Forty years of calcitonin--where are we now? A tribute to the work of Iain Macintyre, FRS.

Calcitonin was discovered as a hypocalcemic principal that was initially thought to originate from the parathyroid gland. This view was corrected subsequently, and an origin from the thyroid C cells was documented. The purification and sequencing of various calcitonins soon followed. Calcitonin is a 32-amino-acid-long peptide with an N-terminal disulfide bridge and a C-terminal prolineamide residue. The peptide was shown to potently inhibit bone resorption; however, a direct osteoclastic action of the peptide was confirmed only in the early 1980s. Several osteoclast calcitonin receptors have subsequently been cloned and sequenced. Specific regions of the receptor necessary for ligand binding and intracellular signaling through cyclic AMP and calcium have been identified through systematic deletion mutagenesis and chimeric receptor studies. Calcitonin's potent antiresorptive effect has led to its use in treating Paget's disease of bone, osteoporosis, and hypercalcemia. This review retraces key aspects of the synthesis and structure of calcitonin, its cellular and molecular actions, and its therapeutic uses as they have emerged over the 40 years since its discovery. The review also examines the implications of these findings for future clinical applications as a tribute to early workers to whom credit must be given for creation of an important and expanding field. Notable are the new approaches currently being used to enhance calcitonin action, including novel allosteric activators of the calcitonin receptor, modulation of the release of endogenous calcitonin by calcimimetic agents, as well as the development of oral calcitonins.

Molecular cloning, expression, and functional characterization of a novel member of the CD38 family of ADP-ribosyl cyclases.

We report the molecular cloning and functional characterization of a novel member of the CD38 family of cyclic ADP-ribose (cADPr)-generating cyclases. We cloned a cDNA insert that encoded a 298-amino-acid-long protein (M(w) approximately 39 kDa). The predicted protein displayed 69, 61, and 58% similarity, respectively, to mouse, rat, and human CD38. Rabbit CD38 was also 28% homologous to Aplysia ADP-ribosyl cyclase and leukocyte CD157 (another ADP-ribosyl cyclase); the three cyclases shared 10 cysteine and 2 adjacent proline residues. We then transfected CD38-negative NIH3T3 cells with cDNA encoding a CD38-EGFP fusion protein. Epifluorescence microscopy showed intense EGFP fluorescence confirming CD38 expression. We finally confirmed the ADP-ribosyl cyclase activity of the expressed CD38 by measuring its ability to catalyze the cyclization of the nicotinamide adenine dinucleotide (NAD(+)) surrogate, NGD(+), to its fluorescent nonhydrolyzable derivative, cGDPr.

Novel biochemical and functional insights into nuclear Ca(2+) transport through IP(3)Rs and RyRs in osteoblasts.

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab(34)) and anti-IP(3)R (Ab(40)) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab(40), anti-RyR-1, anti-RyR-2 (Ab(129)), and anti-RyR-3 (Ab(180)). Only anti-RyR-1 and Ab(40) showed bands corresponding, respectively, to full-length RyR-1 ( approximately 500 kDa) and IP(3)R-1 (approximately 250 kDa). Band intensity was reduced by just approximately 20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca(2+) concentration ([Ca(2+)](np)) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca(2+) via Ca(2+)-ATPase activation (1 mM ATP and approximately 100 nM Ca(2+)). Adequate Ca(2+) loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca(2+)-loaded nuclei to IP(3) or cADP ribose resulted in a rapid and sustained [Ca(2+)](np) elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca(2+) influx in osteoblasts through nuclear membrane-resident IP(3)Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP(3)-Ca(2+) pathway.

A new function for CD38/ADP-ribosyl cyclase in nuclear Ca2+ homeostasis.

Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.

Meloxicam pharmacokinetics in renal impairment.

