1 Department of Nutrition and Foodservice Systems, University of North Carolina at Greensboro, Greensboro, North Carolina 27402; and 2 Mineral Bioavailability Laboratory, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 · well1 · 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 µM) 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.
trifluoperazine; 24-hydroxylase; calbindin D9k
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DYNAMICS OF INTESTINAL calcium absorption have been characterized in a wide variety of species and systems (32), and we have understood for over 50 years that the efficiency of intestinal calcium absorption is related to vitamin D status (31). However, the observation that the efficiency of calcium absorption declines with aging in animals and humans (2, 10) and our limited understanding of the molecular details of intestinal calcium absorption justify the continued mechanistic evaluation of this process. The active hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is thought to regulate intestinal calcium absorption by increasing the transcellular flux of calcium through enterocytes (28). Two models have been proposed to describe the mechanism of vitamin D-mediated calcium absorption. In the facilitated diffusion model 1,25(OH)2D3 stimulates calcium movement at three important steps: 1) entry of calcium into the cell via a putative apical membrane transport protein or channel, 2) intracellular diffusion of calcium through the cytosol via the ferrying action of calbindin D, and 3) extrusion across the basolateral membrane via an ATP-dependent calcium pump (8). In the vesicular transport model calcium enters the cell either through a calcium channel or by endocytosis and is localized into lysosomes; calcium from lysosomes is thought to be extruded from cells by exocytosis (29). Some evidence suggests that calbindin D is also involved in this process (30). Although evidence from various experimental approaches exists to support both of these models of vitamin D-mediated intestinal calcium transport, the development of a cell culture model for examining the molecular details of 1,25(OH)2D3-mediated calcium absorption would be useful for testing details of each model. With this in mind, our group has previously reported on several key characteristics of calcium transport in Caco-2 cells, a human colon adenocarcinoma cell line (17, 20, 24).
In culture, Caco-2 cells spontaneously differentiate and form a polarized epithelial monolayer with tight junctions and express a differentiated cell phenotype consistent with absorptive small intestine-like enterocytes (33, 36). Since its initial characterization as a model for drug transport, Caco-2 cells have been used as an in vitro model to study intestinal transport of micronutrients, including minerals such as zinc (19, 34), iron (1, 3, 25), and calcium (17, 18, 20, 23, 24). In particular, findings from our laboratory show how various characteristics of Caco-2 cells make them a potentially useful model to study vitamin D-induced calcium transport. For example, Caco-2 cells have a functional vitamin D receptor (23) and have calcium transport kinetics that suggest the presence of both a saturable and nonsaturable calcium transport pathway, similar to what has been observed in human and animal intestine. 1,25(OH)2D3 treatment induces the saturable, but not diffusional, component of calcium transport (24) and induces accumulation of calbindin D9k and 24-hydroxylase mRNA (17, 20).
Two studies were recently published that suggest that calcium transport across monolayers of Caco-2 cells is purely diffusional, i.e., nonspecific (7, 12). In a study by Blais et al. (7) no attempt was made to investigate vitamin D-mediated calcium transport, whereas a study by Chirayath et al. (12) found that in a rapidly proliferating, less differentiated clone of Caco-2 cells (AQ), 1,25(OH)2D3 reduced transepithelial electrical resistance (TEER), thus permitting an increased paracellular movement of calcium. In contrast, we now report additional evidence to demonstrate that the regulation of calcium transport in the parent cell line of Caco-2 cells is a transcellular pathway and is due to the specific regulation of cellular events by 1,25(OH)2D3.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Conditions of cell culture. Caco-2 cells (American Type Culture Collection, Rockville, MD) were propagated and maintained in high-glucose DMEM (4.5 g glucose/l), supplemented with 10 mM HEPES, 44 mM sodium bicarbonate, 1 mM sodium pyruvate, 100 µM nonessential amino acids, 50 µg/l gentamicin sulfate, 100 U/l penicillin, 100 U/l streptomycin, 2 mM glutamine, final pH 7.2. For routine passage, medium contained 20% fetal bovine serum (FBS), cells were seeded into T-75 flasks at a density of 1 × 106 cells/flask, and cells were passaged every 3-4 days when the cultures were 80% confluent. For transport studies, 250,000 cells were seeded onto permeable membrane filter inserts (24.5-mm diam, 0.4 µm; Costar, Cambridge, MA). For mRNA studies, 200,000 cells were seeded into six-well dishes (35-mm diam, Costar). Cultures were used for experiments between passage 25 and 60. After cells reached confluence they were fed with DMEM supplemented with 10% FBS every other day until day 12 in culture [the point of maximum expression of enzymatic markers of differentiation (23)]. Cells were subsequently fed every day until the end of the experiment (day 15 in culture). Cell culture medium, nutrients, and antibiotics were purchased from BioWhittaker (Walkersville, MD), FBS was purchased from HyClone Laboratories (Logan, UT), vitamin D compounds were obtained from Bio-Mol (Plymouth Meeting, PA), and chemicals for all other purposes were obtained from Sigma Chemical (St. Louis, MO).
