Selective Transient Receptor Potential Research Articles

Piezo1 stretch-activated channel activity differs between murine bone marrow-derived and cardiac tissue-resident macrophages.

Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical - in particular in a rhythmically contracting organ such as the heart. Cultured MΦ, differentiated in vitro from bone marrow (MΦBM), form a popular research model. This study explores the activity of mechanosensitive ion channels (MSC) in murine MΦBM and compares it to MSC activity in MΦ enzymatically isolated from cardiac tissue (tissue-resident MΦ; MΦTR). We show that MΦBM and MΦTR have stretch-induced currents, indicating the presence of functional MSC in their plasma membrane. The current profiles in MΦBM and in MΦTR show characteristics of cation non-selective MSC such as Piezo1 or transient receptor potential channels. While Piezo1 ion channel activity is detectable in the plasma membrane of MΦBM using the patch-clamp technique, or by measuring cytosolic calcium concentration upon perfusion with the Piezo1 channel agonist Yoda1, no Piezo1 channel activity was observed in MΦTR. The selective transient receptor potential vanilloid 4 (TRPV4) channel agonist GSK1016790A induces calcium entry in MΦTR and in MΦBM. In MΦ isolated from left-ventricular scar tissue 28days after cryoablation, stretch-induced current characteristics are not significantly different compared to non-injured control tissue, even though scarred ventricular tissue is expected to be mechanically remodelled and to contain an altered composition of pre-existing cardiac and circulation-recruited MΦ. Our data suggest that the in vitro differentiation protocols used to obtain MΦBM generate cells that differ from MΦ recruited from the circulation during tissue repair in vivo. Further investigations are needed to explore MSC identity in lineage-traced MΦ in scar tissue, and to compare mechanosensitivity of circulating monocytes with that of MΦBM. KEY POINTS: Bone marrow-derived (MΦBM) and tissue resident (MΦTR) macrophages have stretch-induced currents, indicating expression of functional mechanosensitive channels (MSC) in their plasma membrane. Stretch-activated current profiles show characteristics of cation non-selective MSC; and mRNA coding for MSC, including Piezo1 and TRPV4, is expressed in murine MΦBM and in MΦTR. Calcium entry upon pharmacological activation of TRPV4 confirms functionality of the channel in MΦTR and in MΦBM. Piezo1 ion channel activity is detected in the plasma membrane of MΦBM but not in MΦTR, suggesting that MΦBM may not be a good model to study the mechanotransduction of MΦTR. Stretch-induced currents, Piezo1mRNA expression and response to pharmacological activation are not significantly changed in cardiac MΦ 28days after cryoinjury compared to sham operated mice.

ENDOCANNABINOID SYSTEM IN THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS MODULATES AUTONOMIC AND CARDIOVASCULAR CHANGES BUT NOT VASOPRESSIN RESPONSE IN A RAT HEMORRHAGIC SHOCK MODEL.

We evaluated the participation of the endocannabinoid system in the paraventricular nucleus of the hypothalamus (PVN) on the cardiovascular, autonomic, and plasma vasopressin (AVP) responses evoked by hemorrhagic shock in rats. For this, the PVN was bilaterally treated with either vehicle, the selective cannabinoid receptor type 1 antagonist AM251, the selective fatty acid amide hydrolase amide enzyme inhibitor URB597, the selective monoacylglycerol-lipase enzyme inhibitor JZL184, or the selective transient receptor potential vanilloid type 1 antagonist capsazepine. We evaluated changes on arterial pressure, heart rate, tail skin temperature (ST), and plasma AVP responses induced by bleeding, which started 10 min after PVN treatment. We observed that bilateral microinjection of AM251 into the PVN reduced the hypotension during the hemorrhage and prevented the return of blood pressure to baseline values in the posthemorrhagic period. Inhibition of local 2-arachidonoylglycerol metabolism by PVN treatment with JZL184 induced similar effects in relation to those observed in AM251-treated animals. Inhibition of local anandamide metabolism via PVN treatment with URB597 decreased the depressor effect and ST drop induced by the hemorrhagic stimulus. Bilateral microinjection of capsazepine mitigated the fall in blood pressure and ST. None of the PVN treatments altered the increased plasma concentration of AVP and tachycardia induced by hemorrhage. Taken together, present results suggest that endocannabinoid neurotransmission within the PVN plays a prominent role in cardiovascular and autonomic, but not neuroendocrine, responses evoked by hemorrhage.

