Chemo-nociceptive signalling from the colon is enhanced by mild colitis and blocked by inhibition of transient receptor potential ankyrin 1 channels

Background and purpose:

Transient receptor potential ankyrin 1 (TRPA1) channels are expressed by primary afferent neurones and activated by irritant chemicals including allyl isothiocyanate (AITC). Here we investigated whether intracolonic AITC causes afferent input to the spinal cord and whether this response is modified by mild colitis, morphine or a TRPA1 channel blocker.

Experimental approach:

One hour after intracolonic administration of AITC to female mice, afferent signalling was visualized by expression of c-Fos in laminae I–II_o of the spinal dorsal horn at sacral segment S1. Mild colitis was induced by dextran sulphate sodium (DSS) added to drinking water for 1 week.

Key results:

Relative to vehicle, AITC (2%) increased expression of c-Fos in the spinal cord. Following induction of mild colitis by DSS (2%), spinal c-Fos responses to AITC, but not vehicle, were augmented by 41%. Colonic inflammation was present (increased myeloperoxidase content and disease activity score), whereas colonic histology, locomotion, feeding and drinking remained unchanged. Morphine (10 mg·kg⁻¹) or the TRPA1 channel blocker HC-030031 (300 mg·kg⁻¹) inhibited the spinal c-Fos response to AITC, in control and DSS-pretreated animals, whereas the response to intracolonic capsaicin (5%) was blocked by morphine but not HC-030031.

Conclusions and implications:

Activation of colonic TRPA1 channels is signalled to the spinal cord. Mild colitis enhanced this afferent input that, as it is sensitive to morphine, is most likely of a chemonociceptive nature. As several irritant chemicals can be present in chyme, TRPA1 channels may mediate several gastrointestinal pain conditions.     

Transient receptor potential ankyrin receptor 1 is a novel target for pro-tussive agents

Background and purpose:

The transient receptor potential ankyrin receptor 1 (TRPA1) is a cation channel, co-expressed with the pro-tussive transient receptor potential vanilloid type 1 (TRPV1) channel in primary sensory neurons. TRPA1 is activated by a series of irritant exogenous and endogenous α,β-unsaturated aldehydes which seem to play a role in airway diseases. We investigated whether TRPA1 agonists provoke cough in guinea pigs and whether TRPA1 antagonists inhibit this response.

Experimental approach:

Animals were placed in a Perspex box, and cough sounds were recorded and counted by observers unaware of the treatment used.

Key results:

Inhalation of two selective TRPA1 agonists, allyl isothiocyanate and cinnamaldehyde, dose-dependently caused cough in control guinea pigs, but not in those with airway sensory nerves desensitized by capsaicin. Coughs elicited by TRPA1 agonists were reduced by non-selective (camphor and gentamicin) and selective (HC-030031) TRPA1 antagonists, whereas they were unaffected by the TRPV1 antagonist, capsazepine. Acrolein and crotonaldehyde, two α,β-unsaturated aldehydes recently identified as TRPA1 stimulants and contained in cigarette smoke, air pollution or produced endogenously by oxidative stress, caused a remarkable tussive effect, a response that was selectively inhibited by HC-030031. Part of the cough response induced by cigarette smoke inhalation was inhibited by HC-030031, suggesting the involvement of TRPA1.

Conclusions and implications:

A novel pro-tussive pathway involves the TRPA1 channel, expressed by capsaicin-sensitive airway sensory nerves and is activated by a series of exogenous (cigarette smoke) and endogenous irritants. These results suggest TRPA1 may be a novel target for anti-tussive medicines.     

Effect of TRPA1 activator allyl isothiocyanate (AITC) on rat dural and pial arteries

BackgroundTransient receptor potential ankyrin 1 (TRPA1) channels may have a role in migraine as some substances known to cause headache activate the channel. In the craniovascular system such activation causes a calcitonin gene-related peptide (CGRP)-dependent increase in meningeal blood flow. TRPA1 channels in the endothelium of cerebral arteries cause vasodilation when activated. The headache preventive substance feverfew inhibits activation of TRPA1 channels. In this study we aim to compare and characterize the effect of the TRPA1 agonist allyl isothiocyanate (AITC) on the diameter of rat dural and pial arteries in vivo.MethodsThe genuine closed-cranial window technique in rats was used to examine changes in dural and pial artery diameter and mean arterial blood pressure (MABP) after intracarotid infusion of AITC. Blockade experiments were performed by intravenous infusion of olcegepant, HC-030031, sumatriptan or capsazepine immediately after infusion of AITC, in four different groups of rats.ResultsAITC caused a significant dilation of dural arteries, which was inhibited by HC-030031, olcegepant and sumatriptan, but not by capsazepine.In pial arteries AITC caused a significant dilation, which was not inhibited by any of the pre-treatments, suggesting a poor penetration of the blood-brain barrier or autoregulation due to dimethyl sulfoxide (DMSO) mediated decrease in MABP during HC-030031 infusion.AITC did not cause a significant change in MABP.ConclusionAITC causes dilation of dural arteries via a mechanism dependent on CGRP and TRPA1 that is sensitive to sumatriptan. AITC causes a small but significant dilation of pial arteries.     

Activation of transient receptor potential A1 by a non-pungent capsaicin-like compound, capsiate

BACKGROUND AND PURPOSE

Capsiate is produced by ‘CH-19 Sweet’ (Capsicum annuun L.), a non-pungent cultivar of red pepper. Like capsaicin, capsiate is thought to enhance energy metabolism by activating the sympathetic nervous system and suppressing inflammation, but the underlying mechanisms for this are uncertain. We previously reported that capsiate could activate transient receptor potential vanilloid 1 (TRPV1), a capsaicin receptor. The purpose of the present study is to investigate whether capsinoids activate other TRP channels.

EXPERIMENTAL APPROACH

Using Ca²⁺ imaging and whole-cell patch-clamp methods, we analysed the response of TRP channels to three kinds of capsinoids, capsiate, dihydrocapsiate and nordihydrocapsiate, in HEK293T cells expressing TRP channels or in primary cultures of mouse dorsal root ganglion neurons.

KEY RESULTS

We found that in both cell types TRP ankyrin 1 (TRPA1) had a slightly weaker response to capsinoids compared with TRPV1, with the capsiate EC₅₀ for TRPA1 activation being more than that for TRPV1 activation, and that the capsinoid-evoked action was blocked by a specific TRPA1 antagonist. TRPA1 was activated by capsinoids, but not by their degradation products. Amino acids known to participate in TRPA1 activation following cysteine covalent modification or zinc treatment were not involved in the activation of TRPA1 by capsinoid.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results indicate that capsinoids activate TRPA1 by an as yet unknown mechanism, and TRPA1 could be involved in physiological phenomena associated with capsinoid treatment.     

Statins and fenofibrate affect skeletal muscle chloride conductance in rats by differently impairing ClC-1 channel regulation and expression

Background and purpose:

Statins and fibrates can produce mild to life-threatening skeletal muscle damage. Resting chloride channel conductance (gCl), carried by the ClC-1 channel, is reduced in muscles of rats chronically treated with fluvastatin, atorvastatin or fenofibrate, along with increased resting cytosolic calcium in statin-treated rats. A high gCl, controlled by the Ca²⁺-dependent protein kinase C (PKC), maintains sarcolemma electrical stability and its reduction alters muscle function. Here, we investigated how statins and fenofibrate impaired gCl.

Experimental approach:

In rats treated with fluvastatin, atorvastatin or fenofibrate, we examined the involvement of PKC in gCl reduction by the two intracellular microelectrodes technique and ClC-1 mRNA level by quantitative real time-polymerase chain reaction. Direct drug effects were tested by patch clamp analysis on human ClC-1 channels expressed in human embryonic kidney (HEK) 293 cells.

Key results:

Chelerythrine, a PKC inhibitor, applied in vitro on muscle dissected from atorvastatin-treated rats fully restored gCl, suggesting the involvement of this enzyme in statin action. Chelerythrine partially restored gCl in muscles from fluvastatin-treated rats but not in those from fenofibrate-treated rats, implying additional mechanisms for gCl impairment. Accordingly, a decrease of ClC-1 channel mRNA was found in both fluvastatin-and fenofibrate-treated rat muscles. Fenofibric acid, the in vivo metabolite of fenofibrate, but not fluvastatin, rapidly reduced chloride currents in HEK 293 cells.

Conclusions and implications:

Our data suggest multiple mechanisms underlie the effect of statins and fenofibrate on ClC-1 channel conductance. While statins promote Ca²⁺-mediated PKC activation, fenofibrate directly inhibits ClC-1 channels and both fluvastatin and fenofibrate impair expression of mRNA for ClC-1.     

Tanshinone II-A sodium sulfonate (DS-201) enhances human BKCa channel activity by selectively targeting the pore-forming α subunit

Aim:

Tanshinone II-A sodium sulfonate (DS-201), a water-soluble derivative of Tanshinone II-A, has been found to induce vascular relaxation and activate BK_Ca channels. The aim of this study was to explore the mechanisms underlying the action of DS-201 on BK_Ca channels.

Methods:

Human BK_Ca channels containing α subunit alone or α plus β1 subunits were expressed in HEK293 cells. BK_Ca currents were recorded from the cells using patch-clamp technique. The expression and trafficking of BK_Ca subunits in HEK293 cells or vascular smooth muscle cells (VSMCs) were detected by Western blotting, flow cytometry and confocal microscopy.

Results:

DS-201 (40–160 μmol/L) concentration-dependently increased the total open probability of BK_Ca channels in HEK293 cells, associated with enhancements of Ca²⁺ and voltage dependence as well as a delay in deactivation. Coexpression of β1 subunit did not affect the action of DS-201: the values of EC₅₀ for BK_Ca channels containing α subunit alone and α plus β1 subunit were 66.6±1.5 and 62.0±1.1 μmol/L, respectively. In both HEK293 cells and VSMCs, DS-201 (80 μmol/L) markedly increased the expression of α subunit without affecting β1 subunit. In HEK293 cells, DS-201 enriched the membranous level of α subunit, likely by accelerating the trafficking and suppressing the internalization of α subunit. In both HEK293 cells and VSMCs, DS-201 (≥320 μmol/L) induced significant cytotoxicity.

Conclusion:

DS-201 selectively targets the pore-forming α subunit of human BK_Ca channels, thus enhancing the channel activities and increasing the subunit expression and trafficking, whereas the β1 subunit does not contribute to the action of DS-201.     

Inhibitory effect of cannabichromene, a major non-psychotropic cannabinoid extracted from Cannabis sativa, on inflammation-induced hypermotility in mice

BACKGROUND AND PURPOSE

Cannabichromene (CBC) is a major non-psychotropic phytocannabinoid that inhibits endocannabinoid inactivation and activates the transient receptor potential ankyrin-1 (TRPA1). Both endocannabinoids and TRPA1 may modulate gastrointestinal motility. Here, we investigated the effect of CBC on mouse intestinal motility in physiological and pathological states.

EXPERIMENTAL APPROACH

Inflammation was induced in the mouse small intestine by croton oil. Endocannabinoid (anandamide and 2-arachidonoyl glycerol), palmitoylethanolamide and oleoylethanolamide levels were measured by liquid chromatography-mass spectrometry; TRPA1 and cannabinoid receptors were analysed by quantitative RT-PCR; upper gastrointestinal transit, colonic propulsion and whole gut transit were evaluated in vivo; contractility was evaluated in vitro by stimulating the isolated ileum, in an organ bath, with ACh or electrical field stimulation (EFS).

KEY RESULTS

Croton oil administration was associated with decreased levels of anandamide (but not 2-arachidonoyl glycerol) and palmitoylethanolamide, up-regulation of TRPA1 and CB₁ receptors and down-regulation of CB₂ receptors. Ex vivo CBC did not change endocannabinoid levels, but it altered the mRNA expression of TRPA1 and cannabinoid receptors. In vivo, CBC did not affect motility in control mice, but normalized croton oil-induced hypermotility. In vitro, CBC reduced preferentially EFS- versus ACh-induced contractions. Both in vitro and in vivo, the inhibitory effect of CBC was not modified by cannabinoid or TRPA1 receptor antagonists.

CONCLUSION AND IMPLICATIONS

CBC selectively reduces inflammation-induced hypermotility in vivo in a manner that is not dependent on cannabinoid receptors or TRPA1.     

The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels

BACKGROUND

Depletion of the Ca²⁺ store by ryanodine receptor (RyR) agonists induces store-operated Ca²⁺ entry (SOCE). 4-Chloro-3-ethylphenol (4-CEP) and 4-chloro-m-cresol (4-CmC) are RyR agonists commonly used as research tools and diagnostic reagents for malignant hyperthermia. Here, we investigated the effects of 4-CEP and its analogues on SOCE.

EXPERIMENTAL APPROACH

SOCE and ORAI1-3 currents were recorded by Ca²⁺ imaging and whole-cell patch recordings in rat L6 myoblasts and in HEK293 cells overexpressing STIM1/ORAI1-3.

