RFC-1 gene expression regulates folate absorption in mouse small intestine.
Mediated folate compound transport inward in isolated luminal epithelial cells from mouse small intestine was delineated as pH-dependent and non-pH-dependent components on the basis of their differential sensitivity to the stilbene inhibitor, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid. pH dependence was manifested as higher maximum capacity (Vmax) for influx of l, L-5-CH3-H4folate at acidic pH compared with neutral or alkaline pH with no effect on saturability (Km). The pH-dependent component was relatively insensitive to inhibition by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid and highly saturable (Km or Ki = 2 to 4 microM) in the case of folic acid, folate coenzymes, and 4-aminofolate analogues as permeants or inhibitors. The non-pH-dependent component was highly sensitive to 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid and poorly and variably saturable (Km or Ki = 20 to >2000 microM) with respect to these folate compounds. Only the pH-dependent transport component was developmentally regulated, showing much higher maximum capacity for l,L-5-CH3-H4folate influx in mature absorptive rather than proliferative crypt cells. The increase in pH-dependent influx during maturation was associated with an increase in RFC-1 gene expression in the form of a 2.5-kilobase RNA transcript and 58-kDa brush-border membrane protein detected by folate-based affinity labeling and with anti-mouse RFC-1 peptide antibodies. The size of this protein was the same as that encoded by RFC-1 mRNA. The treatment of mature absorptive cells with either the affinity label or the anti-RFC-1 peptide antibodies inhibited influx of l, L-[3H]-5-CH3-H4folate in a concentration-dependent manner. These results strongly suggest that pH-dependent folate absorption in this tissue is regulated by RFC-1 gene expression.
Chiao
J H
JH
Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Roy
K
K
Tolner
B
B
Yang
C H
CH
Sirotnak
F M
FM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000042:Absorption / Q000187:drug effects; D000345:Affinity Labels; D000630:Aminopterin / Q000031:analogs & derivatives; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005492:Folic Acid / Q000031:analogs & derivatives; D015870:Gene Expression; D007421:Intestine, Small / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D026901:Membrane Transport Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D012333:RNA, Messenger / Q000302:isolation & purification; D013388:Succinimides; D013763:Tetrahydrofolates / Q000378:metabolism*
D000345:Affinity Labels; D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C492292:Slc19a2 protein, mouse; D013388:Succinimides; D013763:Tetrahydrofolates; D005492:Folic Acid; C056458:aminopterin N-hydroxysuccinimide ester; D000630:Aminopterin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C005984:5-methyltetrahydrofolate