Biomedical subjects

J Ferrier

Publications and source records attributed to J Ferrier.

At least 19 recordsLinked to original sources

PubMed1997

Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching.

The actin-dependent sensory and response elements of stromal cells that are involved in mechanical signal transduction are poorly understood. To study mechanotransduction we have described previously a collagen-magnetic bead model in which application of well-defined forces to integrins induces an immediate (< 1 second) calcium influx. In this report we used the model to determine the role of calcium ions and tyrosine-phosphorylation in the regulation of force-mediated actin assembly and the resulting change in membrane rigidity. Collagen-beads were bound to cells through the focal adhesion-associated proteins talin, vinculin, alpha 2-integrin and beta-actin, indicating that force application was mediated through cytoskeletal elements. When force (2 N/m2) was applied to collagen beads, confocal microscopy showed a marked vertical extension of the cell which was counteracted by an actin-mediated retraction. Immunoblotting showed that force application induced F-actin accumulation at the bead-membrane complex but vinculin, talin and alpha 2-integrin remained unchanged. Atomic force microscopy showed that membrane rigidity increased 6-fold in the vicinity of beads which had been exposed to force. Force also induced tyrosine phosphorylation of several cytoplasmic proteins including paxillin. The force-induced actin accumulation was blocked in cells loaded with BAPTA/AM or in cells preincubated with genistein, an inhibitor of tyrosine phosphorylation. Repeated force application progressively inhibited the amplitude of force-induced calcium ion flux. As force-induced actin reorganization was dependent on calcium and tyrosine phosphorylation, and as progressive increases of filamentous actin in the submembrane cortex were correlated with increased membrane rigidity and dampened calcium influx, we suggest that cortical actin regulates stretch-activated cation permeable channel activity and provides a desensitization mechanism for cells exposed to repeated long-term mechanical stimuli. The actin response may be cytoprotective since it counteracts the initial force-mediated membrane extension and potentially strengthens cytoskeletal integrity at force-transfer points.

Actins↗

PubMed1996

Localized calcium signaling in multinucleated osteoclasts.

Localized intracellular Ca2+ ([Ca2+]i) pulses, fluctuations, and repetitive spikes were detected in multinucleated rabbit osteoclasts in the presence of serum and in response to calcitonin using the fluorescent calcium indicator fluo-3 and a laser scanning microscope. We observed that these [Ca2+], changes were often restricted within a region of the cell body or propagated from the initial region of occurrence to other parts of the cell body but not to all parts. These observations suggest the existence of significant barriers to Ca2+ transport between different cytoplasmic regions of the osteoclast. To further investigate this phenomenon, we mechanically perturbed different cellular regions by touching locally with a micropipette. This usually induced a local increase in cytosolic and nuclear free [Ca2+]i. In some cases there was propagation of the [Ca2+]i increase to other regions but with part of the cell body not affected. Those regions of the cell body to which the [Ca2+]i increase did not propagate had a [Ca2+]i response to a direct mechanical perturbation. Our data show that osteoclasts can have different [Ca2+]i activities in apparently equivalent cellular regions, no matter how generated. This suggests that there can be a number of spatially separate Ca2+ regulatory systems within an osteoclast cell body.

Animals↗

PubMed1996

Nuclear versus perinuclear and cytoplasmic calcium in osteoclasts.

We measured fluorescence from the calcium indicator Fluo-3 in multinucleated osteoclasts. In the initial state, each nucleus is surrounded by a ring of bright fluorescence. Following activation of purinergic receptors by 100 microM ATP there is a pulse of cellular fluorescence increase, and nuclear fluorescence intensity becomes greater than that of the cytoplasm. This is followed by a period during which the fluorescence of the cell decreases below that of the initial state. During the pulsed increase following purinergic receptor activation, the perinuclear fluorescence intensity does not increase as much as that in the nuclear centers and, following this pulse, the perinuclear fluorescence intensity decreases more than that in the nuclear centers, relative to the initial state. Measurements in which Mn2+ was introduced into the cell show that the number of Fluo-3 molecules per unit horizontal area in the nuclear centers is slightly greater than that in the perinuclear regions, and more than twice that in the surrounding cytoplasm. These results show that there is a much higher free calcium concentration in the perinuclear regions than in the nuclear centers in the initial state, with a release of free calcium from the perinuclear regions following activation of the purinergic receptors. These data also provide evidence that the free calcium concentration in the nuclear centers is lower than in the cytoplasm in the initial state.

Aniline Compounds↗

PubMed1996

Tissue fixation security in transosseous rotator cuff repairs: a mechanical comparison of simple versus mattress sutures.

The primary purpose of this investigation was to compare tissue fixation security by simple sutures versus mattress sutures in transosseous rotator cuff repair. These two repair techniques were each performed in 17 human cadaver shoulders, with two bone tunnels being used for the repair by two simple sutures and two other bone tunnels being used for the repair by one mattress suture. The repairs were loaded to failure in a servohydraulic materials test system. Rotator cuff repair by simple sutures was found to be significantly stronger than repair by mattress sutures (P = .0007). The average ultimate load to failure for the simple suture construct (189.62 N) was 39.72% greater than that for the mattress suture construct (135.71 N). Most of the failures occurred by suture breakage at the knot. Load-sharing by multiple suture tails and multiple knots in the simple suture configuration likely contributed to its superior strength characteristics compared with the mattress suture configuration.

Biomechanical Phenomena↗

PubMed1995

Calcium signal induced by mechanical perturbation of osteoclasts.

Multinucleated osteoclasts from rabbit long bone, 1-6 days in culture, respond to mechanical perturbation with a transient increase of intracellular calcium concentration ([Ca2+]i), as measured with the fluorescent indicator fluo-3 on a confocal laser scanning microscope. In experiments with different extracellular calcium concentrations (from 11.8 mM to calcium-free), the incidence, the magnitude, and the duration of [Ca2+]i responses decreases with decreasing bathing [Ca2+]. Following mechanical perturbation, a thapsigargin-induced [Ca2+]i response has a lower magnitude than the thapsigargin-induced response without mechanical perturbation. In thapsigargin-pretreated osteoclasts the mechanical perturbation-induced rise in [Ca2+]i is larger and longer than in control cells. Ni2+ inhibits the incidence and decreases both the magnitude and the duration of the responses, while nifedipine, verapamil, and Gd3+ have no effect. These measurements show that rabbit osteoclasts transduce a mechanical perturbation of the cell membrane into a [Ca2+]i signal via both a calcium influx and an internal calcium release.

Animals↗

PubMed1995

Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts.

The ability to apply controlled forces to the cell membrane may enable elucidation of the mechanisms and pathways involved in signal transduction in response to applied physical stimuli. We have developed a magnetic particle-electromagnet model that allows the application of controlled forces to the plasma membrane of substrate-attached fibroblasts. The system allows applied forces to be controlled by the magnitude of the magnetic field and by the surface area of cell membrane covered with collagen-coated ferric beads. Analysis by single-cell ratio fluorimetry of fura 2-loaded cells demonstrated large calcium transients (50-300 nM) in response to the magnetic force applications. Experiments using either the stretch-activated channel blocker gadolinium chloride or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to eliminate external calcium ions, or addition of extracellular manganese ions, indicated that there was a calcium influx through putative stretch-activated channels. The probability of a calcium influx in single cells was increased by higher surface bead loading and the degree of cell spreading. Depolymerization of actin filaments by cytochalasin D increased the amplitude of calcium response twofold. The regulation of calcium flux by filamentous actin content and by cell spreading indicates a possible modulatory role for the cytoskeleton in channel sensitivity. Magnetic force application to beads on single cells provides a controlled model to study mechanisms and heterogeneity in physical force stimulation of cation-permeable channels.

Actins↗

PubMed1995

Osteoclast ATP receptor activation leads to a transient decrease in intracellular pH.

Application of extracellular adenosine triphosphate (ATP) induces a pulsed decrease in osteoclast intracellular pH (pHi), as measured with seminaphthofluorescein (SNAFL)-calcein on a laser scanning confocal microscope. Adenosine diphosphate also produces a pHi decrease, but adenosine monophosphate, uridine triphosphate, 2-methylthio-ATP, and beta, gamma-methylene-ATP have little effect on pHi. The ATP-induced pHi decrease is largely inhibited by suramin, a P2 purinergic receptor blocker. Clamping intracellular free [Ca2+] ([Ca2+]i) with BAPTA/AM does not affect the ATP-induced pHi change, showing that this pHi decrease is not caused by the increased intracellular [Ca2+]i that is produced by activation of osteoclast purinergic receptors. We show that an increase in [Ca2+]i by itself will produce a pHi increase. The ATP effect is not blocked by inhibition of Na+/H+ exchange by either Na(+)-free bathing medium or amiloride. Two inhibitors of the osteoclast cell membrane proton pump, N-ethylmaleimide and vanadate, produce partial inhibition of the ATP-induced pHi decrease. Two other proton pump inhibitors, bafilomycin and N,N'-dicyclohexylcarbodiimide, have no influence on the ATP effect. None of the proton pump inhibitors but vanadate has a direct effect on pHi. Vanadate produces a transient pHi increase upon application to the bathing medium, possibly as a result of its known effect of stimulating the Na+/H+ exchanger. Inhibition of Cl-/HCO3- exchange by decreasing extracellular Cl- gives a pronounced long-term pHi increase, supporting the hypothesis that this exchange has an important role in osteoclast pHi homeostasis. In Cl(-)-free extracellular medium, there is a greatly reduced effect of extracellular ATP on pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1994

Displacement and translocation of osteoblast-like cells by osteoclasts.

Rabbit osteoclasts and rabbit osteoblast-like stroma cells (OB cells) were placed onto plastic surfaces and the migration patterns of individual osteoclasts and osteoclast-OB interactions were analyzed with time-lapse recording. To induce directed migration, the cultures were exposed to an electrical field of 0.01 or 0.1 V/mm. At 0.1 V/mm, osteoclasts moved directly toward the anode in some cases, clearing OB cells from their path of migration. In other cases, osteoclasts migrated toward the anode for part of the time but then changed direction and moved toward groups of OB cells. Observations were made on osteoclasts interacting with single OB cells or small colonies and on osteoclasts interacting with OB monolayers, at both field strengths; the results were independent of field strength. There were several characteristic behaviors. With single OB cells and small OB colonies, retraction of OB cells upon contact with the osteoclast was the predominant mechanism whereby these cells begin to move out of the path of the osteoclast. A pronounced ruffling or blebbing of the OB cell membrane often followed retraction. When osteoclasts displaced OB cells that were part of a monolayer, extension of an osteoclast lamellipodium underneath the edge of the OB cell layer generally preceded partial retraction of the OB cells involved. It sometimes appeared as if the detached or partially detached OB cells were "pushed" by the osteoclast, which in some cases resulted in OB cells being moved hundreds of microns in a period of a few hours, at rates comparable to the normal speed for osteoclast migration (congruent to 100 microns/h), much faster than the normal speed for OB cells (congruent to 10 microns/h).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Mechanisms of ATP-induced Ca2+ signaling in osteoclasts.

We investigate the mechanisms underlying the intracellular calcium pulse that occurs in response to extracellular adenosine triphosphate (ATP) in osteoclasts. We find that pre-loading of GDP-beta-S abolishes the response in Ca(2+)-free medium, demonstrating an internal release of Ca2+ via a pathway that involves a G protein. GDP-beta-S does not block in normal Ca(2+)-containing medium, suggesting that ATP also induces a Ca2+ influx across the cell membrane. We confirmed this using the Mn2+ quenching technique, which shows significant opening of Ca2+ channels. We find a smaller response to adenosine diphosphate (ADP) and 2-methylthio-ATP (2-MeSATP), but no response to beta, gamma-methylene-ATP (AMP-PCP), adenosine monophosphate (AMP) or uridine triphosphate (UTP). Prior application of AMP and UTP, but not AMP-PCP, blocks the response to ATP. Our results indicate that the receptor is a P2 subtype that is not characteristic of any previously reported P2 receptor or combination of P2 receptors.

Adenosine Diphosphate↗

PubMed1993

Osteoblast-like cells have a variable mixed population of purino/nucleotide receptors.

Osteoblast-like UMR 106.06 cells respond to extracellular application of nucleotides with a fast intracellular calcium pulse (latency of about 20 s, half-width of about 10 s), as measured with fluo-3 on a confocal laser scanning system. Cross-inhibition experiments at 50 microM show that, on a cell population basis, adenosine triphosphate (ATP) strongly inhibits the effect of uridine triphosphate (UTP) or 2-methylthio-ATP (2-MeSATP) applied within 2 min after the end of the ATP-induced pulse, while prior application of UTP or 2-MeSATP only weakly inhibits the ATP effect, and UTP and 2-MeSATP weakly inhibit each other. Furthermore, there are clear differences in cross-inhibition between individual cells. Our measurements provide strong evidence that these cells have at least two types of purino/nucleotide receptors, probably P2y and P2u, with a proportion that varies between individual cells.

Adenosine Triphosphate↗

PubMed1993

Interleukin-1 alpha induces a sustained increase in cytosolic free calcium in cultured rabbit osteoclasts.

Using laser confocal microscopy and a Ca2+ indicator, fluo-3, we find that osteoclasts respond to IL-1 alpha with a sustained increase (4 to 40 min) in cytosolic free calcium ([Ca2+]i). The delay time (T1/2) varies from 2 to 8 minutes. These effects cannot be mimicked by application of cAMP and can be blocked by removal of Ca2+ from the bathing medium. Pre-incubation with indomethacin (30 microM) does not block the IL-1 alpha effect on [Ca2+]i. We employ a 'micropipette' method and a 'pieces of cover slip' method to isolate single osteoclasts. Our results provide evidence that IL-1 alpha directly stimulates osteoclasts via a [Ca2+]i signalling pathway. We postulate that this slowly developing, long-lasting increase in [Ca2+]i in osteoclasts may account for the stimulatory effect of IL-1 alpha on osteoclastic bone resorption in conjunction with signalling molecules from osteoblasts.

Animals↗

PubMed1993

ATP induces an intracellular calcium pulse in osteoclasts.

Adenosine triphosphate (ATP; 50 microM) induces a large, fast, transient increase in intracellular calcium ([Ca2+]i) in rabbit osteoclasts, as measured with fluo-3 on a confocal laser scanning system. The [Ca2+]i increase is most intense in the nuclei of these multinuclear cells, indicating that Ca2+ release is occurring just outside the nuclei. ATP produces a much larger effect than adenosine, indicating that these cells have P2 purinergic receptors. Ca(2+)-free bathing medium and the Ca2+ channel blocker Cd2+ both inhibit but do not block the effect, showing that internal Ca2+ release is involved, but that either the steady state Ca2+ influx or a change in influx is important in modulating the effect. Thapsigargin also inhibits the effect. The ATP effect is repeatable with no change in bathing medium, implying that this signalling pathway has a mechanism for adaptation to progressively higher levels of ATP.

Adenosine Triphosphate↗

PubMed1992

Propagation of a calcium pulse between osteoblastic cells.

Using rat calvaria cells in primary culture monolayers and bone-like nodules, and isolated rat osteosarcoma cells, we show via laser scanning confocal microscopy and fluorescent indicator fluo-3/AM, that mechanical perturbation of a cell results in a transient increase (pulse) of measured intracellular calcium concentration that propagates from cell to cell, even between cells connected only by a thin process. The calcium pulse does not occur in the mechanically perturbed cell in calcium-free bathing medium, nor is there pulse propagation under this condition. Halothane, which blocks gap junctions, inhibits propagation. Propagation velocity does not decrease with successive cell to cell steps. These observations suggest the existence of a self-regenerating calcium signaling mechanism that may be based on a form of calcium-induced calcium release.

Aniline Compounds↗

PubMed1992

Hormone responses of in vitro bone nodule cells: studies on changes of intracellular calcium and membrane potential in response to parathyroid hormone and calcitonin.

We used two techniques to study the responses of individual in vitro bone nodule cells to parathyroid hormone (PTH) and calcitonin (CT). These techniques are laser scanning confocal imaging with a fluorescent indicator to measure intracellular free [Ca2+], and microelectrode impalement to measure the electrical potential difference across the cell membrane. We applied these measurement techniques to cells in the top cellular layer of nodules that form in vitro in cultures of cells obtained from fetal rat calvaria. Our measurements showed a transient increase in intracellular free [Ca2+] following application of PTH or CT. The duration of the increase in fluorescent intensity following PTH application varied from about 100 to more than 300 s, and the duration following CT application was from 30 to 80 s. In some measurements we applied both hormones in sequence, and observed that some cells showed an intracellular [Ca2+] response to both hormones, while other cells apparently responded to only one or the other of the hormones, or to neither. We also observed membrane potential changes in response to PTH and to CT. The membrane potential response to CT was quite small. The time courses of these membrane potential changes consisted of a depolarizing phase lasting about 100 s (with both hormones) followed by a hyperpolarizing phase (with PTH). Control measurements using only the vehicle solutions were carried out with both techniques, producing negligible responses.

Animals↗

PubMed1991

Ba(2+)-induced action potentials in osteoblastic cells.

