RESUMEN
The Goldblatt model of hypertension (2K-1C) in rats is characterized by renal sympathetic nerve activity (rSNA). We investigated the effects of unilateral renal denervation of the clipped kidney (DNX) on sodium transporters of the unclipped kidneys and the cardiovascular, autonomic, and renal functions in 2K-1C and control (CTR) rats. The mean arterial pressure (MAP) and rSNA were evaluated in experimental groups. Kidney function and NHE3, NCC, ENaCß, and ENaCγ protein expressions were assessed. The glomerular filtration rate (GRF) and renal plasma flow were not changed by DNX, but the urinary (CTR: 0.0042 ± 0.001; 2K-1C: 0.014 ± 0.003; DNX: 0.005 ± 0.0013 mL/min/g renal tissue) and filtration fractions (CTR: 0.29 ± 0.02; 2K-1C: 0.51 ± 0.06; DNX: 0.28 ± 0.04 mL/min/g renal tissue) were normalized. The Na+/H+ exchanger (NHE3) was reduced in 2K-1C, and DNX normalized NHE3 (CTR: 100 ± 6; 2K-1C: 44 ± 14, DNX: 84 ± 13%). Conversely, the Na+/Cl- cotransporter (NCC) was increased in 2K-1C and was reduced by DNX (CTR: 94 ± 6; 2K-1C: 144 ± 8; DNX: 60 ± 15%). In conclusion, DNX in Goldblatt rats reduced blood pressure and proteinuria independently of GRF with a distinct regulation of NHE3 and NCC in unclipped kidneys.
Asunto(s)
Riñón , Intercambiador 3 de Sodio-Hidrógeno , Animales , Riñón/inervación , Riñón/metabolismo , Ratas , Masculino , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Tasa de Filtración Glomerular , Desnervación , Isquemia/metabolismo , Presión Sanguínea , Ratas Wistar , Hipertensión/metabolismo , Canales Epiteliales de Sodio/metabolismo , Modelos Animales de Enfermedad , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Heterologous expression systems have been used as a powerful experimental strategy to study the function of many proteins, particularly ion transporters. For this experiment, it is fundamental to prepare an expression vector encoding a protein of interest. However, we encountered problems in vector preparation of the voltage sensor domain (VSD) of murine sperm-specific Na+/H+ exchanger (sNHE) due to its severe toxicity to bacteria. We overcame the problems by insertion of an amber stop codon or a synthetic intron into the coding sequence of the VSD in the expression vectors. Both methods allowed us to express the protein of interest in HEK293 cells (combined with a stop codon suppression system for amber codon). The VSD of mouse sNHE generates voltage-dependent outward ionic currents, which is a probable cause of toxicity to bacteria. We propose these two strategies as practical solutions to study the function of any protein toxic to bacteria.
Asunto(s)
Protones , Semen , Animales , Bacterias/metabolismo , Codón de Terminación/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Semen/metabolismo , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Espermatozoides/metabolismoRESUMEN
Vuralia turcica (Fabaceae; Papilionoideae) is a critically endangered endemic plant species in Turkey. This plant grows naturally in saline environments, although the photosynthesis and physiological functions of many plants are affected by salt stress. Molecular control mechanisms and identification of genes involved in these mechanisms constitute the critical field of study in plant science. Trehalose-6-phosphate synthase (TPS) is one of the essential enzyme genes involved in trehalose biosynthesis, which is protective against salt stress. Also, the vacuolar Na+/H+ antiporter gene (NHX) is known to be useful in salt tolerance. In this study, the TPS and NHX-like genes in V. turcica were partially sequenced using degenerate primers for the first time and submitted to the NCBI database (accession numbers MK120983 and MH757417, respectively). Also, the expression levels of the genes encoding TPS and NHX were investigated. The results indicate that the increase in both the level of applied salt and cadmium is coupled with the increase in the expression level of NHX and TPS genes. However, salt exposure significantly affected the expression level of the NHX gene. The findings suggest that the NHX gene might play a crucial role in the salt tolerance ability of V. turcica.
Asunto(s)
Cadmio , Fabaceae , Cadmio/toxicidad , Fabaceae/metabolismo , Regulación de la Expresión Génica de las Plantas , Glucosiltransferasas , Filogenia , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , TurquíaRESUMEN
Free-living amoeba of the genus Acanthamoeba are ubiquitous protozoa involved in opportunistic and non-opportunistic infection in humans, such as granulomatous amoebic encephalitis and amoebic keratitis. Both infections have challenging characteristics such as the formation of the resistant cysts in infected tissues, hampering the treatment and most usual diagnosis depending on time-consuming and/or low sensitivity techniques. The use of monoclonal antibodies presents itself as an opportunity for the development of more effective alternative diagnostic methods, as well as an important and useful tool in the search for new therapeutic targets. This study investigated the possibility of using a previously produced monoclonal antibody (mAb3), as a diagnostic tool for the detection of Acanthamoeba trophozoites by direct and indirect flow cytometry and immunofluorescence. Immunoprecipitation assay and mass spectrometry allowed the isolation of the antibody's target and suggested it is a transporter part of the CPA (cation: proton antiporter) superfamily. In vitro tests indicate an important role of this target in Acanthamoeba's encystment physiology. Our results support the importance of studying the role of CPA2 transporters in the context of acanthamoebiasis, as this may be a way to identify new therapeutic candidates.
Asunto(s)
Acanthamoeba/inmunología , Amebiasis/diagnóstico , Proteínas Protozoarias/genética , Intercambiadores de Sodio-Hidrógeno/genética , Acanthamoeba/genética , Amebiasis/parasitología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Anticuerpos Antiprotozoarios , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Estructura Secundaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Alineación de Secuencia , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/metabolismo , Trofozoítos/genética , Trofozoítos/inmunologíaRESUMEN
Stress responses in teleosts include the release of hormones into the bloodstream. Their effects depend on the species and on the environmental conditions. The Amazon basin collects waters of diverse chemical composition, and some fish are able to inhabit several of them. However, the effects of these waters on the stress axis are still unknown. Here we show how acute air-exposure differently affects stress biomarkers in tambaqui (Colossoma macropomum), a tropical model species, when acclimated to two Amazonian waters (Rio Negro -RN- water rich in humic acids and poor in ions, and groundwater -IG- with no humic acids and higher concentration of ions). This study described primary and secondary stress responses after air exposure including plasma cortisol, energy metabolites, pH and ions, skin mucus energy metabolites, as well as gills and kidney Na+/K+-ATPase and Na+/H+-exchanger (NHE) activities. Several differences were described in these stress biomarkers due to the acclimation water. The most remarkable ones include increased mucus glucose only in RN-fish, and mucus lactate only in IG-fish after air exposure. Moreover, an inverse relationship between plasma cortisol and Na+ concentrations as well as a direct relationship between plasma ammonia and branchial NHE activity were observed only in RN-fish. Our results demonstrate how important is to study stress responses in fish acclimated to different environments, as physiological differences can be magnified during episodes of high energy expenditure. In addition to having a direct application in aquaculture, this study will improve the management of critical ecosystems such as the Amazon.
Asunto(s)
Aclimatación , Characiformes/fisiología , Estrés Fisiológico , Amoníaco/metabolismo , Animales , Brasil , Homeostasis , Intercambiadores de Sodio-Hidrógeno/metabolismo , Agua/químicaRESUMEN
Climatic change is pointed as one of the major challenges for global food security. Based on current models of climate change, reduction in precipitations and in turn, increase in the soil salinity will be a sharp constraint for crops productivity worldwide. In this context, root fungi appear as a new strategy to improve plant ecophysiological performance and crop yield under abiotic stress. In this study, we evaluated the impact of the two fungal endophytes Penicillium brevicompactum and P. chrysogenum isolated from Antarctic plants on nutrients and Na+ contents, net photosynthesis, water use efficiency, yield and survival in tomato and lettuce, facing salinity stress conditions. Inoculation of plant roots with fungal endophytes resulted in greater fresh and dry biomass production, and an enhanced survival rate under salt conditions. Inoculation of plants with the fungal endophytes was related with a higher up/down-regulation of ion homeostasis by enhanced expression of the NHX1 gene. The two endophytes diminished the effects of salt stress in tomato and lettuce, provoked a higher efficiency in photosynthetic energy production and an improved sequestration of Na+ in vacuoles is suggested by the upregulating of the expression of vacuolar NHX1 Na+/H+ antiporters. Promoting plant-beneficial interactions with root symbionts appears to be an environmentally friendly strategy to mitigate the impact of climate change variables on crop production.
