Background and Purpose: Local anesthetics block sodium and a variety of potassium channels. Although previous studies identified a residue in the pore signature sequence together with three residues in the S6 segment as a putative binding site, the precise molecular basis of Kv potassium channel inhibition by local anesthetics remained unknown. Kv crystal structures predict that some of these residues point away from the central cavity and face into a drug binding site called ‘side pockets´. Thus, the question arises whether the binding site of local anesthetics is exclusively located in the central cavity or also involves the ‘side pockets´. Experimental Approach: A systematic functional alanine mutagenesis approach, scanning 58 mutants, in concert with in silico docking experiments and molecular dynamics simulations were utilized to elucidate the binding site of bupivacaine and ropivacaine. Key Results: Kv1.5 inhibition by local anesthetics requires binding to the central cavity and the ‘side pockets´, where the latter requires interactions with residues of the S5 and the backside of the S6 segment. Mutations in the ‘side pockets´ remove stereoselectivity of Kv1.5 inhibition by bupivacaine. Strikingly, while we found that binding to the ‘side pockets´ is conserved for the different local anesthetics, the binding mode in the central cavity and the ‘side pockets´ shows considerable variations. Conclusion and Implications: Local anesthetics bind to the central cavity and the ‘side pockets´ which provides a crucial key for the molecular understanding of their Kv channel affinity and stereoselectivity, as well as their spectrum of side effects.
Diabetes is a chronic metabolic disorder associated with the accelerated development of macrovascular (atherosclerosis, coronary artery disease) and microvascular complications (nephropathy, retinopathy and neuropathy), which remain the principal cause of mortality and morbidity in this population. Current understanding of cellular and molecular pathways of diabetes-driven vascular complications as well as therapeutic interventions have arisen from studying disease pathogenesis in animal models. Diabetes-associated vascular complications are multi-faceted, involving the interaction between various cellular and molecular pathways. Thus, the choice of an appropriate animal model to study vascular pathogenesis is important in our quest to identify innovative and mechanism-based targeted therapies to reduce the burden of diabetic complications. Herein, we provide up-to-date information on available mouse models of both Type 1 and Type 2 diabetic vascular complications as well as experimental analysis and research outputs.
Background and purpose Statins, inhibitors of HMG-CoA reductase, are mainstay treatment for hypercholesterolemia. However, muscle pain and weakness prevent many patients from benefiting from their cardioprotective effects. We previously demonstrated that simvastatin activates skeletal ryanodine receptors (RyR1), an effect that could be important in initiating myopathy. We therefore investigated if RyR1 activation is a standard property of commonly-prescribed statins. Using a range of structurally-diverse statin analogues we examined structural features associated with RyR1 activation, aiming to identify statins lacking this property. Experimental Approach Compounds were screened for RyR1 activity utilising [3H]ryanodine binding. Mechanistic insight into RyR1 activity was studied by incorporating RyR1 channels from sheep, mouse or rabbit skeletal muscle into bilayers. Key Results All UK-prescribed statins activated RyR1 at nanomolar concentrations. Cerivastatin, withdrawn from the market due to life-threatening muscle-related side effects, was more effective than currently-prescribed statins and possessed the unique ability to open RyR1 channels independently of cytosolic Ca2+. We synthesised the statin pharmacophore and it did not activate RyR1. We also identified five analogues retaining potent HMG-CoA reductase inhibition that inhibited RyR1 and four analogues that lacked the ability to activate RyR1. Conclusion and Implications That cervistatin activates RyR1 most strongly supports the hypothesis that RyR1 activation is implicated in statin-induced myopathy. Demonstrating that statin-regulation of RyR1 and HMG-CoA reductase are separable effects allows the role of RyR1 in statin-induced myopathy to be further elucidated by the tool compounds identified, thus paving the way for the development of effective cardioprotective statins with improved patient tolerance.
Background and Purpose Vancomycin is one of the most common antibiotics administered in the hospital setting, yet acute kidney injury is a major limiting factor. Common combinations of antibiotics with vancomycin have been reported to worsen and improve vancomycin-induced kidney injury. We aimed to study the impact of flucloxacillin and imipenem-cilastatin on kidney injury when combined with vancomycin in our translational rat model. Experimental Approach Male Sprague-Dawley rats received allometrically scaled (1) vancomycin (2) flucloxacillin, (3) vancomycin+flucloxacillin, (4) vancomycin+imipenem-cilastatin, or (5) saline for 4 days. Vancomycin was administered intravenously and flucloxacillin or imipenem-cilastatin were administered intraperitoneally. Kidney injury was evaluated via drug accumulation and urinary biomarkers including urinary output, kidney injury molecule-1 (KIM-1), clusterin, and osteopontin. Relationships between vancomycin accumulation in the kidney and urinary kidney injury biomarkers were explored. Key Results Urinary output increased every study day for vancomycin+flucloxacillin; whereas in the vancomycin group it was elevated after the first dose only. In the vancomycin+flucloxacillin group, urinary KIM-1/24h increased on all days compared to vancomycin. In the vancomycin+imipenem-cilastatin group, urinary KIM-1/24h was decreased on days 1 and 2 compared to vancomycin. Similar trends were observed for clusterin. More vancomycin accumulated in the kidney with vancomycin+flucloxacillin compared to vancomycin and vancomycin+imipenem-cilastatin. The accumulation of vancomycin in the kidney tissue correlated with increasing urinary KIM-1 (4-parameter Hill Slope, R2=0.7985). Conclusion and Implications Vancomycin+flucloxacillin caused more kidney injury compared to vancomycin alone and vancomycin+imipenem-cilastatin in a translational rat model as determined by multiple kidney injury biomarkers. The combination of vancomycin+imipenem-cilastatin was nephroprotective.
Background and Purpose: ISX9 is a neurogenesis-promoting small molecule compound which can upregulate the expression of NeuroD1 and induce differentiation of neuronal, cardiac and islet endocrine progenitors. So far, the molecular mechanisms underlying the action of ISX9 still remain elusive. Experimental Approach: To identify a novel agonist of the Wnt/β‐catenin, a cell-based SuperTOPFlash reporter system was used to screen known-compound libraries. An activation effect of ISX9 on the Wnt/β‐catenin pathway was analysed with the SuperTOPFlash or SuperFOPFlash reporter system. Effects of ISX9 on Axin1/LRP6 interaction were examined using a mammalian two-hybrid system, co‐immunoprecipitation, microscale thermophoresis (MST), emission spectra and mass spectroscopy assays. The expression of Wnt target and stemmness marker genes were evaluated with real‐time PCR and immunoblotting. In vivo hair regeneration abilities of ISX9 were analysed by immunohistochemical staining, real‐time PCR and immunoblotting in hair regrowth model using C57BL/6J mice. Key Results: In this study, ISX9 was identified as a novel agonist of the Wnt/β‐catenin pathway. ISX9 targeted Axin1 by covalently binding to its N-terminal region and potentiated the LRP6-Axin1 interaction, thereby resulting in the stabilization of β‐catenin and upregulation of Wnt target genes and stemmness marker genes. Moreover, the topical application of ISX9 markedly promoted hair regrowth in C57BL/6J mice and induced hair follicle transition from telogen to anagen via enhancing Wnt/β‐catenin pathway. Conclusions and Implications: Taken together, our study unraveled that ISX9 could activate Wnt/β‐catenin signaling by potentiating the association between LRP6 and Axin1, and may be a promising therapeutic agent for alopecia treatment
The access of drugs into the central nervous system (CNS) is regulated by the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB). A large body of evidence supports perturbation of these barriers in neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Modifications to the BBB and BSCB are also reported in amyotrophic lateral sclerosis (ALS), albeit these modifications have received less attention relative to those in other neurodegenerative diseases. Such alterations to the BBB and BSCB have the potential to impact on CNS exposure of drugs in ALS, modulating the effectiveness of drugs intended to reach the brain and the toxicity of drugs that are not intended to reach the brain. Given the clinical importance of these phenomena, this review will summarise reported modifications to the BBB and BSCB in ALS, discuss their impact on CNS drug exposure and suggest further research directions so as to optimise medicine use in people with ALS.