Cell treatments. In the first calcium transport experiment cells were treated with 10 nM 1,25(OH)2D3 for 0, 24, 48, or 72 h, and calcium transport was determined in the apicalto-basolateral (A-to-B) and B-to-A direction. In all experiments, treatments were timed so that cell treatment ended on day 15 in culture. In the second experiment cells were treated with vehicle, 10 nM 1,25(OH)2D3, 100 nM 25-hydroxyvitamin D3 [25(OH)D3], or 100 nM 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] for 48 h. The 25(OH)D3 and 24,25(OH)2D3 treatments were also combined with the 10 nM 1,25(OH)2D3 treatment to determine if these compounds altered the cellular response to calcitriol. In the third experiment, vehicle or 1,25(OH)2D3 pretreated cells (10 nM, 48 h) were also treated with trifluoperazine (TFP, 0-50 µM) during the transport study (which includes a 30-min equilibration period followed by a 30-min transport period). Finally, to assess the importance of transcriptional events on vitamin D-inducible calcium transport, cells were treated with 1 µg/ml actinomycin D for 8 h followed by a coincubation with 1,25(OH)2D3 or ethanol vehicle for 40 h. Calcium transport was studied after this period. The effect of 4 µg/ml actinomycin D or 10 µg/ml cycloheximide on 24-hydroxylase and calbindin D9k mRNA levels was assessed after an initial 1-h preincubation with the inhibitor, followed by treatment with 1,25(OH)2D3 for either 8 h (for 24-hydroxylase mRNA) or 48 h (calbindin D9k) in the presence or absence of the inhibitor. Specific mRNA levels were determined by RT-PCR. In all experiments inhibitors and vitamin D compounds were diluted in DMEM plus 5% FBS. Control treatments were comprised of vehicles for the inhibitors or vitamin D compounds diluted to the same extent as the most concentrated inhibitor or vitamin D stock used in a particular experiment (0.1% or less final ethanol concentration).
Calcium transport studies. Transport studies in the A-to-B direction were conducted as previously reported by Fleet and Wood (20). Unless noted otherwise, calcium transport refers to transport in the A-to-B direction. The transport buffer contained 1 µCi 45Ca/ml and 500 µM calcium as calcium chloride. In all experiments, phenol red was included in the transport buffer at 500 µM as a means of measuring diffusional transport through the monolayer (9). The percentage of phenol red transported per minute was calculated, and the equivalent amount of calcium was subtracted from the value for total calcium transport to derive the value for saturable calcium transport as described previously (20). Experiments had six wells per treatment and were conducted three times.
In the experiments where both A-to-B and B-to-A transport were determined, an equal volume of buffer was placed on each side of the monolayer (2 ml), and 500 µM calcium was used on both sides of the monolayer. When A-to-B transport was assessed, 1 µCi/ml 45Ca was placed in the apical reservoir, and movement into the basolateral compartment was followed, whereas when B-to-A transport was assessed the 45Ca was placed in the basolateral compartment and movement into the apical compartment was followed. Each treatment was done in three wells and the experiments were conducted three times. In one experiment, phenol red (500 µM) was used to estimate paracellular diffusion through the Caco-2 cell monolayer in either the A-to-B or B-to-A direction.Effect of differentiation and 1,25(OH)2D3 treatment on TEER. Electrical resistance of the Caco-2 cell monolayer grown on permeable membrane filter supports was measured using a voltohmmeter (World Precision Instruments, Sarasota, FL). Resistance was measured from day 3 in culture (just before confluence) to day 15 in culture. TEER was measured in six wells at each of the time points. This experiment was repeated three times. In addition, the effect of treating 12-day-old cultures for 72 h with 10 nM 1,25(OH)2D3 on TEER was examined (n = 6 wells/treatment, repeated 8 times).
RT-PCR analysis for mRNA levels. After experimental treatments, cells were harvested and RNA was isolated and analyzed for 24-hydroxylase, calbindin D9k, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels by semiquantitative RT-PCR using conditions previously described (17, 19). Primer sets were derived from previously published sequences and are GAPDH, forward primer bases 386-403 and reverse primer bases 561-580; calbindin D9k, forward primer bases 1-18 and reverse primer bases 218-237; 24-hydroxylase, forward primer bases 1633-1653 and reverse primer 1912-1932 (11). To minimize the potential for variability in the reverse transcriptase reaction, cDNA was prepared from total cellular RNA for all samples at the same time, using the same reagents. Specific target messages were detected by amplifying cDNAs for 20 (GAPDH), 22 (24-hydroxylase), or 25 cycles (calbindin D9k). These cycles were previously determined to fall within linear range of amplification efficiency for each of the primer sets. Each target message was detected in a separate PCR reaction. The identity of the three PCR products was confirmed by subcloning the PCR product into the pCR2.1 TA cloning vector (Invitrogen, Carlsbad, CA) followed by sequencing (data not shown). PCR products were subjected to 2.5% agarose gel electrophoresis followed by visualization of ethidium bromide-stained gels under ultraviolet light. PCR product levels were determined by densitometry on photographic negatives of the gels. Data were normalized to the level of GAPDH expression within the sample and then expressed relative to the expression seen in control (calbindin D9k)- or vitamin D (24-hydroxylase)-treated cells.
Analysis of data. Data were analyzed by ANOVA using the Systat statistical package (35). Multiple comparisons were done using Fisher's protected least-significant difference. Significance is at the level of P < 0.05. Values are expressed as means ± SE.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We utilized two approaches to assess the impact of
1,25(OH)2D3
on the permeability of the Caco-2 monolayer:
1) TEER and
2) phenol red transport. TEER
increased as the cells in the monolayer reached full differentiation
over 15 days in culture (Fig. 1). However,
treatment of 12-day-old cultures with 10 nM
1,25(OH)2D3 for 72 h did not significantly reduce TEER [control, 1,044.0 ± 30.8 · cm2;
1,25(OH)2D3,
985.5 ± 28.8
· cm2].
The level of phenol red movement across the monolayer was 0.5% per
hour in both A-to-B and B-to-A directions and was not
altered by time of vitamin D treatment. Phenol red transport was not
increased by calcitriol treatment in any of the other experiments
presented (see Fig. 4 and Table
1).
|
|
|
Our initial calcium transport experiment examined whether there was net
A-to-B calcium transport across a monolayer of fully differentiated
Caco-2 cells. As shown in Fig. 2, total calcium transport in the A-to-B
direction was progressively elevated by increasing time of treatment
with 10 nM
1,25(OH)2D3
so that by 72 h transport it had increased 2.1-fold to 1.006 ± 0.008 nmol · well1 · min
1
(Fig. 2). Initially, B-to-A movement was 2.7% per hour or 70% of the
A-to-B value. However, unlike A-to-B transport, B-to-A transport was
increased by calcitriol treatment only after the initial 24-h period
(from 0.337 ± 0.017 to 0.466 ± 0.008 nmol · well
1 · min
1)
and did not change significantly thereafter. As a result, net calcium
flux (A-to-B minus B-to-A) increased 3.5-fold, from 0.14 ± 0.058 to
0.495 ± 0.013 nmol · well
1 · min
1
with 72 h of
1,25(OH)2D3
treatment (Fig. 2).
