The effects of parathyroid hormone (PTH) on bone collagen synthesis were assessed in organ cultures of fetal rat calvaria by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and non-collagen protein (NCP) using purified bacterial collagenase. 1) PTH decreased the incorporation of labeled proline into CDP at concentrations similar to those which stimulate bone resorption in vitro. 2) This effect was observed in bones treated for 6 h, but not for 3 h; it was maximal at 24 h and was maintained for 96 h. Bones treated with PTH for 48 h and transferred to control media for 48 h showed recovery of CDP labeling to control values. 3) the effect was specific for bone collagen. There was little alteration in the incorporation of proline into NCP, and incorporation into collagen was not inhibited. 4) The effect could be ascribed to decreased collagen synthesis and not to changes in amino acid uptake, precursor pool size, or degradation of newly synthesized CDP. In 3 hour experiments, PTH did increase the labeling of CDP and NCP, but only at tracer concentration of proline in the medium, compatible with an early stimulation of amino acid uptake. 5) Similar inhibition was observed with purified bovine (1-84) PTH and synthetic bovine PTH (1-34) as well as with crude homologous PTH obtained from rat parathyroid gland culture fluid. Human (hCT) and salmon (sCT) calcitonin did not inhibit the effect of PTH on the labeling of CDP nor did they stimulate CDP labeling directly at concentrations which inhibited bone resorption. Dibutyryl cyclic-3',5'-adenosine monophosphate (D3cAMP) inhibited labeling of CDP at concentrations of .03 to .3 mM, thus mimicking the action of PTH. However, in this system DBcAMP inhibited 45Ca release, thus mimicking CT. We conclude that the direct effect of PTH on bone collagen synthesis is a slow reversible inhibition, not opposed by CT. This effect may be mediated by cAMP formation in bone cells.
The effects of vitamin D metabolites on bone collagen synthesis were assessed in organ cultures of fetal rat calvaria by measuring the incorporation of a 2-h pulse of [ :) H]proline into collagenase-digestible (CDP) and non-collagen protein (NCP) by using purified bacterial collagenase. Addition of la,25-dihydroxyvitamin Da (la,25(OH)2D.i) to cultures containing 5% vitamin D-deficient serum produced a dose-related inhibition of incorporation of proline into CDP over the range of 10"" to 10~7 M at 24 h. Similar inhibition was obtained with la,25(OH)2D,) in 4-day cultures by using medium supplemented with bovine serum albumin instead of serum. The effect on the labeling of CDP was selective and in most experiments there was no effect on incorporation of proline into NCP. la,24R,25-trihydroxyvitamin D. t (la,24R,25(OH).,D.,) at K)-9 -10-7 M also inhibited the labeling of CDP, but three other compounds, 25-hydroxy-, 24R,25-dihydroxy-, and 24S,25-dihydroxyvitamin D.i, had no consistent effect. To test whether a metabolite of vitamin D in serum might affect collagen synthesis, bones were cultured with 1-10% serum from vitamin D-deficient and vitamin D-treated animals. There was no greater labeling of CDP and NCP with vitamin D-sufficient serum. We conclude that the vitamin D metabolites which are most active in stimulating bone resorption, la,25(OH) 2 D:t and la,24R,25(OH). } D.), also inhibit bone collagen synthesis in vitro. No evidence for a stimulatory effect on bone collagen synthesis was obtained by using the currently available natural metabolites of vitamin D or by comparing serum from vitamin D-deficient or -treated animals. (Endocrinology 102: 731 , 1978) V ITAMIN D appears to be required for skeletal growth and mineralization. Hence, it seems paradoxical that the active form of the vitamin, la,25-dihydroxyvitamin D3 (la,25(OH) 2 D;3), is a potent stimulator of bone resorption (1). One explanation for this apparent discrepancy is that intestinal absorption of calcium and phosphate is increased to a greater extent by the active metabolites of vitamin D than is bone resorption. This would increase the serum calcium and phosphate concentrations, thus enhancing bone mineralization, while at the same time suppressing parathyroid hormone (PTH) so that bone resorption might not be increased in vivo. It may also be important to stimulate bone resorption in order to maintain skeletal growth because, at least in bone remodeling, new bone formation generally occurs at resorption sites. In either case, vitamin D would have a permissive, rather than a direct, role in bone growth. The initial stimulus to bone formation might be from other growth-regulating hormones such as insulin and somatomedin(s) (2, 3), by local feedback effects of changes in mineralization, or by serum calcium and phosphate concentration themselves (4).An attractive alternative is that vitamin D or its metabolites have a direct stimulatory effect, either upon collagen synthesis by osteoblasts or upon bone mineralization. In vivo experiments (5) and...
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