Sustained release of salmon calcitonin in vivo from lactide: Glycolide copolymer depots

Summary Studies were carried out to determine whether monolithic depot formulations, prepared using lactide:glycolide copolymers, could be used to administer salmon calcitonin (sCT) to rats in vivo. Formulations containing 2, 5, or 10% (w/w) sCT were administered subcutaneously to female Wistar strain rats. Release of sCT was determined by measurement of peptide in plasma using a specific radioimmunoassay and by measurement of residual sCT in the depots after recovery at postmortem. Plasma calcium concentrations and cumulative weight gain of the animals were used to measure pharmacological effects of the released sCT. Release of sCT from the depots was controlled by the copolymer and was sustained for periods up to 10 days. However, the release of sCT from the depots did not significantly alter plasma calcium concentrations, and effects on cumulative weight gain were small and transient. Peptide loading of the formulations was shown to modify sCT release. Maximal release of sCT from depots containing 10% peptide occurred over a 7 to 14-day period postadministration, with 5% sCT release occurred between days 11 and 14, and with 2% sCT, the period of maximal release was between days 11 and 18. Release of peptide from the depots was essentially complete by 21 days postadministration irrespective of the peptide loading. These data suggest that lactide:glycolide copolymer depots may have application for the convenient clinical administration of sCT in metabolic bone diseases.     

Prevention of ovariectomy osteopenia in rats after vaginal administration of Hyaff 11 microspheres containing salmon calcitonin

The benzyl ester of hyaluronic acid (Hyaff 11) is a highly mucoadhesive polymer and can be processed into microspheres that can effectively deliver incorporated drugs by closely adhering to the mucosal surface and by protecting the drug from enzymatic inactivation. In this study, Hyaff 11 microspheres have been investigated as novel delivery system for the vaginal administration of salmon calcitonin (sCT) to ovariectomized rats. Moreover, this particular animal model has been used to evaluate the biological activity of the polypeptide after vaginal and I.M. administration by measuring the skeletal effect of sCT. A total of 66 female rats were used: 6 rats served as the basal group; 12 were subjected to sham operation and left without pharmacological treatment; 48 underwent bilateral ovariectomy and these were divided at random into four groups of 12 animals. One group was not submitted to pharmacological treatment; another was treated daily with sCT I.M.; in the other two groups, the hormone was incorporated into Hyaff 11 microspheres and was daily administered by vaginal route as dry powder or dispersed in pessaries. Sixty days after surgery, rats were sacrificed and the proximal third of the right tibia was removed and prepared for histomorphometry. Treatment with sCT, independent of the administration route, largely prevented the bone volume loss of the nontreated ovariectomized group. Prevention mainly depended on the maintenance of the trabecular number and on the increase of the trabecular thickness. Independent of the administration procedure, sCT greatly reduced the increase of tartrateresistant acid phosphatase-positive poly- and, particularly, mononucleated osteoclasts found in the nontreated ovariectomized group. The increase of mineral appositon rate of the nontreated ovariectomized group was significantly prevented in the sCT treated groups. The results demonstrate that vaginal administration of sCT, as well as I.M. administration, prevent bone loss due to ovariectomy mainly by inhibiting osteoclastic resorption.     

The effects of salmon calcitonin-induced hypocalcemia on bone metabolism in ovariectomized rats

