RESUMEN
BACKGROUND: The carnitine/acylcarnitine carrier (CAC) represents the route of delivering acyl moieties to the mitochondrial matrix for accomplishing the fatty acid ß-oxidation. The CAC has a couple of Cys residues (C136 and C155) most reactive toward ROS and redox signaling compounds such as GSH, NO, and H2S. Among physiological compounds reacting with Cys, itaconate is produced during inflammation and represents the connection between oxidative metabolism and immune responses. The possible interaction between the CAC and itaconate has been investigated. METHODS: the modulatory effects of itaconate on the transport activity of the native and recombinant CAC were tested using the proteoliposome experimental model together with site-directed mutagenesis and computational analysis. RESULTS: Itaconate reacts with the CAC causing irreversible inhibition. Dose-response experiment performed with the native and recombinant protein showed IC50 for itaconate of 11 ± 4.6 mM and 8.4 ± 2.9 mM, respectively. The IC50 decreased to 3.8 ± 1.0 mM by lowering the pH from pH 7.0 to pH 6.5. Inhibition kinetics revealed a non-competitive type of inhibition. C136 is the main target of itaconate, as demonstrated by the increased IC50 of mutants in which this Cys was substituted by Val. The central role of C136 was confirmed by covalent docking. Administration of dimethyl itaconate to HeLa cells inhibited the CAC transport activity, suggesting that itaconate could react with the CAC also in intact cells.
Asunto(s)
Proteínas de Transporte de Membrana , Mitocondrias , Humanos , Carnitina/metabolismo , Cisteína/metabolismo , Células HeLa/efectos de los fármacos , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Moduladores del Transporte de Membrana/farmacologíaRESUMEN
Aim: To separate and evaluate 9-cyclopropylmethoxy-dihydrotetrabenazine (13a) and its stereoisomers for their high affinity for vesicular monoamine transporter-2 (VMAT2). Method: Stereoisomers of 13a were separated and configurations were ascertained by chiral chromatography and crystal diffraction combined with 1H-1H NOESY assay. Possible binding modes of eight stereoisomers and VMAT2 were explored by molecular docking assays. The VMAT2 affinity of the stereoisomers, inhibition in vivo and pharmacokinetics in rats were evaluated. Results: Three stereoisomers were obtained: P1, P2 and P3, and all had similar VMAT2 binding modes. P2 [(2R, 3R, 11bR)-13a] showed the highest potential VMAT2 binding activity (Ki = 0.75 nM), decreased locomotor activity in rats and had an oral absolute bioavailability of 92.0%. Conclusion: P2 has good efficacy and pharmacokinetic properties and warrants further development to treat tardive dyskinesia.
Asunto(s)
Moduladores del Transporte de Membrana/farmacología , Tetrabenazina , Proteínas de Transporte Vesicular de Monoaminas , Animales , Simulación del Acoplamiento Molecular , Ratas , Estereoisomerismo , Tetrabenazina/análogos & derivados , Tetrabenazina/química , Tetrabenazina/farmacología , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
A series of modified N-cyclohexyl-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-methylpyrimidin-4-amine (CyPPA) analogues were synthesized by replacing the cyclohexane moiety with different 4-substituted cyclohexane rings, tyrosine analogues, or mono- and dihalophenyl rings and were subsequently studied for their potentiation of KCa2 channel activity. Among the N-benzene-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine derivatives, halogen decoration at positions 2 and 5 of benzene-substituted 4-pyrimidineamine in compound 2q conferred a â¼10-fold higher potency, while halogen substitution at positions 3 and 4 of benzene-substituted 4-pyrimidineamine in compound 2o conferred a â¼7-fold higher potency on potentiating KCa2.2a channels, compared to that of the parent template CyPPA. Both compounds retained the KCa2.2a/KCa2.3 subtype selectivity. Based on the initial evaluation, compounds 2o and 2q were selected for testing in an electrophysiological model of spinocerebellar ataxia type 2 (SCA2). Both compounds were able to normalize the abnormal firing of Purkinje cells in cerebellar slices from SCA2 mice, suggesting the potential therapeutic usefulness of these compounds for treating symptoms of ataxia.
Asunto(s)
Cerebelo , Moduladores del Transporte de Membrana , Canales de Potasio Calcio-Activados , Células de Purkinje , Pirimidinas , Ataxias Espinocerebelosas , Animales , Femenino , Masculino , Ratones , Cerebelo/efectos de los fármacos , Modelos Animales de Enfermedad , Activación del Canal Iónico , Moduladores del Transporte de Membrana/química , Moduladores del Transporte de Membrana/farmacología , Canales de Potasio Calcio-Activados/agonistas , Canales de Potasio Calcio-Activados/metabolismo , Células de Purkinje/efectos de los fármacos , Pirimidinas/química , Ataxias Espinocerebelosas/tratamiento farmacológico , Ataxias Espinocerebelosas/metabolismo , Ataxias Espinocerebelosas/patología , Relación Estructura-ActividadRESUMEN
Quantitative prediction of drug-drug interactions (DDIs) involving organic anion transporting polypeptide (OATP)1B1/1B3 inhibition is limited by uncertainty in the translatability of experimentally determined in vitro inhibition potency (half-maximal inhibitory concentration (IC50 )). This study used an OATP1B endogenous biomarker-informed physiologically-based pharmacokinetic (PBPK) modeling approach to predict the effect of inhibitor drugs on the pharmacokinetics (PKs) of OATP1B substrates. Initial static analysis with about 42 inhibitor drugs, using in vitro IC50 values and unbound liver inlet concentrations (Iin,max,u ), suggested in vivo OATP1B inhibition risk for drugs with R-value (1+ Iin,max,u /IC50 ) above 1.5. A full-PBPK model accounting for transporter-mediated hepatic disposition was developed for coproporphyrin I (CP-I), an endogenous OATP1B biomarker. For several inhibitors (cyclosporine, diltiazem, fenebrutinib, GDC-0810, itraconazole, probenecid, and rifampicin at 3 different doses), PBPK models were developed and verified against available CP-I plasma exposure data to obtain in vivo OATP1B inhibition potency-which tend to be lower than the experimentally measured in vitro IC50 by about 2-fold (probenecid and rifampicin) to 37-fold (GDC-0810). Models verified with CP-I data are subsequently used to predict DDIs with OATP1B probe drugs, rosuvastatin and pitavastatin. The predicted and observed area under the plasma concentration-time curve ratios are within 20% error in 55% cases, and within 30% error in 89% cases. Collectively, this comprehensive study illustrates the adequacy and utility of endogenous biomarker-informed PBPK modeling in mechanistic understanding and quantitative predictions of OATP1B-mediated DDIs in drug development.
Asunto(s)
Atorvastatina/farmacocinética , Coproporfirinas/sangre , Transportador 1 de Anión Orgánico Específico del Hígado/antagonistas & inhibidores , Hígado/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Modelos Biológicos , Rosuvastatina Cálcica/farmacocinética , Biomarcadores/sangre , Simulación por Computador , Interacciones Farmacológicas , Células HEK293 , Humanos , Hígado/metabolismo , Transportador 1 de Anión Orgánico Específico del Hígado/genética , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Medición de Riesgo , Factores de RiesgoRESUMEN
Gaba-ergic neurons are a diverse cell class with extensive influence over cortical processing, but their role in experience-dependent plasticity is not completely understood. Here we addressed the role of cortical somatostatin- (SOM-INs) and vasoactive intestinal polypeptide- (VIP-INs) containing interneurons in a Pavlovian conditioning where stimulation of the vibrissae is used as a conditioned stimulus and tail shock as unconditioned one. This procedure induces a plastic change observed as an enlargement of the cortical functional representation of vibrissae activated during conditioning. Using layer-targeted, cell-selective DREADD transductions, we examined the involvement of SOM-INs and VIP-INs activity in learning-related plastic changes. Under optical recordings, we injected DREADD-expressing vectors into layer IV (L4) barrels or layer II/III (L2/3) areas corresponding to the activated vibrissae. The activity of the interneurons was modulated during all conditioning sessions, and functional 2-deoxyglucose (2DG) maps were obtained 24 h after the last session. In mice with L4 but not L2/3 SOM-INs suppressed during conditioning, the plastic change of whisker representation was absent. The behavioral effect of conditioning was disturbed. Both L4 SOM-INs excitation and L2/3 VIP-INs inhibition during conditioning did not affect the plasticity or the conditioned response. We found the activity of L4 SOM-INs is indispensable in the formation of learning-induced plastic change. We propose that L4 SOM-INs may provide disinhibition by blocking L4 parvalbumin interneurons, allowing a flow of information into upper cortical layers during learning.