Trains of long-duration "action potentials" were induced by Ba2+ in osteoblast-like rat osteosarcoma cells (ROS 17/2.8), under current clamp and voltage clamp. Large depolarizing pulses were seen in microelectrode measurements at 37 degrees C following the addition of 10 or 20 mM Ba2+ to physiological bathing medium. Application of BAY K 8644 resulted in the onset of the pulses at earlier times and at more negative potentials. The pulses were blocked by nifedipine and Cd2+, but not by Ni2+. Large inward current pulses were seen in whole-cell patch technique voltage-clamp measurements at 37 degrees C in the presence of from 10 to 110 mM Ba2+ in the bathing medium. The current pulses were not seen at 22 degrees C in the presence of 110 mM Ba2+, but could be induced by BAY K 8644. These pulses were not blocked by TTX, but were blocked by nifedipine, Cd2+, Zn2+, Co2+, and by an increase in bathing [Ca2+]. The shape and frequency of the current pulses were the same as for voltage pulses under current clamp. A model that can explain these observations involves opening of L-type Ca2+ channels in a voltage-independent manner by cytosolic Ba2+ via a screening of Ca2+ from sites that produce either inactivation or a lower probability of opening in the activated state. There would be a closing of these channels at higher [Ba2+] as Ba2+ is forced onto these sites. A refractory period is also required to give repeated pulses of openings.

Action Potentials↗

PubMed1991

Continuously active sodium channels in osteoblastic ROS 17/2.8 cells.

Cell-attached patch clamp experiments revealed 13-20 pS Na(+)-conducting channels active at normal resting potentials (-28 +/- 1 mV; +/- SEM; 7 cells) in the rat osteosarcoma cell line, ROS 17/2.8. These channels were not blocked by tetrodotoxin, Cd2+, verapamil, or nifedipine. Replacing all cations in the patch pipette except Ca2+ with tetraethylammonium (TEA+) abolishes channel activity; but adding TEA+ to a pipette solution containing only Na+ does not. Depolarization was not necessary to activate these channels, and the open times were much longer than the millisecond open times characteristic of Na+ channels in excitable cells. Current-voltage curves reconstructed from mean single channel currents and mean channel open times resemble L-type Ca2+ current-voltage curves obtained from whole-cell experiments, with current peaks shifted to resting or more hyperpolarized potentials. The voltage sensitivity of these channels has implications on membrane potential stability and on the hyperpolarizing membrane potential spiking activity exhibited by ROS 17/2.8 cells.

Animals↗

PubMed1991

An experimental test of a model for repeated Ca2+ spikes in osteoblastic cells.

A model for cytosolic Ca2+ spikes is presented that incorporates continual influx of Ca2+, uptake into an intracellular compartment, and Ca(2+)-induced Ca2+ release from the compartment. Two versions are used. In one, release is controlled by explicit thresholds, while in the other, release is a continuous function of cytosolic and compartmental [Ca2+]. Some model predictions are as follows. Starting with low Ca2+ influx and no spikes: (1) induction of spiking when Ca2+ influx is increased. Starting with spikes: (2) increase in magnitude and decrease in frequency when influx is reduced; (3) inhibition of spiking if influx is greatly reduced; (4) decrease in the root-mean-square value when influx is increased; and (5) elimination of spiking if influx is greatly increased. Since there is good evidence that hyperpolarizing spikes reflect cytosolic Ca2+ spikes, we used electrophysiological measurements to test the model. Each model prediction was confirmed by experiments in which Ca2+ influx was manipulated. However, the original spike activity tended to return within 5-30 min, indicating a cellular resetting process.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1989

Osteoblastic cells have L-type calcium channels.

Whole cell patch clamp studies on osteoblast-like rat osteosarcoma cells (ROS 17/2.8) show the existence of L-type calcium channels in the cell membrane. Measurements were carried out at both 21 and 37 degrees C. With isotonic CsCl in the pipette and a bathing medium containing either 110 or 10 mM Ba2+, a strong depolarizing pulse was required to activate an inward current. The current-voltage relationship (I-V) of this inward current showed a maximum amplitude near +30 mV at 21 and 37 degrees C, with 110 mM Ba2+ in the bathing medium, and near +10 mV at 37 degrees C with 10 mM Ba2+. At both 21 and 37 degrees C the dihydropyridine, BAY K 8644 (2 microM), increased this current and shifted the I-V maximum to less positive potentials, while nifedipine (5 microM) reduced the current. Cd2+ (50 microM) and Co2+ (100 microM) blocked the current. At 21 degrees C the measured inward current showed a slow inactivation, with a time constant of some hundreds of milliseconds. At 37 degrees C, inactivation was considerably faster. The current was suppressed by holding the membrane potential more positive than -30 mV. These data are strong evidence that ROS 17/2.8 cells have a significant number of 'L-type' calcium channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