High phosphate level directly stimulates parathyroid hormone secretion and synthesis by human parathyroid tissue in vitro

Phosphate retention plays an important role in the pathogenesis of secondary hyperparathyroidism in patients with renal failure. In in vitro studies, high extracellular phosphate levels directly stimulate PTH secretion in rat and bovine parathyroid tissue. The present study evaluates the effect of high phosphate levels on the secretion of PTH and the production of prepro PTH mRNA in human hyperplastic parathyroid glands. The study includes parathyroid glands obtained from patients with primary adenomas and from hemodialysis and kidney-transplant patients with diffuse and nodular secondary hyperplasia. The experiments were performed in vitro using small pieces of parathyroid tissue. The ability of high calcium levels to decrease PTH secretion was less in adenomas than in secondary hyperplasia; among the secondary hyperplasia, nodular was less responsive to an increase in calcium than diffuse hyperplasia. In diffuse hyperplasia, PTH secretion was increased in response to 3 and 4 mM phosphate compared with 2 mM phosphate, despite a high calcium concentration in the medium; prepro PTH mRNA levels increased after incubation in 4 mM phosphate. Similar results were obtained with nodular hyperplasia, except that the elevation of PTH secretion in response to 3 mM phosphate did not attain statistical significance. In adenomas, high calcium concentrations (1.5 mM) did not result in inhibition of PTH secretion, independent of the phosphate concentration, and the prepro PTH mRNA was not significantly increased by high phosphate levels. In conclusion, first, the PTH secretory response to an increase in calcium concentration is less in nodular than diffuse hyperplasia; second, high phosphate levels directly affect PTH secretion and gene expression in patients with advanced secondary hyperparathyroidism.     

Diltiazem stimulates parathyroid hormone secretion in vivo whereas felodipine does not

The impact of two calcium channel blockers of different structure, diltiazem and felodipine, on PTH secretion was studied under hyper- and hypocalcemic conditions. Six healthy volunteers were investigated before and after treatment with felodipine, then after treatment with diltiazem. Under each of these three conditions, they received first a calcium infusion (0.109 mmol/kg over 130 min). Blood was drawn every 5-10 min for measurements of Ca2+ and intact PTH concentrations, and urine was collected over the infusion periods for measurements of calcium and creatinine. Basal levels of Ca2+ and intact PTH concentrations were similar under the three conditions. During calcium infusion, Ca2+ increased linearly from 1.27 to 1.51 mmol/L during the control period. Based on the whole response curve, Ca2+/time, this rise was less marked (P < 0.002) during each of the calcium channel blocker periods than under control conditions, although the three values of urinary calcium excretion were similar. In addition, PTH secretion was less suppressed on diltiazem than on felodipine therapy or during the control period (P < 0.04). During EDTA infusion, Ca2+ decreased in a linear way from 1.27 to 1.07 mmol/L during the control period. Based on the whole response curve, Ca2+/time, this decrease was more marked during felodipine than during diltiazem treatment or in the control period (P < 0.001). Although Ca2+ concentrations did not differ between the control and diltiazem periods, PTH levels were 1.3-fold higher (P < 0.0001) during diltiazem, but similar in the control and felodipine periods. These data demonstrate that diltiazem, but not felodipine, stimulates PTH secretion in vivo in man, with a maximal effect observed under hypocalcemic conditions.     

Phosphorus deficiency, parathyroid hormone and bone resorption in the growing rat

Sham-operated and parathyroidectomized (PTX) rats were divided into two pair-fed groups, one on a normal mineral intake (0.5% Ca, 0.3% P), the other on a regimen low in phosphorus (0.5% Ca, 0.03% P). P depletion led to a drop in plasma P and urine P, a rise in plasma Ca and a marked rise in urine Ca, a drop in serum magnesium and a rise in urine Mg. The changes were more pronounced in the PTX animals, but final values were the same in both groups. Parallel bone-seeking isotope (85Sr, 177Lu, 237Np) studies in nonablated animals revealed an increase in the urinary nuclide output and in the urine/tibia ratio in P-deficient animals. Normal and primary bone osteocytes decreased and enlarged osteocytes increased as a result of P deficiency; osteoclasts and osteoblasts also increased. Bone composition showed a drop in ash content and a rise in water, with a light decrease in both Ca and P, and a corresponding rise in hydroxyproline and nitrogen in the P-deficient animals. The results are interpreted to mean that P-deficiency in the young growing rat leads to an increase in bone resorption which occurs also in the absence of parathyroid hormone (PTH). The fact that final values were similar in the control and PTX P-deficient animals suggests that steady-state regulation can also occur without PTH. Because P-deficiency leads to rapid hypercalcemia and rapid marked hypercalciuria, there may exist a mechanism for phosphate regulation which would then supersede Ca homeostasis. The change in serum and urine Mg levels may reflect a decrease in tubular Ca and Mg reabsorption associated with P-deficiency.     

In vitro parathyroid hormone release in patients with secondary hyperparathyroidism

BACKGROUND: The purpose of this study was to determine the precise endocrine characteristics of parathyroid function in secondary hyperparathyroidism (sHPT). METHODS: We examined the effects of extracellular ionized calcium (Ca2+) varying from 0.5 to 2.0 mM on parathyroid hormone (PTH) release in parathyroid cell suspensions using a mid-regional PTH assay. Cells were obtained from 26 patients with sHPT who were divided into two groups according to the type of hyperplasia they exhibited, either nodular (n = 16) or diffuse (n = 10). For comparison, we also analyzed data from nine patients with primary hyperparathyroidism (pHPT; adenomas). RESULTS: Significant in vitro suppression of PTH release by Ca2+ was observed in the majority of subjects, regardless of the histologic abnormality. The pHPT group exhibited no significant relationship between clinical and in vitro data. In contrast, in the sHPT group (taken as a whole), suppression of PTH release by Ca2+ exhibited a plateau at a total serum calcium concentration of 2.5 mmol/L, and a parathyroid gland weight of 2 g. CONCLUSIONS: These findings suggest that there is a curvilinear relationship in sHPT, but not pHPT, between the in vitro calcium sensitivity of parathyroid cells and total serum calcium, as well as gland weight. The in vitro calcium sensitivity in sHPT remains constant when the total serum calcium concentration exceeds 2.5 mmol/L, or when the gland weight exceeds 2 g.     

Intermittent parathyroid hormone administration stimulates bone formation in the mandibles of aged ovariectomized rats

OBJECTIVE: To assess the effect of various antirheumatic drugs on cytokine, cytokine inhibitor, and prostaglandin E (PGE) production by normal blood mononuclear cells (MNC) and rheumatoid arthritis (RA) synovial fibroblasts in vitro. METHODS: MNC from healthy donors and RA synovial fibroblasts were preincubated with or without prostaglandin E2 (PGE2), indomethacin, dexamethasone, gold sodium thiomalate (GSTM), methotrexate (MTX), and cyclosporin A (CyA), and then cultured in the absence or presence of interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) for 48 h. We characterized cytokines such as IL-1 beta, IL-8, monocyte chemoattractant protein-1 (MCP-1), and cytokine inhibitors such as IL-1 receptor antagonist (IL-1ra) and soluble TNF receptors (sTNFR p55 + p75) as well as PGE in the cell-free culture supernatants. RESULTS: In MNC and synovial fibroblast cultures dexamethasone, GSTM, and PGE2 most markedly downregulated spontaneous and/or cytokine stimulated production of IL-1 beta, IL-14a, IL-8, and MCP-1, whereas sTNFR shedding was not affected. In contrast, MTX and CyA had only marginal or no effects on mediator release, whereas indomethacin inhibited only PGE production. CONCLUSION: Among several antirheumatic drugs examined, dexamethasone and GSTM exhibited the most potent inhibitory effects on inflammatory cytokine and cytokine inhibitor production by blood mononuclear cells and synovial fibroblasts. These drugs may exert their antiinflammatory actions by unspecific suppression of monocyte and fibroblast secretory function.     

Secretion of parathyroid hormone by abnormal human parathyroid glands in vitro

Radiation survival curves for Lewis lung tumours in the lungs ranging in size from 0-5 to 20 mm3 have been obtained, and a size-dependent variation in hypoxic fraction was found. Cell-survival studies following treatment of various sizes of s.c. tumours indicated that the effects of 60Co gamma-rays and the chemotherapeutic agents 1,3-bas(2-chloroethyl)-1-nitrosourea (BCNU) and cyclophosphamide are all size-dependent. Large pulmonary nodules which had regressed but had not been cured by cyclophosphamide regrew with a radiosensitivity that was characteristic of previously untreated tumours. The results give additional experimental support to the clinical interest in early adjuvant therapy of micrometastases, and sequential combined modality therapy for larger tumours.     

Cytochrome P-450 inhibition blocks bone resorption in vitro and in vivo

BACKGROUND AND METHODS: To find an intra-abdominal pressure (IAP) range for laparoscopic procedures that elicits only moderate splanchnic and pulmonary hemodynamic and metabolic changes, including hepatic and intestinal tissue pH and superficial hepatic blood flow, we installed an IAP of 7 and 14 mm Hg each for 30 minutes in 10 healthy pigs (30 +/- 4 kg). RESULTS: In parallel with the increase of IAP, the mean transmural pulmonary artery pressure increased (from 25 +/- 3 to 27 +/- 4 at 7 mm Hg IAP and 30 +/- 6 mm Hg at 14 mm Hg IAP, p < 0.05); the pulmonary artery-to-pulmonary capillary wedge pressure gradient also increased (from 17 +/- 2.7 to 21 +/- 3 mm Hg at 7 mm Hg IAP and 24 +/- 4.2 mm Hg at 14 mm Hg IAP, p < 0.01), and the arterial oxygenation decreased (p < 0.005). Relevant changes at an IAP of 14 mm Hg were observed in right atrial pressure during inspiration (from 7 +/- 2 to 12 +/- 3 mm Hg, p < 0. 0001) and in abdominal aortic flow (from 1.43 +/- 0.4 to 1.19 +/- 0. 3 L/min, p < 0.01). However, transmural right atrial pressure and cardiac output remained essentially unchanged. Portal and hepatic venous pressure increased in parallel with the IAP (portal: from 12 +/- 3 to 17 +/- 3 at 7 mm Hg IAP and 22 +/- 3 mm Hg at 14 mm Hg IAP, p < 0.01; hepatic venous: from 8 +/- 3 to 14 +/- 6 at 7 mm Hg IAP and 19 +/- 6 mm Hg at 14 mm Hg IAP, p < 0.005), but the transmural portal and hepatic venous pressures decreased (p < 0.01), indicating decreased venous filling. Portal flow was maintained at 7 mm Hg but decreased at 14 mm Hg from 474 +/- 199 to 395 +/- 175 mL/min (p < 0. 01), whereas hepatic arterial flow remained stable. Hepatic superficial blood flow decreased during insufflation and increased after desufflation. Tissue pH fell together with portal and hepatic venous pH (intestinal: from 7.323 +/- 0.05 to 7.217 +/- 0.04; hepatic: from 7.259 +/- 0.04 to 7.125 +/- 0.06, both p < 0.01) at 14 mm Hg. CONCLUSION: The hemodynamic and metabolic derangement in the pulmonary and splanchnic compartments are dependent on the extent of carbon dioxide pneumoperitoneum. The effect of low IAP (7 mm Hg) on splanchnic perfusion is minimal. However, higher IAPs (14 mm Hg) decrease portal and superficial hepatic blood flow and hepatic and intestinal tissue pH.     

Phagocytosis-enhancing effect of lactoferrin on bovine peripheral blood monocytes in vitro and in vivo

The effect of lactoferrin (LF) on in vitro and in vivo phagocytic ability of bovine blood monocytes was studied. It was demonstrated that bovine LF enhanced in vitro phagocytosis of bacteria and ovine erythrocyte-antibody complexes and increased intracellular killing of Staphylococcus albus. Monocytes of colostrum deprived calves, which were intravenously injected with LF, also exhibited elevated phagocytic properties.     

Assay of bone resorption in vivo with 3H-tetracycline

3H-Tetracycline (3H-TC) was used to quantify resorption in whole bones of growing rats and dogs. After repeated isotopic labeling of actively growing embryos or neonates, 3H-TC was observed to be distributed homogeneously and in equilibrium with 45Ca. A rapid and large loss of 3H-TC and a small loss of 45Ca occurred during the early weeks of rapid bone growth, suggesting that absolute amounts of 45Ca resorbed from bone, as reflected by losses of 3H-TC, are five to ten times greater than the net amounts of 45Ca lost from bone. Minimal loss of 3H-TC occurred due to nonspecific physicochemical exchange in vivo or in vitro (5%) except with nonradioactive tetracycline, and 3H-TC was not greatly exchanged or reused (10%) in vivo. The data are considered in terms of local and systemic conservation of calcium.     

The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation

During vertebrate limb development, growth plate chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (-/-) cells, and analyzed cell-cell interactions in the growth plate in vivo. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic chondrocytes. Further, ectopic hypertrophic chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.     

Conversion of proparathyroid hormone to parathyroid hormone: studies in vitro with trypsin

The conversion of proparathyroid hormone to parathyroid hormone (PTH) was studied in vitro employing pancreatic trypsin as a prototype converting enzyme. Digestion of intact radiolabeled bovine prohormone with trypsin (0.1%) (w/w) resulted in release of a peptide comigrating with intact hormone marker in systems resolving both on the basis of charge (urea polyacrylamide gels, pH 4.4) and size (sodium dodecyl sulfate-urea polyacrylamide gels, pH 7.2). Tryptic digestions of a synthetic analogue of bovine prohormone, ProPTH-(-6 + 34), consisting of the prohormone hexapeptide covalently bonded to the NIH2 terminus of the active fragment of the hormone, released in high yield the hexapeptide and the intact active hormone fragment before any other smaller fragments. Analyses of digestions were by: (i) thin-layer chromatography and amino acid analysis of digestion products; (ii) comparison of the biological activity of the prophormone substrate and the products of digestion; and (iii) peptide end-group analysis by the Edman method during progressive tryptic hydrolysis over 22 h. The latter experiments demonstrated cleavage of more than 75% of the hexapeptide-hormone peptide bond before cleavage of other trypsin-sensitive sites within the molecule. It is concluded that the specificity of cleavage at the hexapeptide-hormone bond in the process of intracellular hydrolysis of proparathyroid hormone resides primarily in the sequence and/or conformation of the precursor molecule; inasmuch as conversion of prohormone to hormone can be efficiently accomplished by pancreatic trypsin in vitro, there is, therefore, no need to postulate the existence of an intracellular converting enzyme within the parathyroid cell that possesses unique hydrolytic specificity.     

Effects of hormone replacement therapy in bone resorption, in post-menopausal women

BACKGROUND: The effects of different therapies on bone loss rate can be measured using biochemical markers of bone resorption such as urinary hydroxyproline. AIM: To study the effects of hormone replacement therapy on urinary hydroxyproline in postmenopausal women. PATIENTS AND METHODS: Eighty three postmenopausal women without hormone replacement therapy, 54 postmenopausal women receiving hormone replacement therapy and 16 premenopausal women (considered as the control group) were studied. Hydroxyproline was measured in an early morning urine sample, after one day of diet without meat or gelatin. RESULTS: Urinary hydroxyproline in premenopausal women was 33.7 +/- 7.9 mg/g creatinine. The figure for postmenopausal women with hormonal replacement therapy was 33.7 +/- 5.9 mg/g creatinine. Postmenopausal women without replacement therapy had an urinary hydroxyproline of 47.4 +/- 8.5 mg/g creatinine, significantly higher than that of premenopausal and supplemented women. In 21 postmenopausal women, hydroxyproline was measured before and after three months of replacement therapy, values decreased 35.5 +/- 11% in this period and there was a direct correlation between initial values and the degree of reduction (r = 0.69, p < 0.001). CONCLUSIONS: Postmenopausal women receiving hormone replacement therapy have a urinary hydroxyproline excretion similar to that of premenopausal women.     

In vivo and in vitro macrophage activation by systemic adjuvants

Six systemic adjuvants of immunity were tested for their ability to induce macrophage activation. Four of them: living BCG, hydrosoluble extracts from BCG (HIU II) and from M.smegmatis (IPM), and lipopolysaccharide from E.coli (LPS), when administered to normal mice render macrophages non-specifically cytotoxic for tumor cells in vitro. The intensity of this phenomenon varied according to the route and time of adjuvant administration. In contrast, lentinan extracted from Lentinus edodes, and levamisole which is a synthetic chemical compound, depressed macrophage cytotoxic potential. BCG, IPM and LPS were shown to have a direct action on macrophages. After in vitro exposure to these agents, the cytotoxic potential of normal macrophages was greatly increased. Levamisole was unable to stimulate this macrophage function directly in vitro. On the other hand, such a macrophage activation has been induced in vitro when normal macrophages were cultivated in the presence of MIF coming from the supernatant of human lymphoblastoid cell lines.     

In vitro production of parathyroid hormone-related protein by cholesteatoma and normal skin

Severe inflammation and infection of the middle ear occasionally results in clinical evidence of bone resorption but with the addition of the cholesteatoma epithelium it becomes inevitable. This study examined production by the cholesteatoma epithelium of a bone resorbing factor, namely parathyroid hormone-related protein (PTH-rP), which would not be expected in inflammatory states alone. PTH-rP was detected in the conditioned medium of primary and secondary cell cultures derived from 12 cholesteatoma biopsies. The levels of PTH-rP were significantly greater than in control cultures of cells derived from normal scalp tissue. Production by the cholesteatoma epithelium may explain the increased incidence of bone resorption in this disease, particularly in cases where inflammation is minimal.     

The effect of Kampo formulae on bone resorption in vitro and in vivo. I. Active constituents of Tsu-kan-gan

Four water extracts of Kampo formulae (Yi-kkan-sen, Dai-ho-in-gan, Ni-chi-gan, Tsu-kan-gan) were screened for their inhibitory activities on bone resorption induced by parathyroid hormone (PTH) in organ culture using neonatal mouse parietal bones. Among the Kampo formulae, Tsu-kan-gan (TKG) showed the most potent inhibitory activity. We further fractionated the TKG water extract by monitoring the inhibitory activity on bone resorption stimulated by PTH in vitro. The MeOH fraction of the water extract inhibited PTH-stimulated bone resorption, and its inhibitory activity was more potent than those of other fractions. The MeOH fraction was then subjected to Sephadex LH-20 column chromatography to give fractions I, II and III, which were examined for bone resorption activity. Fraction I inhibited PTH-stimulated bone resorption, and its inhibitory activity was more potent than those of the other fractions. Upon oral administration of the three fractions (100 mg/kg/d) to ovariectomized (OVX) mice, fractions I and III prevented the decrease of bone mineral density (BMD) of the lumbar vertebra. Eleven compounds isolated from the MeOH fraction were examined for their inhibitory effect on PTH-stimulated bone resorption. Among them, berberine (1), syringin (3), limonin (4) and mangiferin (10) showed a significant inhibitory effect on bone resorption. In the formation assay of osteoclast-like cells, these compounds decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs). The inhibitory effect of TKG on bone resorption may be at least partly due to the inhibitory action of these compounds.     

The cellular actions of interleukin-11 on bone resorption in vitro

The pleiotropic cytokine interleukin-11 (IL-11) stimulates osteoclast formation in vitro, but it is not known whether it influences other steps in the bone-resorptive cascade. Using a variety of in vitro model systems for studying bone resorption we have investigated the effects of IL-11 on 1) osteoclast formation, fusion, migration, and activity; and 2) osteoblast-mediated osteoid degradation. The involvement of matrix metalloproteinases (MMPs) and products of arachidonic acid metabolism in IL-11-mediated resorption were also assessed. We first examined the bone-resorptive effects of IL-11 by assessing 45Ca release from neonatal mouse calvarial bones. IL-11 dose-dependently stimulated bone resorption with an EC50 of 10(-10) M. The kinetics of IL-11-mediated 45Ca release demonstrated that it was without effect for the first 48 h of culture, but by 96 h, it stimulated 45Ca release to the same level as that produced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (a hormone that stimulates osteoclast formation and activity). IL-11 also produced a dose-dependent increase in osteoblast-mediated type I collagen degradation with a maximum of 58.0 +/- 6.2% at 5 x 10(-9) M; this effect of IL-11 was less than that produced by 1,25-(OH)2D3 (76.5 +/- 7.1%) and was prevented by an inhibitor of MMPs, but not those blocking arachidonic acid metabolism. We then tested the effects of IL-11 on isolated mouse osteoclasts cultured on ivory slices in the presence and absence of primary mouse osteoblasts. IL-11 had no effect on isolated osteoclast activity even in coculture with primary osteoblasts. We then examined the effects of IL-11 on the formation of osteoclast-like multinucleate cells in mouse bone marrow cultures and the resorptive activity of such cultures using ivory as a substrate. IL-11 dose-dependently increased 1) the number of tartrate-resistant acid phosphatase-positive osteoclast-like multinucleate cells and 2) the surface area of lacunar resorption, although the effects were less than that of 1,25-(OH)2D3. The effect of IL-11 on bone marrow lacunar resorption was prevented by a combination of inhibitors of 5-lipoxygenase and cyclooxygenase. In 17-day-old metatarsal bones, IL-11 prevented the migration of (pre)osteoclasts to future resorption sites, whereas their fusion was unaffected. These results provide strong evidence that IL-11 stimulates bone resorption by enhancing osteoclast formation and osteoblast-mediated osteoid degradation rather than stimulating osteoclast migration and activity. Our data also suggest that the stimulatory effects of IL-11 involve both MMPs and products of arachidonic acid metabolism.     

Osteoid resorption by mononuclear cells in vitro

For the first time, mononuclear cell-mediated ingestion of osteoid in cultures of long bones of fetal rats is described and characterized. The mononuclear cells, located at sites of osteoid deposition, ingest collagen fibrils and clumps of mineral crystals which are segregated within cytoplasmic vacuoles or multivesicular bodies. The ingestion of osteoid continues in cultures treated with agents that normally inhibit osteoclastic bone resorption. Morphologically, the osteoid-containing cells are characterized by a moderate number of mitochondria and short-stranded rough endoplasmic reticulum, a modest Golgi apparatus and variable numbers of vesicles, vacuoles, and multivesicular bodies. The morphologic appearance of the mononuclear cell is consistent with that of a macrophage.