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Background and Purpose: We extend the characterization of the TRPM8 antagonist VBJ103 with tests of selectivity, specificity and distribution, therapeutic efficacy of systemic administration against oxaliplatin-induced cold hyperalgesia and the impact of systemic administration on core body temperature (CBT)., Experimental Approach: Selectivity at human TRPA1 and TRPV1 as well as in vitro safety profiling was determined. Effects of systemic administration of VBJ103 were evaluated in a model of oxaliplatin-induced cold hyperalgesia. Both peripheral and centrally mediated effects of VBJ103 on CBT were assessed with radiotelemetry., Key Results: VBJ103 had no antagonist activity at TRPV1 and TRPA1, but low potency TRPA1 activation. The only safety liability detected was partial inhibition of the dopamine transporter (DAT). VBJ103 delivered subcutaneously dose-dependently attenuated cold hypersensitivity in oxaliplatin-treated mice at 3, 10 and 30 mg·kg -1 (n = 7, P < 0.05). VBJ103 (30 mg·kg -1 ) antinociception was influenced by neither the TRPA1 antagonist HC-030031 nor the DAT antagonist GBR12909. Subcutaneous administration of VBJ103 (3, 10 and 30 mg·kg -1 , but not 100 or 300 mg·kg -1 , n = 7) decreased CBT (2°C). Intraperitoneal (i.p.) administration of VBJ103 (3, 10 and 30 mg·kg -1 ) dose-dependently decreased CBT to an extent larger than that detected with subcutaneous administration. Intracerebroventricular (i.c.v.) administration (306 nmol/1 μL; n = 5) did not alter CBT., Conclusions and Implications: We achieve therapeutic efficacy with subcutaneous administration of a novel TRPM8 antagonist that attenuates deleterious influences on CBT, a side effect that has largely prevented the translation of TRPM8 as a target., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: The mechanistic target of rapamycin (mTOR) signalling pathway is a key regulator of cell growth and metabolism. Its deregulation is implicated in several diseases. The macrolide rapamycin, a specific inhibitor of mTOR, has immunosuppressive, anti-inflammatory and antiproliferative properties. Recently, we identified tacrolimus, another macrolide immunosuppressant, as a novel activator of TRPM8 ion channels, involved in cold temperature sensing, thermoregulation, tearing and cold pain. We hypothesized that rapamycin may also have agonist activity on TRPM8 channels., Experimental Approach: Using calcium imaging and electrophysiology in transfected HEK293 cells and wildtype or Trpm8 KO mouse DRG neurons, we characterized rapamycin's effects on TRPM8 channels. We also examined the effects of rapamycin on tearing in mice., Key Results: Micromolar concentrations of rapamycin activated rat and mouse TRPM8 channels directly and potentiated cold-evoked responses, effects also observed in human TRPM8 channels. In cultured mouse DRG neurons, rapamycin increased intracellular calcium levels almost exclusively in cold-sensitive neurons. Responses were markedly decreased in Trpm8 KO mice or by TRPM8 channel antagonists. Cutaneous cold thermoreceptor endings were also activated by rapamycin. Topical application of rapamycin to the eye surface evokes tearing in mice by a TRPM8-dependent mechanism., Conclusion and Implications: These results identify TRPM8 cationic channels in sensory neurons as novel molecular targets of the immunosuppressant rapamycin. These findings may help explain some of its therapeutic effects after topical application to the skin and the eye surface. Moreover, rapamycin could be used as an experimental tool in the clinic to explore cold thermoreceptors., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: The analgesic action of paracetamol involves K V 7 channels, and its metabolite N-acetyl-p-benzo quinone imine (NAPQI), a cysteine modifying reagent, was shown to increase currents through such channels in nociceptors. Modification of cysteine residues by N-ethylmaleimide, H 2 O 2 , or nitric oxide has been found to modulate currents through K V 7 channels. The study aims to identify whether, and if so which, cysteine residues in neuronal K V 7 channels might be responsible for the effects of NAPQI., Experimental Approach: To address this question, we used a combination of perforated patch-clamp recordings, site-directed mutagenesis, and mass spectrometry applied to recombinant K V 7.1 to K V 7.5 channels., Key Results: Currents through the cardiac subtype K V 7.1 were reduced by NAPQI. Currents through all other subtypes were increased, either by an isolated shift of the channel voltage dependence to more negative values (K V 7.3) or by such a shift combined with increased maximal current levels (K V 7.2, K V 7.4, K V 7.5). A stretch of three cysteine residues in the S2-S3 linker region of K V 7.2 was necessary and sufficient to mediate these effects., Conclusion and Implication: The paracetamol metabolite N-acetyl-p-benzo quinone imine (NAPQI) modifies cysteine residues of K V 7 subunits and reinforces channel gating in homomeric and heteromeric K V 7.2 to K V 7.5, but not in K V 7.1 channels. In K V 7.2, a triple cysteine motif located within the S2-S3 linker region mediates this reinforcement that can be expected to reduce the excitability of nociceptors and to mediate antinociceptive actions of paracetamol., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Neuropathic pain, a debilitating condition with unmet medical needs, can be characterised as hyperexcitability of nociceptive neurons caused by dysfunction of ion channels. Voltage-gated potassium channels type 7 (Kv7), responsible for maintaining neuronal resting membrane potential and thus excitability, reside under tight control of G protein-coupled receptors (GPCRs). Calcium-sensing receptor (CaSR) is a GPCR that regulates the activity of numerous ion channels, but whether CaSR can control Kv7 channel function has been unexplored until now., Experimental Approach: Experiments were conducted in recombinant cell models, mouse dorsal root ganglia (DRG) neurons and human induced pluripotent stem cell (hiPSC)-derived nociceptive-like neurons using patch-clamp electrophysiology and molecular biology techniques., Key Results: Our results demonstrate that CaSR is expressed in recombinant cell models, hiPSC-derived nociceptive-like neurons and mouse DRG neurons, and its activation induced depolarisation via Kv7.2/7.3 channel inhibition. The CaSR-Kv7.2/7.3 channel crosslink was mediated via the G i/o protein-adenylate cyclase-cyclicAMP-protein kinase A signalling cascade. Suppression of CaSR function demonstrated a potential to rescue hiPSC-derived nociceptive-like neurons from algogenic cocktail-induced hyperexcitability., Conclusion and Implications: This study demonstrates that the CaSR-Kv7.2/7.3 channel crosslink, via a G i/o protein signalling pathway, effectively regulates neuronal excitability, providing a feasible pharmacological target for neuronal hyperexcitability management in neuropathic pain., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Receptor activity-modifying proteins (RAMPs) and melanocortin receptor accessory proteins (MRAPs) modulate expression and signalling of calcitonin and melanocortin GPCRs. Interactions with other GPCRs have also been reported. The cannabinoid receptors, CB 1 and CB 2 , and two putative cannabinoid receptors, GPR18 and GPR55, exhibit substantial intracellular expression and there are discrepancies in ligand responsiveness between studies. We investigated whether interactions with RAMPs or MRAPs could explain these phenomena., Experimental Approach: Receptors and accessory proteins were co-expressed in HEK-293 cells. Selected receptors were studied at basal expression levels and also with enhanced expression produced by incorporation of a preprolactin signal sequence/peptide (pplss). Cell surface and total expression of receptors and accessory proteins were quantified using immunocytochemistry. Signalling was measured using cAMP (CAMYEL) and G protein dissociation (TRUPATH Gα 13 ) biosensors., Key Results: MRAP2 enhanced surface and total expression of GPR18. Pplss-GPR18 increased detection of cell surface MRAP2. MRAP1α and MRAP2 reduced GPR55 surface and total expression, correlating with reduced constitutive, but not agonist-induced, signalling. GPR55, pplss-CB 1 and CB 2 reduced detection of MRAP1α at the cell surface. Pplss-CB 1 agonist potency was reduced by MRAP2 in Gα 13 but not cAMP assays, consistent with MRAP2 reducing pplss-CB 1 expression. Some cannabinoid receptors increased RAMP2 or RAMP3 total expression without influencing surface expression., Conclusions and Implications: Mutual influences on expression and/or function for specific accessory protein-receptor pairings raises the strong potential for physiological and disease-relevant consequences. Sequestration and/or hetero-oligomerisation of cannabinoid receptors with accessory proteins is a possible novel mechanism for receptor crosstalk., Linked Articles: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Thromboxane A 2 (TXA 2 ) is a prostanoid produced during platelet activaton, important in enhancing platelet reactivity by activation of TP receptors. However, due to the short half-life, studying TXA 2 signalling is challenging. To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA 2 analogues and explored their pharmacology on heterologous and endogenously expressed TP receptor function., Experimental Approach: Platelet functional and signalling responses were studied using aggregometry, Ca 2+ mobilisation experiments and immunoblotting and compared with an analogue of the TXA 2 precursor prostaglandin H 2 , U46619. Gα q /Gα s receptor signalling was determined using a bioluminescence resonance energy transfer (BRET) assay in a cell line overexpression system., Key Results: BRET studies revealed that F-TXA 2 and F 2 -TXA 2 promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F 2 -TXA 2 caused reversible aggregation in platelets, whereas F-TXA 2 and U46619 induced sustained aggregation. Blocking the IP receptor switched F 2 -TXA 2 -mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F 2 -TXA 2 -mediated IP receptor activation. F 2 -TXA 2 rapidly and potently stimulated platelet TP receptor-mediated protein kinase C/P-pleckstrin, whereas IP-mediated protein kinase A/P-vasodilator-stimulated phosphoprotein was more delayed., Conclusion and Implications: F-TXA 2 is a close analogue to TXA 2 used as a selective tool for TP receptor platelet activation. In contrast, F 2 -TXA 2 acts on both TP and IP receptors differently over time, resulting in an initial wave of TP receptor-mediated platelet aggregation followed by IP receptor-induced reversibility of aggregation. This study reveals the potential difference in the temporal aspects of stimulatory and inhibitory pathways involved in platelet activation., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Activation of CB 1 by exogenous agonists causes adverse effects in vivo. Positive allosteric modulation may offer improved therapeutic potential and a reduced on-target adverse effect profile compared with orthosteric agonists, due to reduced desensitisation/tolerance, but this has not been directly tested. This study investigated the ability of PAMs/ago-PAMs to induce receptor regulation pathways, including desensitisation and receptor internalisation., Experimental Approach: Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and β-arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB 1 in response to the PAMs ZCZ011, GAT229 and ABD1236 alone and in combination with the orthosteric agonists AEA, 2-AG, and AMB-FUBINACA., Key Results: ZCZ011, GAT229 and ABD1236 were allosteric agonists in all pathways tested. The ago-PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time course compared to transient activation by orthosteric agonists. In combination with 2-AG but not AEA or AMB-FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2 phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEA-induced signalling in all pathways tested; however, no notable potentiation of 2-AG or AMB-FUBINACA was observed., Conclusion and Implications: Ago-PAMs can potentiate endocannabinoid CB 1 agonism by AEA to a larger extent compared with 2-AG. However, all compounds were found to be allosteric agonists and induce activation of CB 1 in the absence of endocannabinoid, including β-arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Voltage sensitivity is a common feature of many membrane proteins, including some G-protein coupled receptors (GPCRs). However, the functional consequences of voltage sensitivity in GPCRs are not well understood., Experimental Approach: In this study, we investigated the voltage sensitivity of the post-synaptic metabotropic glutamate receptor mGlu 5 and its impact on synaptic transmission. Using biosensors and electrophysiological recordings in non-excitable HEK293T cells or neurons., Key Results: We found that mGlu 5 receptor function is optimal at resting membrane potentials. We observed that membrane depolarization significantly reduced mGlu 5 receptor activation, Gq-PLC/PKC stimulation, Ca 2+ release and mGlu 5 receptor-gated currents through transient receptor potential canonical, TRPC6, channels or glutamate ionotropic NMDA receptors. Notably, we report a previously unknown activity of the NMDA receptor at the resting potential of neurons, enabled by mGlu 5 ., Conclusions and Implications: Our findings suggest that mGlu 5 receptor activity is directly regulated by membrane voltage which may have a significant impact on synaptic processes and pathophysiological functions., (© 2024 British Pharmacological Society.)

