Biomedical subjects

S J Dixon

Publications and source records attributed to S J Dixon.

At least 19 recordsLinked to original sources

PubMed2006

Analysis of a missense variant of the human N-formyl peptide receptor that is associated with agonist-independent beta-arrestin association and indices of inflammation.

Formyl-Met-Leu-Phe (fMLP) is a potent chemoattractant molecule released from both bacteria and damaged mitochondria that activates fMLP receptors (FPR) leading to neutrophil chemotaxis, degranulation and superoxide production. A common missense single nucleotide polymorphism in the human FPR1 gene at nucleotide c.32C>T results in the amino-acid substitution, p.I11T, in the FPR1 extracellular amino-terminus. The minor (c.32T) allele frequencies were 0.25, 0.27, 0.25, 0.15 and 0.14 in healthy Caucasian, African, East Indian, Chinese and Native Canadian individuals, respectively. In subjects homozygous for the p.T11 allele, we find elevated serum concentrations of C-reactive protein, increased absolute counts of blood leukocytes and neutrophils, and erythrocyte sedimentation rates. When expressed in HEK 293 and RBL-2H3 cells a substantial proportion of FPR1 p.I11T variant is retained intracellularly and agonist-independent internalization of the FPR1 p.I11T variant, but not the wild-type FPR1, is constitutively associated with beta-arrestin2-GFP in vesicles. Moreover, basal N-acetyl-D-glucosaminidase release is increased in primary neutrophils isolated from subjects either heterozygous or homozygous for the FPR1 p.T11 allele. Taken together, the data suggest an increased receptor activity and phenotypic expression of increased inflammatory indices in subjects with the p.T11 allele.

Arrestins↗

PubMed2003

Regulation of osteoclasts by calcitonin and amphiphilic calcitonin conjugates: role of cytosolic calcium.

The peptide hormone calcitonin is a potent inhibitor of osteoclastic resorption, but it is unstable and poorly absorbed following oral administration. Conjugates of salmon calcitonin covalently linked to low-molecular-weight amphiphilic polymers show improved stability and absorption. The purpose of this study was to investigate the biological activity of these conjugates in vitro using rat osteoclasts and HEK-293 cells transfected with the C1a isoform of the calcitonin receptor. Salmon calcitonin or its conjugates (10 pM-10 nM) caused rapid arrest of osteoclast membrane ruffling and subsequent retraction. The same amphiphilic polymer attached to an unrelated protein had no effect on osteoclast morphology or motility. Since calcitonin-induced retraction of osteoclasts is thought to be mediated by Ca2+ signaling, we investigated the effects of calcitonin and its conjugates on cytosolic free Ca2+ concentration ([Ca24]i). In HEK-293 cells transfected with the calcitonin receptor, these agents induced transient elevations of [Ca2+]i. However, the rise of [Ca2+]i in HEK-293 cells occurred at concentrations 100-1000-fold higher than those required to elicit osteoclast retraction. To investigate the role of Ca2+ in osteoclast retraction, we preloaded cells with BAPTA to buffer changes in [Ca2+]i. BAPTA decreased the initial rate of calcitonin-induced osteoclast retraction, but it did not affect the degree of retraction 2-3 hours following calcitonin, indicating that retraction is mediated primarily by Ca(2+)-independent processes. We conclude that calcitonin conjugates cause osteoclast retraction and [Ca2+]i signaling in a manner similar to that elicited by calcitonin. Thus, orally bioavailable calcitonin conjugates show potential for use as antiresorptive agents.

Animals↗

PubMed2001

Activity-dependent development of P2X7 current and Ca2+ entry in rabbit osteoclasts.

Bone remodeling is regulated by local factors and modulated by mechanical stimuli. Mechanical stimulation can cause release of ATP, an agent that stimulates osteoclastic resorption at low concentrations and inhibits at high concentrations. We examined whether osteoclasts express P2X(7) receptors, which are activated by high concentrations of ATP and can behave as ion channels or cause the formation of membrane pores. Rabbit osteoclasts were studied using patch clamp techniques. Successive or prolonged applications of 2'- & 3'-O-(4-benzoylbenzoyl)-ATP (BzATP, a relatively potent P2X(7) agonist) or high concentrations of ATP caused the development of a slowly deactivating inward current. The underlying channel was permeable only to small cations, ruling out pore formation. Divalent cations reduced current magnitude, consistent with the presence of P2X(7) receptors, a finding confirmed in rat osteoclasts by immunocytochemistry. Successive applications of BzATP also elicited [Ca(2+)](i) elevations that required extracellular Ca(2+). The BzATP-induced current and the rise of [Ca(2+)](i) were temporally associated, and both were inhibited by PPADS, a P2X(7) antagonist. This study demonstrates that high concentrations of ATP activate P2X(7) receptors and provides the first functional evidence for an extracellular ligand-gated Ca(2+) influx pathway in osteoclasts. ATP released in response to mechanical stimuli may act through P2X(7) receptors to inhibit osteoclastic resorption.

Adenosine Triphosphate↗

PubMed2001

Calcium inhibits human placental 11beta-hydroxysteroid dehydrogenase type 2 activity.

The effect of Ca2+ on the conversion of cortisol to its inert metabolite cortisone, the reaction catalyzed by the microsomal enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), was investigated in human placental microsomes. Placental microsomal 11beta-HSD2 activity, as determined by the rate of conversion of cortisol to cortisone, was inhibited up to 50% by increasing free Ca2+ concentrations from 22 to 268 nM. The Ca2+-induced inhibition was reversible since chelation of endogenous Ca2+ with EGTA increased 11beta-HSD2 activity up to 200%. Ca2+ decreased the maximal velocity (Vmax) of the 11beta-HSD2 catalyzed conversion of cortisol to cortisone without altering the Km of 11beta-HSD2 for cortisol, indicating that Ca2+ modulates the catalytic efficiency rather than the substrate binding of 11beta-HSD2. Moreover, the Ca2+-induced inhibition does not appear to involve altered cofactor (NAD+) binding since the inhibition of microsomal 11beta-HSD2 activity by a sub-maximal concentration of free Ca2+ was not overcome by increasing the concentration of NAD+. These findings in the microsomes were then extended to an intact cell system, JEG-3 cells, an established model for human placental trophoblasts. In these cells, an increase in cytosolic free Ca2+ concentration ([Ca2+]i) elicited by a known physiological stimulus, PGF(2alpha), was accompanied by a 40% decrease in the level of 11beta-HSD2 activity. Furthermore, the PGF(2alpha)-induced inhibition of 11beta-HSD2 activity was abrogated when increases in [Ca2+]i were blocked with the intracellular Ca2+ chelator, BAPTA. Collectively, these results demonstrate for the first time that Ca2+ inhibits human placental 11beta-HSD2 activity by a post-translational mechanism not involving substrate or cofactor binding.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed2001

Activation of P2Y but not P2X(4) nucleotide receptors causes elevation of [Ca2+]i in mammalian osteoclasts.

Extracellular nucleotides cause elevation of cytosolic free Ca2+ concentration ([Ca2+](i)) in osteoclasts, although the sources of Ca2+ are uncertain. Activation of P2Y receptors causes Ca2+ release from stores, whereas P2X receptors are ligand-gated channels that mediate Ca2+ influx in some cell types. To examine the sources of Ca2+, we studied osteoclasts from rat and rabbit using fura 2 fluorescence and patch clamp. Nucleotide-induced rise of ([Ca2+](i)) persisted on removal of extracellular Ca2+ (Ca), indicating involvement of stores. Inhibition of phospholipase C (PLC) with U-73122 or inhibition of endoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid or thapsigargin abolished the rise of ([Ca2+](i)). After store depletion in the absence of Ca, addition of Ca led to a rise of ([Ca2+](i)) consistent with store-operated Ca2+ influx. Store-operated Ca2+ influx was greater at negative potentials and was blocked by La(3+). In patch-clamp studies where PLC was blocked, ATP induced inward current indicating activation of P2X(4) nucleotide receptors, but with no rise of ([Ca2+](i)). We conclude that nucleotide-induced elevation of [Ca(2+)](i) in osteoclasts arises primarily through activation of P2Y nucleotide receptors, leading to release of Ca2+ from intracellular stores.

Adenosine Triphosphate↗

PubMed2001

Calcitonin (CT) rapidly increases NA(+)/H(+) exchange and metabolic acid production: effects mediated selectively by the C1A CT receptor isoform.

Two isoforms of the calcitonin receptor are expressed in rabbit: the common C1a isoform and the calcitonin receptor Delta e13 isoform, which has a deletion in the seventh transmembrane domain. Using microphysiometry, we investigated the effects of calcitonin on proton efflux from HEK293 cells stably transfected with C1a, calcitonin receptor Delta e13, or empty vector. In C1a-expressing cells only, calcitonin rapidly induced a biphasic elevation in proton efflux consisting of an initial transient and a sustained plateau, accompanied by an increase in lactate efflux. Inhibitors of Na(+)/H(+) exchange abolished only the initial transient, whereas removal of extracellular glucose abolished only the sustained plateau. These data suggest that activation of Na(+)/H(+) exchange mediates the initial transient, whereas increased glucose metabolism underlies the sustained plateau. Because both receptor isoforms activate adenylyl cyclase, the lack of effect of calcitonin on proton efflux from calcitonin receptor Delta e13-expressing cells argued against involvement of cAMP in activating proton efflux. Similarly, studies involving elevation or buffering of cytosolic free Ca(2+) concentration argued against involvement of Ca(2+). Activation of PKC mimicked the plateau phase of calcitonin-induced proton efflux from C1a cells, whereas inhibition or depletion of PKC suppressed it. Activation of proton transport and production are novel cellular responses to calcitonin, mediated selectively by the C1a receptor isoform via a mechanism involving PKC.

Animals↗

PubMed2001

Transforming growth factor-beta induces osteoclast ruffling and chemotaxis: potential role in osteoclast recruitment.

Transforming growth factor-beta (TGF-beta) is released from the matrix during bone resorption and has been implicated in the pathogenesis of giant cell tumors of bone and the expansion of breast cancer metastases in bone. Because osteoclasts mediate tumor-induced osteolysis, we investigated whether TGF-beta stimulates osteoclast recruitment. Osteoclasts were isolated from rat long bones and time-lapse video microscopy was used to monitor their morphology and motility. Within 5 minutes, TGF-beta (0.1 nM) induced dynamic ruffling, with 65% of osteoclasts displaying membrane ruffles compared with 35% in untreated controls. Over a 2-h period, osteoclasts exhibited significant directed migration toward a source of TGF-beta, indicating chemotaxis. echistatin, an alphavbeta3 integrin blocker that inhibits macrophage colony-stimulating factor (M-CSF)-induced osteoclast migration, did not prevent the migration of osteoclasts toward TGF-beta. In contrast, a beta1 integrin blocking antibody inhibited osteoclast chemotaxis toward TGF-beta but not M-CSF. These data indicate the selective use of integrins by osteoclasts migrating in response to different chemotaxins. In addition, wortmannin and U0126 inhibited TGF-beta-induced chemotaxis, suggesting involvement of the phosphatidylinositol 3 (PI 3) kinase and mitogen-activated protein (MAP) kinase signaling pathways. Physiologically, TGF-beta, may coordinate osteoclast activity by recruiting osteoclasts to existing sites of resorption. Pathologically, TGF-beta-induced osteoclast recruitment may be critical for expansion of primary and metastatic tumors in bone.

Androstadienes↗

PubMed2001

Osteoclast ion channels: potential targets for antiresorptive drugs.

This review summarizes the types of ion channels that have been identified in osteoclasts and considers their potential as targets for therapeutic agents aimed at the treatment of osteoporosis and other bone disorders. We focus on channels that have been identified using molecular and electrophysiological approaches. Numerous ion channels have been characterized, including K(+), H(+), Na(+), nonselective cation and Cl(-) channels. K(+) channels include an inward rectifier K(+) channel (Kir2.1) that is regulated by G proteins, and a transient outward rectifier K(+) channel (Kv1.3) that is regulated by cell-matrix interactions and by extracellular cations such as Ca(2+) and H(+). In addition, two classes of Ca(2+)-activated K(+) channels have been described--large and intermediate conductance channels, which are activated by increases of cytosolic Ca(2+) concentration. Other channels include stretch-activated nonselective cation channels and voltage-activated H(+) channels. A recent revelation is the presence of ligand-gated channels in osteoclasts, including P2X nucleotide receptors and glutamate-activated channels. Osteoclasts also exhibit an outwardly rectifying Cl(-) current that is activated by cell swelling. Kir2.1 and Cl(-) channels may be essential for resorptive activity because they provide pathways to compensate for charge accumulation arising from the electrogenic transport of H(+). As in other cell types, osteoclast ion channels also play important roles in setting the membrane potential, signal transduction and cell volume regulation. These channels represent potential targets for the development of antiresorptive drugs.

Amino Acid Sequence↗

PubMed2001

Suppression of analgesia in rats induced by morphine or L-NAME but not both drugs by microTesla, frequency-modulated magnetic fields.

In Experiment I, the nociceptive threshold for a criterion response to thermal stimuli (hot plate) for rats was obtained following injections with one of various dosages of either morphine or the nitric oxide synthase inhibitor, L-NAME. On the bases of these results, rats in Experiment II were injected with either morphine (4 mg/kg), L-NAME (50 mg/kg), both morphine and L-NAME, or saline after baseline measurements for nociception and then exposed to either a 1 microTesla magnetic field or to a sham field for 30 min. The magnetic field, presented once every 4 sec, was a frequency-modulated pattern whose pixel durations, for each of the 837 successive values, were 1 msec. Nociceptive thresholds were measured immediately after the exposure and 30 min later. The results indicated that exposure to this magnetic field abolished the analgesic effects of morphine or L-NAME when injected separately but not when injected together relative to rats that had received these drugs and had been exposed to the sham field.

Analgesia↗

PubMed2000

Sodium-ascorbate cotransport controls intracellular ascorbate concentration in primary astrocyte cultures expressing the SVCT2 transporter.

Expression of the Na(+)-ascorbate cotransporter, SVCT2, was detected in rat brain and in primary cultures of cerebral astrocytes by Northern blot analysis. SVCT2 expression in cultured astrocytes increased in response to the cyclic AMP analog, dibutyryl cyclic AMP. A mathematical model of ascorbic acid transport was developed to evaluate the hypothesis that Na(+)-ascorbate cotransport across the plasma membrane regulates the steady state intracellular concentration of ascorbic acid in these cells. The outcomes predicted by this model were compared to experimental observations obtained with primary cultures of rat cerebral astrocytes exposed to normal and pathologic conditions. Both cotransport activity and intracellular ascorbic acid concentration increased in astrocytes activated by dibutyryl cyclic AMP. Conversely transport activity and ascorbic acid concentration were decreased by hyposmotic cell swelling, low extracellular Na(+) concentration, and depolarizing levels of extracellular K(+). In cells incubated for up to 3 h in medium having an ascorbic acid concentration typical of brain extracellular fluid, the changes in intracellular ascorbic acid concentration actually measured were not significantly different from those predicted by modeling changes in Na(+)-ascorbate cotransport activity. Thus, it was not necessary to specify alterations in vitamin C metabolism or efflux pathways in order to predict the steady state intracellular ascorbic acid concentration. These results establish that SVCT2 regulates intracellular ascorbic acid concentration in primary astrocyte cultures. They further indicate that the intracellular-to-extracellular ratio of ascorbic acid concentration at steady state depends on the electrochemical gradients of Na(+) and ascorbate across the plasma membrane.

Algorithms↗

PubMed2000

Electrophysiological characterization of ion channels in osteoclasts isolated from human deciduous teeth.

Ion channels contribute to several important processes in osteoclasts, including proton transport and volume regulation. Although ion channels have been described in osteoclasts from several species, little is known about their properties in human osteoclasts. We devised a method for isolation of authentic human osteoclasts from deciduous teeth undergoing root resorption, and characterized currents in these cells using patch-clamp techniques. Three types of K(+) current were identified. Hyperpolarization elicited an inwardly rectifying K(+) current in most osteoclasts, which was inhibited by Ba(2+) in a voltage- and time-dependent manner. Depolarization elicited an outwardly rectifying and tetraethylammonium-sensitive current, consistent with a large-conductance Ca(2+)-dependent K(+) channel. In addition to these basal currents, extracellular adenosine 5'-triphosphate (ATP) elicited a linear current that was identified as a Ca(2+)-dependent K(+) current, based on its reversal potential close to that predicted for K(+), its blockade by quinine, and its activation by Ca(2+) ionophore. Last, an outwardly rectifying current was observed to activate spontaneously or in response to ATP, with properties of a swelling-activated Cl(-) current. This current reversed direction close to the Cl(-) equilibrium potential and was blocked by the anion channel blocker, niflumic acid, identifying it as a Cl(-) current. In summary, we have developed a novel method for isolation of authentic human osteoclasts and have characterized K(+) and Cl(-) currents. Cl(-) current mediates charge compensation during electrogenic H(+) transport, so activation of Cl(-) current may contribute to the stimulatory effects of extracellular ATP on bone resorption.

Calcium↗

PubMed2000

Surface effects on ground reaction forces and lower extremity kinematics in running.

INTRODUCTION: Although running surface stiffness has been associated with overuse injuries, all evidence to support this suggestion has been circumstantial. In the present study, the biomechanical response of heel-toe runners to changes in running surface has been investigated. METHODS: Six heel-toe runners performed shod running trials over three surfaces: a conventional asphalt surface, a new rubber-modified asphalt surface, and an acrylic sports surface. The surfaces were categorised according to impact absorbing ability using standard impact test procedures (BS 7044). RESULTS: The rubber-modified asphalt was found to exhibit the greatest amount of mechanical impact absorption, and the conventional asphalt the least. The comparison of peak impact force values across surfaces for the group of subjects demonstrated no significant differences in magnitude of force. However, a significant reduction in loading rate of peak impact force was detected for the rubber-modified surface compared with conventional asphalt (P < 0.1). Although analysis of group data revealed no significant differences in kinematic variables when running on the different surfaces, a varied response to surface manipulation among runners was demonstrated, with marked differences in initial joint angles, peak joint angles, and peak joint angular velocities being observed. DISCUSSION: For some subjects, the maintenance of similar peak impact forces for different running surfaces was explained by observed kinematic adjustments. For example, when running on the surface providing the least impact absorption, an increased initial knee flexion was observed for some subjects, suggesting an increased lower extremity compliance. However, for some subjects, sagittal plane kinematic data were not sufficient for the explanation of peak impact force results. It appears that the mechanism of adaptation varies among runners, highlighting the requirement of individual subject analyses.

Biomechanical Phenomena↗

PubMed1999

The shock attenuation characteristics of four different insoles when worn in a military boot during running and marching.

A study was undertaken to determine if placing shock absorbing insoles in the boots of Royal Marine recruits would attenuate the peak pressure at the foot-boot interface, when marching at 4.8 kph carrying a 32 kg (70 lb) Bergen and running at 12.8 kph in loose order plus webbing weighing 10 kg (22 lb). Four types of insoles were assessed: viscoelastic polymetric insole (Cambion(R)) polymetric foam insole (PPT(R)) Saran insole (military issue) and Sorbothane(R). There was a fifth control condition in which no insoles were used. Pressure measurements during heel strike and forefoot loading were taken using Paratec equipment with pressure measuring insoles placed in the boots. Data were obtained from eleven subjects and indicated that all the insoles significantly (P<0.05) attenuated the peak pressures generated during heel strike and forefoot loading. The performance of the four insoles in terms of peak pressure attenuation ranked in order with the best first were: Sorbothane Cambion PPT Saran. The Sorbothane insole was substantially and significantly (P<0.05) better than the other insoles in terms of attenuating peak pressures during heel strike. During running, mean peak pressure at heel strike was 494 kPa in the control condition, this was reduced to 377 kPa when wearing Sorbothane insoles (a reduction of 27%). When marching the Sorbothane insoles reduced the mean peak pressure at heel strike from 395 kPa (control) to 303 kPa (23% reduction). During forefoot loading the peak pressure attenuation of all four insoles was similar, although on average the Sorbothane insole performed slightly better than the others and was significantly different (P<0. 05) to the Cambion insole. Mean peak forefoot loading pressure in the control condition when running was 413 kPa, with the Sorbothane insole it was 367 kPa, during marching the respective mean peak pressures were 397 and 323 kPa. It is concluded that of the four types of insoles assessed the Sorbothane insoles attenuated the greatest amounts of the peak pressure generated at heel strike and during forefoot loading when running and marching wearing military boots.

Cross-Over Studies↗

PubMed1999

The impact of genetic removal of GFAP and/or vimentin on glutamine levels and transport of glucose and ascorbate in astrocytes.

The importance of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP) and vimentin for astrocyte function was studied by investigating astrocytes prepared from GFAP-/- and/or vimentin-/- mice. The rate of glucose uptake through facilitative hexose transporters was not affected by depletion of GFAP or vimentin. Similarly, the absence of these IF proteins did not affect ascorbate uptake, under control or cyclic AMP-stimulated conditions, or ascorbate efflux through volume-sensitive organic anion channels. However, compared with wild-type astrocytes, glutamine concentrations were increased up to 200% in GFAP-/- astrocytes and up to 150% in GFAP+/- astrocytes and this increase was not dependent on the presence of vimentin. GFAP-/- astrocytes in culture still contain IFs (made of vimentin and nestin), whereas GFAP-/- vim-/- cultured astrocytes lack IFs. Thus, glutamine levels appear to correlate inversely with GFAP, rather than depend on the presence of IFs per se. Furthermore, the effect of GFAP is dose-dependent since the glutamine concentration in GFAP+/- astrocytes falls between those in wild-type and GFAP-/- astrocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Artificial playing surfaces research: a review of medical, engineering and biomechanical aspects.

In this paper, current knowledge of artificial playing surfaces is reviewed. Research status in the fields of sports medicine, engineering and biomechanics is described. A multidisciplinary approach to the study of artificial sports surface properties is recommended. The development of modelling techniques to characterise fundamental material properties is described as the most appropriate method for the unique specification of material properties such as stiffness and damping characteristics. It is suggested that the systematic manipulation of fundamental surface material properties in biomechanics research will allow the identification of subject responses to clearly defined surface variation. It is suggested that subjects should be grouped according to characteristic behaviour on specific sports surfaces. It is speculated that future biomechanics research will identify subject criterion related to differing group responses. The literature evidence of interactions between sports shoes and sports surfaces leads to the suggestion that sports shoe and sports surface companies should work together in the development of ideal shoe - surface combinations for particular groups of subjects.

Athletic Injuries↗

PubMed1999

Insulin-like growth factor I rapidly enhances acid efflux from osteoblastic cells.

Insulin-like growth factor I (IGF-I) is thought to stimulate bone resorption indirectly through a primary effect on osteoblasts, which in turn activate osteoclasts by as-yet-unidentified mechanisms. Small decreases in extracellular pH (pHo) dramatically increase the resorptive activity of osteoclasts. Our purpose was to characterize the effect of IGF-I on acid production by osteoblastic cells. When confluent, UMR-106 osteoblast-like cells and rat calvarial cells acidified the compartment beneath them. Superfusion with IGF-I caused a further decrease in pHo. To investigate the mechanism, we monitored acid efflux from subconfluent cultures. IGF-I rapidly increased net efflux of H+ equivalents in a concentration-dependent manner. IGF-II (10 nM) evoked a smaller response than IGF-I (10 nM). The response to IGF-I was partially dependent on extracellular Na+, but not glucose, and exhibited little if any desensitization. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, abolished the response to IGF-I but not to parathyroid hormone. Thus IGF-I enhances acid efflux from osteoblastic cells, via a signaling pathway dependent on activation of phosphatidylinositol 3-kinase. In vivo, acidification of the compartment between the osteogenic cell layer and the bone matrix may affect diverse processes, including mineralization and osteoclastic bone resorption.

Acids↗

PubMed1999

Calcium signalling via multiple P2 purinoceptor subtypes in rat osteoclasts.

Extracellular nucleotides bind to P2 purinoceptors in many tissues. P2X purinoceptors are intrinsic ion channels that mediate depolarization and influx of Ca(2+), whereas P2Y purinoceptors are coupled through G-proteins to mobilization of intracellular Ca(2+). Previous studies have yielded conflicting information on the responses of osteoclasts to nucleotides. The purpose of this study was to investigate the pathways underlying purinoceptor-mediated Ca(2+) signalling in authentic mammalian osteoclasts. Osteoclasts, isolated from the long bones of neonatal rats, were loaded with the Ca(2+)-sensitive probe fura-2 and [Ca(2+)](i) was monitored by microspectrofluorimetry. ATP (10-100 microM) induced transient elevation of [Ca(2+)](i) in 74% of osteoclasts tested. Similar responses were observed in Ca(2+)-free media, consistent with release of Ca(2+) from intracellular stores. Oscillations in [Ca(2+)](i) were observed only in osteoclasts that had a 'rounded' morphology. Responses to selective P2 agonists were consistent with the presence of multiple purinoceptor subtypes, including members of both the P2Y and P2X families. Alendronate, a bisphosphonate with structural similarities to methylene ATP analogues, neither activated nor blocked the Ca(2+) response mediated by osteoclast purinoceptors. Mechanical stimulation of osteoclasts elicited transient elevation of [Ca(2+)](i) which involved Ca(2+) influx and, in some cases, release from stores. The nucleotidase apyrase did not inhibit deformation-induced elevation of [Ca(2+)](i) in the presence of extracellular Ca(2+), indicating that nucleotide release is not essential for mechanically induced Ca(2+) influx. These findings indicate that osteoclasts exhibit multiple P2 purinoceptor subtypes, linked to elevation of [Ca(2+)](i).

Adenosine Triphosphate↗

PubMed1999

Role of alpha(v)beta(3) integrin in osteoclast migration and formation of the sealing zone.

The alpha(v)beta(3) integrin is abundantly expressed in osteoclasts and has been implicated in the regulation of osteoclast function, especially in cell attachment. However, in vivo studies have shown that echistatin, an RGD-containing disintegrin which binds to alpha(v)beta(3), inhibits bone resorption without changing the number of osteoclasts on the bone surface, suggesting inhibition of osteoclast activity. The objective of this study was to examine how occupancy of alpha(v)beta(3) integrins inhibits osteoclast function, using primary rat osteoclasts and murine pre-fusion osteoclast-like cells formed in a co-culture system. We show that: (1) echistatin inhibits bone resorption in vitro at lower concentrations (IC(50 )= 0.1 nM) than those required to detach osteoclasts from bone (IC(50 ) approximately 1 microM); (2) echistatin (IC(50 )= 0.1 nM) inhibits M-CSF-induced migration and cell spreading of osteoclasts; (3) alpha(v)beta(3) integrins are localized in podosomes at the leading edge of migrating osteoclasts, whereas, with echistatin treatment (0.1 nM), alpha(v)beta(3) disperses randomly throughout the adhesion surface; and (4) when bone resorption is fully inhibited with echistatin, there is visible disruption of the sealing zone (IC(50 )= 13 nM), and alpha(v)beta(3) visualized with confocal microscopy re-distributes from the basolateral membranes to intracellular vesicular structures. Taken together, these findings suggest that alpha(v)beta(3) integrin plays a role in the regulation of two processes required for effective osteoclastic bone resorption: cell migration (IC(50 )= 0.1 nM) and maintenance of the sealing zone (IC(50) approximately 10 nM).

Actins↗