Association of common variants in NPPA and NPPB with circulating natriuretic peptides and blood pressure

We examined the association of common variants at the NPPA-NPPB locus with circulating concentrations of the natriuretic peptides, which have blood pressure-lowering properties. We genotyped SNPs at the NPPA-NPPB locus in 14,743 individuals of European ancestry, and identified associations of plasma atrial natriuretic peptide with rs5068 (P = 8 × 10 -70), rs198358 (P = 8 × 10 -30) and rs632793 (P = 2 × 10 -10), and of plasma B-type natriuretic peptide with rs5068 (P = 3 × 10 -12), rs198358 (P = 1 × 10 -25) and rs632793 (P = 2 × 10 -68). In 29,717 individuals, the alleles of rs5068 and rs198358 that showed association with increased circulating natriuretic peptide concentrations were also found to be associated with lower systolic (P = 2 × 10 -6 and 6 × 10 -5, respectively) and diastolic blood pressure (P = 1 × 10 -6 and 5 × 10 -5), as well as reduced odds of hypertension (OR = 0.85, 95% CI = 0.79-0.92, P = 4 × 10 -5; OR = 0.90, 95% CI = 0.85-0.95, P = 2 × 10 -4, respectively). Common genetic variants at the NPPA-NPPB locus found to be associated with circulating natriuretic peptide concentrations contribute to interindividual variation in blood pressure and hypertension.

Ca2+- and volume-sensitive chloride currents are differentially regulated by agonists and store-operated Ca2+ entry.

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca2+]i in a Ca2+-free medium followed by a secondary [Ca2+]i rise upon Ca2+ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca2+-activated (ICl(Ca)) and volume-regulated (ICl, swell) chloride currents. NPPB and DIDS more efficiently inhibited ICl(Ca) and ICl, swell, respectively. Cell swelling caused by hypotonic solution invariably activated ICl, swell while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling–induced ICl, swell, while its inactive analogue U-73343 had no effect. ICl(Ca) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca2+]i in the pipette solution at 500 nM. In contrast, ICl, swell could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced ICl, swell in a nonadditive manner, suggesting their convergence on a common pathway. ICl, swell and ICl(Ca) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated ICl(Ca), but abolished ICl, swell, thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that ICl, swell can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a "selection" switch between closely localized channels.

Tailored Microarray Platform for the Detection of Marine Toxins

Currently, there are no fast in vitro broad spectrum screening bioassays for the detection of marine toxins. The aim of this study was to develop such an assay. In gene expression profiling experiments 17 marker genes were provisionally selected that were differentially regulated in human intestinal Caco-2 cells upon exposure to the lipophilic shellfish poisons azaspiracid-1 (AZA1) or dinophysis toxin-1 (DTX1). These 17 genes together with two control genes were the basis for the design of a tailored microarray platform for the detection of these marine toxins and potentially others. Five out of the 17 selected marker genes on this dedicated DNA microarray gave dear signals, whereby the resulting fingerprints could be used to detect these toxins. CEACAM1, DDIT4, and TUBB3 were up-regulated by both AZA1 and DTX1, TRIB3 was up-regulated by AZA1 only, and OSR2 by DTX1 only. Analysis by singleplex qRT-PCR revealed the up- and down-regulation of the selected RGS16 and NPPB marker genes by DTX1, that were not envisioned by the new developed dedicated array. The qRT-PCR targeting the DDIT4, RSG16 and NPPB genes thus already resulted in a specific pattern for AZA1 and DTX1 indicating that for this specific case qRT-PCR might a be more suitable approach than a dedicated array.

Functional and pharmacological characterization of volume-regulated anion channels in human normal and cystic fibrosis bronchial and nasal epithelial cells

Volume-regulated anion channels (VRACs) are widely present in various cell types and have important functions ranging from regulatory volume decrease to control of cell proliferation and apoptosis. Here we aimed to compare the biophysical features and pharmacological profiles of VRAC currents in healthy and cystic fibrosis (CF) respiratory epithelial cells in order to characterize these currents both functionally and pharmacologically. Whole-cell electrophysiology was used to characterize the VRAC current in normal (16HBE14o-; HBE) and CF cell lines (CFBE14o-; CFBE), as well as in native human nasal epithelial cells. Application of hypotonic solution produced current responses of similar sizes in both HBE and CFBE cells. Biophysical properties of VRACs, such as instantaneous activation and deactivation upon voltage step, some inactivation at potentials positive to 40 mV and outwardly-rectifying I-V curves, were indistinguishable in both cell types. Extensive pharmacological analysis of the currents revealed a similar pharmacological profile in response to three blockers--NPPB, DCPIB and DIDS. Native primary human nasal epithelial cells from both healthy and CF volunteers also showed typical VRAC responses of comparable sizes. VRACs in these cells were more sensitive to external solution hypotonicity compared to HBE and CFBE cells. In all cell types studied robust VRAC currents could be induced at constant cell volume by G-protein activation with GTPγS infusion. This study provides the first extensive comparative functional and pharmacological analysis of VRAC currents in normal and CF airway epithelial cells and shows that VRACs are unimpaired molecularly or functionally in CF.

Caracterização dos mecanismos de transporte epitelial no intestino da dourada, Sparus auratus

Dissertação de mest., Aquacultura e Pescas, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2010

Localization and regulation by GABA of anion transporter present in pollen grain protoplasts and tube of Arabidopsis thaliana

Tese de doutoramento, Biologia (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Ciências, 2015

Blood pressure loci identified with a gene-centric array.

Raised blood pressure (BP) is a major risk factor for cardiovascular disease. Previous studies have identified 47 distinct genetic variants robustly associated with BP, but collectively these explain only a few percent of the heritability for BP phenotypes. To find additional BP loci, we used a bespoke gene-centric array to genotype an independent discovery sample of 25,118 individuals that combined hypertensive case-control and general population samples. We followed up four SNPs associated with BP at our p < 8.56 × 10(-7) study-specific significance threshold and six suggestively associated SNPs in a further 59,349 individuals. We identified and replicated a SNP at LSP1/TNNT3, a SNP at MTHFR-NPPB independent (r(2) = 0.33) of previous reports, and replicated SNPs at AGT and ATP2B1 reported previously. An analysis of combined discovery and follow-up data identified SNPs significantly associated with BP at p < 8.56 × 10(-7) at four further loci (NPR3, HFE, NOS3, and SOX6). The high number of discoveries made with modest genotyping effort can be attributed to using a large-scale yet targeted genotyping array and to the development of a weighting scheme that maximized power when meta-analyzing results from samples ascertained with extreme phenotypes, in combination with results from nonascertained or population samples. Chromatin immunoprecipitation and transcript expression data highlight potential gene regulatory mechanisms at the MTHFR and NOS3 loci. These results provide candidates for further study to help dissect mechanisms affecting BP and highlight the utility of studying SNPs and samples that are independent of those studied previously even when the sample size is smaller than that in previous studies.

Is GABA involved in regulating plant growth and development? /

Rapid and large accumulation of GABA (y-aminobutyric acid) in response to a number of plant stresses has been well documented. But the role(s) of GABA in plants is not well defined. In recent years, the possibility of GABA involvement in regulating plant growth and development has been raised. In the present study, this possibility was examined. First, to rapidly and accurately determine GABA levels in plant tissues, a spectrometric method for GABA determination was developed based on a commercially available enzyme Gabase. Seventy mM LaCb almost completely removed water-soluble pigments from plant tissues which greatly interfere with the absorbance reading at 340nm. Inactivation of GAD (glutamate decarboxylase) by immediately adding methanol to a frozen plant tissue powder was suggested to prevent GABA production during extraction. The recovery of GABA with this method was approximately 100%. Second, the relationship between GABA levels and hypocotyl elongation in soybean seedlings was analyzed using different approaches to regulate in vivo GABA levels and the elongation of hypocotyls. The following major observations were made. (1) Mechanical stimulation by stroking elevated GABA levels and concurrently induced a rapid and significant reduction in hypocotyl elongation. (2) External GABA was demonstrated to penetrate into the hypocotyls using '*C-GABA. Application of external GABA elevated in vivo GABA levels, but failed to inhibit hypocotyl elongation. (3) LaCla and blue light irradiation caused an inhibition in the elongation of dark-grown hypocotyls, whereas GABA levels were not significantly affected. (4) Ca^was suggested to be involved in the signal transduction pathway leading from mechanical stimulation to GABA production, as indicated by the ability of La'* to inhibit GABA production in stimulated hypocotyls. (5) Bicuculline, saclofen and baclofen (agonists and antagonists of GABA receptors in animals) had no effect on hypocotyl elongation. It might indicate that GABA-binding components which are structurally similar to animal GABA receptors and functionally capable of regulating plant growth may not exist in plants. Therefore, the conclusion was drawn that GABA alone is not sufficient to inhibit hypocotyl elongation. Third, chloride influx in isolated Asparagus cells was enhanced by lOmM GABA during a 3 hour incubation, but the effect was not specific for GABA. Chloride efflux was not influenced by GABA. Both influx and efflux of chloride were significantly inhibited by NPPB, a chloride channel blocker. These results suggest that GABA does not influence the activity of plant chloride channels.

