Calcium Transport In Caco-2 Cells Research Articles

Effects of phenytoin and carbamazepine on calcium transport in Caco-2 cells

Adverse effects of anti-seizure/anti-epileptic medications on bone density have been observed and reported since the early 1960s. Phenytoin and carbamazepine are two commonly prescribed anti-epileptic drugs most frequently associated with osteomalacia including fractures, bone demineralization, and reduced bone formation. The mechanism by which anti-epileptic drugs induce bone loss is not fully explained. We hypothesized that anti-epileptic drugs may impair dietary calcium absorption in the intestine. Using Caco-2 cells, a model transport system for study of the function of the intestinal epithelium, we determined the effects of several anti-epileptic drugs on intestinal epithelial calcium transport. In our system, phenytoin and carbamazepine dose-dependently inhibit active calcium transport from the apical to basolateral side of Caco-2 cells under physiologic calcium conditions. Vitamin D ameliorates the anti-epileptic drug-induced decrease in calcium permeability.

Comparative effects of glucose-lysineversus glucose-methionine Maillard reaction products consumption:in vitro andin vivo calcium availability

The influence of glucose-lysine and glucose-methionine Maillard reaction products (MRPs) on calcium availability was studied in rats and in Caco-2 cells. Equimolar glucose/lysine and glucose/methionine mixtures (40% moisture) were heated (150 degrees C, 30 or 90 min) to prepare samples (GL30, GL90, GM30, and GM90, respectively). For 21 days, the rats were fed the AIN-93G diet (control group) or diets containing separately 3% of the heated mixtures (GL30, GL90, GM30, and GM90 groups, respectively). In the last week of the trial, a calcium balance was performed. On day 21, the animals were sacrificed and their livers and femurs removed for analysis of calcium levels. The GL30 and GM30 samples and the corresponding raw mixtures were used for Caco-2 cells experiments. Fecal excretion of calcium decreased and urinary elimination increased in the GM30 and GM90 groups. In accordance, increased calcium transport in Caco-2 cells was found in the presence of the GM30 sample, compared with the raw sample. Bone calcium concentration was lower among the animals consuming MRP diets, compared with the control group. The possible long-term effects of MRP intake on calcium deposition in the bone should be further studied to ascertain the implications on related diseases.

Effects of heat treatment of casein in the presence of reducing sugars on calcium bioavailability: in vitro and in vivo assays.

Casein-glucose-fructose mixtures unheated (C) or after heating (HC) were added to a solution of ionic calcium to study calcium speciation and included in diets (C-D, HC-D) for rats. Samples and diets were digested in vitro. Supernatants of digested samples were used for transport experiments with Caco-2 cells. Total soluble and ionic calcium levels were lower and precipitated calcium levels higher with HC compared to C. Dialyzed calcium from the diets was highly ionic and lower in HC-D compared to C-D. Nondialyzed soluble calcium was also lower, whereas precipitated calcium was higher, in HC-D. HC increased calcium transport in Caco-2 cells compared to C, but transport efficiency decreased due to lower calcium solubililty after digestion. Urinary calcium increased with HC-D consumption without changes in calcium absorbed and retained. Maillard reaction products in HC decrease calcium solubility, but enterocyte metabolism and calcium absorption and retention seem to be unaffected. Nevertheless, urinary calcium losses increase.

1,25-Dihydroxyvitamin D3 increases the expression of the CaT1 epithelial calcium channel in the Caco-2 human intestinal cell line.

The active hormonal form of vitamin D (1,25-dihydroxyvitamin D) is the primary regulator of intestinal calcium absorption efficiency. In vitamin D deficiency, intestinal calcium absorption is low leading to an increased risk of developing negative calcium balance and bone loss. 1,25-dihydroxyvitamin D has been shown to stimulate calcium absorption in experimental animals and in human subjects. However, the molecular details of calcium transport across the enterocyte are not fully defined. Recently, two novel epithelial calcium channels (CaT1/ECaC2 and ECaC1/CaT2) have been cloned and suggested to be important in regulating intestinal calcium absorption. However, to date neither gene has been shown to be regulated by vitamin D status. We have previously shown that 1,25-dihydroxyvitamin stimulates transcellular calcium transport in Caco-2 cells, a human intestinal cell line. In the current study, we have demonstrated that Caco-2 cells express low but detectable levels of CaT1 mRNA in the absence of 1,25-dihydroxyvitamin D treatment. CaT1 mRNA expression is rapidly up regulated (4-fold increase at 4 h and 10-fold at 24 h) by treatment with 1,25-dihydroxyvitamin D (10(-7) moles/L). Moreover, the increase in CaT1 mRNA expression preceded by several hours the vitamin D induction of calbindin D9K, a putative cytosolic calcium transport protein. These observations are the first to demonstrate regulation of CaT1 expression by vitamin D and are consistent with a new model of intestinal calcium absorption wherein vitamin D-mediated changes in brush border membrane CaT1 levels could be the primary gatekeeper regulating homeostatic modulation of intestinal calcium absorption efficiency.

Specific 1,25(OH)2D3-mediated regulation of transcellular calcium transport in Caco-2 cells

Calcium transport in the apical-to-basolateral (A-to-B) or B-to-A direction was examined in cells treated with 10 nM 1, 25-dihydroxyvitamin D3 [1,25(OH)2D3, calcitriol] for up to 72 h. Net A-to-B calcium transport was positive at all time points and increased from 0.14 +/- 0.06 to 0.50 +/- 0.01 nmol. well-1. min-1 after 72 h of calcitriol treatment. Neither phenol red transport nor transepithelial electrical resistance was altered by calcitriol treatment, suggesting that the increase in net A-to-B calcium transport was not due to paracellular movement. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 (100 nM, 48 h) alters basal or calcitriol-stimulated A-to-B calcium transport. Treatment with the calmodulin antagonist trifluoperazine (50 microM) reduced calcitriol-stimulated A-to-B Ca transport by 56%. The transcription inhibitor actinomycin D inhibited calcitriol-regulated A-to-B calcium transport as well as calbindin D9k and 24-hydroxylase mRNA accumulation. These data demonstrate that calcitriol-mediated A-to-B calcium transport in Caco-2 cells is a specific, transcellular process that requires transcriptional events normally mediated through the vitamin D receptor.

Vitamin D-regulated calcium transport in Caco-2 cells: unique in vitro model

The human colon adenocarcinoma cell line Caco-2 is the only intestinal cell line to differentiate spontaneously in culture exhibiting structural and biochemical characteristics of mature enterocytes and to possess a vitamin D receptor in the fully differentiated state. Transepithelial calcium transport was characterized in differentiated Caco-2 cells grown on permeable filters supports to assess the potential utility of this cell line as an in vitro model to study 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced calcium transport. Calcium transport was increased in a dose-dependent manner by 1,25(OH)2D3. Total calcium transport at different calcium concentrations could be fitted to a modified Michaelis-Menten equation containing a linear transport component. The maximum rate of saturable calcium transport was increased by 4.3-fold (P less than 0.005) in cells treated with 10(-8) M 1,25(OH)2D3. This treatment also increased the apparent buffer calcium concentration that results in half-maximal velocity from 0.4 to 1.3 mM but had no significant effect on nonsaturable calcium transport. Caco-2 cells grown on permeable filter supports provide a unique in vitro human cell culture model to study the mechanism of vitamin D-regulated transepithelial intestinal calcium transport.