Menthol excites dural afferent neurons by inhibiting leak K+ conductance in rats

Menthol—a natural organic compound—is widely used for relieving various pain conditions including migraine. However, a high dose of menthol reportedly decreases pain thresholds and enhances pain responses. Accordingly, in the present study, we addressed the effect of menthol on the excitability of acutely isolated dural afferent neurons, which were identified with a fluorescent dye, using the whole-cell patch-clamp technique. Under a voltage-clamped condition, menthol altered the holding current levels in a concentration-dependent manner. The menthol-induced current (IMenthol) remained unaffected by the addition of selective transient receptor potential melastatin 8 antagonists. Moreover, the reversal potential of IMenthol was similar to the equilibrium potential of K+. IMenthol was accompanied by an increase in input resistance, thereby suggesting that menthol decreases the leak K+ conductance. Under a current-clamped condition, menthol caused depolarization of the membrane potential and decreased the threshold for the generation of action potential. While the IMenthol was substantially inhibited by 10 μM XE-991, a selective KV7 blocker, the M-current mediated by KV7 was not detected in the nociceptive neurons tested in the present study. Moreover, IMenthol decreased under acidic extracellular pH conditions or in the presence of 3 μM A-1899, a selective K2P3.1 and K2P9.1 blocker. The present results suggest that menthol inhibits leak K+ channels, possibly acid-sensitive two-pore domain K+ channels, thereby increasing the excitability of nociceptive sensory neurons. The resultant increase in neuron excitability may partially be responsible for the pronociceptive effect mediated by high menthol doses.

Efficacy of KPR-5714, a selective transient receptor potential melastatin 8 antagonist, on chronic psychological stress-induced bladder overactivity in male rats.

Chronic psychological stress aggravates lower urinary tract symptoms. Among others, water avoidance stress is a chronic psychological stressor that plays a causal role in the exacerbation and development of bladder dysfunction in rats. In this report, the effects of KPR-5714, which is a selective transient receptor potential melastatin 8 (TRPM8) antagonist, on bladder overactivity induced by water avoidance stress were examined. Male rats were subjected to water avoidance stress for 2 h per day for 10 consecutive days. The effects of water avoidance stress on voiding behavior using metabolic cages and histological bladder changes were investigated in rats. The involvement of bladder C-fiber afferent on voiding frequency in rats exposed to water avoidance stress was assessed using capsaicin. The effects of KPR-5714 on storage dysfunction in rats subjected to water avoidance stress were examined. In voiding behavior measurements, water avoidance stress-induced storage dysfunction, causing a decrease in the mean voided volume and increasing voiding frequency. A comparison of bladders from normal rats and rats exposed to water avoidance stress showed no histological differences. Water avoidance stress-induced bladder overactivity was completely inhibited by pretreatment with capsaicin. KPR-5714 showed a tendency to increase the mean voided volume and significantly decreased the voiding frequency without affecting the total voided volume in these rats. The results suggest that KPR-5714 is a promising option for treating chronic psychological stress-induced bladder overactivity.

Functional basis for calmodulation of the TRPV5 calcium channel.

Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+ -selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic lobe-specific CaM regulation. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+ , and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without requiring CaM N-lobe calcification. Moreover, we show a Ca2+ -dependent binding stoichiometry at the single channel level. In conclusion, our study proposes a new model for CaM-dependent regulation - calmodulation - of this uniquely Ca2+ -selective TRP channel TRPV5 that involves apoCaM interaction and lobe-specific actions, which may be of significant physiological relevance given its role as gatekeeper of Ca2+ transport in the kidney. KEY POINTS: The renal Ca2+ channel TRPV5 is an important player in maintenance of the body's Ca2+ homeostasis. Activity of TRPV5 is controlled by a negative feedback loop that involves calmodulin (CaM), a protein with two Ca2+ -binding lobes. We investigated the dynamics of the interaction between TRPV5 and CaM with advanced fluorescence microscopy techniques. Our data support a new model for CaM-dependent regulation of TRPV5 channel activity with CaM lobe-specific actions and demonstrates Ca2+ -dependent binding stoichiometries. This study improves our understanding of the mechanism underlying fast channel inactivation, which is physiologically relevant given the gatekeeper function of TRPV5 in Ca2+ reabsorption in the kidney.