Background and Purpose: Blood-brain barrier (BBB) breakdown is one of the most crucial pathological changes of cerebral ischemia-reperfusion (I/R) injury. Trilobatin (TLB), a naturally occurring food additive, exerts neuroprotective effect against cerebral I/R injury as demonstrated in our previous study. This study was designed to investigate the effect of TLB on disruption of BBB after cerebral I/R injury. Experimental Approach: Rats with focal cerebral ischemia caused by transient middle cerebral artery occlusion (MCAO) and brain microvascular endothelial cells along with human astrocytes to mimic blood brain barrier (BBB) injury caused by oxygen and glucose deprivation (OGD) followed by reoxygenation (OGD/R). Key results: The results showed that TLB effectively maintained the integrity of BBB and inhibited neuronal loss following cerebral I/R challenge. Furthermore, TLB dramatically increased tight junction proteins including ZO-1, occludin and claudin 5, as well as decreased the levels of apolipoprotein E (APOE) 4, cyclophilin A (CypA), and phosphorylated nuclear factor kappa B (NF-κB), thereby reduced proinflammatory cytokines. In addition, TLB also decreased Bax/Bcl-2 ratio and cleaved-caspase 3 level along with reduced the number of apoptotic neurons. Intriguingly, molecular docking and transcriptomics predicted MMP9 was a prominent gene evoked by TLB treatment. Furthermore, the protective effect of TLB on OGD/R-induced the loss of BBB integrity in human brain microvascular endothelial cell and astrocyte co-cultures in vitro was markedly reinforced by knockdown of MMP9. Conclusions and implications: Our findings reveal a novel property of TLB: saving BBB disruption following cerebral I/R via targeting MMP9 and inhibiting APOE4/CypA/NF-κB axis.
Alcohol use disorder (AUD) is one of the most common but still poorly treated psychiatric conditions. Developing new treatments requires a better understanding of the aetiology of symptoms and evaluation of novel therapeutic targets in preclinical studies. Recent developments in our understanding of the reinforcement-based cognitive biases (RBCBs) that contribute to the development of AUD and its treatment offer new opportunities for both clinical and preclinical research. In this review, we first briefly describe psychological and cognitive theories that implicate various aspects of reinforcement sensitivity in the development, maintenance, and recurrence of alcohol addiction. Furthermore, in separate sections, we describe studies investigating RBCBs and their neural, neurochemical, and pharmacological correlates, and we discuss possible interactions between RBCBs and trajectories of AUD. Finally, we describe how recent translational studies using state-of-the-art animal models can facilitate our understanding of the role of reinforcement sensitivity and RBCBs in various aspects of AUD.
Background and purpose: Regulator of G-protein signal 4 (RGS4) is a signal transduction protein that accelerates intrinsic GTPase activity of Gαi/o and Gαq subunits, suppressing GPCR signaling. Here we investigate whether RGS4 modulates nociceptin/orphanin FQ opioid (NOP) receptor signaling and whether 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 N/OFQ or AT-403 in the presence of the RGS4 inhibitor, CCG-203920, and D1-stimulated cAMP or pERK responses were monitored. In vivo, CCG-203920 was co-administered with AT-403 and levodopa 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 levodopa. Levodopa acutely upregulated RGS4 in the lesioned striatum. Conclusions and Implications: RGS4 physiologically inhibits NOP receptor signaling and an RGS4 inhibitor enhances NOP responses. Furthermore, an RGS4 inhibitor improved the antidyskinetic potential of NOP receptor agonists, mitigating the effects of upregulation of striatal RGS4 levels occurring during dyskinesia expression.
Since cell penetrating peptides are promising tools for delivery of cargo into cells, factors limiting or facilitating their cellular uptake are intensely studied. Using labeling with pH-insensitive and pH-sensitive dyes we report that escape of penetratin from acidic endo-lysosomal compartments is retarded compared to its total cellular uptake. The membrane dipole potential, known to alter transmembrane transport of charged molecules, is shown to be negatively correlated with the concentration of penetratin in the cytoplasmic compartment. Treatment of cells with therapeutically relevant concentrations of atorvastatin, an inhibitor of HMG-CoA reductase and cholesterol synthesis, significantly increased endosomal escape of penetratin in two different cell types. This effect of atorvastatin correlated with its ability to decrease the membrane dipole potential. These results highlight the importance of the dipole potential in regulating cellular uptake of cell penetrating peptides and suggest a clinically relevant way of boosting this process.
