Sulfate and chloride transport in Caco-2 cells: differential regulation by thyroxine and the possible role of DRA gene.
The current studies were undertaken to establish an in vitro cellular model to study the transport of SO and Cl(-) and hormonal regulation and to define the possible function of the downregulated in adenoma (DRA) gene. Utilizing a postconfluent Caco-2 cell line, we studied the OH(-) gradient-driven (35)SO and (36)Cl(-) uptake. Our findings consistent with the presence of an apical carrier-mediated (35)SO/OH(-) exchange process in Caco-2 cells include: 1) demonstration of saturation kinetics [Michaelis-Menten constant (K(m)) of 0.2 +/- 0.08 mM for SO and maximum velocity of 1.1 +/- 0.2 pmol x mg protein(-1) x 2 min(-1)]; 2) sensitivity to inhibition by DIDS (K(i) = 0.9 +/- 0.3 microM); and 3) competitive inhibition by oxalate and Cl(-) but not by nitrate and short chain fatty acids, with a higher K(i) (5.95 +/- 1 mM) for Cl(-) compared with oxalate (K(i) = 0.2 +/- 0.03 mM). Our results also suggested that the SO/OH(-) and Cl(-)/OH(-) exchange processes in Caco-2 cells are distinct based on the following: 1) the SO/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 SO/OH(-) exchange activity with a high K(i) compared with the K(m) for SO, indicating a lower affinity for Cl(-); 3) DIDS competitively inhibited the Cl(-)/OH(-) exchange process, whereas it inhibited the SO/OH(-) exchange activity in a mixed-type manner; and 4) utilizing the RNase protection assay, our results showed that 24-h incubation with 100 nM of thyroxine significantly decreased the relative abundance of DRA mRNA along with the SO/OH(-) exchange activity but without any change in Cl(-)/OH(-) exchange process. In summary, these studies demonstrated the feasibility of utilizing Caco-2 cell line as a model to study the apical SO/OH(-) and Cl(-)/OH(-) exchange processes in the human intestine and indicated that the two transporters are distinct and that DRA may be predominantly a SO transporter with a capacity to transport Cl(-) as well.
Alrefai
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Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago and West Side Veterans Affairs Medical Center, Chicago, Illinois 60612, USA.
Tyagi
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Mansour
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Saksena
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Syed
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Ramaswamy
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Dudeja
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D017920:Antiporters / Q000378:metabolism; D001693:Biological Transport, Active / Q000187:drug effects / Q000235:genetics; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000235:genetics*; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides / Q000378:metabolism*; D015536:Down-Regulation / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics*; D023282:Nuclease Protection Assays; D015347:RNA Probes; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000076988:Sulfate Transporters; D013431:Sulfates / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D013974:Thyroxine / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics*
D017920:Antiporters; D002352:Carrier Proteins; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D008565:Membrane Proteins; D015347:RNA Probes; D012333:RNA, Messenger; C081011:SLC26A3 protein, human; D000076988:Sulfate Transporters; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014157:Transcription Factors; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D013974:Thyroxine