Background and Purpose: The TRPM8 ion channel is involved in innocuous cold sensing and has a potent anti-inflammatory action. Its activation by lower temperature or chemical agonists such as menthol and icilin induces analgesic effects, reversing hypersensitivity and reducing chronic pain. On the other hand, prostacyclin (PGI 2 ) enhances pain and inflammation by activating the IP receptors. Due to the critical roles of TRPM8 and IP receptors in the regulation of inflammatory pain, and considering their overlapping expression pattern, we analysed the functional interaction between human TRPM8 and IP receptors., Experimental Approach: We transiently expressed human TRPM8 channels and IP receptors in HEK293T cells and carried out intracellular calcium and cAMP measurements. Additionally, we cultured neurons from the dorsal root ganglia (DRGs) of mice and determined the increase in intracellular calcium triggered by the TRPM8 agonist, icilin, in the presence of the IP receptor agonist cicaprost, the IP receptor antagonist Cay10441, and the Gq/11 inhibitor YM254890., Key Results: Activation of IP receptors by selective agonists (cicaprost, beraprost, and iloprost) inhibited TRPM8 channel function, independently of the Gs-cAMP pathway. The potent inhibition of TRPM8 channels by IP receptor agonists involved Gq/11 coupling. These effects were also observed in neurons isolated from murine DRGs., Conclusions and Implications: Our results demonstrate an unusual signalling pathway of IP receptors by coupling to Gq/11 proteins to inhibit TRPM8 channel function. This pathway may contribute to a better understanding of the role of TRPM8 channels and IP receptors in regulating pain and inflammation., (© 2023 British Pharmacological Society.)

Background and Purpose: Opioid-induced respiratory depression limits the use of μ-opioid receptor agonists in clinical settings and is the main cause of opioid overdose fatalities. The relative potential of different opioid agonists to induce respiratory depression at doses exceeding those producing analgesia is understudied despite its relevance to assessments of opioid safety. Here we evaluated the respiratory depressant and anti-nociceptive effects of three novel opioids and relate these measurements to their in vitro efficacy., Experimental Approach: Respiration was measured in awake, freely moving male CD-1 mice using whole body plethysmography. Anti-nociception was measured using the hot plate test. Morphine, oliceridine and tianeptine were administered intraperitoneally, whereas methadone, oxycodone and SR-17018 were administered orally. Receptor activation and arrestin-3 recruitment were measured in HEK293 cells using BRET assays., Key Results: Across the dose ranges examined, all opioids studied depressed respiration in a dose-dependent manner, with similar effects at the highest doses, and with tianeptine and oliceridine showing reduced duration of effect, when compared with morphine, oxycodone, methadone and SR-17018. When administered at doses that induced similar respiratory depression, all opioids induced similar anti-nociception, with tianeptine and oliceridine again showing reduced duration of effect. These data were consistent with the in vitro agonist activity of the tested compounds., Conclusion and Implications: In addition to providing effective anti-nociception, the novel opioids, oliceridine, tianeptine and SR-17018 depress respiration in male mice. However, the different potencies and kinetics of effect between these novel opioids may be relevant to their therapeutic application in different clinical settings., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: In macrophages, transient receptor potential vanilloid 2 (TRPV2) channel contributes to various cellular processes such as cytokine production, differentiation, phagocytosis and migration. Due to a lack of selective pharmacological tools, its function in immunological processes is not well understood and the identification of novel and selective TRPV2 modulators is highly desirable., Experimental Approach: Novel and selective TRPV2 modulators were identified by screening a compound library using Ca 2+ influx assays with human embryonic kidney 293 (HEK293) cells heterologously expressing rat TRPV2. Hits were further characterized and validated with Ca 2+ influx and electrophysiological assays. Phagocytosis and migration of macrophages were analysed and the contribution of TRPV2 to the generation of Ca 2+ microdomains was studied by total internal reflection fluorescence microscopy (TIRFM)., Key Results: The compound IV2-1, a dithiolane derivative (1,3-dithiolan-2-ylidene)-4-methyl-5-phenylpentan-2-one), is a potent inhibitor of heterologously expressed TRPV2 channels (IC 50  = 6.3 ± 0.7 μM) but does not modify TRPV1, TRPV3 or TRPV4 channels. IV2-1 also inhibits TRPV2-mediated Ca 2+ influx in macrophages. IV2-1 inhibits macrophage phagocytosis along with valdecoxib and after siRNA-mediated knockdown. Moreover, TRPV2 inhibition inhibits lipopolysaccharide-induced migration of macrophages whereas TRPV2 activation promotes migration. After activation, TRPV2 shapes Ca 2+ microdomains predominantly at the margin of macrophages, which are important cellular regions to promote phagocytosis and migration., Conclusions and Implications: IV2-1 is a novel TRPV2-selective blocker and underline the role of TRPV2 in macrophage-mediated phagocytosis and migration. Furthermore, we provide evidence that TRPV2 activation generates Ca 2+ microdomains, which may be involved in phagocytosis and migration of macrophages., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: The illicit use of fentanyl-like drugs (fentanyls), which are μ opioid receptor agonists, and the many overdose deaths that result, has become a major problem. Fentanyls are very potent in vivo, leading to respiratory depression and death. However, the efficacy and possible signalling bias of different fentanyls is not clearly known. Here, we compared the relative efficacy and bias of a series of fentanyls., Experimental Approach: For agonist signalling bias and efficacy measurements, Bioluminescence Resonance Energy Transfer experiments were undertaken in HEK293T cells transiently transfected with μ opioid receptors, to assess Gi protein activation and β-arrestin 2 recruitment. Agonist-induced cell surface receptor loss was assessed using an enzyme-linked immunosorbent assay, whilst agonist-induced G protein-coupled inwardly rectifying potassium channel current activation was measured electrophysiologically from rat locus coeruleus slices. Ligand poses in the μ opioid receptor were determined in silico using molecular dynamics simulations., Key Results: Relative to the reference ligand DAMGO, carfentanil was β-arrestin-biased, whereas fentanyl, sufentanil and alfentanil did not display bias. Carfentanil induced potent and extensive cell surface receptor loss, whilst the marked desensitisation of G protein-coupled inwardly rectifying potassium channel currents in the continued presence of carfentanil in neurones was prevented by a GRK2/3 inhibitor. Molecular dynamics simulations suggested unique interactions of carfentanil with the orthosteric site of the receptor that could underlie the bias., Conclusions and Implications: Carfentanil is a β-arrestin-biased opioid drug at the μ receptor. It is uncertain how such bias influences in vivo effects of carfentanil relative to other fentanyls., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Acute respiratory distress syndrome (ARDS) is a catastrophic pulmonary inflammatory dysfunction with a high mortality rate. An overwhelming immune response by neutrophils is a key feature in infective or sterile ARDS. The formyl peptide receptor 1 (FPR1) is a crucial damage-sensing receptor for inflammatory reactions in the initiation and progression of neutrophil-mediated ARDS. However, effective targets for controlling dysregulated neutrophilic inflammatory injuries in ARDS are limited., Experimental Approach: Human neutrophils were used to explore the anti-inflammatory effects of cyclic lipopeptide anteiso-C13-surfactin (IA-1) from marine Bacillus amyloliquefaciens. The lipopolysaccharide-induced model of ARDS in mice was used to determine the therapeutic potential of IA-1 in ARDS. Lung tissues were harvested for histology analyses., Key Results: The lipopeptide IA-1 inhibited immune responses of neutrophils, including respiratory burst, degranulation, and expression of adhesion molecules. IA-1 inhibited the binding of N-formyl peptides to FPR1 in human neutrophils and in hFPR1-transfected HEK293 cells. We identified IA-1 as a competitive FPR1 antagonist, thus diminishing the downstream signalling pathways involving calcium, mitogen-activated protein kinases and Akt. Furthermore, IA-1 ameliorated the inflammatory damage to lung tissue, by decreasing neutrophil infiltration, reducing elastase release and oxidative stress in endotoxemic mice., Conclusion and Implications: The lipopeptide IA-1 could serve as a therapeutic option for ARDS by inhibiting FPR1-mediated neutrophilic injury., (© 2023 British Pharmacological Society.)

Background and Purpose: Ca V 3.1-3 currents differentially contribute to neuronal firing patterns. Ca V 3 are regulated by G protein-coupled receptors (GPCRs) activity, but information about Ca V 3 as targets of the constitutive activity of GPCRs is scarce. We investigate the impact of D 5 recpetor constitutive activity, a GPCR with high levels of basal activity, on Ca V 3 functionality. D 5 recpetor and Ca V 3 are expressed in the hippocampus and have been independently linked to pathophysiological states associated with epilepsy., Experimental Approach: Our study models were HEK293T cells heterologously expressing D 1 or D 5 receptor and Ca V 3.1-3, and mouse brain slices containing the hippocampus. We used chlorpromazine (D 1 /D 5 inverse agonist) and a D 5 receptor mutant lacking constitutive activity as experimental tools. We measured Ca V 3 currents and excitability parameters using the patch-clamp technique. We completed our study with computational modelling and imaging technique., Key Results: We found a higher sensitivity to TTA-P2 (Ca V 3 blocker) in CA1 pyramidal neurons obtained from chlorpromazine-treated animals compared with vehicle-treated animals. We found that Ca V 3.2 and Ca V 3.3-but not Ca V 3.1-are targets of D 5 receptor constitutive activity in HEK293T cells. Finally, we found an increased firing rate in CA1 pyramidal neurons from chlorpromazine-treated animals in comparison with vehicle-treated animals. Similar changes in firing rate were observed on a neuronal model with controlled Ca V 3 currents levels., Conclusions and Implications: Native hippocampal Ca V 3 and recombinant Ca V 3.2-3 are sensitive to D 5 receptor constitutive activity. Manipulation of D 5 receptor constitutive activity could be a valuable strategy to control neuronal excitability, especially in exacerbated conditions such as epilepsy., (© 2022 British Pharmacological Society.)

