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Pflügers Archiv - European Journal of Physiology

, Volume 455, Issue 5, pp 799–810 | Cite as

NHE1, NHE2, and NHE4 contribute to regulation of cell pH in T84 colon cancer cells

  • Ana Rosa Beltrán
  • Marco Antonio Ramírez
  • Luciene R. Carraro-Lacroix
  • Yumi Hiraki
  • Nancy Amaral Rebouças
  • Gerhard MalnicEmail author
Acid-Base Regulation

Abstract

The isoforms of the Na+/H+ exchanger present in T84 human colon cells were identified by functional and molecular methods. Cell pH was measured by fluorescence microscopy using the probe BCECF. Based on the pH recovery after an ammonium pulse and determination of buffering capacity of these cells, the rate of H+ extrusion (J H) was 3.68 mM/min. After the use of the amiloride derivative HOE-694 at 25 μM, which inhibits the isoforms NHE1 and NHE2, there remained 43% of the above transport rate, the nature of which was investigated. Evidence of the presence of NHE1, NHE2, and NHE4 was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) (mRNA) and Western blot. There was no decrease of J H by the NHE3 inhibitor S3226 (1 μM) and no evidence of this isoform by RT-PCR was found. The following functional evidence for the presence of NHE4 was obtained: 25 μM EIPA abolished J H entirely, but NHE4 was not inhibited at 10 μM; substitution of Na by K increased the remainder, a property of NHE4; hypertonicity also increased this fraction of J H. Cl-dependent NHE was not detected: in 0 Cl solutions J H was increased and not reduced. In 0 Cl cell volume decreased significantly, which was abolished by the Cl channel blocker NPPB, indicating that the 0 Cl effect was because of reduction of cell volume. In conclusion, T84 human colon cells contain three isoforms of the Na+/H+ exchanger, NHE1, NHE2, and NHE4, but not the Cl-dependent NHE.

Keywords

T84 colon cells NHE isoforms NHE4 Cell volume Cell pH 

Notes

Acknowledgements

This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP, Pronex, and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq).

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Ana Rosa Beltrán
    • 1
  • Marco Antonio Ramírez
    • 2
  • Luciene R. Carraro-Lacroix
    • 1
  • Yumi Hiraki
    • 1
  • Nancy Amaral Rebouças
    • 1
  • Gerhard Malnic
    • 1
    Email author
  1. 1.Departamento de Fisiologia e Biofísica, Instituto de Ciências BiomédicasUniversidade de São PauloSão PauloBrazil
  2. 2.Biomedical Department, Faculty of Health SciencesUniversity of AntofagastaAntofagastaChile

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