The ovariectomized rat has proved to be a most useful model for preclinical testing of potential therapies for osteoporosis. We describe the immediate effects of a single treatment with salmon calcitonin (sCT) on calcium homeostasis and bone turnover markers in 6-month-old sham and ovariectomized (ovx) rats at 15 days postovariectomy. Rats were fasted for 24 h prior to and following administration of 0.3 µg/kg body weight sCT. Blood specimens were collected at 0 (pretreatment), 2, 4, and 8 h. Urine samples were collected during the intervening periods. sCT treatment produced a decrease in blood ionized calcium at 2 h posttreatment in sham and ovx rats (P < 0.001), which was exaggerated in the ovx rats (P < 0.001). Increased parathyroid hormone (PTH) levels (P < 0.001) accompanied the hypocalcemia in ovx rats. Furthermore, PTH levels were significantly higher in ovx rats compared with sham rats for the same ionized calcium range of 1.275–1.300 mmol/l (P < 0.05). sCT treatment in sham rats increased urine hydroxyproline (UHyp) at 6 h posttreatment (P < 0.01). In conclusion, the calcitonin-induced hypocalcemia and secondary hyperparathyroidism was more pronounced in the ovariectomized rats, consistent with the actions of calcitonin in states of increased bone turnover induced by estrogen deficiency. This study highlights the importance of considering the actions of PTH and estrogen status when interpreting changes in calcium homeostasis and bone turnover following treatment with calcitonin in rodent models and provides further evidence for a potential role of estrogen in parathyroid function.     

Hypocalcemic effect of salmon calcitonin following single and repeated nasal and intravenous administration in young rabbits

The effect of the polypeptide salmon calcitonin (sCT) on serum calcium concentrations following intranasal and intravenous administration was studied in young rabbits. A small, hypocalcemic effect was observed after nasal administration of sCT without additives, indicating that the nasal sCT absorption was low. The absorption could be improved by addition of an absorption-enhancing adjuvant to the nasal preparation. The absorption, however, was still far from complete as was apparent from the much stronger effect of intravenously injected sCT. When a number of sCT doses were given during a 10-week period, the hypocalcemic effect per sCT dose in the young rabbits decreased after intravenous and, although less pronounced, after nasal administration. The decreased response to sCT is probably not related to the induction of neutralizing antibodies or desensitization of sCT receptors, but is more likely associated with the age-dependent level of bone activity.     

Biological properties of salmon calcitonin IV.

In this study we characterized the biological activity of the recently identified salmon calcitonin (sCT) IV, in order to evaluate its potential therapeutic value. In the rat bioassay, sCT IV exhibited a 30% higher hypocalcemic activity than sCT I. The capacity of the molecule to inhibit bone resorption was assessed in vitro by the bone resorbing assay and the pit assay. An inhibitory effect, similar to that of sCT I, was observed in both assays. The interaction of sCT IV with the rabbit CT receptor was also studied. The affinity of sCT IV for the receptor was similar to that of sCT I, as was the potency for stimulating cAMP production. The antigenicity of the two molecules was not identical. Thus, this new CT could represent a useful novel therapeutic agent for the treatment of bone disorders.     

Effect of intranasal salmon calcitonin therapy on bone mass and bone turnover in early postmenopausal women: A dose-response study

We examine the dose-related effect of intranasal salmon calcitonin (sCT) on the early postmenopausal bone loss and bone turnover; a 2-year, prospective, randomized, double-blind, placebo-controlled study was carried out with 134 healthy women who had passed a natural menopause within 6 months to 3 years. The women were allocated randomly to 2 years of treatment with either 100, 200, or 400 IU of sCT given intranasally or placebo. All groups received a calcium supplement of 500 mg. Twenty-one women left the study before its end and 91 complied with the study criteria throughout. Bone mineral content/density of the distal forearm and lumbar spine and biochemical parameters of bone turnover were measured. Although the measurements after 24 months revealed no significant difference between groups in bone mineral density of the lumbar spine, the average changes over time revealed prevention of bone loss in the groups treated with 200 and 400 IU of sCT (0.2 to-0.6%) and declines of 0.8-1.7% in the groups treated with 100 IU of sCT and placebo (P<0.05–0.01; within-group testing). There was no dose-related response to sCT but there was a significant difference between the pooled groups treated with 200 plus 400 IU of sCT versus the 100 IU sCT and placebo-treated groups (P=0.030–0.005). The same difference between groups was seen for biochemical parameters of bone turnover (P=0.022–0.003). The biochemical parameters of bone turnover revealed decreases of 10–20% (P<0.001; within group testing) in the groups treated with the two highest sCT doses. It was concluded that nasal sCT in doses of 200 and 400 IU has some effect in women soon after the menopause—preventing the bone loss in the spine throughout the first year of therapy and lowering the bone turnover. It may be used as an alternative to hormone replacement when estrogens are contraindicated. The present data indicate that discontinuous strategies should be preferred.     

Human parathyroid hormone (1–34) and salmon calcitonin do not reverse impaired mineralization produced by high doses of 1,25 dihydroxyvitamin D3

Summary We have reported recently that pharmacologic doses of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) stimulated bone matrix formation but impaired mineralization. The objective of this study was to determine if parathyroid hormone (hPTH 1-34) or calcitonin (sCT) would mineralize the osteoid induced by 1,25(OH)₂D₃ in rat long bones. In one experiment, male Sprague-Dawley rats were given daily subcutaneous injections of vehicle: 8 μg hPTH(1-34); 125 ng 1,25(OH)₂D₃; or both 8 μg hPTH and 125 ng 1,25(OH)₂D₃ per 100 g body weight for 12 days. In a second experiment, rats received daily injections of vehicle: 2 U sCT; 125 ng 1,25(OH)₂D₃; or both 2 U sCT and 125 ng 1,25(OH)₂D₃ per 100 g body weight for 18 days. Calcium (Ca), hydroxyproline (Hyp), and dry weight (DW) of the distal femur and serum calcium, phosphate, and serum bone Gla protein (BGP) were measured. In rats given both 1,25(OH)₂D₃ and hPTH, total bone DW and Hyp increased (P<.01) without a corresponding increase in bone Ca so that Ca/Hyp decreased 47% (P<.01) from control and remained comparable to values for rats treated with 1,25(OH)₂D₃ alone. In rats treated with both 1,25(OH)₂D₃ and sCT, total bone DW and Hyp increased while Ca decreased so that Ca/Hyp decreased 38% from control (P<.05), and remained comparable to values for rats treated with 1,25(OH)₂D₃ alone. These results indicate that hPTH or sCT, given by intermittent injection to rats for 12 or 18 days respectively, failed to mineralize the osteoid induced by high doses of 1,25(OH)₂D₃.     

Human calcitonin has the same inhibitory effect on osteoclastic bone resorption by human giant cell tumor cells as salmon calcitonin

Human calcitonin (hCT) has been reported to have a less hypocalcemizing effect on rats and to have a lower binding affinity for the receptor of mouse osteoclasts than salmon CT(sCT). In this study we comparatively examined the effect of hCT and sCT on osteoclastic boneresorbing activity of unfractionated cells obtained from human giant cell tumor of bone and from rabbit and mouse long bones. We found that hCT had the same inhibitory effect as sCT on the bone-resorbing activity of human and rabbit osteoclastic cells, but a different one on that of mouse cells. These results indicate that the activity of drugs should be assayed using human cells if possible.     

Formation of neutralizing antibodies during intranasal synthetic salmon calcitonin treatment of postmenopausal osteoporosis

Nineteen patients with postmenopausal osteoporosis were treated with 200 u (15 nmol) synthetic salmon calcitonin (sCT) intranasally per day for 15 months. Six months after the start of the nasal administration of sCT, antibodies were recognized in 7, and after 15 months in 10 of the 19 patients studied. The half-maximal dilution of serum binding to 60 pmol/1 [¹²⁵I] sCT (dilution-50) ranged from 2 to 490, and half-maximal inhibition of [¹²⁵I]sCT binding (60 pmol/1) from 91 to 221 pmol/l sCT. In a cultured breast cancer cell line (T47D) cAMP production was stimulated by sCT (EC₅₀ 70 pmol/l). Stimulated cAMP production by sCT (50 pmol/1) was reduced to between 4% and 23% in the presence of serum from patients with antibody dilution-50 of [¹²⁵I]sCT binding exceeding 32. In patients with lower titer antibodies cAMP production was only marginally suppressed. The values of patients with postmenopausal osteoporosis were in the range of those of earlier studied patients with Paget's disease and clinical resistance to sCT. There was a linear relation between the antibody dilution-50 and the serum dilution required for half-maximal inhibition of cAMP production (P<0.01). In conclusion, neutralizing antibodies to sCT may contribute to the decreased responsiveness of bone mineral loss during prolonged treatment with sCT.     

The effect of intranasal salmon calcitonin on postmenopausal bone turnover as assessed by biochemical markers: Evidence of maximal effect after 8 weeks of continuous treatment

Although treatment with intranasal salmon calcitonin (sCT) has been shown to effectively inhibit postmenopausal bone loss, there is still controversy over both timing and the duration of its application. In an open prospective study, we therefore assessed the effect of short-term intranasal sCT on postmenopausal bone turnover, employing biochemical markers of bone metabolism. Ten early postmenopausal, previously untreated women (1–5 years after menopause) with biochemical evidence of increased bone resorption and a low bone mineral density at baseline were treated with intranasal sCT (100 IU B.I.D.) for a period of 3 months. Oral calcium (500 mg/day) was administered simultaneously, and during a further 3 month followup interval. Treatment with sCT resulted in a pronounced suppression of bone resorption markers with a maximum effect reached after 8 weeks of therapy: as compared to the respective baseline values, mean levels decreased by −26.2%±3.4% (P<0.001) for pyridinoline, −32.7%±3.5% (P<0.001) for deoxypyridinoline, −32.7%±3.3% (P<0.001) for hydroxyproline, and −24.1%±8.2% (P<0.001) for the amino-terminal telopeptide. In contrast, changes in bone formation markers of osteocalcin (−14.4%±4.8%,P<0.05) and C-terminal procollagen type I propeptide (−7.9%±3.9%, ns) were much less pronounced. Unexpectedly, after week 8 of the study all resorption markers showed a plateau and a trend to increase, although intranasal sCT was continued for a total of 12 weeks. This effect could not be attributed to the formation of anti-sCT antibodies. After cessation of treatment, both bone formation and resorption markers rapidly returned to baseline levels. Bone mineral density of both spine and hip showed no significant change during the observation period. Our results demonstrate that in postmenopausal women with a high bone turnover, intranasal treatment with 200 IU of sCT effectively reduces bone turnover and maintains bone mass, the maximum effect being reached after 8 weeks of treatment.     

Effect of estrogen and calcitonin on vertebral bone density and vertebral height in osteoporotic women

Estrogen and calcitonin increase bone density of osteoporotic women, particularly on the axial skeleton. To verify whether the effect of these drugs might in part be biased by spurious increases in bone density due to radiologically irrelevant microfractures, and consequent subtle decreases in vertebral height, we followed the changes in vertebral bone density (VBD), assessed by quantitative computed tomography, in relation to vertebral height (VH) in 60 osteoporotic women. VH was measured as the sum of anterior, central and posterior heights of L1–L3 vertebral bodies on lateral radiographs. Patients received either salmon calcitonin (sCT: 50 IU subcutaneously three times per week,n=18), hormonal replacement therapy (HRT: conjugated estrogen 0.625 mg/day, days 1–25, plus medroxyprogesterone acetate 10 mg/day, days 16–25 of each month;n=21) or calcium alone (Ca: 1000 mg/day,n=21). After 1 year, VBD increased in the HRT group (+5.0 ± 1.9%,p=0.010), did not change significantly in the sCT group (+3.3 ± 2.3%,p=0.167), and decreased by 6.1 ± 1.0% (p<0.001) in the Ca group. By analysis of variance, the changes induced by HRT and sCT were significantly different from those observed in the Ca group (F=7.982,p<0.001). VH decreased slightly in all three subsets of patients (–0.9 ± 0.5% in sCT, –1.5 ± 0.3% in HRT, –0.1 ± 0.5% in Ca), but these changes were not significantly different between groups (F=2.545,p=0.081). Correcting for this height decrease by analysis of covariance did not affect the significant effect of sCT and HRT on VBD, compared with that of Ca (F=6.801,p=0.001). Furthermore, no correlation was found between changes in bone VBD and VH in any of the groups. These data indicate that microfractures do not significantly account for the increases in bone density during active treatment of osteoporosis with either estrogen or calcitonin.     

Rectal salmon calcitonin for the treatment of postmenopausal osteoporosis

Summary In a 2-year study, we examined bone mass and calcium metabolism in 36 elderly women with moderate osteoporosis. The study period comprised 1 year of observation, during which the women received no treatment affecting calcium metabolism, and 1 year of treatment, during which all participants received daily salmon calcitonin (sCT) 100 IU rectally and calcium 500 mg. During the observational period a significant bone loss of 1.5% was seen in the forearm (P<0.01), whereas the spinal bone mass was virtually unchanged. After institution of treatment, the bone loss was arrested in the forearm and a significant increase of about 2% was seen in the spine (P<0.01). The net effect of treatment revealed a positive outcome in both bone compartments (1.9% and 2.9%, P<0.05–0.01). Correspondingly, the parameters of bone turnover (serum alkaline phosphatase, plasma bone Gla protein, and fasting urinary hydroxyproline/creatinine) did not change during the observational period, but significantly declined, 10–30%, during sCT treatment (P<0.01–0.001). Tolerance was generally good, although in one woman, anoscopy revealed irritative changes in the rectal mucosa. We conclude that, given rectally, sCT is well absorbed and well tolerated and that it has a beneficial effect on calcium metabolism in moderately osteoporotic women.     

Prevention of corticosteroid-induced osteoporosis with salmon calcitonin in sarcoid patients

Summary The aim of this study was to evaluate the usefulness of salmon calcitonin (sCT) in preventing corticosteroid-induced osteoporosis. Three groups of patients with sarcoidosis requiring long-term steroid therapy were followed for 2 years with yearly evaluations of vertebral cancellous mineral content (VCMC) by quantitative computed tomography. The first group (n=18) was treated with intramuscular (i.m.) sCT for the 2-year study period; the second (n=11) with i.m. sCT for the first 4 months and then with sCT nasal spray for 20 months; the third (n=35) received no sCT. We observed a large mineral loss in the third group but a very slight drop of VCMC in the two groups receiving sCT. SCT nasal spray was better tolerated and as effective as i.m. injections. The action of sCT appeared extremely useful, especially in the first year of steroid therapy when corticosteroid-induced mineral loss was maximal. We conclude that sCT nasal spray is a good tool for preventing corticosteroid-induced osteoporosis.     

Salmon calcitonin and cGMP production by human kidney: studies in vivo and in vitro

In order to evaluate whether or not the action of salmon calcitonin (sCT) at the kidney level could be mediated through specific receptors for the hormone, we have studied the effects of sCT infusions on urinary excretion of cyclic nucleotides in humans. Parallel in vitro studies have been conducted by evaluating the effects of sCT on cyclic nucleotide levels in primary cultures of cortical and medullary human kidney cells. In vivo experiments showed that sCT induced an increase in cGMP in human urine, which was rapid and short-lasting, being superimposable on the increase of urinary excretion of calcium and magnesium. The increase of inorganic phosphate urinary excretion was delayed and appeared to parallel that of urinary cAMP. On the other hand, our in vitro experiments showed that sCT stimulated the guanylate cyclase-cGMP system of human kidney cortical cells at nanomolar concentrations, while higher concentrations of the hormone were required to activate the adenylate cyclase-cAMP system. In addition, sCT was not able to significantly modify the cellular levels of either nucleotide in human kidney medullary cells. Present data demonstrated a direct effect of sCT on human kidney cortical cGMP production, while the efficacy of sCT on the kidney cortex adenylate cyclase-cAMP system appears to be delayed and/or reduced.     

An effective regimen of intranasal salmon calcitonin in early postmenopausal bone loss

Summary In order to devise a convenient and effective therapeutic regimen of intranasal salmon calcitonin (sCT) for the treatment of early postmenopausal bone loss, we studied the effects of a 1-year course of sCT nasal spray on vertebral mineral content (VMC), assessed by dual photon densitometry, and bone turnover in 21 early postmenopausal osteoporotic women. Subjects enrolled in the study had a value above the normal average of at least one index of bone turnover: whole body retention (WBR) of ^99mTc-methylenedichloro-bisphosphonate (^99mTc-MDP), serum bone gla protein (BGP), urinary hydroxyproline/creatinine excretion (HOP/Cr). After baseline evaluation, patients were randomized for treatment with either sCT (200 IU every other day) or plabebo. Treatment with sCT significantly increased VMC by 2.7±0.9% at 6 months, and 3.3±0.8% at 1 year, whereas a progressive decline was observed in the placebo group (-2.6±0.5%, and -3.5±0.5% after 6 and 12 months, respectively). These changes were associated with a progressive and significant reduction of all parameters of bone turnover in the sCT-treated patients, whereas no changes were detected in the control group during the study period. The differences between the two groups were significant after 1 year for VMC, BGP, and WBR (P<0.05, one-way analysis of variance). Thus, 200 IU intranasal sCT administered on alternate days is adequate to stop the fast bone loss occurring early after the menopause in women with high bone turnover rates. This therapeutical modality represents an important addition to the available pharmacologic spectrum for the prevention and treatment of postmenopausal osteoporosis.     

Intranasal Delivery of PEGylated Salmon Calcitonins: Hypocalcemic Effects in Rats

To evaluate the hypocalcemic effect of polyethylene gtycol-conjugated salmon calcitonins (PEG-sCT) in rats, mono-PEGylated sCTs (mono-PEG-sCTs) and unmodified sCT were administered via the intranasal route and serum calcium levels were measured by colorimetric assay using o-cresolphthalein. Mono-PEG-sCTs were prepared with different sizes of succinimidyl succinate monomethoxy PEG molecules (PEG_2K, PEG_5K, PEG_12K) and characterized by HPLC and MALDI-TOF mass spectrometry. Nasal instillation of mono-PEG_2K-sCT at a dose of 2 IU/kg resulted in sustained reduction in serum calcium levels over 8 hr, with a maximum reduction (% max_d) of 13% after 6 hr of application. Whereas unmodified sCT showed a transient decrease in serum calcium levels with the maximum reduction (5%) observed after 30 min of administration. The overall reductions in serum calcium levels expressed as the net change in AUC relative to control in 8 hr were 11.9 ± 0.2, 4.6 ± 0.7, and 2.6 ± 0.7% for mono-PEG_2K-, mono-PEG_5K-, and mono-PEG_12K-sCT, respectively, compared to 3.2 ± 0.6% for unmodified sCT. The relative bioavailability of nasally administered 2 IU/kg of mono-PEG_2K-sCT was approximately 4-fold higher than nasally administrated unmodified sCT, and the absolute bioavailability was approximately 91% of intravenously injected sCT in 8 hr. It can be concluded that the intranasal absorption of mono-PEG-sCTs was inversely related to the molecular weights of the PEG attached. Of the PEGylated sCTs examined, mono-PEG_2K-sCT showed the most pronounced hypocalcemic effect. Therefore the intranasal application would probably be an alternative route of administration for mono-PEG-sCTs in achieving sustained calcium-lowering effects.     

