Calcif. Tiss. Res. 19, 1--7 (1975) 9 by Springer-Verlag 1975
Original Papers Net Uptake and Release of Calcium and Phosphate by Bone in vitro: Effects of Medium Calcium and Phosphate Concentrations H. H. Messer*, S. Y. Yuen, a n d D. H. Copp Department of Physiology, University of British Columbia, Vancouver Received January 16, accepted June 8, 1975 The effects of varying the initial calcium and phosphate concentrations of the culture media on bone calcium and phosphate release were examined, using whole calvaria from 3-day-old mice in 48-hour cultures. The initial calcium and phosphate concentrations of the culture media were varied in the range 3-10 mg/100 ml; either calcium or phosphate alone was changed while the other ion was held constant, or the concentrations of both were varied while the Ca:P ratio was held constant. For all combinations, 3 treatment groups were used: i) control (no added hormone); ii) 0.5 U/ml PTH; iii) 50 mU/ml CT. The release of calcium and phosphate from the bones was greatest at low initial calcium or phosphate concentrations in the media, and least at high initial concentrations. High concentrations of both ions together abolished hormonal responses and resulted in extensive uptake of calcium and phosphate by the bones. The response to PTH was lost at a high concentration of either ion alone, while a response to CT was observed under all experimental conditions except simultaneously high calcium and phosphate concentrations.
Key words: Bone culture - - Calcium - - Phosphate - - Parathyroid hormone - - Calcitonin
Introduction T h e b a l a n c e b e t w e e n a c c r e t i o n a n d r e s o r p t i o n in b o n e cultures is v e r y sensitive t o a n u m b e r of factors, including age (Messer et al., 1974b), s t r u c t u r a l i n t e g r i t y of t h e bones (Russell a n d T a l m a g e , 1972), p a r a t h y r o i d h o r m o n e ( P T H ) a n d calcitonin (CT) (Heersche, 1969; Messer et al., 1974a; R e y n o l d s a n d Dingle, 1970), a n d t h e a c t i v e m e t a b o l i t e s of v i t a m i n D (Reynolds et al., 1973, 1974). T h e c o n c e n t r a t i o n s of calcium a n d p h o s p h a t e in t h e c u l t u r e m e d i a m a y also p l a y a n i m p o r t a n t role, since these a r e t h e p r e d o m i n a n t c o n s t i t u e n t s of bone mineral. Also, t h e s e ions m a y be i n v o l v e d in m e d i a t i n g h o r m o n a l effects on bone cells (Rasmussen, 1971). R a i s z a n d N i e m a n n (1969) r e p o r t e d t h a t a high p h o s p h a t e c o n c e n t r a t i o n in t h e c u l t u r e m e d i u m r e d u c e d bone resorption, while changes in calcium c o n c e n t r a t i o n e x e r t e d l i t t l e effect. T h e b o n e culture m e t h o d used in t h e i r s t u d y p r e c l u d e d a n i n v e s t i g a t i o n of calcium u p t a k e . W e h a v e i n v e s t i g a t e d t h e effects of changes in t h e calcium a n d p h o s p h a t e c o n c e n t r a t i o n s of t h e c u l t u r e m e d i a on accretion a n d r e s o r p t i o n in c a l v a r i a from 3 - d a y - o l d mice. I n a previous s t u d y (Messer et al., 1974b), c a l v a r i a of this age
For reprints: D. H. Copp, Department of Physiology, University of British Columbia, Vancouver, B. C., Canada. * Present address: Division of Oral Biology, School of Dentistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
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d e m o n s t r a t e d a close b a l a n c e b e t w e e n accretion a n d r e s o r p t i o n in culture, in t h e absence of a d d e d hormones. These bones m a y t h u s be used effectively t o s t u d y changes in t h e b a l a n c e b e t w e e n t h e t w o processes.
