Prostaglandin receptor EP2 mediates PGE2 stimulated hypercalcemia in mice in vivo

Prostaglandin E2 (PGE2) can stimulate bone resorption by a cyclic AMP-dependent pathway. Two PGE2 receptors, EP2 and EP4 have been shown to play a role in PGE2 stimulation of osteoclast formation. In primary osteoblastic cell cultures from EP2 wild type (EP2 +/+) mice, PGE2 (0.1 microM) increased cyclic AMP production 3.5-fold, but PGE2 had no effect on cells from mice in which the EP2 receptor had been deleted (EP2 -/-). To examine the role of the EP2 receptor in the resorption response in vivo we injected PGE2 in EP2 -/- mice, and compared them with EP2 +/+ mice. Injection of PGE2 (3 mg/kg, four times daily for three days) in 9- to 12-month-old male mice on a 129 SvEv background increased serum calcium from 9.8 +/- 0.5 to 10.7 +/- 0.3 mg/dl (P < 0.01) in EP2 +/+ mice but not in EP2 -/- mice (10.1 +/- 0.3 vs. 10.2 +/- 0.3 mg/dl). PGE2 injection (6 mg/kg twice a day for three days) in 3-4 month old male mice on a C57 BL/6 X 129 SvEv background increased calcium from 8.2 +/- 0.1 to 9.0 +/- 0.3 mg/dl (P < 0.05) in EP2 +/+ mice but had no effect in EP2-/- mice (8.4 +/- 0.1 vs. 8.3 +/- 0.2 mg/dl). Injection of PGE2 over the calvariae of EP2 +/+ and EP2-/- mice increased the expression of receptor activator of nuclear factor kappaB ligand (RANKL) both locally and in the tibia, but RANKL responses were lower in EP2 -/- mice. We conclude that EP2 receptor plays a role in the hypercalcemic response to PGE2. This impaired response in EP2 -/- mice may be due to decreased ability to stimulate cyclic AMP and in part, to a smaller increase in the expression of RANKL mRNA.     

Treatment of multiple myeloma with nasal spray calcitonin: a histomorphometric and biochemical study

To evaluate the effect of calcitonin on the bone lesions of multiple myeloma, we studied 11 patients treated for 3 months with salmon calcitonin in nasal spray (200 IU) and 500 mg of elemental calcium/day. Pre- and post-treatment biochemical and histomorphometric parameters were compared to those of 12 patients treated for the same time with 500 mg elemental calcium alone. Both groups received the same hematological treatment.In the group treated with calcitonin there was a significant increase (P < 0.01) in trabecular bone volume, cortical thickness, osteoid volume and osteoid seam thickness index and the osteoclast resorption surface fell significantly (P < 0.01). There was also a decline (P < 0.001) in corrected serum calcium and OHP/Cr, which accounts for the diminished bone resorption. The group not treated with calcitonin showed only significant changes in OHP/Cr which increase (P < 0.05).Calcitonin was perfectly tolerated by all patients and our results show it to be useful in the treatment of bone lesions of multiple myeloma.     

The effect of calcitonin treatment on plasma nitric oxide levels in post-menopausal osteoporotic patients

Several recent studies have revealed a wide role for nitric oxide (NO) in bone metabolism. Low doses of NO cause bone resorption, but higher doses of NO inhibit bone resorbing activity. Cytokines are potent stimulators of NO production. NO is a very short-lived molecules. It exists for only 6-10 s only before it is converted by oxygen and water into the end-products nitrates and nitrites. Osteoporosis is a metabolic bone disease, characterized by a decreased amount of bone and increased susceptibility to fracture. NO may be involved as a mediator of bone disease such as post-menopausal osteoporosis. Calcitonin is a peptide hormone that inhibits bone resorption. The function of calcitonin in some cells is often unclear. In this study 30 post-menopausal osteoporotic women of ages ranging between 55 and 59 years without systemic diseases and free of any drug therapy were included. Twenty of them, randomly chosen, were treated with calcium (500 mg day(-1))+calcitonin (nasal spray 100 U day(-1)) and the other 10 women (control group) were treated with calcium only. This treatment was applied for 6 months and NO values were measured in each of the two groups before and after treatment. Our findings demonstrate that NO regulates osteoclastic bone resorption activity in association with calcitonin.     

Basal and stimulated calcitonin secretion in primary hyperparathyroidism before and after parathyroidectomy

To investigate calcitonin secretion in primary hyperparathyroidism, basal and stimulated (3 mg Ca++/kg body weight/10 min) immunoreactive calcitonin plasma levels were studied before parathyroidectomy. Plasma calcitonin levels were raised in 50% of patients regardless of sex, but a significant correlation between basal plasma calcium and calcitonin was found only in males. A reduced calcitonin response to calcium infusion was observed in all patients. Parathyroidectomy invariably induced a normalization of calcitonin basal levels. Our findings confirm the existence of a decreased parafollicular cell reserve probably as a consequence of the persistent hypercalcemic state in hyperparathyroid patients and suggest that it is more frequent in females.     

Salmon calcitonin induces pituitary tumor in rats.

Calcitonin is widely used in the treatment of post-menopausal osteoporosis. The present study was designed to investigate the effects of salmon calcitonin (SCT) on the incidence of the pituitary tumors in Sprague-Dawley (SD) rats. Subcutaneous injections of SCT at a dose of 160 IU/kg/day for 6 months reduced body weight and induced one pituitary hyperplasia and three pituitary adenomas in 4 of 5 animals, while 5 controls did not show any changes. Prolactin-positive cells were located at the periphery of the affected pituitaries adjacent to the prolactin-negative adenomas. In addition, serum concentrations of prolactin and TSH were lower than in the controls, although serum calcium or LH levels were not significantly different from the controls. Among 7 animals treated with SCT for 6 months followed by no medication for another 6 months, 5 adenomas were detected, one of which had invasive growth toward the adjacent tissue, whereas only one adenoma was found in 9 controls. These results suggest that SCT administration at a high dose may induce the formation of pituitary adenoma, or may accelerate the development of spontaneous pituitary adenomas, some of which show frequent mitotic figures and invasive growth into the surrounding tissue, possibly resulting in malignant transformation. This indicates the need for caution in considering whether calcitonin injections into patients with osteoporosis as well as Paget's disease may induce such pituitary tumors.     

Glucagon in the Treatment of Paget's Disease of Bone

Glucagon given by intravenous infusion at a dosage of 0·2 to 0·8 mg/hour to four patients with Paget''s disease of bone resulted in a dramatic fall in plasma alkaline phosphatase. This was associated with a fall in 24-hour urinary calcium and in total urinary hydroxyproline excretion and a marked relief of bone pain.Glucagon may induce these changes by three possible mechanisms: (1) by stimulating release of calcitonin; (2) by a direct action of the hormone on bone; and (3) by stimulation of certain bone pyrophosphatases, thus altering the local mechanisms controlling the rate of bone formation and resorption.     

Inhibition of Alveoloar Bone Loss During Maturation of the Rat

The effects of dichloromethylene diphosphonate (C1₂MDP) on previously described maturational bone loss were quantified in 24 Wistar rats. Four groups of six animals each were treated daily from 18 to 28 weeks of age with a subcutaneous injection of a 0.1, 1.0 or 10mg/kg dose of C1₂MDP or were sham treated with 0.9% NaC1. Prior to sacrifice, all animals were double-labeled with tetracycline. The interdental bone between first and second maxillary molars was evaluated on decalcified stained sections and on undecalcified ground sections by digitizing morphometry. No significant differences in periodontal ligament width, bone height, osteoclast numbers or tetracycline labeled surface were detected. Significantly greater bone area (p<0.01) and bone width (p<0.005), and significantly fewer osteoblasts/mm of bone surface (p<0.001) were measured in rats treated with 10 mg/kg doses of C1₂MDP; and a dose response was demonstrated. It was concluded that C1₂MDP prevented interdental bone loss by inhibiting bone resorption, and that bone formation may have been indirectly reduced through a coupling mechanism.     

