[How healthy are German schools? Trends from the years 2002 to 2010]. / Wie gesund sind deutsche Schulen? Entwicklungstrends von 2002 bis 2010.

This study aims to analyse the time trends of several conditions of the school environment (satisfaction with school, school demands, quality of instruction, classmate support) in Germany that are known to affect the health of pupils.We used the national German data of the Health-Behaviour in School-aged Children (HBSC) studies conducted in the years 2002, 2006 und 2010. The time trends of these four var-iables are described by using linear and logistic regression analyses considering survey year, age group (11, 13, 15 years), gender, and school track as independent variables.We found an increase of the perceived quality of instruction and of the student's satisfaction with school from the year 2002 to the year 2010. Furthermore, pupils report slightly less support from their classmates in the present survey compared to 2002. There are no changes in the reported demands.These trend results are discussed in the light of the impact of the PISA study and the efforts to implement settings-based health promoting schools in Germany.

[Mobbing and violence at school. Trends from 2002 to 2010]. / Mobbing und Gewalt an Schulen. Entwicklungstrends von 2002 bis 2010.

The purpose of this study was to undertake an assessment and differentiated examination of the development of bullying and violence in schools between 2002 and 2010 in Germany.We examined the national German data of Health Behaviour in School-aged Children (HBSC) study in 2002, 2006 and 2010. A paper-pencil questionnaire was distributed to a representative sample (N=17 929) of 11-, 13- and 15-year-old school children. The evaluation of the data was done by descriptive statistics and logistic regression analyses, controlled by age, gender, family affluence, school type and survey year.A clear positive trend could be identified: from 2002 to 2010 the number of bullies and bully victims decreased whereas the group of the uninvolved pupils increased. There was a delay in this trend for children with low family affluence.The obvious success in the prevention of violence is shown by the decreasing rate of bullies. The paper discusses whether future prevention should focus more on victims and children with educationally deprived background.

[The HBSC Study in Germany--study design and methodology]. / Die HBSC-Studie in Deutschland--Studiendesign und Methodik.

The aim of the HBSC-Study is to collect data on the physical and mental health and health behaviour of children and adolescents and to gain a deeper insight into their situation and the specific environment they grow up in. The HBSC-study is an international school-based cross-sectional survey conducted in collaboration with the World Health Organization (WHO). The survey takes place every 4 years since 1982 and is based on a standardised protocol. In Germany the survey was first conducted in 1994 as a pilot study in North Rhine-Westphalia. The German sample is based on a random sample of classes in all public schools in Germany. 11-, 13-, and 15-year-old pupils are surveyed by means of a paper and pencil questionnaire. The questionnaire comprises a broad selection of -topics, including sociodemographics, health and risk behaviours, family, school and peers. The reported trends in the supplement are based on the data from surveys in 2002 (N=5.650), 2006 (N=7.274) and 2010 (N=5.005). The representative samples for each of the survey years are defined as follows: in 2002 the data is based on information collected in 4 Federal States (Berlin, Hesse, North Rhine-Westphalia, Saxony); in 2006 5 states define the German data file (Berlin, Hamburg, Hesse, North Rhine-Westphalia, Saxony). The data from the 2010 survey comprises data from 15 Federal States. The HBSC-data contributes towards a better understanding of the relationship between health and living conditions of young people. The papers in this supplement deliver important insights into the living context of young people and in doing this they provide important information about their health and the long-term effectiveness of public-health-measures.

[Bullying, psychosocial health and risk behaviour in adolescence]. / Bullying, psychosoziale Gesundheit und Risikoverhalten im Jugendalter.

BACKGROUND: Bullying as a subform of aggressive behaviour has not received much attention as a specific risk behaviour in adolescence. Especially the adverse health effects in relation to bullying have been barely discussed in Germany. The objective of this study is to present age- and gender-specific prevalences in bullying and to analyse the association between the different bullying roles and subjective health as well as risk behaviour. METHODS: Data were obtained from the German part of the international WHO collaborative study "Health Behaviour in School-aged Children (HBSC)" in 2002. Overall, 5,650 school children aged 11-15 years were interviewed with a standardised questionnaire. Multivariate logistic regression models were used to analyse the association between bullying, psychosocial health and risk behaviour separately for girls and boys. RESULTS: About 17% of the boys and 10% of the girls aged 11-15 years were classified as repeated bullying perpetrators. About 10% of the school children are victims of being bullied several times a month. Another 3-5% of the adolescents belonged to the group of simultaneous victims and perpetrators (bully-victims). Perpetrators as well as victims showed strong associations with psychosocial health and risk behaviour. Independently of gender, victims were significantly more likely to report repeated psychosomatic complaints, adverse mental health and negative self-reported health (boys only), than uninvolved students. Especially for male perpetrators, strong associations with regular tobacco and alcohol use and repeated drunkenness were found, while these behaviour types were significantly less prevalent among victims. The bully-victim group is characterised by high rates of psychosomatic complaints and mental health problems (boys only). CONCLUSIONS: Bullying also seems to be widespread in schools in Germany and is strongly associated with subjective health and substance-related risk behaviour. The results suggest that bullying is a critical issue that requires increasing attention in health research. The unique health problems of victims and perpetrators suggest different intervention strategies for both groups.

A possible role of the junctional face protein JP-45 in modulating Ca2+ release in skeletal muscle.

We investigated the functional role of JP-45, a recently discovered protein of the junctional face membrane (JFM) of skeletal muscle. For this purpose, we expressed JP-45 C-terminally tagged with the fluorescent protein DsRed2 by nuclear microinjection in myotubes derived from the C2C12 skeletal muscle cell line and performed whole-cell voltage-clamp experiments. We recorded in parallel cell membrane currents and Ca(2+) signals using fura-2 during step depolarization. It was found that properties of the voltage-activated Ca(2+) current were not significantly changed in JP-45-DsRed2-expressing C2C12 myotubes whereas the amplitude of depolarization-induced Ca(2+) transient was decreased compared to control myotubes expressing only DsRed2. Converting Ca(2+) transients to Ca(2+) input flux using a model fit approach to quantify Ca(2+) removal, the change could be attributed to an alteration in voltage-activated Ca(2+) permeability rather than to altered removal properties or a lower Ca(2+) content of the sarcoplasmic reticulum (SR). Determining non-linear capacitive currents revealed a reduction of Ca(2+) permeability per voltage-sensor charge. The results may be explained by a modulatory effect of JP-45 related to its reported in vitro interaction with the dihydropyridine receptor and the SR Ca(2+) binding protein calsequestrin (CSQ).

Voltage-controlled Ca2+ release and entry flux in isolated adult muscle fibres of the mouse.

The voltage-activated fluxes of Ca(2+) from the sarcoplasmic reticulum (SR) and from the extracellular space were studied in skeletal muscle fibres of adult mice. Single fibres of the interosseus muscle were enzymatically isolated and voltage clamped using a two-electrode technique. The fibres were perfused from the current-passing micropipette with a solution containing 15 mm EGTA and 0.2 mm of either fura-2 or the faster, lower affinity indicator fura-FF. Electrical recordings in parallel with the fluorescence measurements allowed the estimation of intramembrane gating charge movements and transmembrane Ca(2+) inward current exhibiting half-maximal activation at -7.60 +/- 1.29 and 3.0 +/- 1.44 mV, respectively. The rate of Ca(2+) release from the SR was calculated after fitting the relaxation phases of fluorescence ratio signals with a kinetic model to quantify overall Ca(2+) removal. Results obtained with the two indicators were similar. Ca(2+) release was 2-3 orders of magnitude larger than the flux carried by the L-type Ca(2+) current. At maximal depolarization (+50 mV), release flux peaked at about 3 ms after the onset of the voltage pulse and then decayed in two distinct phases. The slower phase, most likely resulting from SR depletion, indicated a decrease in lumenal Ca(2+) content by about 80% within 100 ms. Unlike in frog fibres, the kinetics of the rapid phase of decay showed no dependence on the filling state of the SR and the results provide little evidence for a substantial increase of SR permeability on depletion. The approach described here promises insight into excitation-contraction coupling in future studies of genetically altered mice.

Voltage-dependent Ca2+ fluxes in skeletal myotubes determined using a removal model analysis.

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the "removal model fit" approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 microM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at -16 and -20 mV, respectively, compared with -2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 microM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.

Voltage-activated calcium signals in myotubes loaded with high concentrations of EGTA.

In the present study we describe the analysis of optically recorded whole cell Ca(2+) transients elicited by depolarization in cultured skeletal myotubes. Myotubes were obtained from the mouse muscle-derived cell line C2C12 and from mouse satellite cells. The cells were voltage-clamped and perfused with an artificial intracellular solution containing 15 mM EGTA to ensure that the bulk of the Ca(2+) mobilized by depolarization is bound to this extrinsic buffer. The apparent on- and off-rate constants of EGTA and the dissociation rate constant of fura-2 in the cell were estimated by investigating the Ca(2+)-dependence of kinetic components of the fluorescence decay after repolarization. These parameters were used to calculate the time course of the total voltage-controlled flux of Ca(2+) to the myoplasmic space (Ca(2+) input flux). The validity of the procedure was confirmed by model simulations using artificial Ca(2+) input fluxes. Both C2C12 and primary-cultured myotubes showed a very similar phasic-tonic time course of the Ca(2+) input flux. In most measurements, the input flux was considerably larger and showed a different time course than the estimated Ca(2+) flux carried by the L-type Ca(2+) channels, indicating that it consists mainly of voltage-controlled Ca(2+) release from the sarcoplasmic reticulum. In cells with extremely small fluorescence transients, the calculated input fluxes matched the kinetic characteristics of the Ca(2+) inward current, indicating that Ca(2+) release was absent. These measurements served as a control for the fidelity of the fluorimetric flux analysis. The procedures promise a deeper insight into alterations of Ca(2+) release gating in studies employing myotube expression systems for mutant or chimeric protein components of excitation-contraction coupling.

Excitation-contraction coupling in skeletal muscle of a mouse lacking the dihydropyridine receptor subunit gamma1.

1. In skeletal muscle, dihydropyridine (DHP) receptors control both Ca(2+) entry (L-type current) and internal Ca(2+) release in a voltage-dependent manner. Here we investigated the question of whether elimination of the skeletal muscle-specific DHP receptor subunit gamma1 affects excitation-contraction (E-C) coupling. We studied intracellular Ca(2+) release and force production in muscle preparations of a mouse deficient in the gamma1 subunit (gamma-/-). 2. The rate of internal Ca(2+) release at large depolarization (+20 mV) was determined in voltage-clamped primary-cultured myotubes derived from satellite cells of adult mice by analysing fura-2 fluorescence signals and estimating the concentration of free and bound Ca(2+). On average, gamma-/- cells showed an increase in release of about one-third of the control value and no alterations in the time course. 3. Voltage of half-maximal activation (V(1/2)) and voltage sensitivity (k) were not significantly different in gamma-/- myotubes, either for internal Ca(2+) release activation or for the simultaneously measured L-type Ca(2+) conductance. The same was true for maximal Ca(2+) inward current and conductance. 4. Contractions evoked by electrical stimuli were recorded in isolated extensor digitorum longus (EDL; fast, glycolytic) and soleus (slow, oxidative) muscles under normal conditions and during fatigue induced by repetitive tetanic stimulation. Neither time course nor amplitudes of twitches and tetani nor force-frequency relations showed significant alterations in the gamma1-deficient muscles. 5. In conclusion, the overall results show that the gamma1 subunit is not essential for voltage-controlled Ca(2+) release and force production.

Malignant hyperthermia and excitation-contraction coupling.

Malignant hyperthermia (MH) is a state of elevated skeletal muscle metabolism that may occur during general anaesthesia in genetically pre-disposed individuals. Malignant hyperthermia results from altered control of sarcoplasmic reticulum (SR) Ca2+ release. Mutations have been identified in MH-susceptible (MHS) individuals in two key proteins of excitation-contraction (EC) coupling, the Ca2+ release channel of the SR, ryanodine receptor type 1 (RyR1) and the alpha1-subunit of the dihydropyridine receptor (DHPR, L-type Ca2+ channel). During EC coupling, the DHPR senses the plasma membrane depolarization and transmits the information to the ryanodine receptor (RyR). As a consequence, Ca2+ is released from the terminal cisternae of the SR. One of the human MH-mutations of RyR1 (Arg614Cys) is also found at the homologous location in the RyR of swine (Arg615Cys). This animal model permits the investigation of physiological consequences of the homozygously expressed mutant release channel. Of particular interest is the question of whether voltage-controlled release of Ca2+ is altered by MH-mutations in the absence of MH-triggering substances. This question has recently been addressed in this laboratory by studying Ca2+ release under voltage clamp conditions in both isolated human skeletal muscle fibres and porcine myotubes.

Malignant hyperthermia mutation Arg615Cys in the porcine ryanodine receptor alters voltage dependence of Ca2+ release.

Ca2+ inward current and fura-2 Ca2+ transients were simultaneously recorded in porcine myotubes. Myotubes from normal pigs and cells from specimens homozygous for the Arg615Cys (malignant hyperthermia) mutation of the skeletal muscle ryanodine receptor RyR1 were investigated. We addressed the question whether this mutation alters the voltage dependence of Ca2+ release from the sarcoplasmic reticulum. The time course of the total flux of Ca2+ into the myoplasm was estimated. Analysis showed that the largest input Ca2+ flux occurred immediately after depolarization. Amplitude and time course of the Ca2+ flux at large depolarizations were not significantly different in the Arg615Cys myotubes. Ca2+ release from the sarcoplasmic reticulum was activated at more negative potentials than the L-type Ca2+ conductance. In the controls, the potentials for half-maximal activation V 1/2 were -9.0mV and 16.5 mV, respectively. In myotubes expressing the Arg615Cys mutation, Ca2+ release was activated at significantly lower depolarizing potentials (V = -23.5 mV) than in control myotubes. In contrast, V of conductance activation (13.5 mV) was not significantly different from controls. The specific shift in the voltage dependence of Ca2+ release caused by this mutation can be well described by altering a voltage-independent reaction of the ryanodine receptor that is coupled to the voltage-dependent transitions of the L-type Ca2+ channel.

[Violence in the school--an analysis and prevention]. / Gewalt in der Schule--Analyse und Prävention.

The "Forschungsgruppe Schulevaluation" (Research Group for School Evaluation, Technical University of Dresden) conducted several empirical investigations which led to rich knowledge concerning the amount of violence in schools, the different forms of violence, roles that actors of and sufferers from violent action play and the causes for violence. Besides the wellknown socialisatory influence of families, media consumption and peers for the formation of violent behaviour of pupils and of behaviour that has an affinity to violence a causal influence of schools was detected. Questions concerning the prevention of violence, the conducting of pilot studies on the latter point in one single school as well as the evaluation of the preventory measures taken were important points in our work. In the present study we will present the most important empirical results and our experience with the prevention of violence.

Effects of the dihydropyridine receptor subunits gamma and alpha2delta on the kinetics of heterologously expressed L-type Ca2+ channels.

Ba2+ currents through L-type Ca2+ channels were measured in tsA201 cells transiently transfected with expression vectors encoding the dihydropyridine (DHP) receptor subunits alpha1C, beta1a-GFP, alpha2delta and gamma. The subunit effect on channel function was studied by omitting either alpha2delta or gamma from the transfection mixture and analyzing the voltage dependence and kinetics of activation, inactivation and recovery from inactivation. Activation could be described by a single exponential function while the time course of inactivation of the Ba2+ current followed a double exponential function. Progressively longer depolarization led to increasingly slower recovery, indicating the successive occupancy of several inactive states. Activation parameters remained largely unaffected in y-deficient cells whereas the voltage dependence of inactivation was shifted by 16 mV to more positive potentials and the larger one of the two inactivation time constants was increased by one-third. On the other hand, alpha2delta-deficient cells showed decreased current density and slowed activation and inactivation. Recovery from inactivation was significantly slowed by gamma coexpression. This and the effect of the gamma subunit on steady-state inactivation were independent of the presence of alpha2delta. We conclude that y stabilizes L-type Ca2+ channel inactivation in a way similar to certain Ca(2+)-antagonistic drugs. Alpha2delta is not needed for this effect.

Tools for flash-photolysis experiments on voltage-clamped muscle fibre segments.

