The effect of age and 1,25-dihydroxyvitamin D3 treatment on the intestinal calcium-binding protein of suckling rats

ARCHIVES OF BIOCHEMISTRY ANDBIOPHYSICS

Vol. 196, No. 2, September, pp. 624-630, 1979

The Effect of Age and 1,25Dihydroxyvitamin D, Treatment on the Intestinal Calcium-Binding Protein of Suckling Rats1 TZUU-HUE1 Department

of Oral Biology,

UENG,* ELLIS E. GOLUB,3 AND FELIX The University

of Connecticut Health

BRONNER4

Center, Farmington,

Connecticut

06052

Received February 22, 1979; revised May 1, 1979 The intestinal level of the vitamin D-dependent duodenal calcium-binding protein was assayed by an equilibrated column technique in rat embryos, neonates, and pups. Calcium-binding protein was undetectable in unborn, newborn, and l- to e-day-old rats, i.e., the level was lower than in severely vitamin D-deficient animals. Calcium-binding protein was detected after the animals were 5-days old and thereafter rose monotonically as a function of body weight. Treatment with 1,25-dihydroxyvitamin D, failed to raise the calcium-binding protein levels of newborn or l-day-old rats, but doubled the level in ll- or 12-day-old pups. Plasma calcium was raised in all treated animals. The failure to detect calcium-binding protein in vitamin D-replete suckling animals provides evidence for a dissociation between calcium absorption and calcium binding protein.

The vitamin D-dependent CaBP5 found in intestinal cells (1, 2) and mucosal scrapings (3-7) is the best-known molecular expression of the hormone-like action of vitamin D (8, 9). It is a small (M, = g-12,000), fairly acidic protein with two calcium-binding sites, a pI of 4.7, and an apparent calCiUIn ZLSSOCiatiOn COrEkant Of IO6 t0 lo’/M (10). Since CaBP can be readily isolated from the 100,OOOgsupernate (5, 6), it is, operationally speaking, a cytosolic protein, though its actual cellular location is still in dispute (10, 11). The amount of CaBP in the intestine appears to be proportional (12) to the amount of circulating 1,25-(OH),-D,, a vitamin D metabolite that is produced in the kidney

and plays a major role in the hormone-like action of vitamin D (13). However, it is not clear whether the presence of circulating 1,25-(OH),-D, will always ensue in the biosynthesis of CaBP, or whether circumstances exist when injection of the metabolite into the circulation will fail to stimulate CaBP biosynthesis. In this study we have examined the effect of age and 1,25-(OH),-D, treatment on intestinal CaBP in young rats. As will be shown, rats do not have detectable CaBP levels in their intestines until they are about 5 days old and until that age do not respond to treatment with 1,25-(OH),-D, with an increase in their CaBP levels. MATERIALS

’ Supported by Grant AM14251, NIAMDD, National Institutes of Health, and the University of Connecticut Research Foundation. * Present address: Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Md. 20014. 3 Present address: Department of Biochemistry, School of Dentistry, University of Pennsylvania, Philadelphia, Pa. 19104. 4 To whom inquiries should be addressed. 5 Abbreviations used: CaBP, vitamin D-dependent duodenal calcium-binding protein; 1,25-(OH),-D,, 1,25dihydroxyvitamin D,. 0003.9861/79/100624-07$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND METHODS

Animals. Sprague-Dawley rats, purchased from ARS, Madison, Wisconsin were used. Pregnancy was established by vaginal smears and the age of the unborn litters was deduced from the date of impregnation. Body weights were found to be a positive function of chronological age (Fig. 1). Since the precise age of a litter was not always known, body weight rather than age was used as a parameter for CaBP levels. The dams were kept on a commercial chow. The pups were allowed to nurse and became weaned at about 21 days, but animals 18 days or older may also have eaten chow in addition to nursing. Plasma samples 624

VITAMIN

D AND INTESTINAL

Ca-BINDING PROTEIN IN SUCKLING RATS

625

RESULTS 0 6o

Figure 2 shows the elution profiles obtained by determining the radioactive cont tent of the eluted fractions. The baseline ii 20represents the calcium content of the buffer P __:i_ (in terms of cpm/ml). The first peak correoJ 20 0 IO sponds to the void volume peak, i.e., calAGE, DAYS cium binders of a size excluded by the gel. FIG. 1. Body weight as a function of age in unborn The second peak of this chromatogram, and suckling rats. designated as peak B (VJV, = 1.4), contains the CaBP ((5); Fig. 2B), as there was no such peak in a similar elution chromatowere obtained by sampling blood from the tail vein with gram of material from vitamin D-deficient the aid of heparinized capillary tubes. In the case of rats (Fig. 2C). Gel electrophoresis (2) also neonates, blood was obtained following decapitation. Treatment with 1,.25-(OH)2-D,. Animals were han- failed to reveal CaBP. As evident from the dled with plastic gloves to minimize rejection by the protein content of the eluted material (Fig. 2A), most of the protein was found in the mother (14). 1,25-(OH),-D, was injected intraperi= 2.6 toneally. The metabolite, initially dissolved in ethanol, void volume peak. The peak at V$V, was further diluted in physiological saline solution. in the protein elution chromatogram (Fig. Control animals received the same volume of saline 2A) contains no protein (16) and showed no only. No adverse effects of the injection were noted. protein bands by polyacrylamide gel elec?E I? AO-

Approximately 16 h after dosing, blood samples were obtained, the pups killed by decapitation or cervical dislocation, and their proximal intestines rapidly removed and processed. Estimation of duodenal CaBP content. CaBP was isolated from mucosal scrapings, essentially as previously described (2, 5, 6). To obtain partially purified CaBP, the freeze-dried initial supernate (S-100) was dissolved in 0.02 M ammonium acetate, pH ‘7.2, containing 7 pM CaCl, (11,000 cpm YWnmol) and its protein content determined. If the sample contained <5 mg protein, it was chromatographed by descending chromatography (flow rate: 6 ml/h) on a Sephadex G-50 (Fine) column (0.65 x 100 cm). If the sample contained 20-30 mg protein, it was chromatographed by ascending chromatography (flow rate: 20 ml/h) on a larger (1.5 x 80 cm) Sephadex G-50 column. Both columns had been equilibrated with the Caz+-containing ammonia acetate solution. CaBP content is expressed as nanomoles Caboundper milligram S-100 protein. Calcium assays and protein determination. Calcium was determined with the aid of atomic absorptiometry. Plasma samples were diluted with 0.1% LaCl, and plasma standards contained appropriate amounts of added sodium (6). Protein content, as assayed spectrophotometrically (15), correlated well (r = 0.88) with the assay by the method of Lowry et al. (16). Measurement of radioactivity. Radioactivity measurements were carried out in a Nuclear-Chicago Unilux IIA counter. 45Cacontent of aqueous samples was determined with 4 ml Bray’s (17) scintillator. Counting efficiency was about 50%, determined with the aid of internal standards.

I

sb

Id0 FRACTION

Ii0

260

NUMBER

FIG. 2. Elution profiles of supernates from intestinal homogenates by the equilibrated column procedure. (A) Protein content (A280n,,,). (B) Calcium content of material from vitamin D-replete animals (+D). The arrow points to the fractions that contain CaBP (peak B, VJV, = 1.4). (C) Calcium content of material from vitamin D-deficient animals (-D). Notice the absence of peak B. Both groups of animals had been kept on a low-calcium diet (0.06% Ca, 0.2% P; (6)) from weaning and were killed about 5 weeks later. The +D group received 2200 IU vitamin D,/kg feed; the -D group received no vitamin D,.

626

UENG ET AL.

trophoresis. The trough in the two calcium elution chromatograms (Figs. 2B and C) represents, as pointed out by Hummel and Dreyer (18), calcium equivalent to that removed by the binders. The area of the trough equals that of the peaks within ? 10% (data not shown). The area of peak B will equal the amount of CaBP, provided the elution buffer calcium concentration exceeds the apparent dissociation constant of binder and this fraction contains no other calcium binding molecules. Counter ion electrophoresis (2) has shown that peak B is about 50% pure in terms of protein, but contains no calcium binders other than CaBP. The apparent dissociation constant of CaBP is about 0.7 x lo+ M (Golub and Bronner, unpublished). This value, obtained by the competitive binding assay with Chelex resin (5), is somewhat smaller than previously reported (5), but in good agreement with that reported by Bruns et al. (‘7). The calcium concentration of the equilibrated column buffer was chosen to be approximately lo-fold this value. Parallel analysis of CaBP from vitamin Dreplete and partially vitamin D-deficient rats by the competitive Chelex assay (5) and by evaluation of the peak B area led to

FIG. 4. The relationship between protein load and recovered calcium-binding activity of peak B (VJV, = 1.4, Fig. 2). (A) Recovery from a column, 0.65 x 100 cm, fraction size 0.2 ml, flow rate 6 ml/h. (B) Recovery from a column, 1.5 X 80 cm, fraction size 1.0 ml, flow rate 20 ml/h. Eighty to one hundred male SpragueDawley rats had been placed on D-replete semisynthetic regimens (6) that contained either 0.06% Ca, 0.2% P (low calcium) or 1.5% Ca, 1.5% P (high calcium). Material from peak B was isolated after the animals had been on the regimens 7 or more days. The slopes for the low calcium diets were 2.1 in both columns; the slope for the high calcium diet for column A was 0.83.

the data displayed in Fig. 3. There was excellent correlation (r = 0.94) between the two procedures. The positive intercept and a slope of less than 1 may have been caused by the inability to detect low levels of CaBP by the Chelex binding assay and by possible contamination of the peak containing CaBP with calcium binders from the void volume. To minimize the latter, columns with better separation between void volume and CaBP peaks were built (Fig. 2) and CaBP values were estimated from the sum of the activities in fractions containing 50% or more of the difference between baseline and maximum peak activity. To compare successive runs on the same chromatographic column, it is necessary either to load the same amount of protein or to recover material in proportion to the FIG. 3. Correlation of two assay methods of CaBP. load. Figure 4 shows that recovery was proValues plotted on the ordinate were obtained by es- portional to the load in both columns emtimating the area of peak B (VJV, = 1.4) in an eluployed in this study. Moreover, the slopes tion profile as shown in Fig. 2. Values obtained by for material from animals on low calcium parallel assays of the same material by the quantitative competitive binding assay with Chelex resin (5) are diets were the same for both columns and plotted on the abscissa. The regression line, calculated were therefore directly comparable. Figure 4 also shows that the recovery by the method of least squares, has a slope of 0.75 and an intercept of 8.7. The correlation coefficient is 0.94. slope of material from animals on a high cal-

VITAMIN

D AND INTESTINAL

Ca-BINDING

cium diet was flatter, i.e., the amount of CaBP recovered per milligram protein applied was less. Since the intestinal content of CaBP is lower in animals on high calcium diets than in animals on low calcium diets (2, 5, 12, 19), the flatter slope obtained with material from animals on high as compared with material from animals on low calcium diets indicates that only CaBP is detected under these conditions and that the presence of other proteins does not lead to erroneous results. This conclusion was also tested by preparing mixtures of S-100 protein containing varying proportions of CaBP. The amount of calcium-binding activity recovered was directly proportional to the known CaBP content (Ueng and Bronner, unpublished). The mean standard error of duplicate column runs (n = 10) was 8%. Figure 5 shows that in the rat duodenal CaBP is not detectable until after the animal has reached a body weight beyond 10 g. According to Fig. 1, this corresponds to about 5 days of age. Thereafter, CaBP increases monotonically with body weight until weaning (Fig. 5). In weaned animals, CaBP levels rise slowly for another 4-6 weeks

PROTEIN IN SUCKLING

0

10

20

30

627

RATS

40

50

60

BODY WEIGHT, 0

FIG. 5. CaBP content of proximal intestine as a funetion of body weight in unborn and suckling rats.

reaching a maximum value of approximately 2.0 (Buckley, Golub, and Bronner, unpublished). They then decline with age, increasing body weight notwithstanding (7, 20). Table I indicates that treatment with 1,25-(OH)2-D3 did not lead to increased CaBP levels in the neonates, but did increase CaBP levels in ll- and 12-day-old rats. Even though treatment failed to increase CaBP in the neonates, plasma cal-

TABLE I EFFECT OF TREATMENT WITH 1,25-(OH)s-D3a Body weight 63)

(days)

7.1

0

7.6 Ed' 26.4 (0.5) 26.6 (0.3) 94.6 (1.9)

Age

1 11

WI 43

1,25-(OH),-D, w

No. animals

Vehicle only 0.125 Vehicle only 0.125 Vehicle only 0.375 Vehicle only 0.375 Vehicle only 0.500

11 14 7 7 5 5 5 6 2 2

Kw (mddl) 11.46 (0.08) 12.11 (0.22)

10.48 (0.16) 11.70 (0.18) 4.92 (0.18) 6.45 (0.14)

CaBP nmol Cab,,,,Jmg S-106 protein Undetectabled Undetectable Undetectable Undetectable 0.48 0.93 0.55 0.90 0.23 1.65

a All injections were administered intraperitoneally 16 h before sacrifice. Animals were littermates. All animals except the 43-day-old ones were being suckled by vitamin D-replete mothers. The 43-day-old group was on a vitamin D-deficient diet from weaning. * Plasma calcium [CaJ values of treated animals are italicized and differ statistically (P < 0.05) from those of their respective controls. c Numbers in parentheses are standard errors of mean. d “Undetectable” means < 0.1 nmol CaJmg S-100 protein. e Numbers in brackets estimated from Fig. 1.

628

UENG ET AL.

cium increased significantly (P < 0.05). Table I also shows that the CaBP level of 6week-old vitamin D-deficient animals was higher than that of the vitamin D-replete neonates and that the deficient animals responded to 1,25-(OH),-D, treatment by attaining much higher CaBP levels than the replete pups. DISCUSSION

The apparent absence of CaBP in unborn and particularly in neonatal rats was a striking and unexpected finding. It may therefore be useful to analyze the sensitivity of the method employed here. The lower limit of detection is about 0.1 nmol Cabound6or 0.05 nmol CaBP, since 1 mol CaBP binds 2 mol calcium (5, 10). This lower limit is somewhat below the value found in severely vitamin D-deficient rats (Table I). With the youngest animals studied here, tissue pools were prepared from five or more animals, yielding samples containing at least l-2 mg S-100 protein. The maximum load was 3-4 mg protein for the smaller and 30-40 mg for the larger columns. In rats over 21 days of age, mucosal scrapings readily yield 20 mg S-100 protein per animal. The equilibrated column procedure is therefore adequate for physiological or nutritional studies involving single animals or cells isolated from such animals (21, 22). The absence of CaBP in rats under 5 days of age raises an important question concerning the function of this molecule, as this is the first reported instance of the absence of CaBP in animals with normal calcium absorption. Calcium absorption was not determined experimentally in the present study, but newborn rats begin suckling within 24 h of birth and continue gaining weight (Fig. 1). Moreover, the specific activity of bone calcium in suckling rats has been shown to be the same as that of the milk of their mothers who had received the radioisotope 6 A typical baseline value is 14,433 ? 27 (SE) cpm/O.2 ml, and the specific activity in that case might be 8000 cpm/nmol Ca. A typical peak might comprise 12 tubes. One-tenth nanomole Ca is equivalent to 800 cpm or 67 cpm/tube (0.2 ml) above the baseline, a count 2.5-fold that of the SE of the baseline.

many weeks before they gave birth (23). A separation between CaBP and calcium absorption has also been reported by Spencer et ~2. (24, 25) and Morrissey et al. (26), who have shown that in the vitamin D-deficient chick, vitamin D administration stimulated calcium absorption before synthesis of CaBP was detected. Edelstein and colleagues (27,28) have reported that they were unable to detect 1,25(OH),-D, in unborn or neonatal rats whose mothers were given a single dose of 1,25(OH),-D,. The same investigators detected only very small quantities when the metabolite was administered continuously to the pregnant mothers. Noff and Edelstein (28) also reported that most of the small amount of 1,25-(OH)z-D, found in the pup was in the form of an ester that is presumed not to be biologically active. These investigators therefore concluded that fetuses or suckling rats do not require 1,25-(OH),-Da. The data reported here would confirm this conclusion, at least as regards the molecular expression of this metabolite in neonatal intestine. Noff and Edelstein (28) also suggest that esterification of 1,25-(OH),-D, protects rat pups against metabolite-induced hypercalcemia and Weisman et al. (29) have proposed that infantile hypercalcemia is a defect in the esterification of the dihydroxyvitamin DS. Our data (Table I) indicate that when neonatal rats were treated with 1,25(OH&D3, their blood calcium rose significantly. Moreover, the degree of hypercalcemia was similar to what had been predicted from other experiments (Eq. 1 in Ref. (6)). This suggests that enough of the metabolite reached some target cells to produce a physiological response and makes all the more significant the failure of the intestinal cells to produce CaBP. Since CaBP was found in rats 5 days of age or older (Fig. 5) and since treatment with 1,25-(OH),-D, of younger rats failed to produce CaBP, it seems reasonable to conclude that the capacity to produce CaBP is programmed independently of the presence of 1,25-(OH),-D,. In turn, the failure to respond to 1,25-(OH)2-D, may be attributed either to a lack of receptors for 1,25-(OH)z-D3 or to a block along the sequence of events initiated by the interac-

VITAMIN

D AND INTESTINAL

Ca-BINDING

PROTEIN IN SUCKLING RATS

629

tion of 1,25-(OH),-D, with the cytosolic and REFERENCES nuclear receptors. FREUND, T., AND BRONNER, F. (1975) Science In most situations it has proved difficult 190, 1300-1302. to distinguish between a response indeUENG, T.-H., AND BRONNER, F. (1979) Arch. Biopendent of and one dependent on 1,25them. Biophys., in press. (OH&D,. Thus Moriuchi and DeLuca (30) KALLFELZ, F. A., AND WASSERMAN, R. H. (1967) treated g-day-old chick embryos with 32.5 Proc. Sot. Exp. Biol. Med. 125, 54-58. nmol 1,25-(OH&!-D, and found a significant DRESCHER, D., AND DELUCA, H. F. (1971) Bioincrease in calcium-binding activity in the chemistry 10, 2308-2312. lgday-old chick which, without treatment, 5. FREUND, T., AND BRONNER, F. (1975) Amer. J. Physiol. 228, 861-869. would have had lower calcium-binding levels. Subsequently, Oku et al. (31) re- 6. BRONNER, F., AND FREUND, T. (1975) Amer. J. Physiol. 229, 689-694. ported that the 3.5 S binding component in the duodenal cytosol of chick embryo, iden- 7. BRUNS, M. E., FLIESHER, E. B., AND AVIOLI, L. V. (1977) J. Biol. Chem. 252, 4145-4150. tified as the receptor for 1,25-(OH),-D,, was 8. HAUSSLER, M. (1974) Nub. Rev. 32, 257-266. detectable in the 15-day-old embryo and had 9. HAUSSLER, M. R., AND MCCAIN, T. A. (1977) N. risen to high levels in the 19-day-old emEngl. J. Med. 297, 1041-1050. bryo. Therefore receptors for 1,25-(OH),-D, 10. WASSERMAN, R. H., AND FEHER, J. J. (1977) in were already present. Oku et al. (31) also Calcium Binding Proteins and Calcium Function suggested that some regulation of vitamin D (Wasserman, R. H., Corradino, R. A., Carafoli, E., Kretsinger, R. H., MacLennan, D. H., and metabolism was the reason why CaBP was Singer, F. L., eds.), pp. 293-302, Northnot produced in the chick until hatching (32). Holland, New York. The situation in the rat seems different. Up to about 5 days of age, the rat appears 11. MORRISSEY, R. L., EMPSON, R. N., ZOLOCK, D. T., BIKLE, D. D., AND BUCCI, T. J. (1978) unable to produce CaBP, even when treated Biochim. Biophys. Acta 538, 34-41. with 1,25-(OH)z-D,. If this inability were EDELSTEIN, S., NOFF, D., SINAI, L., HARELL, A., due to the lack of receptors for 1,25-(OH&- 12. PUSCHETT, J. B., GOLUB, E. E., AND BROND3 or to a block in the sequence of transcripNER, F. (1978) Biochem. J. 170, 227-233. tional, translational, and posttranslational 13. DELUCA, H. F. (1978) Arch. Znt. Med. 138, 836events, then 1,25-(OH)z-D3 would appear to 847. be incapable of either inducing its receptors 14. WIDDOWSON, E. M., AND MCCANCE, R. A. (1960) or overcoming the block to CaBP synthesis. Proc. Royal Sot. B 152, 188-206. 15. MURPHY, J. B., AND KIES, M. W. (1960) Biochim. Table I shows that in the ll- or lZday-old Biophys. Acta 45, 382-384. rats treatment with 1,25-(OH),-D,, at a dose 16. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., level easily 400-fold the amount circulating AND RANDALL, R. J. (1951) J. Biol. Chem. 193, in these animals, barely doubled the amount 265-275. of CaBP, while a comparable (though 17. BRAY, G. A. (1960) Anal. Biochem. 1, 279-285. slightly larger) dose raised the CaBP level 18. HUMMEL, J. P., AND DREYER, W. J. (1962) Biolo-fold in older, vitamin D-deficient rats. chim. Biophys. Acta 63, 530-532. These findings suggest an upper limit to the 19. MORRISSEY, R. L., AND WASSERMAN, R. H. amount of CaBP that can be synthesized, (1971) Amer. J. Physiol. 220, 1509-1515. perhaps because the programmed number 20. PANSU, D., BELLATON, C., AND BRONNER, F. of receptors is limited. Studies exploring (1979) J. Nutr. 109, 508-512. this aspect of CaBP biosynthesis are in 21. GOLUB, E. E., REID, M., BOSSAK, C., WOLPERT, L., GAGLIARDI, L., AND BRONNER, F. (1977) progress. in Calcium-Binding Proteins and Calcium Func-

ACKNOWLEDGMENTS We thank Marilyn Reid and Cynthia Bossak for expert technical assistance. Generous gifts of crystalline 1,25-(OH&-D, by Dr. Milan Uskokovic of HoffmanLaRoche, Inc., are gratefully acknowledged.

tion (Wasserman, R. H., Corradino, R. A., Carafoli, E., Kretsinger, R. H., MacLennan, D. H., and Siegel, F. D., eds.), pp. 364-366, North-Holland, New York. 22. UENG, T.-H., GOLUB, E. E., REID, M., AND BRONNER, F. (1978) Fed. Proc. 37(3), 272. 23. BRONNER, F. (1960) Science 132, 472-473.

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24. SPENCER, R., CHAPMAN, M., WILSON, P., AND LAWSON, E. (1976) Nature (London) 203, 161163. 25. SPENCER, R., CHAPMAN, M., WILSON, P. W., AND LAWSON, D. (1978) Biochem. J. 170, 93-101. 26. MORRISSEY, R. L., ZOLOCK, D. T., BIKLE, D. D., EMPSON, R. N., AND BUCCI, T. J. (1978) Biochim. Biophys. Actu 538, 23-33. 27. WEISMAN, T., SAPIR, R., HARELL, A., AND EDELSTEIN, S. (19’76) Biochim. Biophys. Acta 428,

388-395.

ET AL. 28. NOFF, D., AND EDELSTEIN, S. (1979) Harm. Res., in press. 29. WEISMAN, T., HARELL, A., AND EDELSTEIN, S. (1979) Med. Hypoth. 5, 379-382. 30. MORIUCHI, S., AND DELUCA, H. F. (1974) Arch. Biochem. Biophys. 164, 165-171. 31. OKU, T., SHIMURA, F., MORIUCHI, S., AND HOSOYA, N. (1976)Endocrinology 23,375-381. 32. CORRADINO, R. A., TAYLOR, A. N., AND WASSERMAN, R. H. (1969) Fed. Proc. 28, 1834.