4 gating and discusses key signaling pathways and emerging TRPV4 modulators such as the mechanosensitive Piezo1 ion channel. A collective understanding of how endogenous stimuli can influence TRPV4 function is critical for future therapeutic endeavors to modulate this channel.

Mechanistic discoveries and simulation-guided assay optimization of portable hormone biosensors with cell-free protein synthesis.

Nuclear receptors (NRs) influence nearly every system of the body and our lives depend on correct NR signaling. Thus, a key environmental and pharmaceutical quest is to identify and detect chemicals which interact with nuclear hormone receptors, including endocrine disrupting chemicals (EDCs), therapeutic receptor modulators, and natural hormones. Previously reported biosensors of nuclear hormone receptor ligands facilitated rapid detection of NR ligands using cell-free protein synthesis (CFPS). In this work, the advantages of CFPS are further leveraged and combined with kinetic analysis, autoradiography, and western blot to elucidate the molecular mechanism of this biosensor. Additionally, mathematical simulations of enzyme kinetics are used to optimize the biosensor assay, ultimately lengthening its readable window by five-fold and improving sensor signal strength by two-fold. This approach enabled the creation of an on-demand thyroid hormone biosensor with an observable color-change readout. This mathematical and experimental approach provides insight for engineering rapid and field-deployable CFPS biosensors and promises to improve methods for detecting natural hormones, therapeutic receptor modulators, and EDCs.

Positive allosteric modulation of endogenous delta opioid receptor signaling in the enteric nervous system is a potential treatment for gastrointestinal motility disorders.

Allosteric modulators (AMs) are molecules that can fine-tune signaling by G protein-coupled receptors (GPCRs). Although they are a promising therapeutic approach for treating a range of disorders, allosteric modulation of GPCRs in the context of the enteric nervous system (ENS) and digestive dysfunction remains largely unexplored. This study examined allosteric modulation of the delta opioid receptor (DOR) in the ENS and assessed the suitability of DOR AMs for the treatment of irritable bowel syndrome (IBS) symptoms using mouse models. The effects of the positive allosteric modulator (PAM) of DOR, BMS-986187, on neurogenic contractions of the mouse colon and on DOR internalization in enteric neurons were quantified. The ability of BMS-986187 to influence colonic motility was assessed both in vitro and in vivo. BMS-986187 displayed DOR-selective PAM-agonist activity and orthosteric agonist probe dependence in the mouse colon. BMS-986187 augmented the inhibitory effects of DOR agonists on neurogenic contractions and enhanced reflex-evoked DOR internalization in myenteric neurons. BMS-986187 significantly increased DOR endocytosis in myenteric neurons in response to the weakly internalizing agonist ARM390. BMS-986187 reduced the generation of complex motor patterns in the isolated intact colon. BMS-986187 reduced fecal output and diarrhea onset in the novel environment stress and castor oil models of IBS symptoms, respectively. DOR PAMs enhance DOR-mediated signaling in the ENS and have potential benefit for the treatment of dysmotility. This study provides proof of concept to support the use of GPCR AMs for the treatment of gastrointestinal motility disorders.NEW & NOTEWORTHY This study assesses the use of positive allosteric modulation as a pharmacological approach to enhance opioid receptor signaling in the enteric nervous system. We demonstrate that selective modulation of endogenous delta opioid receptor signaling can suppress colonic motility without causing constipation. We propose that allosteric modulation of opioid receptor signaling may be a therapeutic strategy to normalize gastrointestinal motility in conditions such as irritable bowel syndrome.

Contributions of bile acids to gastrointestinal physiology as receptor agonists and modifiers of ion channels.

Bile acids (BAs) are known to be important regulators of intestinal motility and epithelial fluid and electrolyte transport. Over the past two decades, significant advances in identifying and characterizing the receptors, transporters, and ion channels targeted by BAs have led to exciting new insights into the molecular mechanisms involved in these processes. Our appreciation of BAs, their receptors, and BA-modulated ion channels as potential targets for the development of new approaches to treat intestinal motility and transport disorders is increasing. In the current review, we aim to summarize recent advances in our knowledge of the different BA receptors and BA-modulated ion channels present in the gastrointestinal system. We discuss how they regulate motility and epithelial transport, their roles in pathogenesis, and their therapeutic potential in a range of gastrointestinal diseases.

BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1-ubiquitin clusters.

ATG9A, the only multi-pass transmembrane protein among core ATG proteins, is an essential regulator of autophagy, yet its regulatory mechanisms and network of interactions are poorly understood. Through quantitative BioID proteomics, we identify a network of ATG9A interactions that includes members of the ULK1 complex and regulators of membrane fusion and vesicle trafficking, including the TRAPP, EARP, GARP, exocyst, AP-1, and AP-4 complexes. These interactions mark pathways of ATG9A trafficking through ER, Golgi, and endosomal systems. In exploring these data, we find that ATG9A interacts with components of the ULK1 complex, particularly ATG13 and ATG101. Using knockout/reconstitution and split-mVenus approaches to capture the ATG13-ATG101 dimer, we find that ATG9A interacts with ATG13-ATG101 independently of ULK1. Deletion of ATG13 or ATG101 causes a shift in ATG9A distribution, resulting in an aberrant accumulation of ATG9A at stalled clusters of p62/SQSTM1 and ubiquitin, which can be rescued by an ULK1 binding-deficient mutant of ATG13. Together, these data reveal ATG9A interactions in vesicle-trafficking and autophagy pathways, including a role for an ULK1-independent ATG13 complex in regulating ATG9A.

Shifting attention between modalities: Revisiting the modality-shift effect in autism.

Selective attention to a sensory modality has been observed experimentally in studies of the modality-shift effect - a relative performance benefit for targets preceded by a target in the same modality, compared to a different modality. Differences in selective attention are commonly observed in autism and we investigated whether exogenous (automatic) shift costs between modalities are increased. Autistic adults and neurotypical controls made speeded discrimination responses to simple visual, tactile and auditory targets. Shift costs were observed for each target modality in participant response times and were largest for auditory targets, reflective of fast responses on auditory repeat trials. Critically, shift costs were similar between the groups. However, integrating speed and accuracy data using drift-diffusion modelling revealed that shift costs in drift rates (reflecting the quality of information extracted from the stimulus) were reduced for autistic participants compared with neurotypicals. It may be that, unlike neurotypicals, there is little difference between attention within and between sensory modalities for autistic people. This finding also highlights the benefit of combining reaction time and accuracy data using decision models to better characterise selective attention in autism.

'No idea of time': Parents report differences in autistic children's behaviour relating to time in a mixed-methods study.

LAY ABSTRACT: Many everyday activities require us to organise our behaviours with respect to time. There is some evidence that autistic children have problems with how they perceive and understand time. However, little is currently known about this, or the ways in which behaviours related to time are impacted in daily life. In this study, 113 parents of autistic children and 201 parents of neurotypical children completed a questionnaire and open-ended questions about their child's behaviour relating to time. Questionnaire scores were lower in the autistic group compared with neurotypicals, which suggests that behaviours relating to time are affected in autistic children. The open-ended responses further confirmed that the autistic children struggled with time and that this impacted on them and their family. Three key themes were identified. Theme 1: autistic children have problems with learning about concepts relating to time such as telling the time from a clock and using words to describe time (hours, minutes, etc.) appropriately. Theme 2: autistic children think about the future differently. Planning and working under time pressure were described as a problem. Theme 3: autistic children have strong interests which take up a lot of their attention and worrying about having sufficient time to pursue these interests causes anxiety. This research indicates that behaviours related to time can have a considerable impact on the lives of autistic children and that targeted support may be required.

Serotonin-induced vascular permeability is mediated by transient receptor potential vanilloid 4 in the airways and upper gastrointestinal tract of mice.

Endothelial and epithelial cells form physical barriers that modulate the exchange of fluid and molecules. The integrity of these barriers can be influenced by signaling through G protein-coupled receptors (GPCRs) and ion channels. Serotonin (5-HT) is an important vasoactive mediator of tissue edema and inflammation. However, the mechanisms that drive 5-HT-induced plasma extravasation are poorly defined. The Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel is an established enhancer of signaling by GPCRs that promote inflammation and endothelial barrier disruption. Here, we investigated the role of TRPV4 in 5-HT-induced plasma extravasation using pharmacological and genetic approaches. Activation of either TRPV4 or 5-HT receptors promoted significant plasma extravasation in the airway and upper gastrointestinal tract of mice. 5-HT-mediated extravasation was significantly reduced by pharmacological inhibition of the 5-HT2A receptor subtype, or with antagonism or deletion of TRPV4, consistent with functional interaction between 5-HT receptors and TRPV4. Inhibition of receptors for the neuropeptides substance P (SP) or calcitonin gene-related peptide (CGRP) diminished 5-HT-induced plasma extravasation. Supporting studies assessing treatment of HUVEC with 5-HT, CGRP, or SP was associated with ERK phosphorylation. Exposure to the TRPV4 activator GSK1016790A, but not 5-HT, increased intracellular Ca2+ in these cells. However, 5-HT pre-treatment enhanced GSK1016790A-mediated Ca2+ signaling, consistent with sensitization of TRPV4. The functional interaction was further characterized in HEK293 cells expressing 5-HT2A to reveal that TRPV4 enhances the duration of 5-HT-evoked Ca2+ signaling through a PLA2 and PKC-dependent mechanism. In summary, this study demonstrates that TRPV4 contributes to 5-HT2A-induced plasma extravasation in the airways and upper GI tract, with evidence supporting a mechanism of action involving SP and CGRP release.