Anion specificity of the jejunal folate carrier: effects of reduced folate analogues on folate uptake and efflux.
We previously reported that 3H-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 (folate Km = 0.19 microM) 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 was cis inhibited (greater than 90%) by reduced folate analogues: dihydrofolate (IC50 = 0.40 microM), folinic acid (IC50 = 0.50 microM), 5-methyltetrahydrofolate (IC50 = 0.53 microM), and (+)amethopterin (IC50 = 0.93 microM). In contrast, 10 microM (-)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 PO4(-3] did not inhibit jejunal folate uptake. Furthermore, folate uptake was not inhibited by SO4(-2) (4 mM) or oxalate (4 mM) thereby distinguishing this carrier from the previously described intestinal SO4(-2)/OH- and oxalate/Cl- exchangers. After BBM vesicles were loaded with 3H-folate, the initial velocity of 3H-folate efflux was stimulated by unlabeled folate in the efflux medium. The transstimulation of 3H-folate efflux by unlabeled folate was furosemide (or DIDS) inhibitable and temperature sensitive. Half-maximal stimulation of furosemide-sensitive 3H-folate efflux was observed with 0.25 +/- 0.05 microM unlabeled folate, a concentration similar to the Km for folate uptake. These data suggest that folate-stimulated 3H-folate efflux is mediated by the folate/OH- exchanger.(ABSTRACT TRUNCATED AT 250 WORDS)
Schron
C M
CM
Department of Internal Medicine, University of Cincinnati College of Medicine, Ohio 45267.
Washington
C
C
Blitzer
B L
BL
D000818:Animals; D000838:Anions / Q000378:metabolism*; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D007474:Ion Exchange; D007583:Jejunum / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008871:Microvilli / Q000378:metabolism; D011817:Rabbits; D011956:Receptors, Cell Surface
D000838:Anions; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D005492:Folic Acid