[Ion transporters in the lungs. Use as therapeutic targets]. / Transportadores de iones en pulmón. Uso como dianas terapéuticas.

The chloride channels, sodium and bicarbonate channels, and aquaporin water channels are coordinated to maintain the airway surface liquid that is necessary for mucociliary clearance. The general mechanism for the transport of electrolytes and fluids depends mainly on the differential expression and distribution of ion transporters and pumps. Ions and water move through the paracellular or transcellular pathways. The transcellular route of electrolyte transport requires an active transport (dependent on ATP) or passive (following electrochemical gradients) of ions. The paracellular pathway is a passive process that is ultimately controlled by the predominant transepithelial electrochemical gradients. Cystic fibrosis is a hereditary disease that is produced by mutations in the gene that encode cystic fibrosis transmembrane conductance regulatory protein (CFTR) that acts as a chloride channel and performs functions of hydration of periciliary fluid and maintenance of luminal pH. The dysfunction of the chlorine channel in the respiratory epithelium determines an alteration in the bronchial secretions, with an increase in its viscosity and alteration of the mucociliary clearance and that associated with infectious processes can lead to irreversible lung damage. CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease, and bronchial hyperreactivity in asthma. There are drugs that exploit physiological mechanisms in the transport of ions with a therapeutic objective.

Los canales de cloruros, de sodio, de bicarbonato y los de agua (aquaporinas) se coordinan para mantener la cubierta líquido superficial de las vías respiratorias, que es necesaria para el aclaramiento mucociliar. El mecanismo general para el transporte de electrolitos y agua depende principalmente de la expresión diferencial y distribución de los transportadores y bombas de iones. Los iones y el agua se mueven a través de las vía paracelular o transcelular. La ruta transcelular del transporte de electrolitos requiere un transporte activo (dependiente de ATP) o pasivo (siguiendo gradientes electroquímicos) de iones. La ruta paracelular es un proceso pasivo que está controlado, en última instancia, por los gradientes electroquímicos transepiteliales predominantes. La fibrosis quística es una enfermedad hereditaria que se produce por mutaciones en el gen que codifica la proteína reguladora de la conductibilidad transmembrana de la fibrosis quística (CFTR) que actúa como un canal de cloro y cumple funciones de hidratación del líquido periciliar y mantenimiento del pH luminal. La disfunción del canal de cloro en el epitelio respiratorio determina una alteración en las secreciones bronquiales, con aumento de su viscosidad y alteración de la depuración mucociliar y que asociado a procesos infecciosos puede conducir a daño pulmonar irreversible. La disfunción del CFTR, también se ha visto implicado en la patogénesis de la pancreatitis aguda, en la enfermedad pulmonar obstructiva crónica y la hiperreactividad en el asma. Existen fármacos que aprovechan los mecanismos fisiológicos en el transporte de iones, con un objetivo terapéutico.

Transportadores de iones en pulmón: Uso como dianas terapéuticas / Ion transporters in the lungs. Use as therapeutic targets

Los canales de cloruros, de sodio, de bicarbonato y los de agua (aquaporinas) se coordinan para mantener la cubierta líquido superficial de las vías respiratorias, que es necesaria para el aclaramiento mucociliar. El mecanismo general para el transporte de electrolitos y agua depende principalmente de la expresión diferencial y distribución de los transportadores y bombas de iones. Los iones y el agua se mueven a través de las vía paracelular o transcelular. La ruta transcelular del transporte de electrolitos requiere un transporte activo (dependiente de ATP) o pasivo (siguiendo gradientes electroquímicos) de iones. La ruta paracelular es un proceso pasivo que está controlado, en última instancia, por los gradientes electroquímicos transepiteliales predominantes. La fibrosis quística es una enfermedad hereditaria que se produce por mutaciones en el gen que codifica la proteína reguladora de la conductibilidad transmembrana de la fibrosis quística (CFTR) que actúa como un canal de cloro y cumple funciones de hidratación del líquido periciliar y mantenimiento del pH luminal. La disfunción del canal de cloro en el epitelio respiratorio determina una alteración en las secreciones bronquiales, con aumento de su viscosidad y alteración de la depuración mucociliar y que asociado a procesos infecciosos puede conducir a daño pulmonar irreversible. La disfunción del CFTR, también se ha visto implicado en la patogénesis de la pancreatitis aguda, en la enfermedad pulmonar obstructiva crónica y la hiperreactividad en el asma. Existen fármacos que aprovechan los mecanismos fisiológicos en el transporte de iones, con un objetivo terapéutico.

The chloride channels, sodium and bicarbonate channels, and aquaporin water channels are coordinated to maintain the airway surface liquid that is necessary for mucociliary clearance. The general mechanism for the transport of electrolytes and fluids depends mainly on the differential expression and distribution of ion transporters and pumps. Ions and water move through the paracellular or transcellular pathways. The transcellular route of electrolyte transport requires an active transport (dependent on ATP) or passive (following electrochemical gradients) of ions. The paracellular pathway is a passive process that is ultimately controlled by the predominant transepithelial electrochemical gradients. Cystic fibrosis is a hereditary disease that is produced by mutations in the gene that encode cystic fibrosis transmembrane conductance regulatory protein (CFTR) that acts as a chloride channel and performs functions of hydration of periciliary fluid and maintenance of luminal pH. The dysfunction of the chlorine channel in the respiratory epithelium determines an alteration in the bronchial secretions, with an increase in its viscosity and alteration of the mucociliary clearance and that associated with infectious processes can lead to irreversible lung damage. CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease, and bronchial hyperreactivity in asthma. There are drugs that exploit physiological mechanisms in the transport of ions with a therapeutic objective.

The median eminence as the hypothalamic area involved in rapid transfer of glucose to the brain: functional and cellular mechanisms.

Our data proposes that glucose is transferred directly to the cerebrospinal fluid (CSF) of the hypothalamic ventricular cavity through a rapid "fast-track-type mechanism" that would efficiently stimulate the glucosensing areas. This mechanism would occur at the level of the median eminence (ME), a periventricular hypothalamic zone with no blood-brain barrier. This "fast-track" mechanism would involve specific glial cells of the ME known as ß2 tanycytes that could function as "inverted enterocytes," expressing low-affinity glucose transporters GLUT2 and GLUT6 in order to rapidly transfer glucose to the CSF. Due to the large size of tanycytes, the presence of a high concentration of mitochondria and the expression of low-affinity glucose transporters, it would be expected that these cells accumulate glucose in the endoplasmic reticulum (ER) by sequestering glucose-6-phosphate (G-6-P), in a similar way to that recently demonstrated in astrocytes. Glucose could diffuse through the cells by micrometric distances to be released in the apical region of ß2 tanycytes, towards the CSF. Through this mechanism, levels of glucose would increase inside the hypothalamus, stimulating glucosensing mechanisms quickly and efficiently. KEY MESSAGES: • Glucose diffuses through the median eminence cells (ß2 tanycytes), towards the hypothalamic CSF. • Glucose is transferred through a rapid "fast-track-type mechanism" via GLUT2 and GLUT6. • Through this mechanism, hypothalamic glucose levels increase, stimulating glucosensing.

How can 50 years of solute transport data in articular cartilage inform the design of arthritis therapeutics?

OBJECTIVE: For the last half century, transport of nutrients and therapeutics in articular cartilage has been studied with various in vitro systems that attempt to model in vivo conditions. However, experimental technique, tissue species, and tissue storage condition (fresh/frozen) vary widely and there is debate on the most appropriate model system. Additionally, there is still no clear overarching framework with which to predict solute transport properties based on molecular characteristics. This review aims to develop such a framework, and to assess whether experimental procedure affects trends in transport data. METHODS: Solute data from 31 published papers that investigated transport in healthy articular cartilage were obtained and analyzed for trends. RESULTS: Here, we show that diffusivity of spherical and globular solutes in cartilage can be predicted by molecular weight (MW) and hydrodynamic radius via a power-law relationship. This relationship is robust for many solutes, spanning 5 orders of magnitude in MW and was not affected by variations in cartilage species, age, condition (fresh/frozen), and experimental technique. Traditional models of transport in porous media exhibited mixed effectiveness at predicting diffusivity in cartilage, but were good in predicting solute partition coefficient. CONCLUSION: Ultimately, these robust relationships can be used to accurately predict and improve transport of solutes in adult human cartilage and enable the development of better optimized arthritis therapeutics.

Dietary α-linolenic acid supplementation alters skeletal muscle plasma membrane lipid composition, sarcolemmal FAT/CD36 abundance, and palmitate transport rates.

