RESUMEN

Introduction: Degenerin proteins, such as ßENaC and ASIC2, have been implicated in cardiovascular function. However, their role in metabolic syndrome have not been studied. To begin to assess this interaction, we evaluated the impact of a high fat diet (HFD) on mice lacking normal levels of ASIC2 (ASIC2-/-) and ßENaC (ßENaCm/m). Methods: Twenty-week-old male and female mice were placed on a 60% HFD for 12 weeks. Body weight was measured weekly, and body composition by non-invasive ECHO MRI and fasting blood glucose were measured at 0, 4, 8 and 12 weeks. A glucose tolerance test was administered after 12 weeks. Differences between ASIC2-/-/ßENaCm/m and WT groups were compared using independent t-tests or ANOVA where appropriate within each sex. Data are presented as mean ± SEM and ASIC2-/-/ßENaCm/m vs. WT. Results: At 20 weeks of age, ASIC2-/-/ßENaCm/m mice (n=9F/10M) weighed less and gained less weight than WT (n=12F/16M). Total body fat and lean body masses were reduced in female and male ASIC2-/-/ßENaCm/m mice. Total body fat and lean body masses as % control were identical at the end of 12 weeks. Fasting blood glucoses were lower in female and male ASIC2-/-/ßENaCm/m vs. WT mice after 12 weeks HFD. The area under the curve for the glucose tolerance test was reduced in female and tended (p=.079) to decrease in male ASIC2-/-/ßENaCm/m. Plasma leptin and insulin were reduced in female and male ASIC2-/-/ßENaCm/m vs. WT mice. Plasma insulin in female ASIC2-/-/ßENaCm/m mice remained unchanged throughout the HFD period. Liver and liver fat masses, as well as percent liver fat, were reduced in both female and male ASIC2-/-/ßENaCm/m mice after HFD. Plasma triglycerides, cholesterol, LDL- and HDL-cholesterols were markedly improved in male and/or female ASIC2-/-/ßENaCm/m following the HFD. Discussion: These novel findings suggest that loss of ASIC2 and ßENaC offer a significant protection against HFD-induced metabolic syndrome.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Dieta Alta en Grasa , Síndrome Metabólico , Ratones Noqueados , Animales , Dieta Alta en Grasa/efectos adversos , Síndrome Metabólico/metabolismo , Síndrome Metabólico/etiología , Masculino , Ratones , Femenino , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Composición Corporal , Ratones Endogámicos C57BL , Canales Epiteliales de Sodio/metabolismo , Canales Epiteliales de Sodio/genética , Glucemia/metabolismo , Peso Corporal , Prueba de Tolerancia a la Glucosa

RESUMEN

Glioblastoma (GBM) is the most common malignant primary brain cancer that, despite recent advances in the understanding of its pathogenesis, remains incurable. GBM contains a subpopulation of cells with stem cell-like properties called cancer stem cells (CSCs). Several studies have demonstrated that CSCs are resistant to conventional chemotherapy and radiation thus representing important targets for novel anti-cancer therapies. Proton sensing receptors expressed by CSCs could represent important factors involved in the adaptation of tumours to the extracellular environment. Accordingly, the expression of acid-sensing ion channels (ASICs), proton-gated sodium channels mainly expressed in the neurons of peripheral (PNS) and central nervous system (CNS), has been demonstrated in several tumours and linked to an increase in cell migration and proliferation. In this paper we report that the ASIC3 isoform, usually absent in the CNS and present in the PNS, is enriched in human GBM CSCs while poorly expressed in the healthy human brain. We propose here a novel therapeutic strategy based on the pharmacological activation of ASIC3, which induces a significant GBM CSCs damage while being non-toxic for neurons. This approach might offer a promising and appealing new translational pathway for the treatment of glioblastoma.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Neoplasias Encefálicas , Proliferación Celular , Glioblastoma , Células Madre Neoplásicas , Humanos , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/tratamiento farmacológico , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos

RESUMEN

Acidic microenvironments is a cancer progression driver, unclear core mechanism hinders the discovery of new diagnostic or therapeutic targets. ASIC3 is an extracellular proton sensor and acid-sensitive, but its role in acidic tumor microenvironment of colorectal cancer is not reported. Functional analysis data show that colorectal cancer cells respond to specific concentration of lactate to accelerate invasion and metastasis, and ASIC3 is the main actor in this process. Mechanism reveal de novo lipid synthesis is a regulatory process of ASIC3, down-regulated ASIC3 increases and interacts with ACC1 and SCD1, which are key enzymes in de novo lipid synthesis pathway, this interaction results in increased unsaturated fatty acids, which in turn induce EMT to promote metastasis, and overexpression of ASIC3 reduces acidic TME-enhanced colorectal cancer metastasis. Clinical samples of colorectal cancer also exhibit decreased ASIC3 expression, and low ASIC3 expression is associated with metastasis and stage of colorectal cancer. This study is the first to identify the role of the ASIC3-ACC1/SCD1 axis in acid-enhanced colorectal cancer metastasis. The expression pattern of ASIC3 in colorectal cancer differs significantly from that in other types of cancers, ASIC3 may serve as a novel and reliable marker for acidic microenvironmental in colorectal cancer, and potentially a therapeutic target.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Neoplasias Colorrectales , Transición Epitelial-Mesenquimal , Ácido Láctico , Metástasis de la Neoplasia , Humanos , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Ácido Láctico/metabolismo , Línea Celular Tumoral , Estearoil-CoA Desaturasa/metabolismo , Estearoil-CoA Desaturasa/genética , Microambiente Tumoral , Animales , Lípidos , Regulación Neoplásica de la Expresión Génica

RESUMEN

Insulin has been shown to modulate neuronal processes through insulin receptors. The ion channels located on neurons may be important targets for insulin/insulin receptor signaling. Both insulin receptors and acid-sensing ion channels (ASICs) are expressed in dorsal root ganglia (DRG) neurons. However, it is still unclear whether there is an interaction between them. Therefore, the purpose of this investigation was to determine the effects of insulin on the functional activity of ASICs. A 5 min application of insulin rapidly enhanced acid-evoked ASIC currents in rat DRG neurons in a concentration-dependent manner. Insulin shifted the concentration-response plot for ASIC currents upward, with an increase of 46.2 ± 7.6% in the maximal current response. The insulin-induced increase in ASIC currents was eliminated by the insulin receptor antagonist GSK1838705, the tyrosine kinase inhibitor lavendustin A, and the phosphatidylinositol-3 kinase antagonist wortmannin. Moreover, insulin increased the number of acid-triggered action potentials by activating insulin receptors. Finally, local administration of insulin exacerbated the spontaneous nociceptive behaviors induced by intraplantar acid injection and the mechanical hyperalgesia induced by intramuscular acid injections through peripheral insulin receptors. These results suggested that insulin/insulin receptor signaling enhanced the functional activity of ASICs via tyrosine kinase and phosphatidylinositol-3 kinase pathways. Our findings revealed that ASICs were targets in primary sensory neurons for insulin receptor signaling, which may underlie insulin modulation of pain.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Ganglios Espinales , Insulina , Receptor de Insulina , Células Receptoras Sensoriales , Animales , Canales Iónicos Sensibles al Ácido/metabolismo , Insulina/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/citología , Ratas , Receptor de Insulina/metabolismo , Masculino , Transducción de Señal/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Ratas Sprague-Dawley , Hiperalgesia/metabolismo , Células Cultivadas

RESUMEN

BACKGROUND: A two-way relationship exists between type 2 diabetes (T2DM) and human nonalcoholic steatohepatitis (NASH). Several diabetic NASH models have the disadvantages of long cycles or inconsistent with the actual incidence of human disease, which would be costly and time-consuming to investigate disease pathogenesis and develop drugs. Therefore, there is an urgent need to establish a diabetic NASH mouse model. METHODS: The combination between Fructose-palmitate-cholesterol diet (FPC) and Streptozotocin (STZ) (FPC+STZ) was used to construct diabetic NASH mouse model. The in vivo effects of silencing acid-sensitive Ion Channel 1a (ASIC1a) were examined with an adeno-associated virus 9 (AAV9) carrying ASIC1a short hairpin RNA (shRNA) in FPC+STZ model. RESULTS: The mice fed with FPC for 12 weeks had insulin resistance, hyperinsulinemia, lipid accumulation, and increased hepatic levels of inflammatory factors. However, it still did not develop remarkable liver fibrosis. Most interestingly, noticeable fibrotic scars were observed in the liver of mice from FPC+STZ group. Furthermore, insulin therapy significantly ameliorated FPC+STZ-induced NASH-related liver fibrosis, indicating that hyperglycemia is of great significance in NASH development and progression. Importantly, ASIC1a was found to be involved in the pathogenesis of diabetic NASH as demonstrated that silencing ASIC1a in HSCs significantly ameliorated FPC+STZ-induced NASH fibrosis. Mechanistically, ASIC1a interacted with Poly Adp-adenosine ribose polymerase (PARP1) to promote HSC activation by inducing autophagy. CONCLUSION: A FPC diet combined with an injection of STZ induces a diabetic NASH mouse model in a shorter period. Targeting ASIC1a may provide a novel therapeutic target for the treatment of diabetic NASH.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Diabetes Mellitus Experimental , Enfermedad del Hígado Graso no Alcohólico , Animales , Masculino , Ratones , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Fructosa , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/patología , Insulina/metabolismo , Resistencia a la Insulina , Hígado/patología , Hígado/metabolismo , Hígado/efectos de los fármacos , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Estreptozocina

