1 Geriatric Research, Education, and Clinical Center, St. Louis Veterans Affairs Medical Center, St. Louis 63125; and 2 Division of Geriatric Medicine and 3 Department of Biochemistry and Molecular Biology, St. Louis University Health Sciences Center, St. Louis, Missouri 63104
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ABSTRACT |
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The plasma membrane Ca pump of intestinal absorptive cells has been proposed as a component in the vitamin D-dependent active transport of Ca. Because intestinal Ca transport declines with age, the purpose of this study was to determine if changes in Ca pump expression parallel this decline. Intestinal levels of the plasma membrane Ca pump protein were measured by Western blotting in Fischer 344 rats that were 2, 12, and 24 mo of age. Ca pump protein levels declined by 90% in the duodenum and 65% in the ileum between 2 and 12 mo of age, the time during which active Ca transport declines markedly. The effect of age on the induction of the Ca pump by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D, was determined. Rats were made deficient in 1,25(OH)2D3 by feeding a high-strontium diet, and they were then dosed with 1,25(OH)2D3 or vehicle at 48, 24, and 6 h. In 12-mo-old rats 1,25(OH)2D3 induced duodenal Ca pump protein to only 39% and active Ca transport to 33% of that seen in 2-mo-old animals. These studies demonstrate that decreased expression of the plasma membrane Ca pump protein, along with calbindin protein, parallels the decline in intestinal Ca transport and its response to 1,25(OH)2D3 with age.
intestinal calcium transport; Western blotting; rats; 1,25-dihydroxyvitamin D3
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INTRODUCTION |
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THE ABSORPTION of Ca by the small intestine declines with age in humans (1) and rats (2, 8, 19, 26). The capacity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, to stimulate Ca absorption also declines with age (8, 9, 19, 26). Two important components of the intestinal Ca transport system are the Ca-binding protein calbindin and the plasma membrane Ca pump (24). Calbindin is thought to help translocate Ca from the brush border to the basolateral membrane. The Ca pump localized on the basolateral membrane and then pumps the Ca out of the absorptive cell and onto the serosal side of the intestine. In addition, there is evidence that calbindin itself may stimulate basolateral Ca pump activity (21). In young animals, both calbindin and the Ca pump are stimulated by 1,25(OH)2D3, and this may largely account for the overall stimulation of Ca absorption by 1,25(OH)2D3.
There are a number of correlations between the basolateral Ca pump and vitamin D-dependent active Ca transport. There is a strong correlation between Ca transport and basolateral Ca pump activity with regard to intestinal segment (17), villus-crypt axis (22), and stimulation by 1,25(OH)2D3 (17, 22). The plasma membrane Ca pump protein and mRNA levels are correlated with changes in Ca transport due to 1,25(OH)2D3 administration and the feeding of diets low in Ca or phosphorus (14, 23, 25).
There is also evidence that the Ca pump, along with calbindin, is involved in the age-related changes in Ca absorption. It is the active transport of Ca by the duodenum that declines the most with age (8), and the Ca pump is an energy-requiring step in the active movement of Ca across the intestine (23). In addition, it has previously been shown that the capacity of basolateral membrane vesicles to accumulate Ca in an energy-dependent manner declines with age (6). This is seen in both the duodenum and the ileum and could be due to decreased amounts of the Ca pump protein and/or to decreased functionality of the protein with age.
The first purpose of these studies was to determine whether Ca pump protein levels changed with age in the duodenum and ileum. The protein levels were then correlated with changes in Ca pump activity (6) and Ca pump mRNA levels (5) with age. Calbindin protein levels were also determined for comparison.
The second purpose of the studies was to determine whether the capacity of 1,25(OH)2D3 to increase Ca pump protein declined with age. To determine this, a multiple-dose model was employed. The model showed the expected decline in the capacity of 1,25(OH)2D3 to stimulate Ca transport with age. It was then used to determine the effect of 1,25(OH)2D3 on the expression of the duodenal Ca pump as a function of age. In addition, the responses of intestinal calbindin and the vitamin D-24-hydroxylase protein levels to 1,25(OH)2D3 were determined for comparison. A decline in the capacity of 1,25(OH)2D3 to stimulate key proteins could account for the decreased capacity of 1,25(OH)2D3 to stimulate Ca absorption.
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MATERIALS AND METHODS |
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Animals. Experiments were performed using young (2 mo), adult (12 mo), and old (22 mo) male Fischer 344 rats (F344/NNIA). Animals were obtained from the National Institute on Aging colony maintained by Harlan Industries (Indianapolis, IN). Rats were maintained under microbial barrier conditions and were fed a semisynthetic diet containing 1.2% Ca, 0.8% phosphorus, and 3.3 IU/g vitamin D3 (Purina Rodent Chow, Ralston-Purina, St. Louis, MO).
To study the effect of 1,25(OH)2D3 on vitamin D-dependent gene expression, a multidose model was used. Rats were first made deficient in 1,25(OH)2D3 by feeding them a high-strontium diet (3, 9). The diet consisted of a low-Ca diet (Teklad test diet no. 170120, Harlan Teklad, Madison, WI) containing 0.8% strontium, and it was given for 6 days. This diet has been shown to markedly reduce serum 1,25(OH)2D3 levels, intestinal Ca transport, and intestinal calbindin levels (3, 9). To determine the effect of 1,25(OH)2D3, rats were given intraperitoneal injections of 1,25(OH)2D3 (100 ng/100 g body wt) at 48, 24, and 6 h before killing. Control rats were given ethanol vehicle only. These dosing times were chosen to examine both the long-term and short-term effects of 1,25(OH)2D3 in a single experiment (9). On the day of the experiment, the rats were killed, the abdominal cavity was exposed by midline incision, and the duodenum and ileum were removed. Blood was collected into heparinized tubes, and the plasma was frozen for later analysis of plasma 1,25(OH)2D3, Ca, and phosphorus.Western blotting of intestinal proteins. For Western blotting, intestinal cells were isolated from the proximal duodenum (0-5 cm distal to the pylorus) and the distal ileum (0-10 cm proximal to the cecum). Intestinal cells were isolated by incubation with citrate, as previously described (6). Briefly, duodenal and ileal sacs were filled with the citrate buffer, and the sacs were incubated for 15 min at 37°C. Cells were released from the underlying tissue by gently squeezing the sacs and collecting the cells in sucrose buffer. Cells were collected by low-speed centrifugation and were resuspended in homogenizing buffer (25 mM NaCl, 1 mM HEPES, 0.2 mM phenylmethylsulfonyl fluoride, and 0.2% SDS at pH 8.0). Cells were homogenized using a Polytron (Brinkmann Instruments) for 1 min at the medium setting, and aliquots of the homogenates containing 50 mg protein were subjected to SDS-PAGE using either 7% (Ca pump) or 12% (calbindin and CYP24) gels. Western blotting was performed as previously described (7). After electrophoresis, proteins were transferred electrophoretically to nitrocellulose membranes (Hybond-ECL, Amersham, Arlington Heights, IL). The membrane was incubated with the first antibody for 1 h followed by 1 h with the appropriate second antibody.
