Calcium in osteoblast-enriched bone cells.

The exchangeability, location, and amount of the total calcium in bone cells were studied in relation to their osteoblastic activity. Cells were isolated from neonatal rat calvariae by sequential collagenase digestion and incubated with 45Ca2+ before or after various treatments. 45Ca2+, 40Ca2+, and DNA were determined on each cell sample. Long-term experiments were performed on cultured cells. The cells closest to the forming bone had the highest alkaline phosphatase activity and the highest Ca2+ content (i.e., 17 mm Ca2+/l cell water, as compared to soft tissue cells, which have 2-3 mm Ca2+/l (Borle 1981). About 50% of this Ca2+ exchanges readily with 45Ca2+ and appears to be located almost entirely in the cell membrane. Thirty-five percent exchanges only slowly with a t1/2 of 27 hours, and is located within the cell, principally in the mitochondria as seen in pyroantimonate fixed cells (Neuman et al. 1985). In spite of its slow exchange-ability, this Ca2+ fraction can be mobilized or augmented rapidly when Ca2+ supply is reduced or increased in vitro or in vivo. 1,25(OH)2D3 given in vivo increased this intracellular Ca2+ when the Ca2+ supply was low and released it when the Ca2+ supply was high. About 15% of the Ca2+ in these cells was nonexchangeable. These results suggest that osteoblasts do process more Ca2+ when closer to the mineralization site. Whether this Ca2+ is en route from blood to bone or from bone to blood will require further study.

The calcium-buffering phase of bone mineral: some clues to its form and formation.

Devitalized, neonatal, rat calvariae incubated in a buffered solution release Ca2+ and Pi during the first day into the medium until levels reach 1.2 mM and 2.6 mM, respectively. Thereafter levels gradually decrease to stabilize by the fifth day at 0.5 mM and 2 mM. Comparison of these solubility changes with those of known calcium phosphate salts suggests that calvarial solubility is controlled by a mixture of impure, soluble, apatite precursors, which at cell death transform to an impure apatite. Increasing concentrations of Mg2+ increased the rate of Ca2+ release and, at 3 mM, stabilized Ca2+ by the fifth day at 0.9 mM. Decreasing the pH from 7.4 to 7.2 produced similar solubility changes as did the addition of citrate at 0.3 mM, phosphocitrate at 0.3 mM, ATP at 0.1 mM, and the bisphosphonate, HEBP, at 0.1 mM. Osteocalcin did not increase calvarial solubility but was able to slow the rate of calcium phosphate phase transition if present at the time of in vitro crystal formation. Phosphocitrate and HEBP were also more effective when present during in vitro crystal formation. HEBP was most effective when present during biological crystal formation, as shown by the increased solubility of devitalized calvariae removed after in vivo administration of HEBP. In vivo manipulations of osteoclast activity produced changes in plasma calcium which correlated with the solubility of the corresponding calvariae after removal and devitalization. Low milk intake increased calvarial solubility. Increasing doses of 1,25(OH)2D3 increased plasma calcium and calvarial solubility, both of which were reversed by injection of acetazolamide. It was concluded from this survey of devitalized bone solubility that calcium exchange between bone and body fluids can buffer calcium homeostasis in the young rat. The exchange is passive. The active components appear to be osteoblastic formation of soluble apatite precursors and their stabilizers and, in reverse, osteoclastic transformation of apatite to precursors by H2CO3 secretion.

Blood:bone disequilibrium. VI. Studies of the solubility characteristics of brushite: apatite mixtures and their stabilization by noncollagenous proteins of bone.

Recent evidence of the occurrence of brushite in newly formed bone mineral prompted a study of the solubility properties of brushite:apatite mixtures under physiological conditions and the influence on them of pH, lactate, pyruvate, and, particularly, noncollagenous bone proteins (NCBPs). Brushite alone was surprisingly stable in solution at pH 7.4, 37 degrees C. In the presence of increasing amounts of apatite, hydrolysis of brushite to an insoluble phase occurred. A decrease of 0.1 pH unit or the addition of 1.5 mM pyruvate or 10 mM lactate increased the ion activity product (Ca2+ x HPO4(2-) 3 or more times. However, within the loose envelope of bone such conditions so different from those in the circulation might be only local or temporary. NCBPs, on the other hand, stabilized brushite in solution alone as well as in the presence of apatite for days. They probably act by adsorbing strongly to the crystal surface and preventing nucleation by apatite. This brushite-apatite-bone protein system exhibits solubility characteristics that can resolve the old problems presented by the participation of the skeleton in extracellular calcium homeostasis on the one hand, and by the apparent insolubility of the apatite mineral of bone on the other.

Blood: bone disequilibrium. V. Effects of a diphosphonate (EHDP) on phosphate fluxes in rat calvaria.

Calvaria taken from rats (normal or thyroparathyroidectomized) given a diphosphonate (EHDP 10 mg/kg daily for 7 days) were placed in special Ussing chambers for the measurement of phosphate fluxes to and from bone. When compared with appropriate controls, the experimental calvaria showed a marked decrease in influx, K, a significant increase in the projected equilibrium concentration, E/K, where E is the efflux and a significant increase in the calcium concentration in the medium. It is concluded that this large increase in passive "solubility" is caused by the diphosphonate's ability to stabilize the small amount of regulator phase (brushite or brushite-apatite mixture) present in bone.

A study of bone proteins which can prevent hydroxyapatite formation.

The ability of noncollagenous bone proteins (NCBP) to inhibit calcium phosphate precipitation in vitro raises the question as to the nature and the relative efficiency of such proteins in vivo. To investigate this question NCBP from young adult sheep bones were fractionated using nondegradative techniques. Their relative activity was measured by their efficiency in preventing hydroxyapatite growth. On a weight (of protein recovered) basis, the activity is about equally divided between the Gla-containing protein, osteocalcin, and a phosphorylated protein essentially the same as osteonectin. On a molecular weight basis, the activity of the phosphorylated protein is almost three times higher than that of the Gla-containing protein. Finally, the inhibitory activity of the phosphorylated protein is alkaline phosphatase sensitive. The properties of this protein could provide a means of regulating the solubility of bone mineral and maintaining an equilibrium of calcium between bone and blood.

Studies of diffusion in calvaria.

The rates of diffusion of small ions and neutral molecules through isolated calvaria have been determined. Compared with data published on self-diffusion and diffusion through cartilage, H2O, 3-O-methylglucose, lactate, sulfate, and methylamine diffuse at approximately 2/3 the expected rate. Diffusion of H2O and sulfate was unaffected by the administration of the diphosphonate 1-hydroxyethane-1,1-diphosphonate (EHDP), although phosphate fluxes are markedly diminished. Diffusion of water was nearly doubled by vitamin D deficiency. A 1-week treatment with 1,25(OH)2D3 had no effect on H2O diffusion while returning calcium influx to normal. It is concluded that bone matrix permits a flow of small neutral and ionized molecules nearly comparable to that in cartilage. When changes in calcium and phosphate influx are observed, they can be ascribed to the exchange properties of bone mineral and not to changes in matrix permeability.

Blood/bone disequilibrium: IV. Reciprocal effects of calcium and phosphate concentrations on ion fluxes.

Quantitative measurements were made of the ion fluxes of calcium and phosphate into and from calvaria (mouse or rat) when clamped in specially designed micro-Ussing chambers. The effects of varying concentrations of calcium were examined on the influx and efflux of calcium and of its counterion, phosphate. A comparable series of experiments was performed with varying phosphate concentrations. Both ions, as their concentrations increased, depressed their own influx, increased their own efflux, and significantly increased the equilibrium concentration, E/K, supported by the calvaria. Similarly, both ions, as their concentrations increased, affected the influx or efflux of their counterion only slightly but did depress the counterion's equilibrium level, E/K, significantly. In spite of these changes it was shown that calvaria effectively buffered the medium at physiological concentrations of calcium and phosphate. The buffering capacity, however, was small, and the balance, E/K, was modified by small uptake or loss of either ion. The small size of the interacting mineral pool was confirmed by direct measurement of the rapidly exchanging fractions of both calcium or phosphate. They were only approximately 1% of the total ions present. The significance of these findings is discussed.

Formation and serum disappearance of fragments of parathyroid hormone in the infused dog.

Radioiodinated parathyroid hormone [125I-PTH(1-84)] was infused into intact dogs. At various times venous samples were chromatographed to determine the levels of intact hormone (1-84) and large fragments in the circulation. Both bioactive (electrolytically iodinated) and inactive (chloramine-T-labeled) preparations were used. In both instances, plateau concentrations of intact hormone and of large metabolite(s) were quickly reached. After cessation of infusion the levels of intact hormone and metabolite(s) quickly declined. Clearly, in the dog, the peripheral formation and disappearance of large fragments of exogenous PTH occur at rates comparable to the clearance of the intact hormone itself.

Blood:bone disequilibrium. III. Linkage between cell energetics and Ca fluxes.

Specially designed Ussing chambers were used for the quantitation of fluxes of Ca2+ and lactate ions to and from viable calvaria taken from 2- to 3-day-old rat pups and young adult mice. The latter in vitro support Ca2+ levels comparable to plasma levels in vivo. The effects of various metabolic inhibitors and parathyroid hormone were studied. The results support previous findings that Ca influx into bone is a passive concentration-dependent process not significantly affected by alterations in the energy status of the bone cells. In large measure, the efflux from bone also appears to be passive. However, significant alterations in the calcium levels supported by efflux from bone were observed as acute effects of metabolic inhibitors. Part of the metabolically derived efflux may be attributed to acid production (aerobic glycolysis). However, part must be ascribed to cellular activities not yet defined.

The influx of pyrophosphate ions into calvaria in vitro.

The flux of 32P-labeled inorganic pyrophosphate (PPi) into bone was studied in vitro using Ussing chamber techniques and calvaria from newborn rat pups. Because insignificant hydrolysis and backflux of PPi took place under experimental conditions, it was possible to study the penetration of bone membranes by PP1 unambiguously. At physiological concentrations, the influx was found to be linearly concentration dependent and to follow first order kinetics, apparently a simple diffusion process. In magnitude, the intrinsic constant for influx was found to be approximately one-half that of inorganic phosphate ions under comparable conditions.

The metabolism of labeled parathyroid hormone. VII Attempts to modify hormone deposition.

Using a 125I-labeled parathyroid hormone with apparently full biological activity, a series of experiments was performed on rats to determine the effects of several physiological parameters on the deposition and metabolism of the hormone in the target tissues, liver, kidney and bone. Among the variables studied were: systemic phosphate levels, systemic calcium levels, calcitonin administration, pregnancy, and dosage (at low levels). Some small variations in deposition and tissue metabolism of the hormone were observed but the invariance of the pattern of deposition and the rapid automatic destruction by target tissues was most impressive.

The metabolism of labeled parathyroid hormone. II. Methodological studies.

Further studies on the preparation of 125I-labeled, biologically active parathyroid hormone have resulted in improvements in the procedure, in the storage of the labeled product, and in the establisment of the location of the label in the polypeptide chain. Approximately 95% of the label is on the single tyrosyl residue, amino acid 43, in the bovine hormone. New procedures have also been developed for the isolation and quantitative gel filtration of the labeled hormone deposited in tissues (liver, kidney and bone).

The metabolism of labeled parathyroid hormone. IV. Autoradiographic studies.

Autoradiographs were prepared from tissues of rats sacrificed 10 minutes after injection of biologically active 125I-labeled parathyroid hormone. No radioactivity was seen in intestine and muscle. Deposition in liver was diffuse showing some sinusoidal concentrations. Depostion in kidney was high and, nearly all activity appeared in selected tubules (presumably proximal tubules) in the outer third of the cortex. Specific localization was also seen in bone particularly in the cellular layers of periosteum and endosteum adjacent to bony matrix and to some extent in osteocytes.

The metabolism of labeled parathyroid hormone. III. Studies in rats.

A series of experiments indicated that 125I-parathyroid hormone administered to the rat i.v. was deposited very rapidly in three organ systems only, bone, kidney and the liver. As indicated by increasing solubility in trichloracetic acid, the hormone seemed to be rapidly metabolized at the sites of deposition. The experiments were in part repeated employing quantitative extraction and gel filtration procedures. It was found that intact hormone predominated (82%) in the circulation but was cleared very rapidly: 91, 97, and 99% having left by 10, 30 and 60 minutes respectively after injection. The intact hormone was found at 10 minutes to be principally in kidney, bone, and liver. Although there was evidence of fragmentation preferentially to polypeptides of molecular weights of 6000, 4500 and 2500 these cannot be claimed to represent real molecular entities. By one hour, only a few per cent of the injected hormone remained intact, the bulk having been degraded to fragments of small molecular weight.

The metabolism of labeled parathyroid hormone. V. Collected biological studies.

Biologically active 125I-labeled parathyroid hormone (125I-PTH) was used in a series of studies in dogs and chickens designed to confirm and augment earlier studies in rats. As in rats, a three exponential equation was required to describe disappearance of 125I-PTH from the blood in the dog. The first two "half-lives" (1.8 and 7 min) accounted for the bulk of the dose. Also as in rats, deposition of apparently intact hormone took place rapidly in kidney, liver and bone in both the dog and the chicken. Degradation occurred very rapidly in all three target organs. Three labeled hormones of different biological activities were compared in the rat. Inactive, oxidized hormone was rejected by the liver but showed markedly increased deposition in kidney and the higher the purity of the hormone the higher was its uptake by liver. Exploration of a wide range of dosages revealed few effects on distribution (smaller deposition in liver and kidney at highest dosages, 65 mug/rat). Fresh sera did not degrade hormone rapidly or extensively. There was no deposition of hormone in intestinal mucosa, marrow, and red cells. Nephrectomy increased deposition in liver and bone. Finally, the perfused liver was capable of extensive degradation of the hormone.

The metabolism of labeled parathyroid hormone. VI. Effects of vitamin D status.

Labeled 125I-PTH with full biological activity was utilized to investigate the interrelationship between vitamin D and parathyroid hormone (PTH) function. The distribution of labeled PTH was studied in rats and chickens on diets containing various amounts of vitamin D and calcium. The effects of vitamin D deficiency were found to be minor. There was a slightly delayed (10--30%) blood clearance of the hormone and a smaller deposition in the liver (20--30%) in the D-deficient animals. Deposition in bone and kidney appeared to be essentially normal. It is concluded that any failure of PTH to evoke a bone response in the deficient animal is not due to the failure of binding of hormone at the target organ.