AIMS: The aim of the present study was to determine how the pharmacokinetics of meloxicam are affected by kidney dysfunction and consequently to define the appropriate dose for the use of meloxicam in patients with mild or moderate renal impairment. METHODS: Meloxicam was administered to subjects with mild (creatinine clearance 41-60 ml min-1) to moderate (20-40 ml min-1) renal impairment compared with normal renal function (> 60 ml min-1). Thirty-eight subjects received meloxicam 15 mg once daily over 9 days. Meloxicam plasma concentrations were determined from blood samples taken during the study and pharmacokinetic parameters calculated according to noncompartmental methods. RESULTS: Subjects with no or mild renal impairment showed similar pharmacokinetic profiles (geometric mean AUCSS (%gCV) 55 (33%) vs 55 (38%) micrograms ml-1 h). Subjects with moderate renal impairment demonstrated lower total plasma meloxicam concentrations (AUCSS 35 (50%) micrograms ml-1 h, with corresponding higher plasma clearance (P = 0.013) compared with subjects with no renal impairment. However, this was combined with higher meloxicam free fractions in moderately impaired subjects such that free meloxicam concentrations were similar in all three groups. Meloxicam was well tolerated with few adverse events occurring and no difference in incidence observable between groups. CONCLUSIONS: On the basis of these results there is no necessity for a dosage adjustment when administering meloxicam to patients with mild to moderate renal impairment.

Evaluation of the safety, tolerability, and efficacy of meloxicam tablets in patients with osteoarthritis.

This 12-week, open-label, multicenter study assessed the safety, tolerability, and efficacy of the tablet formulation of meloxicam 15 mg, a new, nonsteroidal antiinflammatory drug (NSAID), in patients with confirmed osteoarthritis (OA) of the hip or knee. Meloxicam differs from established NSAIDs in its preferential activity against cyclooxygenase type 2 compared with cyclooxygenase type 1. One hundred thirty-nine patients were given meloxicam tablets 15 mg once daily. Data were analyzed using an intent-to-treat analysis. Assessments of global tolerability at the end of the study showed meloxicam tablets to be well or very well tolerated by 83% of patients, with only 4% rating the tablets poorly. This finding was supported by the low incidence of adverse events. Only 11% of patients discontinued therapy due to gastrointestinal adverse events, and no serious gastrointestinal adverse events related to meloxicam 15-mg tablets occurred during treatment. Efficacy results showed significant improvement from baseline, with 41% of patients experiencing mild or no pain by the end of the study. In conclusion, the tablet formulation of meloxicam 15 mg is well tolerated and effective in patients with moderate-to-severe, clinical confirmed OA of the hip or knee.

A six-month double-blind trial to compare the efficacy and safety of meloxicam 7.5 mg daily and naproxen 750 mg daily in patients with rheumatoid arthritis.

Meloxicam is a new non-steroidal anti-inflammatory drug (NSAID) which preferentially inhibits cyclooxygenase-2 over cyclooxygenase-1. A double-blind parallel-group trial compared meloxicam 7.5 mg once daily (n = 199) with naproxen 750 mg (n = 180) in rheumatoid arthritis. There was no significant difference between the groups regarding the primary efficacy variables (global efficacy assessment by patient and investigator, number of painful/tender and swollen joints) and eight of the ten secondary efficacy endpoints. Only the swollen joint severity index and the number of discontinuations due to lack of efficacy favoured naproxen 750 mg significantly over meloxicam 7.5 mg. Meloxicam was better tolerated in the gastrointestinal (GI) tract, with fewer GI adverse events in the meloxicam-treated group (30.3%) than in the naproxen-treated group (44.7%), where two patients developed ulcers. No ulcers were seen in meloxicam patients. Significantly more patients discontinued due to GI adverse events in the naproxen group. Additionally, there was a significant decrease in haemoglobin and a significant increase in serum creatinine and urea in the naproxen group compared with the meloxicam group. In conclusion, meloxicam 7.5 mg once daily is a promising treatment in rheumatoid arthritis, with efficacy comparable to naproxen 750 mg. Meloxicam has the advantage of a significantly lower incidence of GI and renal side effects.

An open study to assess the safety and tolerability of meloxicam 15 mg in subjects with rheumatic disease and mild renal impairment.

