Vijai S. Shankar

A ryanodine receptor-like molecule expressed in the osteoclast plasma membrane functions in extracellular Ca2+ sensing.

Ryanodine receptors (RyRs) reside in microsomal membranes where they gate Ca2+ release in response to changes in the cytosolic Ca2+ concentration. In the osteoclast, a divalent cation sensor, the Ca2+ receptor (CaR), located within the cells plasma membrane, monitors changes in the extracellular Ca2+ concentration. Here we show that a RyR-like molecule is a functional component of this receptor. We have demonstrated that [3H] ryanodine specifically binds to freshly isolated rat osteoclasts. The binding was displaced by ryanodine itself, the CaR agonist Ni2+ and the RyR antagonist ruthenium red. The latter also inhibited cytosolic Ca2+ elevations induced by Ni2+. In contrast, the responses to Ni2+ were strongly potentiated by an antiserum Ab129 raised to an epitope located within the channel-forming domain of the type II RyR. The antiserum also stained the surface of intact, unfixed, trypan blue-negative osteoclasts. Serial confocal sections and immunogold scanning electron microscopy confirmed a plasma membrane localization of this staining. Antiserum Ab34 directed to a putatively intracellular RyR epitope expectedly did not stain live osteoclasts nor did it potentiate CaR activation. It did, however, stain fixed, permeabilized cells in a distinctive cytoplasmic pattern. We conclude that an RyR-like molecule resides within the osteoclast plasma membrane and plays in important role in extracellular Ca2+ sensing.

Role of the endothelial cell in osteoclast control: New perspectives

The osteoclast is of central importance in the process of bone remodeling. Its function is regulated by hormones and locally produced factors. Endothelial cells occur in close proximity to the osteoclast. Some endothelial cell-derived products, including endothelins, nitric oxide, and reactive oxygen species, have been recently implicated as modulators of osteoclast function. Endothelins inhibit bone resorption and osteoclast margin ruffling (quiescence or Q effect) at concentrations similar to those effective for their primary vasoconstrictive action. Contrary to expectations, however, it has been shown that endothelin action on the osteoclast is not mediated through an elevation of cytosolic Ca2+. Nitric oxide (NO) produces marked cell retraction (retraction or R effect), but its detailed mode of action is unknown. However, it is clear that the effects of this autocoid are not due to enhanced cyclic guanosine monophosphate (cGMP) production, a transduction system commonly used by NO. Finally, the reactive oxygen species H2O2 has been shown recently to enhance osteoclastic activity. Thus, the reported effects of the endothelial cell-derived products on the osteoclast are generally consistent with a regulatory role for endothelial cells in osteoclast control and suggest the existence of unique activation pathways, well worth exploring further. Unravelling the responsible mechanisms may also help understand the pathophysiology of a range of bone and joint diseases. For example, in rheumatoid arthritis, there is increased H2O2 production from activated neutrophils, and bone resorption is a major pathophysiological feature.

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.

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.

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.

The osteoclast Ca2+ receptor is highly sensitive to activation by transition metal cations

We report changes in the cytosolic Ca2+ concentration ([Ca2+]i) of single rat osteoclasts in response to Ca2+ receptor activation by micromolar concentrations of the transition metal cations, Cd2+ and Ni2+. The extracellular application of Cd2+ or Ni2+ resulted in a concentration-dependent elevation of cytosolic [Ca2+]. Each monophasic [Ca2+]i response consisted of an initial rapid rise of [Ca2+]i to a peak value followed by an exponential decay. Prior application of Cd2+ or Ni2+ induced refractoriness to a second application of the same cation. The results confirm the existence of a divalent cation-sensitive site on the osteoclast showing features of concentration-dependent activation and use-dependent inactivation.

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.

Amylin in bone conservation current evidence and hypothetical Considerations

Amylin, a 37-amino-acid long single-chain polypeptide, is structurally homologous to calcitonin and calcitonin gene-related peptide (CGRP). The peptide is secreted from pancreatic beta cells and is thought to have an anti-insulin action. Here, we review the recently described effects of amylin on calcium homeostasis and discuss its possible role in bone conservation. Amylin is a potent hypocalcemic and antiresorptive peptide. Studies using isolated osteoclasts have revealed that amylin inhibits cell motility (Q effect), without affecting cell spread area or elevating cytosolic [Ca(2+)]. Thus, amylin action is similar to that of calcitonin, but lower in potency. Lower circulating concentrations of amylin in type-1 diabetes may cause the bone loss associated with this condition.

Evidence that a ryanodine receptor triggers signal transduction in the osteoclast.

We have investigated the effect of the alkaloid ryanodine on the release of intracellularly stored Ca2+ in response to activation of the osteoclast Ca2+ receptor by the surrogate agonist, Ni2+, Ni2+ (6 mM) in the presence of ethylene-glycol bis-(aminoethyl ether) tetraacetic acid (EGTA) (1.2 mM) and valinomycin (5 microM) induced a transient elevation of cytosolic [Ca2+] in fura 2-loaded osteoclasts. This transient was superimposed upon a small steady elevation of cytosolic [Ca2+] induced by the initial application of valinomycin alone. Ryanodine (10 microM) completely abolished such responsiveness. However, cytosolic [Ca2+] transients were restored when osteoclasts were depolarized by the extracellular inclusion of 100 mM-[K+] in the same solution. Thus, we demonstrate a sensitivity of the osteoclast signal transduction system to ryanodine for the first time to our knowledge.

Dimensional analysis of osteoclastic bone resorption and the measurement of biologically active calcitonin

Calcitonin inhibits bone resorption through a direct action on the osteoclast. We report a quantitative analysis of bone resorption by disaggregated rat osteoclasts. We then used our findings to develop a formal bioassay for calcitonin. Osteoclasts were mechanically disaggregated from neonatal rat long bones and dispersed at low densities on slices of devitalized bovine cortical bone. The resulting areas of bone excavation were quantified to micrometric precision by scanning electron microscopy together with computer‐assisted image analysis. These findings were correlated with the volumes of bone resorption in the same slices measured by confocal scanning microscopy for the first time. The total planar areas of bone resorption per slice correlated linearly (r = 0.78) with the confocal microscopic measurements of total volume resorbed, provided that volume was expressed to its two‐thirds power. The latter transformation resulted in representations of the determined areas ([length]2) and volumes ([length]3) which were dimensionally consistent. These findings thus demonstrate that osteoclastic bone excavations show a consistent relationship between area and volume and that assessments of the area of excavations accordingly provide an empirical representation of the volume of bone resorbed. Furthermore, in view of the skewed nature of the distributions of area measurements, we assessed the effect of transforming the response variable to derive a metameter, (planar area of resorption)1/2. Such transformed data points, which expressed the data in the dimensions of [length], were more normally distributed than the raw data points and had more stable variances over a wider concentration range. We accordingly determined relative potencies using parallel line analyses on the transformed data. The latter offered a consistent correlation to the volume measurements when these were also converted to dimensions of [length] (r = 0.805). It was confirmed that the inhibition of bone resorption by calcitonins from various species, namely, pig, salmon and eel, was quantitatively dependent upon concentration of the respective peptides. The resulting assay was also found to be sufficiently sensitive to measure picomolar peptide concentrations with a precision, lambda (standard deviation/slope), ranging between 0.3 and 0.8. Finally, we identified factors affecting assay precision and sensitivity.