Tricuspid papillary fibroelastoma causing syncopal episodes.
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Aged↗
Biomedical subjects
V S Shankar
Publications and source records attributed to V S Shankar.
Extracellularly applied ruthenium red and cADP ribose elevate cytosolic Ca2+ in isolated rat osteoclasts.
We demonstrated recently that the divalent cation-sensing receptor on the osteoclast, the Ca2+ receptor (CaR), is a functional component of a cell surface-expressed ryanodine receptor-like molecule (RyR). The objective of the present study was to further characterize this putative RyR by use of the two well-known cell-impermeant RyR modulators, ruthenium red and adenosine 3',5'-cyclic diphosphate ribose (cADPr). We found that, when applied extracellularly, ruthenium red (5 x 10(-8)-10(-4) M) and cADPr (5 x 10(-6) M) triggered an elevation of cytosolic [Ca2+]. Depolarization of the cell membrane by the application of 0.1 M K+ in the presence of 5 x 10(-6) M. valinomycin resulted in a concentration-dependent increase in the magnitude of the cytosolic Ca2+ response to extracellular ruthenium red (5 x 10(-9) and 5 x 10(-5) M), a phenomenon that was not seen when osteoclasts were hyperpolarized using 5 x 10(-3) M K+ with 5 x 10(-6) M valinomycin. In the presence of an intact nonleaky cell membrane, these results would favor a plasma membrane locus of action for the two modulators. Furthermore, pretreatment of osteoclasts with either modulator resulted in a markedly attenuated cytosolic Ca2+ transient elicited in response to the CaR agonist Ni2+, thus confirming an interaction between the cADPr- and ruthenium red-sensitive sites and the osteoclast CaR. The inhibition of the cytosolic Ca2+ response to Ni2+ induced by ruthenium red remained unchanged in the face of membrane potential changes. Finally, the cytosolic Ca2+ response to caffeine (5 x 10(-4) M), another RyR modulator, was also strongly attenuated by pretreatment with 5 x 10(-9) M ruthenium red. We conclude that ruthenium red and cADPr act on plasma membrane-resident sites and that both these sites interact with the process of divalent cation sensing.
Adenosine Diphosphate Ribose↗
Regulation of extracellular calcium sensing in rat osteoclasts by femtomolar calcitonin concentrations.
Certain eukaryotic cells can sense changes in their extracellular Ca2+ concentration through molecular structures termed Ca(2+)-sensing receptors (CaRs). We have shown recently that in the bone-resorbing osteoclast, a unique cell surface-expressed ryanodine receptor (RyR), functions as the CaR. The present study demonstrates that the sensitivity of this receptor is modulated by physiological femtomolar concentrations of the bone-conserving hormone, calcitonin. Calcitonin was found to inhibit cytosolic Ca2+ responses to both Ca2+ and Ni2+. The latter inhibition was mimicked by amylin (10(-12) M), calcitonin gene-related peptide (10(-12) M), cholera toxin (5 micrograms/l) and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) (2.5 x 10(-4) or 5 x 10(-4) M) and was reversed by the protein kinase A phosphorylation inhibitor, IP-20. Finally, using a quench flow module, we showed that cellular cAMP levels rise to a peak within 25 ms of calcitonin application; this is consistent with the peptide's rapid effect on CaR activation. We conclude, therefore, that cAMP plays a critical role in the control of CaR function by calcitonin.
Animals↗
Extracellular cation sensing by the enterocyte: prediction of a novel divalent cation "receptor".
We report that the divalent cation Ni2+ elicits elevations in the cytosolic free Ca2+ concentration ([Ca2+]) in cultured enterocytes. These elevations were monophasic, each response consisting of a rapid initial transient rise of cytosolic [Ca2+] to a peak value followed by an exponential decline. The magnitude of the cytosolic [Ca2+] elevation varied with the concentration of applied Ni2+. In some cells, a single application of Ni2+ induced oscillatory changes in cytosolic [Ca2+]. There was also evidence for use-dependent inactivation: a conditioning application of Ni2+ substantially attenuated the response resulting from its subsequent application. Our findings thus demonstrate the existence of a divalent cation-sensing "receptor" on the enterocyte. This putative receptor may play a role in regulating mineral absorption across the enterocyte membrane.
Calcium↗
Effect of membrane potential on surface Ca2+ receptor activation in rat osteoclasts.