The discovery of (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile, a potent and selective agonist of human transient receptor potential cation channel subfamily m member 5 (TRPM5) and evaluation of as a potential gastrointestinal prokinetic agent

This publication details the discovery of a series of selective transient receptor potential cation channel subfamily M member 5 (TRPM5) agonists culminating with the identification of the lead compound (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile (39). We describe herein our biological rationale for agonism of the target, the examination of the then current literature tool molecules, and finally the process of our discovery starting with a high throughput screening hit through lead development. We also detail the selectivity of the lead compound 39 versus related family members TRPA1, TRPV1, TRPV4, TRPM4 and TRPM8, the drug metabolism and pharmacokinetics (DMPK) profile and in vivo efficacy in a mouse model of gastrointestinal motility.

Klotho Modulates Pro-Fibrotic Activities in Human Atrial Fibroblasts through Inhibition of Phospholipase C Signaling and Suppression of Store-Operated Calcium Entry.

Background: Atrial fibroblasts activation causes atrial fibrosis, which is one major pathophysiological contributor to atrial fibrillation (AF) genesis. Klotho is a pleiotropic protein with remarkable cardiovascular effects, including anti-inflammatory, anti-oxidative, and anti-apoptotic effects. This study investigated whether Klotho can modulate the activity of human atrial fibroblasts and provides an anti-fibrotic effect. Methods: Cell migration assay and proliferation assay were used to investigate fibrogenesis activities in single human atrial fibroblasts with or without treatment of Klotho (10 and 100 pM, 48 h). Calcium fluorescence imaging, the whole-cell patch-clamp, and Western blotting were performed in human atrial fibroblasts treated with and without Klotho (100 pM, 48 h) to evaluate the store-operated calcium entry (SOCE), transient receptor potential (TRP) currents, and downstream signaling. Results: High dose of Klotho (100 pM, 48 h) significantly reduced the migration of human atrial fibroblasts without alternating their proliferation; in addition, treatment of Klotho (100 pM, 48 h) also decreased SOCE and TRP currents. In the presence of BI-749327 (a selective canonical TRP 6 channel inhibitor, 1 μM, 48 h), Klotho (100 pM, 48 h) could not inhibit fibroblast migration nor suppress the TRP currents. Klotho-treated fibroblasts (100 pM, 48 h) had lower phosphorylated phospholipase C (PLC) (p-PLCβ3 Ser537) expression than the control. The PLC inhibitor, U73122 (1 μM, 48 h), reduced the migration, decreased SOCE and TRP currents, and lowered p-PLCβ3 in atrial fibroblasts, similar to Klotho. In the presence of the U73122 (1 μM, 48 h), Klotho (100 pM, 48 h) could not further modulate the migration and collagen synthesis nor suppress the TRP currents in human atrial fibroblasts. Conclusions: Klotho inhibited pro-fibrotic activities and SOCE by inhibiting the PLC signaling and suppressing the TRP currents, which may provide a novel insight into atrial fibrosis and arrhythmogenesis.

KPR-5714, a selective transient receptor potential melastatin 8 antagonist, improves voiding dysfunction in rats with bladder overactivity but does not affect voiding behavior in normal rats.

Transient receptor potential melastatin 8 (TRPM8) has a role in the abnormal sensory transduction of the bladder and is involved in the pathophysiology of hyperactivity bladder disorders. The aim of this study is to examine the effects of KPR-5714, a novel and selective TRPM8 antagonist, on voiding dysfunction induced by bladder afferent hyperactivity via mechanosensitive C-fibers in rats. The effects of intragastric administration of KPR-5714 on bladder overactivity induced by intravesical instillation of 10 mM ATP were investigated using cystometry in conscious female rats. We examined the effects of oral administration of KPR-5714 on voiding behavior using a metabolic cage in normal male rats and rats with an intratesticular injection of 3% acetic acid. In cystometry measurements, the intercontraction interval was decreased by intravesical ATP instillation. KPR-5714 (0.1, 0.3, and 1 mg/kg) dose-dependently prolonged the shortened intercontraction interval provoked by ATP. In voiding behavior measurements, intratesticular injection of acetic acid decreased the mean voided volume and increased voiding frequency. KPR-5714 (0.1 and 0.3 mg/kg) dose-dependently increased the mean voided volume and decreased voiding frequency without affecting the total voided volume in these rats. However, KPR-5714 (1 and 10 mg/kg) did not influence the voiding behavior in normal rats. The present results suggest that KPR-5714 improves voiding dysfunction by inhibiting the enhanced activity of mechanosensitive bladder C-fibers in rats with bladder overactivity and shows no significant change in voiding behavior in normal rats.

Functional Characterization of TRPV-Like Ion Channels Involved in Nematocyst Discharge from the Sea Anemone Diadumene lineata.

AbstractCnidarians require mechanical stimuli to trigger nematocyst discharge and initiate feeding behaviors. The interval from triggering stimulus to response is tens of microseconds, making it likely that mechanically gated ion channels trigger nematocyst discharge. Because many transient receptor potential channels are mechanically gated, we hypothesized that nematocyst discharge involves transient receptor potential channels. We therefore tested various transient receptor potential channel inhibitors to determine whether they inhibit nematocyst discharge and prey killing in the acontiate sea anemone (Actinaria) Diadumene lineata (a.k.a. Haliplanella luciae). Three types of cnidocyte supporting cell complexes regulate nematocyst discharge in anemones: Types C, B, and A. Discharge from Type Cs is directly triggered by stimulation of contact-sensitive mechanoreceptors, while Type Bs require activation of chemoreceptors from prey-derived N-acetylated sugars to sensitize contact-sensitive mechanoreceptors. In Type As, activated chemoreceptors tune vibration-sensitive mechanoreceptors that predispose contact-sensitive mechanoreceptors for triggering. The non-selective transient receptor potential channel blockers lanthanum and gadolinium dose-dependently inhibited about 80% of prey killing and all nematocyst discharge from Type Bs and Type Cs, but not Type As. The selective transient receptor potential vanilloid 4 (TRPV4) blocker GSK2193874 inhibited Type As and Type Bs. However, the selective TRPV4 blockers HC-067047 and RN-1734 inhibited only Type As. Thus, three TRPV4-selective blockers implicate TRPV-like involvement in discharge from Type As, whereas GSK2193874 also affected Type Bs. Our results suggest that a TRPV-like homolog plays an essential role in nematocyst-mediated prey killing from Type As, whereas other transient receptor potential channels are likely involved in discharge from Type B and C cnidocyte supporting cell complexes.

About the Cover

Next article FreeAbout the CoverPDFPDF PLUSFull Text Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinked InRedditEmailQR Code SectionsMoreThis issue’s cover shows clonal sea anemones Diadumene (Haliplanella) lineata. Sea anemones are sessile marine cnidarians. Cnidarians use organelles called nematocysts made and deployed by cnidocytes to capture and kill prey rapidly following sensory stimulation. When triggered, nematocysts deploy an eversible barbed tubule through which a venom is injected into prey. Backward-facing barbs and an anchoring mechanism on the tentacle combine to retain captured prey, while tentacles bearing prey bend toward the mouth for ingestion.Nematocyst discharge is under hierarchal control of two classes of chemoreceptors: vibration-sensitive mechanoreceptors (VSMs) and contact-sensitive mechanoreceptors (CSMs). Chemoreceptors and VSMs on two or more supporting cells adjacent to cnidocytes form cnidocyte-supporting cell complexes (CSCCs), the functional units of nematocyst discharge. Three types of CSCCs regulate nematocyst discharge in D. lineata: (i) Type As have associated NANA chemoreceptors that tune vibration-sensitive mechanoreceptors (VSMs) to the movements of swimming prey that, in turn, predispose CSMs to trigger; (ii) Type Bs are sensitized by N-acetylneuraminic acid (NANA) that predisposes triggering CSMs to physical contact; and (iii) Type C CSCCs are activated by physical stimulation of contact-sensitive mechanoreceptors. The CSMs and VSMs require extracellular calcium, and the delay from triggering to discharge is measured in milliseconds. Such features suggest mechanogated ion channels, features shared by several types of transient receptor potential (TRP) channels.On pages 48–61 of this issue, A. Nam, S. Quarshie, M. Kimble, and D. A. Hessinger test general and selective TRP channel blockers, using a quantitative prey killing assay to implicate a variety of TRP channels involved in nematocyst-mediated prey killing. They found that non-selective TRP blockers, such as gadolinium (Gd+3) and lanthanum (La+3), potently and broadly inhibit both nematocyst discharge and prey killing from Type Bs and Cs, which is consistent with TRP channel involvement. They then demonstrated that three selective TRPV4 blockers inhibited prey killing from vibration-sensitive Type A CSCCs. Their data implicate TRPV4-like channels in nematocyst discharge from vibration-sensitive Type As and pharmacologically distinct TRP channels in Type Bs and Cs.Credits: Photo of Diadumene lineata by David Reed, with permission. Available under Creative Commons License CC BY-NC-SA 2.0. Cover design: Olivia Kinker, University of Chicago Press. Next article DetailsFiguresReferencesCited by The Biological Bulletin Volume 242, Number 1February 2022 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.1086/719750 Views: 936Total views on this site © 2022 The University of Chicago. All rights reserved.PDF download Crossref reports no articles citing this article.

Airway Ciliary Beating Affected by the Pcp4 Dose-Dependent [Ca2+]i Increase in Down Syndrome Mice, Ts1Rhr

In Ts1Rhr, a Down syndrome model mouse, the airway ciliary beatings are impaired; that is, decreases in ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of ciliary beat amplitude)). A resumption to two copies of the Pcp4 gene on the Ts1Rhr trisomic segment (Ts1Rhr:Pcp4+/+/-) rescues the decreases in CBF and CBA that occur in Ts1Rhr. In airway cilia, upon stimulation with procaterol (a β2-agonist), the CBF increase is slower over the time course than the CBA increase because of cAMP degradation by Ca2+/calmodulin-dependent phosphodiesterase 1 (PDE1) existing in the metabolon regulating CBF. In Ts1Rhr, procaterol-stimulated CBF increase was much slower over the time course than in the wild-type mouse (Wt) or Ts1Rhr:Pcp4+/+/-. However, in the presence of 8MmIBMX (8-methoxymethyl isobutylmethyl xanthine, an inhibitor of PDE1) or calmidazolium (an inhibitor of calmodulin), in both Wt and Ts1Rhr, procaterol stimulates CBF and CBA increases over a similar time course. Measurements of cAMP revealed that the cAMP contents were lower in Ts1Rhr than in Wt or in Ts1Rhr:Pcp4+/+/-, suggesting the activation of PDE1A that is present in Ts1Rhr airway cilia. Measurements of the intracellular Ca2+ concentration ([Ca2+]i) in airway ciliary cells revealed that temperature (increasing from 25 to 37 °C) or 4αPDD (a selective transient receptor potential vanilloid 4 (TRPV4) agonist) stimulates a larger [Ca2+]i increase in Ts1Rhr than in Wt or Ts1Rhr:Pcp4+/+/-. In airway ciliary cells of Ts1Rhr, Pcp4-dose dependent activation of TRPV4 appears to induce an increase in the basal [Ca2+]i. In early embryonic day mice, a basal [Ca2+]i increased by PCP4 expressed may affect axonemal regulatory complexes regulated by the Ca2+-signal in Ts1Rhr, leading to a decrease in the basal CBF and CBA of airway cilia.

TRP Channels as Lower Urinary Tract Sensory Targets.

Several members of the transient receptor potential (TRP) superfamily, including TRPV1, TRPV2, TRPV4, TRM4, TRPM8 and TRPA1, are expressed in the lower urinary tract (LUT), not only in neuronal fibers innervating the bladder and urethra, but also in the urothelial and muscular layers of the bladder and urethral walls. In the LUT, TRP channels are mainly involved in nociception and mechanosensory transduction. Animal studies have suggested the therapeutic potential of several TRP channels for the treatment of both bladder over- and underactivity and bladder pain disorders,; however translation of this finding to clinical application has been slow and the involvement of these channels in normal human bladder function, and in various pathologic states have not been established. The development of selective TRP channel agonists and antagonists is ongoing and the use of such agents can be expected to offer new and important information concerning both normal physiological functions and possible therapeutic applications.

Maternal Transient Receptor Potential Vanilloid 6 (Trpv6) Is Involved In Offspring Bone Development.

Embryonic growth and bone development depend on placental Ca2+ transport across the feto-maternal barrier to supply minerals to the fetus. The individual factors and cellular mechanisms that regulate placental Ca2+ transfer, however, are only beginning to emerge. We find that the Ca2+ -selective transient receptor potential vanilloid 6 (TRPV6) channel is expressed in trophoblasts of the fetal labyrinth, in the yolk sac, and in the maternal part of the placenta. Lack of functional TRPV6 channels in the mother leads to a reduced Ca2+ content in both placenta and embryo. Ca2+ uptake in trophoblasts is impaired in the absence of Trpv6. Trpv6-deficient embryos are smaller, have a lower body weight, and shorter and less calcified femurs. The altered cortical bone microarchitecture persists in adulthood. We show that TRPV6's Ca2+ -conducting property causes this embryonic and bone phenotype. Our results show that TRPV6 is necessary for the Ca2+ uptake in trophoblasts and that TRPV6 deficiency in the placenta leads to reduced embryo growth, minor bone calcification, and impaired bone development. © 2019 American Society for Bone and Mineral Research.

Involvement of transient receptor potential melastatin 4 channels in the resting membrane potential setting and cholinergic contractile responses in mouse detrusor and ileal smooth muscles.

Here, we investigated the effects of 9-hydroxyphenanthrene (9-phenanthrol), a potent and selective transient receptor potential melastatin 4 (TRPM4) channel blocker, on the resting membrane potential and cholinergic contractile responses to elucidate the functional role of TRPM4 channels in the contractile activities of mouse detrusor and ileal longitudinal smooth muscles. We observed that, 9-phenanthrol (3–30 µM) did not significantly inhibit high K+-induced contractions in both preparations; however, 9-phenanthrol (10 µM) strongly inhibited cholinergic contractions evoked by electrical field stimulation in detrusor preparations compared to inhibitions in ileal preparations. 9-Phenanthrol (10 µM) significantly inhibited the muscarinic agonist, carbachol-induced contractile responses and slowed the maximum upstroke velocities of the contraction in detrusor preparations. However, the agent (10 µM) did not inhibit the contractions due to intracellular Ca2+ release evoked by carbachol, suggesting that the inhibitory effect of 9-phenanthrol may primarily be due to the inhibition of the membrane depolarization process incurred by TRPM4 channels. On the other hand, 9-phenanthrol (10 µM) did not affect carbachol-induced contractile responses in ileal preparations. Further, 9-phenanthrol (10 µM) significantly hyperpolarized the resting membrane potential and decreased the basal tone in both detrusor and ileal muscle preparations. Taken together, our results suggest that TRPM4 channels are constitutively active and are involved in setting of the resting membrane potential, thereby regulating the basal tone in detrusor and ileal smooth muscles. Thus, TRPM4 channels play a significant role in cholinergic signaling in detrusor, but not ileal, smooth muscles.

Repeated exposure to temperature variation exacerbates airway inflammation through TRPA1 in a mouse model of asthma.

Studies from epidemiology suggest that ambient temperature is one of the underlying triggers and potential causes of asthma. The aim of this study was to examine the impact and the molecular mechanism of temperature-invoked airway inflammation using an experimental model of asthma in BALB/c mice. Mice were exposed to different temperature conditions (steady 26°C, 26°C/18°C cycle, 26°C/10°C cycle) and received sensitization and challenge of ovalbumin (OVA) during a 21-day period. HC030031, a selective transient receptor potential A1 (TRPA1) channel blocker, was used to investigate the underlying mechanism of TRPA1 in 'asthmatic' airways. After the final OVA challenge, in vivo lung function was measured, and bronchoalveolar lavage fluid (BALF) and pulmonary inflammation were assessed. The temperature variations, especially the largest temperature difference (16°C), exacerbated airway inflammation in OVA-induced mice, increasing the levels of serum total-IgE (immunoglobulin E) and IgG1, inflammatory cells and cytokines in BALF. Analysis of histopathological changes and lung function verified that repeated exposure to very cold and changed temperatures aggravated airway hyperresponsiveness (AHR). Significant upregulation of TRPA1 expression was revealed by immunohistochemistry in the presence of the largest temperature variation (26°C/10°C cycle), while administration of HC030031 successfully inhibited TRPA1 expression, thus attenuating the asthma-like pathological features. Repeated exposure to temperature variation exacerbated experimental 'asthma' and TRPA1 mediated this temperature-dependent inflammatory effect.

Focal selective chemo-ablation of spinal cardiac afferent nerve by resiniferatoxin protects the heart from pressure overload-induced hypertrophy

Resiniferatoxin (RTX), a selective transient receptor potential vanilloid 1 (TRPV1) receptor agonist, can eliminate TRPV1+ primary sensory afferents and blunt cardiac sympathetic afferent reflex for a relatively long period. The present study determined the effects of intrathecal RTX administration on transverse aortic constriction (TAC)-induced cardiac dysfunction and cardiac remodeling in rats. Five days before TAC, RTX (2 μg/10 μl) was injected intrathecally into the T2/T3 interspace of rats. Cardiac sympathetic nerve activities (CSNAs) and cardiac structure and function were determined eight weeks after TAC. Intrathecal RTX administration abolished TRPV1 expression in the dorsal horn and reduced over-activated CSNA in the TAC rat model. Hemodynamic analysis revealed that RTX reduced left ventricular end-diastolic pressure, indicating the improvement of cardiac compliance. Histologic analysis, real-time reverse transcription-polymerase chain reaction, and Western blots showed that RTX prevented TAC-induced cardiac hypertrophy, cardiac fibrosis, and cardiac apoptosis and reduced the expression of apoptotic proteins and myocardial mRNAs. In conclusion, these results demonstrate that focal chemo-ablation of TRPV1+ afferents in the spinal cord protects the heart from pressure overload-induced cardiac remodeling and cardiac dysfunction, which suggest a novel promising therapeutic method for cardiac hypertrophy and diastolic dysfunction.

Tadalafil attenuates hypotonicity-induced Ca2+ influx via TRPV2 and TRPV4 in primary rat bladder urothelial cell cultures.

To investigate the localization of phosphodiesterase 5 (PDE5) and the molecular mechanism underlying the effect of the PDE5 inhibitor tadalafil in signal transduction in the bladder urothelium. PDE5 expression in rat bladder tissues and cultured primary rat bladder urothelial cells was evaluated using immunochemistry and western blot assays. Ca2+ influx in cells exposed to isotonic solution, hypotonic solution, a selective transient receptor potential vanilloid 2 (TRPV2) channel agonist (cannabidiol), a selective TRPV4 channel agonist (GSK1016790A), a TRP cation channel melastatin 7 (TRPM7) channel agonist (PIP2), or a purinergic receptor agonist (ATP) in the presence or absence of 10 µM tadalafil was evaluated using calcium imaging techniques. We also evaluated stretch-induced changes in ATP concentration in the mouse bladder in the presence or absence of 100 µM tadalafil. Immunochemistry and western blot analyses demonstrated that PDE5 is abundantly expressed in the bladder urothelium and in primary rat urothelial cells. Ca2+ influx induced by hypotonic stimulation, GSK1016790A, or cannabidiol was significantly inhibited by tadalafil, whereas ATP-induced Ca2+ influx was unaffected by tadalafil. PIP2 did not induce Ca2+ influx. ATP release in tadalafil-pretreated bladders significantly decreased compared to control bladders. Tadalafil attenuates Ca2+ influx via TRPV4 and TRPV2, and inhibits ATP release in the bladder urothelium. These findings indicate that tadalafil functions as an inhibitor of urothelial signal transduction.