Asunto(s)
Interneuronas/fisiología , Aprendizaje , Inhibición Neural , Plasticidad Neuronal , Corteza Somatosensorial/fisiología , Animales , Neuronas GABAérgicas/efectos de los fármacos , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/fisiología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Moduladores del Transporte de Membrana/farmacología , Ratones , Corteza Somatosensorial/citología , Somatostatina/genética , Somatostatina/metabolismo , Vibrisas/inervación , Vibrisas/fisiologíaAsunto(s)
Aminofenoles/efectos adversos , Benzodioxoles/efectos adversos , Indoles/efectos adversos , Moduladores del Transporte de Membrana/efectos adversos , Pirazoles/efectos adversos , Piridinas/efectos adversos , Quinolinas/efectos adversos , Síndrome de Stevens-Johnson/etiología , Adulto , Combinación de Medicamentos , Humanos , Masculino , Síndrome de Stevens-Johnson/diagnósticoRESUMEN
Microbial infections remain a public health problem due to the upsurge of bacterial resistance. In this study, the antibacterial, antibiofilm, and efflux pump inhibitory activities of the stem bark of Acacia macrostachya, an indigenous African medicinal plant, were investigated. In traditional medicine, the plant is used in the treatment of microbial infections and inflammatory conditions. A crude methanol extract obtained by Soxhlet extraction was partitioned by column chromatography to obtain the petroleum ether, ethyl acetate, and methanol fractions. Antibacterial, efflux pump inhibition and antibiofilm formation activities were assessed by the high-throughput spot culture growth inhibition (HT-SPOTi), ethidium bromide accumulation, and the crystal violet retention assay, respectively. The minimum inhibitory concentrations (MICs) of the crude extract and major fractions ranged from 250 to ≥500 µg/mL. At a concentration of 3.9-250 µg/mL, all extracts demonstrated >80% inhibition of biofilm formation in S. aureus. In P. aeruginosa, the EtOAc fraction showed the highest antibiofilm activity (59-69%) while the pet-ether fraction was most active against E. coli biofilms (45-67%). Among the test samples, the crude extract, methanol, and ethyl acetate fractions showed remarkable efflux pump inhibition in S. aureus, E. coli, and P. aeruginosa. At ½ MIC, the methanol fraction demonstrated significant accumulation of EtBr in E. coli having superior efflux inhibition over the standard EPIs: chlorpromazine and verapamil. Tannins, flavonoids, triterpenoids, phytosterols, coumarins, and saponins were identified in preliminary phytochemical studies. Stigmasterol was identified in the EtOAc fraction. This study justifies the use of A. macrostachya in the treatment of infections in traditional medicine and highlights its potential as a source of bioactive compounds that could possibly interact with some resistance mechanisms in bacteria to combat antimicrobial resistance.
Asunto(s)
Acacia , Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Corteza de la Planta , Extractos Vegetales/farmacología , Tallos de la Planta , Antibacterianos/aislamiento & purificación , Biopelículas/crecimiento & desarrollo , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Humanos , Moduladores del Transporte de Membrana/aislamiento & purificación , Moduladores del Transporte de Membrana/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Extractos Vegetales/aislamiento & purificación , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiologíaRESUMEN
Glut1 deficiency syndrome is caused by SLC2A1 mutations on chromosome 1p34.2 that impairs glucose transport across the blood-brain barrier resulting in hypoglycorrhachia and decreased fuel for brain metabolism. Neuroglycopenia causes a drug-resistant metabolic epilepsy due to energy deficiency. Standard treatment for Glut1 deficiency syndrome is the ketogenic diet that decreases the demand for brain glucose by supplying ketones as alternative fuel. Treatment options are limited if patients fail the ketogenic diet. We present a case of successful diazoxide use with continuous glucose monitoring in a patient with Glut1 deficiency syndrome who did not respond to the ketogenic diet.
Asunto(s)
Automonitorización de la Glucosa Sanguínea , Errores Innatos del Metabolismo de los Carbohidratos/diagnóstico , Errores Innatos del Metabolismo de los Carbohidratos/tratamiento farmacológico , Diazóxido/farmacología , Moduladores del Transporte de Membrana/farmacología , Proteínas de Transporte de Monosacáridos/deficiencia , Convulsiones/tratamiento farmacológico , Adolescente , Errores Innatos del Metabolismo de los Carbohidratos/sangre , Diazóxido/administración & dosificación , Femenino , Humanos , Proteínas de Transporte de Monosacáridos/sangre , Convulsiones/etiologíaRESUMEN
Chloride transport across cell membranes is broadly involved in epithelial fluid transport, cell volume and pH regulation, muscle contraction, membrane excitability, and organellar acidification. The human genome encodes at least 53 chloride-transporting proteins with expression in cell plasma or intracellular membranes, which include chloride channels, exchangers, and cotransporters, some having broad anion specificity. Loss-of-function mutations in chloride transporters cause a wide variety of human diseases, including cystic fibrosis, secretory diarrhea, kidney stones, salt-wasting nephropathy, myotonia, osteopetrosis, hearing loss, and goiter. Although impactful advances have been made in the past decade in drug treatment of cystic fibrosis using small molecule modulators of the defective cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, other chloride channels and solute carrier proteins (SLCs) represent relatively underexplored target classes for drug discovery. New opportunities have emerged for the development of chloride transport modulators as potential therapeutics for secretory diarrheas, constipation, dry eye disorders, kidney stones, polycystic kidney disease, hypertension, and osteoporosis. Approaches to chloride transport-targeted drug discovery are reviewed herein, with focus on chloride channel and exchanger classes in which recent preclinical advances have been made in the identification of small molecule modulators and in proof of concept testing in experimental animal models.
Asunto(s)
Antiportadores/efectos de los fármacos , Canales de Cloruro/efectos de los fármacos , Cloruros/metabolismo , Diseño de Fármacos , Descubrimiento de Drogas , Moduladores del Transporte de Membrana/farmacología , Animales , Antiportadores/genética , Antiportadores/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Transporte Iónico , Cinética , Moduladores del Transporte de Membrana/química , Mutación , Transportadores de Sulfato/efectos de los fármacos , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismoRESUMEN
Transient receptor potential ankyrin 1 (TRPA1) channel is a calcium permeable, non-selective cation channel, expressed in the sensory neurons and non-neuronal cells of different tissues. Initially studied for its role in pain and inflammation, TRPA1 has now functionally involved in multiple other physiological functions. TRPA1 channel has been extensively studied for modulation by pungent compounds present in the spices and herbs. In the last decade, the role of TRPA1 agonism in body weight reduction, secretion of hunger and satiety hormones, insulin secretion and thermogenesis, has unveiled the potential of the TRPA1 channel to be used as a preventive target to tackle obesity and associated comorbidities including insulin resistance in type 2 diabetes. In this review, we summarized the recent findings of TRPA1 based dietary/non-dietary modulation for its role in obesity prevention and therapeutics.
Asunto(s)
Productos Biológicos/farmacología , Moduladores del Transporte de Membrana/farmacología , Obesidad/prevención & control , Canal Catiónico TRPA1/agonistas , Canal Catiónico TRPA1/fisiología , Animales , Humanos , Canal Catiónico TRPA1/químicaRESUMEN
Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific transporter mediating the cellular uptake of steroids and numerous drugs. OATP2B1 is abundantly expressed in the intestine and is also present in various tumors. Increased steroid hormone uptake by OATP2B1 has been suggested to promote progression of hormone dependent tumors. 13α-estrones are effective inhibitors of endogenous estrogen formation and are potential candidates to inhibit proliferation of hormone dependent cancers. Recently, we have identified a variety of 13α/ß-estrone-based inhibitors of OATP2B1. However, the nature of this interaction, whether these inhibitors are potential transported substrates of OATP2B1 and hence may be enriched in OATP2B1-overexpressing cells, has not yet been investigated. In the current study we explored the antiproliferative effect of the most effective OATP2B1 inhibitor 13α/ß-estrones in control and OATP2B1-overexpressing A431 carcinoma cells. We found an increased antiproliferative effect of 3-O-benzyl 13α/ß-estrones in both mock transfected and OATP2B1-overexpressing cells. However, C-2 halogenated 13α-estrones had a selective OATP2B1-mediated cell growth inhibitory effect. In order to demonstrate that increased sensitization can be attributed to OATP2B1-mediated cellular uptake, tritium labeled 2-bromo-13α-estrone was synthesized for direct transport measurements. These experiments revealed increased accumulation of [3H]2-bromo-13α-estrone due to OATP2B1 function. Our results indicate that C-2 halogenated 13α-estrones are good candidates in the design of anti-cancer drugs targeting OATP2B1.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Estrona/farmacología , Moduladores del Transporte de Membrana/farmacología , Transportadores de Anión Orgánico/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Antineoplásicos/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Estrona/análogos & derivados , Estrona/metabolismo , Humanos , Moduladores del Transporte de Membrana/metabolismo , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
BACKGROUND: Oliceridine is a biased ligand at the µ-opioid receptor recently approved for the treatment of acute pain. In a thorough QT study, corrected QT (QTc) prolongation displayed peaks at 2.5 and 60 minutes after a supratherapeutic dose. The mean plasma concentration peaked at 5 minutes, declining rapidly thereafter. OBJECTIVE: The purpose of this study was to examine the basis for the delayed effect of oliceridine to prolong the QTc interval. METHODS: Repolarization parameters and tissue accumulation of oliceridine were evaluated in rabbit left ventricular wedge preparations over a period of 5 hours. The effects of oliceridine on ion channel currents were evaluated in human embryonic kidney and Chinese hamster ovary cells. Quinidine was used as a control. RESULTS: Oliceridine and quinidine produced a progressive prolongation of the QTc interval and action potential duration over a period of 5 hours, paralleling slow progressive tissue uptake of the drugs. Oliceridine caused modest prolongation of these parameters, whereas quinidine produced a prominent prolongation of action potential duration and QTc interval as well as development of early afterdepolarization (after 2 hours), resulting in a high torsades de pointes score. The 50% inhibitory concentration values for the oliceridine inhibition of the rapidly activating delayed rectifier current (human ether a-go-go current) and late sodium channel current were 2.2 and 3.45 µM when assessed after traditional acute exposure but much lower after 3 hours of drug exposure. CONCLUSION: Our findings suggest that a gradual increase of intracellular access of drugs to the hERG channels as a result of their intracellular uptake and accumulation can significantly delay effects on repolarization, thus confounding the assessment of QT interval prolongation and arrhythmic risk when studied acutely. The multi-ion channel effects of oliceridine, late sodium channel current inhibition in particular, point to a low risk of devloping torsades de pointes.
Asunto(s)
Arritmias Cardíacas , Canal de Potasio ERG1/antagonistas & inhibidores , Compuestos de Espiro/farmacocinética , Tiofenos/farmacocinética , Analgésicos Opioides/farmacocinética , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Arritmias Cardíacas/prevención & control , Línea Celular , Cricetulus , Humanos , Concentración 50 Inhibidora , Síndrome de QT Prolongado/inducido químicamente , Síndrome de QT Prolongado/metabolismo , Síndrome de QT Prolongado/fisiopatología , Moduladores del Transporte de Membrana/farmacología , Quinidina/farmacocinética , Distribución Tisular , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacocinéticaRESUMEN
Atopic dermatitis (AD) is a multifaceted, chronic relapsing inflammatory skin disease that affects people of all ages. It is characterized by chronic eczema, constant pruritus, and severe discomfort. AD often progresses from mild annoyance to intractable pruritic inflammatory lesions associated with exacerbated skin sensitivity. The T helper-2 (Th2) response is mainly linked to the acute and subacute phase, whereas Th1 response has been associated in addition with the chronic phase. IL-17, IL-22, TSLP, and IL-31 also play a role in AD. Transient receptor potential (TRP) cation channels play a significant role in neuroinflammation, itch and pain, indicating neuroimmune circuits in AD. However, the Th2-driven cutaneous sensitization of TRP channels is underappreciated. Emerging findings suggest that critical Th2-related cytokines cause potentiation of TRP channels, thereby exaggerating inflammation and itch sensation. Evidence involves the following: (i) IL-13 enhances TRPV1 and TRPA1 transcription levels; (ii) IL-31 sensitizes TRPV1 via transcriptional and channel modulation, and indirectly modulates TRPV3 in keratinocytes; (iii) The Th2-cytokine TSLP increases TRPA1 synthesis in sensory neurons. These changes could be further enhanced by other Th2 cytokines, including IL-4, IL-25, and IL-33, which are inducers for IL-13, IL-31, or TSLP in skin. Taken together, this review highlights that Th2 cytokines potentiate TRP channels through diverse mechanisms under different inflammatory and pruritic conditions, and link this effect to distinct signaling cascades in AD. This review strengthens the notion that interrupting Th2-driven modulation of TRP channels will inhibit transition from acute to chronic AD, thereby aiding the development of effective therapeutics and treatment optimization.
Asunto(s)
Citocinas/metabolismo , Dermatitis Atópica/metabolismo , Mediadores de Inflamación/metabolismo , Prurito/metabolismo , Piel/metabolismo , Células Th2/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/genética , Dermatitis Atópica/inmunología , Progresión de la Enfermedad , Humanos , Moduladores del Transporte de Membrana/uso terapéutico , Terapia Molecular Dirigida , Prurito/tratamiento farmacológico , Prurito/genética , Prurito/inmunología , Transducción de Señal , Piel/efectos de los fármacos , Piel/inmunología , Células Th2/inmunología , Activación Transcripcional , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (If) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2 to 5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that ß-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of If Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.
Asunto(s)
Relojes Biológicos/fisiología , Fenómenos Electrofisiológicos , Miocitos Cardíacos/fisiología , Nodo Sinoatrial/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Relojes Biológicos/efectos de los fármacos , Simulación por Computador , Diástole/efectos de los fármacos , Diástole/fisiología , Fenómenos Electrofisiológicos/efectos de los fármacos , Células HEK293 , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Ivabradina/farmacología , Moduladores del Transporte de Membrana/farmacología , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Nodo Sinoatrial/efectos de los fármacosRESUMEN
Recent studies in our lab have shown that the KV7 channel activator, flupirtine, inhibits colonic epithelial Cl- secretion through effects on submucosal neurons of the enteric nervous system (ENS). We hypothesized that flupirtine would also stimulate Na+ absorption as a result of reduced secretory ENS input to the epithelium. To test this hypothesis, unidirectional 22Na+ fluxes were measured under voltage-clamped conditions. Pharmacological approaches using an Ussing-style recording chamber combined with immunofluorescence microscopy techniques were used to determine the effect of flupirtine on active Na+ transport in the rat colon. Flupirtine stimulated electroneutral Na+ absorption in partially seromuscular-stripped colonic tissues, while simultaneously inhibiting short-circuit current (ISC; i.e., Cl- secretion). Both of these effects were attenuated by pretreatment with the ENS inhibitor, tetrodotoxin. The Na+/H+ exchanger isoform 3 (NHE-3)-selective inhibitor, S3226, significantly inhibited flupirtine-stimulated Na+ absorption, whereas the NHE-2-selective inhibitor HOE-694 did not. NHE-3 localization near the apical membranes of surface epithelial cells was also more apparent in flupirtine-treated colon versus control. Flupirtine did not alter epithelial Na+ channel (ENaC)-mediated Na+ absorption in distal colonic tissues obtained from hyperaldosteronaemic rats and had no effect in the normal ileum but did stimulate Na+ absorption in the proximal colon. Finally, the parallel effects of flupirtine on ISC (Cl- secretion) and Na+ absorption were significantly correlated with each other. Together, these data indicate that flupirtine stimulates NHE-3-dependent Na+ absorption, likely as a result of reduced stimulatory input to the colonic epithelium by submucosal ENS neurons.NEW & NOTEWORTHY We present a novel mechanism regarding regulation of epithelial ion transport by enteric neurons. Activation of neuronal KV7 K+ channels markedly stimulates Na+ absorption and inhibits Cl- secretion across the colonic epithelium. This may be useful in developing new treatments for diarrheal disorders, such as irritable bowel syndrome with diarrhea (IBS-D).
Asunto(s)
Aminopiridinas/farmacología , Colon/metabolismo , Sistema Nervioso Entérico/metabolismo , Absorción Intestinal , Sodio/metabolismo , Animales , Colon/efectos de los fármacos , Canales Epiteliales de Sodio/metabolismo , Guanidinas/farmacología , Masculino , Moduladores del Transporte de Membrana/farmacología , Metacrilatos/farmacología , Ratas , Ratas Sprague-Dawley , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/metabolismo , Sulfonas/farmacologíaRESUMEN
Transient receptor potential vanilloid (TRPV) channels are part of the TRP channel superfamily and named after the first identified member TRPV1, that is sensitive to the vanillylamide capsaicin. Their overall structure is similar to the structure of voltage gated potassium channels (Kv) built up as homotetramers from subunits with six transmembrane helices (S1-S6). Six TRPV channel subtypes (TRPV1-6) are known, that can be subdivided into the thermoTRPV (TRPV1-4) and the Ca2+-selective TRPV channels (TRPV5, TRPV6). Contrary to Kv channels, TRPV channels are not primary voltage gated. All six channels have distinct properties and react to several endogenous ligands as well as different gating stimuli such as heat, pH, mechanical stress, or osmotic changes. Their physiological functions are highly diverse and subtype as well as tissue specific. In many tissues they serve as sensors for different pain stimuli (heat, pressure, pH) and contribute to the homeostasis of electrolytes, the maintenance of barrier functions and the development of macrophages. Due to their fundamental role in manifold physiological and pathophysiological processes, TRPV channels are promising targets for drug development. However, drugs targeting specific TRPV channels, that are suitable for drug therapy, are rare. Moreover, selective and potent compounds for further research at TRPV channels are often lacking. In this review different aspects of the structure, the different gating stimuli, the expression pattern, the physiological and pathophysiological roles as well as the modulating mechanisms of synthetic, natural and endogenous ligands are summarized.
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Analgésicos/farmacología , Antineoplásicos/farmacología , Factores Inmunológicos/farmacología , Moduladores del Transporte de Membrana/farmacología , Canales Catiónicos TRPV/metabolismo , Analgésicos/química , Analgésicos/clasificación , Antineoplásicos/química , Antineoplásicos/clasificación , Sitios de Unión , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Humanos , Factores Inmunológicos/química , Factores Inmunológicos/clasificación , Activación del Canal Iónico/efectos de los fármacos , Ligandos , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Moduladores del Transporte de Membrana/química , Moduladores del Transporte de Membrana/clasificación , Modelos Moleculares , Especificidad de Órganos , Unión Proteica , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/clasificaciónRESUMEN
The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca2+ release-activated Ca2+, transient receptor potential, P2X, volume-regulated anion and K+ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.
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Artritis Reumatoide/tratamiento farmacológico , Calcio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Linfocitos T Reguladores/efectos de los fármacos , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Calcio/inmunología , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/inmunología , Señalización del Calcio , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/inmunología , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/inmunología , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Moduladores del Transporte de Membrana/química , Ratones , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X/inmunología , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/inmunologíaRESUMEN
Ion transport modulators are most commonly used to treat various noncommunicable diseases including diabetes and hypertension. They are also known to bind to receptors on various immune cells, but the immunomodulatory properties of most ion transport modulators have not been fully elucidated. We assessed the effects of thirteen FDA-approved ion transport modulators, namely, ambroxol HCl, amiloride HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide, metformin, omeprazole, pantoprazole, phenytoin, verapamil, drug X, and drug Y on superoxide production, nitric oxide production, and cytokine expression by THP-1-derived macrophages that had been stimulated with ethanol-inactivated Mycobacterium bovis BCG. Ambroxol HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide, metformin, pantoprazole, phenytoin, verapamil, and drug Y had an inhibitory effect on nitric oxide production, while all the test drugs had an inhibitory effect on superoxide production. Amiloride HCl, diazoxide, digoxin, furosemide, phenytoin, verapamil, drug X, and drug Y enhanced the expression of IL-1ß and TNF-α. Unlike most immunomodulatory compounds currently in clinical use, most of the test drugs inhibited some inflammatory processes while promoting others. Ion pumps and ion channels could therefore serve as targets for more selective immunomodulatory agents which do not cause overt immunosuppression.
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Inflamación/tratamiento farmacológico , Macrófagos/inmunología , Moduladores del Transporte de Membrana/uso terapéutico , Mycobacterium bovis/inmunología , Ambroxol/uso terapéutico , Células Cultivadas , Humanos , Inmunomodulación , Interleucina-1beta/metabolismo , Transporte Iónico , Macrófagos/efectos de los fármacos , Células THP-1 , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Several years ago, the crystallographic structures of the transient receptor potential vanilloid 1 (TRPV1) in the presence of agonists and antagonists were reported, providing structural information about its chemical activation and inactivation. TRPV1's activation increases the transport of calcium and sodium ions, leading to the excitation of sensory neurons and the perception of pain. On the other hand, its antagonistic inactivation has been explored to design analgesic drugs. The interactions between the antagonists 5,5-diarylpentadienamides (DPDAs) and TRPV1 were studied here to explain why they inactivate TRPV1. The present work identified the structural features of TRPV1-DPDA complexes, starting with a consideration of the orientations of the ligands inside the TRPV1 binding site by using molecular docking. After this, a chemometrics analysis was performed (i) to compare the orientations of the antagonists (by using LigRMSD), (ii) to describe the recurrent interactions between the protein residues and ligand groups in the complexes (by using interaction fingerprints), and (iii) to describe the relationship between topological features of the ligands and their differential antagonistic activities (by using a quantitative structure-activity relationship (QSAR) with 2D autocorrelation descriptors). The interactions between the DPDA groups and the residues Y511, S512, T550, R557, and E570 (with a recognized role in the binding of classic ligands), and the occupancy of isoquinoline or 3-hydroxy-3,4-dihydroquinolin-2(1H)-one groups of the DPDAs in the vanilloid pocket of TRPV1 were clearly described. Based on the results, the structural features that explain why DPDAs inactivate TRPV1 were clearly exposed. These features can be considered for the design of novel TRPV1 antagonists.
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Moduladores del Transporte de Membrana/química , Simulación del Acoplamiento Molecular , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/química , HumanosRESUMEN
Hypertrophic scar (HS) formation is a skin fibroproliferative disease that occurs following a cutaneous injury, leading to functional and cosmetic impairment. To date, few therapeutic treatments exhibit satisfactory outcomes. The mechanical force has been shown to be a key regulator of HS formation, but the underlying mechanism is not completely understood. The Piezo1 channel has been identified as a novel mechanically activated cation channel (MAC) and is reportedly capable of regulating force-mediated cellular biological behaviors. However, the mechanotransduction role of Piezo1 in HS formation has not been investigated. In this work, we found that Piezo1 was overexpressed in myofibroblasts of human and rat HS tissues. In vitro, cyclic mechanical stretch (CMS) increased Piezo1 expression and Piezo1-mediated calcium influx in human dermal fibroblasts (HDFs). In addition, Piezo1 activity promoted HDFs proliferation, motility, and differentiation in response to CMS. More importantly, intradermal injection of GsMTx4, a Piezo1-blocking peptide, protected rats from stretch-induced HS formation. Together, Piezo1 was shown to participate in HS formation and could be a novel target for the development of promising therapies for HS formation.