Renal Effects of Calcitonin

Porcine calcitonin in a slow-release gelatin vehicle was given by intramuscular injection to 10 patients—four with primary hyperparathyroidism, four with Paget''s disease, and two with carcinoma of the breast and hypercalcaemia. All cases showed a fall in serum calcium with an immediate rise in urine calcium. All except three patients with primary hyperparathyroidism showed a fall in serum phosphorus, but an immediate rise in urine phosphorus occurred in all cases. Urine hydroxyproline output fell in three patients with severe Paget''s disease. Urine sodium rose in all cases, but the effects on potassium, magnesium, water, and pH were not appreciably different from results obtained in four control subjects who were given the gelatin vehicle alone.The data suggest that calcitonin caused a decrease in the tubular resorption of calcium and phosphorus. The hypocalcaemic effect appeared to be due to a decrease in bone resorption in the patients with Paget''s disease but in the remaining cases could be accounted for in part or entirely by the rise in urine calcium.     

Overexpression and purification of recombinant eel calcitonin and its phylogenetic analysis

Calcitonin (CT), a peptide hormone that is widely used for the treatment of osteoporosis, Paget's disease, hypercalcemic shock and chronic pain in terminal cancer patients, is produced by the para-follicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish. Fish calcitonin, like eel calcitonin (eCT), is more potent and longer lasting than human CT and is one of the many bioactive peptides that require C-terminal amidation for full biological activity. In this study we describe the over-expression and over-production of C-terminal amidated eCT in recombinant Streptomyces avermitilis. A phylogenetic analysis was performed with all the known CT amino acid sequences.     

Anti-hypercalcemic effect of orally administered recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing salmon calcitonin on hypercalcemic rats

Oral delivery of salmon calcitonin (sCT) to rats via a recombinant Saccharomyces cerevisiae was assessed. A synthetic sCT gene was cloned and expressed in S. cerevisiae yAGA2-sCT. Recombinant salmon calcitonin (rsCT) expression was detected by flow cytometry. The resorption activity of osteoclasts was inhibited by 3 × 10⁻⁶ M rsCT. Oral administration of 5 g lyophilized yAGA2-sCT/kg to hypercalcemic rats decreased serum calcium from 2.8 ± 0.02–2.7 ± 0.02 mM.     

Plasma monomeric calcitonin as a marker of disease activity in multiple myeloma patients with osteolysis.

Circulating monomeric human calcitonin (hCT-M), parathyroid hormone, osteocalcin, alkaline phosphatase, urinary hydroxyproline, corrected serum calcium and inorganic phosphate were measured in 49 multiple myeloma patients and 49 matched controls. In patients with Durie-Salmon stage III disease hCT-M levels (16.9 +/- 5.8 ng/l, mean +/- SD) were significantly higher than controls and stage I patients (P less than 0.01), and correlated directly with corrected serum calcium (r = 0.74; P less than 0.001). In the same subgroup 14 of 15 patients had plasma hCT-M concentrations higher than the mean + 2SD of the controls. The calcium infusion test induced an increase of hCT-M in normocalcemic patients which was significantly greater in patients with advanced disease than in either controls or stage I patients. These findings suggest that hCT-M may be a biochemical index of bone resorption and disease activity in myeloma patients with osteolysis. In fact, its plasma concentrations were elevated in a large proportion (93%) of patients with severe bone involvement, and correlated directly with serum calcium. Moreover, our findings suggest the presence of a calcitonin-dependent calcium homeostatic mechanism, that protects against hypercalcemia due to tumor osteolysis.     

Plasma concentrations of immunoreactive calcitonin in rats during lactation

A mild hypocalcemia (0.5 mg/dl) is observed in rats after 14 days of lactation, but plasma and thyroidal calcitonin (CT) levels are both increased on day 7 of lactation. Plasma CT levels are higher (x2) in lactating females than those found in virgin females from day 7 to the end of lactation (21 days). In vitro, the CT secretion rate after calcium stimulation(3 mM) is not different between lactating and virgin females. The rapid removal of pups after parturition in control females induces a rebound in plasma calcium (+1.4 mg/dl) and plasma phosphate (+1.6 mg/dl) associated with elevated plasma CT values. Our results suggest, that the transient mild hypocalcemia of lactation is preceded by increased plasma CT levels; and that it is not the cause of elevated plasma parathyroid hormone levels already reported by us.     

Effects of electromagnetic stimulation on the functional responsiveness of isolated rat osteoclasts

We report the effects of pulsed electromagnetic fields (PEMFs) on the responsiveness of osteoclasts to cellular, hormonal, and ionic signals. Osteoclasts isolated from neonatal rat long bones were dispersed onto either slices of devitalised cortical bone (for the measurement of resorptive activity) or glass coverslips (for the determination of the cytosolic free Ca²⁺ concentration, [Ca²⁺]). Osteoclasts were also cocultured on bone with osteoblastlike, UMR-106 cells. Bone resorption was quantitated by scanning electron microscopy and computer-assisted morphometry. PEMF application to osteoblast–osteoclast cocultures for 18 hr resulted in a twofold stimulation of bone resorption. In contrast, resorption by isolated osteoclasts remained unchanged in the presence of PEMFs, suggesting that osteoblasts were necessary for the PEMF-induced resorption simulation seen in osteoblast–osteoclast cocultures. Furthermore, the potent inhibitory action of the hormone calcitonin on bone resorption was unaffected by PEMF application. However, PEMFs completely reversed another quite distinct action of calcitonin on the osteoclast: its potent inhibitory effect on the activation of the divalent cation-sensing (or Ca²⁺) receptor. For these experiments, we made fura 2-based measurements of cytosolic [Ca²⁺] in single osteoclasts in response to the application of a known Ca²⁺ receptor agonist, Ni²⁺. We first confirmed that activation of the osteoclast Ca²⁺ receptor by Ni²⁺ (5 mM) resulted in a characteristic monophasic elevation of cytosolic [Ca²⁺]. As shown previously, this response was attenuated strongly by calcitonin at concentrations between 0.03 and 3 nM but remained intact in response to PEMFs. PEMF application, however, prevented the inhibitory effect of calcitonin on Ni²⁺-induced cytosolic Ca²⁺ elevation. This suggested that the fields disrupted the interaction between the calcitonin and Ca²⁺ receptor systems. In conclusion, we have shown that electromagnetic fields stimulate bone resorption through an action on the osteoblast and, by abolishing the inhibitory effects of calcitonin, also restore the responsiveness of osteoclasts to divalent cations. J. Cell. Physiol. 176:537–544, 1998. © 1998 Wiley-Liss, Inc.     

The effect of calcitonin on prolactin secretion during gestation

The relationship between calcitonin (CT) and prolactin (PRL) was studied by means of the injection of salmon calcitonin (SCT) i.p. on day 1 of gestation. An estrogen inhibitor - clomiphene - was also administered to certain groups of animals on day 4 and 5 of gestation. SCT did not affect PRL levels on day 1 of gestation nor on days 5 or 7, but it prevented the rise of PRL levels observed in animals submitted to injection stress on days 4 and 5. In animals treated with clomiphene, the inhibition by SCT on PRL increase after injection stress was partially abolished. SCT while not affecting basal PRL level prevented the rise observed after stress and this effect occurred some days later. Thus SCT could exercise a delayed neuroendocrine control. This action of SCT seemed to be partially dependent upon the presence of estrogens.     

Effect of dichloromethylene diphosphonate (Cl2MDP) on immune function in breast cancer patients with bone metastases

Immune function was studied in normocalcemic breast cancer patients with bone metastases treated with either dichloromethylene diphosphonate (Cl2MDP) or placebo. The results showed no significant difference between the two patient groups. This suggests that Cl2MDP does not markedly impair the host's defense mechanisms, and in this respect can be safely used in the treatment of patients with resorptive bone disease.     

The effects of dichloromethylene biphosphonate on osteoporotic femora of adult castrate male rats

In a previous study we reported that castration of adult male rats resulted in femoral osteoporosis 4 months later. The present study tested the effects of dichloromethylene biphosphonate (Cl2MBP, formerly Cl2MDP), a bone resorption inhibitor, on the development of osteoporosis in femora of adult castrated male rats. One-year-old male Holtzman rats were assigned to three groups: sham-operated controls; castrated-no treatment; castrated + Cl2MBP injections 3 times/week. The animals were sacrificed 4 months later. Femora from untreated castrated rats were osteoporotic, confirming our earlier study. Femora from Cl2MBP-treated castrates did not differ from those of controls and were significantly denser and more robust than those of untreated castrated rats. The results indicated that Cl2MBP treatment started immediately after castration prevented the development of femoral osteoporosis in adult castrated male rats.     

Explanation for Unusual Potency of Salmon Calcitonin

THE calcitonins are polypeptide hormones of thirty-two amino-acids which lower serum calcium in mammals by inhibiting bone resorption¹. During evaluation of these hormones as a means of treating skeletal disorders in man, particularly Paget's disease of bone^{2, 3}, the surprising observation was made that calcitonin from the salmon (SCT) is 20–200 times more potent than porcine calcitonin (PCT) and at least ten times more potent than human calcitonin^{3, 4}. SCT is far more potent than any mammalian calcitonin yet tested in a wide variety of animal species^{5, 6}. This unusual potency of salmon calcitonin could reflect either a greater hormone affinity for receptor sites or a greater resistance to metabolic destruction. We now report evidence which supports the latter possibility, infused SCT disappears from the circulation of the dog much more slowly than does PCT.     

Effects of the calcium antagonist, diltiazem on in vitro and in vivo/in vitro bone resorption

The effect of the calcium antagonist diltiazem on bone resorption in organ culture has been investigated. It was found that diltiazem was ineffective alone but that in concentrations above 5 mumol/l it reduced mineral and organic resorption induced in vitro by 1.25 dihydroxycholecalciferol (1.25 (OH)2D3). No additivity with calcitonin effects was observed. Diltiazem did not significantly affect bone resorbing activity stimulated by 24,25(OH)2D3. Bone resorption was measured by an in vivo/in vitro technique using 45Ca prelabelled mice. Compared with 1.25(OH)2D3 alone treated group (0.480 pmol/g), it was found that diltiazem (100 nmol/g) reduced bone resorption without effect on calcium and phosphorus plasmatic concentrations at death. These data suggest that such a calcium antagonist is able to inhibit 1.25-(OH)2D3-increased-bone resorption either in vitro or in vivo/in vitro.     

A calcitonin-like action of prostaglandin E1

The pharmacological effects of PGE1 (6 and 9 days, 2-1,250 micrograms/kg per day subcutaneously) upon the growth and the bone resorption of mammals were studied using the proximal tibia and upper incisor of immature rats along with lead acetate as a time marker, and upon the serum calcium and inorganic phosphorus levels. The following results were obtained. 1. PGE1 hardly affected the body weight or the weight of organs of the rats but apparently inhibited the longitudinal growth of proximal tibia in a dose related manner. 2. PGE1 clearly inhibited not only the longitudinal growth (incisor growth) but also the appositional growth (dentin formation) of incisal dentin. 3. The grade of the inhibitory effect on the growth was in the order of bone growth greater than dentin formation greater than incisor growth. 4. The occurrence of osteoporosis due to a low calcium diet was inhibited by the simultaneous administration of PGE1, the mechanism being considered to be mainly due to the inhibitory effect on the bone resorption. 5. PGE1 lowered the level of serum calcium and the lowering effect was not observed in the thyro-parathyroidectomized rat. From the facts that the above effects were exactly the same as those of calcitonin (1), the possibility that the subcutaneous injection of PGE1 may induce a calcitonin-like action, a part of which may dependent on the calcitonin secretion is suggested.