Meloxicam is a new non-steroidal anti-inflammatory drug (NSAID) which has shown potent anti-inflammatory properties but good gastrointestinal (GI) renal tolerability. The safety and tolerability profile of orally administered meloxicam 15 mg given once daily over a 28 day treatment period in renally impaired patients with rheumatic disease is presented here. A total of 25 patients (aged 43-78 yr, mean age 70 yr) with rheumatic disease and mild renal impairment were enrolled in this multicentre, open-label study, with 22 patients completing the 28 day treatment period. The median estimated creatinine clearance and N-acetyl-beta-glucosaminidase/creatinine ratios (a marker of renal tubular damage) recorded at day 14, day 28 or 4-7 days after meloxicam treatment was terminated, were not statistically significantly different from baseline values. There was no evidence of accumulation of meloxicam. Overall, meloxicam was well tolerated. The most common adverse events were GI complaints of abdominal pain and dyspepsia. No adverse events related to the urinary system, or increases in serum urea or potassium were recorded. The results suggest that meloxicam, 15 mg once daily, does not further compromise renal function or result in accumulation of meloxicam over this treatment period in patients with pre-existing mild renal impairment.

Osteoclast function and its control.

Bone resorption appears to be dependent on a range of processes. It requires an adequate number of osteoclasts to access bone mineral. These osteoclasts must be activated by a mechanism which is dependent upon prior osteoblastic stimulation. A range of factors then contribute to the formation of a functionally effective resorptive hemivacuole. These entail osteoclast adhesion to the bone surface leading to the formation of a sealing zone. Only then can subsequent processes such as H+ ion transport, enzyme secretion and matrix digestion become effective. Thus, any one process is potentially limiting to resorption and is a potential target for regulation. Long-range regulation takes place through the action of hormones, of which the mode of action of calcitonin has been the subject of recent investigations in isolated osteoclasts. Such studies have shown a possible involvement of distinguishable receptor subtypes, the occupancy of which may activate at least two types of triggering mechanism. It is likely that an eventual influence on motility properties through G protein mediation accounts for the actions of this hormone and of related peptides such as amylin and CGRP at the cellular level. Similar pathways may contribute to shorter range modulation of osteoclast activity by increases in ambient Ca2+. Finally, there is recent evidence for a contribution of endothelial cell-derived product to osteoclast regulation.

Linkage of extracellular and intracellular control of cytosolic Ca2+ in rat osteoclasts in the presence of thapsigargin.

Cytosolic [Ca2+] was measured in single osteoclasts using fura-2 in experiments investigating the effects of Ca2+ "receptor" activation using thapsigargin as a means of depleting intracellular Ca2+ stores. Application of 4 microM thapsigargin to osteoclasts in Ca(2+)-free solutions resulted in an elevation of cytosolic [Ca2+]. Under similar conditions, activation of the osteoclast Ca2+ receptor by the substitute divalent cation agonist, Ni2+, resulted in a transient elevation of cytosolic [Ca2+]. In both instances, restoration of extracellular [Ca2+] to 1.25 mM resulted in an "overshoot" of cytosolic [Ca2+]. Prior depletion of intracellular Ca2+ stores by thapsigargin markedly reduced the magnitude of the cytosolic [Ca2+] response to a subsequent application of 5 mM Ni2+. The application of 2 microM thapsigargin to intercept the falling phase of the Ni(2+)-induced cytosolic Ca2+ signal resulted in a sustained elevation of cytosolic [Ca2+], which was terminated by a second application of the same Ni2+. Furthermore, the sustained elevation of cytosolic [Ca2+] induced by thapsigargin application alone was abolished by late application of Ni2+. We conclude that activation of the surface membrane Ca2+ receptor on the osteoclast results in the cytosolic release of Ca2+ from intracellular storage organelles; the refilling of such stores depends upon a thapsigargin-sensitive Ca(2+)-ATPase; store depletion induces capacitative Ca2+ influx; and the Ca2+ influx pathway is sensitive to blockade by Ni2+.

