RESUMEN
Large-conductance, calcium-activated potassium channels (BK channels) and the Na/K-ATPase are expressed universally in vascular smooth muscle. The Na/K-ATPase may act via changes in the intracellular Ca2+ concentration mediated by the Na/Ca exchanger (NCX) and via Src kinase. Both pathways are known to regulate BK channels. Whether BK channels functionally interact in vascular smooth muscle cells with the Na/K-ATPase remains to be elucidated. Thus, this study addressed the hypothesis that BK channels limit ouabain-induced vasocontraction. Rat mesenteric arteries were studied using isometric myography, FURA-2 fluorimetry and proximity ligation assay. The BK channel blocker iberiotoxin potentiated methoxamine-induced contractions. The cardiotonic steroid, ouabain (10-5 M), induced a contractile effect of IBTX at basal tension prior to methoxamine administration and enhanced the pro-contractile effect of IBTX on methoxamine-induced contractions. These facilitating effects of ouabain were prevented by the inhibition of either NCX or Src kinase. Furthermore, inhibition of NCX or Src kinase reduced the BK channel-mediated negative feedback regulation of arterial contraction. The effects of NCX and Src kinase inhibition were independent of each other. Co-localization of the Na/K-ATPase and the BK channel was evident. Our data suggest that BK channels limit ouabain-induced vasocontraction by a dual mechanism involving the NCX and Src kinase signaling. The data propose that the NCX and the Src kinase pathways, mediating the ouabain-induced activation of the BK channel, act in an independent manner.
Asunto(s)
Canales de Potasio de Gran Conductancia Activados por el Calcio , Arterias Mesentéricas , Músculo Liso Vascular , Ouabaína , Intercambiador de Sodio-Calcio , ATPasa Intercambiadora de Sodio-Potasio , Familia-src Quinasas , Animales , Ouabaína/farmacología , Familia-src Quinasas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Ratas , Masculino , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Vasoconstricción/efectos de los fármacos , Ratas Wistar , Contracción Muscular/efectos de los fármacosRESUMEN
The functioning of the cardiovascular system is critical for embryo survival. Cardiac contractions depend on the sequential activation of different classes of voltage-gated ion channels. Understanding the fundamental features of these interactions is important for identifying the mechanisms of pathologies development in the myocardium. However, at present there is no consensus on which ion channels are involved in the formation of automaticity in the early embryonic stages. The aim of this study was to elucidate the expression of genes encoding various types of ion channels that are involved in the generation of electrical activity chicken heart at different stages of ontogenesis. We analyzed the expression of 14 genes from different families of ion channels. It was revealed that the expression profiles of ion channel genes change depending on the stages of ontogenesis. The HCN4, CACNA1D, SCN1A, SCN5A, KCNA1 genes have maximum expression at the tubular heart stage. In adult, a switch occurs to the higher expression of CACNA1C, KCNH6, RYR and SLC8A1 genes. This data correlated with the results obtained by the microelectrode method. It can be assumed that the automaticity of the tubular heart is mainly due to the mechanism of the «membrane-clock¼ (hyperpolarization-activated current (If), Ca2+-current L-type (ICaL), Na+-current (INa) and the slow component of the delayed rectifier K+-current (IKs)). Whereas in adult birds, the mechanism for generating electrical impulses is determined by both « membrane- clock¼ and «Ca2+-clock¼.
Asunto(s)
Pollos , Regulación del Desarrollo de la Expresión Génica , Corazón , Miocardio , Animales , Embrión de Pollo , Miocardio/metabolismo , Pollos/genética , Corazón/embriología , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismoRESUMEN
Parkinson's disease (PD) is a progressive neurodegenerative disorder that lacks effective treatment strategies to halt or delay its progression. The homeostasis of Ca2+ ions is crucial for ensuring optimal cellular functions and survival, especially for neuronal cells. In the context of PD, the systems regulating cellular Ca2+ are compromised, leading to Ca2+-dependent synaptic dysfunction, impaired neuronal plasticity, and ultimately, neuronal loss. Recent research efforts directed toward understanding the pathology of PD have yielded significant insights, particularly highlighting the close relationship between Ca2+ dysregulation, neuroinflammation, and neurodegeneration. However, the precise mechanisms driving the selective loss of dopaminergic neurons in PD remain elusive. The disruption of Ca2+ homeostasis is a key factor, engaging various neurodegenerative and neuroinflammatory pathways and affecting intracellular organelles that store Ca2+. Specifically, impaired functioning of mitochondria, lysosomes, and the endoplasmic reticulum (ER) in Ca2+ metabolism is believed to contribute to the disease's pathophysiology. The Na+-Ca2+ exchanger (NCX) is considered an important key regulator of Ca2+ homeostasis in various cell types, including neurons, astrocytes, and microglia. Alterations in NCX activity are associated with neurodegenerative processes in different models of PD. In this review, we will explore the role of Ca2+ dysregulation and neuroinflammation as primary drivers of PD-related neurodegeneration, with an emphasis on the pivotal role of NCX in the pathology of PD. Consequently, NCXs and their interplay with intracellular organelles may emerge as potentially pivotal players in the mechanisms underlying PD neurodegeneration, providing a promising avenue for therapeutic intervention aimed at halting neurodegeneration.
Asunto(s)
Calcio , Enfermedades Neuroinflamatorias , Enfermedad de Parkinson , Intercambiador de Sodio-Calcio , Humanos , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Calcio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Animales , Orgánulos/metabolismo , Homeostasis , Mitocondrias/metabolismo , Mitocondrias/patologíaRESUMEN
BACKGROUND: While calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na+/Ca2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were evaluated. RESULTS: Partial and complete blockage of Ca2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation. CONCLUSIONS: Inhibition of outflux and influx of Ca2+ triggered similar effects, thus indicating that both forward and reverse Ca2+ transport through NCX exchangers are essential for sperm capacitation.
Asunto(s)
Calcio , Intercambiador de Sodio-Calcio , Capacitación Espermática , Animales , Masculino , Capacitación Espermática/efectos de los fármacos , Intercambiador de Sodio-Calcio/metabolismo , Intercambiador de Sodio-Calcio/efectos de los fármacos , Calcio/metabolismo , Porcinos , Espermatozoides/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Motilidad Espermática/efectos de los fármacos , Reacción Acrosómica/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacosRESUMEN
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.
Asunto(s)
Calcio , Venenos de Cnidarios , Miocitos Cardíacos , Venas Pulmonares , Intercambiador de Sodio-Calcio , Animales , Venas Pulmonares/metabolismo , Venas Pulmonares/citología , Venas Pulmonares/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Cobayas , Calcio/metabolismo , Venenos de Cnidarios/farmacología , Intercambiador de Sodio-Calcio/metabolismo , Sodio/metabolismo , Masculino , Potenciales de Acción/efectos de los fármacos , Canales de Sodio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Compuestos de Anilina/farmacología , Sulfonamidas/farmacología , Señalización del Calcio/efectos de los fármacos , Isoquinolinas , Éteres FenílicosRESUMEN
BACKGROUND: Hyponatremia, frequently observed in patients with chronic kidney disease, is associated with increased cardiovascular morbidity and mortality. Hyponatremia or low osmolality induces oxidative stress and cell death, both of which accelerate vascular calcification (VC), a critical phenotype in patients with chronic kidney disease. Whether hyponatremia or low osmolality plays a role in the pathogenesis of VC is unknown. METHODS: Human vascular smooth muscle cells (VSMCs) and mouse aortic rings were cultured in various osmotic conditions and calcifying medium supplemented with high calcium and phosphate. The effects of low osmolality on phenotypic change and oxidative stress in the cultured VSMCs were examined. Microarray analysis was conducted to determine the main signaling pathway of osmolality-related VC. The transcellular sodium and calcium ions flux across the VSMCs were visualized by live imaging. Furthermore, the effect of osmolality on calciprotein particles (CPPs) was investigated. Associations between arterial intimal calcification and hyponatremia or low osmolality were examined by a cross-sectional study using human autopsy specimens obtained in the Hisayama Study. RESULTS: Low osmolality exacerbated calcification of the ECM (extracellular matrix) of cultured VSMCs and mouse aortic rings. Oxidative stress and osteogenic differentiation of VSMCs were identified as the underlying mechanisms responsible for low osmolality-induced VC. Microarray analysis showed that low osmolality activated the Rac1 (Ras-related C3 botulinum toxin substrate 1)-Akt (protein kinase B) pathway and reduced NCX1 (Na-Ca exchanger 1) expression. Live imaging showed synchronic calcium ion efflux and sodium ion influx via NCX1 when extracellular sodium ion concentrations were increased. An NCX1 inhibitor promoted calcifying media-induced VC by reducing calcium ion efflux. Furthermore, low osmolality accelerated the generation and maturation steps of CPPs. The cross-sectional study of human autopsy specimens showed that hyponatremia and low osmolality were associated with a greater area of arterial intimal calcification. CONCLUSIONS: Hyponatremia and low osmolality promote VC through multiple cellular processes, including the Rac1-Akt pathway activation.
Asunto(s)
Hiponatremia , Músculo Liso Vascular , Miocitos del Músculo Liso , Estrés Oxidativo , Calcificación Vascular , Animales , Humanos , Hiponatremia/metabolismo , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Concentración Osmolar , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Masculino , Células Cultivadas , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Ratones Endogámicos C57BL , Femenino , Transducción de Señal , Intercambiador de Sodio-Calcio/metabolismo , Anciano , Estudios Transversales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratones , Osteogénesis , Persona de Mediana Edad , Modelos Animales de Enfermedad , Calcio/metabolismo , Fenotipo , Proteína de Unión al GTP rac1RESUMEN
OBJECTIVE: Hepatic Ca2+ signaling has been identified as a crucial key factor in driving gluconeogenesis. The involvement of mitochondria in hormone-induced Ca2+ signaling and their contribution to metabolic activity remain, however, poorly understood. Moreover, the molecular mechanism governing the mitochondrial Ca2+ efflux signaling remains unresolved. This study investigates the role of the Na+/Ca2+ exchanger, NCLX, in modulating hepatic mitochondrial Ca2+ efflux, and examines its physiological significance in hormonal hepatic Ca2+ signaling, gluconeogenesis, and mitochondrial bioenergetics. METHODS: Primary mouse hepatocytes from both an AAV-mediated conditional hepatic-specific and a total mitochondrial Na+/Ca2+ exchanger, NCLX, knockout (KO) mouse models were employed for fluorescent monitoring of purinergic and glucagon/vasopressin-dependent mitochondrial and cytosolic hepatic Ca2+ responses in cultured hepatocytes. Isolated liver mitochondria and permeabilized primary hepatocytes were used to analyze the ion-dependence of Ca2+ efflux. Utilizing the conditional hepatic-specific NCLX KO model, the rate of gluconeogenesis was assessed by first monitoring glucose levels in fasted mice, and subsequently subjecting the mice to a pyruvate tolerance test while monitoring their blood glucose. Additionally, cultured primary hepatocytes from both genotypes were assessed in vitro for glucagon-dependent glucose production and cellular bioenergetics through glucose oxidase assay and Seahorse respirometry, respectively. RESULTS: Analysis of Ca2+ responses in isolated liver mitochondria and cultured primary hepatocytes from NCLX KO versus WT mice showed that NCLX serves as the principal mechanism for mitochondrial calcium extrusion in hepatocytes. We then determined the role of NCLX in glucagon and vasopressin-induced Ca2+ oscillations. Consistent with previous studies, glucagon and vasopressin triggered Ca2+ oscillations in WT hepatocytes, however, the deletion of NCLX resulted in selective elimination of mitochondrial, but not cytosolic, Ca2+ oscillations, underscoring NCLX's pivotal role in mitochondrial Ca2+ regulation. Subsequent in vivo investigation for hepatic NCLX role in gluconeogenesis revealed that, as opposed to WT mice which maintained normoglycemic blood glucose levels when fasted, conditional hepatic-specific NCLX KO mice exhibited a faster drop in glucose levels, becoming hypoglycemic. Furthermore, KO mice showed deficient conversion of pyruvate to glucose when challenged under fasting conditions. Concurrent in vitro assessments showed impaired glucagon-dependent glucose production and compromised bioenergetics in KO hepatocytes, thereby underscoring NCLX's significant contribution to hepatic glucose metabolism. CONCLUSIONS: The study findings demonstrate that NCLX acts as the primary Ca2+ efflux mechanism in hepatocytes. NCLX is indispensable for regulating hormone-induced mitochondrial Ca2+ oscillations, mitochondrial metabolism, and sustenance of hepatic gluconeogenesis.
Asunto(s)
Señalización del Calcio , Calcio , Glucagón , Gluconeogénesis , Hepatocitos , Ratones Noqueados , Intercambiador de Sodio-Calcio , Animales , Ratones , Hepatocitos/metabolismo , Calcio/metabolismo , Glucagón/metabolismo , Masculino , Intercambiador de Sodio-Calcio/metabolismo , Mitocondrias Hepáticas/metabolismo , Hígado/metabolismo , Vasopresinas/metabolismo , Células Cultivadas , Glucosa/metabolismo , Ratones Endogámicos C57BLRESUMEN
The Renin-Angiotensin-Aldosterone System (RAAS) has been implicated in systemic and neurogenic hypertension. The infusion of RAAS inhibitors blunted arterial pressure and efficacy of use-dependent synaptic transmission in sympathetic ganglia. The current investigation aims to elucidate the impact of RAAS-mediated receptors on left ventricular cardiomyocytes and the role of the sarcolemma-bound carrier system in the heart of the hypertensive transgene model. A significant increase in mRNA and the protein expression for angiotensin II (AngII) receptor subtype-1 (AT1R) was observed in (mREN2)27 transgenic compared to the normotensive rodents. Concurrently, there was an upregulation in AT1R and a downregulation in the MAS1 proto-oncogene protein receptor as well as the AngII subtype-2 receptor in hypertensive rodents. There were modifications in the expressions of sarcolemma Na+-K+-ATPase, Na+-Ca2+ exchanger, and Sarcoendoplasmic Reticulum Calcium ATPase in the transgenic hypertensive model. These observations suggest chronic RAAS activation led to a shift in receptor balance favoring augmented cardiac contractility and disruption in calcium handling through modifications of membrane-bound carrier proteins and blood pressure. The study provides insight into mechanisms underlying RAAS-mediated cardiac dysfunction and highlights the potential value of targeting the protective arm of AngII in hypertension.
Asunto(s)
Ventrículos Cardíacos , Hipertensión , Sistema Renina-Angiotensina , Animales , Hipertensión/metabolismo , Ventrículos Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 1/genética , Ratas , Proto-Oncogenes Mas , Presión Sanguínea , Masculino , Ratones , Receptor de Angiotensina Tipo 2/metabolismo , Receptor de Angiotensina Tipo 2/genética , Sarcolema/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Intercambiador de Sodio-Calcio/metabolismo , Intercambiador de Sodio-Calcio/genética , Ratones TransgénicosRESUMEN
The sarcolemmal Ca2+ efflux pathways, Na+-Ca2+-exchanger (NCX) and Ca2+-ATPase (PMCA), play a crucial role in the regulation of intracellular Ca2+ load and Ca2+ transient in cardiomyocytes. The distribution of these pathways between the t-tubular and surface membrane of ventricular cardiomyocytes varies between species and is not clear in human. Moreover, several studies suggest that this distribution changes during the development and heart diseases. However, the consequences of NCX and PMCA redistribution in human ventricular cardiomyocytes have not yet been elucidated. In this study, we aimed to address this point by using a mathematical model of the human ventricular myocyte incorporating t-tubules, dyadic spaces, and subsarcolemmal spaces. Effects of various combinations of t-tubular fractions of NCX and PMCA were explored, using values between 0.2 and 1 as reported in animal experiments under normal and pathological conditions. Small variations in the action potential duration (≤ 2%), but significant changes in the peak value of cytosolic Ca2+ transient (up to 17%) were observed at stimulation frequencies corresponding to the human heart rate at rest and during activity. The analysis of model results revealed that the changes in Ca2+ transient induced by redistribution of NCX and PMCA were mainly caused by alterations in Ca2+ concentrations in the subsarcolemmal spaces and cytosol during the diastolic phase of the stimulation cycle. The results suggest that redistribution of both transporters between the t-tubular and surface membranes contributes to changes in contractility in human ventricular cardiomyocytes during their development and heart disease and may promote arrhythmogenesis.
Asunto(s)
Calcio , Ventrículos Cardíacos , Miocitos Cardíacos , Sarcolema , Intercambiador de Sodio-Calcio , Humanos , Miocitos Cardíacos/metabolismo , Calcio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Ventrículos Cardíacos/metabolismo , Sarcolema/metabolismo , Potenciales de Acción , Señalización del Calcio , Membrana Celular/metabolismo , Modelos Biológicos , Modelos CardiovascularesRESUMEN
Sodium-calcium exchanger (NCX) proteins are ubiquitously expressed and play a pivotal role in cellular calcium homeostasis by mediating uphill calcium efflux across the cell membrane. Intracellular calcium allosterically regulates the exchange activity by binding to two cytoplasmic calcium-binding domains, CBD1 and CBD2. However, the calcium-binding affinities of these domains are seemingly inadequate to sense physiological calcium oscillations. Previously, magnesium binding to either domain was shown to tune their affinity for calcium, bringing it into the physiological range. However, while the magnesium-binding site of CBD2 was identified, the identity of the CBD1 magnesium site remains elusive. Here, using molecular dynamics in combination with differential scanning fluorimetry and mutational analysis, we pinpoint the magnesium-binding site in CBD1. Specifically, among four calcium-binding sites (Ca1-Ca4) in this domain, only Ca1 can accommodate magnesium with an affinity similar to its free intracellular concentration. Moreover, our results provide mechanistic insights into the modulation of the regulatory calcium affinity by magnesium, which allows an adequate NCX activity level throughout varying physiological needs.
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Calcio , Magnesio , Intercambiador de Sodio-Calcio , Intercambiador de Sodio-Calcio/química , Intercambiador de Sodio-Calcio/metabolismo , Intercambiador de Sodio-Calcio/genética , Magnesio/metabolismo , Calcio/metabolismo , Sitios de Unión , Humanos , Regulación Alostérica , Simulación de Dinámica Molecular , Unión Proteica , Dominios ProteicosRESUMEN
We explored physiological effects of the sodium-glucose co-transporter-2 inhibitor empagliflozin on intact experimentally hypertrophic murine hearts following transverse aortic constriction (TAC). Postoperative drug (2-6 weeks) challenge resulted in reduced late Na+ currents, and increased phosphorylated (p-)CaMK-II and Nav1.5 but not total (t)-CaMK-II, and Na+/Ca2+ exchanger expression, confirming previous cardiomyocyte-level reports. It rescued TAC-induced reductions in echocardiographic ejection fraction and fractional shortening, and diastolic anterior and posterior wall thickening. Dual voltage- and Ca2+-optical mapping of Langendorff-perfused hearts demonstrated that empagliflozin rescued TAC-induced increases in action potential durations at 80% recovery (APD80), Ca2+ transient peak signals and durations at 80% recovery (CaTD80), times to peak Ca2+ (TTP100) and Ca2+ decay constants (Decay30-90) during regular 10-Hz stimulation, and Ca2+ transient alternans with shortening cycle length. Isoproterenol shortened APD80 in sham-operated and TAC-only hearts, shortening CaTD80 and Decay30-90 but sparing TTP100 and Ca2+ transient alternans in all groups. All groups showed similar APD80, and TAC-only hearts showed greater CaTD80, heterogeneities following isoproterenol challenge. Empagliflozin abolished or reduced ventricular tachycardia and premature ventricular contractions and associated re-entrant conduction patterns, in isoproterenol-challenged TAC-operated hearts following successive burst pacing episodes. Empagliflozin thus rescues TAC-induced ventricular hypertrophy and systolic functional, Ca2+ homeostatic, and pro-arrhythmogenic changes in intact hearts.
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Compuestos de Bencidrilo , Calcio , Glucósidos , Homeostasis , Animales , Compuestos de Bencidrilo/farmacología , Glucósidos/farmacología , Ratones , Calcio/metabolismo , Homeostasis/efectos de los fármacos , Masculino , Potenciales de Acción/efectos de los fármacos , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/tratamiento farmacológico , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/cirugía , Ratones Endogámicos C57BL , Isoproterenol/farmacología , Modelos Animales de EnfermedadRESUMEN
Consecutive interactions of 3Na+ or 1Ca2+ with the Na+/Ca2+ exchanger (NCX) result in an alternative exposure (access) of the cytosolic and extracellular vestibules to opposite sides of the membrane, where ion-induced transitions between the outward-facing (OF) and inward-facing (IF) conformational states drive a transport cycle. Here, we investigate sub-state populations of apo and ion-bound species in the OF and IF states by analyzing detergent-solubilized and nanodisc-reconstituted preparations of NCX_Mj with 19F-NMR. The 19F probe was covalently attached to the cysteine residues at entry locations of the cytosolic and extracellular vestibules. Multiple sub-states of apo and ion-bound species were observed in nanodisc-reconstituted (but not in detergent-solubilized) NCX_Mj, meaning that the lipid-membrane environment preconditions multiple sub-state populations toward the OF/IF swapping. Most importantly, ion-induced sub-state redistributions occur within each major (OF or IF) state, where sub-state interconversions may precondition the OF/IF swapping. In contrast with large changes in population redistributions, the sum of sub-state populations within each inherent state (OF or IF) remains nearly unchanged upon ion addition. The present findings allow the further elucidation of structure-dynamic modules underlying ion-induced conformational changes that determine a functional asymmetry of ion access/translocation at opposite sides of the membrane and ion transport rates concurring physiological demands.
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Detergentes , Conformación Proteica , Intercambiador de Sodio-Calcio , Detergentes/química , Intercambiador de Sodio-Calcio/química , Intercambiador de Sodio-Calcio/metabolismo , Intercambiador de Sodio-Calcio/genética , Iones/química , Nanoestructuras/química , Solubilidad , Animales , Espectroscopía de Resonancia Magnética/métodosRESUMEN
Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na+/Ca2+ exchanger isoform-3 (NCX3, SLC8A3) was presumed to function as major Ca2+ transporter responsible for Ca2+ extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV-mediated mineralization process and its impact on bone formation. Using differentiated MC3T3-E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca2+ deposition due to reduced Ca2+ entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3-/-) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, µCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3-/- mice, thus supporting the critical role of NCX3 in facilitating Ca2+ uptake into the MVs to initiate osteoblast-mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization-related disorders.
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Calcificación Fisiológica , Calcio , Osteoblastos , Intercambiador de Sodio-Calcio , Animales , Femenino , Masculino , Ratones , Calcio/metabolismo , Diferenciación Celular , Línea Celular , Vesículas Extracelulares/metabolismo , Ratones Noqueados , Osteoblastos/metabolismo , Osteogénesis , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Signs and symptoms of hypernatremia largely indicate central nervous system dysfunction. Acute hypernatremia can cause demyelinating lesions similar to that observed in osmotic demyelination syndrome (ODS). We have previously demonstrated that microglia accumulate in ODS lesions and minocycline protects against ODS by inhibiting microglial activation. However, the direct effect of rapid rise in the sodium concentrations on microglia is largely unknown. In addition, the effect of chronic hypernatremia on microglia also remains elusive. Here, we investigated the effects of acute (6 or 24â¯h) and chronic (the extracellular sodium concentration was increased gradually for at least 7 days) high sodium concentrations on microglia using the microglial cell line, BV-2. We found that both acute and chronic high sodium concentrations increase NOS2 expression and nitric oxide (NO) production. We also demonstrated that the expression of nuclear factor of activated T-cells-5 (NFAT5) is increased by high sodium concentrations. Furthermore, NFAT5 knockdown suppressed NOS2 expression and NO production. We also demonstrated that high sodium concentrations decreased intracellular Ca2+ concentration and an inhibitor of Na+/Ca2+ exchanger, NCX, suppressed a decrease in intracellular Ca2+ concentrations and NOS2 expression and NO production induced by high sodium concentrations. Furthermore, minocycline inhibited NOS2 expression and NO production induced by high sodium concentrations. These in vitro data suggest that microglial activity in response to high sodium concentrations is regulated by NFAT5 and Ca2+ efflux through NCX and is suppressed by minocycline.
Asunto(s)
Hipernatremia , Microglía , Minociclina , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico , Microglía/metabolismo , Microglía/efectos de los fármacos , Animales , Óxido Nítrico/metabolismo , Hipernatremia/metabolismo , Hipernatremia/patología , Hipernatremia/genética , Minociclina/farmacología , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Sodio/metabolismo , Línea Celular , Calcio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Intercambiador de Sodio-Calcio/genética , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/genéticaRESUMEN
The Na+-Ca2+ exchanger (NCX1) is the dominant Ca2+ extrusion mechanism in cardiac myocytes. NCX1 activity is inhibited by intracellular Na+ via a process known as Na+-dependent inactivation. A central question is whether this inactivation plays a physiological role in heart function. Using CRISPR/Cas9, we inserted the K229Q mutation in the gene (Slc8a1) encoding for NCX1. This mutation removes the Na+-dependent inactivation while preserving transport properties and other allosteric regulations. NCX1 mRNA levels, protein expression, and protein localization are unchanged in K229Q male mice. However, they exhibit reduced left ventricular ejection fraction and fractional shortening, while displaying a prolonged QT interval. K229Q ventricular myocytes show enhanced NCX1 activity, resulting in action potential prolongation, higher incidence of aberrant action potentials, a faster decline of Ca2+ transients, and depressed cell shortening. The results demonstrate that NCX1 Na+-dependent inactivation plays an essential role in heart function by affecting both cardiac excitability and contractility.
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Potenciales de Acción , Calcio , Miocitos Cardíacos , Intercambiador de Sodio-Calcio , Sodio , Intercambiador de Sodio-Calcio/metabolismo , Intercambiador de Sodio-Calcio/genética , Animales , Miocitos Cardíacos/metabolismo , Masculino , Sodio/metabolismo , Ratones , Calcio/metabolismo , Contracción Miocárdica/fisiología , Contracción Miocárdica/genética , Corazón/fisiología , Humanos , Mutación , Sistemas CRISPR-CasRESUMEN
Elevated intracellular sodium Nai adversely affects mitochondrial metabolism and is a common feature of heart failure. The reversibility of acute Na induced metabolic changes is evaluated in Langendorff perfused rat hearts using the Na/K ATPase inhibitor ouabain and the myosin-uncoupler para-aminoblebbistatin to maintain constant energetic demand. Elevated Nai decreases Gibb's free energy of ATP hydrolysis, increases the TCA cycle intermediates succinate and fumarate, decreases ETC activity at Complexes I, II and III, and causes a redox shift of CoQ to CoQH2, which are all reversed on lowering Nai to baseline levels. Pseudo hypoxia and stabilization of HIF-1α is observed despite normal tissue oxygenation. Inhibition of mitochondrial Na/Ca-exchange with CGP-37517 or treatment with the mitochondrial ROS scavenger MitoQ prevents the metabolic alterations during Nai elevation. Elevated Nai plays a reversible role in the metabolic and functional changes and is a novel therapeutic target to correct metabolic dysfunction in heart failure.
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Mitocondrias Cardíacas , Sodio , Animales , Ratas , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Sodio/metabolismo , Masculino , Miocardio/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/tratamiento farmacológico , Adenosina Trifosfato/metabolismo , Ciclo del Ácido Cítrico/efectos de los fármacos , Ratas Sprague-Dawley , Compuestos Organofosforados/farmacología , Compuestos Organofosforados/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Ubiquinona/metabolismo , Ubiquinona/análogos & derivados , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Oxidación-Reducción , Ácido Succínico/metabolismoRESUMEN
Although bortezomib (BTZ) is the cornerstone of anti-multiple myeloma (MM) therapy, the inevitable primary and secondary drug resistance still seriously affects the prognosis of patients. New treatment strategies are in need. Sodium-calcium exchanger 1 (NCX1) is a calcium-permeable ion transporter on the membrane, and our previous studies showed that low NCX1 confers inferior viability in MM cells and suppressed osteoclast differentiation. However, the effect of NCX1 on BTZ sensitivity of MM and its possible mechanism remain unclear. In this study, we investigated the effect of NCX1 on BTZ sensitivity in MM, focusing on cellular processes of autophagy and cell viability. Our results provide evidence that NCX1 expression correlates with MM disease progression and low NCX1 expression increases BTZ sensitivity. NCX1/Ca2+ triggered autophagic flux through non-canonical NFκB pathway in MM cells, leading to attenuated the sensitivity of BTZ. Knockdown or inhibition of NCX1 could potentiate the anti-MM activity of BTZ in vitro and vivo, and inhibition of autophagy sensitized NCX1-overexpressing MM cells to BTZ. In general, this work implicates NCX1 as a potential therapeutic target in MM with BTZ resistance and provides novel mechanistic insights into its vital role in combating BTZ resistance.
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Autofagia , Bortezomib , Mieloma Múltiple , Intercambiador de Sodio-Calcio , Intercambiador de Sodio-Calcio/metabolismo , Intercambiador de Sodio-Calcio/genética , Humanos , Autofagia/efectos de los fármacos , Animales , Bortezomib/farmacología , Mieloma Múltiple/patología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/genética , Línea Celular Tumoral , Ratones , Calcio/metabolismo , Resistencia a Antineoplásicos/genética , FN-kappa B/metabolismo , Supervivencia Celular/efectos de los fármacosRESUMEN
Diabetes is commonly associated with an elevated level of reactive carbonyl species due to alteration of glucose and fatty acid metabolism. These metabolic changes cause an abnormality in cardiac Ca2+ regulation that can lead to cardiomyopathies. In this study, we explored how the reactive α-dicarbonyl methylglyoxal (MGO) affects Ca2+ regulation in mouse ventricular myocytes. Analysis of intracellular Ca2+ dynamics revealed that MGO (200 µM) increases action potential (AP)-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load, with a limited effect on L-type Ca2+ channel-mediated Ca2+ transients and SERCA-mediated Ca2+ uptake. At the same time, MGO significantly slowed down cytosolic Ca2+ extrusion by Na+/Ca2+ exchanger (NCX). MGO also increased the frequency of Ca2+ waves during rest and these Ca2+ release events were abolished by an external solution with zero [Na+] and [Ca2+]. Adrenergic receptor activation with isoproterenol (10 nM) increased Ca2+ transients and SR Ca2+ load, but it also triggered spontaneous Ca2+ waves in 27% of studied cells. Pretreatment of myocytes with MGO increased the fraction of cells with Ca2+ waves during adrenergic receptor stimulation by 163%. Measurements of intracellular [Na+] revealed that MGO increases cytosolic [Na+] by 57% from the maximal effect produced by the Na+-K+ ATPase inhibitor ouabain (20 µM). This increase in cytosolic [Na+] was a result of activation of a tetrodotoxin-sensitive Na+ influx, but not an inhibition of Na+-K+ ATPase. An increase in cytosolic [Na+] after treating cells with ouabain produced similar effects on Ca2+ regulation as MGO. These results suggest that protein carbonylation can affect cardiac Ca2+ regulation by increasing cytosolic [Na+] via a tetrodotoxin-sensitive pathway. This, in turn, reduces Ca2+ extrusion by NCX, causing SR Ca2+ overload and spontaneous Ca2+ waves.