Menthol-Induced Cutaneous Vasodilation Is Preserved in Essential Hypertensive Men and Women.

Menthol is a selective transient receptor potential melastatin 8 (TRPM8) channel agonist that induces cutaneous vasodilation in young, normotensive men and women through nitric oxide synthase (NOS)-, endothelium-derived hyperpolarizing factor (EDHF)-, and sensory nerve-mediated mechanisms. Microvascular dysfunction is present in essential hypertension and whether menthol induces vasodilation is men and women with essential hypertension is equivocal. Four intradermal microdialysis fibers were placed in the forearm of 9 essential hypertensive and 10 age-matched normotensive control subjects. Sites were pretreated with lactated Ringer's (control), l-NAME (NOS inhibited), TEA (EDHF inhibited), and lidocaine (sensory nerve inhibited). The microdialysis fibers were then perfused with 7 increasing doses of menthol (0.1-500 mM). Red cell flux in response to menthol was measured with laser Doppler flowmetry. Data were normalized to mean arterial pressure and presented as a percentage of site-specific maximum vasodilation (%CVCmax). At the control site, menthol caused vasodilation in both the normotensive and hypertensive groups (menthol doses 100, 250, and 500 mM; all P < 0.05 compared to baseline). There were no differences between groups (P = 0.58, main effect). There was no effect of either NOS or sensory nerve inhibition on menthol-induced vasodilation in the normotensive group; however, menthol-induced vasodilation was attenuated with NOS and sensory nerve inhibition in the hypertensive group. EDHF inhibition attenuated menthol-induced vasodilation in both groups. Menthol-induced vasodilation has NO, EDHF, and sensory nerve components. Menthol-induced cutaneous vasodilation is preserved in hypertensive subjects. However, the hypertensive subjects exhibited a loss of redundant vasodilator systems.

β2-Microglobulin elicits itch-related responses in mice through the direct activation of primary afferent neurons expressing transient receptor potential vanilloid 1

Uremic pruritus is an unpleasant symptom in patients undergoing hemodialysis, and the underlying mechanisms remain unclear. β2-Microglobulin (β2-MG) is well-known as an MHC class I molecule and its level is increased in the plasma of patients undergoing hemodialysis. In this study, we investigated whether β2-MG was a pruritogen in mice. Intradermal injections of β2-MG into the rostral back induced scratching in a dose-dependent manner. Intradermal injection of β2-MG into the cheek also elicited scratching, but not wiping. β2-MG-induced scratching was inhibited by the μ-opioid receptor antagonist naltrexone hydrochloride. β2-MG-induced scratching was not inhibited by antagonists of itch-related receptors (e.g., H1 histamine receptor (terfenadine), TP thromboxane receptor (DCHCH), BLT1 leukotriene B4 receptor (CMHVA), and proteinase-activated receptor 2 (FSLLRY-NH2)). However, β2-MG-induced scratching was attenuated in mice desensitized by repeated application of capsaicin and also by a selective transient receptor potential vanilloid 1 (TRPV1) antagonist (BCTC). In addition, β2-MG induced phosphorylation of extracellular signal-regulated kinase (a marker of activated neurons) in primary culture of dorsal root ganglion neurons that expressed TRPV1. These results suggest that β2-MG is a pruritogen and elicits itch-related responses, at least in part, through TRPV1-expressing primary sensory neurons.

Pharmacological evaluation of novel (6-aminopyridin-3-yl)(4-(pyridin-2-yl)piperazin-1-yl) methanone derivatives as TRPV4 antagonists for the treatment of pain

A novel series of (6-aminopyridin-3-yl)(4-(pyridin-2-yl)piperazin-1-yl) methanone derivatives were identified as selective transient receptor potential vanilloid 4 (TRPV4) channel antagonist and showed analgesic effect in Freund’s Complete Adjuvant (FCA) induced mechanical hyperalgesia model in guinea pig and rat. Modification of right part based on the compound 16d which was disclosed in our previous communication led to the identification of compound 26i as a flagship compound. In this paper, we described the details about design, synthesis and structure-activity relationship (SAR) analysis at right and left part of these derivatives (Fig. 1).