Background and Purpose Neonatal seizures are a clinical emergency. Current anti-seizure medications, however, fail to resolve seizures in ~50% of infants. The P2X7 receptor (P2X7R) is an important driver of inflammation and evidence suggest P2X7R contributing to seizures and epilepsy in adults. To date, however, no genetic proof has been provided to determine the contribution of the P2X7R to neonatal seizures, its effects on inflammatory signalling during neonatal seizures and the therapeutic potential of P2X7R-based treatments on long-lasting brain excitability. Experimental Approach Neonatal seizures were induced via global hypoxia in 7 day-old mouse pups (P7). The role of P2X7Rs during seizures was analyzed in P2X7R overexpressing and knock-out mice. Treatment of wild-type mice post-hypoxia with the P2X7R antagonist JNJ-47965567 was used to determine the effects of the P2X7R on long-lasting brain hyperexcitability. Cell type-specific P2X7R expression was analyzed via P2X7R-EGFP reporter mice. RNA sequencing was used to monitor P2X7R-dependent hippocampal down-stream signalling. Key Results P2X7R deletion reduced seizure severity whereas P2X7R overexpression exacerbated seizure severity and reduced responsiveness to anti-seizure medication. P2X7R deficiency let to an anti-inflammatory phenotype in microglia and treatment of mice with a P2X7R antagonist reduced long-lasting brain hyperexcitability. RNA sequencing identified several pathways altered in P2X7R knock-out mice after neonatal hypoxia including a down-regulation of genes implicated in inflammation and glutamatergic signalling. Conclusion and Implications Treatments based on targeting the P2X7R may represent a novel therapeutic strategy for neonatal seizures with P2X7Rs contributing to the generation of neonatal seizures, driving inflammatory processes and long-term hyperexcitability states.
Background and Purpose Airway hyperresponsiveness (AHR) is a central abnormality in asthma. Interleukin-5 (IL-5) may modulate AHR in animal models of asthma, but inconsistent data are available on the impact of targeting IL-5 pathway against AHR. The difference between targeting IL-5 or IL-5Rα in modulating AHR remains to be understood in human airways. The aim of this study was to compare the role of the anti-IL-5Rα benralizumab and the anti-IL-5 mepolizumab against AHR, and to assess whether these agents influence the levels of cyclic adenosine monophosphate (cAMP). Experimental Approach Passively sensitized human airways were treated with benralizumab and mepolizumab. The primary endpoint was the inhibition of AHR to histamine; the secondary endpoints were the protective effect against AHR to parasympathetic activation and mechanical stress, and the tissue modulation of cAMP. Key Results Benralizumab and mepolizumab significantly (P<0.001 vs. positive control) prevented the AHR to histamine (maximal effect -134.14±14.93% and -108.29±32.16%, respectively), with benralizumab being 0.73±0.10 logarithm significantly (P<0.05) more potent than mepolizumab. Benralizumab and mepolizumab significantly (P<0.001 vs. positive control) inhibited the AHR to transmural stimulation and mechanical stress. Benralizumab was 0.45±0.16 logarithm significantly (P<0.05) more potent than mepolizumab against AHR to parasympathetic activation. The effect of these agents was significantly correlated (P<0.001) with increased levels of cAMP. Conclusion Targeting the IL-5/IL-5Rα axis is an effective strategy to prevent the AHR. Benralizumab resulted more potent than the mepolizumab and the concentration dependent beneficial effects of both these agents were related with improved levels of cAMP in hyperresponsive airways.
Background and Purpose: Resurgence in the use of chloroquine as a putative treatment for COVID-19 has seen recent cases of fatal toxicity due to unintentional overdoses. Protocols for the management of poisoning recommend diazepam, although there are uncertainties in its pharmacology and efficacy in this context. The aim was to assess the effects of diazepam in experimental models of chloroquine cardiotoxicity. Experimental Approach: In vitro experiments involved cardiac tissues isolated from rats and incubated with chloroquine, alone, or in combination with diazepam. In vivo models of toxicity involved chloroquine administered intravenously to pentobarbitone-anaesthetised rats and rabbits. Randomised, controlled interventional studies in rats assessed diazepam, clonazepam and Ro5-4864 administered: (i) prior, (ii) during, and (iii) after chloroquine; and the effects of diazepam: (iv) at high dose, (v) in urethane-anaesthetised rats, and (vi) co-administered with adrenaline. Key Results: Chloroquine decreased the developed tension of left atria, prolonged the effective refractory period of atria, ventricular tissue and right papillary muscles, and caused dose-dependent impairment of haemodynamic and electrocardiographic parameters. Cardiac arrhythmias indicated impairment of atrioventricular conduction. Studies (i), (ii) and (v) showed no differences between interventions and control. Diazepam increased heart rate in study (iv) and, as with clonazepam, also prolonged the QTc interval in study (iii). Combined administration of diazepam and adrenaline in study (vi) improved cardiac contractility but caused hypokalaemia. Conclusion and Implications: Neither diazepam, nor other ligands for benzodiazepine binding sites, protect against or attenuate chloroquine cardiotoxicity. However, diazepam may augment the effects of positive inotropes in reducing chloroquine cardiotoxicity.
Background and Purpose Ca2+ influx via TRPV4 triggers Ca2+ release from the IP3-sensitive internal store to generate repetitive oscillations. While mitochondria are acknowledged regulators of IP3-mediated Ca2+ release, how TRPV4-mediated Ca2+ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca2+-induced Ca2+ release at IP3 receptors, to being independent of Ca2+ influx and instead mediated by ATP release via pannexins. Experimental Approach TRPV4 evoked Ca2+ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520. Key ResultsTRPV4 activation with GSK1016790A(GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca2+ waves in the absence of external Ca2+. The ATP synthase inhibitor oligomycin did not potentiate TRPV4 mediated Ca2+ signals. GSK-evoked Ca2+ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the phospholipase C (PLC) blocker U73122 and the inositol triphosphate receptor (IP3 R) blocker caffeine. The Ca2+ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid. Conclusion and Implications These results highlight a previously unknown role of mitochondria in shaping TRPV4 mediated Ca2+ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4-mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP3 evoked Ca2+ release.
Migraine is one of the most common of neurological disorders with a global prevalence of up to 15%. One in five migraineurs have frequent episodic or chronic migraine requiring prophylactic treatment. In recent years, specific pharmaceutical treatments targeting calcitonin gene-related peptide (CGRP) signalling molecules have provided safe and effective treatments; monoclonal antibodies for prophylaxis and gepants for acute therapy. Albeit the beneficious impact of these new drugs, it is important to understand the molecular mechanisms involved to better understand migraine pathophysiology and improve the therapy. Here we describe current views on the role of the CGRP family of peptides CGRP, calcitonin (CT), adrenomedullin (AM), amylin (AMY) and their receptors in the trigeminovascular system (TGV). All these molecules are present within the TGV system but differ in expression and localization. It is likely that they have different roles, which can be utilized in providing additional drug targets.
Epilepsy is a common neurological disorder characterized by repeated and spontaneous epileptic seizures, which is not well controlled by current medication. Traditional theory supports that epilepsy results from the imbalance of excitatory glutamate neurons and inhibitory GABAergic neurons. Recently, shreds of evidence from available clinical and preclinical researches suggest that histamine in the central nervous system plays an important role in the modulation of neural excitability and pathogenesis of epilepsy. Many histamine receptor ligands show positive response in animal epilepsy models, among which the H3R antagonist pitolisant even has shown a good anti-epileptic effect in clinical trials. New insights are focusing on the potential action of histamine receptors to control and treat epilepsy. This review summarizes the findings from animal and clinical researches on the role of brain histamine and histamine receptor in epilepsy. Importantly, we further provide perspectives on some possible research directions for future studies.
Background and Purpose: Overexpression of astrocytic lactoferrin (Lf) was observed in the brains of Alzheimer’s disease (AD) patients, whereas the role of astrocytic Lf in AD progression remains unexplored. In this study, we aimed to evaluate the effects of astrocytic Lf on AD progression. Experimental Approach: The APP/PS1 mice with astrocytes overexpressing human Lf were developed to evaluate the effects of astrocytic Lf on AD progression, and the N2a-sw cells were employed to further uncover the mechanism of astrocytic Lf on β-amyloid (Aβ) production. Key Results: Astrocytic Lf overexpression increased protein phosphatase 2A (PP2A) activity, and reduced amyloid precursor protein (APP) phosphorylation, Aβ burden and tau hyperphosphorylation in APP/PS1 mice. Mechanistically, astrocytic Lf overexpression promoted the astrocytic Lf secretion into neurons in APP/PS1 mice, and the conditional medium from astrocytes overexpressing Lf inhibited the p-APP(Thr668) expression in N2a-sw cells. Furthermore, the recombinant human Lf (hLf) also significantly enhanced PP2A activity and inhibited p-APP expression, while inhibitions of p38 or PP2A activities abrogated the hLf-induced p-APP downregulation in N2a-sw cells. Additionally, hLf promoted the interaction of p38 and PP2A via p38 activation, thereby enhancing PP2A activity; and low-density lipoprotein receptor-related protein 1 (LRP1) knockdown significantly reversed the hLf-induced p38 activation and p-APP downregulation. Conclusions and Implications: Our data suggested that astrocytic Lf promoted neuronal p38 activation via targeting to LRP1, subsequently promoting p38 binds to PP2A to enhance PP2A activity, which finally inhibited Aβ production via APP dephosphorylation. Therefore, promoting astrocytic Lf expression may be a potential strategy against AD.