Effects of extracellular iron concentration on calcium absorption and relationship between Ca2+ and cell apoptosis in Caco-2 cells

AIM: 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 mu mol/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 mu mol/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 A(23187) (0.1, 1.0 and 10 mu mol/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 A(23187) 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). CONCLUSION: 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. (c) 2005 The WJG Press and Elsevier Inc. All rights reserved.