RESUMEN

Acid-sensing ion channel 1 (ASIC1) is critical in acidotoxicity and significantly contributes to neuronal death in cerebral stroke. Pharmacological inhibition of ASIC1 has been shown to reduce neuronal death. However, the potential of utilizing exosomes derived from pluripotent stem cells to achieve inhibition of Asic1 remains to be explored. Developing qualified exosome products with precise and potent active ingredients suitable for clinical application is also ongoing. Here, we adopt small RNA-seq to interrogate the miRNA contents in exosomes of pluripotent stem cell induced mesenchymal stem cell (iMSC). RNA-seq was used to compare the oxygen-glucose deprivation-damaged neurons before and after the delivery of exosomes. We used Western blot to quantify the Asic1 protein abundance in neurons before and after exosome treatment. An in vivo test on rats validated the neuroprotective effect of iMSC-derived exosome and its active potent miRNA hsa-mir-125b-5p. We demonstrate that pluripotent stem cell-derived iMSCs produce exosomes with consistent miRNA contents and sustained expression. These exosomes efficiently rescue injured neurons, alleviate the pathological burden, and restore neuron function in rats under oxygen-glucose deprivation stress. Furthermore, we identify hsa-mir-125b-5p as the active component responsible for inhibiting the Asic1a protein and protecting neurons. We validated a novel therapeutic strategy to enhance acidosis resilience in cerebral stroke by utilizing exosomes derived from pluripotent stem cells with specific miRNA content. This holds promise for cerebral stroke treatment with the potential to reduce neuronal damage and improve clinical patient outcomes.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Acidosis , Exosomas , MicroARNs , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Animales , MicroARNs/genética , MicroARNs/metabolismo , Exosomas/metabolismo , Ratas , Acidosis/metabolismo , Neuronas/metabolismo , Neuronas/patología , Masculino , Humanos , Células Madre Mesenquimatosas/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/genética , Ratas Sprague-Dawley , Isquemia Encefálica/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo

RESUMEN

The monocyte-macrophage system plays an important role in phagocytosis of pathogens and cellular debris following infection or tissue injury in several pathophysiological conditions. We examined ENaC/ASIC subunit transcript expression and the importance of select subunits in migration of bone marrow derived monocytes (freshly isolated) and macrophages (monocytes differentiated in culture). We also examined the effect of select subunit deletion on macrophage phenotype. BM monocytes were harvested from the femurs of male and female WT and KO mice (6-12 weeks of age). Our results show that α, ß, γENaC, and ASIC1-5 transcripts are expressed in BM macrophages and monocytes to varying degrees. At least αENaC, ßENaC, and ASIC2 subunits contribute to chemotactic migration responses in BM monocyte-macrophages. Polarization markers (CD86, soluble TNFα) in BM macrophages from mice lacking ASIC2a plus ßENaC were shifted towards the M1 phenotype. Furthermore, select M1 phenotypic markers were recovered with rescue of ßENaC or ASIC2. Taken together, these data suggest that ßENaC and ASIC2 play an important role in BM macrophage migration and loss of ßENaC and/or ASIC2 partially polarizes macrophages to the M1 phenotype. Thus, targeting ENaC/ASIC expression in BM macrophages may regulate their ability to migrate to sites of injury.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Quimiotaxis , Canales Epiteliales de Sodio , Macrófagos , Monocitos , Animales , Canales Epiteliales de Sodio/metabolismo , Canales Epiteliales de Sodio/genética , Macrófagos/metabolismo , Masculino , Ratones , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Femenino , Monocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Células de la Médula Ósea/metabolismo , Células Cultivadas

RESUMEN

Glutamate is traditionally viewed as the first messenger to activate NMDAR (N-methyl-D-aspartate receptor)-dependent cell death pathways in stroke1,2, but unsuccessful clinical trials with NMDAR antagonists implicate the engagement of other mechanisms3-7. Here we show that glutamate and its structural analogues, including NMDAR antagonist L-AP5 (also known as APV), robustly potentiate currents mediated by acid-sensing ion channels (ASICs) associated with acidosis-induced neurotoxicity in stroke4. Glutamate increases the affinity of ASICs for protons and their open probability, aggravating ischaemic neurotoxicity in both in vitro and in vivo models. Site-directed mutagenesis, structure-based modelling and functional assays reveal a bona fide glutamate-binding cavity in the extracellular domain of ASIC1a. Computational drug screening identified a small molecule, LK-2, that binds to this cavity and abolishes glutamate-dependent potentiation of ASIC currents but spares NMDARs. LK-2 reduces the infarct volume and improves sensorimotor recovery in a mouse model of ischaemic stroke, reminiscent of that seen in mice with Asic1a knockout or knockout of other cation channels4-7. We conclude that glutamate functions as a positive allosteric modulator for ASICs to exacerbate neurotoxicity, and preferential targeting of the glutamate-binding site on ASICs over that on NMDARs may be strategized for developing stroke therapeutics lacking the psychotic side effects of NMDAR antagonists.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Isquemia Encefálica , Ácido Glutámico , Animales , Femenino , Humanos , Masculino , Ratones , 2-Amino-5-fosfonovalerato/efectos adversos , 2-Amino-5-fosfonovalerato/metabolismo , 2-Amino-5-fosfonovalerato/farmacología , Canales Iónicos Sensibles al Ácido/química , Canales Iónicos Sensibles al Ácido/deficiencia , Canales Iónicos Sensibles al Ácido/efectos de los fármacos , Canales Iónicos Sensibles al Ácido/genética , Canales Iónicos Sensibles al Ácido/metabolismo , Regulación Alostérica/efectos de los fármacos , Sitios de Unión/genética , Isquemia Encefálica/inducido químicamente , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Ácido Glutámico/análogos & derivados , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Ácido Glutámico/toxicidad , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Protones , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo

RESUMEN

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a. Currently, the specific channel residues responsible for this pharmacological profile, and the mambalgin pharmacophore at ASIC1b remain unknown. Here we identify non-conserved residues at the ASIC1 subunit interface that drive differences in the mambalgin pharmacology from rat ASIC1a to ASIC1b, some of which likely do not make peptide binding interactions. Additionally, an amino acid variation below the core binding site explains potency differences between rat and human ASIC1. Two regions within the palm domain, which contribute to subtype-dependent effects for mambalgins, play key roles in ASIC gating, consistent with subtype-specific differences in the peptides mechanism. Lastly, there is a shared primary mambalgin pharmacophore for ASIC1a and ASIC1b activity, with certain peripheral peptide residues showing variant-specific significance for potency. Through our broad mutagenesis studies across various species and subtype variants, we gain a more comprehensive understanding of the pharmacophore and the intricate molecular interactions that underlie ligand specificity. These insights pave the way for the development of more potent and targeted peptide analogues required to advance our understating of human ASIC1 function and its role in disease.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Venenos Elapídicos , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Canales Iónicos Sensibles al Ácido/química , Animales , Humanos , Ratas , Venenos Elapídicos/química , Venenos Elapídicos/metabolismo , Venenos Elapídicos/farmacología , Venenos Elapídicos/genética , Secuencia de Aminoácidos , Sitios de Unión , Modelos Moleculares , Xenopus laevis , Péptidos

RESUMEN

Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels activated by extracellular acidification. ASICs are involved in learning, fear sensing, pain sensation and neurodegeneration. Increasing the extracellular Ca2+ concentration decreases the H+ sensitivity of ASIC1a, suggesting a competition for binding sites between H+ and Ca2+ ions. Here, we predicted candidate residues for Ca2+ binding on ASIC1a, based on available structural information and our molecular dynamics simulations. With functional measurements, we identified several residues in cavities previously associated with pH-dependent gating, whose mutation reduced the modulation by extracellular Ca2+ of the ASIC1a pH dependence of activation and desensitization. This occurred likely owing to a disruption of Ca2+ binding. Our results link one of the two predicted Ca2+-binding sites in each ASIC1a acidic pocket to the modulation of channel activation. Mg2+ regulates ASICs in a similar way as does Ca2+. We show that Mg2+ shares some of the binding sites with Ca2+. Finally, we provide evidence that some of the ASIC1a Ca2+-binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation-binding sites in ASIC1a shows how Ca2+ affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Calcio , Simulación de Dinámica Molecular , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/química , Canales Iónicos Sensibles al Ácido/genética , Sitios de Unión , Calcio/metabolismo , Animales , Unión Proteica , Concentración de Iones de Hidrógeno , Magnesio/metabolismo , Humanos , Activación del Canal Iónico , Mutación , Conformación Proteica

RESUMEN

Psoriasis is an immune-mediated skin disease associated with neurogenic inflammation, but the underlying molecular mechanism remains unclear. We demonstrate here that acid-sensing ion channel 3 (ASIC3) exacerbates psoriatic inflammation through a sensory neurogenic pathway. Global or nociceptor-specific Asic3 knockout (KO) in female mice alleviates imiquimod-induced psoriatic acanthosis and type 17 inflammation to the same extent as nociceptor ablation. However, ASIC3 is dispensable for IL-23-induced psoriatic inflammation that bypasses the need for nociceptors. Mechanistically, ASIC3 activation induces the activity-dependent release of calcitonin gene-related peptide (CGRP) from sensory neurons to promote neurogenic inflammation. Botulinum neurotoxin A and CGRP antagonists prevent sensory neuron-mediated exacerbation of psoriatic inflammation to similar extents as Asic3 KO. In contrast, replenishing CGRP in the skin of Asic3 KO mice restores the inflammatory response. These findings establish sensory ASIC3 as a critical constituent in psoriatic inflammation, and a promising target for neurogenic inflammation management.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Péptido Relacionado con Gen de Calcitonina , Ratones Noqueados , Psoriasis , Células Receptoras Sensoriales , Animales , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Femenino , Psoriasis/metabolismo , Psoriasis/patología , Psoriasis/genética , Psoriasis/inducido químicamente , Ratones , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/genética , Células Receptoras Sensoriales/metabolismo , Piel/metabolismo , Piel/patología , Imiquimod , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Inflamación/metabolismo , Inflamación Neurogénica/metabolismo , Humanos , Nociceptores/metabolismo , Interleucina-23/metabolismo , Interleucina-23/genética

RESUMEN

AIMS: Activation of central respiratory chemoreceptors provides excitatory drive to both respiratory and sympathetic outputs. The enhanced respiratory-sympathetic coupling contributes to the onset and development of hypertension. However, the specific central targets and molecular mechanisms involved in this process remain elusive. This study aimed to investigate the role of acid-sensing ion channel 1 (ASIC1) in nucleus tractus solitarii (NTS) neurons in CO2-stimulated cardiorespiratory effects in spontaneously hypertensive rats (SHRs). MAIN METHODS: Respiration and blood pressure of conscious rats were recorded by whole-body plethysmography and telemetry, respectively. Western blot was used to detect the expression difference of ASIC1 protein in NTS region between Wistar-Kyoto (WKY) rats and SHRs. Excitability of NTS neurons were assessed by extracellular recordings. KEY FINDINGS: Compared to WKY rats, the enhanced CO2-stimulated cardiopulmonary effect and up-regulation of ASIC1 in the NTS were already observed in 4-week-old prehypertensive SHRs. Furthermore, specific blockade of ASIC1 effectively attenuated the CO2-stimulated increase in firing rate of NTS neurons in anesthetized adult SHRs. Intracerebroventricular injections of the ASIC1a blocker PcTx1 or knockdown Asic1 in NTS neurons significantly reduced the heightened CO2-stimulated ventilatory response, and diminished the CO2-stimulated increase in arterial pressure and heart rate in adult SHRs. SIGNIFICANCE: These findings showed that dysregulated ASIC1 signaling in the NTS contribute to the exaggerated CO2-stimulated cardiorespiratory effects observed in SHRs.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Presión Sanguínea , Dióxido de Carbono , Hipertensión , Neuronas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Núcleo Solitario , Animales , Canales Iónicos Sensibles al Ácido/metabolismo , Núcleo Solitario/metabolismo , Ratas , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Masculino , Dióxido de Carbono/metabolismo , Hipertensión/metabolismo , Hipertensión/fisiopatología , Presión Sanguínea/efectos de los fármacos , Respiración/efectos de los fármacos , Péptidos , Venenos de Araña

RESUMEN

This study carried out to investigate the anti-inflammatory and antinociceptive effect of tropane alkaloid (EB7) isolated from E. bezerrae. It evaluated the toxicity and possible involvement of ion channels in the antinociceptive effect of EB7, as well as its anti-inflammatory effect in adult zebrafish (Zfa). Docking studies with EB7 and COX-1 and 2 were also performed. The tested doses of EB7 (4, 20 and 40 mg/kg) did not show any toxic effect on Zfa during the 96h of analysis (LD50>40 mg/kg). They did not produce any alteration in the locomotor behavior of the animals. Furthermore, EB7 showed promising pharmacological effects as it prevented the nociceptive behavior induced by hypertonic saline, capsaicin, formalin and acid saline. EB7 had its analgesic effect blocked by amiloride involving the neuromodulation of ASICs in Zfa. In evaluating the anti-inflammatory activity, the edema induced by κ-carrageenan 3.5 % was reduced by the dose of 40 mg/kg of EB7 observed after the fourth hour of analysis, indicating an effect similar to that of ibuprofen. Molecular docking results indicated that EB7 exhibited better affinity energy when compared to ibuprofen control against the two evaluated targets binding at different sites in the cocrystallized COX-1 and 2 inhibitors.

Asunto(s)

Analgésicos , Simulación del Acoplamiento Molecular , Pez Cebra , Animales , Analgésicos/farmacología , Analgésicos/química , Analgésicos/aislamiento & purificación , Tropanos/farmacología , Tropanos/aislamiento & purificación , Tropanos/química , Edema/tratamiento farmacológico , Edema/inducido químicamente , Carragenina/farmacología , Ciclooxigenasa 2/metabolismo , Ciclooxigenasa 1/metabolismo , Bignoniaceae/química , Relación Dosis-Respuesta a Droga , Relación Estructura-Actividad , Alcaloides/farmacología , Alcaloides/aislamiento & purificación , Alcaloides/química , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/química , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/aislamiento & purificación , Antiinflamatorios/farmacología , Antiinflamatorios/química , Antiinflamatorios/aislamiento & purificación , Estructura Molecular

RESUMEN

BACKGROUND: Activation of the acid-sensing ion channels (ASICs) by tissue acidosis, a common feature of brain ischemia, contributes to ischemic brain injury, while blockade of ASICs results in protection. Cholestane-3ß,5α,6ß-triol (Triol), a major cholesterol metabolite, has been demonstrated as an endogenous neuroprotectant; however, the mechanism underlying its neuroprotective activity remains elusive. In this study, we tested the hypothesis that inhibition of ASICs is a potential mechanism. METHODS: The whole-cell patch-clamp technique was used to examine the effect of Triol on ASICs heterogeneously expressed in Chinese hamster ovary cells and ASICs endogenously expressed in primary cultured mouse cortical neurons. Acid-induced injury of cultured mouse cortical neurons and middle cerebral artery occlusion-induced ischemic brain injury in wild-type and ASIC1 and ASIC2 knockout mice were studied to examine the protective effect of Triol. RESULTS: Triol inhibits ASICs in a subunit-dependent manner. In Chinese hamster ovary cells, it inhibits homomeric ASIC1a and ASIC3 without affecting ASIC1ß and ASIC2a. In cultured mouse cortical neurons, it inhibits homomeric ASIC1a and heteromeric ASIC1a-containing channels. The inhibition is use-dependent but voltage- and pH-independent. Structure-activity relationship analysis suggests that hydroxyls at the 5 and 6 positions of the A/B ring are critical functional groups. Triol alleviates acidosis-mediated injury of cultured mouse cortical neurons and protects against middle cerebral artery occlusion-induced brain injury in an ASIC1a-dependent manner. CONCLUSIONS: Our study identifies Triol as a novel ASIC inhibitor, which may serve as a new pharmacological tool for studying ASICs and may also be developed as a potential drug for treating stroke.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Acidosis , Cricetulus , Ratones Noqueados , Animales , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Ratones , Células CHO , Acidosis/metabolismo , Acidosis/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Cricetinae , Fármacos Neuroprotectores/farmacología , Colestanoles/farmacología , Ratones Endogámicos C57BL , Bloqueadores del Canal Iónico Sensible al Ácido/farmacología , Masculino , Células Cultivadas

