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  • 1
    ISSN: 1432-2013
    Keywords: Ca2+ influx ; Nystatin perforated patchclamp technique ; Fura-2 ; HT29 ; ATP ; Thapsigargin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Indirect evidence has accumulated indicating a voltage dependence of the agonist-stimulated Ca2+ influx into epithelial cells. Manoeuvres expected to depolarise the membrane voltage during agonist stimulation resulted in: (1) a decrease of the sustained phase of the adenosine triphosphate (ATP, 10−5 mol/l)-induced intracellular Ca2+ transient, (2) a reduced fura-2 Mn2+-quenching rate, and (3) prevention of the refilling of the agonist-sensitive store. To quantify the change in intracellular Ca2+ as a function of membrane voltage, we measured simultaneously the intracellular Ca2+ activity ([Ca2+]i) with fura-2 and the electrical properties using the nystatin perforated patch-clamp technique in single HT29 cells. Ca2+ influx was either stimulated by ATP (10−5 mol/l) or thapsigargin (TG, 10−8 mol/l). After [Ca2+]i reached the sustained plateau phase we clamped the membrane voltage in steps of 10 mV in either direction. A stepwise depolarisation resulted in a stepwise reduction of [Ca2+]i. Similarly a stepwise hyperpolarisation resulted in a stepwise increase of [Ca2+]i (ATP: 27.5±10 nmol/l per 10 mV, n=6; TG: 19 ±7.9 nmol/l per 10 mV, n=12). The summarised data show a linear relationship between the Δ fluorescence ratio 340/380 nm change and the applied holding voltage. In unstimulated cells the same voltage-clamp protocol did not change [Ca2+]i (n=9). Under extracellular Ca2+-free conditions [Ca2+]i remained unaltered when changing the membrane voltage. These data provide direct evidence that the Ca2+ influx in epithelial cells is membrane voltage dependent. Our data indicate that small changes in membrane voltage lead to substantial changes in [Ca2+]i. This may be due either to a change of driving force for Ca2+ into the cell, or may reflect voltage-dependent regulation of the respective Ca2+ entry mechanism.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-2013
    Keywords: Key words BCECF ; Fura-2 ; pHi ; [Ca2+]i ; HT29 ; Carbachol ; Neurotensin ; ATP ; InsP3 ; Cell volume ; Calcein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  In this study we examined the influence of intracellular pH (pHi) on agonist-induced changes of intracellular Ca2+ activity ([Ca2+]i) in HT29 cells. pHi and [Ca2+]i were measured microspectrofluorimetrically using BCECF and fura-2, respectively. Buffers containing trimethylamine (TriMA), NH3/NH4 + and acetate were used to clamp pHi to defined values. The magnitudes of the peak and plateau of [Ca2+]i transients induced by carbachol (CCH, 10–6 mol/l) were greatly enhanced by an acidic pHi and nearly abolished by an alkaline pHi. The relationship between pHi and the [Ca2+]i peak was nearly linear from pHi 7.0 to 7.8. This effect of pHi was also observed at higher CCH concentrations (10–4 and 10–5 mol/l), at which the inhibitory effect of an alkaline pHi was more pronounced than the stimulatory effect of an acidic pHi. An acidic pHi shifted the CCH concentration/response curve to the left, whereas an alkaline pHi led to a rightward shift. The influence of pHi on [Ca2+]i transients induced by neurotensin (10–8 mol/l) or ATP (5 × 10–7 mol/l) was similar to its influence on those induced by CCH, but generally not as pronounced. Measurements of cellular inositol 1,4,5-trisphosphate (InsP 3) showed no changes in response to acidification with acetate (20 mmol/l) or alkalinization with TriMA (20 mmol/l). The InsP 3 increase induced by CCH was unaltered at an acidic pHi, but was augmented at an alkaline pHi. Confocal measurements of cell volume showed no significant changes induced by TriMA or acetate. Slow-whole-cell patch-clamp experiments showed no additional effect of CCH on the membrane voltage (V m) measured after TriMA or acetate application. We conclude that pHi is a physiological modulator of hormonal effects in HT29 cells, as the [Ca2+]i responses to agonists were significantly changed at already slightly altered pHi. The measurements of InsP 3, cell volume and V m show that pHi must act distally to the InsP 3 production, and not via changes of cell volume or V m.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-0932
    Keywords: Biomécanique ; Rachis cervical ; Fracture de l'odontoïde ; Stabilité ; Ostéosynthèse de l'odontoïde par vis ; Biomechanics ; Cervical spine ; Odontoid fracture ; Stability ; Odontoid screw fixation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Fresh odontoid fractures of types II and III and in some cases nonunions of the odontoid can be repaired by direct anterior screw fixation preserving C1–C2 motion. The goal of this experimental study was to investigate the biomechanical stability of the fragment achieved by this direct odontoid osteosynthesis according to Böhler. Sixteen human C1–C2 segments with fractures of type II or type III were biomechanically tested in vitro under standardized conditions. Flexion and extension moments, anterior and posterior shear forces were applied, and the motion of the refixed odontoid fragment relative to C2 was determined. The results show that direct screw fixation of the odontoid under the experimental conditions provides sufficient stability for the dens fragment.
    Notes: Résumé Les fractures fraîches de l'odontoïde de type II et III et certains cas de pseudarthrose de l'odontoïde peuvent être réparés par une ostéosynthèse antérieure directe préservant la mobilité C1–C2. Le but de cette étude expérimentale était d'explorer la stabilité biomécanique permise par cette ostéosynthèse directe de l'odontoïde selon Böhler. 16 segments C1–C2 présentant des fractures de type II et III ont été testés in vitro dans des conditions bien définies. On a appliqué des moments de flexion et d'extension et des forces de cisaillement antérieur et postérieur et l'on a déterminé la mobilité du fragment odontoïdien refixé par rapport à C2. Les résultats ont montré que la fixation directe par vissage de l'odontoïde assure dans les conditions expérimentales une stabilité suffisante au fragment odontoïdien.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-0932
    Keywords: Biomécanique ; Rachis cervical ; Fracture de l'odontoïde ; Instabilité atlanto-axoïdienne ; Arthrodèse atlanto-axoïdienne postérieure ; Biomechanics ; Cervical spine ; Odontoid fracture ; Atlantoaxial instability ; Posterior atlantoaxial fusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Unstable C1-C2 segments are generally treated surgically. Depending on the indication a direct screw fixation of the odontoid or a C1-C2 arthrodesis is a possible technique. In this experimental in vitro study the three different atlantoaxial fusion techniques by Gallie, Brooks, and Magerl were compared biomechanically. Sixteen human C1-C2 segments with odontoid fractures of type II and III were investigated under standardized conditions. Flexion and extension moments, anterior, and posterior shear forces, left and right torsional moments were applied, and the motion of C1 relative to C2 was determined. The results of this investigation show clearly that the segments treated with the technique by Magerl with transarticular screws achieved the highest stiffness, compared to the wiring methods of Brooks and Gallie. These differences were most evident for posterior shear forces and for torsional moments. For these load conditions the ratio of stiffness Magerl: Brooks: Gallie was about 10:2:1. Significant differences for the plastic deformation of the differently fixed C1-C2 segments were found within the first few load/unload cycles, which give information about the relationship between primary and long-term stability.
    Notes: Résumé L'instabilité C1-C2 est le plus souvent traitée chirurgicalement. Selon l'indication, peuvent être proposées soit une ostéosynthèse par vissage direct de l'odontoïde, soit une arthrodèse C1-C2. Dans cette étude expérimentale in vitro, on a comparé au plan biomécanique, les trois différentes techniques d'arthrodèse atlanto-axoïdiennes, selon Gallie, Brooks et Magerl. 16 segments C1-C2 présentant des fractures de type II et III ont été testés in vitro dans des conditions bien définies. On a appliqué des moments de flexion et d'extension, des forces de cisaillement antérieur et postérieur et des moments de torsion vers la droite et vers la gauche, et l'on a analysé la mobilité de C1-C2. Les résultats de cette investigation ont clairement démontré que les segments traités par la technique de Magerl avec des vis transarticulaires, présentaient la plus grande rigidité, en comparaison des méthodes de cerclage de Brooks et de Gallie. Ces différences étaient plus évidentes dans le cas du cisaillement postérieur et de la torsion. Dans ces conditions de contrainte, le rapport de rigidité Magerl/Brooks/Gallie était de 10/2/1. Des différences significatives de déformation plastique ont été retrouvées entre les différentes fixations C1-C2 au cours des premiers cycles de mise en charge et décharge, qui renseignent sur la relation entre la stabilité primaire et à long terme.
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  • 5
    ISSN: 1432-2013
    Keywords: [Ca2+]i export ; Thapsigargin ; fura-2 ; HT29 ; CFPAC-1 ; ATP ; Carbachol ; Neurotensin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract There is increasing evidence that some agonists not only induce intracellular Ca2+ increases, due to store release and transmembranous influx, but also that they stimulate Ca2+ efflux. We have investigated the agonist-stimulated response on the intracellular Ca2+ activity ([Ca2+]i) in the presence of thapsigargin (10−8 mol/l, TG) in HT29 and CFPAC-1 cells. For CFPAC-1 the agonists ATP (10−7–10−3 mol/l, n=9), carbachol (10−6–10−3 mol/l, n=5) and neurotensin (10−10–10−7 mol/l, n=6) all induced a concentration-dependent decrease in [Ca2+]i in the presence of TG. Similar results were obtained with HT29 cells. This decrease of [Ca2+]i could be caused by a reduced Ca2+ influx, either due to a reduced driving force for Ca2+ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca2+ permeability. Using the fura-2 Mn2+ quenching technique we demonstrated that ATP did not slow the TG-induced Mn2+ quench. This indicates that the agonist-induced [Ca2+]i decrease in the presence of TG was not due to a reduced influx of Ca2+ into the cell, but rather due to stimulation of Ca2+ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of −49±3.6 mV (n=9). Within the first 3 min ATP was still able to induce a depolarization which could be attributed to an increase in Cl− conductance. This was expected, since at this time after TG stimulation all Ca2+ agonists still liberated some [Ca2+]i. When TG incubation was prolonged, agonist application led to strongly attenuated or to no electrical responses. Therefore, the agonist-stimulated [Ca2+]i decrease cannot be explained by the reduction of the driving force for Ca2+ into the cell. In the same cells hypotonic swelling (160 mosmol/l, n=15) still induced a further [Ca2+]i increase in the presence of TG and concomitantly induced Cl− and K+ conductances. We conclude that the agonist-induced decrease of [Ca2+]i in the presence of TG probably unmasks a stimulation of [Ca2+]i export.
    Type of Medium: Electronic Resource
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