In our next experiment, we examined whether other vitamin D compounds that were chemically related to 1,25(OH)2D3 could stimulate calcium transport in Caco-2 cells. Neither exposure with 25(OH)D3 nor 24,25(OH)2D3 (100 nM, 48 h) increased total A-to-B calcium movement, whereas 10 nM 1,25(OH)2D3 for 48 h increased transport by 56% (Table 1). Combining 25(OH)D3 or 24,25(OH)2D3 with 1,25(OH)2D3 did not alter the induction of calcium transport by 1,25(OH)2D3.
If
1,25(OH)2D3
were to stimulate transcellular calcium transport, we hypothesized that
inhibition of calmodulin-dependent events (e.g., brush-border calcium
uptake and basolateral membrane calcium extrusion) should limit total
A-to-B calcium transport across the Caco-2 monolayer. Figure
3 shows that increasing amounts of the
calmodulin antagonist TFP progressively inhibited the vitamin D-stimulated portion of calcium transport. The maximal inhibition of
56% was observed at 50 µM. Similar findings were seen in an experiment that examined only the effect of 50 µM TFP on A-to-B calcium transport and phenol red movement (data not shown). In this
experiment calcitriol-mediated A-to-B calcium transport was blocked by
TFP, whereas phenol red movement was increased from 1.0 ± 0.05 to 1.7 ± 0.05%. Thus even as diffusion through the monolayer
was increased, the effect of calcitriol on calcium transport was
blocked.
|
In our final set of experiments, we examined the effect of the
transcription inhibitor actinomycin D on the
1,25(OH)2D3-mediated component of calcium transport. Actinomycin D increased total A-to-B
calcium movement across the monolayer and eliminated the difference in
A-to-B calcium transport between control and vitamin D-treated cells.
The increase in total calcium transport was due to enhanced
paracellular diffusion, as demonstrated by increased phenol red
movement from 0.59 to 1.75% per hour. However, when A-to-B calcium
transport was corrected to account for this increase in diffusional
flux, actinomycin D prevented the
1,25(OH)2D3-mediated increase in transcellular calcium transport (Fig.
4). This coincided with an inhibition of
1,25(OH)2D3-mediated
gene expression as reflected in the loss of calbindin
D9k and 24-hydroxylase mRNA following actinomycin D treatment (Fig. 5).
Although actinomycin D had an inhibitory effect on both calbindin
D9k and 24-hydroxylase mRNA
levels, cycloheximide treatment reduced only the accumulation of
calbindin D9k mRNA (Fig. 5).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In these experiments we observed a net positive A-to-B calcium transport flux that was inducible by 1,25(OH)2D3 (Fig. 2). Moreover, we found that 1,25(OH)2D3 did not affect paracellular movement across Caco-2 cell monolayers, as measured by phenol red transport and changes in TEER. These observations are consistent with our previous reports showing that 1,25(OH)2D3 treatment (10 nM, 72 h) did not increase inulin movement through Caco-2 cell monolayers (19) and that 1,25(OH)2D3 induced the saturable but not the linear component of A-to-B calcium transport in Caco-2 cells (24). We believe that these data support the existence of a regulated A-to-B calcium transport pathway that is usually associated with a transcellular transport process (14).
Another observation that shows vitamin D-inducible A-to-B calcium transport is transcellular in Caco-2 cells is that TFP suppressed the vitamin D-mediated component of calcium transport (Fig. 3). TFP interferes with calmodulin binding to proteins and thereby inhibits both cellular calcium extrusion by the basolateral membrane calcium pump (26) and calcium uptake at the brush-border membrane (4, 5). Thus, regardless of the site of action, the fact that TFP inhibits vitamin D-mediated calcium transport in Caco-2 cells reflects a movement through a transcellular pathway. Our TFP data in Caco-2 cell monolayers is similar to the effect that Favus et al. (16) observed for TFP on vitamin D-mediated calcium absorption in rat intestine.
Finally, the transcription inhibitor actinomycin D blocked vitamin D-mediated A-to-B calcium transport (Fig. 4). This suggests that de novo RNA synthesis is required for 1,25(OH)2D3-mediated intestinal calcium transport. Our data are in contrast to findings by Bikle et al. (6), who showed that treating rachitic chicks with cycloheximide or actinomycin D blocked the production of several 1,25(OH)2D3-inducible proteins in the intestine but did not block 1,25(OH)2D3-stimulated calcium absorption. Although our observations could be due to the prolonged treatment of actinomycin D we used in our study (48 h), a shorter 24-h protocol yielded similar results (data not shown). We have previously noted that 12-16 h of vitamin D treatment are needed to significantly alter calcium transport in Caco-2 cells (20). This suggests the Caco-2 cell system may be different from the rachitic chick and that it adapts to 1,25(OH)2D3 treatment by synthesizing new proteins that are involved in calcium transport. One such protein could be calbindin D9k.
Calbindin D9k is a small-molecular-weight calcium-binding protein proposed to function as either an intracellular calcium buffer or an intracellular ferry protein that facilitates diffusion of calcium across the cell (8, 13). Actinomycin D treatment completely blocked vitamin D-induced accumulation of calbindin D9k mRNA in the Caco-2 cells; thus the inhibition in A-to-B calcium transport might be due to blocking production of this protein. Inhibition of protein translation with cycloheximide also blocked the 1,25(OH)2D3-induced accumulation of calbindin D9k mRNA, but not 24-hydroxylase mRNA, in Caco-2 cells (Fig. 5). This is consistent with previous work that demonstrates posttranscriptional regulation of calbindin D9k message in rat intestine and suggests that vitamin D could induce the synthesis of a protein that stabilizes the calbindin D9k mRNA. Further research is being conducted to more fully understand the mechanisms by which vitamin D results in calbindin D9k mRNA accumulation.
Although our observations are consistent with the presence of a transcellular pathway for calcium transport in Caco-2 cells, two recent studies contradict this notion. Blais et al. (7) found that basal A-to-B calcium transport across Caco-2 monolayers (used between passage 25 and 40) was equal to paracellular transport (assessed with mannitol) at approximately 3% per hour. However, the values reported for mannitol transport by Blais et al. (7) were significantly higher than the 0.5% per hour previously reported in Caco-2 cells by Han et al. (25) and Chirayath et al. (12). This suggests that the integrity of their Caco-2 monolayers used by Blais et al. (7) may have been impaired or altered in some way so as to make them unacceptably leaky for calcium transport studies. In addition, these investigators did not examine the influence of 1,25(OH)2D3 on transport, thus their studies do not specifically address the suitability of the Caco-2 model to study the mechanism of vitamin D-regulated intestinal calcium absorption.
Chirayath et al. (12) have reported data using Caco-2 cells that directly conflict with several of our critical findings. For example, they found that 10 nM 1,25(OH)2D3 treatment for up to 72 h reduced TEER in Caco-2 monolayers. In addition, they did not observe a net A-to-B flux of calcium under basal or 1,25(OH)D3-stimulated conditions nor did they see a reduction in 1,25(OH)2D3-inducible calcium transport following treatment with the calcium pump inhibitor calmidazolium. We believe that these conflicting observations may be due to major differences in the characteristics of the Caco-2 cell population examined in the two studies.
First, Chirayath et al. (12) used a subclone of Caco-2 cells (AQ) derived from an established clone (Caco-2/15) after passage 100. Yu et al. (37) showed that high passage number Caco-2 cells (93-108) have lower carrier-mediated transport, higher TEER, and lower alkaline phosphatase activity (a marker of intestinal cell differentiation) than low passage number cells (28-36 passages). They suggest that this was due to selection of fast-growing subpopulations from the original heterogeneous Caco-2 cell line during repeated passaging. Features of a less-differentiated cell phenotype are also seen in the AQ subclone used by Chirayath et al., e.g., reduced doubling time (24 vs. 36 h) and lower alkaline phosphatase activity (60 vs. 190 mU/mg protein), compared with the Caco-2/15 parent clone and TEER values 50% higher than those we report for the parent stock. These differences in the AQ subclone show how various clonal cell lines of Caco-2 can have important phenotypic differences, such as a reduced ability to fully differentiate, that could render them inappropriate as an experimental model to study vitamin D-mediated calcium transport.
Next, we felt it was noteworthy that Chirayath et al. (12) did not see an inhibition of vitamin D-mediated A-to-B calcium transport after treatment with 100 µmol/l calmidazolium. However, Gietzen et al. (22) previously showed and we confirmed (data not shown) that the maximum concentration of calmidazolium (also called R-24571) in aqueous medium at pH 7.0 is 20 µM. As a result, we believe there is reason to question the validity of the calmidazolium data presented by Chirayath et al. (12) as well as their conclusion that calcium is not transported by a transcellular pathway in Caco-2 cells.
In summary, we have shown that the treatment of Caco-2 cells with 1,25(OH)2D3, but not 25(OH)D3 or 24,25(OH)2D3, causes an increase in net A-to-B calcium transport across Caco-2 cell monolayers. Moreover, treatment with TFP significantly reduced 1,25(OH)2D3-mediated calcium transport. In contrast, we found that 1,25(OH)2D3 did not have a significant effect on the paracellular transport pathway, as estimated by phenol red movement and TEER. These observations, coupled with our additional observation that calcium transport and calbindin D9k mRNA expression were blocked by actinomycin D treatment, are consistent with a genomic effect of 1,25(OH)2D3 on transcellular A-to-B calcium transport in Caco-2 cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jessica Bradley and Kerry Quinn for the technical assistance and contributions of data to the manuscript.
![]() |
FOOTNOTES |
---|
Some data cited in this report were presented at the 77th and 82nd annual meetings of the American Society for Nutrition Science in New Orleans, LA, and Washington, DC, respectively (FASEB J. 7: A497, 7: A87, and 12: A1020).
This project was funded in part with funds from the University of North Carolina at Greensboro, the National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-54111, and the United States Department of Agriculture, Agricultural Research Service under contract 53-3K06-5-10.
The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. C. Fleet, Dept. of Nutrition and Foodservice Systems, Univ. of North Carolina at Greensboro, PO Box 26170, Greensboro, NC 27402-6170 (E-mail: jim_fleet{at}uncg.edu).
Received 25 September 1998; accepted in final form 22 December 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alvarez-Hernandez, X.,
M. Smith,
and
J. Glass.
Regulation of iron uptake and transport by transferrin in Caco-2 cells, an intestinal cell line.
Biochim. Biophys. Acta
1192:
215-222,
1994[Medline].
2.
Armbrecht, H. J.,
T. V. Zenser,
M. E. Bruns,
and
B. B. Davis.
Effect of age on intestinal calcium absorption and adaptation to dietary calcium.
Am. J. Physiol.
236 (Endocrinol. Metab. Gastrointest. Physiol. 5):
E769-E774,
1979
3.
Arredondo, M.,
A. Orellana,
M. A. Garate,
and
M. T. Nunez.
Intracellular iron regulates iron absorption and IRP activity in intestinal epithelial (Caco-2) cells.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G275-G280,
1997
4.
Bikle, D.,
S. Munson,
S. Christakos,
R. Kumar,
and
P. Buckendahl.
Calmodulin binding to the intestinal brush-border membrane: comparison to other calcium-binding proteins.
Biochim. Biophys. Acta
1010:
122-127,
1989[Medline].
5.
Bikle, D. D.,
and
S. Munson.
The villus gradient of brush border membrane calmodulin and the calcium-independent calmodulin-binding protein parallels that of calcium-accumulating ability.
Endocrinology
118:
727-732,
1986[Abstract].
6.
Bikle, D. D.,
D. T. Zolock,
R. L. Morrissey,
and
R. H. Herman.
Independence of 1,25-dihydroxyvitamin D3-mediated calcium transport from de novo RNA and protein synthesis.
J. Biol. Chem.
253:
484-488,
1978[Abstract].
7.
Blais, A.,
P. Aymard,
and
B. Lacour.
Paracellular calcium transport across Caco-2 and HT29 cell monolayers.
Pflügers Arch.
434:
300-305,
1997[Medline].
8.
Bronner, F.,
D. Pansu,
and
W. D. Stein.
An analysis of intestinal calcium transport across the rat intestine.
Am. J. Physiol.
250 (Gastrointest. Liver Physiol. 13):
G561-G569,
1986
9.
Bronner, F.,
and
K. Spence.
Non-saturable Ca transport in the rat intestine is via the paracellular pathway.
In: Cellular Calcium and Phosphate Transport in Health and Disease. Progress in Clinical and Biological Research, edited by F. Bronner,
and M. Peterlink. New York: Liss, 1988, p. 277-283.
10.
Bullamore, J. R.,
J. C. Gallagher,
R. Wilkinson,
B. E. C. Nordin,
and
D. H. Marshall.
Effect of age on calcium absorption.
Lancet
2:
535-537,
1970[Medline].
11.
Chen, K. S.,
J. M. Prahl,
and
H. F. DeLuca.
Isolation and expression of human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA.
Proc. Natl. Acad. Sci. USA
90:
4543-4547,
1993[Abstract].
12.
Chirayath, M. V.,
L. Gajdzik,
W. Hulla,
J. Graf,
H. S. Cross,
and
M. Peterlink.
Vitamin D increases tight-junction conductance and paracellular Ca2+ transport in Caco-2 cell cultures.
Am. J. Physiol.
274 (Gastrointest. Liver Physiol. 37):
G389-G396,
1998
13.
Christakos, S.,
M. Raval-Pandya,
R. P. Wernyj,
and
W. Yang.
Genomic mechanisms involved in the pleiotropic actions of 1,25-dihydroxyvitamin D3.
Biochem. J.
316:
361-371,
1996[Medline].
14.
Duflos, C.,
C. Bellaton,
N. Baghdassarian,
M. Gadoux,
D. Pansu,
and
F. Bronner.
1,25-Dihydroxycholecalciferol regulates rat intestinal calbindin D9k posttranscriptionally.
J. Nutr.
126:
834-841,
1996[Medline].
15.
Dupret, J. M.,
P. Brun,
and
M. Thomasset.
In vivo effects of transcriptional and translational inhibitors on duodenal vitamin D-dependent calcium-binding protein messenger ribonucleic acid stimulation by 1,25-dihydroxycholecalciferol.
Endocrinology
119:
2476-2483,
1986[Abstract].
16.
Favus, M. J.,
E. Angeid-Backman,
M. D. Breyer,
and
F. L. Coe.
Effects of trifluoperazine, ouabain, and ethacrynic acid on intestinal calcium.
Am. J. Physiol.
244 (Gastrointest. Liver Physiol. 7):
G111-G115,
1983
17.
Fleet, J. C.,
J. Bradley,
G. S. Reddy,
R. Ray,
and
R. J. Wood.
1,25-(OH)2-vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro.
Arch. Biochem. Biophys.
329:
228-234,
1996[Medline].
18.
Fleet, J. C.,
M. E. Bruns,
J. M. Hock,
and
R. J. Wood.
Growth hormone and parathyroid hormone stimulate intestinal calcium absorption in aged female rats.
Endocrinology
134:
1755-1760,
1994[Abstract].
19.
Fleet, J. C.,
A. J. Turnbull,
M. Bourcier,
and
R. J. Wood.
Vitamin D-sensitive and quinacrine-sensitive zinc transport in human intestinal cell line Caco-2.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G1037-G1045,
1993
20.
Fleet, J. C.,
and
R. J. Wood.
Identification of calbindin D9k mRNA and its regulation by 1,25 dihydroxyvitamin D3 in Caco-2 cells.
Arch. Biochem. Biophys.
308:
171-174,
1994[Medline].
21.
Fort, P.,
M. Piechaczyk,
S. E. Sabrouty,
C. Dani,
P. Jeanteur,
and
J. M. Blanchard.
Various rat tissues express only one major mRNA species from the glyceraldehyde 3-phosphate-dehydrogenase family.
Nucleic Acids Res.
13:
1431-1442,
1985[Abstract].
22.
Gietzen, K.,
A. Wuthrich,
and
H. Bader.
R 24571: a new powerful inhibitor of red blood cell Ca++-transport ATPase and of calmodulin-regulated functions.
Biochem. Biophys. Res. Commun.
101:
418-425,
1981[Medline].
23.
Giuliano, A. R.,
R. T. Franceschi,
and
R. J. Wood.
Characterization of the vitamin D receptor from the Caco-2 human colon carcinoma cell line: effect of cellular differentiation.
Arch. Biochem. Biophys.
285:
261-269,
1991[Medline].
24.
Giuliano, A. R.,
and
R. J. Wood.
Vitamin D-regulated calcium transport in Caco-2 cells: unique in vitro model.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G207-G212,
1991
25.
Han, O.,
M. L. Failla,
and
J. C. Smith.
Transferrin-iron and proinflammatory cytokines influence iron status and apical iron transport efficiency of Caco-2 intestinal cell line.
J. Nutr. Biochem.
8:
585-591,
1997.
26.
Hinds, T. R.,
B. V. Raess,
and
F. F. Vincenzi.
Plasma membrane Ca2+ transport: antagonism by several potential inhibitors.
J. Membr. Biol.
58:
57-65,
1981[Medline].
27.
Howard, A.,
S. Legon,
N. K. Spurr,
and
J. R. F. Walters.
Molecular cloning and chromosomal assignment of human calbindin-D9k.
Biochem. Biophys. Res. Commun.
185:
663-669,
1992[Medline].
28.
Nellans, H. N.
Intestinal calcium absorption. Interplay of paracellular and cellular pathways.
Miner. Electrolyte Metab.
16:
101-108,
1990[Medline].
29.
Nemere, I.
Vesicular calcium transport in chick intestine.
J. Nutr.
122:
657-661,
1992[Medline].
30.
Nemere, I.,
V. Leathers,
and
A. W. Norman.
1,25 Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysozomes containing calcium and calcium-binding protein (calbindin-D28k).
J. Biol. Chem.
261:
16106-16114,
1986
31.
Nicolaysen, R.
The absorption of calcium as a function of the body saturation with calcium.
Acta Physiol. Scand.
5:
200-211,
1943.
32.
Norman, A. W.
Intestinal calcium absorption: a vitamin D-hormone mediated adaptive response.
Am. J. Clin. Nutr.
51:
290-300,
1990[Abstract].
33.
Pinto, M.,
S. Robine-Leon,
M. D. Appay,
M. Kedinger,
N. Triadow,
E. Dussaulx,
B. Lacroix,
P. Simon-Assmann,
K. Haffer,
J. Fogh,
and
A. Zweibaum.
Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture.
Biol. Cell
47:
323-330,
1983.
34.
Raffaniello, R. D.,
S.-Y. Lee,
S. Teichberg,
and
R. A. Wapnir.
Distinct mechanisms of zinc uptake at the apical and basolateral membranes of Caco-2 cells.
J. Cell. Physiol.
152:
356-361,
1992[Medline].
35.
Wilkinson, L.,
M. Hill,
J. P. Welna,
and
G. K. Birkenbeuel.
Systat for Windows: Statistics, version 5 edition. Evanston, IL: Systat, 1992.
36.
Yee, S.
In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in manfact or myth?
Pharm. Res.
14:
763-766,
1997[Medline].
37.
Yu, H.,
T. J. Cook,
and
P. J. Sinko.
Evidence for diminished functional expression of intestinal transporters in Caco-2 cell monolayers at high passages.
Pharm. Res.
14:
757-762,
1997[Medline].