106
Blochtmwa et Btophysica Acta, 411 (1975) 106--112 © Elsevmr Scientific Pubhshmg Company, Amsterdam -- Printed in The Netherlands
BBA 27753 RESPONSE OF RENAL CALCIUM-BINDING PROTEIN INDEPENDENCE OF KIDNEY VITAMIN D HYDROXYLATION
ARIE BARa, SHMUEL HURWITZa and SAMUEL EDELSTEINb aInstltute of Animal Scwnce, Agrwultural Research Organization, The Volcani Center, Bet Dagan and b Department of Endocrinology, Munzcipal Governmental Medical Center, Ichllov Hospital, Tel Avw (Israel)
(Received May 9th, 1975)
Summary Dietary calcium and dietary phosphorus restriction were studied in chinks fed either cholecalciferol or la-hydroxycholecalciferol. Intestinal calcium absorption and calcium-binding protein of la-hydroxycholecalciferol-treated chicks remained unchanged under dietary calcium restriction, but increased under dietary phosphorus restriction. Kidney calcium-binding protein was not altered by dietary calcium restriction in chicks treated with either cholecalciferol or la-hydroxycholecalciferol, but increased under dietary phosphorus restriction independent of the vitamin D source. In contrast to the intestine, calcium-binding activity of the kidney was found to be poorly related to the calcium-binding protein concentration. It is suggested that kidney calciumbinding protein is regulated by a mechamsm different from that of intestinal calcium-binding protein, and that its concentration in renal tissue is related to renal calcium excretion or plasma calcium level.
Introduction A vitamin D-dependent calcium-binding protein was found in kidneys of chicks [1,2] and other species [3--5] and is believed to be associated with the function of this organ in calcium homeostasis [1,5,6]. In the chick, this protein was found to be the same as or similar to the calcium-binding protein found previously in the intestine [5], since both have the same electrophoretic mobility [2] and react similarly with a specific antiserum against intestinal calciumbinding protein [ 1 ]. Intestinal calcium-binding protein is associated with regulatory changes in calcium absorption [5]. Such changes, as produced by dietary calcium restriction, are probably mediated through corresponding changes in the formation of
107 1,25 0.05) in b o d y weight, plasma calcium, plasma inorganic phosphate and percent bone ash between chicks fed the control diet with 5 pg/kg l~-hydroxycholecalciferol and those fed the same diet with 40 pg/kg cholecalciferol. However, both 4~Ca 2÷ absorption and intestinal calcium binding protein were higher (P 0.01) in the l~-hydroxycholecalciferol controls than in the cholecalciferol controls (Table II). The responses of bone ash and plasma minerals to Ca 2÷ and phosphorus restriction in l~-hydroxycholecalciferol-fed chicks (Table I) are similar to those previously reported for cholecalciferol-fed chicks [13,15]. Duodenal 47Ca2÷ absorption and calcium-binding protein (Table I I ) w e r e significantly higher (P < 0.01) in chicks fed cholecalciferol-supplemented diets, either low in calcium or low in phosphorus, compared with control chicks. The TABLE
I
BODY WEIGHT, PLASMA CALCIUM AND INORGANIC PHOSPHORUS, FED CONTINUOUSLY WITH lc~-HYDROXYCHOLECALCIFEROL Results
Vitamin
are given as mean
AND BONE ASH OF CHICKS
-+ S . E . o f s i x a m r n a l s .
