RESUMEN

BACKGROUND SLC4A4 is differentially expressed in a variety of tumors, but its significance in colon adenocarcinoma has not been determined. MATERIAL AND METHODS Transcriptomes of two cohorts, GSE41258 and GSE32323, contained in The Cancer Genome Atlas (TCGA) were analysed to determine differences in SLC4A4 expression between tumor and normal tissue and their correlations with overall survival. The relationships between SLC4A4 expression and clinical characteristics were determined by COX regression analysis and logistic regression analysis, and correlations of SLC4A4 levels with tumor infiltrating immune cells (TIICs) and genes with high mutation frequency were evaluated by Pearson correlation analysis. Molecular functions and signaling pathways that might be affected by changes in SLC4A4 expression were determined by gene set enrichment analysis (GSEA). The overall distribution of TIICs was determined by two web servers: tumor immune estimation resource (TIMER) and CIBERSORT. RESULTS SLC4A4 expression was lower in colon adenocarcinoma than in normal colon tissue, suggesting that SLC4A4 was associated with poor prognosis. Reduced SLC4A4 expression was also associated with lymph node invasion and distant metastasis and was moderately correlated with increased expression of MUC4 and SMAD4, two genes with high mutation frequency in colon adenocarcinoma. GSEA indicated that changes in SLC4A4 expression affects several biological processes, including mismatch repair, base excision repair, and DNA replication. Eight TIICs in the tumor microenvironment differed significantly in groups with low and high expression of SLC4A4. CONCLUSIONS SLC4A4 may be a novel biomarker predicting prognosis in patients with colon adenocarcinoma. TIICs differed significantly in samples with higher and lower expression of SLC4A4.

Asunto(s)

Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Bases de Datos Factuales , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Simportadores de Sodio-Bicarbonato/biosíntesis , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/genética , Femenino , Humanos , Masculino , Proteínas de Neoplasias/genética , Pronóstico , Simportadores de Sodio-Bicarbonato/genética

RESUMEN

Pancreatic cancer (PC) is a malignant tumor that is difficult to diagnose and treat. Circular RNAs (circRNAs) are biomarkers that may be used to diagnose certain cancers or act as targets for cancer treatment. We aimed to explore the functions of human circular RNA 001587 (hsa_circRNA_001587) on the progression of PC and the underlying mechanism. The expression pattern of hsa_circRNA_001587 and microRNA-223 (miR-223) in PC tissues and cells was determined by RT-qPCR. Dual-luciferase reporter gene assay, RNA-pulldown, Argonaute 2 (AGO2) immunoprecipitation assay, and Northern blot analysis were applied to verify the binding relationships among hsa_circRNA_001587, miR-223 and solute carrier family 4 member 4 (SLC4A4). Further analysis of their roles was performed in PC cell line PANC-1. Moreover, we either downregulated or upregulated the expression of hsa_circRNA_001587, miR-223, and SLC4A4 by transfection in vitro. A mouse xenograft model of PC cells was established to evaluate tumor growth in vivo. hsa_circRNA_001587 was poorly expressed, but miR-223 was highly expressed in PC tissues and cell lines. Upregulation of hsa_circRNA_001587 downregulated the expression of matrix metalloproteinase-2 and-9, minichromosome maintenance 2, and vascular endothelial growth factor, and decreased the proliferation, migration, invasion, angiogenic and tumorigenic abilities of PC cells. MiR-223, which can bind with hsa_circRNA_001587, reversed the effects of hsa_circRNA_001587 on PC cells. In addition, SLC4A4 was identified as a target of miR-223, and its knockdown could counteract the regulatory effects of overexpressed hsa_circRNA_001587 or inhibited miR-223 expression on PC cells. Therefore, hsa_circRNA_001587 inhibits PC cell migration, invasion, angiogenesis and tumorigenesis by impairing miR-223-mediated SLC4A4 inhibition.NEW & NOTEWORTHY Human circular (hsa_circ)RNA_001587 and solute carrier family 4 member 4 (SLC4A4) are poorly expressed but microRNA (miR)R-223 is overexpressed in pancreatic cancer (PC) cells. hsa_circRNA_001587 binds to miR-223. Overexpression of hsa_circRNA_001587 inhibits PC progression. Overexpression of miR-223 downregulates the expression of SLC4A4 and promotes PC cell growth. hsa_circRNA_001587 may be a potential target for PC treatment.

Asunto(s)

Movimiento Celular/genética , MicroARNs/genética , MicroARNs/metabolismo , Invasividad Neoplásica/genética , Neovascularización Patológica/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , ARN Circular/genética , Simportadores de Sodio-Bicarbonato/biosíntesis , Simportadores de Sodio-Bicarbonato/genética , Adulto , Anciano , Animales , Línea Celular Tumoral , Femenino , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Regulación hacia Arriba/genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The high-flux/low-affinity cyanobacterial bicarbonate transporter BicA is a member of sulfate permease/solute carrier 26 (SulP/SLC26) family and plays a major role in cyanobacterial inorganic carbon uptake. In order to study this important membrane protein, robust platforms for over-expression and protocols for purification are required. In this work we have optimized the expression and purification of BicA from strain Synechocystis sp. PCC 6803 (BicA6803) in Escherichia coli. It was determined that expression with C43 (DE3) Rosetta2 at 37 °C produced the highest levels of over-expressed BicA6803 relative to other strains screened, and membrane solubilization with n-dodecyl-ß-d-maltopyranoside facilitated the purification of high levels of stable and homogenous BicA6803. Using these expression and purification strategies, the final yields of purified BicA were 6.5 ± 1.0 mg per liter of culture.