The cellular processes influenced by consuming polyunsaturated fatty acids remains poorly defined. Within skeletal muscle, a rate-limiting step in fatty acid oxidation is the movement of lipids across the sarcolemmal membrane, and therefore, we aimed to determine the effects of consuming flaxseed oil high in α-linolenic acid (ALA), on plasma membrane lipid composition and the capacity to transport palmitate. Rats fed a diet supplemented with ALA (10%) displayed marked increases in omega-3 polyunsaturated fatty acids (PUFAs) within whole muscle and sarcolemmal membranes (approximately five-fold), at the apparent expense of arachidonic acid (-50%). These changes coincided with increased sarcolemmal palmitate transport rates (+20%), plasma membrane fatty acid translocase (FAT/CD36; +20%) abundance, skeletal muscle triacylglycerol content (approximately twofold), and rates of whole body fat oxidation (~50%). The redistribution of FAT/CD36 to the plasma membrane could not be explained by increased phosphorylation of signaling pathways implicated in regulating FAT/CD36 trafficking events (i.e., phosphorylation of ERK1/2, CaMKII, AMPK, and Akt), suggesting the increased n-3 PUFA composition of the plasma membrane influenced FAT/CD36 accumulation. Altogether, the present data provide evidence that a diet supplemented with ALA increases the transport of lipids into resting skeletal muscle in conjunction with increased sarcolemmal n-3 PUFA and FAT/CD36 contents.

Does a voltage-sensitive outer envelope transport mechanism contributes to the chloroplast iron uptake?

MAIN CONCLUSION: Based on the effects of inorganic salts on chloroplast Fe uptake, the presence of a voltage-dependent step is proposed to play a role in Fe uptake through the outer envelope. Although iron (Fe) plays a crucial role in chloroplast physiology, only few pieces of information are available on the mechanisms of chloroplast Fe acquisition. Here, the effect of inorganic salts on the Fe uptake of intact chloroplasts was tested, assessing Fe and transition metal uptake using bathophenantroline-based spectrophotometric detection and plasma emission-coupled mass spectrometry, respectively. The microenvironment of Fe was studied by Mössbauer spectroscopy. Transition metal cations (Cd2+, Zn2+, and Mn2+) enhanced, whereas oxoanions (NO3-, SO42-, and BO33-) reduced the chloroplast Fe uptake. The effect was insensitive to diuron (DCMU), an inhibitor of chloroplast inner envelope-associated Fe uptake. The inorganic salts affected neither Fe forms in the uptake assay buffer nor those incorporated into the chloroplasts. The significantly lower Zn and Mn uptake compared to that of Fe indicates that different mechanisms/transporters are involved in their acquisition. The enhancing effect of transition metals on chloroplast Fe uptake is likely related to outer envelope-associated processes, since divalent metal cations are known to inhibit Fe2+ transport across the inner envelope. Thus, a voltage-dependent step is proposed to play a role in Fe uptake through the chloroplast outer envelope on the basis of the contrasting effects of transition metal cations and oxoaninons.

The Plasma Membrane Protein Nce102 Implicated in Eisosome Formation Rescues a Heme Defect in Mitochondria.

The cellular transport of the cofactor heme and its biosynthetic intermediates such as protoporphyrin IX is a complex and highly coordinated process. To investigate the molecular details of this trafficking pathway, we created a synthetic lesion in the heme biosynthetic pathway by deleting the gene HEM15 encoding the enzyme ferrochelatase in S. cerevisiae and performed a genetic suppressor screen. Cells lacking Hem15 are respiratory-defective because of an inefficient heme delivery to the mitochondria. Thus, the biogenesis of mitochondrial cytochromes is negatively affected. The suppressor screen resulted in the isolation of respiratory-competent colonies containing two distinct missense mutations in Nce102, a protein that localizes to plasma membrane invaginations designated as eisosomes. The presence of the Nce102 mutant alleles enabled formation of the mitochondrial respiratory complexes and respiratory growth in hem15Δ cells cultured in supplemental hemin. Respiratory function in hem15Δ cells can also be restored by the presence of a heterologous plasma membrane heme permease (HRG-4), but the mode of suppression mediated by the Nce102 mutant is more efficient. Attenuation of the endocytic pathway through deletion of the gene END3 impaired the Nce102-mediated rescue, suggesting that the Nce102 mutants lead to suppression through the yeast endocytic pathway.

