RFC-1 GENE-EXPRESSION REGULATES FOLATE ABSORPTION IN MOUSE SMALL-INTESTINE

Mediated folate compound transport inward in isolated luminal epitheli al cells from mouse small intestine was delineated as pH-dependent and non-pH-dependent components on the basis of their differential sensit ivity to the stilbene inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-di sulfonic acid, pH dependence was manifested as higher maximum capacity (V-max) for influx of l,L-5-CH3-H(4)folate at acidic pH compared with neutral or alkaline pH with no effect on saturability (K-m), The pH d ependent component was relatively insensitive to inhibition by 4,4'-di isothiocyanatostilbene-2,2'-disulfonic acid and highly saturable (K-m or K-i = 2 to 4 mu M) in the case of folic acid, folate coenzymes, and 4-aminofolate analogues as permeants or inhibitors, The non-pH-depend ent component was highly sensitive to 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid and poorly and variably saturable (K-m or K-i = 20 to > 2000 mu M) with respect to these folate compounds, Only the pH-de pendent transport component was developmentally regulated, showing muc h higher maximum capacity for l,L-5-CH3-H(4)folate influx in mature ab sorptive rather than proliferative crypt cells, The increase in pH-dep endent 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 l abeling 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-[H-3]-5-CH3-H(4)folate i n a concentration-dependent manner, These results strongly suggest tha t pH-dependent folate absorption in this tissue is regulated by RFC-1 gene expression.