Effects of extracellular iron concentration on calcium absorption and relationship between Ca2+ and cell apoptosis in Caco-2 cells.
OBJECTIVE
To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells.


METHODS
Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry.


RESULTS
Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase XI and dispase I. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 micromol/L) increased the FI of Caco-2 cells from 27.50+/-13.18 (control, n=150) to 35.71+/-13.99 (n=150, P<0.01), 72.19+/-35.40 (n=150, P<0.01) and 211.34+/-29.03 (n=150, P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 micromol/L). The FI value of Caco-2 cells treated by FAC was 185.85+/-33.77 (n=150, P<0.01), 122.73+/-58.47 (n=150, P<0.01), and 53.29+/-19.82 (n=150, P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187 (0.1, 1.0 and 10 micromol/L) increased the FI of Caco-2 cells from 40.45+/-13.95 (control, n=150) to 45.19+/-21.95 (n=150, P<0.01), 89.87+/-43.29 (n=150, P<0.01) and 104.64+/-51.07 (n=150, P<0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell number was positively correlated with [Ca2+]i.


CONCLUSIONS
Ca2+ absorbability is increased with the decrease of extracellular iron concentration Fe3+ and hindered with the increase of Fe3+ consistence out of them. Furthermore, increase of [Ca2+]i can induce apoptosis in Caco-2 cells.
Wang
Li
L
Life Science College, Hebei Normal University, Shijiazhuang 050016, Hebei Province, China.
Li
Qing
Q
Duan
Xiang-Lin
XL
Chang
Yan-Zhong
YZ
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D018938:Caco-2 Cells; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000493:pharmacokinetics*; D018929:Cell Culture Techniques / Q000379:methods; D003676:Deferoxamine / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D005290:Ferric Compounds / Q000494:pharmacology; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology*; D007476:Ionophores / Q000494:pharmacology; D007501:Iron / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology
D005290:Ferric Compounds; D007476:Ionophores; D007502:Iron Chelating Agents; D000644:Quaternary Ammonium Compounds; D000001:Calcimycin; D007501:Iron; D003676:Deferoxamine; D002118:Calcium; C013531:ferric ammonium citrate