Asunto(s)
Productos Agrícolas/metabolismo , Productos Agrícolas/fisiología , Endófitos/fisiología , Raíces de Plantas/metabolismo , Raíces de Plantas/fisiología , Estrés Salino/fisiología , Sodio/metabolismo , Regiones Antárticas , Biomasa , Cambio Climático , Productos Agrícolas/microbiología , Regulación hacia Abajo/fisiología , Homeostasis/fisiología , Iones/metabolismo , Lactuca/metabolismo , Lactuca/microbiología , Lactuca/fisiología , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiología , Solanum lycopersicum/fisiología , Penicillium chrysogenum/fisiología , Fotosíntesis/fisiología , Raíces de Plantas/microbiología , Salinidad , Intercambiadores de Sodio-Hidrógeno/metabolismo , Suelo , Estrés Fisiológico/fisiología , Tasa de Supervivencia , Regulación hacia Arriba/fisiología , Agua/metabolismoRESUMEN
Bactridine 2 (Bact-2) is an antibacterial toxin from Tityus discrepans venom which modifies isoforms 1.2, 1.4 and 1.6 voltage-dependent sodium (Nav) channels. Bactridine-induced Na+ outflow in Yersinia enterocolitica was blocked by amiloride, suggesting that Bact-2 effect was mediated by an amiloride sensitive sodium channel. In this study we show that Bact-2 increases also an outward rectifying current in rat dorsal root ganglia (DRG) sensory neurons; therefore, the nature of the outward rectifying currents was characterized and then the effect of Bact-2 on these currents was studied. These currents are enhanced by amiloride, are decreased by Na+ when an outward pH gradient is present and its reversal potential coincides with that of a Cl-/H+ exchanger, suggesting that rectifying currents are produced by the electrogenic Cl-/H+ exchanger modulated by the Na+/H+ antiporter. Bact-2 also leads to an increase of the outward currents in a similar way to the produced by the inhibition of the Na+/H+ exchanger. Additionally, the subsequent application of Bact-2 after blocking the Na+/H+ exchanger does not produce any further effect, suggesting that Bact-2 modifies the outward current by modulating the activity of the Na+/H+ exchanger. The effect of Bact-2 on pHi regulation was determined using the pH indicator BCECF. The results show that the Na+/H+ exchanger is blocked by amiloride and Na+ free solutions and is modulated by Bact-2 in a similar way as cariporide. This study validates that besides Nav channels, Bact-2 modulates the activity of the Na+/H+ exchanger.
Asunto(s)
Ganglios Espinales/citología , Neuronas/efectos de los fármacos , Venenos de Escorpión/química , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/metabolismo , Amilorida , Animales , Antiportadores/metabolismo , Fenómenos Electrofisiológicos/efectos de los fármacos , Concentración de Iones de Hidrógeno , Masculino , Ratas , Ratas Sprague-Dawley , Escorpiones/fisiología , Sodio , ZincRESUMEN
Alfalfa, usually known as the "Queen of Forages", is the main source of vegetable protein to meat and milk production systems worldwide. This legume is extremely rich in proteins due to its highly efficient symbiotic association with nitrogen-fixing strains. In the last years, alfalfa culture has been displaced to saline environments by other important crops, including major cereals, a fact that has reduced its biomass production and symbiotic nitrogen fixation. In this short communication, we report the high forage production and nutrient quality of alfalfa under saline conditions by alfalfa transformation with the AtNHX1 Na+/H+ antiporter and inoculation with the stress-resistant nitrogen-fixing strain Sinorhizobium meliloti B401. Therefore, the incorporation of transgenic traits into salt-sensitive legumes in association with the inoculation with natural stress-resistant isolates could be a robust approach to improve the productivity and quality of these important nitrogen-fixing crops.
