Mechanism of folate transport across the human colonic basolateral membrane.
Previous studies from our laboratory have demonstrated the existence of a folate transporter in the human colonic apical membranes. The current studies were undertaken to examine the possible presence and function of a folate carrier in the human colonic basolateral membrane vesicles (BLMV). BLMV were purified from mucosal scrapings of colons of organ donors by a Percoll-density gradient centrifugation technique, and uptake studies were performed using a rapid filtration technique. Our results on [(3)H]Pte-Glu uptake are summarized as follows: 1) uptake was sensitive to osmolarity of the incubation medium; 2) Na(+) removal from the incubation medium did not affect folate uptake into BLMV; 3) uptake was significantly increased with decreasing incubation buffer pH from 8 to 4; 4) uptake demonstrated saturation kinetics with an apparent Michaelis constant of 9.6 +/- 0.48 microM and a maximal velocity of 8.10 +/- 0.36 pmol x mg protein(-1) x 10 s(-1); 5) uptake was markedly inhibited by the structural analog methotrexate (inhibitory constant = 8.28 +/- 1.0 microM); 6) uptake into BLMV demonstrated a trans-stimulation phenomenon; 7) anion exchange inhibitors DIDS and SITS significantly inhibited folate uptake; and 8) uptake was potential-insensitive, as voltage clamping of vesicles or making them inside positive with K(+)/valinomycin failed to influence folate uptake. Western blot analysis using purified human colonic basolateral membrane preparations and specific polyclonal antibodies against the human reduced folate carrier (hRFC) has shown expression of the hRFC protein at this membrane domain. These data demonstrate the existence of a pH-dependent, DIDS-sensitive, electroneutral, carrier-mediated mechanism for folate transport across the human colonic basolateral membranes.
Dudeja
P K
PK
Department of Medicine, University of Illinois at Chicago and Westside Veterans Affairs Medical Center, Chicago, Illinois 60612, USA. pkdudeja@uic.edu
Kode
A
A
Alnounou
M
M
Tyagi
S
S
Torania
S
S
Subramanian
V S
VS
Said
H M
HM
D000328:Adult; D000595:Amino Acid Sequence; D000906:Antibodies; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D003106:Colon / Q000378:metabolism*; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000493:pharmacokinetics*; D005493:Folic Acid Antagonists / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007408:Intestinal Absorption / Q000502:physiology*; D007583:Jejunum / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface; D012964:Sodium / Q000494:pharmacology; D022161:Transport Vesicles / Q000378:metabolism; D014316:Tritium
D000906:Antibodies; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D005493:Folic Acid Antagonists; D011956:Receptors, Cell Surface; D014316:Tritium; D005492:Folic Acid; D012964:Sodium; D008727:Methotrexate