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Biomedical subjects

B J Simon

Publications and source records attributed to B J Simon.

At least 19 recordsLinked to original sources
PubMed2003

Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells.

Osteocytes, the predominant cells in bone, are postulated to be responsible for sensing mechanical and electrical stimuli, transducing signals via gap junctions. Osteocytes respond to induced shear by increasing connexin 43 (Cx43) levels, suggesting that they might be sensitive to physical stimuli like low-frequency electromagnetic fields (EMF). Immature osteoblasts exhibit decreased intercellular communication in response to EMF but no change in Cx43. Here, we examined long term effects of pulsed EMF (PEMF) on MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells. In MLO-Y4 cell cultures, PEMF for 8 h/day for one, two or four days increased alkaline phosphatase activity but had no effect on cell number or osteocalcin. Transforming growth factor beta-1 (TGF-beta 1) and prostaglandin E(2) were increased, and NO(2-) was altered. PEMFs effect on TGF-beta1 was via a prostaglandin-dependent mechanism involving Cox-1 but not Cox-2. In ROS 17/2.8 cells, PEMF for 24, 48 or 72 h did not affect cell number, osteocalcin mRNA or osteocalcin protein. PEMF reduced Cx43 protein in both cells. Longer exposures decreased Cx43 mRNA. This indicates that cells in the osteoblast lineage, including well-differentiated osteoblast-like ROS 17/2.8 cells and terminally differentiated osteocyte-like MLO-Y4 cells, respond to PEMF with changes in local factor production and reduced Cx43, suggesting decreased gap junctional signaling.

Alkaline Phosphatase↗
PubMed2001

The efficacy of direct current stimulation for lumbar intertransverse process fusions in an animal model.

STUDY DESIGN: Posterolateral lumbar intertransverse process fusion using a rabbit model with autologous bone graft and direct current stimulation was compared with fusion achieved by using autologous bone graft alone. OBJECTIVES: To determine the efficacy of direct current electrical stimulation for the posterolateral lumbar intertransverse process fusion technique by using a 20-microA current and the more recently developed 60-microA current delivered by an implantable direct current stimulator. SUMMARY OF BACKGROUND DATA: Previous studies have demonstrated a positive effect of direct current electrical stimulation on posterior spinal fusion techniques. However, until recently, the environment of an intertransverse fusion was not well simulated. The current research examined the posterolateral lumbar intertransverse process fusion technique with direct current electrical stimulation using a rabbit model. This appears to parallel human fusion techniques more closely and allows for lower cost and technical ease. METHODS: In this study, 44 adult New Zealand white rabbits underwent an L5-L6 intertransverse process fusion. All the fusions used an autologous bone graft obtained from bilateral posterior iliac crests. A device was implanted in all the rabbits subcutaneously, and they were divided randomly into three groups: a sham or nonfunctioning group, a 20-microA low-current stimulator group, and a 60-microA higher-current stimulator group. Spinal fusion was evaluated radiographically, histologically, and manually as well as by biomechanical testing 5 weeks after surgery. RESULTS: Radiographic grades, manual palpation, biomechanical strength, and stiffness showed an increasing trend from sham or inactive stimulator groups to low-current and then to high-current stimulator groups. Histologic analysis revealed that the higher-current stimulator showed that, statistically, the healing response of the host tissue to the autograft had increased significantly, as compared with the sham. CONCLUSIONS: Direct current electrical stimulation is efficacious in improving both the healing rate and strength in this posterolateral lumbar fusion model. In addition, it appears that this effect is enhanced by increasing the stimulation current from 20 microA to 60 microA.

Animals↗
PubMed2001

Pulsed electromagnetic fields increase growth factor release by nonunion cells.

The mechanisms involved in pulsed electromagnetic field stimulation of nonunions are not known. Animal and cell culture models suggest endochondral ossification is stimulated by increasing cartilage mass and production of transforming growth factor-beta 1. For the current study, the effect of pulsed electromagnetic field stimulation on cells from human hypertrophic (n = 3) and atrophic (n = 4) nonunion tissues was examined. Cultures were placed between Helmholtz coils, and an electromagnetic field (4.5-ms bursts of 20 pulses repeating at 15 Hz) was applied to 1/2 of them 8 hours per day for 1, 2, or 4 days. There was a time-dependent increase in transforming growth factor-beta 1 in the conditioned media of treated hypertrophic nonunion cells by Day 2 and of atrophic nonunion cells by Day 4. There was no effect on cell number, [3H]-thymidine incorporation, alkaline phosphatase activity, collagen synthesis, or prostaglandin E2 and osteocalcin production. This indicates that human nonunion cells respond to pulsed electromagnetic fields in culture and that transforming growth factor-beta 1 production is an early event. The delayed response of hypertrophic and atrophic nonunion cells (> 24 hours) suggests that a cascade of regulatory events is stimulated, culminating in growth factor synthesis and release.