The transcriptional activator GaaR of Aspergillus niger is required for release and utilization of d-galacturonic acid from pectin.

We identified the d-galacturonic acid (GA)-responsive transcriptional activator GaaR of the saprotrophic fungus, Aspergillus niger, which was found to be essential for growth on GA and polygalacturonic acid (PGA). Growth of the ΔgaaR strain was reduced on complex pectins. Genome-wide expression analysis showed that GaaR is required for the expression of genes necessary to release GA from PGA and more complex pectins, to transport GA into the cell, and to induce the GA catabolic pathway. Residual growth of ΔgaaR on complex pectins is likely due to the expression of pectinases acting on rhamnogalacturonan and subsequent metabolism of the monosaccharides other than GA.

Metalloprobes: Fluorescence imaging of multidrug resistance (MDR1) P-Glycoprotein (Pgp)-mediated functional transport activity in cellulo.

Radiolabeled metalloprobes offer sensitive tools for evaluating quantitative accumulation of chemical entities within pooled cell populations. Although beneficial in translational nuclear imaging, this method precludes interrogation of effects resulting from variations at a single cell level, within the same segment of cell population. Compared with radiotracer bioassays, fluorescence imaging offers a cost-efficient technique to assess accumulation of metalloprobes at a single cell level, and determine their intracellular localization under live cell conditions. To evaluate, whether or not radiotracer assay and fluorescence imaging provide complementary information on utility of metalloprobes to assess functional expression of P-glycoprotein (Pgp) on plasma membrane of tumor cells, imaging studies of fluorescent cationic Ga(III)-ENBDMPI (bis(3-ethoxy-2-hydroxy-benzylidene)-N,N'-bis(2,2-dimethyl-3-amino-propyl)ethylenediamine) and its neutral counterpart Zn(II)-ENBDMPI are performed. While the uptake profiles of the cationic metalloprobe are inversely proportional to expression of Pgp in tumor cells, the accumulation profiles of the neutral Zn(II)-ENBDMPI in non-MDR and MDR cells are not significantly impacted. The cationic Ga(III)-ENBDMPI maps with Mito-Tracker Red, thereby confirming localization within mitochondria of non-MDR (Pgp-) cells. Depolarization of both plasmalemmal and mitochondrial potentials decreased retention of the cationic Ga(III)-ENBDMPI within the mitochondria. Additionally, LY335979, an antagonist-induced accumulation of the cationic Ga(III) metalloprobe in MDR (Pgp+) cells indicated specificity of the agent. Compared with traits of Ga(III)-ENBDMPI as a Pgp recognized substrate, Zn(II)-ENBDMPI demonstrated uptake in both MDR and non-MDR cells thus indicating the significance of overall molecular charge in mediating Pgp recognition profiles. Combined data indicate that live cell imaging can offer a cost-effective methodology for monitoring functional Pgp expression.

Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability.

CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with ß-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer.

Inhibition of human GLUT1 and GLUT5 by plant carbohydrate products; insights into transport specificity.

Glucose transporters GLUT1 (transports glucose) and GLUT5 (transports fructose), in addition to their functions in normal metabolism, have been implicated in several diseases including cancer and diabetes. While GLUT1 has several inhibitors, none have been described for GLUT5. By transport activity assays we found two plant products, rubusoside (from Rubus suavissimus) and astragalin-6-glucoside (a glycosylated derivative of astragalin, from Phytolacca americana) that inhibited human GLUT5. These plants are utilized in traditional medicine: R. suavissimus for weight loss and P. americana for cancer treatment, but the molecular interactions of these products are unknown. Rubusoside also inhibited human GLUT1, but astragalin-6-glucoside did not. In silico analysis of rubusoside:protein interactions pinpointed a major difference in substrate cavity between these transporters, a residue that is a tryptophan in GLUT1 but an alanine in GLUT5. Investigation of mutant proteins supported the importance of this position in ligand specificity. GLUT1W388A became susceptible to inhibition by astragalin-6-glucoside and resistant to rubusoside. GLUT5A396W transported fructose and also glucose, and maintained inhibition by rubusoside and astragalin-6-glucoside. Astragalin-6-glucoside can serve as a starting point in the design of specific inhibitors for GLUT5. The application of these studies to understanding glucose transporters and their interaction with substrates and ligands is discussed.

Importance of silicon and mechanisms of biosilica formation in plants.

Silicon (Si) is one of the most prevalent macroelements, performing an essential function in healing plants in response to environmental stresses. The purpose of using Si is to induce resistance to distinct stresses, diseases, and pathogens. Additionally, Si can improve the condition of soils, which contain toxic levels of heavy metals along with other chemical elements. Silicon minimizes toxicity of Fe, Al, and Mn, increases the availability of P, and enhances drought along with salt tolerance in plants through the formation of silicified tissues in plants. However, the concentration of Si depends on the plants genotype and organisms. Hence, the physiological mechanisms and metabolic activities of plants may be affected by Si application. Peptides as well as amino acids can effectively create polysilicic species through interactions with different species of silicate inside solution. The carboxylic acid and the alcohol groups of serine and asparagine tend not to engage in any significant role in polysilicates formation, but the hydroxyl group side chain can be involved in the formation of hydrogen bond with Si(OH)4. The mechanisms and trend of Si absorption are different between plant species. Furthermore, the transportation of Si requires an energy mechanism; thus, low temperatures and metabolic repressors inhibit Si transportation.

Chondrocytic cells express the taurine transporter on their plasma membrane and regulate its expression under anisotonic conditions.

Taurine is a small organic osmolyte which participates in cell volume regulation. Chondrocytes have been shown to accumulate and release taurine; in bone, taurine participates in bone metabolism. However, its role in skeletal cells is poorly understood, especially in chondrocytes. This study investigated the regulation of taurine transporter in chondrocytic cells. We examined the transcriptional regulation of the taurine transporter under anisotonia by reporter gene and real-time RT-PCR assays. The effect of providing supplementary taurine on cell viability was evaluated with the lactate dehydrogenase release assay. The localization of the taurine transporter in human chondrosarcoma cells was studied by overexpressing a taurine transporter-enhanced green fluorescent protein. We observed that the transcription of the taurine transporter gene was up-regulated in hypertonic conditions. Hyperosmolarity-related cell death could be partly abolished by taurine supplementation in the medium. As expected, the fluorescently labeled taurine transporter localized at the plasma membrane. In polarized epithelial MDCK cells, the strongest fluorescence signal was located in the lateral cell membrane area. We also observed that the taurine transporter gene was expressed in several human tissues and malignant cell lines. This is the first study to present information on the transcriptional regulation of taurine transporter gene and the localization of the taurine transporter protein in chondrocytic cells.

Intestinal lipid absorption and lipoprotein formation.

PURPOSE OF REVIEW: To summarize the evidence for the presence of two lipid absorption pathways and their regulation. RECENT FINDINGS: Lipid absorption involves hydrolysis of dietary fat in the lumen of the intestine, followed by the uptake of hydrolyzed products by enterocytes. Lipids are resynthesized in the endoplasmic reticulum and are either secreted with chylomicrons and HDLs or stored as cytoplasmic lipid droplets. Lipids in the droplets are hydrolyzed and are secreted at a later time. Secretion of lipids by the chylomicron and HDL pathways are dependent on microsomal triglyceride transfer protein (MTP) and ATP-binding cassette family A protein 1, respectively, and are regulated independently. Gene-ablation studies showed that MTP function and chylomicron assembly is essential for the absorption of triglycerides. Ablation of MTP abolishes triglyceride absorption and results in massive triglyceride accumulation in enterocytes. Although the majority of phospholipid, cholesterol, and vitamin E are absorbed through the chylomicron pathway, a significant amount of these lipids are also absorbed via the HDL pathway. Chylomicron assembly and secretion is increased by the enhanced availability of fatty acids, whereas the HDL pathway is upregulated by liver X receptor agonists. SUMMARY: Triglycerides are exclusively transported with chylomicrons and this process is critically dependent on MTP. In addition to chylomicrons, absorption of phospholipids, free cholesterol, retinol, and vitamin E also involves HDLs. These two pathways are complementary and are regulated independently. They may be targeted to lower lipid absorption in order to control hyperlipidemia, obesity, metabolic syndrome, steatosis, insulin resistance, atherosclerosis, and other disorders.

Molecular identification and functional characterization of the human colonic thiamine pyrophosphate transporter.

Colonic microbiota synthesize a considerable amount of thiamine in the form of thiamine pyrophosphate (TPP). Recent functional studies from our laboratory have shown the existence of a specific, high-affinity, and regulated carrier-mediated uptake system for TPP in human colonocytes. Nothing, however, is known about the molecular identity of this system. Here we report on the molecular identification of the colonic TPP uptake system as the product of the SLC44A4 gene. We cloned the cDNA of SLC44A4 from human colonic epithelial NCM460 cells, which, upon expression in ARPE19 cells, led to a significant (p < 0.01, >5-fold) induction in [(3)H]TPP uptake. Uptake by the induced system was also found to be temperature- and energy-dependent; Na(+)-independent, slightly higher at acidic buffer pH, and highly sensitive to protonophores; saturable as a function of TPP concentration, with an apparent Km of 0.17 ± 0.064 µM; and highly specific for TPP and not affected by free thiamine, thiamine monophosphate, or choline. Expression of the human TPP transporter was found to be high in the colon and negligible in the small intestine. A cell surface biotinylation assay and live cell confocal imaging studies showed the human TPP transporter protein to be expressed at the apical membrane domain of polarized epithelia. These results show, for the first time, the molecular identification and characterization of a specific and high-affinity TPP uptake system in human colonocytes. The findings further support the hypothesis that the microbiota-generated TPP is absorbable and could contribute toward host thiamine homeostasis, especially toward cellular nutrition of colonocytes.

Advantages and disadvantages of the animal models v. in vitro studies in iron metabolism: a review.

Iron deficiency is the most common nutritional deficiency in the world. Special molecules have evolved for iron acquisition, transport and storage in soluble, nontoxic forms. Studies about the effects of iron on health are focused on iron metabolism or nutrition to prevent or treat iron deficiency and anemia. These studies are focused in two main aspects: (1) basic studies to elucidate iron metabolism and (2) nutritional studies to evaluate the efficacy of iron supplementation to prevent or treat iron deficiency and anemia. This paper reviews the advantages and disadvantages of the experimental models commonly used as well as the methods that are more used in studies related to iron. In vitro studies have used different parts of the gut. In vivo studies are done in humans and animals such as mice, rats, pigs and monkeys. Iron metabolism is a complex process that includes interactions at the systemic level. In vitro studies, despite physiological differences to humans, are useful to increase knowledge related to this essential micronutrient. Isotopic techniques are the most recommended in studies related to iron, but their high cost and required logistic, making them difficult to use. The depletion-repletion of hemoglobin is a method commonly used in animal studies. Three depletion-repletion techniques are mostly used: hemoglobin regeneration efficiency, relative biological values (RBV) and metabolic balance, which are official methods of the association of official analytical chemists. These techniques are well-validated to be used as studies related to iron and their results can be extrapolated to humans. Knowledge about the main advantages and disadvantages of the in vitro and animal models, and methods used in these studies, could increase confidence of researchers in the experimental results with less costs.

Nuclear receptor LXR: a new partner for sodium-dependent phosphate cotransporters.

New pharmaceutical research approaches are focusing on trying to alleviate the perturbed phosphate (Pi) homeostasis associated with the onset of chronic kidney disease; this includes activation of some of the nuclear receptors. We have recently reported the down regulation of the intestinal and renal sodium-phosphate (NaPi) cotransporters by the liver X receptor (LXR) agonists, and the consequent decrease of the serum Pi levels. In this review, we describe our current knowledge of the different proteins involved in the renal and intestinal actions of LXR.

Secreted glyceraldehye-3-phosphate dehydrogenase is a multifunctional autocrine transferrin receptor for cellular iron acquisition.

BACKGROUND: The long held view is that mammalian cells obtain transferrin (Tf) bound iron utilizing specialized membrane anchored receptors. Here we report that, during increased iron demand, cells secrete the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which enhances cellular uptake of Tf and iron. METHODS: These observations could be mimicked by utilizing purified GAPDH injected into mice as well as when supplemented in culture medium of model cell lines and primary cell types that play a key role in iron metabolism. Transferrin and iron delivery was evaluated by biochemical, biophysical and imaging based assays. RESULTS: This mode of iron uptake is a saturable, energy dependent pathway, utilizing raft as well as non-raft domains of the cell membrane and also involves the membrane protein CD87 (uPAR). Tf internalized by this mode is also catabolized. CONCLUSIONS: Our research demonstrates that, even in cell types that express the known surface receptor based mechanism for transferrin uptake, more transferrin is delivered by this route which represents a hidden dimension of iron homeostasis. GENERAL SIGNIFICANCE: Iron is an essential trace metal for practically all living organisms however its acquisition presents major challenges. The current paradigm is that living organisms have developed well orchestrated and evolved mechanisms involving iron carrier molecules and their specific receptors to regulate its absorption, transport, storage and mobilization. Our research uncovers a hidden and primitive pathway of bulk iron trafficking involving a secreted receptor that is a multifunctional glycolytic enzyme that has implications in pathological conditions such as infectious diseases and cancer.

Evaluation of the permeability and P-glycoprotein efflux of carbamazepine and several derivatives across mouse small intestine by the Ussing chamber technique.

PURPOSE: The rational discovery and development of new antiepileptic drugs (AEDs) with safer therapeutic index and better pharmacokinetic properties is still warranted nowadays. Because the long-term management of epilepsy is attained by means of orally administered AEDs, investigation of their potential to be well absorbed at the intestinal level is mandatory. Moreover, involvement of the efflux transport mediated by P-glycoprotein (P-gp) may compromise the systemic and central nervous system disposition of AEDs. Therefore, this study aimed at characterizing mouse jejunal passive transport and the possible active efflux mediated by P-gp of a series of dibenz[b,f]azepine-5-carboxamide derivatives (carbamazepine [CBZ], oxcarbazepine [OXC], S-licarbazepine [S-Lic], R-licarbazepine [R-Lic], carbamazepine-10,11-epoxide [CBZ-E], 10,11-trans-dihydroxy-10,11-dihydro-carbamazepine [trans-diol], and BIA 2-024), which comprise some AEDs and metabolites. METHODS: Permeation studies were performed with freshly excised mouse jejunum segments mounted in Ussing chambers. Absorptive (M-S) and secretive (S-M) transports were analyzed with and without verapamil, which is a P-gp inhibitor widely recognized. Apparent permeability coefficients (P(app) ) in both directions and in absence or presence of verapamil were determined for each test compound. The in vitro method was validated using five controls that included high and low permeable markers with known absorption fraction (Fa) and also well-known P-gp substrates. The integrity of intestinal membrane was guaranteed during the assay by measuring the transepithelial electrical resistance. KEY FINDINGS: The correlation obtained between P(app) (M-S) and Fa of references was high (r(2) = 0.9945), and could be used to classify the derivatives according to Biopharmaceutical Classification System: CBZ and OXC were the only classified as highly permeable. The P(app) (S-M) of OXC, CBZ-E, R-Lic, and BIA 2-024 were significantly higher than their P(app) (M-S). After verapamil addition, their P(app) (S-M) lowered while P(app) (S-M) increased, suggesting the involvement of P-gp on the transport of those compounds across mouse jejunum segments. In opposition, CBZ, S-Lic, and trans-diol presented no statistical differences between the P(app) values reported in both directions, with or without verapamil. The results reported herein suggest that differences in biodisposition of S-Lic and R-Lic might result from their distinct interaction with P-gp. SIGNIFICANCE: The Ussing chamber model used herein showed to be useful for predicting Fa of AEDs and the involvement of efflux transport, namely P-gp, on their absorption. This is an important achievement as compounds that are not transported by P-gp may offer advantages when used in patients with pharmacoresistant epilepsy.

Gle1 is a multifunctional DEAD-box protein regulator that modulates Ded1 in translation initiation.

DEAD-box protein (Dbp) family members are essential for gene expression; however, their precise roles and regulation are not fully defined. During messenger (m)RNA export, Gle1 bound to inositol hexakisphosphate (IP(6)) acts via Dbp5 to facilitate remodeling of mRNA-protein complexes. In contrast, here we define a novel Gle1 role in translation initiation through regulation of a different DEAD-box protein, the initiation factor Ded1. We find that Gle1 physically and genetically interacts with Ded1. Surprisingly, whereas Gle1 stimulates Dbp5, it inhibits Ded1 ATPase activity in vitro, and IP(6) does not affect this inhibition. Functionally, a gle1-4 mutant specifically suppresses initiation defects in a ded1-120 mutant, and ded1 and gle1 mutants have complementary perturbations in AUG start site recognition. Consistent with this role in initiation, Gle1 inhibits translation in vitro in competent extracts. These results indicate that Gle1 has a direct role in initiation and negatively regulates Ded1. Together, the differential regulation of two distinct DEAD-box proteins by a common factor (Gle1) establishes a new paradigm for controlling gene expression and coupling translation with mRNA export.