Osteoclasts are known to possess a divalent cation-sensitive receptor, the Ca2+ receptor (CaR). The latter monitors changes in the local Ca2+ concentration generated as a result of hydroxyapatite dissolution. CaR activation elevates cytosolic [Ca2+] and thereby inhibits osteoclastic bone resorption. Recent studies have used Ni2+ as a surrogate CaR agonist to elicit changes in cytosolic [Ca2+]. This article examines the effects of membrane potential changes on the kinetics of the cytosolic [Ca2+] signal resulting from such Ni(2+)-induced CaR activation. Membrane potential was altered through variations in the extracellular [K] in combination with applications of the K+ ionophore, valinomycin. Membrane potential changes were confirmed by independent electrophysiological patch clamp studies of whole osteoclasts. The application of valinomycin produced a distinct, sustained elevation of cytosolic [Ca2+] in single fura 2-loaded cells, a "primary" response. This response was independent of valinomycin concentration (between 5 nM to 5 microM) and persisted in Ca(2+)-free, EGTA-containing solutions. It also persisted both in high (105 mM) and low (5 mM) extracellular [K+]. A gradual "secondary" elevation of cytosolic [Ca2+] then followed with the continued application of valinomycin, but this was eliminated by sequestering the extracellular [Ca2+] or by increasing extracellular [K+] from 5 to 105 mM. In a separate set of experiments, the presence of 5 microM [valinomycin]-([K+] = 5 mM) prolonged the cytosolic [Ca2+] signal elicited by 50 microM-[Ni2+] application. These prolonged kinetics persisted in low extracellular [Ca2+] (zero-added Ca2+), but reverted to a rapid time-course in the presence of 105 mM-[K+] or at higher [Ni2+] (500 microM and 5 mM). The experiments thus indicate that membrane voltage modifies the kinetics of CaR activation by Ni2+ and therefore suggests that the CaR is an integral protein in the osteoclast surface membrane.
Animals↗
Caffeine modulates Ca2+ receptor activation in isolated rat osteoclasts and induces intracellular Ca2+ release.
A ryanodine-sensitive pathway is involved in intracellular Ca2+ release in response to activation of the osteoclast cell surface Ca2+ receptor. We now report that the ryanodine-receptor modulator, caffeine itself released intracellularly stored Ca2+ and, strongly inhibited Ca2+ release triggered in response to Ca(2+)-receptor activation by Ni2+, a surrogate cation agonist. Caffeine yielded a bell-shaped concentration-response curve (0.005-2 mM) and displayed use-dependent inactivation. Furthermore, responses to caffeine were abolished on prior application of Ni2+ (5 mM). Subthreshold (0.005 mM) caffeine concentrations abolished Ni(2+)-induced elevations in the cytosolic Ca2+ concentration ([Ca2+]). However, in a Ca(2+)-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-containing solution (extracellular [Ca2+] < 10 nM), caffeine (0.5 mM) neither elevated [Ca2+] nor inhibited the response to Ni2+. Finally, when caffeine was applied to intercept the plateau phase of the cytosolic Ca2+ signal triggered by extracellular Ca2+ elevation (10 mM), a rapid but reversible inactivation followed. These studies strongly indicate the existence of a caffeine-sensitive mechanism for the release of intracellularly stored Ca2+ in the osteoclast.
Animals↗
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 cell's 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.
Animals↗
Modulation of the osteoclast Ca2+ receptor by extracellular protons: possible linkage between Ca2+ sensing and extracellular acidification.
We report a sensitivity of the osteoclast cell surface Ca2+ receptor to extracellular protons. Freshly isolated rat osteoclasts were exposed to the known agonists of the Ca2+ receptor, Ca2+ and Ni2+, in extracellular solutions set at different pH values. Decreasing the extracellular pH from 7.8 to 4.0 units markedly potentiated the cytosolic Ca2+ signals elicited in response to Ca2+ receptor activation by either Ni2+ (50 microM, 500 microM or 5 mM) or Ca2+ (5 mM). Each response consisted of a rapid and usually transient elevation of cytosolic [Ca2+]. Maximal cytosolic [Ca2+] responses were obtained at pH values of 6.6 (for 5 mM-[Ni2+]) and 4.0 units (for 5 mM-[Ca2+]). Finally, the effects of extracellular pH persisted in Ca(2+)-free, EGTA-containing solutions, suggesting a modulation of intracellular Ca2+ release.
Animals↗
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.
Animals↗
Calcium influx and release in isolated rat osteoclasts.
Intracellular and extracellular sources of cytosolic [Ca2+] elevation in isolated rat osteoclasts were explored by a comparison of fura-2 signals in response to application of the Ca2+ ionophore, ionomycin, in Ca(2+)-containing and in Ca(2+)-free bathing solutions. Cytosolic [Ca2+] transients persisted in osteoclasts bathed in Ca(2+)-free, EGTA-containing solutions. They consisted of a peak cytosolic [Ca2+bd elevation followed by a full decay to baseline and were refractory to manipulations of surface membrane potential through changes in extracellular [K+]. They disappeared upon intracellular Ca2+ store depletion through repeated ionophore applications. They were therefore attributable solely to intracellularly stored Ca2+. In contrast, the fura-2 peaks in osteoclasts exposed to Ca(2+)-containing solutions decayed to sustained levels. Cytosolic [Ca2+] responses then persisted with repeated ionomycin application. These latter phenomena are accordingly attributable to extracellular Ca2+ entry. Finally, restoration of extracellular [Ca2+] to 1.25 mM following the depletion of intracellular Ca2+ stores by treatment with ionomycin elicited a cytosolic [Ca2+] 'overshoot' consistent with capacitative Ca2+ entry via a cytosolic route. These results demonstrate a refillable intracellular source of cytosolic Ca2+ that could function in osteoclastic regulation.
Animals↗
Quantitative studies on the effect of prostacyclin on freshly isolated rat osteoclasts in culture.
Prostaglandins exert marked but transient inhibitory effects on bone resorption. The present study examines the effects of prostacyclin (0.15 to 25 microM) on the morphology of freshly disaggregated rat osteoclasts. An area descriptor, rho, represented changes in total cell spread area, and a motility descriptor, mu, represented overall changes in cell motility. The application of prostacyclin intercepted the trend of an increasing cell spread area with time and produced a transient reduction of rho, an R effect. Its magnitude depended upon concentration and was marked at 25 microM prostacyclin. The subsequent recovery (+0.8/min) of rho at this concentration resembled the persistent spreading seen in the absence of the agonist. There was also a sustained decrease in mu to approximately 60% of its pretreatment value (a Q effect) following the application of 25 microM prostacyclin. The extracellular application of 20 mM [Ca2+] produced a similarly transient cell retraction preceded by a rise of cytosolic [Ca2+], but without a corresponding decrease in mu. In contrast, prostacyclin did not elevate cytosolic [Ca2+], suggesting the triggering of an alternative transduction pathway. A fully reversible retraction together with incomplete quiescence may explain the transience characteristic of the antiresorptive action of prostacyclin.
Animals↗
Voltage sensitivity of the osteoclast calcium receptor.
We demonstrated previously that osteoclasts possess a divalent cation-sensitive "receptor", the Ca2+ receptor. Activation of the Ca2+ receptor by the surrogate cation Ni2+ was shown to elicit an increase in cytosolic [Ca2+] to a peak value followed by an exponential decline. In the present study we examined the influence of surface membrane voltage on the kinetics of Ca2+ receptor inactivation. The K+ ionophore, valinomycin was applied to intercept the declining phase of the cytosolic [Ca2+] transient elicited by application of between 50 microM- and 5 mM-[Ni2+]. This resulted in a sustained elevation of cytosolic [Ca2+] or even a 'hump' followed by a gradual decline. Such a kinetic alteration persisted in a Ca(2+)-free solution, but was abolished in high extracellular [K+] (105 mM). Thus, we demonstrate for the first time to our knowledge, a modulatory effect of membrane potential on the function of the osteoclast Ca2+ receptor.
Animals↗
Stimulation of a Gs-like G protein in the osteoclast inhibits bone resorption but enhances tartrate-resistant acid phosphatase secretion.
Previous studies have demonstrated that G-protein agonists induce quiescence (Q effect) or retraction (R effect) in isolated osteoclasts. We now report the functional effects of such agonists on osteoclastic bone resorption and enzyme release. Exposure of osteoclasts to tetrafluoro-aluminate anions (AlF4-), a universal G protein stimulator, resulted in a marked concentration-dependent inhibition of bone resorption. This was associated with a dramatic increase in the secretion of the osteoclast-specific enzyme, tartrate-resistant acid phosphatase (TRAP). Cholera toxin, a Gs stimulator and a selective Q effect agonist, similarly abolished bone resorption and enhanced TRAP secretion. In contrast, pertussis toxin, a Gi inhibitor and a selective R effect agonist, inhibited bone resorption significantly, but slightly reduced enzyme release. The results suggest an involvement of a Gs-like G protein in TRAP secretion from the osteoclast, possibly through a cyclic AMP-dependent mechanism.
Acid Phosphatase↗
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+.
Animals↗
Activation of the Ca2+ "receptor" on the osteoclast by Ni2+ elicits cytosolic Ca2+ signals: evidence for receptor activation and inactivation, intracellular Ca2+ redistribution, and divalent cation modulation.
Earlier studies have demonstrated that a high (mM) extracellular Ca2+ concentration triggers intracellular [Ca2+] signals with a consequent inhibition of bone resorptive activity. We now report that micromolar concentrations of the divalent cation, Ni2+, elicited rapid and concentration-dependent elevations of cytosolic [Ca2+]. The peak change in cytosolic [Ca2+] increased monotonically with the application of [Ni2+] in the 50-5,000 microM range in solutions containing 1.25 mM-[Ca2+] and 0.8 mM-[Mg2+]. The resulting concentration-response function suggested Ni(2+)-induced activation of a single class of binding site (Hill coefficient = 1). The triggering process also exhibited a concentration-dependent inactivation in which conditioning Ni2+ applications in the range 5-1,500 microM-[Ni2+] inhibited subsequent responses to a maximally effective [Ni2+] of 5,000 microM. Ni(2+)-induced cytosolic [Ca2+] responses were not dependent on extracellular [Ca2+]. Thus, when 5,000 microM-[Ni2+] was applied to osteoclasts in Ca(2+)-free, ethylene glycol bis-(aminoethyl ether) tetraacetic acid (EGTA)-containing medium (< or = 5 nM-[Ca2+] and 0.8 mM-[Mg2+]), cytosolic [Ca2+] responses resembled those obtained in the presence of 1.25 mM-[Ca2+]. Prior depletion of intracellular Ca2+ stores by ionomycin prevented Ni(2+)-induced cytosolic [Ca2+] responses, suggesting a major role for intracellular Ca2+ redistribution in the response to Ni2+. The effects of Ni2+ were also modulated by the extracellular concentration of the divalent cations, Ca2+ and Mg2+. When these cations were not added to the culture medium (0 microM-[Ca2+] and [Mg2+]), even low [Ni2+] ranging between 5 pM and 50 microM elicited progressively larger cytosolic [Ca2+] transients. However, the response magnitude decreased at higher, 250-5,000 microM-[Ni2+], resulting in a "hooked" concentration-response curve. Furthermore, increasing extracellular [Mg2+] or [Ca2+] (0-1 mM) diminished the response to 50 microM-[Ni2+], a concentration on the rising phase of the "hook." Similar increases (0-10 mM) in extracellular [Mg2+] or [Ca2+] increased the response to 5,000 microM-[Ni2+], a concentration on the falling phase of the "hook". These findings are consistent with the existence of a membrane receptor strongly sensitive to Ni2+ as well as the divalent cations, Ca2+ and Mg2+. Receptor occupancy apparently activates intracellular Ca2+ release followed by inactivation. Furthermore, repriming is independent of intracellular Ca2+ stores, suggesting that such inactivation operates at a transduction step between receptor occupancy and intracellular Ca2+ release.
Animals↗
Tetracyclines modulate cytosolic Ca2+ responses in the osteoclast associated with "Ca2+ receptor" activation.
We report the effects of tetracycline analogues on cytosolic Ca2+ transients resulting from application of ionic nickel (Ni2+), a potent surrogate agonist of the osteoclast Ca2+ "receptor". Preincubation with minocycline (1 mg/l) or a chemically modified tetracycline, 4-dedimethyl-aminotetracycline (CMT-1) (1 or 10 mg/l), resulted in a significant attenuation of the magnitude of the cytosolic [Ca2+] response to an application of 5 mM-[Ni2+]. Preincubation with doxycycline (1 or 10 mg/l) failed to produce similar results. In addition, application of minocycline alone (0.1-100 mg/l) resulted in a 3.5-fold elevation of cytosolic [Ca2+]. The results suggest a novel action of tetracyclines on the osteoclast Ca2+ "receptor".
Animals↗
The effect of tetracyclines on quantitative measures of osteoclast morphology.
We report the effects of the tetracycline analogues 4-dedimethylaminotetracycline (CMT-1) and minocycline on osteoclast spreading and motility. Both agents influenced the morphometric descriptor of cell spread area, rho, producing cellular retraction or an R effect (half-times: 30 and 44 minutes for CMT-1 and minocycline, respectively). At the concentrations employed, the tetracycline-induced R effects were significantly slower than, but were qualitatively similar to, those resulting from Ca2+ "receptor" activation through the application of 15 mM-[Ca2+] (slopes: -1.25, -0.18, and -4.40/minute for 10 mg/l-[CMT-1], 10 mg/l-[minocycline] and 15 mM-[Ca2+], respectively). In contrast, the same tetracycline concentrations did not influence osteoclast margin ruffling activity as described by mu, a motility descriptor known to be influenced by elevations of cellular cyclic AMP. Thus, the tetracyclines exert morphometric effects comparable to changes selectively activated by occupancy of the osteoclast Ca2+ "receptor" which may act through an increase in cytosolic [Ca2+].
Animals↗
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.
Calcium↗