Bone matrix studies Influences of parathyroid extract,calcitonin, and cholecalciferol and of rickets and its treatment

Bones from young rats were incubated with radioactive glucosamine and proline. The concentrations and specific activities of matrix glycosaminoglycan fractions, prepared by a cetylpyridinium chloride method, and the specific activity of insoluble collagen hydroxyproline were determined. Acute parathyroid extract treatment increased labelling of hyaluronic acid and a glycopeptide fraction. These effects were partially blocked by calcitonin treatment which had no effect by itself. Parathyroid extract inhibited collagen synthesis and this effect was not blocked by calcitonin. Effects of these two hormones on labelling of chondroitin sulfate fractions were more variable. Vitamin D-3 caused an increase in labelling of all matrix fractions measured in bone from thyroparathyroidectomized rats, but its stimulating effect upon collagen synthesis was blocked by paratyroid extract.Bones from rats made rachitic on a phosphorus and vitamin D-deficient diet were incubated in vitro with radioactive glucosamine and proline. Over a three-week period rachitic bone exhibited a progressive fall in concentration and labelling of a glycopeptide-hyaluronic acid fraction, while pair-fed animals supplemented either with phosphorus alone or with phosphorus and vitamine D-3 not only remineralized their bones, but the bones showed a pronounced increment in concentration and labelling of this fraction. Both treatment regimens also enhanced chondroitin sulfate and collagen labelling.     

Collagenase, procollagenase and bone resorption. Effects of heparin, parathyroid hormone and calcitonin.

1. The addition of heparin to the culture fluid of mouse tibiae or calvaria did not cause any significant resorption of bone collagen or mineral. However, heparin (or analogue sulfated polyanions), enhanced greatly the amount of latent, trypsin-activatable collagenase (i.e. procollagenase) released by the bones in the medium without influencing that of directly active collagenase which was always very low. Heparin appeared to act by increasing the production of the enzyme which is immediately excreted. Procollagenase and collagenase are not stored in bone tissue, even under conditions where it is in active resorption. 2. Parathyroid hormone induced in the explants a resorption of both mineral and collagen that was inhibited by calcitonin. These hormones, however, had no influence on the release of procollagenase or collagenase either in the presence or in the absence of heparin. 3. Once activated, bone collagenase digested the collagen of the bone explants, and more extensively after their demineralization. Thus the latent collagenase that accumulates around non-resorbing bones has to be considered as a precursor, (and not as a residue), of active enzyme. 4. Active collagenase added to incipient cultures of bones disappeared with a half-life of 24 h. The lost enzyme could, however, not be reactivated by trypsin and thus was not transformed into latent procollagenase.     

Experimental study of changes in osteoblastic shape induced by calcitonin and parathyroid extract in an organ culture system

Summary Neonate rat endocranial osteoblasts were cultured on their bone surfaces in control medium (CC) or medium to which either parathyroid extract (PTE) or calcitonin (CT) had been added for 2, 4, 8 or 24 h. Some were cultured for 24 h in CC, then for 2, 4, 8 or 24 h in either CT or PTE medium; or for 24 h in PTE, then for 2, 4, 8 or 24 h in either CC or CT; or 24 h in CT and 2, 4, 8 or 24 h in CC. The dorsal ruffling of the cells in CC was found to be suppressed by later culturing with PTE and the disoriented cells reorganized to form arrays of parallel cells. The effects of PTE were also reversed by CC or CT: the osteoblasts in the second culture (CC) lost elongation and order, and proceeded through a proliferative phase before exhibiting the ruffling form similar to a single CC 24 h culture. PTE-cultured osteoblasts showed an increase in cell overlap and contact so that a more competent barrier was formed separating the bone from the medium. In control or CT medium, however, intercellular gaps were greater than in vivo.We are grateful for the expert technical assistance of Elaine Bailey, for laboratory facilities kindly provided by Dr. Martin Evans, and for financial support from the Medical Research Council     

Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture.

Rabbit articular chondrocytes in suspension culture synthesize Type II collagen [3alpha1(II)] in the absence of extracellular Ca2+ and Type I collagen [2alpha1(I) - alpha2] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca2+ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl2. If added directly to the suspension culture medium containing no CaCl2, calcitonin stimulates an active efflux of Ca2+ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.     

Rat enterocyte Na+ transport in vitro. Action of parathyroid hormone and calcitonin

Parathyroid hormone (PTH) and calcitonin exert well known effects on the renal tubule which are thought to involve specific hormone receptors and adenyl cyclase. In the intestine, it is not clear whether the action of PTH and calcitonin is only indirect or also direct, and their mechanisms of action are much less well established. In the present study, possible direct effects of PTH and calcitonin on Na+ transport in isolated intestinal epithelial cells of rats were investigated. In the presence of bovine PTH (1.2 I.U/ml) in the incubation medium, the Na+ efflux rate constant (oKNa) of isolated enterocytes was significantly reduced when compared to that in control experiments with the hormone vehicle only. The mean depression of oKNa induced by bovine PTH was 26% as compared to the control (100%) and to that induced by ouabain (4.0 mM) which was 44%. No depressant effect of bovine PTH on oKNa was observed when the isolated enterocytes were incubated with ouabain (4.0 mM). Thus, bovine PTH appeared to inhibit the ouabain-sensitive Na+ pump. When incubating the isolated epithelial cells in an EGTA-containing CA2+-free medium, bovine PTH lost its capacity to inhibit oKNa. Thus, the presence of extracellular Ca2+ appeared necessary for the inhibitory effect of bovine PTH. In contrast to its effect on oKNa, bovine PTH induced no change in net Na+ uptake by isolated enterocytes. Moreover, no significant effect on enterocyte Na+ transport could be demonstrated for salmon or porcine calcitonin at two different concentrations in the incubation medium, Neither bovine PTH nor salmon calcitonin induced significant changes in enterocyte cyclic AMP or cycle GMP concentrations. It was concluded that bovine PTH, but not calcitonin, exerted a directed inhibitory effect on the ouabain-sensitive oKNa of isolated rat enterocytes. The effect of bovine PTH occurred without measurable activation of the cyclic nucleotide system but needed the presence of Ca2+ in the incubation medium to be operative.     

The effect of thyroparathyroidectomy, parathyroid hormone and calcitonin on plasma strontium in rats

The effect of thyroparathyroidectomy (TPTX) on the plasmatic Sr concentrations in rats previously supplemented with this element, has been studied, as well as its effect on the treatment of TPTX rats with hormonal combinations and, finally, the one presenting hormonal excess or defect of the phosphocalcium metabolism regulating hormones: parathormone (PTH) and calcitonin (CT). Twenty four hours after TPTX, the plasmatic Sr concentrations show a pattern similar to those of Ca and Mg and contrary to Pi. The subsequent evolution is different, as the plasmatic concentrations increase, probably due to the maintenance of Sr supplementation. The administration of this element to TPTX rats and the treatment with a hormonal combination with two of the following hormones: PTH, CT and T4 antagonize the hormonal effect on the restoration of the plasmatic concentrations of the elements analyzed. The PTH excess and defect (TPTX treated with CT + T4) show plasmatic increases in Sr; the CT excess provokes decreases while the defect (administration of PTH + T4) causes increases. The T4 administration reproduces the CT effects, but inconsistently. These results suggest that CT may be the hormone that plays a regulating role in the plasmatic Sr concentrations.     

Effect of propranolol and metoprolol on parathyroid hormone and calcitonin secretions in uraemic patients.

Nine uraemic patients not being treated by dialysis received intravenous propranolol 1 microgram/kg/min for 85 minutes after a priming dose of 1 mg. Fifteen days later, six of them received intravenous metoprolol 1.2 microgram/kg/min after a priming dose of 1.2 mg. Plasma concentrations of parathyroid hormone (PTH) and calcitonin fell significantly after propranolol but not after metoprolol, whereas no change in plasma concentrations of ionised calcium and phosphate occurred with either drug. Heart rate fell similarly with both drugs. The fact that propranolol acutely suppressed PTH and calcitonin secretion in uraemic patients indicates that further studies are warranted to assess the long-term effects of the drug on the secretion of these hormones and on renal osteodystrophy. The contrast between the responses to propranolol and metoprolol supports the concept that PTH and calcitonin secretion is modulated through specific beta 2-receptors.     

Identification of calcitonin gene related peptide in ovine hypothalamic extract.

Calcitonin gene-related polypeptide (CGRP) was purified from ovine hypothalamic extracts. Its amino acid sequence was determined as: Ser-(Cys)-Asn-Thr-Ala-Thr-(Cys)-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser- Arg-Ser - Gly-Gly-Val-Val-Lys-Ser-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Gln-Ala-Phe- NH2. This sequence differs from rat CGRP by two amino acid substitutions (Ser for Asp25 and Gln for Glu35). Adenylate cyclase stimulating activity in rat pituitary cell cultures was monitored during the isolation. CGRP had adenylate cyclase stimulating activity comparable to corticotropin-releasing hormone, suggesting a hypophysiotropic role for CGRP. This is the first chemical characterization of CGRP in the brain (hypothalamus).     

Localization of cell groups sensitive to parathyroid hormone and calcitonin in rat skeletal tissue.

The level of cyclic AMP in various fractions of rat skeletal tissue was measured after in vitro or in vivo administration of parathyroid hormone and calcitonin. Incubations of bone fractions prepared from young (5 weeks of age thyroparathyroidectomized rats revealed that both parathyroid hormone and calcitonin increased the cyclic AMP level in fractions of epiphysis, metaphysis and marrow cells. Cyclic AMP accumulation in incubated perisoteum and diaphysis were induced solely by parathyroid hormone. In in vivo experiments the cyclic AMP level in the tibia of the thyroparathyroidectomized rat was increased by infusion of either parathyroid hormone or calcitonin, and the simultaneous administration of each maximally effective dose of the two hormones exhibited an additive effect. Within 2 min, parathyroid hormone infusion caused an elevation of cyclic AMP content in periosteum and metaphysis. Rapid increase of cyclic AMP in the metaphysis was also induced by calcitonin, and the effect of the two hormones on cyclic AMP accumulation in this fraction was additive. Small but significant increase of cyclic AMP in the diaphysis was detected at 5 min after the administration of parathyroid hormone. Calcitonin infusion did not show any consistent effects on periosteum and diaphysis.     

Mapping polypeptide hormone genes in the mouse: somatostatin, glucagon, calcitonin, and parathyroid hormone

Mouse-Chinese hamster hybrids segregating mouse chromosomes were analyzed by Southern hybridization techniques to map the genes for somatostatin (Smst), glucagon (Gcg), calcitonin (Calc), and parathyroid hormone (Pth). The mouse gene for somatostatin, detected on a 20-kb EcoRI fragment, is located on mouse chromosome 16. Glucagon cDNA hybridized to a 14-kb EcoRI fragment residing on chromosome 2. Calcitonin and parathyroid hormone genes, detected on 7.8-kb HindIII and 6.0-kb BamHI fragments, respectively, were on mouse chromosome 7. The calcitonin and parathyroid hormone genes appear to be part of a larger linkage group which has been conserved in mouse and man.     

Calcium-binding proteins in the parathyroid gland. Detailed studies of parathyroid secretory protein

In order to identify calcium (Ca2+)-binding proteins in the parathyroid gland, we used electrophoretic blots of proteins separated by a two-dimensional nondenaturing/denaturing gel system and incubated them with 45Ca2+. Parathyroid secretory protein (PSP) and proteins with approximate molecular weights of 98,000, 88,000, 58,000, and 30,000 were noted to bind Ca2+ in cytosolic fractions from bovine parathyroid, adrenal, and pituitary glands. However, differences in the binding affinity and capacity of the various proteins were observed. PSP displayed a low affinity and high binding capacity for Ca2+. In the presence of 5 mM MgCl2 and 60 mM KCl, native PSP (immobilized on nitrocellulose filters) bound 7.5 mol of Ca2+/mol of protein monomer with an apparent Kd of 1.1 mM. Immunoblotting identified the association of PSP with parathyroid cell membranes in a Ca2+-dependent manner. This property, together with its heat stability, distinguished PSP from other cytosolic Ca2+-binding proteins which were identified. There was also evidence for a Ca2+-dependent protein-protein interaction (aggregation) of PSP present in a Nonidet P-40 extract of cell membranes. The high Ca2+ binding capacity of PSP and its Ca2+-dependent membrane association may be features that make PSP a potentially important protein in secretory cells.     

Cell population dynamics of periodontal ligament stimulated with parathyroid extract.

Young adult rats were injected with parathyroid extract (PTE). Periodontal ligament (PDL) adjacent to a previously resorbing alveolar bone surface was divided into four zones number I to IV, from bone to cementum. Zones I and IV were within 25 mmu of the bone and cementum surfaces, respectively, while a line bisecting the center of the PDL separated Zones II and III. A net increase of about 16 total nuclei in all zones was observed during the first five hours after PTE administration. Since local mitosis accounted for no more than two nuclei, approximately 14 cells apparently migrated into the area. Over the first five hours Zones I and II combined showed a 21-cell increase, being apparently the sole recipients of cells migrating into the field (14) plus approximately seven more from Zone III, which lost cells during the time period. The concurrent histological observation in Zone II, of increased intravascular monocytes and perivascular macrophages during the first five hours, suggests cells are migrating into Zone II via vascular channels. The data suggest two sources for increased PDL cellularity associated with stimulated osteoclast histogenesis: (1) local PDL cellular proliferation and (2) influx of migrating cells (probably through vascular channels) during first five hours after PTE.     

Serum levels of calcitonin, parathyroid hormone and 25-hydroxycholecalciferol in patients with diabetes mellitus

Blood serum levels of calcitonin, parathyroid hormone, calcium, magnesium and inorganic phosphate have been measured in basal condition and following intravenous administration of calcium in 31 patients with diabetes of type I, in 31 patients with diabetes of type II and in 29 healthy subjects. The level of 25-hydroxy cholecalciferol was measured in all these patients in basal condition only. It was found that the basal calcitonin level was significantly higher in patients with both types of diabetes than in healthy subjects. The administration of calcium caused a significantly higher increase in the blood calcitonin level in patients with type I diabetes than in those with type II diabetes. It was found in addition that in women with type II diabetes blood serum level of parathyroid hormone was significantly higher than that in men suffering from diabetes of the same type, suggesting the participation of some sex-related factor in the pathogenesis of the abnormal parathyroid level in these patients.     

In vitro studies of calcitonin release in man.

The influence of various agents on calcitonin release from human thyroid was studied in vitro. Under the condition of this investigation, calcium, magnesium and phosphate did not stimulate calcitonin release from short-term incubated slices of human thyroid. However, pentagastrin and USP glucagon were potent stimulators of calcitonin release. Theophylline and dibutyryl cyclic AMP were also potent stimuli. A highly purified preparation of pancreatic glucagon was without an effect. Those agents which stimulated calcitonin release were associated with augmented cyclic AMP accumulation. Although maximal discharge of calcitonin required the presence of calcium, out in vitro experiments raise the question as to whether a gastrointestinal hormone, rather than calcium, might not be the principal agent affecting calcitonin release.