Wouter Everaerts

TRPA1 acts as a cold sensor in vitro and in vivo

TRPA1 functions as an excitatory ionotropic receptor in sensory neurons. It was originally described as a noxious cold-activated channel, but its cold sensitivity has been disputed in later studies, and the contribution of TRPA1 to thermosensing is currently a matter of strong debate. Here, we provide several lines of evidence to establish that TRPA1 acts as a cold sensor in vitro and in vivo. First, we demonstrate that heterologously expressed TRPA1 is activated by cold in a Ca2+-independent and Ca2+ store-independent manner; temperature-dependent gating of TRPA1 is mechanistically analogous to that of other temperature-sensitive TRP channels, and it is preserved after treatment with the TRPA1 agonist mustard oil. Second, we identify and characterize a specific subset of cold-sensitive trigeminal ganglion neurons that is absent in TRPA1-deficient mice. Finally, cold plate and tail-flick experiments reveal TRPA1-dependent, cold-induced nociceptive behavior in mice. We conclude that TRPA1 acts as a major sensor for noxious cold.

Deletion of the transient receptor potential cation channel TRPV4 impairs murine bladder voiding

Here we provide evidence for a critical role of the transient receptor potential cation channel, subfamily V, member 4 (TRPV4) in normal bladder function. Immunofluorescence demonstrated TRPV4 expression in mouse and rat urothelium and vascular endothelium, but not in other cell types of the bladder. Intracellular Ca2+ measurements on urothelial cells isolated from mice revealed a TRPV4-dependent response to the selective TRPV4 agonist 4alpha-phorbol 12,13-didecanoate and to hypotonic cell swelling. Behavioral studies demonstrated that TRPV4-/- mice manifest an incontinent phenotype but show normal exploratory activity and anxiety-related behavior. Cystometric experiments revealed that TRPV4-/- mice exhibit a lower frequency of voiding contractions as well as a higher frequency of nonvoiding contractions. Additionally, the amplitude of the spontaneous contractions in explanted bladder strips from TRPV4-/- mice was significantly reduced. Finally, a decreased intravesical stretch-evoked ATP release was found in isolated whole bladders from TRPV4-/- mice. These data demonstrate a previously unrecognized role for TRPV4 in voiding behavior, raising the possibility that TRPV4 plays a critical role in urothelium-mediated transduction of intravesical mechanical pressure.

The vanilloid transient receptor potential channel TRPV4: From structure to disease

The Transient Receptor Potential Vanilloid 4 channel, TRPV4, is a Ca(2+) and Mg(2+) permeable non-selective cation channel involved in many different cellular functions. It is activated by a variety of physical and chemical stimuli, including heat, mechano-stimuli, endogenous substances such as arachidonic acid and its cytochrome P450-derived metabolites (epoxyeicosatrienoic acids), endocannabinoids (anandamide and 2-arachidonoylglycerol), as well as synthetic alpha-phorbol derivatives. Recently, TRPV4 has been characterized as an important player modulating osteoclast differentiation in bone remodelling and as a urothelial mechanosensor that controls normal voiding. Several TRPV4 gain-of-function mutations are shown to cause autosomal-dominant bone dysplasias such as brachyolmia and Koszlowski disease. In this review we comprehensively describe the structural, biophysical and (patho)physiological properties of the TRPV4 channel and we summarize the current knowledge about the role of TRPV4 in the pathogenesis of several diseases.

Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis

Reduced functional bladder capacity and concomitant increased micturition frequency (pollakisuria) are common lower urinary tract symptoms associated with conditions such as cystitis, prostatic hyperplasia, neurological disease, and overactive bladder syndrome. These symptoms can profoundly affect the quality of life of afflicted individuals, but available pharmacological treatments are often unsatisfactory. Recent work has demonstrated that the cation channel TRPV4 is highly expressed in urothelial cells and plays a role in sensing the normal filling state of the bladder. In this article, we show that the development of cystitis-induced bladder dysfunction is strongly impaired in Trpv4−/− mice. Moreover, we describe HC-067047, a previously uncharacterized, potent, and selective TRPV4 antagonist that increases functional bladder capacity and reduces micturition frequency in WT mice and rats with cystitis. HC-067047 did not affect bladder function in Trpv4−/− mice, demonstrating that its in vivo effects are on target. These results indicate that TRPV4 antagonists may provide a promising means of treating bladder dysfunction.

Nicotine activates the chemosensory cation channel TRPA1.

Topical application of nicotine, as used in nicotine replacement therapies, causes irritation of the mucosa and skin. This reaction has been attributed to activation of nicotinic acetylcholine receptors (nAChRs) in chemosensory neurons. In contrast with this view, we found that the chemosensory cation channel transient receptor potential A1 (TRPA1) is crucially involved in nicotine-induced irritation. We found that micromolar concentrations of nicotine activated heterologously expressed mouse and human TRPA1. Nicotine acted in a membrane-delimited manner, stabilizing the open state(s) and destabilizing the closed state(s) of the channel. In the presence of the general nAChR blocker hexamethonium, nociceptive neurons showed nicotine-induced responses that were strongly reduced in TRPA1-deficient mice. Finally, TRPA1 mediated the mouse airway constriction reflex to nasal instillation of nicotine. The identification of TRPA1 as a nicotine target suggests that existing models of nicotine-induced irritation should be revised and may facilitate the development of smoking cessation therapies with less adverse effects.

The Capsaicin Receptor TRPV1 Is a Crucial Mediator of the Noxious Effects of Mustard Oil

Mustard oil (MO) is a plant-derived irritant that has been extensively used in experimental models to induce pain and inflammation. The noxious effects of MO are currently ascribed to specific activation of the cation channel TRPA1 in nociceptive neurons. In contrast to this view, we show here that the capsaicin receptor TRPV1 has a surprisingly large contribution to aversive and pain responses and visceral irritation induced by MO. Furthermore, we found that this can be explained by previously unknown properties of this compound. First, MO has a bimodal effect on TRPA1, producing current inhibition at millimolar concentrations. Second, it directly and stably activates mouse and human recombinant TRPV1, as well as TRPV1 channels in mouse sensory neurons. Finally, physiological temperatures enhance MO-induced TRPV1 stimulation. Our results refute the dogma that TRPA1 is the sole nocisensor for MO and motivate a revision of the putative roles of these channels in models of MO-induced pain and inflammation. We propose that TRPV1 has a generalized role in the detection of irritant botanical defensive traits and in the coevolution of multiple mammalian and plant species.

Functional characterization of transient receptor potential channels in mouse urothelial cells.

The bladder urothelium is currently believed to be a sensory structure, contributing to mechano- and chemosensation in the bladder. Transient receptor potential (TRP) cation channels act as polymodal sensors and may underlie some of the receptive properties of urothelial cells. However, the exact TRP channel expression profile of urothelial cells is unclear. In this study, we have performed a systematic analysis of the molecular and functional expression of various TRP channels in mouse urothelium. Urothelial cells from control and trpv4-/- mice were isolated, cultured (12-48 h), and used for quantitative real-time PCR, immunocytochemistry, calcium imaging, and whole cell patch-clamp experiments. At the mRNA level, TRPV4, TRPV2, and TRPM7 were the most abundantly expressed TRP genes. Immunohistochemistry showed a clear expression of TRPV4 in the plasma membrane, whereas TRPV2 was more prominent in the cytoplasm. TRPM7 was detected in the plasma membrane as well as cytoplasmic vesicles. Calcium imaging and patch-clamp experiments using TRP channel agonists and antagonists provided evidence for the functional expression of TRPV4, TRPV2, and TRPM7 but not of TRPA1, TRPV1, and TRPM8. In conclusion, we have demonstrated functional expression of TRPV4, TRPV2, and TRPM7 in mouse urothelial cells. These channels may contribute to the (mechano)sensory function of the urothelial layer and represent potential targets for the treatment of bladder dysfunction.

Where is TRPV1 expressed in the bladder, do we see the real channel?

Transient receptor potential channel-vanilloid subfamily member 1 (TRPV1) is an important target in the treatment of bladder overactivity. This receptor is suggested to function as a mechanosensor in the normal bladder and to mediate the development of bladder overactivity during cystitis. Our aim was to determine the cellular distribution of TRPV1 in mouse and rat bladder tissue. We used three different commercial TRPV1 antibodies to perform immunohistochemistry on bladder tissue from rats and wild-type and TRPV1−/− mice, using trigeminal ganglia as a control tissue for TRPV1 expression. Although two of the antibodies seemed to react specifically in trigeminal ganglion tissue, all the antibodies produced a similar staining pattern in the urothelium of wild-type and TRPV1−/− mice. These data show that TRPV1 antibodies can cause an aspecific immunostaining in bladder tissue, urging for additional research to confirm the exact distribution of TRPV1 in bladder. In conclusion, we think that the use of negative controls on knockout mice, whenever available, is mandatory when conducting immunohistochemical localization studies.

Preservation of the Neurovascular Bundles Is Associated with Improved Time to Continence After Radical Prostatectomy But Not Long-term Continence Rates: Results of a Systematic Review and Meta-analysis

CONTEXT The aetiology of urinary incontinence following radical prostatectomy (RP) is incompletely understood. In particular, it is unclear whether there is a relationship between neurovascular bundle (NVB) sparing and post-RP urinary continence. OBJECTIVE To review systematically the association of NVB sparing in RP with postoperative urinary continence outcomes and synthesise the results in a meta-analysis. EVIDENCE ACQUISITION This study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis statement. PubMed, Medline, and Cochrane Central Register of Controlled Trials were searched (December 2013), yielding 3413 unique records. A total of 27 longitudinal cohort studies were selected for inclusion. Studies were evaluated using a predefined criteria adapted from the Cochrane Tool to Assess Risk of Bias in Cohort Studies. EVIDENCE SYNTHESIS Data from 13 749 participants in 27 studies were synthesised in a meta-analysis. An assessment of the study methodology revealed a high risk of bias due to differences in baseline characteristics, outcome assessment, and the likely presence of unreported confounding factors such as meticulous apical dissection. Meta-analysis demonstrated that nerve sparing (NS) compared with non-nerve sparing (NNS) resulted in improved early urinary continence rates up to 6 mo postoperatively. Beyond this time, no significant difference was observed. This effect was seen most clearly for bilateral NS compared with NNS. A sensitivity analysis of prospective cohort studies revealed consistent results. CONCLUSIONS This analysis demonstrates an association between NS and improved urinary continence outcomes up to 6 mo postoperatively. NS in men with poor preoperative erectile function should be considered in the context of oncologic risk stratification because it may improve time to continence recovery. The underlying cause of the relationship between NS and continence is unknown. It may represent preservation of the intrapelvic somatic nerves supplying the rhabdosphincter or the influence of other confounding factors. Future research should be directed towards improving understanding of the anatomy of urinary continence and the pathophysiology of post-RP incontinence. PATIENT SUMMARY We found that avoiding damage to the nerves around the prostate improves urinary continence in the first 6 mo after surgery. After this time, there is no difference in continence between men who had these nerves removed and those who had them saved. This finding could be due to a true effect of saving these nerves or to a number of other factors affecting the research.

Characterization of upper lamina propria interstitial cells in bladders from patients with neurogenic detrusor overactivity and bladder pain syndrome

The upper lamina propria (ULP) area of interstitial cells (IC) in bladder has been studied for more than a decade in several species including human beings. Nevertheless there is still lack of uniformity in terminology of this cell layer. The aim of the present study was to add new data to the morphological and immunohistochemical phenotype of these cells and to find out whether this phenotype is changed in bladders from patients with neurogenic detrusor overactivity (NDO) and bladder pain syndrome (BPS). Bladder tissue was obtained from a control group and from patients with NDO and BPS. Samples were processed for morphology, electron microscopy and immunohistochemistry. A morphological and immunohistochemical phenotype for the ULP IC was assessed and changes in this phenotype were looked for in samples from patients with NDO and BPS. The ULP IC were characterized ultrastructurally by the presence of actin filaments with densifications, many caveolae and abundant rough endoplasmic reticulum (RER); on immunohistochemistry ULP IC were immunoreactive for α‐sma, vimentin, CD10 and podoplanin and categorized as interstitial Cajal‐like cells (ICLC). In NDO and BPS bladders we found a phenotypical shift towards a fibroblastic phenotype which was even more pronounced in the NDO group. In both groups there was also an increased presence in ULP lymphocytes. The ULP area in the human bladder contains a population of ICLC with distinct ultrastructural morphology and immunohistochemical phenotype. Their unique α‐sma+/desmin–/CD34– phenotype allows studying this population in various bladder disorders. In bladders form patients with BPS and NDO, we observed these ULP ICLC to shift towards a fibroblast phenotype.