For the plasma membrane Ca pump, the 5F10 monoclonal antibody to the erythrocyte Ca pump (kindly supplied by Drs. R. Kumar and J. Penniston, Mayo Clinic, Rochester, MN) was used (11). The 5F10 monoclonal antibody recognizes the plasma membrane Ca pump in a number of tissues and species (12). This antibody has recently been shown to recognize residues in the region of 719-738, which is highly conserved in all forms of the pump (15). In the intestine, it has been previously shown to recognize the Ca pump in the basolateral region of the intestinal absorptive cell in both rat (11) and chicken (23). The intestinal pump recognized by this antibody is stimulated by 1,25(OH)2D3 directly or via increased serum levels in response to diets low in Ca or phosphorus (23). The second antibody for the Ca pump was a sheep anti-mouse IgM antibody linked to horseradish peroxidase. For calbindin, a polyclonal antibody to rat intestinal calbindin D9k (kindly supplied by Dr. Elizabeth Bruns, University of Virginia Medical Center, Charlottesville, VA) was used (3), and a polyclonal antibody was used to detect the vitamin D 24-hydroxylase (CYP24) (7). This antibody was prepared commercially against a synthetic peptide based on the NH2-terminal sequence of the rat cytochrome P-450 CYP24 (Chiron, San Diego, CA). A donkey anti-rabbit IgG antibody linked to horseradish peroxidase was used as the second antibody for calbindin and CYP24. The antigen-antibody complexes were visualized by chemiluminescence using luminol-peroxidase reagents supplied by Amersham (ECL Western blotting kit and Hyperfilm-ECL). Bands were quantitated by densitometry over the linear range of the absorption curve. In preliminary experiments, absorbance was found to be linear between 50 and 10 µg of total protein per lane. Based on actin rehybridization, sample loading was quite uniform. Therefore, absorbance was routinely normalized to total intestinal protein.Measurement of intestinal Ca transport. Intestinal Ca transport was measured in vitro using everted sacs, as previously described (8). Briefly, everted duodenal sacs were formed using the first 5 cm of intestine distal to the pylorus. Sacs were filled with 0.35 ml buffer containing 0.25 mM Ca labeled with 45CaCl2 (ICN, Costa Mesa, CA), and the filled sacs were incubated in flasks containing 10 ml of the same 45Ca-labeled buffer. After 1.5 h, samples of the external incubation medium and the inside fluid from each sac were collected and assayed in triplicate for 45Ca using scintillation counting. The ratio of the inside (serosal) to outside (mucosal) counts was calculated and called the S-to-M ratio (S/M), which is a measure of active Ca transport.
Basolateral Ca pump activity. Ca pump activity was measured as previously described (6). Basolateral membrane vesicles were isolated from the rat duodenum by differential and gradient density centrifugation. Ca uptake was measured in the presence of 5 µM radiolabeled CaCl2 and 5 mM ATP. Ca uptake was terminated at the indicated times by dilution with a cold-stop solution followed by rapid filtration. The filter was then washed, and the amount of radiolabeled Ca remaining on the filter was determined by scintillation counting.
Plasma assays and statistics. Plasma 1,25(OH)2D3 was measured using a commercial kit (ImmunoNuclear, Stillwater, MN). Plasma Ca and phosphorus were measured colorimetrically using commercial kits (Sigma, St. Louis, MO).
The data from these experiments are reported as means ± SE of the number of animals indicated. Statistical analyses were performed using Student's two-tailed t-test. A confidence level of ![]() |
RESULTS |
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Effect of age on expression of intestinal Ca pump and calbindin.
In initial studies, pools of intestinal protein from several animals
were subjected to Western blotting using the antibody to the plasma
membrane Ca pump (Fig. 1). Two major bands
with sizes of 155 and 130 kDa were visible along with a minor band at
90 kDa. The size and number of the bands did not change appreciably with age or segment. However, the intensity of the bands declined markedly with age in both duodenum and ileum. In addition, the intensity in the ileum was much less than in the duodenum of the same
age group.
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Effect of 1,25(OH)2D on vitamin D-dependent protein
expression.
To determine the effect of 1,25(OH)2D3 in
young and adult rats, animals were made deficient in
1,25(OH)2D by feeding a strontium-containing diet.
Animals were then dosed with 1,25(OH)2D3 or
vehicle at 48, 24, and 6 h before killing. The capacity of
1,25(OH)2D3 to stimulate Ca transport was
determined using everted duodenal sacs.
1,25(OH)2D3 stimulated Ca transport in both
young and adult rats (Fig. 5). The maximal
stimulation in the young was three times higher than that seen in the
adult. The 1,25(OH)2D3-stimulated component
in the adult was only one-fourth that of the young.
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Effect of 1,25(OH)2D on basolateral membrane Ca
transport.