Adult↗
PubMed2001

Factors predicting early in-hospital death in blunt thoracic aortic injury.

BACKGROUND: Despite technical advances in the diagnosis and repair of blunt thoracic aortic injury (TAI),1,2 patients continue to die after arrival at trauma centers. Most of these deaths occur consequent to free intrapleural aortic rupture before surgical repair. We sought to identify characteristics of the subpopulation of blunt TAI patients at particular risk for early in-hospital death. METHODS: The records of a Level I trauma center were reviewed for the period from 1/1/90 through 5/1/00. Fifty-one patients with aortic injury were identified. A subgroup was identified with isolated aortic injury. The resultant study group consisted of 26 patients and included 11 cases of free rupture (FR) and 15 cases of contained rupture (CR). Data from both groups were analyzed for mechanism, abnormal vital signs, initial chest radiographic findings, symptoms, physical findings, time course from injury to rupture or treatment, clinical outcome, and pathologic findings. RESULTS: All 11 of the FR group died. In the CR group, 14/15 survived. Mechanism in all cases was "high-speed" or "head-on" motor vehicle collision. All cases with FR had the mediastinal abnormalities of "grossly widened mediastinum" + hemothorax; 10/11 had an episode of transient hypotension that initially responded to fluid resuscitation before their terminal event. In the CR group, there were a variety of chest radiographic findings, but only one "grossly widened mediastinum," no hemothorax, and no incidences of hypotension. However, multivariate analysis revealed that the combination of grossly widened mediastinum + hemothorax + transient hypotension was strongly and uniquely associated with those patients who went on to have free rupture (FR group) (p < 0.00001, Fisher's exact test). CONCLUSION: For patients exposed to a high-speed decelerative mechanism, the constellation of "grossly widened mediastinum + hemothorax with transient hemodynamic instability" appears not only to be highly specific for aortic injury but also to be a marker of impending sudden death from free rupture. This association would seem to warrant immediate surgery in this high-risk group without the traditional diagnostic studies.

Accidents, Traffic↗
PubMed2000

Implantable spinal fusion stimulator: assessment of MR safety and artifacts.

The objective of this investigation was to perform magnetic resonance (MR) imaging safety and artifact testing of an implantable spinal fusion stimulator. Magnetic field interactions, artifacts, and operational aspects of an implantable spinal fusion stimulator were evaluated in association with a 1.5 T MR system. Magnetic field-related translational attraction was measured using the deflection angle test. A special test apparatus was used to determine torque at 4.7 T. Artifacts were characterized using fast multiplanar spoiled gradient-echo, T1-weighted spin-echo, and T1-weighted fast spin-echo sequences. Operational aspects of the implantable spinal fusion stimulator before and after exposure to MR imaging at 1.5 T were assessed. In addition, nine patients (six lumbar spine and three cervical spine) with implantable spinal fusion stimulators underwent MR imaging. The findings indicated that magnetic field interactions were relatively minor, artifacts were well characterized and should not create diagnostic problems, and there were no changes in the operation of the spinal fusion stimulator. The nine patients underwent MR procedures without substantial adverse events or complaints. Based on the results of this investigation and in consideration of the findings from previous studies of MR imaging safety for the implantable spinal fusion stimulator, MR imaging may be performed safely in patients using MR systems operating at 1.5 T or less following specific recommendations and precautions.

Artifacts↗
PubMed1999

In vivo evaluation of coralline hydroxyapatite and direct current electrical stimulation in lumbar spinal fusion.

STUDY DESIGN: An animal model of posterolateral intertransverse process lumbar spinal fusion using autologous bone, coralline hydroxyapatite, and/or direct current electrical stimulation. OBJECTIVES: To evaluate the effect of an osteoconductive bone graft substitute and direct-current electrical stimulation on the rate of pseudarthrosis in a rabbit spinal fusion model. SUMMARY OF BACKGROUND DATA: Conventional techniques for the surgical treatment of degenerative conditions in the lumbar spine have a substantial failure rate and associated morbidity. Bone graft substitutes and electrical stimulation are alternative techniques to enhance fusion rates and limit the morbidity associated with posterolateral intertransverse process fusion using autologous iliac crest bone graft. METHODS: Fifty-three adult female New Zealand White rabbits underwent single-level lumbar posterolateral intertransverse process fusion. Animals were assigned to one of four groups using either autologous bone (Group I), coralline hydroxyapatite with autologous bone marrow aspirate (Group II), coralline hydroxyapatite with a 40-microA implantable direct current electrical stimulator and bone marrow aspirate (Group III), or coralline hydroxyapatite with a 100-microA implantable direct current electrical stimulator and bone marrow aspirate (Group IV). Animals were killed at 8 weeks, and fused motion segments were subjected to manual palpation, mechanical testing, and radiographic and histologic analysis to assess the fusion mass. RESULTS: Successful fusion was achieved in 57% (8/14) of animals in Group I, 25% (3/12) in Group II, 50% (6/12) in Group III, and 87% (13/15) in Group IV. Mean stiffness and ultimate load to failure were significantly higher in Group IV than in all other groups (P < 0.05). Histologic analysis demonstrated a qualitative increase in fusion mass in Group IV versus all other groups. CONCLUSIONS: Direct-current electrical stimulation increased fusion rates in a dose-dependent manner in a rabbit spinal fusion model. Coralline hydroxyapatite is an osteoconductive bone graft substitute, and thus requires an osteoinductive stimulus to ensure reliable fusion rates. Furthermore, coralline hydroxyapatite and direct current electrical stimulation can be used together to increase fusion rates in a rabbit spinal fusion model while avoiding the morbidity associated with harvesting iliac crest bone.

Animals↗
PubMed1998

Effects of electromagnetic stimulation on the functional responsiveness of isolated rat osteoclasts.

We report the effects of pulsed electromagnetic fields (PEMFs) on the responsiveness of osteoclasts to cellular, hormonal, and ionic signals. Osteoclasts isolated from neonatal rat long bones were dispersed onto either slices of devitalised cortical bone (for the measurement of resorptive activity) or glass coverslips (for the determination of the cytosolic free Ca2+ concentration, [Ca2+]). Osteoclasts were also cocultured on bone with osteoblastlike, UMR-106 cells. Bone resorption was quantitated by scanning electron microscopy and computer-assisted morphometry. PEMF application to osteoblast-osteoclast cocultures for 18 hr resulted in a twofold stimulation of bone resorption. In contrast, resorption by isolated osteoclasts remained unchanged in the presence of PEMFs, suggesting that osteoblasts were necessary for the PEMF-induced resorption simulation seen in osteoblast-osteoclast cocultures. Furthermore, the potent inhibitory action of the hormone calcitonin on bone resorption was unaffected by PEMF application. However, PEMFs completely reversed another quite distinct action of calcitonin on the osteoclast: its potent inhibitory effect on the activation of the divalent cation-sensing (or Ca2+) receptor. For these experiments, we made fura 2-based measurements of cytosolic [Ca2+] in single osteoclasts in response to the application of a known Ca2+ receptor agonist, Ni2+. We first confirmed that activation of the osteoclast Ca2+ receptor by Ni2+ (5 mM) resulted in a characteristic monophasic elevation of cytosolic [Ca2+]. As shown previously, this response was attenuated strongly by calcitonin at concentrations between 0.03 and 3 nM but remained intact in response to PEMFs. PEMF application, however, prevented the inhibitory effect of calcitonin on Ni2+-induced cytosolic Ca2+ elevation. This suggested that the fields disrupted the interaction between the calcitonin and Ca2+ receptor systems. In conclusion, we have shown that electromagnetic fields stimulate bone resorption through an action on the osteoblast and, by abolishing the inhibitory effects of calcitonin, also restore the responsiveness of osteoclasts to divalent cations.

Animals↗
PubMed1998

Delayed hemothorax after blunt thoracic trauma: an uncommon entity with significant morbidity.