KEY RESULTS

4-CEP induced a significant release of Ca²⁺ in rat L6 myoblasts, but inhibited SOCE. The inhibitory effect was concentration-dependent and more potent than its analogues 4-CmC and 4-chlorophenol (4-ClP). In the HEK293 T-REx cells overexpressing STIM1/ORAI1-3, 4-CEP inhibited the ORAI1, ORAI2 and ORAI3 currents evoked by thapsigargin. The 2-APB-induced ORAI3 current was also blocked by 4-CEP. This inhibitory effect was reversible and independent of the Ca²⁺ release. The two analogues, 4-CmC and 4-ClP, also inhibited the ORAI1-3 channels. Excised patch and intracellular application of 4-CEP demonstrated that the action site was located extracellularly. Moreover, 4-CEP evoked STIM1 translocation and subplasmalemmal clustering through its Ca²⁺ store-depleting effect via the activation of RyR, but no effect on STIM1 redistribution was observed in cells co-expressing STIM1/ORAI1-3.

CONCLUSION AND IMPLICATIONS

4-CEP not only acts as a RyR agonist to deplete the Ca²⁺ store and trigger STIM1 subplasmalemmal translocation and clustering, but also directly inhibits ORAI1-3 channels. These findings demonstrate a novel pharmacological property for the chlorophenol derivatives that act as RyR agonists.     

Discovery and structural optimization of 1-phenyl-3-(1-phenylethyl)urea derivatives as novel inhibitors of CRAC channel

Aim:

Ca²⁺-release-activated Ca²⁺ (CRAC) channel, a subfamily of store-operated channels, is formed by calcium release-activated calcium modulator 1 (ORAI1), and gated by stromal interaction molecule 1 (STIM1). CRAC channel may be a novel target for the treatment of immune disorders and allergy. The aim of this study was to identify novel small molecule CRAC channel inhibitors.

Methods:

HEK293 cells stably co-expressing both ORAI1 and STIM1 were used for high-throughput screening. A hit, 1-phenyl-3-(1-phenylethyl)urea, was identified that inhibited CRAC channels by targeting ORAI1. Five series of its derivatives were designed and synthesized, and their primary structure-activity relationships (SARs) were analyzed. All derivatives were assessed for their effects on Ca²⁺ influx through CRAC channels on HEK293 cells, cytotoxicity in Jurkat cells, and IL-2 production in Jurkat cells expressing ORAI1-SS-eGFP.

Results:

A total of 19 hits were discovered in libraries containing 32 000 compounds using the high-throughput screening. 1-Phenyl-3-(1-phenylethyl)urea inhibited Ca²⁺ influx with IC₅₀ of 3.25±0.17 μmol/L. SAR study on its derivatives showed that the alkyl substituent on the α-position of the left-side benzylic amine (R1) was essential for Ca²⁺ influx inhibition and that the S-configuration was better than the R-configuration. The derivatives in which the right-side R3 was substituted by an electron-donating group showed more potent inhibitory activity than those that were substituted by electron-withdrawing groups. Furthermore, the free N–H of urea was not necessary to maintain the high potency of Ca²⁺ influx inhibition. The N,N′-disubstituted or N′-substituted derivatives showed relatively low cytotoxicity but maintained the ability to inhibit IL-2 production. Among them, compound 5b showed an improved inhibition of IL-2 production and low cytotoxicity.

Conclusion:

1-Phenyl-3-(1-phenylethyl)urea is a novel CRAC channel inhibitor that specifically targets ORAI1. This study provides a new chemical scaffold for design and development of CRAC channel inhibitors with improved Ca²⁺ influx inhibition, immune inhibition and low cytotoxicity.     

Apigenin, a plant-derived flavone, activates transient receptor potential vanilloid 4 cation channel

BACKGROUND AND PURPOSE

Transient receptor potential vanilloid 4 (TRPV4) is a Ca²⁺-permeable channel with multiple modes of activation. Apigenin is a plant-derived flavone, which has potential preventive effects on the development of cardiovascular disease. We set out to explore the effects of apigenin on TRPV4 channel activity and its role in vasodilatation.

EXPERIMENTAL APPROACH

The effects of apigenin (0.01–30 µM) on TPRV4 channels were investigated in HEK293 cells over-expressing TRPV4, rat primary cultured mesenteric artery endothelial cells (MAECs) and isolated small mesenteric arterial segments using whole-cell patch clamp, fluorescent Ca²⁺ imaging, intracellular recording and pressure myography.

KEY RESULTS

Whole-cell patch clamp and fluorescent Ca²⁺ imaging in HEK cells over-expressing TRPV4 showed that apigenin concentration-dependently stimulated the TRPV4-mediated cation current and Ca²⁺ influx. In MAECs, apigenin stimulated Ca²⁺ influx in a concentration-dependent manner. These increases in cation current and Ca²⁺ influx were markedly inhibited by TRPV4-specific blockers and siRNAs. Furthermore, pressure myography and intracellular recording in small third-order mesenteric arteries showed that apigenin dose-dependently evoked smooth muscle cell membrane hyperpolarization and subsequent vascular dilatation, which were significantly inhibited by TRPV4-specific blockers. TRPV4 blocker or charybdotoxin (200 nM) plus apamin (100 nM) diminished the apigenin-induced dilatation.

CONCLUSION AND IMPLICATIONS

This is the first study to demonstrate the selective stimulation of TRPV4 by apigenin. Apigenin was found to activate TRPV4 channels in a dose-dependent manner in HEK cells over-expressing TRPV4 and in native endothelial cells. In rat small mesenteric arteries, apigenin acts on TRPV4 in endothelial cells to induce EDHF-mediated vascular dilatation.     

Hydrogen sulfide-induced mechanical hyperalgesia and allodynia require activation of both Cav3.2 and TRPA1 channels in mice

BACKGROUND AND PURPOSE

Hydrogen sulfide, a gasotransmitter, facilitates somatic pain signals via activation of Ca_v3.2 T-type calcium channels in rats. Given evidence for the activation of transient receptor potential ankyrin-1 (TRPA1) channels by H₂S, we asked whether TRPA1 channels, in addition to Ca_v3.2 channels, contribute to the H₂S-induced mechanical hyperalgesia and allodynia in mice.

EXPERIMENTAL APPROACH

Mechanical hyperalgesia and allodynia were evaluated by the von Frey test in mice. Ca_v3.2 or TRPA1 channels in the sensory neurons were silenced by repeated intrathecal administration of antisense oligodeoxynucleotides in mice.

KEY RESULTS

Intraplantar administration of NaHS evoked hyperalgesia and allodynia in mice, an effect attenuated or abolished by NNC 55–0396 or mibefradil, T-type calcium channel blockers, and by ascorbic acid or zinc chloride, known to selectively inhibit Ca_v3.2 channels, out of the three isoforms of T-type calcium channels. Silencing of Ca_v3.2 channels in the sensory neurons also prevented the NaHS-induced hyperalgesia and allodynia in mice. The NaHS-induced hyperalgesia and allodynia in mice were significantly suppressed by AP18, a TRPA1 channel blocker, and by silencing of TRPA1 channels in the sensory neurons.

CONCLUSIONS AND IMPLICATIONS

Mechanical hyperalgesia and allodynia induced by NaHS/H₂S required activation of both Ca_v3.2 and TRPA1 channels in mice.     

Mechanisms of zolpidem-induced long QT syndrome: acute inhibition of recombinant hERG K+ channels and action potential prolongation in human cardiomyocytes derived from induced pluripotent stem cells

Background and Purpose

Zolpidem, a short-acting hypnotic drug prescribed to treat insomnia, has been clinically associated with acquired long QT syndrome (LQTS) and torsade de pointes (TdP) tachyarrhythmia. LQTS is primarily attributed to reduction of cardiac human ether-a-go-go-related gene (hERG)/I_Kr currents. We hypothesized that zolpidem prolongs the cardiac action potential through inhibition of hERG K⁺ channels.

Experimental Approach

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record hERG currents from Xenopus oocytes and from HEK 293 cells. In addition, hERG protein trafficking was evaluated in HEK 293 cells by Western blot analysis, and action potential duration (APD) was assessed in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes.

Key Results

Zolpidem caused acute hERG channel blockade in oocytes (IC₅₀ = 61.5 μM) and in HEK 293 cells (IC₅₀ = 65.5 μM). Mutation of residues Y652 and F656 attenuated hERG inhibition, suggesting drug binding to a receptor site inside the channel pore. Channels were blocked in open and inactivated states in a voltage- and frequency-independent manner. Zolpidem accelerated hERG channel inactivation but did not affect I–V relationships of steady-state activation and inactivation. In contrast to the majority of hERG inhibitors, hERG cell surface trafficking was not impaired by zolpidem. Finally, acute zolpidem exposure resulted in APD prolongation in hiPSC-derived cardiomyocytes.

Conclusions and Implications

Zolpidem inhibits cardiac hERG K⁺ channels. Despite a relatively low affinity of zolpidem to hERG channels, APD prolongation may lead to acquired LQTS and TdP in cases of reduced repolarization reserve or zolpidem overdose.     

Functional expression of KCNQ (Kv7) channels in guinea pig bladder smooth muscle and their contribution to spontaneous activity

Background and Purpose

The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility.

Experimental Approach

KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors.

Key Results

KCNQ subtypes 1–5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca²⁺-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and K_ATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20 μM) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity.

Conclusions and Implications

These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder.     

Chemerin C9 peptide induces receptor internalization through a clathrin-independent pathway

Aim： The chemerin receptor CMKLR1 is one type of G protein-coupled receptors abundant in monocyte-derived dendritic cells and macrophages, which plays a key role in the entry of a subset of immunodeficiency viruses including HIV/SlV into lymphocytes and macrophages. The aim of this work was to investigate how CMKLR1 was internalized and whether its internalization affected cell signaling in vitro. Methods： Rat basophilic leukemia RBL-2H3 cells, HEK 293 cells, and HeLa cells were used. CMKLR1 internalization was visualized by confocal microscopy imaging or using a FACScan flow cytometer. Six potential phosphorylation sites （Ser337, Ser343, Thr352, Ser344, Ser347, and Ser350） in CMKLR1 were substituted with alanine using site-directed mutagenesis. Heterologous expression of wild type and mutant CMKLR1 allowed for functional characterization of endocytosis, Ca2＋ flux and extracellular signal-regulated kinase （ERK） phosphorylation. Results： Chemerin and the chemerin-derived nonapeptide （C9） induced dose-dependent loss of cell surface CMKLR1-GFP fusion protein and increased its intracellular accumulation in HEK 293 cells and RBL-2H3 cells stably expressing CMKLRI. Up to 90% of CMKLR1 was internalized after treatment with C9 （1μmol/L）. By using different agents, it was demonstrated that clathrin-independent mechanism was involved in CMKLR1 internalization. Mutations in Ser343 for G protein-coupled receptor kinase phosphorylation and in Ser347 for PKC phosphorylation abrogated CMKLR1 internalization. Loss of CMKLR1 internalization partially enhanced the receptor signaling, as shown by increased Ca2＋ flux and a shorter latency to peak level of ERK phosphorylation. Conclusion： CMKLR1 internalization occurs in a clathrin-independent manner, which negatively regulated the receptor-mediated Ca2~ flux and ERK phosphorylation.     

Inhibition of human recombinant T-type calcium channels by the endocannabinoid N-arachidonoyl dopamine

Background and purpose:

N-arachidonoyl dopamine (NADA) has complex effects on nociception mediated via cannabinoid CB₁ receptors and the transient receptor potential vanilloid receptor 1 (TRPV1). Anandamide, the prototypic CB₁/TRPV1 agonist, also inhibits T-type voltage-gated calcium channel currents (I_Ca). These channels are expressed by many excitable cells, including neurons involved in pain detection and processing. We sought to determine whether NADA and the prototypic arachidonoyl amino acid, N-arachidonoyl glycine (NAGly) modulate T-type I_Ca

Experimental approach:

Human recombinant T-type I_Ca (Ca_V3 channels) expressed in HEK 293 cells and native mouse T-type I_Ca were examined using standard whole-cell voltage clamp electrophysiology techniques.

Key results:

N-arachidonoyl dopamine completely inhibited Ca_V3 channels with a rank order of potency (pEC₅₀) of Ca_V3.3 (6.45) ≥ Ca_V3.1 (6.29) > Ca_V3.2 (5.95). NAGly (10 µmol·L⁻¹) inhibited Ca_V3 I_Ca by approximately 50% or less. The effects of NADA and NAGly were voltage- but not use-dependent, and both compounds produced significant hyperpolarizing shifts in Ca_V3 channel steady-state inactivation relationships. By contrast with anandamide, NADA and NAGly had modest effects on Ca_V3 channel kinetics. Both NAGly and NADA inhibited native T-type I_Ca in mouse sensory neurons.

Conclusions and implications:

N-arachidonoyl dopamine and NAGly increase the steady-state inactivation of Ca_V3 channels, reducing the number of channels available to open during depolarization. These effects occur at NADA concentrations at or below to those affecting CB₁ and TRPV1 receptors. Together with anandamide, the arachidonoyl neurotransmitter amides, NADA and NAGly, represent a new family of endogenous T-type I_Ca modulators.     

A Ca²⁺-dependent chloride current and Ca²⁺ influx via Ca(v)1.2 ion channels play major roles in P2Y receptor-mediated pulmonary vasoconstriction

BACKGROUND AND PURPOSE

ATP, UTP and UDP act at smooth muscle P2X and P2Y receptors to constrict rat intrapulmonary arteries, but the underlying signalling pathways are poorly understood. Here, we determined the roles of the Ca²⁺-dependent chloride ion current (I_Cl,Ca), Ca_v1.2 ion channels and Ca²⁺ influx.

EXPERIMENTAL APPROACH

Isometric tension was recorded from endothelium-denuded rat intrapulmonary artery rings (i.d. 200–500 µm) mounted on a wire myograph.

KEY RESULTS

The I_Cl,Ca blockers, niflumic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and the Ca_v1.2 channel blocker, nifedipine, reduced peak amplitude of contractions evoked by UTP and UDP by ∼45–50% and in a non-additive manner. Ca²⁺-free buffer inhibited responses by ∼70%. Niflumic acid and nifedipine similarly depressed contractions to ATP, but Ca²⁺-free buffer almost abolished the response. After peaking, contractions to UTP and UDP decayed slowly by 50–70% to a sustained plateau, which was rapidly inhibited by niflumic acid and nifedipine. Contractions to ATP, however, reversed rapidly and fully. Tannic acid contracted tissues per se and potentiated nucleotide-evoked contractions.

CONCLUSIONS AND IMPLICATIONS

I_Cl,Ca and Ca²⁺ influx via Ca_v1.2 ion channels contribute substantially and equally to contractions of rat intrapulmonary arteries evoked by UTP and UDP, via P2Y receptors. ATP also activates these mechanisms via P2Y receptors, but the greater dependence on extracellular Ca²⁺ most likely reflects additional influx through the P2X1 receptor pore. The lack of a sustained response to ATP is probably due to it acting at P2 receptor subtypes that desensitize rapidly. Thus multiple signalling mechanisms contribute to pulmonary artery vasoconstriction mediated by P2 receptors.     

The blockade of transient receptor potential ankirin 1 (TRPA1) signalling mediates antidepressant- and anxiolytic-like actions in mice

Background and Purpose

Transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) are involved in many biological processes, including nociception and hyperalgesia. Whereas the involvement of TRPV1 in psychiatric disorders such as anxiety and depression has been reported, little is known regarding the role of TRPA1 in these conditions.

Experimental Approach

We investigated the role of TRPA1 in mice models of depression [forced swimming test (FST)] and anxiety [elevated plus maze (EPM) test].

Key Results

Administration of the TRPA1 antagonist ({"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681","term_text":"HC030031"}}HC030031, 30 nmol in 2 μL, i.c.v.) reduced immobility time in the FST. Similar results were obtained after oral administration of {"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681","term_text":"HC030031"}}HC030031 (30–300 mg·kg⁻¹). The reduction in immobility time in FST induced by {"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681","term_text":"HC030031"}}HC030031 (100 mg·kg⁻¹) was completely prevented by pretreatment with TRPA1 agonist, cinnamaldehyde (50 mg·kg⁻¹, p.o.), which per se was inactive. In the EPM test, pretreatment with cinnamaldehyde (50 mg·kg⁻¹, p.o.), which per se did not affect behaviour response, prevented the anxiolytic-like effect (increased open arm exploration) evoked by TRPA1 blockade ({"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681","term_text":"HC030031"}}HC030031, 100 mg·kg⁻¹, p.o.). Treatment with either cinnamaldehyde or {"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681","term_text":"HC030031"}}HC030031 did not affect spontaneous ambulation. Furthermore, TRPA1-deficient mice showed anxiolytic- and antidepressant-like phenotypes in the FST and EPM test respectively.

