Genomic Organization and Developmental Expression of Aquaporin-5 in Lung M. Douglas Lee, Landon S. King, Søren Nielsen and Peter Agre Chest 1997;111;111S-113S DOI 10.1378/chest.111.6_Supplement.111S The online version of this article, along with updated information and services can be found online on the World Wide Web at: http://chestjournal.chestpubs.org/content/111/6_Supple ment/111S.citation CHEST is the official journal of the American College of Chest Physicians. It has been published monthly since 1935. Copyright 1997 by the American College of Chest Physicians, 3300 Dundee Road, Northbrook, IL 60062. All rights reserved. No part of this article or PDF may be reproduced or distributed without the prior written permission of the copyright holder. (http://chestjournal.chestpubs.org/site/misc/reprints.xhtml) ISSN:0012-3692 Downloaded from chestjournal.chestpubs.org by guest on February 19, 2010 1997 by the American College of Chest Physicians Studies in Human Lung Epithelial Cells Am Rev Respir Dis 1990; 142:1250-57 5 Suzuki S, Zuege D, Berthiaume Y. Sodium-independent mod¬ Human alveolar carcinoma cells (A549) were plated ulation of Na+-K+-ATPase activity bybeta-adrenergic agonist in for 24 h prior to infection with MOIs of 1 to 50 of alveolar type II cells. Am J Physiol 1995; 268:L983-L90 AdMRCMVa!, AdMRCMVpi, and AdMRCMVp-Gal. 6 Olivera W, Ridge K, Wood LDH, et al. Active sodium Cells infected with MOIs of 5 to 50 of AdMRCMVc^ transport and alveolar epithelial Na-K-ATPase increase dur¬ demonstrated twofold to threefold increases in ouabain- ing subacute hyperoxia in rats. Am J Physiol 1994; 266:L577- inhibitable 86Rb+ uptake 24 h after infection. No changes L84 in Na+/K+-ATPase function were noted following infec¬ 7 Schneeberger EE, McCarthy KM. Cytochemical localization tion with AdMRCMVPi or AdMRCMVp-Gal. Northern of Na, K-ATPase in rat type II pneumocytes. J Appl Physiol blot of total RNA a cDNA 1986; 20:1584-89 analysis using rat-specific probe 8 O'Brodovich HM. The role of active revealed the of ax mRNA in cells infected Na+ transport by lung presence only in the clearance of fluid. New Horizons with AdMRCMVcq; no change in px message was noted. epithelium airspace Western blot showed increased 1995; 3:240-47 analysis significantly ax 9 K, Ilekis Factor P, et al. Inhibition of the in cells infected with These find¬ Ridge J, Na,K protein AdMRCMVcq. ATPase by transfection of alveolar type 2 cells with antisense ings suggest that ax subunit may be the rate-limiting RNA to the Na,K ATPase [abstract]. Am Rev Respir Dis subunit in A549 cells and that Na+/K+-ATPase function 1992; 145:A832 can be increased in these cells using gene transfer. 10 McGrory W7J, Bautista DS, Graham FL. A simple technique The human ax isoform is two to three logs more for the rescue of early region 1 mutations into infectious sensitive to ouabain than is the rat a2 isoform. To dem¬ human adenovirus type 5. Virology 1988; 163:614-17 onstrate transgene expression, ouabain sensitivity was determined in A549 cells by ascertaining the concentra¬ tion of ouabain that produced a 50% reduction in 86Rb+ uptake (IC50). A549 cells were infected with AdM- Genomic and RCMVcq at an MOI of 25, and 86Rb+ uptake was Organization measured using 15 different concentrations of ouabain Developmental Expression of (IX10-11 to 1X10~3). IC50 values were determined using Aquaporin-5 in Lung* a computerized nonlinear least squares regression func¬ tion to test for the of one or two designed presence M. Douglas Lee, MD; Landon S. King, MD; isozymes of a receptor with different affinities for a ligand S0ren Nielsen, PhD; and Peter Agre, MD (ouabain). AdMRCMVax-infected cells demonstrated two distinct IC50 values (IC50(1)=3.5X10~ IC50(2)- Sham and (CHEST 1997; 111:111S-113S) 3.2X10"5). AdMRCMVp-Gal infected cells "T\ of the of water transporters one was not iscovery aquaporin family each demonstrated IC50 that different from -"-^ a molecular for the first The identification of a second two provided explanation osmotically ax IC50. a1 IC50 driven water transport across cell membranes of mamma¬ in or logs greater than that found the sham AdMRCMVP- lian and tissues. At there are six known Gal infected cells that of two functional plant present, suggests presence mammalian aquaporins distributed in a wide variety of ax isozymes: the endogenous ouabain-sensitive human ax tissues and cell In the distribution of each and the ouabain-resistant rat types. general, transgenic aY. aquaporin is unique, though there are some scattered sites These results demonstrate that adenoviruses can effi¬ of (reviewed and Recent identifi¬ transfer to alveolar cells and overlap by King Agre1). ciently genes lung epithelial cation and characterization of the genes encoding these augment Na+/K+-ATPase abundance and function. The have enabled to establish¬ to infection with individual subunits differs aquaporins investigators begin response ing a role for aquaporins in the pathogenesis of several between cell types such that the Px subunit could be disease states.23 the wealth of evidence in recent for rat ATII cells and be the rate- Despite rate-limiting a! may years to explain the physiology of salt transport in the lung, limiting subunit for human A549 cells in vitro. Conceiv¬ the molecular mechanisms for transcellular movement of ably, gene transfer to the alveolar epithelium may be water in the lower respiratory tract remain poorly under¬ applicable to the prevention and treatment of pulmonary stood. edema. Four aquaporins have been identified in the lung by Northern blot or References analysis immunoblotting4'6 (Table 1). 1 Sznajder JI, Olivera WG, Ridge KM, et al. Mechanisms of *From the Departments of Pulmonary and Critical Care Medi¬ lung liquid clearance during hyperoxia in isolated rat lungs. cine (Drs. Lee and King), Medicine and Biological Chemistry Am J Respir Crit Care Med 1995; 151:1519-25 (Drs. Lee, King, and Agre), Johns Hopkins University, Balti¬ 2 Rutschman DH, Olivera W, Sznajder JI. Active transport and more; and Cell Biology, Institute of Anatomy (Dr. Nielsen), passive liquid movement in isolated perfused rat lungs. J Appl University of Aarhus, Denmark. 75:1574-80 Supported in part by NIH ROI grants EY11239, HL48268, and Physiol 1993; HL33991 NRSA F32 HL09420 and NRSA 3 Goodman BE, Kim K, Crandall ED. Evidence for active (P.A.), (M.D.L.), F32 HL09119-02 (L.K.); Dr. Lee was recipient of an Allen and sodium transport across alveolar epithelium of isolated rat Hanburys Pulmonary Fellowship Award. lung. J Appl Physiol 1987; 62:2460-66 Reprint requests: Peter Agre, MD, Department ofBiological Chem¬ 4 Matthay M, Wiener-Kronish J. Intact epithelial barrier func¬ istry, Johns Hopkins University School ofMedicine, 725 N Wolfe St, tion is critical for the resolution of alveolar edema in humans. Baltimore, MD 21205-2185; email: [email protected] CHEST/ 111 /6/JUNE, 1997 SUPPLEMENT 111S Downloaded from chestjournal.chestpubs.org by guest on February 19, 2010 1997 by the American College of Chest Physicians Table 1.Aquaporins in Lung induction of AQP1, AQP5 was expressed in rat lung 1 to 2 after with levels into Human days birth, protein steadily increasing Gene adulthood.11 While AQP1 expression was induced in both Protein Location Expression in Lung perinatal and adult rat lung by corticosteroids,4 AQP5 was not induced in rat lung at any age. Immunoelectron Aquaporin-1 7pl4 Peribronchial endothelium, visceral localization demonstrated in the rare alveolar microscopic AQP5 apical pleura, pneumocytes membrane of I was not Aquaporin-3 9p21 -» 12 Basolateral surface of bronchial type pneumocytes; AQP5 present epithelium in type II pneumocytes, airway epithelium, or vascular Aquaporin-4 18qll.2-12.1 Basolateral surface of bronchial structures. and tracheal epithelium These studies have defined the gene structure for Aquaporin-5 12ql3 Type I pneumocytes, trachea, AQPS and localized the cell-specific expression of bronchial submucosa AQPS protein in alveolar epithelium. Characterization of the AQPS gene structure should aid future studies directed at defining the promoter-specific elements is in the in vascular required for AQPS gene regulation during normal Aquaporin-1 (AQP1) expressed lung and in clinical disorders of endothelium, visceral pleura, and a subset of pneumo¬ development respiratory cytes.4 AQP4, the major brain aquaporin,7 localizes to tissue. The abundance of AQP5 on the apical surface of the basolateral membrane of bronchial and tracheal type I pneumocytes suggests a role in alveolar trans- epthelium in lung.5 AQP3 is expressed in the lung membrane water movement, though the lack of a exclusively on the basolateral surface of tracheal epithe¬ subapical or basolateral location for AQP5 may indicate lium.5 The absence of AQPs 1, 3, and 4 in type I that other as yet undefined aquaporins may participate pneumocytes or on the apical surface of airway epithe¬ in the movement of water across the alveolar epithe¬ lial cells suggests the presence of another aquaporin in lium. Additional studies aimed at addressing this issue those locations. Recently, the complementary DNA and determining the precise localization of AQP5 in (cDNA) for the fifth mammalian aquaporin (AQP5) was upper airway epithelium are currently underway. isolated from rat; Northern blot analysis demonstrated AQP5 in rat salivary and lacrimal glands, cornea, and lung.6 Herein we discuss the isolation and characteriza¬ References tion of the human cDNA and as well as the AQP5 gene, 1 P. of the water and distribution of in rat King LS, Agre Pathophysiology aquaporin ontogeny AQP5 lung. channels. Annu Rev 1996; 58:619-48 the rat cDNA as a molecular an Physiol Using AQP5 probe, 2 Deen PMT, Knoers NVAM, et al. cDNA clone was isolated from a human submax- Verdijk MAJ, Require¬ AQPS ment of human renal water channel aquaporin-2 for illary gland library.