Intestinal Ca2+ absorption revisited: A molecular and clinical approach.

Ca2+ has an important role in the maintenance of the skeleton and is involved in the main physiological processes. Its homeostasis is controlled by the intestine, kidney, bone and parathyroid glands. The intestinal Ca2+ absorption occurs mainly via the paracellular and the transcellular pathways. The proteins involved in both ways are regulated by calcitriol and other hormones as well as dietary factors. Fibroblast growth factor 23 (FGF-23) is a strong antagonist of vitamin D action. Part of the intestinal Ca2+ movement seems to be vitamin D independent. Intestinal Ca2+ absorption changes according to different physiological conditions. It is promoted under high Ca2+ demands such as growth, pregnancy, lactation, dietary Ca2+ deficiency and high physical activity. In contrast, the intestinal Ca2+ transport decreases with aging. Oxidative stress inhibits the intestinal Ca2+ absorption whereas the antioxidants counteract the effects of prooxidants leading to the normalization of this physiological process. Several pathologies such as celiac disease, inflammatory bowel diseases, Turner syndrome and others occur with inhibition of intestinal Ca2+ absorption, some hypercalciurias show Ca2+ hyperabsorption, most of these alterations are related to the vitamin D endocrine system. Further research work should be accomplished in order not only to know more molecular details but also to detect possible therapeutic targets to ameliorate or avoid the consequences of altered intestinal Ca2+ absorption.

Lithocholic acid: a new emergent protector of intestinal calcium absorption under oxidant conditions.

LCA and 1,25(OH)2D3 are vitamin D receptor ligands with different binding affinity. The secosteroid stimulates intestinal Ca2+ absorption. Whether LCA alters this process remains unknown. The aim of our work was to determine the effect of LCA on intestinal Ca2+ absorption in the absence or presence of NaDOC, bile acid that inhibits the cation transport. The data show that LCA by itself did not alter intestinal Ca2+ absorption, but prevented the inhibitory effect of NaDOC. The concomitant administration of LCA avoided the reduction of intestinal alkaline phosphatase activity caused by NaDOC. In addition, LCA blocked a decrease caused by NaDOC on gene and protein expression of molecules involved in the transcellular pathway of intestinal Ca2+ absorption. The oxidative stress and apoptosis triggered by NaDOC were abrogated by LCA co-treatment. In conclusion, LCA placed in the intestinal lumen protects intestinal Ca2+ absorption against the inhibitory effects caused by NaDOC. LCA avoids the reduction of the transcellular Ca2+ movement, apparently by blocking the oxidative stress and apoptosis triggered by NaDOC, normalizing the gene and protein expression of molecules involved in Ca2+ movement. Therefore, LCA might become a possible treatment to improve intestinal calcium absorption under oxidant conditions.

Ursodeoxycholic and deoxycholic acids: Differential effects on intestinal Ca(2+) uptake, apoptosis and autophagy of rat intestine.

The aim of this work was to study the effect of sodium deoxycholate (NaDOC) and ursodeoxycholic acid (UDCA) on Ca(2+) uptake by enterocytes and the underlying mechanisms. Rats were divided into four groups: a) controls, b) treated with NaDOC, c) treated with UDCA d) treated with NaDOC and UDCA. Ca(2+) uptake was studied in enterocytes with different degrees of maturation. Apoptosis, autophagy and NO content and iNOS protein expression were evaluated. NaDOC decreased and UDCA increased Ca(2+) uptake only in mature enterocytes. The enhancement of protein expression of Fas, FasL, caspase-8 and caspase-3 activity by NaDOC indicates triggering of the apoptotic extrinsic pathway, which was blocked by UDCA. NO content and iNOS protein expression were enhanced by NaDOC, and avoided by UDCA. The increment of acidic vesicular organelles and LC3 II produced by NaDOC was also prevented by UDCA. In conclusion, the inhibitory effects of NaDOC on intestinal Ca(2+) absorption occur by decreasing the Ca(2+) uptake by mature enterocytes. NaDOC triggers apoptosis and autophagy, in part as a result of nitrosative stress. In contrast, UDCA increases the Ca(2+) uptake by mature enterocytes, and in combination with NaDOC acts as an antiapoptotic and antiautophagic agent normalizing the transcellular Ca(2+) pathway.

Oxidative stress, cell cycle arrest and differentiation contribute toward the antiproliferative action of BSO and calcitriol on Caco-2 cells.

The prognosis and incidence of colon cancer are linked to vitamin D3 serum levels. To evaluate the effects of D,L-buthionine-S,R-sulfoximine (BSO), 1,25(OH)2D3 and their combination on intestinal Caco-2 cell growth, to elucidate the possible cellular mechanisms involved in their antiproliferative action, and to determine whether BSO acts as a sensitizer to 1,25(OH)2D3 treatment, enabling minimization of the toxic effects caused by high doses of the steroid. Human colon cancer Caco-2 cells were treated with 1,25(OH)2D3, BSO, both, or vehicle. Cell proliferation was evaluated by crystal violet staining. Cell cycle and mitochondrial membrane potential were measured by flow cytometry. Total glutathione, catalase, superoxide dismutase, superoxide anion levels, and alkaline phosphatase activities were analyzed by spectrophotometry. DNA fragmentation was evaluated using the terminal dUTP nick end labeling assay. BSO and 1,25(OH)2D3 inhibited Caco-2 cell growth, an effect that was higher with the combined treatment. The antiproliferative effect produced by the combination could be protected by ascorbic acid. BSO plus 1,25(OH)2D3 induced cell cycle arrest and suppressed cell division. Total glutathione decreased and superoxide anion increased with BSO and BSO plus 1,25(OH)2D3. Catalase activity increased with the combined treatment. Mitochondrial membrane potential and alkaline phosphatase activity were altered by 1,25(OH)2D3 alone or plus BSO. The percentage of terminal dUTP nick end labeling-positive cells was increased. BSO increases the antiproliferative effect of 1,25(OH)2D3 on Caco-2 cells through induction of oxidative stress, which occurs simultaneously with DNA breakage. The antioxidant system can partially compensate the damage induced by BSO plus 1,25(OH)2D3. Cell differentiation induction is also involved in the response to the combined treatment.

Effects of quercetin and menadione on intestinal calcium absorption and the underlying mechanisms.

Quercetin (QT) could be considered as a potential therapeutic agent for different diseases due to its antioxidant, anti-inflammatory, antiviral and anticancer properties. This study was designed to investigate the ability of QT to protect the chick intestine against menadione (MEN) induced injury in vivo and in vitro. Four-week old chicks (Gallus gallus) were treated i.p. with 2.5µmol of MEN/kg b.w. or with i.l. 50µM QT or both. QT protected the intestinal Ca(2+) absorption against the inhibition caused by MEN, but QT alone did not modify. Glutathione (GSH) depletion provoked by MEN in chick enterocytes was abolished by QT treatment, whereas QT alone did not modify the intestinal GSH content. The enhancement of GSH peroxidase activity produced by MEN was blocked by QT treatment. In contrast, superoxide dismutase activity remained high after simultaneous treatment of enterocytes with MEN and QT. The flavonol also avoided changes in the mitochondrial membrane permeability (swelling) produced by MEN. The FasL/Fas/caspase-3 pathway was activated by MEN, effect that was abrogated by QT. In conclusion, QT may be useful in preventing inhibition of chick intestinal Ca(2+) absorption caused by MEN or other substances that deplete GSH, by blocking the oxidative stress and the FasL/Fas/caspase-3 pathway activation.

Buthionine sulfoximine and 1,25-dihydroxyvitamin D induce apoptosis in breast cancer cells via induction of reactive oxygen species.

Calcitriol or 1,25(OH)(2)D(3) is a negative growth regulator of breast cancer cells. The aim of this study was to determine whether L-buthionine-S,R-sulfoximine, a glutathione-depleting drug, modifies the antiproliferative effects of 1,25(OH)(2)D(3) on MCF-7 cells. For comparison, we included studies in MCF-7 cells selected for vitamin D resistance and in human mammary epithelial cells transformed with SV40 and ras. Our data indicate that L-buthionine-S,R-sulfoximine enhances the growth inhibition of 1,25(OH)(2)D(3) in all transformed breast cell lines. This effect is mediated by ROS leading to apoptosis. In conclusion, BSO alters redox state and sensitizes breast cancer cells to 1,25(OH)(2)D(3)-mediated apoptosis.

Sodium deoxycholate inhibits chick duodenal calcium absorption through oxidative stress and apoptosis.

High concentrations of sodium deoxycholate (NaDOC) produce toxic effects. This study explores the effect of a single high concentration of NaDOC on the intestinal Ca(2+) absorption and the underlying mechanisms. Chicks were divided into two groups: 1) controls and 2) treated with different concentrations of NaDOC in the duodenal loop for variable times. Intestinal Ca(2+) absorption was measured as well as the gene and protein expressions of molecules involved in the Ca(2+) transcellular pathway. NaDOC inhibited the intestinal Ca(2+) absorption, which was concentration dependent. Ca(2+)-ATPase mRNA decreased by the bile salt and the same occurred with the protein expression of Ca(2+)-ATPase, calbindin D(28k) and Na(+)/Ca(2+) exchanger. NaDOC produced oxidative stress as judged by ROS generation, mitochondrial swelling and glutathione depletion. Furthermore, the antioxidant quercetin blocked the inhibitory effect of NaDOC on the intestinal Ca(2+) absorption. Apoptosis was also triggered by the bile salt, as indicated by the TUNEL staining and the cytochrome c release from the mitochondria. As a compensatory mechanism, enzyme activities of the antioxidant system were all increased. In conclusion, a single high concentration of NaDOC inhibits intestinal Ca(2+) absorption through downregulation of proteins involved in the transcellular pathway, as a consequence of oxidative stress and mitochondria mediated apoptosis.

Molecular mechanisms involved in the enhancement of mitochondrial malate dehydrogenase activity by calcitriol in chick intestine.

Mitochondrial malate dehydrogenase (mMDH) from the intestine is the NAD-linked oxidoreductase of the tricarboxylic acid cycle with the highest activity and response to vitamin D treatment in vitamin D-deficient chicks (-D). The aim of this study was to elucidate potential molecular mechanisms by which cholecalciferol or calcitriol enhances the activity of this enzyme. One group of animals used was composed of -D and -D treated with cholecalciferol or with calcitriol. A second group consisted of -D and -D supplemented with high Ca(2+) diet. A third group included chicks receiving either a normal or a low Ca(2+) diet. In some experiments, animals were injected with cycloheximide. Data showed that either vitamin D (cholecalciferol or calcitriol) or a low Ca(2+) diet increases mMDH activity. High Ca(2+) diet did not modify the intestinal mMDH activity from -D. The mMDH activity from -D remained unaltered when duodenal cells were exposed to 10(-8) mol/L calcitriol for 15 min. The enhancement of mMDH activity by calcitriol was completely abolished by simultaneous cycloheximide injection to -D. mMDH mRNA levels, detected by RT-PCR, indicate that calcitriol did not affect gene expression. In contrast, Western blots show that calcitriol enhanced the protein expression. In conclusion, calcitriol stimulates intestinal mMDH activity by increasing protein synthesis. No response of mMDH activity by rapid effects of calcitriol or activation through increment of serum Ca(2+) was demonstrated. Consequently, ATP production would be increased, facilitating the Ca(2+) exit from the enterocytes via the Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger, which participate in the intestinal Ca(2+) absorption.

Antiproliferative action of menadione and 1,25(OH)2D3 on breast cancer cells.

Calcitriol or 1,25(OH)(2)D(3) is a negative growth regulator of MCF-7 breast cancer cells. The growth arrest is due to apoptosis activation, which involves mitochondrial disruption. This effect is blunted in vitamin D resistant cells (MCF-7(DRes) cells). Menadione (MEN), a glutathione (GSH)-depleting compound, may potentiate antitumoral effects of anticancer drugs. The aim of this study was to investigate whether MEN enhances cellular responsiveness of MCF-7 cells to 1,25(OH)(2)D(3). Cells were cultured and treated with different concentrations of 1,25(OH)(2)D(3)+/-MEN or vehicle for 96 h. GSH levels and the activity of antioxidant enzymes were determined by spectrophotometry and ROS production by flow cytometry. Both drugs decreased growth and enhanced ROS in MCF-7 cells, obtaining the maximal effects when 1,25(OH)(2)D(3) was combined with MEN (P<0.01 vs. Control and vs. each compound alone). MCF-7(DRes) cells were not responsive to 1,25(OH)(2)D(3), but the cell proliferation was slightly inhibited by the combined treatment. Calcitriol and MEN separately enhanced antioxidant enzyme activities, but when they were used in combination, the effect was more pronounced (P<0.05 vs. Control and vs. each compound alone). MEN, calcitriol and the combined treatment decreased GSH levels (P<0.05 vs. Control). The data indicate that MEN potentiates the effect of 1,25(OH)(2)D(3) on growth arrest in MCF-7 cells by oxidative stress and increases the activities of antioxidant enzymes, probably as a compensatory mechanism.

Minireview on regulation of intestinal calcium absorption. Emphasis on molecular mechanisms of transcellular pathway.

An overview of current information on the mechanisms by which intestinal calcium absorption occurs is described in this article. Both paracellular and transcellular pathways are analyzed. Special emphasis focuses on molecules participating in the latter pathway, such as TRPV5 and TRPV6 channels, located in the apical region of the enterocytes, CB(9k) and CB(28k), presumably involved in the cation movement from the apical to the basolateral pole of the cell, and PMCA(1b) and Na(+)/Ca(2+) exchanger, proteins that extrude Ca(2+) from the cells. Current concepts on the relative importance of paracellular and transcellular calcium transport and the vitamin D dependence of each pathway are referred and analyzed showing the contrasting views on this issue. More detailed information is given regarding the stimulatory effect of vitamin D on intestinal Ca(2+) absorption either in animal models or in the human intestine. The possible mechanisms triggered by hormones such as PTH, calcitonin, estrogen, thyroid hormone, glucocorticoids and different nutritional factors on intestinal calcium absorption are also reviewed. Finally, the influence of physiological conditions such as growth, pregnancy, lactation and aging on intestinal calcium absorption are discussed.

Mitochondrial dysfunction is responsible for the intestinal calcium absorption inhibition induced by menadione.

Menadione (MEN) inhibits intestinal calcium absorption by a mechanism not completely understood. The aim of this work was to find out the role of mitochondria in this inhibitory mechanism. Hence, normal chicks treated with one i.p. dose of MEN were studied in comparison with controls. Intestinal calcium absorption was measured by the in situ ligated intestinal segment technique. GSH, oxidoreductase activities from the Krebs cycle and enzymes of the antioxidant system were measured in isolated mitochondria. Mitochondrial membrane potential was measured by a flow cytometer technique. DNA fragmentation and cytochrome c localization were determined by immunocytochemistry. Data indicate that in 30 min, MEN decreases intestinal Ca(2+) absorption, which returns to the control values after 10 h. GSH was only decreased for half an hour, while the activity of malate dehydrogenase and alpha-ketoglutarate dehydrogenase was diminished for 48 h. Mn(2+)-superoxide dismutase activity was increased in 30 min, whereas the activity of catalase and glutathione peroxidase remained unaltered. DNA fragmentation and cytochrome c release were maximal in 30 min, but were recovered after 15 h. In conclusion, MEN inhibits intestinal Ca(2+) absorption by mitochondrial dysfunction as revealed by GSH depletion and alteration of the permeability triggering the release of cytochrome c and DNA fragmentation.

Dietary calcium deficiency increases Ca2+ uptake and Ca2+ extrusion mechanisms in chick enterocytes.

Ca2+ uptake and Ca2+ extrusion mechanisms were studied in enterocytes with different degree of differentiation from chicks adapted to a low Ca2+ diet as compared to animals fed a normal diet. Chicks adapted to a low Ca2+ diet presented hypocalcemia, hypophosphatemia and increased serum 1,25(OH)2D3 and Ca2+ absorption. Low Ca2+ diet increased the alkaline phosphatase (AP) activity, independently of the cellular maturation, but it did not alter gamma-glutamyl-transpeptidase activity. Ca2+ uptake, Ca2+-ATPase and Na(+)/Ca2+ exchanger activities and expressions were increased by the mineral-deficient diet either in mature or immature enterocytes. Western blots analysis shows that vitamin D receptor (VDR) expression was much higher in crypt cells than in mature cells. Low Ca2+ diet decreased the number of vitamin D receptor units in both kinds of cells. In conclusion, changes in Ca2+ uptake and Ca2+ extrusion mechanisms in the enterocytes by a low Ca2+ diet appear to be a result of enhanced serum levels of 1,25(OH)2D3, which would promote cellular differentiation producing cells more efficient to express vitamin D dependent genes required for Ca2+ absorption.

Effects of a single dose of menadione on the intestinal calcium absorption and associated variables.

The effect of a single large dose of menadione on intestinal calcium absorption and associated variables was investigated in chicks fed a normal diet. The data show that 2.5 micro mol of menadione/kg of b.w. causes inhibition of calcium transfer from lumen-to-blood within 30 min. This effect seems to be related to oxidative stress provoked by menadione as judged by glutathione depletion and an increment in the total carbonyl group content produced at the same time. Two enzymes presumably involved in calcium transcellular movement, such as alkaline phosphatase, located in the brush border membrane, and Ca(2+)- pump ATPase, which sits in the basolateral membrane, were also inhibited. The enzyme inhibition could be due to alterations caused by the appearance of free hydroxyl groups, which are triggered by glutathione depletion. Addition of glutathione monoester to the duodenal loop caused reversion of the menadione effect on both intestinal calcium absorption and alkaline phosphatase activity. In conclusion, menadione shifts the balance of oxidative and reductive processes in the enterocyte towards oxidation causing deleterious effects on intestinal Ca(2+) absorption and associated variables, which could be prevented by administration of oral glutathione monoester.