Materials and Methods 1. Culture Media. Medium CMRL 1066 was obtained with calcium and phosphate omitted (Grand Island Biological Company, Grand Island, New York). Calcium and phosphate were added as required, using sterile solutions of CaC12 or Na2HPO 4 (containing 10 mg Ca or P per ml), to yield final concentrations of each ion in the range 3-10 mg per 100 ml. The complete culture medium consisted of 95% CMRL 1066, 5% heat-inactivated horse serum, plus penicillin (50 U/ml) and streptomycin (50 ~g/ml). Hormones were added as appropriate: bovine parathyroid hormone (PTH) 0.5 Unit/ml (parathyroid extract Eli Lilly and Company, Indianapolis, Indiana), or calcitonin (CT), 50 mUnits/ml (pure natural salmon ealeitonin, lot AL1025, Armour Pharmaceutical Company, Kankakee, Illinois). 2. Bone Culture Method. Whole calvaria from 3-day-old mice were incubated for 48 h with 2.5 ml culture medium, as described previously (Messer et al., 1974b). Following incubation, the media and calvaria (ashed at 550 ~ were assayed for calcium by atomic absorption spectrophotometry (Instructional Manual, Series 82-360, Jarrell Ash Company, 1964) and phosphate (Gomori, 1942). The initial calcium and phosphate concentrations of all media, and the final calcium and phosphate concentrations of media incubated without bones were also determined. Concentration of the two ions did not change during culture without added bones. ~qet release or uptake by calvaria was then calculated as a percentage of the total calcium and phosphate originally present in each bone. Three treatment groups, each of 7 or 8 calvaria, were used for all levels of calcium and phosphate tested: i) control (no added hormone), ii) 0.5 U/ml PTH, iii) 50 mU/ml CT. Three levels of calcium or phosphate in the culture medium were used, designated as low (3 rag/ 100 ml), intermediate (5-6 mg/100 ml) and high (10-11 rag/100 ml). When either ion alone was varied, the other was held constant at the intermediate level.
Results 1. Response to Changes in Calcium Concentration
T h e effects of initial calcium c o n c e n t r a t i o n of t h e c u l t u r e m e d i u m were i n v e s t i g a t e d using 3 levels of calcium (3.0, 5.9 a n d 10.8 mg/100 ml) while t h e p h o s p h a t e c o n c e n t r a t i o n was h e l d c o n s t a n t a t 5.6 rag/100 ml. T h e n e t release ( p r e s u m a b l y due to resorption) was s t r o n g l y influenced b y t h e initial calcium c o n c e n t r a t i o n (Fig. 1). T h e p e r c e n t a g e loss of bone m i n e r a l b y c a l v a r i a i n c u b a t e d in control m e d i u m (no a d d e d hormones) was a p p r o x i m a t e l y twice as g r e a t when t h e i n i t i a l calcium c o n c e n t r a t i o n was 3.0 rag/100 ml as w h e n t h e initial level was 5.9 rag/100 ml. T h e high initial calcium c o n c e n t r a t i o n (10.8 rag/100 ml) r e s u l t e d in a small n e t calcium a n d p h o s p h a t e u p t a k e b y t h e bones. CT e x e r t e d a m a r k e d influence a t all calcium concentrations. E x c e p t in t h e low calcium group, CT p r o m o t e d a n e t u p t a k e of calcium a n d p h o s p h a t e b y t h e calvaria. T h e initial c a l c m m c o n c e n t r a t i o n of t h e m e d i u m influenced t h e e x t e n t of t h i s calcium a n d p h o s p h a t e u p t a k e , w i t h a n effect a p p r o x i m a t e l y parallel to t h a t of control cultures. T h e response of c a l v a r i a to P T H a t different calcium c o n c e n t r a t i o n s was m o r e compley. A t t h e i n t e r m e d i a t e calcium concentration, P T H e n h a n c e d calcium release b y a p p r o x i m a t e l y 60% a b o v e control values (p ~ 0.01; S t u d e n t ' s t test). H o w e v e r , a t t h e low calcium concentration, when calcium release b y b o t h control a n d P T H - t r e a t e d bones was m u c h greater, t h e
Medium Ion Concentrations and Bone Metabolism in Culture Calcium
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Initial Ca concentratio~l (mgllOOml) Fig. 1. Effect of initial calcium concentration of the culture medium on calcium and phosphate release by bones in organ culture. Initial phosphate concentration was 5.6 mg 100 ml in all cases. Whole calvaria from 3-day-old mice were cultured for 48 h in 2.5 mI culture medlum. Uptake and release are expressed as a percentage of the calcium and phosphate initially present in each calvarium and each value represents the mean =k S.E.M. for 7 or 8 cultures per treatment group
P T H enllanced t h i s b y o n l y a p p r o x i m a t e l y 25% which was n o t significantly different from t h e corresponding control value. A t t h e high calcium level, t h e response t o P T H was lost, a n d a slight u p t a k e of calcium was recorded. The release or u p t a k e of p h o s p h a t e followed t h e s a m e t r e n d s as t h e calcium, even t h o u g h t h e initial p h o s p h a t e c o n c e n t r a t i o n in t h e m e d i a was h e l d c o n s t a n t a t a n h l t e r m e d i a t e level. T h e r e were small differences in t h e n e t m o v e m e n t of t h e t w o ions u n d e r some conditions. F o r e x a m p l e , a low calcium c o n c e n t r a t i o n d i d n o t m a r k e d l y e n h a n c e t h e release of p h o s p h a t e in response t o P T H ; a t a high calcium c o n c e n t r a t i o n t h e r e was a small n e t release of p h o s p h a t e b y P T H - t r e a t e d bones d e s p i t e a small n e t u p t a k e of calcium. F o r control a n d P T H - t r e a t c d calvaria, t h e p e r c e n t a g e release of p h o s p h a t e was o n l y s l i g h t l y different from that of calcinm, while C T - t r e a t e d c a l v a r i a showed a consistently g r e a t e r u p t a k e of calcium t h a n of p h o s p h a t e .
2. Response to Changes in Phosphate Concentration C a l v a r i a were i n c u b a t e d in m e d i a w i t h initial p h o s p h a t e c o n c e n t r a t i o n s of 3.0, 5.6 or 10.0 ml/100 m l ; t h e calcium c o n c e n t r a t i o n in all cases was 5.9 m l /
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H . H . Messer et al.
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Fig. 2. Effect of initial phosphate concentration on calcium and phosphate uptake or release in bone cultures. Initial calcium concentration was 5.9 mg/100 ml in all cases. Experimental conditions were identical to those for Fig. 1
100 ml. The effect of varying the phosphate concentration in this range was very similar to t h a t of varying the calcium concentration as described above. The extent of mineral loss from control and P T H - t r e a t e d bones was inversely related to initial phosphate levels, while the uptake b y CT-treated bones increased with increasing phosphate concentration (Fig. 2). The stimulatory effect of P T H on Ca and P release, presumably due to enhanced resorption, was abolished by a high phosphate concentration, but not by a low phosphate level. The response to CT persisted throughout the range of phosphate concentrations tested. There was close coupling of calcium release or uptake with phosphate release or uptake. At a low initial phosphate concentration, the loss of phosphate from calvaria exceeded t h a t of calcium on a percentage basis for all treatment groups (p < 0.02 for control and CT groups, p < 0.05 for P T H groups; Student's t test). At intermediate and high phosphate concentrations, the percentage loss or uptake of calcium did not differ significantly from t h a t of phosphate.
3. E//ects o/Constant Calcium: Phosphorus Ratio When both calcium and phosphate concentrations in the culture media were low (Fig. 3), the release of calcium and phosphate b y control and P T H - t r e a t e d bones were not markedly different from values obtained when either ion alone
Medium Ion Concentrations and Bone Metabolism in Culture Calcium
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Fig. 3. Calcium and phosphate uptake or release in bone culture at constant Ca:P ratio in the culture medium. Both calcium and phosphate concentrations were varied in the range 3 to 10 rag/100 ml, at a constant ratio (Ca: P = 1:1). Experimental conditions were identical to those described in Fig. 1
was at a low initial concentration, while the response to CT was reduced. At high concentrations of both calcium and phosphate, all hormonal responses were abolished. Calvaria in all treatment groups took up large quantities of additional mineral from the culture media. The percentage release or uptake of calcium was not significantly different from that of phosphate except for the CT-treated group at intermediate initial calcium and phosphate concentrations (p < 0 . 0 1 ; Student's t test). Discussion
The initial concentrations of calcium and phosphate in the culture media exerted a profound influence on the balance between accretion and resorption of bone in vitro. The two ions were equally effective in modulating resorption and deposition. A low concentiation of either ion appeared to promote a predominance of resorption over accretion, while a high concentration of either ion favored accretion. Hormonal responses were also influenced by the calcium and phosphate concentrations of the culture medium. The most prominent effect was the loss of response to P T H at a high concentration of either calcium or phosphate. The response to CT was much less affected by ion levels in the culture medium. While the extent of calcium and phosphate uptake by CT-treated calvaria was dependent
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H.H. Messer et at.
on the initial levels in the culture media, significant differences from control cultures persisted at all ion levels. The loss of all hormonal responses at high concentrations of both calcium and phosphate may be explained by the extensive precipitation of calcium phosphate in the bones, sufficient to overwhelm any cellular effects. The ability of the bone cells to survive these high concentrations of calcium and phosphate is not k n o ~ , and the deposition of calcium and phosphate in these bones may simply reflect loss of all cellular control. Raisz and Niemann (1969), using fetal rat long bones prelabelled with 45Ca, also reported that a high phosphate concentration in the culture medium inhibited resorption and abolished the response to PTH. However, in contrast to the findings of the present study, they reported that changes in calcium concentration exerted a much lesser effect. I t is unlikely that the differences between the two studies can be explained by differences in culture conditions (animal species, age, choice of bone used, composition of culture medium) or techniques of measuring bone resorption (45Ca release or analytically measured calcium), since the two systems respond similarly in most respects. Since a high plasma calcium concentration in vivo would be expected to lead to reduced bone resorption (via reduced secretion of P T H and increased secretion of CT), a similar result might be expected in vitro, via a local feedback mechanism. Several recent studies have suggested that calcium and phosphate metabolism may be partially independent. I n vivo, CT-induced hypophosphatemia is not necessarily accompanied by hypocalcemia (Messer and Copp, 1974; Talmage et al., 1974). In bone culture studies, it has been shown that calcium may be deposited at the same time as phosphate is being lost (Messer et al., 1974 a; Russell and Talmage, 1972). The extent of such independent movement of ions is clearly limited. When the concentration of either ion alone in the culture media was changed, the loss or uptake of both ions by the calvaria tended to parallel each other closely, although there was some tendency for smaller changes in uptake or release of the ion that was not varied. Except in the case of CT-treated calvaria, which showed a generally greater uptake of calcium than of phosphate, only small differences in the extent of release or uptake between the two ions were observed. This is not surprising since dissolution or precipitation of hydroxyapatite involves both ions in relatively constant proportions, with probably only small deviations from the normal Ca:P ratio being possible. The basis of the greater uptake of calcium than of phosphate by CT-treated calvaria remains unknown. If the in vitro effects of ion concentrations on bone resorption are also operative in vivo, the present findings have possible implications for calcium (and phosphate) homeostatic mechanisms. In the intact animal, fluctuations in plasma calcium concentration are controlled via hormonal responses (PTH and CT secretion), with bone as a major target tissue (Copp, 1969). If the present study reflects the in vivo situation, a direct response of bone to plasma calcium and phosphate concentrations is also suggested. Whether such a mechanism might be mediated via bone cells (as are hormonal effects) or simply by a mass action effect of the ions cannot be determined from these experiments. This possibility must be qualified by the fact that the range of calcium and phosphate concentrations used in this study is greater than that normally encountered in vivo. However, it is
Medium Ion Concentrations and Bone Metabolism in Culture
7
still considered to be within a pathophysiologie range that can be produced in e x p e r i m e n t a l a n i m a l s (Raisz a n d N i e m a n n , 1969). This s~udy was supported by Grant MT 370, Medical Research Council, Canada.
References Copp, D. H. : Endocrine control of calcium homeostasis. J. Endocr. 43, 137-161 (1969) Gomori, G.: A modification of the colorimetric phosphorus determination for use with the photoelectric colorimeter. J. Lab. clin. Med. 27, 955-960 (1942) Heersche, J.N.M.: The effect of thyrocalcitonin and parathyroid hormone on bone metabolism in tissue culture. Proc. kon. ned. Akad. Wet. 72, 286-298 (1969) Messer, H.H., Armstrong, W.D., Singer, L. : Characteristics of calcium uptake by calcit~nintreated mouse calvaria in vitro. Calcif. Tiss. Res. 15, 85-92 (1974a) Messer, H.H., Copp, D. It. : Changes in response to calcitonin following prolonged administration to intact rats. Proc. Soc. exp. Biol. (N.Y.) ]46, 643-647 (1974) Messer, H.H., Yucn, S.Y., Copp, D. H. : Effect of age on bone calcium and phosphate metabolism in organ culture. Calcif. Tiss. Res. 16, 89-92 (1974b) Raisz, L. G., Niemann, I. : Effect of phosphate, calcium and magnesium on bone resorption and hormonal responses in tissue culture. Endocrinology 85, 446-452 (1969) Rasmussen, H. : Ionic and hormonal control of calcium homeostasis. Amer. J. ivied. 50, 567-588 (1971) Reynolds, J.J., Dingle, J.T. : A sensitive in vitro method for studying the induction and inhibition of bone resorption. Calcif. Tiss. Res. 4, 339-349 (1970) Reynolds, J.J., Holick, M.F., DeLuca, II.F.: The role of vitamin D metabolites in bone resorption. Calcif. Tiss. Res. 12, 295-301 (1973) Reynolds, J.J., Holick, M.F., DeLnca, I-I.F. : The effects of vitamin D analogues on bone resorption. Calcif. Tiss. Res. 15, 333-339 (1974) Russell, J.E., Talmage, R. V. : Differential effects of Ca and prior parathyroid state on proline -~H uptake by rat bone in vitro. Amer. J. Physiol. 222, 1604-1608 (1972) Talmage, R.V., Anderson, J.J.B., Kennedy, J.W.: Separation of the hypocalcemic and hypophosphatemic actions of calcitonin with disodium ethane-1-hydroxy-1, 1-diphosphonate. Endocrinology 94, 413-418 (1974)
Original Papers Net Uptake and Release of Calcium and Phosphate by Bone in vitro: Effects of Medium Calcium and Phosphate Concentrations H. H. Messer*, S. Y. Yuen, a n d D. H. Copp Department of Physiology, University of British Columbia, Vancouver Received January 16, accepted June 8, 1975 The effects of varying the initial calcium and phosphate concentrations of the culture media on bone calcium and phosphate release were examined, using whole calvaria from 3-day-old mice in 48-hour cultures. The initial calcium and phosphate concentrations of the culture media were varied in the range 3-10 mg/100 ml; either calcium or phosphate alone was changed while the other ion was held constant, or the concentrations of both were varied while the Ca:P ratio was held constant. For all combinations, 3 treatment groups were used: i) control (no added hormone); ii) 0.5 U/ml PTH; iii) 50 mU/ml CT. The release of calcium and phosphate from the bones was greatest at low initial calcium or phosphate concentrations in the media, and least at high initial concentrations. High concentrations of both ions together abolished hormonal responses and resulted in extensive uptake of calcium and phosphate by the bones. The response to PTH was lost at a high concentration of either ion alone, while a response to CT was observed under all experimental conditions except simultaneously high calcium and phosphate concentrations.
Key words: Bone culture - - Calcium - - Phosphate - - Parathyroid hormone - - Calcitonin
Introduction T h e b a l a n c e b e t w e e n a c c r e t i o n a n d r e s o r p t i o n in b o n e cultures is v e r y sensitive t o a n u m b e r of factors, including age (Messer et al., 1974b), s t r u c t u r a l i n t e g r i t y of t h e bones (Russell a n d T a l m a g e , 1972), p a r a t h y r o i d h o r m o n e ( P T H ) a n d calcitonin (CT) (Heersche, 1969; Messer et al., 1974a; R e y n o l d s a n d Dingle, 1970), a n d t h e a c t i v e m e t a b o l i t e s of v i t a m i n D (Reynolds et al., 1973, 1974). T h e c o n c e n t r a t i o n s of calcium a n d p h o s p h a t e in t h e c u l t u r e m e d i a m a y also p l a y a n i m p o r t a n t role, since these a r e t h e p r e d o m i n a n t c o n s t i t u e n t s of bone mineral. Also, t h e s e ions m a y be i n v o l v e d in m e d i a t i n g h o r m o n a l effects on bone cells (Rasmussen, 1971). R a i s z a n d N i e m a n n (1969) r e p o r t e d t h a t a high p h o s p h a t e c o n c e n t r a t i o n in t h e c u l t u r e m e d i u m r e d u c e d bone resorption, while changes in calcium c o n c e n t r a t i o n e x e r t e d l i t t l e effect. T h e b o n e culture m e t h o d used in t h e i r s t u d y p r e c l u d e d a n i n v e s t i g a t i o n of calcium u p t a k e . W e h a v e i n v e s t i g a t e d t h e effects of changes in t h e calcium a n d p h o s p h a t e c o n c e n t r a t i o n s of t h e c u l t u r e m e d i a on accretion a n d r e s o r p t i o n in c a l v a r i a from 3 - d a y - o l d mice. I n a previous s t u d y (Messer et al., 1974b), c a l v a r i a of this age
For reprints: D. H. Copp, Department of Physiology, University of British Columbia, Vancouver, B. C., Canada. * Present address: Division of Oral Biology, School of Dentistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
2
H . H . Messer et al.
d e m o n s t r a t e d a close b a l a n c e b e t w e e n accretion a n d r e s o r p t i o n in culture, in t h e absence of a d d e d hormones. These bones m a y t h u s be used effectively t o s t u d y changes in t h e b a l a n c e b e t w e e n t h e t w o processes.
Materials and Methods 1. Culture Media. Medium CMRL 1066 was obtained with calcium and phosphate omitted (Grand Island Biological Company, Grand Island, New York). Calcium and phosphate were added as required, using sterile solutions of CaC12 or Na2HPO 4 (containing 10 mg Ca or P per ml), to yield final concentrations of each ion in the range 3-10 mg per 100 ml. The complete culture medium consisted of 95% CMRL 1066, 5% heat-inactivated horse serum, plus penicillin (50 U/ml) and streptomycin (50 ~g/ml). Hormones were added as appropriate: bovine parathyroid hormone (PTH) 0.5 Unit/ml (parathyroid extract Eli Lilly and Company, Indianapolis, Indiana), or calcitonin (CT), 50 mUnits/ml (pure natural salmon ealeitonin, lot AL1025, Armour Pharmaceutical Company, Kankakee, Illinois). 2. Bone Culture Method. Whole calvaria from 3-day-old mice were incubated for 48 h with 2.5 ml culture medium, as described previously (Messer et al., 1974b). Following incubation, the media and calvaria (ashed at 550 ~ were assayed for calcium by atomic absorption spectrophotometry (Instructional Manual, Series 82-360, Jarrell Ash Company, 1964) and phosphate (Gomori, 1942). The initial calcium and phosphate concentrations of all media, and the final calcium and phosphate concentrations of media incubated without bones were also determined. Concentration of the two ions did not change during culture without added bones. ~qet release or uptake by calvaria was then calculated as a percentage of the total calcium and phosphate originally present in each bone. Three treatment groups, each of 7 or 8 calvaria, were used for all levels of calcium and phosphate tested: i) control (no added hormone), ii) 0.5 U/ml PTH, iii) 50 mU/ml CT. Three levels of calcium or phosphate in the culture medium were used, designated as low (3 rag/ 100 ml), intermediate (5-6 mg/100 ml) and high (10-11 rag/100 ml). When either ion alone was varied, the other was held constant at the intermediate level.
Results 1. Response to Changes in Calcium Concentration
T h e effects of initial calcium c o n c e n t r a t i o n of t h e c u l t u r e m e d i u m were i n v e s t i g a t e d using 3 levels of calcium (3.0, 5.9 a n d 10.8 mg/100 ml) while t h e p h o s p h a t e c o n c e n t r a t i o n was h e l d c o n s t a n t a t 5.6 rag/100 ml. T h e n e t release ( p r e s u m a b l y due to resorption) was s t r o n g l y influenced b y t h e initial calcium c o n c e n t r a t i o n (Fig. 1). T h e p e r c e n t a g e loss of bone m i n e r a l b y c a l v a r i a i n c u b a t e d in control m e d i u m (no a d d e d hormones) was a p p r o x i m a t e l y twice as g r e a t when t h e i n i t i a l calcium c o n c e n t r a t i o n was 3.0 rag/100 ml as w h e n t h e initial level was 5.9 rag/100 ml. T h e high initial calcium c o n c e n t r a t i o n (10.8 rag/100 ml) r e s u l t e d in a small n e t calcium a n d p h o s p h a t e u p t a k e b y t h e bones. CT e x e r t e d a m a r k e d influence a t all calcium concentrations. E x c e p t in t h e low calcium group, CT p r o m o t e d a n e t u p t a k e of calcium a n d p h o s p h a t e b y t h e calvaria. T h e initial c a l c m m c o n c e n t r a t i o n of t h e m e d i u m influenced t h e e x t e n t of t h i s calcium a n d p h o s p h a t e u p t a k e , w i t h a n effect a p p r o x i m a t e l y parallel to t h a t of control cultures. T h e response of c a l v a r i a to P T H a t different calcium c o n c e n t r a t i o n s was m o r e compley. A t t h e i n t e r m e d i a t e calcium concentration, P T H e n h a n c e d calcium release b y a p p r o x i m a t e l y 60% a b o v e control values (p ~ 0.01; S t u d e n t ' s t test). H o w e v e r , a t t h e low calcium concentration, when calcium release b y b o t h control a n d P T H - t r e a t e d bones was m u c h greater, t h e
Medium Ion Concentrations and Bone Metabolism in Culture Calcium
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Initial Ca concentratio~l (mgllOOml) Fig. 1. Effect of initial calcium concentration of the culture medium on calcium and phosphate release by bones in organ culture. Initial phosphate concentration was 5.6 mg 100 ml in all cases. Whole calvaria from 3-day-old mice were cultured for 48 h in 2.5 mI culture medlum. Uptake and release are expressed as a percentage of the calcium and phosphate initially present in each calvarium and each value represents the mean =k S.E.M. for 7 or 8 cultures per treatment group
P T H enllanced t h i s b y o n l y a p p r o x i m a t e l y 25% which was n o t significantly different from t h e corresponding control value. A t t h e high calcium level, t h e response t o P T H was lost, a n d a slight u p t a k e of calcium was recorded. The release or u p t a k e of p h o s p h a t e followed t h e s a m e t r e n d s as t h e calcium, even t h o u g h t h e initial p h o s p h a t e c o n c e n t r a t i o n in t h e m e d i a was h e l d c o n s t a n t a t a n h l t e r m e d i a t e level. T h e r e were small differences in t h e n e t m o v e m e n t of t h e t w o ions u n d e r some conditions. F o r e x a m p l e , a low calcium c o n c e n t r a t i o n d i d n o t m a r k e d l y e n h a n c e t h e release of p h o s p h a t e in response t o P T H ; a t a high calcium c o n c e n t r a t i o n t h e r e was a small n e t release of p h o s p h a t e b y P T H - t r e a t e d bones d e s p i t e a small n e t u p t a k e of calcium. F o r control a n d P T H - t r e a t c d calvaria, t h e p e r c e n t a g e release of p h o s p h a t e was o n l y s l i g h t l y different from that of calcinm, while C T - t r e a t e d c a l v a r i a showed a consistently g r e a t e r u p t a k e of calcium t h a n of p h o s p h a t e .
2. Response to Changes in Phosphate Concentration C a l v a r i a were i n c u b a t e d in m e d i a w i t h initial p h o s p h a t e c o n c e n t r a t i o n s of 3.0, 5.6 or 10.0 ml/100 m l ; t h e calcium c o n c e n t r a t i o n in all cases was 5.9 m l /
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100 ml. The effect of varying the phosphate concentration in this range was very similar to t h a t of varying the calcium concentration as described above. The extent of mineral loss from control and P T H - t r e a t e d bones was inversely related to initial phosphate levels, while the uptake b y CT-treated bones increased with increasing phosphate concentration (Fig. 2). The stimulatory effect of P T H on Ca and P release, presumably due to enhanced resorption, was abolished by a high phosphate concentration, but not by a low phosphate level. The response to CT persisted throughout the range of phosphate concentrations tested. There was close coupling of calcium release or uptake with phosphate release or uptake. At a low initial phosphate concentration, the loss of phosphate from calvaria exceeded t h a t of calcium on a percentage basis for all treatment groups (p < 0.02 for control and CT groups, p < 0.05 for P T H groups; Student's t test). At intermediate and high phosphate concentrations, the percentage loss or uptake of calcium did not differ significantly from t h a t of phosphate.
3. E//ects o/Constant Calcium: Phosphorus Ratio When both calcium and phosphate concentrations in the culture media were low (Fig. 3), the release of calcium and phosphate b y control and P T H - t r e a t e d bones were not markedly different from values obtained when either ion alone
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Fig. 3. Calcium and phosphate uptake or release in bone culture at constant Ca:P ratio in the culture medium. Both calcium and phosphate concentrations were varied in the range 3 to 10 rag/100 ml, at a constant ratio (Ca: P = 1:1). Experimental conditions were identical to those described in Fig. 1
was at a low initial concentration, while the response to CT was reduced. At high concentrations of both calcium and phosphate, all hormonal responses were abolished. Calvaria in all treatment groups took up large quantities of additional mineral from the culture media. The percentage release or uptake of calcium was not significantly different from that of phosphate except for the CT-treated group at intermediate initial calcium and phosphate concentrations (p < 0 . 0 1 ; Student's t test). Discussion
The initial concentrations of calcium and phosphate in the culture media exerted a profound influence on the balance between accretion and resorption of bone in vitro. The two ions were equally effective in modulating resorption and deposition. A low concentiation of either ion appeared to promote a predominance of resorption over accretion, while a high concentration of either ion favored accretion. Hormonal responses were also influenced by the calcium and phosphate concentrations of the culture medium. The most prominent effect was the loss of response to P T H at a high concentration of either calcium or phosphate. The response to CT was much less affected by ion levels in the culture medium. While the extent of calcium and phosphate uptake by CT-treated calvaria was dependent
6
H.H. Messer et at.
on the initial levels in the culture media, significant differences from control cultures persisted at all ion levels. The loss of all hormonal responses at high concentrations of both calcium and phosphate may be explained by the extensive precipitation of calcium phosphate in the bones, sufficient to overwhelm any cellular effects. The ability of the bone cells to survive these high concentrations of calcium and phosphate is not k n o ~ , and the deposition of calcium and phosphate in these bones may simply reflect loss of all cellular control. Raisz and Niemann (1969), using fetal rat long bones prelabelled with 45Ca, also reported that a high phosphate concentration in the culture medium inhibited resorption and abolished the response to PTH. However, in contrast to the findings of the present study, they reported that changes in calcium concentration exerted a much lesser effect. I t is unlikely that the differences between the two studies can be explained by differences in culture conditions (animal species, age, choice of bone used, composition of culture medium) or techniques of measuring bone resorption (45Ca release or analytically measured calcium), since the two systems respond similarly in most respects. Since a high plasma calcium concentration in vivo would be expected to lead to reduced bone resorption (via reduced secretion of P T H and increased secretion of CT), a similar result might be expected in vitro, via a local feedback mechanism. Several recent studies have suggested that calcium and phosphate metabolism may be partially independent. I n vivo, CT-induced hypophosphatemia is not necessarily accompanied by hypocalcemia (Messer and Copp, 1974; Talmage et al., 1974). In bone culture studies, it has been shown that calcium may be deposited at the same time as phosphate is being lost (Messer et al., 1974 a; Russell and Talmage, 1972). The extent of such independent movement of ions is clearly limited. When the concentration of either ion alone in the culture media was changed, the loss or uptake of both ions by the calvaria tended to parallel each other closely, although there was some tendency for smaller changes in uptake or release of the ion that was not varied. Except in the case of CT-treated calvaria, which showed a generally greater uptake of calcium than of phosphate, only small differences in the extent of release or uptake between the two ions were observed. This is not surprising since dissolution or precipitation of hydroxyapatite involves both ions in relatively constant proportions, with probably only small deviations from the normal Ca:P ratio being possible. The basis of the greater uptake of calcium than of phosphate by CT-treated calvaria remains unknown. If the in vitro effects of ion concentrations on bone resorption are also operative in vivo, the present findings have possible implications for calcium (and phosphate) homeostatic mechanisms. In the intact animal, fluctuations in plasma calcium concentration are controlled via hormonal responses (PTH and CT secretion), with bone as a major target tissue (Copp, 1969). If the present study reflects the in vivo situation, a direct response of bone to plasma calcium and phosphate concentrations is also suggested. Whether such a mechanism might be mediated via bone cells (as are hormonal effects) or simply by a mass action effect of the ions cannot be determined from these experiments. This possibility must be qualified by the fact that the range of calcium and phosphate concentrations used in this study is greater than that normally encountered in vivo. However, it is
Medium Ion Concentrations and Bone Metabolism in Culture
7
still considered to be within a pathophysiologie range that can be produced in e x p e r i m e n t a l a n i m a l s (Raisz a n d N i e m a n n , 1969). This s~udy was supported by Grant MT 370, Medical Research Council, Canada.
References Copp, D. H. : Endocrine control of calcium homeostasis. J. Endocr. 43, 137-161 (1969) Gomori, G.: A modification of the colorimetric phosphorus determination for use with the photoelectric colorimeter. J. Lab. clin. Med. 27, 955-960 (1942) Heersche, J.N.M.: The effect of thyrocalcitonin and parathyroid hormone on bone metabolism in tissue culture. Proc. kon. ned. Akad. Wet. 72, 286-298 (1969) Messer, H.H., Armstrong, W.D., Singer, L. : Characteristics of calcium uptake by calcit~nintreated mouse calvaria in vitro. Calcif. Tiss. Res. 15, 85-92 (1974a) Messer, H.H., Copp, D. It. : Changes in response to calcitonin following prolonged administration to intact rats. Proc. Soc. exp. Biol. (N.Y.) ]46, 643-647 (1974) Messer, H.H., Yucn, S.Y., Copp, D. H. : Effect of age on bone calcium and phosphate metabolism in organ culture. Calcif. Tiss. Res. 16, 89-92 (1974b) Raisz, L. G., Niemann, I. : Effect of phosphate, calcium and magnesium on bone resorption and hormonal responses in tissue culture. Endocrinology 85, 446-452 (1969) Rasmussen, H. : Ionic and hormonal control of calcium homeostasis. Amer. J. ivied. 50, 567-588 (1971) Reynolds, J.J., Dingle, J.T. : A sensitive in vitro method for studying the induction and inhibition of bone resorption. Calcif. Tiss. Res. 4, 339-349 (1970) Reynolds, J.J., Holick, M.F., DeLuca, II.F.: The role of vitamin D metabolites in bone resorption. Calcif. Tiss. Res. 12, 295-301 (1973) Reynolds, J.J., Holick, M.F., DeLnca, I-I.F. : The effects of vitamin D analogues on bone resorption. Calcif. Tiss. Res. 15, 333-339 (1974) Russell, J.E., Talmage, R. V. : Differential effects of Ca and prior parathyroid state on proline -~H uptake by rat bone in vitro. Amer. J. Physiol. 222, 1604-1608 (1972) Talmage, R.V., Anderson, J.J.B., Kennedy, J.W.: Separation of the hypocalcemic and hypophosphatemic actions of calcitonin with disodium ethane-1-hydroxy-1, 1-diphosphonate. Endocrinology 94, 413-418 (1974)