Cellular biology of bone resorption.

Past knowledge and the recent developments on the formation, activation and mode of action of osteoclasts, with particular reference to the regulation of each individual step, have been reviewed. The following conclusions of consensus have emerged. 1. The resorption of bone is the result of successive steps that can be regulated individually. 2. Osteoclast progenitors are formed in bone marrow. This is followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone. 3. The exact pathways of differentiation of the osteoclast progenators to mature osteoclasts are debatable, but there is clear evidence that stromal cells support osteoclast generation. 4. Osteoclasts are activated following contact with mineralized bone. This appears to be controlled by osteoblasts that expose mineral to osteoclasts and/or release a factor that activates these cells. 5. Activated osteoclasts dissolve the bone mineral and digest the organic matter of bone by the action of agents secreted in the segregated microcompartments underlying their ruffled borders. The mineral is solubilized by protons generated from CO2 by carbonic anhydrase and secreted by an ATP-driven vacuolar H(+)-K(+)-ATPase located at the ruffled border. The organic matrix of the bone is removed by acid proteinases, particularly cysteine-proteinases that are secreted together with other lysosomal enzymes in the acid environment of the resorption zone. 6. Osteoclastic bone resorption is directly regulated by a polypeptide hormone, calcitonin (CT), and locally, by ionized calcium (Ca2+) generated as a result of osteoclastic bone resorption. 7. There is new evidence that osteoclast activity may also be influenced by the endothelial cells via generation of products including PG, NO and endothelin.

Extracellular Ca2+ sensing by the osteoclast.

An increasing number of cell types appear to detect changes in the extracellular Ca2+ concentration and and accordingly modify their function. We review recent evidence for the existence and function of such a mechanism in the osteoclast. Elevated external [Ca2+] in the mM range reduces bone resorption and results in motile changes in the cells. These changes may partly result from elevations of cytosolic [Ca2+] triggered through activation of a surface Ca2+ receptor. Closer analyses of the increases in cytosolic [Ca2+] associated with receptor activation are hindered by the action of this ion both as extracellular agonist and intracellular second messenger. Variations in the peak cytosolic [Ca2+] response to external Ca2+ with changes in cell membrane potential by K+ and valinomycin establish a contribution from extracellular Ca2+. Use of CIO4-, Ni2+ and Cd2+ as surrogate activators in low extracellular [Ca2+] indicate a contribution from Ca2+ release from intracellular stores as well. Such agonists also modify Ca2+ redistribution in other systems, such as skeletal muscle. Thus, we may gain insights into osteoclast extracellular Ca2+ detection and transduction from known features of more well-characterised cell systems.

Amylin inhibits bone resorption by a direct effect on the motility of rat osteoclasts.

We have performed a set of independent studies on the effects of the circulating pancreatic polypeptide, amylin, on rat osteoclast function, in vitro. Time-lapse video observations, measuring cell protrusions and retraction, showed that 250 nmol l-1 amylin or 250 nmol l-1 beta-calcitonin gene-related peptide (beta-CGRP) inhibited osteoclast motility (quiescence or Q effect). Both amylin and beta-CGRP produced inhibitory responses with a significant first-order regression over time (half-times, 19 and 28 min respectively). In contrast, 250 nmol l-1 amylin or 250 nmol l-1 beta-CGRP produced no change of osteoclast spread area, whilst 300 pmol l-1 calcitonin (CT) application resulted in cell retraction (R effect). Forskolin (10 mumol l-1) mimicked amylin and CGRP in inhibiting osteoclast motility (half-time, 8.6 min), and similarly lacked an effect on cell spread area. Neither amylin nor beta-CGRP (62.5-1250 nmol l-1) elevated cytosolic free calcium levels ([Ca2+]i) in single osteoclasts whilst 300 pmol l-1 salmon calcitonin (sCT) produced a rapid phasic elevation of [Ca2+]i, confirming previous results with asusuberic (1-7) eel calcitonin. The osteoclast-bone resorption assay revealed the following potency difference in direct comparison of the area of resorption per bone slice: beta-CGRP/amylin, 0.1; sCT/amylin, 800 and human CT/amylin, 12. The potency of deamidated amylin approached that of beta-CGRP. Assay precision ranged between 0.3 and 0.8. Amylin (250 nmol l-1) also significantly (P < 0.05) reduced supernatant (tartrate-resistant) acid phosphatase in the bone-osteoclast cultures. These measures independently indicate an effect of amylin on osteoclast motility through mechanisms distinct from those of calcitonin, possibly through different selectivities for receptor subtypes, the cyclic AMP-linked 'amylin subtype' and the [Ca2+]i-linked 'calcitonin subtype'.

