RESUMO
Sea urchin eggs are covered with layers of extracellular matrix, namely, the vitelline layer (VL) and jelly coat (JC). It has been shown that sea urchin eggs' JC components serve as chemoattractants or ligands for the receptor on the fertilizing sperm to promote the acrosome reaction. Moreover, the egg's VL provides receptors for conspecific sperm to bind, and, to date, at least two sperm receptors have been identified on the surface of sea urchin eggs. Interestingly, however, according to our previous work, denuded sea urchin eggs devoid of the JC and VL do not fail to become fertilized by sperm. Instead, they are bound and penetratedby multiple sperm, raising the possibility that an alternative pathway independent of the VL-residing sperm receptor may be at work. In this research, we studied the roles of the JC and VL using intact and denuded eggs and the synthetic polyamine BPA-C8. BPA-C8 is known to bind to the negatively charged macromolecular complexes in the cells, such as the JC, VL, and the plasma membrane of echinoderm eggs, as well as to the actin filaments in fibroblasts. Our results showed that, when added to seawater, BPA-C8 significantly repressed the Ca2+ wave in the intact P. lividus eggs at fertilization. In eggs deprived of the VL and JC, BPA-C8 binds to the plasma membrane and increases fibrous structures connecting microvilli, thereby allowing the denuded eggs to revert towards monospermy at fertilization. However, the reduced Ca2+ signal in denuded eggs was nullified compared to the intact eggs because removing the JC and VL already decreased the Ca2+ wave. BPA-C8 does not cross the VL and the cell membrane of unfertilized sea urchin eggs to diffuse into the cytoplasm at variance with the fibroblasts. Indeed, the jasplakinolide-induced polymerization of subplasmalemmal actin filaments was inhibited in the eggs microinjected with BPA-C8, but not in the ones bath-incubated with the same dose of BPA-C8.
Assuntos
Fertilização , Óvulo , Ouriços-do-Mar , Animais , Fertilização/efeitos dos fármacos , Ouriços-do-Mar/efeitos dos fármacos , Ouriços-do-Mar/metabolismo , Óvulo/metabolismo , Óvulo/efeitos dos fármacos , Masculino , Poliaminas/metabolismo , Poliaminas/farmacologia , Feminino , Espermatozoides/metabolismo , Espermatozoides/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Cálcio/metabolismoRESUMO
Autism spectrum disorder (ASD) is known as a group of neurodevelopmental conditions including stereotyped and repetitive behaviors, besides social and sensorimotor deficits. Anatomical and functional evidence indicates atypical maturation of the striatum. Astrocytes regulate the maturation and plasticity of synaptic circuits, and impaired calcium signaling is associated with repetitive behaviors and atypical social interaction. Spontaneous calcium transients (SCT) recorded in the striatal astrocytes of the rat were investigated in the preclinical model of ASD by prenatal exposure to valproic acid (VPA). Our results showed sensorimotor delay, augmented glial fibrillary acidic protein -a typical intermediate filament protein expressed by astrocytes- and diminished expression of GABAA-ρ3 through development, and increased frequency of SCT with a reduced latency that resulted in a diminished amplitude in the VPA model. The convulsant picrotoxin, a GABAA (γ-aminobutyric acid type A) receptor antagonist, reduced the frequency of SCT in both experimental groups but rescued this parameter to control levels in the preclinical ASD model. The amplitude and latency of SCT were decreased by picrotoxin in both experimental groups. Nipecotic acid, a GABA uptake inhibitor, reduced the mean amplitude only for the control group. Nevertheless, nipecotic acid increased the frequency but diminished the latency in both experimental groups. Thus, we conclude that striatal astrocytes exhibit SCT modulated by GABAA-mediated signaling, and prenatal exposure to VPA disturbs this tuning.
Assuntos
Astrócitos , Corpo Estriado , Animais , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/efeitos dos fármacos , Feminino , Gravidez , Ratos , Ácido Valproico/farmacologia , Ratos Wistar , Picrotoxina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Modelos Animais de Doenças , Masculino , Cálcio/metabolismo , Transtorno do Espectro Autista/metabolismo , Transtorno Autístico/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismoRESUMO
ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca2+ concentration ([Ca2+]i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca2+i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca2+]i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca2+ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10-6 M), which depletes ER Ca2+ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10-5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca2+]i was significantly lower after chelating Ca2+i with 2 mM EGTA in Ca2+-free buffer. The peak [Ca2+]i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10-5 M and BzATP at 10-4 M. Incubating CGCs with dantrolene alone decreased [Ca2+]i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca2+i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca2+]i. This study contributes to the understanding of diseases arising from defective Ca2+ signaling in nonexcitable cells.NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca2+]i) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca2+]i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca2+]i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca2+]i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca2+i homeostasis.
Assuntos
Trifosfato de Adenosina , Cálcio , Células Caliciformes , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Cálcio/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Ratos , Células Cultivadas , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/efeitos dos fármacos , Agonistas Purinérgicos/farmacologia , Ratos Sprague-Dawley , Sinalização do Cálcio/efeitos dos fármacos , Humanos , Masculino , Fosfolipases Tipo C/metabolismoRESUMO
Sodium butyrate (NaB) improves ß-cell function in preclinical models of diabetes; however, the mechanisms underlying these beneficial effects have not been fully elucidated. In this study, we investigated the impact of NaB on ß-cell function and calcium (Ca2+) signaling using ex vivo and in vitro models of diabetes. Our results show that NaB significantly improved glucose-stimulated insulin secretion in islets from human organ donors with type 2 diabetes and in cytokine-treated INS-1 ß cells. Consistently, NaB improved glucose-stimulated Ca2+ oscillations in mouse islets treated with proinflammatory cytokines. Because the oscillatory phenotype of Ca2+ in the ß cell is governed by changes in endoplasmic reticulum (ER) Ca2+ levels, we explored the relationship between NaB and store-operated calcium entry (SOCE), a rescue mechanism that acts to refill ER Ca2+ levels through STIM1-mediated gating of plasmalemmal Orai channels. We found that NaB treatment preserved basal ER Ca2+ levels and restored SOCE in IL-1ß-treated INS-1 cells. Furthermore, we linked these changes with the restoration of STIM1 levels in cytokine-treated INS-1 cells and mouse islets, and we found that NaB treatment was sufficient to prevent ß-cell death in response to IL-1ß treatment. Mechanistic experiments revealed that NaB mediated these beneficial effects in the ß-cell through histone deacetylase (HDAC) inhibition, iNOS suppression, and modulation of AKT-GSK-3 signaling. Taken together, these data support a model whereby NaB treatment promotes ß-cell function and Ca2+ homeostasis under proinflammatory conditions through pleiotropic effects that are linked with maintenance of SOCE. These results also suggest a relationship between ß-cell SOCE and gut microbiome-derived butyrate that may be relevant in the treatment and prevention of diabetes.
Assuntos
Ácido Butírico , Cálcio , Células Secretoras de Insulina , Molécula 1 de Interação Estromal , Animais , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Molécula 1 de Interação Estromal/metabolismo , Camundongos , Humanos , Ácido Butírico/farmacologia , Cálcio/metabolismo , Citocinas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Retículo Endoplasmático/metabolismo , Diabetes Mellitus Tipo 2/metabolismoRESUMO
Ovarian clear cell carcinoma (OCCC) is a subtype of ovarian cancer and is highly malignant with high chemoresistance. CACNA1H is pivotal in tumor development. However, the role of CACNA1H in the acquisition process of chemotherapeutic resistance in OCCC cells is rarely reported. Therefore, this study aimed to explore the role of CACNA1H in chemotherapy resistance of OCCC cells and its related mechanism. Based on bioinformatics analysis, we found that CACNA1H was downregulated in chemoresistant OCCC patients compared to chemosensitive OCCC patients. Comparing DDP-resistant and sensitive OCCC cell lines, the resistant strain showed lower CACNA1H mRNA expression. CACNA1H expression was associated with calcium signaling pathways in chemoresistant OCCC patients. CACNA1H mRNA expression was significantly downregulated in OCCC cells compared to normal ovarian epithelial cells. When CACNA1H was overexpressed, intracellular Ca2+ concentration and protein levels of p-CaMKII and p-Akt were significantly upregulated, while protein levels of LC3-II/LC3-I and Beclin1 were downregulated, indicating a repression of autophagy. The rescue experiment revealed that CACNA1H overexpression in drug-resistant OCCC cells reduced autophagy-induced DDP resistance via CaMKII/Akt signaling. Overall, CACNA1H increased intracellular Ca2+ concentration and activated CaMKII/Akt signaling pathway in OCCC, thereby repressing autophagy to maintain the sensitivity of OCCC cells to DDP.
Assuntos
Adenocarcinoma de Células Claras , Autofagia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas , Feminino , Humanos , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/tratamento farmacológico , Adenocarcinoma de Células Claras/patologia , Adenocarcinoma de Células Claras/metabolismo , Autofagia/genética , Autofagia/efeitos dos fármacos , Cálcio/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
The effects of enhanced late INa, a persistent component of the Na+ channel current, on the intracellular ion dynamics and the automaticity of the pulmonary vein cardiomyocytes were studied with fluorescent microscopy. Anemonia viridis toxin II (ATX- II), an enhancer of late INa, caused increases in the basal Na+ and Ca2+ concentrations, increases in the number of Ca2+ sparks and Ca2+ waves, and the generation of repetitive Ca2+ transients. These phenomena were inhibited by eleclazine, a blocker of the late INa; SEA0400, an inhibitor of the Na+/Ca2+ exchanger (NCX); H89, a protein kinase A (PKA) inhibitor; and KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results suggest that enhancement of late INa in the pulmonary vein cardiomyocytes causes disturbance of the intracellular ion environment through activation of the NCX and Ca2+-dependent enzymes. Such mechanisms are probably involved in the ectopic electrical activity of the pulmonary vein myocardium.
Assuntos
Cálcio , Venenos de Cnidários , Miócitos Cardíacos , Veias Pulmonares , Trocador de Sódio e Cálcio , Animais , Veias Pulmonares/metabolismo , Veias Pulmonares/citologia , Veias Pulmonares/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Cobaias , Cálcio/metabolismo , Venenos de Cnidários/farmacologia , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Masculino , Potenciais de Ação/efeitos dos fármacos , Canais de Sódio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Compostos de Anilina/farmacologia , Sulfonamidas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Isoquinolinas , Éteres FenílicosRESUMO
Seizures are increasingly being recognized as the hallmark of Alzheimer's disease (AD). Neuronal hyperactivity can be a consequence of neuronal damage caused by abnormal amyloid ß (Aß) depositions. However, it can also be a cell-autonomous phenomenon causing AD by Aß-independent mechanisms. Various studies using animal models have shown that Ca2+ is released from the endoplasmic reticulum (ER) via type 1 inositol triphosphate receptors (InsP3R1s) and ryanodine receptors (RyRs). To investigate which is the main pathophysiological mechanism in human neurons, we measured Ca2+ signaling in neural cells derived from three early-onset AD patients harboring Presenilin-1 variants (PSEN1 p.A246E, p.L286V, and p.M146L). Of these, it has been reported that PSEN1 p.A246E and p.L286V did not produce a significant amount of abnormal Aß. We found all PSEN1-mutant neurons, but not wild-type, caused abnormal Ca2+-bursts in a manner dependent on the calcium channel, Ryanodine Receptor 2 (RyR2). Indeed, carvedilol, an RyR2 inhibitor, and VK-II-86, an analog of carvedilol without the ß-blocking effects, sufficiently eliminated the abnormal Ca2+ bursts. In contrast, Dantrolene, an inhibitor of RyR1 and RyR3, and Xestospongin c, an IP3R inhibitor, did not attenuate the Ca2+-bursts. The Western blotting showed that RyR2 expression was not affected by PSEN1 p.A246E, suggesting that the variant may activate the RyR2. The RNA-Seq data revealed that ER-stress responsive genes were increased, and mitochondrial Ca2+-transporter genes were decreased in PSEN1A246E cells compared to the WT neurons. Thus, we propose that aberrant Ca2+ signaling is a key link between human pathogenic PSEN1 variants and cell-intrinsic hyperactivity prior to deposition of abnormal Aß, offering prospects for the development of targeted prevention strategies for at-risk individuals.
Assuntos
Doença de Alzheimer , Sinalização do Cálcio , Cálcio , Carvedilol , Neurônios , Presenilina-1 , Canal de Liberação de Cálcio do Receptor de Rianodina , Feminino , Humanos , Masculino , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Presenilina-1/genética , Presenilina-1/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Carvedilol/farmacologiaRESUMO
Hydrogen sulfide (H2S) is a gaseous signaling molecule that influences digestive and nervous system functions. Enteric glial cells (EGCs) are integral to the enteric nervous system and play a role in regulating gastrointestinal motility. This study explored the dual effects of exogenous H2S on EGCs and the influence of apoptosis-related pathways and ion channels in EGCs. We also administered honokiol for further interventional studies. The results revealed that low-concentration H2S increased the mitochondrial membrane potential (MMP) of EGCs, decreased the whole-cell membrane potential, downregulated BAX and caspase-3, upregulated Bcl2 expression, reduced apoptosis, and promoted cell proliferation. The Ca2+ concentration, Cx43 mRNA, and protein expression were also increased. A high concentration of H2S had the opposite effect. In addition, GFAP mRNA expression was upregulated in the test-low group, downregulated in the test-high group, and upregulated in the test-high + Hon group. Honokiol treatment increased MMP, reduced whole-cell membrane potential, inhibited BAX and caspase-3 expression, increased Bcl2 expression, decreased cell apoptosis, and increased cell proliferation. The Ca2+ concentration, Cx43 mRNA, and protein expression were also upregulated. In conclusion, our study showed that exogenous H2S can bidirectionally regulate EGC proliferation and apoptosis by affecting MMP and cell membrane potential via the Bcl2/BAX/caspase-3 pathway and modulate Cx43-mediated Ca2+ responses in EGCs to regulate colonic motility bidirectionally. Honokiol can ameliorate the damage to EGCs induced by high H2S concentrations through the Bcl2/BAX/caspase-3 pathway and improve colon motility by increasing Cx43 expression and Ca2+ concentration.
Assuntos
Apoptose , Compostos de Bifenilo , Sinalização do Cálcio , Proliferação de Células , Conexina 43 , Sulfeto de Hidrogênio , Lignanas , Neuroglia , Ratos Sprague-Dawley , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Lignanas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Ratos , Conexina 43/metabolismo , Conexina 43/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Cálcio/metabolismo , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/metabolismo , Células Cultivadas , Compostos Alílicos , FenóisRESUMO
Atopic dermatitis (AD) is a common allergic inflammatory skin condition marked by severe itching, skin lichenification, and chronic inflammation. AD results from a complex immune response, primarily driven by T lymphocytes and environmental triggers, leading to a disrupted epidermal barrier function. Traditional treatments, such as topical corticosteroids, have limitations due to long-term side effects, highlighting the need for safer alternatives. Here, we aimed to show that Agrimonia coreana extract (ACext) can be used in treating AD-related dermatologic symptoms. ACext could inhibit CRAC (Calcium Release-Activated Calcium) channel activity, reducing Orai1/CRAC currents and decreasing intracellular calcium signaling. This inhibition was further confirmed by the reduced IL-2 levels and T cell proliferation upon ACext treatment. In a mouse model of AD, ACext significantly ameliorates symptoms, improves histological parameters, and enhances skin barrier function, demonstrating its potential for treating AD.
Assuntos
Agrimonia , Dermatite Atópica , Extratos Vegetais , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Animais , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Camundongos , Agrimonia/química , Modelos Animais de Doenças , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Humanos , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Sinalização do Cálcio/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/imunologiaRESUMO
Uncarboxylated osteocalcin (ucOC) is a hormone secreted by osteoblasts that strengthens bone during mineralization and is a biomarker for ongoing bone formation. It also regulates glucose homeostasis by stimulating insulin secretion from pancreatic ß-cells. However, its effect on ß-cells under hyperglycemic diabetic conditions is unclear. The objective of this study was to investigate ucOC's effect on insulin secretion in ß-cells maintained under high glucose conditions. We hypothesized that hyperglycemia potentiates insulin secretion in response to ucOC stimulation. Using INS-1 cells, we performed insulin secretion experiments, intracellular calcium recordings, and RT-qPCR to determine ucOC's effect on glucose-stimulated insulin secretion (GSIS)-related genes. The results reveal that ucOC significantly increased insulin secretion under hyperglycemic conditions compared to lower glucose levels. High glucose conditions also potentiated the effect of ucOC on calcium signals, which enhanced insulin secretion. The increase in intracellular calcium was due to an influx from the extracellular space via voltage-dependent calcium channels (VDCCs). Interestingly, the treatment of cells with NPS-2143, a GPRC6A blocker, failed to abolish the calcium signals. Uncarboxylated osteocalcin upregulated the expression of GSIS-related genes under high glucose conditions (450 mg/dL) compared to cells under standard culture conditions (200 mg/dL). In conclusion, hyperglycemia potentiates ucOC-induced insulin secretion in ß-cells by opening VDCCs and upregulating GSIS genes. These findings provide a better understanding of ucOC's mechanism in the diabetic state and could lead to alternative treatments to stimulate insulin secretion.
Assuntos
Hiperglicemia , Secreção de Insulina , Células Secretoras de Insulina , Osteocalcina , Animais , Osteocalcina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Hiperglicemia/metabolismo , Ratos , Secreção de Insulina/efeitos dos fármacos , Insulina/metabolismo , Glucose/metabolismo , Cálcio/metabolismo , Linhagem Celular , Sinalização do Cálcio/efeitos dos fármacosRESUMO
TMEM16 proteins, which function as Ca2+activated Cl channels are involved in regulating a wide variety of cellular pathways and functions. The modulators of Cl channels can be used for the moleculebased treatment of respiratory diseases, cystic fibrosis, tumors, cancer, osteoporosis and coronavirus disease 2019. The TMEM16 proteins link Ca2+ signaling, cellular electrical activity and lipid transport. Thus, deciphering these complex regulatory mechanisms may enable a more comprehensive understanding of the physiological functions of the TMEM16 proteins and assist in ascertaining the applicability of these proteins as potential pharmacological targets for the treatment of a range of diseases. The present review examined the structures, functions and characteristics of the different types of TMEM16 proteins, their association with the pathogenesis of various diseases and the applicability of TMEM16 modulatorbased treatment methods.
Assuntos
Anoctaminas , Proteínas de Transferência de Fosfolipídeos , Humanos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Anoctaminas/metabolismo , Anoctaminas/genética , Animais , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , COVID-19/metabolismo , SARS-CoV-2/metabolismo , Terapia de Alvo Molecular , Sinalização do Cálcio/efeitos dos fármacosRESUMO
To assess the toxicity of Hexabromocyclododecane (HBCD), the population, individual, and cellular biochemical parameters of the rotifer Brachionus plicatilis exposed to different concentrations of HBCD were investigated. The results showed that the population growth rate, reproductive period, and offspring number in B. plicatilis significantly decreased under 324 µg/L and 648 µg/L HBCD. Antioxidant enzyme activity and mRNA expression of CAT and Mn-SOD were promoted at low concentrations (32 µg/L and 64 µg/L) and inhibited at high concentrations (324 µg/L and 648 µg/L), while MDA content accumulated continuously with increasing HBCD concentrations, indicating that HBCD induced oxidation imbalance in rotifers. Further evidence was provided by the correlation between DNA fragmentation and physiological changes. The increased intercellular concentration of Ca2+ and the expression of CaM mRNA suggested that HBCD activated pathways related to calcium signaling. In summary, the excessive production of ROS induced by HBCD was considered to be the main cause of reproductive toxicity.
Assuntos
Hidrocarbonetos Bromados , Espécies Reativas de Oxigênio , Reprodução , Rotíferos , Poluentes Químicos da Água , Animais , Rotíferos/efeitos dos fármacos , Rotíferos/genética , Espécies Reativas de Oxigênio/metabolismo , Poluentes Químicos da Água/toxicidade , Hidrocarbonetos Bromados/toxicidade , Reprodução/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacosRESUMO
Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the inositol (1,4,5)-trisphosphate (IP3) signaling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET)-based cytosolic cyclic adenosine monophosphate (cAMP) sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, the addition of the α1-agonist, phenylephrine (PE, 3 µM), resulted in a FRET change of 21.20 ± 7.43%, and the addition of membrane-permeant IP3 derivative 2,3,6-tri-O-butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 µM) resulted in a peak of 20.31 ± 6.74%. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-aminoethyl diphenylborinate (2-APB, 2.5 µM) or Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs was not inhibited by 2-APB or Xestospongin-C. Finally, the localization of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin-C. These data support further investigation of the proarrhythmic nature and components of IP3-induced cAMP signaling to identify potential pharmacological targets.NEW & NOTEWORTHY This study shows that indirect activation of the IP3 pathway in atrial myocytes using phenylephrine and direct activation using IP3-AM leads to an increase in cAMP and is in part localized to the cell membrane. These changes can be pharmacologically inhibited using IP3R inhibitors. However, the cAMP rise in ventricular myocytes is independent of IP3R calcium release. Our data support further investigation into the proarrhythmic nature of IP3-induced cAMP signaling.
Assuntos
AMP Cíclico , Citosol , Transferência Ressonante de Energia de Fluorescência , Átrios do Coração , Receptores de Inositol 1,4,5-Trifosfato , Miócitos Cardíacos , Animais , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , AMP Cíclico/metabolismo , Átrios do Coração/metabolismo , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/citologia , Citosol/metabolismo , Ratos , Ratos Sprague-Dawley , Células Cultivadas , Animais Recém-Nascidos , Compostos de Boro/farmacologia , Fenilefrina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Inositol 1,4,5-Trifosfato/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacosRESUMO
Calcium ions (Ca2+) play crucial roles in sperm motility and fertilization. The copine (CPNE) family comprises several Ca2+-dependent phospholipid-binding proteins. Of these, CPNE1 is extensively expressed in mammalian tissues; however, its precise role in testicular development and spermatogenesis is yet to be fully characterized. In this study, we used proteomics to analyze testicular biopsies and found that levels of CPNE1 were significantly reduced in patients with non-obstructive azoospermia (defective spermatogenesis) compared to those in patients with obstructive azoospermia (physiological spermatogenesis). In mice, CPNE1 is expressed at various stages of germ cell development and is associated with the Golgi apparatus. Ultimately, CPNE1 is expressed in the flagella of mature sperms. To further examine the role of CPNE1, we developed a Cpne1 knockout mouse model. Analysis showed that the loss of Cpne1 did not impair testicular development, spermatogenesis, or sperm morphology and motility in physiological conditions. When treated with gadolinium (III) chloride or 2-aminoethoxydiphenyl borate, known inhibitors of store-operated Ca2+ entry, Ca2+ signals and sperm motility were significantly compromised in wild-type mice; however, both mechanisms were conserved in KO mice. These results suggested that CPNE1 is dispensable for testicular development, spermatogenesis or sperm motility in physiological conditions. In addition, CPNE1 may represent a target of Ca2+ channel inhibitors and may therefore be implicated in the regulation of Ca2+ signaling and sperm motility.
Assuntos
Azoospermia , Camundongos Knockout , Motilidade dos Espermatozoides , Espermatogênese , Espermatozoides , Masculino , Animais , Motilidade dos Espermatozoides/efeitos dos fármacos , Camundongos , Espermatogênese/efeitos dos fármacos , Azoospermia/metabolismo , Azoospermia/genética , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Humanos , Testículo/metabolismo , Testículo/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Sinalização do Cálcio/efeitos dos fármacosRESUMO
Pharmacological vitamin C (VC) has gained attention for its pro-oxidant characteristics and selective ability to induce cancer cell death. However, defining its role in cancer has been challenging due to its complex redox properties. In this study, using a human osteosarcoma (OS) model, we show that the redox-active property of VC is critical for inducing non-apoptotic cancer cell death via intracellular reactive oxygen species (ROS)-iron-calcium crosstalk and mitochondrial dysfunction. In both 2D and 3D OS cell culture models, only the oxidizable form of VC demonstrated potent dose-dependent cytotoxicity, while non-oxidizable and oxidized VC derivatives had minimal effects. Live-cell imaging showed that only oxidizable VC caused a surge in cytotoxic ROS, dependent on iron rather than copper. Inhibitors of ferroptosis, a form of iron-dependent cell death, along with classical apoptosis inhibitors, were unable to completely counteract the cytotoxic effects induced by VC. Further pharmacological and genetic inhibition analyses showed that VC triggers calcium release through inositol 1,4,5-trisphosphate receptors (IP3Rs), leading to mitochondrial ROS production and eventual cell death. RNA sequencing revealed down-regulation of genes involved in the mitochondrial electron transport chain and oxidative phosphorylation upon pharmacological VC treatment. Consistently, high-dose VC reduced mitochondrial membrane potential, oxidative phosphorylation, and ATP levels, with ATP reconstitution rescuing VC-induced cytotoxicity. In vivo OS xenograft studies demonstrated reduced tumor growth with high-dose VC administration, concomitant with the altered expression of mitochondrial ATP synthase (MT-ATP). These findings emphasize VC's potential clinical utility in osteosarcoma treatment by inducing mitochondrial metabolic dysfunction through a vicious intracellular ROS-iron-calcium cycle.
Assuntos
Ácido Ascórbico , Sinalização do Cálcio , Ferro , Mitocôndrias , Osteossarcoma , Oxirredução , Espécies Reativas de Oxigênio , Osteossarcoma/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Humanos , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Ferro/metabolismo , Animais , Oxirredução/efeitos dos fármacos , Camundongos , Linhagem Celular Tumoral , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologiaRESUMO
Aberrant Ca2+ signaling is an early hallmark of multiple neurodegenerative syndromes including Alzheimer's and Parkinson's disease (AD and PD) as well as classes of rare genetic disorders such as Spinocebellar Ataxias. Therapeutic strategies that target aberrant Ca2+ signals whilst allowing normal neuronal Ca2+ signals have been a challenge. In a recent study Princen et al., performed a screen in the tauP301L cell model of AD for drugs that could specifically ameliorate the excess Ca2+ entry observed. They identified a class of compounds referred to as ReS19-T that interact with Septins, previously identified as regulators of the Store-operated Ca2+ entry channel Orai. Drug treatment of the cellular model, a mouse model and human iPSC derived neurons alleviate cellular and systemic deficits associated with tauP301L. Comparison of Septin filament architecture in disease conditions with and without the drug treatment indicate that excess Ca2+ entry is a consequence of abnormal Septin filament architecture resulting in aberrant ER-PM contacts. The importance of membrane contacts for maintaining precise cellular signaling has been recognized previously. However, the molecular mechanism by which Septin filaments organize the ER-PM junctions to regulate Ca2+ entry through Orai remains to be fully understood.
Assuntos
Cálcio , Septinas , Animais , Humanos , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Proteína ORAI1/metabolismo , Septinas/metabolismoRESUMO
The gut microbiota and their metabolites are closely linked to obesity-related diseases, such as type 2 diabetes, but their causal relationship and underlying mechanisms remain largely elusive. Here, we found that dysbiosis-induced tyramine (TA) suppresses high-fat diet (HFD)-mediated insulin resistance in both Drosophila and mice. In Drosophila, HFD increases cytosolic Ca2+ signaling in enterocytes, which, in turn, suppresses intestinal lipid levels. 16 S rRNA sequencing and metabolomics revealed that HFD leads to increased prevalence of tyrosine decarboxylase (Tdc)-expressing bacteria and resulting tyramine production. Tyramine acts on the tyramine receptor, TyrR1, to promote cytosolic Ca2+ signaling and activation of the CRTC-CREB complex to transcriptionally suppress dietary lipid digestion and lipogenesis in enterocytes, while promoting mitochondrial biogenesis. Furthermore, the tyramine-induced cytosolic Ca2+ signaling is sufficient to suppress HFD-induced obesity and insulin resistance in Drosophila. In mice, tyramine intake also improves glucose tolerance and insulin sensitivity under HFD. These results indicate that dysbiosis-induced tyramine suppresses insulin resistance in both flies and mice under HFD, suggesting a potential therapeutic strategy for related metabolic disorders, such as diabetes.
Assuntos
Sinalização do Cálcio , Dieta Hiperlipídica , Microbioma Gastrointestinal , Resistência à Insulina , Tiramina , Animais , Tiramina/metabolismo , Tiramina/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Camundongos , Sinalização do Cálcio/efeitos dos fármacos , Obesidade/metabolismo , Obesidade/microbiologia , Obesidade/etiologia , Masculino , Drosophila/metabolismo , Disbiose/metabolismo , Disbiose/microbiologia , Camundongos Endogâmicos C57BL , Drosophila melanogaster/microbiologia , Drosophila melanogaster/metabolismo , Enterócitos/metabolismo , Enterócitos/efeitos dos fármacosRESUMO
Nitric oxide (NO) is widely recognized for its role in regulating renal function and blood pressure. However, the precise mechanisms by which NO affects renal epithelial cells remain understudied. Our previous research has shown that NO signaling in glomerular podocytes can be initiated by Angiotensin II (ANG II) but not by ATP. This study aims to elucidate the crucial interplay between the renin-angiotensin system (RAS) and NO production in podocytes. To conduct our research, we used cultured human podocytes and freshly isolated rat glomeruli. A variety of RAS peptides were used, alongside confocal microscopy, to detect NO production and NO/Ca2+ cross talk. Dynamic changes in the podocyte cytoskeleton, mediated by RAS-NO intracellular signaling, were observed using fluorescent labeling for F-actin and scanning probe microscopy. The experiments demonstrated that ANG II and ANG III generated high levels of NO by activating the angiotensin II type 2 receptor (AT2R). We did not detect functional MAS receptor presence in podocytes, and the moderate NO response to ANG 1-7 was also mediated through AT2R. Furthermore, NO production impacted intracellular Ca2+ signaling and correlated with an increase in podocyte volume and growth. Scanning probe experiments revealed that AT2R activation and the corresponding NO generation are responsible for the protrusion of podocyte lamellipodia. Taken together, our data indicate that AT2R activation enhances NO production in podocytes and subsequently mediates changes in Ca2+ signaling and podocyte volume dynamics. These mechanisms may play a significant role in both physiological and pathophysiological interactions between the RAS and podocytes.NEW & NOTEWORTHY The renin-angiotensin system plays a crucial role in the production of intracellular nitric oxide within podocytes. This mechanism operates through the activation of the angiotensin II type 2 receptor, leading to dynamic modifications in intracellular calcium levels and the actin filament network. This intricate process is vital for linking the activity of angiotensin receptors to podocyte function.
Assuntos
Angiotensina II , Óxido Nítrico , Podócitos , Receptor Tipo 2 de Angiotensina , Sistema Renina-Angiotensina , Transdução de Sinais , Podócitos/metabolismo , Óxido Nítrico/metabolismo , Sistema Renina-Angiotensina/fisiologia , Animais , Humanos , Angiotensina II/farmacologia , Receptor Tipo 2 de Angiotensina/metabolismo , Células Cultivadas , Angiotensina I/metabolismo , Ratos , Sinalização do Cálcio/efeitos dos fármacos , Masculino , Angiotensina III/metabolismo , Angiotensina III/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologiaRESUMO
Heart Failure with preserved ejection fraction (HFpEF) has a high rate of sudden cardiac death (SCD) and empirical treatment is ineffective. We developed a novel preclinical model of metabolic HFpEF that presents with stress-induced ventricular tachycardia (VT). Mechanistically, we discovered arrhythmogenic changes in intracellular Ca2+ handling distinct from the changes pathognomonic for heart failure with reduced ejection fraction. We further show that dantrolene, a stabilizer of the ryanodine receptor Ca2+ channel, attenuates HFpEF-associated arrhythmogenic Ca2+ handling in vitro and suppresses stress-induced VT in vivo. We propose ryanodine receptor stabilization as a mechanistic approach to mitigation of malignant VT in metabolic HFpEF.
Assuntos
Arritmias Cardíacas , Cálcio , Dantroleno , Modelos Animais de Doenças , Insuficiência Cardíaca , Canal de Liberação de Cálcio do Receptor de Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/tratamento farmacológico , Dantroleno/farmacologia , Volume Sistólico/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Humanos , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/tratamento farmacológico , Camundongos , Masculino , Sinalização do Cálcio/efeitos dos fármacosRESUMO
Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases such as thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 G-protein coupled receptors-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and transient receptor potential canonical 3 (TRPC3) channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells, contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation.