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Sample records for active na transport

  1. Active urea transport independent of H+ and Na+ transport in frog skin epithelium.

    PubMed

    Lacoste, I; Dunel-Erb, S; Harvey, B J; Laurent, P; Ehrenfeld, J

    1991-10-01

    We investigated the relationship between H+ secretion (JH), Na+ absorption (JNa), and urea transport (Ju) in skin of frogs (Rana esculenta) adapted to running tap water, NaCl (100 mM), and KCl (100 mM). In addition, cell morphological changes, particularly in the mitochondria-rich cells (MRC), were followed. NaCl adaptation stimulated an active Ju, reduced JNa and JH, and caused a decrease in the apical surface of MRC. After KCl adaptation, JNa and JH were increased and highly correlated, with a twofold increase in Ju, whereas the numerous MRC developed infoldings on their apical membranes. No correlation was found between JH and Ju. Clamping the skins in a range of +/- 50 mV or changing the external pH from 7.4 to 5.4 (at high cellular buffering power) had no effect on Ju. Depolarization of the basolateral membranes (serosal KCl-Ringer) had no effect on Ju. Ju was reversibly blocked by acidification of the cells by oxygen-free solution and sulfhydryl reagents (Hg2+, p-chloromercuribenzenesulfonic acid, and N-ethylmaleimide). Diethylstilbestrol, a proton transport blocker, had no effect on Ju. Apical addition of amiloride and derivatives (phenamil and ethylisopropyl amiloride) reversibly blocked Ju, whereas ouabain had no effect. We conclude that a cation (Na+ or H+)-dependent process is unlikely to exist in R. esculenta skin. A primary active transport in a two-step process is the simplest hypothesis to account for the energy-dependent Ju that develops in NaCl-adapted frogs.

  2. Prolactin increases hepatic Na+/taurocholate co-transport activity and messenger RNA post partum.

    PubMed Central

    Ganguly, T C; Liu, Y; Hyde, J F; Hagenbuch, B; Meier, P J; Vore, M

    1994-01-01

    We have shown that Na+/taurocholate co-transport activity is decreased in pregnancy, but rebounds post partum relative to non-pregnant controls, and that activity can be increased by treatment with ovine prolactin [Ganguly, Hyde and Vore (1993) J. Pharmacol. Exp. Ther. 267, 82-87]. To determine the basis for these effects, Na+/taurocholate co-transport was determined in purified basolateral liver plasma-membrane (bLPM) vesicles and compared with steady-state mRNA levels encoding the Na+/taurocholate-co-transporting polypeptide (Ntcp) in non-pregnant controls, pregnant rats (19-20 days pregnant), rats post partum (48 h post partum) and rats post partum treated with bromocriptine to inhibit prolactin secretion. Na+/taurocholate co-transport activity (nmol/5 s per mg of protein) in bLPM was decreased from 10.4 +/- 1.8 in non-pregnant controls to 7.9 +/- 0.6 in bLPM in pregnant rats, but rebounded to 17.5 +/- 1.3 post partum; treatment of rats post partum with bromocriptine to inhibit prolactin secretion decreased activity to 14.1 +/- 0.9. Northern and slot-blot analyses revealed similar changes in mRNA for Ntcp, so that a positive correlation was observed between Na+/taurocholate co-transport activity and Ntcp mRNA. Furthermore, treatment of ovariectomized rats with ovine prolactin increased Ntcp mRNA 10-fold compared with solvent-treated controls, consistent with the 2-fold increase in Vmax, for Na+/taurocholate co-transport in isolated hepatocytes. These data are the first to demonstrate endogenous physiological regulation by prolactin of Ntcp mRNA in parallel with Na+/taurocholate co-transport activity. Images Figure 2 PMID:7945260

  3. The Na+/I symporter (NIS) mediates electroneutral active transport of the environmental pollutant perchlorate.

    PubMed

    Dohán, Orsolya; Portulano, Carla; Basquin, Cécile; Reyna-Neyra, Andrea; Amzel, L Mario; Carrasco, Nancy

    2007-12-18

    The Na(+)/I(-) symporter (NIS) is a key plasma membrane protein that mediates active I(-) uptake in the thyroid, lactating breast, and other tissues with an electrogenic stoichiometry of 2 Na(+) per I(-). In the thyroid, NIS-mediated I(-) uptake is the first step in the biosynthesis of the iodine-containing thyroid hormones, which are essential early in life for proper CNS development. In the lactating breast, NIS mediates the translocation of I(-) to the milk, thus supplying this essential anion to the nursing newborn. Perchlorate (ClO(4)(-)) is a well known competitive inhibitor of NIS. Exposure to food and water contaminated with ClO(4)(-) is common in the U.S. population, and the public health impact of such exposure is currently being debated. To date, it is still uncertain whether ClO(4)(-) is a NIS blocker or a transported substrate of NIS. Here we show in vitro and in vivo that NIS actively transports ClO(4)(-), including ClO(4)(-) translocation to the milk. A simple mathematical fluxes model accurately predicts the effect of ClO(4)(-) transport on the rate and extent of I(-) accumulation. Strikingly, the Na(+)/ ClO(4)(-) transport stoichiometry is electroneutral, uncovering that NIS translocates different substrates with different stoichiometries. That NIS actively concentrates ClO(4)(-) in maternal milk suggests that exposure of newborns to high levels of ClO(4)(-) may pose a greater health risk than previously acknowledged because ClO(4)(-) would thus directly inhibit the newborns' thyroidal I(-) uptake.

  4. Cardiac glycoside activities link Na(+)/K(+) ATPase ion-transport to breast cancer cell migration via correlative SAR.

    PubMed

    Magpusao, Anniefer N; Omolloh, George; Johnson, Joshua; Gascón, José; Peczuh, Mark W; Fenteany, Gabriel

    2015-02-20

    The cardiac glycosides ouabain and digitoxin, established Na(+)/K(+) ATPase inhibitors, were found to inhibit MDA-MB-231 breast cancer cell migration through an unbiased chemical genetics screen for cell motility. The Na(+)/K(+) ATPase acts both as an ion-transporter and as a receptor for cardiac glycosides. To delineate which function is related to breast cancer cell migration, structure-activity relationship (SAR) profiles of cardiac glycosides were established at the cellular (cell migration inhibition), molecular (Na(+)/K(+) ATPase inhibition), and atomic (computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a similar profile, a decrease in potency when the parent cardiac glycoside structure was modified, for each activity investigated. Since assays were done at the cellular, molecular, and atomic levels, correlation of SAR profiles across these multiple assays established links between cellular activity and specific protein-small molecule interactions. The observed antimigratory effects in breast cancer cells are directly related to the inhibition of Na(+)/K(+) transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation path formed by transmembrane helices αM1-M6 correlates with the Na(+) pump activity and cell migration. Other Na(+)/K(+) ATPase inhibitors that are structurally distinct from cardiac glycosides also exhibit antimigratory activity, corroborating the conclusion that the antiport function of Na(+)/K(+) ATPase and not the receptor function is important for supporting the motility of MDA-MB-231 breast cancer cells. Correlative SAR can establish new relationships between specific biochemical functions and higher-level cellular processes, particularly for proteins with multiple functions and small molecules with unknown or various modes of action.

  5. Stimulation of Na{sup +}/K{sup +} ATPase activity and Na{sup +} coupled glucose transport by {beta}-catenin

    SciTech Connect

    Sopjani, Mentor; Alesutan, Ioana; Wilmes, Jan; Dermaku-Sopjani, Miribane; Lam, Rebecca S.; Jakupi, Muharrem; Foeller, Michael; Lang, Florian

    2010-11-19

    Research highlights: {yields} The oncogenic transcription factor {beta}-catenin stimulates the Na{sup +}/K{sup +}-ATPase. {yields} {beta}-Catenin stimulates SGLT1 dependent Na{sup +}, glucose cotransport. {yields} The effects are independent of transcription. {yields} {beta}-Catenin sensitive transport may contribute to properties of proliferating cells. -- Abstract: {beta}-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. {beta}-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that {beta}-catenin influences membrane transport. To this end, {beta}-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of {beta}-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na{sup +}/K{sup +}-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of {beta}-catenin on the endogenous Na{sup +}/K{sup +}-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of {beta}-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of {beta}-catenin expression. The stimulating effect of {beta}-catenin on both Na{sup +}/K{sup +} ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of {beta}-catenin, i.e. the regulation of transport.

  6. Na+ recirculation and isosmotic transport.

    PubMed

    Larsen, E H; Møbjerg, N

    2006-01-01

    The Na(+) recirculation theory for solute-coupled fluid absorption is an expansion of the local osmosis concept introduced by Curran and analyzed by Diamond & Bossert. Based on studies on small intestine the theory assumes that the observed recirculation of Na(+) serves regulation of the osmolarity of the absorbate. Mathematical modeling reproducing bioelectric and hydrosmotic properties of small intestine and proximal tubule, respectively, predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1 (-/-) mice, and the inverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow. The model reproduces the volume responses of cells and lateral intercellular space (lis) following replacement of luminal NaCl by sucrose as well as the linear dependence of volume absorption on luminal NaCl concentration. Analysis of solvent drag on Na(+) in tight junctions provides explanation for the surprisingly high metabolic efficiency of Na(+) reabsorption. The model predicts and explains low metabolic efficiency in diluted external baths. Hyperosmolarity of lis is governed by the hydraulic permeability of the apical plasma membrane and tight junction with 6-7 mOsm in small intestine and < or = 1 mOsm in proximal tubule. Truly isosmotic transport demands a Na(+) recirculation of 50-70% in small intestine but might be barely measurable in proximal tubule. The model fails to reproduce a certain type of observations: The reduced volume absorption at transepithelial osmotic equilibrium in AQP1 knockout mice, and the stimulated water absorption by gallbladder in diluted external solutions. Thus, it indicates cellular regulation of apical Na(+) uptake, which is not included in the mathematical treatment.

  7. NMR studies on Na+ transport in Synechococcus PCC 6311

    NASA Technical Reports Server (NTRS)

    Nitschmann, W. H.; Packer, L.

    1992-01-01

    The freshwater cyanobacterium Synechococcus PCC 6311 is able to adapt to grow after sudden exposure to salt (NaCl) stress. We have investigated the mechanism of Na+ transport in these cells during adaptation to high salinity. Na+ influx under dark aerobic conditions occurred independently of delta pH or delta psi across the cytoplasmic membrane, ATPase activity, and respiratory electron transport. These findings are consistent with the existence of Na+/monovalent anion cotransport or simultaneous Na+/H+ +anion/OH- exchange. Na+ influx was dependent on Cl-, Br-, NO3-, or NO2-. No Na+ uptake occurred after addition of NaI, NaHCO3, or Na2SO4. Na+ extrusion was absolutely dependent on delta pH and on an ATPase activity and/or on respiratory electron transport. This indicates that Na+ extrusion via Na+/H+ exchange is driven by primary H+ pumps in the cytoplasmic membrane. Cells grown for 4 days in 0.5 m NaCl medium, "salt-grown cells," differ from control cells by a lower maximum velocity of Na+ influx and by lower steady-state ratios of [Na+]in/[Na+]out. These results indicate that cells grown in high-salt medium increase their capacity to extrude Na+. During salt adaptation Na+ extrusion driven by respiratory electron transport increased from about 15 to 50%.

  8. Down-regulation of Na+-coupled glutamate transporter EAAT3 and EAAT4 by AMP-activated protein kinase.

    PubMed

    Sopjani, Mentor; Alesutan, Ioana; Dërmaku-Sopjani, Miribane; Fraser, Scott; Kemp, Bruce E; Föller, Michael; Lang, Florian

    2010-06-01

    The glutamate transporters EAAT3 and EAAT4 are expressed in neurons. They contribute to the cellular uptake of glutamate and aspartate and thus to the clearance of the excitatory transmitters from the extracellular space. During ischemia, extracellular accumulation of glutamate may trigger excitotoxicity. Energy depletion leads to activation of the AMP-activated protein kinase (AMPK), a kinase enhancing energy production and limiting energy expenditure. The present study thus explored the possibility that AMPK regulates EAAT3 and/or EAAT4. To this end, EAAT3 or EAAT4 were expressed in Xenopus oocytes with or without AMPK and electrogenic glutamate transport determined by dual electrode voltage clamp. In EAAT3- and in EAAT4-expressing oocytes glutamate generated a current (I(g)), which was half maximal (K(M)) at 74 microM (EAAT3) or at 4 microM (EAAT4) glutamate. Co-expression of constitutively active (gammaR70Q)AMPK or of wild type AMPK did not affect K(M) but significantly decreased the maximal I(g) in both EAAT3- (by 34%) and EAAT4- (by 49%) expressing oocytes. Co-expression of the inactive mutant (alphaK45R)AMPK [alpha1(K45R)beta1gamma1] did not appreciably affect I(g). According to confocal microscopy and chemiluminescence co-expression of (gammaR70Q)AMPK or of wild type AMPK reduced the membrane abundance of EAAT3 and EAAT4. The observations show that AMPK down-regulates Na(+)-coupled glutamate transport.

  9. Glial Na(+) -dependent ion transporters in pathophysiological conditions.

    PubMed

    Boscia, Francesca; Begum, Gulnaz; Pignataro, Giuseppe; Sirabella, Rossana; Cuomo, Ornella; Casamassa, Antonella; Sun, Dandan; Annunziato, Lucio

    2016-10-01

    Sodium dynamics are essential for regulating functional processes in glial cells. Indeed, glial Na(+) signaling influences and regulates important glial activities, and plays a role in neuron-glia interaction under physiological conditions or in response to injury of the central nervous system (CNS). Emerging studies indicate that Na(+) pumps and Na(+) -dependent ion transporters in astrocytes, microglia, and oligodendrocytes regulate Na(+) homeostasis and play a fundamental role in modulating glial activities in neurological diseases. In this review, we first briefly introduced the emerging roles of each glial cell type in the pathophysiology of cerebral ischemia, Alzheimer's disease, epilepsy, Parkinson's disease, Amyotrophic Lateral Sclerosis, and myelin diseases. Then, we discussed the current knowledge on the main roles played by the different glial Na(+) -dependent ion transporters, including Na(+) /K(+) ATPase, Na(+) /Ca(2+) exchangers, Na(+) /H(+) exchangers, Na(+) -K(+) -Cl(-) cotransporters, and Na(+) - HCO3- cotransporter in the pathophysiology of the diverse CNS diseases. We highlighted their contributions in cell survival, synaptic pathology, gliotransmission, pH homeostasis, and their role in glial activation, migration, gliosis, inflammation, and tissue repair processes. Therefore, this review summarizes the foundation work for targeting Na(+) -dependent ion transporters in glia as a novel strategy to control important glial activities associated with Na(+) dynamics in different neurological disorders. GLIA 2016;64:1677-1697.

  10. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

  11. Pyrophosphate-Fueled Na+ and H+ Transport in Prokaryotes

    PubMed Central

    Malinen, Anssi M.; Luoto, Heidi H.

    2013-01-01

    SUMMARY In its early history, life appeared to depend on pyrophosphate rather than ATP as the source of energy. Ancient membrane pyrophosphatases that couple pyrophosphate hydrolysis to active H+ transport across biological membranes (H+-pyrophosphatases) have long been known in prokaryotes, plants, and protists. Recent studies have identified two evolutionarily related and widespread prokaryotic relics that can pump Na+ (Na+-pyrophosphatase) or both Na+ and H+ (Na+,H+-pyrophosphatase). Both these transporters require Na+ for pyrophosphate hydrolysis and are further activated by K+. The determination of the three-dimensional structures of H+- and Na+-pyrophosphatases has been another recent breakthrough in the studies of these cation pumps. Structural and functional studies have highlighted the major determinants of the cation specificities of membrane pyrophosphatases and their potential use in constructing transgenic stress-resistant organisms. PMID:23699258

  12. Characteristics and pharmacological regulation of epithelial Na+ channel (ENaC) and epithelial Na+ transport.

    PubMed

    Marunaka, Yoshinori

    2014-01-01

    Epithelial Na(+) transport participates in control of various body functions and conditions: e.g., homeostasis of body fluid content influencing blood pressure, control of amounts of fluids covering the apical surface of alveolar epithelial cells at appropriate levels for normal gas exchange, and prevention of bacterial/viral infection. Epithelial Na(+) transport via the transcellular pathway is mediated by the entry step of Na(+) across the apical membrane via Epithelial Na(+) Channel (ENaC) located at the apical membrane, and the extrusion step of Na(+) across the basolateral membrane via the Na(+),K(+)-ATPase located at the basolateral membrane. The rate-limiting step of the epithelial Na(+) transport via the transcellular pathway is generally recognized to be the entry step of Na(+) across the apical membrane via ENaC. Thus, up-/down-regulation of ENaC essentially participates in regulatory systems of blood pressure and normal gas exchange. Amount of ENaC-mediated Na(+) transport is determined by the number of ENaCs located at the apical membrane, activity (open probability) of individual ENaC located at the apical membrane, single channel conductance of ENaC located at the apical membrane, and driving force for the Na(+) entry via ENaCs across the apical membrane. In the present review article, I discuss the characteristics of ENaC and how these factors are regulated.

  13. Impact of mechanical stress on ion transport in native lung epithelium (Xenopus laevis): short-term activation of Na+, Cl (-) and K+ channels.

    PubMed

    Bogdan, Roman; Veith, Christine; Clauss, Wolfgang; Fronius, Martin

    2008-09-01

    Epithelia, in general, and the lung epithelium, in particular, are exposed to mechanical forces, but little is known about their impact on pulmonary ion transport. In our present study, we employed transepithelial ion transport measurements on Xenopus lung preparations using custom-built Ussing chambers. Tissues were exposed to mechanical stress by increasing the water column (5 cm) at one side of the tissues. Apical exposure to hydrostatic pressure significantly decreased the short circuit current (I (SC): 24 +/- 1%, n = 152), slightly decreased the transepithelial resistance (R (T): 7 +/- 2%, n = 152), but increased the apical membrane capacitance (C (M): 16 +/- 6%, n = 9). The pressure-induced effect was sensitive to Na+ (amiloride), Cl(-) (DIDS, NFA, NPPB) and K+ channel blockers (Ba2+), glibenclamide). Further on, it was accompanied by increased extracellular ATP levels. The results show that mechanical stress leads to an activation of Na+, Cl(-), and K+ conductances in a native pulmonary epithelium resulting in a net decrease of ion absorption. This could be of considerable interest, since an altered ion transport may contribute to pathophysiological conditions, e.g., the formation of pulmonary edema during artificial ventilation.

  14. Acidic pH and short-chain fatty acids activate Na+ transport but differentially modulate expression of Na+/H+ exchanger isoforms 1, 2, and 3 in omasal epithelium.

    PubMed

    Lu, Zhongyan; Yao, Lei; Jiang, Zhengqian; Aschenbach, Jörg R; Martens, Holger; Shen, Zanming

    2016-01-01

    Low sodium content in feed and large amounts of salivary sodium secretion are essential requirements to efficient sodium reabsorption in the dairy cow. It is already known that Na(+)/H(+) exchange (NHE) of the ruminal epithelium plays a key role in Na(+) absorption, and its function is influenced by the presence of short-chain fatty acids (SCFA) and mucosal pH. By contrast, the functional role and regulation of NHE in omasal epithelium have not been completely understood. In the present study, we used model studies in small ruminants (sheep and goats) to investigate NHE-mediated Na(+) transport and the effects of pH and SCFA on NHE activity in omasal epithelium and on the expression of NHE isoform in omasal epithelial cells. Conventional Ussing chamber technique, primary cell culture, quantitative PCR, and Western blot were used. In native omasal epithelium of sheep, the Na(+) transport was electroneutral, and it was inhibited by the specific NHE3 inhibitor 3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride, which decreased mucosal-to-serosal, serosal-to-mucosal, and net flux rates of Na(+) by 80% each. The application of low mucosal pH (6.4 or 5.8) in the presence of SCFA activated the Na(+) transport across omasal epithelium of sheep compared with that at pH 7.4. In cultured omasal epithelial cells of goats, mRNA and protein of NHE1, NHE2, and NHE3 were detected. The application of SCFA increased NHE1 mRNA and protein expression, which was most prominent when the culture medium pH decreased from 7.4 to 6.8. At variance, the mRNA and protein expression of NHE2 and NHE3 were decreased with low pH and SCFA, which was contrary to the published data from ruminal epithelial studies. In conclusion, this paper shows that (1) NHE1, NHE2, and NHE3 are expressed in omasal epithelium; (2) NHE3 mediates the major portion of transepithelial Na(+) transport in omasal epithelium; and (3) SCFA and acidic pH acutely

  15. The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3- salvage transporter human Na+-HCO3- cotransport isoform 3.

    PubMed

    Park, Meeyoung; Ko, Shigeru B H; Choi, Joo Young; Muallem, Gaia; Thomas, Philip J; Pushkin, Alexander; Lee, Myeong-Sok; Kim, Joo Young; Lee, Min Goo; Muallem, Shmuel; Kurtz, Ira

    2002-12-27

    Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO(3)(-) secretion and HCO(3)(-) salvage in secretory epithelia. At least two luminal transporters mediate HCO(3)(-) salvage, the Na(+)/H(+) exchanger (NHE3) and the Na(+)-HCO(3)(-) cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na(+)-dependent, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO(3)(-) transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO(3)(-)-transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO(3)(-) transport by regulating the activity of all luminal HCO(3)(-) secretion and salvage mechanisms of secretory epithelial cells.

  16. Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-α-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis

    PubMed Central

    Yokomichi, Tomonobu; Morimoto, Kyoko; Oshima, Nana; Yamada, Yuriko; Fu, Liwei; Taketani, Shigeru; Ando, Masayoshi; Kataoka, Takao

    2011-01-01

    Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-α-induced ICAM-1 protein expression almost completely, whereas the TNF-α-induced ICAM-1 mRNA expression and NF-κB signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis. PMID:24970122

  17. Na+ Tolerance and Na+ Transport in Higher Plants

    PubMed Central

    TESTER, MARK; DAVENPORT, ROMOLA

    2003-01-01

    Tolerance to high soil [Na+] involves processes in many different parts of the plant, and is manifested in a wide range of specializations at disparate levels of organization, such as gross morphology, membrane transport, biochemistry and gene transcription. Multiple adaptations to high [Na+] operate concurrently within a particular plant, and mechanisms of tolerance show large taxonomic variation. These mechanisms can occur in all cells within the plant, or can occur in specific cell types, reflecting adaptations at two major levels of organization: those that confer tolerance to individual cells, and those that contribute to tolerance not of cells per se, but of the whole plant. Salt‐tolerant cells can contribute to salt tolerance of plants; but we suggest that equally important in a wide range of conditions are processes involving the management of Na+ movements within the plant. These require specific cell types in specific locations within the plant catalysing transport in a coordinated manner. For further understanding of whole plant tolerance, we require more knowledge of cell‐specific transport processes and the consequences of manipulation of transporters and signalling elements in specific cell types. PMID:12646496

  18. Na(+)-I- symport activity is present in membrane vesicles from thyrotropin-deprived non-I(-)-transporting cultured thyroid cells.

    PubMed Central

    Kaminsky, S M; Levy, O; Salvador, C; Dai, G; Carrasco, N

    1994-01-01

    The active accumulation of I- in the thyroid gland is mediated by the Na(+)-I- symporter and driven by the Na+ gradient generated by the Na+/K(+)-ATPase. Thyrotropin (TSH) stimulates thyroidal I- accumulation. Rat thyroid-derived FRTL-5 cells require TSH to accumulate I-. TSH withdrawal for over 7 days results in complete loss of Na(+)-I-symport activity in these cells [Weiss, S. J., Philp, N. J. and Grollman, E. F. (1984) Endocrinology 114, 1090-1098]. Surprisingly, membrane vesicles prepared from FRTL-5 cells maintained in TSH-free medium [TSH(-)cells]accumulate I-, suggesting that the absence of Na(+)-I- symport activity in TSH(-) cells cannot be due solely to a decrease in the biosynthesis of either the symporter or a putative activating factor. This finding indicates that the Na(+)-I- symporter is present, probably in an inactive state, in TSH(-) cells despite their lack of Na(+)-I- symport activity. Na(+)-I- symport activity in thyroid membrane vesicles is enhanced when conditions for vesicle preparation favor proteolysis. Subcellular fractionation studies in both TSH(+) and TSH(-) cells show that Na(+)-I- symport activity is mostly associated with fractions enriched in plasma membrane rather than in intracellular membranes, suggesting that the Na(+)-I- symporter may constitutively reside in the plasma membrane and may be activated by TSH. Images PMID:8170988

  19. Membrane Na+-pyrophosphatases can transport protons at low sodium concentrations.

    PubMed

    Luoto, Heidi H; Nordbo, Erika; Baykov, Alexander A; Lahti, Reijo; Malinen, Anssi M

    2013-12-06

    Membrane-bound Na(+)-pyrophosphatase (Na(+)-PPase), working in parallel with the corresponding ATP-energized pumps, catalyzes active Na(+) transport in bacteria and archaea. Each ~75-kDa subunit of homodimeric Na(+)-PPase forms an unusual funnel-like structure with a catalytic site in the cytoplasmic part and a hydrophilic gated channel in the membrane. Here, we show that at subphysiological Na(+) concentrations (<5 mM), the Na(+)-PPases of Chlorobium limicola, four other bacteria, and one archaeon additionally exhibit an H(+)-pumping activity in inverted membrane vesicles prepared from recombinant Escherichia coli strains. H(+) accumulation in vesicles was measured with fluorescent pH indicators. At pH 6.2-8.2, H(+) transport activity was high at 0.1 mM Na(+) but decreased progressively with increasing Na(+) concentrations until virtually disappearing at 5 mM Na(+). In contrast, (22)Na(+) transport activity changed little over a Na(+) concentration range of 0.05-10 mM. Conservative substitutions of gate Glu(242) and nearby Ser(243) and Asn(677) residues reduced the catalytic and transport functions of the enzyme but did not affect the Na(+) dependence of H(+) transport, whereas a Lys(681) substitution abolished H(+) (but not Na(+)) transport. All four substitutions markedly decreased PPase affinity for the activating Na(+) ion. These results are interpreted in terms of a model that assumes the presence of two Na(+)-binding sites in the channel: one associated with the gate and controlling all enzyme activities and the other located at a distance and controlling only H(+) transport activity. The inherent H(+) transport activity of Na(+)-PPase provides a rationale for its easy evolution toward specific H(+) transport.

  20. Na+-independent phosphate transport in Caco2BBE cells.

    PubMed

    Candeal, Eduardo; Caldas, Yupanqui A; Guillén, Natalia; Levi, Moshe; Sorribas, Víctor

    2014-12-15

    Pi transport in epithelia has both Na(+)-dependent and Na(+)-independent components, but so far only Na(+)-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na(+)-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na(+)-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi DMEM (4 mM) from 1 day to several days. No response to low-Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4 mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells in 1 mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO4 (2-), HCO3 (-), and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1 mM Pi. Phosphate transport from cells maintained at 4 mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that multifunctional anion exchangers of the Slc26 family as well as members of Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropic retrovirus receptor 1 are not involved.

  1. Na+-independent phosphate transport in Caco2BBE cells

    PubMed Central

    Candeal, Eduardo; Caldas, Yupanqui A.; Guillén, Natalia; Levi, Moshe

    2014-01-01

    Pi transport in epithelia has both Na+-dependent and Na+-independent components, but so far only Na+-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na+-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na+-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi DMEM (4 mM) from 1 day to several days. No response to low-Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4 mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells in 1 mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO42−, HCO3−, and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1 mM Pi. Phosphate transport from cells maintained at 4 mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that multifunctional anion exchangers of the Slc26 family as well as members of Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropic retrovirus receptor 1 are not involved. PMID:25298422

  2. Sex-specific effects of sex steroids on alveolar epithelial Na(+) transport.

    PubMed

    Haase, Melanie; Laube, Mandy; Thome, Ulrich H

    2017-03-01

    Alveolar fluid clearance mediates perinatal lung transition to air breathing in newborn infants, which is accomplished by epithelial Na(+) channels (ENaC) and Na-K-ATPase. Male sex represents a major risk factor for developing respiratory distress, especially in preterm infants. We previously showed that male sex is associated with reduced epithelial Na(+) transport, possibly contributing to the sexual dimorphism in newborn respiratory distress. This study aimed to determine sex-specific effects of sex steroids on epithelial Na(+) transport. The effects of testosterone, 5α-dihydrotestosterone (DHT), estradiol, and progesterone on Na(+) transport and Na(+) channel expression were determined in fetal distal lung epithelial (FDLE) cells of male and female rat fetuses by Ussing chamber and mRNA expression analyses. DHT showed a minor effect only in male FDLE cells by decreasing epithelial Na(+) transport. However, flutamide, an androgen receptor antagonist, did not abolish the gender imbalance, and testosterone lacked any effect on Na(+) transport in male and female FDLE cells. In contrast, estradiol and progesterone increased Na(+) transport and Na(+) channel expression especially in females, and prevented the inhibiting effect of DHT in males. Estrogen receptor inhibition decreased Na(+) channel expression and eliminated the sex differences. In conclusion, female sex steroids stimulate Na(+) transport especially in females and prevent the inhibitory effect of DHT in males. The ineffectiveness of testosterone suggests that Na(+) transport is largely unaffected by androgens. Thus, the higher responsiveness of female cells to female sex steroids explains the higher Na(+) transport activity, possibly leading to a functional advantage in females.

  3. Na+/glucose co-transporter abundance and activity in the small intestine of lambs: enhancement by abomasal infusion of casein.

    PubMed

    Mabjeesh, Sameer J; Guy, Dafna; Sklan, David

    2003-05-01

    The purpose of the present study was to determine the effect of abomasal casein infusion on glucose uptake and abundance of the Na+/glucose co-transporter (SGLT1) 1 in the ovine small intestine. Lambs (body weight 35 (sem 1.0) kg) were surgically fitted with abomasal infusion catheters and were fed diets containing equal portions of wheat hay and cracked maize. Lambs were infused with either 500 g water/d or with 500 g water containing 35 g casein/d. The infusion period lasted 10 d, after which lambs were killed, exsanguinated and eviscerated. Brush border membrane vesicles (BBMV) were prepared using mucosa from different small intestinal regions. Intake and total tract digestibility of nutrients were similar between treatments and averaged 1134, 1142 and 486 g/d and 67, 70, and 94 % for DM, organic matter and non-structural carbohydrates respectively. Crude protein (Nx6.25) digestibility was 15 % greater in the casein-infused than control lambs. Glucose uptake to BBMV ranged from 101 to 337 pmol/mg protein per s along the small intestine and was greatest in the mid-section of the small intestine. In the mid-jejunum, glucose uptake was greater (P<0.07) in lambs infused with casein and averaged 120 pmol/mg protein per s compared with 68 pmol/mg protein per s in the control group. SGLT1 affinity was similar between treatments and averaged 104 microm in the different segments of the small intestine of lambs. However, lambs infused with casein exhibited similar values along the small intestine and affinity averaged 106 microm, while in the control group a greater affinity (85 microm) was measured in the mid-jejunum. SGLT1 protein abundance was correlated with glucose uptake in the BBMV in the casein-treated lambs, but not in the control group. These results suggest that glucose uptake along the small intestine of lambs is influenced by casein or its derivatives in the small intestine via SGLT1 affinity and activity at the brush border membrane, and that SGLT1 activity

  4. Cation Transport in Li+ and Na+ Rich Antiperovskites

    NASA Astrophysics Data System (ADS)

    Howard, John; Daemen, Luke; Zhao, Yusheng; LANL Team; UNLV Team

    2014-03-01

    A large number of compounds possessing the perovskite crystal structure demonstrate interesting properties such as ferroelectricity, magnetoresistance, and superconductivity. In this study, we present findings on a new class of materials, namely Li+ and Na+ rich antiperovskites, with emphasis on cation transport for solid state battery applications. The electrolytes have the general formula A3 BX where A is a Li+ or Na+ cation, B is an O2- or S2- anion, and X is a Cl- or Br- anion; mixed compositions were also studied. X-ray diffraction techniques were used for phase identification, sample purity, and unit cell refinement. In each case, the materials crystallize in a cubic unit cell with space group Pm 3 m . The ionic conductivity was determined for each material as a function of temperature using impedance spectroscopy methods. Activation energies for cation diffusion were determined by fitting the conductivity data to the Arrhenius equation σ = σ0/T e -Ea /kB T .

  5. The Physiological Relevance of Na+-Coupled K+-Transport.

    PubMed Central

    Maathuis, FJM.; Verlin, D.; Smith, F. A.; Sanders, D.; Fernandez, J. A.; Walker, N. A.

    1996-01-01

    Plant roots utilize at least two distinct pathways with high and low affinities to accumulate K+. The system for high-affinity K+ uptake, which takes place against the electrochemical K+ gradient, requires direct energization. Energization of K+ uptake via Na+ coupling has been observed in algae and was recently proposed as a mechanism for K+ uptake in wheat (Triticum aestivum L.). To investigate whether Na+ coupling has general physiological relevance in energizing K+ transport, we screened a number of species, including Arabidopsis thaliana L. Heynh. ecotype Columbia, wheat, and barley (Hordeum vulgare L.), for the presence of Na+-coupled K+ uptake. Rb+-flux analysis and electrophysiological K+-transport assays were performed in the presence and absence of Na+ and provided evidence for a coupling between K+ and Na+ transport in several aquatic species. However, all investigated terrestrial species were able to sustain growth and K+ uptake in the absence of Na+. Furthermore, the addition of Na+ was either without effect or inhibited K+ absorption. The latter characteristic was independent of growth conditions with respect to Na+ status and pH. Our results suggest that in terrestrial species Na+-coupled K+ transport has no or limited physiological relevance, whereas in certain aquatic angiosperms and algae this type of secondary transport energization plays a significant role. PMID:12226467

  6. Regulators of Slc4 bicarbonate transporter activity

    PubMed Central

    Thornell, Ian M.; Bevensee, Mark O.

    2015-01-01

    The Slc4 family of transporters is comprised of anion exchangers (AE1-4), Na+-coupled bicarbonate transporters (NCBTs) including electrogenic Na/bicarbonate cotransporters (NBCe1 and NBCe2), electroneutral Na/bicarbonate cotransporters (NBCn1 and NBCn2), and the electroneutral Na-driven Cl-bicarbonate exchanger (NDCBE), as well as a borate transporter (BTR1). These transporters regulate intracellular pH (pHi) and contribute to steady-state pHi, but are also involved in other physiological processes including CO2 carriage by red blood cells and solute secretion/reabsorption across epithelia. Acid-base transporters function as either acid extruders or acid loaders, with the Slc4 proteins moving HCO−3 either into or out of cells. According to results from both molecular and functional studies, multiple Slc4 proteins and/or associated splice variants with similar expected effects on pHi are often found in the same tissue or cell. Such apparent redundancy is likely to be physiologically important. In addition to regulating pHi, a HCO−3 transporter contributes to a cell's ability to fine tune the intracellular regulation of the cotransported/exchanged ion(s) (e.g., Na+ or Cl−). In addition, functionally similar transporters or splice variants with different regulatory profiles will optimize pH physiology and solute transport under various conditions or within subcellular domains. Such optimization will depend on activated signaling pathways and transporter expression profiles. In this review, we will summarize and discuss both well-known and more recently identified regulators of the Slc4 proteins. Some of these regulators include traditional second messengers, lipids, binding proteins, autoregulatory domains, and less conventional regulators. The material presented will provide insight into the diversity and physiological significance of multiple members within the Slc4 gene family. PMID:26124722

  7. Transport to Rhebpress activity.

    PubMed

    Garrido, Amanda; Brandt, Marta; Djouder, Nabil

    2016-01-01

    The small GTPases from the rat sarcoma (Ras) superfamily are a heterogeneous group of proteins of about 21 kDa that act as molecular switches, modulating cell signaling pathways and controlling diverse cellular processes. They are active when bound to guanosine triphosphate (GTP) and inactive when bound to guanosine diphosphate (GDP). Ras homolog enriched in brain (Rheb) is a member of the Ras GTPase superfamily and a key activator of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1). We recently determined that microspherule protein 1 (MCRS1) maintains Rheb at lysosomal surfaces in an amino acid-dependent manner. MCRS1 depletion promotes the formation of the GDP-bound form of Rheb, which is then delocalized from the lysosomal platform and transported to endocytic recycling vesicles, leading to mTORC1 inactivation. During this delocalization process, Rheb-GDP remains farnesylated and associated with cellular endomembranes. These findings provide new insights into the regulation of small GTPases, whose activity depends on both their GTP/GDP switch state and their capacity to move between different cellular membrane-bound compartments. Dynamic spatial transport between compartments makes it possible to alter the proximity of small GTPases to their activatory sites depending on the prevailing physiological and cellular conditions.

  8. Osmolyte and Na+ transport balances of rat hepatocytes as a function of hypertonic stress.

    PubMed

    Wehner, F; Tinel, H

    2000-11-01

    The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an influx of Na+ that is then exchanged for K+ via stimulation of Na+/K+-adenosine triphosphatase (ATPase). In this study, we analysed the activation pattern of the Na+ transporters underlying RVI as a function of the degree of hypertonic stress. In confluent primary cultures, four hypertonic conditions were tested (changes from 300 to 327, 360, 400 or 450 mosmol/l) and the activities of Na+ conductance, Na+/H+ antiport, Na+-K+-2Cl- symport and Na+/K+-ATPase were quantified using intracellular microelectrodes, microfluorometry and time-dependent, furosemide- or ouabain-sensitive 86Rb+ uptake, respectively. Neither Na+ conductance nor Na+-K+-2Cl- symport responded to 327 mosmol/A. At 360, 400 and 450 mosmol/l, uptake via these transporters would lead to increases of cell Na+ by 33.0, 49.0 and 49.0 and by 4.5, 10.4 and 9.2 mmol/l per 10 min, respectively. In contrast, Na+/H+ antiport exhibited 65% of its maximal activation already at 327 mosmol/l. At the four osmolarities tested, this transporter would augment cell Na+ by 6.9, 8.9, 9.8 and 10.6 mmol/l per 10 min. The sums of Na+ import were consistent with the amounts of Na+ exported via Na+/K+-ATPase plus the actual increases of cell Na+ (21.2, 58.5, 63.6 and 68.3 mmol/l per 10 min and 2.2, 4.0, 6.3 and 8.2 mmol/l, respectively). In addition, these elevations of cell Na+ plus the increases of cell K+ (via Na+/K+-ATPase) that amounted to 5.0, 6.5, 17.5 and 18.4 mmol/l were consistent with the increases of intracellular osmotic (cationic) activity of 2.5, 11.5, 21.0 and 28.5 mmol/l, respectively, computed from RVI data. It is concluded that the principle of rat hepatocyte RVI, i.e. an initial uptake of Na+ that is then exchanged for K+ via Na+/K+-ATPase, is realized over the entire range of 9-50% hypertonicity tested. The set-point for the activation of RVI clearly lies below 327 mosmol/l. Na+/H+ antiport is the most sensitive Na+ importer

  9. Expression of apical Na(+)-L-glutamine co-transport activity, B(0)-system neutral amino acid co-transporter (B(0)AT1) and angiotensin-converting enzyme 2 along the jejunal crypt-villus axis in young pigs fed a liquid formula

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gut apical amino acid (AA) transport activity is high at birth and during suckling, thus being essential to maintain luminal nutrient-dependent mucosal growth through providing AA as essential metabolic fuel, substrates and nutrient stimuli for cellular growth. Because system-B(0) Na(+)-neutral AA c...

  10. The gasotransmitter hydrogen sulphide decreases Na+ transport across pulmonary epithelial cells

    PubMed Central

    Althaus, M; Urness, KD; Clauss, WG; Baines, DL; Fronius, M

    2012-01-01

    BACKGROUND AND PURPOSE The transepithelial absorption of Na+ in the lungs is crucial for the maintenance of the volume and composition of epithelial lining fluid. The regulation of Na+ transport is essential, because hypo- or hyperabsorption of Na+ is associated with lung diseases such as pulmonary oedema or cystic fibrosis. This study investigated the effects of the gaseous signalling molecule hydrogen sulphide (H2S) on Na+ absorption across pulmonary epithelial cells. EXPERIMENTAL APPROACH Ion transport processes were electrophysiologically assessed in Ussing chambers on H441 cells grown on permeable supports at air/liquid interface and on native tracheal preparations of pigs and mice. The effects of H2S were further investigated on Na+ channels expressed in Xenopus oocytes and Na+/K+-ATPase activity in vitro. Membrane abundance of Na+/K+-ATPase was determined by surface biotinylation and Western blot. Cellular ATP concentrations were measured colorimetrically, and cytosolic Ca2+ concentrations were measured with Fura-2. KEY RESULTS H2S rapidly and reversibly inhibited Na+ transport in all the models employed. H2S had no effect on Na+ channels, whereas it decreased Na+/K+-ATPase currents. H2S did not affect the membrane abundance of Na+/K+-ATPase, its metabolic or calcium-dependent regulation, or its direct activity. However, H2S inhibited basolateral calcium-dependent K+ channels, which consequently decreased Na+ absorption by H441 monolayers. CONCLUSIONS AND IMPLICATIONS H2S impairs pulmonary transepithelial Na+ absorption, mainly by inhibiting basolateral Ca2+-dependent K+ channels. These data suggest that the H2S signalling system might represent a novel pharmacological target for modifying pulmonary transepithelial Na+ transport. PMID:22352810

  11. Na+-dependent and Na+-independent betaine transport across the apical membrane of rat renal epithelium.

    PubMed

    Cano, Mercedes; Calonge, María L; Ilundáin, Anunciación A

    2015-10-01

    The low renal excretion of betaine indicates that the kidney efficiently reabsorbs the betaine filtered by the glomeruli but the mechanisms involved in such a process have been scarcely investigated. We have detected concentrative and non-concentrative betaine transport activity in brush-border membrane vesicles (BBMV) from rat renal cortex and medulla. The concentrative system is the Sodium/Imino-acid Transporter 1 (SIT1) because it is Na+- and Cl--dependent, electrogenic and is inhibited by an anti-SIT1 antibody. Its apparent affinity constant for betaine, Kt, is 1.1±0.5 mM and its maximal transport velocity, Vmax, 0.5±0.1 nmol betaine/mg protein/s. Inhibitors of the Na+/Cl-/betaine uptake are L-proline (75%) and cold betaine, L-carnitine and choline (40-60%). Neither creatine, TEA, taurine, β-alanine, GABA nor glycine significantly inhibited Na+/Cl-/betaine uptake. The non-concentrative betaine transport system is Na+- and H+-independent, electroneutral, with a Kt for betaine of 47±7 μM and a Vmax of 7.8±1 pmol betaine/mg protein/s. Its transport activity is nearly abolished by betaine, followed by L-carnitine (70-80%) and proline (40-50%), but a difference from the Na+/Cl-/betaine transport is that it is inhibited by TEA (approx. 50%) and unaffected by choline. The underlying carrier functions as an antiporter linking betaine entry into the BBMV with the efflux of either L-carnitine or betaine, an exchange unaffected by the anti-SIT1 antibody. As far as we know this is the first work reporting that betaine crosses the apical membrane of rat renal epithelium by SIT1 and by a Na+- and H+-independent transport system.

  12. INTEX-NA: Intercontinental Chemical Transport Experiment - North America

    NASA Technical Reports Server (NTRS)

    Singh, Hanwant B.; Jacob, D.; Pfister, L.; Hipskind, R. Stephen (Technical Monitor)

    2002-01-01

    INTEX-NA is an integrated atmospheric chemistry field experiment to be performed over North America using the NASA DC-8 and P-3B aircraft as its primary platforms. It seeks to understand the exchange of chemicals and aerosols between continents and the global troposphere. The constituents of interest are ozone and its precursors (hydrocarbons, NOX and HOX), aerosols, and the major greenhouse gases (CO2, CH4, N2O). INTEX-NA will provide the observational database needed to quantify inflow, outflow, and transformations of chemicals over North America. INTEX-NA is to be performed in two phases. Phase A will take place during the period of May-August 2004 and Phase B during March-June 2006. Phase A is in summer when photochemistry is most intense and climatic issues involving aerosols and carbon cycle are most pressing, and Phase B is in spring when Asian transport to North America is at its peak. INTEX-NA will coordinate its activities with concurrent measurement programs including satellites (e. g. Terra, Aura, Envisat), field activities undertaken by the North American Carbon Program (NACP), and other U.S. and international partners. However, it is being designed as a 'stand alone' mission such that its successful execution is not contingent on other programs. Synthesis of the ensemble of observation from surface, airborne, and space platforms, with the help of global/regional models is an important It is anticipated that approximately 175 flight hours for each of the aircraft (DC-8 and P-3B) will be required for each Phase. Principal operational sites are tentatively selected to be Bangor, ME; Wallops Island, VA; Seattle, WA; Rhinelander, WI; Lancaster, CA; and New Orleans, LA. These coastal and continental sites can support large missions and are suitable for INTEX-NA objectives. The experiment will be supported by forecasts from meteorological and chemical models, satellite observations, surface networks, and enhanced O3,-sonde releases. In addition to

  13. Astrocytic GABA transporter activity modulates excitatory neurotransmission

    PubMed Central

    Boddum, Kim; Jensen, Thomas P.; Magloire, Vincent; Kristiansen, Uffe; Rusakov, Dmitri A.; Pavlov, Ivan; Walker, Matthew C.

    2016-01-01

    Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus. PMID:27886179

  14. Expression of apical Na(+)-L-glutamine co-transport activity, B(0)-system neutral amino acid co-transporter (B(0)AT1) and angiotensin-converting enzyme 2 along the jejunal crypt-villus axis in young pigs fed a liquid formula.

    PubMed

    Yang, Chengbo; Yang, Xiaojian; Lackeyram, Dale; Rideout, Todd C; Wang, Zirong; Stoll, Barbara; Yin, Yulong; Burrin, Douglas G; Fan, Ming Z

    2016-06-01

    Gut apical amino acid (AA) transport activity is high at birth and during suckling, thus being essential to maintain luminal nutrient-dependent mucosal growth through providing AA as essential metabolic fuel, substrates and nutrient stimuli for cellular growth. Because system-B(0) Na(+)-neutral AA co-transporter (B(0)AT1, encoded by the SLC6A19 gene) plays a dominant role for apical uptake of large neutral AA including L-Gln, we hypothesized that high apical Na(+)-Gln co-transport activity, and B(0)AT1 (SLC6A19) in co-expression with angiotensin-converting enzyme 2 (ACE2) were expressed along the entire small intestinal crypt-villus axis in young animals via unique control mechanisms. Kinetics of Na(+)-Gln co-transport activity in the apical membrane vesicles, prepared from epithelial cells sequentially isolated along the jejunal crypt-villus axis from liquid formula-fed young pigs, were measured with the membrane potential being clamped to zero using thiocyanate. Apical maximal Na(+)-Gln co-transport activity was much higher (p < 0.05) in the upper villus cells than in the middle villus (by 29 %) and the crypt (by 30 %) cells, whereas Na(+)-Gln co-transport affinity was lower (p < 0.05) in the upper villus cells than in the middle villus and the crypt cells. The B(0)AT1 (SLC6A19) mRNA abundance was lower (p < 0.05) in the crypt (by 40-47 %) than in the villus cells. There were no significant differences in B(0)AT1 and ACE2 protein abundances on the apical membrane among the upper villus, the middle villus and the crypt cells. Our study suggests that piglet fast growth is associated with very high intestinal apical Na(+)-neutral AA uptake activities via abundantly co-expressing B(0)AT1 and ACE2 proteins in the apical membrane and by transcribing the B(0)AT1 (SLC6A19) gene in the epithelia along the entire crypt-villus axis.

  15. Insulin and phorbol ester stimulate conductive Na/sup +/ transport through a common pathway

    SciTech Connect

    Civan, M.M.; Peterson-Yantorno, K.; O'Brien, T.G.

    1988-02-01

    Insulin stimulates Na/sup +/ transport across frog skin, toad urinary bladder, and the distal renal nephron. This stimulation reflects an increase in apical membrane Na/sup +/ permeability and a stimulation of the basolateral membrane Na,K-exchange pump. Considerable indirect evidence has suggested that the apical natriferic effect of insulin is mediated by activation of protein kinase C. However, no direct information has been available documenting that insulin and protein kinase C indeed share a common pathway in stimulating Na/sup +/ transport across frog skin. In the present work, the authors have studied the interaction of insulin and phorbol 12-myristate 13-acetate (PMA), a documented activator of protein kinase C. Preincubation of skins with 1,2-dioctanoylglycerol, another activator of protein kinase C, increases baseline Na/sup +/ transport and reduces the subsequent natriferic response to PMA. Preincubation with PMA markedly reduces the subsequent natriferic action of insulin. This effect does not appear to primarily reflect PMA-induced internalization of insulin receptors. The insulin receptors are localized on the basolateral surface of frog skin, but the application of PMA to this surface is much less effective than mucosal treatment in reducing the response to insulin. The current results provide documentation that insulin and protein kinase C share a common pathway in stimulating Na/sup +/ transport across frog skin. The data are consistent with the concept that the natriferic effect of insulin on frog skin is, at least in part, mediated by activation of protein kinase C.

  16. A Histidine Cluster in the Cytoplasmic Domain of the Na-H Exchanger NHE1 Confers pH-sensitive Phospholipid Binding and Regulates Transporter Activity.

    PubMed

    Webb, Bradley A; White, Katharine A; Grillo-Hill, Bree K; Schönichen, André; Choi, Changhoon; Barber, Diane L

    2016-11-11

    The Na-H exchanger NHE1 contributes to intracellular pH (pHi) homeostasis in normal cells and the constitutively increased pHi in cancer. NHE1 activity is allosterically regulated by intracellular protons, with greater activity at lower pHi However, the molecular mechanism for pH-dependent NHE1 activity remains incompletely resolved. We report that an evolutionarily conserved cluster of histidine residues located in the C-terminal cytoplasmic domain between two phosphatidylinositol 4,5-bisphosphate binding sites (PI(4,5)P2) of NHE1 confers pH-dependent PI(4,5)P2 binding and regulates NHE1 activity. A GST fusion of the wild type C-terminal cytoplasmic domain of NHE1 showed increased maximum PI(4,5)P2 binding at pH 7.0 compared with pH 7.5. However, pH-sensitive binding is abolished by substitutions of the His-rich cluster to arginine (RXXR3) or alanine (AXXA3), mimicking protonated and neutral histidine residues, respectively, and the RXXR3 mutant had significantly greater PI(4,5)P2 binding than AXXA3. When expressed in cells, NHE1 activity and pHi were significantly increased with NHE1-RXXR3 and decreased with NHE1-AXXA3 compared with wild type NHE1. Additionally, fibroblasts expressing NHE1-RXXR3 had significantly more contractile actin filaments and focal adhesions compared with fibroblasts expressing wild type NHE1, consistent with increased pHi enabling cytoskeletal remodeling. These data identify a molecular mechanism for pH-sensitive PI(4,5)P2 binding regulating NHE1 activity and suggest that the evolutionarily conserved cluster of four histidines in the proximal cytoplasmic domain of NHE1 may constitute a proton modifier site. Moreover, a constitutively activated NHE1-RXXR3 mutant is a new tool that will be useful for studying how increased pHi contributes to cell behaviors, most notably the biology of cancer cells.

  17. Molecular motions involved in Na-K-Cl cotransporter-mediated ion transport and transporter activation revealed by internal cross-linking between transmembrane domains 10 and 11/12.

    PubMed

    Monette, Michelle Y; Somasekharan, Suma; Forbush, Biff

    2014-03-14

    We examined the relationship between transmembrane domain (TM) 10 and TM11/12 in NKCC1, testing homology models based on the structure of AdiC in the same transporter superfamily. We hypothesized that introduced cysteine pairs would be close enough for disulfide formation and would alter transport function: indeed, evidence for cross-link formation with low micromolar concentrations of copper phenanthroline or iodine was found in 3 of 8 initially tested pairs and in 1 of 26 additionally tested pairs. Inhibition of transport was observed with copper phenanthroline and iodine treatment of P676C/A734C and I677C/A734C, consistent with the proximity of these residues and with movement of TM10 during the occlusion step of ion transport. We also found Cu(2+) inhibition of the single-cysteine mutant A675C, suggesting that this residue and Met(382) of TM3 are involved in a Cu(2+)-binding site. Surprisingly, cross-linking of P676C/I730C was found to prevent rapid deactivation of the transporter while not affecting the dephosphorylation rate, thus uncoupling the phosphorylation and activation steps. Consistent with this, (a) cross-linking of P676C/I730C was dependent on activation state, and (b) mutants lacking the phosphoregulatory domain could still be activated by cross-linking. These results suggest a model of NKCC activation that involves movement of TM12 relative to TM10, which is likely tied to movement of the large C terminus, a process somehow triggered by phosphorylation of the regulatory domain in the N terminus.

  18. Ameloblast Modulation and Transport of Cl−, Na+, and K+ during Amelogenesis

    PubMed Central

    Bronckers, A.L.J.J.; Lyaruu, D.; Jalali, R.; Medina, J.F.; Zandieh-Doulabi, B.; DenBesten, P.K.

    2015-01-01

    Ameloblasts express transmembrane proteins for transport of mineral ions and regulation of pH in the enamel space. Two major transporters recently identified in ameloblasts are the Na+K+-dependent calcium transporter NCKX4 and the Na+-dependent HPO42– (Pi) cotransporter NaPi-2b. To regulate pH, ameloblasts express anion exchanger 2 (Ae2a,b), chloride channel Cftr, and amelogenins that can bind protons. Exposure to fluoride or null mutation of Cftr, Ae2a,b, or Amelx each results in formation of hypomineralized enamel. We hypothesized that enamel hypomineralization associated with disturbed pH regulation results from reduced ion transport by NCKX4 and NaPi-2b. This was tested by correlation analyses among the levels of Ca, Pi, Cl, Na, and K in forming enamel of mice with null mutation of Cftr, Ae2a,b, and Amelx, according to quantitative x-ray electron probe microanalysis. Immunohistochemistry, polymerase chain reaction analysis, and Western blotting confirmed the presence of apical NaPi-2b and Nckx4 in maturation-stage ameloblasts. In wild-type mice, K levels in enamel were negatively correlated with Ca and Cl but less negatively or even positively in fluorotic enamel. Na did not correlate with P or Ca in enamel of wild-type mice but showed strong positive correlation in fluorotic and nonfluorotic Ae2a,b- and Cftr-null enamel. In hypomineralizing enamel of all models tested, 1) Cl− was strongly reduced; 2) K+ and Na+ accumulated (Na+ not in Amelx-null enamel); and 3) modulation was delayed or blocked. These results suggest that a Na+K+-dependent calcium transporter (likely NCKX4) and a Na+-dependent Pi transporter (potentially NaPi-2b) located in ruffle-ended ameloblasts operate in a coordinated way with the pH-regulating machinery to transport Ca2+, Pi, and bicarbonate into maturation-stage enamel. Acidification and/or associated physicochemical/electrochemical changes in ion levels in enamel fluid near the apical ameloblast membrane may reduce the transport

  19. Energetics of active transport processes.

    PubMed

    Essig, A; Caplan, S R

    1968-12-01

    Discussions of active transport usually assume stoichiometry between the rate of transport J(+) and the metabolic rate J(r). However, the observation of a linear relationship between J(+) and J(r) does not imply a stoichiometric relationship, i.e., complete coupling. Since coupling may possibly be incomplete, we examine systems of an arbitrary degree of coupling q, regarding stoichiometry as a limiting case. We consider a sodium pump, with J(+) and J(r) linear functions of the electrochemical potential difference, -X(+), and the chemical affinity of the metabolic driving reaction, A. The affinity is well defined even for various complex reaction pathways. Incorporation of a series barrier and a parallel leak does not affect the linearity of the composite observable system. The affinity of some region of the metabolic chain may be maintained constant, either by large pools of reactants or by regulation. If so, this affinity can be evaluated by two independent methods. Sodium transport is conveniently characterized by the open-circuit potential (Deltapsi)(I=0) and the natural limits, level flow (J(+))(X+=0), and static head X(0) (+) = (X(+))(J+=0). With high degrees of coupling -X(0) (+)/F approaches the electromotive force E(Na) (Ussing); -X(0) (+)/F cannot be identified with ((RT/F) ln f)(X+=0), where f is the flux ratio. The efficiency eta = -J(+)X(+)/J(r)A is of significance only when appreciable energy is being converted from one form to another. When either J(+) or -X(+) is small eta is low; the significant parameters are then the efficacies epsilon(J+) = J(+)/J(r)A and epsilon(X+) = -X(+)/J(r)A, respectively maximal at level flow and static head. Leak increases both J(+) and epsilon(J+) for isotonic saline reabsorption, but diminishes -X(0) (+) and epsilon(Xfemale symbol). Electrical resistance reflects both passive parameters and metabolism. Various fundamental relations are preserved despite coupling of passive ion and water flows.

  20. Laboratory Exercise on Active Transport.

    ERIC Educational Resources Information Center

    Stalheim-Smith, Ann; Fitch, Greg K.

    1985-01-01

    Describes a laboratory exercise which demonstrates qualitatively the specificity of the transport mechanism, including a consideration of the competitive inhibition, and the role of adenosine triphosphate (ATP) in active transport. The exercise, which can be completed in two to three hours by groups of four students, consistently produces reliable…

  1. Activated transport in AMTEC electrodes

    NASA Technical Reports Server (NTRS)

    Williams, R. M.; Jeffries-Nakamura, B.; Ryan, M. A.; Underwood, M. L.; O'Connor, D.; Kikkert, S.

    1992-01-01

    Transport of alkali, metal atoms through porous cathodes of alkali metal thermal-to-electric converter (AMTEC) cells is responsible for significant reducible losses in the electrical performance of these cells. Experimental evidence for activated transport of metal atoms at grain surfaces and boundaries within some AMTEC electrodes has been derived from temperature dependent studies as well as from analysis of the detailed frequency dependence of ac impedance results for other electrodes, including thin, mature molybdenum electrodes which exhibit transport dominated by free molecular flow of sodium gas at low frequencies or dc conditions. Activated surface transport will almost always exist in parallel with free molecular flow transport, and the process of alkali atom adsorption/desorption from the electrode surface will invariably be part of the transport process, and possibly a dominant part in some cases. The temperature dependence of the diffusion coefficient of the alkali metal through the electrode in several cases provides an activation energy and preexponential, but at least two activated processes may be operative, and the activation parameters should be expected to depend on the alkali metal activity gradient that the electrode experiences. In the case of Pt/W/Mn electrodes operated for 2500 hours, limiting currents varied with electrode thickness, and the activation parameters could be assigned primarily to the surface/grain boundary diffusion process.

  2. Effects of purinergic stimulation, CFTR and osmotic stress on amiloride-sensitive Na+ transport in epithelia and Xenopus oocytes.

    PubMed

    Schreiber, R; König, J; Sun, J; Markovich, D; Kunzelmann, K

    2003-03-15

    Both stimulation of purinergic receptors by ATP and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibit amiloride-sensitive Na+ transport and activate Cl- secretion. These changes in ion transport may well affect cell volume. We therefore examined whether cell shrinkage or cell swelling do affect amiloride-sensitive Na+ transport in epithelial tissues or Xenopus oocytes and whether osmotic stress interferes with regulation of Na+ transport by ATP or CFTR. Stimulation of purinergic receptors by ATP/UTP or activation of CFTR by IBMX and forskolin inhibited amiloride-sensitive transport in mouse trachea and colon, respectively, by a mechanism that was Cl- dependent. When exposed to a hypertonic but not hypotonic bath solution, amiloride-sensitive Na+ transport was inhibited in mouse trachea and colon, independent of the extracellular Cl- concentration. Both inhibition of Na+ transport by hypertonic bath solution and ATP were additive. When coexpressed in Xenopus oocytes, activation of CFTR by IBMX and forskolin inhibited the epithelial Na+ channel (ENaC) in a Cl- dependent fashion. However, both hypertonic and hypotonic bath solutions showed only minor effects on amiloride-sensitive conductance, independent of the bath Cl- concentration. Moreover, CFTR-induced inhibition of ENaC could be detected in oocytes even after exposure to hypertonic or hypotonic bath solutions. We conclude that amiloride-sensitive Na+ absorption in mouse airways and colon is inhibited by cell shrinkage by a mechanism that does not interfere with purinergic and CFTR-mediated inhibition of ENaC.

  3. A general method for determining secondary active transporter substrate stoichiometry.

    PubMed

    Fitzgerald, Gabriel A; Mulligan, Christopher; Mindell, Joseph A

    2017-01-25

    The number of ions required to drive substrate transport through a secondary active transporter determines the protein's ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter's mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na(+)-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures.

  4. Quantification of Na+,K+ pumps and their transport rate in skeletal muscle: functional significance.

    PubMed

    Clausen, Torben

    2013-10-01

    During excitation, muscle cells gain Na(+) and lose K(+), leading to a rise in extracellular K(+) ([K(+)]o), depolarization, and loss of excitability. Recent studies support the idea that these events are important causes of muscle fatigue and that full use of the Na(+),K(+)-ATPase (also known as the Na(+),K(+) pump) is often essential for adequate clearance of extracellular K(+). As a result of their electrogenic action, Na(+),K(+) pumps also help reverse depolarization arising during excitation, hyperkalemia, and anoxia, or from cell damage resulting from exercise, rhabdomyolysis, or muscle diseases. The ability to evaluate Na(+),K(+)-pump function and the capacity of the Na(+),K(+) pumps to fill these needs require quantification of the total content of Na(+),K(+) pumps in skeletal muscle. Inhibition of Na(+),K(+)-pump activity, or a decrease in their content, reduces muscle contractility. Conversely, stimulation of the Na(+),K(+)-pump transport rate or increasing the content of Na(+),K(+) pumps enhances muscle excitability and contractility. Measurements of [(3)H]ouabain binding to skeletal muscle in vivo or in vitro have enabled the reproducible quantification of the total content of Na(+),K(+) pumps in molar units in various animal species, and in both healthy people and individuals with various diseases. In contrast, measurements of 3-O-methylfluorescein phosphatase activity associated with the Na(+),K(+)-ATPase may show inconsistent results. Measurements of Na(+) and K(+) fluxes in intact isolated muscles show that, after Na(+) loading or intense excitation, all the Na(+),K(+) pumps are functional, allowing calculation of the maximum Na(+),K(+)-pumping capacity, expressed in molar units/g muscle/min. The activity and content of Na(+),K(+) pumps are regulated by exercise, inactivity, K(+) deficiency, fasting, age, and several hormones and pharmaceuticals. Studies on the α-subunit isoforms of the Na(+),K(+)-ATPase have detected a relative increase in their

  5. Activated transport in AMTEC electrodes

    SciTech Connect

    Williams, R.M.; Jeffries-Nakamura, B.; Ryan, M.A.; Underwood, M.L.; O`Connor, D.; Kikkert, S.

    1992-07-01

    Transport of alkali metal atoms through porous cathodes of alkali metal thermal-to-electric converter (AMTEC) cells is responsible for significant, reducible losses in the electrical performance of these cells. Experimental evidence for activated transport of metal atoms at grain surfaces and boundaries within some AMTEC electrodes has been derived from temperature dependent studies as well as from analysis of the detailed frequency dependence of ac impedance results for other electrodes, including thin, mature molybdenum electrodes which exhibit transport dominated by free molecular flow of sodium gas at low frequencies or dc conditions. Activated surface transport will almost always exist in parallel with free molecular flow transport, and the process of alkali atom adsorption/desorption from the electrode surface will invariably be part of the transport process, and possibly a dominant part in some cases. Little can be learned about the detailed mass transport process from the ac impedance or current voltage curves of an electrode at one set of operating parameters, because the transport process includes a number of important physical parameters that are not all uniquely determined by one experiment. The temperature dependence of diffusion coefficient of the alkali metal through the electrode in several cases provides an activation energy and pre-exponential, but at least two activated processes may be operative, and the activation parameters should be expected to depend on the alkali metal activity gradient that the electrode experiences. In the case of Pt/W/Mn electrodes operated for 2500 hours, limiting currents varied with electrode thickness, and the activation parameters could be assigned primarily to the surface/grain boundary diffusion process. 17 refs.

  6. Activated transport in AMTEC electrodes

    SciTech Connect

    Williams, R.M.; Jeffries-Nakamura, B.; Ryan, M.A.; Underwood, M.L.; O'Connor, D.; Kikkert, S.

    1992-01-01

    Transport of alkali metal atoms through porous cathodes of alkali metal thermal-to-electric converter (AMTEC) cells is responsible for significant, reducible losses in the electrical performance of these cells. Experimental evidence for activated transport of metal atoms at grain surfaces and boundaries within some AMTEC electrodes has been derived from temperature dependent studies as well as from analysis of the detailed frequency dependence of ac impedance results for other electrodes, including thin, mature molybdenum electrodes which exhibit transport dominated by free molecular flow of sodium gas at low frequencies or dc conditions. Activated surface transport will almost always exist in parallel with free molecular flow transport, and the process of alkali atom adsorption/desorption from the electrode surface will invariably be part of the transport process, and possibly a dominant part in some cases. Little can be learned about the detailed mass transport process from the ac impedance or current voltage curves of an electrode at one set of operating parameters, because the transport process includes a number of important physical parameters that are not all uniquely determined by one experiment. The temperature dependence of diffusion coefficient of the alkali metal through the electrode in several cases provides an activation energy and pre-exponential, but at least two activated processes may be operative, and the activation parameters should be expected to depend on the alkali metal activity gradient that the electrode experiences. In the case of Pt/W/Mn electrodes operated for 2500 hours, limiting currents varied with electrode thickness, and the activation parameters could be assigned primarily to the surface/grain boundary diffusion process. 17 refs.

  7. Intracellular [Na+], Na+ pathways, and fluid transport in cultured bovine corneal endothelial cells.

    PubMed

    Kuang, Kunyan; Li, Yansui; Yiming, Maimaiti; Sánchez, José M; Iserovich, Pavel; Cragoe, E J; Diecke, Friedrich P J; Fischbarg, Jorge

    2004-07-01

    The mechanism of fluid transport across corneal endothelium remains unclear. We examine here the relative contributions of cellular mechanisms of Na+ transport and the homeostasis of intracellular [Na+] in cultured bovine corneal endothelial cells, and the influence of ambient Na+ and HCO3- on the deturgescence of rabbit cornea. Bovine corneal endothelial cells plated on glass coverslips were incubated for 60 min with 10 microm of the fluorescent Na+ indicator SBFI precursor in HCO3- HEPES (BH) Ringer's solution. After loading, cells were placed in a perfusion chamber. Indicator fluorescence (490 nm) was determined with a Chance-Legallais time-sharing fluorometer. Its voltage output was the ratio of the emissions excited at 340 and 380 nm. For calibration, cells were treated with gramicidin D. For fluid transport measurements, rabbit corneas were mounted in a Dikstein-Maurice chamber, and stromal thickness was measured with a specular microscope. The steady-state [Na+]i in BH was 14.36+/-0.38 mM (n = mean+/-s.e.). Upon exposure to Na+ -free BH solution (choline substituted), [Na+]i decreased to 1.81+/-0.20mM (n = 19). When going from Na+ -free plus 100 microm ouabain to BH plus ouabain, [Na+]i increased to 46.17+/-2.50 (n = 6) with a half time of 1.26+/-0.04 min; if 0.1 microm phenamil plus ouabain were present, it reached only 21.78+/-1.50mm. The exponential time constants (min-1) were: 0.56+/-0.04 for the Na+ pump; 0.39+/-0.01 for the phenamil sensitive Na+ channel; and 0.17+/-0.02 for the ouabain-phenamil-insensitive pathways. In HCO3- free medium (gluconate substituted), [Na+]i was 14.03+/-0.11mM; upon changing to BH medium, it increased to 30.77+/-0.74 mm. This last [Na+]i increase was inhibited 66% by 100 microm DIDS. Using BH medium, corneal thickness remained nearly constant, increasing at a rate of only 2.9+/-0.9 microm hr-1 during 3 hr. However, stromal thickness increased drastically (swelling rate 36.1+/-2.6 microm hr-1) in corneas superfused with BH

  8. Sodium ion transport mechanisms in antiperovskite electrolytes Na3OBr and Na4OI2: An in Situ neutron diffraction study

    DOE PAGES

    Zhu, Jinlong; Wang, Yonggang; Li, Shuai; ...

