Hyperphosphataemia and bone resorption in histamine injected laying hens

The effects of histamine on bone resorption during eggshell formation, were studied. The study included the effect of histamine on the clearance of 45Ca and 32P from plasma, and on the release from the femur or uptake by the egg shell of 45Ca and 32P injected at the time of the first experiment or 10 hr prior to the experiment (experiment 2). Histamine led to a decrease in the disappearance rates of 45Ca and 32P, without any modification of the femur and egg shell levels. However, the increase in the amount of plasma 32P and the decrease in the 32P specific activity (SA) in bone, both elicited by histamine, argues in favour of the skeletal origin of hyperphosphataemia induced by histamine during egg shell formation.     

Urinary excretion of pyridinium crosslinks: a new marker of bone resorption in metabolic bone disease

The pyridinium derivatives hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are intermolecular crosslinking compounds of collagen which are only present in its mature form. Contrasting to the wide distribution of type I and II rollagens, HP and LP are absent from skin, ligament and fascia, and their major sources are bone and cartilage. Using a specific HPLC assay, we have determined the 24-h excretion of HP and LP crosslinks in normal adults of both sexes, in patients with primary hyperparathyroidism and in patients with Paget's disease of bone before and after intravenous treatment with aminopropylidene bisphosphonate (APB). Mean adult normal values were 33 ± 13 pmol/μmol creatinine for HP and 6.3 ± 3.4 pmol/μmol creatinine for LP. In women, menopause induced a 2–3-fold increase of HP and LP reflecting the well documented postmenopausal increase of bone turnover. In the urine of patients with primary hyperparathyroidism and of patients with active Paget's disease of bone, urinary crosslinks were significantly higher than in age-matched controls, with a mean 3- and 12-fold increase, respectively. Urinary excretion of hydroxyproline is a well recognized but poorly sensitive marker of bone turnover, reflecting resorption. In the same patients, the effect of menopause and disease state on hydroxyproline excretion was much less dramatic than on HP and LP. During intravenous APB treatment of pagetic patients, there was an early decrease of HP and LP, which was significant after 24 h and reached 62% at 4 days, contrasting with a late and milder decrease of urinary hydroxyproline. Because APB is a potent inhibitor of resorption which does not have a direct short-term effect on bone formation, these data also indicate that urinary excretion of HP and LP reflect only coilagen degradation occurring during osteoclastic resorption and not the degradation of newly synthesized collagen. We conclude that urinary HP and LP excretion represents the firs sensitive and specific marker of bone resorption. Its use should be valuable in the clinical investigation of metabolic bone diseases, especially osteoporosis.     

The effect of calcium supplements on plasma alkaline phosphatase and urinary hydroxyproline in postmenopausal women

Although calcium supplements are widely used in the treatment of osteoporosis, their beneficial effect is not conclusively established. We now report some effects of a calcium supplement (1 g/day) given for 6 to 12 weeks to 15 postmenopausal osteoporotic women. The mean fasting urinary hydroxyproline/creatinine ratio decreased from 0.021 +/- 0.002 to 0.015 +/- 0.001 (P less than 0.0025), indicating a significant reduction in bone resorption. The mean plasma alkaline phosphatase fell from 123 +/- 5 U/l to 104 +/- 3.1 U/l (P less than 0.01), probably representing some secondary reduction in bone formation following the inhibition of bone resorption. These results support the concept that calcium supplementation is useful in the treatment of postmenopausal osteoporosis.     

High sodium chloride intake exacerbates immobilization-induced bone resorption and protein losses

We examined, in immobilization, the effect of a diet high in sodium chloride (NaCl) on bone markers, nitrogen balance, and acid-base status. Eight healthy male test subjects participated in a 14-day head-down-tilt bed rest (HDBR) study. During the bed rest period they received, in a randomized crossover design, a high (7.7 meq Na(+)/kg body wt per day) and a low (0.7 meq Na(+)/kg body wt per day) NaCl diet. As expected, 24-h excretion of urinary calcium was significantly greater in the high-NaCl-intake HDBR phase than in the low-NaCl-intake HDBR phase (P < 0.001). High NaCl intake caused a 43-50% greater excretion of the bone resorption markers COOH- (CTX) and NH(2)- (NTX) terminal telopeptide of type I collagen in HDBR than low NaCl in HDBR (CTX/NTX: P < 0.001). Serum concentrations of the bone formation markers bone-specific alkaline phosphatase (bAP) and NH(2)-terminal propeptide of type I procollagen (PINP) were identical in both NaCl intake phases. High NaCl intake led to a more negative nitrogen balance in HDBR (P < 0.001). Changes were accompanied by increased serum chloride concentration (P = 0.008), reduced blood bicarbonate (P = 0.017), and base excess (P = 0.009) whereas net acid excretion was lower during high than during low NaCl intake in immobilization (P < 0.001). High NaCl intake during immobilization exacerbates disuse-induced bone and muscle loss by causing further protein wasting and an increase in bone resorption. Changes in the acid-base status, mainly caused by disturbances in electrolyte metabolism, seem to determine NaCl-induced degradation processes.     

JunD suppresses bone formation and contributes to low bone mass induced by estrogen depletion

JunD is an activator protein-1 (AP-1) component though its function in skeletal system is still not fully understood. To elucidate the role of JunD in the regulation of bone metabolism, we analyzed JunD-deficient mice. JunD deficiency significantly increased bone mass and trabecular number. This bone mass enhancement was due to JunD deficiency-induced increase in bone formation activities in vivo. Such augmentation of bone formation was associated with simultaneous increase in bone resorption while the former was dominant over the latter as accumulation of bone mass occurred in JunD-deficient mice. In a pathological condition relevant to postmenopausal osteoporosis, ovariectomy reduced bone mass in wild type (WT) mice as known before. Interestingly, JunD deficiency suppressed ovariectomy-induced increase in bone resorption and kept high bone mass. In addition, JunD deficiency also enhanced new bone formation after bone marrow ablation. Examination of molecular bases for these observations revealed that JunD deficiency enhanced expression levels of c-jun, fra-1, and fra-2 in bone in conjunction with elevated expression levels of runx2, type I collagen, and osteocalcin. Thus, JunD is involved in estrogen depletion-induced osteopenia via its action to suppress bone formation and to enhance bone resorption.     

The influence of nitric oxide synthase inhibitor L-NAME on bones of male rats with streptozotocin-induced diabetes

The pathophysiological processes underlying the development of diabetic osteopenia has not hitherto been elucidated. Induction of streptozotocin diabetes leads in our experiments to decrease of bone density, ash, mineral content and to thinner cortical width compared to control male rats. In order to investigate the pathogenetic role of bone resorption by osteoclasts in streptozotocin-induced diabetes, we determined the circulating levels of tartrate-resistant acid phosphatase (TRAP), a biochemical marker for bone resorption. Plasma TRAP values in diabetic rats did not differ from their corresponding controls. Streptozotocin diabetes by itself did not have any effect on the weight of seminal vesicles which are highly testosterone-dependent. Low doses of nitric oxide cause bone resorption, but higher doses of NO inhibit bone resorbing activity. We examined the effect of L-NAME (inhibitor of nitric oxide production) after six weeks of administration to diabetic rats. There was no further significant loss of bone mineral density, ash and mineral content or tibia weight in diabetic rats treated with L-NAME. L-NAME itself did not decrease bone metabolism. In our study no evidence of an increased bone resorption was found. Our results have indicated that a predominance of bone resorption over bone formation is not involved in the pathogenesis of diabetes-associated osteopenia. Inhibition of NO neither increased osteoclastic activity (TRAP) nor induced osteopenia in L-NAME-treated rats. This suggests a possibility that NO is not involved in the pathogenesis of diabetic osteopenia.     

Fluoride reduces the rate of dissolution of bone

Recently, fluoride has been used in the treatment of osteoporosis in an attempt to rebuild bone lost due to this disease. Fluoride has been shown to have a profound effect on osteoblasts and bone formation. Studies have shown that synthetic fluorapatite is more stable and less soluble than hydroxyapatite. This study was designed to determine the dissolution properties of bones from rats fed a normal diet versus rats fed a high fluoride diet. Intact and deproteinized bones were dissolved in buffered solutions, containing physiological amounts of inorganic Ca and P, at pH 3.4, 4.4 and 5.4 The data demonstrate that fluoridated bones dissolve slower than normal bones, deproteinized bones dissolve faster than intact bones and that the initial rate of dissolution of fluoridated bone is always significantly lower than that of normal bone. This may help to explain the reduced rate of osteoclastic resorption in patients undergoing fluoride therapy.     

Blood coagulation and bone metabolism: some characteristics of the bone resorptive effect of thrombin in mouse calvarial bones in vitro

Chronic inflammatory processes are often associated with bone resorption. Stimulated by the current great interest in the role of coagulation factors in inflammation and immune injury, we have studied the effect of thrombin on mouse calvarial bones in vitro. Thrombin caused a dose-dependent (0.1-7 U/ml) stimulation of 45Ca release from neonatal mouse calvarial bones. Thrombin also stimulated the mobilization of stable calcium and inorganic phosphate, the release of 3H from [3H]proline-labelled calvaria, the production of lactate and the release of the lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase. Thrombin also enhanced 45Ca release from fetal rat long bones, although this bone resorption assay was less sensitive to thrombin than the mouse calvarial system. The bone resorption stimulatory activity of thrombin in mouse calvaria could be inhibited by calcitonin and an increased concentration of phosphate in the culture medium. Thrombin-induced 45Ca release in mouse calvaria was sensitive to inhibition by hydrocortisone and dexamethasone. By contrast, 45Ca release response to parathyroid hormone was insensitive to corticosteroids. The prostaglandin synthetase inhibitors indomethacin, meclofenamic acid and naproxen and 5,8,11,14-eicosatetraynoic acid reduced 45Ca release from thrombin-stimulated calvaria. However, significant stimulation by thrombin could be achieved also in bones treated with inhibitors of arachidonate metabolism. The results obtained suggest that thrombin can stimulate cell-mediated bone resorption by an osteoclast-dependent mechanism. The mechanism of action may involve both prostaglandin-dependent and prostaglandin-independent pathways. Our findings indicate that thrombin may contribute to the bone resorptive processes seen in periodontal disease and rheumatoid arthritis.     

Cortical bone dynamics, strength, and densitometry after induction of emphysema in hamsters.

Recent evidence suggests that patients suffering from chronic obstructive pulmonary disease are also at an increased risk of developing osteoporosis. The pathophysiological mechanism(s) linking these progressive diseases is unknown. The goal of this investigation was to determine whether there were alterations in bone mineral density and content, cortical bone structure and strength, and indexes of bone formation and resorption in the elastase-induced emphysematous hamster. At 3 wk after induction of emphysema, the femoral bone mineral content was 8% less (P = 0.026) and the femoral fracture strength was 6% less (P = 0.032) in the emphysematous hamster than in controls. The cortical area was 8.4% less (P = 0.013) and the periosteal mineral appositional rate was 27% less (P = 0.05) than in controls. Additionally, the endocortical eroded surface in the emphysematous group was about twice that in the control group (P = 0.003). Differences in some indexes of bone formation and resorption, paralleled by differences in bone structure and strength, were observed 3 wk after induction of emphysema. These differences in skeletal metabolism and strength may help explain some of the skeletal changes associated with chronic obstructive pulmonary disease in humans.     

Influence of age and sex on the urinary excretion of total and non-dializable hydroxyproline

Urinary excretion of total OHPr, an index of bone resorption, was evaluated in 68 normal subjects (25 males and 43 females) aged 19-83 years. In 49 of them non-dialyzable OHPr(ndOHPr), which reflects bone matrix formation, was also determined. Total urinary OHPr, expressed as mg/24 h, significantly decreased with advancing age in both sexes: however by means of multiple regression analysis no correlation was found after correction of OHPr behaviour for changes in creatinine clearance. On the contrary ndOHPr was inversely correlated with age (r = -0,56, p less than 0,001) even when creatinine clearance was held constant (p less than 0,05). The findings obtained seem to show that a decrease in osteogenetic activity is also responsible for the physiological ageing bone loss.     

Dosing-time dependent effect of dexamethasone on bone density in rats

AimsWhile glucocorticoids are widely used to treat patients with various diseases, they often cause adverse effects such as bone fractures. In this study, we investigated whether the decrease in bone density induced by glucocorticoid therapy was ameliorated by optimizing a dosing-time.Main methodsRats were administered with dexamethasone (Dex) orally (1 mg/kg/day) for 6 weeks at a resting or an active period. After the end of the treatment, bone density of femur, biomarkers of bone formation and resorption, and other biomedical variables were measured.Key findingsBone density of femur was significantly decreased by the 6-week treatment with Dex, and the degree of decrease in the 14 HALO (hours after light on) dosing group (an active period) was larger than that in the 2 HALO dosing group (a resting period). Although urinary calcium excretion was accelerated by Dex treatment, secondary hyperparathyroidism was not detected. Histomorphometry analysis showed that Dex suppressed bone resorption, which was larger in the 2 HALO than in the 14 HALO groups. These data indicate that Dex equally suppressed bone formation in the 2 and 14 HALO groups, but inhibited bone resorption more in the 2 HALO than in the 14 HALO groups.SignificanceThis study shows that the decrease in bone density induced by Dex was changed by its dosing-time.     

Effects of 1,25-dihydroxyvitamin D3 on bone resorption and 45Ca2+ efflux in bone cells

1,25-dihydroxyvitamin D3[1,25(OH)2D3] effects on bone resorption in organ culture and on 45Ca2+ efflux rates in bone cells were measured in presence of a calcium channel inhibitor, diltiazem. Though, diltiazem reduced the 45Ca release from mice calvaria it did not act at the same Ca compartment as 1,25(OH)2D3 to alter Ca2+ flux parameters. It therefore seems difficult to hypothesize a simple relationship between bone resorption and Ca2+ movements in bone cells.     

Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin

Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.     

Effect of protein malnutrition on the metabolism of dermal collagen in the rat

The effect of protein malnutrition on the metabolism of collagen was studied in young female albino rats after a single injection of 3H-proline by determining the specific as well as total activities of 3H-hydroxyproline in the skin collagen fractions and in the urine. a) Compared to controls, the total activity of 3H-hydroxyproline in the soluble collagen and in the urine was significantly lower in the deficient group at 12 hrs. after the administration of 3H-proline. b) The urinary excretion of hydroxyproline and the total activity of urinary 3H-hydroxyproline measured after four weeks of labelled proline injection were also considerably decreased in the protein-deficient animals. c) When the total radioactivities of both soluble and insoluble collagen are expressed as a percentage of the sum of both, the recorded activity was more in soluble and less in insoluble collagen at 12 and 120 hrs. after the administration of 3H-proline, due to the influence of protein malnutrition. The results of the present investigation therefore clearly indicate that the synthesis of collagen is decreased and accompanied by a retardation in the maturation of soluble to insoluble collagen in the protein-deficient animals compared to controls. In addition, protein deficiency is accompanied by decreased rates of catabolism of both soluble and insoluble collagen.     

Effects of chronic NH4Cl dosage and swimming exercise on bone metabolic turnover in rats

To determine the effects of ammonium chloride (NH4Cl) dosage and swimming exercise training during 4 weeks on bone metabolic turnover in rats, seven-week-old female 24 Wister-Kyoto (WKY) rats were investigated by bone status including bone mineral density (BMD) and biomechanical markers from blood and urine. Twenty-four rats (initial weight: 191.2+/-7.6 g) were randomly divided into four groups: baseline (8 weeks old) control group (n=6, BC), 4-week control group (n=6, Con), 4-week swimming exercise loading group (n=6, Swim) and 4-week chronic NH4Cl dosage group (n=6, Acid). All rats were fed an AIN93M diet (Ca: 0.5%, P: 0.3%), and both Con and Swim groups were pair-fed by feeding volume of the NH4Cl dosage group. The acid group only received 0.25 M NH4Cl distilled water ad libitum. At the end of the experimental period, rats were sacrificed with blood drawn and femur and tibia were removed for analysis of bone mineral density (BMD) by dual energy X-ray absorptiometry (DEXA). In the Swim group, 24-hour urinary deoxypiridinoline (Dpd) excretion, reflecting bone resorption, was significantly increased (p<0.05) with a tendency towards decrease of BMD (N.S.), and body weight and abdominal fat weight were decreased in approximately 7% (p<0.05) and 58% (p<0.001), as compared with age matched Con rats. In the Acid group, 24-hour urinary calcium (Ca) and phosphorus (P) excretion were increased approximately 2.1-fold (p<0.05) and 2.0-fold (p<0.01), respectively, with increase of kidney weight as much as in the Con groups. Serum Ca and P concentration, as well as urinary Dpd excretion were, however, not significantly changed. These results suggest that blood Ca and P concentrations in the chronic acidosis condition during the 4-weeks might be maintained by hypercalciuria and hyperphosphaturia with kidney disorder, and swimming exercise training leads to decrease in BMD with stimulation of bone resorption and reduction of body fat.     

Effects of selective prostaglandin EP2 and EP4 receptor agonists on bone resorption and formation in fetal rat organ cultures

Prostaglandin E2 (PGE2) can stimulate bone resorption and formation through receptors which activate adenylyl cyclase. We examined the effects of selective EP2 and EP4 agonists (EP2A and EP4A) on the release of previously incorporated 45Ca from fetal rat long bones and the incorporation of [3H]-proline or [3H]-thymidine (TdR) in fetal rat calvaria to assess the relative effects of these selective agonists on bone formation and resorption. Only EP4A was effective in increasing 45Ca release. Both agonists increased [3H]-TdR incorporation and [3H]-proline incorporation into calvariae, particularly in the presence of cortisol.     

Bending properties, porosity, and ash fraction of black bear (Ursus americanus) cortical bone are not compromised with aging despite annual periods of disuse

In many species, including humans, disuse causes an imbalance in bone remodeling that leads to increased bone porosity as a result of increased bone resorption and decreased bone formation. However, black bears (Ursus americanus) may not develop disuse osteopenia, to the extent that other animals do, during long periods of disuse (i.e. hibernation) because they maintain osteoblastic bone formation during hibernation, even though bone resorption is increased during hibernation. Black bears may also have a mechanism to rapidly and completely recover the bone lost (by increased resorption during hibernation) during their remobilization period. Our findings suggest that cortical bone bending strength (211-328 MPa), bending modulus (16.0-29.5 MPa), fracture energy (0.0118-0.0205 J mm(-2)), porosity (2.3-7.1%), and ash fraction (0.638-0.672) are not compromised with age in black bears, despite annual periods of disuse. In fact, the ultimate strength (p=0.01), modulus (p=0.04), and ash fraction (p=0.03) of cortical bone were shown to significantly increase with age (2-14 yrs). Female bears give birth and nurse during hibernation; however, we found no significant (p>0.16) differences between male and female bone properties. Other animals require remobilization periods 2-3 times longer than the immobilization period to recover the bone lost during disuse. Our findings support the idea that black bears, which hibernate 5-7 months annually, have evolved a biological mechanism to mitigate the adverse effects of disuse on bone porosity and mechanical behavior.     

IL-6 is produced by osteoblasts and induces bone resorption

To examine the possible involvement of IL-6 in bone metabolism, a mouse osteoblastic cell line (MC3T3-E1) and primary osteoblast-like cells from fetal mouse calvaria were cultured with several systemic and local bone-resorbing agents and their expression of IL-6 mRNA was determined. Local bone-resorbing agents such as IL-1 alpha, IL-1 beta, TNF-alpha, and LPS greatly induced IL-6 mRNA expression in both MC3T3-E1 cells and primary osteoblast-like cells. Parathyroid hormone slightly increased expression of IL-6 mRNA in primary osteoblast-like cells but not in MC3T3-E1 cells. Neither IL-6 nor 1 alpha,25-dihydroxyvitamin D3 increased expression of IL-6 mRNA in either of the osteoblast-like cells. In agreement with the expression of IL-6 mRNA, biologically active IL-6 was produced in response to the treatment with IL-1 alpha, TNF-alpha, and LPS in MC3T3-E1 cells. Adding IL-6 dose dependently stimulated the release of 45Ca from prelabeled fetal mouse calvaria. Simultaneously adding suboptimal concentrations of IL-6 and IL-1 alpha induced bone resorption cooperatively. In accord with the increase in the release of 45Ca by IL-6, there were three times as many osteoclasts in the bone sections of calvaria cultured with IL-6 for 5 days as in the controls. IL-6 slightly suppressed alkaline phosphatase activity and collagen synthesis in MC3T3-E1 cells. These results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone-resorbing agents.     

Immobilization on the day 14th does not disrupts the basic diurnal rhythm of bone resorption

Weight bearing and physical activity are important mechanical stimuli to bone growth and metabolism, and microgravity, such a space flight and/or bed rest, induces bone resorption and bone loss. An increased excretion of urinary Ca, an increased bone resorption and a decreased bone mineral density (BMD) have been observed in bed rest experiment of healthy subjects. Bone resorption markers show the specific circadian rhythms in human. Cross-linked carboxyl-terminal telopeptide of type I collagen (ICTP) and the urinary excretion of deoxypyridinoline (Dpy) are the highest in the early morning and the lowest late at night. Bed rest immobilization might influence these rhythms, due to no mechanical loading with loss of daily life activity. Bone resorption markers in healthy subjects had been compared between before and during bed rest to determine disruption of diurnal rhythms of bone resorption.     

Control of bone resorption by semaphorin 4D is dependent on ovarian function

Osteoporosis is one of the most common bone pathologies, which are characterized by a decrease in bone mass. It is well established that bone mass, which results from a balanced bone formation and bone resorption, is regulated by many hormonal, environmental and genetic factors. Here we report that the immune semaphorin 4D (Sema4D) is a novel factor controlling bone resorption. Sema4D-deficient primary osteoclasts showed impaired spreading, adhesion, migration and resorption due to altered ß3 integrin sub-unit downstream signaling. In apparent accordance with these in vitro results, Sema4D deletion in sexually mature female mice led to a high bone mass phenotype due to defective bone resorption by osteoclasts. Mutant males, however, displayed normal bone mass and the female osteopetrotic phenotype was only detected at the onset of sexual maturity, indicating that, in vivo, this intrinsic osteoclast defect might be overcome in these mice. Using bone marrow cross transplantation, we confirmed that Sema4D controls bone resorption through an indirect mechanism. In addition, we show that Sema4D −/− mice were less fertile than their WT littermates. A decrease in Gnrh1 hypothalamic expression and a reduced number of ovarian follicles can explain this attenuated fertility. Interestingly, ovariectomy abrogated the bone resorption phenotype in Sema4D −/− mice, providing the evidence that the observed high bone mass phenotype is strictly dependent on ovarian function. Altogether, this study reveals that, in vivo, Sema4D is an indirect regulator of bone resorption, which acts via its effect on reproductive function.