Conclusion and Implications

The present findings indicate that genetic deletion or pharmacological blockade of TRPA1 produces inhibitory activity in mouse models of anxiety and depression. These results imply that TRPA1 exerts tonic control, promoting anxiety and depression, and that TRPA1 antagonism has potential as an innovative strategy for the treatment of anxiety and mood disorders.     

Natural and synthetic modulators of SK (K(ca)2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7

BACKGROUND AND PURPOSE

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells.

EXPERIMENTAL APPROACH

We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments.

KEY RESULTS

TRPM7 currents were inhibited by modulators of small conductance Ca²⁺-activated K⁺ channels (K_Ca2.1–2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC₅₀ 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg²⁺-dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

This study reveals a tight overlap in the pharmacological profiles of TRPM7 and K_Ca2.1–2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7.     

Genistein potentiates activity of the cation channel TRPC5 independently of tyrosine kinases

Background and purpose:

TRPC5 is a Ca²⁺-permeable channel with multiple modes of activation. We have explored the effects of genistein, a plant-derived isoflavone, on TRPC5 activity, and the mechanism(s) involved.

Experimental approach:

Effects of genistein on TRPC5 channels were investigated in TRPC5-over-expressing human embryonic kidney 293 (HEK) cells and bovine aortic endothelial cells (BAECs) using fluorescent Ca²⁺ imaging and electrophysiological techniques.

Key results:

In TRPC5-over-expressing HEK cells, genistein stimulated TRPC5-mediated Ca²⁺ influx, concentration dependently (EC₅₀= 93 µM). Genistein and lanthanum activated TRPC5 channels synergistically. Effects of genistein on TRPC5 channels were mimicked by daidzein (100 µM), a genistein analogue inactive as a tyrosine kinase inhibitor, but not by known tyrosine kinase inhibitors herbimycin (2 µM), PP2 (20 µM) and lavendustin A (10 µM). Action of genistein on TRPC5 channels was not affected by an oestrogen receptor inhibitor ICI-182780 (50 µM) or a phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (10 µM), suggesting genistein did not act through oestrogen receptors or phospholipase C. In BAECs, genistein (100 µM) stimulated TRPC5-mediated Ca²⁺ influx. In patch clamp studies, both genistein (50 µM) and daidzein (50 µM) augmented TRPC5-mediated whole-cell cation current in TRPC5 over-expressing HEK cells. Genistein stimulated TRPC5 channel activity in excised inside-out membrane patch, suggesting that its action was relatively direct and did not require cytosolic factors.

Conclusions and implications:

The present study is the first to demonstrate stimulation of a TRP channel by isoflavones. Genistein is a lipophilic compound able to stimulate TRPC5 activity in TRPC5-over-expressing HEK cells and in native vascular endothelial cells.     

Smooth muscle relaxation and activation of the large conductance Ca++ – activated K+ (BKCa) channel by novel oestrogens

Background and Purpose

Oestrogens can interact directly with membrane receptors and channels and can activate vascular BK_Ca channels. We hypothesized that novel oestrogen derivatives could relax smooth muscle by an extracllular effect on the α and β₁ subunits of the BK_Ca channel, rather than at an intracellular site.

Experimental Approach

We studied the effects of novel oestrogens on the tension of pre-contracted isolated rat aortic rings, and on the electrophysiological properties of HEK 293 cells expressing the hSloα or hSloα+β₁ subunits. Two of the derivatives incorporated a quaternary ammonium side-chain making them membrane impermeable.

Key Results

Oestrone, oestrone oxime and Quat DME-oestradiol relaxed pre-contracted rat aorta, but only Quat DME-oestradiol-induced relaxation was iberiotoxin sensitive. However, only potassium currents recorded in HEK 293 cells over-expressing both hSloα and hSloβ₁ were activated by oestrone, oestrone oxime and Quat DME-oestradiol.

Conclusion and Implications

The novel oestrogens were able to relax smooth muscle, but through different mechanisms. In particular, oestrone oxime required the presence of the endothelium to exert much of its effect, whilst Quat DME-oestradiol depended both on NO and BK_Ca channel activation. The activation of BK_Ca currents in HEK 293 cells expressing hSloα+β₁ by Quat DME-oestradiol is consistent with an extracellular binding site between the two subunits. The binding site resides between the extracellular N terminal of the α subunit and the extracellular loop between TM1 and 2 of the β₁ subunit. Membrane-impermeant Quat DME-oestradiol lacks an exchangeable hydrogen on the A ring obviating antioxidant activity.