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Endocrinology Vol. 144, No. 5 1726-1735
Copyright © 2003 by The Endocrine Society

Effect of Growth and Maturation on Membrane-Initiated Actions of 1,25-Dihydroxyvitamin D3. I. Calcium Transport, Receptor Kinetics, and Signal Transduction in Intestine of Male Chickens

Birgitta Larsson and Ilka Nemere

Department of Nutrition and Food Sciences and Biotechnology and Genomic Research Center, Utah State University, Logan, Utah 84322-8700

Address all correspondence and requests for reprints to: Dr. Ilka Nemere, Department of Nutrition and Food Sciences, Utah State University, Logan, Utah 84322-8700. E-mail: nemere{at}cc.usu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To study the physiological relevance of membrane-initiated steroid signaling, we investigated the correlation of age in male chickens with the magnitude of responses to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in duodena from 7-, 14-, 28-, and 58-wk-old birds. Measurements of 1,25-(OH)2D3 (130 pM) responsiveness as a function of age, showed a decreased intestinal Ca2+ transport. Western analyses of isolated basal lateral membranes indicated a decreased expression of the membrane-associated rapid response binding protein with increasing age. Saturation analyses of [3H]1,25-(OH)2D3 binding to basal lateral membranes, revealed an allosteric interaction identified as cooperative binding. A significant increase in Kd was observed with increasing age, indicating decreasing affinity. Determinations of the number of binding sites yielded a binding capacity of 190–250 fmol/mg protein during growth and maturation, whereas in adulthood (58 wk) saturable binding was no longer observed. Data obtained in parallel analyses of binding of [3H]1,25-(OH)2D3 to nuclear fraction vitamin D receptor, in contrast, indicated an absence of cooperative binding and an absence of significant changes in Kd or binding capacity with age. Membrane-initiated signal transduction by 1,25-(OH)2D3 was assessed by determination of protein kinase C and A activities. Stimulation of protein kinase C activity in response to 1,25-(OH)2D3 decreased with age, whereas no age-correlated changes in steroid-stimulated protein kinase A activities were observed. Thus, in conclusion, our experiments demonstrate that there is a decrease in responsiveness to exogenous 1,25-(OH)2D3 as a function of age in duodena of male chickens, which can be correlated to a decreased affinity for 1,25-(OH)2D3, a reduced expression of membrane-associated rapid response binding protein, and a decreased protein kinase C activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WIDELY accepted that the hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] exerts a portion of its effects through nuclear-initiated steroid signal transduction (NISS) (1). In intestine, the NISS actions of the hormone are responsible for synthesis of the cellular components necessary for transcellular calcium and phosphate transport (2). In normal, vitamin D-replete animals, 1,25-(OH)2D3 is also capable of activating membrane-initiated steroid signaling (MISS) to promote the rapid onset of enhanced calcium and phosphate transport (2).

To assess the physiological role of the 1,25-(OH)2D3-MISS in intestine, we chose to evaluate it within the context of aging. Previous studies have reported age-related malabsorption of calcium that may be due to decreased levels of 1,25-(OH)2D3 (3, 4), and responsiveness of the intestine to the steroid hormone (5, 6). Although both 1,25-(OH)2D3-NISS and -MISS have been implicated in age-related decreases in intestinal responsiveness (7, 8, 9, 10, 11, 12, 13), none of these studies has addressed the roles of both pathways within the same system.

In the present study we investigated the effect of growth and development on the rapid intestinal responsiveness to 1,25-(OH)2D3. Proximal alterations in responsiveness could be due to altered binding parameters of the nuclear vitamin D receptor (nVDR), the 1,25-(OH)2D3 membrane-associated, rapid response steroid binding protein (1,25D3-MARRS bp), and/or signal transduction. Our results suggest pleiotropic effects of maturation on both 1,25D3-MARRS bp binding parameters and 1,25-(OH)2D3 MISS in relation to the acute stimulation of duodenal calcium transport by hormone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
White leghorn cockerels were obtained from Merrill Poultry (Poul, ID), the vitamin D-supplemented diet (1.0% calcium and 1.0% phosphorus) was purchased from Nutrena Feeds (Murray, UT), chloropent was obtained from Fort Dodge Laboratories, (Fort Dodge, IA), 45CaCl2 and [3H]1,25-(OH)2D3 were obtained from NEN Life Science Products (Boston, MA). Immobilon-P polyvinylidene difluoride membranes were obtained from Millipore Corp. (Bedford, MA), kits for protein kinase C (PKC) and protein kinase A (PKA) determinations were obtained from Life Technologies, Inc. (Waverly, MA), Bradford dye was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA) and [{gamma}-32P]ATP was purchased from NEN Life Science Products. All other chemicals were of the highest grade available and were obtained from Sigma-Aldrich (St. Louis, MO).

Animals
White leghorn cockerels were obtained on the day of hatching and raised on a vitamin D-supplemented diet before experiment. Animals of 7, 14, 28, and 58 wk of age were studied. On the day of the experiments, chickens were anesthetized with 0.3 ml/100 g body weight chloropent. All protocols were approved by the Utah State University institutional animal care and use committee.

Perfusion studies
Perfusion studies were performed as described previously (14). After cannulation of the celiac artery, vascular perfusion was initiated with Gey’s balanced salt solution (GBSS) containing 0.125% BSA and the vehicle ethanol (0.005%, final concentration). The celiac vein (for collection of transported radionuclide) and lumen were also cannulated. At -20 min, luminal perfusion was initiated with GBSS lacking bicarbonate and containing 1 µCi/ml 45CaCl2. Each perfusion experiment was divided into three periods. During the first period (-20 to -10 min), vascular perfusion with aerated GBSS-control medium (containing 0.005% ethanol) was conducted to allow the system to reach a steady state. During the second period (-10 to 0 min) samples of venous effluent were collected for assessment of basal 45Ca transport. Throughout the third period, the preparation was exposed to either GBSS-control medium or 130 pM 1,25-(OH)2D3 for 40 min, and radioactivity was assessed in the collected fractions. The transport during the third period was normalized to the corresponding average basal transport rate.

Preparation of crude nuclei and basal lateral membranes (BLM)
Subcellular fractions were prepared (n = 3 for each age group) as reported previously (15, 16). Intestinal epithelium was disrupted in 40 ml homogenization medium (250 mM sucrose, 5 mM histidine-imidazole, and 2 mM EGTA, pH 7.0) with a Dounce homogenizer (Kontes Co., Vineland, NJ) and a Teflon pestle. The epithelium was fractioned by differential centrifugation, resulting in a crude nuclear fraction, and an intracellular organelle fraction containing the BLM. Intracellular organelles were separated from BLM by centrifugation in Percoll. Eighteen fractions were collected from the gradient (52 drops each). Fractions 16–18 containing BLM (15, 16) were pooled, and the Percoll was removed by ultracentrifugation (15). Marker enzyme activities in BLM fractions indicated a 12-fold enrichment for Na+,K+-ATPase over whole homogenate, and 0.7- and 1-fold enrichments for acid phosphatase and succinate dehydrogenase, respectively, relative to corresponding specific activities in whole homogenates. The BLM fractions contained 36% of gradient Na+,K+-ATPase specific activity, 8% of gradient acid phosphatase activity, and 4% of gradient succinate dehydrogenase activity. Crude nuclei and BLM were stored at -20 C until used.

Saturation analysis
BLM fractions were adjusted to 50 µg protein/tube and incubated in TED buffer (10 mM Tris, 2 mM EDTA, and 1 mM dithiothreitol, pH 7.4) overnight (0 C) with 0.5, 1.0, 2.0, or 4.0 nM [3H]1,25-(OH)2D3 in the absence (total binding) or presence of a 200-fold molar excess of unlabeled 1,25-(OH)2D3 (nonspecific binding). Each sample was assayed in triplicate for total and nonspecific binding. Nuclear fractions from each group were incubated in the same way as BLMs, but with [3H]1,25-(OH)2D3 concentrations of 0.25, 0.5, 1.0, or 2.0 nM.

After incubation overnight, bound and free ligands were separated by either perchloric acid precipitation for binding to BLM (15) or by a hydroxylapatite (HAP) assay for binding to nuclear fractions (17).

For BLM fractions, perchloric acid and carrier protein ({gamma}-globulin) were added to each tube, the mixture was incubated on ice for 20 min, and the precipitated protein was pelleted by centrifugation. The pellets containing the seco-steroid binding moiety, were solubilized in guanidinium/HCl, decanted into vials, liquid scintillation cocktail was added, and the amount of [3H]1,25-(OH)2D3 was assessed.

Although the perchloric acid method is suitable for hydrophobic membrane proteins (1,25D3-MARRS bp), it fails to detect the nVDR (15). Saturation analysis of [3H]1,25-(OH)2D3 binding was therefore assessed in crude preparations of nuclei by the use of a HAP assay as reported previously (17). Briefly, after incubation with different concentrations of [3H]1,25-(OH)2D3 overnight (see above), 200 µl HAP and 800 µl TED containing 0.5% Triton X-100 were added to each tube, and the solutions were mixed. The HAP-bound receptor was pelleted by centrifugation, washed twice with 800 µl TED containing 0.5% Triton X-100, treated with 1 ml ethanol to extract ligand, and centrifuged. The resulting supernatant was transferred to glass scintillation vials, the ethanol was evaporated, and the amount [3H]1,25-(OH)2D3 was measured after the addition of fluor.

SDS-PAGE and Western analysis
SDS-PAGE and Western blot analysis were used to determine immunoreactive levels of 1,25D3-MARRS bp. Proteins (15 µg/well; Ref. 18) were separated on an 8% SDS-PAGE gel with a 5% stacking gel. After separation on SDS-PAGE, proteins were transferred to a polyvinylidene difluoride membrane by the use of a Trans-Blot SD Semidry transfer cell (Bio-Rad Laboratories, Inc.), and Western analyses were performed according to the Millipore Corp. protocol as follows. To avoid nonspecific binding, the membrane was soaked for 1 h at 37 C in blocking solution [0.5% nonfat dry milk in PBS (0.9% NaCl and 10 mM Na2HPO4, pH 7.4], followed by washing three times for 5 min each time with washing solution [0.1% (wt/vol) BSA in Tris-buffered saline (TBS; 0.9% NaCl in 20 mM Tris-HCl, pH 7.4)], and incubation with primary antibody Ab099 (rabbit anti-1,25D3-MARRS bp N-terminal peptide; dilution, 1:5000 in 1% BSA and 0.05% Tween 20 in TBS) (18) overnight at 4 C. After three additional washes, the membrane was incubated with secondary antibody (alkaline phosphatase-conjugated goat antirabbit IgG) in 1% BSA and 0.05% Tween 20 in TBS for 2 h at room temperature and then washed as before. Immunoreactive bands were visualized with the chromogens 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium, and relative amounts of 1,25D3-MARRS bp were quantitated by densitometry with an Alpha Imager 2200 using the Documentation and Analysis System (version 5.5, Alpha Innotech Corp., San Leandro, CA).

Determination of PKC and PKA activities and protein
Intestinal epithelial cells from two male chicken duodena per experiment (n = 3 per age group) were isolated by citrate chelation as reported previously (19). After removing the duodenum, chilling it for 15 min in saline, and excising the pancreas, the duodenum was everted and rinsed in saline, and the intestinal epithelial cells were isolated by chelation. The isolated cells were combined and centrifuged at 500 x g at 4 C for 5 min, and the resulting pellet was resuspended in 20 ml GBSS-BSA [119 mM NaCl, 4.96 mM KCl, 0.22 mM KH2PO4, 0.84 mM Na2HPO4, 1.03 mM MgCl2, 0.28 mM MgSO4, 0.9 mM CaCl2 (pH 7.4), and 0.125% BSA]. Cells were treated at room temperature with either ethanol or 130 pM 1,25-(OH)2D3 at time zero. For PKC, cells were incubated for 5 min; for PKA, the incubation lasted for 7 min (20). After incubation, the cells were centrifuged at 1000 x g at 4 C for 10 min, the supernatant was decanted, and the pellet was stored at -20 C until used.

PKC and PKA activities were analyzed using commercially available assay systems (Invitrogen, Carlsbad, CA). For PKC activity, the pellets were extracted with buffer [20 mM Tris (pH 7.5), 0.5 mM EDTA. 0.5 mM EGTA, 0.5% Triton X-100, and 25 µg/ml each of aprotinin and leupeptin] by homogenization, followed by 2 min at 14,000 rpm in a microcentrifuge. Samples were adjusted to 10 µg protein/tube with buffer [20 mM Tris (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 10 mM ß-mercaptoethanol, and 200 mM NaCl] and preincubated in the presence of activator (10 µM phorbol 12-myristate 13-acetate, 2.8 mg/ml phosphatidyl serine, and Triton X-100 mixed micelles) or inhibitor [20 µM PKC-(19–36)] for 20 min at 23 C before the addition of substrate solution [25 µM acetylated myelin basic protein-(4–14), [{gamma}-32P]ATP (20–25 µCi/ml), 20 µM ATP, 1 mM CaCl2, 20 mM MgCl2, and 20 mM Tris, pH 7.5] for 5 min at 30 C. Aliquots of the incubation mixtures were spotted onto phosphocellulose disks; the disks were washed and placed in scintillation vials for assessment of incorporated radioactivity. For PKA activity, proteins (5 µg) from the supernatant fractions (extracted by homogenization with 5 mM EDTA and 50 mM Tris, pH 7.5, and centrifugation as described above) were incubated at room temperature for 20 min in the presence of activator (10 µM cAMP) or inhibitor [1 µM protein kinase inhibitor-(6–22) amide] only, in the presence of both activator and inhibitor, and in the absence of both. A synthetic heptapeptide substrate in solution (50 µM Kemptide, 100 µM ATP, 10 mM MgCl2, 1 mg/ml BSA, and 20–25 µCi/ml [{gamma}-32P]ATP) was added to each tube, the mixture was incubated for 5 min at 30 C, and 20 µl of the mixture were spotted onto a phosphocellulose disc. The discs were washed and transferred into scintillation vials, 32P was assessed, and PKA activity was calculated. Protein concentrations were determined using the Bradford dye according to the manufacturer’s instructions (Bio-Rad Laboratories, Inc.).

Statistics and data analysis
The specific binding of [3H]1,25-(OH)2D3 to receptor was calculated and plotted against the corresponding concentration of [3H]1,25-(OH)2D3 The data were analyzed by nonlinear regression analysis by fitting to either a three-parameter sigmoid equation or a hyperbolic function. The sigmoid equation was as follows:

(1)
where Y is specifically bound [3H]1,25-(OH)2D3 (femtomoles per milligram of protein), x is the nanomolar concentration of 1,25-(OH)2D3 in the incubation mixture, a is the maximum specifically bound [3H]1,25-(OH)2D3 (Bmax), b is the minimum specifically bound [3H]1,25-(OH)2D3, and x0 is the concentration of 1,25-(OH)2D3 in the incubation mixture at half-maximum bound [3H]1,25-(OH)2D3 (Kd). For the hyperbolic function, the equation was as follows:

(2)
where Y is specifically bound [3H]1,25-(OH)2D3 (femtomoles per milligram of protein), x is the incubation nanomolar concentration of 1,25-(OH)2D3 in the incubation mixture, a is the maximum specifically bound [3H]1,25-(OH)2D3 (Bmax), and b is the concentration of 1,25-(OH)2D3 in the incubation mixture at half-maximum bound [3H]1,25-(OH)2D3 (Kd).

Data from Hill analysis, perfusion studies, and SDS-PAGE/Western blots were analyzed by linear regression. The coefficient of variation (R2) and the adjusted variation (adjR2) were used as a measure of how well the regression model describes the data. A one-way ANOVA with F statistics was used to gauge the contribution of the independent variable to predict the dependent variable. The significance level was set at P < 0.05, and data are presented as the mean ± SEM.

When comparing Bmax, Kd, Hill coefficients, and changes in calcium transport over time within and between different age groups, an unpaired t test was used. When variables were used in more than one comparison, a sequentially rejective Bonferroni test was used (21). Thus, for comparison of three groups, significance was obtained at 0.05/3 = 0.016. In the PKC and PKA measurements, statistical comparisons were performed using one-way ANOVA, followed by Student-Newman-Keuls post hoc test when comparing a factor with more than two levels. The tests used were two-tailed, and the significance level was set at P < 0.05. Data are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of age on intestinal calcium transport
Male chickens of 7, 14, 28, and 58 wk of age with average weights (mean ± SEM) of 0.56 ± 0.02, 0.96 ± 0.03, 1.28 ± 0.03, and 2.53 ± 0.05 kg, respectively, were used. As shown in Fig. 1Go, A–D, vascular perfusion with 1,25-(OH)2D3 resulted in a linear increase in intestinal calcium transport for all ages tested. Calcium transport in duodena perfused with steroid hormone was significantly (P < 0.0125) higher than that in vehicle controls for all ages. A significant (P < 0.0125) age-dependent decrease in stimulated calcium transport was also observed when comparing slope coefficients for the different ages (Fig. 1Go, A–D). At 40 min, stimulated calcium transport in duodena from 7-, 14-, 28-, and 58-wk-old birds went from 240% of the control value to 190%, 180%, and 150%, respectively. In an additional series of perfusions with 58-wk-old birds, 650 pM 1,25-(OH)2D3 elicited an equivalent increase of 150% over vehicle controls (data not shown).



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  		Figure 1. Effects of age on the rapid stimulation of intestinal calcium transport by 1,25-(OH)2D3. Isolated duodena of male chickens 7 (A), 14 (B), 28 (C), and 58 (D) wk of age were vascularly perfused with the vehicle ethanol (0.005%, final concentration) during the basal phase and again in the treated phase for controls ({circ}) or with 130 pM 1,25-(OH)2D3 during the treated phase ({bullet}). Values represent 45Ca in the venous effluent during the treatment period normalized to the corresponding average basal transport determined at -10 to 0 min. Data were analyzed by linear regression, and the slopes for 1,25-(OH)2D3-treated (S1,25) and vehicle-treated (Sc) duodena were calculated. Significant differences in the slope coefficients between S1,25 and Sc within each age group and between S1,25 in different age groups were determined by one-way ANOVA, followed by t test (P < 0.0125). All age groups showed a significant (P < 0.0125) increase in calcium transport after treatment with 1,25-(OH)2D3 compared with control treatment. Lowercase letters indicate significant differences (P < 0.0125) when comparing S1,25 and Sc between age groups. Data are expressed as the mean ± SEM (n = 3–5).

 
Saturation analysis of [3H]1,25-(OH)2D3 binding to nVDR
Figure 2Go, A–D, illustrates the results of specific binding activity as a function of increasing concentrations of ligand in nuclear fractions prepared from intestinal mucosa of 7-, 14-, 28-, and 58-wk-old male birds. At 1 nM [3H]1,25-(OH)2D3 average values (total and nonspecific binding, respectively) for the age groups were 2880 and 316, 7920 and 4420, 7820 and 5420, and 2330 and 710 cpm/µg protein. The higher values for both total and nonspecific binding in crude nuclei from intestinal epithelium of 14- and 28-wk-old birds reflect the use of different protein levels. However, specific binding remained linear over this wide range of protein concentrations (Nemere, I., unpublished observations).



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  		Figure 2. Saturation analysis of 1,25-(OH)2D3 binding to nVDR. Crude nuclear fractions enriched in nVDR isolated from male chickens 7 (A), 14 (B), 28 (C), and 58 (D) wk of age were incubated with 0.25, 0.5, 1.0, or 2.0 nM [3H]1,25-(OH)2D3 in the absence or presence of a 200-fold molar excess of unlabeled steroid. Bound radioactivity was assessed by the HAP assay. Data were fitted to a three-parameter hyperbolic function and tested by nonlinear regression. No significant differences in Bmax or Kd with increasing age were observed when determined by one-way ANOVA, followed by t test. P < 0.016 was considered significant. Data are presented as the mean ± SEM for three independent preparations in each age group.

 
Specific binding data obtained in the analyses were fitted to a hyperbolic function (P < 0.05 for all ages; 7 wk: F = 84.5; df = 11; R2 = 0.89; adjR2 = 0.88; 14 wk: F = 62.1; df = 9; R2 = 0.89; adjR2 = 0.87; 28 wk: F = 39.7; df = 11; R2 = 0.80; adjR2 = 0.78; 58 wk: F = 110.1; df =7; R2 = 0.95; adjR2 = 0.94). The Bmax values for 7-, 14-, 28-, and 58-wk-old birds were 31.1 ± 3.9, 36.4 ± 5.2, 31.0 ± 5.1, and 22.7 ± 2.6 fmol/mg protein, respectively. In the same age groups, specific binding became half-saturable at Kd values of 0.35 ± 0.12, 0.38 ± 0.17, 0.29 ± 0.18, and 0.30 ± 0.11 nM, respectively. No significant differences in Bmax or Kd with increasing age were observed.

The data described in Fig. 2Go, A–D, were recalculated for Hill analysis, which yielded curves that could be described by a linear regression (P < 0.05 for all ages; 7 wk: F = 19.6; df = 11; R2 = 0.66; adjR2 = 0.63; 14 wk: F = 5.4; df = 7; R2 = 0.48; adjR2 = 0.39; 28 wk: F = 9.2; df = 9; R2 = 0.53; adjR2 = 0.48; 58 wk: F = 35.7; df =7; R2 = 0.86; adjR2 = 0.83). The resultant apparent Hill coefficients (napp) were 1.02 ± 0.23, 0.78 ± 0.33, 0.97 ± 0.32, and 1.49 ± 0.25 for 7-, 14-, 28-, and 58-wk-old birds, respectively (Fig. 3Go, A–D). No significant differences in napp were observed for the different ages. Taken together, both the hyperbolic distribution and the slope in the Hill analysis indicate the absence of cooperative binding. With regard to maximal binding and affinity of nVDR for 1,25-(OH)2D3, no changes were observed with increasing age.



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  		Figure 3. Hill analysis of 1,25-(OH)2D3 binding to nVDR. Data presented in Fig. 2Go, A–D, were recalculated for Hill analysis. Seven- (A), 14- (B), 28- (C), and 58- (D) wk-old birds were studied. [Sb], Specifically bound fraction of the administered total free ([Sf]) [3H]1,25-(OH)2D3. No significant differences in napp were observed with increasing age when compared by one-way ANOVA, followed by t test. An napp equal to 1 shows the presence of noncooperative binding for 1,25-(OH)2D3 to nVDR. Data are presented as the mean ± SEM (n = 3/tested [3H]1,25-(OH)2D3 concentration). P < 0.016 was considered significant.

 
Saturation analysis of 1,25-(OH)2D3 binding to 1,25D3-MARRS bp and Western analysis
Figure 4Go, A–D, shows specific binding activity as a function of increasing concentrations of ligand in BLM fractions prepared from intestinal mucosa of 7-, 14-, 28-, and 58-wk-old male birds. At 1 nM [3H]1,25-(OH)2D3 average values (total and nonspecific binding, respectively) for the age groups were 12,150 and 10,930, 8,660 and 8,140, 7,900 and 7,460, and 7,720 and 7,670 cpm/50 µg protein. Curve-fitting analyses of the data resulted in a better fit to a sigmoid function compared with a hyperbolic function (P < 0.05 for all ages; 7 wk: F = 20.0; df = 24; R2 = 0.65; adjR2 = 0.61; 14 wk: F = 99.5; df = 14; R2 = 0.94; adjR2 = 0.93; 28 wk: F = 71.9; df = 14; R2 = 0.92; adjR2 = 0.91; 58 wk: F = 79.1; df = 14, R2 = 0.93; adjR2 = 0.92), indicating the presence of allosteric binding. Kd values were 0.59 ± 0.13, 1.03 ± 0.10, 0.99 ± 0.07, and 2.65 ± 0.84 nM for 7-, 14-, 28-, and 58-wk-old birds, respectively. A significant (P < 0.016) increase in Kd was observed between 7- and 14-wk-old birds. This decreased affinity was sustained through the subsequent ages tested. Bmax values for 7-, 14-, 28-, and 58-wk-old birds were 189 ± 18, 250 ± 13, 186 ± 11, and 640 ± 160 fmol/mg protein, respectively, although the last value cannot actually be used as a measure of binding sites, because saturation was not achieved at the concentrations tested (Fig. 4DGo). A statistically significant difference (P < 0.016) was observed between Bmax values for 7-, 14-, and 58-wk-old birds. The 28-wk-old birds showed a significant difference in Bmax compared with 14- and 58-wk-old birds, whereas there was no significant difference between 28- and 7-wk-old birds (Fig. 4Go, A–D).



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  		Figure 4. Saturation analysis of 1,25-(OH)2D3 binding to 1,25D3-MARRS bp. BLM isolated from male chickens 7 (A), 14 (B), 28 (C), and 58 (D) wk of age were incubated with 0.05, 1.0, 2.0, or 4.0 nM [3H]1,25-(OH)2D3 in the absence or presence of a 200-fold molar excess of unlabeled steroid. Bound radioactivity was assessed by precipitation with perchloric acid in the presence of carrier bovine {gamma}-globulin. Data were fitted to a three-parameter sigmoid function and tested by nonlinear regression. Significant differences in maximum binding capacity, Bmax, and Kd, with increasing age were determined by one-way ANOVA, followed by t test. Lowercase letters indicate significant differences (P < 0.016) between ages. Data are presented as the mean ± SEM (n = 3 independent preparations in each age group).

 
A recalculation of the data in Fig. 4Go, A–D, for Hill analysis resulted in curves that were described by linear regression (P < 0.05 for all ages; 7 wk: F = 8.08; df = 12; R2 = 0.24; adjR2 = 0.37; 14 wk: F = 52.9; df = 9; R2 = 0.87; adjR2 = 0.85; 28 wk: F = 23.3; df = 9; R2 = 0.74; adjR2 = 0.71; 58 wk: F = 113; df = 9; R2 = 0.93; adjR2 = 0.93). This analysis yielded Hill coefficients greater than 1 for all ages, indicating the presence of positive cooperativity in the binding of 1,25-(OH)2D3 to 1,25D3-MARRS bp. The napp were 1.22 ± 0.43, 2.11 ± 0.29, 2.47 ± 0.51, and 3.19 ± 0.30 for 7-, 14-, 28-, and 58-wk-old birds, respectively (Fig. 5Go, A–D). Statistical comparison of napp among the age groups revealed a significant (P < 0.016) difference between 7- and 58-wk-old birds and between 14- and 58-wk-old birds.



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  		Figure 5. Hill analysis of 1,25-(OH)2D3 binding to 1,25D3-MARRS bp. Data presented in Fig. 4Go, A–D, were recalculated for Hill analysis. Seven- (A), 14- (B), 28- (C), and 58- (D) wk-old birds were studied. [Sb], Specifically bound fraction of the administered total free ([Sf]) [3H]1,25-(OH)2D3. The napp were larger than 1 in all age groups, indicating positive cooperative binding for 1,25-(OH)2D3 to 1,25D3-MARRS bp. Lowercase letters indicate significant (P < 0.016) differences determined by one-way ANOVA, followed by t test. Data are presented as the mean ± SEM.

 
For each age group, three independent BLM preparations were subjected to Western blotting with Ab099 for analyses of 1,25D3-MARRS bp levels. Representative blots are shown in Fig. 6AGo. Multiple blots were subjected to densitometric analyses for quantitation. Linear regression analysis of the data obtained revealed a significant (P < 0.05) decrease in the expression of 1,25D3-MARRS bp with increasing age (Fig. 6BGo).



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  		Figure 6. Expression of 1,25D3-MARRS bp in BLM. The expression of 1,25D3-MARRS bp in BLM isolated from duodenal epithelium of male chickens aged 7, 14, 28, and 58 wk was studied by separation of proteins on 8% SDS-PAGE, followed by Western blot analysis. For Western analysis, Ab099 (rabbit anti-1,25D3-MARRS bp N-terminal peptide) was used as primary antibody, and alkaline phosphatase-conjugated goat antirabbit IgG was used as secondary antibody. Immunoreactive bands were visualized with the chromogens 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (A), and relative amounts of 1,25D3-MARRS bp were quantitated using densitometry and computer software (B). A linear regression analysis of the data revealed a significant (P < 0.05) decrease in 1,25D3-MARRS bp expression with increasing age. Data are presented as the mean ± SEM (n = 8–12) from four separate blots, each containing samples from all age groups.

 
Thus, the results demonstrate a decrease in both the expression of 1,25D3-MARRS bp and the affinity of the receptor with increasing age, and show the presence of positive cooperativity in the binding of 1,25-(OH)2D3 to 1,25D3-MARRS bp.

Effects of age on 1,25-(OH)2D3-induced PKC and PKA activities
Despite the significant changes in 1,25D3-MARRS bp affinity and levels, it was recognized that differences in intestinal responsiveness to steroid might be due to pleiotropic changes at the cellular level between growing and mature animals. Thus, the effects of 130 pM 1,25-(OH)2D3 on PKC and PKA activities in isolated intestinal cells from male chickens of different ages were tested.

Figure 7Go illustrates the results of PKC activity determinations in parallel incubations of control and hormone-treated cells from 7-, 14-, 28-, and 58-wk-old birds. Extracts from all age groups were assayed concomitantly to validate comparisons of basal (control) levels. As shown in Fig. 7Go, PKC activity was affected by age in both vehicle-treated (control) and 1,25-(OH)2D3-treated cells. PKC activity in vehicle-treated enterocytes exhibited an age-related decrease, yielding values of 138 ± 9.9, 52.9 ± 5.9, 43.0 ± 8.4, and 19.6 ± 2.9 pmol/mg protein·min (n = 3–14) for 7-, 14-, 28-, and 58-wk-old birds, respectively. Statistical analyses indicated a significant (P < 0.05) decrease in basal values between 7-wk-old animals and increasing ages (Fig. 7Go). PKC activity after treatment with 1,25-(OH)2D3 was 205 ± 12, 51.3 ± 9.2, 55.1 ± 6.4, and 6.7 ± 1.7 pmol/mg protein·min (n = 4–14), respectively, for the tested ages. Upon comparison of treatment and corresponding controls within each age group, a significant (P < 0.05) increase in activity was observed after treatment with hormone in isolated cells from 7-wk-old animals. There were no changes in PKC activity in similar preparations from 14 and 28-wk-old animals, whereas enterocytes from 58-wk-old birds showed a significant (P < 0.05) decrease in PKC activity after hormone treatment (Fig. 7Go).



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  		Figure 7. Age-related changes in PKC activity. Intestinal epithelial cells from 7-, 14-, 28-, and 58-wk-old male chickens were isolated and treated with either 130 pM 1,25-(OH)2D3 (D3) or ethanol (C) for 5 min. The cells were collected by centrifugation and extracted, and the supernatant fractions were analyzed for PKC activity. Significant differences between D3 and C within each age group (*) and differences between C for the different age groups (lowercase letters) were determined by one-way ANOVA, followed by Student-Newman-Keuls post hoc test (P < 0.05). Data are presented as the mean ± SEM (n = 3–14).

 
The lack of PKC stimulation was further tested in the perfusion system. Vascular perfusion of duodena from 58-wk-old birds with 100 nM phorbol ester (n = 2) failed to increase transport above levels observed in vehicle controls (0.125% dimethylsulfoxide, final concentration; n = 2; data not shown).

The results of analyses of PKA activity in vehicle- and 1,25-(OH)2D3-treated cells from 7-, 14-, 28-, and 58-wk-old male chickens are shown in Fig. 8Go. The activities in vehicle-treated cells from 7-, 14-, 28-, and 58-wk-old birds were 26 ± 2.5%, 31.3 ± 8.5%, 29.4 ± 0.3%, and 21.2 ± 8.2% activated PKA/mg protein (n = 3–6), respectively, and those for 1,25-(OH)2D3-treated cells in the same age groups were 54.9 ± 6.3%, 49.5 ± 4.7%, 57.5 ± 12%, and 57.6 ± 2.2% activated PKA/mg protein (n = 3–5), respectively (Fig. 8Go). No significant effect of age was observed in PKA activity when comparing vehicle-treated cells. A significant (P < 0.05) 1,25-(OH)2D3-stimulated increase in PKA activity, however, was found for 7- and 58-wk-old birds relative to corresponding controls.



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  		Figure 8. Age-related changes in PKA activity. Intestinal epithelial cells from 7-, 14-, 28-, and 58-wk-old male chickens were isolated and treated with either 130 pM 1,25-(OH)2D3 (D3) or ethanol (C) for 7 min, the cells were harvested by centrifugation and extracted, and the supernatant fractions were analyzed for PKA activity. Significant differences between D3 and C within each age group (*) and differences between C for the different age groups (lowercase letters) were determined by one-way ANOVA, followed by Student-Newman-Keuls post hoc test (P < 0.05). Data are presented as the mean ± SEM (n = 3–14).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results indicate that the rapid stimulation of intestinal calcium transport by 1,25-(OH)2D3 is of the greatest magnitude in young, growing chicks. The magnitude of the response drops gradually at 14 and 28 wk of age until it is approximately 60% of initial values at 58 wk of age. These findings agree well with a physiologically important role for 1,25-(OH)2D3 MISS in enhancing intestinal calcium transport for the mineralization of new bone formed in growing animals. Although this response is diminished in adults, it is not abolished, suggesting that the rapid uptake of dietary calcium continues to benefit the animal.

Two readily apparent explanations for such diminished responsiveness are altered receptor binding parameters and/or signal transduction. In our studies of the nVDR, no significant changes in affinity for hormone were detected in the age groups studied. Moreover, no alterations in the number of binding sites were detected between 7- and 28-wk-old birds, whereas a decline in responsiveness was observed. An apparent decrease in the number of hormone-binding sites was observed at 58 wk, although the decline was not statistically significant. It is conceivable that in older age groups, diminished levels of nVDR occur (7, 8, 9, 10), although in other studies this was not the case (6, 22).

In the current work it is likely that the nVDR remained functionally active in the age groups studied, as there was no detectable loss of basal PKA activity. Adenylate cyclase, which produces cAMP to activate PKA, apparently requires the nuclear actions of 1,25-(OH)2D3 for expression, because it is diminished in vitamin D-deficient animals (23, 24). Thus, the transcriptional effects of 1,25-(OH)2D3 provide a biochemically competent cell, in this case by inducing the expression of the adenylate cyclase enzyme, which, in turn, allows its rapid stimulation by membrane-initiated actions.

The lack of correlation between nVDR levels and calcium transport indicated by our results is in agreement with the recent report by Fleet et al. (25). In contrast, Erben et al. (26) argued that only the nVDR is necessary for both nuclear and membrane-initiated effects. These workers have reported studies of mice engineered to contain an nVDR lacking the DNA-binding domain, but retaining the ligand-binding domain, which is phenotypically equivalent to VDR-/- mice (26). However, the researchers did not consider the earlier observations that the nuclear actions of 1,25-(OH)2D3 are required to induce components of the calcium transport pathway (14), which, in turn, allow ligand binding to the 1,25D3-MARRS bp to produce an effect.

Transformation of data obtained in saturation binding studies indicated major differences between the nVDR and the 1,25D3-MARRS bp with respect to affinity, number of binding sites, and cooperativity. The 1,25D3-MARRS bp exhibited positive cooperativity, as has been found in other systems (27, 28) as well as for other steroids binding to their cognate membrane receptors (29, 30, 31, 32, 33). Our data indicate that the nVDR and 1,25D3-MARRS bp are biochemically distinct entities or separate proteins. However, we currently cannot determine whether the positive cooperativity of the 1,25D3-MARRS bp is due to homodimerization/oligomerization or interaction with other proteins.

Analyses of 1,25D3-MARRS bp binding parameters with age revealed a decrease in affinity that paralleled decreased intestinal responsiveness, as judged by calcium transport. Calculated values of Bmax remained fairly constant between 7–28 wk, whereas saturation was not observed in BLM from 58-wk-old birds. Western analyses indicated a gradual, but steady, decline in the level of 1,25D3-MARRS bp with age. The decrease in the high affinity binding component most likely allows ligand to interact with high capacity, low affinity moieties, leading to a lack of saturation. Two additional points must be taken into consideration in gauging the contributions of these binding parameters: although circulating levels of 1,25-(OH)2D3 decline from 300 to 50 pM steroid in young vs. adult birds (34), the positive cooperativity of binding by the 1,25D3-MARRS bp increased with age in our studies. Thus, a number of maturation-related changes contribute to the binding of 1,25-(OH)2D3 and its efficacy in eliciting a physiological response. With respect to membrane-initiated calcium transport, an inability of relatively high levels of steroid hormone in the vascular perfusate to overcome the maturation-related decrease in affinity of the 1,25D3-MARRS bp most likely indicates the existence of either an additional age-sensitive binding partner or a component of a signal transduction pathway.

A dramatic age-induced alteration in PKC activity was observed in chick intestinal epithelial cells. To our knowledge, this is the first report of decreasing basal values of PKC as a function of age. In rat duodenum, Balogh et al. (13) reported an increase in basal PKC activity with age. These divergent observations may be related to species differences. In rats, the same group of workers (12, 13) also noted an age-related decline in 1,25-(OH)2D3 stimulation of PKC activity as well as a change in subcellular distribution of PKC isozymes (35).

In the present investigation the observation that basal PKC activity declines with age may be related to the cessation of growth. Earlier studies have reported that GH is permissive for 1,25-(OH)2D3-stimulated mineral transport in the intestine (36, 37). Thus, declining levels of GH may be responsible for the decreased PKC levels that are involved in 1,25-(OH)2D3-mediated transport.

By comparison, the decreased level of steroid hormone-enhanced PKC activity with maturation may in part be related to the parallel decrease in affinity of the 1,25D3-MARRS bp for ligand. We previously reported (18) that pretreatment of cells with Ab099 directed against the 1,25D3-MARRS bp prevents the secosteroid-mediated activation of PKC, whereas equivalent incubations with antibody against the nVDR did not. However, the finding that not even phorbol ester can stimulate transport in older birds indicates a fundamental change in PKC. In addition, this pathway represents an auxiliary means of stimulating calcium transport, rather than the primary signal transduction route.

The activation of PKA has also been implicated in the stimulation of intestinal ion transport (20, 38). However, our current observations in age groups encompassing growth and adulthood indicated no decline in 1,25-(OH)2D3 stimulation of PKA activity. This signal transduction pathway may contribute to the continued existence of a rapid response to 1,25-(OH)2D3 in mature birds, whereas the additional stimulation of PKC activity in young birds may account for a more robust transport response. In addition, the persistence of PKA activation by hormone even at 58 wk suggests that this pathway is regulated differently by 1,25-(OH)2D3 than the PKC pathway. The nature of this difference in the interaction with the 1,25D3-MARRS bp remains to be determined. In contrast to our findings, studies in rats revealed an age-related decline in PKA activity (11). Such interspecies variations, in addition to the multiple maturation-related changes reported in the current work, indicate the pleiotropic effects of the aging process.

The combined data indicate that the rapid MISS response to 1,25-(OH)2D3 in the intestine plays a physiologically important role in growing animals by boosting intestinal mineral absorption to meet the demands of bone formation. As such, the relevant pathways may provide new therapeutic targets for the correction of calcium and phosphate malabsorption syndromes.


   Footnotes
 
This work was supported by the Utah Agricultural Experiment Station, Utah State University (Logan, UT). Approved as Journal Paper 7528. In addition we acknowledge support from a CURI grant (to I.N.) and the Sweden America Foundation (to B.L.). Portions of these results were presented by B.L. in partial fulfillment for the degree of Doctor of Philosophy.

Abbreviations: adjR2, Adjusted coefficient of variance; BLM, basal lateral membrane; Bmax, binding capacity; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; 1,25D3-MARRS bp, membrane-associated rapid response binding protein; GBSS, Gey’s balanced salt solution; HAP, hydroxylapatite; MISS, membrane-initiated steroid signaling; napp, apparent Hill coefficient; NISS, nuclear-initiated steroid signal transduction; nVDR, nuclear vitamin D receptor; PKA, protein kinase A; PKC, protein kinase C; TBS, Tris-buffered saline.

Received October 25, 2002.

Accepted for publication January 8, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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