RESUMEN
The surface expression of Na/K-ATPase α1 (NKA) is significantly reduced in primary prostate tumors and further decreased in bone metastatic lesions. Here, we show that the loss of cell surface expression of NKA induces epithelial-mesenchymal transition (EMT) and promotes metastatic potential and tumor growth of prostate cancer (PCa) by decreasing the expression of E-cadherin and increasing c-Myc expression via the activation of Src/FAK pathways. Mechanistically, reduced surface expression of NKA in PCa is due to increased endocytosis through the activation of NKA/Src receptor complex. Using a high-throughput NKA ligand-screening platform, we have discovered MB5 as an inverse agonist of the NKA/Src receptor complex, capable of blocking the endocytosis of NKA. MB5 treatment increased NKA expression and E-cadherin in PCa cells, which reversed EMT and consequently decreased the invasion and growth of spheroid models and tumor xenografts. Thus, we have identified a hitherto unrecognized mechanism that regulates EMT and invasiveness of PCa and demonstrated for the first time the feasibility of identifying inverse agonists of receptor NKA/Src complex and their potential utility as anticancer drugs. We, therefore, conclude that cell surface expression of α1 NKA can be targeted for the development of new therapeutics against aggressive PCa and that MB5 may serve as a prototype for drug development against EMT in metastatic PCa.
Asunto(s)
Agonismo Inverso de Drogas , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/fisiología , Neoplasias de la Próstata/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ouabaína/farmacología , Tiamina/análogos & derivados , Tiamina/farmacología , Tiamina/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Successful drug repurposing relies on the understanding of molecular mechanisms of the target compound. Cardiac glycosides have demonstrated potent anticancer activities; however, the pharmacological mechanisms underlying their anticancer effects remained elusive, which has restricted their further development in cancer treatment. A bottleneck is the lack of comprehensive understanding about genes and signaling pathways that are altered at the early stage of drug treatment, which is key to understand how they inhibit cancer. To address this issue, we first investigated the anticancer effects of a panel of 68 naturally isolated cardiac glycosides. Our results illustrate critical structure activity relationship of these compounds on cancer cell survival. We confirmed the anticancer effect of cardiac glycoside in mouse tumor xenografts. Through RNA sequencing, quantitative PCR and immunoblotting, we show that cardiac glycoside first activated autophagy and then induced apoptosis. Further activating autophagy by rapamycin or inhibiting apoptosis by caspase inhibitor mitigated cardiac glycoside-induced cell death, whereas inhibiting autophagy by RNA interference-mediated depletion of critical autophagy genes enhanced cell death. While depletion of Na/K-ATPase, the protein target of cardiac glycosides, by RNA interference inhibited both autophagy activation and apoptosis induction by cardiac glycoside, expression of human, but not rodent Na/K-ATPase, increased cell sensitivity to cardiac glycoside. In conclusion, our analyses reveal sequential activation of autophagy and apoptosis during early stages of cardiac glycoside treatment and indicate the importance of Na/K-ATPase in their anticancer effects.
Asunto(s)
Antineoplásicos/uso terapéutico , Glicósidos Cardíacos/uso terapéutico , Neoplasias/enzimología , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Células A549 , Animales , Antineoplásicos/farmacología , Glicósidos Cardíacos/farmacología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/fisiología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Neuronal Na+/K+-ATPase is responsible for the maintenance of ionic gradient across plasma membrane. In doing so, in a healthy brain, Na+/K+-ATPase activity accounts for nearly half of total brain energy consumption. The α3-subunit containing Na+/K+-ATPase expression is restricted to neurons. Heterozygous mutations within α3-subunit leads to Rapid-onset Dystonia Parkinsonism, Alternating Hemiplegia of Childhood and other neurological and neuropsychiatric disorders. Additionally, proteins such as α-synuclein, amyloid-ß, tau and SOD1 whose aggregation is associated to neurodegenerative diseases directly bind and impair α3-Na+/K+-ATPase activity. The review will provide a summary of neuronal α3-Na+/K+-ATPase functional properties, expression pattern, protein-protein interactions at the plasma membrane, biophysical properties (distribution and lateral diffusion). Lastly, the role of α3-Na+/K+-ATPase in neurological and neurodegenerative disorders will be discussed. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.
Asunto(s)
Trastornos Mentales/enzimología , Trastornos Mentales/genética , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/genética , Neuronas/enzimología , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Animales , Trastornos Distónicos/diagnóstico , Trastornos Distónicos/enzimología , Trastornos Distónicos/genética , Hemiplejía/diagnóstico , Hemiplejía/enzimología , Hemiplejía/genética , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Trastornos Mentales/diagnóstico , Mutación/genética , Enfermedades Neurodegenerativas/diagnóstico , Neuronas/patologíaRESUMEN
OBJECTIVES: Temozolomide (TMZ) is one of the most commonly used clinical drugs for glioblastoma (GBM) treatment, but its drug sensitivity needs to be improved. Gamabufotalin (CS-6), the primary component of the traditional Chinese medicine "ChanSu," was shown to have strong anti-cancer activity. However, more efforts should be directed towards reducing its toxicity or effective treatment doses. METHODS: Target fishing experiment, Western blotting, PCR, confocal immunofluorescence and molecular cloning techniques were performed to search for possible downstream signalling pathways. In addition, GBM xenografts were used to further determine the potential molecular mechanisms of the synergistic effects of CS-6 and TMZ in vivo. RESULTS: Mechanistic research revealed a negative feedback loop between ATP1A3 and AQP4 through which CS-6 inhibited GBM growth and mediated the synergistic treatment effect of CS-6 and TMZ. In addition, by mutating potential amino acid residues of ATP1A3, which were predicted by modelling and docking to interact with CS-6, we demonstrated that abrogating hydrogen bonding of the amino acid Thr794 interferes with the activation of ATP1A3 by CS-6 and that the Thr794Ala mutation directly affects the synergistic treatment efficacy of CS-6 and TMZ. CONCLUSIONS: As the main potential target of CS-6, ATP1A3 activation critically depends on the hydrogen bonding of Thr794 with CS-6. The combination of CS-6 and TMZ could significantly reduce the therapeutic doses and promote the anti-cancer efficacy of CS-6/TMZ monotherapy.
Asunto(s)
Acuaporina 4/metabolismo , Bufanólidos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Proteínas de Neoplasias/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Temozolomida/farmacología , Animales , Acuaporina 4/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Proteínas de Neoplasias/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by progressive lung scarring and excessive extracellular matrix depositon. When stimulated, alveolar epithelial cells (AECs) are aberrantly activated, the expression of profibrotic molecules is enhanced, and lung fibrosis is promoted, but the mechanism for this is unclear. It has been reported that a downregulation of the Na,KATPase ß1 subunit in renal epithelial cells is involved in renal fibrosis development, but the role of this protein in lung fibrosis remains unknown. In the present study, the expression of the Na,KATPase ß1 subunit was revealed to be markedly decreased in AECs of patients with IPF and a bleomycininduced pulmonary fibrosis mouse model. Treatment with transforming growth factor ß1 led to significantly downregulation of the Na,KATPase ß1 subunit in lung adenocarcioma A549 cells. Furthermore, the knockdown of the Na,KATPase ß1 subunit in A549 cells resulted in the upregulation of profibrotic molecules, activation of the neurogenic locus notch homolog protein 1 and extracellular signalregulated kinase 1/2 signaling pathways and induction of endoplasmic reticulum stress. These findings reveal that the downregulation of the Na,KATPase ß1 subunit enhances the expression of profibrotic molecules in AECs and may contribute to IPF pathogenesis.
Asunto(s)
Células Epiteliales Alveolares/enzimología , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Fibrosis Pulmonar Idiopática/enzimología , Sistema de Señalización de MAP Quinasas , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Células A549 , Adulto , Células Epiteliales Alveolares/patología , Animales , Estrés del Retículo Endoplásmico , Humanos , Fibrosis Pulmonar Idiopática/patología , Masculino , Ratones , Persona de Mediana EdadRESUMEN
OBJECTIVE: Cardiomyocyte hypertrophy is considered to be a compensatory process of heart suffering from pathological damages. This study aimed to evaluate effects of Na+/K+ APTaseα2 (NAKα2) on isoprenaline (ISO) induced cardiomyocyte hypertrophy. MATERIALS AND METHODS: Mouse atrial cardiomyocytes were cultured and treated with ISO to establish cardiomyocyte hypertrophy model. NAKα2 over-expression and small interfere RNA (siRNA) plasmids were constructed and transfected to cardiomyocytes. Influx Ca2+ ([Ca2+]i) was measured using flow cytometry method. Fibrosis formation was examined with Masson staining. Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining was used to examine apoptosis. Major histocompatibility complex ß (ß-MHC), atrial natriuretic peptides (ANP), B-type natriuretic peptides (BNP) were evaluated with quantitative Real-time PCR (qRT-PCR). Western blot was used to detect ß-MHC, ANP, BNP, Na+/Ca2+ exchanger (NCX) and L-type calcium channel (LTCC). RESULTS: NAKα2 significantly inhibited NCX and LTCC expression compared to that in ISO-treated cardiomyocytes (p<0.05). NAKα2 significantly suppressed expression of ß-MHC, ANP and BNP compared to that in ISO-treated cardiomyocytes (p<0.05). NAKα2 significantly alleviated fibrosis formation and inhibited apoptosis compared to that in ISO-treated cardiomyocytes (p<0.05). NAKα2 reduced intracellular calcineurin and activated phosphorylation of calcineurin-nuclear factor of activated T cells (NFAT) compared to ISO-treated cardiomyocytes (p<0.05). NAKα2 significantly strengthened effects of Klotho on ISO-induced up-regulation of hypertrophy associated molecules (p<0.05) by activating LTCC and NCX. Comparing to ISO-treated cardiomyocytes, NAKα2 combining Klotho treatment exhibited significantly better improvement of Ca2+ influx, alleviation of fibrosis and reduction of apoptosis by triggering LTCC/NCX signaling pathway. CONCLUSIONS: Over-expression of NKAα2 suppressed fibrosis formation and protected against cardiomyocyte hypertrophy by inhibiting hypertrophy associated molecules, alleviating apoptosis and activating LTCC/NCX signaling pathway.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Cardiomegalia/metabolismo , Miocitos Cardíacos/metabolismo , Transducción de Señal/fisiología , Intercambiador de Sodio-Calcio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Cardiomegalia/genética , Cardiomegalia/patología , Línea Celular , Fibrosis/genética , Fibrosis/metabolismo , Fibrosis/patología , Ratones , Miocitos Cardíacos/patología , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
The sodium potassium ATPase (Na+/K+ ATPase) is essential for the maintenance of a low intracellular Na+ and a high intracellular K+ concentration. Loss of function of the Na+/K+ ATPase due to mutations in Na+/K+ ATPase genes, anoxic conditions, depletion of ATP or inhibition of the Na+/K+ ATPase function using cardiac glycosides such as digitalis, causes a depolarization of the resting membrane potential. While in non-excitable cells, the uptake of glucose and amino acids is decreased if the function of the Na+/K+ ATPase is compromised, in excitable cells the symptoms range from local hyper-excitability to inactivating depolarization. Although several studies have demonstrated the differential expression of the various Na+/K+ ATPase alpha and beta isoforms in the brain tissue of rodents, their expression profile during development has yet to be thoroughly investigated. An immunohistochemical analysis of postnatal day 19 mouse brain showed ubiquitous expression of Na+/K+ ATPase isoforms α1, ß1 and ß2 in both neurons and glial cells, whereas α2 was expressed mostly in glial cells and the α3 and ß3 isoforms were expressed in neurons. Furthermore, we examined potential changes in the relative expression of the different Na+/K+ ATPase isoforms in different brain areas of postnatal day 6 and in adult 9 months old animals using immunoblot analysis. Our results show a significant up-regulation of the α1 isoform in cortex, hippocampus and cerebellum, whereas, the α2 isoform was significantly up-regulated in midbrain. The ß3 isoform showed a significant up-regulation in all brain areas investigated. The up-regulation of the α3 isoform matched that of the ß2 isoform which were both significantly up-regulated in cortex, hippocampus and midbrain, suggesting that the increased maturation of the neuronal network is accompanied by an increase in expression of α3/ß2 complexes in these brain structures.
Asunto(s)
Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Regulación Enzimológica de la Expresión Génica , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Factores de Edad , Animales , Animales Recién Nacidos , Isoenzimas/biosíntesis , Isoenzimas/genética , Ratones , Ratones Endogámicos C57BL , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Although antiretroviral therapy is highly effective in suppressing human immunodeficiency virus type-1 (HIV) replication, treatment has failed to eliminate viral reservoirs and discontinuation of treatment results in viral reactivation. Here, we demonstrate that peptides Tat-vFLIP-α2 and Tat-Beclin 1/BECN1 which have been shown to induce a Na+/K+-ATPase- and a macroautophagy/autophagy-dependent form of cell death, autosis, can preferentially kill HIV-infected macrophages while preventing virological rebound. To improve bioavailability and drug delivery, Tat-vFLIP-α2 was encapsulated into biodegradable PLGA (poly lactic-co-glycolic acid)-lipid-PEG (polyethylene glycol) nanoparticles for long-lasting intracellular delivery. After a single dose of NP-vFLIP-α2, HIV-infected macrophages were preferentially killed in a dose-dependent manner compared to uninfected or untreated HIV-infected cells with complete inhibition of HIV infection at 10 µM of peptide. HIV-infected macrophages treated with NP-vFLIP-α2 exhibited increased markers of autophagy including LC3B lipidation, SQSTM1/p62 degradation and Na+/K+-ATPase expression compared to untreated uninfected or infected cells. Moreover, the increased cell death observed in HIV-infected cells was not altered by treatment with bafilomycin A1 (BAF) or the caspase inhibitor Z-VAD-FMK, but could be reversed following treatment with the Na+/K+-ATPase inhibitor, digoxin, or knockdown of ATG5 or ATG7. NP-vFLIP-α2 induced preferential killing was also detected in HIV-infected macrophages under antiretroviral suppression without inducing viral reactivation. Additionally, we found that Na+/K+-ATPase was upregulated in HIV-infected cells, which enhanced NP-vFLIP-α2 induced cell death. These findings provide a novel strategy to eradicate HIV-infected macrophages by selectively killing infected cells through the induction of Na+/K+-ATPase dependent autophagy, while preventing reactivation of virus and new infection of uninfected bystander cells.
Asunto(s)
Autofagia , Infecciones por VIH/enzimología , VIH-1/fisiología , Macrófagos/virología , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Antivirales/farmacología , Autofagia/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Infecciones por VIH/patología , VIH-1/efectos de los fármacos , Humanos , Lípidos/química , Modelos Biológicos , Nanopartículas/química , Nanopartículas/ultraestructura , Péptidos/farmacología , Interferencia de ARN , Replicación Viral/efectos de los fármacosRESUMEN
LLC-PK1 cells, an immortalized epithelial cell line derived from pig renal proximal tubules, express all the major players of the endocannabinoid system (ECS) such as CB1, CB2 and TRPV1 receptors, as well as the main enzymes involved in the biosynthesis and degradation of the major endocannabinoids named 2-arachidonoylglycerol, 2-AG and anandamide, AEA. Here we investigated whether the damages caused by ischemic insults either in vitro using LLC-PK1 cells exposed to antimycin A (an inductor of ATP-depletion) or in vivo using Wistar rats in a classic renal ischemia and reperfusion (IR) protocol, lead to changes in AEA and 2-AG levels, as well as altered expression of genes from the main enzymes involved in the regulation of the ECS. Our data show that the mRNA levels of the CB1 receptor gene were downregulated, while the transcript levels of monoacylglycerol lipase (MAGL), the main 2-AG degradative enzyme, were upregulated in LLC-PK1 cells after IR model. Accordingly, IR was accompanied by a significant reduction in the levels of 2-AG and AEA, as well as of the two endocannabinoid related molecules, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in LLC-PK1 cells. In kidney cortex homogenates, only AEA levels were significantly decreased. In addition, we found that in both the in vitro and in vivo model IR caused a reduction in the expression and activity of the Na+/K+ ATPase. These changes were reversed by the CB1/CB2 agonist WIN55,212, in a CB1-receptor dependent manner in the LLC-PK1 IR model. In conclusion, the ECS and Na+/K+ ATPase are down-regulated following IR in LLC-PK1 cells and rat kidney. We suggest that CB1 agonists might represent a potential strategy to reverse the consequences of IR injury in kidney tissues.