Intracerebroventricular calcitonin prevents stress-induced gastric dysfunction

BACKGROUND: Restraint stress produces gastric hypercontractility and acidity leading to stress ulceration. Intracerebroventricular (ICV) salmon calcitonin (sCT) decreases restraint injury and acidity, but its effects on restraint-induced hypercontractility are unknown. METHODS: Using stereotactic guidance, ICV catheters were placed into the lateral ventricle of adult male rats and calibrated gastric strain gauge transducers were implanted 5 days prior to restraint stress. sCT rats (n = 8) were pretreated with 5 microg of calcitonin ICV (10 microl volume), while controls (n = 10) received 10 microl of ICV saline prior to restraint for 2 h at 20 degrees C followed by 2 h at 4 degrees C. Gastric motility data were collected with AT-CODAS and analyzed with ADVANCED CODAS. Gastric volume, pH, and lesions were recorded following the stress. RESULTS: ICV calcitonin prevented gastric mucosal injury in all animals (0% vs 100%, P <.01) and elevated pH slightly (2.5 +/-.3 vs 1.6 +/-.1, P <.05). Stress caused the force of contractions to increase from 0.35 +/-.1 to 1.38 +/-.4 g in controls (P <.01), while treated animal's force fell from.42 +/-.1 to 0.2 +/-.05 g (P <.01 vs control). Stress did not affect contractions/min (3.4 +.6 vs 3.5 +.3), but sCT increased frequency (2.5 +.4 to 5.0 +.2, P <.01). Stress prolonged contraction duration (11.5 + 1 to 16.5 + 1.7 s, P <.01), but stress's effect was prevented by sCT (11.0 +.5 to 11.2 +.3, P <.01 vs control). CONCLUSIONS: Pretreatment with 5 microg central sCT prevents the increased amplitude and duration of gastric contractions produced by restraint stress for 2 h, in association with gastroprotection.     

Expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals

Metatarsal bones of 15-day-old mouse embryos contain proliferative tartrate-resistant acid phosphatase (TRAP) negative (-) osteoclast progenitors that progressively differentiate into multinucleated TRAP positive (+) osteoclasts. Using histochemical and autoradiographic techniques, we have examined the expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals. Fresh mouse metatarsals from embryos aged 14-17 days and metatarsals from 15-day-old embryos cultured for 1, 2, 3, and 6 days were stained for TRAP. Calcitonin binding to osteoclasts and their precursors was studied by incubating metatarsals with [125I]salmon calcitonin (sCT) and quantitating grain counts from autoradiographs of tissue sections. Calcitonin receptors first appear on nonproliferating osteoclast precursors, most often just after or simultaneously with the development of TRAP activity. The effect of sCT on the development of TRAP+ mononuclear preosteoclasts was examined by culturing 15-day-old metatarsals in the continuous presence of 5 mU sCT for periods of up to 3 days and quantitating the number of TRAP+ mononuclear preosteoclasts that develop. Calcitonin did not affect the differentiation of osteoclasts up to the stage of the TRAP+ mononuclear preosteoclast.     

RNA synthesis in isolated rat osteoclasts: inhibitory effect of calcitonin.

The metabolism of RNA has not been studied in the osteoclast (OC) because these bone-resorbing cells are only available in small numbers and cultures are always contaminated with other cells. Using two single-cell assay techniques, tritiated uridine (3H-UdR) autoradiography and gallocyanin quantitative cytophotometry, we have examined RNA synthesis in OCs isolated from neonatal rats. Oligo-nuclear OCs showed greater nuclear uptake of 3H-UdR than cells with many nuclei, and the variance of nuclear labeling within polykarya was greater in the latter, possibly because they contain nuclei of various ages. Salmon calcitonin (sCT) was a potent (ED50 approximately 5 x 10(-12) M) and rapid (40% reduction in 2 h, 75% reduction in 6 h) inhibitor of 3H-UdR uptake, and also reduced cytochemical total cellular RNA by 22% within 4 h. Forskolin (10(-5) M) inhibited nuclear uptake of 3H-UdR, suggesting that the sCT response may be mediated by cyclic AMP. Following a short (30 min) exposure to sCT, there was a progressive decline in labeling, followed by complete recovery by 4.5 h, a response possibly related to the phenomenon of calcitonin-induced persistent activation of adenylate cyclase. Inhibition of OC RNA synthesis may be an important component of its anti-resorptive action.