    2016-06-02

    Na-rich antiperovskites are recently developed solid electrolytes with enhanced sodium ionic conductivity and show promising functionality as a novel solid electrolyte in an all solid-stat battery. In this work, the sodium ionic transport pathways of the parent compound Na3OBr, as well as the modified layered antiperovskite Na4OI2, were studied and compared through temperature dependent neutron diffraction combined with the maximum entropy method. In the cubic Na3OBr antiperovskite, the nuclear density distribution maps at 500 K indicate that sodium ions ho within and among oxygen octahedra, and Br- ions are not involved in the tetragonal Na4OI2 antiperovskite, Na ions, which connectmore » octahedra in the ab plane, have the lowest activation energy barrier. In conclusion, the transport of sodium ions along the c axis is assisted by I- ions.« less

  9. Sodium Ion Transport Mechanisms in Antiperovskite Electrolytes Na3OBr and Na4OI2: An in Situ Neutron Diffraction Study.

    PubMed

    Zhu, Jinlong; Wang, Yonggang; Li, Shuai; Howard, John W; Neuefeind, Jörg; Ren, Yang; Wang, Hui; Liang, Chengdu; Yang, Wenge; Zou, Ruqiang; Jin, Changqing; Zhao, Yusheng

    2016-06-20

    Na-rich antiperovskites are recently developed solid electrolytes with enhanced sodium ionic conductivity and show promising functionality as a novel solid electrolyte in an all solid-state battery. In this work, the sodium ionic transport pathways of the parent compound Na3OBr, as well as the modified layered antiperovskite Na4OI2, were studied and compared through temperature-dependent neutron diffraction combined with the maximum entropy method. In the cubic Na3OBr antiperovskite, the nuclear density distribution maps at 500 K indicate that sodium ions hop within and among oxygen octahedra, and Br(-) ions are not involved. In the tetragonal Na4OI2 antiperovskite, Na ions, which connect octahedra in the ab plane, have the lowest activation energy barrier. The transport of sodium ions along the c axis is assisted by I(-) ions.

  10. Solute transport and oxygen consumption along the nephrons: effects of Na+ transport inhibitors.

    PubMed

    Layton, Anita T; Laghmani, Kamel; Vallon, Volker; Edwards, Aurélie

    2016-12-01

    Sodium and its associated anions are the major determinant of extracellular fluid volume, and the reabsorption of Na(+) by the kidney plays a crucial role in long-term blood pressure control. The goal of this study was to investigate the extent to which inhibitors of transepithelial Na(+) transport (TNa) along the nephron alter urinary solute excretion and TNa efficiency and how those effects may vary along different nephron segments. To accomplish that goal, we used the multinephron model developed in the companion study (28). That model represents detailed transcellular and paracellular transport processes along the nephrons of a rat kidney. We simulated the inhibition of the Na(+)/H(+) exchanger (NHE3), the bumetanide-sensitive Na(+)-K(+)-2Cl(-) transporter (NKCC2), the Na(+)-Cl(-) cotransporter (NCC), and the amiloride-sensitive Na(+) channel (ENaC). Under baseline conditions, NHE3, NKCC2, NCC, and ENaC reabsorb 36, 22, 4, and 7%, respectively, of filtered Na(+) The model predicted that inhibition of NHE3 substantially reduced proximal tubule TNa and oxygen consumption (QO2 ). Whole-kidney TNa efficiency, as reflected by the number of moles of Na(+) reabsorbed per moles of O2 consumed (denoted by the ratio TNa/QO2 ), decreased by ∼20% with 80% inhibition of NHE3. NKCC2 inhibition simulations predicted a substantial reduction in thick ascending limb TNa and QO2 ; however, the effect on whole-kidney TNa/QO2 was minor. Tubular K(+) transport was also substantially impaired, resulting in elevated urinary K(+) excretion. The most notable effect of NCC inhibition was to increase the excretion of Na(+), K(+), and Cl(-); its impact on whole-kidney TNa and its efficiency was minor. Inhibition of ENaC was predicted to have opposite effects on the excretion of Na(+) (increased) and K(+) (decreased) and to have only a minor impact on whole-kidney TNa and TNa/QO2 Overall, model predictions agree well with measured changes in Na(+) and K(+) excretion in response to

  11. Interaction of α-Lipoic Acid with the Human Na+/Multivitamin Transporter (hSMVT).

    PubMed

    Zehnpfennig, Britta; Wiriyasermkul, Pattama; Carlson, David A; Quick, Matthias

    2015-06-26

    The human Na(+)/multivitamin transporter (hSMVT) has been suggested to transport α-lipoic acid (LA), a potent antioxidant and anti-inflammatory agent used in therapeutic applications, e.g. in the treatment of diabetic neuropathy and Alzheimer disease. However, the molecular basis of the cellular delivery of LA and in particular the stereospecificity of the transport process are not well understood. Here, we expressed recombinant hSMVT in Pichia pastoris and used affinity chromatography to purify the detergent-solubilized protein followed by reconstitution of hSMVT in lipid bilayers. Using a combined approach encompassing radiolabeled LA transport and equilibrium binding studies in conjunction with the stabilized R-(+)- and S-(-)-enantiomers and the R,S-(+/-) racemic mixture of LA or lipoamide, we identified the biologically active form of LA, R-LA, to be the physiological substrate of hSMVT. Interaction of R-LA with hSMVT is strictly dependent on Na(+). Under equilibrium conditions, hSMVT can simultaneously bind ~2 molecules of R-LA in a biphasic binding isotherm with dissociation constants (Kd) of 0.9 and 7.4 μm. Transport of R-LA in the oocyte and reconstituted system is exclusively dependent on Na(+) and exhibits an affinity of ~3 μm. Measuring transport with known amounts of protein in proteoliposomes containing hSMVT in outside-out orientation yielded a catalytic turnover number (kcat) of about 1 s(-1), a value that is well in agreement with other Na(+)-coupled transporters. Our data suggest that hSMVT-mediated transport is highly specific for R-LA at our tested concentration range, a finding with wide ramifications for the use of LA in therapeutic applications.

  12. Sodium and proton effects on inward proton transport through Na/K pumps.

    PubMed

    Mitchell, Travis J; Zugarramurdi, Camila; Olivera, J Fernando; Gatto, Craig; Artigas, Pablo

    2014-06-17

    The Na/K pump hydrolyzes ATP to export three intracellular Na (Nai) as it imports two extracellular K (Ko) across animal plasma membranes. Within the protein, two ion-binding sites (sites I and II) can reciprocally bind Na or K, but a third site (site III) exclusively binds Na in a voltage-dependent fashion. In the absence of Nao and Ko, the pump passively imports protons, generating an inward current (IH). To elucidate the mechanisms of IH, we used voltage-clamp techniques to investigate the [H]o, [Na]o, and voltage dependence of IH in Na/K pumps from ventricular myocytes and in ouabain-resistant pumps expressed in Xenopus oocytes. Lowering pHo revealed that Ho both activates IH (in a voltage-dependent manner) and inhibits it (in a voltage-independent manner) by binding to different sites. Nao effects depend on pHo; at pHo where no Ho inhibition is observed, Nao inhibits IH at all concentrations, but when applied at pHo that inhibits pump-mediated current, low [Na]o activates IH and high [Na]o inhibits it. Our results demonstrate that IH is a property inherent to Na/K pumps, not linked to the oocyte expression environment, explains differences in the characteristics of IH previously reported in the literature, and supports a model in which 1), protons leak through site III; 2), binding of two Na or two protons to sites I and II inhibits proton transport; and 3), pumps with mixed Na/proton occupancy of sites I and II remain permeable to protons.

  13. The connexion between active cation transport and metabolism in erythrocytes

    PubMed Central

    Whittam, R.; Ager, Margaret E.

    1965-01-01

    1. A study has been made of the dependence on the concentrations of internal Na+ and external K+ of lactate and phosphate production in human erythrocytes. 2. Lactate production was stimulated by Na+ and K+ but only when they were internal and external respectively. The stimulation was counteracted by ouabain. The production of phosphate was affected in the same way. 3. There is a quantitative correlation between these effects and those previously found for cation movements and the membrane adenosine triphosphatase. 4. It is concluded that the rate of energy production in glycolysis is partly controlled by the magnitude of active transport; the extent of this regulation is shown to vary from 25 to 75% of a basal rate that is independent of active transport. 5. The activity of the membrane adenosine triphosphatase was also compared with rates of Na+ and K+ transport. The latter were varied by altering the concentrations of internal Na+ and external K+, and by inhibiting with ouabain. 6. A threefold variation of active transport rate was accompanied by a parallel change in the membrane adenosine-triphosphatase activity. The results show a constant stoicheiometry for the number of ions moved/mol. of ATP hydrolysed, independent of the electrochemical gradient against which the ions were moved. 7. Calculations show that the amount of ATP hydrolysed would provide enough energy for the osmotic work. The results are discussed in relation to possible mechanisms for active transport. PMID:16749106

  14. Activation of ion transport systems during cell volume regulation

    SciTech Connect

    Eveloff, J.L.; Warnock, D.G.

    1987-01-01

    This review discusses the activation of transport pathways during volume regulation, including their characteristics, the possible biochemical pathways that may mediate the activation of transport pathways, and the relations between volume regulation and transepithelial transport in renal cells. Many cells regulate their volume when exposed to an anisotonic medium. The changes in cell volume are caused by activation of ion transport pathways, plus the accompanying osmotically driven water movement such that cell volume returns toward normal levels. The swelling of hypertonically shrunken cells is termed regulatory volume increase (RVI) and involves an influx of NaCl into the cell via either activation of Na-Cl, Na-K-2Cl cotransport systems, or Na/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchangers. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease (RVD) and involves an efflux of KCl and water from the cell by activation of either separate K/sup +/ and Cl/sup -/ conductances, a K-Cl cotransport system, or parallel K/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchangers. The biochemical mechanisms involved in the activation of transport systems are largely unknown, however, the phosphoinositide pathway may be implicated in RVI; phorbol esters, cGMP, and Ca/sup 2 +/ affect the process of volume regulation. Renal tubular cells, as well as the blood cells that transverse the medulla, are subjected to increasing osmotic gradients from the corticomedullary junction to the papillary tip, as well as changing interstitial and tubule fluid osmolarity, depending on the diuretic state of the animal. Medullary cells from the loop of Henle and the papilla can volume regulate by activating Na-K-2Cl cotransport or Na/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchange systems.

  15. Intracellular Requirements for Passive Proton Transport through the Na(+),K(+)-ATPase.

    PubMed

    Stanley, Kevin S; Meyer, Dylan J; Gatto, Craig; Artigas, Pablo

    2016-12-06

    The Na(+),K(+)-ATPase (NKA or Na/K pump) hydrolyzes one ATP to exchange three intracellular Na+ (Na(+)i) for two extracellular K+ (K(+)o) across the plasma membrane by cycling through a set of reversible transitions between phosphorylated and dephosphorylated conformations, alternately opening ion-binding sites externally (E2) or internally (E1). With subsaturating [Na(+)]o and [K(+)]o, the phosphorylated E2P conformation passively imports protons generating an inward current (IH), which may be exacerbated in NKA-subunit mutations associated with human disease. To elucidate the mechanisms of IH, we studied the effects of intracellular ligands (transported ions, nucleotides, and beryllium fluoride) on IH and, for comparison, on transient currents measured at normal Na(+)o (QNa). Utilizing inside-out patches from Xenopus oocytes heterologously expressing NKA, we observed that 1) in the presence of Na(+)i, IH and QNa were both activated by ATP, but not ADP; 2) the [Na(+)]i dependence of IH in saturating ATP showed K0.5,Na = 1.8 ± 0.2 mM and the [ATP] dependence at saturating [Na(+)]i yielded K0.5,ATP = 48 ± 11 μM (in comparison, Na(+)i-dependent QNa yields K0.5,Na = 0.8 ± 0.2 mM and K0.5,ATP = 0.43 ± 0.03 μM; 3) ATP activated IH in the presence of K(+)i (∼15% of the IH observed in Na(+)i) only when Mg(2+)i was also present; and 4) beryllium fluoride induced maximal IH even in the absence of nucleotide. These data indicate that IH occurs when NKA is in an externally open E2P state with nucleotide bound, a conformation that can be reached through forward Na/K pump phosphorylation of E1, with Na(+)i and ATP, or by backward binding of K(+)i to E1, which drives the pump to the occluded E2(2K), where free Pi (at the micromolar levels found in millimolar ATP solutions) promotes external release of occluded K(+) by backdoor NKA phosphorylation. Maximal IH through beryllium-fluorinated NKA indicates that this complex mimics ATP-bound E2P states.

  16. Kinetic properties and Na+ dependence of rheogenic Na(+)-HCO3- co-transport in frog retinal pigment epithelium.

    PubMed Central

    la Cour, M

    1991-01-01

    1. Na(+)-HCO3- co-transport across the retinal membrane of the frog retinal pigment epithelium was studied by means of double-barrelled pH-selective microelectrodes. Transient changes in the intracellular pH were monitored in response to abrupt changes in the Na+ concentration on the retinal side of the epithelium. 2. The experiments were performed as follows. The Na(+)-HCO3- co-transport was inhibited by perfusing the retinal side of the epithelium with a Na(+)-free solution. The co-transport was then stimulated by changing the perfusate from the Na(+)-free solution to a solution which contained from 5 to 110 mM-Na+. The resulting inward Na(+)-HCO3- co-transport produced an intracellular alkalinization, the initial rate of which was used to calculate the initial rate of Na(+)-HCO3- co-transport, JHCO3-. 3. The Na+ dependence of the Na(+)-HCO3- co-transport was studied at two different values of extracellular pH (7.40 and 7.10), at constant extracellular HCO3- concentration (27.5 mM) and at two different extracellular HCO3- concentrations (27.5 mM and 55 mM) at constant extracellular pH (7.40). In these experiments, the calculated values of JHCO3- followed single Michaelis-Menten kinetics with respect to the extracellular Na+ concentration. 4. The data are consistent with a model in which the co-transporter has a single binding site for the Na+ ion with an apparent affinity constant (apparent Km) of 37 mM. The apparent affinity constant for Na+ was independent of the extracellular concentration of CO3(2-) in the range of 16-65 microM, and of the extracellular HCO3- concentration in the range 27.5-55 mM. 5. The NaCO3- ion-pair hypothesis, in which sodium binds to the co-transporter and is translocated across the cell membrane as the NaCO3- ion pair, was analysed. For stoichiometries 1:2 and 1:3 of the Na(+)-HCO3- co-transport, the NaCO3- ion-pair hypothesis was found incompatible with the data. 6. The intracellular buffer capacity as measured by the CO2 method was

  17. Brain Na+, K+-ATPase Activity In Aging and Disease

    PubMed Central

    de Lores Arnaiz, Georgina Rodríguez; Ordieres, María Graciela López

    2014-01-01

    Na+/K+ pump or sodium- and potassium-activated adenosine 5’-triphosphatase (Na+, K+-ATPase), its enzymatic version, is a crucial protein responsible for the electrochemical gradient across the cell membranes. It is an ion transporter, which in addition to exchange cations, is the ligand for cardenolides. This enzyme regulates the entry of K+ with the exit of Na+ from cells, being the responsible for Na+/K+ equilibrium maintenance through neuronal membranes. This transport system couples the hydrolysis of one molecule of ATP to exchange three sodium ions for two potassium ions, thus maintaining the normal gradient of these cations in animal cells. Oxidative metabolism is very active in brain, where large amounts of chemical energy as ATP molecules are consumed, mostly required for the maintenance of the ionic gradients that underlie resting and action potentials which are involved in nerve impulse propagation, neurotransmitter release and cation homeostasis. Protein phosphorylation is a key process in biological regulation. At nervous system level, protein phosphorylation is the major molecular mechanism through which the function of neural proteins is modulted in response to extracellular signals, including the response to neurotransmitter stimuli. It is the major mechanism of neural plasticity, including memory processing. The phosphorylation of Na+, K+-ATPase catalytic subunit inhibits enzyme activity whereas the inhibition of protein kinase C restores the enzyme activity. The dephosphorylation of neuronal Na+, K+-ATPase is mediated by calcineurin, a serine / threonine phosphatase. The latter enzyme is involved in a wide range of cellular responses to Ca2+ mobilizing signals, in the regulation of neuronal excitability by controlling the activity of ion channels, in the release of neurotransmitters and hormones, as well as in synaptic plasticity and gene transcription. In the present article evidence showing Na+, K+-ATPase involvement in signaling pathways

  18. Development of Na/sup +/-dependent hexose transport in cultured renal epithelial cells (LLC-PK/sub 1/)

    SciTech Connect

    Weiss, E.R.; Amsler, K.; Dawson, W.D.; Cook, J.S.

    1984-01-01

    A number of factors were explored to analyze how they interact to yield the increasing transport capacity in differentiating cell populations. These factors include the number of functional transporters in the population, the distribution of these transporters among the individual cells, the Na/sup +/ chemical gradient, the transmembrane potential, the pathways and activities of these pathways for efflux of glucoside, and cell-cell coupling between accumulating and non-accumulating cells. 35 references, 9 figures, 2 tables. (ACR)

  19. The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport*

    PubMed Central

    Felts, Bruce; Pramod, Akula Bala; Sandtner, Walter; Burbach, Nathan; Bulling, Simon; Sitte, Harald H.; Henry, L. Keith

    2014-01-01

    Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na+, Cl−, and K+ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na+-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca2+ (but not other cations) to functionally replace Na+ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca2+ and Na+ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca2+ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na+-binding sites. PMID:24293367

  20. The two Na+ sites in the human serotonin transporter play distinct roles in the ion coupling and electrogenicity of transport.

    PubMed

    Felts, Bruce; Pramod, Akula Bala; Sandtner, Walter; Burbach, Nathan; Bulling, Simon; Sitte, Harald H; Henry, L Keith

    2014-01-17

    Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na(+), Cl(-), and K(+) gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na(+)-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca(2+) (but not other cations) to functionally replace Na(+) for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca(2+) and Na(+) illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca(2+) promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na(+)-binding sites.

  1. Regulation of airway surface liquid volume and mucus transport by active ion transport.

    PubMed

    Tarran, Robert

    2004-01-01

    Mucus clearance is an important component of the lung's innate defense against disease, and the ability of the airways to clear mucus is strongly dependent on the volume of liquid on airway surfaces. Whether airway surface liquid (ASL) volume is maintained by passive surface forces or by active ion transport is controversial yet crucial to the understanding of how this system operates in both health and disease. In support of active ion transport being the major determinant of ASL volume, we have demonstrated that normal airway epithelia sense and autoregulate ASL height (volume) by adjusting the rates of Na+ absorption and Cl- secretion to maintain mucus transport.

  2. Scanning ion-selective electrode technique and X-ray microanalysis provide direct evidence of contrasting Na+ transport ability from root to shoot in salt-sensitive cucumber and salt-tolerant pumpkin under NaCl stress.

    PubMed

    Lei, Bo; Huang, Yuan; Sun, Jingyu; Xie, Junjun; Niu, Mengliang; Liu, Zhixiong; Fan, Molin; Bie, Zhilong

    2014-12-01

    Grafting onto salt-tolerant pumpkin rootstock can increase cucumber salt tolerance. Previous studies have suggested that this can be attributed to pumpkin roots with higher capacity to limit the transport of Na(+) to the shoot than cucumber roots. However, the mechanism remains unclear. This study investigated the transport of Na(+) in salt-tolerant pumpkin and salt-sensitive cucumber plants under high (200 mM) or moderate (90 mM) NaCl stress. Scanning ion-selective electrode technique showed that pumpkin roots exhibited a higher capacity to extrude Na(+), and a correspondingly increased H(+) influx under 200 or 90 mM NaCl stress. The 200 mM NaCl induced Na(+)/H(+) exchange in the root was inhibited by amiloride (a Na(+)/H(+) antiporter inhibitor) or vanadate [a plasma membrane (PM) H(+) -ATPase inhibitor], indicating that Na(+) exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na(+)/H(+) antiporter across the PM, and the Na(+)/H(+) antiporter system in salt stressed pumpkin roots was sufficient to exclude Na(+) X-ray microanalysis showed higher Na(+) in the cortex, but lower Na(+) in the stele of pumpkin roots than that in cucumber roots under 90 mM NaCl stress, suggesting that the highly vacuolated root cortical cells of pumpkin roots could sequester more Na(+), limit the radial transport of Na(+) to the stele and thus restrict the transport of Na(+) to the shoot. These results provide direct evidence for pumpkin roots with higher capacity to limit the transport of Na(+) to the shoot than cucumber roots.

  3. Inhibition of epithelial Na sup + transport by atriopeptin, protein kinase c, and pertussis toxin

    SciTech Connect

    Mohrmann, M.; Cantiello, H.F.; Ausiello, D.A. )

    1987-08-01

    The authors have recently shown the selective inhibition of an amiloride-sensitive, conductive pathway for Na{sup +} by atrial natriuretic peptide and 8-bromoguanosine 3{prime},5{prime}-cyclic monophosphate (8-BrcGMP) in the renal epithelial cell line, LLC-PK{sub i}. Using {sup 22}Na{sup +} fluxes, they further investigated the modulation of Na{sup +} transport by atrial natriuretic peptide and by agents that increase cGMP production, activate protein kinase c, or modulate guanine nucleotide regulatory protein function. Sodium nitroprusside increases intracellular cGMP concentrations without affecting cAMP concentrations and completely inhibits amiloride-sensitive Na{sup +} uptake in a time- and concentration-dependent manner. Oleoyl 2-acetylglycerol and phorbol 12-myristate 13-acetate, activators of protein kinase c, inhibit Na{sup +} uptake by 93 {plus minus} 13 and 51 {plus minus} 10%, respectively. Prolonged incubation with phorbol ester results in the downregulation of protein kinase c activity and reduces the inhibitory effect of atrial natriuretic peptide, suggesting that the action of this peptide involves stimulation of protein kinase c. Pertussis toxin, which induces the ADP-ribosylation of a 41-kDa guanine nucleotide regulatory protein in LLC-PK{sub i} cells, inhibits {sup 22}Na{sup +} influx to the same extent as amiloride. Thus, increasing cGMP, activating protein kinase c, and ADP-ribosylating a guanine nucleotide regulatory protein all inhibit Na{sup +} uptake. These events may be sequentially involved in the action of atrial natriuretic peptide.

  4. NaBC1 is a ubiquitous electrogenic Na+ -coupled borate transporter essential for cellular boron homeostasis and cell growth and proliferation.

    PubMed

    Park, Meeyoung; Li, Qin; Shcheynikov, Nikolay; Zeng, Weizong; Muallem, Shmuel

    2004-11-05

    Boron is a vital micronutrient in plants and may be essential for animal growth and development. Whereas the role of boron in the life cycle of plants is well documented, nothing is known about boron homeostasis and function in animal cells. NaBC1, the mammalian homolog of AtBor1, is a borate transporter. In the absence of borate, NaBC1 conducts Na(+) and OH(-) (H(+)), while in the presence of borate, NaBC1 functions as an electrogenic, voltage-regulated, Na(+)-coupled B(OH)(4)(-) transporter. At low concentrations, borate activated the MAPK pathway to stimulate cell growth and proliferation, and at high concentrations, it was toxic. Accordingly, overexpression of NaBC1 shifted both effects of borate to the left, whereas knockdown of NaBC1 halted cell growth and proliferation. These findings may reveal a previously unrecognized role for NaBC1 in borate homeostasis and open the way to better understanding of the many presumed physiological roles of borate in animals.

  5. Common folds and transport mechanisms of secondary active transporters.

    PubMed

    Shi, Yigong

    2013-01-01

    Secondary active transporters exploit the electrochemical potential of solutes to shuttle specific substrate molecules across biological membranes, usually against their concentration gradient. Transporters of different functional families with little sequence similarity have repeatedly been found to exhibit similar folds, exemplified by the MFS, LeuT, and NhaA folds. Observations of multiple conformational states of the same transporter, represented by the LeuT superfamily members Mhp1, AdiC, vSGLT, and LeuT, led to proposals that structural changes are associated with substrate binding and transport. Despite recent biochemical and structural advances, our understanding of substrate recognition and energy coupling is rather preliminary. This review focuses on the common folds and shared transport mechanisms of secondary active transporters. Available structural information generally supports the alternating access model for substrate transport, with variations and extensions made by emerging structural, biochemical, and computational evidence.

  6. Political activity for physical activity: health advocacy for active transport

    PubMed Central

    2011-01-01

    Effective health advocacy is a priority for efforts to increase population participation in physical activity. Local councils are an important audience for this advocacy. The aim of the current study was to describe features of advocacy for active transport via submissions to city council annual plans in New Zealand, and the impact of an information sheet to encourage the health sector to be involved in this process. Written submissions to city council's annual consultation process were requested for 16 city councils over the period of three years (2007/08, 2008/09, and 2009/10). Submissions were reviewed and categories of responses were created. An advocacy information sheet encouraging health sector participation and summarising some of the evidence-base related to physical activity, active transport and health was released just prior to the 2009/10 submission time. Over the period of the study, city councils received 47,392 submissions, 17% of which were related to active transport. Most submissions came from city residents, with a small proportion (2%) from the health sector. The largest category of submissions was in support of pedestrian and cycling infrastructure, design and maintenance of facilities and additional features to support use of these transport modes. Health arguments featured prominently in justifications for active transport initiatives, including concerns about injury risk, obesity, physical inactivity, personal safety and facilities for people with disabilities. There was evidence that the information sheet was utilised by some health sector submitters (12.5%), providing tentative support for initiatives of this nature. In conclusion, the study provides novel information about the current nature of health advocacy for active transport and informs future advocacy efforts about areas for emphasis, such as health benefits of active transport, and potential alliances with other sectors such as environmental sustainability, transport and urban

  7. Activity-Dependent Adenosine Release May Be Linked to Activation of Na+-K+ ATPase: An In Vitro Rat Study

    PubMed Central

    Sims, Robert Edward; Dale, Nicholas

    2014-01-01

    In the brain, extracellular adenosine increases as a result of neuronal activity. The mechanisms by which this occurs are only incompletely understood. Here we investigate the hypothesis that the Na+ influxes associated with neuronal signalling activate the Na+-K+ ATPase which, by consuming ATP, generates intracellular adenosine that is then released via transporters. By measuring adenosine release directly with microelectrode biosensors, we have demonstrated that AMPA-receptor evoked adenosine release in basal forebrain and cortex depends on extracellular Na+. We have simultaneously imaged intracellular Na+ and measured adenosine release. The accumulation of intracellular Na+ during AMPA receptor activation preceded adenosine release by some 90 s. By removing extracellular Ca2+, and thus preventing indiscriminate neuronal activation, we used ouabain to test the role of the Na+-K+ ATPase in the release of adenosine. Under conditions which caused a Na+ influx, brief applications of ouabain increased the accumulation of intracellular Na+ but conversely rapidly reduced extracellular adenosine levels. In addition, ouabain greatly reduced the amount of adenosine released during application of AMPA. Our data therefore suggest that activity of the Na+-K+ ATPase is directly linked to the efflux of adenosine and could provide a universal mechanism that couples adenosine release to neuronal activity. The Na+-K+ ATPase-dependent adenosine efflux is likely to provide adenosine-mediated activity-dependent negative feedback that will be important in many diverse functional contexts including the regulation of sleep. PMID:24489921

  8. Development of a label-free assay for sodium-dependent phosphate transporter NaPi-IIb.

    PubMed

    Wong, Soo-Hang; Gao, Alice; Ward, Sabrina; Henley, Charles; Lee, Paul H

    2012-07-01

    The most widely used assay format for characterizing plasma membrane transporter activity measures accumulation of radiolabeled substrates in tissues or cells expressing the transporters. This assay format had limitations and disadvantages; therefore, there was an unmet need for development of a homogeneous, nonradioactive assay for membrane transporter proteins. In this report, the authors describe the development of a label-free homogeneous assay for the sodium-dependent phosphate transporter NaPi-IIb using the Epic system. The addition of phosphate stimulated a dynamic mass redistribution (DMR) profile unique to cells expressing NaPi-IIb but not on parental cells. This DMR profile was phosphate specific because sulfate or buffer alone did not elicit the same response. Furthermore, the DMR response observed was phosphate and sodium dependent, with Km values in the micromolar and millimolar range, respectively. A known NaPi-IIb noncompetitive inhibitor was shown to completely inhibit the phosphate-stimulated DMR response, suggesting that this observed DMR response is an NaPi-IIb-mediated cellular event. The results demonstrate that a novel label-free assay was developed for studying transporter-mediated cellular activity, and this DMR assay platform could be applicable to other membrane transporter proteins.

  9. Cation transport coupled to ATP hydrolysis by the (Na, K)-ATPase: An integrated, animated model.

    PubMed

    Leone, Francisco A; Furriel, Rosa P M; McNamara, John C; Horisberger, Jean D; Borin, Ivana A

    2010-07-01

    An Adobe® animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na(+) and K(+) translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P(2c) -type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E(1) /E(2) -ATPase as it undergoes conformational changes between the E(1) and E(2) forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger's scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure-function relationship. The movements of the various domains within the (Na, K)-ATPase α-subunit illustrate the conformational changes that occur during Na(+) and K(+) translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the "core engine" of the pump, with respect to ATP binding, cation transport, and ADP and P(i) release.

  10. Determinants of substrate and cation transport in the human Na+/dicarboxylate cotransporter NaDC3.

    PubMed

    Schlessinger, Avner; Sun, Nina N; Colas, Claire; Pajor, Ana M

    2014-06-13

    Metabolic intermediates, such as succinate and citrate, regulate important processes ranging from energy metabolism to fatty acid synthesis. Cytosolic concentrations of these metabolites are controlled, in part, by members of the SLC13 gene family. The molecular mechanism underlying Na(+)-coupled di- and tricarboxylate transport by this family is understood poorly. The human Na(+)/dicarboxylate cotransporter NaDC3 (SLC13A3) is found in various tissues, including the kidney, liver, and brain. In addition to citric acid cycle intermediates such as α-ketoglutarate and succinate, NaDC3 transports other compounds into cells, including N-acetyl aspartate, mercaptosuccinate, and glutathione, in keeping with its dual roles in cell nutrition and detoxification. In this study, we construct a homology structural model of NaDC3 on the basis of the structure of the Vibrio cholerae homolog vcINDY. Our computations are followed by experimental testing of the predicted NaDC3 structure and mode of interaction with various substrates. The results of this study show that the substrate and cation binding domains of NaDC3 are composed of residues in the opposing hairpin loops and unwound portions of adjacent helices. Furthermore, these results provide a possible explanation for the differential substrate specificity among dicarboxylate transporters that underpin their diverse biological roles in metabolism and detoxification. The structural model of NaDC3 provides a framework for understanding substrate selectivity and the Na(+)-coupled anion transport mechanism by the human SLC13 family and other key solute carrier transporters.

  11. Upregulation of Na-coupled glucose transporter SGLT1 by Tau tubulin kinase 2.

    PubMed

    Alesutan, Ioana; Sopjani, Mentor; Dërmaku-Sopjani, Miribane; Munoz, Carlos; Voelkl, Jakob; Lang, Florian

    2012-01-01

    The Tau-tubulin-kinase 2 (TTBK2) is a serine/threonine kinase expressed in various tissues including tumors. Up-regulation of TTBK2 increases resistance of tumor cells against antiangiogenic treatment and confers cell survival. Tumor cell survival critically depends on cellular uptake of glucose, which is partially accomplished by SGLT1 (SLC5A1) mediated Na(+)-coupled glucose transport. The present study explored whether TTBK2 participates in the regulation of SGLT1 activity. To this end, electrogenic glucose transport was determined in Xenopus oocytes expressing SGLT1 with or without wild-type TTBK2, truncated TTBK2([1-450]) or kinase inactive mutants TTBK2-KD and TTBK2-KD([1-450]). TTBK2, but not TTBK2([1-450]), TTBK2-KD or TTBK2-KD([1-450]), increased membrane carrier protein abundance and electrogenic glucose transport capacity in SGLT1-expressing Xenopus oocytes. Thus TTBK2 is a completely novel regulator of Na(+)-coupled glucose transport.

  12. Characteristics of injury and recovery of net NO3- transport of barley seedlings from treatments of NaCl

    NASA Technical Reports Server (NTRS)

    Klobus, G.; Ward, M. R.; Huffaker, R. C.

    1988-01-01

    The nature of the injury and recovery of nitrate uptake (net uptake) from NaCl stress in young barley (Hordeum vulgare L, var CM 72) seedlings was investigated. Nitrate uptake was inhibited rapidly by NaCl, within 1 minute after exposure to 200 millimolar NaCl. The duration of exposure to saline conditions determined the time of recovery of NO3- uptake from NaCl stress. Recovery was dependent on the presence of NO3- and was inhibited by cycloheximide, 6-methylpurine, and cerulenin, respective inhibitors of protein, RNA, and sterol/fatty acid synthesis. These inhibitors also prevented the induction of the NO3- uptake system in uninduced seedlings. Uninduced seedlings exhibited endogenous NO3- transport activity that appeared to be constitutive. This constitutive activity was also inhibited by NaCl. Recovery of constitutive NO3- uptake did not require the presence of NO3-.

  13. Intestinal ammonia transport in freshwater and seawater acclimated rainbow trout (Oncorhynchus mykiss): evidence for a Na+ coupled uptake mechanism.

    PubMed

    Rubino, Julian G; Zimmer, Alex M; Wood, Chris M

    2015-05-01

    In vitro gut sac experiments were performed on freshwater and 60% seawater acclimated trout (Oncorhynchus mykiss) under treatments designed to discern possible mechanisms of intestinal ammonia transport. Seawater acclimation increased ammonia flux rate into the serosal saline (Jsamm) in the anterior intestine, however it did not alter Jsamm in the mid- or posterior intestine suggesting similar mechanisms of ammonia handling in freshwater and seawater fish. Both fluid transport rate (FTR) and Jsamm were inhibited in response to basolateral ouabain treatment, suggesting a linkage of ammonia uptake to active transport, possibly coupled to fluid transport processes via solvent drag. Furthermore, decreases in FTR and Jsamm caused by low Na(+) treatment indicated a Na(+) linked transport mechanism. Mucosal bumetanide (10(-4) M) had no impact on FTR, yet decreased Jsamm in the anterior and mid-intestine, suggesting NH4(+) substitution for K(+) on an apical NKCC, and at least a partial uncoupling of ammonia transport from fluid transport. Additional treatments (amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), phenamil, bafilomycin, 4',6-diamidino-2-phenylindole (DAPI), high sodium) intended to disrupt alternative routes of Na(+) uptake yielded no change in FTR or Jsamm, suggesting the absence of direct competition between Na(+) and ammonia for transport. Finally, [(14)C]methylamine permeability (PMA) measurements indicated the likely presence of an intestinal Rh-mediated ammonia transport system, as increasing NH4Cl (0, 1, 5 mmol l(-1)) concentrations reduced PMA, suggesting competition for transport through Rh proteins. Overall, the data presented in this paper provide some of the first insights into mechanisms of teleost intestinal ammonia transport.

  14. Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2

    PubMed Central

    Subramanian, Nandhitha; Scopelitti, Amanda J.; Carland, Jane E.; Ryan, Renae M.; O’Mara, Megan L.; Vandenberg, Robert J.

    2016-01-01

    The Na+/Cl- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na+ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na+ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na+-dependent leucine transporter, LeuTAa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na+ binding sites in GlyT2, but the physical location of the third Na+ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuTAa. Although betaine transport by BetP is coupled to the co-transport of two Na+ ions, the first Na+ site is not conserved between BetP and LeuTAa, the so called Na1' site. We hypothesized that the third Na+ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na+ placed at the location consistent with the Na1' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na+ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10. PMID:27337045

  15. Red blood cell cation transports in uraemic anaemia: evidence for an increased K/Cl co-transport activity. Effects of dialysis and erythropoietin treatment.

    PubMed

    De Franceschi, L; Olivieri, O; Girelli, D; Lupo, A; Bernich, P; Corrocher, R

    1995-10-01

    This study examines the role of uraemia and the effect of different dialysis treatments on red cell cation transport. We evaluated the main cation transport systems in erythrocytes of non-dialysed end-stage renal disease (ESRD) subjects, of patients undergoing haemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD), as well as the changes induced by human recombinant erythropoietin (r-HuEPO) administration. In uraemic undialysed and dialysed patients, we observed an increase in K/Cl co-transport activity and in shrinkage-induced amiloride-sensitive (HMA-sensitive) Na efflux (Na/H exchange) and a decrease in Na/K pump and Na/K/Cl co-transport activity, while Na/Li exchange was increased only in dialysed patients. In uraemic erythrocytes, we showed for the first time an increased K/Cl co-transport activity, which was cell age independent. Generally, the different method of dialysis (CAPD or HD) did not modify the cation transport abnormalities observed. During the treatment with r-HuEPO, all the systems, with the exception of the Na/K pump and Na/K/Cl co-transport, increased their activities following the increase of circulating young red cells. The changes produced under r-HuEPO administration were transient and cation transports returned to the baseline values within 100 days of treatment, indicating a primary and prominent pathogenetic role of uraemia in modulating the red cell membrane cation transport activities.

  16. Active sodium transport and the electrophysiology of rabbit colon.

    PubMed

    Schultz, S G; Frizzell, R A; Nellans, H N

    1977-05-12

    The electrophysiologic properties of rabbit colonic epithelial cells were investigated employing microelectrode techniques. Under open-circuit conditions, the transepithelial electrical potential difference (PD) averaged 20 mV, serosa positive, and the intracellular electrical potential (psimc) averaged -32 mV, cell interior negative with respect to the mucosal solution; under short-circuit conditions, psimc averaged -46 mV. The addition of amiloride to the mucosal solution abolishes the transepithelial PD and active Na transport, and psimc is hyperpolarized to an average value of -53 mV. These results indicate that Na entry into the mucosal cell is a conductive process which, normally, depolarized psimc. The data obtained were interpreted using a double-membrane equivalent electrical circuit model of the "active Na transport pathway" involving two voltage-independent electromotive forces (emf's) and two voltage-independent resistances arrayed in series. Our observations are consistent with the notions that: (a) The emf's and resistances across the mucosal and baso-lateral membranes are determined predominantly by the emf (64 mV) and resistance of the Na entry process and the emf (53 mV) and resistance of the process responsible for active Na extrusion across the baso-lateral membranes: that is, the electrophysiological properties of the cell appear to be determined solely by the properties and processes responsible for transcellular active Na transport. The emf of the Na entry process is consistent with the notion that the Na activity in the intracellular transport pool is approximately one-tenth that in the mucosal solution or about 14 mM. (b) In the presence of amiloride, the transcellular conductance is essentially abolished and the total tissue conductance is the result of ionic diffusion through paracellular pathways. (c) The negative intracellular potential (with respect to the mucosal solution) is due primarily to the presence of a low resistance

  17. A general method for determining secondary active transporter substrate stoichiometry

    PubMed Central

    Fitzgerald, Gabriel A; Mulligan, Christopher; Mindell, Joseph A

    2017-01-01

    The number of ions required to drive substrate transport through a secondary active transporter determines the protein’s ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter’s mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na+-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures. DOI: http://dx.doi.org/10.7554/eLife.21016.001 PMID:28121290

  18. Na-Stimulated Transport of l-Methionine in Brevibacterium linens CNRZ 918.

    PubMed

    Ferchichi, M; Hemme, D; Nardi, M

    1987-09-01

    The transport of l-methionine by the gram-positive species Brevibacterium linens CNRZ 918 is described. The one transport system (K(m) = 55 muM) found is constitutive for l-methionine, stereospecific, and pH and temperature dependent. Entry of l-methionine into cells is controlled by the internal methionine pool. Competition studies indicate that l-methionine and alpha-aminobutyric acid share a common carrier for their transport. Neither methionine derivatives substituted on the amino or carboxyl groups nor d-methionine was an inhibitor, whereas powerful inhibition was shown by l-cysteine, s-methyl-l-cysteine, dl-selenomethionine and dl-homocysteine. Sodium plays important and varied roles in l-methionine transport by B. linens CNRZ 918: (i) it stimulates transport without affecting the K(m), (ii) it increases the specific activity (on a biomass basis) of the l-methionine transport system when present with methionine in the medium, suggesting a coinduction mechanism. l-Methionine transport requires an exogenous energy source, which may be succinic, lactic, acetic, or pyruvic acid but not glucose or sucrose. The fact that l-methionine transport was stimulated by potassium arsenate and to a lesser extent by potassium fluoride suggests that high-energy phosphorylated intermediates are not involved in the process. Monensin eliminates stimulation by sodium. Gramicidin and carbonyl cyanide-m-chlorophenylhydrazone act in the presence or absence of Na. N-Ethylmaleimide, p-chloromercurobenzoate, valinomycin, sodium azide, and potassium cyanide have no or only a partial inhibitory effect. These results tend to indicate that the proton motive force reinforced by the Na gradient is involved in the mechanism of energy coupling of l-methionine transport by B. linens CNRZ 918. Thus, this transport is partially similar to the well-described systems in gram-negative bacteria, except for the role of sodium, which is very effective in B. linens, a species adapted to the high sodium

  19. Thermodynamic and Transport Properties of H2O + NaCl from Polarizable Force Fields.

    PubMed

    Jiang, Hao; Mester, Zoltan; Moultos, Othonas A; Economou, Ioannis G; Panagiotopoulos, Athanassios Z

    2015-08-11

    Molecular dynamics and Monte Carlo simulations were performed to obtain thermodynamic and transport properties of the binary H2O + NaCl system using the polarizable force fields of Kiss and Baranyai ( J. Chem. Phys. 2013 , 138 , 204507 and 2014 , 141 , 114501 ). In particular, liquid densities, electrolyte and crystal chemical potentials of NaCl, salt solubilities, mean ionic activity coefficients, vapor pressures, vapor-liquid interfacial tensions, and viscosities were obtained as functions of temperature, pressure, and salt concentration. We compared the performance of the polarizable force fields against fixed-point-charge (nonpolarizable) models. Most of the properties of interest are better represented by the polarizable models, which also remain physically realistic at elevated temperatures.

  20. Fluid dilution and efficiency of Na+ transport in a mathematical model of a thick ascending limb cell

    PubMed Central

    Clausen, Chris; Marcano, Mariano; Layton, Anita T.; Layton, Harold E.; Moore, Leon C.

    2013-01-01

    Thick ascending limb (TAL) cells are capable of reducing tubular fluid Na+ concentration to as low as ∼25 mM, and yet they are thought to transport Na+ efficiently owing to passive paracellular Na+ absorption. Transport efficiency in the TAL is of particular importance in the outer medulla where O2 availability is limited by low blood flow. We used a mathematical model of a TAL cell to estimate the efficiency of Na+ transport and to examine how tubular dilution and cell volume regulation influence transport efficiency. The TAL cell model represents 13 major solutes and the associated transporters and channels; model equations are based on mass conservation and electroneutrality constraints. We analyzed TAL transport in cells with conditions relevant to the inner stripe of the outer medulla, the cortico-medullary junction, and the distal cortical TAL. At each location Na+ transport efficiency was computed as functions of changes in luminal NaCl concentration ([NaCl]), [K+], [NH4+], junctional Na+ permeability, and apical K+ permeability. Na+ transport efficiency was calculated as the ratio of total net Na+ transport to transcellular Na+ transport. Transport efficiency is predicted to be highest at the cortico-medullary boundary where the transepithelial Na+ gradient is the smallest. Transport efficiency is lowest in the cortex where luminal [NaCl] approaches static head. PMID:23097469

  1. Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1.

    PubMed Central

    Pajor, A M; Kahn, E S; Gangula, R

    2000-01-01

    The role of cationic amino acids in the Na(+)/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased K(m) for succinate of 1.8 mM, compared with 0.3 mM in the wild-type NaDC-1. The R349A mutant had Na(+) and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding. PMID:10970779

  2. Transepithelial glucose transport and Na+/K+ homeostasis in enterocytes: an integrative model

    PubMed Central

    Drengstig, Tormod; Ruoff, Peter

    2014-01-01

    The uptake of glucose and the nutrient coupled transcellular sodium traffic across epithelial cells in the small intestine has been an ongoing topic in physiological research for over half a century. Driving the uptake of nutrients like glucose, enterocytes must have regulatory mechanisms that respond to the considerable changes in the inflow of sodium during absorption. The Na-K-ATPase membrane protein plays a major role in this regulation. We propose the hypothesis that the amount of active Na-K-ATPase in enterocytes is directly regulated by the concentration of intracellular Na+ and that this regulation together with a regulation of basolateral K permeability by intracellular ATP gives the enterocyte the ability to maintain ionic Na+/K+ homeostasis. To explore these regulatory mechanisms, we present a mathematical model of the sodium coupled uptake of glucose in epithelial enterocytes. Our model integrates knowledge about individual transporter proteins including apical SGLT1, basolateral Na-K-ATPase, and GLUT2, together with diffusion and membrane potentials. The intracellular concentrations of glucose, sodium, potassium, and chloride are modeled by nonlinear differential equations, and molecular flows are calculated based on experimental kinetic data from the literature, including substrate saturation, product inhibition, and modulation by membrane potential. Simulation results of the model without the addition of regulatory mechanisms fit well with published short-term observations, including cell depolarization and increased concentration of intracellular glucose and sodium during increased concentration of luminal glucose/sodium. Adding regulatory mechanisms for regulation of Na-K-ATPase and K permeability to the model show that our hypothesis predicts observed long-term ionic homeostasis. PMID:24898586

  3. Na+-dependent transporters mediate HCO3– salvage across the luminal membrane of the main pancreatic duct

    PubMed Central

    Lee, Min Goo; Ahn, Wooin; Choi, Joo Young; Luo, Xiang; Seo, Jeong Taeg; Schultheis, Patrick J.; Shull, Gary E.; Kim, Kyung Hwan; Muallem, Shmuel

    2000-01-01

    To study the roles of Na+-dependent H+ transporters, we characterized H+ efflux mechanisms in the pancreatic duct in wild-type, NHE2–/–, and NHE3–/– mice. The pancreatic duct expresses NHE1 in the basolateral membrane, and NHE2 and NHE3 in the luminal membrane, but does not contain NHE4 or NHE5. Basolateral Na+-dependent H+ efflux in the microperfused duct was inhibited by 1.5 μM of the amiloride analogue HOE 694, consistent with expression of NHE1, whereas the luminal activity required 50 μM HOE 694 for effective inhibition, suggesting that the efflux might be mediated by NHE2. However, disruption of NHE2 had no effect on luminal transport, while disruption of the NHE3 gene reduced luminal Na+-dependent H+ efflux by ∼45%. Notably, the remaining luminal Na+-dependent H+ efflux in ducts from NHE3–/– mice was inhibited by 50 μM HOE 694. Hence, ∼55% of luminal H+ efflux (or HCO3– influx) in the pancreatic duct is mediated by a novel, HOE 694–sensitive, Na+-dependent mechanism. H+ transport by NHE3 and the novel transporter is inhibited by cAMP, albeit to different extents. We propose that multiple Na+-dependent mechanisms in the luminal membrane of the pancreatic duct absorb Na+ and HCO3– to produce a pancreatic juice that is poor in HCO3– and rich in Cl– during basal secretion. Inhibition of the transporters during stimulated secretion aids in producing the HCO3–-rich pancreatic juice. PMID:10841524

  4. Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog.

    PubMed

    Tavoulari, Sotiria; Margheritis, Eleonora; Nagarajan, Anu; DeWitt, David C; Zhang, Yuan-Wei; Rosado, Edwin; Ravera, Silvia; Rhoades, Elizabeth; Forrest, Lucy R; Rudnick, Gary

    2016-01-15

    In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na(+) stabilizes outward-open conformational states. We examined how each of the two LeuT Na(+) binding sites contributes to Na(+)-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na(+), suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na(+) responsiveness. However, mutation of Na1 residues also influenced the Na(+)-dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na(+) and transmembrane helices 1 and 8, whereas Na(+) binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening.

  5. Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog*

    PubMed Central

    Tavoulari, Sotiria; Margheritis, Eleonora; Nagarajan, Anu; DeWitt, David C.; Zhang, Yuan-Wei; Rosado, Edwin; Ravera, Silvia; Rhoades, Elizabeth; Forrest, Lucy R.; Rudnick, Gary

    2016-01-01

    In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na+ stabilizes outward-open conformational states. We examined how each of the two LeuT Na+ binding sites contributes to Na+-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na+, suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na+ responsiveness. However, mutation of Na1 residues also influenced the Na+-dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na+ and transmembrane helices 1 and 8, whereas Na+ binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening. PMID:26582198

  6. Effect of chaotropic anions on the sodium transport by the Na,K-ATPase.

    PubMed

    Ayuyan, Artem G; Sokolov, Valerij S; Lenz, Alexander A; Apell, Hans-Jürgen

    2006-02-01

    The effect of choline iodide, bromide and chloride on the kinetics of the electrogenic sodium transport by the Na,K-ATPase was investigated in a model system of ATPase-containing membrane fragments adsorbed on the lipid bilayer membrane. The kinetic parameters of Na(+) transport were determined from short circuit currents after fast release of ATP from its caged precursor. The falling phase of the current transients could be fitted by a single exponential with the time constant, tau (2). Its temperature dependence allowed an estimation of the activation energy of the rate-limiting reaction step, the conformation transition E(1)/E(2). Choline iodide and bromide caused a decrease of the activation energy as well as the overall rate of the process expressed as the pre-exponential factor A of the Arrhenius equation. If choline iodide or bromide were present on the cytoplasmic and extracellular sides of the protein, the temperature dependent changes were more pronounced than when present on the cytoplasmic side only. These results can be explained by an effect of the anions on water structure on the extracellular surface of the protein, where a deep access channel connects the ion-binding sites with the solution. Chloride ions also caused a deceleration of the electrogenic transport, however, in contrast to iodide or bromide, they did not affect the activation energy, and were more effective when added on the cytoplasmic side. This effect can be explained by asymmetric screening of the negative surface charges which leads to a transmembrane electric potential that modifies the ion transfer.

  7. Thyroid thermogenesis. Relationships between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in rat skeletal muscle.

    PubMed

    Asano, Y; Liberman, U A; Edelman, I S

    1976-02-01

    The effect of thyroid status on QO2, QO2 (t) and NaK-ATPase activity was examined in rat skeletal muscle. QO2(t) (i.e. Na+-transport-dependent respiration) was estimated with ouabain or Na+-free media supplemented with K+. In contrast to the effects of ouabain on ion composition, intracellular K+ was maintained at about 125 meq/liter, and intracellular Na+ was almost nil in the Na+-free media. The estimates of QO2(t) were independent of the considerable differences in tissue ion concentrations. The increase in QO2(t) account for 47% of the increase in QO2 in the transition from the hypothyroid to the euthyroid state and 84% of the increase in the transition from the euthyroid to the hyperthyroid state. Surgical thyroidectomy lowered NaK-ATPase activity of the microsomal fraction (expressed per milligram protein) 32%; injections of triodothyronine (T3) increased this activity 75% in initially hypothyroid rats and 26% in initially euthyroid rats. Thyroidectomy was attended by significant falls in serum Ca and Pi concentrations. Administration of T3 resulted in further declines in serum Ca and marked increases in serum Ps concentrations. Similar effects were seen in 131I-treated rats, but the magnitude of the declines in serum Ca were less. The effects of T3 on QO2, QO2(t), and NaK-ATPase activity of skeletal muscle were indistinguishable in the 131I-ablated and surgically thyroidectomized rats. In thyroidectomized or euthyroid rats given repeated doses of T3, QO2(t) and NaA-ATPase activity increased proportionately. In thyroidectomized rats injected with single doses of T3, either 10, 50, or 250 mug/100 g body wt, QO2(t) increased linearly with NaK-ATPase activity. The kinetics of the NaK-ATPase activity was assessed with an ATP-generating system. T3 elicited a significant increase in Vmax with no change in Km for ATP.

  8. A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform[OPEN

    PubMed Central

    Nagarajan, Yagnesh; Rongala, Jay; Luang, Sukanya; Shadiac, Nadim; Sutton, Tim; Tyerman, Stephen D.; McPhee, Gordon; Voelcker, Nicolas H.; Lee, Jung-Goo

    2016-01-01

    Plant growth and survival depend upon the activity of membrane transporters that control the movement and distribution of solutes into, around, and out of plants. Although many plant transporters are known, their intrinsic properties make them difficult to study. In barley (Hordeum vulgare), the root anion-permeable transporter Bot1 plays a key role in tolerance to high soil boron, facilitating the efflux of borate from cells. However, its three-dimensional structure is unavailable and the molecular basis of its permeation function is unknown. Using an integrative platform of computational, biophysical, and biochemical tools as well as molecular biology, electrophysiology, and bioinformatics, we provide insight into the origin of transport function of Bot1. An atomistic model, supported by atomic force microscopy measurements, reveals that the protein folds into 13 transmembrane-spanning and five cytoplasmic α-helices. We predict a trimeric assembly of Bot1 and the presence of a Na+ ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology of Bot1 detects Na+-dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using alanine scanning, molecular dynamics simulations, and transport measurements, we show that conductance by Bot1 is abolished by removal of the Na+ ion binding site. Our data enhance the understanding of the permeation functions of Bot1. PMID:26672067

  9. Plasma membrane H-ATPase activity is involved in adaptation of tomato calli to NaCl.

    PubMed

    Kerkeb, Loubna; Donaire, Juan Pedro; Rodríguez-Rosales, María Pilar

    2001-04-01

    A tomato (Lycopersicon esculentum Mill. cv. Pera) callus culture tolerant to NaCl was obtained by successive subcultures of NaCl-sensitive calli in medium supplemented with 50 mM NaCl. NaCl-tolerant calli grew better than NaCl-sensitive calli in media supplemented with 50 and 100 mM NaCl. Analysis of callus ion content showed a strong increase in Na+ and Cl- both in NaCl-tolerant and -sensitive calli grown in media containing NaCl for one subculture. Cells from NaCl-tolerant calli showed a higher H+ extrusion activity than those from NaCl-sensitive calli grown for one subculture in the presence of NaCl. The inhibition of H+ extrusion by NaCl-sensitive cells was correlated with an inhibition of microsomal vanadate-sensitive H+-ATPase (EC 3.6.1.35) and ATP-dependent H+ transport, while the stimulation of H+ extrusion by cells tolerant to 50 mM NaCl was correlated with an increase in plasma membrane ATP-dependent H+ transport. The increase of ATP-dependent H+ extrusion in plasma membranes isolated from 50 mM NaCl-tolerant calli was not a result of stimulation of a vanadate-sensitive ATP hydrolytic activity or an increase in passive permeability to H+. Relative to NaCl-sensitive calli, plasma membrane H+-ATPase from calli tolerant to 50 mM NaCl showed a lower Km for Mg2+-ATP. Our results indicate that tolerance of tomato calli to 50 mM NaCl increases the affinity of plasma membrane H+-ATPase for the substrate ATP and stimulates the H+-pumping activity of this enzyme without modifying its phosphohydrolytic activity.

  10. Renal brush-border Na/sup +/-H/sup +/ exchange activity in the aging rat

    SciTech Connect

    Kinsella, J.L.; Sacktor, B.

    1987-04-01

    Amiloride-sensitive Na/sup +/-H/sup +/ exchange activity in brush-border membrane vesicles isolated from male rat proximal tubules was decreased in the senescent rat (24 mo) compared with the young adult (6 mo). There was no significant loss in Na/sup +/-H/sup +/ exchange activity in the kidneys of animals between 6 and 18 mo of age. Amiloride-insensitive /sup 22/Na/sup +/ uptake and the rate of pH gradient dissipation were not altered during aging. The decrease in sodium-dependent (/sup 32/P) phosphate transport preceded the decline in Na/sup +/-H/sup +/ exchange activity by at least 6 mo. Sodium-dependent D-(/sup 3/H) glucose transport was not significantly altered during aging. Thus various renal plasma membrane transport functions were affected differently in the aging rat. The decrease in Na/sup +/-H/sup +/ exchange activity during aging contrasted with the increase in exchange activity reported previously in acute ablation models of chronic renal failure.

  11. Substrate regulation of ascorbate transport activity in astrocytes

    SciTech Connect

    Wilson, J.X.; Jaworski, E.M.; Kulaga, A.; Dixon, S.J. )

    1990-10-01

    Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent L-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular L-ascorbate on the activity of this transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-(14C)ascorbate for 1 min at 37 degrees C. We observed that the apparent maximal rate of uptake (Vmax) increased rapidly (less than 1 h) when cultured cells were deprived of L-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for L-(14C)ascorbate. The increase in Vmax was reversed by addition of L-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-(3H(G))glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.

  12. The transport properties of activated carbon fibers

    SciTech Connect

    di Vittorio, S.L. . Dept. of Materials Science and Engineering); Dresselhaus, M.S. . Dept. of Electrical Engineering and Computer Science Massachusetts Inst. of Tech., Cambridge, MA . Dept. of Physics); Endo, M. . Dept. of Electrical Engineering); Issi, J-P.; Piraux, L.

    1990-07-01

    The transport properties of activated isotropic pitch-based carbon fibers with surface area 1000 m{sup 2}/g have been investigated. We report preliminary results on the electrical conductivity, the magnetoresistance, the thermal conductivity and the thermopower of these fibers as a function of temperature. Comparisons are made to transport properties of other disordered carbons. 19 refs., 4 figs.

  13. The Transport Properties of Activated Carbon Fibers

    DOE R&D Accomplishments Database

    di Vittorio, S. L.; Dresselhaus, M. S.; Endo, M.; Issi, J-P.; Piraux, L.

    1990-07-01

    The transport properties of activated isotropic pitch-based carbon fibers with surface area 1000 m{sup 2}/g have been investigated. We report preliminary results on the electrical conductivity, the magnetoresistance, the thermal conductivity and the thermopower of these fibers as a function of temperature. Comparisons are made to transport properties of other disordered carbons.

  14. Thyroid-induced alterations in myocardial sodium-potassium-activated adenosine triphosphatase, monovalent cation active transport, and cardiac glycoside binding.

    PubMed Central

    Curfman, G D; Crowley, T J; Smith, T W

    1977-01-01

    The effects of thyroid hormone on guinea pig myocardial NaK-ATPase activity, transmembrane monovalent cation active transport, and cardiac glycoside binding were were examined. NaK-ATPase activities of left atrial and left ventricular homogenates of control and triiodothyronine (T3)-treated animals were determined, and compared to activities of skeletal muscle and liver. T3 administration was associated with a significant increase of 18% in left atrial and left ventricular NaK-ATPase specific activities. This increment was less than that noted in skeletal muscle (+42%) and liver (+30%). To determine if enhanced NaK-ATPase activity was accompanied by increased monovalent cation active transport, in vitro 86Rb+ uptake by left atrial strips and hemidiaphragms was measured. Transition from the euthyroid to the hyperthyroid state resulted in a 68% increase in active 86Rb+ uptake by left atrium, and a 62% increase in active uptake by diaphragm. Passive 86Rb+ uptake was not affected in either tissue. Ouabain binding by atrial and ventricular homogenates of T3-treated animals was increased by 19 and 17%, respectively, compared to controls, in close agreement with thyroid-induced increments in NaK-ATPase activiey. Taken together, these results are consistent with enhanced myocardial NaK-ATPase activity and monovalent cation activt transport due to an increase in the number of functional enzyme complexes. PMID:138689

  15. Na+ dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) in primary astrocyte cultures: effect of oxidative stress.

    PubMed

    Miralles, V J; Martínez-López, I; Zaragozá, R; Borrás, E; García, C; Pallardó, F V; Viña, J R

    2001-12-13

    The Na+ -dependent L-glutamate transporters EAAT1(GLAST), EAAT2 (GLT-1) and EAAT3 (EAAC1) are expressed in primary astrocyte cultures, showing that the EAAT3 transporter is not neuron-specific. The presence of these three transporters was evaluated by RT-PCR, immunoblotting, immunocytochemical techniques, and transport activity. When primary astrocyte cultures were incubated with L-buthionine-(S,R)-sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, the GSH concentration was significantly lower than in control cultures, but the expression and amount of protein of EAAT1, EAAT2 and EAAT3 and transport of L-glutamate was unchanged. Oxidative stress was created by adding H(2)O(2) or tert.-butyl hydroperoxide (t-bOOH) to the primary astrocyte cultures and cell damage was evaluated by measuring activity of lactate dehydrogenase. Under oxidative stress, GSH levels were significantly lower than in control astrocytes; but the expression and the amount of protein of the three transporters remained unchanged. However, L-glutamate uptake was significantly lower in astrocytes under oxidative conditions when compared to controls. L-Glutamate uptake was not changed in the presence of ascorbate, but was partially recovered in the presence of DTT and GSH ethyl ester. This report emphasizes that oxidative stress and not GSH depletion alters transporter activity without changing transporter expression.

  16. Structure and mechanism of a Na+-independent amino acid transporter.

    PubMed

    Shaffer, Paul L; Goehring, April; Shankaranarayanan, Aruna; Gouaux, Eric

    2009-08-21

    Amino acid, polyamine, and organocation (APC) transporters are secondary transporters that play essential roles in nutrient uptake, neurotransmitter recycling, ionic homeostasis, and regulation of cell volume. Here, we present the crystal structure of apo-ApcT, a proton-coupled broad-specificity amino acid transporter, at 2.35 angstrom resolution. The structure contains 12 transmembrane helices, with the first 10 consisting of an inverted structural repeat of 5 transmembrane helices like the leucine transporter LeuT. The ApcT structure reveals an inward-facing, apo state and an amine moiety of lysine-158 located in a position equivalent to the sodium ion site Na2 of LeuT. We propose that lysine-158 is central to proton-coupled transport and that the amine group serves the same functional role as the Na2 ion in LeuT, thus demonstrating common principles among proton- and sodium-coupled transporters.

  17. Nax loci affect SOS1-like Na+/H+ exchanger expression and activity in wheat

    PubMed Central

    Zhu, Min; Shabala, Lana; Cuin, Tracey A; Huang, Xin; Zhou, Meixue; Munns, Rana; Shabala, Sergey

    2016-01-01

    Salinity stress tolerance in durum wheat is strongly associated with a plant’s ability to control Na+ delivery to the shoot. Two loci, termed Nax1 and Nax2, were recently identified as being critical for this process and the sodium transporters HKT1;4 and HKT1;5 were identified as the respective candidate genes. These transporters retrieve Na+ from the xylem, thus limiting the rates of Na+ transport from the root to the shoot. In this work, we show that the Nax loci also affect activity and expression levels of the SOS1-like Na+/H+ exchanger in both root cortical and stelar tissues. Net Na+ efflux measured in isolated steles from salt-treated plants, using the non-invasive ion flux measuring MIFE technique, decreased in the sequence: Tamaroi (parental line)>Nax1=Nax2>Nax1:Nax2 lines. This efflux was sensitive to amiloride (a known inhibitor of the Na+/H+ exchanger) and was mirrored by net H+ flux changes. TdSOS1 relative transcript levels were 6–10-fold lower in Nax lines compared with Tamaroi. Thus, it appears that Nax loci confer two highly complementary mechanisms, both of which contribute towards reducing the xylem Na+ content. One enhances the retrieval of Na+ back into the root stele via HKT1;4 or HKT1;5, whilst the other reduces the rate of Na+ loading into the xylem via SOS1. It is suggested that such duality plays an important adaptive role with greater versatility for responding to a changing environment and controlling Na+ delivery to the shoot. PMID:26585227

  18. Ouabain Regulates CFTR-Mediated Anion Secretion and Na,K-ATPase Transport in ADPKD Cells

    PubMed Central

    Jansson, Kyle; Venugopal, Jessica; Sánchez, Gladis; Magenheimer, Brenda S.; Reif, Gail A.; Wallace, Darren P.; Calvet, James P.

    2015-01-01

    Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) requires the transepithelial secretion of fluid into the cyst lumen. We previously showed that physiological amounts of ouabain enhance cAMP-dependent fluid secretion and cyst growth of human ADPKD cyst epithelial cells in culture and formation of cyst-like dilations in metanephric kidneys from Pkd1 mutant mice. Here, we investigated the mechanisms by which ouabain promotes cAMP-dependent fluid secretion and cystogenesis. Ouabain (3 nM) enhanced cAMP-induced cyst-like dilations in embryonic kidneys from Pkd1m1Bei mice, but had no effect on metanephroi from Pkd1m1Bei mice that lack expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, ouabain stimulation of cAMP-induced fluid secretion and in vitro cyst growth of ADPKD cells were abrogated by CFTR inhibition, showing that CFTR is required for ouabain effects on ADPKD fluid secretion. Moreover, ouabain directly enhanced the cAMP-dependent Cl− efflux mediated by CFTR in ADPKD monolayers. Ouabain increased the trafficking of CFTR to the plasma membrane and upregulated the expression of the CFTR activator PDZK1. Finally, ouabain decreased plasma membrane expression and activity of the Na,K-ATPase in ADPKD cells. Altogether, these results show that ouabain enhances net fluid secretion and cyst formation by activating apical anion secretion via CFTR and decreasing basolateral Na+ transport via Na,K-ATPase. These results provide new information on the mechanisms by which ouabain affects ADPKD cells and further highlight the importance of ouabain as a non-genomic stimulator of cystogenesis in ADPKD. PMID:26289599

  19. Measurement of mitochondrial Ca2+ transport mediated by three transport proteins: VDAC1, the Na+/Ca2+ exchanger, and the Ca2+ uniporter.

    PubMed

    Ben-Hail, Danya; Palty, Raz; Shoshan-Barmatz, Varda

    2014-02-01

    Ca(2+) is a ubiquitous cellular signal, with changes in intracellular Ca(2+) concentration not only stimulating a number of intercellular events but also triggering cell death pathways, including apoptosis. Mitochondrial Ca(2+) uptake and release play pivotal roles in cellular physiology by regulating intracellular Ca(2+) signaling, energy metabolism and cell death. Ca(2+) transport across the inner and outer mitochondrial membranes is mediated by several proteins, including channels, antiporters, and a uniporter. In this article, we present the background to several methods now established for assaying mitochondrial Ca(2+) transport activity across both mitochondrial membranes. The first of these is Ca(2+) transport mediated by the outer mitochondrial protein, the voltage-dependent anion-selective channel protein 1 (VDAC1, also known as porin 1), both as a purified protein reconstituted into a planar lipid bilayer (PLB) or into liposomes and as a mitochondrial membrane-embedded protein. The second method involves isolated mitochondria for assaying the activity of an inner mitochondrial membrane transport protein, the mitochondrial Ca(2+) uniporter (MCU) that transports Ca(2+) and is powered by the steep mitochondrial membrane potential. In the event of Ca(2+) overload, this leads to opening of the mitochondrial permeability transition pore (MPTP) and cell death. The third method describes how Na(+)-dependent mitochondrial Ca(2+) efflux mediated by mitochondrial NCLX, a member of the Na(+)/Ca(2+) exchanger superfamily, can be assayed in digitonin-permeabilized HEK-293 cells. The Ca(2+)-transport assays can be performed under various conditions and in combination with inhibitors, allowing detailed characterization of the transport activity of interest.