OBJECTIVE: To describe the nature of delayed hemothorax occurring after blunt thoracic trauma and to identify the population at risk for this complication. METHODS: A retrospective review was conducted of 36 consecutive patients with hemothorax consequent to blunt trauma. Criteria for the definition of delayed hemothorax were established involving normal interval chest radiographs or computed tomographic scans during hospitalization. RESULTS: Twelve cases of delayed development of hemothorax were identified. Ninety-two percent of cases occurred in patients with multiple or displaced rib fractures. Presentation occurred from 18 hours to 6 days after injury. Eleven of the 12 cases were heralded by a prodrome of new pleuritic chest pain and dyspnea that occurred from 4 to 19 hours before treatment. CONCLUSION: Delayed hemothorax after blunt trauma is a unique entity occurring in patients with multiple or displaced rib fractures. Vigilance for the recognizable prodrome in the high-risk population should allow early remediation of this complication.

Aged↗
PubMed1996

Extracellularly applied ruthenium red and cADP ribose elevate cytosolic Ca2+ in isolated rat osteoclasts.

We demonstrated recently that the divalent cation-sensing receptor on the osteoclast, the Ca2+ receptor (CaR), is a functional component of a cell surface-expressed ryanodine receptor-like molecule (RyR). The objective of the present study was to further characterize this putative RyR by use of the two well-known cell-impermeant RyR modulators, ruthenium red and adenosine 3',5'-cyclic diphosphate ribose (cADPr). We found that, when applied extracellularly, ruthenium red (5 x 10(-8)-10(-4) M) and cADPr (5 x 10(-6) M) triggered an elevation of cytosolic [Ca2+]. Depolarization of the cell membrane by the application of 0.1 M K+ in the presence of 5 x 10(-6) M. valinomycin resulted in a concentration-dependent increase in the magnitude of the cytosolic Ca2+ response to extracellular ruthenium red (5 x 10(-9) and 5 x 10(-5) M), a phenomenon that was not seen when osteoclasts were hyperpolarized using 5 x 10(-3) M K+ with 5 x 10(-6) M valinomycin. In the presence of an intact nonleaky cell membrane, these results would favor a plasma membrane locus of action for the two modulators. Furthermore, pretreatment of osteoclasts with either modulator resulted in a markedly attenuated cytosolic Ca2+ transient elicited in response to the CaR agonist Ni2+, thus confirming an interaction between the cADPr- and ruthenium red-sensitive sites and the osteoclast CaR. The inhibition of the cytosolic Ca2+ response to Ni2+ induced by ruthenium red remained unchanged in the face of membrane potential changes. Finally, the cytosolic Ca2+ response to caffeine (5 x 10(-4) M), another RyR modulator, was also strongly attenuated by pretreatment with 5 x 10(-9) M ruthenium red. We conclude that ruthenium red and cADPr act on plasma membrane-resident sites and that both these sites interact with the process of divalent cation sensing.

Adenosine Diphosphate Ribose↗
PubMed1995

A ryanodine receptor-like molecule expressed in the osteoclast plasma membrane functions in extracellular Ca2+ sensing.

Ryanodine receptors (RyRs) reside in microsomal membranes where they gate Ca2+ release in response to changes in the cytosolic Ca2+ concentration. In the osteoclast, a divalent cation sensor, the Ca2+ receptor (CaR), located within the cell's plasma membrane, monitors changes in the extracellular Ca2+ concentration. Here we show that a RyR-like molecule is a functional component of this receptor. We have demonstrated that [3H] ryanodine specifically binds to freshly isolated rat osteoclasts. The binding was displaced by ryanodine itself, the CaR agonist Ni2+ and the RyR antagonist ruthenium red. The latter also inhibited cytosolic Ca2+ elevations induced by Ni2+. In contrast, the responses to Ni2+ were strongly potentiated by an antiserum Ab129 raised to an epitope located within the channel-forming domain of the type II RyR. The antiserum also stained the surface of intact, unfixed, trypan blue-negative osteoclasts. Serial confocal sections and immunogold scanning electron microscopy confirmed a plasma membrane localization of this staining. Antiserum Ab34 directed to a putatively intracellular RyR epitope expectedly did not stain live osteoclasts nor did it potentiate CaR activation. It did, however, stain fixed, permeabilized cells in a distinctive cytoplasmic pattern. We conclude that an RyR-like molecule resides within the osteoclast plasma membrane and plays in important role in extracellular Ca2+ sensing.

Animals↗
PubMed1994

Vehicular trauma triage by mechanism: avoidance of the unproductive evaluation.

An instrument was developed using routinely available field data to identify the sizable subgroup of stable vehicular trauma victims initially triaged to the trauma center by mechanism indicators alone who are in reality at minimal risk for serious injury. The six most common vehicular mechanism indicators seen at a level I trauma center were evaluated: rollover, head-on greater than 30 mph, intrusion, prolonged extrication, other death in same vehicle, and ejection. Review of 1235 consecutive trauma team activations yielded 349 victims with a qualifying vehicular mechanism. Outcome indicators were used to classify patients into two groups: Minor Injury (MI) and Severe Injury (SI). Nineteen common field data elements routinely reported on arrival by the regional Emergency Medical Service (EMS) personnel were then reviewed. Data patterns associated only with the MI group were sought. A checklist was developed for Mechanism vehicular trauma utilizing physiologic, anatomic, and neurologic elements. A single positive element would define trauma team activations. Retrospectively, use of this instrument would have excluded 56% of the MI group from unproductive trauma team referral, but nearly none of the SI group. We conclude that an identifiable subset of trauma patients referred by vehicular mechanism criteria alone could be safely evaluated on arrival in the emergency department as a form of secondary triage rather than by referral to the trauma team. The use of an appropriate exclusionary instrument can still preserve the sensitivity of trauma team activation for severely injured victims.

Accidents, Traffic↗
PubMed1992

Characterization of an ATP-driven H+ pump in human placental brush-border membrane vesicles.

The presence of an ATP-driven H+ pump as measured by H+ uptake upon addition of ATP was not demonstrable in human placental brush-border membrane vesicles when used in their native form, owing to their right-side-out orientation. However, the presence of the H+ pump in these membranes became evident when the membrane vesicles were transiently exposed to 1% cholate, with subsequent removal of the detergent to re-form the vesicles. Apparently, cholate pretreatment reoriented the H+ pump from an inward-facing configuration to outward-facing. Consequently, H+ uptake in response to externally added ATP was easily demonstrable in these cholate-pretreated vesicles by using the delta pH indicator Acridine Orange. In addition, bafilomycin A1-sensitive ATPase activity was measurable in cholate-pretreated vesicles, but not in native intact vesicles, indicating reorientation of the H+ pump. The reoriented H+ pump was electrogenic because H+ uptake was stimulated by an inside-negative anion-diffusion potential or when the vesicles were voltage-clamped. ATP supported H+ uptake with an apparent Km of 260 microM. ITP and GTP supported the pump activity partially, whereas CTP and UTP did not. Mg2+ and Mn2+ were the most preferred bivalent cations. Co2+ and Zn2+ showed partial activity, whereas Ca2+ and Ba2+ showed little or no activity. The pump was inhibited by nanomolar concentrations of bafilomycin A1 and micromolar concentrations of N-ethylmaleimide, p-chloromercuribenzenesulphonate, NN-dicyclohexylcarbodi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was relatively insensitive to oligomycin, vanadate and NaN3. The inhibition by N-ethylmaleimide was protectable by ATP. It is concluded that human placental brush-border membranes possess an ATP-driven H+ pump and that, on the basis of its characteristics, it belongs to the class of vacuolar (V-type) H+ pumps.

Acridine Orange↗
PubMed1992

Charge movement and SR calcium release in frog skeletal muscle can be related by a Hodgkin-Huxley model with four gating particles.

Charge movement currents (IQ) and calcium transients (delta[Ca2+]) were measured simultaneously in frog skeletal muscle fibers, voltage clamped in a double vaseline gap chamber, using Antipyrylazo III as the calcium indicator. The rate of release of calcium from the SR (Rrel) was calculated from the calcium transients using the removal model of Melzer, W., E. Rios, and M. F. Schneider (1987. Biophys. J. 51:849-863.). IQ and delta [Ca2+] were calculated for 100 ms depolarizing test pulses to membrane potentials from -30 to +20 mV. To eliminate an inactivating component of Rrel, each test pulse was preceded by a large, fixed prepulse to +20 mV. The resulting Rrel records, which represent the noninactivating component of Rrel, were compared with integral of IQdt.(Q), the total charge that moves. The voltage dependence of the steady state Rrel was steeper then that of Q and shifted to the right. During depolarization, the Rrel waveform was similar to that of Q but was delayed by several ms, while, during repolarization, Rrel preceded Q. All of these results could be explained with a Hodgkin-Huxley type model for E-C coupling in which four voltage sensors in the t-tubule membrane which give rise to IQ must all be in their activating positions for the calcium release channel in the SR membrane to open. his model is consistent with the structural architecture of the triadic junction in which four dihydropyridine receptors (the voltage sensors for E-C coupling) in the t-tubule membrane are closely associated with each ryanodine receptor(the calcium release channel) in the SR membrane [Block, B. A., T. Imagawa, K. P. Campbell, and C. Franzini-Armstrong. 1988. J.Cell. Biol. 107:2587-2600.]). Some aspects of this work have appeared in abstract form (Simon, B. J., and D. Hill. 1991. Biophys. J.59:64a. ([Abstr.]).

Animals↗
PubMed1992

Effects of procaine and caffeine on calcium release from the sarcoplasmic reticulum in frog skeletal muscle.

1. Resting myoplasmic free [Ca2+] and [Ca2+] transients (delta [Ca2+]) were measured in single voltage-clamped frog skeletal muscle fibres in the presence and absence of procaine, caffeine or procaine plus caffeine using Fura-2 fluorescence and antipyrylazo III (Ap III) absorbance signals. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum (SR) was calculated from the calcium transients and corrected for the relatively small decline due to depletion of calcium from the SR. 2. Procaine (1 mM) reversibly suppressed delta [Ca2+] and the corresponding Rrel by about 40% for 60-100 ms depolarizing steps to -40 to +20 mV. Procaine had little effect on either the waveform or voltage dependence of the Rrel records. 3. [Ca2+] transients calculated from Fura-2 fluorescence changes in the presence or absence of procaine had similar time courses and amplitudes as those calculated from the Ap III absorbance changes suggesting that 1 mM-procaine did not interfere with the ability of Ap III or Fura-2 to monitor delta [Ca2+]. 4. Although 1 mM-procaine depressed Rrel it had no effect on intramembrane charge movements (IQ) calculated from membrane currents recorded simultaneously with delta [Ca2+]. 5. Procaine (1 mM) reversibly inhibited the potentiating effect of 0.5 mM-caffeine on delta [Ca2+]. The amplitude and waveform of the Rrel records were similar in control fibres and in the presence of 1 mM-procaine plus 0.5 mM-caffeine. 6. In the presence of 0.5 mM-caffeine delta [Ca2+] after 10-20 ms voltage steps exhibited an increase in the time to peak and a slower decay time course compared with caffeine-free controls, suggestive of significant calcium-induced calcium release in the presence of caffeine. These effects of caffeine were completely and reversibly blocked by 1 mM-procaine. 7. In the absence of caffeine, 1 mM-procaine caused a small decrease in time to peak of delta [Ca2+] after 10-30 ms duration voltage steps compared to the bracketing control and wash runs without procaine. Rrel turned off faster after 10 ms pulses in procaine than in the absence of procaine, but the turn-off of release was about equally fast with or without procaine after pulses of 20 ms or longer. The effect of procaine after 10 ms pulses in the absence of caffeine may indicate suppression of a component of calcium-induced calcium release in control that inactivates during the pulse.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

The bulk of Ca2+ released to the myoplasm is free in the sarcoplasmic reticulum and does not unbind from calsequestrin.

Calsequestrin (CS) is the major Ca2+ binding protein contained in the lumen of sarcoplasmic reticulum (SR). Ca2+ binding properties and tissue concentration of CS of frog skeletal muscle were measured. At equilibrium, maximal Ca2+ binding capacity of purified CS was about 1.2 mumol Ca2+/mg protein. Apparent Kds for Ca2+ were around 50 microM in the absence of salts, around 0.9 mM in the presence of 100 mM KCl, and around 1.1 mM under 'physiological' conditions. Quantitation of CS in homogenates was accomplished by three methods (Stains-all staining, immunoblotting and 45Ca ligand overlay). Frog muscle contained about 0.5 mg of CS/g wet weight, that is 6.1 mM CS inside the SR. At rest the in situ free [Ca2+] of SR was calculated to be 3.6 mM, and, thus, CS is largely saturated with Ca2+. Moreover, computer simulations of Ca2+ release indicated that about 75% of Ca2+ released during a twitch is free in the SR and does not unbind from CS.

Animals↗
PubMed1991

Calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers.

The steady-state calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum was studied in voltage-clamped, cut segments of frog skeletal muscle fibers containing two calcium indicators, fura-2 and anti-pyrylazo III (AP III). Fura-2 fluorescence was used to monitor resting calcium and relatively small calcium transients during small depolarizations. AP III absorbance signals were used to monitor larger calcium transients during larger depolarizations. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum was calculated from the calcium transients. The equilibrium calcium dependence of inactivation of calcium release was determined using 200-ms prepulses of various amplitudes to elevate [Ca2+] to various steady levels. Each prepulse was followed by a constant test pulse. The suppression of peak Rrel during the test pulse provided a measure of the extent of inactivation of release at the end of the prepulse. The [Ca2+] dependence of inactivation indicated that binding of more than one calcium ion was required to inactivate each release channel. Half-maximal inactivation was produced at a [Ca2+] of approximately 0.3 microM. Variation of the prepulse duration and amplitude showed that the suppression of peak release was consistent with calcium-dependent inactivation of calcium release but not with calcium depletion. The same calcium dependence of inactivation was obtained using different amplitude test pulses to determine the degree of inactivation. Prepulses that produced near maximal inactivation of release during the following test pulse produced no suppression of intramembrane charge movement during the test pulse, indicating that inactivation occurred at a step beyond the voltage sensor for calcium release. Three alternative set of properties that were assumed for the rapidly equilibrating calcium-binding sites intrinsic to the fibers gave somewhat different Rrel records, but gave very similar calcium dependence of inactivation. Thus, equilibrium inactivation of calcium release appears to be produced by rather modest increases in [Ca2+] above the resting level and in a steeply calcium-dependent manner. However, the inactivation develops relatively slowly even during marked elevation of [Ca2+], indicating that a calcium-independent transition appears to occur after the initial calcium-binding step.

Animals↗
PubMed1991

Decline of myoplasmic Ca2+, recovery of calcium release and sarcoplasmic Ca2+ pump properties in frog skeletal muscle.

1. The two calcium indicators Antipyrylazo III (AP III) and Fura-2 were used simultaneously to monitor free myoplasmic [Ca2+] in voltage-clamped cut segments of frog skeletal muscle fibres (8-10 degrees C). Antipyrylazo III was used for the relatively large [Ca2+] transients during 100-200 ms depolarizing pulses to -20 to 0 mV and for the rapid decline of [Ca2+] during the 200 ms after the pulses. Fura-2 was used to follow the slow decline of the small remaining elevation of [Ca2+] during the following 16 s (slow recovery period) and to monitor resting [Ca2+]. 2. From 1 to 16 s of the slow recovery period [Ca2+] declined with two exponential components, having time constants of 1.9 +/- 0.3 and 13.5 +/- 1.5 s (these and all other values are means +/- S.E.M. of eleven runs from seven fibres). At 1.2 s after the end of the pulses the amplitudes of the fast and slow exponential components of decline of [Ca2+] were 34 +/- 7 and 31 +/- 4 nM, respectively. The resting [Ca2+] in these runs was 40 +/- 4 nM. 3. The time course of calcium bound to parvalbumin [( Ca-Parv]) was calculated from the [Ca2+] records using literature values for the parvalbumin kinetic constants. From 1 to 16 s of the slow recovery period the total calcium [Ca]T outside the sarcoplasmic reticulum (SR) was assumed to equal [Ca-Parv] + [Ca-Fura]. During this period [Ca]T declined with two exponential components having time constants of 1.7 +/- 0.2 and 14.2 +/- 1.4 s, the same as those for [Ca2+]. Assuming the total concentration of parvalbumin cation binding sites to be 1000 microM, the fast and slow components of [Ca]T had amplitudes of 117 +/- 21 and 147 +/- 16 microM, respectively, at 1.2 s after the pulses. 4. The rate of decline of [Ca]T, -d[Ca]T/dt, was used as a measure of the net rate of removal of calcium from the myoplasm by the SR. From 3 to 16 s of the slow recovery period and in the resting fibre -d[Ca]T/dt varied with [Ca2+] according to A[Ca2+]n-L. The term A[Ca2+]n represents the pump rate and L represents a constant rate of calcium leak from the SR. 5. For 40 nM less than or equal to [Ca2+] less than or equal to 80 nM, the power n for the [Ca2+] dependence of pump rate was 3.9 +/- 0.6.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