Further studies on the mode of action of calcitonin on isolated rat osteoclasts: pharmacological evidence for a second site mediating intracellular Ca2+ mobilization and cell retraction.

Calcitonin is a circulating polypeptide that inhibits bone resorption by inducing both quiescence (Q effect) and retraction (R effect) in osteoclasts. Two structurally related members of the calcitonin gene peptide family, calcitonin gene-related peptide (CGRP) and amylin, inhibit osteoclastic bone resorption selectively via the Q effect. In the present study, we have made measurements of cell spread area in response to the application of amylin, CGRP and a peptide fragment of CGRP, CGRP-(Val8Phe37). We found that, over a wide concentration range (50 pmol/l to 2.5 mumol/l), the selective Q effect agonists did not produce an R effect. Furthermore, the peptides, when used at a 50-fold higher molar concentration than calcitonin, did not antagonize calcitonin-induced cell retraction. Additionally, experiments designed to measure changes in the intracellular free calcium concentration ([Ca2+]i) in single osteoclasts revealed that, unlike calcitonin, the non-calcitonin Q effect agonists did not produce a rise in [Ca2+]i. The peptides were also unable to attenuate the peak rise in [Ca2+]i induced by calcitonin. The results support our hypothesis that the inhibitory activity of calcitonin on osteoclastic bone resorption is mediated by two sites which may or may not be part of the same receptor complex. One of these is the classical Q effect site coupled to adenylate cyclase via a cholera toxin-sensitive Gs. This site can be activated by nanomolar concentrations of calcitonin, amylin, CGRP or CGRP-(Val8Phe37). A novel R effect site, possibly coupled via a pertussis toxin-sensitive G protein to a [Ca2+]i elevating mechanism is predicted from this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of osteoclastic bone resorption by hydrogen peroxide.

The molecular mechanisms underlying the pathophysiology of bone destruction still remain poorly understood. We have found that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is a potent stimulator of osteoclastic bone resorption and cell motility. A marked enhancement of bone resorption was noted when rat osteoclasts, cultured on devitalised bovine cortical bone, were exposed to 10 nM [H2O2]. Apart from exposing osteoclasts to a low extracellular pH, which is known to enhance osteoclastic bone resorption, we provide first evidence for a molecule that stimulates osteoclastic bone resorption in osteoclast cultures that do not respond to parathyroid hormone and 1, 25 dihydroxyvitamin D3. We envisage that both basic biological and practical clinical implications may eventually follow from these studies.

Regulation of cytosolic free calcium in isolated rat osteoclasts by calcitonin.