  20. Effects of glial glutamate transporter inhibitors on intracellular Na+ in mouse astrocytes.

    PubMed

    Chatton, J Y; Shimamoto, K; Magistretti, P J

    2001-03-02

    The effects of inhibitors of the glial Na+/glutamate co-transporter on the intracellular Na+ concentration ([Na+](i)) were investigated in mouse cortical astrocytes. [Na+](i) was monitored by fluorescence microscopy on single astrocytes using the Na+-sensitive probe sodium-binding benzofuran isophtalate. Application of the competitive inhibitors threo-beta-hydroxyaspartate (THA) and trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC) resulted in robust and reversible increases in [Na+](i) that were comparable in shape to the response to glutamate but about twice lower in amplitude. As previously observed with glutamate, the amplitude of the [Na+](i) response to these compounds was concentration-dependent with EC(50) values of 11.1 microM (THA) and 7.6 microM (t-PDC), as was the initial rate of [Na+](i) rise (EC(50) values of 14.8 microM for THA and 11.5 microM for t-PDC). Both compounds diminished the response to subsequent glutamate applications, possibly because of an inhibitory effect of the intracellularly-accumulated compounds. In comparison, the newly-developed compound threo-beta-benzyloxyaspartate (TBOA) alone did not cause any significant alteration of [Na+](i) up to a concentration of 500 microM . TBOA inhibited the [Na+](i) response evoked by 200 microM glutamate in a concentration-dependent manner with IC(50) values of 114 and 63 microM, as measured on the amplitude and the initial rate, respectively. The maximum inhibition of glutamate-evoked [Na+](i) increase by TBOA was approximately 70%. The residual response persisted in the presence of a non-NMDA receptor antagonist or the inhibitor of the GLT-1 glutamate transporters, dihydrokainate (DHK). In view of the complete reversibility of its effects, TBOA represents a very useful pharmacological tool for studies of glutamate transporters.

  1. Ratchet transport powered by chiral active particles

    PubMed Central

    Ai, Bao-quan

    2016-01-01

    We numerically investigate the ratchet transport of mixtures of active and passive particles in a transversal asymmetric channel. A big passive particle is immersed in a ‘sea’ of active particles. Due to the chirality of active particles, the longitudinal directed transport is induced by the transversal asymmetry. For the active particles, the chirality completely determines the direction of the ratchet transport, the counterclockwise and clockwise particles move to the opposite directions and can be separated. However, for the passive particle, the transport behavior becomes complicated, the direction is determined by competitions among the chirality, the self-propulsion speed, and the packing fraction. Interestingly, within certain parameters, the passive particle moves to the left, while active particles move to the right. In addition, there exist optimal parameters (the chirality, the height of the barrier, the self-propulsion speed and the packing fraction) at which the rectified efficiency takes its maximal value. Our findings could be used for the experimental pursuit of the ratchet transport powered by chiral active particles. PMID:26795952

  2. Na(+) transport, and the E(1)P-E(2)P conformational transition of the Na(+)/K(+)-ATPase.

    PubMed Central

    Babes, A; Fendler, K

    2000-01-01

    We have used admittance analysis together with the black lipid membrane technique to analyze electrogenic reactions within the Na(+) branch of the reaction cycle of the Na(+)/K(+)-ATPase. ATP release by flash photolysis of caged ATP induced changes in the admittance of the compound membrane system that are associated with partial reactions of the Na(+)/K(+)-ATPase. Frequency spectra and the Na(+) dependence of the capacitive signal are consistent with an electrogenic or electroneutral E(1)P <--> E(2)P conformational transition which is rate limiting for a faster electrogenic Na(+) dissociation reaction. We determine the relaxation rate of the rate-limiting reaction and the equilibrium constants for both reactions at pH 6.2-8.5. The relaxation rate has a maximum value at pH 7.4 (approximately 320 s(-1)), which drops to acidic (approximately 190 s(-1)) and basic (approximately 110 s(-1)) pH. The E(1)P <--> E(2)P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant approximately 0.8) but moves more to the E(1)P side at basic pH 8.5 (equilibrium constant approximately 0.4). The Na(+) affinity at the extracellular binding site decreases from approximately 900 mM at pH 6.2 to approximately 200 mM at pH 8.5. The results suggest that during Na(+) transport the free energy supplied by the hydrolysis of ATP is mainly used for the generation of a low-affinity extracellular Na(+) discharge site. Ionic strength and lyotropic anions both decrease the relaxation rate. However, while ionic strength does not change the position of the conformational equilibrium E(1)P <--> E(2)P, lyotropic anions shift it to E(1)P. PMID:11053130

  3. Stochastic steps in secondary active sugar transport.

    PubMed

    Adelman, Joshua L; Ghezzi, Chiara; Bisignano, Paola; Loo, Donald D F; Choe, Seungho; Abramson, Jeff; Rosenberg, John M; Wright, Ernest M; Grabe, Michael

    2016-07-05

    Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.

  4. Stochastic steps in secondary active sugar transport

    PubMed Central

    Adelman, Joshua L.; Ghezzi, Chiara; Bisignano, Paola; Loo, Donald D. F.; Choe, Seungho; Abramson, Jeff; Rosenberg, John M.; Wright, Ernest M.; Grabe, Michael

    2016-01-01

    Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state. PMID:27325773

  5. The endocannabinoid system in renal cells: regulation of Na+ transport by CB1 receptors through distinct cell signalling pathways

    PubMed Central

    Sampaio, L S; Taveira Da Silva, R; Lima, D; Sampaio, C L C; Iannotti, F A; Mazzarella, E; Di Marzo, V; Vieyra, A; Reis, R A M; Einicker-Lamas, M

    2015-01-01

    Background and Purpose The function of the endocannabinoid system (ECS) in renal tissue is not completely understood. Kidney function is closely related to ion reabsorption in the proximal tubule, the nephron segment responsible for the re-absorption of 70–80% of the filtrate. We studied the effect of compounds modulating the activity of cannabinoid (CB) receptors on the active re-absorption of Na+ in LLC-PK1 cells. Experimental Approach Changes in Na+/K+-ATPase activity were assessed after treatment with WIN55,212-2 (WIN), a non-selective lipid agonist, and haemopressin (HP), an inverse peptide agonist at CB1 receptors. Pharmacological tools were used to investigate the signalling pathways involved in the modulation of Na+ transport. Key Results In addition to CB1 and CB2 receptors and TRPV1 channels, the mRNAs encoding for enzymes of the ECS were also expressed in LLC-PK1. WIN (10−7 M) and HP (10−6 M) altered Na+ re-absorption in LLC-PK1 in a dual manner. They both acutely (after 1 min) increased Na+/K+-ATPase activity in a TRPV1 antagonist-sensitive way. WIN's stimulating effect persisted for 30 min, and this effect was partially blocked by a CB1 antagonist or a PKC inhibitor. In contrast, HP inhibited Na+/K+-ATPase after 30 min incubation, and this effect was attenuated by a CB1 antagonist or a PKA inhibitor. Conclusion and Implications The ECS is expressed in LLC-PK1 cells. Both CB1 receptors and TRPV1 channels regulate Na+/K+-ATPase activity in these cells, and are modulated by lipid and peptide CB1 receptor ligands, which act via different signalling pathways. PMID:25537261

  6. In rat hepatocytes, the hypertonic activation of Na(+) conductance and Na(+)-K(+)-2Cl(-) symport--but not Na(+)-H(+) antiport--is mediated by protein kinase C.

    PubMed

    Heinzinger, H; van den Boom, F; Tinel, H; Wehner, F

    2001-11-01

    1. The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an import of extracellular Na(+) via Na(+) conductance, Na(+)-K(+)-2Cl(-) symport, and Na(+)-H(+) antiport. 2. Here, the protein kinase inhibitors staurosporine (100 nmol l(-1)) and bis-indolyl-maleimide I (400 nmol l(-1)) were used to test for a possible contribution of protein kinase C (PKC) to the hypertonic activation of these transporters in confluent primary cultures. 3. Stimulation of Na(+) conductance was monitored: (i) by use of a differential approach based on Na(+) fluxes, (ii) by means of cable analysis, and (iii) in experiments with low Na(+) pulses. All three experimental protocols in concert demonstrated a block of the activation of Na(+) conductance by staurosporine and bis-indolyl-maleimide I. 4. In addition, both compounds significantly reduced the hypertonic activation of Na(+)-K(+)-2Cl(-) symport (quantified on the basis of furosemide-sensitive (86)Rb(+) uptake) to approximately 30 %. 5. In contrast, neither staurosporine nor bis-indolyl-maleimide I had any detectable effect on the hypertonicity-induced alkalinization of cell pH via Na(+)-H(+) antiport (determined fluorometrically). 6. Staurosporine and bis-indolyl-maleimide I completely blocked the RVI of rat hepatocytes (quantified by means of confocal laser-scanning microscopy). The high efficiency of the block suggests an additional inhibitory effect of both compounds on the activity of Na(+)/K(+)-ATPase (determined as ouabain-sensitive (86)Rb(+) uptake). 7. It is concluded that the hypertonic activation of rat hepatocyte Na(+) conductance and Na(+)-K(+)-2Cl(-) symport--but not Na(+)-H(+) antiport--is probably mediated by PKC.

  7. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter.

    PubMed

    Lu, Min; Symersky, Jindrich; Radchenko, Martha; Koide, Akiko; Guo, Yi; Nie, Rongxin; Koide, Shohei

    2013-02-05

    Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na(+) or H(+) gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na(+)-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs(+) (a Na(+) congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation-π interaction in the Na(+)-binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate- and cation-binding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na(+)-induced drug export. Based on our structural and functional analyses, we suggest that Na(+) triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport.

  8. Carbonic anhydrase II increases the activity of the human electrogenic Na+/HCO3- cotransporter.

    PubMed

    Becker, Holger M; Deitmer, Joachim W

    2007-05-04

    Several acid/base-coupled membrane transporters, such as the electrogenic sodium-bicarbonate cotransporter (NBCe1), have been shown to bind to different carbonic anhydrase isoforms to create a "transport metabolon." We have expressed NBCe1 derived from human kidney in oocytes of Xenopus leavis and determined its transport activity by recording the membrane current in voltage clamp, and the cytosolic H(+) and Na(+) concentrations using ion-selective microelectrodes. When carbonic anhydrase isoform II (CAII) had been injected into oocytes, the membrane current and the rate of cytosolic Na(+) rise, indicative for NBCe1 activity, increased significantly with the amount of injected CAII (2-200 ng). The CAII inhibitor ethoxyzolamide reversed the effects of CAII on the NBCe1 activity. Co-expressing wild-type CAII or NH(2)-terminal mutant CAII together with NBCe1 provided similar results, whereas co-expressing the catalytically inactive CAII mutant V143Y had no effect on NBCe1 activity. Mass spectrometric analysis and the rate of cytosolic H(+) change following addition of CO(2)/HCO(3)(-) confirmed the catalytic activity of injected and expressed CAII in oocytes. Our results show that the transport capacity of NBCe1 is enhanced by the catalytic activity of CAII, in line with the notion that CAII forms a transport metabolon with NBCe1.

  9. Long-term alcohol exposure elicits hippocampal nonsynaptic epileptiform activity changes associated with expression and functional changes in NKCC1, KCC2 co-transporters and Na(+)/K(+)-ATPase.

    PubMed

    Santos, Luiz E C; Rodrigues, Antônio M; Lopes, Mariana R; Costa, Victor D C; Scorza, Carla A; Scorza, Fulvio A; Cavalheiro, Esper A; Almeida, Antônio-Carlos G

    2017-01-06

    Nonsynaptic mechanism changes, particularly the enhancement of NKCC1 expression in the dentate gyrus (DG) after 4weeks of ethanol consumption, motivate the present work, in which rats were submitted to a period of chronic consumption (12weeks). Four groups of six animals (6-week-old male Wistar rats) were formed, including the control (C), ethanol 1 (E1), ethanol 2 (E2) and ethanol 3 (E3) groups. The rats in the E1, E2 and E3 groups were treated daily with a 30% v/v solution of ethanol, administered via oral gavage (1.0, 2.0 and 3.0g/kg, respectively). Nonsynaptic epileptiform activities (NEA) were induced by means of the zero-Ca(2+) and high-K(+) model using hippocampal slices and were recorded in the DG. The presence of NKCC1, KCC2, α1-Na(+)/K(+)-ATPase and GFAP immunoreactivity was analyzed. The results demonstrate that alcohol consumption changes NEA, and these changes are more prominent at the lower dosage. An increase in the DC shifts associated with epileptiform discharges was present with the low dose. This increase was correlated with the increment of NKCC1 expression. Confocal microscopy images indicate the NKCC1 increase was pronounced in the initial axonal segment of granule cells. The blockage of these cotransporters during NEA induction with bumetanide suppressed the DC shift increase and diminished all parameters of NEA that were quantified for all groups treated with ethanol. Therefore, the increase in NKCC1 expression and the effective activity of this cotransporter, which were observed in the treated groups, suggest that drugs that act for block NKCC1 represent promising strategies for diminishing the effects of alcohol damage on the brain.

  10. Development of novel active transport membrande devices

    SciTech Connect

    Laciak, D.V.

    1994-11-01

    Air Products has undertaken a research program to fabricate and evaluate gas separation membranes based upon promising ``active-transport`` (AT) materials recently developed in our laboratories. Active Transport materials are ionic polymers and molten salts which undergo reversible interaction or reaction with ammonia and carbon dioxide. The materials are useful for separating these gases from mixtures with hydrogen. Moreover, AT membranes have the unique property of possessing high permeability towards ammnonia and carbon dioxide but low permeability towards hydrogen and can thus be used to permeate these components from a gas stream while retaining hydrogen at high pressure.

  11. Sodium-coupled and electrogenic transport of B-complex vitamin nicotinic acid by slc5a8, a member of the Na/glucose co-transporter gene family

    PubMed Central

    Gopal, Elangovan; Fei, You-Jun; Miyauchi, Seiji; Zhuang, Lina; Prasad, Puttur D.; Ganapathy, Vadivel

    2005-01-01

    SMCT (sodium-coupled monocarboxylate transporter; slc5a8) is a Na+-coupled transporter for lactate, pyruvate and short-chain fatty acids. Similar to these already known substrates of SMCT, the water-soluble B-complex vitamin nicotinic acid also exists as a monocarboxylate anion (nicotinate) under physiological conditions. Therefore we evaluated the ability of SMCT to mediate the uptake of nicotinate. In mammalian cells, the cloned mouse SMCT (slc5a8) induced the uptake of nicotinate. The SMCT-induced uptake was Na+-dependent. The Michaelis constant for the uptake process was 296±88 μM. The Na+-activation kinetics indicated that at least two Na+ ions are involved in the process. Among the various structural analogues tested, nicotinate was the most effective substrate. Nicotinamide and methylnicotinate were not recognized by the transporter. 2-Pyrazine carboxylate and isonicotinate interacted with the transporter to a moderate extent. SMCT-mediated uptake of nicotinate was inhibited by lactate and pyruvate. In the Xenopus laevis oocyte expression system, SMCT-mediated nicotinate transport was electrogenic, as evident from the nicotinate-induced inward currents under voltage-clamp conditions. Substrate-induced currents in this expression system corroborated the substrate specificity determined in the mammalian cell expression system. The kinetic parameters with regard to the affinity of the transporter for nicotinate and the Hill coefficient for Na+ activation, determined by using the oocyte expression system, were also similar to those obtained from the mammalian cell expression system. We conclude that SMCT functions not only as a Na+-coupled transporter for short-chain fatty acids and lactate but also as a Na+-coupled transporter for the water-soluble vitamin nicotinic acid. PMID:15651982

  12. Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae

    PubMed Central

    Mulligan, Christopher; Fitzgerald, Gabriel A.; Wang, Da-Neng

    2014-01-01

    The SLC13 transporter family, whose members play key physiological roles in the regulation of fatty acid synthesis, adiposity, insulin resistance, and other processes, catalyzes the transport of Krebs cycle intermediates and sulfate across the plasma membrane of mammalian cells. SLC13 transporters are part of the divalent anion:Na+ symporter (DASS) family that includes several well-characterized bacterial members. Despite sharing significant sequence similarity, the functional characteristics of DASS family members differ with regard to their substrate and coupling ion dependence. The publication of a high resolution structure of dimer VcINDY, a bacterial DASS family member, provides crucial structural insight into this transporter family. However, marrying this structural insight to the current functional understanding of this family also demands a comprehensive analysis of the transporter’s functional properties. To this end, we purified VcINDY, reconstituted it into liposomes, and determined its basic functional characteristics. Our data demonstrate that VcINDY is a high affinity, Na+-dependent transporter with a preference for C4- and C5-dicarboxylates. Transport of the model substrate, succinate, is highly pH dependent, consistent with VcINDY strongly preferring the substrate’s dianionic form. VcINDY transport is electrogenic with succinate coupled to the transport of three or more Na+ ions. In contrast to succinate, citrate, bound in the VcINDY crystal structure (in an inward-facing conformation), seems to interact only weakly with the transporter in vitro. These transport properties together provide a functional framework for future experimental and computational examinations of the VcINDY transport mechanism. PMID:24821967

  13. Upregulation of Na+ transporter abundances in response to chronic thiazide or loop diuretic treatment in rats.

    PubMed

    Na, Ki Young; Oh, Yoon Kyu; Han, Jin Suk; Joo, Kwon Wook; Lee, Jung Sang; Earm, Jae-Ho; Knepper, Mark A; Kim, Gheun-Ho

    2003-01-01

    Furosemide and hydrochlorothiazide (HCTZ) exert their diuretic actions by binding to apical Na(+) transporters, viz., the Na(+)-K(+)-2Cl(-) cotransporter in the thick ascending limb and the Na(+)-Cl(-) cotransporter in the distal convoluted tubule, respectively. We carried out semiquantitative immunoblotting and immunohistochemistry of rat kidneys to investigate whether chronic administration of furosemide or HCTZ is associated with compensatory changes in the abundance of Na(+) transporters downstream from the primary site of action. Osmotic minipumps were implanted into Sprague-Dawley rats to deliver furosemide (12 mg/day) or HCTZ (3.75 mg/day) for 7 days. To prevent volume depletion, all animals were offered tap water and a solution containing 0.8% NaCl and 0.1% KCl as drinking fluid. The diuretic/natriuretic response was quantified in response to both agents by using quantitative urine collections. Semiquantitative immunoblotting revealed that the abundances of thick ascending limb Na(+)-K(+)-2Cl(-) cotransporter and all three subunits of the epithelial Na(+) channel (ENaC) were increased by furosemide infusion. HCTZ infusion increased the abundances of thiazide-sensitive Na(+)-Cl(-) cotransporter and beta-ENaC in the cortex and beta- and gamma-ENaC in the outer medulla. Consistent with these results, beta-ENaC immunohistochemistry showed a remarkable increase in immunoreactivity in the principal cells of collecting ducts with either diuretic treatment. These increases in the abundance of Na(+) transporters in response to chronic diuretic treatment may account for the generation of diuretic tolerance associated with long-term diuretic use.

  14. Health Impacts of Active Transportation in Europe

    PubMed Central

    Rojas-Rueda, David; de Nazelle, Audrey; Andersen, Zorana J.; Braun-Fahrländer, Charlotte; Bruha, Jan; Bruhova-Foltynova, Hana; Desqueyroux, Hélène; Praznoczy, Corinne; Ragettli, Martina S.; Tainio, Marko; Nieuwenhuijsen, Mark J.

    2016-01-01

    Policies that stimulate active transportation (walking and bicycling) have been related to heath benefits. This study aims to assess the potential health risks and benefits of promoting active transportation for commuting populations (age groups 16–64) in six European cities. We conducted a health impact assessment using two scenarios: increased cycling and increased walking. The primary outcome measure was all-cause mortality related to changes in physical activity level, exposure to fine particulate matter air pollution with a diameter <2.5 μm, as well as traffic fatalities in the cities of Barcelona, Basel, Copenhagen, Paris, Prague, and Warsaw. All scenarios produced health benefits in the six cities. An increase in bicycle trips to 35% of all trips (as in Copenhagen) produced the highest benefits among the different scenarios analysed in Warsaw 113 (76–163) annual deaths avoided, Prague 61 (29–104), Barcelona 37 (24–56), Paris 37 (18–64) and Basel 5 (3–9). An increase in walking trips to 50% of all trips (as in Paris) resulted in 19 (3–42) deaths avoided annually in Warsaw, 11(3–21) in Prague, 6 (4–9) in Basel, 3 (2–6) in Copenhagen and 3 (2–4) in Barcelona. The scenarios would also reduce carbon dioxide emissions in the six cities by 1,139 to 26,423 (metric tonnes per year). Policies to promote active transportation may produce health benefits, but these depend of the existing characteristics of the cities. Increased collaboration between health practitioners, transport specialists and urban planners will help to introduce the health perspective in transport policies and promote active transportation. PMID:26930213

  15. Shoot Na+ Exclusion and Increased Salinity Tolerance Engineered by Cell Type–Specific Alteration of Na+ Transport in Arabidopsis[W][OA

    PubMed Central

    Møller, Inge S.; Gilliham, Matthew; Jha, Deepa; Mayo, Gwenda M.; Roy, Stuart J.; Coates, Juliet C.; Haseloff, Jim; Tester, Mark

    2009-01-01

    Soil salinity affects large areas of cultivated land, causing significant reductions in crop yield globally. The Na+ toxicity of many crop plants is correlated with overaccumulation of Na+ in the shoot. We have previously suggested that the engineering of Na+ exclusion from the shoot could be achieved through an alteration of plasma membrane Na+ transport processes in the root, if these alterations were cell type specific. Here, it is shown that expression of the Na+ transporter HKT1;1 in the mature root stele of Arabidopsis thaliana decreases Na+ accumulation in the shoot by 37 to 64%. The expression of HKT1;1 specifically in the mature root stele is achieved using an enhancer trap expression system for specific and strong overexpression. The effect in the shoot is caused by the increased influx, mediated by HKT1;1, of Na+ into stelar root cells, which is demonstrated in planta and leads to a reduction of root-to-shoot transfer of Na+. Plants with reduced shoot Na+ also have increased salinity tolerance. By contrast, plants constitutively expressing HKT1;1 driven by the cauliflower mosaic virus 35S promoter accumulated high shoot Na+ and grew poorly. Our results demonstrate that the modification of a specific Na+ transport process in specific cell types can reduce shoot Na+ accumulation, an important component of salinity tolerance of many higher plants. PMID:19584143

  16. Single Na+ channels activated by veratridine and batrachotoxin

    PubMed Central

    1987-01-01

    Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded. PMID:2435846

  17. NaDC3 Induces Premature Cellular Senescence by Promoting Transport of Krebs Cycle Intermediates, Increasing NADH, and Exacerbating Oxidative Damage.

    PubMed

    Ma, Yuxiang; Bai, Xue-Yuan; Du, Xuan; Fu, Bo; Chen, Xiangmei

    2016-01-01

    High-affinity sodium-dependent dicarboxylate cotransporter 3 (NaDC3) is a key metabolism-regulating membrane protein responsible for transport of Krebs cycle intermediates. NaDC3 is upregulated as organs age, but knowledge regarding the underlying mechanisms by which NaDC3 modulates mammalian aging is limited. In this study, we showed that NaDC3 overexpression accelerated cellular senescence in young human diploid cells (MRC-5 and WI-38) and primary renal tubular cells, leading to cell cycle arrest in G1 phase and increased expression of senescent biomarkers, senescence-associated β-galactosidase and p16. Intracellular levels of reactive oxygen species, 8-hydroxy-2'-deoxyguanosine, malondialdehyde, and carbonyl were significantly enhanced, and activities of respiratory complexes I and III and ATP level were significantly decreased in NaDC3-infected cells. Stressful premature senescent phenotypes induced by NaDC3 were markedly ameliorated via treatment with the antioxidants Tiron and Tempol. High expression of NaDC3 caused a prominent increase in intracellular levels of Krebs cycle intermediates and NADH. Exogenous NADH and NAD(+) may aggravate and attenuate the aging phenotypes induced by NaDC3, respectively. These results suggest that NaDC3 can induce premature cellular senescence by promoting the transport of Krebs cycle intermediates, increasing generation of NADH and reactive oxygen species and leading to oxidative damage. Our results clarify the aging signaling pathway regulated by NaDC3.

  18. Regional differences in rat conjunctival ion transport activities.

    PubMed

    Yu, Dongfang; Thelin, William R; Rogers, Troy D; Stutts, M Jackson; Randell, Scott H; Grubb, Barbara R; Boucher, Richard C

    2012-10-01

    Active ion transport and coupled osmotic water flow are essential to maintain ocular surface health. We investigated regional differences in the ion transport activities of the rat conjunctivas and compared these activities with those of cornea and lacrimal gland. The epithelial sodium channel (ENaC), sodium/glucose cotransporter 1 (Slc5a1), transmembrane protein 16 (Tmem16a, b, f, and g), cystic fibrosis transmembrane conductance regulator (Cftr), and mucin (Muc4, 5ac, and 5b) mRNA expression was characterized by RT-PCR. ENaC proteins were measured by Western blot. Prespecified regions (palpebral, fornical, and bulbar) of freshly isolated conjunctival tissues and cell cultures were studied electrophysiologically with Ussing chambers. The transepithelial electrical potential difference (PD) of the ocular surface was also measured in vivo. The effect of amiloride and UTP on the tear volume was evaluated in lacrimal gland excised rats. All selected genes were detected but with different expression patterns. We detected αENaC protein in all tissues, βENaC in palpebral and fornical conjunctiva, and γENaC in all tissues except lacrimal glands. Electrophysiological studies of conjunctival tissues and cell cultures identified functional ENaC, SLC5A1, CFTR, and TMEM16. Fornical conjunctiva exhibited the most active ion transport under basal conditions amongst conjunctival regions. PD measurements confirmed functional ENaC-mediated Na(+) transport on the ocular surface. Amiloride and UTP increased tear volume in lacrimal gland excised rats. This study demonstrated that the different regions of the conjunctiva exhibited a spectrum of ion transport activities. Understanding the specific functions of distinct regions of the conjunctiva may foster a better understanding of the physiology maintaining hydration of the ocular surface.

  19. Regional differences in rat conjunctival ion transport activities

    PubMed Central

    Yu, Dongfang; Thelin, William R.; Rogers, Troy D.; Stutts, M. Jackson; Randell, Scott H.; Grubb, Barbara R.

    2012-01-01

    Active ion transport and coupled osmotic water flow are essential to maintain ocular surface health. We investigated regional differences in the ion transport activities of the rat conjunctivas and compared these activities with those of cornea and lacrimal gland. The epithelial sodium channel (ENaC), sodium/glucose cotransporter 1 (Slc5a1), transmembrane protein 16 (Tmem16a, b, f, and g), cystic fibrosis transmembrane conductance regulator (Cftr), and mucin (Muc4, 5ac, and 5b) mRNA expression was characterized by RT-PCR. ENaC proteins were measured by Western blot. Prespecified regions (palpebral, fornical, and bulbar) of freshly isolated conjunctival tissues and cell cultures were studied electrophysiologically with Ussing chambers. The transepithelial electrical potential difference (PD) of the ocular surface was also measured in vivo. The effect of amiloride and UTP on the tear volume was evaluated in lacrimal gland excised rats. All selected genes were detected but with different expression patterns. We detected αENaC protein in all tissues, βENaC in palpebral and fornical conjunctiva, and γENaC in all tissues except lacrimal glands. Electrophysiological studies of conjunctival tissues and cell cultures identified functional ENaC, SLC5A1, CFTR, and TMEM16. Fornical conjunctiva exhibited the most active ion transport under basal conditions amongst conjunctival regions. PD measurements confirmed functional ENaC-mediated Na+ transport on the ocular surface. Amiloride and UTP increased tear volume in lacrimal gland excised rats. This study demonstrated that the different regions of the conjunctiva exhibited a spectrum of ion transport activities. Understanding the specific functions of distinct regions of the conjunctiva may foster a better understanding of the physiology maintaining hydration of the ocular surface. PMID:22814399

  20. Fluid transport by active elastic membranes

    NASA Astrophysics Data System (ADS)

    Evans, Arthur A.; Lauga, Eric

    2011-09-01

    A flexible membrane deforming its shape in time can self-propel in a viscous fluid. Alternatively, if the membrane is anchored, its deformation will lead to fluid transport. Past work in this area focused on situations where the deformation kinematics of the membrane were prescribed. Here we consider models where the deformation of the membrane is not prescribed, but instead the membrane is internally forced. Both the time-varying membrane shape and the resulting fluid motion result then from a balance between prescribed internal active stresses, internal passive resistance, and external viscous stresses. We introduce two specific models for such active internal forcing: one where a distribution of active bending moments is prescribed, and one where active inclusions exert normal stresses on the membrane by pumping fluid through it. In each case, we asymptotically calculate the membrane shape and the fluid transport velocities for small forcing amplitudes, and recover our results using scaling analysis.

  1. Phospholemman Overexpression Inhibits Na+-K+-ATPase in Adult Rat Cardiac Myocytes: Relevance to Decreased Na+ pump Activity in Post-Infarction Myocytes

    PubMed Central

    Zhang, Xue-Qian; Moorman, J. Randall; Ahlers, Belinda A.; Carl, Lois L.; Lake, Douglas E.; Song, Jianliang; Mounsey, J. Paul; Tucker, Amy L.; Chan, Yiu-mo; Rothblum, Lawrence I.; Stahl, Richard C.; Carey, David J.; Cheung, Joseph Y.

    2005-01-01

    Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postinfarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na+-K+-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by 2- and 4-fold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine68 of PLM. Na+-K+-ATPase activity was measured as ouabain-sensitive Na+-K+ pump current (Ip). Compared to control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly (P<0.0001) lower at similar membrane voltages, pipette Na+ ([Na+]pip) and extracellular K+ concentrations ([K+]o). From −70 to +60 mV, neither [Na+]pip nor [K+]o required to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased Vmax without appreciable changes in Km for Na+ and K+ in PLM overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na+-K+-ATPase expression since there were no changes in either protein or messenger RNA levels of either α1 or α2 isoforms of Na+-K+-ATPase. In native rat cardiac myocytes, PLM co-immunoprecipitated with α-subunits of Na+-K+-ATPase. Inhibition of Na+-K+-ATPase by PLM overexpression, in addition to previously reported decrease in Na+-K+-ATPase expression, may explain altered Vmax but not Km of Na+-K+-ATPase in postinfarction rat myocytes. PMID:16195392

  2. Transport mechanism and pH regulation of the Na+/H+ antiporter NhaA from Escherichia coli: an electrophysiological study.

    PubMed

    Mager, Thomas; Rimon, Abraham; Padan, Etana; Fendler, Klaus

    2011-07-01

    Using an electrophysiological assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na(+) or H(+) gradients as the driving force. Under symmetrical pH conditions with a Na(+) gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the K(m) for Na(+) at acidic pH. Under symmetrical Na(+) concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na(+) and H(+) binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na(+) induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na(+) translocation was found to be associated with the displacement of a negative charge. In conclusion, the electrophysiological assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation.

  3. Functional characterization in Xenopus oocytes of Na+ transport systems from durum wheat reveals diversity among two HKT1;4 transporters.

    PubMed

    Ben Amar, Siwar; Brini, Faiçal; Sentenac, Hervé; Masmoudi, Khaled; Véry, Anne-Aliénor

    2014-01-01

    Plant tolerance to salinity constraint involves complex and integrated functions including control of Na(+) uptake, translocation, and compartmentalization. Several members of the high-affinity K(+) transporter (HKT) family, which comprises plasma-membrane transporters permeable to K(+) and Na(+) or to Na(+) only, have been shown to play major roles in plant Na(+) and K(+) homeostasis. Among them, HKT1;4 has been identified as corresponding to a quantitative trait locus (QTL) of salt tolerance in wheat but was not functionally characterized. Here, we isolated two HKT1;4-type cDNAs from a salt-tolerant durum wheat (Triticum turgidum L. subsp. durum) cultivar, Om Rabia3, and investigated the functional properties of the encoded transporters using a two-electrode voltage-clamp technique, after expression in Xenopus oocytes. Both transporters displayed high selectivity for Na(+), their permeability to other monovalent cations (K(+), Li(+), Cs(+), and Rb(+)) being ten times lower than that to Na(+). Both TdHKT1;4-1 and TdHKT1;4-2 transported Na(+) with low affinity, although the half-saturation of the conductance was observed at a Na(+) concentration four times lower in TdHKT1;4-1 than in TdHKT1;4-2. External K(+) did not inhibit Na(+) transport through these transporters. Quinine slightly inhibited TdHKT1;4-2 but not TdHKT1;4-1. Overall, these data identified TdHKT1;4 transporters as new Na(+)-selective transporters within the HKT family, displaying their own functional features. Furthermore, they showed that important differences in affinity exist among durum wheat HKT1;4 transporters. This suggests that the salt tolerance QTL involving HKT1;4 may be at least in part explained by functional variability among wheat HKT1;4-type transporters.

  4. 39K, 23Na, and 31P NMR Studies of Ion Transport in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Ogino, T.; den Hollander, J. A.; Shulman, R. G.

    1983-09-01

    The relationship between efflux and influx of K+, Na+, and intracellular pH (pHin) in yeast cells upon energizing by oxygenation was studied by using the noninvasive technique of 39K, 23Na, and 31P NMR spectroscopy. By introducing an anionic paramagnetic shift reagent, Dy3+(P3O105-)2, into the medium, NMR signals of intra- and extracellular K+ and Na+ could be resolved, enabling us to study ion transport processes by NMR. Measurements showed that 40% of the intracellular K+ and Na+ in yeast cells contributed to the NMR intensities. By applying this correction factor, the intracellular ion concentrations were determined to be 130-170 mM K+ and 2.5 mM Na+ for fresh yeast cells. With the aid of a home-built solenoidal coil probe for 39K and a double-tuned probe for 23Na and 31P, we could follow time courses of K+ and Na+ transport and of pHin with a time resolution of 1 min. It was shown that H+ extrusion is correlated with K+ uptake and not with Na+ uptake upon energizing yeast cells by oxygenation. When the cells were deenergized after the aerobic period, K+ efflux, H+ influx, and Na+ influx were calculated to be 1.6, 1.5, and 0.15 μ mol/min per ml of cell water, respectively. Therefore, under the present conditions, K+ efflux is balanced by exchange for H+ with an approximate stoichiometry of 1:1.

  5. Molecular dynamics simulations of Na{sup +} and leucine transport by LeuT

    SciTech Connect

    Chen, Rong Chung, Shin-Ho

    2015-08-14

    Molecular dynamics simulations are used to gain insight into the binding of Na{sup +} and leucine substrate to the bacterial amino acid transporter LeuT, focusing on the crystal structures of LeuT in the outward-open and inward-open states. For both conformations of LeuT, a third Na{sup +} binding site involving Glu290 in addition to the two sites identified from the crystal structures is observed. Once the negative charge from Glu290 in the inward-open LeuT is removed, the ion bound to the third site is ejected from LeuT rapidly, suggesting that the protonation state of Glu290 regulates Na{sup +} binding and release. In Cl{sup −}-dependent transporters where Glu290 is replaced by a neutral serine, a Cl{sup −} ion would be required to replace the role of Glu290. Thus, the simulations provide insights into understanding Na{sup +} and substrate transport as well as Cl{sup −}-independence of LeuT. - Highlights: • Ion binding site involving Glu290 is identified in the outward- and inward-open LeuT. • Sodium is released from inward-open LeuT once the side chain of Glu290 is protonated. • Protonation state of Glu290 regulates sodium binding and transport in LeuT.

  6. Fast Na/+/-ion transport in skeleton structures. [solid electrolyte applications

    NASA Technical Reports Server (NTRS)

    Goodenough, J. B.; Hong, H. Y.-P.; Kafalas, J. A.

    1976-01-01

    The skeleton structures considered in the investigations consist of a rigid subarray with an interconnected interstitial space in which ions move in three dimensions. The classes of skeleton structures investigated include the Im3 phase of high-pressure KSbO3, the defect-pyrochlore structure illustrated by RbMgAlF6, and the carnegieite structure of high-temperature NaAlSiO4. A description is given of the results obtained in transport measurements involving dense polycrystalline ceramic disks. Results obtained in the case of the Na(+)-ion transport in Na3Zr2PSi2O12 appear particularly promising concerning the possible use of such substances in solid-electrolyte applications.

  7. Drug Transporters and Na+/H+ Exchange Regulatory Factor PSD-95/Drosophila Discs Large/ZO-1 Proteins

    PubMed Central

    Walsh, Dustin R.; Nolin, Thomas D.

    2015-01-01

    Drug transporters govern the absorption, distribution, and elimination of pharmacologically active compounds. Members of the solute carrier and ATP binding-cassette drug transporter family mediate cellular drug uptake and efflux processes, thereby coordinating the vectorial movement of drugs across epithelial barriers. To exert their physiologic and pharmacological function in polarized epithelia, drug transporters must be targeted and stabilized to appropriate regions of the cell membrane (i.e., apical versus basolateral). Despite the critical importance of drug transporter membrane targeting, the mechanisms that underlie these processes are largely unknown. Several clinically significant drug transporters possess a recognition sequence that binds to PSD-95/Drosophila discs large/ZO-1 (PDZ) proteins. PDZ proteins, such as the Na+/H+ exchanger regulatory factor (NHERF) family, act to stabilize and organize membrane targeting of multiple transmembrane proteins, including many clinically relevant drug transporters. These PDZ proteins are normally abundant at apical membranes, where they tether membrane-delimited transporters. NHERF expression is particularly high at the apical membrane in polarized tissue such as intestinal, hepatic, and renal epithelia, tissues important to drug disposition. Several recent studies have highlighted NHERF proteins as determinants of drug transporter function secondary to their role in controlling membrane abundance and localization. Mounting evidence strongly suggests that NHERF proteins may have clinically significant roles in pharmacokinetics and pharmacodynamics of several pharmacologically active compounds and may affect drug action in cancer and chronic kidney disease. For these reasons, NHERF proteins represent a novel class of post-translational mediators of drug transport and novel targets for new drug development. PMID:26092975

  8. Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model

    ERIC Educational Resources Information Center

    Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

    2010-01-01

    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

  9. NEUTRALIZATION OF THE ASPARTIC ACID RESIDUE D367, BUT NOT D454, INHIBITS BINDING OF NA+ TO THE GLUTAMATE-FREE FORM AND CYCLING OF THE GLUTAMATE TRANSPORTER EAAC1

    PubMed Central

    Tao, Zhen; Zhang, Zhou; Grewer, Christof

    2008-01-01

    Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na+ ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, D367 and D454, in Na+ cotransport. In order to test the effect of charge neutralization mutations in these positions on Na+ binding to the glutamate-free transporter, we recorded the Na+-induced anion leak current to determine the Km of EAAC1 for Na+. For EAAC1WT, this Km was determined as 120 mM. When the negative charge of D367 was neutralized by mutagenesis to asparagine, Na+ activated the anion leak current with a Km of about 2 M, indicating dramatically impaired Na+ binding to the mutant transporter. In contrast, the Na+ affinity of EAAC1D454N was virtually unchanged compared to the wild type transporter (Km = 90 mM). The reduced occupancy of the Na+ binding site of EAAC1D367N resulted in a dramatic reduction in glutamate affinity (Km = 3.6 mM, 140 mM [Na+]), which could be partially overcome by increasing extracellular [Na+]. In addition to impairing Na+ binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which D367, but not D454 is involved in coordinating the bound Na+ in the glutamate-free transporter form. PMID:16478724

  10. FCCP depolarizes plasma membrane potential by activating proton and Na+ currents in bovine aortic endothelial cells.

    PubMed

    Park, Kyu-Sang; Jo, Inho; Pak, Kim; Bae, Sung-Won; Rhim, Hyewhon; Suh, Suk-Hyo; Park, Jin; Zhu, Hong; So, Insuk; Kim, Ki Whan

    2002-01-01

    We investigated the effects of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore and uncoupler of mitochondrial oxidative phosphorylation in mitochondria, on plasma membrane potential and ionic currents in bovine aortic endothelial cells (BAECs). The membrane potential and ionic currents of BAECs were recorded using the patch-clamp technique in current-clamp and voltage-clamp modes, respectively. FCCP activated ionic currents and depolarized the plasma membrane potential in a dose-dependent manner. Neither the removal of extracellular Ca2+ nor pretreatment with BAPTA/AM affected the FCCP-induced currents, implying that the currents are not associated with the FCCP-induced intracellular [Ca2+]i increase. FCCP-induced currents were significantly influenced by the changes in extracellular or intracellular pH; the increased proton gradient produced by lowering the extracellular pH or intracellular alkalinization augmented the changes in membrane potential and ionic currents caused by FCCP. FCCP-induced currents were significantly reduced under extracellular Na+-free conditions. The reversal potentials of FCCP-induced currents under Na+-free conditions were well fitted to the calculated equilibrium potential for protons. Interestingly, FCCP-induced Na+ transport (subtracted currents, I(control)- I(Na+-free) was closely dependent on extracellular pH, whereas FCCP-induced H+transport was not significantly affected by the absence of Na+. These results suggest that the FCCP-induced ionic currents and depolarization, which are strongly dependent on the plasmalemmal proton gradient, are likely to be mediated by both H+ and Na+ currents across the plasma membrane. The relationship between H+ and Na+ transport still needs to be determined.

  11. Phosphorylation and activation of the plasma membrane Na+/H+ exchanger (NHE1) during osmotic cell shrinkage.

    PubMed

    Rigor, Robert R; Damoc, Catalina; Phinney, Brett S; Cala, Peter M

    2011-01-01

    The Na(+)/H(+)Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na(+)/H(+) exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na(+)/H(+) exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na(+) transport capacity without affecting transport affinity (K(m)=44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ(32)P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na(+) transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.

  12. Synthesis and Electronic Transport of Hydrothermally Synthesized p-Type Na-Doped SnSe

    NASA Astrophysics Data System (ADS)

    Yang, Zong-Ren; Chen, Wei-Hao; Liu, Chia-Jyi

    2016-11-01

    A series of polycrystalline Sn1-x Na x Se with x = 0.00, 0.02, 0.04 and 0.10 were fabricated using hydrothermal synthesis followed by evacuated-and-encapsulated sintering. The as-fabricated materials were characterized using powder x-ray diffraction and electronic transport. The resulting materials were single phase. Partial replacement of Na for Sn leads to a simultaneous increase of electrical conductivity and thermopower. The x = 0.04 sample has the largest power factor among the series of the samples. Upon partial replacement of Na for Sn, the power factor is significantly enhanced as compared to the undoped SnSe. The maximum ZT value of ˜0.4 was achieved for Sn0.96Na0.04Se at 550 K.

  13. Na(+)-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal.

    PubMed

    O'Kane, R L; Martínez-López, I; DeJoseph, M R; Viña, J R; Hawkins, R A

    1999-11-05

    Na(+)-dependent transporters for glutamate exist on astrocytes (EAAT1 and EAAT2) and neurons (EAAT3). These transporters presumably assist in keeping the glutamate concentration low in the extracellular fluid of brain. Recently, Na(+)-dependent glutamate transport was described on the abluminal membrane of the blood-brain barrier. To determine whether the above-mentioned transporters participate in glutamate transport of the blood-brain barrier, total RNA was extracted from bovine cerebral capillaries. cDNA for EAAT1, EAAT2, and EAAT3 was observed, indicating that mRNA was present. Western blot analysis demonstrated all three transporters were expressed on abluminal membranes, but none was detectable on luminal membranes of the blood-brain barrier. Measurement of transport kinetics demonstrated voltage dependence, K(+)-dependence, and an apparent K(m) of 14 microM (aggregate of the three transporters) at a transmembrane potential of -61 mV. Inhibition of glutamate transport was observed using inhibitors specific for EAAT2 (kainic acid and dihydrokainic acid) and EAAT3 (cysteine). The relative activity of the three transporters was found to be approximately 1:3:6 for EAAT1, EAAT2, and EAAT3, respectively. These transporters may assist in maintaining low glutamate concentrations in the extracellular fluid.

  14. Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells.

    PubMed Central

    Felipe, A; Valdes, R; Santo, B; Lloberas, J; Casado, J; Pastor-Anglada, M

    1998-01-01

    Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the 'epithelial-specific isoform' called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization. PMID:9480921

  15. Bursts of Active Transport in Living Cells

    NASA Astrophysics Data System (ADS)

    Wang, Bo; Kuo, James; Granick, Steve

    2013-11-01

    We show, using a large new data set, that the temporally resolved speed of active cargo transport in living cells follows a scaling law over several decades of time and length. The statistical regularities display a time-averaged shape that we interpret to reflect stress buildup, followed by rapid release. The scaling power law agrees quantitatively with those reported in inanimate systems (jammed colloids and granular media, and magnetic Barkhausen noise), suggesting a common origin in pushing through a crowded environment in a weak force regime. The implied regulation of the speed of active cellular transport due to environmental obstruction results in bursts of speed and acceleration. These findings extend the classical notion of molecular crowding.

  16. Bursts of active transport in living cells.

    PubMed

    Wang, Bo; Kuo, James; Granick, Steve

    2013-11-15

    We show, using a large new data set, that the temporally resolved speed of active cargo transport in living cells follows a scaling law over several decades of time and length. The statistical regularities display a time-averaged shape that we interpret to reflect stress buildup, followed by rapid release. The scaling power law agrees quantitatively with those reported in inanimate systems (jammed colloids and granular media, and magnetic Barkhausen noise), suggesting a common origin in pushing through a crowded environment in a weak force regime. The implied regulation of the speed of active cellular transport due to environmental obstruction results in bursts of speed and acceleration. These findings extend the classical notion of molecular crowding.

  17. Regulation of ion channels and transporters by AMP-activated kinase (AMPK)

    PubMed Central

    Lang, Florian; Föller, Michael

    2014-01-01

    The energy-sensing AMP-activated kinase AMPK ensures survival of energy-depleted cells by stimulating ATP production and limiting ATP utilization. Both energy production and energy consumption are profoundly influenced by transport processes across the cell membane including channels, carriers and pumps. Accordingly, AMPK is a powerful regulator of transport across the cell membrane. AMPK regulates diverse K+ channels, Na+ channels, Ca2+ release activated Ca2+ channels, Cl- channels, gap junctional channels, glucose carriers, Na+/H+-exchanger, monocarboxylate-, phosphate-, creatine-, amino acid-, peptide- and osmolyte-transporters, Na+/Ca2+-exchanger, H+-ATPase and Na+/K+-ATPase. AMPK activates ubiquitin ligase Nedd4–2, which labels several plasma membrane proteins for degradation. AMPK further regulates transport proteins by inhibition of Rab GTPase activating protein (GAP) TBC1D1. It stimulates phosphatidylinositol 3-phosphate 5-kinase PIKfyve and inhibits phosphatase and tensin homolog (PTEN) via glycogen synthase kinase 3β (GSK3β). Moreover, it stabilizes F-actin as well as downregulates transcription factor NF-κB. All those cellular effects serve to regulate transport proteins. PMID:24366036

  18. Characterization of Na(+) transport to gain insight into the mechanism of acid-base and ion regulation in white sturgeon (Acipenser transmontanus).

    PubMed

    Shartau, Ryan B; Brix, Kevin V; Brauner, Colin J

    2017-02-01

    Freshwater fish actively take up ions via specific transporters to counter diffusive losses to their hypotonic environment. While much is known about the specific mechanisms employed by teleosts, almost nothing is known about the basal fishes, such as white sturgeon (Acipenser transmontanus) which may offer insight into the evolution of osmo- and ionoregulation in fishes. We investigated Na(+) uptake in juvenile white sturgeon in the presence and absence of transporter inhibitors. We found that sturgeon acclimated to 100μmoll(-1) Na(+) have Na(+) uptake kinetics typical of teleosts and that a Na(+)/H(+) exchanger (NHE) is the predominant transporter for Na(+) uptake. White sturgeon are tolerant to hypercarbia-induced respiratory acidoses and recover blood pH (pHe) at 1.5kPa PCO2 but not at higher PCO2 (6kPa PCO2) where they preferentially regulate intracellular pH (pHi). It was hypothesized that during exposure to hypercarbia Na(+) uptake would increase at CO2 tensions at which fish were capable of pHe regulation but decrease at higher tensions when they were preferentially regulating pHi. We found that Na(+) uptake did not increase at 1.5kPa PCO2, but at 6kPa PCO2 Na(+) uptake was reduced by 95% while low water pH equivalent to 6kPa PCO2 reduced Na(+) uptake by 71%. Lastly, we measured net acid flux during hypercarbia, which indicates that net acid flux is not associated with Na(+) uptake. These findings indicate Na(+) uptake in sturgeon is not different from freshwater teleosts but is sensitive to hypercarbia and is not associated with pHe compensation during hypercarbia.

  19. [Effect of nitrates on active transport of iodine].

    PubMed

    Szökeová, E; Tajtáková, M; Mirossay, L; Mojzis, J; Langer, P; Marcinová, E; Petrovicová, J; Zemberová, E; Bodnár, J

    2001-11-01

    Active iodine transport into the thyrocyte is catalyzed by the transmembrane transport protein Na+/J- symport (NIS) Nitrates can expel iodine from the bond with this transport protein which was found not only in the thyrocyte membrane but also in the cell membrane of the gastric mucosa. The weight of the thyroid gland in mg was significantly greater even when calculated in relation to body weight in the NIT group of rats who were given for 6 days nitrate by gastric tube (100 mg/kg/day) as compared with controls (CON) 17.56 +/- 8.4, 0.07 +/- 0.03/12.10 +/- 9.57, 0.05 +/- 0.03, P < or = 0.01. A lower thyroid activity in per cent calculated per 1 mg of its weight (1.39 +/- 1.0/2.22 +/- 0.9, P < or = 0.01), a higher activity in blood before removal of the thyroid gland (8.54 +/- 4.09/5.45 +/- 2.78) and a lower one after removal of the thyroid gland (1.09 +/- 0.05/0.21 +/- 0.10) before oral administration of I131 in group NIT, suggests a negative effect of nitrates on active iodine transport not only at the level of the thyrocyte but also possible interaction with iodine at the level of the digestive tract. A significantly higher serum level of TT3 in group NIT (0.66 +/- 0.27/0.44 +/- 0.21, P < or = 0.01 regardless of the TSH serum level (2.31 +/- 1.83/2.64 +/- 1.52) and T4 (22.72 +/- 8.2/25 +/- 11.0) suggests a qualitative change in thyroid hormone production in favour of T3 caused even by short-term nitrate administration.

  20. Synthesis and high temperature transport properties of new quaternary layered selenide NaCuMnSe{sub 2}

    SciTech Connect

    Pavan Kumar, V.; Varadaraju, U.V.

    2014-04-01

    Synthesis and high temperature transport properties of NaCu{sub 1+x}Mn{sub 1−x}Se{sub 2}, (x=0−0.75) a new quaternary layered selenide, are reported. NaCuMnSe{sub 2} crystallizes in a trigonal unit cell with space group of P-3m1 (a=4.1276 Å, c=7.1253 Å). The isovalent substitution of Mn{sup 2+} by Cu{sup 2+} is carried out. All the compositions show semiconducting nature, whereas the Seebeck coefficient increases gradually over the entire measured temperature range. Compositions with x=0 and 0.025 follow thermally activated behavior. With increase in copper concentration the conduction mechanism transforms to 2D variable range hopping (VRH) for x=0.05 and 0.075. - Graphical abstract: Crystal structure of NaCuMnSe{sub 2}. - Highlights: • A new quaternary layered selenide NaCuMnSe{sub 2} is synthesized. • All the compositions show semiconducting nature, whereas the Seebeck coefficient increases gradually over the entire measured temperature range. • Conduction mechanism transforms from thermally activated behavior to 2D variable range hopping with increase in copper concentration.

  1. Energy cost of NaCl transport in isolated gills of cutthroat trout.

    PubMed

    Morgan, J D; Iwama, G K

    1999-09-01

    Few studies have made direct estimates of the energy required for ion transport in gills of freshwater (FW) and seawater (SW) fish. Oxygen consumption was measured in excised gill tissue of FW-adapted cutthroat trout (Oncorhynchus clarki clarki) to estimate the energy cost of NaCl transport in that osmoregulatory organ. Ouabain (0.5 mM) and bafilomycin A1 (1 microM) were used to inhibit the Na+-K+ and H+ pumps, respectively. Both inhibitors significantly decreased gill tissue oxygen consumption, accounting for 37% of total tissue respiration. On a whole mass basis, the cost of NaCl uptake in the FW trout gill was estimated to be 1.8% of whole animal oxygen uptake. An isolated, saline-perfused gill arch preparation was also used to compare gill energetics in FW- and SW-adapted trout. The oxygen consumption of FW gills was significantly (33%) higher than SW gills. On a whole animal basis, total gill oxygen consumption in FW and SW trout accounted for 3.9 and 2.4% of resting metabolic rate, respectively. The results of both experiments suggest that the energy cost of NaCl transport in FW and SW trout gills represents a relatively small (<4%) portion of the animal's total energy budget.

  2. Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1

    PubMed Central

    Kazmier, Kelli; Sharma, Shruti; Islam, Shahidul M.; Roux, Benoît; Mchaourab, Hassane S.

    2014-01-01

    Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuT-fold has been captured in outward-facing, occluded, and inward-facing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na+-coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion- and substrate-dependent conformational equilibria. In contrast to the Na+/leucine transporter LeuT, our results suggest that Na+ binding at the conserved second Na+ binding site does not change the energetics of the inward- and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class. PMID:25267652

  3. Molecular dynamics simulations of Na(+) and leucine transport by LeuT.

    PubMed

    Chen, Rong; Chung, Shin-Ho

    2015-08-14

    Molecular dynamics simulations are used to gain insight into the binding of Na(+) and leucine substrate to the bacterial amino acid transporter LeuT, focusing on the crystal structures of LeuT in the outward-open and inward-open states. For both conformations of LeuT, a third Na(+) binding site involving Glu290 in addition to the two sites identified from the crystal structures is observed. Once the negative charge from Glu290 in the inward-open LeuT is removed, the ion bound to the third site is ejected from LeuT rapidly, suggesting that the protonation state of Glu290 regulates Na(+) binding and release. In Cl(-)-dependent transporters where Glu290 is replaced by a neutral serine, a Cl(-) ion would be required to replace the role of Glu290. Thus, the simulations provide insights into understanding Na(+) and substrate transport as well as Cl(-)-independence of LeuT.

  4. Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1.

    PubMed

    Kazmier, Kelli; Sharma, Shruti; Islam, Shahidul M; Roux, Benoît; Mchaourab, Hassane S

    2014-10-14

    Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuT-fold has been captured in outward-facing, occluded, and inward-facing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na(+)-coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion- and substrate-dependent conformational equilibria. In contrast to the Na(+)/leucine transporter LeuT, our results suggest that Na(+) binding at the conserved second Na(+) binding site does not change the energetics of the inward- and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.

  5. Molecular Cloning and Functional Analysis of a Na(+)-Insensitive K(+) Transporter of Capsicum chinense Jacq.

    PubMed

    Ruiz-Lau, Nancy; Bojórquez-Quintal, Emanuel; Benito, Begoña; Echevarría-Machado, Ileana; Sánchez-Cach, Lucila A; Medina-Lara, María de Fátima; Martínez-Estévez, Manuel

    2016-01-01

    High-affinity K(+) (HAK) transporters are encoded by a large family of genes and are ubiquitous in the plant kingdom. These HAK-type transporters participate in low- and high-affinity potassium (K(+)) uptake and are crucial for the maintenance of K(+) homeostasis under hostile conditions. In this study, the full-length cDNA of CcHAK1 gene was isolated from roots of the habanero pepper (Capsicum chinense). CcHAK1 expression was positively regulated by K(+) starvation in roots and was not inhibited in the presence of NaCl. Phylogenetic analysis placed the CcHAK1 transporter in group I of the HAK K(+) transporters, showing that it is closely related to Capsicum annuum CaHAK1 and Solanum lycopersicum LeHAK5. Characterization of the protein in a yeast mutant deficient in high-affinity K(+) uptake (WΔ3) suggested that CcHAK1 function is associated with high-affinity K(+) uptake, with Km and Vmax for Rb of 50 μM and 0.52 nmol mg(-1) min(-1), respectively. K(+) uptake in yeast expressing the CcHAK1 transporter was inhibited by millimolar concentrations of the cations ammonium ([Formula: see text]) and cesium (Cs(+)) but not by sodium (Na(+)). The results presented in this study suggest that the CcHAK1 transporter may contribute to the maintenance of K(+) homeostasis in root cells in C. chinense plants undergoing K(+)-deficiency and salt stress.

  6. Identification of a chloride ion binding site in Na+/Cl -dependent transporters.

    PubMed

    Forrest, Lucy R; Tavoulari, Sotiria; Zhang, Yuan-Wei; Rudnick, Gary; Honig, Barry

    2007-07-31

    The recent determination of the crystal structure of the leucine transporter from Aquifex aeolicus (aaLeuT) has provided significant insights into the function of neurotransmitter:sodium symporters. Transport by aaLeuT is Cl(-) independent, whereas many neurotransmitter:sodium symporters from higher organisms depend on Cl(-) ions. However, the only Cl(-) ion identified in the aaLeuT structure interacts with nonconserved residues in extracellular loops, and thus the relevance of this binding site is unclear. Here, we use calculations of pK(A)s and homology modeling to predict the location of a functionally important Cl(-) binding site in serotonin transporter and other Cl(-)-dependent transporters. We validate our model through the site-directed mutagenesis of residues predicted to coordinate the Cl(-) ion and through the observation of sequence conservation patterns in other Cl(-)-dependent transporters. The proposed site is located midway across the membrane and is formed by residues from transmembrane helices 2, 6, and 7. It is close to the Na1 sodium binding site, thus providing an explanation for the coupling of Cl(-) and Na(+) ions during transport. Other implications of the model are also discussed.

  7. Molecular Cloning and Functional Analysis of a Na+-Insensitive K+ Transporter of Capsicum chinense Jacq

    PubMed Central

    Ruiz-Lau, Nancy; Bojórquez-Quintal, Emanuel; Benito, Begoña; Echevarría-Machado, Ileana; Sánchez-Cach, Lucila A.; Medina-Lara, María de Fátima; Martínez-Estévez, Manuel

    2016-01-01

    High-affinity K+ (HAK) transporters are encoded by a large family of genes and are ubiquitous in the plant kingdom. These HAK-type transporters participate in low- and high-affinity potassium (K+) uptake and are crucial for the maintenance of K+ homeostasis under hostile conditions. In this study, the full-length cDNA of CcHAK1 gene was isolated from roots of the habanero pepper (Capsicum chinense). CcHAK1 expression was positively regulated by K+ starvation in roots and was not inhibited in the presence of NaCl. Phylogenetic analysis placed the CcHAK1 transporter in group I of the HAK K+ transporters, showing that it is closely related to Capsicum annuum CaHAK1 and Solanum lycopersicum LeHAK5. Characterization of the protein in a yeast mutant deficient in high-affinity K+ uptake (WΔ3) suggested that CcHAK1 function is associated with high-affinity K+ uptake, with Km and Vmax for Rb of 50 μM and 0.52 nmol mg−1 min−1, respectively. K+ uptake in yeast expressing the CcHAK1 transporter was inhibited by millimolar concentrations of the cations ammonium (NH4+) and cesium (Cs+) but not by sodium (Na+). The results presented in this study suggest that the CcHAK1 transporter may contribute to the maintenance of K+ homeostasis in root cells in C. chinense plants undergoing K+-deficiency and salt stress. PMID:28083010

  8. NaI detector neutron activation spectra for PGNAA applications

    PubMed

    Gardner; El; Zheng; Hayden; Mayo

    2000-10-01

    When NaI detectors are used in prompt gamma-ray neutron activation analysis devices, they are activated by neutrons that penetrate the detector. While thermal neutron filters like boron or lithium can be used to reduce this activation, it can never be completely eliminated by this approach since high energy neutrons can penetrate the detector and thermalize inside it. This activation results in the emission of prompt gamma rays from both the I and Na and the production of the radioisotopes 128I and 24Na that subsequently decay and emit their characteristic beta particles and gamma rays. The resulting three spectra represent a background for this measurement. An experimental method for obtaining these three spectra is described and results are reported for 2" x 2", 5" x 5", 6" x 6", and 1" x 6" NaI detectors using the thermal neutron beam of the NCSU PULSTAR nuclear reactor. In addition, Monte Carlo simulation programs have been developed and used for simulating these spectra. Good results have been obtained by the Monte Carlo method for the two radioisotope spectra, and it is anticipated that good results will also be obtained for the prompt gamma-ray spectrum when the I and Na coincidence schemes are known.

  9. Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

    PubMed

    Schewe, Bettina; Blenau, Wolfgang; Walz, Bernd

    2012-04-15

    Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.

  10. Basolateral Na+/HCO3– cotransport activity is regulated by the dissociable Na+/H+ exchanger regulatory factor

    PubMed Central

    Bernardo, Angelito A.; Kear, Felicidad T.; Santos, Anna V.P.; Ma, Jianfei; Steplock, Debra; Robey, R. Brooks; Weinman, Edward J.

    1999-01-01

    In the renal proximal tubule, the activities of the basolateral Na+/HCO3– cotransporter (NBC) and the apical Na+/H+ exchanger (NHE3) uniformly vary in parallel, suggesting that they are coordinately regulated. PKA-mediated inhibition of NHE3 is mediated by a PDZ motif–containing protein, the Na+/H+ exchanger regulatory factor (NHE-RF). Given the common inhibition of these transporters after protein kinase A (PKA) activation, we sought to determine whether NHE-RF also plays a role in PKA-regulated NBC activity. Renal cortex immunoblot analysis using anti-peptide antibodies directed against rabbit NHE-RF demonstrated the presence of this regulatory factor in both brush-border membranes (BBMs) and basolateral membranes (BLMs). Using a reconstitution assay, we found that limited trypsin digestion of detergent solubilized rabbit renal BLM preparations resulted in NBC activity that was unaffected by PKA activation. Co-reconstitution of these trypsinized preparations with a recombinant protein corresponding to wild-type rabbit NHE-RF restored the inhibitory effect of PKA on NBC activity in a concentration-dependent manner. NBC activity was inhibited 60% by 10–8M NHE-RF; this effect was not observed in the absence of PKA. Reconstitution with heat-denatured NHE-RF also failed to attenuate NBC activity. To establish further a physiologic role for NHE-RF in NBC regulation, the renal epithelial cell line B-SC-1, which lacks detectable endogenous NHE-RF expression, was engineered to express stably an NHE-RF transgene. NHE-RF–expressing B-SC-1 cells (B-SC-RF) exhibited markedly lower basal levels of NBC activity than did wild-type controls. Inhibition of NBC activity in B-SC-RF cells was enhanced after 10 μM of forskolin treatment, consistent with a postulated role for NHE-RF in mediating the inhibition of NBC activity by PKA. These findings not only suggest NHE-RF involvement in PKA-regulated NBC activity, but also provide a unique molecular mechanism whereby

  11. Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue.

    PubMed

    Zhao, Yongfang; Terry, Daniel S; Shi, Lei; Quick, Matthias; Weinstein, Harel; Blanchard, Scott C; Javitch, Jonathan A

    2011-06-02

    Neurotransmitter/Na(+) symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na(+) electrochemical gradient. NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine, as well as by antidepressants. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na(+) are bound deep within the protein. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold. Subsequently, an 'outward-open' conformation was determined in the presence of the inhibitor tryptophan, and the Na(+)-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy. In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle. We investigated how substrate binding is coupled to structural transitions in LeuT during Na(+)-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ∼10-fold faster than leucine, we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule. Substrate binding in this S2 site appears to act cooperatively

  12. Tubuloglomerular and connecting tubuloglomerular feedback during inhibition of various Na transporters in the nephron.

    PubMed

    Wang, Hong; D'Ambrosio, Martin A; Ren, YiLin; Monu, Sumit R; Leung, Pablo; Kutskill, Kristopher; Garvin, Jeffrey L; Janic, Branislava; Peterson, Edward L; Carretero, Oscar A

    2015-05-01

    Afferent (Af-Art) and efferent arterioles resistance regulate glomerular capillary pressure. The nephron regulates Af-Art resistance via: 1) vasoconstrictor tubuloglomerular feedback (TGF), initiated in the macula densa via Na-K-2Cl cotransporters (NKCC2) and 2) vasodilator connecting tubuloglomerular feedback (CTGF), initiated in connecting tubules via epithelial Na channels (ENaC). Furosemide inhibits NKCC2 and TGF. Benzamil inhibits ENaC and CTGF. In vitro, CTGF dilates preconstricted Af-Arts. In vivo, benzamil decreases stop-flow pressure (PSF), suggesting that CTGF antagonizes TGF; however, even when TGF is blocked, CTGF does not increase PSF, suggesting there is another mechanism antagonizing CTGF. We hypothesize that in addition to NKCC2, activation of Na/H exchanger (NHE) antagonizes CTGF, and when both are blocked CTGF dilates Af-Arts and this effect is blocked by a CTGF inhibitor benzamil. Using micropuncture, we studied the effects of transport inhibitors on TGF responses by measuring PSF while increasing nephron perfusion from 0 to 40 nl/min. Control TGF response (-7.9 ± 0.2 mmHg) was blocked by furosemide (-0.4 ± 0.2 mmHg; P < 0.001). Benzamil restored TGF in the presence of furosemide (furosemide: -0.2 ± 0.1 vs. furosemide+benzamil: -4.3 ± 0.3 mmHg; P < 0.001). With furosemide and NHE inhibitor, dimethylamiloride (DMA), increase in tubular flow increased PSF (furosemide+DMA: 2.7 ± 0.5 mmHg, n = 6), and benzamil blocked this (furosemide+DMA+benzamil: -1.1 ± 0.2 mmHg; P < 0.01, n = 6). We conclude that NHE in the nephron decreases PSF (Af-Art constriction) when NKCC2 and ENaC are inhibited, suggesting that in the absence of NKCC2, NHE causes a TGF response and that CTGF dilates the Af-Art when TGF is blocked with NKCC2 and NHE inhibitors.

  13. Active learning in transportation engineering education

    NASA Astrophysics Data System (ADS)

    Weir, Jennifer Anne

    The objectives of this research were (1) to develop experimental active-based-learning curricula for undergraduate courses in transportation engineering and (2) to assess the effectiveness of an active-learning-based traffic engineering curriculum through an educational experiment. The researcher developed a new highway design course as a pilot study to test selected active-learning techniques before employing them in the traffic engineering curriculum. Active-learning techniques, including multiple-choice questions, short problems completed by individual students or small groups, and group discussions, were used as active interludes within lectures. The researcher also collected and analyzed student performance and attitude data from control and experimental classes to evaluate the relative effectiveness of the traditional lecture (control) approach and the active-learning (experimental) approach. The results indicate that the active-learning approach adopted for the experimental class did have a positive impact on student performance as measured by exam scores. The students in the experimental class also indicated slightly more positive attitudes at the end of the course than the control class, although the difference was not significant. The author recommends that active interludes similar to those in the experimental curricula be used in other courses in civil engineering.

  14. Intracellular Na+, K+ and Cl- activities in Acheta domesticus Malpighian tubules and the response to a diuretic kinin neuropeptide.

    PubMed

    Coast, Geoffrey M

    2012-08-15

    The mechanism of primary urine production and the activity of a diuretic kinin, Achdo-KII, were investigated in malpighian tubules of Acheta domesticus by measuring intracellular Na(+), K(+) and Cl(-) activities, basolateral membrane voltage (V(b)), fluid secretion and transepithelial ion transport. Calculated electrochemical gradients for K(+) and Cl(-) across the basolateral membrane show they are actively transported into principal cells, and basolateral Ba(2+)-sensitive K(+) channels do not contribute to net transepithelial K(+) transport and fluid secretion. A basolateral Cl(-) conductance was revealed after the blockade of K(+) channels with Ba(2+), and a current carried by the passive outward movement of Cl(-) accounts for the hyperpolarization of V(b) in response to Ba(2+). Ion uptake via Na(+)/K(+)/2Cl(-) cotransport, driven by the inwardly directed Na(+) electrochemical gradient, is thermodynamically feasible, and is consistent with the actions of bumetanide, which reduces fluid secretion and both Na(+) and K(+) transport. The Na(+) gradient is maintained by its extrusion across the apical membrane and by a basolateral ouabain-sensitive Na(+)/K(+)-ATPase. Achdo-KII has no significant effect on the intracellular ion activities or V(b). Electrochemical gradients across the apical membrane were estimated from previously published values for the levels of Na(+), K(+) and Cl(-) in the secreted fluid. The electrochemical gradient for Cl(-) favours passive movement into the lumen, but falls towards zero after stimulation by Achdo-KII. This coincides with a twofold increase in Cl(-) transport, which is attributed to the opening of an apical Cl(-) conductance, which depolarises the apical membrane voltage.

  15. Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport.

    PubMed

    Lytton, Jonathan

    2007-09-15

    Mammalian Na+/Ca2+ exchangers are members of three branches of a much larger family of transport proteins [the CaCA (Ca2+/cation antiporter) superfamily] whose main role is to provide control of Ca2+ flux across the plasma membranes or intracellular compartments. Since cytosolic levels of Ca2+ are much lower than those found extracellularly or in sequestered stores, the major function of Na+/Ca2+ exchangers is to extrude Ca2+ from the cytoplasm. The exchangers are, however, fully reversible and thus, under special conditions of subcellular localization and compartmentalized ion gradients, Na+/Ca2+ exchangers may allow Ca2+ entry and may play more specialized roles in Ca2+ movement between compartments. The NCX (Na+/Ca2+ exchanger) [SLC (solute carrier) 8] branch of Na+/Ca2+ exchangers comprises three members: NCX1 has been most extensively studied, and is broadly expressed with particular abundance in heart, brain and kidney, NCX2 is expressed in brain, and NCX3 is expressed in brain and skeletal muscle. The NCX proteins subserve a variety of roles, depending upon the site of expression. These include cardiac excitation-contraction coupling, neuronal signalling and Ca2+ reabsorption in the kidney. The NCKX (Na2+/Ca2+-K+ exchanger) (SLC24) branch of Na+/Ca2+ exchangers transport K+ and Ca2+ in exchange for Na+, and comprises five members: NCKX1 is expressed in retinal rod photoreceptors, NCKX2 is expressed in cone photoreceptors and in neurons throughout the brain, NCKX3 and NCKX4 are abundant in brain, but have a broader tissue distribution, and NCKX5 is expressed in skin, retinal epithelium and brain. The NCKX proteins probably play a particularly prominent role in regulating Ca2+ flux in environments which experience wide and frequent fluctuations in Na+ concentration. Until recently, the range of functions that NCKX proteins play was generally underappreciated. This situation is now changing rapidly as evidence emerges for roles including photoreceptor

  16. Investigation of influence of NaOH and NaCl activating solutions on bentonite stabilization in suspension fertilizers

    NASA Astrophysics Data System (ADS)

    Hoffmann, Krystyna; Hoffmann, Józef; Mikła, Daniel; Huculak-Mä Czka, Marta; Skut, Jakub

    2010-05-01

    1. INTRODUCTION Regular plants growth and their metabolic activity are determined by the macro- (C, H, O, N, P, S, K, Ca, Mg) and micronutrients (Fe, Mn, Zn, Cu, B, Mo, Cl, Ni). The role of these elements is very important, the excess as well as the deficiency have the negative influence on their development [1]. In order to increase yields and quality of crops a mineral, organic and mineral-organic fertilizers are applied. In the last years suspension fertilizers have been of great significance, taking the agricultural benefits into consideration. Suspension fertilizers are products of a new generation on account of higher nutrients concentrations than in the majority of other fertilizers, what makes them more efficient. Suspension fertilizers differ from solid fertilizers in more regular distribution on field. Nutrients are more concentrated what is economically relevant on account of the facilitated transportation. Examinations indicated, that nutrients from suspension fertilizers are more available than from solid fertilizers. The high concentration of nutrients in fertilizer is obtained by introducing a substance which holds them regularly in the suspension. Bentonites are the substances used for stabilization of suspension fertilizers most often [2,3]. Bentonites belong to ore of clay minerals, primarily made from minerals of smectite group, montmorillonite especially [4]. Bentonite loams were formulated as a result of Aluminium Silicate-bearing Rocks weathering and subsequent sedimentation in the aqueous environment. Characteristic features of rocks of the smectite group are their ability to absorb water (swelling), to form thixotrophic suspensions which aren't undergoing sedimentation process for a long time; as well as susceptibility to absorb cations and organic substances [4,5]. Therefore investigations have been carried out in order to evaluate the possibility of application of diverse loamy raw materials as suspension stabilizers for fertilizer

  17. An Na(+)-independent short-chain fatty acid transporter contributes to intracellular pH regulation in murine colonocytes

    PubMed Central

    1995-01-01

    Short-chain fatty acids (SCFAs) are the major anions in the colonic lumen. Experiments studied how intracellular pH (pHi) of isolated colonocytes was affected by exposure to SCFAs normally found in the colon. Isolated crypt fragments were loaded with SNARF-1 (a fluorescent dye with pH-sensitive excitation and emission spectra) and studied in a digital imaging microscope. Intracellular pH was measured in individual colonocytes as the ratio of fluorescence intensity in response to alternating excitation wavelengths (575/505 nm). After exposure to 65 mM acetate, propionate, n-butyrate, or iso-butyrate in isosmotic Na(+)- free media (substituted with tetramethylammonia), all colonocytes acidified rapidly and then > 90% demonstrated a pHi alkalinization (Na(+)-independent pHi recovery). Upon subsequent removal of the SCFA, pHi alkalinized beyond the starting pHi (a pHi overshoot). Using propionate as a test SCFA, experiments demonstrate that the acidification and pHi overshoot are explained by transmembrane influx and efflux of nonionized SCFA, respectively. The basis for the pHi overshoot is shown to be accumulation of propionate during pHi alkalinization. The Na(+)-independent pHi recovery (a) demonstrates saturable propionate activation kinetics; (b) demonstrates substrate specificity for unmodified aliphatic carbon chains; (c) occurs after exposure to SCFAs of widely different metabolic activity, (d) is electroneutral; and (e) is not inhibited by changes in the K+ gradient, Cl- gradient or addition of the anion transport inhibitors DIDS (1 mM), SITS (1 mM), alpha-cyano-4-hydroxycinnamate (4 mM), or probenicid (1 mM). Results suggest that most mouse colonocytes have a previously unreported SCFA transporter which mediates Na(+)-independent pHi recovery. PMID:7658194

  18. Lotus tenuis tolerates combined salinity and waterlogging: maintaining O2 transport to roots and expression of an NHX1-like gene contribute to regulation of Na+ transport.

    PubMed

    Teakle, Natasha L; Amtmann, Anna; Real, Daniel; Colmer, Timothy D

    2010-08-01

    Salinity and waterlogging interact to reduce growth for most crop and pasture species. The combination of these stresses often cause a large increase in the rate of Na(+) and Cl(-) transport to shoots; however, the mechanisms responsible for this are largely unknown. To identify mechanisms contributing to the adverse interaction between salinity and waterlogging, we compared two Lotus species with contrasting tolerances when grown under saline (200 mM NaCl) and O(2)-deficient (stagnant) treatments. Measurements of radial O(2) loss (ROL) under stagnant conditions indicated that more O(2) reaches root tips of Lotus tenuis, compared with Lotus corniculatus. Better internal aeration would contribute to maintaining Na(+) and Cl(-) transport processes in roots of L. tenuis exposed to stagnant-plus-NaCl treatments. L. tenuis root Na(+) concentrations after stagnant-plus-NaCl treatment (200 mM) were 17% higher than L. corniculatus, with 55% of the total plant Na(+) being accumulated in roots, compared with only 39% for L. corniculatus. L. tenuis accumulated more Na(+) in roots, presumably in vacuoles, thereby reducing transport to the shoot (25% lower than L. corniculatus). A candidate gene for vacuole Na(+) accumulation, an NHX1-like gene, was cloned from L. tenuis and identity established via sequencing and yeast complementation. Transcript levels of NHX1 in L. tenuis roots under stagnant-plus-NaCl treatment were the same as for aerated NaCl, whereas L. corniculatus roots had reduced transcript levels. Enhanced O(2) transport to roots enables regulation of Na(+) transport processes in L. tenuis roots, contributing to tolerance to combined salinity and waterlogging stresses.

  19. Regulation of renal Na -K-ATPase in the rat: role of increased potassium transport

    SciTech Connect

    Mujais, S.K.; Chekal, M.A.; Hayslett, J.P.; Katz, A.I.

    1986-08-01

    The purpose of this study was to characterize the alterations in collecting tubule Na -K -ATPase activity produced by sustained increments in dietary potassium in the rat and to evaluate the role of aldosterone in their generation. In adrenal-intact animals, feeding a high-potassium diet or administration of a high physiological dose of aldosterone, which simulates the delivery rate of this hormone during potassium loading, caused marked increments in Na -K -ATPase activity in the cortical collecting tubule (CCT) but had no effect on the enzyme in the inner stripe of the medullary collecting tubule (MCT). A significant increase in enzyme activity was also observed after smaller dietary potassium increments and after 4 days of dietary potassium load. In adrenalectomized rats provided with physiological replacement doses of corticosterone and aldosterone, Na -K -ATPase activity in both CCT and MCT was similar to that of adrenal-intact controls but remained unchanged after 7 days on the potassium-enriched (10-fold) diet. In contrast, adrenalectomized animals receiving the high physiological dose of aldosterone displayed an increase in Na -K -ATPase activity of CCT comparable with that of adrenal-intact animals, whereas the enzyme activity in the MCT was unaffected. In conclusion, 1) following chronic potassium loading Na -K -ATPase activity increases significantly in the CCT with no change in its activity in the inner stripe of the MCT; 2) this increase in enzyme activity occurs in a time-dependent fashion and in proportion to the potassium load; and 3) the stimulation of Na -K -ATPase activity in adrenal-replaced rats is facilitated by augmented levels of aldosterone, such as those actually observed in adrenal-intact rats subjected to chronic potassium loading.

  20. Effects of chlorpromazine on Na+-K+-ATPase pumping and solute transport in rat hepatocytes

    SciTech Connect

    Van Dyke, R.W.; Scharschmidt, B.F.

    1987-11-01

    Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible /sup 86/Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by /sup 36/Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.1-2 mM), but not chlorpromazine sulfoxide, produced a concentration-dependent decrease in Na+-K+-ATPase cation pumping and an increase in intracellular sodium content. Chlorpromazine (100 microM) and ouabain (0.75 mM) also modestly decreased hepatocyte membrane potential. In further studies, chlorpromazine (75 and 100 microM) and ouabain (0.1, 0.5, and 0.75 mM) decreased initial sodium-dependent uptake rates of taurocholate and alanine by 18-63%. Although the steady-state intracellular content of alanine was decreased 25-53% by both agents, chlorpromazine increased the steady-state content of taurocholate by 171% and decreased taurocholate efflux, apparently related to partitioning of taurocholate into a large, slowly turning over intracellular pool. These studies provide direct evidence that chlorpromazine inhibits Na+-K+-ATPase cation pumping in intact cells and that partial inhibition of Na+-K+-ATPase cation pumping is associated with a reduction of both the electrochemical sodium gradient and sodium-dependent solute transport. These effects of chlorpromazine may contribute to chlorpromazine-induced cholestasis in animals and humans.

  1. Effects of chlorpromazine on Na+-K+-ATPase pumping and solute transport in rat hepatocytes.

    PubMed

    Van Dyke, R W; Scharschmidt, B F

    1987-11-01

    Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible 86Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by 36Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.1-2 mM), but not chlorpromazine sulfoxide, produced a concentration-dependent decrease in Na+-K+-ATPase cation pumping and an increase in intracellular sodium content. Chlorpromazine (100 microM) and ouabain (0.75 mM) also modestly decreased hepatocyte membrane potential. In further studies, chlorpromazine (75 and 100 microM) and ouabain (0.1, 0.5, and 0.75 mM) decreased initial sodium-dependent uptake rates of taurocholate and alanine by 18-63%. Although the steady-state intracellular content of alanine was decreased 25-53% by both agents, chlorpromazine increased the steady-state content of taurocholate by 171% and decreased taurocholate efflux, apparently related to partitioning of taurocholate into a large, slowly turning over intracellular pool. These studies provide direct evidence that chlorpromazine inhibits Na+-K+-ATPase cation pumping in intact cells and that partial inhibition of Na+-K+-ATPase cation pumping is associated with a reduction of both the electrochemical sodium gradient and sodium-dependent solute transport. These effects of chlorpromazine may contribute to chlorpromazine-induced cholestasis in animals and humans.

  2. Adaptive response of equine intestinal Na+/glucose co-transporter (SGLT1) to an increase in dietary soluble carbohydrate.

    PubMed

    Dyer, Jane; Al-Rammahi, Miran; Waterfall, Louise; Salmon, Kieron S H; Geor, Ray J; Bouré, Ludovic; Edwards, G Barrie; Proudman, Christopher J; Shirazi-Beechey, Soraya P

    2009-06-01

    Experimental and epidemiological evidence suggests that consumption of hydrolyzable carbohydrate, hCHO (grain), by horses is an important risk factor for colic, a common cause of equine mortality. It is unknown whether the small intestinal capacity to digest hCHO and/or to absorb monosaccharides is limiting, or even if horses can adapt to increased carbohydrate load. We investigated changes in the brush-border membrane carbohydrate digestive enzymes and glucose absorptive capacity of horse small intestine in response to increased hCHO. Expression of the Na(+)/glucose co-transporter, SGLT1, was assessed by Western blotting, immunohistochemistry, Northern blotting, QPCR, and Na(+)-dependent D-glucose transport. Glucose transport rates, SGLT1 protein, and mRNA expression were all 2-fold higher in the jejunum and 3- to 5-fold higher in the ileum of horses maintained on a hCHO-enriched diet compared to pasture forage. Activity of the disaccharidases was unaltered by diet. In a well-controlled study, we determined SGLT1 expression in the duodenal and ileal biopsies of horses switched, gradually over a 2-month period, from low (<1.0 g/kg bwt/day) to high hCHO (6.0 g/kg bwt/day) diets of known composition. We show that SGLT1 expression is enhanced, with time, 2-fold in the duodenum and 3.3-fold in the ileum. The study has important implications for dietary management of the horse.

  3. Volume regulation of intestinal cells of echinoderms: Putative role of ion transporters (Na(+)/K(+)-ATPase and NKCC).

    PubMed

    Castellano, Giovanna C; Souza, Marta M; Freire, Carolina A

    2016-11-01

    Echinoderms are exclusively marine osmoconformer invertebrates. Some species occupy the challenging intertidal region. Upon salinity changes, the extracellular osmotic concentration of these animals also varies, exposing tissues and cells to osmotic challenges. Cells and tissues may then respond with volume regulation mechanisms, which involve transport of ions and water into and/or out of the cells, through ion transporters, such as the Na(+)/K(+)-ATPase and NKCC. The goal of this study was to relate the cell volume regulation capacity of echinoderm intestinal cells Na(+)/K(+)-ATPase and NKCC activities, in three echinoderm species: Holothuria grisea, Arbacia lixula, and Echinometra lucunter. Isolated cells of these species displayed some control of their cell volume upon exposure to anisosmotic media (isolated intestinal cells, calcein fluorescence as indicator of volume change), with a distinct higher capacity shown by H. grisea, which did not swell even upon 50% hyposmotic shock. The holothuroid cells showed indirect evidence (effect of furosemide) of the participation of NKCC in this process, with a secretory function, and of a secondary role by the NKA (effect of ouabain). Other mechanisms are probably responsible for this function in the urchins. Variable expression of these transporters, and others not examined here, may to some extent account for the variability in cell volume regulation capacity in echinoderm cells.

  4. [Mechanism of coupling of ion transport and ATP hydrolysis in the Na-pump].

    PubMed

    Tverdislov, V A; Iakovenko, L V; Rezaeva, M N

    1979-01-01

    A generalized scheme of the reaction pathways during activation of the Na,K-ATPase by sodium and potassium ions and a relevant molecular model of the Na-pump are proposed. The model suggests light and heavy enzyme subunits possessing cavities with ion exchange sites. The cavities are of limited size and can contain only 3 sodium or 2 potassium ions. Free energy of ATP hydrolysis is expended on the formation of a special transient nonequilibrium enzyme conformation. In this conformation ion exchange between the subunit cavities becames possible. Na-pump operates as an enthropy machine: the ion movement across the membrane is provided by thermal oscillations of the subunits.

  5. Single-molecule detection with active transport

    NASA Astrophysics Data System (ADS)

    Ball, David Allan

    A glass capillary is used near the focal region of a custom-built confocal microscope to investigate the use of active transport for single-molecule detection in solution, with both one and two-photon laser excitation. The capillary tip has a diameter of several microns and is carefully aligned nearby to the sub-micron laser beam waist, collinear to the optical axis, so that a negative pressure-difference causes molecules to be drawn into the capillary, along the laser beam axis. The flow of solution, which is characterized by fluorescence correlation spectroscopy (FCS), can increase the single-molecule detection rate for slowly diffusing proteins by over a factor of 100, while the mean rate of photons during each burst is similar to that for random diffusional transport. Also, the flow is along the longest axis of the ellipsoidally-shaped confocal volume, which results in more collected photons per molecule than that for transverse flow at the same speed. When transport is dominated by flow, FCS can no longer distinguish molecules with differing translational diffusion, and hence a fluorescence fluctuation spectroscopy method based on differences in fluorescence brightness is investigated as a means for assaying different solution components, for applications in pharmaceutical drug discovery. Multi-channel fluctuation spectroscopy techniques can also be used for assays with the flow system and hence this dissertation also reports the characterization of a prototype 4-channel single-photon detector with a two-wavelength polarization-resolved optical set-up.

  6. In Situ Investigation of Li and Na Ion Transport with Single Nanowire Electrochemical Devices.

    PubMed

    Xu, Xu; Yan, Mengyu; Tian, Xiaocong; Yang, Chuchu; Shi, Mengzhu; Wei, Qiulong; Xu, Lin; Mai, Liqiang

    2015-06-10

    In the past decades, Li ion batteries are widely considered to be the most promising rechargeable batteries for the rapid development of mobile devices and electric vehicles. There arouses great interest in Na ion batteries, especially in the field of static grid storage due to their much lower production cost compared with Li ion batteries. However, the fundamental mechanism of Li and Na ion transport in nanoscale electrodes of batteries has been rarely experimentally explored. This insight can guide the development and optimization of high-performance electrode materials. In this work, single nanowire devices with multicontacts are designed to obtain detailed information during the electrochemical reactions. This unique platform is employed to in situ investigate and compare the transport properties of Li and Na ions at a single nanowire level. To give different confinement for ions and electrons during the electrochemical processes, two different configurations of nanowire electrode are proposed; one is to fully immerse the nanowire in the electrolyte, and the other is by using photoresist to cover the nanowire with only one end exposed. For both configurations, the conductivity of nanowire decreases after intercalation/deintercalation for both Li and Na ions, indicating that they share the similar electrochemical reaction mechanisms in layered electrodes. However, the conductivity degradation and structure destruction for Na ions is more severe than those of Li ions during the electrochemical processes, which mainly results from the much larger volume of Na ions and greater energy barrier encountered by the limited layered spaces. Moreover, the battery performances of coin cells are compared to further confirm this conclusion. The present work provides a unique platform for in situ electrochemical and electrical probing, which will push the fundamental and practical research of nanowire electrode materials for energy storage applications.

  7. Chloride conductance and Pi transport are separate functions induced by the expression of NaPi-1 in Xenopus oocytes.

    PubMed

    Bröer, S; Schuster, A; Wagner, C A; Bröer, A; Forster, I; Biber, J; Murer, H; Werner, A; Lang, F; Busch, A E

    1998-07-01

    Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl- conductance (GCl), organic anion transport and Na+-dependent Pi-uptake. In the present study we investigated the relation between the NaPi-1 induced GCl and Pi-induced currents and transport. NaPi-1 expression induced Pi-transport, which was not different at 1-20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1-2 days after cRNA injection. In contrast, GCl was augmented at increased amounts of cRNA injection (1-20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. Pi-induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of Pi (>/= 1 mm Pi). The amplitudes of Ip correlated well with GCl. Ip was blocked by the Cl- channel blocker NPPB, partially Na+-dependent and completely abolished in Cl- free solution. In contrast, Pi-transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na+ and weakly affected by Cl- substitution. Endogenous Pi-uptake in water-injected oocytes amounted in all experiments to 30-50% of the Na+-dependent Pi-transport observed in NaPi-1 expressing oocytes. The properties of the endogenous Pi-uptake system (Km for Pi > 1 mM; partial Na+- and Cl--dependence; lack of NPPB block) were similar to the NaPi-1 induced Pi-uptake, but no Ip could be recorded at Pi-concentrations

  8. Ca(2+)-dependent heat production by rat skeletal muscle in hypertonic media depends on Na(+)-Cl- co-transport stimulation.

    PubMed Central

    Chinet, A

    1993-01-01

    1. The rate of energy dissipation (E) in isolated, superfused soleus muscles from young rats was continuously measured under normosmotic and 100-mosM hyperosmotic conditions. The substantial increase of E with respect to basal level in hyperosmolarity (excess E), which is entirely dependent on the presence of extracellular sodium, was largely prevented or inhibited by bumetanide, a potent inhibitor of Na(+)-Cl- co-transport system, or by the removal of chloride from the superfusate (isethionate substitution). Bumetanide or the removal of chloride also acutely decreased basal E, by about 7%. 2. Bumetanide almost entirely suppressed the major, Ca(2+)-dependent part of excess E in hyperosmolarity, as well as the concomitant increase of 45Ca2+ efflux and small increase in resting muscle tension; in contrast, the part of excess E associated with stimulation of Na(+)-H+ exchange in hyperosmolarity was left unmodified. 3. Reduction of 22Na+ influx by bumetanide was more marked in hyperosmolarity than under control conditions, although stimulation of total 22Na+ influx by a 100-mosM stress was not statistically significant. Inhibition of Ca2+ release into the sarcoplasm using dantrolene sodium did not prevent the stimulation of bumetanide-sensitive 22Na+ influx, but rather increased it about fourfold. 4. It is concluded that the largest part of excess E in hyperosmolarity, which is Ca(2+)-dependent energy expenditure, is suppressed when steady-state stimulation of a Na(+)-Cl- co-transport system is inhibited either directly by bumetanide or the removal of extracellular chloride, or indirectly by the blocking of active Na(+)-K+ transport. How the stimulation of Na(+)-Cl- co-transport, by as little as 1 nmol s-1 (g wet muscle weight)-1 during a 100-mosM stress, enhances Ca(2+)-dependent heat by as much as 2.5 mW (g wet muscle weight)-1 remains to be clarified. PMID:8394429

  9. Regulation of the Na(+)-coupled glutamate transporter EAAT3 by PIKfyve.

    PubMed

    Klaus, Fabian; Gehring, Eva-Maria; Zürn, Agathe; Laufer, Joerg; Lindner, Ricco; Strutz-Seebohm, Nathalie; Tavaré, Jeremy M; Rothstein, Jeffrey D; Boehmer, Christoph; Palmada, Monica; Gruner, Ivonne; Lang, Undine E; Seebohm, Guiscard; Lang, Florian

    2009-01-01

    The Na(+), glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end,EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (I(glu)) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I(glu). Coexpression of either, SGK1 orPIKfyve, significantly enhanced I(glu) in EAAT3 expressing oocytes. The increased I(glu) was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I(glu) and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressingEAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter I(glu) in EAAT3 expressing oocytes and completely reversed the stimulating effect ofPIKfyve coexpression on I(glu). The stimulating effect of PIKfyve on I(glu) was abolished by replacement of the serine by alanine in the SGK consensus sequence ((S318A)PIKfyve). Moreover, additional coexpression of(S318A)PIKfyve significantly blunted I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.

  10. Tissue kallikrein activation of the epithelial Na channel

    PubMed Central

    Patel, Ankit B.; Chao, Julie

    2012-01-01

    Epithelial Na Channels (ENaC) are responsible for the apical entry of Na+ in a number of different epithelia including the renal connecting tubule and cortical collecting duct. Proteolytic cleavage of γ-ENaC by serine proteases, including trypsin, furin, elastase, and prostasin, has been shown to increase channel activity. Here, we investigate the ability of another serine protease, tissue kallikrein, to regulate ENaC. We show that excretion of tissue kallikrein, which is secreted into the lumen of the connecting tubule, is stimulated following 5 days of a high-K+ or low-Na+ diet in rats. Urinary proteins reconstituted in a low-Na buffer activated amiloride-sensitive currents (INa) in ENaC-expressing oocytes, suggesting an endogenous urinary protease can activate ENaC. We next tested whether tissue kallikrein can directly cleave and activate ENaC. When rat ENaC-expressing oocytes were exposed to purified tissue kallikrein from rat urine (RTK), ENaC currents increased threefold in both the presence and absence of a soybean trypsin inhibitor (SBTI). RTK and trypsin both decreased the apparent molecular mass of cleaved cell-surface γ-ENaC, while immunodepleted RTK produced no shift in apparent molecular mass, demonstrating the specificity of the tissue kallikrein. A decreased effect of RTK on Xenopus ENaC, which has variations in the putative prostasin cleavage sites in γ-ENaC, suggests these sites are important in RTK activation of ENaC. Mutating the prostasin site in mouse γ-ENaC (γRKRK186QQQQ) abolished ENaC activation and cleavage by RTK while wild-type mouse ENaC was activated and cleaved similar to that of the rat. We conclude that tissue kallikrein can be a physiologically relevant regulator of ENaC activity. PMID:22622459

  11. Contributions of different NaPi cotransporter isoforms to dietary regulation of P transport in the pyloric caeca and intestine of rainbow trout.

    PubMed

    Sugiura, Shozo H; Ferraris, Ronaldo P

    2004-05-01

    The anatomical proximity and embryological relationship of the pyloric caeca (PC) and small intestine of rainbow trout has led to the frequent assumption, on little evidence, that they have the same enzymes and transporters. In trout, the PC is an important absorptive organ for dietary nutrients, but its role in dietary P absorption has not been reported. We found that apical inorganic phosphate (Pi) transport in PC comprises carrier-mediated and diffusive components. Carrier-mediated uptake was energy- and temperature-dependent, competitively inhibited and Na(+)-independent, and greater than the Na(+)-dependent intestinal uptake. Pi uptake in PC was pH-sensitive in the presence of Na(+). Despite the active Pi transport system in PC, high postprandial luminal Pi concentrations ( approximately 20 mmol l(-1)) indicate that diffusive uptake represents approximately 92% of total Pi uptake in PC of fed fish. The nucleotide sequence of a sodium-phosphate cotransporter (NaPi-II) isoform isolated from PC was approximately 8% different from the intestinal NaPi cotransporter. PC-NaPi mRNA was abundant in PC but rare in the intestine, whereas intestinal NaPi mRNA was abundant in the intestine but scarce in PC. Dietary P restriction reduced serum and bone P concentrations, increased intestine-type, but not PC-type, NaPi mRNA in PC, and increased Pi uptake in intestine but not in PC. Intestine-type NaPi expression may be useful for predicting dietary P deficiency.

  12. Cyclic nucleotide gated channel 10 negatively regulates salt tolerance by mediating Na+ transport in Arabidopsis.

    PubMed

    Jin, Yakang; Jing, Wen; Zhang, Qun; Zhang, Wenhua

    2015-01-01

    A number of cyclic nucleotide gated channel (CNGC) genes have been identified in plant genomes, but their functions are mainly undefined. In this study, we identified the role of CNGC10 in the response of Arabidopsis thaliana to salt stress. The cngc10 T-DNA insertion mutant showed greater tolerance to salt than wild-type A. thaliana during seed germination and seedling growth. The cngc10 mutant accumulated less Na(+) and K(+), but not less Ca(2+), in shoots in response to salt stress. By contrast, overexpression of CNGC10 resulted in greater sensitivity to salt stress, and complementation of this gene recovered salt sensitivity. In response to salt stress, heterologous expression of CNGC10 in the Na(+) sensitive yeast mutant strain B31 inhibited growth due to accumulation of Na(+) at a rate greater than that of yeast transformed with an empty vector. Quantitative RT-PCR analysis demonstrated that CNGC10 was expressed mainly in roots and flowers. GUS analysis of a root cross section indicated that CNGC10 was expressed mainly in the endodermis and epidermis. Furthermore, the expression of CNGC10 in roots was dramatically inhibited by exposure to 200 mM NaCl for 6 h. These data suggest that CNGC10 negatively regulates salt tolerance in A. thaliana and may be involved in mediating Na(+) transport.

  13. Transport in active systems crowded by obstacles

    NASA Astrophysics Data System (ADS)

    Huang, Mu-Jie; Schofield, Jeremy; Kapral, Raymond

    2017-02-01

    The reactive and diffusive dynamics of a single chemically powered Janus motor in a crowded medium of moving but passive obstacles is investigated using molecular simulation. It is found that the reaction rate of the catalytic motor reaction decreases in a crowded medium as the volume fraction of obstacles increases as a result of a reduction in the Smoluchowski diffusion-controlled reaction rate coefficient that contributes to the overall reaction rate. A continuum model is constructed and analyzed to interpret the dependence of the steady-state reaction rate observed in simulations on the volume fraction of obstacles in the system. The steady-state concentration fields of reactant and product are shown to be sensitive to the local structure of obstacles around the Janus motor. It is demonstrated that the active motor exhibits enhanced diffusive motion at long times with a diffusion constant that decreases as the volume fraction of crowding species increases. In addition, the dynamical properties of a passive tracer particle in a system containing many active Janus motors is studied to investigate how an active environment influences the transport of non-active species. The diffusivity of a passive tracer particle in an active medium is found to be enhanced in systems with forward-moving Janus motors due to the cooperative dynamics of these motors.

  14. Hormonal modulation of Na+ transport in rat fetal distal lung epithelial cells

    PubMed Central

    Ramminger, S J; Inglis, S K; Olver, R E; Wilson, S M

    2002-01-01

    Isolated rat fetal distal lung epithelial (FDLE) cells were cultured (≈48 h) on permeable supports in medium devoid of hormones and growth factors whilst PO2 was maintained at the level found in either the fetal (23 mmHg) or the postnatal (100 mmHg) alveolar regions. The cells became incorporated into epithelial layers that generated a basal short-circuit current (ISC) attributable to spontaneous Na+ absorption. Cells at neonatal PO2 generated larger currents than did cells at fetal PO2, indicating that this Na+ transport process is oxygen sensitive. Irrespective of PO2, isoprenaline failed to elicit a discernible change in ISC, demonstrating that β-adrenoceptor agonists do not stimulate Na+ transport under these conditions. However, isoprenaline did elicit cAMP accumulation in these cells, indicating that functionally coupled β-adrenoceptors are present. Further experiments showed that isoprenaline did increase ISC in cells treated (24 h) with a combination of tri-iodothyronine (T3, 10 nm) and dexamethasone (200 nm). Studies of basolaterally permeabilised cells showed that these hormones are essential for the isoprenaline-evoked increase in the apical membrane's Na+ conductance (GNa), whereas isoprenaline-evoked changes in apical Cl− conductance (GCl) can occur in both control and hormone-treated cells. Irrespective of their hormonal status, FDLE cells thus express β-adrenoceptors that are functionally coupled to adenylate cyclase, and allow β-adrenoceptor agonists to modulate the apical membrane's anion conductance. However, T3 and dexamethasone are needed if these receptors are to exert control over GNa. These hormones may thus play an important role in the functional maturation of the lung by allowing β-adrenoceptor-mediated control over epithelial Na+ channels in the apical plasma membrane. PMID:12381827

  15. Sodium ion transport mechanisms in antiperovskite electrolytes Na3OBr and Na4OI2: An in Situ neutron diffraction study

    SciTech Connect

    Zhu, Jinlong; Wang, Yonggang; Li, Shuai; Howard, John W.; Neuefeind, Jorg; Ren, Yang; Wang, Hui; Liang, Chengdu; Yang, Wenge; Zou, Ruqiang; Jin, Changqing; Zhao, Yusheng

    2016-06-02

    Na-rich antiperovskites are recently developed solid electrolytes with enhanced sodium ionic conductivity and show promising functionality as a novel solid electrolyte in an all solid-stat battery. In this work, the sodium ionic transport pathways of the parent compound Na3OBr, as well as the modified layered antiperovskite Na4OI2, were studied and compared through temperature dependent neutron diffraction combined with the maximum entropy method. In the cubic Na3OBr antiperovskite, the nuclear density distribution maps at 500 K indicate that sodium ions ho within and among oxygen octahedra, and Br- ions are not involved in the tetragonal Na4OI2 antiperovskite, Na ions, which connect octahedra in the ab plane, have the lowest activation energy barrier. In conclusion, the transport of sodium ions along the c axis is assisted by I- ions.

  16. ATP is a coupling modulator of parallel Na,K-ATPase-K-channel activity in the renal proximal tubule.

    PubMed Central

    Tsuchiya, K; Wang, W; Giebisch, G; Welling, P A

    1992-01-01

    A fundamental and essential property of nearly all salt-transporting epithelia is the tight parallel coupling between the magnitude of the K-conductive pathway at the basolateral membrane and the activity of the Na,K-dependent ATPase (Na,K-ATPase). In the present study, we demonstrate that the coupling response in the renal proximal tubule is governed, at least in part, through the interaction between ATP-sensitive K channels and Na,K-ATPase-mediated changes in intracellular ATP levels. First, we identified a K-selective channel at the basolateral membrane, which is inhibited by the cytosolic addition of ATP. Second, conventional microelectrode analysis in the isolated perfused proximal straight tubule revealed that these channels are the major determinant of the macroscopic K conductance so that ATP-mediated changes in the open probability of the K channel could alter the extent of K recycling. Indeed, the increase in the macroscopic K conductance upon stimulation of transcellular Na transport and pump activity was found to be paralleled by a decrease in intracellular ATP. Finally, a causal link between parallel Na,K-ATPase-K-channel activity and ATP was established by the finding that intracellular ATP loading uncoupled the response. With our recent observations that similar ATP-sensitive K channels are expressed abundantly in other epithelia, we postulate that ATP may act as a universal coupling modulator of parallel Na,K-ATPase-K-channel activity. PMID:1321439

  17. Involvement of disulphide bonds in the renal sodium/phosphate co-transporter NaPi-2.

    PubMed Central

    Xiao, Y; Boyer, C J; Vincent, E; Dugré, A; Vachon, V; Potier, M; Béliveau, R

    1997-01-01

    The rat renal brush border membrane sodium/phosphate co-transporter NaPi-2 was analysed in Western blots with polyclonal antibodies raised against its N-terminal and C-terminal segments. Under reducing conditions, proteins of 45-49 and 70-90 kDa (p45 and p70) were detected with N-terminal antibodies, and proteins of 40 and 70-90 kDa (p40 and p70) were detected with C-terminal antibodies. p40 and p45 apparently result from a post-translational cleavage of NaPi-2 but remain linked through one or more disulphide bonds. Glycosidase digestion showed that both polypeptides are glycosylated; the cleavage site could thus be located between Asn-298 and Asn-328, which have been shown to constitute the only two N-glycosylated residues in NaPi-2. In the absence of reducing agents, both N-terminal and C-terminal antibodies detected p70 and a protein of 180 kDa (p180), suggesting the presence of p70 dimers. Much higher concentrations of beta-mercaptoethanol were required to produce a given effect in intact membrane vesicles than in solubilized proteins, indicating that the affected disulphide bonds are not exposed at the surface of the co-transporter. Phosphate transport activity decreased with increasing concentrations of reducing agents [beta-mercaptoethanol, dithiothreitol and tris-(2-carboxyethyl)phosphine] and was linearly correlated with the amount of p180 detected. The target sizes estimated from the radiation-induced loss of intensity of p40, p70 and p180 were all approx. 190 kDa, suggesting that NaPi-2 exists as an oligomeric protein in which the subunits are sufficiently close to one another to allow substantial energy transfer between the monomers. When protein samples were pretreated with beta-mercaptoethanol [2.5% and 5% (v/v) to optimize the detection of p40 and p70] before irradiation, target sizes estimated from the radiation-induced loss of intensity of p40 and p70 were 74 and 92 kDa respectively, showing the presence of disulphide bridges in the molecular

  18. Dose evaluation based on 24Na activity in the human body at the JCO criticality accident in Tokai-mura.

    PubMed

    Momose, T; Tsujimura, N; Tasaki, T; Kanai, K; Kurihara, O; Hayashi, N; Shinohara, K

    2001-09-01

    24Na in the human body, activated by neutrons emitted at the JCO criticality accident, was observed for 62 subjects, where 148 subjects were measured by the whole body counter of JNC Tokai Works. The 148 subjects, including JCO employees and the contractors, residents neighboring the site and emergency service officers, were measured by the whole-body counter. The neutron-energy spectrum around the facility was calculated using neutron transport codes (ANISN and MCNP), and the relation between an amount of activated sodium in human body and neutron dose was evaluated from the calculated neutron energy spectrum and theoretical neutron capture probability by the human body. The maximum 24Na activity in the body was 7.7 kBq (83 Bq(24Na)/g(23Na)) and the relevant effective dose equivalent was 47 mSv.

  19. Na/sup +/-dependent transport of /sup 14/C-L-lysine across bullfrog alveolar epithelium

    SciTech Connect

    Kim, K.J.; Crandall, E.D.

    1986-03-01

    Transepithelial transport of the basic amino acid L-lysine has been studied utilizing the isolated intact bullfrog lung mounted in the Ussing chamber. Lungs were excised from doubly pithed bullfrogs and sandwiched between two hemichambers. /sup 14/C-(U)-L-lysine was added to the upstream reservoir of amphibian Ringer solution, while the tissue was short-circuited. Two lungs from the same animal were used simultaneously to determine the two opposite unidirectional fluxes. Downstream and upstream radioactivities were assayed and used to estimate the apparent permeability (P) of the labeled lysine. Results indicate that the apparent P of /sup 14/C-L-lysine measured in the alveolar (M) to the pleural (S) direction is 19.06 (+- 2.84) x 10/sup -7/ cm/s and P in the S to M direction is 3.29 (+- 0.02) x 10/sup -7/ cm/s. When the 100 mM NaCl in the bath was replaced by 110 mM choline chloride, the flux of /sup 14/C-L-lysine from the alveolar to the pleural side decreased to the same value as that in the opposite direction. The flux from the pleural to the alveolar direction in the absence of Na/sup +/ did not change. These results suggest that the alveolar epithelium exhibits Na/sup +/-dependent amino acid (L-lysine) transport in the M->S, but not in the S->M, direction.

  20. Refinement of the Central Steps of Substrate Transport by the Aspartate Transporter GltPh: Elucidating the Role of the Na2 Sodium Binding Site

    PubMed Central

    Venkatesan, SanthoshKannan; Saha, Kusumika; Sohail, Azmat; Sandtner, Walter; Freissmuth, Michael; Ecker, Gerhard F.; Sitte, Harald H.; Stockner, Thomas

    2015-01-01

    Glutamate homeostasis in the brain is maintained by glutamate transporter mediated accumulation. Impaired transport is associated with several neurological disorders, including stroke and amyotrophic lateral sclerosis. Crystal structures of the homolog transporter GltPh from Pyrococcus horikoshii revealed large structural changes. Substrate uptake at the atomic level and the mechanism of ion gradient conversion into directional transport remained enigmatic. We observed in repeated simulations that two local structural changes regulated transport. The first change led to formation of the transient Na2 sodium binding site, triggered by side chain rotation of T308. The second change destabilized cytoplasmic ionic interactions. We found that sodium binding to the transiently formed Na2 site energized substrate uptake through reshaping of the energy hypersurface. Uptake experiments in reconstituted proteoliposomes confirmed the proposed mechanism. We reproduced the results in the human glutamate transporter EAAT3 indicating a conserved mechanics from archaea to humans. PMID:26485255

  1. Relationship between sodium-dependent phosphate transporter (NaPi-IIc) function and cellular vacuole formation in opossum kidney cells.

    PubMed

    Shiozaki, Yuji; Segawa, Hiroko; Ohnishi, Saori; Ohi, Akiko; Ito, Mikiko; Kaneko, Ichiro; Kido, Shinsuke; Tatsumi, Sawako; Miyamoto, Ken-ichi

    2015-01-01

    NaPi-IIc/SLC34A3 is a sodium-dependent inorganic phosphate (Pi) transporter in the renal proximal tubules and its mutations cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). In the present study, we created a specific antibody for opossum SLC34A3, NaPi-IIc (oNaPi-IIc), and analyzed its localization and regulation in opossum kidney cells (a tissue culture model of proximal tubular cells). Immunoreactive oNaPi-IIc protein levels increased during the proliferative phase and decreased during differentiation. Moreover, stimulating cell growth upregulated oNaPi-IIc protein levels, whereas suppressing cell proliferation downregulated oNaPi-IIc protein levels. Immunocytochemistry revealed that endogenous and exogenous oNaPi-IIc proteins localized at the protrusion of the plasma membrane, which is a phosphatidylinositol 4,5-bisphosphate (PIP2) rich-membrane, and at the intracellular vacuolar membrane. Exogenous NaPi-IIc also induced cellular vacuoles and localized in the plasma membrane. The ability to form vacuoles is specific to electroneutral NaPi-IIc, and not electrogenic NaPi-IIa or NaPi-IIb. In addition, mutations of NaPi-IIc (S138F and R468W) in HHRH did not cause cellular PIP2-rich vacuoles. In conclusion, our data anticipate that NaPi-IIc may regulate PIP2 production at the plasma membrane and cellular vesicle formation.

  2. Berberine acutely activates the glucose transport activity of GLUT1.

    PubMed

    Cok, Alexandra; Plaisier, Christina; Salie, Matthew J; Oram, Daniel S; Chenge, Jude; Louters, Larry L

    2011-07-01

    Berberine, which has a long history of use in Chinese medicine, has recently been shown to have efficacy in the treatment of diabetes. While the hypoglycemic effect of berberine has been clearly documented in animal and cell line models, such as 3T3-L1 adipocytes and L6 myotube cells, the mechanism of action appears complex with data implicating activation of the insulin signaling pathway as well as activation of the exercise or AMP kinase-mediated pathway. There have been no reports of the acute affects of berberine on the transport activity of the insulin-insensitive glucose transporter, GLUT1. Therefore, we examined the acute effects of berberine on glucose uptake in L929 fibroblast cells, a cell line that express only GLUT1. Berberine- activated glucose uptake reaching maximum stimulation of five-fold at >40 μM. Significant activation (P < 0.05) was measured within 5 min reaching a maximum by 30 min. The berberine effect was not additive to the maximal stimulation by other known stimulants, azide, methylene blue or glucose deprivation, suggesting shared steps between berberine and these stimulants. Berberine significantly reduced the K(m) of glucose uptake from 6.7 ± 1.9 mM to 0.55 ± 0.08 mM, but had no effect on the V(max) of uptake. Compound C, an inhibitor of AMP kinase, did not affect berberine-stimulated glucose uptake, but inhibitors of downstream kinases partially blocked berberine stimulation. SB203580 (inhibitor of p38 MAP kinase) did not affect submaximal berberine activation, but did lower maximal berberine stimulation by 26%, while PD98059 (inhibitor of ERK kinase) completely blocked submaximal berberine activation and decreased the maximal stimulation by 55%. It appears from this study that a portion of the hypoglycemic effects of berberine can be attributed to its acute activation of the transport activity of GLUT1.

  3. Induction of the high-affinity Na(+)-dependent glutamate transport system XAG- by hypertonic stress in the renal epithelial cell line NBL-1.

    PubMed Central

    Ferrer-Martinez, A; Felipe, A; Nicholson, B; Casado, J; Pastor-Anglada, M; McGivan, J

    1995-01-01

    The high-affinity Na(+)-dependent glutamate transport system XAG- is induced (threefold increase in Vmax. with no change in Km) by hypertonicity in the renal epithelial cell line NBL-1. This effect is dependent on protein synthesis and glycosylation and is accompanied by an increase in EAAC1 mRNA levels. Other Na(+)-dependent transport systems in this cell line do not respond to hypertonic stress. In contrast to recent findings [Ruiz-Montasell, Gomez-Angelats, Casado, Felipe, McGivan and Pastor-Anglada (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9569-9573] showing that increased system A activity after hyperosmotic shock results from induction of a regulatory protein, this is the first demonstration that hypertonicity may increase the expression of the gene for an amino acid transport protein itself. Images Figure 4 PMID:7654212

  4. Sugar-activated ion transport in canine lingual epithelium. Implications for sugar taste transduction

    PubMed Central

    1988-01-01

    There is good evidence indicating that ion-transport pathways in the apical regions of lingual epithelial cells, including taste bud cells, may play a role in salt taste reception. In this article, we present evidence that, in the case of the dog, there also exists a sugar- activated ion-transport pathway that is linked to sugar taste transduction. Evidence was drawn from two parallel lines of experiments: (a) ion-transport studies on the isolated canine lingual epithelium, and (b) recordings from the canine chorda tympani. The results in vitro showed that both mono- and disaccharides in the mucosal bath stimulate a dose-dependent increase in the short-circuit current over the concentration range coincident with mammalian sugar taste responses. Transepithelial current evoked by glucose, fructose, or sucrose in either 30 mM NaCl or in Krebs-Henseleit buffer (K-H) was partially blocked by amiloride. Among current carriers activated by saccharides, the current response was greater with Na than with K. Ion flux measurements in K-H during stimulation with 3-O-methylglucose showed that the sugar-evoked current was due to an increase in the Na influx. Ouabain or amiloride reduced the sugar-evoked Na influx without effect on sugar transport as measured with tritiated 3-O-methylglucose. Amiloride inhibited the canine chorda tympani response to 0.5 M NaCl by 70-80% and the response to 0.5 M KCl by approximately 40%. This agreed with the percent inhibition by amiloride of the short-circuit current supported in vitro by NaCl and KCl. Amiloride also partially inhibited the chorda tympani responses to sucrose and to fructose. The results indicate that in the dog: (a) the ion transporter subserving Na taste also subserves part of the response to K, and (b) a sugar-activated, Na- preferring ion-transport system is one mechanism mediating sugar taste transduction. Results in the literature indicate a similar sweet taste mechanism for humans. PMID:3171536

  5. MAP17 Is a Necessary Activator of Renal Na+/Glucose Cotransporter SGLT2.

    PubMed

    Coady, Michael J; El Tarazi, Abdulah; Santer, René; Bissonnette, Pierre; Sasseville, Louis J; Calado, Joaquim; Lussier, Yoann; Dumayne, Christopher; Bichet, Daniel G; Lapointe, Jean-Yves

    2017-01-01

    The renal proximal tubule reabsorbs 90% of the filtered glucose load through the Na(+)-coupled glucose transporter SGLT2, and specific inhibitors of SGLT2 are now available to patients with diabetes to increase urinary glucose excretion. Using expression cloning, we identified an accessory protein, 17 kDa membrane-associated protein (MAP17), that increased SGLT2 activity in RNA-injected Xenopus oocytes by two orders of magnitude. Significant stimulation of SGLT2 activity also occurred in opossum kidney cells cotransfected with SGLT2 and MAP17. Notably, transfection with MAP17 did not change the quantity of SGLT2 protein at the cell surface in either cell type. To confirm the physiologic relevance of the MAP17-SGLT2 interaction, we studied a cohort of 60 individuals with familial renal glucosuria. One patient without any identifiable mutation in the SGLT2 coding gene (SLC5A2) displayed homozygosity for a splicing mutation (c.176+1G>A) in the MAP17 coding gene (PDZK1IP1). In the proximal tubule and in other tissues, MAP17 is known to interact with PDZK1, a scaffolding protein linked to other transporters, including Na(+)/H(+) exchanger 3, and to signaling pathways, such as the A-kinase anchor protein 2/protein kinase A pathway. Thus, these results provide the basis for a more thorough characterization of SGLT2 which would include the possible effects of its inhibition on colocalized renal transporters.

  6. Integration of a 'proton antenna' facilitates transport activity of the monocarboxylate transporter MCT4.

    PubMed

    Noor, Sina Ibne; Pouyssegur, Jacques; Deitmer, Joachim W; Becker, Holger M

    2017-01-01

    Monocarboxylate transporters (MCTs) mediate the proton-coupled transport of high-energy metabolites like lactate and pyruvate and are expressed in nearly every mammalian tissue. We have shown previously that transport activity of MCT4 is enhanced by carbonic anhydrase II (CAII), which has been suggested to function as a 'proton antenna' for the transporter. In the present study, we tested whether creation of an endogenous proton antenna by introduction of a cluster of histidine residues into the C-terminal tail of MCT4 (MCT4-6xHis) could facilitate MCT4 transport activity when heterologously expressed in Xenopus oocytes. Our results show that integration of six histidines into the C-terminal tail does indeed increase transport activity of MCT4 to the same extent as did coexpression of MCT4-WT with CAII. Transport activity of MCT4-6xHis could be further enhanced by coexpression with extracellular CAIV, but not with intracellular CAII. Injection of an antibody against the histidine cluster into MCT4-expressing oocytes decreased transport activity of MCT4-6xHis, while leaving activity of MCT4-WT unaltered. Taken together, these findings suggest that transport activity of the proton-coupled monocarboxylate transporter MCT4 can be facilitated by integration of an endogenous proton antenna into the transporter's C-terminal tail.

  7. Human, rat and chicken small intestinal Na+-Cl−-creatine transporter: functional, molecular characterization and localization

    PubMed Central

    Peral, M J; García-Delgado, M; Calonge, M L; Durán, J M; De La Horra, M C; Wallimann, T; Speer, O; Ilundáin, A A

    2002-01-01

    In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [14C]Creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription-polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na+- and Cl−-dependent, with a probable stoichiometry of 2 Na+: 1 Cl−: 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a Km for creatine of 29 μm. [14C]Creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, β-alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full-length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high-affinity, Na+- and Cl−-dependent, apical creatine uptake. PMID:12433955

  8. The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.

    PubMed

    Bao, Huan; Dalal, Kush; Wang, Victor; Rouiller, Isabelle; Duong, Franck

    2013-08-01

    The coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency.

  9. The Na+ transport in gram-positive bacteria defect in the Mrp antiporter complex measured with 23Na nuclear magnetic resonance.

    PubMed

    Górecki, Kamil; Hägerhäll, Cecilia; Drakenberg, Torbjörn

    2014-01-15

    (23)Na nuclear magnetic resonance (NMR) has previously been used to monitor Na(+) translocation across membranes in gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na(+). In this work, the (23)Na NMR method was adapted for measurements of internal Na(+) concentration in the gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na(+) translocation activity of the Mrp (multiple resistance and pH) antiporter complex, a member of the cation proton antiporter-3 (CPA-3) family. The sodium-sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted could indeed be correlated to the inability of this strain to maintain a lower internal Na(+) concentration than an external one.

  10. Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

    PubMed Central

    Dürr, Katharina L.; Tavraz, Neslihan N.; Spiller, Susan; Friedrich, Thomas

    2013-01-01

    Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting

  11. A Simple Laboratory Exercise Illustrating Active Transport in Yeast Cells.

    ERIC Educational Resources Information Center

    Stambuk, Boris U.

    2000-01-01

    Describes a simple laboratory activity illustrating the chemiosmotic principles of active transport in yeast cells. Demonstrates the energy coupling mechanism of active a-glucoside uptake by Saccaromyces cerevisiae cells with a colorimetric transport assay using very simple equipment. (Contains 22 references.) (Author/YDS)

  12. Reverse osmosis transport of alkali halides and nickel salts through cellulose triacetate membranes. Performance prediction from NaCl experiments

    SciTech Connect

    Nirmal, J.D.; Pandya, V.P.; Desai, N.V.; Rangarajan, R. )

    1992-10-01

    The separation of alkali metal halides, nickel chloride, and nickel sulfate was determined for cellulose triacetate reverse osmosis (CTA RO) membranes. From transport analysis, the relative free energy parameters for transport of these salts through CTA membranes were determined. From these relative free energy parameters of salts, the solute separation by CTA membranes could be predicted from RO experiment with NaCl solution. The transport analysis and an illustration of how the concept is useful are presented in this paper.

  13. Na+/Ca2+ exchanger contributes to stool transport in mice with experimental diarrhea

    PubMed Central

    NISHIYAMA, Kazuhiro; TANIOKA, Kohta; AZUMA, Yasu-Taka; HAYASHI, Satomi; FUJIMOTO, Yasuyuki; YOSHIDA, Natsuho; KITA, Satomi; SUZUKI, Sho; NAKAJIMA, Hidemitsu; IWAMOTO, Takahiro; TAKEUCHI, Tadayoshi

    2016-01-01

    The Na+/Ca2+ exchanger (NCX) is a bidirectional transporter that is controlled by membrane potential and transmembrane gradients of Na+ and Ca2+. To reveal the functional role of NCX on gastrointestinal motility, we have previously used NCX1 and NCX2 heterozygote knockout mice (HET). We found that NCX1 and NCX2 play important roles in the motility of the gastric fundus, ileum and distal colon. Therefore, we believed that NCX1 and NCX2 play an important role in transport of intestinal contents. Here, we investigated the role of NCX in a mouse model of drug-induced diarrhea. The fecal consistencies in NCX1 HET and NCX2 HET were assessed using a diarrhea induced by magnesium sulfate, 5-hydroxytryptamine (5-HT) and prostaglandin E2 (PGE2). NCX2 HET, but not NCX1 HET, exacerbated magnesium sulfate-induced diarrhea by increasing watery fecals. Likewise, 5-HT-induced diarrheas were exacerbated in NCX2 HET, but not NCX1 HET. However, NCX1 HET and NCX2 HET demonstrated PGE2 induced diarrhea similar to those of wild-type mice (WT). As well as the result of the distal colon shown previously, in the proximal and transverse colons of WT, the myenteric plexus layers and the longitudinal and circular muscle layers were strongly immunoreactive to NCX1 and NCX2. In this study, we demonstrate that NCX2 has important roles in development of diarrhea. PMID:27928109

  14. Transportation as a "Related Service": Issues that Involve Transition Activities.

    ERIC Educational Resources Information Center

    Missouri Univ., Columbia. Missouri LINC.

    The paper discusses transportation as a related service for students with disabilities expecially as related to school-to-work transition activities. First, the legislative and legal basis for providing transportation services is discussed in the form of answers to frequently asked questions: why provide transportation? what is the basis for…

  15. Chill activation of compatible solute transporters in Corynebacterium glutamicum at the level of transport activity.

    PubMed

    Ozcan, Nuran; Krämer, Reinhard; Morbach, Susanne

    2005-07-01

    The gram-positive soil bacterium Corynebacterium glutamicum harbors four osmoregulated secondary uptake systems for compatible solutes, BetP, EctP, LcoP, and ProP. When reconstituted in proteoliposomes, BetP was shown to sense hyperosmotic conditions via the increase in luminal K(+) and to respond by instant activation. To study further putative ways of stimulus perception and signal transduction, we have investigated the responses of EctP, LcoP, and BetP, all belonging to the betaine-carnitine-choline transporter family, to chill stress at the level of activity. When fully activated by hyperosmotic stress, they showed the expected increase of activity at increasing temperature. In the absence of osmotic stress, EctP was not activated by chill and LcoP to only a very low extent, whereas BetP was significantly stimulated at low temperature. BetP was maximally activated at 10 degrees C, reaching the same transport rate as that observed under hyperosmotic conditions at this temperature. A role of cytoplasmic K(+) in chill-dependent activation of BetP was ruled out, since (i) the cytoplasmic K(+) concentration did not change significantly at lower temperatures and (ii) a mutant BetP lacking the C-terminal 25 amino acids, which was previously shown to have lost the ability to be activated by luminal K(+), was fully competent in chill sensing. When heterologously expressed in Escherichia coli, BetP did not respond to chill stress. This may indicate that the membrane in which BetP is inserted plays an important role in chill activation and thus in signal transduction by BetP, different from the previously established K(+)-mediated process.

  16. (Na,K)-ATPase-mediated cation pumping in cultured rat hepatocytes. Rapid modulation by alanine and taurocholate transport and characterization of its relationship to intracellular sodium concentration.

    PubMed

    Van Dyke, R W; Scharschmidt, B F

    1983-11-10

    (Na,K)-ATPase is thought to maintain the transmembrane electrochemical sodium gradient which powers secondary active sodium-coupled transport of a variety of solutes including amino acids and bile acids. However, little is known regarding the effect of sodium-coupled solute transport on intracellular sodium concentration ( [Na]ic) and on (Na,K)-ATPase-mediated cation pumping in the intact cell. In order to address this question, we have measured 22Na uptake rate, steady state 22Na content, and ouabain-suppressible 86Rb uptake rate in primary cultures of adult rat hepatocytes under a variety of conditions. Compared with control conditions (sodium uptake rate = 6.00 +/- 0.40 nmol X min-1 X mg-1; [Na]ic = 11.96 +/- 0.54 mM; cation pumping = 2.53 +/- 0.18 nmol X min-1 X mg-1), cation pumping was increased by taurocholate (less than or equal to 158%), alanine (less than or equal to 246%), monensin (less than or equal to 400%), and cold exposure (less than or equal to 525%), and this increase was accompanied by increases in Na uptake and [Na]ic. In contrast, preincubation in low sodium medium decreased all three variables. These changes in cation pumping were blocked in the absence of extracellular sodium and were not accompanied by changes in ouabain-suppressible ATP hydrolysis measured in cell homogenate. An overall plot of cation pumping versus [Na]ic yielded a sigmoid-shaped curve. Values for KNa (17.8 +/- 1.4 mM) and Vmax (8.98 +/- 0.62 nmol X min-1 X mg-1) for cation pumping were estimated assuming three sodium sites per pump unit. These findings indicate that: 1) uptake of alanine and taurocholate is associated with a rapid increase in (Na,K)-ATPase cation pumping; 2) this increase probably results from an increase in pumping per pump unit rather than an increase in the total number of pump units, and it appears to be mediated via an increase in sodium influx and [Na]ic; 3) [Na]ic under control conditions is close to the apparent KNa of cation pumping, implying

  17. Bulk liquid pertraction of NaCl from aqueous solution using carrier-mediated transport.

    PubMed

    Naim, M M; El-Shafei, A A; Moneer, A A; Elewa, M M; Kandeel, W G

    2015-10-06

    In the present work, removal of NaCl using the bulk liquid membrane (BLM) technique has been investigated, using a simple apparatus for conducting the experiments. Variables investigated were volume ratio of donor phase (DP) to receptor phase (RP), presence of sequestering agent (SA) in RP, type of organic liquid membrane (LM), quantity of mobile carrier (MC) in the LM. Stirring speed and volume of LM were kept constant at 100 rpm and 130 ml, respectively. The mass transfer of NaCl was analysed based on kinetic laws of two consecutive irreversible first-order reactions, and kinetic parameters (k1d, k2m, k2r, [Formula: see text], tmax, [Formula: see text], and [Formula: see text]) for the transport of NaCl were investigated. The values obtained demonstrate that the process is diffusionally controlled. Results indicate that the membrane entrance and exit rate constants (k1, k2) increase with decreasing DP:RP ratio and with decrease in quantity of MC, and quantity of SA, and the presence of dichloroethane (DCE) is preferred to chloroform (CF) as LM.

  18. Dopamine transporter availability in motor subtypes of de novo drug-naïve Parkinson's disease.

    PubMed

    Moccia, Marcello; Pappatà, Sabina; Picillo, Marina; Erro, Roberto; Coda, Anna Rita Daniela; Longo, Katia; Vitale, Carmine; Amboni, Marianna; Brunetti, Arturo; Capo, Giuseppe; Salvatore, Marco; Barone, Paolo; Pellecchia, Maria Teresa

    2014-11-01

    Tremor dominant (TD) and akinetic-rigid type (ART) are two motor subtypes of Parkinson's disease associated with different disease progression and neurochemical/neuropathological features. The role of presynaptic nigrostriatal dopaminergic damage is still controversial, poorly explored, and only assessed in medicated patients. In this study, we investigated with FP-CIT SPECT the striatal dopamine transporter (DAT) availability in drug-naïve PD patients with ART and TD phenotypes. Fifty-one de novo, drug-naïve patients with PD underwent FP-CIT SPECT studies. Patients were evaluated with Unified Parkinson's Disease Rating Scale (UPDRS) part III and Hoehn and Yahr scale (H&Y) and divided into ART (24/51) and TD (27/51) according to UPDRS part III. ART and TD patients were not different with regard to age, gender, and disease duration. However, compared to TD, ART patients presented higher UPDRS part III (p = 0.01) and H&Y (p = 0.02) and lower DAT availability in affected and unaffected putamen (p = 0.008 and p = 0.007, respectively), whereas no differences were found in caudate. Moreover, in the whole group of patients, rigidity and bradykinesia, but not tremor scores of UPDRS part III were significantly related to FP-CIT binding in the putamen. These results suggest that in newly diagnosed drug-naïve PD patients DAT availability might be different between ART and TD in relation to different disease severity.

  19. Air pollution exposure: An activity pattern approach for active transportation

    NASA Astrophysics Data System (ADS)

    Adams, Matthew D.; Yiannakoulias, Nikolaos; Kanaroglou, Pavlos S.

    2016-09-01

    In this paper, we demonstrate the calculation of personal air pollution exposure during trips made by active transportation using activity patterns without personal monitors. We calculate exposure as the inhaled dose of particulate matter 2.5 μg or smaller. Two modes of active transportation are compared, and they include cycling and walking. Ambient conditions are calculated by combining mobile and stationary monitoring data in an artificial neural network space-time model. The model uses a land use regression framework and has a prediction accuracy of R2 = 0.78. Exposure is calculated at 10 m or shorter intervals during the trips using inhalation rates associated with both modes. The trips are children's routes between home and school. The average dose during morning cycling trips was 2.17 μg, during morning walking trips was 3.19 μg, during afternoon cycling trips was 2.19 μg and during afternoon walking trips was 3.23 μg. The cycling trip dose was significantly lower than the walking trip dose. The air pollution exposure during walking or cycling trips could not be strongly predicted by either the school or household ambient conditions, either individually or in combination. Multiple linear regression models regressing both the household and school ambient conditions against the dose were only able to account for, at most, six percent of the variance in the exposure. This paper demonstrates that incorporating activity patterns when calculating exposure can improve the estimate of exposure compared to its calculation from ambient conditions.

  20. Voltage coupling of primary H+ V-ATPases to secondary Na+- or K+-dependent transporters.

    PubMed

    Harvey, William R

    2009-06-01

    and other evidence provide convincing support for Kell's electrodic view yet Mitchell's chemiosmotic theory is the one that is accepted by most bioenergetics experts today. First we discuss the interaction between H(+) V-ATPase and the K(+)/2H(+) antiporter that forms the caterpillar K(+) pump, and use the Kell electrodic view to explain how the H(+)s at the outer fluid-membrane interface can drive two H(+) from lumen to cell and one K(+) from cell to lumen via the antiporter even though the pH in the bulk fluid of the lumen is highly alkaline. Exchange of outer bulk fluid K(+) (or Na(+)) with outer interface H(+) in conjunction with (K(+) or Na(+))/2H(+) antiport, transforms the hydrogen ion electrochemical potential difference, mu(H), to a K(+) electrochemical potential difference, mu(K) or a Na(+) electrochemical potential difference, mu(Na). The mu(K) or mu(Na) drives K(+)- or Na(+)-coupled nutrient amino acid transporters (NATs), such as KAAT1 (K(+) amino acid transporter 1), which moves Na(+) and an amino acid into the cell with no H(+)s involved. Examples in which the voltage coupling model is used to interpret ion and amino acid transport in caterpillar and larval mosquito midgut are discussed.

  1. Inhibitory Effect of Fluoride on Na+,K+ ATPase Activity in Human Erythrocyte Membrane.

    PubMed

    A, Shashi; G, Meenakshi

    2015-12-01

    The present study was performed to evaluate the role of long-term consumption of excessive fluoride on electrolyte homeostasis and their transporting mechanisms in erythrocytes of subjects afflicted with dental and skeletal fluorosis. A total of 620 adult (20-50 years) Indian residents participated in this study: 258 men and 242 women exposed to high concentrations of fluoride and 120 age and gender-matched control subjects. Erythrocytes were isolated from blood samples, washed, and used for the estimation of intraerythrocyte sodium and potassium concentrations. Na+,K+ ATPase activity was determined spectrophotometrically from a ghost erythrocyte membrane prepared by osmotic lysis. Erythrocyte analytes were correlated with the water and serum fluoride concentrations by Pearson's bivariate correlation and regression analysis. Results indicated a significant increase in intraerythrocyte sodium (F=14306.265, P<0.0001) in subjects from endemic fluorosis study groups as compared to controls. A significant (P<0.05) positive correlation of intracellular sodium was found with water and serum fluoride concentrations. Mean concentration of intraerythrocytic potassium ions showed significant reduction (F=9136.318, P<0.0001) in subjects exposed to fluoride. A significant (P<0.05) negative correlation of potassium ions was noted with water and serum fluoride concentrations. Na+,K+ ATPase activity was significantly declined (F=1572.763, P<0.0001) in subjects exposed to fluoride. A significant (P<0.05) inverse relationship of Na+,K+ ATPase activity was revealed with water and serum fluoride concentrations.

  2. Sequential depolarization of root cortical and stelar cells induced by an acute salt shock - implications for Na(+) and K(+) transport into xylem vessels.

    PubMed

    Wegner, Lars H; Stefano, Giovanni; Shabala, Lana; Rossi, Marika; Mancuso, Stefano; Shabala, Sergey

    2011-05-01

    Early events in NaCl-induced root ion and water transport were investigated in maize (Zea mays L) roots using a range of microelectrode and imaging techniques. Addition of 100 mm NaCl to the bath resulted in an exponential drop in root xylem pressure, rapid depolarization of trans-root potential and a transient drop in xylem K(+) activity (A(K+) ) within ∼1 min after stress onset. At this time, no detectable amounts of Na(+) were released into the xylem vessels. The observed drop in A(K+) was unexpected, given the fact that application of the physiologically relevant concentrations of Na(+) to isolated stele has caused rapid plasma membrane depolarization and a subsequent K(+) efflux from the stelar tissues. This controversy was explained by the difference in kinetics of NaCl-induced depolarization between cortical and stelar cells. As root cortical cells are first to be depolarized and lose K(+) to the environment, this is associated with some K(+) shift from the stelar symplast to the cortex, resulting in K(+) being transiently removed from the xylem. Once Na(+) is loaded into the xylem (between 1 and 5 min of root exposure to NaCl), stelar cells become more depolarized, and a gradual recovery in A(K+) occurs.

  3. Close Association of Carbonic Anhydrase (CA2a and CA15a), Na+/H+ Exchanger (Nhe3b), and Ammonia Transporter Rhcg1 in Zebrafish Ionocytes Responsible for Na+ Uptake

    PubMed Central

    Ito, Yusuke; Kobayashi, Sayako; Nakamura, Nobuhiro; Miyagi, Hisako; Esaki, Masahiro; Hoshijima, Kazuyuki; Hirose, Shigehisa

    2013-01-01

    Freshwater (FW) fishes actively absorb salt from their environment to tolerate low salinities. We previously reported that vacuolar-type H+-ATPase/mitochondrion-rich cells (H-MRCs) on the skin epithelium of zebrafish larvae (Danio rerio) are primary sites for Na+ uptake. In this study, in an attempt to clarify the mechanism for the Na+ uptake, we performed a systematic analysis of gene expression patterns of zebrafish carbonic anhydrase (CA) isoforms and found that, of 12 CA isoforms, CA2a and CA15a are highly expressed in H-MRCs at larval stages. The ca2a and ca15a mRNA expression were salinity-dependent; they were upregulated in 0.03 mM Na+ water whereas ca15a but not ca2a was down-regulated in 70 mM Na+ water. Immunohistochemistry demonstrated cytoplasmic distribution of CA2a and apical membrane localization of CA15a. Furthermore, cell surface immunofluorescence staining revealed external surface localization of CA15a. Depletion of either CA2a or CA15a expression by Morpholino antisense oligonucleotides resulted in a significant decrease in Na+ accumulation in H-MRCs. An in situ proximity ligation assay demonstrated a very close association of CA2a, CA15a, Na+/H+ exchanger 3b (Nhe3b), and Rhcg1 ammonia transporter in H-MRC. Our findings suggest that CA2a, CA15a, and Rhcg1 play a key role in Na+uptake under FW conditions by forming a transport metabolon with Nhe3b. PMID:23565095

  4. Na+/H+ and Na+/NH4+ exchange activities of zebrafish NHE3b expressed in Xenopus oocytes

    PubMed Central

    Ito, Yusuke; Kato, Akira; Hirata, Taku; Hirose, Shigehisa

    2014-01-01

    Zebrafish Na+/H+ exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na+ is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na+ absorption but not leakage. However, zNHE3b-mediated Na+ absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na+-free media resulted in significant decrease in intracellular pH (pHi) and intracellular Na+ activity (aNai). aNai increased significantly when the cytoplasm was acidified by media containing CO2-HCO3− or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of ∼50 mV, voltage-clamp experiments showed that Na+/H+ exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH3/NH4+ resulted in significant decreases in pHi and aNai and significant increase in intracellular NH4+ activity, indicating that zNHE3b mediates the Na+/NH4+ exchange. In low-Na+ (0.5 mM) media, zNHE3b oocytes maintained aNai of 1.3 mM, and Na+-influx was observed when pHi was decreased by media containing CO2-HCO3− or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na+ absorption from ion-poor fresh water by its Na+/H+ and Na+/NH4+ exchange activities. PMID:24401990

  5. Freshwater transport forms of Na, Mg, and Ca in streams of adjacent headwater catchments composed of differing vegetation

    NASA Astrophysics Data System (ADS)

    Terajima, T.; Moriizumi, M.

    2012-04-01

    To understand the freshwater transport forms of major metals, concentrations of Na, Mg, Ca, Si, and fulvic acid-like materials (FAM) were measured in streams of headwater catchments with differing vegetation (coniferous and deciduous forests). The proportion of non-ionic forms (NIF) relative to total elements in the coniferous and deciduous catchments ranged from 0% to 40% and from 0% to 70%, respectively, in baseflows, and from 5% to 60% and from 20% to 60%, respectively, in stormflows. In the baseflows, NIF and total Si (T-Si) were highly correlated (r > 0.9) in both catchments. In contrast, in the stormflows, T-Si and FAM showed a good correlation (r > 0.8) in both catchments, implying that stormflow may have enhanced organic-inorganic binding. However, in the coniferous catchment, good correlations (r > 0.8) between NIF and T-Si or FAM were associated with only the rising limb of the hydrograph, whereas in the deciduous catchment, good correlations (r > 0.8) were associated with both the rising and falling limbs. These results indicate that: (1) under low-flow conditions, major metals may form binding with clay minerals and thus be transported as NIF, (2) storm events may enhance the binding of clay minerals with humic substances, (3) in the coniferous catchment, the complexation of NIF with the organic-inorganic binding and their transport in stormflows are associated with the rising limb of the storm hydrograph, whereas NIF transport during the falling limb may reflect the effect of other materials, and (4) in the deciduous catchment, NIF transport may occur mainly in association with organic-inorganic binding throughout a storm event. These findings show that active binding of Na, Mg, and Ca in freshwater environments with organic and inorganic substances, under the effect of differing vegetation on that binding, should be carefully examined in studies of chemical hydrology in headwater catchments. Key words: fulvic acid, major metal, complexation, humic

  6. Evaluation of photocatalytic activities of supported catalysts on NaX zeolite or activated charcoal.

    PubMed

    de Brites-Nóbrega, Fernanda F; Polo, Aldino N B; Benedetti, Angélica M; Leão, Mônica M D; Slusarski-Santana, Veronice; Fernandes-Machado, Nádia R C

    2013-12-15

    This study aimed to evaluate the photocatalytic activity of ZnO and Nb2O5 catalysts, both supported on NaX zeolite and activated charcoal (AC). The synergistic effect between oxide and support and the influence of solution pH (3, 7 and 9) on photocatalytic degradation of reactive blue 5G (C.I. 222) were analyzed. The catalysts Nb2O5/NaX, Nb2O5/AC and ZnO/NaX, ZnO/AC with 5 and 10% (wt%) were prepared by wet impregnation. The results showed that the catalysts exhibit quite different structural and textural properties. The synergic effect between ZnO and NaX support was higher than that with the activated charcoal, showing that these catalysts were more efficient. The most photoactive catalyst was 10% ZnO/NaX which showed 100% discoloration of the dye solution at pH 3, 7 and 9 after 0.5, 5 and 2h of irradiation, respectively. The hydrolytic nature of zeolite favored the formation of surface hydroxyl radicals, which increased the activity of the photocatalyst. Thus, catalysts supported on NaX zeolite are promising for use in photocatalysis.

  7. Na+ and K+ levels in living cells: do they depend on the rate of outward transport of Na+?

    PubMed

    Ling, G N; Ochsenfeld, M M

    1976-01-01

    At 25 degrees C, frog sartorius muslces rapidly gained Na+ and lost K+ in iodoacetamide and pure nitrogen. Beginning at normal levels, the concentrations of these ions in the cells reached those in the surrounding Ringer solution in 140 min. Yet during that time the Na+ efflux rate showed no sign of the slowing down demanded by Na-pump theory. The data support the view that maintenance and alterations of N1+ levels in frog muslce cells reflect adsorption on protein sites and the solubility property of bulk phase water and are independent of the rate at which Na+ leaves the cell surface.

  8. Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model.

    PubMed

    Vaquer, Sergi; Cuyàs, Elisabet; Rabadán, Arnau; González, Albert; Fenollosa, Felip; de la Torre, Rafael

    2014-01-01

    Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay (®) (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and

  9. Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model

    PubMed Central

    Vaquer, Sergi; Cuyàs, Elisabet; Rabadán, Arnau; González, Albert; Fenollosa, Felip; de la Torre, Rafael

    2014-01-01

    Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay ® (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and

  10. Proteinases inhibit H(+)-ATPase and Na+/H+ exchange but not water transport in apical and endosomal membranes from rat proximal tubule.

    PubMed

    Sabolić, I; Shi, L B; Brown, D; Ausiello, D A; Verkman, A S

    1992-01-10

    A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H(+)-pump, passive H+ conductance, and Na+/H+ exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H+ transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. As measured by potential-driven H+ accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15-30%, Na+/H+ exchange by 20-55%, and H+ conductance was unchanged. Osmotic water permeability (Pf) was 570 microns/s and was inhibited 85-90% by 0.6 mM HgCl2; proteinase treatment did not affect Pf or the HgCl2 inhibition. In EV, NEM-sensitive H+ accumulation and ATPase activity were inhibited by greater than 95%. Pf (140 microns/s) and HgCl2 inhibition (75-85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na+/H+ exchange by proteinase digestion. The lack of effect of proteinases on water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H(+)-ATPase and Na+/H+ exchanger, has no functionally important membrane domains that are sensitive to proteolysis.

  11. Transporting Radioactive Waste: An Engineering Activity. Grades 5-12.

    ERIC Educational Resources Information Center

    HAZWRAP, The Hazardous Waste Remedial Actions Program.

    This brochure contains an engineering activity for upper elementary, middle school, and high school students that examines the transportation of radioactive waste. The activity is designed to inform students about the existence of radioactive waste and its transportation to disposal sites. Students experiment with methods to contain the waste and…

  12. Airway surface liquid volume expansion induces rapid changes in amiloride-sensitive Na+ transport across upper airway epithelium-Implications concerning the resolution of pulmonary edema

    PubMed Central

    Azizi, Fouad; Arredouani, Abdelilah; Mohammad, Ramzi M

    2015-01-01

    During airway inflammation, airway surface liquid volume (ASLV) expansion may result from the movement of plasma proteins and excess liquid into the airway lumen due to extravasation and elevation of subepithelial hydrostatic pressure. We previously demonstrated that elevation of submucosal hydrostatic pressure increases airway epithelium permeability resulting in ASLV expansion by 500 μL cm−2 h−1. Liquid reabsorption by healthy airway epithelium is regulated by active Na+ transport at a rate of 5 μL cm−2 h−1. Thus, during inflammation the airway epithelium may be submerged by a large volume of luminal liquid. Here, we have investigated the mechanism by which ASLV expansion alters active epithelial Na+ transport, and we have characterized the time course of the change. We used primary cultures of tracheal airway epithelium maintained under air interface (basal ASLV, depth is 7 ± 0.5 μm). To mimic airway flooding, ASLV was expanded to a depth of 5 mm. On switching from basal to expanded ASLV conditions, short-circuit current (Isc, a measure of total transepithelial active ion transport) declined by 90% with a half-time (t1/2) of 1 h. 24 h after the switch, there was no significant change in ATP concentration nor in the number of functional sodium pumps as revealed by [3H]-ouabain binding. However, amiloride-sensitive uptake of 22Na+ was reduced by 70% upon ASLV expansion. This process is reversible since after returning cells back to air interface, Isc recovered with a t1/2 of 5–10 h. These results may have important clinical implications concerning the development of Na+ channels activators and resolution of pulmonary edema. PMID:26333829

  13. Ascorbate transport in pig coronary artery smooth muscle: Na(+) removal and oxidative stress increase loss of accumulated cellular ascorbate.

    PubMed

    Holmes, M E; Samson, S E; Wilson, J X; Dixon, S J; Grover, A K

    2000-01-01

    Pig deendothelialized coronary artery rings and smooth muscle cells cultured from them accumulated ascorbate from medium containing Na(+). The accumulated material was determined to be ascorbate using high-performance liquid chromatography. We further characterized ascorbate uptake in the cultured cells. The data fitted best with a Hill coefficient of 1 for ascorbate (K(asc) = 22 +/- 2 microM) and 2 for Na(+) (K(Na) = 84 +/- 10 mM). The anion transport inhibitors sulfinpyrazone and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) inhibited the uptake. Transferring cultured cells loaded with (14)C-ascorbate into an ascorbate-free solution resulted in a biphasic loss of radioactivity - an initial sulfinpyrazone-insensitive faster phase and a late sulfinpyrazone-sensitive slower phase. Transferring loaded cells into a Na(+)-free medium increased the loss in the initial phase in a sulfinpyrazone-sensitive manner, suggesting that the ascorbate transporter is bidirectional. Including peroxide or superoxide in the solution increased the loss of radioactivity. Thus, ascorbate accumulated in coronary artery smooth muscle cells by a Na(+)-dependent transporter was lost in an ascorbate-free solution, and the loss was increased by removing Na(+) from the medium or by oxidative stress.

  14. Characterization and antibacterial activity of silver exchanged regenerated NaY zeolite from surfactant-modified NaY zeolite.

    PubMed

    Salim, Mashitah Mad; Malek, Nik Ahmad Nizam Nik

    2016-02-01

    The antibacterial activity of regenerated NaY zeolite (thermal treatment from cetyltrimethyl ammonium bromide (CTAB)-modified NaY zeolite and pretreatment with Na ions) loaded with silver ions were examined using the broth dilution minimum inhibitory concentration (MIC) method against Escherichia coli (E. coli ATCC 11229) and Staphylococcus aureus (S. aureus ATCC 6538). X-ray diffraction (XRD), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, field emission scanning electron microscopy (FESEM), energy dispersive X-ray (EDX) and chemical elemental analyses were used to characterize the regenerated NaY and AgY zeolites. The XRD patterns indicated that the calcination and addition of silver ions on regenerated NaY zeolite did not affect the structure of the regenerated NaY zeolite as the characteristic peaks of the NaY zeolite were retained, and no new peaks were observed. The regenerated AgY zeolite showed good antibacterial activity against both bacteria strains in distilled water, and the antibacterial activity of the samples increased with increasing Ag loaded on the regenerated AgY zeolite; the regenerated AgY zeolite was more effective against E. coli than S. aureus. However, the antibacterial activity of the regenerated AgY was not effective in saline solution for both bacteria. The study showed that CTAB-modified NaY zeolite materials could be regenerated to NaY zeolite using thermal treatment (550°C, 5h) and this material has excellent performance as an antibacterial agent after silver ions loading.

  15. Heat transport in active harmonic chains

    SciTech Connect

    Zheng, Mei C.; Ellis, Fred M.; Kottos, Tsampikos; Fleischmann, Ragnar; Geisel, Theo; Prosen, Tomaz

    2011-08-15

    We show that a harmonic lattice model with amplifying and attenuating elements, when coupled to two thermal baths, exhibits unique heat transport properties. Some of these novel features include anomalous nonequilibrium steady-state heat currents, negative differential thermal conductance, as well as nonreciprocal heat transport. We find that when these elements are arranged in a PT-symmetric manner, the domain of existence of the nonequilibrium steady state is maximized. We propose an electronic experimental setup based on resistive-inductive-capacitive (RLC) transmission lines, where our predictions can be tested.

  16. Preparation of activated carbon from wet sludge by electrochemical-NaClO activation.

    PubMed

    Miao, Chen; Ye, Caihong; Zhu, Tianxing; Lou, Ziyang; Yuan, Haiping; Zhu, Nanwen

    2014-01-01

    Activated carbon (AC) from sludge is one potential solution for sewage sludge disposal, while the drying sludge is cost and time consuming for preparation. AC preparation from the wet sludge with electrochemical-NaClO activation was studied in this work. Three pretreatment processes, i.e. chemical activation, electrolysis and electrochemical-reagent reaction, were introduced to improve the sludge-derived AC properties, and the optimum dosage of reagent was tested from the 0.1:1 to 1:1 (mass rate, reagent:dried sludge). It was shown that the electrochemical-NaClO preparation is the best method under the test conditions, in which AC has the maximum Brunauer, Emmett and Teller area of 436 m²/g at a mass ratio of 0.7. Extracellular polymeric substances in sludge can be disintegrated by electrochemical-NaClO pretreatment, with a disintegration degree of more than 45%. The percentage of carbon decreased from 34.16 to 8.81 after treated by electrochemical-NaClO activation. Fourier transform infrared spectra showed that a strong C-Cl stretching was formed in electrochemical-NaClO prepared AC. The maximum adsorption capacity of AC reaches 109 mg/g on MB adsorption experiment at pH 10 and can be repeated for three times with high removal efficiency after regeneration.

  17. Transport and biological activities of bile acids.

    PubMed

    Zwicker, Brittnee L; Agellon, Luis B

    2013-07-01

    Bile acids have emerged as important biological molecules that support the solubilization of various lipids and lipid-soluble compounds in the gut, and the regulation of gene expression and cellular function. Bile acids are synthesized from cholesterol in the liver and eventually released into the small intestine. The majority of bile acids are recovered in the distal end of the small intestine and then returned to the liver for reuse. The components of the mechanism responsible for the recycling of bile acids within the enterohepatic circulation have been identified whereas the mechanism for intracellular transport is less understood. Recently, the ileal lipid binding protein (ILBP; human gene symbol FABP6) was shown to be needed for the efficient transport of bile acids from the apical side to the basolateral side of enterocytes in the distal intestine. This review presents an overview of the transport of bile acids between the liver and the gut as well as within hepatocytes and enterocytes. A variety of pathologies is associated with the malfunction of the bile acid transport system.

  18. Coupling of active sodium transport to oxidative metabolism in the rabbit distal colon.

    PubMed Central

    Durand, J; Durand-Arczynska, W; Wankmiller, D

    1988-01-01

    1. Active transepithelial Na+ transport (Ji) and O2 consumption (Jr) were measured simultaneously in rabbit distal colon, under standard (control) incubation conditions and after various manoeuvres, known to inhibit Na+ transport. 2. The determination of Jr was complicated by the presence of fluctuations of the PO2 in the incubation solution and by spontaneous variations of the tissue respiration, which usually declined slowly with time. 3. The control values of Ji and Jr after 2 h incubation were 55 +/- 4 nequiv min-1 cm-2 and 16 +/- 1 nmol O2 min-1 cm-2, respectively (n = 44). The electrical resistance was 386 +/- 23 omega cm2; it was stable over 6 h. 4. Ji was reduced to a very low level with either amiloride, ouabain or Na+ substitution with choline. In all instances, Jr decreased concomitantly by 15-30%. 5. A plot of the change in Jr versus the change in Ji gave a straight line for all situations, i.e. for the spontaneous decline of Na+ transport and respiration and for the effects of the inhibitors. 6. The linearity between Jr and Ji allows for the determination of a stoichiometric ratio. It is of similar magnitude, when calculated either with the data of spontaneous variations or with those obtained by the action of any inhibitor tested. It is 15-20 Na+ ions per O2 molecule, a value close to that reported previously for amphibian epithelia and also close to the maximum theoretical value of 18 Na+ ions per O2 molecule. PMID:3411504

  19. Interleukin 18 function requires both interleukin 18 receptor and Na-Cl co-transporter

    PubMed Central

    Wang, Jing; Sun, Chongxiu; Gerdes, Norbert; Liu, Conglin; Liao, Mengyang; Liu, Jian; Shi, Michael A.; He, Aina; Zhou, Yi; Sukhova, Galina K.; Chen, Huimei; Cheng, Xianwu; Kuzuya, Masafumi; Murohara, Toyoaki; Zhang, Jie; Cheng, Xiang; Jiang, Mengmeng; Shull, Gary E.; Rogers, Shaunessy; Yang, Chao-Ling; Ke, Qiang; Jelen, Sabina; Bindels, René; Ellison, David H.; Jarolim, Petr; Libby, Peter; Shi, Guo-Ping

    2015-01-01

    Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms1,2. Interruption of IL18 action reduces atherosclerosis in mice3,4. This study shows that the absence of IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E-deficient (Apoe−/−) mice, nor does it affect IL18 cell surface binding or signaling. IL18 antibody-mediated immunoprecipitation identified an interaction between IL18 and Na-Cl co-transporter (NCC), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney5. Yet, we find NCC expression and colocalization with IL18r in atherosclerotic lesions and both molecules form a complex. IL18 also binds to the cell surface and induces cell signaling and down-stream cytokine expression in NCC-transfected COS-7 cells that do not express IL18r. In Apoe−/− mice, combined deficiency of IL18r and NCC, but not single deficiency, protects mice from atherosclerosis. Peritoneal macrophages from Apoe−/− mice or those lacking IL18r or NCC respond to IL18 binding or IL18 induction of cell signaling and cytokine and chemokine production, but those with combined deficiency of IL18r and NCC do not. This study identifies NCC as an IL18-binding protein that coordinates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis. PMID:26099046

  20. Na+ coordination at the Na2 site of the Na+/I- symporter.

    PubMed

    Ferrandino, Giuseppe; Nicola, Juan Pablo; Sánchez, Yuly E; Echeverria, Ignacia; Liu, Yunlong; Amzel, L Mario; Carrasco, Nancy

    2016-09-13

    The sodium/iodide symporter (NIS) mediates active I(-) transport in the thyroid-the first step in thyroid hormone biosynthesis-with a 2 Na(+): 1 I(-) stoichiometry. The two Na(+) binding sites (Na1 and Na2) and the I(-) binding site interact allosterically: when Na(+) binds to a Na(+) site, the affinity of NIS for the other Na(+) and for I(-) increases significantly. In all Na(+)-dependent transporters with the same fold as NIS, the side chains of two residues, S353 and T354 (NIS numbering), were identified as the Na(+) ligands at Na2. To understand the cooperativity between the substrates, we investigated the coordination at the Na2 site. We determined that four other residues-S66, D191, Q194, and Q263-are also involved in Na(+) coordination at this site. Experiments in whole cells demonstrated that these four residues participate in transport by NIS: mutations at these positions result in proteins that, although expressed at the plasma membrane, transport little or no I(-) These residues are conserved throughout the entire SLC5 family, to which NIS belongs, suggesting that they serve a similar function in the other transporters. Our findings also suggest that the increase in affinity that each site displays when an ion binds to another site may result from changes in the dynamics of the transporter. These mechanistic insights deepen our understanding not only of NIS but also of other transporters, including many that, like NIS, are of great medical relevance.

  1. Study of active cooling for supersonic transports

    NASA Technical Reports Server (NTRS)

    Brewer, G. D.; Morris, R. E.

    1975-01-01

    The potential benefits of using the fuel heat sink of hydrogen fueled supersonic transports for cooling large portions of the aircraft wing and fuselage are examined. The heat transfer would be accomplished by using an intermediate fluid such as an ethylene glycol-water solution. Some of the advantages of the system are: (1) reduced costs by using aluminum in place of titanium, (2) reduced cabin heat loads, and (3) more favorable environmental conditions for the aircraft systems. A liquid hydrogen fueled, Mach 2.7 supersonic transport aircraft design was used for the reference uncooled vehicle. The cooled aircraft designs were analyzed to determine their heat sink capability, the extent and location of feasible cooled surfaces, and the coolant passage size and spacing.

  2. Modeling of active transmembrane transport in a mixture theory framework.

    PubMed

    Ateshian, Gerard A; Morrison, Barclay; Hung, Clark T

    2010-05-01

    This study formulates governing equations for active transport across semi-permeable membranes within the framework of the theory of mixtures. In mixture theory, which models the interactions of any number of fluid and solid constituents, a supply term appears in the conservation of linear momentum to describe momentum exchanges among the constituents. In past applications, this momentum supply was used to model frictional interactions only, thereby describing passive transport processes. In this study, it is shown that active transport processes, which impart momentum to solutes or solvent, may also be incorporated in this term. By projecting the equation of conservation of linear momentum along the normal to the membrane, a jump condition is formulated for the mechano-electrochemical potential of fluid constituents which is generally applicable to nonequilibrium processes involving active transport. The resulting relations are simple and easy to use, and address an important need in the membrane transport literature.

  3. [Model of active peristaltic transport in biosystems].

    PubMed

    Klochkov, B N; Romanov, A S

    2013-01-01

    A nonlinear distributed mathematical model of soft vessel with the nonmonotonous static characteristic is proposed and considered. The model describes space-time dynamics of vessel clearance change. Wave phenomena in vessels of different nature and the possibility of peristaltic fluid pumping are discussed and analyzed. The model is rather common in character and represents a description of the whole class of transport phenomena. Lymphatic vessels are particularly considered.

  4. Discovery and characterization of novel inhibitors of the sodium-coupled citrate transporter (NaCT or SLC13A5)

    PubMed Central

    Huard, Kim; Brown, Janice; Jones, Jessica C.; Cabral, Shawn; Futatsugi, Kentaro; Gorgoglione, Matthew; Lanba, Adhiraj; Vera, Nicholas B.; Zhu, Yimin; Yan, Qingyun; Zhou, Yingjiang; Vernochet, Cecile; Riccardi, Keith; Wolford, Angela; Pirman, David; Niosi, Mark; Aspnes, Gary; Herr, Michael; Genung, Nathan E.; Magee, Thomas V.; Uccello, Daniel P.; Loria, Paula; Di, Li; Gosset, James R.; Hepworth, David; Rolph, Timothy; Pfefferkorn, Jeffrey A.; Erion, Derek M.

    2015-01-01

    Citrate is a key regulatory metabolic intermediate as it facilitates the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake, by blocking the sodium-coupled citrate transporter (NaCT or SLC13A5), has been suggested as a potential therapeutic approach to treat metabolic disorders. NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions. The studies herein report the identification and characterization of a novel small dicarboxylate molecule (compound 2) capable of selectively and potently inhibiting citrate transport through NaCT, both in vitro and in vivo. Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT. The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints. PMID:26620127

  5. Insulin-stimulated Na/sup +/ transport in a model renal epithelium: protein synthesis dependence and receptor interactions

    SciTech Connect

    Blazer-Yost, B.L.; Cox, M.

    1987-05-01

    The urinary bladder of the toad, Bufo marinus, is a well characterized model of the mammalian distal nephron. Porcine insulin (approx. 0.5-5.0 ..mu..M) stimulates net mucosal to serosal Na/sup +/ flux within 10 minutes of hormone addition. The response is maintained for at least 5 hr and is completely abolished by low doses (10..mu..M) of the epithelial Na/sup +/ channel blocker amiloride. Insulin-stimulated Na/sup +/ transport does not require new protein synthesis since it is actinomycin-D (10..mu..g/ml) insensitive. Also in 3 separate experiments in which epithelial cell proteins were examined by /sup 35/S-methionine labeling, 2-dimensional polyacrylamide gel electrophoresis/autoradiography, no insulin induced proteins were observed. Equimolar concentrations of purified porcine proinsulin and insulin (0.64..mu..M) stimulate Na/sup +/ transport to the same extent. Thus, the putative toad insulin receptor may have different affinity characteristics than those demonstrated for insulin and proinsulin in mammalian tissues. Alternatively, the natriferic action of insulin in toad urinary bladders may be mediated by occupancy of another receptor. Preliminary experiments indicating that nanomolar concentrations of IGF/sub 1/ stimulate Na/sup +/ transport in this tissue support the latter contention.

  6. In vitro synthesis of a Major Facilitator Transporter for specific active transport across Droplet Interface Bilayers

    PubMed Central

    Findlay, Heather E.; Harris, Nicola J.; Booth, Paula J.

    2016-01-01

    Nature encapsulates reactions within membrane-bound compartments, affording sequential and spatial control over biochemical reactions. Droplet Interface Bilayers are evolving into a valuable platform to mimic this key biological feature in artificial systems. A major issue is manipulating flow across synthetic bilayers. Droplet Interface Bilayers must be functionalised, with seminal work using membrane-inserting toxins, ion channels and pumps illustrating the potential. Specific transport of biomolecules, and notably transport against a concentration gradient, across these bilayers has yet to be demonstrated. Here, we successfully incorporate the archetypal Major Facilitator Superfamily transporter, lactose permease, into Droplet Interface Bilayers and demonstrate both passive and active, uphill transport. This paves the way for controllable transport of sugars, metabolites and other essential biomolecular substrates of this ubiquitous transporter superfamily in DIB networks. Furthermore, cell-free synthesis of lactose permease during DIB formation also results in active transport across the interface bilayer. This adds a specific disaccharide transporter to the small list of integral membrane proteins that can be synthesised via in vitro transcription/translation for applications of DIB-based artificial cell systems. The introduction of a means to promote specific transport of molecules across Droplet Interface Bilayers against a concentration gradient gives a new facet to droplet networks. PMID:27996025

  7. Solid phase transitions and fast ion transport in LiNaSO[sub 4]: LiCl: Na[sub 2]WO[sub 4] mixed systems

    SciTech Connect

    Prabaharan, S.R.S.; Muthusubramanian, P. ); Kulandainathan, M.A.; Kapali, V. )

    1993-10-01

    LiNaSO[sub 4]: LiCl: Na[sub 2]WO[sub 4] composites of a few different compositions have been prepared by quenching the melt and studied for the first time with a view to improve the ionic conductivity of LiNaSO[sub 4] at the lowest possible temperature. The phase formations of the composites have been analyzed by means of X-ray powder diffraction technique. The transport properties have been studied by DSC and complex ac impedance analysis (to extract the dc electrical conductivity, [sigma][sub dc]). The X-ray diffractograms show evidence for solid solutions (ss) as well as second dispersed phase due to indissolved excess compound (LiCl). The [sigma] enhancement may be attributed to the increase in interfacial conductivity due to the increase in concentration of the charge carriers (ions or vacancies) forming a diffuse space charge layer between the two ion conductors, i.e., the solid solution of LiNaSO[sub 4] with dissolved chloride and tungstate fractions and a chloride phase with dissolved sulfate fraction. DSC measurements show improved thermal properties with respect to [alpha]-LiNaSO[sub 4]. The present composite mixtures offer the choice of lower transition temperatures, but these are accompanied by lower transition enthalpies.

  8. Na-coupled bicarbonate transporters of the Slc4 family in the nervous system: function, localization, and relevance to neurologic function

    PubMed Central

    Majumdar, Debeshi; Bevensee, Mark O.

    2010-01-01

    Many cellular processes including neuronal activity are sensitive to changes in intracellular and/or extracellular pH— both of which are regulated by acid-base transporter activity. HCO3−-dependent transporters are particularly potent regulators of intracellular pH in neurons and astrocytes, and also contribute to the composition of the cerebrospinal fluid (CSF). The molecular physiology of HCO3− transporters has advanced considerably over the past ~14 years as investigators have cloned and characterized the function and localization of many Na-Coupled Bicarbonate Transporters of the Slc4 family (NCBTs). In this review, we provide an updated overview of the function and localization of NCBTs in the nervous system. Multiple NCBTs are expressed in neurons and astrocytes in various brain regions, as well as in epithelial cells of the choroid plexus. Characteristics of human patients with SLC4 gene mutations/deletions and results from recent studies on mice with Slc4 gene disruptions highlight the functional importance of NCBTs in neuronal activity, somatosensory function, and CSF production. Furthermore, energy-deficient states (e.g., hypoxia and ischemia) lead to altered expression and activity of NCBTs. Thus, recent studies expand our understanding of the role of NCBTs in regulating the pH and ionic composition of the nervous system that can modulate neuronal activity. PMID:20884330

  9. An Abiotic Glass-Bead Collector Exhibiting Active Transport

    NASA Astrophysics Data System (ADS)

    Goto, Youhei; Kanda, Masato; Yamamoto, Daigo; Shioi, Akihisa

    2015-09-01

    Animals relocate objects as needed by active motion. Active transport is ubiquitous in living organisms but has been difficult to realize in abiotic systems. Here we show that a self-propelled droplet can gather scattered beads toward one place on a floor and sweep it clean. This is a biomimetic active transport with loadings and unloadings, because the transport was performed by a carrier and the motion of the carrier was maintained by the energy of the chemical reaction. The oil droplet produced fluctuation of the local number density of the beads on the floor, followed by its autocatalytic growth. This mechanism may inspire the technologies based on active transport wherein chemical and physical substances migrate as in living organisms.

  10. Energy metabolism of rabbit retina as related to function: high cost of Na+ transport.

    PubMed

    Ames, A; Li, Y Y; Heher, E C; Kimble, C R

    1992-03-01

    Experiments designed to examine the energy requirements of neurophysiological function were performed on isolated rabbit retina. Function was altered by photic stimulation or by function-specific drugs, and the response of energy metabolism was assessed by simultaneous measurements of O2 consumption and lactate production. In other experiments, the supply of O2 or glucose was reduced and the effect on energy metabolism and electrophysiological function was observed. Energy requirements under control conditions in darkness were high, with O2 consumption (per gm dry wt) at 11.3 mumol min-1, with lactate production at 14.8 mumol min-1, and with the derived value for glucose consumption at 9.3 mumol min-1 and for high-energy phosphate (approximately P) generation at 82.6 mumol min-1. Energy reserves were small. Removing glucose abolished the b-wave of the electroretinogram (ERG) with a t1/2 of 1 min, but did not immediately affect O2 consumption or the PIII of the ERG. Removing O2 caused increases of up to 2.7-fold in glycolysis (Pasteur effect) and caused both PIII and b-wave to fail, with a t1/2 of about 5 min. Neurotransmission through the inner retina was supported almost entirely by glycolysis, as evidenced by large increases in lactate production in response to flashing light and decreases in response to transmitter blockers (2.3-fold overall change), with no change in O2 consumption. Phototransduction, on the other hand, was normally supported by oxidative metabolism. The dark current accounted for 41% of the retina's O2 consumption. With O2 reduced, the dark current was partially supported by glycolysis, which accounts (at least in part) for the large Pasteur effect. Na+ transport by NaK ATPase accounted for about half of all energy used, as evidenced by the response to strophanthidin, that is, for 49% of the oxidative energy and 58% of the glycolytic energy. The t1/2 for the turnover of intracellular Na+ was calculated from these data to be less than 1 min

  11. Molecular simulation of thermodynamic and transport properties for the H2O+NaCl system.

    PubMed

    Orozco, Gustavo A; Moultos, Othonas A; Jiang, Hao; Economou, Ioannis G; Panagiotopoulos, Athanassios Z

    2014-12-21

    Molecular dynamics and Monte Carlo simulations have been carried out to obtain thermodynamic and transport properties of the binary mixture H2O+NaCl at temperatures from T = 298 to 473 K. In particular, vapor pressures, liquid densities, viscosities, and vapor-liquid interfacial tensions have been obtained as functions of pressure and salt concentration. Several previously proposed fixed-point-charge models that include either Lennard-Jones (LJ) 12-6 or exponential-6 (Exp6) functional forms to describe non-Coulombic interactions were studied. In particular, for water we used the SPC and SPC/E (LJ) models in their rigid forms, a semiflexible version of the SPC/E (LJ) model, and the Errington-Panagiotopoulos Exp6 model; for NaCl, we used the Smith-Dang and Joung-Cheatham (LJ) parameterizations as well as the Tosi-Fumi (Exp6) model. While none of the model combinations are able to reproduce simultaneously all target properties, vapor pressures are well represented using the SPC plus Joung-Cheathem model combination, and all LJ models do well for the liquid density, with the semiflexible SPC/E plus Joung-Cheatham combination being the most accurate. For viscosities, the combination of rigid SPC/E plus Smith-Dang is the best alternative. For interfacial tensions, the combination of the semiflexible SPC/E plus Smith-Dang or Joung-Cheatham gives the best results. Inclusion of water flexibility improves the mixture densities and interfacial tensions, at the cost of larger deviations for the vapor pressures and viscosities. The Exp6 water plus Tosi-Fumi salt model combination was found to perform poorly for most of the properties of interest, in particular being unable to describe the experimental trend for the vapor pressure as a function of salt concentration.

  12. Molecular simulation of thermodynamic and transport properties for the H2O+NaCl system

    NASA Astrophysics Data System (ADS)

    Orozco, Gustavo A.; Moultos, Othonas A.; Jiang, Hao; Economou, Ioannis G.; Panagiotopoulos, Athanassios Z.

    2014-12-01

    Molecular dynamics and Monte Carlo simulations have been carried out to obtain thermodynamic and transport properties of the binary mixture H2O+NaCl at temperatures from T = 298 to 473 K. In particular, vapor pressures, liquid densities, viscosities, and vapor-liquid interfacial tensions have been obtained as functions of pressure and salt concentration. Several previously proposed fixed-point-charge models that include either Lennard-Jones (LJ) 12-6 or exponential-6 (Exp6) functional forms to describe non-Coulombic interactions were studied. In particular, for water we used the SPC and SPC/E (LJ) models in their rigid forms, a semiflexible version of the SPC/E (LJ) model, and the Errington-Panagiotopoulos Exp6 model; for NaCl, we used the Smith-Dang and Joung-Cheatham (LJ) parameterizations as well as the Tosi-Fumi (Exp6) model. While none of the model combinations are able to reproduce simultaneously all target properties, vapor pressures are well represented using the SPC plus Joung-Cheathem model combination, and all LJ models do well for the liquid density, with the semiflexible SPC/E plus Joung-Cheatham combination being the most accurate. For viscosities, the combination of rigid SPC/E plus Smith-Dang is the best alternative. For interfacial tensions, the combination of the semiflexible SPC/E plus Smith-Dang or Joung-Cheatham gives the best results. Inclusion of water flexibility improves the mixture densities and interfacial tensions, at the cost of larger deviations for the vapor pressures and viscosities. The Exp6 water plus Tosi-Fumi salt model combination was found to perform poorly for most of the properties of interest, in particular being unable to describe the experimental trend for the vapor pressure as a function of salt concentration.

  13. Presynaptic Na+-dependent transport and exocytose of GABA and glutamate in brain in hypergravity.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Pozdnyakova, N.; Krisanova, N.; Himmelreich, N.

    γ-Aminobutyric acid (GABA) and L-glutamate are the most widespread neurotransmitter amino acids in the mammalian central nervous system. GABA is now widely recognized as the major inhibitory neurotransmitter. L-glutamate mediates the most of excitatory synaptic neurotransmission in the brain. They involved in the main aspects of normal brain function. The nerve terminals (synaptosomes) offer several advantages as a model system for the study of general mechanisms of neurosecretion. Our data allowed to conclude that exposure of animals to hypergravity (centrifugation of rats at 10G for 1 hour) had a profound effect on synaptic processes in brain. Comparative analysis of uptake and release of GABA and glutamate have demonstrated that hypergravity loading evokes oppositely directed alterations in inhibitory and excitatory signal transmission. We studied the maximal velocities of [^3H]GABA reuptake and revealed more than twofold enhancement of GABA transporter activity (Vmax rises from 1.4 |pm 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for animals exposed to hypergravity (P ≤ 0.05)). Recently we have also demonstrated the significant lowering of glutamate transporter activity (Vmax of glutamate reuptake decreased from 12.5 ± 3.2 nmol/min/mg of protein in the control group to 5.6 ± 0.9 nmol/min/mg of protein in the group of animals, exposed to the hypergravity stress (P ≤ 0.05)). Significant changes occurred in release of neurotransmitters induced by stimulating exocytosis with the agents, which depolarized nerve terminal plasma membrane. Depolarization-evoked Ca2+-stimulated release was more abundant for GABA (7.2 ± 0.54% and 11,74 ±1,2 % of total accumulated label for control and hypergravity, respectively (P≤0.05)) and was essentially less for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%) after exposure of animals to centrifuge induced artificial gravity. Changes observed in depolarization-evoked exocytotic release

  14. Active transportation safety features around schools in Canada.

    PubMed

    Pinkerton, Bryn; Rosu, Andrei; Janssen, Ian; Pickett, William

    2013-10-31

    The purpose of this study was to describe the presence and quality of active transportation safety features in Canadian school environments that relate to pedestrian and bicycle safety. Variations in these features and associated traffic concerns as perceived by school administrators were examined by geographic status and school type. The study was based on schools that participated in 2009/2010 Health Behaviour in School-aged Children (HBSC) survey. ArcGIS software version 10 and Google Earth were used to assess the presence and quality of ten different active transportation safety features. Findings suggest that there are crosswalks and good sidewalk coverage in the environments surrounding most Canadian schools, but a dearth of bicycle lanes and other traffic calming measures (e.g., speed bumps, traffic chokers). Significant urban/rural inequities exist with a greater prevalence of sidewalk coverage, crosswalks, traffic medians, and speed bumps in urban areas. With the exception of bicycle lanes, the active transportation safety features that were present were generally rated as high quality. Traffic was more of a concern to administrators in urban areas. This study provides novel information about active transportation safety features in Canadian school environments. This information could help guide public health efforts aimed at increasing active transportation levels while simultaneously decreasing active transportation injuries.

  15. Active Transportation Safety Features around Schools in Canada

    PubMed Central

    Pinkerton, Bryn; Rosu, Andrei; Janssen, Ian; Pickett, William

    2013-01-01

    The purpose of this study was to describe the presence and quality of active transportation safety features in Canadian school environments that relate to pedestrian and bicycle safety. Variations in these features and associated traffic concerns as perceived by school administrators were examined by geographic status and school type. The study was based on schools that participated in 2009/2010 Health Behaviour in School-aged Children (HBSC) survey. ArcGIS software version 10 and Google Earth were used to assess the presence and quality of ten different active transportation safety features. Findings suggest that there are crosswalks and good sidewalk coverage in the environments surrounding most Canadian schools, but a dearth of bicycle lanes and other traffic calming measures (e.g., speed bumps, traffic chokers). Significant urban/rural inequities exist with a greater prevalence of sidewalk coverage, crosswalks, traffic medians, and speed bumps in urban areas. With the exception of bicycle lanes, the active transportation safety features that were present were generally rated as high quality. Traffic was more of a concern to administrators in urban areas. This study provides novel information about active transportation safety features in Canadian school environments. This information could help guide public health efforts aimed at increasing active transportation levels while simultaneously decreasing active transportation injuries. PMID:24185844

  16. Entropic Ratchet transport of interacting active Brownian particles

    SciTech Connect

    Ai, Bao-Quan; He, Ya-Feng; Zhong, Wei-Rong

    2014-11-21

    Directed transport of interacting active (self-propelled) Brownian particles is numerically investigated in confined geometries (entropic barriers). The self-propelled velocity can break thermodynamical equilibrium and induce the directed transport. It is found that the interaction between active particles can greatly affect the ratchet transport. For attractive particles, on increasing the interaction strength, the average velocity first decreases to its minima, then increases, and finally decreases to zero. For repulsive particles, when the interaction is very weak, there exists a critical interaction at which the average velocity is minimal, nearly tends to zero, however, for the strong interaction, the average velocity is independent of the interaction.

  17. Active urea transport in lower vertebrates and mammals.

    PubMed

    Bankir, Lise

    2014-01-01

    Some unicellular organisms can take up urea from the surrounding fluids by an uphill pumping mechanism. Several active (energy-dependent) urea transporters (AUTs) have been cloned in these organisms. Functional studies show that active urea transport also occurs in elasmobranchs, amphibians, and mammals. In the two former groups, active urea transport may serve to conserve urea in body fluids in order to balance external high ambient osmolarity or prevent desiccation. In mammals, active urea transport may be associated with the need to either store and/or reuse nitrogen in the case of low nitrogen supply, or to excrete nitrogen efficiently in the case of excess nitrogen intake. There are probably two different families of AUTs, one with a high capacity able to establish only a relatively modest transepithelial concentration difference (renal tubule of some frogs, pars recta of the mammalian kidney, early inner medullary collecting duct in some mammals eating protein-poor diets) and others with a low capacity but able to maintain a high transepithelial concentration difference that has been created by another mechanism or in another organ (elasmobranch gills, ventral skin of some toads, and maybe mammalian urinary bladder). Functional characterization of these transporters shows that some are coupled to sodium (symports or antiports) while others are sodium-independent. In humans, only one genetic anomaly, with a mild phenotype (familial azotemia), is suspected to concern one of these transporters. In spite of abundant functional evidence for such transporters in higher organisms, none have been molecularly identified yet.

  18. /sup 22/Na+ and /sup 86/Rb+ transport in vascular smooth muscle of SHR, Wistar Kyoto, and Wistar rats

    SciTech Connect

    Kuriyama, S.; Denny, T.N.; Aviv, A.

    1988-06-01

    To gain further insight into differences in cellular Na+ and K+ regulation between the spontaneously hypertensive rat (SHR), Wistar Kyoto (WKY), and American Wistar (W) rats, 22Na+ and 86Rb+ washouts were performed under steady-state conditions in cultured vascular smooth muscle cells from the three rat strains. SHR vascular smooth muscle cells showed significantly higher bumetanide sensitive 86Rb+ washout rate constant (x 10(-4)/min; mean +/- SEM) than WKY cells (-38.6 +/- 2.84 and -23.8 +/- 3.58, respectively; p less than 0.005). SHR vascular smooth muscle cells also exhibited significantly higher values than WKY cells in the total 22Na+ washout rate constant (x 10(-2)/min) (-61.0 +/- 1.57 vs. -53.8 +/- 1.24; p less than 0.005). The amiloride sensitive component of the 22Na+ washout rate constant accounted for these differences (-18.6 +/- 1.04 for SHR and -12.1 +/- 2.00 for WKY; p less than 0.05). There were no apparent differences in cellular Na+ concentrations between WKY and SHR cells. In general, the 86Rb+ and 22Na+ washout parameters of W rat cells were quite similar to those of cells from SHR. We conclude that the bumetanide-sensitive 86Rb+ washout (the Na+ K+-cotransport), the overall, and the amiloride-sensitive 22Na+ washout (the latter primarily represents the Na+/H+ antiport) are higher in SHR than WKY rat vascular smooth muscle cells. These findings indicate innate differences in cellular Na+ and K+ transport in vascular smooth muscle cells of the SHR and WKY rat. The mechanisms responsible for these differences are yet to be determined.

  19. Light-activated amino acid transport in Halobacterium halobium envelope vesicles

    NASA Technical Reports Server (NTRS)

    Macdonald, R. E.; Lanyi, J. K.

    1977-01-01

    Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na+ gradient. On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids: arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.

  20. Caulis Sinomenii extracts activate DA/NE transporter and inhibit 5HT transporter.

    PubMed

    Zhao, Gang; Bi, Cheng; Qin, Guo-Wei; Guo, Li-He

    2009-08-01

    Caulis Sinomenii (QFT) has analgesic, sedative, and anxiolytic-like actions, and is proven effective for improving drug dependence that is known to be associated with abnormal monoaminergic transmission. We assessed whether QFT would be biologically active in functionally regulating monoamine transporters using CHO cells expressing dopamine transporter (DAT), norepinephrine transporter (NET), or serotonin transporter (SERT) (i.e. D8, N1, or S6 cells, respectively). Here, we showed that its primary extracts, such as QA, QC, QE, QD, and QB (QFT ethanol, chloroform, ethyl acetate, alkaloid-free chloroform, and alkaloid-containing chloroform extract, respectively), and secondary extracts, such as QE-2, - 3, - 5, - 7, QD-1, - 2, - 3, - 4, - 5, and QB-1, - 2, - 3, - 4, - 5 (fractioned from QE, QD, and QB, respectively), in differing degrees, either increased DA/ NE uptake by corresponding D8/N1 cells or decreased 5HT uptake by S6 cells; wherein, QE-2, QD-3, and QE-7 were potent DA/NE uptake activators while both QE-7 and QB-5 were potent 5HT uptake inhibitors. Furthermore, the enhancement of DA/NE uptake was dependent of DAT/NET activity, and the inhibition of 5HT uptake was typical of competition. Thus, QFT extracts, especially QE-2 and QE-7 (both with stronger potencies), are novel monoamine transporter modulators functioning as DAT/ NET activators and/or SERT inhibitors, and would likely improve neuropsychological disorders through regulating monoamine transporters.

  1. Electronic Transport Properties of New 2-D Materials GeH and NaSn2As2

    NASA Astrophysics Data System (ADS)

    He, Bin; Cultrara, Nicholas; Arguilla, Maxx; Goldberger, Joshua; Heremans, Joseph

    2-D materials potentially have superior thermoelectric properties compared to traditional 3-D materials due to their layered structure. Here we present electrical and thermoelectric transport properties of 2 types of 2-D materials, GeH and NaSn2As2. GeH is a graphane analog which is prepared using chemical exfoliation of CaGe2 crystals. Intrinsic GeH is proven to be a highly resistive material at room temperature. Resistance and Seebeck coefficient of Ga doped GeH are measured in a cryostat with a gating voltage varying from -100V to 100V. NaSn2As2 is another 2-D system, with Na atom embedded between nearly-2D Sn-As layers. Unlike GeH, NaSn2As2 is a metal based of Hall measurements, with p-type behavior, and with van der Pauw resistances on the order of 5m Ω/square. Thermoelectric transport properties of NaSn2As2 will be reported. This work is support by the NSF EFRI-2DARE project EFRI-1433467.

  2. Overexpression of a novel soybean gene modulating Na+ and K+ transport enhances salt tolerance in transgenic tobacco plants.

    PubMed

    Chen, Huatao; He, Hui; Yu, Deyue

    2011-01-01

    Salt is an important factor affecting the growth and development of soybean in saline soil. In this study, a novel soybean gene encoding a transporter (GmHKT1) was identified and its function analyzed using transgenic plants. GmHKT1 encoded a protein of 419 amino acids, with a potential molecular mass of 47.06 kDa and a predicted pI value of 8.59. Comparison of the genomic and cDNA sequences of GmHKT1 identified no intron. The deduced amino acid sequence of GmHKT1 showed 38-49% identity with other plant HKT-like sequences. RT-PCR analysis showed that the expression of GmHKT1 was upregulated by salt stress (150 mM NaCl) in roots and leaves but not in stems. Overexpression of GmHKT1 significantly enhanced the tolerance of transgenic tobacco plants to salt stress, compared with non-transgenic plants. To investigate the role of GmHKT1 in K(+) and Na(+) transport, we compared K(+) and Na(+) accumulation in roots and shoots of wild-type and transgenic tobacco plants. The results suggested that GmHKT1 is a transporter that affected K(+) and Na(+) transport in roots and shoots, and regulated Na(+) /K(+) homeostasis in these organs. Our findings suggest that GmHKT1 plays an important role in response to salt stress and would be useful in engineering crop plants for enhanced tolerance to salt stress.

  3. Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder.

    PubMed

    Eaton, D C; Frace, A M; Silverthorn, S U

    1982-01-01

    The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na+ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal 22Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na+ flux was a linear function of mucosal Na+ concentration. Ouabain-sensitive Na+ flux and ouabain-sensitive, Na+-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na+ with the basolateral transport process is assumed to involve the interaction of some number of Na+ ions, n, with a maximal flux, MMAX, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value for MMAX of 287.8 pM cm-2 sec-1 with an intracellular Na concentration of 2.0 mM Na+ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na+-induced current, we calculate a Na+ to K+ coupling ratio of 1.40 +/- 0.07 for the transport process.

  4. Classroom Activities in Transportation: Technology Education.

    ERIC Educational Resources Information Center

    Wisconsin State Dept. of Public Instruction, Madison.

    This curriculum supplement was designed to correlate directly with "A Guide to Curriculum Planning in Technology Education," published by the Wisconsin Department of Public Instruction. It is also a companion book to three other classroom activity compilations, one in each of the other three major systems of technology--manufacturing,…

  5. Promoting physical activity and reducing climate change: opportunities to replace short car trips with active transportation.

    PubMed

    Maibach, Edward; Steg, Linda; Anable, Jillian

    2009-10-01

    Automobile use is a significant contributor to climate change, local air pollution, pedestrian injuries and deaths, declines in physical activity and obesity. A significant proportion of car use is for short trips that can relatively easily be taken with active transportation options--walking or cycling--or with public transportation. In this commentary, we review a number of immediate, practical opportunities to implement policies and programs that reduce short car trips and increase active transportation.

  6. Cosmogenic 22Na as a steady-state tracer of solute transport and water age in first-order catchments

    NASA Astrophysics Data System (ADS)

    Kaste, James M.; Lauer, Nancy E.; Spaetzel, Alana B.; Goydan, Claire

    2016-12-01

    Naturally-occurring cosmogenic 22Na (T1/2 = 2.6 yr) is a potentially powerful tracer of solute and water movement in catchments. However, due to its low abundance in precipitation (∼10-20 molL-1), there are only a handful of datasets documenting cosmogenic 22Na atmospheric fluxes and concentrations in surface waters. Here we present the first record of cosmogenic 22Na fallout to North America and test its use as a radiometric tracer of water age in three small catchments in the Eastern United States. We show that 22Na deposition to southeastern Virginia, USA during 2012-2014 was 187 ± 10 mBqm-2yr-1 and that flux is largely additive with precipitation amounts. Our measurements of fallout combined with previous 22Na deposition data from other regions indicate that approximately 77% of the variability in the annual global 22Na atmospheric flux is controlled by precipitation. Export of 22Na in drainage waters from three first-order forested catchments ranged from 12.5 to 174 mBq m-2 yr-1 and can be explained by a flux-based radioactive decay model, indicating that the watersheds are in steady-state with respect to cosmogenic 22Na on annual timescales. We conclude that in temperate climates with no systematic changes in rainfall amounts at the annual timescale, 22Na may be useful for quantifying the recharge age of relatively young (<20 yr) surface waters and groundwaters and for tracing solute transport at the watershed scale.

  7. Organophosphate inhibition of avian salt gland Na, K-ATPase activity

    USGS Publications Warehouse

    Eastin, W.C.; Fleming, W.J.; Murray, H.C.

    1982-01-01

    1. Adult black ducks (Anas rubripes) were given freshwater or saltwater (1.5% NaCl) for 11 days and half of each group was also given an organophosphate (17 p.p.m. fenthion) in the diet on days 6-11. 2. After 11 days, ducks drinking saltwater had lost more weight and had higher plasma Na and uric acid concentrations and osmolalities than birds drinking freshwater. 3. Saltwater treatment stimulated the salt gland to increased weight and Na, K-ATPase activity. 4. Fenthion generally reduced plasma and brain cholinesterase activity and depressed cholinesterase and Na, K-ATPase activities in salt glands of birds drinking saltwater.

  8. Active Transportation to School: Findings from a National Survey

    ERIC Educational Resources Information Center

    Fulton, Janet E.; Shisler, Jessica L.; Yore, Michelle M.; Caspersen, Carl J.

    2005-01-01

    In the past, active transportation to school offered an important source of daily physical activity for youth; more recently, however, factors related to distance, safety, or physical or social environments may have contributed to the proportion of children who travel to school by motorized vehicle. The authors examine the characteristics of…

  9. β3 subunit of Na,K ATPase regulates T cell activation with no involvement of Na,K ATPase activity.

    PubMed

    Chruewkamlow, Nuttapol; Pata, Supansa; Mahasongkram, Kodchakorn; Laopajon, Witida; Kasinrerk, Watchara; Chiampanichayakul, Sawitree

    2015-05-01

    Na,K ATPase plays an important role in the regulation of Na(+) and K(+) ions that are required for normal resting membrane potential and various cellular functions. Na,K ATPase is composed of two subunits, α and β subunits. Engagement of the β subunit by an agonistic monoclonal antibody (mAb) P-3E10 inhibited T cell activation and induced the G0/G1 cell cycle arrest. In addition, mAb P-3E10 decreased CD25 expression. The mAb P-3E10, however, did not inhibit the proliferation of cell lines and the phagocytosis activity of phagocytes, and did not interfere with the Na,K ATPase activity. These results indicate that mAb P-3E10 reacts to the β subunit and, as a consequence, brings about the regulation of the T cell activation without disturbing the Na,K pump activity. By sequential immunoprecipitation, we demonstrated the expression of the β3 subunit free form apart from the α subunit. In this study, we propose that the β3 subunits of Na,K ATPase are expressed separately from the α subunit, and play a role in regulation of the immune response.

  10. Na,K-ATPase activity in mouse muscle is regulated by AMPK and PGC-1α.

    PubMed

    Ingwersen, Maria S; Kristensen, Michael; Pilegaard, Henriette; Wojtaszewski, Jørgen F P; Richter, Erik A; Juel, Carsten

    2011-07-01

    Na,K-ATPase activity, which is crucial for skeletal muscle function, undergoes acute and long-term regulation in response to muscle activity. The aim of the present study was to test the hypothesis that AMP kinase (AMPK) and the transcriptional coactivator PGC-1α are underlying factors in long-term regulation of Na,K-ATPase isoform (α,β and PLM) abundance and Na(+) affinity. Repeated treatment of mice with the AMPK activator AICAR decreased total PLM protein content but increased PLM phosphorylation, whereas the number of α- and β-subunits remained unchanged. The K(m) for Na(+) stimulation of Na,K-ATPase was reduced (higher affinity) after AICAR treatment. PLM abundance was increased in AMPK kinase-dead mice compared with control mice, but PLM phosphorylation and Na,K-ATPase Na(+) affinity remained unchanged. Na,K-ATPase activity and subunit distribution were also measured in mice with different degrees of PGC-1α expression. Protein abundances of α1 and α2 were reduced in PGC-1α +/- and -/- mice, and the β(1)/β(2) ratio was increased with PGC-1α overexpression (TG mice). PLM protein abundance was decreased in TG mice, but phosphorylation status was unchanged. Na,K-ATPase V (max) was decreased in PCG-1α TG and KO mice. Experimentally in vitro induced phosphorylation of PLM increased Na,K-ATPase Na(+) affinity, confirming that PLM phosphorylation is important for Na,K-ATPase function. In conclusion, both AMPK and PGC-1α regulate PLM abundance, AMPK regulates PLM phosphorylation and PGC-1α expression influences Na,K-ATPase α(1) and α(2) content and β(1)/β(2) isoform ratio. Phosphorylation of the Na,K-ATPase subunit PLM is an important regulatory mechanism.

  11. Effect of anaerobiosis, dinitrophenol and fluoride on the active intestinal transport of galactose in snail.

    PubMed

    Barber, A; Jordana, R; Ponz, F

    1975-06-01

    The active transport of galactose across the intestinal wall (everted sacs) of the snail Cryptomphalus hortensis Müller has been studied in vitro, under several metabolic conditions. Anaerobiosis does not change the serosal/mucosal galactose gradients which are developed in oxygen atmosphere. Dinitrophenol (10(-4) M) greatly increased the O2 uptake by the tissue and clearly inhibits the sugar transport. At 5 times 10(-4) M concentration, DNP totally prevents the uphill transport while the O2 uptake is normal. The inhibition produced by DNP does not increase by anaerobiosis. Fluoride inhibits the galactose transport and also the O2 uptake. It is deduced that in snail intestine the energy for the active transport of galactose can be supplied by aerobic as much as by anaerobic metabolism. The inhibition by dinitrophenol seems to be independent of its uncoupling action on the oxidative phosphorylation. The inhibitory effect of NaF may be due both to glycolisis inhibition and to alteration of the digestive epithelium.

  12. Locally formed dopamine inhibits Na sup + -K sup + -ATPase activity in rat renal cortical tubule cells

    SciTech Connect

    Seri, I.; Kone, B.C.; Gullans, S.R.; Aperia, A.; Brenner, B.M.; Ballermann, B.J. Karolinska Institute, Stockholm )

    1988-10-01

    Dopamine, generated locally from L-dopa, inhibits Na{sup +}-K{sup +}-ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na{sup +}-K{sup +}-ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate ({dot Q}o{sub 2}) and {sup 86}Rb uptake in renal cortical tubule cell suspensions. L-Dopa did not affect ouabain-insensitive {dot Q}o{sub 2} or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive {dot Q}o{sub 2} in a concentration-dependent manner, with half-maximal inhibition (K{sub 0.5}) of 5 {times} 10{sup {minus}7} M and a maximal inhibition of 14.1 {plus minus} 1.5% at 10{sup {minus}4}M. L-Dopa also blunted the nystatin-stimulated {dot Q}o{sub 2} in a concentration-dependent manner, indicating the L-dopa directly inhibits Na{sup +}-K{sup +}-ATPase activity and not sodium entry. Ouabain-sensitive {sup 86}Rb uptake was also inhibited by L-dopa. Carbidopa, an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} and {sup 86}Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased. By studying the effect of L-dopa on ouabain-sensitive {dot Q}o{sub 2} at increasing extracellular sodium concentrations in the presence of nystatin, the authors demonstrated that the inhibitory effect of locally formed dopamine on the Na{sup +}-K{sup +}-ATPase is indeed dependent on the sodium available for the enzyme and occurs in an uncompetitive manner.

  13. Methylphenidate Decreases ATP Levels and Impairs Glutamate Uptake and Na(+),K(+)-ATPase Activity in Juvenile Rat Hippocampus.

    PubMed

    Schmitz, Felipe; Pierozan, Paula; Rodrigues, André F; Biasibetti, Helena; Grings, Mateus; Zanotto, Bruna; Coelho, Daniella M; Vargas, Carmen R; Leipnitz, Guilhian; Wyse, Angela T S

    2016-11-14

    The study of the long-term neurological consequences of early exposure with methylphenidate (MPH) is very important since this psychostimulant has been widely misused by children and adolescents who do not meet full diagnostic criteria for ADHD. The aim of this study was to examine the effect of early chronic exposure with MPH on amino acids profile, glutamatergic and Na(+),K(+)-ATPase homeostasis, as well as redox and energy status in the hippocampus of juvenile rats. Wistar male rats received intraperitoneal injections of MPH (2.0 mg/kg) or saline solution (controls), once a day, from the 15th to the 45th day of age. Results showed that MPH altered amino acid profile in the hippocampus, decreasing glutamine levels. Glutamate uptake and Na(+),K(+)-ATPase activity were decreased after chronic MPH exposure in the hippocampus of rats. No changes were observed in the immunocontents of glutamate transporters (GLAST and GLT-1), and catalytic subunits of Na(+),K(+)-ATPase (α1, α2, and α3), as well as redox status. Moreover, MPH provoked a decrease in ATP levels in the hippocampus of chronically exposed rats, while citrate synthase, succinate dehydrogenase, respiratory chain complexes activities (II, II-III, and IV), as well as mitochondrial mass and mitochondrial membrane potential were not altered. Taken together, our results suggest that chronic MPH exposure at early age impairs glutamate uptake and Na(+),K(+)-ATPase activity probably by decreasing in ATP levels observed in rat hippocampus.

  14. Ouabain on active transepithelial sodium transport in frog skin: studies with microelectrodes

    PubMed Central

    1979-01-01

    Studies were done with isolated frog skin to determine the effects of 10(-4) M ouabain on the electrophysiological parameters of outer and inner barriers of the Na-transporting cells. Microelectrodes were used to impale the skins from the outer surface to determine the intracellular voltages (Vsco) under conditions of short-circuiting and under conditions where a voltage clamp was used to vary the transepithelial voltage, VT. From this, the electrical resistances of outer (Rfo) and inner (RI) barriers were estimated. In addition, the driving force for active transepithelial Na transport (ENa = E'1) was estimated from the values of VT when the Vo = 0 mV (Helman and Fisher. 1977. J. Gen. Physiol. 69: 571-604). Studies were done with skins bathed with the usual 2.4 meq/liter [K]i in the inner solution as well as with reduced [K]i of 0.5 and 0 meq/liter. Characteristically, the responses to ouabain could be described by an initial rapid phase (5-10 min) during which time the Ri was increased markedly and the E'1 was decreased from control values. Thereafter, during the slow phases of the response, the resistances of both outer and inner barriers increased continuously and markedly with time leading ultimately to essentially complete inhibition of the short-circuit current. Similar studies were done with skins exposed to 10(-4) M amiloride in the outer solution. Although estimates of Ri could not be obtained under these conditions, the effects on the Vsco and E'1 were similar to those observed for the Na-transporting skins. However, the magnitudes of the effects were less and relatively slower than observed for the Na- transporting skins. The results of these studies were analyzed within the context of a proposed electrical model that takes into account the observation that the magnitude of the voltage at the inner barrier appears to exceed the equilibrium potential for K especially when transepithelial Na transport is inhibited at the apical barrier of the cells. PMID

  15. Role of conserved Arg40 and Arg117 in the Na+/proline transporter of Escherichia coli.

    PubMed

    Quick, M; Stölting, S; Jung, H

    1999-10-12

    The Na+/proline transporter of Escherichia coli (PutP) is a member of a large family of Na+/solute symporters. To investigate the role of Arg residues which are conserved within this family, Arg40 at the cytoplasmic end of transmembrane domain (TM) II and Arg117 in cytoplasmic loop 4 of PutP are subjected to amino acid substitution analysis. Removal of the positive charge at position 40 (PutP-R40C, Q, E) leads to a dramatic decrease of the V(max) of Na(+)-coupled proline uptake (1-10% of PutP-wild-type). The reduced transport rates are accompanied by decreased apparent affinities of the transporter for Na+ and Li+ while the apparent affinity for proline is only slightly altered. Furthermore, single Cys PutP-R40C reacts with N-ethylmaleimide (NEM), and this reaction is partially inhibited by proline and more efficiently by Na+ ions. Remarkably, NEM modification of Cys40 inhibits Na(+)-driven proline uptake almost completely while facilitated influx of proline into deenergized cells is stimulated by this reaction, suggesting an at least partially uncoupled phenotype under these conditions. These results suggest that Arg40 is located close to the site of ion binding and is important for the coupling of ion and proline transport. The observations confirm the functional importance of TM II described in earlier studies [M. Quick and H. Jung (1997) Biochemistry 36, 4631-4636]. In contrast to Arg40, Arg117 is apparently not important for function of the mature protein. The low transport rates observed upon substitution of Arg117 (PutP-R117C, K, Q) can at least partially be attributed to reduced amounts of PutP in the membrane. However, once inserted into the membrane, PutP containing Arg117 replacements shows a stability comparable to the wild-type as indicated by pulse-chase experiments. These observations suggest that Arg117 plays a crucial role at a stage prior to complete functional insertion of PutP into the membrane, i. e., by stabilizing a folding intermediate.

  16. Rapid activation of gill Na+,K+-ATPase in the euryhaline teleost Fundulus heteroclitus

    USGS Publications Warehouse

    Mancera, J.M.; McCormick, S.D.

    2000-01-01

    The rapid activation of gill Na+,K+-ATPase was analyzed in the mummichog (Fundulus heteroclitus) and Atlantic salmon (Salmo salar) transferred from low salinity (0.1 ppt) to high salinity (25-35 ppt). In parr and presmolt, Salmo salar gill Na+,K+-ATPase activity started to increase 3 days after transfer. Exposure of Fundulus heteroclitus to 35 ppt seawater (SW) induced a rise in gill Na+,K+-ATPase activity 3 hr after transfer. After 12 hr, the values dropped to initial levels but showed a second significant increase 3 days after transfer. The absence of detergent in the enzyme assay resulted in lower values of gill Na+,K+-ATPase, and the rapid increase after transfer to SW was not observed. Na+,K+-ATPase activity of gill filaments in vitro for 3 hr increased proportionally to the osmolality of the culture medium (600 mosm/kg > 500 mosm/kg > 300 mosm/kg). Osmolality of 800 mosm/kg resulted in lower gill Na+,K+-ATPase activity relative to 600 mosm/kg. Increasing medium osmolality to 600 mosm/kg with mannitol also increased gill Na+,K+-ATPase. Cycloheximide inhibited the increase in gill Na+,K+-ATPase activity observed in hyperosmotic medium in a dose-dependent manner (10-4 M > 10-5 M > 10-6 M). Actinomycin D or bumetanide in the culture (doses of 10-4 M, 10-5 M, and 10-6 M) did not affect gill Na+,K+-ATPase. Injection of fish with actinomycin D prior to gill organ culture, however, prevented the increase in gill Na+,K+-ATPase activity in hyperosmotic media. The results show a very rapid and transitory increase in gill Na+,K+-ATPase activity in the first hours after the transfer of Fundulus heteroclitus to SW that is dependent on translational and transcriptional processes. (C) 2000 Wiley-Liss, Inc.

  17. Advocacy for active transport: advocate and city council perspectives

    PubMed Central

    2010-01-01

    Background Effective advocacy is an important part of efforts to increase population participation in physical activity. Research about effective health advocacy is scarce, however, the health sector can learn from the experiences and knowledge of community advocates and those who are on the receiving end of this advocacy. The aim of this study is to explore advocacy for active transport from the perspectives of community advocates and representatives from City councils. Methods Cycling and walking advocates were identified from the local contact list of Cycling Advocates Network and Living Streets Aotearoa. Semi-structured telephone interviews were conducted with cycle and walking advocates from throughout New Zealand. Advocates also nominated a suitable council officer at their local City council to be interviewed. Interviews were recorded and transcribed and categories of responses for each of the questions created. Results Several processes were used by advocates to engage with council staff, including formal council submissions, meetings, stakeholder forums and partnership in running community events promoting active transport. Several other agencies were identified as being influential for active transport, some as potential coalition partners and others as potential adversaries. Barriers to improving conditions for active transport included a lack of funding, a lack of will-power among either council staff or councillors, limited council staff capacity (time or training) and a culture of providing infrastructure for motor vehicles instead of people. Several suggestions were made about how the health sector could contribute to advocacy efforts, including encouraging political commitment, engaging the media, communicating the potential health benefits of active transport to the general public and being role models in terms of personal travel mode choice and having workplaces that support participation in active transport. Conclusions There is potential for the

  18. Effective contractile response to voltage-gated Na+ channels revealed by a channel activator.

    PubMed

    Ho, W-S Vanessa; Davis, Alison J; Chadha, Preet S; Greenwood, Iain A

    2013-04-15

    This study investigated the molecular identity and impact of enhancing voltage-gated Na(+) (Na(V)) channels in the control of vascular tone. In rat isolated mesenteric and femoral arteries mounted for isometric tension recording, the vascular actions of the Na(V) channel activator veratridine were examined. Na(V) channel expression was probed by molecular techniques and immunocytochemistry. In mesenteric arteries, veratridine induced potent contractions (pEC(50) = 5.19 ± 0.20, E(max) = 12.0 ± 2.7 mN), which were inhibited by 1 μM TTX (a blocker of all Na(V) channel isoforms, except Na(V)1.5, Na(V)1.8, and Na(V)1.9), but not by selective blockers of Na(V)1.7 (ProTx-II, 10 nM) or Na(V)1.8 (A-80347, 1 μM) channels. The responses were insensitive to endothelium removal but were partly (~60%) reduced by chemical destruction of sympathetic nerves by 6-hydroxydopamine (2 mM) or antagonism at the α1-adrenoceptor by prazosin (1 μM). KB-R7943, a blocker of the reverse mode of the Na(+)/Ca(2+) exchanger (3 μM), inhibited veratridine contractions in the absence or presence of prazosin. T16A(inh)-A01, a Ca(2+)-activated Cl(-) channel blocker (10 μM), also inhibited the prazosin-resistant contraction to veratridine. Na(V) channel immunoreactivity was detected in freshly isolated mesenteric myocytes, with apparent colocalization with the Na(+)/Ca(2+) exchanger. Veratridine induced similar contractile effects in the femoral artery, and mRNA transcripts for Na(V)1.2 and Na(V)1.3 channels were evident in both vessel types. We conclude that, in addition to sympathetic nerves, NaV channels are expressed in vascular myocytes, where they are functionally coupled to the reverse mode of Na(+)/Ca(2+) exchanger and subsequent activation of Ca(2+)-activated Cl(-) channels, causing contraction. The TTX-sensitive Na(V)1.2 and Na(V)1.3 channels are likely involved in vascular control.

  19. Oxygen consumption and active sodium and chloride transport in bovine tracheal epithelium.

    PubMed Central

    Durand, J; Durand-Arczynska, W; Schoenenweid, F

    1986-01-01

    The O2 consumption (Jr) and the short-circuit current (Ji) were measured simultaneously in bovine tracheal epithelium in vitro. In this tissue, Ji is the sum of two active transport processes, Cl- secretion and Na+ absorption. Jr was determined from the decrease of PO2 in the incubation solution, at 37 +/- 0.05 degrees C and at a PO2 around 600 torr. Microbial contamination and leaks of dissolved O2 from the solution never exceeded 4% of the rate of PO2 decrease due to the O2 consumption of the tissue. Ji and Jr were stable over 5 h of incubation under standard conditions. Ji was 106 +/- 4 nequiv min-1 cm-2 and Jr was 39.8 +/- 1.1 nmol O2 min-1 cm-2 (mean +/- S.E., n = 46). Ji was varied with several agents known to affect ion transport across the tracheal epithelium. Na+ absorption was inhibited partly with amiloride or completely following Na+ substitution with choline. Cl- secretion was selectively suppressed by furosemide. Ji was also reduced to a very low level, using ouabain or K+ suppression to inhibit the Na+-K+-ATPase. All these manoeuvres resulted in significant reductions of both Ji and Jr. Basal Jr was not affected when Ji was modified. A plot of the relative change in suprabasal Jr versus the relative change of Ji gave a straight line (r = 0.98, n = 60). A plot using absolute values yielded a stoichiometric ratio of 13.9 ions per O2 molecule, for Na+ as well as for Cl-. The stoichiometric ratio was also calculated for each experiment. Its mean value was 14.9 ions per O2 molecule. The population of the ratios was widely dispersed, but this was explained as a predictable statistical phenomenon. PMID:3723416

  20. Macula densa Na(+)/H(+) exchange activities mediated by apical NHE2 and basolateral NHE4 isoforms.

    PubMed

    Peti-Peterdi, J; Chambrey, R; Bebok, Z; Biemesderfer, D; St John, P L; Abrahamson, D R; Warnock, D G; Bell, P D

    2000-03-01

    Functional and immunohistochemical studies were performed to localize and identify Na(+)/H(+) exchanger (NHE) isoforms in macula densa cells. By using the isolated perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney, intracellular pH (pH(i)) was measured with fluorescence microscopy by using 2',7'-bis-(2-carboxyethyl)-5-(and -6) carboxyfluorescein. NHE activity was assayed by measuring the initial rate of Na(+)-dependent pH(i) recovery from an acid load imposed by prior lumen and bath Na(+) removal. Removal of Na(+) from the bath resulted in a significant, DIDS-insensitive, ethylisopropyl amiloride (EIPA)-inhibitable decrease in pH(i). This basolateral transporter showed very low affinity for EIPA and Hoechst 694 (IC(50) = 9.0 and 247 microM, respectively, consistent with NHE4). The recently reported apical NHE was more sensitive to inhibition by these drugs (IC(50) = 0.86 and 7.6 microM, respectively, consistent with NHE2). Increasing osmolality, a known activator of NHE4, greatly stimulated basolateral NHE. Immunohistochemical studies using antibodies against NHE1-4 peptides demonstrated expression of NHE2 along the apical and NHE4 along the basolateral, membrane, whereas NHE1 and NHE3 were not detected. These results suggest that macula densa cells functionally and immunologically express NHE2 at the apical membrane and NHE4 at the basolateral membrane. These two isoforms likely participate in Na(+) transport, pH(i), and cell volume regulation and may be involved in tubuloglomerular feedback signaling by these cells.

  1. Fructose stimulates Na/H exchange activity and sensitizes the proximal tubule to angiotensin II.

    PubMed

    Cabral, Pablo D; Hong, Nancy J; Hye Khan, Md Abdul; Ortiz, Pablo A; Beierwaltes, William H; Imig, John D; Garvin, Jeffrey L

    2014-03-01

    The proximal nephron reabsorbs 60% to 70% of the fluid and sodium and most of the filtered bicarbonate via Na/H exchanger 3. Enhanced proximal nephron transport is implicated in hypertension. Our findings show that a fructose-enriched diet causes salt sensitivity. We hypothesized that fructose stimulates luminal Na/H exchange activity and sensitizes the proximal tubule to angiotensin II. Na/H exchange was measured in rat proximal tubules as the rate of intracellular pH (pHi) recovery in fluorescent units/s. Replacing 5 mmol/L glucose with 5 mmol/L fructose increased the rate of pHi recovery (1.8±0.6 fluorescent units/s; P<0.02; n=8). Staurosporine, a protein kinase C inhibitor, blocked this effect. We studied whether this effect was because of the addition of fructose or removal of glucose. The basal rate of pHi recovery was first tested in the presence of a 0.6-mmol/L glucose and 1, 3, or 5 mmol/L fructose added in a second period. The rate of pHi recovery did not change with 1 mmol/L but it increased with 3 and 5 mmol/L of fructose. Adding 5 mmol/L glucose caused no change. Removal of luminal sodium blocked pHi recovery. With 5.5 mmol/L glucose, angiotensin II (1 pmol/L) did not affect the rate of pHi recovery (change, -1.1±0.5 fluorescent units/s; n=9) but it increased the rate of pHi recovery with 0.6 mmol/L glucose/5 mmol/L fructose (change, 4.0±2.2 fluorescent units/s; P<0.02; n=6). We conclude that fructose stimulates Na/H exchange activity and sensitizes the proximal tubule to angiotensin II. This mechanism is likely dependent on protein kinase C. These results may partially explain the mechanism by which a fructose diet induces hypertension.

  2. Neuroinflammation activates efflux transport by NFκB

    PubMed Central

    Yu, Chuanhui; Argyropoulos, George; Zhang, Yan; Kastin, Abba J.; Hsuchou, Hung; Pan, Weihong

    2009-01-01

    Background/aims Although it is known that drug delivery across the blood-brain barrier (BBB) may be hampered by efflux transport activity of the multidrug resistance (mdr) gene product P-glycoprotein, it is not clear how inflammation regulates efflux transporters. In rat brain endothelial (RBE4) cells of BBB origin, the proinflammatory cytokine TNF mainly induces transcriptional upregulation of mdr1b, and to a lesser extent mdr1a, resulting in greater efflux of the substrates (Yu C et al., Cell Physiol Biochem, 2007). This study further determined the mechanisms by which TNF activates mdr1b promoter activity. Methods/Results Luciferase reporter assays and DNA binding studies show that (a) maximal basal promoter activity was conferred by a 476 bp sequence upstream to the mdr1b transcriptional initiation site; (2) TNF induced upregulation of promoter activity by NFkB nuclear translocation; and (3) the NFκB binding site of the mdr1b promoter was solely responsible for basal and TNF-activated gene transcription, whereas the p53 binding site was not involved. Binding of the p65 subunit of NFκB to nuclear DNA from RBE4 cells was shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. Conclusion Thus, NFκB mediated TNF-induced upregulation of mdr1b promoter activity, illustrating how inflammation activates BBB efflux transport. PMID:19088456

  3. Expression of the surface antigen 4F2hc affects system-L-like neutral-amino-acid-transport activity in mammalian cells.

    PubMed Central

    Bröer, S; Bröer, A; Hamprecht, B

    1997-01-01

    Mammalian cells possess a variety of amino acid-transport systems with overlapping substrate specificity. System L is one of the major amino acid-transport systems of non-epithelial cells. By expression cloning we have recently demonstrated that the surface antigen 4F2hc (CD98) is a necessary component for expression of system-L-like amino acid-transport activity in C6-BU-1 rat glioma cells [Bröer, Bröer and Hamprecht (1995) Biochem. J. 312, 863-870]. 4F2hc mRNA was detected in CHO cells, COS cells, activated lymphocytes isolated from mouse spleen and primary cultures of astrocytes. In all these cell types, Na+-independent isoleucine transport was mediated by system L. No contribution of system y+L to isoleucine or arginine transport was detected in C6-BU-1 cells. In lymphocytes, both system-L-like amino acid-transport activity and 4F2hc mRNA levels increased after treatment with phorbol ester plus ionomycin. Antisense oligonucleotides caused modest inhibition of Na+-independent isoleucine transport in C6-BU-1 cells and primary cultures of astroglial cells, whereas arginine transport was unaffected. Overexpression of 4F2hc cDNA in CHO cells resulted in an increase in Na+-independent isoleucine transport. PMID:9182715

  4. Mesopore control of high surface area NaOH-activated carbon.

    PubMed

    Tseng, Ru-Ling

    2006-11-15

    Activated carbon with BET surface areas in a narrow range from 2318 to 2474 m2/g was made by soaking the char made from corncob in a concentrated NaOH solution at NaOH/char ratios from 3 to 6; the mesopore volumes of the activated carbon were significantly changed from 21 to 58%. The relationships between pore properties (Sp, Vpore, Vmicro/Vpore, Dp) and NaOH dosage were investigated. Comparisons between the methods of NaOH and KOH activation revealed that NaOH activation can suitably control the mesopore specific volume of the activated carbon. Elemental analysis revealed that the H/C and O/C values of the activated carbons of NaOH/char ratios from 3 to 6 were significantly lower. SEM observation of surface hole variation of the activated carbon ascertained that the reaction process was inner pore etching. Based on the above three measurements and experimental investigations, the assumption made by previous researchers, namely that NaOH and KOH produce similar results, was challenged. Furthermore, the adsorption kinetics was used to investigate the adsorption rate of an Elovich equation to determine the relationships between the adsorption behavior on larger molecules (dyes) and smaller molecules (phenols) and the pore structure of the activated carbon.

  5. Dopamine transporter occupancy by RTI-55, inhibition of dopamine transport and stimulation of locomotor activity

    SciTech Connect

    Gatley, S.J.; Gifford, A.N.; Volkow, N.D.

    1997-05-01

    Cocaine analogs such as RTI-55 (or {beta}CIT) with a higher affinity for the DAT are potentially useful as therapeutic drugs in cocaine abuse as well as for radiopharmaceutical use. Previously we showed that in mice RTI-55 (2 mg/Kg, i/p) reduced H-3 cocaine striatum-to-cerebellum ratios (St/Cb, {lg_bullet}) from 1.6 to 1.2 at 3 h after administration, with recovery by 12 h. In the present study we demonstrate a very similar time-course for transport {triangle} measured in striatal homo within 2 min of sacrifice. The maximum inhibition of uptake at about 1 h corresponded to about 80% of the control uptake rate, similar to the percent reduction in St/Cb. The time-course of the effect of this dose of RTI-55 on locomotor activity ({sq_bullet}) was complex, with a drop in the activity measure at 7 h, after a further injection of RTI-55, but activity remained higher than in saline controls. In spite of this complexity, which may be associated with stereotypies and/or exhaustion, the duration of increased activity is consistent with the duration of transporter blockade. These experiments support the notion that PET/SPECT measures of transporter occupancy accurately reflect transporter inhibition.

  6. Mouse SGLT3a generates proton-activated currents but does not transport sugar.

    PubMed

    Barcelona, Stephanie; Menegaz, Danusa; Díez-Sampedro, Ana

    2012-04-15

    Sodium-glucose cotransporters (SGLTs) are secondary active transporters belonging to the SLC5 gene family. SGLT1, a well-characterized member of this family, electrogenically transports glucose and galactose. Human SGLT3 (hSGLT3), despite sharing a high amino acid identity with human SGLT1 (hSGLT1), does not transport sugar, although functions as a sugar sensor. In contrast to humans, two different genes in mice and rats code for two different SGLT3 proteins, SGLT3a and SGLT3b. We previously cloned and characterized mouse SGLT3b (mSGLT3b) and showed that, while it does transport sugar like SGLT1, it likely functions as a physiological sugar sensor like hSGLT3. In this study, we cloned mouse SGLT3a (mSGLT3a) and characterized it by expressing it in Xenopus laevis oocytes and performing electrophysiology and sugar transport assays. mSGLT3a did not transport sugar, and sugars did not induce currents at pH 7.4, though acidic pH induced inward currents that increased in the presence of sugar. Moreover, mutation of residue 457 from glutamate to glutamine resulted in a Na(+)-dependent transport of sugar that was inhibited by phlorizin. To corroborate our results in oocytes, we expressed and characterized mSGLT3a in mammalian cells and confirmed our findings. In addition, we cloned, expressed, and characterized rat SGLT3a in oocytes and found characteristics similar to mSGLT3a. In summary, acidic pH induces currents in mSGLT3a, and sugar-induced currents are increased at acidic pH, but wild-type SGLT3a does not transport sugar.

  7. Molecular physiology of the insect K-activated amino acid transporter 1 (KAAT1) and cation-anion activated amino acid transporter/channel 1 (CAATCH1) in the light of the structure of the homologous protein LeuT.

    PubMed

    Castagna, M; Bossi, E; Sacchi, V F

    2009-06-01

    K-activated amino acid transporter 1 (KAAT1) and cation-anion-activated amino acid transporter/channel 1 (CAATCH1) are amino acid cotransporters, belonging to the Na/Cl-dependent neurotransmitter transporter family (also called SLC6/NSS), that have been cloned from Manduca sexta midgut. They have been thoroughly studied by expression in Xenopus laevis oocytes, and structure/function analyses have made it possible to identify the structural determinants of their cation and amino acid selectivity. About 40 mutants of these proteins have been studied by measuring amino acid uptake and current/voltage relationships. The results obtained since the cloning of KAAT1 and CAATCH1 are here discussed in the light of the 3D model of the first crystallized member of the family, the leucine transporter LeuT.

  8. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1990-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells. 10 figs., 2 tabs.

  9. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1991-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells.

  10. Transport of active ellipsoidal particles in ratchet potentials

    SciTech Connect

    Ai, Bao-Quan Wu, Jian-Chun

    2014-03-07

    Rectified transport of active ellipsoidal particles is numerically investigated in a two-dimensional asymmetric potential. The out-of-equilibrium condition for the active particle is an intrinsic property, which can break thermodynamical equilibrium and induce the directed transport. It is found that the perfect sphere particle can facilitate the rectification, while the needlelike particle destroys the directed transport. There exist optimized values of the parameters (the self-propelled velocity, the torque acting on the body) at which the average velocity takes its maximal value. For the ellipsoidal particle with not large asymmetric parameter, the average velocity decreases with increasing the rotational diffusion rate, while for the needlelike particle (very large asymmetric parameter), the average velocity is a peaked function of the rotational diffusion rate. By introducing a finite load, particles with different shapes (or different self-propelled velocities) will move to the opposite directions, which is able to separate particles of different shapes (or different self-propelled velocities)

  11. Na+ gradient-dependent Mg2+ transport in smooth muscle cells of guinea pig tenia cecum.

    PubMed Central

    Tashiro, M; Konishi, M

    1997-01-01

    Thin strips of guinea pig tenia cecum were loaded with the Mg2+ indicator furaptra, and the indicator fluorescence signals measured in Ca2+-free condition were converted to cytoplasmic-free Mg2+ concentration ([Mg2+]i). Lowering the extracellular Na+ concentration ([Na+]o) caused a reversible increase in [Mg2+]i, consistent with the inhibition of Na+ gradient-dependent extrusion of cellular Mg2+ (Na+-Mg2+ exchange). Curve-fitting analysis indicated that the relation between [Na+]o and the rate of rise in [Mg2+], had a Hill coefficient of approximately 3, a [Na+]o at the half-maximal rate of rise of approximately 30 mM, and a maximal rate of 0.16 +/- 0.01 microM/s (mean +/- SE, n = 6). Depolarization with 56 mM K+ shifted the curve slightly toward higher [Na+]o without significantly changing the maximal rate, suggesting that the Na+-Mg2+ exchange was inhibited by depolarization. The maximal rate would correspond to a flux of 0.15-0.4 pmol/cm2/s, if cytoplasmic Mg2+ buffering power (defined as the ratio of the changes in total Mg2+ and free Mg2+ concentrations) is assumed to be 2-5. Ouabain (1-5 microM) increased the intracellular Na+ concentration, as assessed with fluorescence of SBFI (sodium-binding benzofuran isophthalate, a Na+ indicator), and elevated [Mg2+]i. In ouabain-treated preparations, removal of extracellular Na+ rapidly increased [Mg2+]i, with an initial rate of rise roughly proportional to the degree of the Mg2+ load, and, probably, to the Na+ load caused by ouabain. The enhanced rate of rise in [Mg2+]i (up to approximately 1 microM/s) could be attributed to the Mg2+ influx as a result of the reversed Na+-Mg2+ exchange. Our results support the presence of a reversible and possibly electrogenic Na+-Mg2+ exchange in the smooth muscle cells of tenia cecum. PMID:9414247

  12. Identification of an ovarian voltage-activated Na+-channel type: hints to involvement in luteolysis.

    PubMed

    Bulling, A; Berg, F D; Berg, U; Duffy, D M; Stouffer, R L; Ojeda, S R; Gratzl, M; Mayerhofer, A

    2000-07-01

    An endocrine type of voltage-activated sodium channel (eNaCh) was identified in the human ovary and human luteinized granulosa cells (GC). Whole-cell patch-clamp studies showed that the eNaCh in GC is functional and tetrodotoxin (TTX) sensitive. The luteotrophic hormone human CG (hCG) was found to decrease the peak amplitude of the sodium current within seconds. Treatment with hCG for 24-48 h suppressed not only eNaCh mRNA levels, but also mean Na+ peak currents and resting membrane potentials. An unexpected role for eNaChs in regulating cell morphology and function was indicated after pharmacological modulation of presumed eNaCh steady-state activity in GC cultures for 24-48 h using TTX (NaCh blocker) and veratridine (NaCh activator). TTX preserved a highly differentiated cellular phenotype. Veratridine not only increased the number of secondary lysosomes but also led to a significantly reduced progesterone production. Importantly, endocrine cells of the nonhuman primate corpus luteum (CL), which represent in vivo counterparts of luteinized GC, also contain eNaCh mRNA. Although the mechanism of channel activity under physiological conditions is not clear, it may include persistent Na+ currents. As observed in GC in culture, abundant secondary lysosomes were particularly evident in the regressing CL, suggesting a functional link between eNaCh activity and this form of cellular regression in vivo. Our results identify eNaCh in ovarian endocrine cells and demonstrate that their expression is under the inhibitory control of hCG. Activation of eNaChs in luteal cells, due to loss of gonadotropin support, may initiate a cascade of events leading to decreased CL function, a process that involves lysosomal activation and autophagy. These results imply that ovarian eNaChs are involved in the physiological demise of the temporary endocrine organ CL in the primate ovary during the menstrual cycle. Because commonly used drugs, including phenytoin, target NaChs, these results

  13. A Na+-activated K+ current (IK,Na) is present in guinea-pig but not rat ventricular myocytes.

    PubMed

    Lawrence, C; Rodrigo, G C

    1999-05-01

    The effects of removing extracellular Ca2+ and Mg2+ on the membrane potential, membrane current and intracellular Na+ activity (aiNa) were investigated in guinea-pig and rat ventricular myocytes. Membrane potential was recorded with a patch pipette and whole-cell membrane currents using a single-electrode voltage clamp. Both guinea-pig and rat cells depolarize when the bathing Ca2+ and Mg2+ are removed and the steady-state aiNa increases rapidly from a resting value of 6.4+/- 0.6 mM to 33+/-3.8 mM in guinea-pig (n=9) and from 8.9+/-0.8 mM to 29.3+/-3.0 mM (n=5) in rat ventricular myocytes. Guinea-pig myocytes partially repolarized when, in addition to removal of the bathing Ca2+ and Mg2+, K+ was also removed, however rat cells remained depolarized. A large diltiazem-sensitive inward current was recorded in guinea-pig and rat myocytes, voltage-clamped at -20 mV, when the bathing divalent cations were removed. When the bathing K+ was removed after Ca2+ and Mg2+ depletion, a large outward K+ current developed in guinea-pig, but not in rat myocytes. This current had a reversal potential of -80+/-0.7 mV and was not inhibited by high Mg2+ or glybenclamide indicating that it is not due to activation of non-selective cation or adenosine triphosphate (ATP)-sensitive K channels. The current was not activated when Li+ replaced the bathing Na+ and was blocked by R-56865, suggesting that it was due to the activation of KNa channels.

  14. Suppression of Na+/K+-ATPase activity during estivation in the land snail Otala lactea.

    PubMed

    Ramnanan, Christopher J; Storey, Kenneth B

    2006-02-01

    Entry into the hypometabolic state of estivation requires a coordinated suppression of the rate of cellular ATP turnover, including both ATP-generating and ATP-consuming reactions. As one of the largest consumers of cellular ATP, the plasma membrane Na+/K+-ATPase is a potentially key target for regulation during estivation. Na+/K+-ATPase was investigated in foot muscle and hepatopancreas of the land snail Otala lactea, comparing active and estivating states. In both tissues enzyme properties changed significantly during estivation: maximal activity was reduced by about one-third, affinity for Mg.ATP was reduced (Km was 40% higher), and activation energy (derived from Arrhenius plots) was increased by approximately 45%. Foot muscle Na+/K+-ATPase from estivated snails also showed an 80% increase in Km Na+ and a 60% increase in Ka Mg2+ as compared with active snails, whereas hepatopancreas Na+/K+-ATPase showed a 70% increase in I50 K+ during estivation. Western blotting with antibodies recognizing the alpha subunit of Na+/K+-ATPase showed no change in the amount of enzyme protein during estivation. Instead, the estivation-responsive change in Na+/K+-ATPase activity was linked to posttranslational modification. In vitro incubations manipulating endogenous kinase and phosphatase activities indicated that Na+/K+-ATPase from estivating snails was a high phosphate, low activity form, whereas dephosphorylation returned the enzyme to a high activity state characteristic of active snails. Treatment with protein kinases A, C or G could all mediate changes in enzyme properties in vitro that mimicked the effect of estivation, whereas treatments with protein phosphatase 1 or 2A had the opposite effect. Reversible phosphorylation control of Na+/K+-ATPase can provide the means of coordinating ATP use by this ion pump with the rates of ATP generation by catabolic pathways in estivating snails.

  15. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 1 2013-10-01 2013-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  16. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 1 2014-10-01 2014-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  17. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 1 2012-10-01 2012-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  18. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 1 2011-10-01 2011-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  19. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  20. Intracellular Na(+) modulates large conductance Ca(2+)-activated K (+) currents in human umbilical vein endothelial cells.

    PubMed

    Liang, Guo Hua; Kim, Moon Young; Park, Seonghee; Kim, Ji Aee; Choi, Shinkyu; Suh, Suk Hyo

    2008-10-01

    We studied the effects of Na(+) influx on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na(+) replacement by NMDG(+) or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na(+) concentration ([Na(+)](i)) and vice versa. In addition, extracellular Na(+) depletion decreased [Na(+)](i). In voltage-clamped cells, BK(Ca) currents were markedly increased by extracellular Na(+) depletion. In inside-out patches, increasing [Na(+)](i) from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK(Ca) channels and decreasing intracellular K(+) concentration ([K(+)](i)) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na(+)](i) gradually from 0 to 70 mM, by replacing K(+), markedly reduced single channel conductance and NPo. The Na(+)-Ca(2+) exchange blocker Ni(2+) or KB-R7943 decreased [Na(+)](i) and increased BK(Ca) currents simultaneously, and the Na(+) ionophore monensin completely inhibited BK(Ca) currents. BK(Ca) currents were significantly augmented by increasing extracellular K(+) concentration ([K(+)](o)) from 6 to 12 mM and significantly reduced by decreasing [K(+)](o) from 12 or 6 to 0 mM or applying the Na(+)-K(+) pump inhibitor ouabain. These results suggest that intracellular Na(+) inhibit single channel conductance of BK(Ca) channels and that intracellular K(+) increases single channel conductance and NPo.

  1. Na(+) doping induced changes in the reduction and charge transport characteristics of Al2O3-stabilized, CuO-based materials for CO2 capture.

    PubMed

    Imtiaz, Q; Abdala, P M; Kierzkowska, A M; van Beek, W; Schweiger, S; Rupp, J L M; Müller, C R

    2016-04-28

    Chemical looping combustion (CLC) and chemical looping with oxygen uncoupling (CLOU) are emerging CO2 capture technologies that could reduce appreciably the costs associated with the capture of CO2. In CLC and CLOU, the oxygen required to combust a hydrocarbon is provided by a solid oxygen carrier. Among the transition metal oxides typically considered for CLC and CLOU, copper oxide (CuO) stands out owing to its high oxygen carrying capacity, exothermic reduction reactions and fast reduction kinetics. However, the low Tammann (sintering) temperature of CuO is a serious drawback. In this context, it has been proposed to support CuO on high Tammann temperature and low cost alumina (Al2O3), thus, reducing the morphological changes occurring over multiple CLC or CLOU redox cycles and stabilizing, in turn, the high activity of CuO. However, in CuO-Al2O3 systems, phase stabilization and avoiding the formation of the CuAl2O4 spinel is key to obtaining a material with a high redox stability and activity. Here, we report a Na(+) doping strategy to phase stabilize Al2O3-supported CuO, yielding in turn an inexpensive material with a high redox stability and CO2 capture efficiency. We also demonstrate that doping CuO-Al2O3 with Na(+) improves the oxygen uncoupling characteristics and coke resistance of the oxygen carriers. Utilizing in situ and ex situ X-ray absorption spectroscopy (XAS), the local structure of Cu and the reduction pathways of CuO were determined as a function of the Na(+) content and cycle number. Finally, using 4-point conductivity measurements, we confirm that doping of Al2O3-supported CuO with Na(+) lowers the activation energy for charge transport explaining conclusively the improved redox characteristics of the new oxygen carriers developed.

  2. Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica.

    PubMed

    Dulermo, Rémi; Gamboa-Meléndez, Heber; Ledesma-Amaro, Rodrigo; Thévenieau, France; Nicaud, Jean-Marc

    2015-09-01

    Fatty acid (FA) transport and activation have been extensively studied in the model yeast species Saccharomyces cerevisiae but have rarely been examined in oleaginous yeasts, such as Yarrowia lipolytica. Because the latter begins to be used in biodiesel production, understanding its FA transport and activation mechanisms is essential. We found that Y. lipolytica has FA transport and activation proteins similar to those of S. cerevisiae (Faa1p, Pxa1p, Pxa2p, Ant1p) but mechanism of FA peroxisomal transport and activation differs greatly with that of S. cerevisiae. While the ScPxa1p/ScPxa2p heterodimer is essential for growth on long-chain FAs, ΔYlpxa1 ΔYlpxa2 is not impaired for growth on FAs. Meanwhile, ScAnt1p and YlAnt1p are both essential for yeast growth on medium-chain FAs, suggesting they function similarly. Interestingly, we found that the ΔYlpxa1 ΔYlpxa2 ΔYlant1 mutant was unable to grow on short-, medium-, or long-chain FAs, suggesting that YlPxa1p, YlPxa2p, and YlAnt1p belong to two different FA degradation pathways. We also found that YlFaa1p is involved in FA storage in lipid bodies and that FA remobilization largely depended on YlFat1p, YlPxa1p and YlPxa2p. This study is the first to comprehensively examine FA intracellular transport and activation in oleaginous yeast.

  3. Engineering intracellular active transport systems as in vivo biomolecular tools.

    SciTech Connect

    Bachand, George David; Carroll-Portillo, Amanda

    2006-11-01

    Active transport systems provide essential functions in terms of cell physiology and metastasis. These systems, however, are also co-opted by invading viruses, enabling directed transport of the virus to and from the cell's nucleus (i.e., the site of virus replication). Based on this concept, fundamentally new approaches for interrogating and manipulating the inner workings of living cells may be achievable by co-opting Nature's active transport systems as an in vivo biomolecular tool. The overall goal of this project was to investigate the ability to engineer kinesin-based transport systems for in vivo applications, specifically the collection of effector proteins (e.g., transcriptional regulators) within single cells. In the first part of this project, a chimeric fusion protein consisting of kinesin and a single chain variable fragment (scFv) of an antibody was successfully produced through a recombinant expression system. The kinesin-scFv retained both catalytic and antigenic functionality, enabling selective capture and transport of target antigens. The incorporation of a rabbit IgG-specific scFv into the kinesin established a generalized system for functionalizing kinesin with a wide range of target-selective antibodies raised in rabbits. The second objective was to develop methods of isolating the intact microtubule network from live cells as a platform for evaluating kinesin-based transport within the cytoskeletal architecture of a cell. Successful isolation of intact microtubule networks from two distinct cell types was demonstrated using glutaraldehyde and methanol fixation methods. This work provides a platform for inferring the ability of kinesin-scFv to function in vivo, and may also serve as a three-dimensional scaffold for evaluating and exploiting kinesin-based transport for nanotechnological applications. Overall, the technology developed in this project represents a first-step in engineering active transport system for in vivo applications. Further

  4. Morphine Induces Ubiquitin-Proteasome Activity and Glutamate Transporter Degradation*

    PubMed Central

    Yang, Liling; Wang, Shuxing; Sung, Backil; Lim, Grewo; Mao, Jianren

    2008-01-01

    Glutamate transporters play a crucial role in physiological glutamate homeostasis, neurotoxicity, and glutamatergic regulation of opioid tolerance. However, how the glutamate transporter turnover is regulated remains poorly understood. Here we show that chronic morphine exposure induced posttranscriptional down-regulation of the glutamate transporter EAAC1 in C6 glioma cells with a concurrent decrease in glutamate uptake and increase in proteasome activity, which were blocked by the selective proteasome inhibitor MG-132 or lactacystin but not the lysosomal inhibitor chloroquin. At the cellular level, chronic morphine induced the PTEN (phosphatase and tensin homolog deleted on chromosome Ten)-mediated up-regulation of the ubiquitin E3 ligase Nedd4 via cAMP/protein kinase A signaling, leading to EAAC1 ubiquitination and proteasomal degradation. Either Nedd4 or PTEN knockdown with small interfering RNA prevented the morphine-induced EAAC1 degradation and decreased glutamate uptake. These data indicate that cAMP/protein kinase A signaling serves as an intracellular regulator upstream to the activation of the PTEN/Nedd4-mediated ubiquitin-proteasome system activity that is critical for glutamate transporter turnover. Under an in vivo condition, chronic morphine exposure also induced posttranscriptional down-regulation of the glutamate transporter EAAC1, which was prevented by MG-132, and transcriptional up-regulation of PTEN and Nedd4 within the spinal cord dorsal horn. Thus, inhibition of the ubiquitin-proteasome-mediated glutamate transporter degradation may be an important mechanism for preventing glutamate overexcitation and may offer a new strategy for treating certain neurological disorders and improving opioid therapy in chronic pain management. PMID:18539596

  5. Enhance the Er3+ Upconversion Luminescence by Constructing NaGdF4:Er3+@NaGdF4:Er3+ Active-Core/Active-Shell Nanocrystals

    NASA Astrophysics Data System (ADS)

    Du, Xiaoyu; Wang, Xiangfu; Meng, Lan; Bu, Yanyan; Yan, Xiaohong

    2017-03-01

    NaGdF4:12%Er3+@NaGdF4: x%Er3+ ( x = 0, 6, 8, 10, and 12) active-core/active-shell nanoparticles (NPs) were peculiarly synthesized via a delayed nucleation pathway with procedures. The phase, shape, and size of the resulting core-shell NPs are confirmed by transmission electron microscopy and X-ray diffraction. Coated with a NaGdF4:10%Er3+ active shell around the NaGdF4:12%Er3+ core NPs, a maximum luminescent enhancement of about 336 times higher than the NaGdF4:12%Er3+ core-only NPs was observed under the 1540 nm excitation. The intensity ratio of green to red was adjusted through the construction of the core-shell structure and the change of Er3+ concentration in the shell. By analyzing the lifetimes of emission bands and exploring the energy transition mechanism, the giant luminescence enhancement is mainly attributed to the significant increase in the near-infrared absorption at 1540 nm and efficient energy migration from the shell to core.

  6. Enhance the Er(3+) Upconversion Luminescence by Constructing NaGdF4:Er(3+)@NaGdF4:Er(3+) Active-Core/Active-Shell Nanocrystals.

    PubMed

    Du, Xiaoyu; Wang, Xiangfu; Meng, Lan; Bu, Yanyan; Yan, Xiaohong

    2017-12-01

    NaGdF4:12%Er(3+)@NaGdF4:x%Er(3+) (x = 0, 6, 8, 10, and 12) active-core/active-shell nanoparticles (NPs) were peculiarly synthesized via a delayed nucleation pathway with procedures. The phase, shape, and size of the resulting core-shell NPs are confirmed by transmission electron microscopy and X-ray diffraction. Coated with a NaGdF4:10%Er(3+) active shell around the NaGdF4:12%Er(3+) core NPs, a maximum luminescent enhancement of about 336 times higher than the NaGdF4:12%Er(3+) core-only NPs was observed under the 1540 nm excitation. The intensity ratio of green to red was adjusted through the construction of the core-shell structure and the change of Er(3+) concentration in the shell. By analyzing the lifetimes of emission bands and exploring the energy transition mechanism, the giant luminescence enhancement is mainly attributed to the significant increase in the near-infrared absorption at 1540 nm and efficient energy migration from the shell to core.

  7. Effects of internal and external pH on amiloride-blockable Na transport across toad urinary bladder vesicles

    SciTech Connect

    Garty, H.; Civan, E.D.; Civan, M.M.

    1985-01-01

    The authors have examined the effect of internal and external pH on Na+ transport across toad bladder membrane vesicles. Of the total SSNa uptake measured 0.5-2.0 min after introducing tracer, 80 +/- 4% (mean +/- SE, n = 9) is blocked by the diuretic with a KI of 2 X 10(-8) M. Thus, this amiloride-sensitive flux is mediated by the apical sodium-selective channels. Varying the internal (cytosolic) pH over the physiologic range 7.0-8.0 had no effect on sodium transport; this result suggests that variation of intracellular pH in vivo has no direct apical effect on modulating sodium uptake. On the other hand, SSNa was directly and monotonically dependent on external pH. External acidification also reduced the amiloride-sensitive efflux across the walls of the vesicles. This inhibition of 22Na efflux was noted at external Na concentrations of both 0.2 microM and 53 mM. These results are different from those reported with whole toad bladder. A number of possible bases for these differences are considered and discussed. They suggest that the natriferic response induced by mucosal acidification of whole toad urinary bladder appears to operate indirectly through one or more factors, presumably cytosolic, present in whole cells and absent from the vesicles.

  8. Thyroid hormones increase Na -H exchange activity in renal brush border membranes

    SciTech Connect

    Kinsella, J.; Sacktor, B.

    1985-06-01

    Na -H exchange activity, i.e., amiloride-sensitive Na and H flux, in renal proximal tubule brush border (luminal) membrane vesicles was increased in the hyperthyroid rat and decreased in the hypothyroid rat, relative to the euthyroid animal. A positive correlation was found between Na -H exchange activity and serum concentrations of thyroxine (T4) and triiodothyronine (T3). The thyroid status of the animal did not alter amiloride-insensitive Na uptake. The rate of passive pH gradient dissipation was higher in membrane vesicles from hyperthyroid rats compared to the rate in vesicles from hypothyroid animals, a result which would tend to limit the increase in Na uptake in vesicles from hyperthyroid animals. Na -dependent phosphate uptake was increased in membrane vesicles from hyperthyroid rats; Na -dependent D-glucose and L-proline uptakes were not changed by the thyroid status of the animal. The effect of thyroid hormones in increasing the uptake of Na in the brush border membrane vesicle is consistent with the action of the hormones in enhancing renal Na reabsorption.

  9. Gill Na{sup +}, K{sup +}-ATPase activity in largemouth bass (Micropterus salmoides) inhabiting reservoirs contaminated with mercury

    SciTech Connect

    Brundage, S.; Jagoe, C.H.; Shaw-Allen, P.

    1995-12-31

    Active transport of Na{sup +} and K{sup +} for osmoregulation in fish involves gill Na{sup +}, K{sup +}-ATPase, a membrane-bound enzyme powered by hydrolysis of ATP. Na{sup +}, K{sup +}-ATPase is inhibited by many dissolved metals including Al, Cd, Cu and Hg, resulting in ionoregulatory dysfunction. However, dissolved Hg concentrations are quite low in most aquatic systems, and dietary sources are the most important contributors to Hg burdens in fish. One recent study demonstrated relationships between muscle Hg concentration and gill Na{sup +}, K{sup +}-ATPase in a marine fish, suggesting that Hg accumulated via diet can affect osmoregulation. The authors tested for such a relationship in several age-classes of a freshwater fish (Micropterus salmoides) collected from three reservoirs. Fish from Par Pond and L Lake, on the USDOE Savannah River Site in South Carolina had relatively high Hg content: for Par Pond, muscle and liver ranged from 1.58--12.01 and 1.46--23.22 {micro}g Hg/g dry mass, respectively, and for L Lake muscle and liver ranged from 3.11--5.16 and 1.28--12.59 {micro}g Hg/g dry mass, respectively. Bass from an offsite location, Thurmond Lake, had significantly (P <0.05 by Kruskal-Wallis test) less Hg (muscle and liver range 0.61--2.39 and 0.28--2.32 {micro}g Hg/g dry mass, respectively). In all reservoirs, liver Hg varied more among individuals than muscle Hg. Water chemistry was similar in all reservoirs. Fish from the three reservoirs did not differ significantly in gill ATPase activity, and a correlation between tissue Hg and Na{sup +}, K{sup +}-ATPase activity was not evident.

  10. Na+K+-ATPase activity as a biomarker of toxaphene toxicity in Unio tumidus.

    PubMed

    Pałecz, Danuta; Komuński, Robert; Gabryelak, Teresa

    2005-08-01

    In this study, the effect of toxaphene (camphechlor) on ATPase activity in the microsomal fraction of the Unio tumidus's digestive gland was determined. Toxaphene is a man-made mixture consisting of polychlorinated monoterpens, predominantly bornanes. This compound was primarily used as an insecticide, but in 1982 was officially banned because of its destructive effects on human and animal health. Toxaphene can be transported in the air at long distances and can persist in air, soil and water for years revealing acute and chronic toxicity towards aquatic organisms and wildlife, the increasing risk of cancer in both humans and animals. The microsomal fraction isolated from digestive glands was exposed to 1 x 10(-3) M, 1 x 10(-5) M and 1 x 10(-7) M of toxaphene. The obtained data showed that toxaphene induced a loss of ATPase activity in all used concentrations. The Lineweaver-Burk plots for microsomal Na+K+-ATPase in the presence or the absence of toxaphene as an inhibitor indicated a competitive type of inhibition.

  11. Na+ dependent acid-base transporters in the choroid plexus; insights from slc4 and slc9 gene deletion studies

    PubMed Central

    Christensen, Henriette L.; Nguyen, An T.; Pedersen, Fredrik D.; Damkier, Helle H.

    2013-01-01

    The choroid plexus epithelium (CPE) is located in the ventricular system of the brain, where it secretes the majority of the cerebrospinal fluid (CSF) that fills the ventricular system and surrounds the central nervous system. The CPE is a highly vascularized single layer of cuboidal cells with an unsurpassed transepithelial water and solute transport rate. Several members of the slc4a family of bicarbonate transporters are expressed in the CPE. In the basolateral membrane the electroneutral Na+ dependent Cl−/HCO3− exchanger, NCBE (slc4a10) is expressed. In the luminal membrane, the electrogenic Na+:HCO3− cotransporter, NBCe2 (slc4a5) is expressed. The electroneutral Na+:HCO3− cotransporter, NBCn1 (slc4a7), has been located in both membranes. In addition to the bicarbonate transporters, the Na+/H+ exchanger, NHE1 (slc9a1), is located in the luminal membrane of the CPE. Genetically modified mice targeting slc4a2, slc4a5, slc4a7, slc4a10, and slc9a1 have been generated. Deletion of slc4a5, 7 or 10, or slc9a1 has numerous impacts on CP function and structure in these mice. Removal of the transporters affects brain ventricle size (slc4a5 and slc4a10) and intracellular pH regulation (slc4a7 and slc4a10). In some instances, removal of the proteins from the CPE (slc4a5, 7, and 10) causes changes in abundance and localization of non-target transporters known to be involved in pH regulation and CSF secretion. The focus of this review is to combine the insights gathered from these knockout mice to highlight the impact of slc4 gene deletion on the CSF production and intracellular pH regulation resulting from the deletion of slc4a5, 7 and 10, and slc9a1. Furthermore, the review contains a comparison of the described human mutations of these genes to the findings in the knockout studies. Finally, the future perspective of utilizing these proteins as potential targets for the treatment of CSF disorders will be discussed. PMID:24155723

  12. Fluctuation driven active molecular transport in passive channel proteins

    NASA Astrophysics Data System (ADS)

    Kosztin, Ioan

    2006-03-01

    Living cells interact with their extracellular environment through the cell membrane, which acts as a protective permeability barrier for preserving the internal integrity of the cell. However, cell metabolism requires controlled molecular transport across the cell membrane, a function that is fulfilled by a wide variety of transmembrane proteins, acting as either passive or active transporters. In this talk it is argued that, contrary to the general belief, in active cell membranes passive and spatially asymmetric channel proteins can act as active transporters by consuming energy from nonequilibrium fluctuations fueled by cell metabolism. This assertion is demonstrated in the case of the E. coli aquaglyceroporin GlpF channel protein, whose high resolution crystal structure is manifestly asymmetric. By calculating the glycerol flux through GlpF within the framework of a stochastic model, it is found that, as a result of channel asymmetry, glycerol uptake driven by a concentration gradient is enhanced significantly in the presence of non-equilibrium fluctuations. Furthermore, the enhancement caused by a ratchet-like mechanism is larger for the outward, i.e., from the cytoplasm to the periplasm, flux than for the inward one, suggesting that the same non-equilibrium fluctuations also play an important role in protecting the interior of the cell against poisoning by excess uptake of glycerol. Preliminary data on water and sugar transport through aquaporin and maltoporin channels, respectively, are indicative of the universality of the proposed nonequilibrium-fluctuation-driven active transport mechanism. This work was supported by grants from the Univ. of Missouri Research Board, the Institute for Theoretical Sciences and the Department of Energy (DOE Contract W-7405-ENG-36), and the National Science Foundation (FIBR-0526854).

  13. Increasing influence of the glutamate transporter inhibitor on glutamate release in low [Na +] media under extremal conditions.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Krisanova, N.; Himmelreich, N.

    The effect of the competitive nontransportable inhibitor DL-threo-beta-benzyloxyaspartate DL-TBOA on the release of glutamate in Ca 2 -free Na - and NMDG-supplemented media was evaluated after exposure of rats to extremal conditions 6 min incubation of synaptosomes with 10 mu M DL-TBOA in low Na media resulted in the increase in extracellular L- 14 C glutamate level for control animals by 2 0 pm 0 5 of total accumulated label and 100 mu M DL-TBOA - 3 5 pm 0 5 respectively The experimental data for animals subjected to centrifuge-induced hypergravity showed 4 0 pm 1 0 and 9 0 pm 2 0 increase in L- 14 C glutamate level for 10 mu M and 100 mu M DL-TBOA respectively D le 0 05 The enhancement of the extracellular level of L- 14 C glutamate after application of DL-TBOA would be expected to connect with the inhibition of L- 14 C glutamate uptake process It appears that DL-TBOA inhibited uptake more potently after hypergravity The effect of DL-TBOA on depolarization-induced carrier-mediated L- 14 C glutamate release increased after hypergravity loading in Na - and low Na NMDG- supplemented media 10 mu M DL-TBOA-induced decrease in L- 14 C glutamate release in Na - supplemented medium was 15 2 pm 2 2 in the control experiments and 26 2 pm 3 9 after loading D le 0 05 and in low Na medium was 37 0 pm 2 5 and 45 0 pm 3 4 respectively DL-TBOA was demonstrated to better inhibit the transporter-mediated

  14. Acute inhibition of NCC does not activate distal electrogenic Na+ reabsorption or kaliuresis

    PubMed Central

    Craigie, Eilidh; Homer, Natalie Z. M.; Mullins, John J.; Bailey, Matthew A.

    2014-01-01

    Na+ reabsorption from the distal renal tubule involves electroneutral and electrogenic pathways, with the latter promoting K+ excretion. The relative activities of these two pathways are tightly controlled, participating in the minute-to-minute regulation of systemic K+ balance. The pathways are interdependent: the activity of the NaCl cotransporter (NCC) in the distal convoluted tubule influences the activity of the epithelial Na+ channel (ENaC) downstream. This effect might be mediated by changes in distal Na+ delivery per se or by molecular and structural adaptations in the connecting tubule and collecting ducts. We hypothesized that acute inhibition of NCC activity would cause an immediate increase in Na+ flux through ENaC, with a concomitant increase in renal K+ excretion. We tested this using renal clearance methodology in anesthetized mice, by the administration of hydrochlorothiazide (HCTZ) and/or benzamil (BZM) to exert specific blockade of NCC and ENaC, respectively. Bolus HCTZ elicited a natriuresis that was sustained for up to 110 min; urinary K+ excretion was not affected. Furthermore, the magnitude of the natriuresis was no greater during concomitant BZM administration. This suggests that ENaC-mediated Na+ reabsorption was not normally limited by Na+ delivery, accounting for the absence of thiazide-induced kaliuresis. After dietary Na+ restriction, HCTZ elicited a kaliuresis, but the natiuretic effect of HCTZ was not enhanced by BZM. Our findings support a model in which inhibition of NCC activity does not increase Na+ reabsorption through ENaC solely by increasing distal Na+ delivery but rather by inducing a molecular and structural adaptation in downstream nephron segments. PMID:24402096

  15. Ca2+ signaling evoked by activation of Na+ channels and Na+/Ca2+ exchangers is required for GABA-induced NG2 cell migration

    PubMed Central

    Tong, Xiao-ping; Li, Xiang-yao; Zhou, Bing; Shen, Wanhua; Zhang, Zhi-jun; Xu, Tian-le

    2009-01-01

    NG2 cells originate from various brain regions and migrate to their destinations during early development. These cells express voltage-gated Na+ channels but fail to produce typical action potentials. The physiological role of Na+ channels in these cells is unclear. We found that GABA induces membrane depolarization and Ca2+ elevation in NG2 cells, a process requiring activation of GABAA receptors, Na+ channels, and Na+/Ca2+ exchangers (NCXs), but not Ca2+ channels. We have identified a persistent Na+ current in these cells that may underlie the GABA-induced pathway of prolonged Na+ elevation, which in turn triggers Ca2+ influx via NCXs. This unique Ca2+ signaling pathway is further shown to be involved in the migration of NG2 cells. Thus, GABAergic signaling mediated by sequential activation of GABAA receptors, noninactivating Na+ channels, and NCXs may play an important role in the development and function of NG2 glial cells in the brain. PMID:19596850

  16. Ab-initio Density Functional Theory (DFT) Studies of Electronic, Transport, and Bulk Properties of Sodium Oxide (Na2O)

    NASA Astrophysics Data System (ADS)

    Polin, Daniel; Ziegler, Joshua; Malozovsky, Yuriy; Bagayoko, Diola

    We present the findings of ab-initio calculations of electronic, transport, and structural properties of cubic sodium oxide (Na2O). These results were obtained using density functional theory (DFT), specifically a local density approximation (LDA) potential, and the linear combination of Gaussian orbitals (LCGO). Our implementation of LCGO followed the Bagayoko, Zhao, and Williams method as enhanced by the work of Ekuma and Franklin (BZW-EF). We describe the electronic band structure of Na2O with a direct band gap of 2.22 eV. Our results include predicted values for the electronic band structure and associated energy eigenvalues, the total and partial density of states (DOS and pDOS), the equilibrium lattice constant of Na2O, and the bulk modulus. We have also calculated the electron and holes effective masses in the Γ to L, Γ to X, and Γ to K directions. Acknowledgments: This work was funded in part by the National Science Foundation (NSF) and the Louisiana Board of Regents, through LASiGMA [Award Nos. EPS- 1003897, NSF (2010-15)-RII-SUBR] and NSF HRD-1002541, the US Department of Energy - National, Nuclear Security Administration (NNSA) (Award No. DE- NA0002630), LaSPACE, and LONI-SUBR.

  17. Prolonged Intracellular Na+ Dynamics Govern Electrical Activity in Accessory Olfactory Bulb Mitral Cells

    PubMed Central

    Zylbertal, Asaph; Kahan, Anat; Ben-Shaul, Yoram; Yarom, Yosef; Wagner, Shlomo

    2015-01-01

    Persistent activity has been reported in many brain areas and is hypothesized to mediate working memory and emotional brain states and to rely upon network or biophysical feedback. Here, we demonstrate a novel mechanism by which persistent neuronal activity can be generated without feedback, relying instead on the slow removal of Na+ from neurons following bursts of activity. We show that mitral cells in the accessory olfactory bulb (AOB), which plays a major role in mammalian social behavior, may respond to a brief sensory stimulation with persistent firing. By combining electrical recordings, Ca2+ and Na+ imaging, and realistic computational modeling, we explored the mechanisms underlying the persistent activity in AOB mitral cells. We found that the exceptionally slow inward current that underlies this activity is governed by prolonged dynamics of intracellular Na+ ([Na+]i), which affects neuronal electrical activity via several pathways. Specifically, elevated dendritic [Na+]i reverses the Na+-Ca2+ exchanger activity, thus modifying the [Ca2+]i set-point. This process, which relies on ubiquitous membrane mechanisms, is likely to play a role in other neuronal types in various brain regions. PMID:26674618

  18. Regulatory activation is accompanied by movement in the C terminus of the Na-K-Cl cotransporter (NKCC1).

    PubMed

    Monette, Michelle Y; Forbush, Biff

    2012-01-13

    The Na-K-Cl cotransporter (NKCC1) is expressed in most vertebrate cells and is crucial in the regulation of cell volume and intracellular chloride concentration. To study the structure and function of NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins at two sites within the C terminus and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Both singly and doubly tagged NKCC1s were appropriately produced, trafficked to the plasma membrane, and exhibited (86)Rb transport activity. When both fluorescent probes were placed within the same C terminus of an NKCC1 transporter, we recorded an 11% FRET decrease upon activation of the transporter. This result clearly demonstrates movement of the C terminus during the regulatory response to phosphorylation of the N terminus. When we introduced CFP and YFP separately in different NKCC1 constructs and cotransfected these in HEK cells, we observed FRET between dimer pairs, and the fractional FRET decrease upon transporter activation was 46%. Quantitatively, this indicates that the largest FRET-signaled movement is between dimer pairs, an observation supported by further experiments in which the doubly tagged construct was cotransfectionally diluted with untagged NKCC1. Our results demonstrate that regulation of NKCC1 is accompanied by a large movement between two positions in the C termini of a dimeric cotransporter. We suggest that the NKCC1 C terminus is involved in transport regulation and that dimerization may play a key structural role in the regulatory process. It is anticipated that when combined with structural information, our findings will provide a model for understanding the conformational changes that bring about NKCC1 regulation.

  19. Gamma-ray spectrometer experiment, Apollo 17: NaI(T1) detector crystal activation

    NASA Technical Reports Server (NTRS)

    Trombka, J. I.; Schmadebeck, R. L.; Bielefeld, M.; Okelley, G. D.; Eldridge, J. S.; Northcutt, K. J.; Metzger, A. E.; Schonfeld, E.; Peterson, L. E.; Arnold, J. R.

    1973-01-01

    An attempt was made to obtain experimental data on proton induced activity and its effect on gamma ray spectral measurements. A NaI(T1) crystal flown in Apollo 17 command module was used for the experiment.

  20. Cell volume regulation by trout erythrocytes: characteristics of the transport systems activated by hypotonic swelling.

    PubMed

    Garcia-Romeu, F; Cossins, A R; Motais, R

    1991-01-01

    1. An osmolality reduction of the suspending medium leads to osmotic swelling of trout erythrocytes, which is followed by a volume readjustment towards the original level. The regulatory volume decrease (RVD) was not complete after 1 h. 2. During RVD the cells lost K+ and Cl- but gained Na+. This entry of Na+, which is about half the K+ loss, explains the incomplete volume recovery (it was complete when Na+ was replaced by impermeant N-methyl-D-glucamine). The cells also lose large quantities of taurine, which accounts for about 53% of the volume recovery. In addition RVD is accompanied by the activation of a pathway allowing some large organic cations which are normally impermeant, such as choline or tetramethyl-ammonium, to rapidly penetrate the cells. 3. The swelling-activated K+ loss is not significantly affected by replacement of Cl- by NO3-, indicating that K+ moves through a Cl(-)-independent K+ pathway. Furosemide, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and niflumic acid inhibit the K+ loss. From experiments performed in high-K(+)-containing media, it appears that these compounds block the K+ flux, not by inhibiting Cl- movements but by interfering with the K+ pathway. 4. All the volume-activated pathways (K+, Na+, taurine, choline) are fully inhibited by furosemide and by inhibitors of the anion exchanger such as DIDS and niflumic acid. The concentration required for 50% inhibition (IC50) of both inorganic cations and taurine appears to be similar. It is proposed that DIDS interacts with a unique target which controls all the volume-sensitive transport systems.

  1. Na-KATPase activity and intracellular ion concentrations in the lactating guinea pig mammary gland. Studies on Na-K activated adenosine triphosphatase, XXXVI.

    PubMed

    Vreeswijk, J H; de Pont, J J; Bonting, S L

    1975-01-01

    The intracellular sodium, potassium and chloride concentrations in slices of lactating guinea pig mammary gland have been determined by chemical analysis and the use of appropriate values for extracellular space. These ion concentrations after 1 hr incubation at 37 degrees C in a Krebs-Ringer bicarbonate solution are 45mM Na+, 138 mM K+ and 44 mM Cl-, which values are in agreement with those found in fresh mammary gland slices. Inhibition of the NaK activated ATPase cation pump system of the tissue by 10(-4)M ouabain, anoxia or cooling to 0 degrees C Causes a gain of Na+ and an equimolar loss of K+ without a significant change in chloride concentration. The effect of cooling (0 degrees C) is reversible by reincubation at 37 degrees C. Water content of the tissue (76.5% of wet weight) and extracellular space (40.5%) do not change under these conditions. The results permit the conclusion that the NaK activated ATPase system is responsible for the maintenance of the intracellular Na+ and K+ concentrations, but do not support the presence of a chloride pump.

  2. Highly porous activated carbons prepared from carbon rich Mongolian anthracite by direct NaOH activation

    NASA Astrophysics Data System (ADS)

    Byamba-Ochir, Narandalai; Shim, Wang Geun; Balathanigaimani, M. S.; Moon, Hee

    2016-08-01

    Highly porous activated carbons (ACs) were prepared from Mongolian raw anthracite (MRA) using sodium hydroxide as an activation agent by varying the mass ratio (powdered MRA/NaOH) as well as the mixing method of chemical agent and powdered MRA. The specific BET surface area and total pore volume of the prepared MRA-based activated carbons (MACs) are in the range of 816-2063 m2/g and of 0.55-1.61 cm3/g, respectively. The pore size distribution of MACs show that most of the pores are in the range from large micropores to small mesopores and their distribution can be controlled by the mass ratio and mixing method of the activating agent. As expected from the intrinsic property of the MRA, the highly graphitic surface morphology of prepared carbons was confirmed from Raman spectra and transmission electron microscopy (TEM) studies. Furthermore the FTIR and XPS results reveal that the preparation of MACs with hydrophobic in nature is highly possible by controlling the mixing conditions of activating agent and powdered MRA. Based on all the results, it is suggested that the prepared MACs could be used for many specific applications, requiring high surface area, optimal pore size distribution, proper surface hydrophobicity as well as strong physical strength.

  3. The Asymmetric Active Coupler: Stable Nonlinear Supermodes and Directed Transport

    PubMed Central

    Kominis, Yannis; Bountis, Tassos; Flach, Sergej

    2016-01-01

    We consider the asymmetric active coupler (AAC) consisting of two coupled dissimilar waveguides with gain and loss. We show that under generic conditions, not restricted by parity-time symmetry, there exist finite-power, constant-intensity nonlinear supermodes (NS), resulting from the balance between gain, loss, nonlinearity, coupling and dissimilarity. The system is shown to possess non-reciprocal dynamics enabling directed power transport functionality. PMID:27640818

  4. 77 FR 71430 - New Agency Information Collection Activity Under OMB Review: Public Transportation Baseline...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-30

    ... SECURITY Transportation Security Administration New Agency Information Collection Activity Under OMB Review: Public Transportation Baseline Assessment for Security Enhancement (BASE) Program AGENCY: Transportation Security Administration, DHS. ACTION: 30-day notice. SUMMARY: This notice announces that the...

  5. Purinergic Effects on Na,K-ATPase Activity Differ in Rat and Human Skeletal Muscle

    PubMed Central

    Juel, Carsten; Nordsborg, Nikolai B.; Bangsbo, Jens

    2014-01-01

    Background P2Y receptor activation may link the effect of purines to increased maximal in vitro activity of the Na,K-ATPase in rat muscle. The hypothesis that a similar mechanism is present in human skeletal muscle was investigated with membranes from rat and human skeletal muscle. Results Membranes purified from rat and human muscles were used in the Na,K-ATPase assay. Incubation with ADP, the stable ADP analogue MeS-ADP and UDP increased the Na+ dependent Na,K-ATPase activity in rat muscle membranes, whereas similar treatments of human muscle membranes lowered the Na,K-ATPase activity. UTP incubation resulted in unchanged Na,K-ATPase activity in humans, but pre-incubation with the antagonist suramin resulted in inhibition with UTP, suggesting that P2Y receptors are involved. The Na,K-ATPase in membranes from both rat and human could be stimulated by protein kinase A and C activation. Thus, protein kinase A and C activation can increase Na,K-ATPase activity in human muscle but not via P2Y receptor stimulation. Conclusion The inhibitory effects of most purines (with the exception of UTP) in human muscle membranes are probably due to mass law inhibition of ATP hydrolysis. This inhibition could be blurred in rat due to receptor mediated activation of the Na,K-ATPase. The different effects could be related to a high density of ADP sensitive P2Y1 and P2Y13 receptors in rat, whereas the UTP sensitive P2Y11 could be more abundant in human. Alternatively, rat could possesses a mechanism for protein-protein interaction between P2Y receptors and the Na,K-ATPase, and this mechanism could be absent in human skeletal muscle (perhaps with the exception of the UTP sensitive P2Y11 receptor). Perspective Rat muscle is not a reliable model for purinergic effects on Na,K-ATPase in human skeletal muscle. PMID:24614174

  6. Charge transport in thin layer Na x CoO2 (x ˜ 0.63) studied by terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    Němec, H.; Knížek, K.; Jirák, Z.; Hejtmánek, J.; Soroka, M.; Buršík, J.

    2016-09-01

    Charge transport in Na0.63CoO2 thin film deposited by a spin-coating method was investigated experimentally by time-domain terahertz spectroscopy and theoretically using Monte Carlo calculations of charge response in nano-structured materials. The dominating type of transport mechanism over the entire investigated range of temperatures (20-300 K) is a metallic-like conductivity of charges partly confined in constituting nano-sized grains. Due to the granular character of our thin film, the scattering time at low temperatures is limited by scattering on grain boundaries and the conductivity is strongly suppressed due to capture of a major fraction of charge carriers in deep traps. Nevertheless, our experimental setup and the applied model allowed us to distinguish the parameters related to the grain interior from those influenced by grain boundaries, and to conclude that the metallic type of conductivity is the intrinsic property relevant to single crystal materials.

  7. Substrate-bound outward-open state of the betaine transporter BetP provides insights into Na+ coupling

    NASA Astrophysics Data System (ADS)

    Perez, Camilo; Faust, Belinda; Mehdipour, Ahmad Reza; Francesconi, Kevin A.; Forrest, Lucy R.; Ziegler, Christine

    2014-07-01

    The Na+-coupled betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary transporters, for example, with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.

  8. Synaptic activation modifies microtubules underlying transport of postsynaptic cargo.

    PubMed

    Maas, Christoph; Belgardt, Dorthe; Lee, Han Kyu; Heisler, Frank F; Lappe-Siefke, Corinna; Magiera, Maria M; van Dijk, Juliette; Hausrat, Torben J; Janke, Carsten; Kneussel, Matthias

    2009-05-26

    Synaptic plasticity, the ability of synapses to change in strength, requires alterations in synaptic molecule compositions over time, and synapses undergo selective modifications on stimulation. Molecular motors operate in sorting/transport of neuronal proteins; however, the targeting mechanisms that guide and direct cargo delivery remain elusive. We addressed the impact of synaptic transmission on the regulation of intracellular microtubule (MT)-based transport. We show that increased neuronal activity, as induced through GlyR activity blockade, facilitates tubulin polyglutamylation, a posttranslational modification thought to represent a molecular traffic sign for transport. Also, GlyR activity blockade alters the binding of the MT-associated protein MAP2 to MTs. By using the kinesin (KIF5) and the postsynaptic protein gephyrin as models, we show that such changes of MT tracks are accompanied by reduced motor protein mobility and cargo delivery into neurites. Notably, the observed neurite targeting deficits are prevented on functional depletion or gene expression knockdown of neuronal polyglutamylase. Our data suggest a previously undescribed concept of synaptic transmission regulating MT-dependent cargo delivery.

  9. Cellular cation transport studied by 6/7Li and 23Na NMR in a porous Mo132 Keplerate type nano-capsule as model system.

    PubMed

    Rehder, Dieter; Haupt, Erhard T K; Müller, Achim

    2008-01-01

    Li+ ions can interplay with other cations intrinsically present in the intra- and extra-cellular space (i.e. Na+, K+, Mg2+ and Ca2+) have therapeutic effects (e.g. in the treatment of bipolar disorder) or toxic effects (at higher doses), likely because Li+ interferes with the intra-/extra-cellular concentration gradients of the mentioned physiologically relevant cations. The cellular transmembrane transport can be modelled by molybdenum-oxide-based Keplerates, i.e. nano-sized porous capsules containing 132 Mo centres, monitored through 6/7Li as well as 23Na NMR spectroscopy. The effects on the transport of Li+ cations through the 'ion channels' of these model cells, caused by variations in water amount, temperature, and by the addition of organic cationic 'plugs' and the shift reagent [Dy(PPP)2](7-) are reported. In the investigated solvent systems, water acts as a transport mediator for Li+. Likewise, the counter-transport (Li+/Na+, Li+/K+, Li+/Cs+ and Li+/Ca2+) has been investigated by 7Li NMR and, in the case of Li+/Na+ exchange, by 23Na NMR, and it has been shown that most (in the case of Na+ and K+, all (Ca2+) or almost none (Cs+) of the Li cations is extruded from the internal sites of the artificial cell to the extra-cellular medium, while Na+, K+ and Ca2+ are partially incorporated.

  10. Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity*

    PubMed Central

    Lay, Fung T.; Mills, Grant D.; Poon, Ivan K. H.; Cowieson, Nathan P.; Kirby, Nigel; Baxter, Amy A.; van der Weerden, Nicole L.; Dogovski, Con; Perugini, Matthew A.; Anderson, Marilyn A.; Kvansakul, Marc; Hulett, Mark D.

    2012-01-01

    The plant defensin, NaD1, from the flowers of Nicotiana alata, is a member of a family of cationic peptides that displays growth inhibitory activity against several filamentous fungi, including Fusarium oxysporum. The antifungal activity of NaD1 has been attributed to its ability to permeabilize membranes; however, the molecular basis of this function remains poorly defined. In this study, we have solved the structure of NaD1 from two crystal forms to high resolution (1.4 and 1.58 Å, respectively), both of which contain NaD1 in a dimeric configuration. Using protein cross-linking experiments as well as small angle x-ray scattering analysis and analytical ultracentrifugation, we show that NaD1 forms dimers in solution. The structural studies identified Lys4 as critical in formation of the NaD1 dimer. This was confirmed by site-directed mutagenesis of Lys4 that resulted in substantially reduced dimer formation. Significantly, the reduced ability of the Lys4 mutant to dimerize correlated with diminished antifungal activity. These data demonstrate the importance of dimerization in NaD1 function and have implications for the use of defensins in agribiotechnology applications such as enhancing plant crop protection against fungal pathogens. PMID:22511788

  11. Activation of DOR Attenuates Anoxic K+ Derangement via Inhibition of Na+ Entry in Mouse Cortex

    PubMed Central

    Chao, Dongman; Bazzy-Asaad, Alia; Balboni, Gianfranco; Salvadori, Severo

    2008-01-01

    We have recently found that in the mouse cortex, activation of δ-opioid receptor (DOR) attenuates the disruption of K+ homeostasis induced by hypoxia or oxygen–glucose deprivation. This novel observation suggests that DOR may protect neurons from hypoxic/ischemic insults via the regulation of K+ homeostasis because the disruption of K+ homeostasis plays a critical role in neuronal injury under hypoxic/ischemic stress. The present study was performed to explore the ionic mechanism underlying the DOR-induced neuroprotection. Because anoxia causes Na+ influx and thus stimulates K+ leakage, we investigated whether DOR protects the cortex from anoxic K+ derangement by targeting the Na+-based K+ leakage. By using K+-sensitive microelectrodes in mouse cortical slices, we showed that 1) lowering Na+ concentration and substituting with impermeable N-methyl-D-glucamine caused a concentration-dependent attenuation of anoxic K+ derangement; 2) lowering Na+ concentration by substituting with permeable Li+ tended to potentiate the anoxic K+ derangement; and 3) the DOR-induced protection against the anoxic K+ responses was largely abolished by low-Na+ perfusion irrespective of the substituted cation. We conclude that external Na+ concentration greatly influences anoxic K+ derangement and that DOR activation likely attenuates anoxic K+ derangement induced by the Na+-activated mechanisms in the cortex. PMID:18203692

  12. Na+/K+-ATPase activity during early development and growth of an Antarctic sea urchin.

    PubMed

    Leong, P K; Manahan, D T

    1999-08-01

    In Antarctic environments, the physiological bases for long larval life spans under natural conditions of limited food availability are not understood. The Na+ pump is likely to be involved with hypometabolic regulation in such cold environments. Changes in the activity and metabolic importance of Na+/K+-ATPase were measured in embryos of the Antarctic sea urchin Sterechinus neumayeri and in larvae reared under different feeding conditions. The rate of increase of total Na+/K+-ATPase activity was 3.9 times faster in fed than in unfed larvae. During development and growth, there was an increase in the percentage of total, potential Na+/K+-ATPase activity that was physiologically utilized. In early (10-day-old) gastrulae, 17 % was utilized in vivo, increasing to 77 % in six-arm pluteus (48-day-old) larvae. The metabolic importance of in vivo Na+/K+-ATPase activity also increased during development, accounting for 12 % of metabolic rate at day 10 and 84 % at day 48. When compared at the same enzyme assay temperature (15 degrees C), the protein-specific total Na+/K+-ATPase activities for late embryonic (prism) and early larval (pluteus) stages of S. neumayeri were 2.6 times lower than those for comparable developmental stages of two temperate sea urchin species (Strongylocentrotus purpuratus and Lytechinus pictus).

  13. Na+ coordination at the Na2 site of the Na+/I− symporter

    PubMed Central

    Ferrandino, Giuseppe; Nicola, Juan Pablo; Sánchez, Yuly E.; Echeverria, Ignacia; Liu, Yunlong; Amzel, L. Mario; Carrasco, Nancy

    2016-01-01

    The sodium/iodide symporter (NIS) mediates active I− transport in the thyroid—the first step in thyroid hormone biosynthesis—with a 2 Na+: 1 I− stoichiometry. The two Na+ binding sites (Na1 and Na2) and the I− binding site interact allosterically: when Na+ binds to a Na+ site, the affinity of NIS for the other Na+ and for I− increases significantly. In all Na+-dependent transporters with the same fold as NIS, the side chains of two residues, S353 and T354 (NIS numbering), were identified as the Na+ ligands at Na2. To understand the cooperativity between the substrates, we investigated the coordination at the Na2 site. We determined that four other residues—S66, D191, Q194, and Q263—are also involved in Na+ coordination at this site. Experiments in whole cells demonstrated that these four residues participate in transport by NIS: mutations at these positions result in proteins that, although expressed at the plasma membrane, transport little or no I−. These residues are conserved throughout the entire SLC5 family, to which NIS belongs, suggesting that they serve a similar function in the other transporters. Our findings also suggest that the increase in affinity that each site displays when an ion binds to another site may result from changes in the dynamics of the transporter. These mechanistic insights deepen our understanding not only of NIS but also of other transporters, including many that, like NIS, are of great medical relevance. PMID:27562170

  14. Physical and chemical properties and adsorption type of activated carbon prepared from plum kernels by NaOH activation.

    PubMed

    Tseng, Ru-Ling

    2007-08-25

    Activated carbon was prepared from plum kernels by NaOH activation at six different NaOH/char ratios. The physical properties including the BET surface area, the total pore volume, the micropore ratio, the pore diameter, the burn-off, and the scanning electron microscope (SEM) observation as well as the chemical properties, namely elemental analysis and temperature programmed desorption (TPD), were measured. The results revealed a two-stage activation process: stage 1 activated carbons were obtained at NaOH/char ratios of 0-1, surface pyrolysis being the main reaction; stage 2 activated carbons were obtained at NaOH/char ratios of 2-4, etching and swelling being the main reactions. The physical properties of stage 2 activated carbons were similar, and specific area was from 1478 to 1887m(2)g(-1). The results of reaction mechanism of NaOH activation revealed that it was apparently because of the loss ratio of elements C, H, and O in the activated carbon, and the variations in the surface functional groups and the physical properties. The adsorption of the above activated carbons on phenol and three kinds of dyes (MB, BB1, and AB74) were used for an isotherm equilibrium adsorption study. The data fitted the Langmuir isotherm equation. Various kinds of adsorbents showed different adsorption types; separation factor (R(L)) was used to determine the level of favorability of the adsorption type. In this work, activated carbons prepared by NaOH activation were evaluated in terms of their physical properties, chemical properties, and adsorption type; and activated carbon PKN2 was found to have most application potential.

  15. Theoretical investigation on local structure and transport properties of NaFsbnd AlF3 molten salts under electric field environment

    NASA Astrophysics Data System (ADS)

    Lv, Xiaojun; Xu, Zhenming; Li, Jie; Chen, Jiangan; Liu, Qingsheng

    2016-08-01

    The effect of electric field and molecular ratio CR (NaF/AlF3) on basic structure and transport properties of NaFsbnd AlF3 molten salts were investigated by molecular dynamics simulations with the Buckingham potential model. The [AlF6]3- groups are the dominant specie in NaFsbnd AlF3 molten salts at CR ≥ 2.6, and followed by the [AlF5]2- groups, while CR ≤ 2.4, [AlF5]2- groups are the protagonists up to 40%. In NaFsbnd AlF3 system, with the increase of CR, the proportion of Fb decreases slightly and the percentage of Ff increases dramatically. The Alsbnd F bonds have ionic characters as well as partial covalently characters due to the hybridization of F-2p and Al-3s, 3p orbitals. The order of ion diffusion ability follows as Na+ > F- > Al3+. Adding more NaF can break some F bridges of structure networks and decrease the polymerization degree of NaFsbnd AlF3 molten salts, the viscosity reduces and ionic conductivity increases as a consequence. The calculated results of ionic conductivity are in agreement with the experimental results. Electric field has no significant impact on the local structure characters, while transport properties are not. The change of CR (NaF/AlF3) can significantly affect these characters of both the structure and transport.

  16. Conserved tyrosine in the first transmembrane segment of solute:sodium symporters is involved in Na+-coupled substrate co-transport.

    PubMed

    Mazier, Sonia; Quick, Matthias; Shi, Lei

    2011-08-19

    Solute:sodium symporters (SSSs) transport vital molecules across the plasma membrane of all living organisms. vSGLT, the Na(+)/galactose transporter of Vibrio parahemeolyticus, is the only SSS for which high resolution structural information is available, revealing a LeuT-like fold and a Na(+)-binding site analogous to the Na2 site of LeuT. Whereas the core transmembrane segments (TMs) of SSSs share high structural similarity with other transporters of LeuT-like fold, TM1 does not correspond to any TM in those structural homologs and was only resolved for the backbone atoms in the initial vSGLT structure (Protein Data Bank code 3DH4). To assess the role of TM1 in Na(+)-coupled substrate symport by the SSSs, here we have studied the role of a conserved residue in TM1 by computational modeling in conjunction with radiotracer transport and binding studies. Based on our sequence alignment and much topological data for homologous PutP, the Na(+)/proline transporter, we have simulated a series of vSGLT models with shifted TM1 residue assignments. We show that in two converged vSGLT models that retained the original TM1 backbone conformation, a conserved residue, Tyr-19, is associated with the Na(+) binding interaction network. In silico and in vitro mutagenesis of homologous Tyr-14 in PutP revealed the involvement of this conserved residue in Na(+)-dependent substrate binding and transport. Thus, our combined computational and experimental data provide the first clues about the importance of a conserved residue in TM1, a unique TM in the proteins with LeuT-like fold, in the Na(+)-coupled symport mechanism of SSSs.

  17. Examining Changes in Radioxenon Isotope Activity Ratios during Subsurface Transport

    NASA Astrophysics Data System (ADS)

    Annewandter, Robert

    2014-05-01

    The Non-Proliferation Experiment (NPE) has demonstrated and modelled the usefulness of barometric pumping induced gas transport and subsequent soil gas sampling during On-Site inspections. Generally, gas transport has been widely studied with different numerical codes. However, gas transport of radioxenons and radioiodines in the post-detonation regime and their possible fractionation is still neglected in the open peer-reviewed literature. Atmospheric concentrations of the radioxenons Xe-135, Xe-133m, Xe-133 and Xe-131m can be used to discriminate between civilian releases (nuclear power plants or medical isotope facilities), and nuclear explosion sources. It is based on the multiple isotopic activity ratio method. Yet it is not clear whether subsurface migration of the radionuclides, with eventual release into the atmosphere, can affect the activity ratios due to fractionation. Fractionation can be caused by different mass diffusivities due to mass differences between the radionuclides. Cyclical changes in atmospheric pressure can drive subsurface gas transport. This barometric pumping phenomenon causes an oscillatoric flow in upward trending fractures or highly conductive faults which, combined with diffusion into the porous matrix, leads to a net transport of gaseous components - a so-called ratcheting effect. We use a general purpose reservoir simulator (Complex System Modelling Platform, CSMP++) which is recognized by the oil industry as leading in Discrete Fracture-Matrix (DFM) simulations. It has been applied in a range of fields such as deep geothermal systems, three-phase black oil simulations, fracture propagation in fractured, porous media, and Navier-Stokes pore-scale modelling among others. It is specifically designed to account for structurally complex geologic situation of fractured, porous media. Parabolic differential equations are solved by a continuous Galerkin finite-element method, hyperbolic differential equations by a complementary finite

  18. Differences in associations between active transportation and built environmental exposures when expressed using different components of individual activity spaces.

    PubMed

    van Heeswijck, Torbjorn; Paquet, Catherine; Kestens, Yan; Thierry, Benoit; Morency, Catherine; Daniel, Mark

    2015-05-01

    This study assessed relationships between built environmental exposures measured within components of individual activity spaces (i.e., travel origins, destinations and paths in-between), and use of active transportation in a metropolitan setting. Individuals (n=37,165) were categorised as using active or sedentary transportation based on travel survey data. Generalised Estimating Equations analysis was used to test relationships with active transportation. Strength and significance of relationships between exposures and active transportation varied for different components of the activity space. Associations were strongest when including travel paths in expression of the built environment. Land use mix and greenness were negatively related to active transportation.

  19. Immunohistochemical localization of the Na-K-Cl co-transporter (NKCC1) in the gerbil inner ear.

    PubMed

    Crouch, J J; Sakaguchi, N; Lytle, C; Schulte, B A

    1997-06-01

    We mapped the cellular and subcellular distribution of the Na-K-Cl co-transporter (NKCC) in the adult gerbil inner ear by immunostaining with a monoclonal antibody (MAb T4) generated against human colon NKCC. Heavy immunolabeling was seen in the basolateral plasma membrane of marginal cells in the stria vascularis and dark cells in the vestibular system. Subpopulations of fibrocytes in the cochlear spiral ligament and limbus and underlying the vestibular neurosensory epithelium also stained with moderate to strong intensity, apparently along their entire plasmalemma. Because MAb T4 recognizes both the basolateral secretory (NKCC1) and the apical absorptive (NKCC2) isoforms of the co-transporter, we employed reverse transcription and the polymerase chain reaction (RT-PCR) to explore isoform diversity in inner ear tissues. Using NKCC1 and NKCC2 isoform-specific PCR primers based on mouse and human sequences, only transcripts for NKCC1 were detected in the gerbil inner ear. The presence of abundant NKCC1 in the basolateral plasmalemma of strial marginal and vestibular dark cells confirms conclusions drawn from pharmacological and physiological data. The co-expression of NKCC1 and Na,K-ATPase in highly specialized subpopulations of cochlear and vestibular fibrocytes provides further evidence for their role in recycling K+ leaked or effluxed through hair cells into perilymph back to endolymph, as postulated in current models of inner ear ion homeostasis.

  20. Active and passive transport of drugs in the human placenta.

    PubMed

    Włoch, Stanisław; Pałasz, Artur; Kamiński, Marcin

    2009-10-01

    The human placenta, characterized by the processes of passive transport and facilitated diffusion, contains numerous active transport proteins, usually located in the microvilli of the syncytiotrophoblast or in the endothelium of the capillaries of the villi. These proteins use either the energy from ATP hydrolysis or other mechanisms resulting, among others, from the formation of the maternofetal ion gradient, which facilitates the transfer of various endogenous substances or xenobiotics across the body membranes. The proteins either trigger the efflux of these substances from the fetal tissues via the placenta into the maternal bloodstream, or conversely they accumulate them in the fetal tissues. Both the placenta and the fetus are equipped with independent systems of enzymes of 1st and 2nd phase of substrate metabolism, such as CYP450, glucuronyltransferase or sulphatase. An active therapy with a wide range of drugs, often at high toxicity levels, either shortly before or during pregnancy, has naturally posed a question concerning the degree of impermeability of the placental barrier and how effectively it can be crossed, including any possible negative embryotoxic or teratogenic consequences. Such hazards seem to be quite real, as many drugs are substrates for ABC transporters. Also the placenta itself, including its structure, is subject to vast transformations during pregnancy which may be observed as the thinning of the barrier separating the maternal blood from the fetal one, from 20-30 microm in the first trimester of gestation down to 2-4 microm in the third trimester of gestation.

  1. Comparative Localization and Functional Activity of the Main Hepatobiliary Transporters in HepaRG Cells and Primary Human Hepatocytes

    PubMed Central

    Bachour-El Azzi, Pamela; Sharanek, Ahmad; Burban, Audrey; Li, Ruoya; Guével, Rémy Le; Abdel-Razzak, Ziad; Stieger, Bruno; Guguen-Guillouzo, Christiane; Guillouzo, André

    2015-01-01

    The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na+-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis. PMID:25690737

  2. Comparative Transport Activity of Intact Cells, Membrane Vesicles, and Mesosomes of Bacillus licheniformis

    PubMed Central

    MacLeod, Robert A.; Thurman, Paul; Rogers, H. J.

    1973-01-01

    Sodium ion was shown to stimulate strongly the transport of l-glutamic acid into cells of Bacillus licheniformis 6346 His−. Lithium ion had a slight capacity to replace Na+ in this capacity, but K+ was without effect. Three of five amino acids tested. l-glutamic acid, l-aspartic acid, and l-alanine, were concentrated against a gradient in the cells. Intracellular pools of these amino acids were extractable with 5% trichloroacetic acid. Pools of l-histidine and l-lysine could not be detected. No evidence of active transport of lysine into cells could be detected, and histidine was taken up in the absence of chloramphenicol but not in its presence. The uptake of glutamic acid by membrane vesicle preparations was strongly stimulated by reduced nicotinamide adenine dinucleotide (NADH) and to a lesser extent by succinate. The presence of phenazine methosulfate increased uptake in the presence of succinate. Either l- or d-lactate and adenosine triphosphate were without effect. None of these compounds stimulated the uptake of glutamic acid by mesosomes, although some mesosome preparations contained separable membrane which was very active. NADH strongly stimulated the uptake of aspartic acid and alanine by membrane vesicles but had only a slight effect on the uptake of histidine and lysine. No evidence of active transport of any of the amino acids into mesosomes could be detected either in the presence or absence of NADH. NADH stimulation of the uptake of glutamic acid by membrane vesicles was destroyed by exposure to light of 360 nm; this inactivation was reversible by vitamin K2(5) or K2(10). Sodium ion stimulated transport of glutamic acid by membrane vesicles. PMID:4347247

  3. N-Glycosylation is required for Na{sup +}-dependent vitamin C transporter functionality

    SciTech Connect

    Subramanian, Veedamali S. Marchant, Jonathan S.; Reidling, Jack C.; Said, Hamid M.

    2008-09-12

    The human sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2) mediate cellular uptake of ascorbic acid. Both these transporters contain potential sites for N-glycosylation in their extracellular domains (Asn-138, Asn-144 [hSVCT1]; Asn-188, Asn-196 [hSVCT2]), however the role of N-glycosylation in transporter function is unexplored. On the basis of the result that tunicamycin decreased {sup 14}C-ascorbic acid uptake in HepG2 cells, we systematically ablated all consensus N-glycosylation sites in hSVCT1 and hSVCT2 to resolve any effects on ascorbic acid uptake, transporter expression and targeting. We show that removal of individual N-glycosylation sites significantly impairs protein expression and consequently ascorbic acid uptake for hSVCT1 mutants (N138Q is retained intracellularly) and for hSVCT2 mutants (all of which reach the cell surface). N-Glycosylation is therefore essential for vitamin C transporter functionality.

  4. Serotonin transporter activity in platelets and canine aggression.

    PubMed

    Rosado, Belén; García-Belenguer, Sylvia; Palacio, Jorge; Chacón, Gema; Villegas, Ainara; Alcalde, Ana I

    2010-10-01

    Several studies have suggested an inhibitory action of the serotonergic system in the regulation of canine aggression, but the role of the serotonin (5-HT) transporter (5-HTT) has not been investigated. Platelet 5-HT uptake has been proposed as a peripheral marker of brain 5-HTT. The aim of the study was to investigate the relationship between platelet 5-HTT activity and canine aggression by measuring the rate of 5-HT uptake mediated by 5-HTT in platelets and serum concentrations of 5-HT in both aggressive (n=14) and non-aggressive dogs (n=17). Aggressive dogs showed significantly higher 5-HT uptake by 5-HTT in platelets and lower serum concentrations of 5-HT, compared with the control group. These results suggested an association between an alteration in the serotonergic system and canine aggression, possibly mediated by an increased 5-HT transport.

  5. Platelet Serotonin Transporter Function Predicts Default-Mode Network Activity

    PubMed Central

    Kasess, Christian H.; Meyer, Bernhard M.; Hofmaier, Tina; Diers, Kersten; Bartova, Lucie; Pail, Gerald; Huf, Wolfgang; Uzelac, Zeljko; Hartinger, Beate; Kalcher, Klaudius; Perkmann, Thomas; Haslacher, Helmuth; Meyer-Lindenberg, Andreas; Kasper, Siegfried; Freissmuth, Michael; Windischberger, Christian; Willeit, Matthäus; Lanzenberger, Rupert; Esterbauer, Harald; Brocke, Burkhard; Moser, Ewald; Sitte, Harald H.; Pezawas, Lukas

    2014-01-01

    Background The serotonin transporter (5-HTT) is abundantly expressed in humans by the serotonin transporter gene SLC6A4 and removes serotonin (5-HT) from extracellular space. A blood-brain relationship between platelet and synaptosomal 5-HT reuptake has been suggested, but it is unknown today, if platelet 5-HT uptake can predict neural activation of human brain networks that are known to be under serotonergic influence. Methods A functional magnetic resonance study was performed in 48 healthy subjects and maximal 5-HT uptake velocity (Vmax) was assessed in blood platelets. We used a mixed-effects multilevel analysis technique (MEMA) to test for linear relationships between whole-brain, blood-oxygen-level dependent (BOLD) activity and platelet Vmax. Results The present study demonstrates that increases in platelet Vmax significantly predict default-mode network (DMN) suppression in healthy subjects independent of genetic variation within SLC6A4. Furthermore, functional connectivity analyses indicate that platelet Vmax is related to global DMN activation and not intrinsic DMN connectivity. Conclusion This study provides evidence that platelet Vmax predicts global DMN activation changes in healthy subjects. Given previous reports on platelet-synaptosomal Vmax coupling, results further suggest an important role of neuronal 5-HT reuptake in DMN regulation. PMID:24667541

  6. Sodium deficiency effect on the transport properties of La0.8Na0.2-x□xMnO3 manganites

    NASA Astrophysics Data System (ADS)

    Elghoul, N.; Wali, M.; Kraiem, S.; Rahmouni, H.; Dhahri, E.; Khirouni, K.

    2015-12-01

    Effect of sodium deficiency on the transport properties of La0.8Na0.2-x□xMnO3 manganites is investigated using impedance spectroscopy technique. In the whole explored temperature range (77-700 K), conductivity measurements show the appearance of a metal-semiconductor transition for all investigated samples. Also, a saturation region is observed in σ (T) curves. It is found that conduction mechanism is governed by hopping process. The conductivity of the material decreases with increasing sodium deficiency. The transition temperature and the activation energy values inferred from grain boundary resistance and conductivity analysis are closed to each other. Such result confirms the contribution of grain boundary on the electrical conductivity. The variation of the Average Normalized Change (ANC) and its derivative with temperature gives important information about the available density of trapped charge states. The obtained results explain the observed saturation region in conductivity at high temperature region.

  7. Curcumin directly inhibits the transport activity of GLUT1.

    PubMed

    Gunnink, Leesha K; Alabi, Ola D; Kuiper, Benjamin D; Gunnink, Stephen M; Schuiteman, Sam J; Strohbehn, Lauren E; Hamilton, Kathryn E; Wrobel, Kathryn E; Louters, Larry L

    2016-06-01

    Curcumin, a major ingredient in turmeric, has a long history of medicinal applications in a wide array of maladies including treatment for diabetes and cancer. Seemingly counterintuitive to the documented hypoglycemic effects of curcumin, however, a recent report indicates that curcumin directly inhibits glucose uptake in adipocytes. The major glucose transporter in adipocytes is GLUT4. Therefore, this study investigates the effects of curcumin in cell lines where the major transporter is GLUT1. We report that curcumin has an immediate inhibitory effect on basal glucose uptake in L929 fibroblast cells with a maximum inhibition of 80% achieved at 75 μM curcumin. Curcumin also blocks activation of glucose uptake by azide, glucose deprivation, hydroxylamine, or phenylarsine oxide. Inhibition does not increase with exposure time and the inhibitory effects reverse within an hour. Inhibition does not appear to involve a reaction between curcumin and the thiol side chain of a cysteine residue since neither prior treatment of cells with iodoacetamide nor curcumin with cysteine alters curcumin's inhibitory effects. Curcumin is a mixed inhibitor reducing the Vmax of 2DG transport by about half with little effect on the Km. The inhibitory effects of curcumin are not additive to the effects of cytochalasin B and 75 μM curcumin actually reduces specific cytochalasin B binding by 80%. Taken together, the data suggest that curcumin binds directly to GLUT1 at a site that overlaps with the cytochalasin B binding site and thereby inhibits glucose transport. A direct inhibition of GLUT proteins in intestinal epithelial cells would likely reduce absorption of dietary glucose and contribute to a hypoglycemic effect of curcumin. Also, inhibition of GLUT1 activity might compromise cancer cells that overexpress GLUT1 and be another possible mechanism for the documented anticancer effects of curcumin.

  8. Chloride transport in functionally active phagosomes isolated from Human neutrophils

    PubMed Central

    Aiken, Martha L.; Painter, Richard G.; Zhou, Yun; Wang, Guoshun

    2012-01-01

    Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC–dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport

  9. Curcumin directly inhibits the transport activity of GLUT1

    PubMed Central

    Gunnink, Leesha K.; Alabi, Ola D.; Kuiper, Benjamin D.; Gunnink, Stephen M.; Schuiteman, Sam J.; Strohbehn, Lauren E.; Hamilton, Kathryn E.; Wrobel, Kathryn E.; Louters, Larry L.

    2016-01-01

    Curcumin, a major ingredient in turmeric, has a long history of medicinal applications in a wide array of maladies including treatment for diabetes and cancer. Seemingly counterintuitive to the documented hypoglycemic effects of curcumin, however, a recent report indicates that curcumin directly inhibits glucose uptake in adipocytes. The major glucose transporter in adipocytes is GLUT4. Therefore, this study investigates the effects of curcumin in cell lines where the major transporter is GLUT1. We report that curcumin has an immediate inhibitory effect on basal glucose uptake in L929 fibroblast cells with a maximum inhibition of 80% achieved at 75 μM curcumin. Curcumin also blocks activation of glucose uptake by azide, glucose deprivation, hydroxylamine, or phenylarsine oxide. Inhibition does not increase with exposure time and the inhibitory effects reverse within an hour. Inhibition does not appear to involve a reaction between curcumin and the thiol side chain of a cysteine residue since neither prior treatment of cells with iodoacetamide nor curcumin with cysteine alters curcumin’s inhibitory effects. Curcumin is a mixed inhibitor reducing the Vmax of 2DG transport by about half with little effect on the Km. The inhibitory effects of curcumin are not additive to the effects of cytochalasin B and 75 μM curcumin actually reduces specific cytochalasin B binding by 80%. Taken together, the data suggest that curcumin binds directly to GLUT1 at a site that overlaps with the cytochalasin B binding site and thereby inhibits glucose transport. A direct inhibition of GLUT proteins in intestinal epithelial cells would likely reduce absorption of dietary glucose and contribute to a hypoglycemic effect of curcumin. Also, inhibition of GLUT1 activity might compromise cancer cells that overexpress GLUT1 and be another possible mechanism for the documented anticancer effects of curcumin. PMID:27039889

  10. Measurement and Modeling of Mean Activity Coefficients of NaCl in an Aqueous Mixed Electrolyte Solution Containing Glycine

    NASA Astrophysics Data System (ADS)

    Sheikholeslami, Paniz; Dehghani, M. R.; Safahieh, Tina

    2016-08-01

    An electrochemical cell with two ion-selective electrodes (Na+ glass) and (Cl- solid state) was used to measure the mean ionic activity coefficient of NaCl in an aqueous mixture containing NaCl, glycine, and NaNO3 at 308.15 K. The experiments were conducted at fixed molality of NaNO3 (0.1 m) and various molalities of glycine (0-1 m) and NaCl (up to 0.8 m). The experimental data were modeled using a modified version of the Pitzer equation. Finally the activity coefficient ratio of glycine was determined based on the Maxwell equation.

  11. Complex Response of Epithelial Cells to Inhibition of Na(+) Transport by Amiloride

    DTIC Science & Technology

    1988-01-01

    Physiol. 23): C297-C303, occurring within milliseconds (1, 12), it would be ex- 1988. ’When toad urinary bladder or frog skin epithelia are pected that I...amiloride indicated in response to inhibition of Na+ entry into epithelial that the magnitude of the secondary decline increased in frog cells. We suggest...electrophysiology; cytoskele- ton; short-circuit current Experiments were performed using isolated frog skin and toad urinary bladders. Frogs (Rana

  12. Evaluation of the physical activity biography: sport and transport.

    PubMed

    Rogen, Sandra; Hofmann, Peter; Bauernhofer, Thomas; Müller, Wolfram

    2014-05-01

    Beside the genetic disposition, physical activity (PA) is one of the major health factors and can play a large role in the prevention and therapy of many diseases (cardiovascular diseases, cancer, obesity-related diseases etc.). In contrast to the genetic disposition, PA can be deliberately influenced by lifestyle. Therefore, it is of high importance to assess PA patterns. In order to assess PA reliably and validly, a new questionnaire (Physical Activity Biography, PAB) was created. The PAB assesses recreational PA (sport and transport) and enables to distinguish between endurance intensity levels and considers strength and high speed activity patterns throughout life. This study aims to evaluate the PAB by means of item analysis, retest-reliability and validity (criteria were physical fitness assessed by the questionnaire FFB-mot and by exercise tests). 141 participants answered the PAB. For deriving retest-reliability, 81 participants completed the PAB after a retest-interval of one month again. 55 participated in exercise tests and answered the FFB-mot to determine construct validity. Retest-reliability (ICC) above 0.7 was found for most items. For the items assessing recent PA, the criteria of convergent and discriminant validity were given. Despite the complexity of the question under study, the results fulfilled the expectations concerning reliability and validity. The PAB enables to assess the amount of sport and locomotion a person has accomplished during different life time frames and, because of the protective effects of PA on various diseases, may become an important tool for risk assessment. Key pointsThe risk of chronic diseases depends largely on physical activity biography.A new questionnaire (PAB) assessing recent and lifetime physical activity was created.The PAB assesses physical activity during sports and transport.The results of the evaluation of the PAB fulfilled the expectations.The PAB enables to determine a person's amount of recreational

  13. Evaluation of the Physical Activity Biography: Sport and Transport

    PubMed Central

    Rogen, Sandra; Hofmann, Peter; Bauernhofer, Thomas; Müller, Wolfram

    2014-01-01

    Beside the genetic disposition, physical activity (PA) is one of the major health factors and can play a large role in the prevention and therapy of many diseases (cardiovascular diseases, cancer, obesity-related diseases etc.). In contrast to the genetic disposition, PA can be deliberately influenced by lifestyle. Therefore, it is of high importance to assess PA patterns. In order to assess PA reliably and validly, a new questionnaire (Physical Activity Biography, PAB) was created. The PAB assesses recreational PA (sport and transport) and enables to distinguish between endurance intensity levels and considers strength and high speed activity patterns throughout life. This study aims to evaluate the PAB by means of item analysis, retest-reliability and validity (criteria were physical fitness assessed by the questionnaire FFB-mot and by exercise tests). 141 participants answered the PAB. For deriving retest-reliability, 81 participants completed the PAB after a retest-interval of one month again. 55 participated in exercise tests and answered the FFB-mot to determine construct validity. Retest-reliability (ICC) above 0.7 was found for most items. For the items assessing recent PA, the criteria of convergent and discriminant validity were given. Despite the complexity of the question under study, the results fulfilled the expectations concerning reliability and validity. The PAB enables to assess the amount of sport and locomotion a person has accomplished during different life time frames and, because of the protective effects of PA on various diseases, may become an important tool for risk assessment. Key points The risk of chronic diseases depends largely on physical activity biography. A new questionnaire (PAB) assessing recent and lifetime physical activity was created. The PAB assesses physical activity during sports and transport. The results of the evaluation of the PAB fulfilled the expectations. The PAB enables to determine a person’s amount of

  14. Modeling and dynamics of the inward-facing state of a Na+/Cl- dependent neurotransmitter transporter homologue.

    PubMed

    Shaikh, Saher Afshan; Tajkhorshid, Emad

    2010-08-26

    The leucine transporter (LeuT) has recently commanded exceptional attention due mainly to two distinctions; it provides the only crystal structures available for a protein homologous to the pharmacologically relevant neurotransmitter: sodium symporters (NSS), and, it exhibits a hallmark 5-TM inverted repeat ("LeuT-fold"), a fold recently discovered to also exist in several secondary transporter families, underscoring its general role in transporter function. Constructing the transport cycle of "LeuT-fold" transporters requires detailed structural and dynamic descriptions of the outward-facing (OF) and inward-facing (IF) states, as well as the intermediate states. To this end, we have modeled the structurally unknown IF state of LeuT, based on the known crystal structures of the OF state of LeuT and the IF state of vSGLT, a "LeuT-fold" transporter. The detailed methodology developed for the study combines structure-based alignment, threading, targeted MD and equilibrium MD, and can be applied to other proteins. The resulting IF-state models maintain the secondary structural features of LeuT. Water penetration and solvent accessibility calculations show that TM1, TM3, TM6 and TM8 line the substrate binding/unbinding pathway with TM10 and its pseudosymmetric partner, TM5, participating in the extracellular and intracellular halves of the lumen, respectively. We report conformational hotspots where notable changes in interactions occur between the IF and OF states. We observe Na2 exiting the LeuT-substrate- complex in the IF state, mainly due to TM1 bending. Inducing a transition in only one of the two pseudosymmetric domains, while allowing the second to respond dynamically, is found to be sufficient to induce the formation of the IF state. We also propose that TM2 and TM7 may be facilitators of TM1 and TM6 motion. Thus, this study not only presents a novel modeling methodology applied to obtain the IF state of LeuT, but also describes structural elements involved in

  15. Ca2+ and Zn2+ are transported by the electrogenic 2Na+/1H+ antiporter in echinoderm gastrointestinal epithelium

    PubMed

    Zhuang; Duerr; Ahearn

    1995-01-01

    45Ca2+ uptake by purified brush-border membrane vesicles of starfish (Pycnopodia helianthoides) pyloric ceca was stimulated by an outwardly directed H+ gradient and this stimulation was enhanced by the simultaneous presence of an induced membrane potential (inside negative; K+/valinomycin). External amiloride (competitive inhibitor; Ki=660 µmol l-1) and a monoclonal antibody raised against proteins associated with the lobster (Homarus americanus) electrogenic 2Na+/1H+ antiporter both inhibited approximately half of the proton-gradient-stimulated 45Ca2+ uptake. These results suggested that Ca2+ might be transported by the electrogenic antiporter and that the crustacean antibody was inhibitory to the exchange function in echinoderms, as was recently shown in crustacean epithelial brush-border membrane vesicles. Carrier-mediated 45Ca2+ influx by amiloride-sensitive and amiloride-insensitive systems displayed the following kinetic constants: (amiloride-sensitive) Kt=66±2 µmol l-1; Jmax=0.173±0.002 pmol µg-1 protein 8 s-1; (amiloride-insensitive) Kt=18±0.3 µmol l-1; Jmax=0.100±0.001 pmol µg-1 protein 8 s-1. Zn2+ was a mixed inhibitor of 45Ca2+ influx by carrier-mediated transport, displaying a Ki of 920 µmol l-1. Mn2+, Cu2+, Fe2+ and Mg2+ also inhibited 45Ca2+ uptake, but the mechanism(s) of inhibition by these other cations was not disclosed. An equilibrium shift experiment showed that both Na+ and Zn2+ were able to exchange with equilibrated 45Ca2+ in these vesicles, suggesting that both monovalent and divalent cations were able to enter pyloric cecal cells through a common carrier-mediated transport system. In addition, the echinoderm electrogenic system appeared to exhibit a molecular component recognized by the crustacean antibody that may imply a similar epitope in the two animals.

  16. Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation

    PubMed Central

    Gosselin, Romain-Daniel; Meylan, Patrick; Decosterd, Isabelle

    2013-01-01

    Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release. PMID:24368897

  17. Saharan Dust, Transport Processes, and Possible Impacts on Hurricane Activities

    NASA Technical Reports Server (NTRS)

    Lau, William K. M.; Kim, K. M.

    2010-01-01

    In this paper, we present observational evidence of significant relationships between Saharan dust outbreak, and African Easterly wave activities and hurricane activities. We found two dominant paths of transport of Saharan dust: a northern path, centered at 25degN associated with eastward propagating 6-19 days waves over northern Africa, and a southern path centered at 15degN, associated with the AEW, and the Atlantic ITCZ. Seasons with stronger dust outbreak from the southern path are associated with a drier atmosphere over the Maximum Development Region (MDR) and reduction in tropical cyclone and hurricane activities in the MDR. Seasons with stronger outbreak from the northern path are associated with a cooler N. Atlantic, and suppressed hurricane in the western Atlantic basin.

  18. Conserved Aspartic Acid Residues Lining the Extracellular Loop I of Sodium-coupled Bile Acid Transporter ASBT Interact with Na+ and 7α-OH Moieties on the Ligand Cholestane Skeleton*

    PubMed Central

    Hussainzada, Naissan; Da Silva, Tatiana Claro; Zhang, Eric Y.; Swaan, Peter W.

    2008-01-01

    Functional contributions of residues Val-99—Ser-126 lining extracellular loop (EL) 1 of the apical sodium-dependent bile acid transporter were determined via cysteine-scanning mutagenesis, thiol modification, and in silico interpretation. Despite membrane expression for all but three constructs (S112C, Y117C, S126C), most EL1 mutants (64%) were inactivated by cysteine mutation, suggesting a functional role during sodium/bile acid co-transport. A negative charge at conserved residues Asp-120 and Asp-122 is required for transport function, whereas neutralization of charge at Asp-124 yields a functionally active transporter. D124A exerts low affinity for common bile acids except deoxycholic acid, which uniquely lacks a 7α-hydroxyl (OH) group. Overall, we conclude that (i) Asp-122 functions as a Na+ sensor, binding one of two co-transported Na+ ions, (ii) Asp-124 interacts with 7α-OH groups of bile acids, and (iii) apolar EL1 residues map to hydrophobic ligand pharmacophore features. Based on these data, we propose a comprehensive mechanistic model involving dynamic salt bridge pairs and hydrogen bonding involving multiple residues to describe sodium-dependent bile acid transporter-mediated bile acid and cation translocation. PMID:18508772

  19. Molecular dynamics simulations of Na+/Cl(-)-dependent neurotransmitter transporters in a membrane-aqueous system.

    PubMed

    Jørgensen, Anne Marie; Tagmose, Lena; Jørgensen, Anne Marie M; Bøgesø, Klaus P; Peters, Günther H

    2007-06-01

    We have performed molecular dynamics simulations of a homology model of the human serotonin transporter (hSERT) in a membrane environment and in complex with either the natural substrate 5-HT or the selective serotonin reuptake inhibitor escitalopram. We have also included a transporter homologue, the Aquifex aeolicus leucine transporter (LeuT), in our study to evaluate the applicability of a simple and computationally attractive membrane system. Fluctuations in LeuT extracted from simulations are in good agreement with crystallographic B factors. Furthermore, key interactions identified in the X-ray structure of LeuT are maintained throughout the simulations indicating that our simple membrane system is suitable for studying the transmembrane protein hSERT in complex with 5-HT or escitalopram. For these transporter complexes, only relatively small fluctuations are observed in the ligand-binding cleft. Specific interactions responsible for ligand recognition, are identified in the hSERT-5HT and hSERT-escitalopram complexes. Our findings are in good agreement with predictions from mutagenesis studies.

  20. Active urea transport by the skin of Bufo viridis: Amiloride- and phloretin-sensitive transport sites

    SciTech Connect

    Rapoport, J.; Abuful, A.; Chaimovitz, C.; Noeh, Z.; Hays, R.M. Albert Einstein College of Medicine, New York, NY )

    1988-09-01

    Urea is actively transported inwardly (J{sub i}) across the skin of the green toad Bufo viridis. J{sub i} is markedly enhanced in toads adapted to hypertonic saline. The authors studied urea transport across the skin of Bufo viridis under a variety of experimental conditions, including treatment with amiloride and phloretin, agents that inhibit urea permeability in the bladder of Bufo marinus. Amiloride (10{sup {minus}4} M) significantly inhibited J{sub i} in both adapted and unadapted animals and was unaffected by removal of sodium from the external medium. Phloretin (10{sup {minus}4} M) significantly inhibited J{sub i} in adapted animals by 23-46%; there was also a reduction in J{sub i} in unadapted toads at 10{sup {minus}4} and 5 {times} 10{sup {minus}4} M phloretin. A dose-response study revealed that the concentration of phloretin causing half-maximal inhibition (K{sub {1/2}}) was 5 {times} 10{sup {minus}4} M for adapted animals. J{sub i} was unaffected by the substitution of sucrose for Ringer solution or by ouabain. They conclude (1) the process of adaptation appears to involve an increase in the number of amiloride- and phloretin-inhibitable urea transport sites in the skin, with a possible increase in the affinity of the sites for phloretin; (2) the adapted skin resembles the Bufo marinus urinary bladder with respect to amiloride and phloretin-inhibitable sites; (3) they confirm earlier observations that J{sub i} is independent of sodium transport.

  1. Dopamine Transporter Activity Is Modulated by α-Synuclein.

    PubMed

    Butler, Brittany; Saha, Kaustuv; Rana, Tanu; Becker, Jonas P; Sambo, Danielle; Davari, Paran; Goodwin, J Shawn; Khoshbouei, Habibeh

    2015-12-04

    The duration and strength of the dopaminergic signal are regulated by the dopamine transporter (DAT). Drug addiction and neurodegenerative and neuropsychiatric diseases have all been associated with altered DAT activity. The membrane localization and the activity of DAT are regulated by a number of intracellular proteins. α-Synuclein, a protein partner of DAT, is implicated in neurodegenerative disease and drug addiction. Little is known about the regulatory mechanisms of the interaction between DAT and α-synuclein, the cellular location of this interaction, and the functional consequences of this interaction on the basal, amphetamine-induced DAT-mediated dopamine efflux, and membrane microdomain distribution of the transporter. Here, we found that the majority of DAT·α-synuclein protein complexes are found at the plasma membrane of dopaminergic neurons or mammalian cells and that the amphetamine-mediated increase in DAT activity enhances the association of these proteins at the plasma membrane. Further examination of the interaction of DAT and α-synuclein revealed a transient interaction between these two proteins at the plasma membrane. Additionally, we found DAT-induced membrane depolarization enhances plasma membrane localization of α-synuclein, which in turn increases dopamine efflux and enhances DAT localization in cholesterol-rich membrane microdomains.

  2. Discovery of a Biological Mechanism of Active Transport through the Tympanic Membrane to the Middle Ear

    PubMed Central

    Kurabi, Arwa; Pak, Kwang K.; Bernhardt, Marlen; Baird, Andrew; Ryan, Allen F.

    2016-01-01

    Otitis media (OM) is a common pediatric disease for which systemic antibiotics are often prescribed. While local treatment would avoid the systemic treatment side-effects, the tympanic membrane (TM) represents an impenetrable barrier unless surgically breached. We hypothesized that the TM might harbor innate biological mechanisms that could mediate trans-TM transport. We used two M13-bacteriophage display biopanning strategies to search for mediators of trans-TM transport. First, aliquots of linear phage library displaying 1010th 12mer peptides were applied on the TM of rats with active bacterial OM. The middle ear (ME) contents were then harvested, amplified and the preparation re-applied for additional rounds. Second, the same naïve library was sequentially screened for phage exhibiting TM binding, internalization and then transit. Results revealed a novel set of peptides that transit across the TM to the ME in a time and temperature dependent manner. The peptides with highest transport capacities shared sequence similarities. Historically, the TM was viewed as an impermeable barrier. However, our studies reveal that it is possible to translocate peptide-linked small particles across the TM. This is the first comprehensive biopanning for the isolation of TM transiting peptidic ligands. The identified mechanism offers a new drug delivery platform into the ME. PMID:26946957

  3. Methylphenidate treatment increases Na(+), K (+)-ATPase activity in the cerebrum of young and adult rats.

    PubMed

    Scherer, Emilene B S; Matté, Cristiane; Ferreira, Andréa G K; Gomes, Karin M; Comim, Clarissa M; Mattos, Cristiane; Quevedo, João; Streck, Emilio L; Wyse, Angela T S

    2009-12-01

    Methylphenidate is a central nervous system stimulant used for the treatment of attention-deficit hyperactivity disorder. Na(+), K(+)-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that methylphenidate effects on central nervous system metabolism are poorly known and that Na(+), K(+)-ATPase is essential to normal brain function, the purpose of this study was to evaluate the effect of this drug on Na(+), K(+)-ATPase activity in the cerebrum of young and adult rats. For acute administration, a single injection of methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline was given to rats on postnatal day 25 or postnatal day 60, in the young and adult groups, respectively. For chronic administration, methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline injections were given to young rats starting at postnatal day 25 once daily for 28 days. In adult rats, the same regimen was performed starting at postnatal day 60. Our results showed that acute methylphenidate administration increased Na(+), K(+)-ATPase activity in hippocampus, prefrontal cortex, and striatum of young and adult rats. In young rats, chronic administration of methylphenidate also enhanced Na(+), K(+)-ATPase activity in hippocampus and prefrontal cortex, but not in striatum. When tested in adult rats, Na(+), K(+)-ATPase activity was increased in all cerebral structures studied. The present findings suggest that increased Na(+), K(+)-ATPase activity may be associated with neuronal excitability caused by methylphenidate.

  4. Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+.

    PubMed

    DiFranco, Marino; Hakimjavadi, Hesamedin; Lingrel, Jerry B; Heiny, Judith A

    2015-10-01

    The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K+ concentration in the T-tubules, through its K+ substrate affinity. Apparent K+ affinity was determined from measurements of the K1/2 for K+ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K+ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (-90 to -30 mV) and increased with depolarization in the subthreshold range, -90 to -50 mV. The Q10 was 2.1 over the range of 22-37°C. The K1/2,K of Ip was 4.3±0.3 mM at -90 mV and was relatively voltage independent. This K+ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K+ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K+ increases in proportion to the frequency and duration of action potential firing. This K1/2,K predicts a low fractional occupancy of K+ substrate sites at the resting extracellular K+ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K+ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles.

  5. CFD Model of Water Droplet Transport for ISS Hygiene Activity

    NASA Technical Reports Server (NTRS)

    Son, Chang H.

    2011-01-01

    The goal of the study is to assess the impacts of free water propagation in the Waste and Hygiene Compartment (WHC). Free water can be generated inside the WHC in small quantities due to crew hygiene activity. To mitigate potential impact of free water in Node 3 cabin the WHC doorway is enclosed by a waterproof bump-out, Kabin, with openings at the top and bottom. At the overhead side of the rack, there is a screen that prevents large drops of water from exiting. However, as the avionics fan in the WHC causes airflow toward the deck side of the rack, small quantities of free water may exit at the bottom of the Kabin. A Computational Fluid Dynamics (CFD) analysis of Node 3 cabin airflow made possible to identify the paths of water transport. The Node 3 airflow was computed for several ventilation scenarios. To simulate the droplet transport the Lagrangian discrete phase approach was used. Various initial droplet distributions were considered in the study. The droplet diameter was varied in the range of 2-20 mm. The results of the computations showed that most of the drops fall to the rack surface not far from the WHC curtain. The probability of the droplet transport to the adjacent rack surface with electronic equipment was predicted.

  6. Dopamine Stimulation of Active Na and Cl Absorption in Rabbit Ileum

    PubMed Central

    Donowitz, Mark; Cusolito, Sheila; Battisti, Laurie; Fogel, Ronald; Sharp, Geoffrey W. G.

    1982-01-01

    The effects of dopamine on active intestinal ion transport have been evaluated. An epithelial sheet preparation of rabbit ileum was used in vitro with the Ussing chamber-voltage clamp technique. Dopamine, in the presence of 1 mM ascorbic acid, added to the serosal bathing solution caused a dose-dependent decrease in short-circuit current, with a half-maximal effect at 1.2 μM and maximal effect of −50 μA/cm2 at 50 μM; dopamine decreased the potential difference, and increased the conductance and net Na and net Cl absorption. There was no effect on the residual ion flux. Dopamine did not alter the change in short-circuit current caused by mucosal glucose (10 mM) or serosal theophylline (10 mM). Mucosal dopamine had no effect. The effect of dopamine on short-circuit current was inhibited by the dopamine antagonists haloperidol and domperidone and the α2-adrenergic antagonist yohimbine; there was no effect of the α1-antagonist prazosin and the β-antagonist propranolol. In addition, the α2-adrenergic agonist clonidine, but not the α1-agonist methoxamine caused a dose-dependent decrease in short-circuit current. The ileal effects of dopamine did not occur via conversion into norepinephrine or release of norepinephrine from the peripheral nerves since “peripheral sympathectomy” with 6-hydroxydopamine did not alter the dopamine-induced change in ileal short-circuit current. The dopamine effects were not associated with a change in basal ileal cyclic AMP content but were associated with a decrease in total ileal calcium content as measured by atomic absorption spectrometry and as estimated by 45Ca++ uptake. The decrease in calcium content could be attributed to a dopamine-induced decrease in 45Ca++ influx from the serosal surface. Because of the presence of dopamine in ileal mucosa and these effects on ileal electrolyte transport, it is possible that dopamine may be involved in the physiologic regulation of active intestinal electrolyte absorption. PMID

  7. Activation of Na(+)-K(+)-ATPase with DRm217 attenuates oxidative stress-induced myocardial cell injury via closing Na(+)-K(+)-ATPase/Src/Ros amplifier.

    PubMed

    Yan, Xiaofei; Xun, Meng; Dou, Xiaojuan; Wu, Litao; Zhang, Fujun; Zheng, Jin

    2017-04-01

    Reduced Na(+)-K(+)-ATPase activity has close relationship with cardiomyocyte death. Reactive oxygen species (ROS) also plays an important role in cardiac cell damage. It has been proved that Na(+)-K(+)-ATPase and ROS form a feed-forward amplifier. The aim of this study was to explore whether DRm217, a proved Na(+)/K(+)-ATPase's DR-region specific monoclonal antibody and direct activator, could disrupt Na(+)-K(+)-ATPase/ROS amplifier and protect cardiac cells from ROS-induced injury. We found that DRm217 protected myocardial cells against hydrogen peroxide (H2O2)-induced cardiac cell injury and mitochondrial dysfunction. DRm217 also alleviated the effect of H2O2 on inhibition of Na(+)-K(+)-ATPase activity, Na(+)-K(+)-ATPase cell surface expression, and Src phosphorylation. H2O2-treatment increased intracellular ROS, mitochondrial ROS and induced intracellular Ca(2+), mitochondrial Ca(2+) overload. DRm217 closed Na(+)-K(+)-ATPase/ROS amplifier, alleviated Ca(2+) accumulation and finally inhibited ROS and mitochondrial ROS generation. These novel results may help us to understand the important role of the Na(+)-K(+)-ATPase in oxidative stress and oxidative stress-related disease.

  8. Changes in Na+, K+-ATPase activity and alpha 3 subunit expression in CNS after administration of Na+, K+-ATPase inhibitors.

    PubMed

    Bersier, María Geraldina; Peña, Clara; Arnaiz, Georgina Rodríguez de Lores

    2011-02-01

    The expression of Na(+), K(+)-ATPase α3 subunit and synaptosomal membrane Na(+), K(+)-ATPase activity were analyzed after administration of ouabain and endobain E, respectively commercial and endogenous Na(+), K(+)-ATPase inhibitors. Wistar rats received intracerebroventricularly ouabain or endobain E dissolved in saline solution or Tris-HCl, respectively or the vehicles (controls). Two days later, animals were decapitated, cerebral cortex and hippocampus removed and crude and synaptosomal membrane fractions were isolated. Western blot analysis showed that Na(+), K(+)-ATPase α3 subunit expression increased roughly 40% after administration of 10 or 100 nmoles ouabain in cerebral cortex but remained unaltered in hippocampus. After administration of 10 μl endobain E (1 μl = 28 mg tissue) Na(+), K(+)-ATPase α3 subunit enhanced 130% in cerebral cortex and 103% in hippocampus. The activity of Na(+), K(+)-ATPase in cortical synaptosomal membranes diminished or increased after administration of ouabain or endobain E, respectively. It is concluded that Na(+), K(+)-ATPase inhibitors modify differentially the expression of Na(+), K(+)-ATPase α3 subunit and enzyme activity, most likely involving compensatory mechanisms.

  9. Estimation of Na-24 activity concentration in BAEC TRIGA Research Reactor

    NASA Astrophysics Data System (ADS)

    Ajijul Hoq, M.; Malek Soner, M. A.; Salam, M. A.; Khanom, Salma; Fahad, S. M.

    The Bangladesh Atomic Energy Commission (BAEC) TRIGA Research Reactor is a unique nuclear installation of the country generally implemented for a wide variety of research applications and serves as an excellent source of neutron. During reactor operation it is necessary to measure and control the activity concentration of the pool water for fuel element failure detection and for the determination of contamination. The present study deals with the estimation of activity concentration for Na-24 present in water coolant produced as a result of 23Na (n, γ) 24Na reaction. Several governing equations have been employed to estimate the Na-24 activity concentrations theoretically at different reactor power levels including maximum reactor power of 2.4 MW. From the obtained result it is ensured that the estimated Na-24 activity of 8.83 × 10-3 μCi /cm3 is not significant enough for any radiological hazard. Thus for ensuring radiological safety issues of the research reactor the assessment performed under the present study has an implication.

  10. Active transport of vesicles in neurons is modulated by mechanical tension

    NASA Astrophysics Data System (ADS)

    Ahmed, Wylie W.; Saif, Taher A.

    2014-03-01

    Effective intracellular transport of proteins and organelles is critical in cells, and is especially important for ensuring proper neuron functionality. In neurons, most proteins are synthesized in the cell body and must be transported through thin structures over long distances where normal diffusion is insufficient. Neurons transport subcellular cargo along axons and neurites through a stochastic interplay of active and passive transport. Mechanical tension is critical in maintaining proper function in neurons, but its role in transport is not well understood. To this end, we investigate the active and passive transport of vesicles in Aplysia neurons while changing neurite tension via applied strain, and quantify the resulting dynamics. We found that tension in neurons modulates active transport of vesicles by increasing the probability of active motion, effective diffusivity, and induces a retrograde bias. We show that mechanical tension modulates active transport processes in neurons and that external forces can couple to internal (subcellular) forces and change the overall transport dynamics.

  11. Particle Transport Simulation for Spaceborne, NaI Gamma-Ray Spectrometers

    DTIC Science & Technology

    1988-11-01

    fluxes . Accession For N II GRA&I DTIC TAB Unannounced Justificatlo By Distribution / Copyright Availability Codes ControlZer HMSO London Dist Speoial 1988 3... fluxes . Introduction Radioactivity induced in scintillation detectors by cosmic rays, trapped protons and secondary neutrons produces an important source...which utilises multigroup scattering and absorption cross-section data. It is used here to transport neutrons over the energy range from 15 MeV down

  12. Impact of humic acid fouling on membrane performance and transport of pharmaceutically active compounds in forward osmosis.

    PubMed

    Xie, Ming; Nghiem, Long D; Price, William E; Elimelech, Menachem

    2013-09-01

    The impact of humic acid fouling on the membrane transport of two pharmaceutically active compounds (PhACs) - namely carbamazepine and sulfamethoxazole - in forward osmosis (FO) was investigated. Deposition of humic acid onto the membrane surface was promoted by the complexation with calcium ions in the feed solution and the increase in ionic strength at the membrane surface due to the reverse transport of NaCl draw solute. The increase in the humic acid deposition on the membrane surface led to a substantial decrease in the membrane salt (NaCl) permeability coefficient but did not result in a significant decrease in the membrane pure water permeability coefficient. As the deposition of humic acid increased, the permeation of carbamazepine and sulfamethoxazole decreased, which correlated well with the decrease in the membrane salt (NaCl) permeability coefficient. It is hypothesized that the hydrated humic acid fouling layer hindered solute diffusion through the membrane pore and enhanced solute rejection by steric hindrance, but not the permeation of water molecules. The membrane water and salt (NaCl) permeability coefficients were fully restored by physical cleaning of the membrane, suggesting that humic acid did not penetrate into the membrane pores.

  13. Simulation of Cl− Secretion in Epithelial Tissues: New Methodology Estimating Activity of Electro-Neutral Cl− Transporter

    PubMed Central

    Sasamoto, Kouhei; Niisato, Naomi; Taruno, Akiyuki; Marunaka, Yoshinori

    2015-01-01

    Transcellular Cl− secretion is, in general, mediated by two steps; (1) the entry step of Cl− into the cytosolic space from the basolateral space across the basolateral membrane by Cl− transporters, such as Na+-K+-2Cl− cotransporter (NKCC1, an isoform of NKCC), and (2) the releasing step of Cl− from the cytosolic space into the luminal (air) space across the apical membrane via Cl− channels, such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Transcellular Cl− secretion has been characterized by using various experimental techniques. For example, measurements of short-circuit currents in the Ussing chamber and patch clamp techniques provide us information on transepithelial ion movements via transcellular pathway, transepithelial conductance, activity (open probability) of single channel, and whole cell currents. Although many investigators have tried to clarify roles of Cl− channels and transporters located at the apical and basolateral membranes in transcellular Cl− secretion, it is still unclear how Cl− channels/transporters contribute to transcellular Cl− secretion and are regulated by various stimuli such as Ca2+ and cAMP. In the present study, we simulate transcellular Cl− secretion using mathematical models combined with electrophysiological measurements, providing information on contribution of Cl− channels/transporters to transcellular Cl− secretion, activity of electro-neutral ion transporters and how Cl− channels/transporters are regulated. PMID:26779025

  14. Ionic regulation of Na absorption in proximal colon: cation inhibition of electroneutral Na absorption

    SciTech Connect

    Sellin, J.H.; De Soignie, R.

    1987-01-01

    Active Na absorption (J/sub net//sup NA/) in rabbit proximal colon in vitro is paradoxically stimulated as (Na) in the bathing media is lowered with constant osmolarity. J/sub m..-->..s//sup Na/ increases almost linearly from 0 to 50 mM (Na)/sub 0/ but then plateaus and actually decreases from 50 to 140 mM (Na)/sub 0/, consistent with inhibition of an active transport process. Both lithium and Na are equally effective inhibitors of J/sub net//sup Na/, whereas choline and mannitol do not block the high rate of J/sub net//sup Na/ observed in decreased (Na)/sub 0/. Either gluconate or proprionate replacement of Cl inhibits J/sub net//sup Na/. J/sub net//sup Na/ at lowered (Na)/sub 0/ is electrically silent and is accompanied by increased Cl absorption; it is inhibited by 10/sup -3/ M amiloride and 10/sup -3/ theophylline but not by 10/sup -4/ M bumetanide. Epinephrine is equally effective at stimulating Na absorption at 50 and 140 mM (Na). Na gradient experiments are consistent with a predominantly serosal effect of the decreased (Na)/sub 0/. These results suggest that 1) Na absorption in rabbit proximal colon in vitro is stimulated by decreased (Na); 2) the effect is cation specific, both Na and Li blocking the stimulatory effect; 3) the transport is mediated by Na-H exchange and is Cl dependent but 4) is under different regulatory mechanisms than the epinephrine-sensitive Na-Cl cotransport previously described in proximal colon. Under the appropriate conditions, proximal colon absorbs Na extremely efficiently. Na-H exchange in this epithelium is cation inhibitable, either directly or by a secondary regulatory process.

  15. Adult Active Transport in the Netherlands: An Analysis of Its Contribution to Physical Activity Requirements

    PubMed Central

    Fishman, Elliot; Böcker, Lars; Helbich, Marco

    2015-01-01

    Introduction Modern, urban lifestyles have engineered physical activity out of everyday life and this presents a major threat to human health. The Netherlands is a world leader in active travel, particularly cycling, but little research has sought to quantify the cumulative amount of physical activity through everyday walking and cycling. Methods Using data collected as part of the Dutch National Travel Survey (2010 – 2012), this paper determines the degree to which Dutch walking and cycling contributes to meeting minimum level of physical activity of 150 minutes of moderate intensity aerobic activity throughout the week. The sample includes 74,465 individuals who recorded at least some travel on the day surveyed. As physical activity benefits are cumulative, all walking and cycling trips are analysed, including those to and from public transport. These trips are then converted into an established measure of physical activity intensity, known as metabolic equivalents of tasks. Multivariate Tobit regression models were performed on a range of socio-demographic, transport resources, urban form and meteorological characteristics. Results The results reveal that Dutch men and women participate in 24 and 28 minutes of daily physical activity through walking and cycling, which is 41% and 55% more than the minimum recommended level. It should be noted however that some 57% of the entire sample failed to record any walking or cycling, and an investigation of this particular group serves as an important topic of future research. Active transport was positively related with age, income, bicycle ownership, urban density and air temperature. Car ownership had a strong negative relationship with physically active travel. Conclusion The results of this analysis demonstrate the significance of active transport to counter the emerging issue of sedentary lifestyle disease. The Dutch experience provides other countries with a highly relevant case study in the creation of

  16. Active migration and passive transport of malaria parasites.

    PubMed

    Douglas, Ross G; Amino, Rogerio; Sinnis, Photini; Frischknecht, Freddy

    2015-08-01

    Malaria parasites undergo a complex life cycle between their hosts and vectors. During this cycle the parasites invade different types of cells, migrate across barriers, and transfer from one host to another. Recent literature hints at a misunderstanding of the difference between active, parasite-driven migration and passive, circulation-driven movement of the parasite or parasite-infected cells in the various bodily fluids of mosquito and mammalian hosts. Because both active migration and passive transport could be targeted in different ways to interfere with the parasite, a distinction between the two ways the parasite uses to get from one location to another is essential. We discuss the two types of motion needed for parasite dissemination and elaborate on how they could be targeted by future vaccines or drugs.

  17. Directed transport of active particles over asymmetric energy barriers.

    PubMed

    Koumakis, N; Maggi, C; Di Leonardo, R

    2014-08-21

    We theoretically and numerically investigate the transport of active colloids to target regions, delimited by asymmetric energy barriers. We show that it is possible to introduce a generalized effective temperature that is related to the local variance of particle velocities. The stationary probability distributions can be derived from a simple diffusion equation in the presence of an inhomogeneous effective temperature resulting from the action of external force fields. In particular, transition rates over asymmetric energy barriers can be unbalanced by having different effective temperatures over the two slopes of the barrier. By varying the type of active noise, we find that equal values of diffusivity and persistence time may produce strongly varied effective temperatures and thus stationary distributions.

  18. Coupled ATPase-adenylate kinase activity in ABC transporters

    PubMed Central

    Kaur, Hundeep; Lakatos-Karoly, Andrea; Vogel, Ramona; Nöll, Anne; Tampé, Robert; Glaubitz, Clemens

    2016-01-01

    ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters. PMID:28004795

  19. Active Transport of Nanomaterials Using Motor Proteins -Final Report

    SciTech Connect

    Hess, Henry

    2005-09-01

    During the six months of funding we have focused first on the completion of the research begun at the University of Washington in the previous funding cycle. Specifically, we developed a method to polymerize oriented networks of microtubules on lithographically patterned surfaces (M.S. thesis Robert Doot). The properties of active transport have been studied detail, yielding insights into the dispersion mechanisms (Nitta et al.). The assembly of multifunctional structures with a microtubule core has been investigated (Ramachandran et al.). Isaac Luria (B.S. in physics, U. of Florida 2005) worked on the directed assembly of nanoscale, non-equilibrium structures as a summer intern. He is now a graduate student in my group at the University of Florida. T. Nitta and H. Hess: Dispersion in Active Transport by Kinesin-Powered Molecular Shuttles, Nano Letters, 5, 1337-1342 (2005) S. Ramachandran, K.-H. Ernst, G. D. Bachand, V. Vogel, H. Hess*: Selective Loading of Kinesin-Powered Molecular Shuttles with Protein Cargo and its Application to Biosensing, submitted to Small (2005)

  20. Socioeconomic and regional differences in active transportation in Brazil

    PubMed Central

    de Sá, Thiago Hérick; Pereira, Rafael Henrique Moraes; Duran, Ana Clara; Monteiro, Carlos Augusto

    2016-01-01

    ABSTRACT OBJECTIVE To present national estimates regarding walking or cycling for commuting in Brazil and in 10 metropolitan regions. METHODS By using data from the Health section of 2008’s Pesquisa Nacional por Amostra de Domicílio (Brazil’s National Household Sample Survey), we estimated how often employed people walk or cycle to work, disaggregating our results by sex, age range, education level, household monthly income per capita, urban or rural address, metropolitan regions, and macro-regions in Brazil. Furthermore, we estimated the distribution of this same frequency according to quintiles of household monthly income per capita in each metropolitan region of the country. RESULTS A third of the employed men and women walk or cycle from home to work in Brazil. For both sexes, this share decreases as income and education levels rise, and it is higher among younger individuals, especially among those living in rural areas and in the Northeast region of the country. Depending on the metropolitan region, the practice of active transportation is two to five times more frequent among low-income individuals than among high-income individuals. CONCLUSIONS Walking or cycling to work in Brazil is most frequent among low-income individuals and the ones living in less economically developed areas. Active transportation evaluation in Brazil provides important information for public health and urban mobility policy-making PMID:27355465

  1. Examining Changes in Radioxenon Isotope Activity Ratios during Subsurface Transport

    NASA Astrophysics Data System (ADS)

    Annewandter, R.

    2013-12-01

    The Non-Proliferation Experiment (NPE) has demonstrated and modelled the usefulness of barometric pumping induced soil gas sampling during On-Site inspections. Gas transport has been widely studied with different numerical codes. However, gas transport of all radioxenons in the post-detonation regime and their possible fractionation is still neglected in the open literature. Atmospheric concentrations of the radioxenons Xe-135, Xe-133m, Xe-133 and Xe-131m can be used to discriminate between civilian releases (nuclear power plants or medical isotope facilities), and nuclear explosion sources. It is based on the isotopic activity ratio method. Yet it is not clear whether subsurface migration of the radioxenons, with eventual release into the atmosphere, can affect the activity ratios due to fractionation. Fractionation can be caused by different diffusivities due to mass differences between the radioxenons. A previous study showed surface arrival time of a chemically inert gaseous tracer is affected by its diffusivity. They observed detectable amount for SF6 50 days after detonation and 375 days for He-3. They predict 50 and 80 days for Xe-133 and Ar-37 respectively. Cyclical changes in atmospheric pressure can drive subsurface gas transport. This barometric pumping phenomenon causes an oscillatoric flow in upward trending fractures which, combined with diffusion into the porous matrix, leads to a net transport of gaseous components - a ratcheting effect. We use a general purpose reservoir simulator (Complex System Modelling Platform, CSMP++) which has been applied in a range of fields such as deep geothermal systems, three-phase black oil simulations , fracture propagation in fractured, porous media, Navier-Stokes pore-scale modelling among others. It is specifically designed to account for structurally complex geologic situation of fractured, porous media. Parabolic differential equations are solved by a continuous Galerkin finite-element method, hyperbolic

  2. Mutation of the Na-K-Cl co-transporter gene Slc12a2 results in deafness in mice.

    PubMed

    Dixon, M J; Gazzard, J; Chaudhry, S S; Sampson, N; Schulte, B A; Steel, K P

    1999-08-01

    Hearing impairment is a common human condition, but we know little about the molecular basis of cochlear function. Shaker-with-syndactylism (sy) is a classic deaf mouse mutant and we show here that a second allele, sy(ns), is associated with abnormal production of endolymph, the fluid bathing sensory hair cells. Using a positional candidate approach, we demonstrate that mutations in the gene encoding the basolateral Na-K-Cl co-transporter Slc12a2 (Nkcc1, mBSC2) cause the deafness observed in sy and sy(ns) mice. This finding provides the molecular basis of another link in the chain of K+recycling in the cochlea, a process essential for normal cochlear function.

  3. Angiotensin II stimulates renal proximal tubule Na(+)-ATPase activity through the activation of protein kinase C.

    PubMed

    Rangel, L B A; Caruso-Neves, C; Lara, L S; Lopes, A G

    2002-08-31

    Recently, our group described an AT(1)-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na(+)-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na(+)-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na(+)-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na(+)-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.

  4. Transient receptor potential vanilloid type 4 channels mediate Na-K-Cl-co-transporter-induced brain edema after traumatic brain injury.

    PubMed

    Lu, Kwok-Tung; Huang, Tai-Chun; Tsai, Ya-Hsin; Yang, Yi-Ling

    2017-03-01

    Na(+) -K(+) -2Cl(-) co-transporter (NKCC1) plays an important role in traumatic brain injury (TBI)-induced brain edema via the MAPK cascade. The transient receptor potential vanilloid type 4 (TRPV4) channel participates in neurogenic inflammation, pain transmission, and edema. In this study, we investigated the relationship between NKCC1 and TRPV4 and the related signaling pathways in TBI-induced brain edema and neuronal damage. TBI was induced by the calibrated weight-drop device. Adult male Wistar rats were randomly assigned into sham and experimental groups for time-course studies of TRPV4 expression after TBI. Hippocampal TRPV4, NKCC1, MAPK, and PI-3K cascades were analyzed by western blot, and brain edema was also evaluated among the different groups. Expression of hippocampal TRPV4 peaked at 8 h after TBI, and phosphorylation of the MAPK cascade and Akt was significantly elevated. Administration of either the TRPV4 antagonist, RN1734, or NKCC1 antagonist, bumetanide, significantly attenuated TBI-induced brain edema through decreasing the phosphorylation of MEK, ERK, and Akt proteins. Bumetanide injection inhibited TRPV4 expression, which suggests NKCC1 activation is critical to TRPV4 activation. Our results showed that hippocampal NKCC1 activation increased TRPV4 expression after TBI and then induced severe brain edema and neuronal damage through activation of the MAPK cascade and Akt-related signaling pathway.

  5. Electronic Properties, Screening, and Efficient Carrier Transport in NaSbS2

    NASA Astrophysics Data System (ADS)

    Sun, Jifeng; Singh, David J.

    2017-02-01

    NaSbS2 is a semiconductor that was recently shown to have remarkable efficacy as a solar absorber indicating efficient charge collection even in material containing defects. We report first-principles calculations of properties that show (1) an indirect gap only slightly smaller than the direct gap, which may impede the recombination of photoexcited carriers, (2) highly anisotropic electronic and optical properties reflecting a layered crystal structure, (3) a pushed-up valence-band maximum due to repulsion from the Sb 5 s states, and (4) cross-gap hybridization between the S p —derived valence bands and the Sb 5 p states. This latter feature leads to enhanced Born effective charges that can provide local screening and, therefore, defect tolerance. These features are discussed in relation to the performance of the compound as a semiconductor with efficient charge collection.

  6. Engineering Protein Allostery: 1.05 Å Resolution Structure and Enzymatic Properties of a Na[superscript +]-activated Trypsin

    SciTech Connect

    Page, Michael J.; Carrell, Christopher J.; Di Cera, Enrico

    2008-05-28

    Some trypsin-like proteases are endowed with Na{sup +}-dependent allosteric enhancement of catalytic activity, but this important mechanism has been difficult to engineer in other members of the family. Replacement of 19 amino acids in Streptomyces griseus trypsin targeting the active site and the Na{sup +}-binding site were found necessary to generate efficient Na{sup +} activation. Remarkably, this property was linked to the acquisition of a new substrate selectivity profile similar to that of factor Xa, a Na{sup -} activated protease involved in blood coagulation. The X-ray crystal structure of the mutant trypsin solved to 1.05 {angstrom} resolution defines the engineered Na{sup +} site and active site loops in unprecedented detail. The results demonstrate that trypsin can be engineered into an efficient allosteric protease, and that Na+ activation is interwoven with substrate selectivity in the trypsin scaffold.

  7. "JCE" Classroom Activity Connections: NaCl or CaCl[subscript 2], Smart Polymer Gel Tells More

    ERIC Educational Resources Information Center

    Chen, Yueh-Huey; Lin, Jia-Ying; Wang, Yu-Chen; Yaung, Jing-Fun

    2010-01-01

    This classroom activity connection demonstrates the differences between the effects of NaCl (a salt of monovalent metal ions) and CaCl[subscript 2] (a salt of polyvalent metal ions) on swollen superabsorbent polymer gels. Being ionic compounds, NaCl and CaCl[subscript 2] both collapse the swollen polymer gels. The gel contracted by NaCl reswells…

  8. Mechanism of Na(+)-dependent citrate transport from the structure of an asymmetrical CitS dimer.

    PubMed

    Wöhlert, David; Grötzinger, Maria J; Kühlbrandt, Werner; Yildiz, Özkan

    2015-12-04

    The common human pathogen Salmonella enterica takes up citrate as a nutrient via the sodium symporter SeCitS. Uniquely, our 2.5 Å x-ray structure of the SeCitS dimer shows three different conformations of the active protomer. One protomer is in the outside-facing state. Two are in different inside-facing states. All three states resolve the substrates in their respective binding environments. Together with comprehensive functional studies on reconstituted proteoliposomes, the structures explain the transport mechanism in detail. Our results indicate a six-step process, with a rigid-body 31° rotation of a helix bundle that translocates the bound substrates by 16 Å across the membrane. Similar transport mechanisms may apply to a wide variety of related and unrelated secondary transporters, including important drug targets.

  9. Mechanism of Na+-dependent citrate transport from the structure of an asymmetrical CitS dimer

    PubMed Central

    Wöhlert, David; Grötzinger, Maria J; Kühlbrandt, Werner; Yildiz, Özkan

    2015-01-01

    The common human pathogen Salmonella enterica takes up citrate as a nutrient via the sodium symporter SeCitS. Uniquely, our 2.5 Å x-ray structure of the SeCitS dimer shows three different conformations of the active protomer. One protomer is in the outside-facing state. Two are in different inside-facing states. All three states resolve the substrates in their respective binding environments. Together with comprehensive functional studies on reconstituted proteoliposomes, the structures explain the transport mechanism in detail. Our results indicate a six-step process, with a rigid-body 31° rotation of a helix bundle that translocates the bound substrates by 16 Å across the membrane. Similar transport mechanisms may apply to a wide variety of related and unrelated secondary transporters, including important drug targets. DOI: http://dx.doi.org/10.7554/eLife.09375.001 PMID:26636752

  10. The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na[superscript +]/sugar symport

    SciTech Connect

    Faham, S.; Watanabe, A.; Besserer, G.M.; Cascio, D.; Specht, A.; Hirayama, B.A.; Wright, E.M.; Abramson, J.

    2009-08-27

    Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The -3.0 angstrom structure contains 14 transmembrane (TM) helices in an inward-facing conformation with a core structure of inverted repeats of 5 TM helices (TM2 to TM6 and TM7 to TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to that of the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.

  11. Amphetamine activates calcium channels through dopamine transporter-mediated depolarization.

    PubMed

    Cameron, Krasnodara N; Solis, Ernesto; Ruchala, Iwona; De Felice, Louis J; Eltit, Jose M

    2015-11-01

    Amphetamine (AMPH) and its more potent enantiomer S(+)AMPH are psychostimulants used therapeutically to treat attention deficit hyperactivity disorder and have significant abuse liability. AMPH is a dopamine transporter (DAT) substrate that inhibits dopamine (DA) uptake and is implicated in DA release. Furthermore, AMPH activates ionic currents through DAT that modify cell excitability presumably by modulating voltage-gated channel activity. Indeed, several studies suggest that monoamine transporter-induced depolarization opens voltage-gated Ca(2+) channels (CaV), which would constitute an additional AMPH mechanism of action. In this study we co-express human DAT (hDAT) with Ca(2+) channels that have decreasing sensitivity to membrane depolarization (CaV1.3, CaV1.2 or CaV2.2). Although S(+)AMPH is more potent than DA in transport-competition assays and inward-current generation, at saturating concentrations both substrates indirectly activate voltage-gated L-type Ca(2+) channels (CaV1.3 and CaV1.2) but not the N-type Ca(2+) channel (CaV2.2). Furthermore, the potency to achieve hDAT-CaV electrical coupling is dominated by the substrate affinity on hDAT, with negligible influence of L-type channel voltage sensitivity. In contrast, the maximal coupling-strength (defined as Ca(2+) signal change per unit hDAT current) is influenced by CaV voltage sensitivity, which is greater in CaV1.3- than in CaV1.2-expressing cells. Moreover, relative to DA, S(+)AMPH showed greater coupling-strength at concentrations that induced relatively small hDAT-mediated currents. Therefore S(+)AMPH is not only more potent than DA at inducing hDAT-mediated L-type Ca(2+) channel currents but is a better depolarizing agent since it produces tighter electrical coupling between hDAT-mediated depolarization and L-type Ca(2+) channel activation.

  12. Effect of TGFβ on Na{sup +}/K{sup +} ATPase activity in megakaryocytes

    SciTech Connect

    Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina; Laufer, Stefan; Borst, Oliver; Gawaz, Meinrad; Lang, Florian

    2014-09-26

    Highlights: • TGFß1 markedly up-regulates Na{sup +}/K{sup +} ATPase in megakaryocytes. • The effect is abrogated by p38-MAP kinase inhibitor skepinone. • The effect is abrogated by SGK inhibitor EMD638683. • The effect is abrogated by NF-κB inhibitor wogonin. - Abstract: The Na{sup +}/K{sup +} ATPase generates the Na{sup +} and K{sup +} concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na{sup +}/K{sup +} ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ influences Na{sup +}/K{sup +} ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na{sup +}/K{sup +} ATPase activity determined from K{sup +} induced current utilizing whole cell patch clamp. The pump current (I{sub pump}) was determined in the absence and presence of Na{sup +}/K{sup +} ATPase inhibitor ouabain (100 μM). TGFß1 (60 ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) or NF-κB inhibitor wogonin (50 nM). As a result, the I{sub pump} was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24 h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion

  13. Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris--functional and kinetic asymmetry revealed in proteoliposomes.

    PubMed

    Pingitore, Piero; Pochini, Lorena; Scalise, Mariafrancesca; Galluccio, Michele; Hedfalk, Kristina; Indiveri, Cesare

    2013-09-01

    The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni(2+)-chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [(3)H]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na(+). Internal Na(+) slightly stimulated the antiport. Optimal activity was found at pH7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (≥70%) and by mercurials and methanethiosulfonates (≥80%). Heterologous antiport of [(3)H]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [(3)H]glutamine/glutamine antiport Km values of 0.097 and 1.8mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na(+) on the external side was 32mM. The homology structural model of the hASCT2 protein was built using the GltPh of Pyrococcus horikoshii as template. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism.

  14. Nicotine decreases the activity of glutamate transporter type 3.

    PubMed

    Yoon, Hea-Jo; Lim, Young-Jin; Zuo, Zhiyi; Hur, Wonseok; Do, Sang-Hwan

    2014-02-10

    Nicotine, the main ingredient of tobacco, elicits seizures in animal models and cigarette smoking is regarded as a behavioral risk factor associated with epilepsy or seizures. In the hippocampus, the origin of nicotine-induced seizures, most glutamate uptake could be performed primarily by excitatory amino acid transporter type 3 (EAAT3). An association between temporal lobe epilepsy and EAAT3 downregulation has been reported. Therefore, we hypothesized that nicotine may elicit seizures through the attenuation of EAAT3 activity. We investigated chronic nicotine exposure (72 h) cause reduction of the activity of EAAT3 in a Xenopus oocyte expression system using a two-electrode voltage clamp. The roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) were also determined. Nicotine (0.001-1 μM) resulted in a time- and dose-dependent decrease in EAAT3 activity with maximal inhibition at nicotine concentrations of 0.03 μM or higher and at an exposure time of 72 h. Vmax on the glutamate response was significantly reduced in the nicotine group (0.03 μM for 72 h), but the Km value of EAAT3 for glutamate was not altered. When nicotine-exposed oocytes (0.03 μM for 72 h) were pretreated with phorbol-12-myristate-13-acetate (PMA, a PKC activator), the nicotine-induced reduction in EAAT3 activity was abolished. PKC inhibitors (staurosporine, chelerythrine, and calphostin C) significantly reduced basal EAAT3 activity, but there were no significant differences among the PKC inhibitors, nicotine, and PKC inhibitors+nicotine groups. Similar response patterns were observed among PI3K inhibitors (wortmannin and LY294002), nicotine, and PI3K inhibitors+nicotine. In conclusion, this study suggests that nicotine decreases EAAT3 activity, and that this inhibition seems to be dependent on PKC and PI3K. Our results may provide an additional mechanism for nicotine-induced seizure.

  15. The modulation of erythrocyte Na(+)/K(+)-ATPase activity by curcumin.

    PubMed

    Singh, Prabhakar; Kesharwani, Rajesh Kumar; Misra, Krishna; Rizvi, Syed Ibrahim

    2015-11-01

    Curcumin, an active biphenolic molecule present in turmeric (Curcuma longa), has been reported to elicit plethora of health protective effects. The present study was carried out in vitro, in vivo and in silico to investigate the modulatory effects of curcumin on erythrocyte membrane Na(+)/K(+)-ATPase activity. In vitro curcumin (10(-) (5) M to 10(-) (8) M) was incubated with human erythrocytes membrane. In vivo curcumin (340 mg/kg b.w. and 170 mg/kg b.w.) was supplemented to wistar rats for 21 days. In silico, catalytic unit α of Na(+)/K(+)-ATPase (3b8e.pdb) protein was used as a receptor for the natural ligand ATP to study curcumin-mediated docking simulation using AutoDock4. The in vitro effect of curcumin on the Na(+)/K(+)-ATPase activity in human erythrocytes was biphasic. An inhibitory response was observed at 10(-) (5) M (p < 0.001). An activation of the Na(+)/K(+)-ATPase activity was observed at 10(-) (7) and 10(-) (8) M (p < 0.001 and p < 0.01). In vivo, curcumin supplementation to rats increased the Na(+)/K(+)-ATPase activity at doses 340 mg/kg b.w. (p < 0.001) as well as at 170 mg/kg b.w., (p < 0.01). AutoDock4 docking simulation study showed that both ligands curcumin and ATP actively interacted with amino acids Glu214, Ser215, Glu216, Thr371, Asn377, Arg378, Met379, Arg438, Val440, Ala444, Lys451 and Asp586 at the catalytic cavity of Na+/K+-ATPase. ATP had more H bonding and hydrophobic interaction with active site amino acid residues compared to curcumin. These finding may explain some of the health beneficial properties of curcumin associated with deregulated Na(+)/K(+)-ATPase activity or ions homeostasis.

  16. Ileal apical Na+-dependent bile acid transporter ASBT is upregulated in rats with diabetes mellitus induced by low doses of streptozotocin.

    PubMed

    Annaba, Fadi; Ma, Ke; Kumar, Pradeep; Dudeja, Amish K; Kineman, Rhonda D; Shneider, Benjamin L; Saksena, Seema; Gill, Ravinder K; Alrefai, Waddah A

    2010-10-01

    Increased intestinal bile acid absorption and expansion of the bile acid pool has been implicated in the hypercholesterolemia associated with diabetes mellitus. However, the molecular basis of the increase in bile acid absorption in diabetes mellitus is not fully understood. The ileal apical Na(+)-dependent bile acid transporter (ASBT) is primarily responsible for active reabsorption of the majority of bile acids. Current studies were designed to investigate the modulation of ASBT function and expression in streptozotocin (STZ)-induced diabetes mellitus in rats and to examine the effect of insulin on rat ASBT promoter by insulin. Diabetes mellitus was induced in Sprague-Dawley rats by intraperitoneal injection of low doses of STZ (20 mg/kg body wt) on five consecutive days. Human insulin (10 U/day) was given to a group of diabetic rats for 3 days before euthanasia. RNA and protein were extracted from mucosa isolated from the small intestine and ASBT expression was assessed by real-time quantitative RT-PCR and Western blotting. Our data showed that ASBT mRNA and protein expression were significantly elevated in diabetic rats. Insulin treatment of diabetic rats reversed the increase in ASBT protein expression to control levels. Consistently, ileal Na(+)-dependent [(3)H]taurocholic uptake in isolated intestinal epithelial cells was significantly increased in diabetic rats. In vitro studies utilizing intestinal epithelial Caco-2 cells demonstrated that ASBT expression and promoter activity were significantly decreased by insulin. These studies demonstrated that insulin directly influences ASBT expression and promoter activity and that ASBT function and expression are increased in rats with STZ-induced diabetes mellitus. The increase in ASBT expression may contribute to disturbances in cholesterol homeostasis associated with diabetes mellitus.

  17. Urinary proteolytic activation of renal epithelial Na+ channels in chronic heart failure

    PubMed Central

    Zheng, Hong; Liu, Xuefei; Sharma, Neeru M.; Li, Yulong; Pliquett, Rainer U; Patel, Kaushik P.

    2015-01-01

    One of the key mechanisms involved in renal Na+ retention in chronic heart failure (CHF) is activation of epithelial Na+ channels (ENaC) in collecting tubules. Proteolytic cleavage has an important role in activating ENaC. We hypothesized that enhanced levels of proteases in renal tubular fluid activate ENaC resulting in renal Na+ retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF rats compared to sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06 and plasmin 3.57 vs. sham). Similar increases were observed in urinary samples from patients with CHF. Whole-cell patch-clamp was conducted in cultured renal collecting duct M-1 cells to record Na+ currents. Protease-rich urine (from rats and patients with CHF) significantly increased the Na+ inward current in M-1 cells. Two weeks of protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Increased podocyte lesions were observed in the kidneys of rats with CHF by transmission electron microscopy. Consistent with these results, podocyte damage markers desmin and podocin expressions were also increased in rats with CHF (increased ~2 folds). These findings suggest that podocyte damage may lead to increased proteases in the tubular fluid which in turn contributes to the enhanced renal ENaC activity, providing a novel mechanistic insight for Na+ retention commonly observed in CHF. PMID:26628676

  18. Exercise-induced increase in maximal in vitro Na-K-ATPase activity in human skeletal muscle.

    PubMed

    Juel, Carsten; Nordsborg, Nikolai B; Bangsbo, Jens

    2013-06-15

    The present study investigated whether maximal in vitro Na-K-ATPase activity in human skeletal muscle is changed with exercise and whether it was altered by acute hypoxia. Needle biopsies from 14 subjects were obtained from vastus lateralis before and after 4 min of intense muscle activity. In addition, six subjects exercised also in hypoxia (12.5% oxygen). The Na-K-ATPase assay revealed a 19% increase (P < 0.05) in maximal velocity (Vmax) for Na⁺-dependent Na-K-ATPase activity after exercise and a tendency (P < 0.1) toward a decrease in Km for Na⁺ (increased Na⁺ affinity) in both normoxia and hypoxia. In contrast, the in vitro Na-K-ATPase activity determined with the 3-O-MFPase technique was 11-32% lower after exercise in normoxia (P < 0.05) and hypoxia (P < 0.1). Based on the different results obtained with the Na-K-ATPase assay and the 3-O-MFPase technique, it was suggested that the 3-O-MFPase method is insensitive to changes in Na-K-ATPase activity. To test this possibility, changes in Na-K-ATPase activity was induced by protein kinase C activation. The changes quantified with the Na-K-ATPase assay could not be detected with the 3-O-MFPase method. In addition, purines stimulated Na-K-ATPase activity in rat muscle membranes; these changes could not be detected with the 3-O-MFPase method. Therefore, the 3-O-MFPase technique is not sensitive to changes in Na⁺ sensitivity, and the method is not suited to detecting changes in Na-K-ATPase activity with exercise. In conclusion, muscle activity in humans induces an increased in vitro Na⁺-dependent Na-K-ATPase activity, which contributes to the upregulation of the Na-K-ATPase in association with exercise both in normoxia and hypoxia.

  19. NBCe1 as a Model Carrier for Understanding the Structure-Function Properties of Na+-Coupled SLC4 Transporters in Health and Disease

    PubMed Central

    Kurtz, Ira

    2014-01-01

    SLC4 transporters are membrane proteins that in general mediate the coupled transport of bicarbonate (carbonate) and share amino acid sequence homology. These proteins differ as to whether they also transport Na+ and/or Cl−, in addition to their charge transport stoichiometry, membrane targeting, substrate affinities, developmental expression, regulatory motifs, and protein-protein interactions. These differences account in part for the fact that functionally, SLC4 transporters have various physiological roles in mammals including transepithelial bicarbonate transport, intracellular pH regulation, transport of Na+ and/or Cl−, and possibly water. Bicarbonate transport is not unique to the SLC4 family since the structurally unrelated SLC26 family has at least three proteins that mediate Cl−-HCO3− exchange. The present review focuses on the first of the sodium-dependent SLC4 transporters that was identified whose structure has been most extensively studied: the electrogenic Na+-base cotransporter NBCe1. Mutations in NBCe1 cause proximal renal tubular acidosis (pRTA) with neurologic and ophthalmologic extrarenal manifestations. Recent studies have characterized important structure-function properties of the transporter and how they are perturbed as a result of mutations that cause pRTA. It has become increasingly apparent that the structure of NBCe1 differs in several key features from the SLC4 Cl−-HCO3− exchanger AE1 whose structural properties have been well-studied. In this review, the structure-function properties and regulation of NBCe1 will be highlighted and its role in health and disease will be reviewed in detail. PMID:24515290

  20. Active transport and obesity prevention - A transportation sector obesity impact scoping review and assessment for Melbourne, Australia.

    PubMed

    Brown, V; Moodie, M; Mantilla Herrera, A M; Veerman, J L; Carter, R

    2017-03-01

    Given the alarming prevalence of obesity worldwide and the need for interventions to halt the growing epidemic, more evidence on the role and impact of transport interventions for obesity prevention is required. This study conducts a scoping review of the current evidence of association between modes of transport (motor vehicle, walking, cycling and public transport) and obesity-related outcomes. Eleven reviews and thirty-three primary studies exploring associations between transport behaviours and obesity were identified. Cohort simulation Markov modelling was used to estimate the effects of body mass index (BMI) change on health outcomes and health care costs of diseases causally related to obesity in the Melbourne, Australia population. Results suggest that evidence for an obesity effect of transport behaviours is inconclusive (29% of published studies reported expected associations, 33% mixed associations), and any potential BMI effect is likely to be relatively small. Hypothetical scenario analyses suggest that active transport interventions may contribute small but significant obesity-related health benefits across populations (approximately 65 health adjusted life years gained per year). Therefore active transport interventions that are low cost and targeted to those most amenable to modal switch are the most likely to be effective and cost-effective from an obesity prevention perspective. The uncertain but potentially significant opportunity for health benefits warrants the collection of more and better quality evidence to fully understand the potential relationships between transport behaviours and obesity. Such evidence would contribute to the obesity prevention dialogue and inform policy across the transportation, health and environmental sectors.

  1. The F130S point mutation in the Arabidopsis high-affinity K+ transporter AtHAK5 increases K+ over Na+ and Cs+ selectivity and confers Na+ and Cs+ tolerance to yeast under heterologous expression

    PubMed Central

    Alemán, Fernando; Caballero, Fernando; Ródenas, Reyes; Rivero, Rosa M.; Martínez, Vicente; Rubio, Francisco

    2014-01-01

    Potassium (K+) is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K+ acquisition by plant roots at low external K+ concentrations. Certain abiotic stress conditions such as salinity or Cs+-polluted soils may jeopardize plant K+ nutrition because HAK5-mediated K+ transport is inhibited by Na+ and Cs+. Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na+ tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs+ tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K+ and reduces the Ki values for Na+ and Cs+, suggesting that the F130 residue may contribute to the structure of the pore region involved in K+ binding. In addition, this mutation increases the Vmax for K+. All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter. PMID:25228905

  2. Thermally activated charge transport in microbial protein nanowires

    NASA Astrophysics Data System (ADS)

    Lampa-Pastirk, Sanela; Veazey, Joshua P.; Walsh, Kathleen A.; Feliciano, Gustavo T.; Steidl, Rebecca J.; Tessmer, Stuart H.; Reguera, Gemma

    2016-03-01

    The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors.

  3. Thermally activated charge transport in microbial protein nanowires.

    PubMed

    Lampa-Pastirk, Sanela; Veazey, Joshua P; Walsh, Kathleen A; Feliciano, Gustavo T; Steidl, Rebecca J; Tessmer, Stuart H; Reguera, Gemma

    2016-03-24

    The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors.

  4. Thermally activated charge transport in microbial protein nanowires

    PubMed Central

    Lampa-Pastirk, Sanela; Veazey, Joshua P.; Walsh, Kathleen A.; Feliciano, Gustavo T.; Steidl, Rebecca J.; Tessmer, Stuart H.; Reguera, Gemma

    2016-01-01

    The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors. PMID:27009596

  5. Active ingredients in Chinese medicines promoting blood circulation as Na+/K+-ATPase inhibitors

    PubMed Central

    Chen, Ronald JY; Jinn, Tzyy-rong; Chen, Yi-ching; Chung, Tse-yu; Yang, Wei-hung; Tzen, Jason TC

    2011-01-01

    The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na+/K+-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na+/K+-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na+/K+-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na+/K+-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na+/K+-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na+/K+-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na+/K+-ATPase in the brain could be potential drugs for the treatment of ischemic stroke. PMID:21293466

  6. Astrocytes generate Na+-mediated metabolic waves.

    PubMed

    Bernardinelli, Yann; Magistretti, Pierre J; Chatton, Jean-Yves

    2004-10-12

    Glutamate-evoked Na+ increase in astrocytes has been identified as a signal coupling synaptic activity to glucose consumption. Astrocytes participate in multicellular signaling by transmitting intercellular Ca2+ waves. Here we show that intercellular Na+ waves are also evoked by activation of single cultured cortical mouse astrocytes in parallel with Ca2+ waves; however, there are spatial and temporal differences. Indeed, maneuvers that inhibit Ca2+ waves also inhibit Na+ waves; however, inhibition of the Na+/glutamate cotransporters or enzymatic degradation of extracellular glutamate selectively inhibit the Na+ wave. Thus, glutamate released by a Ca2+ wave-dependent mechanism is taken up by the Na+/glutamate cotransporters, resulting in a regenerative propagation of cytosolic Na+ increases. The Na+ wave gives rise to a spatially correlated increase in glucose uptake, which is prevented by glutamate transporter inhibition. Therefore, astrocytes appear to function as a network for concerted neurometabolic coupling through the generation of intercellular Na+ and metabolic waves.

  7. Cryo-EM structure of the Slo2.2 Na+-activated K+ channel

    PubMed Central

    Hite, Richard; Yuan, Peng; Li, Zongli; Hsuing, Yichun; Walz, Thomas; MacKinnon, Roderick

    2015-01-01

    Na+-activated K+ channels are members of the Slo family of large conductance K+ channels that are widely expressed in the brain, where their opening regulates neuronal excitability. These channels are fascinating for the biological roles they fulfill as well as for their intriguing biophysical properties, including conductance levels ten times most other K+ channels and gating sensitivity to intracellular Na+. Here we present the structure a complete Na+-activated K+ channel, Slo2.2, in the Na+-free state, determined by cryo-electron microscopy at a nominal resolution of 4.5 Å. The channel is composed of a large cytoplasmic gating ring within which resides the Na+-binding site and a transmembrane domain that closely resembles voltage-gated K+ channels. In the structure, the cytoplasmic domain adopts a closed conformation and the ion conduction pore is also closed. The structure provides a first view of a member of the Slo K+ channel family, which reveals features explaining their high conductance and gating mechanism. PMID:26436452

  8. Biomass aggregation influences NaN3 short-term effects on anammox bacteria activity.

    PubMed

    Pedrouso, A; Val Del Río, A; Campos, J L; Méndez, R; Mosquera-Corral, A

    2017-03-01

    The main bottleneck to maintain the long-term stability of the partial nitritation-anammox processes, especially those operated at low temperatures and nitrogen concentrations, is the undesirable development of nitrite oxidizing bacteria (NOB). When this occurs, the punctual addition of compounds with the capacity to specifically inhibit NOB without affecting the process efficiency might be of interest. Sodium azide (NaN3) is an already known NOB inhibitor which at low concentrations does not significantly affect the ammonia oxidizing bacteria (AOB) activity. However, studies about its influence on anammox bacteria are unavailable. For this reason, the objective of the present study was to evaluate the effect of NaN3 on the anammox activity. Three different types of anammox biomass were used: granular biomass comprising AOB and anammox bacteria (G1), anammox enriched granules (G2) and previous anammox granules disaggregated (F1). No inhibitory effect of NaN3 was measured on G1 sludge. However, the anammox activity decreased in the case of G2 and F1. Granular biomass activity was less affected (IC50 90 mg/L, G2) than flocculent one (IC50 5 mg/L, F1). Summing up, not only does the granular structure protect the anammox bacteria from the NaN3 inhibitory effect, but also the AOB act as a barrier decreasing the inhibition.

  9. Theory of activated transport in bilayer quantum Hall systems.

    PubMed

    Roostaei, B; Mullen, K J; Fertig, H A; Simon, S H

    2008-07-25

    We analyze the transport properties of bilayer quantum Hall systems at total filling factor nu=1 in drag geometries as a function of interlayer bias, in the limit where the disorder is sufficiently strong to unbind meron-antimeron pairs, the charged topological defects of the system. We compute the typical energy barrier for these objects to cross incompressible regions within the disordered system using a Hartree-Fock approach, and show how this leads to multiple activation energies when the system is biased. We then demonstrate using a bosonic Chern-Simons theory that in drag geometries current in a single layer directly leads to forces on only two of the four types of merons, inducing dissipation only in the drive layer. Dissipation in the drag layer results from interactions among the merons, resulting in very different temperature dependences for the drag and drive layers, in qualitative agreement with experiment.

  10. Na(+)/H(+) exchanger 3 inhibitor diminishes hepcidin-enhanced duodenal calcium transport in hemizygous β-globin knockout thalassemic mice.

    PubMed

    Charoenphandhu, Narattaphol; Kraidith, Kamonshanok; Lertsuwan, Kornkamon; Sripong, Chanakarn; Suntornsaratoon, Panan; Svasti, Saovaros; Krishnamra, Nateetip; Wongdee, Kannikar

    2017-03-01

    Recent investigation has shown that the liver-derived iron-regulating hormone, hepcidin, can potentiate intestinal calcium absorption in hemizygous β-globin knockout thalassemic (BKO) mice. Since the upregulation of Fe(2+) and H(+) cotransporter, divalent metal transporter (DMT)-1, has been shown to correlate with thalassemia-induced intestinal calcium absorption impairment, the inhibition of the apical Na(+)/H(+) exchanger (NHE)-3 that is essential for cytoplasmic pH regulation and transepithelial sodium absorption was hypothesized to negatively affect hepcidin action. Herein, the positive effect of hepcidin on the duodenal calcium transport was evaluated using Ussing chamber technique. The results showed that BKO mice had lower absorptive surface area and duodenal calcium transport than wild-type mice. Besides, paracellular transport of zinc in BKO mice was compromised. Hepcidin administration completely restored calcium transport. Since this hepcidin action was totally abolished by inhibitors of the basolateral calcium transporters, Na(+)/Ca(2+) exchanger (NCX1) and plasma membrane Ca(2+)-ATPase (PMCA1b), the enhanced calcium flux potentially occurred through the transcellular pathway rather than paracellular pathway. Interestingly, the selective NHE3 inhibitor, 100 nM tenapanor, markedly inhibited hepcidin-enhanced calcium transport. Accordingly, hepcidin is one of the promising therapeutic agents for calcium malabsorption in β-thalassemia. It mainly stimulates the transcellular calcium transport across the duodenal epithelium in an NHE3-dependent manner.

  11. The relative roles of passive surface forces and active ion transport in the modulation of airway surface liquid volume and composition.

    PubMed

    Tarran, R; Grubb, B R; Gatzy, J T; Davis, C W; Boucher, R C

    2001-08-01

    Two hypotheses have been proposed recently that offer different views on the role of airway surface liquid (ASL) in lung defense. The "compositional" hypothesis predicts that ASL [NaCl] is kept low (<50 mM) by passive forces to permit antimicrobial factors to act as a chemical defense. The "volume" hypothesis predicts that ASL volume (height) is regulated isotonically by active ion transport to maintain efficient mechanical mucus clearance as the primary form of lung defense. To compare these hypotheses, we searched for roles for: (1) passive forces (surface tension, ciliary tip capillarity, Donnan, and nonionic osmolytes) in the regulation of ASL composition; and (2) active ion transport in ASL volume regulation. In primary human tracheobronchial cultures, we found no evidence that a low [NaCl] ASL could be produced by passive forces, or that nonionic osmolytes contributed substantially to ASL osmolality. Instead, we found that active ion transport regulated ASL volume (height), and that feedback existed between the ASL and airway epithelia to govern the rate of ion transport and volume absorption. The mucus layer acted as a "reservoir" to buffer periciliary liquid layer height (7 microm) at a level optimal for mucus transport by donating or accepting liquid to or from the periciliary liquid layer, respectively. These data favor the active ion transport/volume model hypothesis to describe ASL physiology.

  12. Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter

    PubMed Central

    Szollosi, Andras; Vieira-Pires, Ricardo S.; Teixeira-Duarte, Celso M.; Rocha, Rita; Morais-Cabral, João H.

    2016-01-01

    KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K+ and Na+) transport proteins that closely resemble K+ channels. These proteins underlie a plethora of cellular functions that are crucial for environmental adaptation in plants, fungi, archaea, and bacteria. The activation mechanism of the Trk/Ktr/HKT proteins remains unknown. It has been shown that ATP stimulates the activity of KtrAB while ADP does not. Here, we present X-ray structural information on the KtrAB complex with bound ADP. A comparison with the KtrAB-ATP structure reveals conformational changes in the ring and in the membrane protein. In combination with a biochemical and functional analysis, we uncover how ligand-dependent changes in the KtrA ring are propagated to the KtrB membrane protein and conclude that, despite their structural similarity, the activation mechanism of KtrAB is markedly different from the activation mechanism of K+ channels. PMID:26771197

  13. Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter.

    PubMed

    Szollosi, Andras; Vieira-Pires, Ricardo S; Teixeira-Duarte, Celso M; Rocha, Rita; Morais-Cabral, João H

    2016-01-01

    KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K+ and Na+) transport proteins that closely resemble K+ channels. These proteins underlie a plethora of cellular functions that are crucial for environmental adaptation in plants, fungi, archaea, and bacteria. The activation mechanism of the Trk/Ktr/HKT proteins remains unknown. It has been shown that ATP stimulates the activity of KtrAB while ADP does not. Here, we present X-ray structural information on the KtrAB complex with bound ADP. A comparison with the KtrAB-ATP structure reveals conformational changes in the ring and in the membrane protein. In combination with a biochemical and functional analysis, we uncover how ligand-dependent changes in the KtrA ring are propagated to the KtrB membrane protein and conclude that, despite their structural similarity, the activation mechanism of KtrAB is markedly different from the activation mechanism of K+ channels.

  14. Thyroid hormone stimulates the renal Na/H exchanger NHE3 by transcriptional activation

    PubMed Central

    CANO, ADRIANA; BAUM, MICHEL; MOE, ORSON W.

    2014-01-01

    Thyroid hormone stimulates renal proximal tubule NaCl and NaHCO3 absorption in part by activating the apical membrane Na/H exchanger NHE3. We used a renal epithelial cell line, the opossum kidney (OK) cell, to define the mechanism by which 3,5,3′-triiodothyronine (T3) increases NHE3 activity. T3 stimulated NHE3 activity, an effect that was blocked by inhibition of cellular transcription or translation. The increase in activity was associated with increases in steady-state cell surface and total cellular NHE3 protein and NHE3 transcript abundance. T3 stimulated transcription of the NHE3 gene and had no effect on NHE3 transcript stability. The transcriptional activity of the 5′-flanking region of the rat NHE3 gene was stimulated by T3 when expressed in OK cells. When heterologously expressed rat NHE3 transcript levels were clamped constant with a constitutive promoter in OK cells, T3 has no effect on rat NHE3 protein abundance, suggesting the absence of regulation of NHE3 protein stability or translation. These studies demonstrate that T3 stimulates NHE3 activity by activating NHE3 gene transcription and increasing NHE3 transcript and protein abundance. PMID:9886925

  15. The effects of dietary sodium loading on the activity and expression of Na, K-ATPase in the rectal gland of the European dogfish (Scyliorhinus canicula).

    PubMed

    MacKenzie, S; Cutler, C P; Hazon, N; Cramb, G

    2002-02-01

    cDNA fragments of both the alpha- and beta-subunits of the Na, K-ATPase and a cDNA fragment of the secretory form of Na-K-Cl cotransporter from the European dogfish (Scyliorhinus canicula) were amplified and cloned using degenerate primers in RT-PCR. These clones were used along with a sCFTR cDNA from the related dogfish shark, Squalus acanthias to characterise the expression of mRNAs for these ion transporters in the dogfish rectal gland subsequent to an acute feeding episode. Following a single feeding event where starved dogfish were fed squid portions (20 g squid/kg fish), there was a delayed and transient 40-fold increase in the activity of Na, K-ATPase in crude rectal gland homogenates. Increases in enzyme activity were apparent 3 h after the feeding event and peaked at 9 h before returning to control values within 24 h. These increases in activity were accompanied by small and transient decreases in plasma sodium and chloride concentrations lasting up to 3 days. Significant increases in the expression of mRNAs for alpha- and beta-subunits of the Na, K-ATPase, the Na-K-Cl cotransporter and CFTR chloride channel were detected but not until 1-2 days after the feeding event. It is concluded that the transient increase in Na, K-ATPase activity is not attributable to increases in the abundance of alpha- and beta-subunit mRNAs but must be associated with some, as yet unknown, post-transcriptional activation mechanism.

  16. Complement activation of electrogenic ion transport in isolated rat colon.

    PubMed

    McCole, D F; Otti, B; Newsholme, P; Baird, A W

    1997-11-15

    The complement cascade is an important component in many immune and inflammatory reactions and may contribute to both the diarrhoea and inflammation associated with inflammatory bowel disease. Isolated rat colonic mucosae were voltage clamped in Ussing chambers. Basolateral addition of zymosan-activated whole human serum (ZAS) induced a rapid onset, transient inward short circuit current (SCC). This response was concentration dependent and was significantly attenuated by pre-heating ZAS at 60 degrees C for 30 min. Depletion of complement from normal human serum with cobra venom factor (CVF) significantly lowered SCC responses. Chloride was the primary charge carrying ion as responses to ZAS were abolished in the presence of the loop diuretic bumetanide. The complement component C3a stimulated ion transport but not to the same extent as whole serum. Exogenous C5 was without effect. The cyclooxygenase inhibitor piroxicam significantly attenuated the response to ZAS. These findings support the possibility that complement activation may contribute to the pathophysiology of secretory diarrhoea since activation of electrogenic chloride secretion converts intestinal epithelia to a state of net fluid secretion.

  17. Serotonin transporter genotype modulates amygdala activity during mood regulation

    PubMed Central

    Rao, Hengyi; Wang, Jiongjiong; Detre, John A.; Breland, Jessica; Sankoorikal, Geena Mary V.; Brodkin, Edward S.; Farah, Martha J.

    2010-01-01

    Recent studies have implicated the short allele of the serotonin transporter-linked polymorphic region (5-HTTLPR) in depression vulnerability, particularly in the context of stress. Several neuroimaging studies have shown that 5-HTTLPR genotype predicts amygdala reactivity to negatively valenced stimuli, suggesting a mechanism whereby the short allele confers depression risk. The current study investigated whether 5-HTTLPR genotype similarly affects neural activity during an induced sad mood and during recovery from sad mood. Participants were 15 homozygous short (S) and 15 homozygous long (L) individuals. Regional cerebral blood flow was measured with perfusion functional magnetic resonance imaging during four scanning blocks: baseline, sad mood, mood recovery and following return to baseline. Comparing mood recovery to baseline, both whole brain analyses and template-based region-of-interest analyses revealed greater amygdala activity for the S vs the L-group. There were no significant amygdala differences found during the induced sad mood. These results demonstrate the effect of the S allele on amygdala activity during intentional mood regulation and suggest that amygdala hyperactivity during recovery from a sad mood may be one mechanism by which the S allele confers depression risk. PMID:19858108

  18. Hypoxia leads to Na,K-ATPase downregulation via Ca(2+) release-activated Ca(2+) channels and AMPK activation.

    PubMed

    Gusarova, Galina A; Trejo, Humberto E; Dada, Laura A; Briva, Arturo; Welch, Lynn C; Hamanaka, Robert B; Mutlu, Gökhan M; Chandel, Navdeep S; Prakriya, Murali; Sznajder, Jacob I

    2011-09-01

    To maintain cellular ATP levels, hypoxia leads to Na,K-ATPase inhibition in a process dependent on reactive oxygen species (ROS) and the activation of AMP-activated kinase α1 (AMPK-α1). We report here that during hypoxia AMPK activation does not require the liver kinase B1 (LKB1) but requires the release of Ca(2+) from the endoplasmic reticulum (ER) and redistribution of STIM1 to ER-plasma membrane junctions, leading to calcium entry via Ca(2+) release-activated Ca(2+) (CRAC) channels. This increase in intracellular Ca(2+) induces Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ)-mediated AMPK activation and Na,K-ATPase downregulation. Also, in cells unable to generate mitochondrial ROS, hypoxia failed to increase intracellular Ca(2+) concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca(2+) signaling. Furthermore, inhibition of CRAC channel function in rat lungs prevented the impairment of alveolar fluid reabsorption caused by hypoxia. These data suggest that during hypoxia, calcium entry via CRAC channels leads to AMPK activation, Na,K-ATPase downregulation, and alveolar epithelial dysfunction.

  19. Intracellular sodium ion activity and sodium transport in rabbit urinary bladder.

    PubMed Central

    Eaton, D C

    1981-01-01

    1. Intracellular potentials and the intracellular activities of Na+ and K+ were examined using conventional and ion-selective micro-electrodes. 2. In animals on a normal diet, the intracellular Na+ activity was 8.6 +/- 2.9 mM (mean +/- S.D.) with a mean short-circuit current of 2.8 +/- 0.9 microA/cm2. 3. In animals on a low-Na+ diet, the intracellular Na+ activity was 18.5 +/- 9.9 mM with a short-circuit current of 4.5 +/- 1.3 microA/cm2 (mean +/- S.D.). 4. There was a correlation between short-circuit current and intracellular Na+ activity which could be fitted by a saturating hyperbolic relationship. 5. Treatment of the issue with ouabain and amiloride produced an increase and a decrease, respectively, in the intracellular Na+ activity. 6. Treatment with aldosterone produced a large increase in short-circuit current with a substantial increase in intracellular Na+ activity. 7. Intracellular Na+ activity does not seem to affect apical membrane permeability directly. PMID:7320880

  20. Na+, K+-activated-ATPase inhibition in rainbow trout: A site for organochlorine pesticide toxicity?

    USGS Publications Warehouse

    Davis, Paul W.; Wedemeyer, Gary A.

    1971-01-01

    1. The Na+, K+-activated, Mg2+-dependent-ATPase enzyme system in a heavy microsomal fraction of rainbow trout (Salmo gairdneri) brain was inhibited in vitro by chlorinated hydrocarbon pesticides.2. T50 (concentration at 50 per cent inhibition) values for dicofol, endosulfan and DDT were 5 × 10−6, 3 × 10−5 and 1 × 10−4 M respectively. Similar inhibition by these pesticides occurred in kidney and gill ATPase preparations.3. An unexpected finding was a failure of the classic inhibitor, ouabain, to block the Na+, K+-activated component of ATPase activity in the gill.4. It is suggested that inhibition of ATPase activity may be a causal factor in the toxic effects of organochlorine pesticides in fishes.

  1. Physical Activity Energy Expenditure in Dutch Adolescents: Contribution of Active Transport to School, Physical Education, and Leisure Time Activities

    ERIC Educational Resources Information Center

    Slingerland, Menno; Borghouts, Lars B.; Hesselink, Matthijs K. C.

    2012-01-01

    Background: Detailed knowledge about physical activity energy expenditure (PAEE) can guide the development of school interventions aimed at reducing overweight in adolescents. However, relevant components of PAEE have never been objectively quantified in this population. This study investigated the contribution of active transport to and from…

  2. Compromising KCC2 transporter activity enhances the development of continuous seizure activity

    PubMed Central

    Kelley, Matthew R.; Deeb, Tarek Z.; Brandon, Nicholas J.; Dunlop, John; Davies, Paul A.; Moss, Stephen J.

    2016-01-01

    Impaired neuronal inhibition has long been associated with the increased probability of seizure occurrence and heightened seizure severity. Fast synaptic inhibition in the brain is primarily mediated by the type A γ-aminobutyric acid receptors (GABAARs), ligand-gated ion channels that can mediate Cl− influx resulting in membrane hyperpolarization and the restriction of neuronal firing. In most adult brain neurons, the K+/Cl− co-transporter-2 (KCC2) establishes hyperpolarizing GABAergic inhibition by maintaining low [Cl−]i. In this study, we sought to understand how decreased KCC2 transport function affects seizure event severity. We impaired KCC2 transport in the 0-Mg2+ ACSF and 4-aminopyridine in vitro models of epileptiform activity in acute mouse brain slices. Experiments with the selective KCC2 inhibitor VU0463271 demonstrated that reduced KCC2 transport increased the duration of SLEs, resulting in non-terminating discharges of clonic-like activity. We also investigated slices obtained from the KCC2-Ser940Ala (S940A) point-mutant mouse, which has a mutation at a known functional phosphorylation site causing behavioral and cellular deficits under hyperexcitable conditions. We recorded from the entorhinal cortex of S940A mouse brain slices in both 0-Mg2+ ACSF and 4-aminopyridine, and demonstrated that loss of the S940 residue increased the susceptibility of continuous clonic-like discharges, an in vitro form of status epilepticus. Our experiments revealed KCC2 transport activity is a critical factor in seizure event duration and mechanisms of termination. Our results highlight the need for therapeutic strategies that potentiate KCC2 transport function in order to decrease seizure event severity and prevent the development of status epilepticus. PMID:27108931

  3. Activation of the epithelial Na+ channel in the collecting duct by vasopressin contributes to water reabsorption.

    PubMed

    Bugaj, Vladislav; Pochynyuk, Oleh; Stockand, James D

    2009-11-01

    We used patch-clamp electrophysiology on isolated, split-open murine collecting ducts (CD) to test the hypothesis that regulation of epithelial sodium channel (ENaC) activity is a physiologically important effect of vasopressin. Surprisingly, this has not been tested directly before. We ask whether vasopressin affects ENaC activity distinguishing between acute and chronic effects, as well as, parsing the cellular signaling pathway and molecular mechanism of regulation. In addition, we quantified possible synergistic regulation of ENaC by vasopressin and aldosterone associating this with a requirement for distal nephron Na+ reabsorption during water conservation vs. maintenance of Na+ balance. We find that vasopressin significantly increases ENaC activity within 2-3 min by increasing open probability (P(o)). This activation was dependent on adenylyl cyclase (AC) and PKA. Water restriction (18-24 h) and pretreatment of isolated CD with vasopressin (approximately 30 min) resulted in a similar increase in P(o). In addition, this also increased the number (N) of active ENaC in the apical membrane. Similar to P(o), increases in N were sensitive to inhibitors of AC. Stressing animals with water and salt restriction separately and jointly revealed an important effect of vasopressin: conservation of water and Na+ each independently increased ENaC activity and jointly had a synergistic effect on channel activity. These results demonstrate a quantitatively important action of vasopressin on ENaC suggesting that distal nephron Na+ reabsorption mediated by this channel contributes to maintenance of water reabsorption. In addition, our results support that the combined actions of vasopressin and aldosterone are required to achieve maximally activated ENaC.

  4. Role of H{sub 2}O{sub 2} on the kinetics of low-affinity high-capacity Na{sup +}-dependent alanine transport in SHR proximal tubular epithelial cells

    SciTech Connect

    Pinto, Vanda; Pinho, Maria Joao; Jose, Pedro A.; Soares-da-Silva, Patricio

    2010-07-30

    Research highlights: {yields} H{sub 2}O{sub 2} in excess is required for the presence of a low-affinity high-capacity component for the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake in SHR PTE cells only. {yields} It is suggested that Na{sup +} binding in renal ASCT2 may be regulated by ROS in SHR PTE cells. -- Abstract: The presence of high and low sodium affinity states for the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake in immortalized renal proximal tubular epithelial (PTE) cells was previously reported (Am. J. Physiol. 293 (2007) R538-R547). This study evaluated the role of H{sub 2}O{sub 2} on the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake of ASCT2 in immortalized renal PTE cells from Wistar Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). Na{sup +} dependence of [{sup 14}C]-L-alanine uptake was investigated replacing NaCl with an equimolar concentration of choline chloride in vehicle- and apocynin-treated cells. Na{sup +} removal from the uptake solution abolished transport activity in both WKY and SHR PTE cells. Decreases in H{sub 2}O{sub 2} levels in the extracellular medium significantly reduced Na{sup +}-K{sub m} and V{sub max} values of the low-affinity high-capacity component in SHR PTE cells, with no effect on the high-affinity low-capacity state of the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake. After removal of apocynin from the culture medium, H{sub 2}O{sub 2} levels returned to basal values within 1 to 3 h in both WKY and SHR PTE cells and these were found stable for the next 24 h. Under these experimental conditions, the Na{sup +}-K{sub m} and V{sub max} of the high-affinity low-capacity state were unaffected and the low-affinity high-capacity component remained significantly decreased 1 day but not 4 days after apocynin removal. In conclusion, H{sub 2}O{sub 2} in excess is required for the presence of a low-affinity high-capacity component for the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake in SHR PTE cells only

  5. The I427T neuraminidase (NA) substitution, located outside the NA active site of an influenza A(H1N1)pdm09 variant with reduced susceptibility to NA inhibitors, alters NA properties and impairs viral fitness.

    PubMed

    Tu, Véronique; Abed, Yacine; Barbeau, Xavier; Carbonneau, Julie; Fage, Clément; Lagüe, Patrick; Boivin, Guy

    2017-01-01

    Emergence of pan neuraminidase inhibitor (NAI)-resistant variants constitutes a serious clinical concern. An influenza A(H1N1)pdm09 variant containing the I427T/Q313R neuraminidase (NA) substitutions was previously identified in a surveillance study. Although these changes are not part of the NA active site, the variant showed reduced susceptibility to many NAIs. In this study, we investigated the mechanism of resistance for the I427T/Q313R substitution and its impact on the NA enzyme and viral fitness. Recombinant wild-type (WT), I427T/Q313R and I427T A(H1N1)pdm09 viruses were generated by reverse genetics and tested for their drug susceptibilities, enzymatic properties and replication kinetics in vitro as well as their virulence in mice. Molecular dynamics (MD) simulations were performed for NA structural analysis. The I427T substitution, which was responsible for the resistance phenotype observed in the double (I427T/Q313R) mutant, induced 17-, 56-, 7-, and 14-fold increases in IC50 values against oseltamivir, zanamivir, peramivir and laninamivir, respectively. The I427T substitution alone or combined to Q313R significantly reduced NA affinity. The I427T/Q313R and to a lesser extent I427T recombinant viruses displayed reduced viral titers vs WT in vitro. In experimentally-infected mice, the mortality rates were 62.5%, 0% and 14.3% for the WT, I417T/Q313R and I427T viruses, respectively. There were about 2.5- and 2-Log reductions in mean lung viral titers on day 5 post-infection for the I427T/Q313R and I427T mutants, respectively, compared to WT. Results from simulations revealed that the I427T change indirectly altered the stability of the catalytic R368 residue of the NA enzyme causing its reduced binding to the substrate/inhibitor. This study demonstrates that the I427T/Q313R mutant, not only alters NAI susceptibility but also compromises NA properties and viral fitness, which could explain its infrequent detection in clinic.

  6. Spontaneously active NaV1.5 sodium channels may underlie odor sensitivity.

    PubMed

    Dionne, Vincent E

    2016-08-01

    The olfactory system is remarkably sensitive to airborne odor molecules, but precisely how very low odor concentrations bordering on just a few molecules per olfactory sensory neuron can trigger graded changes in firing is not clear. This report reexamines signaling in olfactory sensory neurons in light of the recent account of NaV1.5 sodium channel-mediated spontaneous firing. Using a model of spontaneous channel activity, the study shows how even submillivolt changes in membrane potential elicited by odor are expected to cause meaningful changes in NaV1.5-dependent firing. The results suggest that the random window currents of NaV1.5 channels may underpin not only spontaneous firing in olfactory sensory neurons but the cellular response to odor as well, thereby ensuring the robustness and sensitivity of signaling that is especially important for low odor concentrations.

  7. Bactericidal and Fungicidal Activity in the Gas Phase of Sodium Dichloroisocyanurate (NaDCC).

    PubMed

    Proto, Antonio; Zarrella, Ilaria; Cucciniello, Raffaele; Pironti, Concetta; De Caro, Francesco; Motta, Oriana

    2016-08-01

    Sodium dichloroisocyanurate (NaDCC) is usually employed as a disinfectant for the treatment of water, environmental surfaces and medical equipment principally for its effectiveness as a microbicide agent. In this study, we explore the possibility of a new use for NaDCC by investigating the microbicidal activity of chlorine, which derives from the hydrolysis of NaDCC mediated by air humidity, and by testing its effect on the neutralization of microbes present in domestic waste. NaDCC was inserted in a plastic garbage can where LB agar plates, with different dilutions of a known title of four different microorganisms (Escherichia coli, Staphylococcus aureus, Debaryomyces hansenii and Aspergillus brasiliensis), were weakly inserted. The molecular chlorine (Cl2) levels present in the garbage can were quantified using an iodometric titration. The gas emitted in the garbage can presented a strong microbicide effect, inhibiting the proliferation of all four microorganisms and for four consecutive weeks, thus showing that NaDCC hydrolysis, mediated by air humidity, is able to ensure the decontamination of restricted environments, avoiding the proliferation of both Gram-positive and Gram-negative bacteria as well as fungi.

  8. Structural lipid changes and Na(+)/K(+)-ATPase activity of gill cells' basolateral membranes during saltwater acclimation in sea lamprey (Petromyzon marinus, L.) juveniles.

    PubMed

    Lança, Maria João; Machado, Maria; Ferreira, Ana Filipa; Quintella, Bernardo Ruivo; de Almeida, Pedro Raposo

    2015-11-01

    Seawater acclimation is a critical period for anadromous species and a process yet to be understood in lampreys. Considering that changes in lipid composition of the gill cells' basolateral membranes may disrupt the major transporter Na(+)K(+)-ATPase, the goal of this study was to detect changes at this level during juvenile sea lamprey seawater acclimation. The results showed that saltwater acclimation has a direct effect on the fatty acid composition of gill cells basolateral membrane's phospholipids. When held in full-strength seawater, the fatty acid profile of basolateral membrane's phospholipids suffered a restructure by increasing either saturation or the ratio between oleic acid and eicosapentaenoic acid. Simultaneously, the activity of Na(+)K(+)-ATPase revealed a significant and positive correlation with basolateral membrane's cholesterol content in the presence of highest salinity. Our results pointed out for lipid adjustments involving the functional transporter present on the gill cell basolateral membranes to ensure the role played by branchial Na(+)K(+)-ATPase in ion transport during saltwater acclimation process. The responses observed contributed to the strategy adopted by gill cell's basolateral membranes to compensate for osmotic and ionic stressors, to ensure the success of the process of seawater acclimation associated with the downstream trophic migration of juvenile sea lamprey.

  9. Molecular mechanism of ion-ion and ion-substrate coupling in the Na+-dependent leucine transporter LeuT.

    PubMed

    Caplan, David A; Subbotina, Julia O; Noskov, Sergei Yu

    2008-11-15

    Ion-coupled transport of neurotransmitter molecules by neurotransmitter:sodium symporters (NSS) play an important role in the regulation of neuronal signaling. One of the major events in the transport cycle is ion-substrate coupling and formation of the high-affinity occluded state with bound ions and substrate. Molecular mechanisms of ion-substrate coupling and the corresponding ion-substrate stoichiometry in NSS transporters has yet to be understood. The recent determination of a high-resolution structure for a bacterial homolog of Na(+)/Cl(-)-dependent neurotransmitter transporters, LeuT, offers a unique opportunity to analyze the functional roles of the multi-ion binding sites within the binding pocket. The binding pocket of LeuT contains two metal binding sites. The first ion in site NA1 is directly coupled to the bound substrate (Leu) with the second ion in the neighboring site (NA2) only approximately 7 A away. Extensive, fully atomistic, molecular dynamics, and free energy simulations of LeuT in an explicit lipid bilayer are performed to evaluate substrate-binding affinity as a function of the ion load (single versus double occupancy) and occupancy by specific monovalent cations. It was shown that double ion occupancy of the binding pocket is required to ensure substrate coupling to Na(+) and not to Li(+) or K(+) cations. Furthermore, it was found that presence of the ion in site NA2 is required for structural stability of the binding pocket as well as amplified selectivity for Na(+) in the case of double ion occupancy.

  10. Interplay between disulfide bonding and N-glycosylation defines SLC4 Na+-coupled transporter extracellular topography.

    PubMed

    Zhu, Quansheng; Kao, Liyo; Azimov, Rustam; Abuladze, Natalia; Newman, Debra; Kurtz, Ira

    2015-02-27

    The extracellular loop 3 (EL-3) of SLC4 Na(+)-coupled transporters contains 4 highly conserved cysteines and multiple N-glycosylation consensus sites. In the electrogenic Na(+)-HCO3(-) cotransporter NBCe1-A, EL-3 is the largest extracellular loop and is predicted to consist of 82 amino acids. To determine the structural-functional importance of the conserved cysteines and the N-glycosylation sites in NBCe1-A EL-3, we analyzed the potential interplay between EL-3 disulfide bonding and N-glycosylation and their roles in EL-3 topological folding. Our results demonstrate that the 4 highly conserved cysteines form two intramolecular disulfide bonds, Cys(583)-Cys(585) and Cys(617)-Cys(642), respectively, that constrain EL-3 in a folded conformation. The formation of the second disulfide bond is spontaneous and unaffected by the N-glycosylation state of EL-3 or the first disulfide bond, whereas formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected by N-glycosylation. Importantly, EL-3 from each monomer is adjacently located at the NBCe1-A dimeric interface. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. This unique adjacent parallel location of two symmetrically folded EL-3 loops from each monomer resembles a domain-like structure that is potentially important for NBCe1-A function in vivo. Moreover, the formation of this unique structure is critically dependent on the finely tuned interplay between disulfide bonding and N-glycosylation in the membrane processed NBCe1-A dimer.

  11. Molecular simulation of thermodynamic and transport properties for the H{sub 2}O+NaCl system

    SciTech Connect

    Orozco, Gustavo A.; Jiang, Hao; Panagiotopoulos, Athanassios Z.; Moultos, Othonas A.; Economou, Ioannis G.

    2014-12-21

    Molecular dynamics and Monte Carlo simulations have been carried out to obtain thermodynamic and transport properties of the binary mixture H{sub 2}O+NaCl at temperatures from T = 298 to 473 K. In particular, vapor pressures, liquid densities, viscosities, and vapor-liquid interfacial tensions have been obtained as functions of pressure and salt concentration. Several previously proposed fixed-point-charge models that include either Lennard-Jones (LJ) 12-6 or exponential-6 (Exp6) functional forms to describe non-Coulombic interactions were studied. In particular, for water we used the SPC and SPC/E (LJ) models in their rigid forms, a semiflexible version of the SPC/E (LJ) model, and the Errington-Panagiotopoulos Exp6 model; for NaCl, we used the Smith-Dang and Joung-Cheatham (LJ) parameterizations as well as the Tosi-Fumi (Exp6) model. While none of the model combinations are able to reproduce simultaneously all target properties, vapor pressures are well represented using the SPC plus Joung-Cheathem model combination, and all LJ models do well for the liquid density, with the semiflexible SPC/E plus Joung-Cheatham combination being the most accurate. For viscosities, the combination of rigid SPC/E plus Smith-Dang is the best alternative. For interfacial tensions, the combination of the semiflexible SPC/E plus Smith-Dang or Joung-Cheatham gives the best results. Inclusion of water flexibility improves the mixture densities and interfacial tensions, at the cost of larger deviations for the vapor pressures and viscosities. The Exp6 water plus Tosi-Fumi salt model combination was found to perform poorly for most of the properties of interest, in particular being unable to describe the experimental trend for the vapor pressure as a function of salt concentration.

  12. Reversible emission evolution from Ag activated zeolite Na-A upon dehydration/hydration

    SciTech Connect

    Lin, Hui E-mail: fujii@eedept.kobe-u.ac.jp; Imakita, Kenji; Fujii, Minoru E-mail: fujii@eedept.kobe-u.ac.jp

    2014-11-24

    Reversible emission evolution of thermally treated Ag activated zeolite Na-A upon dehydration/hydration in vacuum/water vapor was observed. The phenomenon was observed even for the sample with low Ag{sup +}-Na{sup +} exchanging (8.3%), indicating that the emission from Ag activated zeolites may not come from Ag clusters while from the surrounding coordinated Ag{sup +} ions or Ag{sup 0} atoms. It was disclosed that the characteristic yellow-green emission at ∼560 ± 15 nm is strongly associated with the coordinating water molecules to the Ag{sup +} ions or Ag{sup 0} atoms, which is clear evidence for that the efficient emission from Ag activated zeolites may not originate from the quantum confinement effect.

  13. TRANSPORT

    EPA Science Inventory

    Presentation outline: transport principles, effective solubility; gasoline composition; and field examples (plume diving).
    Presentation conclusions: MTBE transport follows from - phyiscal and chemical properties and hydrology. Field examples show: MTBE plumes > benzene plu...

  14. Role of cell volume variations in Na(+)-K(+)-ATPase recruitment and/or activation in cortical collecting duct.

    PubMed

    Coutry, N; Farman, N; Bonvalet, J P; Blot-Chabaud, M

    1994-05-01

    The aim of this study was to examine whether cell volume variations could play a role in the previously reported Na(+)-K(+)-ATPase pump recruitment and/or activation induced by an increase in intracellular Na concentration (Nai) in cortical collecting ducts (CCD). Isolated CCD from kidneys of aldosterone-repleted mice were incubated in hyper-, hypo-, or isosmotic solutions with and without Na to modify Nai and cell volume independently. Nai, cell volume, and the number of basolateral pumps were measured using 22Na, image analysis, and specific [3H]ouabain binding, respectively. Ouabain-sensitive 86Rb uptake was also measured. In CCD with high Nai, pump recruitment and/or activation was observed only when an increase in tubular volume was associated with Na load. Pump recruitment and/or activation was also induced by cell swelling in the absence of Na load. Recruited and/or activated pumps display an affinity for ouabain and a specific activity (ouabain-sensitive Rb uptake per pump unit) similar to basal pumps. We conclude that 1) cell swelling is implied in the process of Nai-dependent pump recruitment and/or activation, 2) cell swelling can promote pump recruitment and/or activation independently of Na load, 3) basal and recruited and/or activated pumps probably correspond to the same Na(+)-K(+)-ATPase isoform.

  15. CD8+ T cells stimulate Na-Cl co-transporter NCC in distal convoluted tubules leading to salt-sensitive hypertension

    PubMed Central

    Liu, Yunmeng; Rafferty, Tonya M.; Rhee, Sung W.; Webber, Jessica S.; Song, Li; Ko, Benjamin; Hoover, Robert S.; He, Beixiang; Mu, Shengyu

    2017-01-01

    Recent studies suggest a role for T lymphocytes in hypertension. However, whether T cells contribute to renal sodium retention and salt-sensitive hypertension is unknown. Here we demonstrate that T cells infiltrate into the kidney of salt-sensitive hypertensive animals. In particular, CD8+ T cells directly contact the distal convoluted tubule (DCT) in the kidneys of DOCA-salt mice and CD8+ T cell-injected mice, leading to up-regulation of the Na-Cl co-transporter NCC, p-NCC and the development of salt-sensitive hypertension. Co-culture with CD8+ T cells upregulates NCC in mouse DCT cells via ROS-induced activation of Src kinase, up-regulation of the K+ channel Kir4.1, and stimulation of the Cl− channel ClC-K. The last event increases chloride efflux, leading to compensatory chloride influx via NCC activation at the cost of increasing sodium retention. Collectively, these findings provide a mechanism for adaptive immunity involvement in the kidney defect in sodium handling and the pathogenesis of salt-sensitive hypertension. PMID:28067240

  16. (56)Mn, (60)Co, (18)F and (22)Na activity measurements by coincidence technique at VNIIM.

    PubMed

    Evgeny, Tereshchenko; Nikolay, Moiseev; Alexander, Kolodka

    2016-03-01

    For modernization of the Russian national activity standard at the Ionizing Radiation Department of the D.I. Mendeleyev Institute for Metrology, a prototype 4π(LS)-γ(NaI) coincidence arrangement was created, and applied to the standardization of long-lived (60)Co and (22)Na and short-lived (56)Mn and (18)F. Efficiency variation was performed using "grey filters", high voltage variation and variation of low threshold. The main metrological characteristics of the setup were determined: long-term stability, background, dead time, resolution time and temperature dependence. The results obtained have practical applications. The (18)F solution with well-known activity is required for calibration of ionizing chamber used in nuclear medicine. The (56)Mn is used for calibration of manganese bath equipment used in neutron laboratory. The results obtained are in good agreement with 4πγ(NaI)-counting and 4πβ(PC)-γ(NaI) methods of Russian national radioactivity standard. The combined uncertainty (k=2) of results was estimated in the range 1-2%.

  17. Presynaptic Control of Glycine Transporter 2 (GlyT2) by Physical and Functional Association with Plasma Membrane Ca2+-ATPase (PMCA) and Na+-Ca2+ Exchanger (NCX)*

    PubMed Central

    de Juan-Sanz, Jaime; Núñez, Enrique; Zafra, Francisco; Berrocal, María; Corbacho, Isaac; Ibáñez, Ignacio; Arribas-González, Esther; Marcos, Daniel; López-Corcuera, Beatriz; Mata, Ana M.; Aragón, Carmen

    2014-01-01

    Fast inhibitory glycinergic transmission occurs in spinal cord, brainstem, and retina to modulate the processing of motor and sensory information. After synaptic vesicle fusion, glycine is recovered back to the presynaptic terminal by the neuronal glycine transporter 2 (GlyT2) to maintain quantal glycine content in synaptic vesicles. The loss of presynaptic GlyT2 drastically impairs the refilling of glycinergic synaptic vesicles and severely disrupts neurotransmission. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans. Here, we show a novel endogenous regulatory mechanism that can modulate GlyT2 activity based on a compartmentalized interaction between GlyT2, neuronal plasma membrane Ca2+-ATPase (PMCA) isoforms 2 and 3, and Na+/Ca2+-exchanger 1 (NCX1). This GlyT2·PMCA2,3·NCX1 complex is found in lipid raft subdomains where GlyT2 has been previously found to be fully active. We show that endogenous PMCA and NCX activities are necessary for GlyT2 activity and that this modulation depends on lipid raft integrity. Besides, we propose a model in which GlyT2·PMCA2–3·NCX complex would help Na+/K+-ATPase in controlling local Na+ increases derived from GlyT2 activity after neurotransmitter release. PMID:25315779

  18. The Association between Access to Public Transportation and Self-Reported Active Commuting

    PubMed Central

    Djurhuus, Sune; Hansen, Henning S.; Aadahl, Mette; Glümer, Charlotte

    2014-01-01

    Active commuting provides routine-based regular physical activity which can reduce the risk of chronic diseases. Using public transportation involves some walking or cycling to a transit stop, transfers and a walk to the end location and users of public transportation have been found to accumulate more moderate physical activity than non-users. Understanding how public transportation characteristics are associated with active transportation is thus important from a public health perspective. This study examines the associations between objective measures of access to public transportation and self-reported active commuting. Self-reported time spent either walking or cycling commuting each day and the distance to workplace were obtained for adults aged 16 to 65 in the Danish National Health Survey 2010 (n = 28,928). Access to public transportation measures were computed by combining GIS-based road network distances from home address to public transit stops an integrating their service level. Multilevel logistic regression was used to examine the association between access to public transportation measures and active commuting. Distance to bus stop, density of bus stops, and number of transport modes were all positively associated with being an active commuter and with meeting recommendations of physical activity. No significant association was found between bus services at the nearest stop and active commuting. The results highlight the importance of including detailed measurements of access to public transit in order to identify the characteristics that facilitate the use of public transportation and active commuting. PMID:25489998

  19. The association between access to public transportation and self-reported active commuting.

    PubMed

    Djurhuus, Sune; Hansen, Henning S; Aadahl, Mette; Glümer, Charlotte

    2014-12-05

    Active commuting provides routine-based regular physical activity which can reduce the risk of chronic diseases. Using public transportation involves some walking or cycling to a transit stop, transfers and a walk to the end location and users of public transportation have been found to accumulate more moderate physical activity than non-users. Understanding how public transportation characteristics are associated with active transportation is thus important from a public health perspective. This study examines the associations between objective measures of access to public transportation and self-reported active commuting. Self-reported time spent either walking or cycling commuting each day and the distance to workplace were obtained for adults aged 16 to 65 in the Danish National Health Survey 2010 (n = 28,928). Access to public transportation measures were computed by combining GIS-based road network distances from home address to public transit stops an integrating their service level. Multilevel logistic regression was used to examine the association between access to public transportation measures and active commuting. Distance to bus stop, density of bus stops, and number of transport modes were all positively associated with being an active commuter and with meeting recommendations of physical activity. No significant association was found between bus services at the nearest stop and active commuting. The results highlight the importance of including detailed measurements of access to public transit in order to identify the characteristics that facilitate the use of public transportation and active commuting.

  20. Dietary P regulates phosphate transporter expression, phosphatase activity, and effluent P partitioning in trout culture.

    PubMed

    Coloso, R M; King, K; Fletcher, J W; Weis, P; Werner, A; Ferraris, R P

    2003-08-01

    Phosphate utilization by fish is an important issue because of its critical roles in fish growth and aquatic environmental pollution. High dietary phosphorus (P) levels typically decrease the efficiency of P utilization, thereby increasing the amount of P excreted as metabolic waste in effluents emanating from rainbow trout aquaculture. In mammals, vitamin D3 is a known regulator of P utilization but in fish, its regulatory role is unclear. Moreover, the effects of dietary P and vitamin D3 on expression of enzymatic and transport systems potentially involved in phosphate utilization are little known. We therefore monitored production of effluent P, levels of plasma vitamin D3 metabolites, as well as expression of phosphatases and the sodium phosphate cotransporter (NaPi2) in trout fed semipu diets that varied in dietary P and vitamin D3 levels. Mean soluble P concentrations varied markedly with dietary P but not with vitamin D3, and constituted 40-70% of total effluent P production by trout. Particulate P concentrations accounted for 25-50% of effluent P production, but did not vary with dietary P or vitamin D3. P in settleable wastes accounted for <10% of effluent P. The stronger effect of dietary P on effluent P levels is paralleled by its striking effects on phosphatases and NaPi2. The mRNA abundance of the intestinal and renal sodium phosphate transporters increased in fish fed low dietary P; vitamin D3 had no effect. Low-P diets reduced plasma phosphate concentrations. Intracellular phytase activity increased but brushborder alkaline phosphatase activity decreased in the intestine, pyloric caeca, and gills of trout fed diets containing low dietary P. Vitamin D3 had no effect on enzyme activities. Moreover, plasma concentrations of 25-hydroxyvitamin D3 and of 1,25-dihydroxyvitamin D3 were unaffected by dietary P and vitamin D3 levels. The major regulator of P metabolism, and ultimately of levels of P in the effluent from trout culture, is dietary P.