Asunto(s)
Endocannabinoides/metabolismo , Túbulos Renales Proximales/metabolismo , Daño por Reperfusión/metabolismo , Transducción de Señal/fisiología , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Benzoxazinas/farmacología , Benzoxazinas/uso terapéutico , Endocannabinoides/agonistas , Túbulos Renales Proximales/efectos de los fármacos , Células LLC-PK1 , Masculino , Morfolinas/farmacología , Morfolinas/uso terapéutico , Naftalenos/farmacología , Naftalenos/uso terapéutico , Ratas , Ratas Wistar , Daño por Reperfusión/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , PorcinosRESUMEN
Rapid eye movement sleep deprivation-associated elevated noradrenaline increases and decreases neuronal and glial Na-K ATPase activity, respectively. In this study, using C6 cell-line as a model, we investigated the possible intracellular molecular mechanism of noradrenaline-induced decreased glial Na-K ATPase activity. The cells were treated with noradrenaline in the presence or absence of adrenoceptor antagonists, modulators of extra- and intracellular Ca++ and modulators of intracellular signalling pathways. We observed that noradrenaline acting on ß-adrenoceptor decreased Na-K ATPase activity and mRNA expression of the catalytic α2-Na-K ATPase subunit in the C6 cells. Further, cAMP and protein kinase-A mediated release of intracellular Ca++ played a critical role in such decreased α2-Na-K ATPase expression. In contrast, noradrenaline acting on ß-adrenoceptor up-regulated the expression of regulatory ß2-Na-K ATPase subunit, which although was cAMP and Ca++ dependent, was independent of protein kinase-A and protein kinase-C. Combining these with previous findings (including ours) we have proposed a working model for noradrenaline-induced suppression of glial Na-K ATPase activity and alteration in its subunit expression. The findings help understanding noradrenaline-associated maintenance of brain excitability during health and altered states, particularly in relation to rapid eye movement sleep and its deprivation when the noradrenaline level is naturally altered.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Líquido Intracelular/enzimología , Receptores Adrenérgicos beta/fisiología , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/genética , Animales , Carbazoles/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Líquido Intracelular/efectos de los fármacos , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Pirroles/farmacología , Ratas , Sodio/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
The functional roles of bioelectrical signals (ES) created by the flow of specific ions at the mammalian lens equator are poorly understood. We detected that mature, denucleated lens fibers expressed high levels of the α1 and ß1 subunits of Na+ /K+ -ATPase (ATP1A1 and ATP1B1 of the sodium pump) and had a hyperpolarized membrane potential difference (Vmem ). In contrast, differentiating, nucleated lens fiber cells had little ATP1A1 and ATP1B1 and a depolarized Vmem . Mimicking the natural equatorial ES with an applied electrical field (EF) induced a striking reorientation of lens epithelial cells to lie perpendicular to the direction of the EF. An EF also promoted the expression of ß-crystallin, aquaporin-0 (AQP0) and the Beaded Filament Structural Protein 2 (BFSP2) in lens epithelial cells (LECs), all of which are hallmarks of differentiation. In addition, applied EF activated the AKT and CDC2 and inhibition of AKT reduced the activation of CDC2. Our results indicate that the endogenous bioelectrical signal at the lens equator promotes differentiation of LECs into denucleated lens fiber cells via depolarization of Vmem. Development of methods and devices of EF application or amplification in vivo may supply a novel treatment for lens diseases and even promote regeneration of a complete new lens following cataract surgery.
Asunto(s)
Conductividad Eléctrica , Células Epiteliales/citología , Cristalino/citología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Acuaporinas/biosíntesis , Proteína Quinasa CDC2/metabolismo , Bovinos , Diferenciación Celular/fisiología , Línea Celular , Activación Enzimática/fisiología , Proteínas del Ojo/biosíntesis , Humanos , Proteínas de Filamentos Intermediarios/biosíntesis , Potenciales de la Membrana/fisiología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , beta-Cristalinas/biosíntesisRESUMEN
Some Macrobrachium shrimps (Caridea, Palaemonidae) are diadromous; freshwater adults are truly euryhaline, while larvae need saline water for development. Branchial Na+/K+-ATPase (NKA) and carbonic anhydrase (CA) are involved in NaCl absorption in freshwater. This study aimed at verifying the time course of the osmoregulatory response of adult Macrobrachium acanthurus to high salinity brackish water (20), from the first 30min to 5days. The goal was to detect possible transition from hyper- to hyporegulation, the putative involvement of branchial NKA and CA, or the induction of muscular HSP70 expression. Hemolymph osmotic and ionic concentrations remained relatively stable and close to control levels until ~9h of exposure, but later increased consistently (~50%). A fast reduction in NKA activity (3-6h) was observed; these shrimps seem to shut off salt absorption already in the first hours. Later on, especially after 24h, hemolymph concentrations rise but HSP70 expression is not induced, possibly because constitutive levels are already sufficient to prevent protein damage. Time-dependent response mechanisms effective in high salinity brackish water, resulting in salt loading avoidance and suggestive of hyporegulation should be further investigated in decapods that evolutionary invaded freshwater.
Asunto(s)
Proteínas de Artrópodos/biosíntesis , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/biosíntesis , Hemolinfa/metabolismo , Músculos/metabolismo , Palaemonidae/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Concentración OsmolarRESUMEN
The aim of this study was to investigate whether the presence of endogenous estradiol alters the effects of a high-fat (HF) diet on activity/expression of the cardiac Na+/K+-ATPase, via PI3K/IRS and RhoA/ROCK signalling cascades in female rats. For this study, female Wistar rats (8 weeks old, 150-200 g) were fed a standard diet or a HF diet (balanced diet for laboratory rats enriched with 42% fat) for 10 weeks. The results show that rats fed a HF diet exhibited a decrease in phosphorylation of the α1 subunit of Na+/K+-ATPase by 30% (p < 0.05), expression of total α1 subunit of Na+/K+-ATPase by 31% (p < 0.05), and association of IRS1 with p85 subunit of PI3K by 42% (p < 0.05), while the levels of cardiac RhoA and ROCK2 were significantly increased by 84% (p < 0.01) and 62% (p < 0.05), respectively. Our results suggest that a HF diet alters cardiac Na+/K+-ATPase expression via molecular mechanisms involving RhoA/ROCK and IRS-1/PI3K signalling in female rats.
Asunto(s)
Grasas de la Dieta/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Miocardio/enzimología , Transducción de Señal/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Femenino , Ratas , Ratas WistarRESUMEN
Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K+ transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.
Asunto(s)
Hipertensión/genética , Túbulos Renales Proximales/metabolismo , Fosfoproteínas/biosíntesis , Intercambiadores de Sodio-Hidrógeno/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Presión Sanguínea/genética , Línea Celular , Modelos Animales de Enfermedad , Dopamina/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Hipertensión/metabolismo , Hipertensión/patología , Riñón/metabolismo , Riñón/patología , Túbulos Renales Proximales/patología , Masculino , Fosfoproteínas/genética , Ratas , Ratas Endogámicas SHR , Transducción de Señal/genética , Intercambiadores de Sodio-Hidrógeno/genética , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
We evaluated the individual and combined effects of salinity and alkalinity on gill Na+/K+-ATPase enzyme activity, plasma ion concentration, and osmotic pressure in Luciobarbus capito. Increasing salinity concentrations (5, 8, 11, and 14 g/L) were associated with an initial increase and then decrease in L. capito gill Na+/K+-ATPase activity. Activity was affected by the difference between internal and external Na+ ion concentrations and osmotic pressure (P < 0.05). Both plasma ion (Na+, K+, and Cl-) concentration and osmotic pressure increased significantly (P < 0.05). An increase in alkalinity (15, 30, 45, and 60 mM) caused a significant increase in plasma K+ and urea nitrogen concentrations (P < 0.05) but had no effect on either plasma osmotic pressure or gill filament ATPase activity. In the two-factor experiment, the saline-alkaline interaction caused a significant increase in plasma ion (Na+, Cl-, and urea nitrogen) and osmotic pressure (P < 0.05). Variance analysis revealed that salinity, alkalinity, and their interaction significantly affected osmotic pressure, with salinity being most affected, followed by alkalinity, and their interaction. Gill filament ATPase activity increased at first and then decreased; peak values were observed in the orthogonal experiment group at a salinity of 8 g/L and alkalinity of 30 mM.
Asunto(s)
Iones/sangre , Presión Osmótica , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Sodio/sangre , Álcalis/química , Animales , Cyprinidae/metabolismo , Branquias/enzimología , Concentración de Iones de Hidrógeno , Salinidad , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
BACKGROUND: Major depressive disorder (MDD) frequently co-occurs with other psychiatric problems. Our previous study showed that ATP1A1 gene expression level was significantly decreased in MDD patients. This research explores the potential correlations between the ATP1A1 expression level reduction and MDD patients' clinical manifestation. METHODS: All participant patients were diagnosed by Diagnostic and Statistical Manual of Mental Disorders - 4th edition (DSM-IV). Hamilton rating scale for depression (HAM-D) and anxiety (HAM-A) were applied to group patients into different categories. ATP1A1 expression level was measured by reverse transcript real-time polymerase chain reaction. RESULTS: ATP1A1 expression levels of all MDD subgroups showed significant reduction compared to the control group (p<0.01). Further, the trend of ATP1A1 expression level reduction is significantly related to MDD patients' HAM-A scores (p<0.01). However, there was no significance between ATP1A1 level and HAM-D scores (p>0.05). CONCLUSIONS: ATP1A1 expression level reduction is related to MDD anxiety score, which may be an explanation for the clinical manifestations and the underlining physiological mechanisms.
Asunto(s)
Ansiedad/diagnóstico , Ansiedad/metabolismo , Trastorno Depresivo Mayor/diagnóstico , Trastorno Depresivo Mayor/metabolismo , Índice de Severidad de la Enfermedad , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Adulto , Ansiedad/genética , Trastorno Depresivo Mayor/genética , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
A low concentration of boron (B) accelerates the proliferation and differentiation of mammalian osteoblasts. The aim of this study was to investigate the effects of 0.1 mM of B on the membrane function of osteoblastic cells in vitro. Genes involved in cell activity were investigated using gene expression microarray analyses. The Ca2+ influx and efflux were evaluated to demonstrate the activation of L-type Ca2+ channel for the Ca2+ influx, and that of Na+/K+-ATPase for the Ca2+ efflux. A real-time PCR analysis revealed that the messenger RNA (mRNA) expression of four mineralization-related genes was clearly increased after 3 days of culture with a B-supplemented culture medium. Using microarray analyses, five genes involved in cell proliferation and differentiation were upregulated compared to the control group. Regarding the Ca2+ influx, in the nifedipine-pretreated group, the relative fluorescence intensity for 1 min after adding B solution did not increase compared with that for 1 min before addition. In the control group, the relative fluorescence intensity was significantly increased compared with the experimental group (P < 0.05). Regarding the Ca2+ efflux, in the experimental group cultured in 0.1 mM of B-supplemented medium, the relative fluorescence intensity for 10 min after ouabain treatment revealed a significantly lower slope value compared with the control group (P < 0.01). This is the first study to demonstrate the acceleration of Ca2+ flux by B supplementation in osteoblastic cells. Cell membrane stability is related to the mechanism by which a very low concentration of B promotes the proliferation and differentiation of mammalian osteoblastic cells in vitro.
Asunto(s)
Boro/farmacología , Calcio/metabolismo , Osteoblastos/metabolismo , Canales de Calcio Tipo L/biosíntesis , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ATPasa Intercambiadora de Sodio-Potasio/biosíntesisRESUMEN
The α4 Na,K-ATPase is a sperm-specific protein essential for sperm motility and fertility yet little is known about the mechanisms that regulate its expression in germ cells. Here, the potential involvement of DNA methylation in regulating the expression of this sperm-specific protein is explored. A single, intragenic CpG island (Mα4-CGI) was identified in the gene encoding the mouse α4 Na,K-ATPase (Atp1a4), which displayed reduced methylation in mouse sperm (cells that contain α4) compared to mouse kidney (tissue that lacks α4 expression). Unlike the intragenic CGI, the putative promoter (the -700 to +200 region relative to the transcriptional start site) of Atp1a4 did not show differential methylation between kidney and sperm nevertheless it did drive methylation-dependent reporter gene expression in the male germ cell line GC-1spg. Furthermore, treatment of GC-1spg cells with 5-aza2-deoxycytidine led to upregulation of the α4 transcript and decreased methylation of both the Atp1a4 promoter and the Mα4-CGI. In addition, Atp1a4 expression in mouse embryonic stem cells deficient in DNA methyltransferases suggests that both maintenance and de novo methylation are involved in regulating its expression. In an attempt to define the regulatory function of the Mα4-CGI, possible roles of the Mα4-CGI in regulating Atp1a4 expression via methylation-dependent transcriptional elongation inhibition in somatic cells and via its ability to repress promoter activity in germ cells were uncovered. In all, our data suggests that both the promoter and the intragenic CGI could combine to provide multiple modes of regulation for optimizing the Atp1a4 expression level in a cell type-specific manner.
Asunto(s)
Metilación de ADN/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Regiones Promotoras Genéticas/fisiología , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Espermatozoides/enzimología , Testículo/enzimología , Animales , Islas de CpG/fisiología , Masculino , Ratones , Especificidad de Órganos/fisiología , ATPasa Intercambiadora de Sodio-Potasio/genética , Espermatozoides/citología , Testículo/citologíaRESUMEN
In this study, the effects of tributyltin (TBT) on biochemical parameters (antioxidant responses and Na(+) -K(+) -ATPase) in different tissues were investigated by using juvenile common carp (Cyprinus Carpio) as well as growth and ion regulation-related genes expression and stress-related proteins profiling in fish liver. Oxidative stress indices and Na(+) -K(+) -ATPase showed tissues-specific responses in fish exposed to different TBT concentrations. All tested genes related to GH/IGF-I axis and ion-regulation were significantly induced in the TBT group with lower concentrations (except for the igfbp3 in 10 µg/L) and were inhibited in 20 µg/L. In addition, the profiling of two proteins Hsp 70 and MT were increasing in a dose-dependent manner under TBT stress. In short, TBT-induced biochemical and molecular responses in different tissues were reflected in the measured parameters in the test. On the basis of TBT residue levels in the natural environment, more long-term experiments at lower concentrations will be necessary in the future.
Asunto(s)
Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Compuestos de Trialquiltina/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Antioxidantes/metabolismo , Carpas/crecimiento & desarrollo , Regulación de la Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/biosíntesis , Proteínas de Choque Térmico/biosíntesis , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Hígado/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/biosíntesisRESUMEN
CONTEXT: Hypertension is one of the most important risk factors in cardiovascular disease and is the most common chronic disease. Mineralocorticoid receptor (MR) antagonists have been successfully used in clinic for the treatment of hypertension. OBJECTIVE: Our study aims to investigate whether Arctigenin can antagonize MR and inhibit the transcription of Na/K-ATPase. MATERIALS AND METHODS: The yeast two-hybrid assay was used to screen natural products and Arctigenin was identified as an MR antagonist. The direct binding of Arctigenin to MR was determined using assays based on surface plasmon resonance, differential scanning calorimetry and fluorescence quenching. Furthermore, results from mammalian one-hybrid and transcriptional activation experiments also confirmed that Arctigenin can potently antagonize MR in cells. We demonstrated that Arctigenin can decrease the level of Na/K-ATPase mRNA by antagonizing MR in HK-2 cells. RESULTS: Our findings show that Arctigenin can effectively decrease Na/K-ATPase transcription; thus highlight its potential as an anti-hypertensive drug lead compound. DISCUSSION AND CONCLUSION: Our current findings demonstrate that Arctigenin is an antagonist of MR and effectively decreases the Na/K-ATPase 1 gene expression. Our work provides a hint for the drug discovery against cardiovascular disease.