NHE1, NHE2, and NHE4 contribute to regulation of cell pH in T84 colon cancer cells.
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 (JH) was 3.68 mM/min. After the use of the amiloride derivative HOE-694 at 25 microM, 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 JH by the NHE3 inhibitor S3226 (1 microM) and no evidence of this isoform by RT-PCR was found. The following functional evidence for the presence of NHE4 was obtained: 25 microM EIPA abolished JH entirely, but NHE4 was not inhibited at 10 microM; substitution of Na by K increased the remainder, a property of NHE4; hypertonicity also increased this fraction of JH. Cl--dependent NHE was not detected: in 0 Cl- solutions JH 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.
Beltrán
Ana Rosa
AR
Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1524, 05508-900 São Paulo, Sao Paulo, Brazil.
Ramírez
Marco Antonio
MA
Carraro-Lacroix
Luciene R
LR
Hiraki
Yumi
Y
Rebouças
Nancy Amaral
NA
Malnic
Gerhard
G
D000136:Acid-Base Equilibrium / Q000187:drug effects / Q000502:physiology; D000143:Acids / Q000494:pharmacology; D015153:Blotting, Western; D002021:Buffers; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002712:Chlorides / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism; D008954:Models, Biological; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000074082:Sodium-Hydrogen Exchanger 1; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*
D000143:Acids; D002021:Buffers; D027682:Cation Transport Proteins; D002712:Chlorides; D000644:Quaternary Ammonium Compounds; C490998:SLC9A1 protein, human; C081517:SLC9A2 protein, human; C000613385:SLC9A3 protein, human; C000613387:Slc9a3 protein, rat; C081518:Slc9a4 protein, rat; D000074082:Sodium-Hydrogen Exchanger 1; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers