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M Pazianas

Publications and source records attributed to M Pazianas.

At least 37 records · Page 2Linked to original sources
PubMed1994

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↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1993

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↗
PubMed1993

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↗
PubMed1993

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↗
PubMed1993

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↗
PubMed1993

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↗
PubMed1993

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↗
PubMed1993

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.

Animals↗
PubMed1993

Functional consequences of the interaction of Ni2+ with the osteoclast Ca2+ 'receptor'.

Ni2+ was used as an extracellular activator of the Ca2+ 'receptor' in order to study the regulation of osteoclast function in vitro. Application of different micromolar concentrations of Ni2+ to osteoclasts bathed in 1.25 mM [Ca2+] and 0.8 mM [Mg2+] caused a concentration-dependent elevation of cytosolic [Ca2+] measured in single cells using fura-2 fluorescence. Cytosolic [Ca2+] responses to 5 mM [Ni2+] showed a rapidly developing and use-dependent inactivation, unlike those induced by the application of 10 mM [Ca2+]. Pre-treatment with 5 mM [Ni2+] reduced the magnitude of responses to a subsequent extracellular application of 10 mM [Ca2+] and vice versa. Ni2+ treatment elicited a number of functional effects. It produced an inhibition of osteoclastic bone resorption which was sustained over hours. This was associated with a pronounced cell retraction or R effect over the 40 min period following Ni2+ exposure as observed by time-lapse video image analysis. Both these effects varied with concentration. In contrast, granule movement, cell migration, and quantitative indicators of margin ruffling were all unchanged. These findings are consistent with the initiation of a causally related set of specific functional and morphometric events following activation of a specific membrane receptor sensitive to divalent cations.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

Camelford water poisoning accident: serial neuropsychological assessments and further observations on bone aluminium.

The serial cognitive assessment of ten individuals made between 8 and 26 months after the water at Camelford in Cornwall was accidentally contaminated with aluminium sulphate, showed consistent evidence of impairment of information processing and memory. There was no obvious relationship between these impairments and measurements of anxiety and depression. Serial bone biopsies in two individuals showed that the aluminium which was present 6 and 7 months after the accident had disappeared by 19 months. In the eight individuals biopsied 12-17 months after the accident the bone showed no stainable aluminium. Thus, aluminium deposited in the bone of normal individuals can disappear within 18 months. After an accident such as that at Camelford important evidence of toxicity is likely to be missed if an investigation is delayed. The abnormal neuropsychological findings indicate cognitive impairment, but whether this was caused by an acute episode of brain damage, or other causes such as the psychological effects of stress resulting from the accident, is uncertain.

Accidents↗
PubMed1993

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)

Amyloid↗
PubMed1992

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.

Animals↗
PubMed1992

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+.

Animals↗
PubMed1992

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)

Amyloid↗