It is now established that calcium is a second messenger mediating the action of calcitonin on the osteoclast. We have demonstrated that an increase in the concentration of intracellular free calcium ([Ca2+]i) is associated with (and possibly mediates) the functional effects of calcitonin, including an acute reduction of cell spread area (the R effect) and, in the longer term, a reduction in enzyme release. The present study addresses questions relating to mechanisms of calcitonin action on osteoclast [Ca2+]i. We have used asusuberic(1-7) eel and human calcitonin as agonists, and an indo-1-based dual-emission microspectrofluorimetric method for the measurement of [Ca2+]i in single osteoclasts. Whilst asusuberic(1-7) eel calcitonin caused a biphasic increase in [Ca2+]i, human calcitonin produced only a monophasic [Ca2+]i response of a much lower magnitude. Each biphasic response consisted of a rapid initial transient increase, occurring within seconds of exposure, followed by a sustained increase in [Ca2+]i. The magnitude of the latter response was more variable, but was consistently below the peak value of [Ca2+]i. The sustained phase of the calcitonin effect was abolished in extracellular Ca(2+)-free medium. This phase is therefore dependent on extracellular [Ca2+] ([Ca2+]e) whilst the rapid transient increase appeared to be dependent on Ca2+i redistribution. The effects of calcitonin on [Ca2+]i were concentration-dependent, with neither latency nor oscillations. Repetitive 30-s exposures to calcitonin failed to produce subsequent responses. There was a marked concentration-dependent correlation between changes in osteoclast [Ca2+]i and the magnitude of the R effect. Thus the likely components of the biphasic [Ca2-]i response are a rapid redistribution followed by the transmembrane flux of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

A quantitative description of components of in vitro morphometric change in the rat osteoclast model: relationships with cellular function.

We describe the in vitro morphometric changes shown by rat osteoclasts that accompany their functional responses to the application of a range of regulatory agents of known physiological importance. We introduce a cellular motility parameter, mu, which was defined through a quantification of retraction-protrusion behaviour. This was used in conjunction with a net cell retraction, rho, which is derived from the change in total cell area following the application of an agent. These terms were used together for the description of cellular motility changes in response to specific cellular regulatory agents. The definition of retraction-protrusion was normalised against control cell area, to give a dimensionless variable independent of the net cell retraction. Thus, mutual terms present in either descriptor cancelled when the complementary parameter was held constant. Furthermore, the descriptor, mu remained time-invariant for extended intervals (around 20 min) even when rho was varying following cell introduction into culture. Interventions also with substances known to modify osteoclast function, were capable of altering each descriptor, to different extents. Thus elevation of the extracellular Ca2+ concentration ([Ca2+]e) at the osteoclast calcium "receptor" altered rho without changes in mu. In contrast, the polypeptide amylin (250 nM), within 20 minutes of application, elicited a marked change in mu, but only a relatively small change in rho. Finally, human calcitonin treatment (300 pM) influenced both descriptors. When combined together, these morphometric findings accordingly offer complementary descriptions of visible cellular changes in response to added agents of physiological relevance.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlates of osteoclast function in the presence of perchlorate ions in the rat.

The effects of perchlorate anion (ClO4-) on osteoclast properties were investigated through a number of independent in vivo and in vitro procedures. Intravenous infusion of ClO4- significantly reduced plasma [Ca] in young (50 g) Wistar rats, in the absence of changes in plasma [Mg] or [albumin]. This effect was maximal at 20 min after administration, and at a dose of 600 mumol/rat. Scanning electron-microscope images suggested that the presence of 10 mM-perchlorate reduced both the total area of cortical bone resorbed by freshly disaggregated rat osteoclasts, and the number of osteoclastic excavations in vitro. Similar effects were observed in the presence of 5 mM [Ca2+]. The effects of Ca2+ were potentiated by otherwise ineffective (1 mM) doses of perchlorate. Indo-1 dual-emission microspectrofluorimetry indicated a transient sixfold elevation of cytosolic free [Ca2+], in isolated cultured osteoclasts, with addition of 10 mM-perchlorate. Records of time-lapse video images indicated that this was followed by a marked and sustained cell retraction, by up to 70% of control cell area. Such effects were not observed at thiocyanate concentrations (10 mM) that would have produced comparable lyotropic effects as perchlorate. However, perchlorate did not alter morphometric measures for pseudopodial motility and cell migration. Nor did it influence supernatant concentrations of tartrate-resistant (osteoclastic) acid phosphatase in cultures of resorbing osteoclasts. These findings suggest that perchlorate is a potent inhibitor of osteoclast function, and acts through an influence on intracellular [Ca2+], and in turn upon the degree of cell retraction.