NHE1, NHE2, and NHE4 contribute to regulation of cell pH in T84 colon cancer cells
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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.
KeywordsT84 colon cells NHE isoforms NHE4 Cell volume Cell pH
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).
- 1.Biemesderfer D, Pizzonia J, Abu-Alfa A, Exner M, Reilly R, Igarashi P, Aronson PS (1993) NHE3: a Na+/H+ exchanger isoform of renal brush border. Am J Physiol Renal Fluid Electrolyte Physiol 265:F736–F742Google Scholar
- 4.Bookstein C, Xie Y, Rabenau K, Musch MW, McSwine RL, Rao MC, Chang EB (1997) Tissue distribution of Na+/H+ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney. Am J Physiol Cell Physiol 273:C1496–C1505Google Scholar
- 9.Chambrey R, Achard JM, St John PL, Abrahamson DR, Warnock DG (1997) Evidence for an amiloride-insensitive Na+/H+ exchanger in rat renal cortical tubules. Am J Physiol Cell Physiol 273:C1064–C1074Google Scholar
- 10.Chambrey R, Achard JM, Warnock DG (1997) Heterologous expression of rat NHE4: A highly amiloride-resistant Na+/H+ exchanger isoform. Am J Physiol Cell Physiol 272:C90–C98Google Scholar
- 12.de Silva MG, Elliott K, Dahl HH, Fitzpatrick E, Wilcox S, Delatycki M, Williamson R, Efron D, Lynch M, Forrest S (2003) Disruption of a novel member of a sodium/hydrogen exchanger family and DOCK3 is associated with an attention deficit hyperactivity disorder-like phenotype. J Med Genet 40:733–740PubMedCrossRefGoogle Scholar
- 18.Ikuma M, Kashgarian M, Binder HJ, Rajendran VM (1999) Differential regulation of NHE isoforms by sodium depletion in proximal and distal segments of rat colon. Am J Physiol Gastrointest Liver Physiol 276:G539–G549Google Scholar
- 31.Ritter M, Schratzberger P, Rossmann H, Wöll E, Seiler K, Seidler U, Reinisch N, Kahler CM, Zwierzina H, Lang HJ, Lang F, Paulmichl M, Wiedermann CJ (1998) Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes. Br J Pharmacol 124:627–638PubMedCrossRefGoogle Scholar