Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption

The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose- dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.     

Activation and inactivation of the osteoclast Ca2+ receptor by the trivalent cation, La3+.

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 lanthanide metal cation, La3+. The extracellular application of La3+ induced a concentration-dependent elevation of cytosolic [Ca2+]. Prior conditioning of osteoclasts with La3+ resulted in a concentration-dependent reduction of the response to a subsequent application of a maximally effective concentration of Ni2+, a known agonist of the osteoclast Ca2+ receptor. The results establish that the osteoclast Ca2+ receptor is highly sensitive to activation and inactivation by the trivalent cation, La3+.     

Ga3+ inhibits parathyroid hormone release without interacting with the Ca2+ receptor of the parathyroid cell.

Gallium nitrate is an antihypercalcemic agent with established actions on bone. The effects of Ga(NO3)3 on parathyroid hormone (PTH) release, cytoplasmic Ca2+ concentration ([Ca2+]i) and cAMP production of enzymatically dispersed parathyroid cells from bovine as well as normal and pathological human parathyroid glands have now been studied. Ga3+ at 200 microM inhibited PTH release whereas 600 microM NO3- had no effect. The inhibition was additive to that obtained by elevating extracellular Ca2+. Unlike Ca2+, Ga3+ failed to increase [Ca2+]i or reduce cAMP formation. The results indicate that Ga3+ inhibits PTH release by a mechanism other than activation of the cation receptor of the parathyroid cells. This mechanism may contribute also to inhibition by other cations.     

The calcium-sensing receptor is involved in strontium ranelate-induced osteoclast apoptosis. New insights into the associated signaling pathways

Strontium ranelate exerts both an anti-catabolic and an anabolic effect on bone cells. To further investigate the molecular mechanism whereby strontium ranelate inhibits bone resorption, we focused our attention on the effects of strontium ranelate on osteoclast apoptosis and on the underlying mechanism(s). Using primary mature rabbit osteoclasts, we demonstrated that strontium (Sro2+) dose-dependently stimulates the apoptosis of mature osteoclasts. As shown previously for calcium (Cao2+), the Sro2+-induced effect on mature osteoclasts is mediated by the Cao2+-sensing receptor, CaR, which in turn stimulates a phospholipase C-dependent signaling pathway and nuclear translocation of NF-kappaB. Unlike Cao2+, however, Sro2+-induced osteoclast apoptosis was shown to depend on PKCbetaII activation and to be independent of inositol 1,4,5-trisphosphate action. As a consequence of these differences in their intracellular signaling pathways, Sro2+ and Cao2+ in combination were shown to exert a greater effect on mature osteoclast apoptosis than did either divalent cation by itself. Altogether, our results show that Sro2+ acts through the CaR and induces osteoclast apoptosis through a signaling pathway similar to but different in certain respects from that of Cao2+. This difference in the respective signaling cascades enables Sro2+ to potentiate Cao2+-induced osteoclast apoptosis and vice versa. In this manner, it is conceivable that Sro2+ and Cao2+ act together to inhibit bone resorption in strontium ranelate-treated patients.     

Nitric oxide regulation of cGMP production in osteoclasts

Bone resorption by osteoclasts is modified by agents that affect cyclic guanosine monophosphate (cGMP), but their relative physiological roles, and what components of the process are present in osteoclasts or require accessory cells such as osteoblasts, are unclear. We studied cGMP regulation in avian osteoclasts, and in particular the roles of nitric oxide and natriuretic peptides, to clarify the mechanisms involved. C-type natriuretic peptide drives a membrane guanylate cyclase, and increased cGMP production in mixed bone cells. However, C-type natriuretic peptide did not increase cGMP in purified osteoclasts. By contrast, osteoclasts did produce cGMP in response to nitric oxide (NO) generators, sodium nitroprusside or 1-hydroxy-2-oxo-3,3-bis(3-aminoethyl)-1-triazene. These findings indicate that C-type natriuretic peptide and NO modulate cGMP in different types of bone cells. The activity of the osteoclast centers on HCI secretion that dissolves bone mineral, and both NO generators and hydrolysis-resistant cGMP analogues reduced bone degradation, while cGMP antagonists increased activity. NO synthase agonists did not affect activity, arguing against autocrine NO production. Osteoclasts express NO-activated guanylate cyclase and cGMP-dependent protein kinase (G-kinase). G-kinase reduced membrane HCI transport activity in a concentration-dependent manner, and phosphorylated a 60-kD osteoclast membrane protein, which immunoprecipitation showed is not an H+-ATPase subunit. We conclude that cGMP is a negative regulator of osteoclast activity. cGMP is produced in response to NO made by other cells, but not in response to C-type natriuretic peptide. G-kinase modulates osteoclast membrane HCI transport via intermediate protein(s) and may mediate cGMP effects in osteoclasts.     

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.     

Evidence of reciprocal regulation between the high extracellular calcium and RANKL signal transduction pathways in RAW cell derived osteoclasts

During bone resorption, osteoclasts are exposed to high Ca2+ concentrations (up to 40 mM). The role of high extracellular Ca2+ in receptor activator of NF-kappaB ligand (RANKL)-mediated osteoclast survival and their functional interrelationship is unclear. In this study, we show that RANKL enhances osteoclast tolerance to high extracellular Ca2+ by protecting the cell from cell death in a dose dependent manner. We have provided evidence that RANKL does this by attenuating high extracellular Ca2+-induced Ca2+ elevations. Moreover, we have found that high extracellular Ca2+-induced cell death was partially inhibited by a caspase-3 inhibitor, suggesting caspase-3-mediated apoptosis is involved. Conversely, using reporter gene assays and Western blot analysis, we have demonstrated that high extracellular Ca2+ desensitizes the RANKL-induced activation of NF-kappaB and c-Jun N-terminal kinase (JNK), and inhibits constitutive and RANKL-stimulated ERK phosphorylation, indicating a negative feed-back mechanism via specific RANKL signaling pathways. Taken together, this study provides evidence for a reciprocal regulation between high extracellular Ca2+ and RANKL signaling in RAW cell derived osteoclasts. Our data imply a cross talk mechanism of extracellular Ca2+ on osteoclast survival through the regulation of RANKL.     

Functional implications of Ca2+ mobilizing properties for nitric oxide production in aortic endothelium

We have investigated the relationship between Ca2+ mobilization and the cellular production of nitric oxide (NO) by using fura-2 and diaminofluorescein-2 (DAF-2), an NO-sensitive dye, in bovine aortic endothelial cells (BAEC). High concentrations of ATP (100 microM) or thapsigargin (1 micro M) depleted intracellular Ca2+ store sites with a single Ca2+ transient, and induced an increase in DAF-2 fluorescence even in Ca2+-free solution, thereby indicating that store depletion leads to NO production. The same level of increase in DAF-2 fluorescence was elicited by low concentrations of ATP (1 micro M), which induced Ca2+ oscillations but did not deplete store sites, only in the presence of extracellular Ca2+. Furthermore, inhibition of ATP (1 micro M)-induced Ca2+ entry with La3+ suppressed DAF-2 fluorescence. ATP (0.3 micro M), applied in Ca2+-free, Mn2+-containing solution induced Mn2+ entry-coupled fura-2 quenching, repeating shortly after each oscillation peak. These results indicate that NO is produced preferentially by entered Ca2+, and that Ca2+ oscillations, which are induced by low levels of stimulation, play a significant role in NO production by strongly modulating Ca2+ entry.     

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.     

Peroxynitrite formed by mitochondrial NO synthase promotes mitochondrial Ca2+ release

Mitochondria contribute to the maintenance of the intracellular Ca2+ homeostasis by taking up and releasing the cation via separate and specific pathways. The molecular details of the release pathway are elusive but its stimulation by the cross-linking of some vicinal thiols and consequently NAD+ hydrolysis are known. Thiol cross-linking and NAD+ hydrolysis can be achieved by addition of peroxynitrite (ONOO-), the product of the reaction between superoxide (O2-) and nitric oxide (nitrogen monoxide, NO*) to mitochondria. Mitochondria contain an NO synthase (mtNOS), which is stimulated by Ca2+, and are a copious source of O2-. We show here that intramitochondrially formed ONOO- stimulates the specific, NAD+-linked Ca2+ release from mitochondria. Our findings that upon Ca2+ uptake mtNOS is stimulated, that ONOO- is formed, and that Ca2+ is subsequently released from intact mitochondria suggest the existence of a feedback loop, which prevents overloading of mitochondria with Ca2+.     

Divalent cations mimic the inhibitory effect of extracellular ionised calcium on bone resorption by isolated rat osteoclasts: further evidence for a "calcium receptor".

Osteoclast activity is thought to be regulated by calcitonin, as well as by the level of ionised calcium generated locally as a result of bone resorption. The exposure of isolated osteoclasts to elevated ambient calcium levels has been shown to lower resorptive activity and to reduce rates of enzyme release. We have attempted to determine whether these effects are mediated by a divalent cation-sensitive "calcium receptor," as has been reported for the parathyroid chief cells. Thus, we compared the effect of alkaline earth metal cations on osteoclast function using a morphometric measure of bone resorption and a spectrophotometric method for measuring the activity of the released enzyme, acid phosphatase. The exposure of resorbing osteoclasts to between 5 and 20 mM extracellular ionised calcium ([Ca2+]e) inhibited bone resorption and enzyme release to an extent similar to that seen with 0.1 to 10 microM ionomycin. The effect of combining submaximal concentrations of [Ca2+]e (15 mM) and ionomycin (0.1 microM) resulted in additivity, suggesting that the influence of [Ca2+]e on bone resorption was mediated by elevated intracellular calcium levels ([Ca2+]i). The other cations studied (Mg2+, Ba2+) were effective and elicited similar effects, although some required higher concentrations. Thus, whilst Ca2+ and Mg2+ were effective at 10 to 15 mM levels, Ba2+ was effective only at high (20 mM) concentrations. These findings are consistent with an influence of [Ca2+]e on osteoclast activity through an action on a surface membrane "calcium receptor" that can also bind other divalent cations, rather than by passive changes of [Ca2+]i with [Ca2+]e elevation.     

Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes.

Following receptor activation in Xenopus oocytes, spiral waves of intracellular Ca2+ release were observed. We have identified key molecular elements in the pathway that give rise to Ca2+ excitability. The patterns of Ca2+ release produced by GTP-gamma-S and by inositol 1,4,5-trisphosphate (IP3) are indistinguishable from receptor-induced Ca2+ patterns. The regenerative Ca2+ activity is critically dependent on the presence of IP3 and on the concentration of intracellular Ca2+, but is independent of extracellular Ca2+. Broad regions of the intracellular milieu can be synchronously excited to initiate Ca2+ waves and produce pulsating foci of Ca2+ release. By testing the temperature dependence of wavefront propagation, we provide evidence for an underlying process limited by diffusion, consistent with the elementary theory of excitable media. We propose a model for intracellular Ca2+ signaling in which wave propagation is controlled by IP3-mediated Ca2+ release from internal stores, but is modulated by the cytoplasmic concentration and diffusion of Ca2+.     

Neuropeptide inhibition of voltage-gated calcium channels mediated by mobilization of intracellular calcium.

Many neurotransmitters and hormones regulate secretion from endocrine cells and neurons by modulating voltage-gated Ca2+ channels. One proposed mechanism of neurotransmitter inhibition involves protein kinase C, activated by diacylglycerol, a product of phosphatidyl-inositol inositol hydrolysis. Here we show that thyrotropin-releasing hormone (TRH), a neuropeptide that modulates hormone secretion from pituitary tumor cells, inhibits Ca2+ channels via the other limb of the phosphatidylinositol signaling system: TRH causes inositol trisphosphate-triggered Ca2+ release from intracellular organelles, thus causing Ca2(+)-dependent inactivation of Ca2+ channels. Elevation of intracellular Ca2+ concentration is coincident with the onset of TRH-induced inhibition and is necessary and sufficient for its occurrence. The inhibition is blocked by introducing Ca2+ buffers into cells and mimicked by a variety of agents that mobilize Ca2+. Treatments that suppress protein kinase C have no effect on the inhibition. Hence inactivation of Ca2+ channels occurs not only as a result of Ca2+ influx through plasma membrane channels, but also via neurotransmitter-induced Ca2+ mobilization. This phenomenon may be common but overlooked because of the routine use of Ca2+ buffers in patch-clamp electrodes.     

Silver ion triggers Ca2+ release from intracellular store sites in saponin-treated HL-60 cells

We examined the effect of silver ion on Ca2+ mobilization from intracellular stores in permeabilized HL-60 cells using a filtration method and 45Ca2+. In HL-60 cells preloaded with Ca2+ in the presence of ATP, micromolar concentrations of AgNO3 elicited marked Ca2+ release within 1 min. The AgNO3-induced Ca2+ release was not affected by the free Ca2+ concentration in the medium. Equivalent concentrations of AgNO3 inhibited energy-dependent Ca2+ uptake as well as oxalate-supported Ca2+ uptake. In passive Ca2+ release experiments when ATP was completely depleted in the solution, AgNO3 also triggered Ca2+ release. Sulfhydryl protecting agents such as 2-mercaptoethanol, dithiothreitol, and glutathione (reduced form) blocked the AgNO3-induced Ca2+ release. From these results, we conclude that the apparent Ca2+ release induced by AgNO3 is mainly due to inhibition of the Ca2+ pump with increased permeability for Ca2+ and partly due to a direct effect on the Ca2+ release channel, probably by modification of sulfhydryl groups on these proteins.     

Cadmium-induced insulin release does not involve changes in intracellular handling of calcium

A possible interaction between Cd2+ and Ca2+ as a component in Cd2+-induced insulin release was investigated in beta cells isolated from obese hyperglycemic mice. The glucose stimulated Cd2+ uptake was dependent on the concentration of sugar. This uptake was sigmoidal with a Km for glucose of about 5 mM and was suppressed by both 50 microM of the voltage-activated Ca2+ channel blocker D-600 and 12 mM Mg2+. In the presence of 8 mM glucose 5 microM Cd2+ evoked a prompt and sustained stimulatory response, corresponding to about 3-fold of the insulin release obtained in the absence of the ion. Whereas 5 microM Cd2+ was without effect on the glucose-stimulated 45Ca efflux in the presence of extracellular Ca2+, 40 microM inhibited it. At a concentration of 5 microM, Cd2+ had no effect on the resting membrane potential or the depolarization evoked by either glucose or K+. In the absence of extracellular Ca2+ there was only a modest stimulation of 45Ca efflux by 5 microM Cd2+. Studies of the ambient free Ca2+ concentration maintained by permeabilized cells also indicate that 5 microM Cd2+ do not mobilize intracellularly bound Ca2+ to any great extent. On the contrary, at this concentration, Cd2+ even suppressed inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. The present study suggests that Cd2+ stimulates insulin release by a direct mechanism which does not involve an increase in cytoplasmic free Ca2+ concentration.     

Galanin inhibits glucose-stimulated insulin release by a mechanism involving hyperpolarization and lowering of cytoplasmic free Ca2+ concentration

The intrapancreatic neuropeptide galanin has been demonstrated to lower plasma insulin levels in vivo. The effects of this peptide on insulin secretion, cytoplasmic free Ca2+ concentration and membrane potential have now been studied in vitro. Glucose-stimulated insulin secretion was inhibited by galanin under these conditions, indicating a direct effect of the peptide on the beta-cells. The neuropeptide reversed both the increase in membrane potential and cytoplasmic free Ca2+ in response to glucose stimulation. At a non-stimulatory concentration of the sugar, galanin induced a slight hyperpolarization without any effect on cytoplasmic free Ca2+. Galanin did not affect K+-induced increase in cytoplasmic free Ca2+, excluding a direct inhibitory effect on the voltage-activated Ca2+ channels. The results indicate that galanin inhibition of glucose-stimulated insulin release involves hyperpolarization with a subsequent decrease in cytoplasmic free Ca2+.