Anion specificity of the jejunal folate carrier: Effects of reduced folate analogues on folate uptake and efflux

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Abstract
We previously reported that3H-folate uptake by rabbit jejunal brush-border membrane (BBM) vesicles was markedly stimulated by an outwardly directed OH− gradient (pHin 7.7, pHout 5.5), inhibited by anion exchange inhibitors (DIDS, SITS, furosemide), and saturable (folateK m=0.19 μm) suggesting carrier-mediated folate/OH− exchange (or H+/folate cotransport). In the present study, the anion specificity of this transport process was examined. Under conditions of an outwardly directed OH− gradient, DIDS-sensitive folate uptake wascis inhibited (>90%) by reduced folate analogues: dihydrofolate (IC50=0.40 μm), folinic acid (IC50=0.50 μm), 5-methyltetrahydrofolate (IC50=0.53 μm), and (+)amethopterin (IC50=0.93 μM). In contrast, 10 μm (−)amethopterin had only a modest effect on folate uptake (18% inhibition) suggesting stereospecificity of the folate/OH− exchanger. The nonpteridine compounds which are transported by the folate carrier in L1210 leukemic cells (phthalate, thiamine pyrophosphate, and PO 4 −3 ) did not inhibit jejunal folate uptake. Furthermore, folate uptake was not inhibited by SO 4 −2 (4mm) or oxalate (4mm) thereby distinguishing this carrier from the previously described intestinal SO 4 −2 /OH− and oxalate/Cl− exchangers. After BBM vesicles were loaded with3H-folate, the initial velocity of3H-folate efflux was stimulated by unlabeled folate in the efflux medium. The transstimulation of3H-folate efflux by unlabeled folate was furosemide (or DIDS) inhibitable and temperature sensitive. Half-maximal stimulation of furosemide-sensitive3H-folate efflux was observed with 0.25±0.05 μm unlabeled folate, a concentration similar to theK m for folate uptake. These data suggest that folate-stimulated3H-folate efflux is mediated by the folate/OH− exchanger. With the exception of (−) amethopterin, reduced folate analogues also transstimulated furosemide-sensitive3H-folate efflux in a concentration-dependent manner suggesting stereospecific transport of these analogues by the folate/OH− exchanger. In summary, folate transport by the jejunal folate/OH− exchanger demonstrates bothcis inhibition and transstimulation by reduced folate analogues, but not by other inorganic or organic anions suggesting bidirectional transport of folate and a high degree of anion specificity.

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