RESUMEN

Fast growing solid tumors are frequently surrounded by an acidic microenvironment. Tumor cells employ a variety of mechanisms to survive and proliferate under these harsh conditions. In that regard, acid-sensitive membrane receptors constitute a particularly interesting target, since they can affect cellular functions through ion flow and second messenger cascades. Our knowledge of these processes remains sparse, however, especially regarding medulloblastoma, the most common pediatric CNS malignancy. In this study, using RT-qPCR, whole-cell patch clamp, and Ca2+-imaging, we uncovered several ion channels and a G protein-coupled receptor, which were regulated directly or indirectly by low extracellular pH in DAOY and UW228 medulloblastoma cells. Acidification directly activated acid-sensing ion channel 1a (ASIC1a), the proton-activated Cl- channel (PAC, ASOR, or TMEM206), and the proton-activated G protein-coupled receptor OGR1. The resulting Ca2+ signal secondarily activated the large conductance calcium-activated potassium channel (BKCa). Our analyses uncover a complex relationship of these transmembrane proteins in DAOY cells that resulted in cell volume changes and induced cell death under strongly acidic conditions. Collectively, our results suggest that these ion channels in concert with OGR1 may shape the growth and evolution of medulloblastoma cells in their acidic microenvironment.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Meduloblastoma , Receptores Acoplados a Proteínas G , Humanos , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Meduloblastoma/metabolismo , Meduloblastoma/patología , Línea Celular Tumoral , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Concentración de Iones de Hidrógeno , Tamaño de la Célula , Muerte Celular , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Calcio/metabolismo , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología

RESUMEN

Slow transit constipation (STC) is one of the most common gastrointestinal disorders in children and adults worldwide. Paeoniflorin (PF), a monoterpene glycoside compound extracted from the dried root of Paeonia lactiflora, has been found to alleviate STC, but the mechanisms of its effect remain unclear. The present study aimed to investigate the effects and mechanisms of PF on intestinal fluid metabolism and visceral sensitization in rats with compound diphenoxylate-induced STC. Based on the evaluation of the laxative effect, the abdominal withdrawal reflex test, enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry were used to detect the visceral sensitivity, fluid metabolism-related proteins, and acid-sensitive ion channel 3/extracellular signal-regulated kinase (ASIC3/ERK) pathway-related molecules. PF treatment not only attenuated compound diphenoxylate-induced constipation symptoms and colonic pathological damage in rats but also ameliorated colonic fluid metabolic disorders and visceral sensitization abnormalities, as manifested by increased colonic goblet cell counts and mucin2 protein expression, decreased aquaporin3 protein expression, improved abdominal withdrawal reflex scores, reduced visceral pain threshold, upregulated serum 5-hydroxytryptamine, and downregulated vasoactive intestinal peptide levels. Furthermore, PF activated the colonic ASIC3/ERK pathway in STC rats, and ASIC3 inhibition partially counteracted PF's modulatory effects on intestinal fluid and visceral sensation. In conclusion, PF alleviated impaired intestinal fluid metabolism and abnormal visceral sensitization in STC rats and thus relieved their symptoms through activation of the ASIC3/ERK pathway.

Asunto(s)

Canales Iónicos Sensibles al Ácido , Estreñimiento , Glucósidos , Sistema de Señalización de MAP Quinasas , Monoterpenos , Animales , Glucósidos/farmacología , Monoterpenos/farmacología , Monoterpenos/uso terapéutico , Canales Iónicos Sensibles al Ácido/metabolismo , Estreñimiento/tratamiento farmacológico , Estreñimiento/metabolismo , Ratas , Masculino , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratas Sprague-Dawley , Colon/metabolismo , Colon/efectos de los fármacos , Colon/patología , Tránsito Gastrointestinal/efectos de los fármacos , Acuaporina 3/metabolismo , Acuaporina 3/genética , Serotonina/metabolismo , Dolor Visceral/tratamiento farmacológico , Dolor Visceral/metabolismo

RESUMEN

This is the first study to analyze the anti-inflammatory and antinociceptive effect of withanicandrin, isolated from Datura Ferox leaves, and the possible mechanism of action involved in adult zebrafish (ZFa). To this end, the animals were treated intraperitoneally (i. p.) with withanicandrin (4; 20 and 40 mg/kg; 20 µL) and subjected to locomotor activity and acute toxicity. Nociception tests were also carried out with chemical agents, in addition to tests to evaluate inflammatory processes induced by κ-Carrageenan 1.5 % and a Molecular Docking study. As a result, withanicandrin reduced nociceptive behavior by capsaicin at a dose of 40 mg/kg and by acid saline at doses of 4 and 40 mg/kg, through neuromodulation of TRPV1 channels and ASICs, identified through blocking the antinociceptive effect of withanicandrin by the antagonists capsazepine and naloxone. Furthermore, withanicandrin caused an anti-inflammatory effect through the reduction of abdominal edema, absence of leukocyte infiltrate in the liver tissue and reduction of ROS in thel liver tissue and presented better affinity energy compared to control morphine (TRPV1) and ibuprofen (COX-1 and COX-2).

Asunto(s)

Analgésicos , Pez Cebra , Animales , Analgésicos/farmacología , Analgésicos/química , Analgésicos/aislamiento & purificación , Canales Iónicos Sensibles al Ácido/metabolismo , Simulación del Acoplamiento Molecular , Antiinflamatorios/farmacología , Antiinflamatorios/química , Antiinflamatorios/aislamiento & purificación , Carragenina , Relación Estructura-Actividad , Relación Dosis-Respuesta a Droga , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismo , Edema/tratamiento farmacológico , Edema/inducido químicamente , Hojas de la Planta/química , Estructura Molecular

RESUMEN

Acid-sensing ion channels (ASICs) play a key role in the perception and response to extracellular acidification changes. These proton-gated cation channels are critical for neuronal functions, like learning and memory, fear, mechanosensation and internal adjustments like synaptic plasticity. Moreover, they play a key role in neuronal degeneration, ischemic neuronal injury, seizure termination, pain-sensing, etc. Functional ASICs are homo or heterotrimers formed with (ASIC1-ASIC3) homologous subunits. ASIC1a, a major ASIC isoform in the central nervous system (CNS), possesses an acidic pocket in the extracellular region, which is a key regulator of channel gating. Growing data suggest that ASIC1a channels are a potential therapeutic target for treating a variety of neurological disorders, including stroke, epilepsy and pain. Many studies were aimed at identifying allosteric modulators of ASIC channels. However, the regulation of ASICs remains poorly understood. Using all available crystal structures, which correspond to different functional states of ASIC1, and a molecular dynamics simulation (MD) protocol, we analyzed the process of channel inactivation. Then we applied a molecular docking procedure to predict the protein conformation suitable for the amiloride binding. To confirm the effect of its sole active blocker against the ASIC1 state transition route we studied the complex with another MD simulation run. Further experiments evaluated various compounds in the Enamine library that emerge with a detectable ASIC inhibitory activity. We performed a detailed analysis of the structural basis of ASIC1a inhibition by amiloride, using a combination of in silico approaches to visualize its interaction with the ion pore in the open state. An artificial activation (otherwise, expansion of the central pore) causes a complex modification of the channel structure, namely its transmembrane domain. The output protein conformations were used as a set of docking models, suitable for a high-throughput virtual screening of the Enamine chemical library. The outcome of the virtual screening was confirmed by electrophysiological assays with the best results shown for three hit compounds.

Asunto(s)

Amilorida , Benzamidinas , Humanos , Simulación del Acoplamiento Molecular , Canales Iónicos Sensibles al Ácido , Dolor

RESUMEN

In this issue of Neuron, Yamada et al.1 show that fast excitatory neurotransmission by protons acting at acid-sensing ion channels (ASICs) mediates mechanical force-evoked signaling at the Merkel cell-neurite complex, contributing to mammalian tactile discrimination.

Asunto(s)

Células de Merkel , Neuronas , Animales , Neuronas/metabolismo , Protones , Neuritas/metabolismo , Transmisión Sináptica , Canales Iónicos Sensibles al Ácido/metabolismo , Mamíferos/metabolismo

RESUMEN

A novel Lycopodium alkaloid, lycocasine A (1), and seven known Lycopodium alkaloids (2-8), were isolated from Lycopodiastrum casuarinoides. Their structures were determined through NMR, HRESIMS, and X-ray diffraction analysis. Compound 1 features an unprecedented 5/6/6 tricyclic skeleton, highlighted by a 5-aza-tricyclic[6,3,1,02,6]dodecane motif. In bioactivity assays, compound 1 demonstrated weak inhibitory activity against acid-sensing ion channel 1a.

Asunto(s)

Alcaloides , Lycopodiaceae , Lycopodium , Canales Iónicos Sensibles al Ácido , Alcaloides/farmacología , Azacitidina