Asunto(s)

Proteínas Bacterianas , Expresión Génica , Simportadores de Sodio-Bicarbonato , Synechocystis/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Simportadores de Sodio-Bicarbonato/biosíntesis , Simportadores de Sodio-Bicarbonato/química , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/aislamiento & purificación , Synechocystis/metabolismo

RESUMEN

We analyzed the expression of molecular targets of natriuretic action of prolactin in different layers of the kidney in the rat model of cholestasis of pregnancy. Sodium bicarbonate cotransporter NBCe1 was most sensitive to the conditions of cholestasis and cholestasis of pregnancy: the expression NBCe1 mRNA and protein in the renal outer medulla decreased in comparison with the normal. All forms of cholestasis affected the mRNA expression of sodium-potassium chloride co-transporter NCC, α-subunit of the ENaCα epithelial sodium channel, and Nedd4-2 ubiquitin ligase in different layers of the kidney. The obtained data suggest that prolactin provides fine tuning of various sodium transporters in different parts of the nephron under pathological conditions.

Asunto(s)

Colestasis/patología , Transporte Iónico/fisiología , Médula Renal/metabolismo , Prolactina/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Animales , Modelos Animales de Enfermedad , Canales Epiteliales de Sodio/biosíntesis , Canales Epiteliales de Sodio/genética , Femenino , Ubiquitina-Proteína Ligasas Nedd4/biosíntesis , Ubiquitina-Proteína Ligasas Nedd4/genética , Embarazo , ARN Mensajero/biosíntesis , Ratas , Simportadores de Sodio-Bicarbonato/biosíntesis , Simportadores de Sodio-Bicarbonato/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/biosíntesis , Miembro 3 de la Familia de Transportadores de Soluto 12/genética

RESUMEN

The electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), is the major bicarbonate transporter expressed in astrocytes. It is highly sensitive for bicarbonate and the main regulator of intracellular, extracellular, and synaptic pH, thereby modulating neuronal excitability. However, despite these essential functions, the molecular mechanisms underlying NBCe1-mediated astrocytic response to extracellular pH changes are mostly unknown. Using primary mouse cortical astrocyte cultures, we investigated the effect of long-term extracellular metabolic alkalosis on regulation of NBCe1 and elucidated the underlying molecular mechanisms by immunoblotting, biotinylation of surface proteins, intracellular H+ recording using the H+ -sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein, and phosphoproteomic analysis. The results showed significant downregulation of NBCe1 activity following metabolic alkalosis without influencing protein abundance or surface expression of NBCe1. During alkalosis, the rate of intracellular H+ changes upon challenging NBCe1 was decreased in wild-type astrocytes, but not in cortical astrocytes from NBCe1-deficient mice. Alkalosis-induced decrease of NBCe1 activity was rescued after activation of mTOR signaling. Moreover, mass spectrometry revealed constitutively phosphorylated S255-257 and mutational analysis uncovered these residues being crucial for NBCe1 transport activity. Our results demonstrate a novel mTOR-regulated mechanism by which NBCe1 functional expression is regulated. Such mechanism likely applies not only for NBCe1 in astrocytes, but in epithelial cells as well.

Asunto(s)

Astrocitos/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Simportadores de Sodio-Bicarbonato/biosíntesis , Serina-Treonina Quinasas TOR/fisiología , Alcalosis/metabolismo , Alcalosis/patología , Animales , Células Cultivadas , Femenino , Expresión Génica , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/fisiología , Simportadores de Sodio-Bicarbonato/genética

RESUMEN

Genome-wide association studies have revealed an association between variation at the SLC4A7 locus and blood pressure. SLC4A7 encodes the electroneutral Na+/HCO3- co-transporter NBCn1 which regulates intracellular pH (pHi). We conducted a functional study of variants at this locus in primary cultures of vascular smooth muscle and endothelial cells. In both cell types, we found genotype-dependent differences for rs13082711 in DNA-nuclear protein interactions, where the risk allele is associated with increased SLC4A7 expression level, NBCn1 availability and function as reflected in elevated steady-state pHi and accelerated recovery from intracellular acidosis. However, in the presence of Na+/H+ exchange activity, the SLC4A7 genotypic effect on net base uptake and steady-state pHi persisted only in vascular smooth muscle cells but not endothelial cells. We found no discernable effect of the missense polymorphism resulting in the amino acid substitution Glu326Lys. The finding of a genotypic influence on SLC4A7 expression and pHi regulation in vascular smooth muscle cells provides an insight into the molecular mechanism underlying the association of variation at the SLC4A7 locus with blood pressure.

Asunto(s)

Hipertensión/metabolismo , Músculo Liso Vascular/metabolismo , Simportadores de Sodio-Bicarbonato/genética , Alelos , Sustitución de Aminoácidos/genética , Animales , Presión Sanguínea/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Concentración de Iones de Hidrógeno , Hipertensión/genética , Hipertensión/patología , Músculo Liso Vascular/patología , Mutación , Ratas , Sodio/metabolismo , Simportadores de Sodio-Bicarbonato/biosíntesis

RESUMEN

The mechanisms regulating proximal tubule ammonia metabolism are incompletely understood. The present study addressed the role of the proximal tubule basolateral electrogenic Na(+)-coupled bicarbonate cotransporter (NBCe1; Slc4a4) in renal ammonia metabolism. We used mice with heterozygous and homozygous NBCe1 gene deletion and compared these mice with their wild-type littermates. Because homozygous NBCe1 gene deletion causes 100% mortality before day 25, we studied mice at day 8 (±1 day). Both heterozygous and homozygous gene deletion caused a gene dose-related decrease in serum bicarbonate. The ability to lower urinary pH was intact, and even accentuated, with NBCe1 deletion. However, in contrast to the well-known effect of metabolic acidosis to increase urinary ammonia excretion, NBCe1 deletion caused a gene dose-related decrease in ammonia excretion. There was no identifiable change in proximal tubule structure by light microscopy. Examination of proteins involved in renal ammonia metabolism showed decreased expression of phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase, key enzymes in proximal tubule ammonia generation, and increased expression of glutamine synthetase, which recycles intrarenal ammonia and regenerates glutamine. Expression of key proteins involved in ammonia transport outside of the proximal tubule (rhesus B glycoprotein and rhesus C glycoprotein) was not significantly changed by NBCe1 deletion. We conclude from these findings that NBCe1 expression is necessary for normal proximal tubule ammonia metabolism.

Asunto(s)

Amoníaco/metabolismo , Riñón/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Animales , Bicarbonatos/sangre , Western Blotting , Eliminación de Gen , Inmunohistoquímica , Túbulos Renales Colectores/metabolismo , Túbulos Renales Proximales/metabolismo , Ratones , Ratones Noqueados , Potasio/sangre , Sodio/sangre , Simportadores de Sodio-Bicarbonato/biosíntesis , Simportadores de Sodio-Bicarbonato/genética , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Intercambiadores de Sodio-Hidrógeno/genética

RESUMEN

UNLABELLED: Despite the frequent expression of N-terminally truncated ErbB2 (ΔNErbB2/p95HER2) in breast cancer and its association with Herceptin resistance and poor prognosis, it remains poorly understood how ΔNErbB2 affects chemotherapy-induced cell death. Previously it was shown that ΔNErbB2 upregulates acid extrusion from MCF-7 breast cancer cells and that inhibition of the Na(+)/H(+) exchanger (SLC9A1/NHE1) strongly sensitizes ΔNErbB2-expressing MCF-7 cells to cisplatin chemotherapy. The aim of this study was to identify the mechanism through which ΔNErbB2 regulates cisplatin-induced breast cancer cell death, and determine how NHE1 regulates this process. Cisplatin treatment elicited apoptosis, ATM phosphorylation, upregulation of p53, Noxa (PMAIP1), and PUMA (BBC3), and cleavage of caspase-9, -7, fodrin, and PARP-1 in MCF-7 cells. Inducible ΔNErbB2 expression strongly reduced cisplatin-induced ATM- and p53-phosphorylation, augmented Noxa upregulation and caspase-9 and -7 cleavage, doubled p21(WAF1/Cip1) (CDKN1A) expression, and nearly abolished Bcl-2 expression. LC3-GFP analysis demonstrated that autophagic flux was reduced by cisplatin in a manner augmented by ΔNErbB2, yet did not contribute to cisplatin-induced death. Using knockdown approaches, it was shown that cisplatin-induced caspase-7 cleavage in ΔNErbB2-MCF-7 cells was Noxa- and caspase-9 dependent. This pathway was augmented by NHE1 inhibition, while the Na(+)/HCO3 (-) cotransporter (SLC4A7/NBCn1) was internalized following cisplatin exposure. IMPLICATIONS: This work reveals that ΔNErbB2 strongly affects several major pro- and antiapoptotic pathways and provides mechanistic insight into the role of NHE1 in chemotherapy resistance. These findings have relevance for defining therapy regimens in breast cancers with ΔNErbB2 and/or NHE1 overexpression.

Asunto(s)

Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Cisplatino/administración & dosificación , Resistencia a Antineoplásicos/genética , Receptor ErbB-2/biosíntesis , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Proteínas de Transporte de Catión/biosíntesis , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Fosforilación , Receptor ErbB-2/genética , Simportadores de Sodio-Bicarbonato/biosíntesis , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Intercambiadores de Sodio-Hidrógeno/genética

RESUMEN

Corneal endothelial dysfunction remains a major indication for corneal transplantation. Both corneal endothelial cells and stromal cells originate from the neural crest, but have distinct phenotypes and function in the adult cornea. We previously reported that stem cells isolated from the adult corneal stroma [cornea-derived precursors (COPs)] show characteristics of multipotent neural crest-derived stem cells. In this study, we report the induction of functional tissue-engineered corneal endothelium (TECE) from mouse and human COPs. TECE was engineered from Wnt1-Cre/Floxed EGFP mouse COPs in a medium containing retinoic acid and glycogen synthase kinase (GSK) 3ß inhibitor (activator of Wnt/ß-catenin signaling). The expression levels of major markers characterizing corneal endothelial function (Atp1a1, Slc4a4, Car2, Col4a2, Col8a2, and Cdh2) were significantly upregulated. Both retinoic acid and GSK 3ß inhibitor upregulated the expression of Pitx2, a homeobox gene involved in the development of the anterior segment of the eye. GSK 3ß inhibitor increased Atp1a1 expression and Na,K-ATPase pump activity of TECE, which was significantly higher than COPs or control 3T3 cells, and 2.6-fold higher than cultured mouse corneal endothelial cells. Mouse TECE transplanted into rabbit corneas maintained transparency and corneal thickness, whereas control corneas without TECE showed marked edema and increased corneal thickness. Furthermore, we successfully induced TECE from human COPs, and human TECE transplanted into rabbit corneas also maintained corneal transparency and thickness. This protocol enables efficient production of corneal endothelium from corneal stromal stem cells by direct induction, which may lead to a novel stem cell therapy for corneal endothelial dysfunction.

Asunto(s)

Sustancia Propia/metabolismo , Trasplante de Córnea , Endotelio Corneal/citología , Endotelio Corneal/fisiología , Cresta Neural/citología , Ingeniería de Tejidos/métodos , Animales , Antígenos CD/biosíntesis , Cadherinas/biosíntesis , Comunicación Celular , Células Cultivadas , Colágeno Tipo IV/biosíntesis , Colágeno Tipo VIII/biosíntesis , Sustancia Propia/citología , Proteínas de Homeodominio/biosíntesis , Humanos , Ratones , Células Madre Multipotentes , Proteínas del Tejido Nervioso/farmacología , Células-Madre Neurales , Conejos , Simportadores de Sodio-Bicarbonato/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Factores de Transcripción/biosíntesis , Tretinoina/farmacología , Regulación hacia Arriba , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Proteína del Homeodomínio PITX2

RESUMEN

The pituitary hormone prolactin is a pleiotropic endocrine factor that plays a major role in the regulation of ion balance in fish, with demonstrated actions mainly in the gills and kidney. The role of prolactin in intestinal ion transport remains little studied. In marine fish, which have high drinking rates, epithelial bicarbonate secretion in the intestine produces luminal carbonate aggregates believed to play a key role in water and ion homeostasis. The present study was designed to establish the putative role of prolactin in the regulation of intestinal bicarbonate secretion in a marine fish. Basolateral addition of prolactin to the anterior intestine of sea bream mounted in Ussing chambers caused a rapid (<20 min) decrease of bicarbonate secretion measured by pH-stat. A clear inhibitory dose-response curve was obtained, with a maximal inhibition of 60-65% of basal bicarbonate secretion. The threshold concentration of prolactin for a significant effect on bicarbonate secretion was 10 ng ml(-1), which is comparable with putative plasma levels in seawater fish. The effect of prolactin on apical bicarbonate secretion was independent of the generation route for bicarbonate, as shown in a preparation devoid of basolateral HCO(3)(-)/CO(2) buffer. Specific inhibitors of JAK2 (AG-490, 50 µmol l(-1)), PI3K (LY-294002, 75 µmol l(-1)) or MEK (U-012610, 10 µmol l(-1)) caused a 50-70% reduction in the effect of prolactin on bicarbonate secretion, and demonstrated the involvement of prolactin receptors. In addition to rapid effects, prolactin has actions at the genomic level. Incubation of intestinal explants of anterior intestine of the sea bream in vitro for 3 h demonstrated a specific effect of prolactin on the expression of the Slc4a4A Na(+)-HCO(3)(-) co-transporter, but not on the Slc26a6A or Slc26a3B Cl(-)/HCO(3)(-) exchanger. We propose a new role for prolactin in the regulation of bicarbonate secretion, an essential function for ion/water homeostasis in the intestine of marine fish.

Asunto(s)

Bicarbonatos/metabolismo , Mucosa Intestinal/metabolismo , Prolactina/fisiología , Dorada/metabolismo , Animales , Antiportadores de Cloruro-Bicarbonato/biosíntesis , Cromonas/farmacología , Transporte Iónico , Janus Quinasa 2/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Prolactina/administración & dosificación , Prolactina/farmacología , Transducción de Señal , Simportadores de Sodio-Bicarbonato/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Tirfostinos/farmacología , Equilibrio Hidroelectrolítico

RESUMEN

Mutational changes of one transporter can have deleterious effects on epithelial function leaving the cells with the options of either compensating for the loss of function or dedifferentiating. Previous studies have shown that the choroid plexus epithelium (CPE) from mice lacking the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger (NCBE) encoded by Slc4a10 leads to retargeting of the Na(+)/H(+) exchanger 1 (NHE1) from the luminal to the basolateral plasma membrane. We hypothesized that disruption of NCBE, the main basolateral Na(+) importer in the CPE, would lead to a compensatory increase in the abundance of other important transport proteins in this tissue. Aquaporin-1 (AQP1) abundance was 42.7% lower and Na,K-ATPase 36.4% lower in the CPE of Slc4a10 knockout mice, respectively. The NHE1 binding ezrin cytoskeleton appeared disrupted in Slc4a10 knockout mice, whereas no changes were observed in cellular polarization with respect to claudin-2 and appearance of luminal surface microvilli. The renal proximal tubule constitutes a leaky epithelium with high transport rate similar to CPE. Here, Slc4a10 knockout did not affect Na,K-ATPase or AQP1 expression. CPE from AQP1 knockout mice has a secretory defect similar to Slc4a10 mice. However, neither NCBE nor Na,K-ATPase expression was affected in CPE from AQP1 knockout mice. By contrast, the abundance of Na,K-ATPase and NBCe1 was decreased by 23 and 31.7%, respectively, in AQP1 knockout proximal tubules, while the NHE3 abundance was unchanged. In conclusion, CPE lacking NCBE seems to spare the molecular machinery involved in CSF secretion rather than compensate for the loss of the Na(+) loader. Slc4a10 knockout seems to be more deleterious to CPE than AQP1 knockout.

Asunto(s)

Acuaporina 1/genética , Antiportadores de Cloruro-Bicarbonato/deficiencia , Plexo Coroideo/metabolismo , Regulación hacia Abajo/genética , Eliminación de Gen , Regulación de la Expresión Génica/genética , Simportadores de Sodio-Bicarbonato/deficiencia , Animales , Acuaporina 1/biosíntesis , Antiportadores de Cloruro-Bicarbonato/biosíntesis , Antiportadores de Cloruro-Bicarbonato/genética , Femenino , Masculino , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Simportadores de Sodio-Bicarbonato/biosíntesis , Simportadores de Sodio-Bicarbonato/genética

RESUMEN

BACKGROUND: Disturbances in pH affect artery function, but the mechanistic background remains controversial. We investigated whether Na(+), HCO3- transporter NBCn1, by regulating intracellular pH(pH1), influences artery function and blood pressure regulation. METHODS AND RESULTS: Knockout of NBCn1 in mice eliminated Na+, HCO3⁻ cotransport and caused a lower steady-state pH(i) in mesenteric artery smooth muscle and endothelial cells in situ evaluated by fluorescence microscopy. Using myography, arteries from NBCn1 knockout mice showed reduced acetylcholine-induced NO-mediated relaxations and lower rho-kinase-dependent norepinephrine-stimulated smooth muscle Ca²âº sensitivity. Acetylcholine-stimulated NO levels (electrode measurements) and N-nitro-l-arginine methyl ester-sensitive l-arginine conversion (radioisotope measurements) were reduced in arteries from NBCn1 knockout mice, whereas relaxation to NO-donor S-nitroso-N-acetylpenicillamine, acetylcholine-induced endothelial Ca²âº responses (fluorescence microscopy), and total and Ser-1177 phosphorylated endothelial NO-synthase expression (Western blot analyses) were unaffected. Reduced NO-mediated relaxations in arteries from NBCn1 knockout mice were not rescued by superoxide scavenging. Phosphorylation of myosin phosphatase targeting subunit at Thr-850 was reduced in arteries from NBCn1 knockout mice. Evaluated by an in vitro assay, rho-kinase activity was reduced at low pH. Without CO2/HCO3⁻, no differences in pH(i), contraction or relaxation were observed between arteries from NBCn1 knockout and wild-type mice. Based on radiotelemetry and tail-cuff measurements, NBCn1 knockout mice were mildly hypertensive at rest, displayed attenuated blood pressure responses to NO-synthase and rho-kinase inhibition and were resistant to developing hypertension during angiotensin-II infusion. CONCLUSIONS: Intracellular acidification of smooth muscle and endothelial cells after knockout of NBCn1 inhibits NO-mediated and rho-kinase-dependent signaling in isolated arteries and perturbs blood pressure regulation.

Asunto(s)

Calcio/fisiología , Hipertensión/prevención & control , Músculo Liso Vascular/fisiopatología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/fisiología , Simportadores de Sodio-Bicarbonato/deficiencia , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Vasodilatación/fisiología , Animales , Señalización del Calcio/genética , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Hipertensión/genética , Hipertensión/metabolismo , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , Arterias Mesentéricas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Técnicas de Cultivo de Órganos , Simportadores de Sodio-Bicarbonato/biosíntesis , Simportadores de Sodio-Bicarbonato/genética , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Intercambiadores de Sodio-Hidrógeno/genética , Vasodilatación/efectos de los fármacos

RESUMEN

The maintenance of intracellular pH is important in neuronal function. Na(+)/HCO(3)(-) cotransporter (NBC), a bicarbonate-dependent acid-base transport protein, may contribute to cellular acid-base homeostasis in pathophysiological processes. We examined the alterations of NBC immunoreactivity and its protein levels in the hippocampal CA1 region after transient cerebral ischemia in gerbils. In the sham-operated group, moderate NBC immunoreactivity was detected in CA1 pyramidal neurons, and, 12 h after I/R, the immunoreactivity in the pyramidal neurons was markedly increased over controls. Three days after I/R, NBC immunoreactivity nearly disappeared in the CA1 pyramidal neurons. However, NBC immunoreactivity was detected in the non-pyramidal neurons of the ischemic CA1 region at 3 days after I/R. From double immunofluorescence study with glial markers, NBC immunoreactivity was detected in astrocytes, not in microglia, at 4 days after I/R. NBC protein level in the CA1 region was significantly increased at 12 h post-ischemia and significantly decreased at 2 days post-ischemia. Thereafter, NBC protein level was again increased and returned to the level of the sham-operated group at 4 days post-ischemia. On the other hand, treatment with 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), an inorganic anion exchanger blocker including Cl-bicarbonate exchanger, protected CA1 pyramidal neurons from I/R injury at 4 days post-ischemia. These results indicate that changes in NBC expressions may play an important role in neuronal damage and astrocytosis induced by transient cerebral ischemia.

Asunto(s)

Región CA1 Hipocampal/metabolismo , Daño por Reperfusión/metabolismo , Simportadores de Sodio-Bicarbonato/inmunología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Astrocitos/metabolismo , Región CA1 Hipocampal/patología , Gerbillinae , Ataque Isquémico Transitorio/fisiopatología , Masculino , Neuronas/inmunología , Neuronas/metabolismo , Daño por Reperfusión/inmunología , Simportadores de Sodio-Bicarbonato/biosíntesis

RESUMEN

Cariporide, an Na/H exchanger inhibitor, is a drug with cardioprotective properties. However, chronic treatment with cariporide may modify the protein phenotype of the cardiomyocytes. Disruption of the equilibrium between a cariporide-modified phenotype and the supply of cariporide could be deleterious. The aim of this study was to test the effects of this equilibrium rupture (EqR) on cardiac function at baseline and acute ischemia reperfusion. Rats were chronically treated with cariporide (2.5 mg·kg·d) or with placebo for 21 days, after which isolated Langendorff-mode heart perfusion experiments utilized cariporide-free buffer. During this type of perfusion, the drug is rapidly cleared from the cellular environment. After 30 minutes of stabilization, the hearts were subjected to global zero-flow ischemia (25 minutes) followed by reperfusion (45 minutes). Measures of mechanical function, oxygen consumption, lactate plus pyruvate, CO2 and proton release into the coronary effluent were determined. The gene and protein expression of proton extruders was also evaluated. Chronic cariporide administration followed by EqR reduced the expression of the Na/H exchanger, increased the expression of the HCO3 or Na exchanger, decreased monocarboxylate/H carrier expression, reduced the lactate plus pyruvate release but did not change the glucose oxidation rate and mechanical function compared with baseline conditions. The resulting low glycolytic rate was associated with a stronger contracture during ischemia. During reperfusion, the early release of acidic forms was higher and redirected toward the use of the Na/H and HCO3 /Na exchangers to the detriment of the safe monocarboxylate/H carrier. Both phenomena were assumed to increase the Na uptake and activate the Na/Ca exchanger, resulting in Na and Ca overload and further cellular damage. This explains the impaired recovery of the contractile function observed in the EqR group during reperfusion. In conclusion, although cariporide is usually cardioprotective, a disruption of its chronic treatment followed by an ischemia/reperfusion event can become deleterious.

Asunto(s)

Antiarrítmicos/farmacología , Arritmias Cardíacas/prevención & control , Cardiotónicos/farmacología , Guanidinas/farmacología , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Sulfonas/farmacología , Animales , Antiarrítmicos/metabolismo , Arritmias Cardíacas/tratamiento farmacológico , Análisis Químico de la Sangre , Cardiotónicos/metabolismo , Esquema de Medicación , Guanidinas/metabolismo , Pruebas de Función Cardíaca , Homeostasis/efectos de los fármacos , Masculino , Transportadores de Ácidos Monocarboxílicos/biosíntesis , Transportadores de Ácidos Monocarboxílicos/genética , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/fisiopatología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Simportadores de Sodio-Bicarbonato/biosíntesis , Simportadores de Sodio-Bicarbonato/genética , Intercambiadores de Sodio-Hidrógeno/genética , Sulfonas/metabolismo , Simportadores/biosíntesis , Simportadores/genética , Factores de Tiempo

RESUMEN

The SLC4A10 gene, which is highly expressed in the mammalian brain, contains two known alternative splicing units, inserts A and B, and is theoretically capable of producing four NBCn2 splice variants: NBCn2-A, -B, -C, and -D. By immunoprecipitation and western blotting, a previous study showed the putative NBCn2-D to be expressed predominantly in the subcortex (SCX) and medulla (MD) of mouse brain. However, no evidence has been provided, in any species, for the existence of a full-length transcript encoding NBCn2-D. In the present study, we report for the first time the cloning of the full-length cDNAs encoding NBCn2-D from mouse SCX and MD. Based on the frequency of bacterial colonies obtained after PCR, we conclude that in SCX, the NBCn2-A transcript is dominant, whereas in MD, NBCn2-B is dominant. NBCn2-D is the least abundant transcript in each of these two brain regions. An analysis based upon the present PCR data as well as the previous immunoprecipitation/western-blot data suggests the following prevalence of NBCn2 variants in total mouse brain: NBCn2-A (~83%), NBCn2-B (~10%), NBCn2-C (~5%), and NBCn2-D (~2%). We also estimate the prevalence of each variant in each of the five brain regions (i.e., cerebral cortex, SCX, cerebellum, hippocampus, and MD). We hypothesize that the expression of different NBCn2 splice variants is characteristic of specific tissue/cells.

Asunto(s)

Química Encefálica/genética , Antiportadores de Cloruro-Bicarbonato/genética , Regulación de la Expresión Génica , Empalme del ARN , Simportadores de Sodio-Bicarbonato/genética , Transcripción Genética , Animales , Corteza Cerebral/metabolismo , Antiportadores de Cloruro-Bicarbonato/biosíntesis , Clonación Molecular/métodos , ADN Complementario/biosíntesis , Variación Genética/genética , Bulbo Raquídeo/metabolismo , Ratones , Ratones Endogámicos C57BL , Simportadores de Sodio-Bicarbonato/biosíntesis , Xenopus

RESUMEN

In rodent incisors two distinct stages of enamel formation can be identified visually based on cell morphology: the secretory stage and the maturation stage. The expression profiles of many genes characterize both stages, including the bicarbonate transport protein NBCe1. Bicarbonate is a requirement for the mineralizing enamel matrix to buffer excessive protons that form as a consequence of hydroxyapatite formation. NBCe1-B mRNA is up-regulated during the maturation stage of amelogenesis, where hydroxyapatite formation predominates. In this study, a presumed 572-bp NBCe1-B promoter region was subcloned into a reporter construct, and within this 572-bp region of DNA we characterized a 285-bp segment that shows an increase of ≈ 2.3-fold in gene-transcription activity when transfected into ameloblast-like cells and cultured in medium maintained at pH 6.8 (vs. pH 7.4). A presumed pH-responsive transcriptional factor-binding domain(s) thus resides in the 285-bp NBCe1-B promoter region where candidate domains include the nuclear factor of kappa light polypeptide gene enhancer in B-cells1(NFKB1), jun proto-oncogene (JUN), and tumor protein p53(TP53)-binding sites. Mutagenesis studies identify that both the NFKB1- and TP53-binding sites are responsive to changes in the extracellular pH. These data help to explain how ameloblasts respond to the altered extracellular milieu of protons by changing their gene-expression profile throughout the stages of amelogenesis.

Asunto(s)

Ameloblastos/metabolismo , Amelogénesis/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regiones Promotoras Genéticas , Simportadores de Sodio-Bicarbonato/genética , Sitio de Iniciación de la Transcripción , Sitios de Unión , Células Cultivadas , Clonación Molecular , Durapatita/metabolismo , Genes Reporteros , Humanos , Concentración de Iones de Hidrógeno , Subunidad p50 de NF-kappa B/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-jun/genética , Análisis de Secuencia de ADN , Simportadores de Sodio-Bicarbonato/biosíntesis , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba

RESUMEN

The five known Na-coupled HCO(3)(-) transporters (NCBTs) of the solute carrier 4 (SLC4) family play important roles in pH regulation and transepithelial HCO(3)(-) transport. Nearly all of the NCBTs have multiple splice variants. One particular NCBT, the electroneutral Na/HCO(3)(-) cotransporter NBCn2 (SLC4A10), which is predominantly expressed in brain, has three known splice variants-NBCn2-A, -B, and -C-as well as a potential variant-D. It is important to know the tissue-specific expression of the splice variants for understanding the physiological roles of NBCn2 in central nervous system. In the present study, we developed three novel rabbit polyclonal antibodies against NBCn2: (1) anti-ABCD, which recognizes all four variants; (2) anti-BD, which recognizes NBCn2-B and -D; (3) anti-CD, which recognizes NBCn2-C and -D. By western blotting, we examined the expression and distribution of NBCn2 splice variants in five brain regions: cerebral cortex, subcortex, cerebellum, hippocampus, and medulla. The expression pattern revealed with anti-ABCD is distinct from those revealed with anti-BD and anti-CD. Moreover, by using immunoprecipitation in combination with western blotting, we demonstrate that NBCn2-D does indeed exist and that it is predominantly expressed in subcortex, to a lesser extent in medulla, but at very low levels in cortex, cerebellum, and hippocampus. NBCn2-A may be the dominant variant in mouse brain as a whole, and may also dominate in cerebral cortex, cerebellum, and hippocampus. Immunohistochemistry with anti-ABCD shows that NBCn2 is highly expressed in choroid plexus, cortex, molecular layer of cerebellum, hippocampus, and some specific regions of the brainstem.

Asunto(s)

Empalme Alternativo , Encéfalo/metabolismo , Antiportadores de Cloruro-Bicarbonato/biosíntesis , Simportadores de Sodio-Bicarbonato/biosíntesis , Animales , Proteínas de Transporte de Anión/biosíntesis , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/inmunología , Especificidad de Anticuerpos , Antiportadores/biosíntesis , Antiportadores/genética , Antiportadores/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Conejos , Xenopus

RESUMEN

Organic anion transporter 1 (OAT1) and OAT3 in the proximal tubules (PT) of the kidney play important roles in the elimination of harmful endogenous compounds and xenobiotics from the body. We investigated the temporal and spatial expression of OAT1 and OAT3 in the differentiating PT in mouse kidney. Ontogenic expression of OAT1 and OAT3 was investigated by immunohistochemical analysis. The S1, S2, and S3 segments of the PT were identified using antibodies to aquaporin 1 (AQP1), Na+-HCO3- cotransporter 1 (kNBC1), and AQP4. OAT1 immunoreactivity was first detected at PT in the inner cortex of 15-day-old fetuses (F15) and in the outer cortex of 7-day old pups. OAT3 was first observed in the distal tubule of F14 and in S2 segment of the PT of F16 and in S1 and S3 segments around the time of birth; expression increased through postpartum day 21. The ontogenic pattern of expression of OAT1 and OAT3 in the differentiating PT suggests that both transporters may function in the S2 segment in the fetus, but not until after birth in S1 and S3 segments.

Asunto(s)

Túbulos Renales Proximales/metabolismo , Proteína 1 de Transporte de Anión Orgánico/biosíntesis , Transportadores de Anión Orgánico Sodio-Independiente/biosíntesis , Animales , Especificidad de Anticuerpos , Acuaporina 1/biosíntesis , Acuaporina 4/biosíntesis , Diferenciación Celular , Técnica del Anticuerpo Fluorescente , Técnicas para Inmunoenzimas , Inmunohistoquímica , Túbulos Renales Proximales/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Simportadores de Sodio-Bicarbonato/biosíntesis

RESUMEN

PURPOSE: Unilateral ureteral obstruction is a common clinical problem that is often associated with a urinary acidification defect caused by decreased net H(+) secretion and/or HCO(3)(-) reabsorption. To clarify the molecular mechanisms of these defects we examined expression levels of key acid-base transporters along the renal nephron segments and collecting duct. MATERIALS AND METHODS: Wistar rats (Møllegard Breeding Centre, Eiby, Denmark) underwent 24-hour unilateral ureteral obstruction, unilateral ureteral obstruction release followed for 4 days or unilateral ureteral obstruction release followed for 4 days plus experimental acidosis induced by NH(4)Cl oral administration. After sacrifice kidneys were processed for immunoblotting and immunohistochemistry. RESULTS: Semiquantitative immunoblotting revealed that unilateral ureteral obstruction caused significant mean +/- SE down-regulation of type 3 Na(+)/H(+) exchanger to 53% +/- 9%, electrogenic Na(+)/HCO(3)(-) cotransporter to 60% +/- 9%, type 1 bumetanide sensitive Na(+)-K(+)(NH(4)(+)) -2Cl(-) cotransporter to 64% +/- 7%, electroneutral Na(+)/HCO(3)(-) cotransporter to 43% +/- 4% and anion exchanger (pendrin) to 53% +/- 10% in the obstructed kidney, which was confirmed by immunohistochemistry. After release of unilateral ureteral obstruction down-regulation of these transporters persisted together with marked down-regulation of H(+)-adenosine triphosphatase in the obstructed kidney. In rats with unilateral ureteral obstruction release followed for 4 days with experimental acidosis induced by NH(4)Cl oral administration plasma pH and HCO(3)(-) were dramatically decreased in response to NH(4)Cl for 2 days compared with those in sham operated rats with acid loading, indicating a defect in H(+) excretion and HCO(3)(-) reabsorption after obstruction release. Expression of these transporters did not change in the contralateral nonobstructed kidney of rats with unilateral ureteral obstruction and unilateral ureteral obstruction release followed for 4 days. CONCLUSIONS: The expression of renal acid-base transporters is markedly decreased in the obstructed kidney, which may be responsible for the contribution of impaired renal H(+) excretion and HCO(3)(-) reabsorption to the urinary acidification defect in response to unilateral ureteral obstruction.

Asunto(s)

Riñón/metabolismo , Obstrucción Ureteral/metabolismo , Animales , Proteínas de Transporte de Membrana/biosíntesis , ATPasas de Translocación de Protón/biosíntesis , Ratas , Ratas Wistar , Simportadores de Sodio-Bicarbonato/biosíntesis , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Simportadores de Cloruro de Sodio-Potasio/biosíntesis , Miembro 1 de la Familia de Transportadores de Soluto 12 , Transportadores de Sulfato

RESUMEN

Congenital obstructive nephropathy accounts for a major proportion of renal insufficiency in infancy and childhood. In an earlier investigation we demonstrated that bilateral complete ureteral obstruction (BUO) in rats is associated with inadequate urinary acidification [Am J Physiol Renal Physiol. 295(2):F497-506, 2008]. The aim of the study reported here was to determine whether this defect is also associated with unilateral ureteral obstruction (UUO), which is clinically more common than BUO. The time-course of the changes in protein expression levels of major renal acid-base transporters was examined at 7 and 14 weeks in rats with neonatally induced partial unilateral ureteral obstruction (PUUO), which was performed within the first 48 h of life. We observed that protein expression of the renal acid-base transporters NHE3, NBC1, NBCn1, pendrin and Na(+)-K(+)-ATPase was increased in both obstructed and non-obstructed kidneys 7 weeks after the induction of neonatal PUUO. This was confirmed by immunocytochemistry. In contrast, 14 weeks after the induction of PUUO, there was a significant downregulation of the renal acid-base transporters NBC1, NBCn1 and Na(+)-K(+)-ATPase in the obstructed kidneys. These time/age-dependent changes in protein expression were associated with parallel changes in renal function resulting in urine acidification in response to exogenous acid loading. In conclusion, these results show that downregulation of protein expression is a time/age-dependent response to PUUO, which could contribute to the decreased net acid excretion and development of metabolic acidosis in neonatal rats with PUUO.

Asunto(s)

Bombas Iónicas/biosíntesis , Riñón/metabolismo , Obstrucción Ureteral/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Masculino , Ratas , Ratas Wistar , Simportadores de Sodio-Bicarbonato/biosíntesis , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis