DOCSLIB.ORG

A Possible Role for Na',K+-Atpase in Regulating ATP-Dependent

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Possible Role for Na',K+-Atpase in Regulating ATP-Dependent Proc. Nati. Acad. Sci. USA Vol. 86, pp. 539-543, January 1989 Cell Biology A possible role for Na',K+-ATPase in regulating ATP-dependent endosome acidification RENATE FUCHS, SANDRA SCHMID, AND IRA MELLMAN* Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, P.O. Box 3333, New Haven, CT 06510 Communicated by Robert W. Berliner, September 16, 1988 ABSTRACT Endosomes maintain a slightly acidic internal (CHO) cells by free flow electrophoresis, we have recently pH, which is directly responsible for their ability to ensure demonstrated that, even in vitro, kinetically "early" endo- proper sorting of incoming receptors and ligands during somes-i.e., those involved in rapid receptor recycling-are endocytosis. At least two distinct subpopulations of endosomes less capable of ATP-dependent acidification than are "late" can be distinguished, designated "early" and "late" on the endosomes, a distinct subpopulation associated with the basis of their kinetics of labeling with endocytic tracers. The transport of internalized macromolecules to lysosomes (10). subpopulations differ not only in their functions (rapid receptor Since the factors that regulate endosomal pH remain recycling and transport to lysosomes, respectively) but also in unknown, we have sought to investigate the possible basis for their capacities for acidification in intact cells and in vitro. To pH regulation in early and late endosomes and lysosomes. investigate the possible basis for pH regulation in endosomes, Our results indicate that ATP-dependent proton transport in we have studied the transport properties and ion permeabilities endosomes is electrogenic and is accompanied by the gen- of early and late endosomes isolated from Chinese hamster eration of an interior-positive membrane potential that op- ovary cells. Using endosomes selectively labeled with pH- poses continued proton translocation. Specifically in early sensitive endocytic tracers, we found that ATP-dependent endosomes, acidification appears to be further modulated by acidification is electrogenic, being accompanied by the gener- a second electrogenic ion transport system-namely, the ation of an interior-positive membrane potential which opposes Na',K+-ATPase, which is present in the same membrane. further acidification. While membrane potential and, conse- Since the activity ofthe Na' ,K+-ATPase would also result in quently, acidification was controlled by the influx of permeant the generation ofan interior-positive potential, its presence in anions and efflux of protons and alkali cations, acidification early endosomes from CHO cells might explain the limited was further modulated in Na' and K+-containing buffers by capacity of this subpopulation for acidification in vitro and in the ouabain- and vanadate-sensitive Na',K+-ATPase, which vivo. Indeed, recent measurements ofendosome acidification appears to be a functional component of the endosomal in intact cells have suggested that early endosome acidifica- membrane. The data suggest that electrogenic Na' transport tion is enhanced in cells grown in the Na',K+-ATPase due to Na',K+-ATPase activity contributes to the interior- inhibitor ouabain (11). positive membrane potential, thereby reducing ATP-depen- dent H' transport. Importantly, inhibition of acidification by Na',K+-ATPase activity was found only in early endosomes, MATERIALS AND METHODS consistent with their limited acidification capacity relative to Cells and Cell Culture. CHO cells were maintained in late endosomes and lysosomes. suspension culture in a-MEM supplemented with 5% fetal calf serum (J. R. Scientific, Woodland, CA) and penicillin/ It is now well established that acidic pH in endocytic streptomycin. For experiments, cells were plated in complete organelles plays a critical role in maintaining the orderly medium on 15-cm tissue culture dishes (Falcon) 2-3 days traffic and processing of receptors and ligands internalized before use, after which time the monolayers were slightly during endocytosis (for review, see ref. 1). This is particularly subconfluent. true in endosomes, where the low pH facilitates the disso- Endosome Labeling. Endosomes were labeled with either ciation of many receptor-ligand complexes and, accordingly, of two pH-sensitive endocytic tracers, fluorescein isothio- the recycling offree receptors back to the cell surface. While cyanate (FITC)-conjugated dextran (FITC-dextran) (Mr, endosomes, like lysosomes, are known to lower their internal 70,000; Sigma) (2) or FITC-conjugated transfemn (FITC- pH by an ATP-driven proton pump (2, 3), the mechanisms Tfn) prepared as described (12). For labeling with FITC-Tfn, that regulate acidification in either organelle are unclear. which selectively labels early endosomes (10, 12), the mono- Lysosomes are typically the most acidic organelle in mam- layers were washed three times with phosphate-buffered malian cells and generally maintain their internal pH at 4.7- saline (PBS), incubated in serum-free medium for 30 min at 4.8 (4). Endosomes exhibit a greater range of pH values that 37TC, and then in serum-free medium containing FITC-Tfn can vary between 5.0 and 6.5 within a single cell (5, 6). (20 ,ug/ml) for an additional 30-60 min. Inclusion of uncon- Given the role played by endosomal pH in regulating jugated (250 ktg/ml) during uptake decreased the amount of endocytic membrane traffic, it is likely that the observed cell-associated FITC to undetectable levels, indicating that variations in pH are functionally significant. In intact cells, FITC-Tfn endocytosis was specific for the Tfn receptor. For material internalized during endocytosis encounters endo- some experiments, surface-bound FITC-Tfn was removed somes of increasingly acidic pH en route to lysosomes (3, 5- prior to harvest by incubation in pH 5 and then in pH 7 9). This gradient in pH reflects the existence of distinct medium containing the iron chelator desferoxamine (12). subpopulations of endosomes that differ not only in their Labeling with FITC-dextran (5-10 mg/ml) was performed capacity for acidification but also in composition and function in complete medium at 370C for 5 min to label early endo- (10). Using endosomes isolated from Chinese hamster ovary somes, 5 min followed by a 10-min chase in FITC- dextran-free medium to label late endosomes, and 5 min The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: FITC, fluorescein isothiocyanate; Tfn, transferrin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 539 Downloaded by guest on September 25, 2021 540 Cell Biology: Fuchs et al. Proc. Natl. Acad. Sci. USA 86 (1989) followed by a 45-min chase to label lysosomes, as described tion. Fig. 1A illustrates the ATP-dependent acidification of (10). After the labeling period, the monolayers were washed endosomes from CHO cells equilibrated in isosmotic KCl several times with cold PBS and harvested with a rubber buffer. Addition of 2.5 mM ATP caused a rapid quenching of scraper. FITC fluorescence reflecting intravesicular acidification. Isolation of Enriched Endosome Fractions. FITC-Tfn (or When Cl- was replaced with a relatively impermeant organic FITC-dextran)-labeled cells were washed three times by anion (gluconate), acidification was partially inhibited (Fig. centrifugation (350 x g, 5 min at 40C) and resuspended in cold 1B). This suggests that external Cl- helps to dissipate an PBS. The final pellet was suspended in cold TEA buffer (0.25 interior-positive membrane potential formed by the electro- M sucrose/10 mM triethanolamine/10 mM NaOAc/1 mM genic translocation of protons into the endosome. Indeed, EDTA/1 mM phenylmethylsulfonyl fluoride, pH 7.4) and acidification in potassium gluconate buffer was stimulated by disrupted with a stainless steel Dounce homogenizer (Kontes). the K+ ionophore valinomycin (Fig. 1B) by permitting the Enriched endosomal fractions were prepared from the post- rapid efflux of internal K+ down the electrical gradient. nuclear supernatants by centrifugation in discontinuous su- Addition of valinomycin to endosomes in KCI buffer had no crose gradients (40,000 rpm, 1.5 hr, 40C; Beckman SW40 rotor) effect (Fig. 1A). Thus, ATP-driven endosome acidification is (12). The gradients were formed by adjusting the postnuclear electrogenic and, like acidification of isolated lysosome (16, supernatant to 1.15 M sucrose and overlaying with 1 M, 0.86 17), coated vesicle (18, 19), and Golgi fractions (20), it is M, and 0.25 M sucrose in TEA buffer; most of the FITC- partly dependent on the membrane's permeability to Cl-. containing endosomes were recovered at the 0.86/1.0 M In addition, the endosomes were found to have a significant interface. As described (10), cell fractionation by free flow conductance for H', which could also be involved in regu- electrophoresis and Percoll density-gradient centrifugation lating H' accumulation via an electrogenic ATPase. Follow- confirmed that under the labeling conditions used, the en- ing acidification of KCI-equilibrated endosomes, removal of docytic tracers were confined to early and/or late endosomes ATP (by adding hexokinase and glucose) resulted in a as indicated. dissipation of the transmembrane pH gradient (Fig. 1C). The Cell-Free Assay of ATP-Dependent Acidification. ATP- rate of H' efflux after removal of ATP (ti,, -5.8 min) was dependent acidification of isolated endosomes was deter- almost half
Load more

Recommended publications
  • Molecular Regulation and Physiology of the H ,K -Atpases in Kidney
    Molecular Regulation and Physiology of the H؉,K؉-ATPases in Kidney Juan Codina and Thomas D. DuBose Jr Two H؉,K؉-adenosine triphosphatase (ATPase) proteins participate in K؉ absorption and ؉ ؉ ؉ H secretion in the renal medulla. Both the gastric (HK␣1) and colonic (HK␣2)H,K - ATPases have been localized and characterized by a number of techniques, and are known to be highly regulated in response to acid-base and electrolyte disturbances. Both ATPases are dimers of composition ␣/␤ that localize to the apical membrane and both interact with the tetraspanin protein CD63. Although CD63 interacts with the carboxy-terminus of the subunit of the colonic H؉,K؉-ATPase, it interacts with the ␤-subunit of the gastric-␣ H؉,K؉-ATPase. Pharmacologically, both ATPases are distinct; for example, the gastric H؉,K؉-ATPase is inhibited by Sch-28080, but the colonic H؉,K؉-ATPase is inhibited by .ouabain (a classic inhibitor of the Na؉-pump) and is completely insensitive to Sch-28080 The ␣-subunit of the colonic H؉,K؉-ATPase is the only subunit of the X؉,K؉-ATPase superfamily that has 3 different splice variants that emerge by deletion or elongation of the amino-terminus. The messenger RNA and protein of one of these splice variants (HK␣2C)is specifically up-regulated in newborn rats and becomes undetectable in adult rats. There- fore, HK␣2, in addition to its role in potassium and acid-base homeostasis, appears to play a significant role in early growth and development. Finally, because chronic hypokalemia appears to be the most potent stimulus for upregulation of HK␣2, we propose that the HK␣2 participates importantly in the maintenance of chronic metabolic alkalosis.
    [Show full text]
  • SERCA in Genesis of Arrhythmias: What We Already Know and What Is New?
    Review 43 SERCA in genesis of arrhythmias: what we already know and what is new? Nilüfer Erkasap Department of Physiology, Medical Faculty, Eskiflehir Osmangazi University, Eskiflehir, Turkey ABSTRACT This review mainly focuses on the structure, function of the sarco(endo)plasmic reticulum calcium pump (SERCA) and its role in genesis of arrhythmias. SERCA is a membrane protein that belongs to the family of P-type ion translocating ATPases and pumps free cytosolic calcium into intracellular stores. Active transport of Ca2+ is achieved, according to the E1-E2 model, changing of SERCA structure by Ca2+. The affinity of Ca2+ -binding sites varies from high (E1) to low (E2). Three different SERCA genes were identified-SERCA1, SERCA2, and SERCA3. SERCA is mainly represented by the SERCA2a isoform in the heart. In heart muscle, during systole, depolarization triggers the release of Ca2+ from the sarcoplasmic reticulum (SR) and starts contraction. During diastole, muscle relaxation occurs as Ca2+ is again removed from cytosol, predominantly by accumulation into SR via the action of SERCA2a. The main regulator of SERCA2a is phospholamban and another regulator proteolipid of SERCA is sarcolipin. There are a lot of studies on the effect of decreased and/or increased SERCA activity in genesis of arrhythmia. Actually both decrease and increase of SERCA activity in the heart result in some pathological mechanisms such as heart failure and arrhythmia. (Anadolu Kardiyol Derg 2007: 7 Suppl 1; 43-6) Key words: sarco(endo)plasmic reticulum, SERCA, arrhythmia, calcium channels Introduction from cytosol, predominantly by accumulation into sarcoplasmic reticulum via the action of sarco(endo)plasmic reticulum Cardiac physiology is a major area of research in basic and Ca ATPase (SERCA).
    [Show full text]
  • Specific Copb Transporter: Revising P1B-Type Atpase Classification
    + Cu -specific CopB transporter: Revising P1B-type ATPase classification Rahul Purohita,b, Matthew O. Rossa,b, Sharon Bateluc, April Kusowskic,d, Timothy L. Stemmlerc,d, Brian M. Hoffmana,b, and Amy C. Rosenzweiga,b,1 aDepartment of Molecular Biosciences, Northwestern University, Evanston, IL 60208; bDepartment of Chemistry, Northwestern University, Evanston, IL 60208; cDepartment of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48201; and dSchool of Medicine, Wayne State University, Detroit, MI 48201 Contributed by Amy C. Rosenzweig, January 12, 2018 (sent for review December 14, 2017; reviewed by Megan M. McEvoy and Gabriele Meloni) The copper-transporting P1B-ATPases, which play a key role in cellu- metal specificities of the P1B-5 (PCP motif), P1B-6 (SCA motif), lar copper homeostasis, have been divided traditionally into two and P1B-7-ATPases (CSC motif) remain unclear, although some 2+ 2+ subfamilies, the P1B-1-ATPases or CopAs and the P1B-3-ATPases or evidence links the P1B-5-ATPases to Ni and Fe (20, 21). The + CopBs. CopAs selectively export Cu whereas previous studies and remaining two groups are the copper transporters. The P1B-1- + bioinformatic analyses have suggested that CopBs are specific for ATPases, which include ATP7A and ATP7B, transport Cu 2+ 2+ Cu export. Biochemical and spectroscopic characterization of (9, 22), whereas the P1B-3-ATPases are proposed to transport Cu Sphaerobacter thermophilus CopB (StCopB) show that, while it does (23, 24). These two subfamilies differ from one another in several 2+ bind Cu , the binding site is not the prototypical P1B-ATPase trans- ways. First, the TM helix 4 motif is CPC in the P1B-1-ATPases and membrane site and does not involve sulfur coordination as proposed CPH in the P1B-3-ATPases.
    [Show full text]
  • Ca2+-Atpase Molecules As a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum
    International Journal of Molecular Sciences Article Ca2+-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum Jun Nakamura 1,*, Yuusuke Maruyama 1, Genichi Tajima 2, Yuto Komeiji 1, Makiko Suwa 3 and Chikara Sato 1,* 1 Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan; [email protected] (Y.M.); [email protected] (Y.K.) 2 Institute for Excellence in Higher Education, Tohoku University, 41 Kawauchi, Aoba-ku, Sendai, Miyagi 980-8576, Japan; [email protected] 3 Biological Science Course, Graduate School of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuou-ku, Sagamihara, Kanagawa 252-5258, Japan; [email protected] * Correspondence: [email protected] (J.N.); [email protected] (C.S.) Abstract: The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs.
    [Show full text]
  • ATP Synthase: Structure,Abstract: Functionlet F Denote a Eld and and Let Inhibitionv Denote a Vector Space Over F with Nite Positive Dimension
    Spec. Matrices 2019; 7:1–19 Research Article Open Access Kazumasa Nomura* and Paul Terwilliger BioMol Concepts 2019; 10: 1–10 Self-dual Leonard pairs Research Article Open Access https://doi.org/10.1515/spma-2019-0001 Prashant Neupane*, Sudina Bhuju, Nita Thapa,Received Hitesh May 8, 2018; Kumar accepted Bhattarai September 22, 2018 ATP Synthase: Structure,Abstract: FunctionLet F denote a eld and and let InhibitionV denote a vector space over F with nite positive dimension. Consider a pair A, A∗ of diagonalizable F-linear maps on V, each of which acts on an eigenbasis for the other one in an irreducible tridiagonal fashion. Such a pair is called a Leonard pair. We consider the self-dual case in which https://doi.org/10.1515/bmc-2019-0001 there exists an automorphism of the endomorphism algebra of V that swaps A and A∗. Such an automorphism phosphate (Pi), along with considerable release of energy. received September 18, 2018; accepted December 21, 2018. is unique, and called the duality A A∗. In the present paper we give a comprehensive description of this ADP can absorb energy and regain↔ the group to regenerate duality. In particular, we display an invertible F-linear map T on V such that the map X TXT− is the duality Abstract: Oxidative phosphorylation is carried out by an ATP molecule to maintain constant ATP concentration. → A A∗. We express T as a polynomial in A and A∗. We describe how T acts on ags, decompositions, five complexes, which are the sites for electron transport↔ Other than supporting almost all the cellular and ATP synthesis.
    [Show full text]
  • Gastric H,K-Atpase As a Drug Target
    UCLA UCLA Previously Published Works Title Gastric H,K-ATPase as a drug target Permalink https://escholarship.org/uc/item/1g5606x9 Journal Digestive Diseases and Sciences, 51(5) ISSN 0163-2116 Authors Shin, Jai M Sachs, G Publication Date 2006-05-01 Peer reviewed eScholarship.org Powered by the California Digital Library University of California The Gastric H,K-ATPase as a Drug Target Jai Moo Shin and George Sachs* Department of Physiology and Medicine, University of California at Los Angeles, and VA Greater Los Angeles Healthcare System, Los Angeles, California, CA90073, USA * To whom correspondence should be addressed: at Membrane Biology Laboratory, VA Greater Los Angeles Healthcare System, 11301 Wilshire Blvd., Bldg. 113, Rm. 324, Los Angeles, CA 90073 Tel: (310) 268-4672 Fax: (310) 312-9478 e-mail: [email protected] 1 Introduction Gastric acid is secreted by parietal cells in the stomach. These have two known acid stimulatory receptors the H2-receptor and the muscarinic M3 receptor. Gastrin, the major endocrine activator of acid secretion, exerts its action via release of histamine from the ECL cell as does pituitary adenylate cyclase activating peptide (PACAP), a neural mediator of activation of acid secretion. Antagonists of the former two stimulants inhibit gastric acid secretion. Cholinergic receptor antagonists have many side effects and are relatively weak inhibitors at therapeutic doses as compared toH2-receptor antagonists. These drugs were widely developed in the 1970’s and 1980’s and became the first really useful medications for healing of peptic ulcers. However, although good for healing peptic ulcers, they were less effective in treatment of erosive esophagitis.
    [Show full text]
  • Inhibin, Activin, Follistatin and FSH Serum Levels and Testicular
    REPRODUCTIONRESEARCH Expression of Atp8b3 in murine testis and its characterization as a testis specific P-type ATPase Eun-Yeung Gong, Eunsook Park, Hyun Joo Lee and Keesook Lee School of Biological Sciences and Technology, Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea Correspondence should be addressed to K Lee; Email: [email protected] Abstract Spermatogenesis is a complex process that produces haploid motile sperms from diploid spermatogonia through dramatic morphological and biochemical changes. P-type ATPases, which support a variety of cellular processes, have been shown to play a role in the functioning of sperm. In this study, we isolated one putative androgen-regulated gene, which is the previously reported sperm-specific aminophospholipid transporter (Atp8b3, previously known as Saplt), and explored its expression pattern in murine testis and its biochemical characteristics as a P-type ATPase. Atp8b3 is exclusively expressed in the testis and its expression is developmentally regulated during testicular development. Immunohistochemistry of the testis reveals that Atp8b3 is expressed only in germ cells, especially haploid spermatids, and the protein is localized in developing acrosomes. As expected, from its primary amino acid sequence, ATP8B3 has an ATPase activity and is phosphorylated by an ATP-producing acylphosphate intermediate, which is a signature property of the P-Type ATPases. Together, ATP8B3 may play a role in acrosome development and/or in sperm function during fertilization. Reproduction (2009) 137 345–351 Introduction the removal of testosterone by hypophysectomy or ethane dimethanesulfonate (EDS) treatment in rats leads to the Spermatogenesis is one of most complex processes of cell failure of spermatogenesis (Russell & Clermont 1977, differentiation in animals, in which diploid spermatogo- Bartlett et al.
    [Show full text]
  • Structure and Mechanism of the Atpase That Powers Viral Genome
    Structure and mechanism of the ATPase that powers PNAS PLUS viral genome packaging Brendan J. Hilberta,1, Janelle A. Hayesa,1, Nicholas P. Stonea, Caroline M. Duffya, Banumathi Sankaranb, and Brian A. Kelcha,2 aDepartment of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605; and bBerkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Edited by Wei Yang, National Institutes of Health, Bethesda, MD, and approved June 5, 2015 (received for review April 9, 2015) Many viruses package their genomes into procapsids using an with portal; (ii) consequently, it was proposed that the active site ATPase machine that is among the most powerful known biological residues that catalyze ATP hydrolysis are derived from one sub- motors. However, how this motor couples ATP hydrolysis to DNA unit exclusively; (iii) the pore of the ring has a net negative charge translocation is still unknown. Here, we introduce a model system and is largely unconserved; and (iv) DNA was proposed to be with unique properties for studying motor structure and mecha- bound primarily by the nuclease domain. This model is sub- nism. We describe crystal structures of the packaging motor ATPase stantially different from models observed for other nucleic acid domain that exhibit nucleotide-dependent conformational changes translocases of the ASCE family, in which the pore is largely involving a large rotation of an entire subdomain. We also identify conserved and positively charged, and ATP hydrolysis is cata- the arginine finger residue that catalyzes ATP hydrolysis in a neigh- lyzed in trans (11–15). Resolving these disparities between the boring motor subunit, illustrating that previous models for motor proposed assembly and conserved features of the ASCE family is structure need revision.
    [Show full text]
  • Sarcoplasmic Reticulum Ca2+-Atpase (SERCA) Activity During the Transition to Endothermy in an Altricial Bird Edwin R
    © 2019. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2019) 222, jeb201111. doi:10.1242/jeb.201111 RESEARCH ARTICLE Sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity during the transition to endothermy in an altricial bird Edwin R. Price§, Tushar S. Sirsat*, Sarah K. G. Sirsat‡ and Edward M. Dzialowski ABSTRACT endothermic response is dependent upon the maturation of multiple Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a physiological systems, including the neural thermoregulatory centers transmembrane pump critical to muscle calcium cycling during and networks, oxygen and substrate supply pathways, and the contraction, and SERCA has also been proposed as the basis for a oxidative and contractile machinery of skeletal muscles (reviewed by non-shivering thermogenesis mechanism in birds. Despite its potential Price and Dzialowski, 2018). 2+ importance to both shivering and non-shivering thermogenesis, the Sarco(endo)plasmic reticulum Ca -ATPase (SERCA) is a activity of this transporter has rarely been studied in altricial birds, membrane-bound transporter that pumps calcium ions from the and never during the developmental transition from ectothermy to cytosol into the lumen of the sarcoplasmic reticulum (SR) following endothermy. Here, we describe SERCA activity in the pectoralis muscle muscle contraction. High SERCA activity is crucial for rapid and heart ventricle of red-winged blackbird (Agelaius phoeniceus) contraction rates in muscles (Rome and Lindstedt, 1998), as the nestlings, fledglings and adults. Additionally, using a diet manipulation, transporter restores both cytosolic and SR calcium levels that are we tested the hypothesis that muscle SERCA activity is affected by necessary for signaling contraction. Maturation of SERCA activity ’ dietary fatty acid composition, as has been shown in some previous could therefore be important for developing the muscle scapacityfor studies.
    [Show full text]
  • Mechanism of Na-K-Atpase Inhibition by PGE2 in Intestinal Epithelial Cells
    cells Article Mechanism of Na-K-ATPase Inhibition by PGE2 in Intestinal Epithelial Cells Niraj Nepal , Subha Arthur, Jennifer Haynes, Balasubramanian Palaniappan and Uma Sundaram * Department of Clinical and Translational Sciences, Appalachian Clinical and Translational Science Institute, Joan C. Edwards School of Medicine, Marshall University, 1600 Medical Center Drive, Huntington, WV 25701, USA; [email protected] (N.N.); [email protected] (S.A.); [email protected] (J.H.); [email protected] (B.P.) * Correspondence: [email protected]; Tel.: +1-304-691-1841 Abstract: The primary means of intestinal absorption of nutrients by villus cells is via Na-dependent nutrient co-transporters located in the brush border membrane (BBM). These secondary active co- transport processes require a favorable transcellular Na gradient that is provided by Na-K-ATPase. In chronic enteritis, malabsorption of essential nutrients is partially due to inhibition of villus Na- K-ATPase activity mediated by specific immune inflammatory mediators that are known to be elevated in the inflamed mucosa. However, how Prostaglandin E2 (PGE2), a specific mediator of nutrient malabsorption in the villus BBM, may mediate the inhibition of Na-K-ATPase is not known. Therefore, this study aimed to determine the effect of PGE2 on Na-K-ATPase in villus cells and define its mechanism of action. In vitro, in IEC-18 cells, PGE2 treatment significantly reduced Na-K-ATPase activity, accompanied by a significant increase in the intracellular levels of cyclic Adenosine Monophosphate (cAMP). The treatment with cAMP analog 8-Bromo-cAMP mimicked the PGE2-mediated effect on Na-K-ATPase activity, while Rp-cAMP (PKA inhibitor) pretreatment Citation: Nepal, N.; Arthur, S.; reversed the same.
    [Show full text]
  • THE ROLE of V-Atpase in NEURONAL and ENDOCRINE SYSTEMS
    J. exp. Biol. 172, 171-178 (1992) j-y j Printed in Great Britain © The Company of Biologists Limited 1992 THE ROLE OF V-ATPase IN NEURONAL AND ENDOCRINE SYSTEMS BY YOSHINORI M0R1YAMA*, MASATOMO MAEDA AND MASAMITSU FUTAI Department of Organic Chemistry and Biochemistry, Institute of Scientific and Industrial Research, Osaka University, Ibaraki Osaka 567, Japan Summary Synaptic vesicles have important roles in the neural transmission at nerve terminals: the storage and the controlled exocytosis of neurotransmitters. At least two different factors are responsible for the concentration process: the vacuolar-type H+-ATPase (V- ATPase), establishing an electrochemical gradient of protons, and specific transport systems for transmitters. We will discuss our recent progress on the energy-transducing systems in synaptic vesicles: (1) structural aspects of V-ATPase; (2) energy coupling of transport of transmitters; (3) reconstitution of transporters; (4) effects of neurotoxins and neuron blocking agents; (5) function of synaptic-vesicle-like microvesicles from endocrine tissues. Introduction Synaptic vesicles play an important role in neuronal transmission in nerve termini. They accumulate high concentrations of neurotransmitters and, upon receiving the proper signal, secrete them by exocytosis. Similarly, secretory granules in endocrine cells accumulate biogenic amines and secrete them by exocytosis (Ueda, 1986; Johnson, 1988; Njus et al. 1987). We have been interested in the bioenergetics of the accumulation process, which is an initial step in neuronal transmission or biogenic amine secretion. Our results are consistent with a chemiosmotic energy coupling mechanism for the accumulation: a V-ATPase forms an electrochemical gradient of protons across vesicular membranes and transmitters are taken up through specific transporters coupled with this gradient (Fig.
    [Show full text]
  • Characterization of Plasmodium Falciparum ATP-Dependent DNA
    Limudomporn et al. Malar J (2016) 15:526 DOI 10.1186/s12936-016-1573-2 Malaria Journal RESEARCH Open Access Characterization of Plasmodium falciparum ATP‑dependent DNA helicase RuvB3 Paviga Limudomporn1, Saengduen Moonsom1, Ubolsree Leartsakulpanich2, Pattra Suntornthiticharoen3, Songsak Petmitr4, Michael Weinfeld5 and Porntip Chavalitshewinkoon‑Petmitr1* Abstract Background: Malaria is one of the most serious and widespread parasitic diseases affecting humans. Because of the spread of resistance in both parasites and the mosquito vectors to anti-malarial drugs and insecticides, controlling the spread of malaria is becoming difficult. Thus, identifying new drug targets is urgently needed. Helicases play key roles in a wide range of cellular activities involving DNA and RNA transactions, making them attractive anti-malarial drug targets. Methods: ATP-dependent DNA helicase gene (PfRuvB3) of Plasmodium falciparum strain K1, a chloroquine and pyrimethamine-resistant strain, was inserted into pQE-TriSystem His-Strep 2 vector, heterologously expressed and affinity purified. Identity of recombinant PfRuvB3 was confirmed by western blotting coupled with tandem mass spectrometry. Helicase and ATPase activities were characterized as well as co-factors required for optimal function. Results: Recombinant PfRuvB3 has molecular size of 59 kDa, showing both DNA helicase and ATPase activities. Its 2 2 2 2 helicase activity is dependent on divalent cations (Cu +, Mg +, Ni+ or Zn+ ) and ATP or dATP but is inhibited by high NaCl concentration (>100 mM). PfPuvB3 is unable to act on blunt-ended duplex DNA, but manifests ATPase activity in the presence of either single- or double-stranded DNA. PfRuvB3.is inhibited by doxorubicin, daunorubicin and netrop‑ sin, known DNA helicase inhibitors.
    [Show full text]