Asunto(s)
Alimentación Animal , Bacterias/genética , Medicago sativa/genética , Plantas Modificadas Genéticamente/genética , Plantas Tolerantes a la Sal/genética , Simbiosis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Bacterias/metabolismo , Biomasa , Medicago sativa/metabolismo , Fijación del Nitrógeno/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Tolerantes a la Sal/metabolismo , Cloruro de Sodio , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Cardiotonic steroids (CS) are known as modulators of sodium and water homeostasis. These compounds contribute to the excretion of sodium under overload conditions due to its natriuretic property related to the inhibition of the renal Na+/K+-ATPase (NKA) pump α1 isoform. NHE3, the main route for Na+ reabsorption in the proximal tubule, depends on the Na+ gradient generated by the NKA pump. In the present study we aimed to investigate the effects of marinobufagin (MBG) and telocinobufagin (TBG) on the renal function of isolated perfused rat kidney and on the inhibition of NKA activity. Furthermore, we investigated the mechanisms for the cardiotonic steroid-mediated natriuretic effect, by evaluating and comparing the effects of bufalin (BUF), ouabain (OUA), MBG and TBG on NHE3 activity in the renal proximal tubule in vivo. TBG significantly increased GFR, UF, natriuresis and kaliuresis in isolated perfused rat kidney, and inhibits the activity of NKA at a much higher rate than MBG. By stationary microperfusion technique, the perfusion with BUF, OUA, TBG or MBG promoted an inhibitory effect on NHE3 activity, whereas BUF was the most effective agent, and demonstrated a dose-dependent response, with maximal inhibition at 50nM. Furthermore, our data showed the role of NKA-Src kinase pathway in the inhibition of NHE3 by CS. Finally, a downstream step, MEK1/2-ERK1/2 was also investigated, and, similar to Src inhibition, the MEK1/2 inhibitor (U0126) suppressed the BUF effect. Our findings indicate the involvement of NKA-SRc-Kinase-Ras-Raf-ERK1/2 pathway in the downregulation of NHE3 by cardiotonic steroids in the renal proximal tubule, promoting a reduction of proximal sodium reabsorption and natriuresis.
Asunto(s)
Bufanólidos/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Riñón/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Técnicas In Vitro , Túbulos Renales Proximales/metabolismo , Masculino , Ratas , Ratas Wistar , Intercambiador 3 de Sodio-Hidrógeno , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Familia-src Quinasas/fisiologíaRESUMEN
An effective strategy for re-establishing K+ and Na+ homeostasis is a challenge for the improvement of plant performance in saline soil. Specifically, attempts to understand the mechanisms of Na+ extrusion from plant cells, the control of Na+ loading in the xylem and the partitioning of the accumulated Na+ between different plant organs are ongoing. Our goal was to provide insight into how an external nitrogen source affects Na+ accumulation in Sorghum bicolor under saline conditions. The NH4+ supply improved the salt tolerance of the plant by restricting Na+ accumulation and improving the K+/Na+ homeostasis in shoots, which was consistent with the high activity and expression of Na+/H+ antiporters and proton pumps in the plasma membrane and vacuoles in the roots, resulting in low Na+ loading in the xylem. Conversely, although NO3--grown plants had exclusion and sequestration mechanisms for Na+, these responses were not sufficient to reduce Na+ accumulation. In conclusion, NH4+ acts as an efficient signal to activate co-ordinately responses involved in the regulation of Na+ homeostasis in sorghum plants under salt stress, which leads to salt tolerance.
Asunto(s)
Compuestos de Amonio/metabolismo , Raíces de Plantas/metabolismo , Bombas de Protones/metabolismo , Tolerancia a la Sal/fisiología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Sodio/metabolismo , Sorghum/metabolismo , Adenosina Trifosfatasas/análisis , Antiportadores/genética , Antiportadores/metabolismo , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Homeostasis , Nitrógeno/metabolismo , Potasio/metabolismo , Cloruro de Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Vacuolas/metabolismo , Xilema/metabolismoRESUMEN
Presympathetic neurons in the rostral ventrolateral medulla (RVLM) including the adrenergic cell groups play a major role in the modulation of several reflexes required for the control of sympathetic vasomotor tone and blood pressure (BP). Moreover, sympathetic vasomotor drive to the kidneys influence natriuresis and diuresis by inhibiting the cAMP/PKA pathway and redistributing the Na+/H+ exchanger isoform 3 (NHE3) to the body of the microvilli in the proximal tubules. In this study we aimed to evaluate the effects of renal afferents stimulation on (1) the neurochemical phenotype of Fos expressing neurons in the medulla oblongata and (2) the level of abundance and phosphorylation of NHE3 in the renal cortex. We found that electrical stimulation of renal afferents increased heart rate and BP transiently and caused activation of tyrosine hydroxylase (TH)-containing neurons in the RVLM and non-TH neurons in the NTS. Additionally, activation of the inhibitory renorenal reflex over a 30-min period resulted in increased natriuresis and diuresis associated with increased phosphorylation of NHE3 at serine 552, a surrogate for reduced activity of this exchanger, in the contralateral kidney. This effect was not dependent of BP changes considering that no effects on natriuresis or diuresis were found in the ipsilateral-stimulated kidney. Therefore, our data show that renal afferents leads to activation of catecholaminergic and non-catecholaminergic neurons in the medulla oblongata. When renorenal reflex is induced, NHE3 exchanger activity appears to be decreased, resulting in decreased sodium and water reabsorption in the contralateral kidney.
Asunto(s)
Catecolaminas/metabolismo , Riñón/inervación , Riñón/metabolismo , Bulbo Raquídeo/metabolismo , Neuronas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vías Aferentes/citología , Vías Aferentes/metabolismo , Animales , Presión Sanguínea/fisiología , Estimulación Eléctrica , Frecuencia Cardíaca/fisiología , Inmunohistoquímica , Riñón/citología , Masculino , Bulbo Raquídeo/citología , Neuronas/citología , Fosforilación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas Wistar , Reflejo/fisiología , Intercambiador 3 de Sodio-Hidrógeno , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The multisubunit cation/proton antiporter 3 family, also called Mrp, is widely distributed in all three phylogenetic domains (Eukarya, Bacteria, and Archaea). Investigations have focused on Mrp complexes from the domain Bacteria to the exclusion of Archaea, with a consensus emerging that all seven subunits are required for Na+/H+ antiport activity. The MrpA subunit from the MrpABCDEFG Na+/H+ antiporter complex of the archaeon Methanosarcina acetivorans was produced in antiporter-deficient Escherichia coli strains EP432 and KNabc and biochemically characterized to determine the role of MrpA in the complex. Both strains containing MrpA grew in the presence of up to 500 mM NaCl and pH values up to 11.0 with no added NaCl. Everted vesicles from the strains containing MrpA were able to generate a NADH-dependent pH gradient (ΔpH), which was abated by the addition of monovalent cations. The apparent Km values for Na+ and Li+ were similar and ranged from 31 to 63 mM, whereas activity was too low to determine the apparent Km for K+ Optimum activity was obtained between pH 7.0 and 8.0. Homology molecular modeling identified two half-closed symmetry-related ion translocation channels that are linked, forming a continuous path from the cytoplasm to the periplasm, analogous to the NuoL subunit of complex I. Bioinformatics analyses revealed genes encoding homologs of MrpABCDEFG in metabolically diverse methane-producing species. Overall, the results advance the biochemical, evolutionary, and physiological understanding of Mrp complexes that extends to the domain Archaea IMPORTANCE: The work is the first reported characterization of an Mrp complex from the domain Archaea, specifically methanogens, for which Mrp is important for acetotrophic growth. The results show that the MrpA subunit is essential for antiport activity and, importantly, that not all seven subunits are required, which challenges current dogma for Mrp complexes from the domain Bacteria A mechanism is proposed in which an MrpAD subcomplex catalyzes Na+/H+ antiport independent of an MrpBCEFG subcomplex, although the activity of the former is modulated by the latter. Properties of MrpA strengthen proposals that the Mrp complex is of ancient origin and that subunits were recruited to evolve the ancestral complex I. Finally, bioinformatics analyses indicate that Mrp complexes function in diverse methanogenic pathways.
Asunto(s)
Proteínas Arqueales/metabolismo , Regulación de la Expresión Génica Arqueal/fisiología , Methanosarcina/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Arqueales/genética , Transporte Biológico , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Litio/metabolismo , Methanosarcina/genética , Modelos Moleculares , Filogenia , Conformación Proteica , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
We have previously shown in renal cells that expression of the water channel Aquaporin 2 (AQP2) increases the rate of cell proliferation by shortening the transit time through the S and G2 /M phases of the cell cycle. This acceleration is due, at least in part, to a down-regulation of regulatory volume decrease (RVD) mechanisms when volume needs to be increased in order to proceed into the S phase. We hypothesize that in order to increase cell volume, RVD mechanisms may be overtaken by regulatory volume increase mechanisms (RVI). In this study, we investigated if the isoform 2 of the Na+ /H+ exchanger (NHE2), the main ion transporter involved in RVI responses, contributed to the AQP2-increased renal cell proliferation. Three cortical collecting duct cell lines were used: WT-RCCD1 (not expressing AQPs), AQP2-RCCD1 (transfected with AQP2), and mpkCCDc14 (with inducible AQP2 expression). We here demonstrate, for the first time, that both NHE2 protein activity and expression were increased in AQP2-expressing cells. NHE2 inhibition decreased cell proliferation and delayed cell cycle progression by slowing S and G2 /M phases only if AQP2 was expressed. Finally, we observed that only in AQP2-expressing cells a NHE2-dependent RVI response was activated in the S phase. These observations suggest that the AQP2-increased proliferation involves the activation of a regulatory volume increase mechanism dependent on NHE2. Therefore, we propose that the accelerated proliferation of AQP2-expressing cells requires a coordinated modulation of the RVD/RVI activity that contributes to cell volume changes during cell cycle progression. J. Cell. Biochem. 118: 967-978, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Acuaporina 2/metabolismo , Corteza Renal/citología , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Acuaporina 2/genética , Ciclo Celular , Línea Celular , Proliferación Celular , Tamaño de la Célula , Células Epiteliales/citología , Células Epiteliales/metabolismo , Corteza Renal/metabolismo , RatasRESUMEN
The placenta is a vital organ whose function in diseases of pregnancy is altered, resulting in an abnormal supply of nutrients to the foetus. The lack of placental vasculature homeostasis regulation causes endothelial dysfunction and altered vascular reactivity. The proper distribution of acid- (protons (H(+))) and base-equivalents through the placenta is essential to achieve physiological homeostasis. Several membrane transport mechanisms that control H(+) distribution between the extracellular and intracellular spaces are expressed in the human placenta vascular endothelium and syncytiotrophoblast, including sodium (Na(+))/H(+) exchangers (NHEs). One member of the NHEs family is NHE isoform 1 (NHE1), whose activity results in an alkaline intracellular pH (high intracellular pH (pHi)) and an acidic extracellular pH (pHo). Increased NHE1 expression, maximal transport activity, and turnover are reported in human syncytiotrophoblasts and lymphocytes from patients with diabetes mellitus type I (DMT1), and a positive correlation between NHEs activity and plasma factors, such as that between thrombin and platelet factor 3, has been reported in diabetes mellitus type II (DMT2). However, gestational diabetes mellitus (GDM) could result in a higher sensitivity of the human placenta to acidic pHo. We summarized the findings on pHi and pHo modulation in the human placenta with an emphasis on pregnancies in which the mother diagnosed with diabetes mellitus. A potential role of NHEs, particularly NHE1, is proposed regarding placental dysfunction in DMT1, DMT2, and GDM.
Asunto(s)
Diabetes Gestacional/metabolismo , Placenta/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Femenino , Humanos , Concentración de Iones de Hidrógeno , Embarazo , Trofoblastos/metabolismoRESUMEN
To sustain tumor growth, the cancer cells need to adapt to low levels of oxygen (i.e., hypoxia) in the tumor tissue and to the tumor-associated acidic microenvironment. In this phenomenon, the activation of the sodium/proton exchanger 1 (NHE1) at the plasma membrane and the hypoxia-inducible factor (HIF) are critical for the control of the intracellular pH (pHi) and for hypoxia adaptation, respectively. Interestingly, both of these mechanisms end in sustaining cancer cell proliferation. However, regulatory mechanisms of pHi in human ovary tissue and in malignant ascites are unknown. Additionally, a potential role of NHE1 in the modulation of H(+) efflux in human ovarian cancer cells is unknown. In this review, we discussed the characteristics of tumor microenvironment of primary human ovarian tumors and tumor ascites, in terms of pHi regulatory mechanisms and oxygen level. The findings described in the literature suggest that NHE1 may likely play a role in pHi regulation and cell proliferation in human ovarian cancer, potentially involving HIF2α activation. Since ovarian cancer is the fifth cause of prevalence of women cancer in Chile and is usually of late diagnosis, i.e., when the disease jeopardizes peritoneal cavity and other organs, resulting in reduced patient survival, new efforts are required to improve patient-life span and for a better understanding of the pathophysiology of the disease. The potential advantage of the use of amiloride and amiloride-derivatives for cancer treatment in terms of NHE1 expression and activity is also discussed as a therapeutic approach in human ovarian cancer.
Asunto(s)
Neoplasias Ováricas/metabolismo , Animales , Proliferación Celular/fisiología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Oxígeno/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Microambiente Tumoral/fisiologíaRESUMEN
The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 µmol/L STa (30 minutes), 25 µmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 µmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 µmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 µmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144-7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.
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AMP Cíclico/metabolismo , Enterotoxinas/farmacología , Células Epiteliales/efectos de los fármacos , Escherichia coli , Calor , Intestinos/citología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Adulto , Línea Celular Tumoral , GMP Cíclico/metabolismo , Estabilidad de Medicamentos , Enterotoxinas/química , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Cinética , Masculino , Protones , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
The intracellular pH (pHi) of syncytiotrophoblasts is regulated, in part, by Na(+)/H(+) exchanger (NHE)-1, NHE-2, and NHE-3. Failures in pHi homeostasis could alter critical cellular functions such as water transport and cell volume. Here, we evaluated whether alterations in syncytiotrophoblast pHi could modify water uptake mediated by aquaporins (AQPs) and the contribution of NHEs to this mechanism. We showed that changes in syncytiotrophoblast pHi did not affect water uptake in the presence of functional NHEs. However, inhibition of NHEs alters transcellular water transport mediated by AQPs in acidosis. These results suggest an interaction between placental AQPs and NHEs in the regulation of water uptake during acidotic states.
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Acuaporinas/metabolismo , Placenta/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Trofoblastos/metabolismo , Agua/metabolismo , Transporte Biológico/fisiología , Femenino , Humanos , EmbarazoRESUMEN
The excitability of neuronal networks is strongly modulated by changes in pH. The origin of these changes, however, is still under debate. The high complexity of neural systems justifies the use of computational simulation to investigate mechanisms that are possibly involved. Simulated neuronal activity includes non-synaptic epileptiform events (NEA) induced in hippocampal slices perfused with high-K(+) and zero-Ca(2+), therefore in the absence of the synaptic circuitry. A network of functional units composes the NEA model. Each functional unit represents one interface of neuronal/extracellular space/glial segments. Each interface contains transmembrane ionic transports, such as ionic channels, cotransporters, exchangers and pumps. Neuronal interconnections are mediated by gap-junctions, electric field effects and extracellular ionic fluctuations modulated by extracellular electrodiffusion. Mechanisms investigated are those that change intracellular and extracellular ionic concentrations and are able to affect [H(+)]. Our simulations suggest that the intense fluctuations in intra and extracellular concentrations of Na(+), K(+) and Cl(-) that accompany NEA are able to affect the combined action of the Na(+)/H(+) exchanger (NHE), [HCO(-)(3)]/Cl(-) exchanger (HCE), H(+) pump and the catalytic activity of intra and extracellular carbonic anhydrase. Cellular volume changes and extracellular electrodiffusion are responsible for modulating pH.
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Simulación por Computador , Hipocampo/citología , Hipocampo/fisiología , Modelos Neurológicos , Red Nerviosa/citología , Red Nerviosa/fisiología , Tamaño de la Célula , Antiportadores de Cloruro-Bicarbonato/metabolismo , Cloro/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Modelos Biológicos , Neuronas/citología , Neuronas/fisiología , Potasio/metabolismo , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
INTRODUCTION: Aldosterone can induce changes in the expression or activity of Na(+)/H(+) exchanger isoform 1 (NHE-1) in vascular smooth muscle cells. We aimed to clarify whether chronic mineralocorticoid receptor activation exerts an effect on the activity of NHE-1 in the aorta of mineralocorticoid-induced hypertensive rats. METHODS: Uninephrectomized male Sprague-Dawley rats received subcutaneously 10 mg/week of desoxycorticosterone (DOCA) with or without 20 mg/kg of spironolactone, or vehicle alone (n = 20). After four weeks of treatment, the animals were sacrificed; the aorta was excised for subsequent studies, including histological analysis, RT-PCR, Western blot, measurement of NHE-1 activity and vascular contractility in the presence or absence of the selective NHE-1 inhibitor ethyl-isopropyl amiloride (EIPA). RESULTS: Chronic DOCA treatment increased the NHE-1 activity, systolic and diastolic blood pressure, and aortic wall thickness. All these effects were prevented by co-treatment with Spironolactone (p < 0.05). Phenylephrine-induced vascular contractility was significantly reduced in the DOCA group when EIPA was added in the media (p < 0.05). No significant differences in NHE-1 mRNA or protein levels were detected between groups. CONCLUSIONS: Chronic DOCA administration induced functional and morphological alterations in the rat aorta that are partially explained by enhanced NHE-1 activity and prevented by spironolactone. However, we did not observe changes in the NHE-1 transcript or protein levels, suggesting that the effect may be due to post-transcriptional modifications induced by mineralocorticoid receptor activation.
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Aorta/metabolismo , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Espironolactona/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Desoxicorticosterona/farmacología , Hipertensión/fisiopatología , Masculino , Mineralocorticoides , Fenilefrina/farmacología , Ratas Sprague-Dawley , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Coloración y Etiquetado , Vasoconstricción/efectos de los fármacosRESUMEN
Human sperm has several mechanisms to control its ionic milieu, such as the Na,K-ATPase (NKA), the Ca-ATPase of the plasma membrane (PMCA), the Na(+)/Ca(2) (+)-exchanger (NCX) and the Na(+)/H(+)-exchanger (NHE). On the other hand, the dynein-ATPase is the intracellular motor for sperm motility. In this work, we evaluated NKA, PMCA, NHE, NCX and dynein-ATPase activities in human sperm and investigated their correlation with sperm motility. Sperm motility was measured by Computer Assisted Semen Analysis. It was found that the NKA activity is inhibited by ouabain with two Ki (7.9 × 10(-9) and 9.8 × 10(-5)âM), which is consistent with the presence of two isoforms of α subunit of the NKA in the sperm plasma membranes (α1 and α4), being α4 more sensitive to ouabain. The decrease in NKA activity is associated with a reduction in sperm motility. In addition, sperm motility was evaluated in the presence of known inhibitors of NHE, PMCA and NCX, such as amiloride, eosin, and KB-R7943, respectively, as well as in the presence of nigericin after incubation with ouabain. Amiloride, eosin and KB-R7943 significantly reduced sperm motility. Nigericin reversed the effect of ouabain and amiloride on sperm motility. Dynein-ATPase activity was inhibited by acidic pH and micromolar concentrations of Ca(2) (+). We explain our results in terms of inhibition of the dynein-ATPase in the presence of higher cytosolic H(+) and Ca(2) (+), and therefore inhibition of sperm motility.