Electrogenic divalent cation transport in cultured human intestinal Caco-2 epithelia

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Abstract

Intestinal absorption of divalent metal ions (Fe2+, Zn2+, etc.) occurs via the transporter, DMT1, expressed in the apical membrane of villus enterocytes (Gunshin et al. 1997). This transport is coupled to the inward movement of protons, the source of which is the acid microclimate, maintained at the brush border of the absorbing enterocytes (McEwan et al. 1988). This study set out to investigate potential electrogenic transport associated with proton-coupled Fe2+ and Zn2+ transport in cultured human intestinal Caco-2 epithelia. Short-circuit current (ISC) determinations were performed on voltage-clamped Caco-2 epithelia (passages 38-45), grown to confluence on permeable supports (Anotec, Nunc). Cells were bathed with normal Krebs/Hepes or isotonic mannitol/Hepes buffer (37 °C; pH 7.4). At the onset of the experiment, the apical medium was adjusted to pH 6.0. FeSO4 (in solution with ascorbate, 1:10 molar ratio) or ZnSO4 (in solution with histidine, 1:5 molar ratio) were added to the apical medium. In experiments using ouabain (1 mM), this was added to the basolateral medium 1 h prior to the onset of experiment. With the apical perfusate at pH 6.0, apical addition of 100 µM Fe2+ in normal Krebs/Hepes buffer resulted in a transient inward ISC of 0.62 ± 0.11 µA cm-2 (5) (mean ± S.E.M. (n)). Raising apical pH to 7.4 resulted in a significant (P < 0.002; Student's unpaired t test) reduction of this ISC to 0.10 ± 0.00 µA cm-2 (5). This electrogenic response to apically applied Fe2+ (pH 6.0) was unaffected by prolonged exposure to basolateral ouabain (1 mM) (0.52 ± 0.10 µA cm-2 (7)) or isotonic mannitol/Hepes buffer (0.53 ± 0.10 µA cm-2 (5)). Under the latter condition, increasing [Fe2+] from 1 µM to 1 mM resulted in a saturable increase in ISC, with KM = 193 ± 48 µM (6) and Vmax = 63 ± 16 nmol cm-2 h-1. A similar pH-dependent inward ISC was obtained following apical exposure to Zn2+ (100 µM) (ISC pH 6.0 = 0.34 ± 0.04 µA cm-2 (5); ISC pH 7.4 = 0.10 ± 0.03 µA cm-2 (5); P < 0.002). These data demonstrate that Fe2+ and Zn2+ transport in Caco-2 epithelia is associated with an electrogenic process. Transport is pH dependent and does not require the presence of any ion species other than H+ and OH-. The results of this study support the view that DMT1-mediated Fe2+ and Zn2+ transport are both proton coupled and electrogenic.
Original languageEnglish
Title of host publicationProceedings of The Physiological Society
PagesSO76
Number of pages1
Volume539P
ISBN (Electronic)1749-6187
Publication statusPublished - 2002
EventPhysiological Society Meeting 2002 - University of York, York, United Kingdom
Duration: 1 Jan 2002 → …

Conference

ConferencePhysiological Society Meeting 2002
CountryUnited Kingdom
CityYork
Period1/01/02 → …

Keywords

  • iron
  • zinc
  • intestine
  • Caco-2
  • electrogenic
  • DMT-1
  • human
  • epithelia
  • divalent
  • cation

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