The Journal of Membrane Biology

, Volume 197, Issue 2, pp 123–134 | Cite as

Functional Characterization of Na+/H+ Exchangers in Primary Cultures of Prairie Dog Gallbladder

  • S. C. Narins
  • E. H. Park
  • R. Ramakrishnan
  • F. U. Garcia
  • J. N. Diven
  • B. J. Balin
  • C. J. Hammond
  • B. R. Sodam
  • P. R. Smith
  • M. Z. Abedin


Gallbladder Na+ absorption is linked to gallstone formation in prairie dogs. We previously reported Na+/H+ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, Isc, (11.6 ± 0.5 µA · cm−2), potential differences, Vt (2.1 ± 0.2 mV), and resistance, Rt (169 ± 12 Ω · cm2). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable 22Na+ uptake under a H+ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of 22Na+ uptake was 21.4 ± 1.3 nmol · mg prot−1 · min−1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs’ total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (Vmax 9.2 ± 0.3 nmol · mg prot−1 · min−1; Km 11.4 ± 1.4 mM Na+). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na+ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.


Sodium/hydrogen antiporter Epithelial sodium transport Electrophysiology Ussing chambers Primary cultures Gallstones 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Abedin, M.Z., Giurgiu, D.I.N., Abedin, Z.R., Peck, E.A., Su, X., Smith, P.R. 2001Characterization of Na+/H+ exchanger isoform (NHE1, NHE2, and NHE3) expression in prairie dog gallbladder.J. Membrane Biol.182123134CrossRefGoogle Scholar
  2. 2.
    Baerentsen, H., Giraldez, F., Zeuthen, T. 1983Influx mechanism for Na+ and Cl across the brush border membrane of leaky epithelia: a model and microelectrode study.J. Membrane Biol.75205218Google Scholar
  3. 3.
    Bradford, M.M. 1976A rapid and sensitive method for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding.Anal. Biochem.72248254CrossRefPubMedGoogle Scholar
  4. 4.
    Brenneman, D.E., Connor, W.E., Forker, E.L., DenBesten, L. 1972The formation of abnormal bile and cholesterol gallstones from dietary cholesterol in the prairie dog.J. Clin. Invest.5114951503PubMedGoogle Scholar
  5. 5.
    Cates, J.A., Abedin, M.Z., Saunders-Kirkwood, K.D., Moser, A.J., Giurgiu, D.I., Roslyn, J.J. 1995Protein kinase C regulates prairie dog gallbladder ion transport.Surgery117206212PubMedGoogle Scholar
  6. 6.
    Cavet, M.E., Akhter, S., De Medina, F.S., Donowitz, M., Tse, C.M. 1999Na+/H+ exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface.Am. J. Physiol.277C1111C1121PubMedGoogle Scholar
  7. 7.
    Chapman, W.C., Fisk, J., Schot, D., Debelak, J.P., Washington, M.K., Bluth, R.F., Pierce, D., Williams, L.F. 1998Establishment and characterization of primary gallbladder epithelial cell cultures in the prairie dog.J. Surg. Res.8035435CrossRefPubMedGoogle Scholar
  8. 8.
    Cho, J.H., Musch, M.W., Bookstein, C.M., McSwine, R.L, Rabenau, R., Chang, E.B. 1998Aldosterone stimulates intestinal Na+ absorption in rats by increasing NHE3 expression of the proximal colon.Am. J. Physiol.274C586C594PubMedGoogle Scholar
  9. 9.
    Chomczynski, P., Sacchi, N. 1987Single-step method of RNA isolation by acid guanidinium thiocyanante-phenol-chloroform extraction.Anal. Biochem.162156159CrossRefPubMedGoogle Scholar
  10. 10.
    Conter, R.L., Roslyn, J.J., Porter-Fink, V., DenBesten, L. 1986Gallbladder absorption increases during early cholesterol gallstone formation.Am. J. Surg.151184191PubMedGoogle Scholar
  11. 11.
    Counillon, L., Scholz, W., Lang, H.J., Pouyssegur, J. 1993Pharmacological characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties.Mol. Pharmacol.4410411045PubMedGoogle Scholar
  12. 12.
    Cremaschi, D., Meyer, G., Rosetti, C., Botta, G., Palestini, P. 1987The nature of the neutral Na+-Cl -coupled entry at the apical membrane of rabbit gallbladder epithelium I. Na+/H+, Cl/HCO3 double exchange and Na+-Cl symport.J. Membrane Biol.95209218Google Scholar
  13. 13.
    DenBesten, L., Safaie-Shirazi, S., Connor, W.E., Bell, S. 1974Early changes in bile composition and gallstone formation induced by a high cholesterol diet in prairie dogs.Gastroenterology6610361045PubMedGoogle Scholar
  14. 14.
    Diamond, J.M. 1964Transport of salt and water in rabbit and guinea pig gallbladder.J. Gen. Physiol.48114PubMedGoogle Scholar
  15. 15.
    Donowitz, M., De La Horra, C.D., Calonge, M.L., Wood, I.S., Dyer, J., Gribble, S.M., Sanchez De Medina, F., Tse, C.M., Shirazi-Beechey, S.P., llundain, A.A. 1998In birds, NHE2 is the major brush-border Na+/H+ exchanger in colon and is increased by a low-NaCl diet.Am. J. Physiol.274R1659R1669PubMedGoogle Scholar
  16. 16.
    Dudeja, P.K., Rao, D.D., Syed, I., Joshi, V., Dahdal, R.Y., Gardner, C., Risk, M.C., Schmidt, L., Bavishi, D., Kim, K.E., Harig, J.M., Goldstein, J.L., Layden, T., Ramaswamy, K. 1996Intestinal distribution of human N+/H+ exchanger isoforms NHE-1, NHE-2, and NHE-3 mRNA.Am. J. Physiol.271G483G493PubMedGoogle Scholar
  17. 17.
    Giurgiu, D.I., Saunders-Kirkwood, K.D., Roslyn, J.J., Abedin, M.Z. 1997Sequential changes in biliary lipids and gallbladder ion transport during gallstone formation.Ann. Surg.25457465Google Scholar
  18. 18.
    Gunter-Smith, P.J., Abdulkader, O., Hammonds-Odi, L., Scanlon, M., Terrel, R. 2000A primary culture of guinea pig gallladder epithelial cells that is responsive to secretagogues.Am. J. Physiol.279G866G874Google Scholar
  19. 19.
    Gurll, N., DenBesten, L. 1978Animal models of human cholesterol gallstone disease: a review.Lab. Animal Sci.28428432Google Scholar
  20. 20.
    Holzbach, R.T. 1984Animal models of cholesterol gallstone disease.Hepatology4191S198SPubMedGoogle Scholar
  21. 21.
    Holzbach, R.T., Corbusier, C., Marsh, M., Naito, H.K. 1976The process of cholesterol cholelithiasis induced by diet in the prairie dog: a physicochemical characterization.J. Lab. Clin. Med.87987998PubMedGoogle Scholar
  22. 22.
    Ikuma, M., Kashgarian, M., Binder, H.J., Rajendran, V.M. 1999Differential regulation of NHE isoforms by sodium depletion in proximal and distal segments of rat colon.Am. J. Physiol.39G539G549Google Scholar
  23. 23.
    Kandasamy, R.A., Yu, F.H., Harris, R., Boucher, A., Hanrahan, J.W., Orlowski, J. 1995Plasma membrane Na+-H+ exchanger isoforms (NHE-1, -2, -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways.J. Biol. Chem.2702920929216CrossRefPubMedGoogle Scholar
  24. 24.
    Kuver, R., Savard, C., Nguyen, T.D., Osborne, W.R., Lee, S.P. 1997Isolation and long-term culture of gallbladder cells from wild-type and mice.In Vitro Cell Dev. Biol. Anim.33104109PubMedGoogle Scholar
  25. 25.
    LaMorte, W., O’Leary, D., Booker, M., Scott, T. 1993Increased dietary fat content accelerates cholesterol gallstone formation in the cholesterol-fed prairie dog.Hepatology1814981503PubMedGoogle Scholar
  26. 26.
    Lee, S.P. 1978Enhanced fluid transport across gallbladder mucosa in experimental cholelithiasis.Am. J. Physiol.234E575E578PubMedGoogle Scholar
  27. 27.
    Lee, S.P., LaMont, J.T., Carey, M.C. 1981Role of gallbladder mucus hypersecretion in the evolution of cholesterol gallstones:studies in the prairie dog.J. Clin. Invest.6717121723PubMedGoogle Scholar
  28. 28.
    Marti, U., Elsing, C., Renner, E.L., Liechti-Gallati, S., Reichen, J. 1996Differential expression of Na+/H+ antiporter mRNA in biliary epithelial cells and hepatocytes.J. Hepatol.24489502CrossRefGoogle Scholar
  29. 29.
    McSwine, R.L, Musch, M.W., Bookstein, C., Xie, Y., Rao, M., Chang, E.B. 1998Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe.Am. J. Physiol.275C693C701PubMedGoogle Scholar
  30. 30.
    Moser, A.J., Abedin, M.Z., Abedin, Z.R., Roslyn, J.J. 1993Ca2+ calmodulin regulates basal gallbladder absorption.Surgery114300307PubMedGoogle Scholar
  31. 31.
    Oda, D., Lee, S.P., Hayshi, A. 1991Long-term culture and partial characterization of dog gallbladder epithelial cells.Lab. Invest.682682692Google Scholar
  32. 32.
    Plevris, J.N., Walker, S.W., Harrison, D.J., Dhariwal, A., Hayes, P.C., Bouchier, I.A. 1993Primary culture of bovine gall bladder epithelial cells.Gut3416121615PubMedGoogle Scholar
  33. 33.
    Purdum, P.P.O., Ulissi, A., Hylemon, P.B., Shiftman, M.L., Moore, E.W. 1993Cultured human gallbladder epithelia. Methods and partial characterization of a carcinoma-derived model.Lab. Invest.68345353PubMedGoogle Scholar
  34. 34.
    Reuss, L. 1984Independence of apical membrane Na+ and Cl entry in Necturus gallbladder epithelium.J. Gen. Physiol.84423445PubMedGoogle Scholar
  35. 35.
    Rose, R.C., Gelarden, R.T., Narhwold, D.L. 1973Electrical properties of isolated human gallbladder.Am. J. Physiol.22413201326PubMedGoogle Scholar
  36. 36.
    Roslyn, J.J., Abedin, M.Z., Saunders, K.D., Cates, J.A., Strichartz, S.D., Alperin, M., Fromm, M., Palant, C.E. 1991Uncoupled basal sodium absorption and chloride secretion in prairie dog (Cynomys ludovicianus) gallbladder.Comp. Biochem. Physiol.100A335341CrossRefGoogle Scholar
  37. 37.
    Silviani, V., Colombani, V., Heyries, L., Gerolami, A., Cartouzou, G., Marteau, C. 1996Role of the NHE3 isoform of the Na+/H+ by the rabbit gallbladder.Pfluegers Arch. Eur. J. Physiol.432791796CrossRefGoogle Scholar
  38. 38.
    Silviani, V., Gastaldi, M., Planells, R., Marteau, C., Massacrier, A., Cohen, P., Cau, P., Gerolami, A. 1997NHE-3 isoform of the Na+/H+ exchanger in human gallbladder: Localization of specific mRNA by in situ hybridization.J. Hepatol.2612811286CrossRefPubMedGoogle Scholar
  39. 39.
    Strichartz, S.D., Abedin, M.Z., Abdou, M.S., Roslyn, J.J. 1989The effects of amiloride on biliary calcium and cholesterol gallstone formation.Ann. Surg.209152156PubMedGoogle Scholar
  40. 40.
    Weinman, S.A., Reuss, L. 1984Na+/H+ exchange and Na+ entry across the apical membrane of Necturus gallbladder.J. Gen. Physiol.835774PubMedGoogle Scholar
  41. 41.
    Winterhager, J.M., Stewart, C.P., Heintze, K., Petersen, K.U. 1986Electroneutral secretion of bicarbonate by guinea pig gallbladder epithelium.Am. J. Physiol.250C617C628PubMedGoogle Scholar
  42. 42.
    Wormmeester, L., De Medina, F.S., Kokke, F., Tse, C.M., Khurana, S., Bowser, J., Cohen, M.E., Donowitz, M. 1998Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange.Am. J. Physiol.274C1261C1272PubMedGoogle Scholar

Copyright information

© Springer-Verlag New York Inc. 2004

Authors and Affiliations

  • S. C. Narins
    • 1
  • E. H. Park
    • 1
  • R. Ramakrishnan
    • 1
  • F. U. Garcia
    • 1
  • J. N. Diven
    • 1
  • B. J. Balin
    • 3
  • C. J. Hammond
    • 3
  • B. R. Sodam
    • 1
  • P. R. Smith
    • 2
  • M. Z. Abedin
    • 1
  1. 1.Department of Surgery and Department of Pathology and Laboratory MedicineDrexel University College of Medicine, Philadelphia, PAUSA
  2. 2.Department of Physiology and BiophysicsUniversity of Alabama at Birmingham, Birmingham, ALUSA
  3. 3.Department of PathologyMicrobiology & Immunology, Philadelphia College of Osteopathic Medicine, Philadelphia, PAUSA

Personalised recommendations