Diversité fonctionnelle du facteur de transcription Tbx5 dans le coeur

Le cœur des vertébrés est un organe modulaire qui requiert le " patterning " complexe des champs morphogénétiques cardiogènes et la convergence coordonnée des diverses sous-populations de progéniteurs cardiogéniques. Au moins 7 facteurs de transcription de la famille T-box coopèrent au sein de ces nombreuses sous-populations de progéniteurs cardiogéniques afin de réguler la morphogenèse et l’agencement de multiples structures le long de l’ébauche cardiaque, ce qui explique que les mutations humaines de ces gènes engendrent diverses malformations congénitales cardiaques (MCCs). L’un de ces gènes T-box, Tbx5, dont l’haploinsuffisance génère le syndrome de Holt-Oram (SHO), intervient dans une grande variété de réseaux de régulation géniques (RRGs) qui orchestrent la morphogenèse des oreillettes, du ventricule gauche, de la valve mitrale, des septums inter-auriculaire et inter-ventriculaire, ainsi que du système de conduction cardiaque. La diversité des RRGs impliqués dans la formation de ces structures cardiaques suggère que Tbx5 détient une profusion de fonctions qui ne seront identifiables qu’en répertoriant ses activités moléculaires dans chaque lignée cardiaque examinée isolément. Afin d’aborder cette problématique, une ablation génétique de Tbx5 dans l’endocarde a été réalisée. Cette expérience a démontré le rôle crucial de Tbx5 dans la survie des cellules endocardiques bordant le septum primum et des cardiomyocytes au sein de cette structure embryonnaire qui contribuera à la morphogenèse du septum inter-auriculaire. En outre, cette étude a révélé l’existence d’une communication croisée entre la sous-population de cellules endocardiques Tbx5+ et le myocarde au niveau du septum primum, afin d’assurer la survie des cardiomyocytes, et ultimement de garantir la maturation du septum inter-auriculaire. Nos résultats confirment aussi l’importance de l’interdépendance génétique (Tbx5 et Gata4 ainsi que Tbx5 et Nos3) entre différents loci dans la morphogenèse de la cloison inter-auriculaire, et particulièrement de l’influence que peut avoir l’environnement sur la pénétrance et l’expressivité des communications inter-auriculaires (CIAs) dans le SHO. En outre, puisque les fonctions d’un gène dépendent ordinairement des différents isoformes qu’il peut générer, une deuxième étude a focalisé davantage sur l’aspect transcriptionnel de Tbx5. Cette approche a mené à la découverte de 6 transcrits alternatifs exhibant des fonctions à la fois communes et divergentes. La caractérisation de 2 de ces isoformes a révélé le rôle de l’isoforme long (Tbx5_v1) dans la régulation de la croissance des cardiomyocytes durant la cardiogénèse, tandis que l’isoforme court (Tbx5_v2), préférentiellement exprimé dans le cœur mature, réprime la croissance cellulaire. Il est donc entièrement concevable que les mutations de TBX5 entraînant une troncation de la région C-terminale accroissent la concentration d’une protéine mutée qui, à l’instar de Tbx5_v2, interfère avec la croissance de certaines structures cardiaques. En revanche, la divergence de fonctions de ces isoformes, caractérisée par les disparités de localisation subcellulaire et de d’interaction avec d’autres cofacteurs cardiaques, suggère que les mutations affectant davantage un isoforme favoriseraient l’émergence d’un type particulier de MCC. Finalement, un dernier objectif était d’identifier le ou les mécanisme(s) moléculaire(s) par le(s)quel(s) Tbx5 régule son principal gène cible, Nppa, et d’en extraire les indices qui éclairciraient sa fonction transcriptionnelle. Cet objectif nécessitait dans un premier lieu d’identifier les différents modules cis-régulateurs (MCRs) coordonnant la régulation transcriptionnelle de Nppa et Nppb, deux gènes natriurétiques dont l’organisation en tandem et le profil d’expression durant la cardiogénèse sont conservés dans la majorité des vertébrés. L’approche d’empreinte phylogénétique employée pour scanner le locus Nppb/Nppa a permis d’identifier trois MCRs conservés entre diverses espèces de mammifères, dont un (US3) est spécifique aux euthériens. Cette étude a corroboré que la régulation de l’expression du tandem génique Nppb/Nppa requérait l’activité transcriptionnelle d’enhancers en complément aux promoteurs de Nppa et Nppb. La concordance quasiment parfaite entre les profils d’expression de Tbx5 et de ces deux gènes natriurétiques chez les mammifères, suggère que le gradient d’expression ventriculaire de Tbx5 est interprété par le recrutement de ce facteur au niveau des différents enhancers identifiés. En somme, les études présentées dans cette thèse ont permis de clarifier la profusion de fonctions cardiaques que possède Tbx5. Certaines de ces fonctions émanent de l’épissage alternatif de Tbx5, qui favorise la synthèse d’isoformes dotés de propriétés spécifiques. Les diverses interactions combinatoires entre ces isoformes et d’autres facteurs cardiaques au sein des diverses sous-populations de progéniteurs cardiogènes contribuent à l’émergence de RRGs cardiaques divergents.

