RESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and represents the main cause of liver cirrhosis and hepatocellular carcinoma. Cav3.2 is a T-type calcium channel that is widely present in tissues throughout the body and plays a vital role in energy and metabolic balance. However, the effects of Cav3.2 on the NFALD remain unclear. Here, we investigated the role of Cav3.2 channel in the development and progression of NAFLD. After 16 weeks on a high-fat diets (HFD), Cav3.2 knockout (Cav3.2 KO) improved hepatic steatosis, liver injury and metabolic syndrome in an NAFLD mouse model. We provided evidence that Cav3.2 KO inhibited HFD-induced hepatic oxidative stress, inflammation and hepatocyte apoptosis. In addition, Cav3.2 KO also attenuated hepatic lipid accumulation, oxidative stress, inflammation and hepatocyte apoptosis in palmitic acid/oleic acid (PAOA)-treated primary hepatocytes. These results suggest that therapeutic approaches targeting Cav3.2 provide effective approaches for treating NAFLD.
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Apoptosis , Canales de Calcio Tipo T , Dieta Alta en Grasa , Hepatocitos , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico , Estrés Oxidativo , Animales , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Ratones , Dieta Alta en Grasa/efectos adversos , Hepatocitos/metabolismo , Hepatocitos/patología , Masculino , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Hígado/metabolismo , Hígado/patología , Inflamación/genética , Inflamación/metabolismoRESUMEN
Cerebral ischemia disrupts calcium homeostasis in the brain causing excitotoxicity, oxidative stress, inflammation, and neuronal cell apoptosis. During ischemic conditions, T-type calcium channel channels contribute to increase in intracellular calcium ions in both neurons and glial cells therefore, the current study hypothesizes the antagonism of these channels using ML218, a novel specific T-Type inhibitor in experimental model of cerebral ischemia-reperfusion (CI/R) brain injury. CI/R injury was induced in Swiss Albino mice by occlusion of common carotid arteries followed by reperfusion. Animals were assessed for learning and memory (MWM), motor coordination (Rota rod), neurological function (neurological deficit score), cerebral infarction, edema, and histopathological alterations. Biochemical assessments were made for calcium binding proteins (Calmodulin- CaM, calcium/calmodulin-dependent protein kinase II-CaMKII, S100B), oxidative stress (4-hydroxy 2-nonenal-4-HNE, glutathione-GSH, inflammation (nuclear factor kappa-light-chain-enhancer of activated B-p65-NF-kB, tumor necrosis factor-TNF-α, interleukin-IL-10) inducible nitric oxide synthase (iNOS) levels, and acetylcholinesterase activity (AChE) in brain supernatants. Furthermore, serum levels of NF-kB, iNOS, and S100B were also assessed. CI/R animals showed impairment in learning, memory, motor coordination, and neurological function along with increase in cerebral infarction, edema, and histopathological alterations. Furthermore, increase in brain calcium binding proteins, oxidative stress, inflammation, and AChE activity along with serum NF-kB, iNOS, and S100B levels were recorded in CI/R animals. Administration of ML218 (5 mg/kg and 10 mg/kg; i.p.) was observed to recuperate CI/R induced impairments in behavioral, biochemical, and histopathological analysis. Hence, it may be concluded that ML218 mediates neuroprotection during CI/R via decreasing brain and serum calcium binding proteins, inflammation, iNOS, and oxidative stress markers.
Asunto(s)
Proteínas de Unión al Calcio , Estrés Oxidativo , Daño por Reperfusión , Animales , Estrés Oxidativo/efectos de los fármacos , Ratones , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/complicaciones , Masculino , Proteínas de Unión al Calcio/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Inflamación/patología , Inflamación/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Encéfalo/patología , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Acetilcolinesterasa/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/metabolismo , Modelos Animales de EnfermedadRESUMEN
Most neurons are not replaced after injury and thus possess robust intrinsic mechanisms for repair after damage. Axon injury triggers a calcium wave, and calcium and cAMP can augment axon regeneration. In comparison to axon regeneration, dendrite regeneration is poorly understood. To test whether calcium and cAMP might also be involved in dendrite injury signaling, we tracked the responses of Drosophila dendritic arborization neurons to laser severing of axons and dendrites. We found that calcium and subsequently cAMP accumulate in the cell body after both dendrite and axon injury. Two voltage-gated calcium channels (VGCCs), L-Type and T-Type, are required for the calcium influx in response to dendrite injury and play a role in rapid initiation of dendrite regeneration. The AC8 family adenylyl cyclase, Ac78C, is required for cAMP production after dendrite injury and timely initiation of regeneration. Injury-induced cAMP production is sensitive to VGCC reduction, placing calcium upstream of cAMP generation. We propose that two VGCCs initiate global calcium influx in response to dendrite injury followed by production of cAMP by Ac78C. This signaling pathway promotes timely initiation of dendrite regrowth several hours after dendrite damage.
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Adenilil Ciclasas , Canales de Calcio Tipo L , Calcio , AMP Cíclico , Dendritas , Animales , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/genética , Axones/metabolismo , Axones/fisiología , Calcio/metabolismo , Canales de Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/genética , Señalización del Calcio/genética , AMP Cíclico/metabolismo , Dendritas/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regeneración Nerviosa/fisiología , Regeneración Nerviosa/genética , Neuronas/metabolismo , Regeneración/genética , Regeneración/fisiología , Transducción de SeñalRESUMEN
Cisplatin remains the unchallenged standard therapy for NSCLC. However, it is not completely curative due to drug resistance and oxidative stress-induced toxicity. Drug resistance is linked to overexpression of matrix metalloproteinases (MMPs) and aberrant calcium signalling. We report synthesis of novel thiazole-triazole hybrids as MMP-9 inhibitors with T-type calcium channel blocking and antioxidant effects to sensitise NSCLC to cisplatin and ameliorate its toxicity. MTT and whole cell patch clamp assays revealed that 6d has a balanced profile of cytotoxicity (IC50 = 21 ± 1 nM, SI = 12.14) and T-type calcium channel blocking activity (â60% at 10 µM). It exhibited moderate ROS scavenging activity and nanomolar MMP-9 inhibition (IC50 = 90 ± 7 nM) surpassing NNGH with MMP-9 over -2 and MMP-10 over -13 selectivity. Docking and MDs simulated its receptor binding mode. Combination studies confirmed that 6d synergized with cisplatin (CI = 0.69 ± 0.05) lowering its IC50 by 6.89 folds. Overall, the study introduces potential lead adjuvants for NSCLC platinum-based therapy.
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Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Proliferación Celular , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Pulmonares , Metaloproteinasa 9 de la Matriz , Tiazoles , Triazoles , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Triazoles/química , Triazoles/farmacología , Triazoles/síntesis química , Relación Estructura-Actividad , Metaloproteinasa 9 de la Matriz/metabolismo , Tiazoles/química , Tiazoles/farmacología , Tiazoles/síntesis química , Estructura Molecular , Proliferación Celular/efectos de los fármacos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/síntesis química , Cisplatino/farmacología , Cisplatino/química , Canales de Calcio Tipo T/metabolismoRESUMEN
Stimulation of the dorsal half of the rat periaqueductal gray (DPAG) with 60-Hz pulses of increasing intensity, 30-µA pulses of increasing frequency, or increasing doses of an excitatory amino acid elicits sequential defensive responses of exophthalmia, immobility, trotting, galloping, and jumping. These responses may be controlled by voltage-gated calcium channel-specific firing patterns. Indeed, a previous study showed that microinjection of the DPAG with 15 nmol of verapamil, a putative blocker of L-type calcium channels, attenuated all defensive responses to electrical stimulation at the same site as the injection. Accordingly, here we investigated the effects of microinjection of lower doses (0.7 and 7 nmol) of both verapamil and mibefradil, a preferential blocker of T-type calcium channels, on DPAG-evoked defensive behaviors of the male rat. Behaviors were recorded either 24 h before or 10 min, 24 h, and 48 h after microinjection. Effects were analyzed by both threshold logistic analysis and repeated measures analysis of variance for treatment by session interactions. Data showed that the electrodes were all located within the dorsolateral PAG. Compared to the effects of saline, verapamil significantly attenuated exophthalmia, immobility, and trotting. Mibefradil significantly attenuated exophthalmia and marginally attenuated immobility while facilitating trotting. While galloping was not attenuated by either antagonist, jumping was unexpectedly attenuated by 0.7 nmol verapamil only. These results suggest that T-type calcium channels are involved in the low-threshold freezing responses of exophthalmia and immobility, whereas L-type calcium channels are involved in the trotting response that precedes the full-fledged escape responses of galloping and jumping.
Asunto(s)
Bloqueadores de los Canales de Calcio , Canales de Calcio Tipo L , Canales de Calcio Tipo T , Estimulación Eléctrica , Mibefradil , Sustancia Gris Periacueductal , Verapamilo , Animales , Sustancia Gris Periacueductal/efectos de los fármacos , Sustancia Gris Periacueductal/fisiología , Masculino , Canales de Calcio Tipo T/fisiología , Canales de Calcio Tipo T/efectos de los fármacos , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo L/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Mibefradil/farmacología , Verapamilo/farmacología , Ratas , Ratas Wistar , Microinyecciones , Relación Dosis-Respuesta a DrogaRESUMEN
BACKGROUND: Interleukin 24 (IL-24) has been implicated in the nociceptive signaling. However, direct evidence and the precise molecular mechanism underlying IL-24's role in peripheral nociception remain unclear. METHODS: Using patch clamp recording, molecular biological analysis, immunofluorescence labeling, siRNA-mediated knockdown approach and behavior tests, we elucidated the effects of IL-24 on sensory neuronal excitability and peripheral pain sensitivity mediated by T-type Ca2+ channels (T-type channels). RESULTS: IL-24 enhances T-type channel currents (T-currents) in trigeminal ganglion (TG) neurons in a reversible and dose-dependent manner, primarily by activating the interleukin-22 receptor 1 (IL-22R1). Furthermore, we found that the IL-24-induced T-type channel response is mediated through tyrosine-protein kinase Lyn, but not its common downstream target JAK1. IL-24 application significantly activated protein kinase A; this effect was independent of cAMP and prevented by Lyn antagonism. Inhibition of PKA prevented the IL-24-induced T-current response, whereas inhibition of protein kinase C or MAPK kinases had no effect. Functionally, IL-24 increased TG neuronal excitability and enhanced pain sensitivity to mechanical stimuli in mice, both of which were suppressed by blocking T-type channels. In a trigeminal neuropathic pain model induced by chronic constriction injury of the infraorbital nerve, inhibiting IL-22R1 signaling alleviated mechanical allodynia, which was reversed by blocking T-type channels or knocking down Cav3.2. CONCLUSION: Our findings reveal that IL-24 enhances T-currents by stimulating IL-22R1 coupled to Lyn-dependent PKA signaling, leading to TG neuronal hyperexcitability and pain hypersensitivity. Understanding the mechanism of IL-24/IL-22R1 signaling in sensory neurons may pave the way for innovative therapeutic strategies in pain management.
