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

Abstract

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 μmol/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 μmol/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 μmol/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.

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.

Key Words: Iron calcium absorption, Cell apoptosis, Caco-2 cells

INTRODUCTION

Calcium is one of the most important and necessary element which maintains the physiological function. When the absorption of calcium is obstructed, many diseases are induced, such as hypertension, hyperkinesias, colorectal carcinoma, cardiovascular disease and osteoporosis, etc. The iron is one of the most common metals existing in our environment. In the last decade, a number of studies[1,2] suggested that the ferric absorption was inhibited by calcium. However, little is known about the effect of iron concentration change on calcium absorption. The purpose of the present study was to investigate the effect of extracellular iron concentration ([Fe³⁺]) on calcium absorption, and to study the effect of rising intracellular calcium concentration ([Ca²⁺]_i) on the apoptosis of Caco-2 cells.

MATERIALS AND METHODS

Materials

All chemicals were of analytical grade. Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were purchased from Gibco. FDA, A₂₃₁₈₇ and Fluo-3/AM were purchased from the Laboratory of Molecular Cell Biology of Hebei Normal University. Desferrioxamine (DFO), ferric ammonium citrate (FAC) and A23187 were dissolved in Ca²⁺/Mg²⁺-free phosphate-buffered saline containing 0.40 g KCl, 0.06 g KH₂PO₄, 8 g NaCl, 0.35 g NaHCO₃, and 0.09 g Na₂HPO₄·7H₂O in 1-L liquid.

Cell preparation

Caco-2 cells, from cell store room of Shanghai Cell Academic Institution of CAS, were cultured in DMEM supplemented with 10% fetal bovine serum. Culture medium was changed every 2-3 d. The 20-30^th generation of Caco-2 cells cultured for 3 d at 37 °C was digested with trypsin and the cell concentration was fixed to about 1×10⁶/mL, they were then seeded in six-well plates and continuously cultured. The culture liquid was made up of φ (FCS) = 10%, DMEM φ (high sugar) = 85%, double antibiotics in which the final concentration of penicillin was 100 IU/mL and that of streptomycin was 100 μg/mL.

Fluo3-AM loading

The Caco-2 cells were incubated with Fluo3-AM working solution containing 0.03% Pluronic F-127 (the final concen-tration of Fluo3-AM was 20 μmol/L) at 37 °C for 40 min. After incubation, the cells were washed thrice at 25 °C with Ca²⁺/Mg²⁺-free phosphate-buffered saline to remove extrac-ellular Fluo3-AM.

Measurement of [Ca²⁺]_i

After Fluo3-AM loading, the cells were mounted on the small pool of Teflon printed slice, and covered with cover glass. Only the cells with rod shaped and visible striations were used for experiments. The fluorescence signals were detected with a confocal laser scanning system (Biorad lasersharp MRA2, Oxfordshire, UK), which was equipped with a Nikon E-600 eclipse microscope. An argon laser was used to excite Fluo3 at 488 nm and emit at 530 nm. [Ca²⁺]_i changes were represented with fluorescence intensity (FI).

Experimental protocols

The experiments consisted of three groups: (1) Effect of DFO on intracellular calcium concentration ([Ca²⁺]_i): FI was measured after 100, 200 and 300 μmol/L DFO were added to normal phosphate-buffered saline containing Ca²⁺ and Mg²⁺ for 20 min (n = 150). Ca²⁺/Mg²⁺-free phosphate-buffered saline was used as a control (n = 150). (2) Effect of FAC on [Ca²⁺]_i: FI was measured after 10, 50 and 100 μmol/L FAC were added to normal phosphate-buffered saline containing Ca²⁺ and Mg²⁺ for 20 min (n = 150). Ca²⁺/Mg²⁺-free phosphate-buffered saline was used as a control (n = 150). (3) Effect of calcium ionophore A₂₃₁₈₇ on [Ca²⁺]_i: FI was measured after 0.1, 1.0, and 10 μmol/L A₂₃₁₈₇ were added to normal phosphate-buffered saline containing Ca²⁺ and Mg²⁺ for 20 min (n = 150). Ca²⁺/Mg²⁺-free phosphate-buffered saline was used as a control (n = 150).

FCM examination method

The Caco-2 cells of the 20-30^th generation were planted to plastic culture flasks after being digested by trypsin and the number of the cells was 1×10⁶/mL, and cultured for 3 d. The nutrition liquid for growth was discarded after the cells developed into a conflux monolayer, and the course of medication was the same as the previous one. PBS (0.01 mol/L) was added to the cells after they were digested, the cells were then centrifuged and the supernatant fluid was discarded. The cells were fixed with cold ethanol of 70%, and then stored at -4 °C overnight for flow cytometry (FCM) analysis.

Data processing

The cells were dealt with confocal assistant software, and 150 cells were disposed for each sample. The data were analyzed with ANOVA and LSD examination by STAT software, and the examination results were expressed as mean±SD.

The coherence examination of χ²-test was used for the test of apoptotic percentage, and the test method of df = 1 and f_t>5 for two groups was used. The calculating formula was χ² = Σ(f_o-f_t)2/f_t, where practice frequency is denoted with f_o, theoretical frequency is denoted with f_t, and the summation is denoted with Σ. The test level of χ²-test was α = 0.05, and P<0.05 was considered statistically significant.

RESULTS

Primary culture of epithelial cells

The intestinal mucosa suspension after being digested was constituted of recess epithelial cells observed under an inverted microscope. The cell livability was more than 95.7% digested by the mixture of collagenase IX and neutral proteinase I after trypan blue staining. There were undispersed epithelial cells and elastic fiber in the discarded depositions (Figure 1A). Primary cultured cells of intestinal mucosa were adhered after 24-48 h and then converged into groups of cell colonies after 4-6 d (Figure 1B), and the cells joined into pieces after 10-12 d. They were monolayer and polygon cells and pavement-like (Figure 1C). Cell borderlines were clear and not overlapped. The cytoplasm was abundant and their nuclei were round and olivary, the chromatin in the nuclei was very sparse with 1-2 nucleoli. There were few smooth muscle cells, fibroblasts and glial cells when observed under the microscope. The cells cultured in this experiment were not subcultured.

Figure 1 Epithelial cells in primary culture. A: Ingredients in the sediment after centrifugation (×200); B: colonies formed in cells cultured for 6 d (×100); C: pavement-like cells formed in cells cultured for 9 d (×200); D: immune positive cells (↑) (×400); E: no staining in negative group (×100); F: cell movement, adherent cells (▲) and just adherent cells (↓) (×100).

The cell keratin was the characteristic antigen ingredient of epithelial tissue. The antibody of cell keratin marked with biotin was used in this experiment to show qualitatively the rat intestinal epithelial cells. The immunocytochemistry result showed that the intestinal epithelial cells were positive brown (Figure 1D), and that the negative group was not stained (Figure 1E).

Effect of DFO on [Ca²⁺]_i

Fe³⁺ was chelated by adding different concentrations of DFO into the culture liquid. DFO (100, 200 and 300 μmol/L) increased [Ca²⁺]_i of Caco-2 cells in a concentration-dependent manner. The FI value of Caco-2 cells was 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), respectively. The FI value of control was 27.50±13.18 (control, n = 150). There was a significant difference among the treatments (P<0.01, Figure 2A). It showed that the transportation of Ca²⁺ into the cells was increased with the decrease of Fe³⁺ concentration in the cells.

Figure 2 Effect of different concentrations of DFO (A), FAC (B), and A₂₃₁₈₇ (C) on Ca²⁺ concentration in Caco-2 cells. A: 1a: Control, ×200; 1b: the final consistence of DFO was 100 μmol/L, ×200; 1c: the final consistence of DFO was 200 μmol/L, ×200; 1d: the final consistence of DFO was 300 μmol/L, ×200. B: 2a: control, ×200; 2b: the final consistence of FAC was 10 μmol/L, ×200; 2c: the final consistence of FAC was 50 μmol/L, ×200; 2d: the final consistence of FAC was 100 μmol/L, ×200. C: 3a (1) the photograph of transmission light, showing the place of cells, ×200; (2) the photograph of fluorescence, showing the active cells, ×200; 3b: blank control, ×200; 3c: negative control, ×200; 3d: the final consistence of A₂₃₁₈₇ was 0.1 μmol/L, ×200; 3e: the final consistence of A₂₃₁₈₇ was 1.0 μmol/L, ×200; 3f: the final consistence of A₂₃₁₈₇ was 10 μmol/L, ×200.

Effect of ferric ammonium citrate (FAC) on [Ca²⁺]_i

Fe³⁺ was increased by adding different concentrations of Fe³⁺ donor FAC into the culture liquid. The FI value of Caco-2 cells was examined, the result showed that FAC (10, 50 and 100 μmol/L) decreased the FI from 44.43±14.14 (control, n = 150) to 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. There was a significant difference among the treatments (P<0.01, Figure 2B). It showed that the transportation of Ca²⁺ into the cells could be induced by the slight increase of [Fe³⁺], but the transportation of Ca²⁺ into the cells was hindered with the continuous increase of [Fe³⁺] in the cells.

Effect of calcium ionophore A23187 on [Ca²⁺]_i

The cell livability was examined with FDA, and its final concentration was 10 mg/L. FDA could be decomposed and take on green fluorescence when it enters the live cells, but it cannot be decomposed when it enters the dead cells and take on fluorescence. The live nature of cells was observed by confocal laser scanning microscope, and the cell livability was more than 90%.

The FI of blank control without A₂₃₁₈₇ and Fluo-3/AM was very feeble (25.47±6.48, n = 150), and that of negative control without A₂₃₁₈₇ was relatively feeble (40.45±13.95, n = 150). The FI of negative control was obviously stronger than that of blank control (P<0.01). Calcium ionophore A₂₃₁₈₇ (0.1, 1.0 and 10 μmol/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. There was a significant difference among the treatments (P<0.01, Figure 2C).

Effect of [Ca²⁺]_i on the apoptosis of Caco-2 cells

There was a sub-duple body apex, namely apoptotic apex, in front of the G₀/G₁ phase cells of all groups in the PI fluorescent histograms examined with FCM. The result showed that the positive apoptotic rate of control was 0.32%, and the AP apex was very low. The apoptotic percentage of each treatment was 0.69%, 0.90% and 1.10%, respectively. The cell apoptotic percentage of treatment group with A₂₃₁₈₇ (0.1 μmol/L) was obviously higher than that of negative control (P<0.01) examined with χ²-test. The cell apoptotic percentage of treatment group with A₂₃₁₈₇ (10 μmol/L) was also not obviously different from that of treatment group with A₂₃₁₈₇ (1 μmol/L) (P>0.05). But the cell apoptotic percentage of treatment group with A₂₃₁₈₇ (10 μmol/L) was obviously increased compared to that of treatment with A₂₃₁₈₇ (0.1 μmol/L) (P<0.01), suggesting that the cell apoptotic percentage was related with the increase of Ca²⁺ concentration in Caco-2 cells, that is to say, the increase of Ca²⁺ concentration in the Caco-2 cells was positively correlated to its apoptosis.

DISCUSSION

Iron is one of the most abundant micronutrients. Excessive iron also has cell toxicity and induces cell damnification. So there are rigid regulating mechanisms in the body to keep the balance of iron metabolism. Duodenum and jejunum are the main place of iron absorption and the hinge position in regulating the balance of iron metabolism. It is known that iron in the enteric cavity is carried into the small intestine epithelia by divalent metal transporter 1 in its membrane with Fe²⁺ formation. It is commonly considered that calcium hinders iron absorption[1-5]. Iron absorption was hindered by adding CaCl₂ and this function was in a dose-dependent manner by separating rat gastrointestinal loop and observing the iron absorption after the rat was supplied with different doses of calcium (CaCl₂)[6-10]. At present, the mechanism of reciprocity between calcium and iron has not yet been elucidated. Although the absorption methods of heme iron and nonheme iron were different, their absorption was also hindered by calcium. Hallberg et al[6], thought that the same transport carrier was used by calcium and iron, and there existed competition and inhibition between them in the transport process from mucous membrane cells to blood plasma. This track of nonheme iron was the same to heme iron. There were only few reports on the calcium absorption under the effect of iron. Calcium absorption was not influenced by the increase of Fe³⁺ concentration, but it was increased by iron only in the instance of the ratio of 20 to 1 of iron to calcium under the effect of different Fe³⁺ in the brush border vesicle[11-16]. In the present study, we demonstrated that the Fe³⁺ chelating agent DFO increased the Ca²⁺ concentration of Caco-2 cells in a concentration-dependent manner and the FAC decreased the Ca²⁺ concentration of Caco-2 cells, suggesting that calcium absorption is influenced by [Fe³⁺]. But the idiographic mechanism of calcium absorption increased by a low dose of Fe³⁺ and inhibited by a high dose of Fe³⁺ remains to be further established.

In the present study, calcium ionophore A₂₃₁₈₇ increased the Ca²⁺ concentration of Caco-2 cells in a concentration-dependent manner. Ca²⁺ being the important second messenger in cells is the signal of subsistence and death, and almost all physiological activities are regulated by Ca²⁺, for instance, flop of heart, secretion of hormone and transfer and reserve of information in cerebrum. The foundation at the start of life and the process of cells developing into special type cells are touched and controlled by Ca²⁺, and cell physiological activity is regulated by Ca²⁺, and then finally cell apoptosis is ongoing under the function of Ca²⁺. Intracellular calcium concentration is commonly between 0.1 and 10 mmol/L, while extracellular calcium concentration is about 0.1 μmol/L. It is the concentration difference that becomes the base of the physiological function of Ca²⁺. The concentration of dissociating Ca²⁺ in cell cytoplasts is the pivotal factor to regulate various responses, and there are different kinds of calcium-regulating mechanism to keep its balance. Some studies showed that the transitory increase of Ca²⁺ concentration in cells could be reduced by ATP which can stimulate cells[17-22]. Biological function can be regulated by mobilization of Ca²⁺ in cells including temporary reaction of muscle constriction, nerve conduction and cell secretion, etc., and permanent reaction of cell differentiation and proliferation. All in all, cell function is regulated by Ca²⁺, and it could pass the stimulating signals of excitomotors onto the enzyme reaction system and functional protein[23-26].

Excessive [Ca²⁺]_i is harmful to cells. Many damaging factors such as insufficient oxygen, toxin, oxidative stress, defective blood perfusion, blood poisoning, ionizing radiation and enteritis can induce the rising of intracellular calcium, which results in cell apoptosis. A₂₃₁₈₇ is a moving ionic carrier which carries bivalent positive ions of Ca²⁺, Mg²⁺, etc. into cells and two H⁺s are carried out of cells at the same time. Ca²⁺ can step into cell cytoplasts immediately if A₂₃₁₈₇ is added into live cell culture liquid. Therefore, A₂₃₁₈₇ is widely used to increase the extranuclear dissociated Ca²⁺ concentration in cells. The extranuclear Ca²⁺ concentration can be immediately increased by calcium carrier of A₂₃₁₈₇ when touched with cells, and then the endogeneity endoprotease, which can make DNA rupture among the nucleosomes into segments between 180 and 200 bp or its diploid segments taking on ladder atlas in the gel electrophoresis, is aroused and cell apoptosis is increased. Inhibition of oxidative phosphorylation process in mitochondria, decrease of mitochondrial membrane electric potential and ATP content in tissues, activation of phosphatidase and proteinase and irreversible cell damage have been induced by calcium overload[27-32].