Background and Aim: Standard pharmacological analysis of agonist activity utilises measurements of receptor-mediated responses at a set time-point, or at the peak response level, to characterise ligands. However, the occurrence of non-equilibrium conditions may dramatically impact the properties of the response being measured. Here we have analysed the initial kinetic phases of cAMP responses to β 2 -adrenoceptor agonists in HEK293 cells expressing the endogenous β 2 -adrenoceptor at extremely low levels., Experimental Approach: The kinetics of β 2 -adrenoceptor agonist-stimulated cAMP responses were monitored in real-time, in the presence and absence of antagonists, in HEK293 cells expressing the cAMP GloSensor™ biosensor. Potency (EC 50 ) and efficacy (E max ) values were determined at the peak of the agonist GloSensor™ response and compared to kinetic parameters L 50 and IR max values derived from initial response rates., Key Results: The partial agonists salbutamol and salmeterol displayed reduced relative IR max values (with respect to isoprenaline) when compared with their E max values. Except for the fast dissociating bisoprolol, preincubation with β 2 -adrenoceptor antagonists produced a large reduction in the isoprenaline peak response due to a state of hemi-equilibrium in this low receptor reserve system. This effect was exacerbated when IR max parameters were measured. Furthermore, bisoprolol produced a large reduction in isoprenaline IR max consistent with its short residence time., Conclusions and Implications: Kinetic analysis of real-time signalling data can provide valuable insights into the hemi-equilibria that can occur in low receptor reserve systems with agonist-antagonist interactions, due to incomplete dissociation of antagonist whilst the peak agonist response is developing., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Regulator of G-protein signalling 4 (RGS4) is a signal transduction protein that accelerates intrinsic GTPase activity of Gα i/o and Gα q subunits, suppressing GPCR signalling. Here, we investigate whether RGS4 modulates nociceptin/orphanin FQ (N/OFQ) opioid (NOP) receptor signalling and if this modulation has relevance for l-Dopa-induced dyskinesia., Experimental Approach: HEK293T cells transfected with NOP, NOP/RGS4 or NOP/RGS19 were challenged with N/OFQ and the small-molecule NOP agonist AT-403, using D1-stimulated cAMP levels as a readout. Primary rat striatal neurons and adult mouse striatal slices were challenged with either N/OFQ or AT-403 in the presence of the experimental RGS4 chemical probe, CCG-203920, and D1-stimulated cAMP or phosphorylated extracellular signal regulated kinase 1/2 (pERK) responses were monitored. In vivo, CCG-203920 was co-administered with AT-403 and l-Dopa to 6-hydroxydopamine hemilesioned rats, and dyskinetic movements, striatal biochemical correlates of dyskinesia (pERK and pGluR1 levels) and striatal RGS4 levels were measured., Key Results: RGS4 expression reduced NOFQ and AT-403 potency and efficacy in HEK293T cells. CCG-203920 increased N/OFQ potency in primary rat striatal neurons and potentiated AT-403 response in mouse striatal slices. CCG-203920 enhanced AT-403-mediated inhibition of dyskinesia and its biochemical correlates, without compromising its motor-improving effects. Unilateral dopamine depletion caused bilateral reduction of RGS4 levels, which was reversed by l-Dopa. l-Dopa acutely up-regulated RGS4 in the lesioned striatum., Conclusions and Implications: RGS4 physiologically inhibits NOP receptor signalling. CCG-203920 enhanced NOP responses and improved the antidyskinetic potential of NOP receptor agonists, mitigating the effects of striatal RGS4 up-regulation occurring during dyskinesia expression., Linked Articles: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Before advancing to clinical trials, new drugs are screened for their pro-arrhythmic potential using a method that is overly conservative and provides limited mechanistic insight. The shortcomings of this approach can lead to the mis-classification of beneficial drugs as pro-arrhythmic., Experimental Approach: An in silico-in vitro pipeline was developed to circumvent these shortcomings. A computational human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model was used as part of a genetic algorithm to design experiments, specifically electrophysiological voltage clamp (VC) protocols, to identify which of several cardiac ion channels were blocked during in vitro drug studies. Such VC data, along with dynamically clamped action potentials (AP), were acquired from iPSC-CMs before and after treatment with a control solution or a low- (verapamil), intermediate- (cisapride or quinine) or high-risk (quinidine) drug., Key Results: Significant AP prolongation (a pro-arrhythmia marker) was seen in response to quinidine and quinine. The VC protocol identified block of I Kr (a source of arrhythmias) by all strong I Kr blockers, including cisapride, quinidine and quinine. The protocol also detected block of I CaL by verapamil and I to by quinidine. Further demonstrating the power of the approach, the VC data uncovered a previously unidentified I f block by quinine, which was confirmed with experiments using a HEK-293 expression system and automated patch-clamp., Conclusion and Implications: We developed an in silico-in vitro pipeline that simultaneously identifies pro-arrhythmia risk and mechanism of ion channel-blocking drugs. The approach offers a new tool for evaluating cardiotoxicity during preclinical drug screening., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: T-type Ca channels (I Ca ) regulate neuronal excitability and contribute to neurotransmitter release. The phytocannabinoids Δ 9 -tetrahydrocannabinol and cannabidiol effectively modulate T-type I Ca , but effects of other biologically active phytocannabinoids on these channels are unknown. We thus investigated the modulation of T-type I Ca by low abundance phytocannabinoids., Experimental Approach: A fluorometric (fluorescence imaging plate reader [FLIPR]) assay was used to investigate modulation of human T-type I Ca (Ca V 3.1, 3.2 and 3.3) stably expressed in FlpIn-TREx HEK293 cells. The biophysical effects of some compounds were examined using whole-cell patch clamp recordings., Key Results: In the FLIPR assay, all 11 phytocannabinoids tested modulated T-type I Ca , with most inhibiting Ca V 3.1 and Ca V 3.2 more effectively than Ca V 3.3. Cannabigerolic acid was the most potent inhibitor of Ca V 3.1 (pIC 50 6.1 ± 0.6) and Ca V 3.2 (pIC 50 6.4 ± 0.4); in all cases, phytocannabinoid acids were more potent than their corresponding neutral forms. In patch clamp recordings, cannabigerolic acid inhibited Ca V 3.1 and 3.2 with similar potency to the FLIPR assay; the inhibition was associated with significant hyperpolarizing shift in activation and steady-state inactivation of these channels. In contrast, cannabidiol, cannabidivarin, and cannabigerol only affected channel inactivation., Conclusion and Implications: Modulation of T-type calcium channels is a common property of phytocannabinoids, which all increase steady-state inactivation at physiological membrane potentials, with some also affecting channel activation. Thus, T-type I Ca may be a common site of action for phytocannabinoids, and the diverse actions of phytocannabinoids on channel gating may provide insight into structural requirement for selective T-type I Ca modulators., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: The volume regulated anion channel (VRAC) is known to be involved in different aspects of cancer cell behaviour and response to therapies. For this reason, we investigated the effect of DCPIB, a presumably specific blocker of VRAC, in two types of cancer: pancreatic duct adenocarcinoma (PDAC) and melanoma., Experimental Approach: We used patch-clamp electrophysiology, supported by Ca 2+ imaging, gene expression analysis, docking simulation and mutagenesis. We employed two PDAC lines (Panc-1 and MiaPaCa-2), as well as a primary (IGR39) and a metastatic (IGR37) melanoma line., Key Results: DCPIB markedly increased whole-cell currents in Panc-1, MiaPaca2 and IGR39, but not in IGR37 cells. The currents were mostly mediated by K Ca 1.1 channels, commonly known as BK channels. We confirmed DCPIB activation of BK channels also in HEK293 cells transfected with α subunits of this channel. Further experiments showed that in IGR39, and to a smaller degree also in Panc-1 cells, DCPIB induced a rapid Ca 2+ influx. This, in turn, indirectly potentiated BK channels and, in IGR39 cells, additionally activated other Ca 2+ -dependent channels. However, Ca 2+ influx was not required for activation of BK channels by DCPIB, as such activation involved the extracellular part of the protein and we have identified a residue crucial for binding., Conclusion and Implications: DCPIB directly targeted BK channels and, also, acutely increased intracellular Ca 2+ . Our findings extend the list of DCPIB effects that should be taken into consideration for future development of DCPIB-based modulators of ion channels and other membrane proteins., (© 2022 The British Pharmacological Society.)

Background and Purpose: Nitric oxide (NO) activates soluble guanylyl cyclase (sGC) for cGMP production, but in disease, sGC becomes insensitive towards NO activation. What changes occur to sGC during its inactivation in cells is not clear., Experimental Approach: We utilized HEK293 cells expressing sGC proteins to study the changes that occur regarding its haem content, heterodimer status and sGCβ protein partners when the cells were given the oxidant ODQ or the NO donor NOC12 to inactivate sGC. Haem content of sGCβ was monitored in live cells through use of a fluorescent-labelled sGCβ construct, whereas sGC heterodimer status and protein interactions were studied by Western blot analysis. Experiments with purified proteins were also performed., Key Results: ODQ- or NOC12-driven inactivation of sGC in HEK293 cells was associated with haem oxidation (by ODQ), S-nitrosation of the sGCβ subunit (by NOC12), sGC heterodimer breakup and association of the freed sGCβ subunit with cell chaperone Hsp90. These changes occurred without detectable loss of haem from the sGCβ reporter construct. Treating a purified ferrous haem-containing sGCβ with ODQ or NOC12 caused it to bind with Hsp90 without showing any haem loss., Conclusion and Implications: Oxidative (ODQ) or nitrosative (NOC12) inactivation of cell sGC involves sGC heterodimer dissociation and rearrangement of the sGCβ protein partners without any haem loss from sGCβ. Clarifying what changes do and do not occur to sGC during its inactivation in cells may direct strategies to preserve or recover NO-dependent cGMP signalling in health and disease., Linked Articles: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc., (© 2021 The British Pharmacological Society.)

Background and Purpose: QT prolongation and intracellular Ca 2+ loading with diastolic Ca 2+ release via ryanodine receptors (RyR2) are the predominant mechanisms underlying hypokalaemia-induced ventricular arrhythmia. We investigated the antiarrhythmic actions of two RyR2 inhibitors: dantrolene and VK-II-86, a carvedilol analogue lacking antagonist activity at β-adrenoceptors, in hypokalaemia., Experimental Approach: Surface ECG and ventricular action potentials (APs) were recorded from whole-heart murine Langendorff preparations. Ventricular arrhythmia incidence was compared in hearts perfused with low [K + ], and those pretreated with dantrolene or VK-II-86. Whole-cell patch clamping was used in murine and canine ventricular cardiomyocytes to study effects of dantrolene and VK-II-86 on AP parameters in low [K + ] and effects of VK-II-86 on the inward rectifier current (I K1 ), late sodium current (I Na&#95;L ) and the L-type Ca 2+ current (I Ca ). Effects of VK-II-86 on I Kr were investigated in transfected HEK-293 cells. A fluorogenic probe quantified the effects of VK-II-86 on oxidative stress in hypokalaemia., Key Results: Dantrolene reduced the incidence of ventricular arrhythmias induced by low [K + ] in explanted murine hearts by 94%, whereas VK-II-86 prevented all arrhythmias. VK-II-86 prevented hypokalaemia-induced AP prolongation and depolarization but did not alter AP parameters in normokalaemia. Hypokalaemia was associated with decreased I K1 and I Kr , and increased I Na-L , and I Ca . VK-II-86 prevented all hypokalaemia-induced changes in ion channel activity and oxidative stress., Conclusions and Implications: VK-II-86 prevents hypokalaemia-induced arrhythmogenesis by normalizing calcium homeostasis and repolarization reserve. VK-II-86 may provide an effective treatment in hypokalaemia and other arrhythmias caused by delayed repolarization or Ca 2+ overload., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: The cannabinoid CB 2 receptor (CB 2 ) is a promising therapeutic target for modulating inflammation. However, little is known surrounding the mechanisms underpinning CB 2 desensitisation and regulation, particularly the role of GPCR kinases (GRKs). Here, we evaluated the role of six GRK isoforms in β-arrestin recruitment to CB 2 . Mutagenesis of several distal C-terminal aspartic acid residues was also performed in an attempt to delineate additional structural elements involved in the regulation of CB 2 ., Experimental Approach: In CB 2 -expressing HEK 293 cells, β-arrestin translocation was measured using real-time BRET assays. G protein dissociation BRET assays were performed to assess the activation and desensitisation of CB 2 in the presence of β-arrestin 2., Key Results: Overexpression of GRK isoforms 1-6 failed to considerably improve translocation of either β-arrestin 1 or β-arrestin 2 to CB 2 . Consistent with this, inhibition of endogenous GRK2/3 did not substantially reduce β-arrestin 2 translocation. Mutagenesis of C-terminal aspartic acid residues resulted in attenuation of β-arrestin 2 translocation, which translated to a reduction in desensitisation of G protein activation., Conclusion and Implications: Our findings suggest that CB 2 does not adhere to the classical GPCR regulatory paradigm, entailing GRK-mediated and β-arrestin-mediated desensitisation. Instead, C-terminal aspartic acid residues may act as phospho-mimics to induce β-arrestin activation. This study provides novel insights into the regulatory mechanisms of CB 2 , which may aid in our understanding of drug tolerance and dependence., (© 2021 The British Pharmacological Society.)

Background and Purpose: Glucosylsphingosine (GS), an endogenous sphingolipid, is highly accumulated in the epidermis of patients with atopic dermatitis (AD) due to abnormal ceramide metabolism. More importantly, GS can evoke scratching behaviours. However, the precise molecular mechanism by which GS induces pruritus has been elusive. Thus, the present study aimed to elucidate the molecular signalling pathway of GS, especially at the peripheral sensory neuronal levels., Experimental Approach: Calcium imaging was used to investigate the responses of HEK293T cells or mouse dorsal root ganglion (DRG) neurons to application of GS. Scratching behaviour tests were also performed with wild-type and Trpv4 knockout mice., Key Results: GS activated DRG neurons in a manner involving both the 5-HT 2A receptor and TRPV4. Furthermore, GS-induced responses were significantly suppressed by various inhibitors, including ketanserin (5-HT 2A receptor antagonist), YM254890 (Gα q/11 inhibitor), gallein (Gβγ complex inhibitor), U73122 (phospholipase C inhibitor), bisindolylmaleimide I (PKC inhibitor) and HC067047 (TRPV4 antagonist). Moreover, DRG neurons from Trpv4 knockout mice exhibited significantly reduced responses to GS. Additionally, GS-evoked scratching behaviours were greatly decreased by pretreatment with inhibitors of either 5-HT 2A receptor or TRPV4. As expected, GS-evoked scratching behaviour was also significantly decreased in Trpv4 knockout mice., Conclusion and Implications: Overall, the present study provides evidence for a novel molecular signalling pathway for GS-evoked pruritus, which utilizes both 5-HT 2A receptor and TRPV4 in mouse sensory neurons. Considering the high accumulation of GS in the epidermis of patients with AD, GS could be another pruritogen in patients with AD., (© 2021 The British Pharmacological Society.)

Background and Purpose: Despite the availability of a variety of treatment options, many asthma patients have poorly controlled disease with frequent exacerbations. Proteinase-activated receptor-2 (PAR2) has been identified in preclinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 raises intracellular Ca 2+ , inducing MAPK and β-arrestin signalling in the airway, leading to inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and trypsin-induced PAR2 signalling in vitro as well as reducing inflammatory PAR2-associated pain in vivo. We hypothesized that PAR2 antagonism by C391 would attenuate allergen-induced acutely expressed asthma indicators in murine models., Experimental Approach: We evaluated the ability of C391 to alter Alternaria alternata-induced PAR2 signalling pathways in vitro using a human airway epithelial cell line that naturally expresses PAR2 (16HBE14o-) and a transfected embryonic cell line (HEK 293). We next evaluated the ability for C391 to reduce A. alternata-induced acutely expressed asthma indicators in vivo in two murine strains., Key Results: C391 blocked A. alternata-induced, PAR2-dependent Ca 2+ and MAPK signalling in 16HBE14o- cells, as well as β-arrestin recruitment in HEK 293 cells. C391 effectively attenuated A. alternata-induced inflammation, mucus production, mucus cell hyperplasia and airway hyperresponsiveness in acute allergen-challenged murine models., Conclusions and Implications: To our best knowledge, this is the first demonstration of pharmacological intervention of PAR2 to reduce allergen-induced asthma indicators in vivo. These data support further development of PAR2 antagonists as potential first-in-class allergic asthma drugs., (© 2021 The British Pharmacological Society.)

Background and purpose:The aim of the present study was to investigate whether the endogenous metabotropic P2Y receptors modulate ionotropic P2X3 receptor-channels.Experimental approach:Whole-cell patch-clamp experiments were carried out on HEK293 cells permanently transfected with human P2X3 receptors (HEK293-hP2X3 cells) and rat dorsal root ganglion (DRG) neurons.Key results:In both cell types, the P2Y1,12,13 receptor agonist, ADP-β-S, inhibited P2X3 currents evoked by the selective agonist, α,β-methylene ATP (α,β-meATP). This inhibition could be markedly counteracted by replacing in the pipette solution the usual GTP with GDP-β-S, a procedure known to block all G protein heterotrimers. P2X3 currents evoked by ATP, activating both P2Y and P2X receptors, caused a smaller peak amplitude and desensitized faster than those currents evoked by the selective P2X3 receptor agonist α,β-meATP. In the presence of intracellular GDP-β-S, ATP- and α,β-meATP-induced currents were identical. Recovery from P2X3 receptor desensitization induced by repetitive ATP application was slower than the recovery from α,β-meATP-induced desensitization. When G proteins were blocked by intracellular GDP-β-S, the recovery from the ATP- and α,β-meATP-induced desensitization were of comparable speed.Conclusions and Implications:Our results suggest that the activation of P2Y receptors G protein-dependently facilitates the desensitization of P2X3 receptors and suppresses the recovery from the desensitized state. Hence, the concomitant stimulation of P2X3 and P2Y receptors of DRG neurons by ATP may result both in an algesic effect and a partly counterbalancing analgesic activity.British Journal of Pharmacology (2007) 151, 226–236. doi:10.1038/sj.bjp.0707217; published online 12 March 2007 [ABSTRACT FROM AUTHOR]

β-Blockers are widely used in the treatment of cardiovascular diseases. However, their effects on HERG channels at comparable conditions remain to be defined. We investigated the direct acute effects of β-blockers on HERG current and the molecular basis of drug binding to HERG channels with mutations of putative common binding site (Y652A and F656C).β-Blockers were selected based on the receptor subtype. Wild-type, Y652A and F656C mutants of HERG channel were stably expressed in HEK293 cells, and the current was recorded by using whole-cell patch-clamp technique (23°C).Carvedilol (nonselective), propranolol (nonselective) and ICI 118551 (β2-selective) inhibited HERG current in a concentration-dependent manner (IC50 0.51, 3.9 and 9.2 μM, respectively). The IC50 value for carvedilol was a clinically relevant concentration. High metoprolol (β1-selective) concentrations were required for blockade (IC50 145 μM), and atenolol (β1-selective) did not inhibit the HERG current.Inhibition of HERG current by carvedilol, propranolol and ICI 118551 was partially but significantly attenuated in Y652A and F656C mutant channels. Affinities of metoprolol to Y652A and F656C mutant channels were not different compared with the wild-type.HERG current block by all β-blockers was not frequency-dependent.Drug affinities to HERG channels were different in β-blockers. Our results provide additional strategies for clinical usage of β-blockers. Atenolol and metoprolol may be preferable for patients with type 1 and 2 long QT syndrome. Carvedilol has a class III antiarrhythmic effect, which may provide the rationale for a favourable clinical outcome compared with other β-blockers as suggested in the recent COMET (Carvedilol Or Metoprolol European Trial) substudy.British Journal of Pharmacology (2006) 147, 642–652. doi:10.1038/sj.bjp.0706508; published online 28 November 2005 [ABSTRACT FROM AUTHOR]

1 The beta-secretase BACE1 is one of the enzymes that contribute to the production of the Abeta peptide, in vitro and in vivo. JMV1195 was previously shown to inhibit BACE activity in vitro but was unable to block cellular BACE activity. We have designed a new permeable inhibitor, JMV2764 that corresponds to a derivative of JMV1195 to which a penetratin sequence had been added at its N-terminus. We have assessed the ability of JMV2764 to affect BACE1 activity in vitro, and to modify Abeta production in various cell systems. 2 Endogenous beta-secretase or BACE1 activities were monitored in vitro by means of two distinct fluorimetric substrates in HEK293 extracts of cells expressing either wild-type betaAPP, Swedish mutated betaAPP or SPA4CT constructs. Abeta40 recovery was monitored by immunoprecipitation and Western blot analysis. 3 JMV2764 and JMV1195 inhibited endogenous beta-secretase activity of HEK293 cellular homogenates with IC(50)s of 0.8 and 6.6 microM, respectively. Interestingly, JMV2764 also inhibited beta-secretase activity after preincubation with intact cells while JMV1195 was inactive, indicating that unlike JMV1195, JMV2764 could penetrate into the cells. 4 JMV2764 but not JMV1195 also prevented Abeta production by HEK293 cells overexpressing wild-type and Swedish-mutated betaAPP. However, JMV2764 was unable to affect Abeta production from cells expressing SPA4CT, a betaAPP-derived sequence that does not need beta-secretase to produce Abeta. 5 Altogether, we have designed a new cell-permeable BACE1 inhibitor that allows to envision to prevent Abeta production in vivo. Work is in progress to assess the potential of these compounds to prevent Abeta production in transgenic mice overproducing Abeta. [ABSTRACT FROM AUTHOR]

Background and Purpose: Activation of GIRK channels via G protein-coupled GABA B receptors has been shown to attenuate nociceptive transmission. The analgesic α-conotoxin Vc1.1 activates GABA B receptors resulting in inhibition of Ca v 2.2 and Ca v 2.3 channels in mammalian primary afferent neurons. Here, we investigated the effects of analgesic α-conotoxins on recombinant and native GIRK-mediated K + currents and on neuronal excitability., Experimental Approach: The effects of analgesic α-conotoxins, Vc1.1, RgIA, and PeIA, were investigated on inwardly-rectifying K + currents in HEK293T cells recombinantly co-expressing either heteromeric human GIRK1/2 or homomeric GIRK2 subunits, with GABA B receptors. The effects of α-conotoxin Vc1.1 and baclofen were studied on GIRK-mediated K + currents and the passive and active electrical properties of adult mouse dorsal root ganglion neurons., Key Results: Analgesic α-conotoxins Vc1.1, RgIA, and PeIA potentiate inwardly-rectifying K + currents in HEK293T cells recombinantly expressing human GIRK1/2 channels and GABA B receptors. GABA B receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen occurs via a pertussis toxin-sensitive G protein and is inhibited by the selective GABA B receptor antagonist CGP 55845. In adult mouse dorsal root ganglion neurons, GABA B receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen hyperpolarizes the cell membrane potential and reduces excitability., Conclusions and Implications: This is the first report of GIRK channel potentiation via allosteric α-conotoxin Vc1.1-GABA B receptor agonism, leading to decreased neuronal excitability. Such action potentially contributes to the analgesic effects of Vc1.1 and baclofen observed in vivo., (© 2021 The British Pharmacological Society.)

Background and Purpose: Melanogenesis is essential for pigmentation and deregulated melanogenesis causes pigmentary diseases. Psoralen and ultraviolet A (PUVA) therapy strongly stimulates pigmentation, but the underlying molecular mechanisms are elusive., Experimental Approach: Melanin content of cultured human melanocytes was spectrophotometrically measured. Patch-clamp recordings were made in human melanocytes or HEK 293 cells transiently expressing wild type or mutant human TRPV1 and TRPA1 channels. Endogenous expression of TRPV1 and TRPA1 in melanocytes was analysed by western blotting and was knocked down with siRNA. In vivo pigmentary responses were measured by a colorimeter in mouse ear skin. The expression of TRPV1 and TRPA1 in human pigmented lesions was examined by immunohistochemical staining., Key Results: PUVA strongly stimulated melanogenesis and PUVA-induced TRPV1 and TRPA1 channel activation in melanocytes and the resulting Ca 2+ influx were required for the stimulated melanogenesis both in vitro and in vivo. Agonists-induced TRPV1 and TRPA1 activation alone did not stimulate melanogenesis, but it synergized UVA or intrinsic cAMP and NO signalling pathways to stimulate UV-dependent or UV-independent melanogenesis. Moreover, the expressions of TRPV1 and TRPA1 were increased in human melanocytic lesions and inhibition of both channels decreased melanin content in melanoma cells., Conclusion and Implications: TRPV1 and TRPA1 are key molecular sensors and enhancers of extrinsic and intrinsic melanogenic signals in both physiological and pathological conditions, and activation of both channels in melanocytes contributes to PUVA therapy-induced pigmentation. Our work provides a common mechanism of melanogenic regulation and highlights TRPV1 and TRPA1 as potential therapeutic targets for pigmentary disorders., (© 2021 The British Pharmacological Society.)

Background and Purpose: Neurosteroids influence neuronal function and have multiple promising clinical applications. Direct modulation of postsynaptic neurotransmitter receptors by neurosteroids is well characterized, but presynaptic effects remain poorly understood. Here, we report presynaptic glutamate release potentiation by neurosteroids pregnanolone and pregnanolone sulfate and compare their mechanisms of action to phorbol 12,13-dibutyrate (PDBu), a mimic of the second messenger DAG., Experimental Approach: We use whole-cell patch-clamp electrophysiology and pharmacology in rat hippocampal microisland cultures and total internal reflection fluorescence (TIRF) microscopy in HEK293 cells expressing GFP-tagged vesicle priming protein Munc13-1, to explore the mechanisms of neurosteroid presynaptic modulation., Key Results: Pregnanolone sulfate and pregnanolone potentiate glutamate release downstream of presynaptic Ca 2+ influx, resembling the action of a phorbol ester PDBu. PDBu partially occludes the effect of pregnanolone, but not of pregnanolone sulfate. Calphostin C, an inhibitor that disrupts DAG binding to its targets, reduces the effect PDBu and pregnanolone, but not of pregnanolone sulfate, suggesting that pregnanolone might interact with a well-known DAG/phorbol ester target Munc13-1. However, TIRF microscopy experiments found no evidence of pregnanolone-induced membrane translocation of GFP-tagged Munc13-1, suggesting that pregnanolone may regulate Munc13-1 indirectly or interact with other DAG targets., Conclusion and Implications: We describe a novel presynaptic effect of neurosteroids pregnanolone and pregnanolone sulfate to potentiate glutamate release downstream of presynaptic Ca 2+ influx. The mechanism of action of pregnanolone, but not of pregnanolone sulfate, partly overlaps with that of PDBu. Presynaptic effects of neurosteroids may contribute to their therapeutic potential in the treatment of disorders of the glutamate system., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Ca 2+ -activated Cl - channels (Ano1 channels) contribute to the pathogenesis of colorectal cancer. Honokiol is known to inhibit cell proliferation and tumour growth in colorectal cancer. However, the molecular target of honokiol remains unclear. This study aimed to investigate whether honokiol inhibited cell proliferation of colorectal cancer by targeting Ano1 channels., Experimental Approach: Patch-clamp techniques were performed to study the effect of honokiol on Ca 2+ -activated Cl - currents in HEK293 cells overexpressing Ano1- or Ano2-containing plasmids or in human colorectal carcinoma SW620 cells. Site-directed mutagenesis was used to identify the critical residues for honokiol-induced Ano1 inhibition. Proliferation of SW620 cells or human intestinal epithelial NCM460 cells by CCK-8 assays., Key Results: Honokiol blocked Ano1 currents in Ano1-overexpressing HEK293 cells and SW620 cells. Honokiol more potently inhibited Ano1 currents than Ano2 currents. Three amino acids (R429, K430 and N435) were critical for honokiol-induced Ano1 inhibition. The R429A/K430L/N435G mutation reduced the sensitivity of Ano1 to honokiol. Honokiol inhibited SW620 cell proliferation, and this effect was reduced by Ano1-shRNAs. Furthermore, Ano1 overexpression promoted proliferation in NCM460 cells with low Ano1 endogenous expression and resulted in an increased sensitivity to honokiol. Overexpression of the R429A/K430L/N435G mutation reduced WT Ano1-induced increase in the sensitivity of NCM460 cells to honokiol., Conclusion and Implications: We identified a new anticancer mechanism of honokiol, through the inhibition of cell proliferation, by targeting Ano1 Ca 2+ -activated Cl - channels., (© 2021 The British Pharmacological Society.)

Background and Purpose: Quercetin is a well-known plant flavonoid with neuroprotective properties. Earlier work suggested it may relieve psychiatric disorders, cognition deficits and memory dysfunction through anti-oxidant and/or radical scavenging mechanisms. In addition, quercetin modulated the physiological function of some ion channels. However, the detailed ionic mechanisms of the bioeffects of quercetin remain unknown., Experimental Approach: Effects of quercetin on neuronal activities in the prefrontal cortex (PFC) and its ionic mechanisms were analysed by calcium imaging using mice bearing a green fluorescent protein, calmodulin, and M13 fusion protein and patch clamp in acute brain slices from C57BL/6 J mice and in HEK 293 cells. The possible ionic mechanism of action of quercetin on D-amphetamine-induced manic-like effects in mice was explored with c-fos staining and the open field behaviour test., Key Results: Quercetin reduced calcium influx triggered by PFC pyramidal neuronal activity. This effect involved increasing the rheobase of neuronal firing through decreasing membrane resistance following quercetin treatment. Spadin, a blocker of TREK-1 potassium channels, also blocked the effect of quercetin on the membrane resistance and neuronal firing. Further, spadin blocked the neuroprotective effects of quercetin. The effects of quercetin on TREK-1 channels could be mimicked by GF109203X, a protein kinase C inhibitor. In vivo, injection of quercetin relieved the manic hyperlocomotion in mice, induced by D-amphetamine. This action was partly alleviated by spadin., Conclusion and Implications: TREK-1 channels are a novel target for quercetin, by inhibiting PKC. This action could contribute to both the neuroprotective and anti-manic-like effects., (© 2021 The British Pharmacological Society.)

Background and Purpose: Cocamide monoethanolamide (CMEA) is commonly used as a surfactant-foam booster in cosmetic formulations. Upon contact with the eye or other sensitive skin areas, CMEA elicits stinging and lasting irritation. We hypothesized a specific molecular interaction with TRPV1 channels by which CMEA caused eye irritation., Experimental Approach: Eye irritancy was evaluated using eye-wiping tests in rabbits and mice. Intracellular Ca 2+ concentrations and action potentials were measured using Ca 2+ imaging and current clamp respectively. Voltage clamp, site-direct mutagenesis and molecular modelling were used to identify binding pockets for CMEA on TRPV1 channels., Key Results: CMEA-induced eye irritation is ameliorated by selective ablation of TRPV1 channels.Rodents exhibit much stronger responses to CMEA than rabbits. In trigeminal ganglion neurons, CMEA induced Ca 2+ influx and neuronal excitability, effects mitigated by a TRPV1 channel inhibition and absent in TRPV1 knockout neurons. In HEK-293 cells expressing TRPV1 channels, CMEA increased whole-cell currents by increasing channel open probability (EC 50  = 10.2 μM), without affecting TRPV2, TRPV3, TRPV4, and TRPA1 channel activities. Lauric acid monoethanolamide (LAMEA), the most abundant constituent of CMEA, was the most efficacious and potent TRPV1 channel activator, binding to the capsaicin-binding pocket of the channel. The T550I mutants of rabbit and human TRPV1 channels exhibit much lower sensitivity to LAMEA., Conclusions and Implication: CMEA directly activates TRPV1 channels to produce eye irritation. Rabbits, the standard animal used for eye irritancy tests are poor models for evaluating human eye irritants structurally related to CMEA. Our study identifies potential alternatives to CMEA as non-irritating surfactants., (© 2021 The British Pharmacological Society.)

Background and Purpose: TGFβ1-mediated myofibroblast activation contributes to pathological fibrosis in many diseases including idiopathic pulmonary fibrosis (IPF), where myofibroblast resistance to oxidant-mediated apoptosis is also evident. We therefore investigated the involvement of redox-sensitive TRPA1 ion channels on human lung myofibroblasts (HLMFs) cell death and TGFβ1-mediated pro-fibrotic responses., Experimental Approach: The effects of TGFβ1 stimulation on TRPA1 expression and cell viability was studied in HLMFs derived from IPF patients and non-fibrotic patients. We also examined a model of TGFβ1-dependent fibrogenesis in human lung. We used qRT-PCR, immunofluorescent assays, overexpression with lentiviral vectors and electrophysiological methods., Key Results: TRPA1 mRNA, protein and ion currents were expressed in HLMFs derived from both non-fibrotic patient controls and IPF patients, and expression was reduced by TGFβ1. TRPA1 mRNA was also down-regulated by TGFβ1 in a model of lung fibrogenesis in human lung. TRPA1 over-expression or activation induced HLMF apoptosis, and activation of TRPA1 channel activation by H 2 O 2 induced necrosis. TRPA1 inhibition following TGFβ1 down-regulation or pharmacological inhibition, protected HLMFs from both apoptosis and necrosis. Lentiviral vector mediated TRPA1 expression was also found to induce sensitivity to H 2 O 2 induced cell death in a TRPA1-negative HEK293T cell line., Conclusion and Implications: TGFβ1 induces resistance of HLMFs to TRPA1 agonist- and H 2 O 2 -mediated cell death via down-regulation of TRPA1 channels. Our data suggest that therapeutic strategies which prevent TGFβ1-dependent down-regulation of TRPA1 may reduce myofibroblast survival in IPF and therefore improve clinical outcomes., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: The slowly activating delayed rectifier K + channel (I Ks ), composed of pore-forming KCNQ1 α-subunits and ancillary KCNE1 β-subunits, regulates ventricular repolarization in human heart. Propofol, at clinically used concentrations, modestly inhibits the intact (wild-type) I Ks channels and is therefore unlikely to appreciably prolong QT interval in ECG during anaesthesia. However, little information is available concerning the inhibitory effect of propofol on I Ks channel associated with its gene variants implicated in QT prolongation. The KCNE1 single nucleotide polymorphism leading to D85N is associated with drug-induced QT prolongation and therefore regarded as a clinically important genetic variant. This study examined whether KCNE1-D85N affects the sensitivity of I Ks to inhibition by propofol., Experimental Approach: Whole-cell patch-clamp and immunostaining experiments were conducted in HEK293 cells and/or mouse cardiomyocyte-derived HL-1 cells, transfected with wild-type KCNQ1, wild-type or variant KCNE1 cDNAs., Key Results: Propofol inhibited KCNQ1/KCNE1-D85N current more potently than KCNQ1/KCNE1 current in HEK293 cells and HL-1 cells. Immunostaining experiments in HEK293 cells revealed that pretreatment with propofol (10 μM) did not appreciably affect cell membrane expression of KCNQ1 and KCNE1 proteins in KCNQ1/KCNE1 and KCNQ1/KCNE1-D85N channels., Conclusion and Implications: The KCNE1 polymorphism D85N significantly elevates the sensitivity of I Ks to inhibition by propofol. This study detects a functionally important role of KCNE1-D85N polymorphism in conferring genetic susceptibility to propofol-induced QT prolongation and further suggests the possibility that the inhibitory action of anaesthetics on ionic currents becomes exaggerated in patients carrying variants in genes encoding ion channels., (© 2021 The British Pharmacological Society.)

Background and Purpose: VEGF-A is a key mediator of angiogenesis, primarily signalling via VEGF receptor 2 (VEGFR2). Endothelial cells also express the co-receptor neuropilin-1 (NRP1) that potentiates VEGF-A/VEGFR2 signalling. VEGFR2 and NRP1 had distinct real-time ligand binding kinetics when monitored using BRET. We previously characterised fluorescent VEGF-A isoforms tagged at a single site with tetramethylrhodamine (TMR). Here, we explored differences between VEGF-A isoforms in living cells that co-expressed both receptors., Experimental Approach: Receptor localisation was monitored in HEK293T cells expressing both VEGFR2 and NRP1 using membrane-impermeant HaloTag and SnapTag technologies. To isolate ligand binding pharmacology at a defined VEGFR2/NRP1 complex, we developed an assay using NanoBiT complementation technology whereby heteromerisation is required for luminescence emissions. Binding affinities and kinetics of VEGFR2-selective VEGF 165 b-TMR and non-selective VEGF 165 a-TMR were monitored using BRET from this defined complex., Key Results: Cell surface VEGFR2 and NRP1 were co-localised and formed a constitutive heteromeric complex. Despite being selective for VEGFR2, VEGF 165 b-TMR had a distinct kinetic ligand binding profile at the complex that largely remained elevated in cells over 90 min. VEGF 165 a-TMR bound to the VEGFR2/NRP1 complex with kinetics comparable to those of VEGFR2 alone. Using a binding-dead mutant of NRP1 did not affect the binding kinetics or affinity of VEGF 165 a-TMR., Conclusion and Implications: This NanoBiT approach enabled real-time ligand binding to be quantified in living cells at 37°C from a specified complex between a receptor TK and its co-receptor for the first time., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Sodium channel inhibitors can be used to treat hyperexcitability-related diseases, including epilepsies, pain syndromes, neuromuscular disorders and cardiac arrhythmias. The applicability of these drugs is limited by their nonspecific effect on physiological function. They act mainly by sodium channel block and in addition by modulation of channel kinetics. While channel block inhibits healthy and pathological tissue equally, modulation can preferentially inhibit pathological activity. An ideal drug designed to target the sodium channels of pathological tissue would act predominantly by modulation. Thus far, no such drug has been described., Experimental Approach: Patch-clamp experiments with ultra-fast solution exchange and photolabeling-coupled electrophysiology were applied to describe the unique mechanism of riluzole on Nav1.4 sodium channels. In silico docking experiments were used to study the molecular details of binding., Key Results: We present evidence that riluzole acts predominantly by non-blocking modulation. We propose that, being a relatively small molecule, riluzole is able to stay bound to the binding site, but nonetheless stay off the conduction pathway, by residing in one of the fenestrations. We demonstrate how this mechanism can be recognized., Conclusions and Implications: Our results identify riluzole as the prototype of this new class of sodium channel inhibitors. Drugs of this class are expected to selectively prevent hyperexcitability, while having minimal effect on cells firing at a normal rate from a normal resting potential., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Acute kidney injury is a common clinical problem with no definitive or specific treatment. Therefore, the molecular mechanisms of acute kidney injury must be fully understood to develop novel treatments. Nuciferine, a major bioactive compound isolated from the lotus leaf, possesses extensive pharmacological activities. Its effect on folic acid-induced acute kidney injury, however, remains unknown. Here, we aimed to clarify the pharmacological effects of nuciferine and its mechanisms of action in acute kidney injury., Experimental Approach: The effects of nuciferine on folic acid-induced acute kidney injury in mice were investigated. HK-2 human proximal tubular epithelial cells and HEK293T HEK cells were used to evaluate the protective effect of nuciferine on RSL3-induced ferroptosis., Key Results: Nuciferine treatment mitigated the pathological alterations, ameliorated inflammatory cell infiltration and improved kidney dysfunction in mice with folic acid-induced acute kidney injury. In HK-2 and HEK293T cells, nuciferine significantly prevented RSL3-induced ferroptotic cell death. Mechanistically, nuciferine significantly inhibited ferroptosis by preventing iron accumulation and lipid peroxidation in vitro and in vivo. Moreover, knockdown of glutathione (GSH) peroxidase 4 (GPX4) abolished the protective effect of nuciferine against ferroptosis., Conclusion and Implications: Nuciferine ameliorated renal injury in mice with acute kidney injury, perhaps by inhibiting the ferroptosis. Nuciferine may represent a novel treatment that improves recovery from acute kidney injury by targeting ferroptosis., (© 2021 The British Pharmacological Society.)

Background and Purpose: Aminoglycoside antibiotics are positively charged molecules that are known to inhibit several ion channels. In this study, we have shown that aminoglycosides also inhibit the activity of Kir4.1 channels. Aminoglycosides inhibit Kir4.1 channels by a pore-blocking mechanism, plugging the central vestibule of the channel., Experimental Approach: Patch-clamp recordings were made in HEK-293 cells transiently expressing Kir4.1 channels to analyse the effects of gentamicin, neomycin and kanamycin. In silico modelling followed by mutagenesis were realized to identify the residues critical for aminoglycosides binding to Kir4.1., Key Results: Aminoglycoside antibiotics block Kir4.1 channels in a concentration- and voltage-dependent manner, getting access to the protein from the intracellular side of the plasma membrane. Aminoglycosides block Ki4.1 with a rank order of potency as follows: gentamicin ˃ neomycin ˃ kanamycin. The residues T128 and principally E158, facing the central cavity of Kir4.1, are important structural determinants for aminoglycosides binding to the channel, as determined by our in silico modelling and confirmed by mutagenesis experiments., Conclusion and Implications: Kir4.1 channels are also target of aminoglycoside antibiotics, which could affect potassium transport in several tissues., (© 2020 The British Pharmacological Society.)

Background and Purpose: Various GPCRs have been described as being modulated in a voltage-dependent manner. Opioid analgesics act via activation of μ receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on μ receptor signalling., Experimental Approach: We investigated potential voltage sensitivity of μ receptors in heterologous expression systems (HEK293T cells) using electrophysiology in combination with Förster resonance energy transfer-based assays. Depolarization-induced changes in signalling were also tested in physiological rat tissue containing locus coeruleus neurons. We applied depolarization steps across the physiological range of membrane potentials., Key Results: Studying μ receptor function and signalling in cells, we discovered that morphine-induced signalling was strongly dependent on the membrane potential (V M ). This became apparent at the level of G-protein activation, G-protein coupled inwardly rectifying potassium channel (K ir 3.X) currents and binding of GPCR kinases and arrestin3 to μ receptors by a robust increase in signalling upon membrane depolarization. The pronounced voltage sensitivity of morphine-induced μ receptor activation was also observed at the level of K ir 3.X currents in rat locus coeruleus neurons. The efficacy of peptide ligands to activate μ receptors was not (Met-enkephalin) or only moderately ([D-Ala 2 , N-Me-Phe 4 , Gly 5 -ol]-enkephalin) enhanced upon depolarization. In contrast, depolarization reduced the ability of the analgesic fentanyl to activate μ receptors., Conclusion and Implications: Our results indicate a strong ligand-dependent modulation of μ receptor activity by the membrane potential, suggesting preferential activity of morphine in neurons with high neuronal activity., (© 2020 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: NALCN is a Na + leak, GPCR-activated channel that regulates the resting membrane potential and neuronal excitability. Despite numerous possible roles for NALCN in both normal physiology and disease processes, lack of specific blockers hampers further investigation., Experimental Approach: The effect of N-benzhydryl quinuclidine compounds on NALCN channels was demonstrated using whole-cell patch-clamp recordings in HEK293T cells overexpressing NALCN and acutely isolated nigral dopaminergic neurons that express NALCN endogenously. Src kinase activity was measured using a Src kinase assay kit, and voltage and current-clamp recordings from nigral dopaminergic neurons were used to measure NALCN currents and membrane potentials., Key Results: N-benzhydryl quinuclidine compounds inhibited NALCN channels without affecting TRPC channels, another important route for Na + leak. In HEK293T cells overexpressing NALCN, N-benzhydryl quinuclidine compounds potently suppressed muscarinic M 3 receptor-activated NALCN currents. Structure-function relationship studies suggest that the quinuclidine ring with a benzhydryl group imparts the ability to inhibit NALCN currents regardless of Src family kinases. Moreover, N-benzhydryl quinuclidine compounds inhibited not only GPCR-activated NALCN currents but also background Na + leak currents and hyperpolarized the membrane potential in native midbrain dopaminergic neurons that express NALCN endogenously., Conclusion and Implications: These findings suggest that N-benzhydryl quinuclidine compounds have a pharmacological potential to directly inhibit NALCN channels and could be a useful tool to investigate functions of NALCN channels., (© 2020 The British Pharmacological Society.)

Background and Purpose: Th17 cells play critical roles in chronic inflammation, including fibrosis. Histone acetyltransferase p300, a bromodomain-containing protein, acetylates RORγt and promotes Th17 cell development. The bromodomain inhibitor JQ1 was shown to alleviate Th17-mediated pathologies, but the underlying mechanism remains unclear. We hypothesized that JQ1 suppresses the response of Th17 cells by impairing p300-mediated acetylation of RORγt., Experimental Approach: The effect of JQ1 on p300-mediated acetylation of RORγt was investigated in HEK293T (overexpressing Flag-p300 and Myc-RORγt) and human Th17 cells through immunoprecipitation and western blotting. To determine the regions of p300 responsible for JQ1-mediated suppression of HAT activity, we performed HAT assays on recombinant p300 fragments with/without the bromodomain, after exposure to JQ1. Additionally, the effect of JQ1 on p300-mediated acetylation of RORγt and Th17 cell function was verified in vivo, using murine Schistosoma-induced fibrosis models. Liver injury was assessed by histopathological examination and measurement of serum enzyme levels. Expression of Th17 effectors was detected by qRT-PCR, whereas IL-17- and RORγt-positive granuloma cells were detected by FACS., Key Results: JQ1 impaired p300-mediated RORγt acetylation in human Th17 and HEK293T cells. JQ1 failed to suppress the acetyltransferase activity of p300 fragments lacking the bromodomain. JQ1 treatment attenuated Schistosoma-induced fibrosis in mice, by inhibiting RORγt acetylation and IL-17 expression., Conclusions and Implications: JQ1 impairs p300-mediated RORγt acetylation, thus reducing the expression of RORγt target genes, including Th17-specific cytokines. JQ1-mediated inhibition of p300 acetylase activity requires the p300 bromodomain. Strategies targeting p300 may provide new therapeutic approaches for controlling Th17-related diseases., (© 2020 The British Pharmacological Society.)