Wa. Alrefai et al., Sulfate and chloride transport in Caco-2 cells: differential regulation bythyroxine and the possible role of DRA gene, AM J P-GAST, 280(4), 2001, pp. G603-G613
Citations number
40
Categorie Soggetti
da verificare
Journal title
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
The current studies were undertaken to establish an in vitro cellular model
to study the transport of SO42- and Cl- and hormonal regulation and to def
ine the possible function of the downregulated in adenoma (DRA) gene. Utili
zing a postconfluent Caco-2 cell line, we studied the OH- gradient-driven (
SO42-)-S-35 and Cl-36(-) uptake. Our findings consistent with the presence
of an apical carrier-mediated (SO42-)-S-35/OH- exchange process in Caco-2 c
ells include: 1) demonstration of saturation kinetics [Michaelis-Menten con
stant (K-m) of 0.2 +/- 0.08 mM for SO42- and maximum velocity of 1.1 +/- 0.
2 pmol.mg protein(-1).2 min(-1)]; 2) sensitivity to inhibition by DIDS (K-i
= 0.9 +/- 0.3 muM); and 3) competitive inhibition by oxalate and Cl 2 but
not by nitrate and short chain fatty acids, with a higher K-i (5.95 +/- 1 m
M) for Cl- compared with oxalate (K-i = 0.2 +/- 0.03 mM). Our results also
suggested that the SO42-/OH- and Cl-/OH- exchange processes in Caco-2 cells
are distinct based on the following: 1) the SO42-/OH- exchange was highly
sensitive to inhibition by DIDS compared with Cl-/ OH- exchange activity (K
-i for DIDS of 0.3 +/- 0.1 mM); 2) Cl- competitively inhibited the SO42-/OH
- exchange activity with a high K-i compared with the K-m for SO42-, indica
ting a lower affinity for Cl-; 3) DIDS competitively inhibited the Cl-/OH-
exchange process, whereas it inhibited the SO42-/ OH- exchange activity in
a mixed-type manner; and 4) utilizing the RNase protection assay, our resul
ts showed that 24-h incubation with 100 nM of thyroxine significantly decre
ased the relative abundance of DRA mRNA along with the SO42-/OH- exchange a
ctivity but without any change in Cl-/OH- exchange process. In summary, the
se studies demonstrated the feasibility of utilizing Caco-2 cell line as a
model to study the apical SO42-/OH- and Cl-/OH- exchange processes in the h
uman intestine and indicated that the two transporters are distinct and tha
t DRA may be predominantly a SO42- transporter with a capacity to transport
Cl- as well.