Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1998 Oct 1;102(7):1421–1430. doi: 10.1172/JCI3214

Augmentation of lung liquid clearance via adenovirus-mediated transfer of a Na,K-ATPase beta1 subunit gene.

P Factor 1, F Saldias 1, K Ridge 1, V Dumasius 1, J Zabner 1, H A Jaffe 1, G Blanco 1, M Barnard 1, R Mercer 1, R Perrin 1, J I Sznajder 1
PMCID: PMC508990  PMID: 9769335

Abstract

Previous studies have suggested that alveolar Na,K-ATPases play an important role in active Na+ transport and lung edema clearance. We reasoned that overexpression of Na,K-ATPase subunit genes could increase Na,K-ATPase function in lung epithelial cells and edema clearance in rat lungs. To test this hypothesis we produced replication deficient human type 5 adenoviruses containing cDNAs for the rat alpha1 and beta1 Na,K-ATPase subunits (adMRCMValpha1 and adMRCMVbeta1, respectively). As compared to controls, adMRCMVbeta1 increased beta1 subunit expression and Na,K-ATPase function by 2. 5-fold in alveolar type 2 epithelial cells and rat airway epithelial cell monolayers. No change in Na,K-ATPase function was noted after infection with adMRCMValpha1. Rat lungs infected with adMRCMVbeta1, but not adMRCMValpha1, had increased beta1 protein levels and lung liquid clearance 7 d after tracheal instillation. Alveolar epithelial permeability to Na+ and mannitol was mildly increased in animals infected with adMRCMVbeta1 and a similar Escherichia coli lacZ-expressing virus. Our data shows, for the first time, that transfer of the beta1 Na,K-ATPase subunit gene augments Na,K-ATPase function in epithelial cells and liquid clearance in rat lungs. Conceivably, overexpression of Na,K-ATPases could be used as a strategy to augment lung liquid clearance in patients with pulmonary edema.

Full Text

The Full Text of this article is available as a PDF (4.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amin R., Wilmott R., Schwarz Y., Trapnell B., Stark J. Replication-deficient adenovirus induces expression of interleukin-8 by airway epithelial cells in vitro. Hum Gene Ther. 1995 Feb;6(2):145–153. doi: 10.1089/hum.1995.6.2-145. [DOI] [PubMed] [Google Scholar]
  2. Barnard M. L., Olivera W. G., Rutschman D. M., Bertorello A. M., Katz A. I., Sznajder J. I. Dopamine stimulates sodium transport and liquid clearance in rat lung epithelium. Am J Respir Crit Care Med. 1997 Sep;156(3 Pt 1):709–714. doi: 10.1164/ajrccm.156.3.9610013. [DOI] [PubMed] [Google Scholar]
  3. Berencsi K., Uri A., Valyi-Nagy T., Valyi-Nagy I., Meignier B., Peretz F. V., Rando R. F., Plotkin S. A., Gönczöl E. Early region 3-replacement adenovirus recombinants are less pathogenic in cotton rats and mice than early region 3-deleted viruses. Lab Invest. 1994 Sep;71(3):350–358. [PubMed] [Google Scholar]
  4. Berthiaume Y., Staub N. C., Matthay M. A. Beta-adrenergic agonists increase lung liquid clearance in anesthetized sheep. J Clin Invest. 1987 Feb;79(2):335–343. doi: 10.1172/JCI112817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blanco G., Koster J. C., Mercer R. W. The alpha subunit of the Na,K-ATPase specifically and stably associates into oligomers. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8542–8546. doi: 10.1073/pnas.91.18.8542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brody S. L., Crystal R. G. Adenovirus-mediated in vivo gene transfer. Ann N Y Acad Sci. 1994 May 31;716:90–103. doi: 10.1111/j.1749-6632.1994.tb21705.x. [DOI] [PubMed] [Google Scholar]
  7. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  8. Chow D. C., Forte J. G. Functional significance of the beta-subunit for heterodimeric P-type ATPases. J Exp Biol. 1995 Jan;198(Pt 1):1–17. doi: 10.1242/jeb.198.1.1. [DOI] [PubMed] [Google Scholar]
  9. Crystal R. G., McElvaney N. G., Rosenfeld M. A., Chu C. S., Mastrangeli A., Hay J. G., Brody S. L., Jaffe H. A., Eissa N. T., Danel C. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet. 1994 Sep;8(1):42–51. doi: 10.1038/ng0994-42. [DOI] [PubMed] [Google Scholar]
  10. DeTomaso A. W., Blanco G., Mercer R. W. The alpha and beta subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme. J Cell Biol. 1994 Oct;127(1):55–69. doi: 10.1083/jcb.127.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeTomaso A. W., Xie Z. J., Liu G., Mercer R. W. Expression, targeting, and assembly of functional Na,K-ATPase polypeptides in baculovirus-infected insect cells. J Biol Chem. 1993 Jan 15;268(2):1470–1478. [PubMed] [Google Scholar]
  12. Dobbs L. G., Gonzalez R., Williams M. C. An improved method for isolating type II cells in high yield and purity. Am Rev Respir Dis. 1986 Jul;134(1):141–145. doi: 10.1164/arrd.1986.134.1.141. [DOI] [PubMed] [Google Scholar]
  13. Ewart H. S., Klip A. Hormonal regulation of the Na(+)-K(+)-ATPase: mechanisms underlying rapid and sustained changes in pump activity. Am J Physiol. 1995 Aug;269(2 Pt 1):C295–C311. doi: 10.1152/ajpcell.1995.269.2.C295. [DOI] [PubMed] [Google Scholar]
  14. Factor P., Senne C., Dumasius V., Ridge K., Jaffe H. A., Uhal B., Gao Z., Sznajder J. I. Overexpression of the Na+,K+-ATPase alpha1 subunit increases Na+,K+-ATPase function in A549 cells. Am J Respir Cell Mol Biol. 1998 Jun;18(6):741–749. doi: 10.1165/ajrcmb.18.6.2918. [DOI] [PubMed] [Google Scholar]
  15. Factor P., Senne C., Ridge K., Jaffe H. A., Sznajder J. I. Differential effects of adenoviral-mediated transfer of Na+/K(+)-ATPase subunit genes in lung epithelial cells. Chest. 1997 Jun;111(6 Suppl):110S–111S. doi: 10.1378/chest.111.6_supplement.110s. [DOI] [PubMed] [Google Scholar]
  16. Fambrough D. M., Lemas M. V., Hamrick M., Emerick M., Renaud K. J., Inman E. M., Hwang B., Takeyasu K. Analysis of subunit assembly of the Na-K-ATPase. Am J Physiol. 1994 Mar;266(3 Pt 1):C579–C589. doi: 10.1152/ajpcell.1994.266.3.C579. [DOI] [PubMed] [Google Scholar]
  17. Goodman B. E., Kim K. J., Crandall E. D. Evidence for active sodium transport across alveolar epithelium of isolated rat lung. J Appl Physiol (1985) 1987 Jun;62(6):2460–2466. doi: 10.1152/jappl.1987.62.6.2460. [DOI] [PubMed] [Google Scholar]
  18. Jaffe H. A., Danel C., Longenecker G., Metzger M., Setoguchi Y., Rosenfeld M. A., Gant T. W., Thorgeirsson S. S., Stratford-Perricaudet L. D., Perricaudet M. Adenovirus-mediated in vivo gene transfer and expression in normal rat liver. Nat Genet. 1992 Aug;1(5):372–378. doi: 10.1038/ng0892-372. [DOI] [PubMed] [Google Scholar]
  19. Jaisser F., Jaunin P., Geering K., Rossier B. C., Horisberger J. D. Modulation of the Na,K-pump function by beta subunit isoforms. J Gen Physiol. 1994 Apr;103(4):605–623. doi: 10.1085/jgp.103.4.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jewell E. A., Lingrel J. B. Comparison of the substrate dependence properties of the rat Na,K-ATPase alpha 1, alpha 2, and alpha 3 isoforms expressed in HeLa cells. J Biol Chem. 1991 Sep 5;266(25):16925–16930. [PubMed] [Google Scholar]
  21. Katkin J. P., Husser R. C., Langston C., Welty S. E. Exogenous surfactant enhances the delivery of recombinant adenoviral vectors to the lung. Hum Gene Ther. 1997 Jan 20;8(2):171–176. doi: 10.1089/hum.1997.8.2-171. [DOI] [PubMed] [Google Scholar]
  22. Lemas M. V., Hamrick M., Takeyasu K., Fambrough D. M. 26 amino acids of an extracellular domain of the Na,K-ATPase alpha-subunit are sufficient for assembly with the Na,K-ATPase beta-subunit. J Biol Chem. 1994 Mar 18;269(11):8255–8259. [PubMed] [Google Scholar]
  23. Lescale-Matys L., Hensley C. B., Crnkovic-Markovic R., Putnam D. S., McDonough A. A. Low K+ increases Na,K-ATPase abundance in LLC-PK1/Cl4 cells by differentially increasing beta, and not alpha, subunit mRNA. J Biol Chem. 1990 Oct 15;265(29):17935–17940. [PubMed] [Google Scholar]
  24. Lescale-Matys L., Putnam D. S., McDonough A. A. Na(+)-K(+)-ATPase alpha 1- and beta 1-subunit degradation: evidence for multiple subunit specific rates. Am J Physiol. 1993 Mar;264(3 Pt 1):C583–C590. doi: 10.1152/ajpcell.1993.264.3.C583. [DOI] [PubMed] [Google Scholar]
  25. Lingrel J. B., Young R. M., Shull M. M. Multiple forms of the Na,K-ATPase: their genes and tissue specific expression. Prog Clin Biol Res. 1988;268B:105–112. [PubMed] [Google Scholar]
  26. Mastrangeli A., Danel C., Rosenfeld M. A., Stratford-Perricaudet L., Perricaudet M., Pavirani A., Lecocq J. P., Crystal R. G. Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer. J Clin Invest. 1993 Jan;91(1):225–234. doi: 10.1172/JCI116175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Matalon S., Kirk K. L., Bubien J. K., Oh Y., Hu P., Yue G., Shoemaker R., Cragoe E. J., Jr, Benos D. J. Immunocytochemical and functional characterization of Na+ conductance in adult alveolar pneumocytes. Am J Physiol. 1992 May;262(5 Pt 1):C1228–C1238. doi: 10.1152/ajpcell.1992.262.5.C1228. [DOI] [PubMed] [Google Scholar]
  28. Matthay M. A., Folkesson H. G., Verkman A. S. Salt and water transport across alveolar and distal airway epithelia in the adult lung. Am J Physiol. 1996 Apr;270(4 Pt 1):L487–L503. doi: 10.1152/ajplung.1996.270.4.L487. [DOI] [PubMed] [Google Scholar]
  29. Matthay M. A., Landolt C. C., Staub N. C. Differential liquid and protein clearance from the alveoli of anesthetized sheep. J Appl Physiol Respir Environ Exerc Physiol. 1982 Jul;53(1):96–104. doi: 10.1152/jappl.1982.53.1.96. [DOI] [PubMed] [Google Scholar]
  30. McGrory W. J., Bautista D. S., Graham F. L. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology. 1988 Apr;163(2):614–617. doi: 10.1016/0042-6822(88)90302-9. [DOI] [PubMed] [Google Scholar]
  31. Munzer J. S., Daly S. E., Jewell-Motz E. A., Lingrel J. B., Blostein R. Tissue- and isoform-specific kinetic behavior of the Na,K-ATPase. J Biol Chem. 1994 Jun 17;269(24):16668–16676. [PubMed] [Google Scholar]
  32. Nici L., Dowin R., Gilmore-Hebert M., Jamieson J. D., Ingbar D. H. Upregulation of rat lung Na-K-ATPase during hyperoxic injury. Am J Physiol. 1991 Oct;261(4 Pt 1):L307–L314. doi: 10.1152/ajplung.1991.261.4.L307. [DOI] [PubMed] [Google Scholar]
  33. O'Brodovich H. M. The role of active Na+ transport by lung epithelium in the clearance of airspace fluid. New Horiz. 1995 May;3(2):240–247. [PubMed] [Google Scholar]
  34. Olivera W. G., Ridge K. M., Sznajder J. I. Lung liquid clearance and Na,K-ATPase during acute hyperoxia and recovery in rats. Am J Respir Crit Care Med. 1995 Oct;152(4 Pt 1):1229–1234. doi: 10.1164/ajrccm.152.4.7551375. [DOI] [PubMed] [Google Scholar]
  35. Olivera W., Ridge K., Wood L. D., Sznajder J. I. Active sodium transport and alveolar epithelial Na-K-ATPase increase during subacute hyperoxia in rats. Am J Physiol. 1994 May;266(5 Pt 1):L577–L584. doi: 10.1152/ajplung.1994.266.5.L577. [DOI] [PubMed] [Google Scholar]
  36. Pilewski J. M., Sott D. J., Wilson J. M., Albelda S. M. ICAM-1 expression on bronchial epithelium after recombinant adenovirus infection. Am J Respir Cell Mol Biol. 1995 Feb;12(2):142–148. doi: 10.1165/ajrcmb.12.2.7865213. [DOI] [PubMed] [Google Scholar]
  37. Price E. M., Rice D. A., Lingrel J. B. Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit. J Biol Chem. 1990 Apr 25;265(12):6638–6641. [PubMed] [Google Scholar]
  38. Ridge K. M., Rutschman D. H., Factor P., Katz A. I., Bertorello A. M., Sznajder J. L. Differential expression of Na-K-ATPase isoforms in rat alveolar epithelial cells. Am J Physiol. 1997 Jul;273(1 Pt 1):L246–L255. doi: 10.1152/ajplung.1997.273.1.L246. [DOI] [PubMed] [Google Scholar]
  39. Rosenfeld M. A., Chu C. S., Seth P., Danel C., Banks T., Yoneyama K., Yoshimura K., Crystal R. G. Gene transfer to freshly isolated human respiratory epithelial cells in vitro using a replication-deficient adenovirus containing the human cystic fibrosis transmembrane conductance regulator cDNA. Hum Gene Ther. 1994 Mar;5(3):331–342. doi: 10.1089/hum.1994.5.3-331. [DOI] [PubMed] [Google Scholar]
  40. Rutschman D. H., Olivera W., Sznajder J. I. Active transport and passive liquid movement in isolated perfused rat lungs. J Appl Physiol (1985) 1993 Oct;75(4):1574–1580. doi: 10.1152/jappl.1993.75.4.1574. [DOI] [PubMed] [Google Scholar]
  41. Sakuma T., Folkesson H. G., Suzuki S., Okaniwa G., Fujimura S., Matthay M. A. Beta-adrenergic agonist stimulated alveolar fluid clearance in ex vivo human and rat lungs. Am J Respir Crit Care Med. 1997 Feb;155(2):506–512. doi: 10.1164/ajrccm.155.2.9032186. [DOI] [PubMed] [Google Scholar]
  42. Saldías F., Lecuona E., Friedman E., Barnard M. L., Ridge K. M., Sznajder J. I. Modulation of lung liquid clearance by isoproterenol in rat lungs. Am J Physiol. 1998 May;274(5 Pt 1):L694–L701. doi: 10.1152/ajplung.1998.274.5.L694. [DOI] [PubMed] [Google Scholar]
  43. Saumon G., Basset G., Bouchonnet F., Crone C. cAMP and beta-adrenergic stimulation of rat alveolar epithelium. Effects on fluid absorption and paracellular permeability. Pflugers Arch. 1987 Nov;410(4-5):464–470. doi: 10.1007/BF00586526. [DOI] [PubMed] [Google Scholar]
  44. Schneeberger E. E., McCarthy K. M. Cytochemical localization of Na+-K+-ATPase in rat type II pneumocytes. J Appl Physiol (1985) 1986 May;60(5):1584–1589. doi: 10.1152/jappl.1986.60.5.1584. [DOI] [PubMed] [Google Scholar]
  45. Shanbaky N. M., Pressley T. A. Transfection of Na,K-ATPase alpha-subunit: regulation of enzyme abundance. Biochem Cell Biol. 1995 May-Jun;73(5-6):261–268. doi: 10.1139/o95-032. [DOI] [PubMed] [Google Scholar]
  46. Skou J. C., Esmann M. The Na,K-ATPase. J Bioenerg Biomembr. 1992 Jun;24(3):249–261. doi: 10.1007/BF00768846. [DOI] [PubMed] [Google Scholar]
  47. Sznajder J. I., Olivera W. G., Ridge K. M., Rutschman D. H. Mechanisms of lung liquid clearance during hyperoxia in isolated rat lungs. Am J Respir Crit Care Med. 1995 May;151(5):1519–1525. doi: 10.1164/ajrccm.151.5.7735609. [DOI] [PubMed] [Google Scholar]
  48. Tibayan F. A., Chesnutt A. N., Folkesson H. G., Eandi J., Matthay M. A. Dobutamine increases alveolar liquid clearance in ventilated rats by beta-2 receptor stimulation. Am J Respir Crit Care Med. 1997 Aug;156(2 Pt 1):438–444. doi: 10.1164/ajrccm.156.2.9609141. [DOI] [PubMed] [Google Scholar]
  49. Voilley N., Lingueglia E., Champigny G., Mattéi M. G., Waldmann R., Lazdunski M., Barbry P. The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):247–251. doi: 10.1073/pnas.91.1.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wilson J. M. Adenoviruses as gene-delivery vehicles. N Engl J Med. 1996 May 2;334(18):1185–1187. doi: 10.1056/NEJM199605023341809. [DOI] [PubMed] [Google Scholar]
  51. Yang Y., Li Q., Ertl H. C., Wilson J. M. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol. 1995 Apr;69(4):2004–2015. doi: 10.1128/jvi.69.4.2004-2015.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zabner J., Wadsworth S. C., Smith A. E., Welsh M. J. Adenovirus-mediated generation of cAMP-stimulated Cl- transport in cystic fibrosis airway epithelia in vitro: effect of promoter and administration method. Gene Ther. 1996 May;3(5):458–465. [PubMed] [Google Scholar]
  53. Zabner J., Zeiher B. G., Friedman E., Welsh M. J. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time. J Virol. 1996 Oct;70(10):6994–7003. doi: 10.1128/jvi.70.10.6994-7003.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhang X. L., Danto S. I., Borok Z., Eber J. T., Martín-Vasallo P., Lubman R. L. Identification of Na(+)-K(+)-ATPase beta-subunit in alveolar epithelial cells. Am J Physiol. 1997 Jan;272(1 Pt 1):L85–L94. doi: 10.1152/ajplung.1997.272.1.L85. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES