Background Idiopathic rhinitis (IR) is a prevalent condition for which capsaicin nasal spray is the most effective treatment. However, the mechanisms underlying IR and the therapeutic action of ...capsaicin remain unknown. Objective We sought to investigate the molecular and cellular bases of IR and the therapeutic action of capsaicin. Methods Fourteen patients with IR and 12 healthy control subjects (HCs) were treated with intranasal capsaicin. The therapeutic effect was assessed in patients with IR by using visual analog scale and therapeutic response evaluation scores, and nasal hyperreactivity was evaluated by means of cold dry air provocation. Nasal samples served to measure the levels of neuromediators and expression of chemosensory cation channels, protein gene product 9.5 (PGP 9.5), and the mast cell marker c-kit. The effects of capsaicin were also tested in vitro on human nasal epithelial cells and mast cells. Results Patients with IR had higher baseline transient receptor potential cation channel subfamily V, receptor 1 (TRPV1) expression in the nasal mucosa and higher concentrations of substance P (SP) in nasal secretions than HCs. Symptomatic relief was observed in 11 of 14 patients with IR after capsaicin treatment. Expression of TRPV1; transient receptor potential cation channel subfamily M, receptor 8 (TRPM8); and PGP 9.5 was only reduced in patients with IR after capsaicin treatment. Capsaicin did not alter c-KIT expression or nasal epithelial morphology in patients with IR and HCs nor did it induce apoptosis or necrosis in cultured human nasal epithelial cells and mast cells. Conclusion IR features an overexpression of TRPV1 in the nasal mucosa and increased SP levels in nasal secretions. Capsaicin exerts its therapeutic action by ablating the TRPV1-SP nociceptive signaling pathway in the nasal mucosa.
The Transient Receptor Potential Melastatin 3 (TRPM3) is a Ca2+-permeable non-selective cation channel activated by the neurosteroid pregnenolone sulfate (PS). This compound was previously shown to ...contract mouse aorta by activating TRPM3 in vascular smooth muscle cells (VSMC), and proposed as therapeutic modulator of vascular functions. However, PS effects and the role of TRPM3 in resistance arteries remain unknown. Thus, we aimed at determining the localization and physiological role of TRPM3 in mouse mesenteric arteries. Real-time qPCR experiments, anatomical localization using immunofluorescence microscopy and patch-clamp recordings in isolated VSMC showed that TRPM3 expression in mesenteric arteries is restricted to perivascular nerves. Pressure myography experiments in wild type (WT) mouse arteries showed that PS vasodilates with a concentration-dependence that was best fit by two Hill components (effective concentrations, EC50, of 14 and 100 μM). The low EC50 component was absent in preparations from Trpm3 knockout (KO) mice and in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096. TRPM3-dependent vasodilation was partially inhibited by a cocktail of K+ channel blockers, and not mediated by β-adrenergic signaling. We conclude that, contrary to what was found in aorta, PS dilates mesenteric arteries, partly via an activation of TRPM3 that triggers CGRP release from perivascular nerve endings and a subsequent activation of K+ channels in VSMC. We propose that TRPM3 is implicated in the regulation of the tone of resistance arteries and that its activation by yet unidentified endogenous damage-associated molecules lead to protective vasodilation responses in mesenteric arteries.
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•TRPM3 expression is restricted to sensory nerve endings in mesenteric arteries.•TRPM3 activation dilates mesenteric arteries via CGRP release from nerve endings.•Pregnenolone sulfate is a specific TRPM3 aganist at concentrations below 10 μM.•TRPM3-mediated vasodilation is partly driven by activation of KV channels in VSMC.•Endogenous TRPM3 agonists can contribute to vascular tone regulation.
Silica nanoparticles (SiNPs) have numerous beneficial properties and are extensively used in cosmetics and food industries as anti-caking, densifying and hydrophobic agents. However, the increasing ...exposure levels experienced by the general population and the ability of SiNPs to penetrate cells and tissues have raised concerns about possible toxic effects of this material. Although SiNPs are known to affect the function of the airway epithelium, the molecular targets of these particles remain largely unknown. Given that SiNPs interact with the plasma membrane of epithelial cells we hypothesized that they may affect the function of Transient Receptor Potential Vanilloid 4 (TRPV4), a cation-permeable channel that regulates epithelial barrier function. The main aims of this study were to evaluate the effects of SiNPs on the activation of TRPV4 and to determine whether these alter the positive modulatory action of this channel on the ciliary beat frequency in airway epithelial cells.
Using fluorometric measurements of intracellular Ca
concentration (Ca
) we found that SiNPs inhibit activation of TRPV4 by the synthetic agonist GSK1016790A in cultured human airway epithelial cells 16HBE and in primary cultured mouse tracheobronchial epithelial cells. Inhibition of TRPV4 by SiNPs was confirmed in intracellular Ca
imaging and whole-cell patch-clamp experiments performed in HEK293T cells over-expressing this channel. In addition to these effects, SiNPs were found to induce a significant increase in basal Ca
, but in a TRPV4-independent manner. SiNPs enhanced the activation of the capsaicin receptor TRPV1, demonstrating that these particles have a specific inhibitory action on TRPV4 activation. Finally, we found that SiNPs abrogate the increase in ciliary beat frequency induced by TRPV4 activation in mouse airway epithelial cells.
Our results show that SiNPs inhibit TRPV4 activation, and that this effect may impair the positive modulatory action of the stimulation of this channel on the ciliary function in airway epithelial cells. These findings unveil the cation channel TRPV4 as a primary molecular target of SiNPs.
TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in ...inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.
Further insight into the structural biology of TRP channels is crucial to explain molecular mechanisms of channel function.
We purified TRPV3, demonstrated its functional integrity, and used fluorescence spectroscopy to study ligand binding.
TRPV3 ligands induce different conformational changes as observed by tryptophan fluorescence quenching.
Availability of purified TRPV3 allows functional assays outside the cellular context and facilitates future structural studies.
The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for ...prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion. After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions. Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.
Abstract It is often observed in intracellular Ca2+ imaging experiments that the amplitudes of the Ca2+ signals elicited by newly characterized TRP agonists do not correlate with the amplitudes of ...the responses evoked subsequently by a specific potent agonist. We investigated this rather controversial phenomenon by first testing whether it is inherent to the comparison of the effects of weak and strong stimuli. Using five well-characterized TRP channel agonists in commonly used heterologous expression systems we found that the correlation between the amplitudes of the Ca2+ signals triggered by two sequentially applied stimuli is only high when both stimuli are strong. Using mathematical simulations of intracellular Ca2+ dynamics we illustrate that the innate heterogeneity in expression and functional properties of Ca2+ extrusion (e.g. plasma membrane Ca2+ ATPase) and influx (TRP channels) pathways across a cellular population is a sufficient condition for low correlation between the amplitude of Ca2+ signals elicited by weak and strong stimuli. Taken together, our data demonstrate that this phenomenon is an expected outcome of intracellular Ca2+ imaging experiments that cannot be taken as evidence for lack of specificity of low-efficacy stimuli, or as an indicator of the need of other cellular components for channel stimulation.
It is often observed in intracellular Ca(2+) imaging experiments that the amplitudes of the Ca(2+) signals elicited by newly characterized TRP agonists do not correlate with the amplitudes of the ...responses evoked subsequently by a specific potent agonist. We investigated this rather controversial phenomenon by first testing whether it is inherent to the comparison of the effects of weak and strong stimuli. Using five well-characterized TRP channel agonists in commonly used heterologous expression systems we found that the correlation between the amplitudes of the Ca(2+) signals triggered by two sequentially applied stimuli is only high when both stimuli are strong. Using mathematical simulations of intracellular Ca(2+) dynamics we illustrate that the innate heterogeneity in expression and functional properties of Ca(2+) extrusion (e.g. plasma membrane Ca(2+) ATPase) and influx (TRP channels) pathways across a cellular population is a sufficient condition for low correlation between the amplitude of Ca(2+) signals elicited by weak and strong stimuli. Taken together, our data demonstrate that this phenomenon is an expected outcome of intracellular Ca(2+) imaging experiments that cannot be taken as evidence for lack of specificity of low-efficacy stimuli, or as an indicator of the need of other cellular components for channel stimulation.
Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced ...activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of
Trpa1
is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca
2+
channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca
2+
currents in mouse ventricular cardiomyocytes (VCM, with Ca
2+
as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba
2+
as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca
2+
channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca
2+
channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.
Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a Ca(2+)-activated nonselective cation channel involved in the transduction of sweet, bitter, and umami tastes. We ...previously showed that TRPM5 is a locus for the modulation of taste perception by temperature changes, and by quinine and quinidine, 2 bitter compounds that suppress gustatory responses. Here, we determined whether other bitter compounds known to modulate taste perception also affect TRPM5. We found that nicotine inhibits TRPM5 currents with an effective inhibitory concentration of ~1.3mM at -50 mV. This effect may contribute to the inhibitory effect of nicotine on gustatory responses in therapeutic and experimental settings, where nicotine is often employed at millimolar concentrations. In addition, it implies the existence of a TRPM5-independent pathway for the detection of nicotine bitterness. Nicotine seems to act from the extracellular side of the channel, reducing the maximal whole-cell conductance and inducing an acceleration of channel closure that leads to a negative shift of the activation curve. TRPM5 currents were unaffected by nicotine's metabolite cotinine, the intensive sweetener saccharin or by the bitter xanthines caffeine, theobromine, and theophylline. We also tested the effects of bitter compounds on another essential element of the sweet taste transduction pathway, the type 3 IP3 receptor (IP3R3). We found that IP3R3-mediated Ca(2+) flux is slightly enhanced by nicotine, not affected by saccharin, modestly inhibited by caffeine, theobromine, and theophylline, and strongly inhibited by quinine. Our results demonstrate that bitter compounds have differential effects on key elements of the sweet taste transduction pathway, suggesting for heterogeneous mechanisms of bitter-sweet taste interactions.
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