Because there were differences in the effect of
1,25(OH)2D3 on Ca pump protein (Fig. 6), the
effect of 1,25(OH)2D3 on basolateral Ca pump
activity was examined. 1,25(OH)2D3
significantly stimulated Ca uptake by basolateral membrane vesicles
prepared from the duodenum of young animals (Fig.
8). However, there was no significant
stimulation of Ca uptake by 1,25(OH)2D3 in
vesicles prepared from adult animals.
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DISCUSSION |
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These studies show that plasma membrane Ca pump protein in the duodenum declines by 90% between 2 and 12 mo of age (Figs. 1 and 2). There is little further decline between 12 and 24 mo of age. Previous studies in the F344 rat have shown a 73% decrease in duodenal Ca transport between 2 and 12 mo of age with little decline thereafter (8). This decline in Ca pump protein correlates with a decline in the capacity of the basolateral membrane to actively pump Ca (Fig. 4). This is not surprising, because immunohistochemical studies have shown that most of the staining by this antibody is in the basolateral rather than the apical portion of the intestinal absorptive cell in both rat (11) and chick (23). The 5F10 antibody recognizes all forms of the plasma membrane Ca pump (15). This would then suggest that in the intestinal absorptive cell a large percentage of the Ca pumps are mobilized for transcellular Ca transport.
With the 5F10 antibody, the Western blots in these studies showed two major bands at 155 and 130 kDa (Fig. 1). To obtain high-molecular-mass bands, preliminary studies showed that it was necessary to first isolate intestinal cells and then homogenize them in detergent. Direct homogenization of mucosal scrapings resulted in multiple bands on Western blots with molecular masses ranging from 10 to 100 kDa. These bands probably represent proteolytic products because the Ca pump is sensitive to proteolysis. Two high-molecular-mass bands have been reported previously in the intestine using this antibody. In the chick intestine, two bands at 141 and 175 kDa have been reported (25), and bands at 125 and 135 kDa have been reported in the rat intestine (11).
The age-related decline in Ca pump protein parallels the previously reported decline in mRNA levels for the PMCA1 form of the Ca pump (Fig. 4). Another study in male Sprague-Dawley rats found only a slight decline in Ca pump mRNA between 3 and 12 mo of age (20). This could be due to the fact that the present and previous study (5) in F344 rats used 2-mo-old rats rather than 3-mo-old rats. The previous study in F344 rats correlated the decline in Ca pump mRNA with the decline in serum 1,25(OH)2D3 (5). This is consistent with the fact that Ca pump mRNA has been shown to be regulated by 1,25(OH)2D3 in the rat (27) and the chicken (14). Taken together, these results suggest that the age-related decline in serum 1,25(OH)2D3 results in a decrease in Ca pump mRNA, which in turn results in a decrease in Ca pump protein. The major form of the Ca pump in the intestine is PMCA1 (18). It is this form that is stimulated by vitamin D in the rat (27) and that declines with age (5). Thus a large percentage of the PMCA1 form of the Ca pump appears to be involved in Ca absorption in the intestine.
If the age-related decline in serum 1,25(OH)2D3 were the sole contributor to decreased Ca absorption, then administration of 1,25(OH)2D3 would be expected to fully reverse the decline in the adult and old animal. However, even multiple doses of 1,25(OH)2D3 over 48 h did not fully stimulate active Ca transport in the adult rat (Fig. 5). This was not due to differences in plasma 1,25(OH)2D3, Ca, or phosphorus after 1,25(OH)2D3 treatment (Table 1). These results suggest that with age the intestine develops a refractoriness to 1,25(OH)2D3. Consistent with this is the fact that the capacity of 1,25(OH)2D3 to increase Ca pump protein is decreased by 61% in the adult compared with young animals (Fig. 6). This correlates with the decreased capacity of 1,25(OH)2D3 in adults to stimulate Ca transport (Fig. 5).
At the membrane level, there were also differences between young and adult animals (Fig. 8). 1,25(OH)2D3 significantly stimulated basolateral Ca uptake in young but not in adult animals. Based on the fact that 1,25(OH)2D3 modestly increased Ca pump protein in the adult animal (Fig. 6), one might have expected that 1,25(OH)2D3 would also increase Ca pump activity in the adult basolateral membranes. The fact that no increase was seen may be due to a relative insensitivity of the Ca uptake assay to small changes. There was a high level of basal Ca uptake by the vesicles, and the increase by 1,25(OH)2D3 in the young was only 74%.
The expression of intestinal calbindin, which has also been implicated in Ca transport (24), tended to parallel that of the plasma membrane Ca pump in these studies. Calbindin protein levels declined markedly between 2 and 12 mo of age with little change thereafter (Fig. 3). This has been previously shown to correlate with a decline in calbindin mRNA levels (4), which is secondary to the decrease in serum 1,25(OH)2D3 with age. This marked decline in both calbindin and the Ca pump, two major components of the Ca active transport system, could explain the marked decline in duodenal Ca transport between 2 and 12 mo (8, 10). The relative contribution of calbindin and the Ca pump to the overall transport of Ca by the intestine is difficult to estimate and is still an open question. It has been argued that in the rat duodenum the Ca pump may not be rate limiting (13), whereas in the chick duodenum it may be (23). The induction of calbindin protein by 1,25(OH)2D3 was also greatly reduced in adult rats compared with young ones (Fig. 7). Again, a combination of decreased induction of the Ca pump and calbindin by 1,25(OH)2D3 could account for the decreased induction of active transport in the adult (Fig. 5). This would be especially true if calbindin were important for the activation of the Ca pump (21).
It is of interest that the induction of some vitamin D-dependent genes does not decline with age. 1,25(OH)2D3 markedly increases CYP24 protein levels to similar levels in both young and adult rats (Fig. 7). The fact that 1,25(OH)2D3 markedly increases mRNA levels of these proteins in both young and adult rats would suggest that there is no defect with respect to vitamin D receptor function. There have been conflicting reports as to whether vitamin D receptor number decreases (19) or stays the same (26) with age in the rat intestine.
The question arises as to why the capacity of 1,25(OH)2D3 to increase Ca pump and calbindin protein in the intestine declines with age. With regard to this, two studies that examined the effect of age on the induction of mRNA levels of the Ca pump and calbindin are relevant. These studies showed that 1,25(OH)2D3 increases the mRNA levels of the Ca pump (5) and of calbindin (3) to similar levels in young and adult animals. This would suggest that the problem is not decreased transcription of the Ca pump and calbindin gene but rather decreased translation of the mRNA into protein. It has been suggested that intestinal calbindin is regulated posttranscriptionally by 1,25(OH)2D3 (16). This decreased translation could be due to either a decreased capacity of adult ribosomes to translate the mRNA or to a decreased translatability of the adult message itself.
In summary, the decrease in intestinal Ca pump, along with parallel changes in calbindin, may largely account for the age-related decline in Ca transport in the intestine. This decline in protein levels is probably secondary to a decline in Ca pump and calbindin mRNA levels, which, in turn, reflect the age-related decline in serum 1,25(OH)2D3 levels. However, multiple doses of 1,25(OH)2D3 to the adult do not increase Ca transport, the Ca pump, and calbindin to levels seen in the young, despite equivalent mRNA levels. The correlation of Ca pump and calbindin protein levels with Ca transport across age and vitamin D status suggests that these proteins play a major role in the movement of Ca across the intestine.
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ACKNOWLEDGEMENTS |
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We thank Drs. R. Kumar and J. Penniston (Mayo Clinic, Rochester, MN) for the 5F10 Ca pump antibody and Dr. Elizabeth Bruns (University of Virginia School of Medicine, Charlottesville, VA) for the antibody to calbindin D-9k.
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FOOTNOTES |
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This work was supported by the St. Louis Geriatric Research, Education, and Clinical Center, by the Medical Research Service of the Department of Veterans Affairs, and by National Institutes of Health Grant AG-12587.
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: H. J. Armbrecht, Geriatric Center (11G-JB), St. Louis VA Medical Center, St. Louis, MO 63125.
Received 23 October 1998; accepted in final form 25 March 1999.
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