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Canales de Calcio Tipo T , Proteínas Quinasas Dependientes de AMP Cíclico , Receptores de Interleucina , Células Receptoras Sensoriales , Transducción de Señal , Ganglio del Trigémino , Familia-src Quinasas , Animales , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/genética , Familia-src Quinasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ganglio del Trigémino/metabolismo , Masculino , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Receptores de Interleucina/metabolismo , Ratones , Ratones Endogámicos C57BL , Interleucinas/metabolismoRESUMEN
Pheochromocytoma (PCC) and abdominal paraganglioma (aPGL) (together abbreviated PPGL) frequently present with an underlying genetic event in a PPGL driver gene, and additional susceptibility genes are anticipated. Here, we re-analyzed whole-exome sequencing data for PCC patients and identified two patients with rare missense variants in the calcium voltage-gated channel subunit 1H gene (CACNA1H). CACNA1H variants were also found in the clinical setting in PCC patients using targeted sequencing and from analysis of The Cancer Genome Atlas database. In total, CACNA1H variants were found in six PCC cases. Three of these were constitutional, and two are known to have functional consequences on hormone production and gene expression in primary aldosteronism and aldosterone-producing adrenocortical adenoma. In general, PPGL exhibited reduced CACNA1H mRNA expression as compared to normal adrenal. Immunohistochemistry showed strong CACNA1H (CaV3.2) staining in adrenal medulla while PPGL typically had weak or negative staining. Reduced CACNA1H gene expression was especially pronounced in PCC compared to aPGL and in PPGL with cluster 2 kinase signaling phenotype. Furthermore, CACNA1H levels correlated with HIF1A and HIF2A. Moreover, TCGA data revealed a correlation between CACNA1H methylation density and gene expression. Expression of rCacna1h in PC12 cells induced differential protein expression profiles, determined by mass spectrometry, as well as a shift in the membrane potential where maximum calcium currents were observed, as determined by electrophysiology. The findings suggest the involvement of CACNA1H/CaV3.2 in pheochromocytoma development and establish a potential link between the etiology of adrenomedullary and adrenocortical tumor development.
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Neoplasias de las Glándulas Suprarrenales , Regulación hacia Abajo , Feocromocitoma , Feocromocitoma/genética , Feocromocitoma/metabolismo , Humanos , Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/metabolismo , Femenino , Masculino , Animales , Persona de Mediana Edad , Adulto , Ratas , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Células PC12RESUMEN
BACKGROUND AND AIM: Colorectal cancer (CRC) originates from pre-existing polyps in the colon. The development of different subtypes of CRC is influenced by various genetic and epigenetic characteristics. CpG island methylator phenotype (CIMP) is found in about 15-20% of sporadic CRCs and is associated with hypermethylation of certain gene promoters. This study aims to find prognostic genes and compare their expression and methylation status as potential biomarkers in patients with serrated sessile adenomas/polyps (SSAP) and CRC, in order to evaluate which, one is a better predictor of disease. METHOD: This study employed a multi-phase approach to investigate genes associated with CRC and SSAP. Initially, two gene expression datasets were analyzed using R and Limma package to identify differentially expressed genes (DEGs). Venn diagram analysis further refined the selection, revealing four genes from the Weissenberg panel with significant changes. These genes, underwent thorough in silico evaluations. Once confirmed, they proceeded to wet lab experimentation, focusing on expression and methylation status. This comprehensive methodology ensured a robust examination of the genes involved in CRC and SSAP. RESULT: This study identified cancer-specific genes, with 8,351 and 1,769 genes specifically down-regulated in SSAP and CRC tissues, respectively. The down-regulated genes were associated with cell adhesion, negative regulation of cell proliferation, and drug response. Four highly downregulated genes in the Weissenberg panel, including CACNA1G, IGF2, MLH1, and SOCS1. In vitro analysis showed that they are hypermethylated in both SSAP and CRC samples while their expressions decreased only in CRC samples. CONCLUSION: This suggests that the decrease in gene expression could help determine whether a polyp will become cancerous. Using both methylation status and gene expression status of genes in the Weissenberg panel in prognostic tests may lead to better prognoses for patients.
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Neoplasias Colorrectales , Islas de CpG , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina , Homólogo 1 de la Proteína MutL , Proteína 1 Supresora de la Señalización de Citocinas , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas/genética , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Metilación de ADN/genética , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Islas de CpG/genética , Femenino , Pólipos del Colon/genética , Pólipos del Colon/metabolismo , Pólipos del Colon/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Masculino , Regulación hacia Abajo/genética , Simulación por Computador , Persona de Mediana Edad , Adenoma/genética , Adenoma/metabolismo , Adenoma/patología , Regiones Promotoras Genéticas/genética , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Perfilación de la Expresión Génica/métodos , Anciano , PronósticoRESUMEN
BACKGROUND: T-type calcium channels, characterized as low-voltage activated (LVA) calcium channels, play crucial physiological roles across a wide range of tissues, including both the neuronal and nonneuronal systems. Using in situ hybridization and RNA interference (RNAi) techniques in vitro, we previously identified the tissue distribution and physiological function of the T-type calcium channel α1 subunit (DdCα1G) in the plant-parasitic nematode Ditylenchus destructor. METHODS AND RESULTS: To further characterize the functional role of DdCα1G, we employed a combination of immunohistochemistry and fungus-mediated RNAi and found that DdCα1G was clearly distributed in stylet-related tissue, oesophageal gland-related tissue, secretory-excretory duct-related tissue and male spicule-related tissue. Silencing DdCα1G led to impairments in the locomotion, feeding, reproductive ability and protein secretion of nematodes. To confirm the defects in behavior, we used phalloidin staining to examine muscle changes in DdCα1G-RNAi nematodes. Our observations demonstrated that defective behaviors are associated with related muscular atrophy. CONCLUSION: Our findings provide a deeper understanding of the physiological functions of T-type calcium channels in plant-parasitic nematodes. The T-type calcium channel can be considered a promising target for sustainable nematode management practices.
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Actinas , Canales de Calcio Tipo T , Interferencia de ARN , Animales , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/genética , Actinas/metabolismo , Actinas/genética , Masculino , Hongos/genética , Silenciador del GenRESUMEN
Reactive sulfur species including sulfides, polysulfides and cysteine hydropersulfide play extensive roles in health and disease, which involve modification of protein functions through the interaction with metals bound to the proteins, cleavage of cysteine disulfide (S-S) bonds and S-persulfidation of cysteine residues. Sulfides over a wide micromolar concentration range enhance the activity of Cav3.2 T-type Ca2+ channels by eliminating Zn2+ bound to the channels, thereby promoting somatic and visceral pain. Cav3.2 is under inhibition by Zn2+ in physiological conditions, so that sulfides function to reboot Cav3.2 from Zn2+ inhibition and increase the excitability of nociceptors. On the other hand, polysulfides generated from sulfides activate TRPA1 channels via cysteine S-persulfidation, thereby facilitating somatic, but not visceral, pain. Thus, Cav3.2 function enhancement by sulfides and TRPA1 activation by polysulfides, synergistically accelerate somatic pain signals. The increased activity of the sulfide/Cav3.2 system, in particular, appears to have a great impact on pathological pain, and may thus serve as a therapeutic target for treatment of neuropathic and inflammatory pain including visceral pain.
Asunto(s)
Canales de Calcio Tipo T , Sulfuros , Canal Catiónico TRPA1 , Sulfuros/farmacología , Canal Catiónico TRPA1/metabolismo , Humanos , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/fisiología , Animales , Zinc/metabolismo , Dolor/metabolismo , Dolor/tratamiento farmacológico , Nociceptores/metabolismo , Nociceptores/efectos de los fármacosRESUMEN
Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.
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Canales de Calcio Tipo T , Modelos Animales de Enfermedad , Hiperalgesia , Dolor Postoperatorio , Venenos de Escorpión , Animales , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/química , Ratones , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Dolor Postoperatorio/tratamiento farmacológico , Dolor Postoperatorio/metabolismo , Calcio/metabolismo , Masculino , Humanos , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/químicaRESUMEN
Ca2+ influx through Cav3.3 T-type channel plays crucial roles in neuronal excitability and is subject to regulation by various signaling molecules. However, our understanding of the partners of Cav3.3 and the related regulatory pathways remains largely limited. To address this quest, we employed the rat Cav3.3 C-terminus as bait in yeast-two-hybrid screenings of a cDNA library, identifying rat Gß2 as an interaction partner. Subsequent assays revealed that the interaction of Gß2 subunit was specific to the Cav3.3 C-terminus. Through systematic dissection of the C-terminus, we pinpointed a 22 amino acid sequence (amino acids 1789-1810) as the Gß2 interaction site. Coexpression studies of rat Cav3.3 with various Gßγ compositions were conducted in HEK-293 cells. Patch clamp recordings revealed that coexpression of Gß2γ2 reduced Cav3.3 current density and accelerated inactivation kinetics. Interestingly, the effects were not unique to Gß2γ2, but were mimicked by Gß2 alone as well as other Gßγ dimers, with similar potencies. Deletion of the Gß2 interaction site abolished the effects of Gß2γ2. Importantly, these Gß2 effects were reproduced in human Cav3.3. Overall, our findings provide evidence that Gß(γ) complexes inhibit Cav3.3 channel activity and accelerate the inactivation kinetics through the Gß interaction with the Cav3.3 C-terminus.
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Canales de Calcio Tipo T , Subunidades beta de la Proteína de Unión al GTP , Animales , Humanos , Ratas , Canales de Calcio Tipo R , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/química , Proteínas de Transporte de Catión , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/química , Células HEK293 , Cinética , Técnicas de Placa-Clamp , Unión ProteicaRESUMEN
BACKGROUND: Neuromedin B (Nmb) plays a pivotal role in the transmission of neuroinflammation, particularly during spinal cord ischemia-reperfusion injury (SCII). However, the detailed molecular mechanisms underlying this process remain elusive. METHODS: The SCII model was established by clamping the abdominal aorta of male Sprague-Dawley (SD) rats for 60 min. The protein expression levels of Nmb, Cav3.2, and IL-1ß were detected by Western blotting, while miR-214-3p expression was quantified by qRT-PCR. The targeted regulation between miR-214-3p and Nmb was investigated using a dual-luciferase reporter gene assay. The cellular localization of Nmb and Cav3.2 with cell-specific markers was visualized by immunofluorescence staining. The specific roles of miR-214-3p on the Nmb/Cav3.2 interactions in SCII-injured rats were explored by intrathecal injection of Cav3.2-siRNA, PD168368 (a specific NmbR inhibitor) and synthetic miR-214-3p agomir and antagomir in separate experiments. Additionally, hind-limb motor function was evaluated using the modified Tarlov scores. RESULTS: Compared to the Sham group, the protein expression levels of Nmb, Cav3.2, and the proinflammatory factor Interleukin(IL)-1ß were significantly elevated at 24 h post-SCII. Intrathecal injection of PD168368 and Cav3.2-siRNA significantly suppressed the expression of Cav3.2 and IL-1ß compared to the SCII group. The miRDB database and dual-luciferase reporter gene assay identified Nmb as a direct target of miR-214-3p. As expected, in vivo overexpression of miR-214-3p by agomir-214-3p pretreatment significantly inhibited the increases in Nmb, Cav3.2 and IL-1ß expression and improved lower limb motor function in SCII-injured rats, while antagomiR-214-3p pretreatment reversed these effects. CONCLUSIONS: Nmb protein levels positively correlated with Cav3.2 expression in SCII rats. Upregulating miR-214-3p ameliorated hind-limb motor function and protected against neuroinflammation via inhibiting the aberrant Nmb/Cav3.2 interactions and downstream IL-1ß release. These findings provide novel therapeutic targets for clinical prevention and treatment of SCII.
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Canales de Calcio Tipo T , MicroARNs , Enfermedades Neuroinflamatorias , Daño por Reperfusión , Isquemia de la Médula Espinal , Animales , Masculino , Ratas , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Modelos Animales de Enfermedad , Interleucina-1beta/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Transducción de Señal , Médula Espinal/metabolismo , Isquemia de la Médula Espinal/metabolismo , Isquemia de la Médula Espinal/genéticaRESUMEN
The Cav3.2 subtype of T-type calcium channels has been targeted for developing analgesics and anti-epileptics for its role in pain and epilepsy. Here we present the cryo-EM structures of Cav3.2 alone and in complex with four T-type calcium channel selective antagonists with overall resolutions ranging from 2.8 Å to 3.2 Å. The four compounds display two binding poses. ACT-709478 and TTA-A2 both place their cyclopropylphenyl-containing ends in the central cavity to directly obstruct ion flow, meanwhile extending their polar tails into the IV-I fenestration. TTA-P2 and ML218 project their 3,5-dichlorobenzamide groups into the II-III fenestration and place their hydrophobic tails in the cavity to impede ion permeation. The fenestration-penetrating mode immediately affords an explanation for the state-dependent activities of these antagonists. Structure-guided mutational analysis identifies several key residues that determine the T-type preference of these drugs. The structures also suggest the role of an endogenous lipid in stabilizing drug binding in the central cavity.
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Bloqueadores de los Canales de Calcio , Canales de Calcio Tipo T , Microscopía por Crioelectrón , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/química , Humanos , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/farmacología , Sitios de Unión , Unión Proteica , Modelos Moleculares , Células HEK293RESUMEN
Endothelial dysfunction develops with age and may precede cardiovascular disease. Animal data suggest that T-type calcium channels play an important role in endothelial function, but data from humans are lacking. This study included 15 healthy, sedentary, elderly males for a double blinded, randomized controlled trial. For 8 weeks, they were given 40 mg/day of either efonidipine (L- and T-type calcium channel blocker (CCB)) or nifedipine (L-type CCB). Vascular function was evaluated by graded femoral arterial infusions of acetylcholine (ACh; endothelium-dependent vasodilator) and sodium nitroprusside (endothelium-independent vasodilator) both with and without co-infusion of N-acetylcysteine (NAC; antioxidant). We measured leg blood flow and mean arterial pressure and calculated leg vascular conductance to evaluate the leg vascular responses. Despite no significant change in blood pressure in either group, we observed higher leg blood flow responses (Δ 0.43 ± 0.45 l/min, P = 0.006) and leg vascular conductance (Δ 5.38 ± 5.67 ml/min/mmHg, P = 0.005) to intra-arterial ACh after efonidipine, whereas there was no change in the nifedipine group, and no differences between groups. We found no upregulation of endothelial nitric oxide synthase in vastus lateralis muscle biopsies within or between groups. Smooth muscle cell responsiveness was unaltered by efonidipine or nifedipine. Intravenous co-infusion of NAC did not affect endothelium-dependent vasodilatation in either of the CCB groups. These results suggest that 8 weeks' inhibition of T- and L-type calcium channels augments endothelium-dependent vasodilatory function in healthy elderly males. Further studies are required to elucidate if T-type calcium channel inhibition can counteract endothelial dysfunction.
Asunto(s)
Bloqueadores de los Canales de Calcio , Canales de Calcio Tipo T , Endotelio Vascular , Nifedipino , Nitrofenoles , Humanos , Masculino , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/efectos de los fármacos , Anciano , Bloqueadores de los Canales de Calcio/farmacología , Nifedipino/farmacología , Proyectos Piloto , Método Doble Ciego , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Dihidropiridinas/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Vasodilatadores/farmacología , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Flujo Sanguíneo Regional/efectos de los fármacos , Flujo Sanguíneo Regional/fisiología , Compuestos Organofosforados/farmacología , Acetilcolina/farmacología , Pierna/irrigación sanguínea , Nitroprusiato/farmacología , Persona de Mediana EdadRESUMEN
Cav3.2 T-type calcium channels are important targets for pain relief in rodent models of inflammatory and neuropathic pain. Even though many T-type channel blockers have been tested in mice, only one molecule, ABT-639, has been tested in phase II clinical studies and did not produce analgesic effects over placebo. Here we examined the effects of ABT-639 on Cav3.2 channel activity in tsA-201 cells and dorsal root ganglion (DRG) neurons, in comparison with another established Cav3.2 inhibitor Z944. These experiments revealed that Z944 mediated â¼100-fold more potent inhibition of Cav3.2 currents than ABT-639, with the latter blocking channel activity by less than 15 percent when applied at a concentration of 30 µM. A slight increase in ABT-639 potency was observed at more depolarized holding potentials, suggesting that this compound may act preferentially on inactivated channels. We tested the effects of both compounds in the Complete Freund's Adjuvant (CFA) model of chronic inflammatory pain, and in partial sciatic nerve injury model of neuropathic pain in mice. In the neuropathic pain model, both Z944 and ABT-639 reversed mechanical hypersensitivity to similar degrees when delivered systemically, but remarkably, when delivered intrathecally, only Z944 was effective. In the CFA model, both compounds reversed thermal hyperalgesia upon systemic delivery, but only Z944 mediated pain relief upon intrathecal delivery, indicating that ABT-639 acts primarily at peripheral sites. ABT-639 lost its analgesic effects in CFA treated Cav3.2 null mice, indicating that these channels are essential for ABT-639-mediated pain relief despite its poor inhibition of Cav3.2 currents.
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Bencenosulfonamidas , Canales de Calcio Tipo T , Dolor Crónico , Compuestos Heterocíclicos con 2 Anillos , Neuralgia , Ratones , Animales , Neuralgia/tratamiento farmacológico , Analgésicos/farmacología , Analgésicos/uso terapéutico , Hiperalgesia/tratamiento farmacológico , Modelos Animales de Enfermedad , Dolor Crónico/tratamiento farmacológico , Bloqueadores de los Canales de Calcio/farmacologíaRESUMEN
AIM: Bradyarrhythmias result from inhibition of automaticity, prolonged repolarization, or slow conduction in the heart. The ERG channels mediate the repolarizing current IKr in the cardiac action potential, whereas T-type calcium channels (TTCC) are involved in the sinoatrial pacemaker and atrioventricular conduction in mammals. Zebrafish have become a valuable research model for human cardiac electrophysiology and disease. Here, we investigate the contribution of ERG channels and TTCCs to the pacemaker and atrioventricular conduction in zebrafish larvae and determine the mechanisms causing atrioventricular block. METHODS: Zebrafish larvae expressing ratiometric fluorescent Ca2+ biosensors in the heart were used to measure Ca2+ levels and rhythm in beating hearts in vivo, concurrently with contraction and hemodynamics. The atrioventricular delay (the time between the start of atrial and ventricular Ca2+ transients) was used to measure impulse conduction velocity and distinguished between slow conduction and prolonged refractoriness as the cause of the conduction block. RESULTS: ERG blockers caused bradycardia and atrioventricular block by prolonging the refractory period in the atrioventricular canal and in working ventricular myocytes. In contrast, inhibition of TTCCs caused bradycardia and second-degree block (Mobitz type I) by slowing atrioventricular conduction. TTCC block did not affect ventricular contractility, despite being highly expressed in cardiomyocytes. Concomitant measurement of Ca2+ levels and ventricular size showed mechano-mechanical coupling: increased preload resulted in a stronger heart contraction in vivo. CONCLUSION: ERG channels and TTCCs influence the heart rate and atrioventricular conduction in zebrafish larvae. The zebrafish lines expressing Ca2+ biosensors in the heart allow us to investigate physiological feedback mechanisms and complex arrhythmias.
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Bloqueo Atrioventricular , Canales de Calcio Tipo T , Marcapaso Artificial , Humanos , Animales , Pez Cebra , Frecuencia Cardíaca/fisiología , Bradicardia , Canales de Calcio Tipo T/fisiología , Canales de Potasio Éter-A-Go-Go , Miocitos Cardíacos , Mamíferos , Regulador Transcripcional ERGRESUMEN
T-type calcium channels perform crucial physiological roles across a wide spectrum of tissues, spanning both neuronal and non-neuronal system. For instance, they serve as pivotal regulators of neuronal excitability, contribute to cardiac pacemaking, and mediate the secretion of hormones. These functions significantly hinge upon the intricate interplay of T-type channels with interacting proteins that modulate their expression and function at the plasma membrane. In this review, we offer a panoramic exploration of the current knowledge surrounding these T-type channel interactors, and spotlight certain aspects of their potential for drug-based therapeutic intervention.