D source
Diet
Cholecalclferol
Control
la-Hydroxycholecalclferol
Control L o w C a 2+ Low P
* Slgnifmant difference
Body wt (g)
224 243 239 229
± 15 -+ 1 2 ± 8 -+ 4
Plasma calcmm (mg/lO0 9.7 9.6 9.2 11.6
ml)
-+ 0 . 1 ± 0.2 ± 0.4 ± 0.4*
(P ~ 0.01) from la-hydroxycholecalciferol
Plasma inorgamc phosphorus (mg/ml)
ash (% d r y matter)
5.6 5.5 4.8 2.2
38.1 38.1 29.5 30.2
± ± t ±
0.2 0.2 0.5 0.1"
control
group
Bone
± 0.4 +- 0 . 5 ± 0.5* ± 0.3*
109 T A B L E II D U O D E N A L 4 7 C A L C I U M A B S O R P T I O N A N D C A L C I U M - B I N D I N G P R O T E I N AS I N F L U E N C E D BY DIETARY MINERAL CONTENT AND SOURCE OF VITAMIN D ACTIVITY I n t r m l 2 , 47Ca2+ a b s o r p t i o n a n d c a l c i u m - b i n d i n g p r o t e i n w e r e 2 6 . 4 ± 2.5 a n d 1.07 ± 0 . 0 8 m c h i c k s f e d t h e c o n t r o l d t e t a n d c h o l e c a l c f f e r o l , r e s p e c t i v e l y . R e s u l t s g i v e n are m e a n -+ S.E. of six a n i m a l s . Vitamin D source
Cholecalcfferol (trial 1)
l~-Hydroxycholecalcfferol (trial 2)
Parameter
47Ca2+ a b s o r p t i o n , % dose Calcmm-bmding protein, m g / g tissue Calcmm-bmding activity, c p m • 10 -5 p e r g h s s u e 47Ca 2+ a b s o r p t m n , % d o s e Calcmm-bindmg protein, m g / g tissue Calcium-binding achvlty,
Dietary treatment Control
L o w Ca 2+
Low P
2 7 . 0 ± 2.0 0.93 ± 0.05
6 3 . 2 ± 3.9* 2.40 + 0.13"
6 1 . 5 ± 3.4* 2.31 ± 0 . 1 2 "
12.6
11.7
6.5
+- 0 . 4
36.9 ± 2.2 1.64 ± 0.08 6.5
± 0.9*
4 0 . 0 ± 2.0 1 . 4 9 -+ 0 . 0 6
± 0.3
6.2
± 0.4
± 0.8*
46.1 -+ 1 . 3 " 2.27 ± 0 . 1 1 " 8.8
± 0.3*
c p m • 10 -5 p e r g tissue * Significant difference f r o m the respective control group.
same parameters were not influenced in la-hydroxycholecalciferol-fed chicks by dietary calcium restriction (P > 0.05), but were significantly higher (P < 0.01) in chicks fed the low-phosphorus diet. The response to the low-phosphorus diet was, however, smaller with la-hydroxycholecalciferol than with cholecalciferol. Kidney calcium-binding protein concentration was not significantly influenced (P > 0.05) by the low-calcium treatment (Table III) in chicks fed cholecalciferol or la-hydroxycholecalciferol. The low-phosphorus treatment resulted in an increased (P < 0.01) concentration of calcium-binding protein in kidneys obtained from either cholecalciferol or la-hydroxycholecalciferol-fed chicks. The immunological measurements of kidney calcium-binding protein are supTABLE III T H E C O N C E N T R A T I O N O F K I D N E Y C A L C I U M - B I N D I N G P R O T E I N AS I N F L U E N C E D BY D I E T A R Y M I N E R A L C O N T E N T A N D SOURCE OF V I T A M I N D A C T I V I T Y R e s u l t s are g i v e n as m e a n +- S.E. of six animals. Vitamin D source
Cholecalciferol (trial 1)
l~-Hydroxycholecalclferol (trial 2)
Parameter
Calcium-binding protein, m g / g tissue Calcium-binding activity, cpm • 10 -5 p e r g tissue Calcium-binding protein, m g / g tissue Calcium-binding activity, cpm • 10 -$ p e r g tissue
Dietary treatment Control
L o w Ca 2+
Low P
0.38 ± 0.04
0.39 ± 0.02
0.70 ± 0.07*
4.8
3.9
5.7
± 0.3
± 0.3
-+ 0 . 4
0.40 ± 0.05
0 . 3 0 ± 0.01
0.62 ± 0.04*
5.0
5.4
6.0
± 0.4
* S i g n i f i c a n t d i f f e r e n c e (P ~ 0 . 0 1 ) f r o m t h e r e s p e c t i v e c o n t r o l .
-+ 0 . 2
± 0.4
110
,A
B
.C.
D
~ = ii
Fzg. l T h e e l e c t r o p h o r e t l c p a t t e r n of i n t e s t i n a l and k i d n e y s u p e r n a t a n t s A r r o w i n d i c a t e s p o s i t i o n of c a l c m m - b m d m g p r o t e i n A, i n t e s t i n a l s u p e r n a t a n t s ( l o w - c a l c m m c h i c k s ) , B, k i d n e y s u p e r n a t a n t s , c o n t r o l chicks, C, l o w - c a l c m m c h i c k s , D~ l o w - p h o s p h a t e c h i c k s T h e s a m e q u a n t i t y of p r o t e i n w a s a p p h e d to each of t h e d i f f e r e n t gels
ported by the electrophoretic patterns shown m Fig. 1. A rapidly migrating protein fraction in kidney material, with a n R F similar to chick intestinal calcium-binding protein, is apparent. The density of this fraction is higher in material obtained from chicks fed the low-phosphorus diet. Calcium-binding activity of kidney supernatants did not always reflect the calcium-binding protern concentration measured by the immunological or electrophoretic techniques, possibly owing to differences in calcium concentration of supernatants prepared from chicks of the different dietary treatments (unpublished data). Discussion la-hydroxycholecalciferol, at the concentration used in this study, proved to be as effective as higher concentrations of cholecalciferol in supporting body weight, bone ash, plasma calcium and inorganic phosphate. Moreover, it promoted higher 47Ca2. absorption and duodenal calcium-binding protein than that obtained in birds fed cholecalciferol. This finding, in agreement with other studies [22 24], demonstrates that la-hydroxycholecalciferol is a more potent source of vitamin D activity than the vitamin itself.
111
The increase in calcium absorption and intestinal calcium-binding protein in response to a low calcium intake is well recognized for cholecalciferol-fed chicks [13 15]. Such a response to a low-calcium intake is not apparent in chicks fed the la-hydroxycholecalciferol (Table II). A similar observation was also reported for chicks fed either dihydrotachysterol3 [15] or 1,25-dlhydroxycholecalclferol [8]. None of the three compounds, dihydrotachysterol~, la-hydroxycholecalciferol or 1,25-dihydroxycholecalciferol, reqmres kidney hydroxylation in position 1 for the expression of its biological activity [7,8,17,22]. Furthermore, an increase m intestinal 1,25-dihydroxycholecalclferol was observed by Edelstein et al. [10], in response to a low-calcium intake. Thus, the present results support the previous hypothesis [7,8,9] that the intestinal adaptation to a low-calcium intake is regulated by modulation of 1,25-dihydroxycholecalciferol synthesis in the kidney. Results given in Table II show that cholecalcIferol-fed chinks respond to a low-phosphorus diet similarly to those fed the low-calcium diet, e.g. by increased 47 Ca2. absorption and intestinal calcium-binding protein; another similarity is the elevated 1,25-dihydroxycholecalciferol in their intestinal mucosa [10]. A partial response of calcmm-binding protein and calcium absorption to a low-phosphorus intake was obtained in the presence of la-hydroxycholecalciferol, In similarity to previous observations with dlhydrotachysterol3-fed chicks [15]. It may thus be concluded that at least part of the response of 4 ~Ca2+ absorption and calcium-binding protein to low-phosphorus diet is ladependent of changes in the rate of vitamin D h y d r o x y l a t i o n In the kidney. At present, the responsible regulatory system cannot be identified, although it remains to be shown whether stimulation of liver h y d r o x y l a t i o n at position 25 of the vitamin D metabolite may occur under conditions of phosphorus dehclency. In the kidney, both low-calcium and low-phosphorus intake result in an increased concentration of 1,25-dihydroxycholeealeiferol [ 10 ]. If the kidney is considered to be a target organ for this hormonal form of vitamin D, and if indeed renal calcium-binding concentration IS regulated by the level of 1,25dihydroxycholeealeiferol as in the intestine, this calcium-binding protein would be expected to increase in response to low-calcium and low-phosphorus treatments. The results (Table III), however, show that renal calcium-binding protein remained unchanged, or even decreased slightly under conditions of low calcium intake, but increased in response to low phosphorus intake. Furthermore, the response of renal calcium-binding protein was similar for chicks fed either cholecalciferol or la-hydroxycholecaleiferol. Thus it may be suggested that, unlike intestinal calcium-binding protein, the renal protein is regulated by means additional to or other than the level of 1,25-dihydroxycholeealciferol, despite the chemical similarity of the two proteins and their vitamin D-dependence [1,21. The physiological role of renal calcium-binding protein has not been elucidated. Preliminary results from this laboratory (Hurwitz et al., unpublished data) show that the urinary calcium concentrations were 2.3 ± 0.6, 0.5 ± 0.1 and 17.0 ± 1.9 mM in chicks fed the control, low-calcmm and low-phosphorus diets, respectively. The high excretion of calcium by the birds fed the lowphosphorus diet is expected in view of their hypercaleemia and their inability
112
to calcify their bones properly, due to the deficiency in phosphorus. Thus, increase in renal calcium-binding protein in this group appears to be associated with either hypercalcemia or increased calcium excretion. The present results regarding renal calcium-binding protein are reminiscent of findings with uterine calcium-binding protein in laying hens [25]. In this organ also, calcium-binding protein did not increase under conditions of calcium deficiency, whereas a marked increase in calcium-binding protein was observed in the intestine. Thus, calcium-binding protein, although identical or similar in various organs, appears to be controlled by different physiological means, according to the function of the respective organ. Acknowledgements Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. 1975 Series, No. 150-E. Supported by the Poultry Marketing Board of Israel. The authors thank Professor R.H. Wasserman of Cornell University, Ithaca, New York, for the generous supply of the specific antiserum and Professor Y. Mazor of the Weizmann Institute of Science, Rehovot, Israel, for the synthetic la-hydroxycholecalciferol. The technical assistance of Mrs M. Cotter is gratefully acknowledged. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Taylor, A N and Wasserman, R H (1972) Am J. Physlol. 223 110--114 Taylor, A.N. and Wasserman, R.H (1967) Arch. B1ochem. Bmphys. 1 1 9 , 5 3 6 - - 5 4 0 Piazolo, P., Schleyer, M and Franz, H.E (1971) Hoppe-Seyler's Z. Physlol. Chem. 352, 1 4 8 0 - - 1 4 8 6 Fu]Imer, C.S. and Wasserman, R.H. (1973) Blochim. B1ophys. Acta 3 1 7 , 1 7 2 186 Wasserman, R H., Taylor A.N. and Fullmer, C.S. (1974) B1ochem. Soc. Spec. Publ. 3, 55 --74 Larkms, R.G., MacAuley, S.J. and MacIntyre, I. (1974) Nature 2 5 2 , 4 1 2 - - 4 1 4 Norman, A.W. and Henry, H. (1974) Recent Progress in Nucleic Acld Research 3 0 , 4 3 1 - - 4 5 0 DeLuea, H F. (1974) Biochem. Soc. Spec. Publ. 3, 5 26 Kodlcek, D. (1974) Lancet 1, 325---329 Edelsteln, S., Harell, A., Bar, A and Hurwltz, S. (1975) B1ochim. B1ophys. Acta 3 8 5 , 4 3 8 4 4 3 Emtage, J S., Lawson, D.E.M. and Kodicek, E. (1974) B1ochem. J. 1 4 0 , 2 3 9 - - 2 4 7 Carlsson, A (1953) Acta Pharmacol. Toxicol. 93, 32--40 Morrissey, R.L. and Wasserman, R.H. (1971) Am. J. Physlol. 220, 1509--1515 Httrwltz, S., Bar, A. and Cohen, I. (1973) Am. J Physlol. 2 2 5 , 1 5 0 - - 1 5 4 Bar, A. and Wasserman, R.H. (1973) Biochem. Biophys. Res. Commun. 54, 191--196 Omdahl, J.L and Thornton, P.A. (1972) Proc. Soe. Exp. Biol. Med. 1 3 9 , 9 7 5 - - 9 8 0 Hallick, R.B and DeLuca, H.F. (1972) J. Biol, Chem. 247,91---97 Hurwltz, S. and Bar, A. (1972) Am. J. Physlol. 2 2 2 , 7 6 1 - - 7 6 7 Corradmo, R.A (1973) J Cell Biol. 58, 64--78 Gomori, G. (1942) J. Lab. Chn. Med. 2 7 , 9 5 5 - - 9 6 0 Snedecor, G.W and Cochran, W.G. (1968) Statlstlcal Methods, 6th ed., Iowa State University, Ames, Iowa Hohck, M F., Semmler, E~J., Schnoes, H.K and DeLuca, H.F. (1973) Science 1 8 0 , 1 9 0 - - 1 9 1 Zerwekh, J E., Brumbaugh, P.F.. Haussler, D H., Cork, DA. and Haussler, M.R. (1974) B1ochemlstry 13 4 097--4102 Cork, D J . , Haussler, M.R , Pitt, M~I., Rlzzardo E., Hesse, R.H. and Pechet, M.M (1974) Endocrinology 94, 1337--1345 Bar, A. and Hurwitz. S. (1973) Comp. Blochem. Physiol. 45A, 579---586
Blochtmwa et Btophysica Acta, 411 (1975) 106--112 © Elsevmr Scientific Pubhshmg Company, Amsterdam -- Printed in The Netherlands
BBA 27753 RESPONSE OF RENAL CALCIUM-BINDING PROTEIN INDEPENDENCE OF KIDNEY VITAMIN D HYDROXYLATION
ARIE BARa, SHMUEL HURWITZa and SAMUEL EDELSTEINb aInstltute of Animal Scwnce, Agrwultural Research Organization, The Volcani Center, Bet Dagan and b Department of Endocrinology, Munzcipal Governmental Medical Center, Ichllov Hospital, Tel Avw (Israel)
(Received May 9th, 1975)
Summary Dietary calcium and dietary phosphorus restriction were studied in chinks fed either cholecalciferol or la-hydroxycholecalciferol. Intestinal calcium absorption and calcium-binding protein of la-hydroxycholecalciferol-treated chicks remained unchanged under dietary calcium restriction, but increased under dietary phosphorus restriction. Kidney calcium-binding protein was not altered by dietary calcium restriction in chicks treated with either cholecalciferol or la-hydroxycholecalciferol, but increased under dietary phosphorus restriction independent of the vitamin D source. In contrast to the intestine, calcium-binding activity of the kidney was found to be poorly related to the calcium-binding protein concentration. It is suggested that kidney calciumbinding protein is regulated by a mechamsm different from that of intestinal calcium-binding protein, and that its concentration in renal tissue is related to renal calcium excretion or plasma calcium level.
Introduction A vitamin D-dependent calcium-binding protein was found in kidneys of chicks [1,2] and other species [3--5] and is believed to be associated with the function of this organ in calcium homeostasis [1,5,6]. In the chick, this protein was found to be the same as or similar to the calcium-binding protein found previously in the intestine [5], since both have the same electrophoretic mobility [2] and react similarly with a specific antiserum against intestinal calciumbinding protein [ 1 ]. Intestinal calcium-binding protein is associated with regulatory changes in calcium absorption [5]. Such changes, as produced by dietary calcium restriction, are probably mediated through corresponding changes in the formation of
107 1,25 0.05) in b o d y weight, plasma calcium, plasma inorganic phosphate and percent bone ash between chicks fed the control diet with 5 pg/kg l~-hydroxycholecalciferol and those fed the same diet with 40 pg/kg cholecalciferol. However, both 4~Ca 2÷ absorption and intestinal calcium binding protein were higher (P 0.01) in the l~-hydroxycholecalciferol controls than in the cholecalciferol controls (Table II). The responses of bone ash and plasma minerals to Ca 2÷ and phosphorus restriction in l~-hydroxycholecalciferol-fed chicks (Table I) are similar to those previously reported for cholecalciferol-fed chicks [13,15]. Duodenal 47Ca2÷ absorption and calcium-binding protein (Table I I ) w e r e significantly higher (P < 0.01) in chicks fed cholecalciferol-supplemented diets, either low in calcium or low in phosphorus, compared with control chicks. The TABLE
I
BODY WEIGHT, PLASMA CALCIUM AND INORGANIC PHOSPHORUS, FED CONTINUOUSLY WITH lc~-HYDROXYCHOLECALCIFEROL Results
Vitamin
are given as mean
AND BONE ASH OF CHICKS
-+ S . E . o f s i x a m r n a l s .
D source
Diet
Cholecalclferol
Control
la-Hydroxycholecalclferol
Control L o w C a 2+ Low P
* Slgnifmant difference
Body wt (g)
224 243 239 229
± 15 -+ 1 2 ± 8 -+ 4
Plasma calcmm (mg/lO0 9.7 9.6 9.2 11.6
ml)
-+ 0 . 1 ± 0.2 ± 0.4 ± 0.4*
(P ~ 0.01) from la-hydroxycholecalciferol
Plasma inorgamc phosphorus (mg/ml)
ash (% d r y matter)
5.6 5.5 4.8 2.2
38.1 38.1 29.5 30.2
± ± t ±
0.2 0.2 0.5 0.1"
control
group
Bone
± 0.4 +- 0 . 5 ± 0.5* ± 0.3*
109 T A B L E II D U O D E N A L 4 7 C A L C I U M A B S O R P T I O N A N D C A L C I U M - B I N D I N G P R O T E I N AS I N F L U E N C E D BY DIETARY MINERAL CONTENT AND SOURCE OF VITAMIN D ACTIVITY I n t r m l 2 , 47Ca2+ a b s o r p t i o n a n d c a l c i u m - b i n d i n g p r o t e i n w e r e 2 6 . 4 ± 2.5 a n d 1.07 ± 0 . 0 8 m c h i c k s f e d t h e c o n t r o l d t e t a n d c h o l e c a l c f f e r o l , r e s p e c t i v e l y . R e s u l t s g i v e n are m e a n -+ S.E. of six a n i m a l s . Vitamin D source
Cholecalcfferol (trial 1)
l~-Hydroxycholecalcfferol (trial 2)
Parameter
47Ca2+ a b s o r p t i o n , % dose Calcmm-bmding protein, m g / g tissue Calcmm-bmding activity, c p m • 10 -5 p e r g h s s u e 47Ca 2+ a b s o r p t m n , % d o s e Calcmm-bindmg protein, m g / g tissue Calcium-binding achvlty,
Dietary treatment Control
L o w Ca 2+
Low P
2 7 . 0 ± 2.0 0.93 ± 0.05
6 3 . 2 ± 3.9* 2.40 + 0.13"
6 1 . 5 ± 3.4* 2.31 ± 0 . 1 2 "
12.6
11.7
6.5
+- 0 . 4
36.9 ± 2.2 1.64 ± 0.08 6.5
± 0.9*
4 0 . 0 ± 2.0 1 . 4 9 -+ 0 . 0 6
± 0.3
6.2
± 0.4
± 0.8*
46.1 -+ 1 . 3 " 2.27 ± 0 . 1 1 " 8.8
± 0.3*
c p m • 10 -5 p e r g tissue * Significant difference f r o m the respective control group.
same parameters were not influenced in la-hydroxycholecalciferol-fed chicks by dietary calcium restriction (P > 0.05), but were significantly higher (P < 0.01) in chicks fed the low-phosphorus diet. The response to the low-phosphorus diet was, however, smaller with la-hydroxycholecalciferol than with cholecalciferol. Kidney calcium-binding protein concentration was not significantly influenced (P > 0.05) by the low-calcium treatment (Table III) in chicks fed cholecalciferol or la-hydroxycholecalciferol. The low-phosphorus treatment resulted in an increased (P < 0.01) concentration of calcium-binding protein in kidneys obtained from either cholecalciferol or la-hydroxycholecalciferol-fed chicks. The immunological measurements of kidney calcium-binding protein are supTABLE III T H E C O N C E N T R A T I O N O F K I D N E Y C A L C I U M - B I N D I N G P R O T E I N AS I N F L U E N C E D BY D I E T A R Y M I N E R A L C O N T E N T A N D SOURCE OF V I T A M I N D A C T I V I T Y R e s u l t s are g i v e n as m e a n +- S.E. of six animals. Vitamin D source
Cholecalciferol (trial 1)
l~-Hydroxycholecalclferol (trial 2)
Parameter
Calcium-binding protein, m g / g tissue Calcium-binding activity, cpm • 10 -5 p e r g tissue Calcium-binding protein, m g / g tissue Calcium-binding activity, cpm • 10 -$ p e r g tissue
Dietary treatment Control
L o w Ca 2+
Low P
0.38 ± 0.04
0.39 ± 0.02
0.70 ± 0.07*
4.8
3.9
5.7
± 0.3
± 0.3
-+ 0 . 4
0.40 ± 0.05
0 . 3 0 ± 0.01
0.62 ± 0.04*
5.0
5.4
6.0
± 0.4
* S i g n i f i c a n t d i f f e r e n c e (P ~ 0 . 0 1 ) f r o m t h e r e s p e c t i v e c o n t r o l .
-+ 0 . 2
± 0.4
110
,A
B
.C.
D
~ = ii
Fzg. l T h e e l e c t r o p h o r e t l c p a t t e r n of i n t e s t i n a l and k i d n e y s u p e r n a t a n t s A r r o w i n d i c a t e s p o s i t i o n of c a l c m m - b m d m g p r o t e i n A, i n t e s t i n a l s u p e r n a t a n t s ( l o w - c a l c m m c h i c k s ) , B, k i d n e y s u p e r n a t a n t s , c o n t r o l chicks, C, l o w - c a l c m m c h i c k s , D~ l o w - p h o s p h a t e c h i c k s T h e s a m e q u a n t i t y of p r o t e i n w a s a p p h e d to each of t h e d i f f e r e n t gels
ported by the electrophoretic patterns shown m Fig. 1. A rapidly migrating protein fraction in kidney material, with a n R F similar to chick intestinal calcium-binding protein, is apparent. The density of this fraction is higher in material obtained from chicks fed the low-phosphorus diet. Calcium-binding activity of kidney supernatants did not always reflect the calcium-binding protern concentration measured by the immunological or electrophoretic techniques, possibly owing to differences in calcium concentration of supernatants prepared from chicks of the different dietary treatments (unpublished data). Discussion la-hydroxycholecalciferol, at the concentration used in this study, proved to be as effective as higher concentrations of cholecalciferol in supporting body weight, bone ash, plasma calcium and inorganic phosphate. Moreover, it promoted higher 47Ca2. absorption and duodenal calcium-binding protein than that obtained in birds fed cholecalciferol. This finding, in agreement with other studies [22 24], demonstrates that la-hydroxycholecalciferol is a more potent source of vitamin D activity than the vitamin itself.
111
The increase in calcium absorption and intestinal calcium-binding protein in response to a low calcium intake is well recognized for cholecalciferol-fed chicks [13 15]. Such a response to a low-calcium intake is not apparent in chicks fed the la-hydroxycholecalciferol (Table II). A similar observation was also reported for chicks fed either dihydrotachysterol3 [15] or 1,25-dlhydroxycholecalclferol [8]. None of the three compounds, dihydrotachysterol~, la-hydroxycholecalciferol or 1,25-dihydroxycholecalciferol, reqmres kidney hydroxylation in position 1 for the expression of its biological activity [7,8,17,22]. Furthermore, an increase m intestinal 1,25-dihydroxycholecalclferol was observed by Edelstein et al. [10], in response to a low-calcium intake. Thus, the present results support the previous hypothesis [7,8,9] that the intestinal adaptation to a low-calcium intake is regulated by modulation of 1,25-dihydroxycholecalciferol synthesis in the kidney. Results given in Table II show that cholecalcIferol-fed chinks respond to a low-phosphorus diet similarly to those fed the low-calcium diet, e.g. by increased 47 Ca2. absorption and intestinal calcium-binding protein; another similarity is the elevated 1,25-dihydroxycholecalciferol in their intestinal mucosa [10]. A partial response of calcmm-binding protein and calcium absorption to a low-phosphorus intake was obtained in the presence of la-hydroxycholecalciferol, In similarity to previous observations with dlhydrotachysterol3-fed chicks [15]. It may thus be concluded that at least part of the response of 4 ~Ca2+ absorption and calcium-binding protein to low-phosphorus diet is ladependent of changes in the rate of vitamin D h y d r o x y l a t i o n In the kidney. At present, the responsible regulatory system cannot be identified, although it remains to be shown whether stimulation of liver h y d r o x y l a t i o n at position 25 of the vitamin D metabolite may occur under conditions of phosphorus dehclency. In the kidney, both low-calcium and low-phosphorus intake result in an increased concentration of 1,25-dihydroxycholeealeiferol [ 10 ]. If the kidney is considered to be a target organ for this hormonal form of vitamin D, and if indeed renal calcium-binding concentration IS regulated by the level of 1,25dihydroxycholeealeiferol as in the intestine, this calcium-binding protein would be expected to increase in response to low-calcium and low-phosphorus treatments. The results (Table III), however, show that renal calcium-binding protein remained unchanged, or even decreased slightly under conditions of low calcium intake, but increased in response to low phosphorus intake. Furthermore, the response of renal calcium-binding protein was similar for chicks fed either cholecalciferol or la-hydroxycholecaleiferol. Thus it may be suggested that, unlike intestinal calcium-binding protein, the renal protein is regulated by means additional to or other than the level of 1,25-dihydroxycholeealciferol, despite the chemical similarity of the two proteins and their vitamin D-dependence [1,21. The physiological role of renal calcium-binding protein has not been elucidated. Preliminary results from this laboratory (Hurwitz et al., unpublished data) show that the urinary calcium concentrations were 2.3 ± 0.6, 0.5 ± 0.1 and 17.0 ± 1.9 mM in chicks fed the control, low-calcmm and low-phosphorus diets, respectively. The high excretion of calcium by the birds fed the lowphosphorus diet is expected in view of their hypercaleemia and their inability
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to calcify their bones properly, due to the deficiency in phosphorus. Thus, increase in renal calcium-binding protein in this group appears to be associated with either hypercalcemia or increased calcium excretion. The present results regarding renal calcium-binding protein are reminiscent of findings with uterine calcium-binding protein in laying hens [25]. In this organ also, calcium-binding protein did not increase under conditions of calcium deficiency, whereas a marked increase in calcium-binding protein was observed in the intestine. Thus, calcium-binding protein, although identical or similar in various organs, appears to be controlled by different physiological means, according to the function of the respective organ. Acknowledgements Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. 1975 Series, No. 150-E. Supported by the Poultry Marketing Board of Israel. The authors thank Professor R.H. Wasserman of Cornell University, Ithaca, New York, for the generous supply of the specific antiserum and Professor Y. Mazor of the Weizmann Institute of Science, Rehovot, Israel, for the synthetic la-hydroxycholecalciferol. The technical assistance of Mrs M. Cotter is gratefully acknowledged. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
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