NHE1, NHE2, and NHE4 contribute to regulation of cell pH in T84 colon cancer cells

The isoforms of the Na+/H+ exchanger present in T84 human colon cells were identified by functional and molecular methods. Cell pH was measured by fluorescence microscopy using the probe BCECF. Based on the pH recovery after an ammonium pulse and determination of buffering capacity of these cells, the rate of H+ extrusion (J(H)) was 3.68 mM/min. After the use of the amiloride derivative HOE-694 at 25 mu M, which inhibits the isoforms NHE1 and NHE2, there remained 43% of the above transport rate, the nature of which was investigated. Evidence of the presence of NHE1, NHE2, and NHE4 was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) (mRNA) and Western blot. There was no decrease of J(H) by the NHE3 inhibitor S3226 (1 mu M) and no evidence of this isoform by RT-PCR was found. The following functional evidence for the presence of NHE4 was obtained: 25 mu M EIPA abolished J(H) entirely, but NHE4 was not inhibited at 10 mu M; substitution of Na by K increased the remainder, a property of NHE4; hypertonicity also increased this fraction of J(H). Cl--dependent NHE was not detected: in 0 Cl- solutions J(H) was increased and not reduced. In 0 Cl- cell volume decreased significantly, which was abolished by the Cl- channel blocker NPPB, indicating that the 0 Cl- effect was because of reduction of cell volume. In conclusion, T84 human colon cells contain three isoforms of the Na+/H+ exchanger, NHE1, NHE2, and NHE4, but not the Cl-dependent NHE.

Role of CFTR and ClC-5 in Modulating Vacuolar H(+)-ATPase Activity in Kidney Proximal Tubule

Background/Aims: It has been widely accepted that chloride ions moving along chloride channels act to dissipate the electrical gradient established by the electrogenic transport of H(+) ions performed by H(+)-ATPase into subcellular vesicles. Largely known in intracellular compartments, this mechanism is also important at the plasma membrane of cells from various tissues, including kidney. The present work was performed to study the modulation of plasma membrane H(+)-ATPase by chloride channels, in particular, CFTR and ClC-5 in kidney proximal tubule. Methods and Results: Using in vivo stationary microperfusion, it was observed that ATPase-mediated HCO(3)(-) reabsorption was significantly reduced in the presence of the Cl(-) channels inhibitor NPPB. This effect was confirmed in vitro by measuring the cell pH recovery rates after a NH(4)Cl pulse in immortalized rat renal proximal tubule cells, IRPTC. In these cells, even after abolishing the membrane potential with valinomycin, ATPase activity was seen to be still dependent on Cl(-). siRNA-mediated CFTR channels and ClC-5 chloride-proton exchanger knockdown significantly reduced H(+)-ATPase activity and V-ATPase B2 subunit expression. Conclusion: These results indicate a role of chloride in modulating plasma membrane H(+)-ATPase activity in proximal tubule and suggest that both CFTR and ClC-5 modulate ATPase activity. Copyright (C) 2010 S. Karger AG, Basel

O Circuito espacial de produção do biodiesel: configuração no território brasileiro e uso do território Norte-Rio-Grandense

This research aims to understand the use of the territory from the Rio Grande do Norte to the circuit spatial of production from the Biodiesel understanding it as part of the national context. The introduction of biodiesel into the Brazilian energy matrix begins to take shape in the year 2005 with the implementation of the National Program for Production and Use of Biodiesel (NPPB). This is anchored on three pillars: social inclusion (through family agriculture), environmental sustainability and economic viability. The NPPB consists of a set of standards, which turned into shares and distributed by almost all the national territory. Our reflection assumes that the places accommodating different forms of productive activities and, thus, the performance of the circuit space of biodiesel production depends on several factors, including the configuration of the territory they receive this new nexus economic. Understanding that the places by their technical content determine the realization of productive activities, it was found that the inclusion of this circuit space production in Rio Grande do Norte, reveals the reality expressed in the national territory, where some places are endowed with a privileged technical content and other extremely poor infrastructure. As our research could confirm the circuit space of biodiesel production that is expressed in the way plants are distributed within the country (mostly in South-Central), in more significant participation of small farmers in the South in the supply of raw material for the production of biodiesel and the main raw material used for the production of agrofuels (soybeans, whose production sector is highly consolidated and technified). Already the project materialization of NPPB regarding the settlements and communities of the Rio Grande do Norte State, we observed that the "event" or advent of realization encountered a technical means lacking in infrastructure that need to be molded to the needs of production, or is to cultivate castor beans or sunflower would be needed inputs and implements that farmers don´t have. Given the above, the research concludes that, as proposed previously, the use of the territory of Rio Grande do Norte to this circuit has summarized the performance of experiments, either with regard to the participation of family farmers as suppliers of raw materials, including the development of related research in the stage of production fuel oil by Petrobras. This finding came from research done from the concept of "spatial circuits of production," which allowed the verification of the circuit that steps outlined in Potiguar territory. Regarding the inclusion of family farmers in the productive circuit in RN, research reveals that, in the manner as has been done, it is doomed to failure. Furthermore, we dare say that this persistence in trying to put these family farmers, the way is being made, and the resulting successive failures, indicating that social inclusion advocated by NPPB not take place, and that this production circuit relies on the same logic circuit concentrated and exclusive space for ethanol production

A release mechanism for stored ATP in ocular ciliary epithelial cells

Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the fluorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl−-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.

Anion Channels and the Stimulation of Anthocyanin Accumulation by Blue Light in Arabidopsis Seedlings1

Activation of anion channels by blue light begins within seconds of irradiation in seedlings and is related to the ensuing growth inhibition. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a potent, selective, and reversible blocker of these anion channels in Arabidopsis thaliana. Here we show that 20 μm NPPB blocked 72% of the blue-light-induced accumulation of anthocyanin pigments in seedlings. Feeding biosynthetic intermediates to wild-type and tt5 seedlings provided evidence that NPPB prevented blue light from up-regulating one or more steps between and including phenylalanine ammonia lyase and chalcone isomerase. NPPB was found to have no significant effect on the blue-light-induced increase in transcript levels of PAL1, CHS, CHI, or DFR, which are genes that encode anthocyanin-biosynthetic enzymes. Immunoblots revealed that NPPB also did not inhibit the accumulation of the chalcone synthase, chalcone isomerase, or flavanone-3-hydroxylase proteins. This is in contrast to the reduced anthocyanin accumulation displayed by a mutant lacking the HY4 blue-light receptor, as hy4 displayed reduced expression of the above enzymes. Taken together, the data indicate that blue light acting through HY4 leads to an increase in the amount of biosynthetic enzymes, but blue light must also act through a separate, anion-channel-dependent system to create a fully functional biosynthetic pathway.

An anion channel in Arabidopsis hypocotyls activated by blue light.

A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition.