Comparative Biochemistry and Physiology, Part A 139 (2004) 133 – 141 www.elsevier.com/locate/cbpa
Dietary calcium deficiency increases Ca2+ uptake and Ca2+ extrusion mechanisms in chick enterocytes Viviana A. Centenoa, Gabriela E. Dı´az de Barbozaa, Ana M. Marchionattia, Arturo E. Alisioa, Maria E. Dallorsob, Rene´e Nasif a, Nori G. Tolosa de Talamonia,* a
Laboratorio de Metabolismo Fosfoca´lcico y Vitamina D bDr. F. Can˜asQ, Ca´tedra de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias Me´dicas, Universidad Nacional de Co´rdoba, Co´rdoba, Argentina b Facultad de Ciencias Agrarias, Universidad de Lomas de Zamora, Argentina Received 18 December 2003; received in revised form 2 August 2004; accepted 4 August 2004
Abstract 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 g-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. D 2004 Elsevier Inc. All rights reserved. Keywords: Ca2+ pump; Ca2+ uptake; Chick; Na+/Ca2+ exchanger; Villus tip/crypt axis; Low Ca2+ diet; Calcitriol; Alkaline phosphatase; Vitamin D receptor
1. Introduction Low Ca2+ intake causes hypocalcemia, bone mass loss and increment in the risk of osteoporosis development either in humans or in experimental animals. In order to conserve calcium required for bone mineral content, adaptive mechanisms have evolved, which include increase
Abbreviations: AP, alkaline phosphatase; BBM, brush border membrane; BLM, basolateral membrane; BLMV, basolateral membrane vesicles; Ca2+-ATPase, plasma membrane calcium pump; 1,25(OH)2D3, 1a,25dihydroxyvitamin D3; DTT, dithiothreitol; PTH, parathyroid hormone; TBS, Tris-buffered saline solution; VDR, vitamin D receptor. * Corresponding author. Tel.: +54 351 4333024; fax: +54 351 4333072. E-mail address:
[email protected] (N.G. Tolosa de Talamoni). 1095-6433/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2004.08.002
in the efficiency of intestinal Ca2+ absorption and decrease in urinary calcium losses. The mechanisms of adaptation to a low Ca2+ diet depend on the vitamin D status, mainly on the synthesis rate of 1a,25-dihydroxyvitamin D3(1,25(OH)2D3). Low Ca2+ diets affect not only vitamin D metabolism, but also parathyroid cell proliferation, regulation of parathyroid hormone (PTH) gene expression and PTH secretion (Silver et al., 2002; Moallem et al., 1998). Ileum seems to play a major role in the adaptation of rats to low Ca2+ diet by stimulating the transcellular Ca2+ pathway, which is negligible under physiological conditions with usual Ca2+ intakes (Auchere et al., 1998). However, others (Bronner and Pansu, 1999; Bronner, 2003) showed that a low calcium intake produces an up regulation of active transcellular Ca2+ transport in the duodenum, but not in the passive paracellular process occurring in the jejunum and ileum.
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Consistent with the requirements for an increment in intestinal Ca2+ absorption during dietary Ca2+ restriction, an induction of the intestinal Ca2+ pump as well as of calbindin D9k mRNA has been detected in rat intestine (Matkovits and Christakos, 1995) and increases in the amount of Ca2+ pump and calbindin D28k in chick duodenum (Wasserman et al.,1992). Although epithelial Ca2+ channel has been demonstrated to be present at the apical side of the intestine to introduce calcium into the cell, the effect of 1,25(OH)2D3 administration or dietary calcium deficiency on its mRNA levels is controversial (Peng et al., 1999; Song et al., 2003; Slepchenko and Bronner, 2001). The lining mucosa is a complex epithelial layer formed mainly by enterocytes, which are formed from stem cells in the villus crypt that differentiate along the villus to finally be extruded as senescent cells into the lumen (Freeman, 1995). Calcium uptake and responses to 1,25(OH)2D3 depend on the degree of maturation of those cells (Weiser and Zelinski, 1990; Bikle et al., 1984). Alkaline phosphatase (AP), marker enzyme of differentiation, presents higher activity in cells from the villus tip than in those from the crypt, which are considered more immature. The amount of proteins involved in the transcellular Ca2+ pathway, such as calbindin and Ca2+ pump, is apparently higher in mature cells than in crypt enterocytes (Wu et al., 1992), while Na+/ Ca2+ exchanger content is higher in immature enterocytes (Van Corven et al., 1985). The basolateral membrane (BLM) from the enterocyte was previously shown to contain two proteins involved in the Ca2+ extrusion from the cell to the lamina propria: the plasma membrane Ca2+-ATPase or calcium pump and the Na+/Ca2+ exchanger. The aim of this study was to determine Ca2+ uptake and the activities of Ca2+-ATPase and Na+/Ca2+ exchanger in enterocytes with different degree of differentiation in chicks adapted to a low Ca2+ diet as compared to normal ones. The activity of intestinal AP and g-glutamyl transpeptidase, marker enzymes of enterocyte differentiation, was measured under the same experimental conditions. A possible involvement of the vitamin D receptor (VDR) on the mechanisms triggered by the low Ca2+ diet was also studied.
previously described (Mykkanen and Wasserman, 1981) and both diets contained 1200 IU of cholecalciferol per kilogram. The protocol was conducted according to the Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and their suffering. Blood samples were taken by cardiac puncture for determination of plasma calcium by atomic spectrometry and to measure phosphate (Henry et al., 1980) and 1,25(OH)2D3 levels (Reinhardt et al., 1984). Afterwards, the animals were killed by cervical dislocation, and the excised duodenae were rinsed with cold 0.15 mol/l NaCl, and cells or membrane fractions were isolated as described below. 2.2. Lumen-to-plasma Ca2+ transfer The animals were laparotomized under ether anesthesia and a 10-cm segment of duodenum was ligated following the technique previously described (Marchionatti et al., 2001). One milliliter of 150 mmol/l NaCl, 1 mmol/l CaCl2, containing 1.85105 Bq 45Ca2+, pH 7.2, was introduced into the lumen of the ligated intestinal segment. After 30 min, blood was withdrawn by cardiac puncture, centrifuged and the plasma 45Ca2+ was determined in a liquid scintillation counter. Absorption was defined as appearance of 45Ca2+ in the blood. 2.3. Duodenal villus-crypt cell isolation The method of Walters and Weiser (1987) for isolation of small intestinal cells was used. Five fractions were collected, which were progressively enriched for villus tip to crypt cells (fractions 1 and 2, tip cells, fractions 3 and 4, intermediate cells, fraction 5, crypt cells). The cells were collected by centrifugation at 200g for 5 min, resuspended in 140 mmol/l KCl (or 140 mmol/l NaCl depending of the study), 10 mmol/l HEPES, pH 7.4, and washed twice by centrifugation. Alkaline phosphatase (EC 3.1.3.1.) (Walter and Schu¨tt, 1974), and g-glutamyl-transpeptidase (EC 2.3.2.2) (Meister et al., 1981) activities were measured as marker enzymes of cell maturation. Cell viability was determined by the Trypan blue exclusion technique. 2.4. Calcium uptake by villus tip/crypt cells
2. Materials and methods 2.1. Animals and diets One-day-old Cobb Harding chicks (Gallus domesticus) were obtained from Indacor (Rı´o Ceballos, Co´rdoba, Argentina) and were fed a chick starter diet (Cargill, S.A.) for 3 weeks. At this time, the chicks were divided into two groups and each group was fed for 10 additional days one of the following diets: (1) control diet (1% Ca, 0.8% P), (2) low Ca2+ diet (0.1% Ca, 0.8% P) as
The procedure of Ca2+ uptake by intestinal epithelial cells described by Liang et al. (1986) was used with slight variations. An aliquot of 300 Al of cell suspension (about 5 g of protein/l), was incubated with an uptake solution composed of 140 mmol/l KCl, 10 mmol/l HEPES, 2 mmol/l CaCl2 pH 7.4 plus 1.85108 Bq/l of 45CaCl2. At different times, the Ca2+ uptake was stopped with 1 ml of the same solution free of 45Ca plus 2 mmol/l EGTA. The mixture was centrifuged 1 min at 10,000g, which was done twice. The final pellet was resuspended in 1 mol/l
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NaOH. Protein quantitation was performed as reported earlier (Marchionatti et al., 2001). 2.5. Preparation of basolateral membrane vesicles (BLMV) from villus tip/crypt cell BLMV from either villus tip or crypt cells were prepared according to the procedure of Scalera et al. (1980). The enrichment factors of these preparations were determined by measuring the activity of Na+/K+ ATPase (EC 3.6.1.3) (Scharschmidt et al., 1979) and sucrase (EC 3.2.1.26) (Dahlqvist, 1974) as markers of BLM and brush border membranes (BBM), respectively. 2.6. Determination of intestinal Na +/Ca2+ exchanger activity The method described by Smith and Smith (1987) was used with slight modifications. Purified BLMV were resuspended in a medium containing 140 mmol/l NaCl, 5 mmol/l KCl, 20 mmol/l HEPES, 1 mmol/l CaCl2, 1 mmol/l MgCl2 and 10 mmol/l glucose pH 7.4. An aliquot of 2 ml from each sample was incubated with 100 Al of 0.5 mmol/l ouabain for 60 min at 37 8C in a stirring bath. Ca2+ uptake was initiated by adding 10 Al of BLMV to 200 Al of uptake solution plus sodium (140 mmol/l NaCl, 20 mmol/l HEPES, 1 mmol/l CaCl2) or uptake solution minus sodium (190 mmol/l N-methylglucamine, 20 mmol/l HEPES, 1 mmol/l CaCl2), both containing 1.85108 Bq/l of 45CaCl2. The uptake was stopped at 5, 10 or 15 s by adding 100 mmol/l MgCl2, 2 mmol/l EGTA and 20 mmol/l HEPES, pH 7.4. An aliquot of 50 Al was removed from the incubation mixture, was filtered in a Millipore HAWP 0.25 membrane of 0.45 Am of pore size and washed three times with the stop
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solution. Radioactivity retained in the filters was measured in a liquid scintillation counter Beckman LS6500 (Fullerton, CA). 2.7. Determination of intestinal plasma membrane Ca2+-ATPase activity The procedure was based on the technique of Ghijsen et al. (1982). BLMV were resuspended in a medium containing 120 mmol/l KCl, 5 mmol/l MgCl2, 20 mmol/l HEPES– Tris, pH 7.4. Ca2+ uptake was initiated by adding 10 Al of sample to 350 Al of uptake medium composed of 120 mmol/ l KCl, 5 mmol/l MgCl2, 3010 6 mol/l CaCl2, 3.7108 Bq/ l 45CaCl2, 20 mmol/l HEPES–Tris, pH 7.4 in the presence or absence of 5 mmol/l ATP. At 15, 30 or 60 s, the uptake was stopped by adding 120 mmol/l KCl, 5 mmol/l MgCl2, 0.5 mmol/l EGTA and 20 mmol/l HEPES–Tris pH 7.4. An aliquot was removed from the incubation mixture, was filtered with the stop solution, and the radioactivity retained in the filters was measured. 2.8. Immunoblots The expression of calcium pump, Na+/Ca2+ exchanger and VDR was studied by Western blots analysis. Proteins (the amount is specified in each figure) from BLMV or enterocyte homogenates were separated by SDS-PAGE with 8% acrylamide and 0.1% SDS (Laemmli, 1970), running in the same gel protein standards of known molecular weight (Amersham Int., Little Chalfont, England). The proteins were transferred to nitrocellulose sheets (Towbin et al., 1979). The blots were incubated with the anti-calcium pump antibody 5F10 (a gift from Dr. John Penniston, Mayo Clinic, Rochester, MN), 1:500 dilution, or anti Na+/Ca2+ exchanger
Fig. 1. Effect of low calcium diet on serum Ca2+ (A), serum P (B), serum 1,25(OH)2D3 (C) and lumen to plasma Ca2+ transfer (D). Values are meansFS.E. from six to nine determinations. *Pb0.01.
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2.10. Statistical analysis The data are expressed as meansFstandard errors. Student’s t-test was used to compare the means. Differences were considered significant at Pb0.05.
3. Results Serum calcium and phosphorus were decreased by the low Ca2+ diet (Fig. 1A and B), whereas lumen-to-plasma Ca2+ transfer was increased (Fig. 1D), indicating that the chicks were well adapted to the dietary Ca2+ restriction, as reported earlier (Tolosa de Talamoni, 1996). Serum 1,25(OH)2D3 levels were four fold in chicks adapted to
Fig. 2. Effect of the low calcium diet on the alkaline phosphatase (A), and g-glutamyl transpeptidase (B) activities by enterocytes with different degree of maturation. Cells were collected in five fractions: F1 and F2 correspond to tip cells, F3 and F4 to intermediate cells and F5 to crypt cells. The activity of both enzymes was assayed as described in Material and methods. Values are meansFS.E. from six to nine experiments. *Pb0.05 vs. Normal diet. **Pb0.01 vs. Normal diet.
antibody NB 300-127 (NCX-1, Novus Biologicals, Littleton, CO), 1:200 dilution, or anti-VDR monoclonal antibody 9A7 (Affinity Bioreagents, Golden, CO, USA), 1:500 dilution, to detect Ca2+ pump, Na+/Ca2+ exchanger and VDR, respectively. The secondary antibody, anti-mouse or anti-rat Ig G (whole molecule) peroxidase conjugate (Sigma, St. Louis, MO, USA), was incubated at room temperature for 2 h. Nonspecific binding on the nitrocellulose was blocked with nonfat dry milk in Tris-buffered saline solution (TBS) and washes between incubation steps were accomplished with TBS. The detection was performed by autoradiography after using ECL Western blotting detection reagents (Amersham Int.). 2.9. Chemicals 45
Ca2+ was purchased from Dupont NEN (Boston, MA). All other chemicals were of reagent grade and obtained from Sigma.
Fig. 3. Effect of the low calcium diet on the time course of 45Ca uptake by isolated villus tip (A) and crypt (B) cells. Ca uptake solution was composed of 140 mmol/l KCl, 10 mmol/l HEPES, 2 mmol/l CaCl2 pH 7.4 plus 1.85108 Bq/l of 45Ca plus 2 mmol/l EGTA. At different times, the Ca2+ uptake was stopped with 1 ml of the same solution free of 45Ca plus 2 mmol/l EGTA. The mixture was centrifuged, the pellet was resuspended and the radioactivity was read in a liquid scintillation counter. Values are meansFS.E. of six experiments. *Pb0.05 vs. Normal diet. **Pb0.01 vs. Normal diet.
V.A. Centeno et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 133–141 Table 1 Purity of basolateral membranes vesicles of duodenal villus tip and crypt cells Na+/K+ ATPase activity
Sucrase activity
Purification factor
Tip cells Crypt cells
Normal diet
Low Ca2+ diet
Normal diet
Low Ca2+ diet
9.01F1.10 3.40F1.20
9.63F1.80 3.86F1.80
0.97F0.40 1.31F0.40
1.40F0.60 1.21F0.30
The purification factor was calculated as the ratio between the specific activities of the final membrane preparation and the initial homogenate. Values are meansFstandard error of three to four experiments.
the low Ca2+ diet in comparison with the control chicks (Fig. 1C), which means that vitamin D metabolism had been modified by hypocalcemia, high serum PTH or both, as demonstrated earlier (Weisinger et al., 1989). Cells from the intestinal villus were collected in five fractions. The first two fractions corresponded to the mature cells (tip cells), the third and four fractions contained the intermediate cells whereas the crypt cells were in the last fraction. The enzyme activities of AP and g-glutamyl-transpeptidase were higher in intestinal epithelial cells from mid to tip villus than those from crypt villus, an indication that enterocytes from the villus tip were more mature than those from the crypt. Low Ca2+ diet increased the AP activity, independently of the cellular maturation, but it did not alter the g-glutamyl- transpeptidase activity (Fig. 2A and B).
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Ca2+ uptake by chick enterocytes with different degree of maturation was increased by the low Ca2+ diet either in cells from the villus tip or from the villus crypt as compared to the normal controls. With both diets, Ca2+ uptake by enterocytes from the villus tip was higher than that by the crypt cells (Fig. 3A and B). The characterization of BLMV was accomplished by enrichment factors determinations of marker enzymes, which were calculated as a ratio between the activity of the enzyme in the BLMV and that of the initial homogenate. As expected, the enrichment factor of Na+/K+-ATPase activity, a marker enzyme of BLM, was higher in the tip cells than in the crypt cells and the low Ca2+ diet did not affect those values. The enrichment factor of sucrase activity, a marker enzyme of BBM, was not altered by the mineral deficiency either in tip cells or crypt cells (Table 1). These data suggested that the purity of BLMV was adequate for transport studies and contamination of BLMV with BBM was negligible. Ca2+-ATPase activity was increased by the low Ca2+ diet in BLMV from either villus tip (Fig. 4A) or from villus crypt (Fig. 4B). The activity of the enzyme was lower in crypt cells than in tip cells from chicks fed either low or normal Ca2+ diet. Western blot analysis shows that the low Ca2+ diet increased the protein expression in BLMV either from the villus tip or from villus crypt cells (Fig. 4C). Na+-dependent Ca2+ uptake by BLMV from villus tip and crypt cells was increased by the low Ca2+ diet (Fig. 5A and B). Protein expression was also higher in BLMV from
Fig. 4. Effect of the low calcium diet on the activity of Ca2+-ATPase by BLMV from villus tip (A) and crypt (B) duodenal cells. Duodenal BLMV preparations and Ca2+ uptake conditions were described in Material and methods. Values are meansFS.E. of four experiments. *Pb0.05 vs. Normal diet. (C) One representative Western blot analysis of duodenal BLMV from chicks fed Normal or Low calcium diets, using anti-Ca2+ pump monoclonal antibody 5F10 (1:500). The protein amount applied to the gel was 30 Ag to each lane. The densitometric values for Ca pump, expressed in arbitrary units, in the Western blots were as follows: Normal diet, BLMV from tip cells, n=4, 155.86F6.09 vs. BLMV from tip cells of Low calcium diet, n=4, 183.94*F2.49, *Pb0.05; Normal diet, BLMV from crypt cells, n=4, 81.30F3.23 vs. BLMV from crypt cells of Low calcium diet, n=4, 132.72*F7.61, *Pb0.01.
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Fig. 5. Effect of the low calcium diet on the activity of Na+/Ca2+ exchanger by BLMV from villus tip (A) and crypt (B) duodenal cells. Duodenal BLMV preparations and Ca2+ uptake conditions were described in Material and methods. Values are meansFS.E. of four experiments. *Pb0.05 vs. Normal diet. (C) One representative Western blot analysis of duodenal BLMV from chicks fed Normal or Low calcium diets, using anti- Na+/Ca2+ exchanger monoclonal antibody NB 300-127 (1:200). The protein amount applied to the gel was 50 Ag to each lane. The densitometric values for Na+/Ca2+ exchanger, expressed in arbitrary units, in the Western blots were as follows: Normal diet, BLMV from tip cells, n=4, 25.01F0.54 vs. BLMV from tip cells of Low calcium diet, n=4, 84.83*F0.57, *Pb0.001; Normal diet, BLMV from crypt cells, n=4, 26.71F0.74 vs. BLMV from crypt cells of Low calcium diet, n=4, 30.74*F0.61, *Pb0.001).
mature and immature enterocytes of low Ca2+ chicks as compared to the control ones (Fig. 5C). As expected, VDR expression was higher in the crypt cells than in the villus tip cells. The low Ca2+ diet decreased VDR expression in the intestinal epithelial cells, this effect being more noticeable in mature cells (Fig. 6).
4. Discussion The higher levels of serum 1,25(OH)2D3 and lumen-toplasma Ca2+ transfer in Ca2+ deficient animals as compared to the normal group indicated that they had indeed adapted
to the dietary Ca2+ restriction. Further, the serum Ca2+ and P values were lower than those in the normal animals, as we have previously demonstrated (Tolosa de Talamoni, 1996). AP activity, that is normally higher in the villus tip cells and gradually decreases down the crypt (Walters and Weiser, 1987), is enhanced by the mineral deficiency maintaining the highest values in the villus tip cells. gGlutamyl-transpeptidase activity remains completely unaltered by the same dietary treatment. Interestingly, vitamin D or calcitriol administration to vitamin D-deficient animals has been shown to increase AP activity (Bikle et al., 1984) and not to modify g-glutamyl-transpeptidase activity (Do Thanh et al., 1996). Jeng et al. (1994) have demonstrated
Fig. 6. Western blot analysis of chicken vitamin D receptor (VDR) from enterocytes with different degree of maturation. Eighty micrograms of total cell protein was applied in each lane and probed with the anti-vitamin D receptor monoclonal antibody 9A7 (1:500). Villus tip cells (lanes 1 and 2), crypt cells (lanes 3 and 4). The densitometric values for VDR, expressed in arbitrary units, in the Western blots were as follows: Normal diet, BLMV from tip cells n=4, 29.60F0.27 vs. BLMV from tip cells of Low calcium diet, n=4, 23.79*F0.22, *Pb0.001; Normal diet, BLMV from crypt cells, n=4, 243.65F0.57 vs. BLMV from crypt cells of Low calcium diet, n=4, 238.84*F0.95, *Pb0.01).
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that 1,25(OH)2D3 regulates the activity and the mRNA levels of intestinal AP. They showed that AP isoform in IEC-6 cells, a rat small intestine crypt cell line, is different from that of the tip cells. That AP isoform is also present in other tissues, not all of them involved in the mineral homeostasis. The authors think that the regulation of the AP isoform by calcitriol in the crypt cells could be related to the intestinal development and not directly to the calcium metabolism. Therefore, it is quite possible that in our study, the increment in the AP activity by the low Ca2+ diet could stimulate the intestinal cell differentiation. As expected, Ca2+ uptake by enterocytes was higher in the villus tip than in the villus crypt, supporting the idea that mature enterocytes have the greatest capacity for transcellular Ca2+ movement (Walters and Weiser, 1987). Low Ca2+ diet increased the Ca2+ uptake either in mature or immature enterocytes. However, Ca 2+ -deficient diet decreased VDR expression in both types of cells. An inverse relationship between 1,25(OH) 2D3 and VDR expression has been also shown in rats fed a low Ca2+ diet (Meyer et al., 1992). Since VDR levels are low (this paper) or it has not been detected in mature enterocytes (Colston et al., 1994), the most active in calcium transport, the increased levels of 1,25(OH)2D3 caused by the low Ca2+ diet do not regulate the intestinal function by up-modulation of its nuclear receptor but promoting differentiation, which would produce cells more capable to express vitamin Ddependent genes required for calcium absorption. It has been suggested that dietary Ca2+ might have dual effects on VDR gene expression because homologous stimulation of VDR gene expression by calcitriol does not occur on a low Ca2+ diet, as a result of transcriptional repression by concomitant increased PTH (Ferrari et al., 1998). Two main molecules are involved in Ca2+ exit from the enterocytes: plasma membrane Ca2+-ATPase or Ca2+ pump and Na+/Ca2+ exchanger. Our data show that dietary Ca2+ deficiency increases Ca2+-ATPase activity of BLMV from enterocytes with different degree of maturation as compared to that of the enterocytes from normal chicks. These results are in agreement with previous data showing that either calcitriol or low Ca2+ and low P diets increase Ca2+ pump units of chicken intestine (Wasserman et al., 1992). Since Ca2+-ATPase mRNA of chick intestine is increased by a low Ca2+ diet (Cai et al., 1993), it is possible to think that the mineral-deficient diet increases Ca2+ pump gene expression in enterocytes, independently of their degree of maturation. Na+/Ca2+ exchanger activity of intestinal BLMV was also increased by dietary Ca2+ restriction either in mature or immature enterocytes. Hoenderop et al. (2000) were not able to show by immunocytochemistry the presence of Na+/ Ca2+ exchanger in rabbit intestine. This apparent discrepancy with our data could be due to species differences or to the presence of an isoform of Na+/Ca2+ exchanger that may not be detected with the antibody that they employed as suggested by the same authors. Ghijsen et al. (1983) have also demonstrated Na+/Ca2+ exchanger activity in rat
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intestine, but they did not detect any effect of vitamin D. On the contrary, Lytton et al. (1996) have demonstrated that parathyroidectomized rats treated with a diet deficient in phosphate, which increases the calcitriol levels, showed an increase in the transcript for kidney Na+/Ca2+ exchanger. The stimulation of intestinal Ca2+-ATPase and Na+/Ca2+ exchanger activities by the low Ca2+ diet are mainly attributed to the high levels of serum 1,25(OH)2D3, whose synthesis is triggered by hypocalcemia and/or PTH. A direct effect of PTH on the intestinal function could not be disregarded. Picotto et al. (1997) demonstrated that PTH stimulates rapidly Ca2+ influx into rat duodenal cells. However, the direct effects of PTH on intestine have been shown after short periods of time, seconds to minutes, and the presence of PTH receptor in chick intestine has not been reported yet. Watson et al. (2000) have recently detected nuclear localization of PTH/PTHrP receptor in rat intestine but its role remains unclear. Another possibility might be that the lipid composition and fluidity changes caused in intestinal BLMV by the low Ca2+ diet (Alisio et al., 1997; Tolosa de Talamoni, 1996) could alter the activities of Ca2+ pump and Na+/Ca2+ exchanger, which are known to be sensitive to their lipidic environments (Lee, 1998). Moreover, these changes might vary according to the degree of cell maturation. To our knowledge, this is the first demonstration that a low Ca2+ diet increases Ca2+ uptake and the activities of the Ca2+ pump and Na+/Ca2+ exchanger, the two main molecules involved in the Ca2+ extrusion, either in mature or immature enterocytes from chick duodenum. On the contrary, the low Ca2+ diet decreases VDR expression in both kinds of cells being VDR expression much higher in the crypt than in the tip cells. These results indicate that the intestinal mechanism of adaptation to the low Ca2+ diet involves the two proteins located at the BLM that extrude Ca2+ out of the cell, whose activity appears 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.
Acknowledgements This work was supported by grants from FONCYT (PICT 9805-03126), SECYT (UNC) and CONICET (PIP 98), Argentina. Dr. Nori Tolosa de Talamoni is a member of the Investigator Career from the CONICET. Viviana Centeno is a recipient fellowship from FONCYT.
References Alisio, A.E., Can˜as, F.M., Bronia, D.H., Pereira, R.D., Tolosa de Talamoni, N.G., 1997. Effect of vitamin D deficiency on lipid composition and calcium transport in basolateral membrane vesicles from chick intestine. Biochem. Mol. Biol. Int. 42, 339 – 347.
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Auchere, D., Tardivel, S., Gounelle, J., Drueke, T., Lacour, B., 1998. Role of transcellular pathway in ileal Ca2+ absorption: stimulation by low Ca2+ diet. Am. J. Physiol. 275, G951 – G956. Bikle, D.D., Zolock, D.T., Munson, S., 1984. Differential response of duodenal epithelial cells to 1,25-dihydroxyvitamin D3 according to position on the villus: a comparison of calcium uptake, calcium-binding protein, and alkaline phosphatase activity. Endocrinology 115, 2077 – 2084. Bronner, F., Pansu, D., 1999. Nutritional aspects of calcium absorption. J. Nutr. 129, 9 – 12. Bronner, F., 2003. Mechanisms of intestinal calcium absorption. J. Cell. Biochem. 88, 387 – 393. Cai, Q., Chandler, J.S., Wasserman, R.H., Kumar, R., Penniston, J.T., 1993. Vitamin D and adaptation to dietary calcium and phosphate deficiencies increase intestinal plasma membrane calcium pump gene expression. Proc. Natl. Acad. Sci. U. S. A. 90, 1345 – 1349. Colston, K.W., Mackay, A.G., Finlayson, C., Wu, J.C., Maxwell, J.D., 1994. Localization of vitamin D receptor in normal human duodenum and in patients with coeliac disease. Gut 35, 1219 – 1225. Dahlqvist, A., 1974. Disaccharidases. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 916 – 922. Do Thanh, X., Massicot, F., Do, B., Breget, R., Nivet, V., Durand, D., Warnet, J.M., Claude, J.R., Clot, J.P., 1996. 1 alpha, 25-dihydroxyvitamin D3 stimulated alkaline phosphatase activity in cultured pig kidney epithelial LLC-PK1 cells. Acta Physiol. Scand. 158, 107 – 111. Ferrari, S.L., Bonjour, J.P., Rizzoli, R., 1998. The vitamin D receptor gene and calcium metabolism. Trends Endocrinol. Metab. 9, 259 – 265. Freeman, T.C., 1995. Parallel patterns of cell-specific gene expression during enterocyte differentiation and maturation in the small intestine of the rabbit. Differentiation 59, 179 – 192. Ghijsen, W.E., De Jong, M.D., Van Os, C.H., 1982. ATP-dependent calcium transport and its correlation with Ca2+-ATPase activity in basolateral plasma membranes of rat duodenum. Biochim. Biophys. Acta 689, 327 – 336. Ghijsen, W.E., De Jong, M.D., Van Os, C.H., 1983. Kinetic properties of Na+/Ca2+ exchange in basolateral plasma membranes of rat small intestine. Biochim. Biophys. Acta 730, 85 – 94. Henry, R.J., Cannon, D.C., Winkelman, J.W., 1980. Inorganic phosphorus assays. Quı´mica Clı´nica. Bases y te´cnicas. JIMS, Barcelona, pp. 732 – 735. Hoenderop, J.G., Hartog, A., Stuiver, M., Doucet, A., Willems, P.H., Bindels, R.J., 2000. Localization of the epithelial Ca2+ channel in rabbit kidney and intestine. J. Am. Soc. Nephrol. 11, 1171 – 1178. Jeng, Y.J., Watson, C.S., Thomas, M.L., 1994. Identification of vitamin Dstimulated alkaline phosphatase in IEC-6 cells, a rat small intestine crypt cell line. Exp. Cell Res. 212, 338 – 343. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lee, A.G., 1998. How lipids interact with an intrinsic membrane protein: the case of the calcium pump. Biochim. Biophys. Acta 1376, 381 – 390. Liang, C.T., Barnes, J., Balakir, R.A., Sacktor, B., 1986. Characterization of 1,25-dihydroxyvitamin D3-dependent calcium uptake in isolated chick duodenal cells. J. Membr. Biol. 90, 145 – 156. Lytton, J., Lee, S.L., Lee, W.S., Baal, J., Bindels, R.J., Kilav, R., NavehMany, T., Silver, J., 1996. The kidney sodium–calcium exchanger. Ann. N.Y. Acad. Sci. 779, 58 – 72. Marchionatti, A.M., Alisio, A.E., Dı´az de Barboza, G.E., Baudino, V., Tolosa de Talamoni, N.G., 2001. d-l-Buthionine-S,R-sulfoximine affects intestinal alkaline phosphatase activity. Comp. Biochem. Physiol., C 129, 85 – 91. Matkovits, T., Christakos, S., 1995. Variable in vivo regulation of rat vitamin D-dependent genes (osteopontin, Ca,Mg-adenosine triphosphatase, and 25-hydroxyvitamin D3 24-hydroxylase): implications for differing mechanisms of regulation and involvement of multiple factors. Endocrinology 136, 3971 – 3982. Meister, A., Tate, S.S., Griffith, O.W., 1981. Gamma-glutamyltranspeptidase. Methods Enzymol. 77, 237 – 253.
Meyer, J., Fullmer, C.S., Wasserman, R.H., Komm, B.S., Haussler, M.R., 1992. Dietary restriction of calcium, phosphorus, and vitamin D elicits differential regulation of the mRNAs for avian intestinal calbindin D28k and the 1,25-dihydroxyvitamin D3 receptor. J. Bone Miner. Res. 7, 441 – 448. Moallem, E., Kilav, R., Silver, J., Naveh-Many, T., 1998. RNA–protein binding and post-transcriptional regulation of PTH gene expression by calcium and phosphate. J. Biol. Chem. 273, 5253 – 5259. Mykkanen, H.M., Wasserman, R.H., 1981. Gastrointestinal absorption of lead (203Pb) in chicks: influence of lead, calcium and age. J. Nutr. 111, 1757 – 1765. Peng, J.B., Chen, X.Z., Berger, U.V., Vassilev, P.M., Tsukaguchi, H., Brown, E.M., Hediger, M.A., 1999. Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. J. Biol. Chem. 274, 22739 – 22746. Picotto, G., Massheimer, V., Boland, R.L., 1997. Parathyroid hormone stimulates calcium influx and the cAMP messenger system in rat enterocytes. Am. J. Physiol. 273, C1349 – C1353. Reinhardt, T.A., Horst, R.L., Orf, J.W., Hollis, B.W., 1984. A microassay for 1,25-dihydroxyvitamin D not requiring high performance liquid chromatography: application to clinical studies. J. Clin. Endocrinol. Metab. 58, 91 – 98. Scalera, V., Storelli, C., Storelli-Joss, C., Hasse, W., Murer, H., 1980. A simple and fast method for the isolation of basolateral plasma membranes from rat small intestinal epithelial cells. Biochem. J. 186, 177 – 181. Scharschmidt, B.F., Keeffe, E.B., Blankenship, N.M., Ockner, R.K., 1979. Validation of a recording spectrophotometric method for measurement of membrane-associated Mg- and NaK-ATPase activity. J. Lab. Clin. Med. 93, 790 – 799. Silver, J., Kilav, R., Naveh-Many, T., 2002. Mechanisms of secondary hyperparathyroidism. Am. J. Physiol., Renal Physiol. 283, F367 – F376. Slepchenko, B.M., Bronner, F., 2001. Modeling of transcellular Ca transport in rat duodenum points to coexistence of two mechanisms of apical entry. Am. J. Physiol. 281, C270 – C281. Smith, J.B., Smith, L., 1987. Extracellular Na+ dependence of changes in free Ca2+, 45Ca2+ efflux, and total cell Ca2+ produced by angiotensin II in cultured arterial muscle cells. J. Biol. Chem. 262, 17455 – 17460. Song, Y., Kato, S., Fleet, J.C., 2003. Vitamin D receptor (VDR) knockout mice reveal VDR-independent regulation of intestinal calcium absorption and ECaC2 and Calbindin D9k mRNA. J. Nutr. 133, 374 – 380. Tolosa de Talamoni, N.G., 1996. Calcium and phosphorus deficiencies alter the lipid composition and fluidity of intestinal basolateral membranes. Comp. Biochem. Physiol., A 115, 309 – 315. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U. S. A. 76, 4350 – 4354. Van Corven, E.J., Roche, C., Van Os, C.H., 1985. Distribution of Ca2+ATPase, ATP-dependent Ca2+-transport, calmodulin and vitamin Ddependent Ca2+-binding protein along the villus-crypt axis in rat duodenum. Biochim. Biophys. Acta 820, 274 – 282. Walter, K., Schqtt, C., 1974. Acid and alkaline phosphatase in serum (Twopoint method). In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 856 – 860. Walters, J.R., Weiser, M.M., 1987. Calcium transport by rat duodenal villus and crypt basolateral membranes. Am. J. Physiol. 252, G170 – G177. Wasserman, R.H., Smith, C.A., Brindak, M.E., de Talamoni, N.G., Fullmer, C.S., Penniston, J.T., Kumar, R., 1992. Vitamin D and mineral deficiencies increase the plasma membrane calcium pump of chicken intestine. Gastroenterology 102, 886 – 894. Watson, P.H., Fraher, L.J., Hendy, G.N., Chung, U.I., Kisiel, M., Natale, B.V., Hodsman, A.B., 2000. Nuclear localization of the type 1 PTH/ PTHrP receptor in rat tissues. J. Bone Miner. Res. 15, 1033 – 1044. Weiser, M.M., Zelinski, J.M., 1990. Calcium absorption. Vitamin D effects on rat intestinal calcium transport systems depend on the state of enterocyte differentiation along the crypt-villus axis. In: Pansu, D.,
V.A. Centeno et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 133–141 Bronner, F. (Eds.), Calcium Transport and Intracellular Calcium Homeostasis. Springer-Verlag, Berlin Heidelberg, pp. 249 – 256. Weisinger, J.R., Favus, M.J., Langman, C.B., Bushinsky, D.A., 1989. Regulation of 1,25-dihydroxyvitamin D3 by calcium in the parathyroidectomized, parathyroid hormone-replete rat. J. Bone Miner. Res. 4, 929 – 935.
141
Wu, J.C., Smith, M.W., Lawson, D.E., 1992. Time dependency of 1,25(OH)2D3 induction of calbindin mRNA and calbindin expression in chick enterocytes during their differentiation along the crypt-villus axis. Differentiation 51, 195 – 200.