An experimental set-up is described that allows the combination of rapid transmembrane voltage changes and photometric calcium recording with the fast photochemical turnover of substances applied externally or intracellularly to cut skeletal muscle fibres. It consists of a double-vaseline-gap system, designed for use with a xenon-flash-lamp device and a dual-wavelength microscope photometer. The pools of the vaseline gap chamber that contain the solutions surrounding the cut ends and the voltage-clamped segment of the muscle fibre are closed and have volumes of 20-50 microl. Thin tubes allow rapid solution change or continuous perfusion in the chamber compartments. Accessory tools were constructed to simplify focussing and measuring the flash-light intensity. A pilot light delivered from a red laser diode is used as a guide beam to target the ultraviolet (UV) flash to the preparation. The light distribution in the focal region and the relative changes in flash intensity with increasing numbers of flashes were quantified with an instrument that integrates the photo-current of a UV-sensitive silicon diode. The function of the set-up was demonstrated by measuring the efficiency of Ca2+ release from DM-nitrophen in quartz capillaries using the Ca(2+)-sensitive dye antipyrylazo III and by recording the flash-induced recovery of L-type calcium currents in muscle fibres blocked by the light-sensitive dihydropyridine drug nifedipine.

Kinetics of inactivation and restoration from inactivation of the L-type calcium current in human myotubes.

1. Inactivation and recovery kinetics of L-type calcium currents were measured in myotubes derived from satellite cells of human skeletal muscle using the whole cell patch clamp technique. 2. The time course of inactivation at potentials above the activation threshold was obtained from the decay of the current during 15 s depolarizing pulses. At subthreshold potentials, prepulses of different durations, followed by +20 mV test pulses, were used. The time course could be well described by single exponential functions of time. The time constant decreased from 17.8 +/- 7.5 s at -30 mV to 1.78 +/- 0.15 s at +50 mV. 3. Restoration from inactivation caused by 15 s depolarization to +20 mV was slowed by depolarization in the restoration interval. The time constant increased from 1.11 +/- 0.17 s at -90 mV to 7.57 +/- 2.54 s at -10 mV. 4. Restoration showed different kinetics depending on the duration of the conditioning depolarization. While the time constant was similar at restoration potentials of -90 and -50 mV after a 1 s conditioning prepulse, it increased with increasing prepulse duration at -50 mV and decreased at -90 mV. 5. The experiments showed that the rates of inactivation and restoration of the L-type calcium current in human myotubes were not identical when observed at the same potential. The results indicate the presence of more than one inactivated state and point to different voltage-dependent pathways for inactivation and restoration.

Modification of excitation-contraction coupling by 4-chloro-m-cresol in voltage-clamped cut muscle fibres of the frog (R. pipiens).

1. The effect of 5 microM 4-chloro-m-cresol (4-CmC) on voltage-controlled Ca2+ release was studied in cut muscle fibres of the frog loaded with internal solutions containing 15 mM EGTA. Fibres were voltage clamped using a double Vaseline gap system, and Ca2+ signals were recorded with the fluorescent indicator dye fura-2 2. Resting intracellular free Ca2+ concentration increased from 61 to 100 nM upon application of 4-CmC. 3. Both peak rate of release of intracellularly stored Ca2+ and the steady level attained after 50 ms of depolarization increased, but the potentiation of the latter was more pronounced (by a factor of 1.7 versus 1.3). The voltage of half-maximal activation remained unchanged. 4. Non-linear intramembranous charge movements showed no significant change in voltage dependence while the maximal charge displaced by depolarization increased by 25 %. 5. The dependence of peak release flux on total intramembranous charge was not different in 4-CmC, but for the steady level of release the steepness of the relation increased by a factor of 1.3. 6. The stimulating effect of 5 microM 4-CmC on depolarization-induced Ca2+ release resembled the potentiation by 0.5 mM caffeine. However, 0.5 mM caffeine increased the peak and steady levels of the release rate by a similar factor and caused no increase in the resting free calcium concentration, indicating different modes of action of the two substances. 7. Neither 5 microM 4-CmC nor 0.5 mM caffeine led to a loss of voltage control of Ca2+ release during repolarization after short depolarizations, as has been reported previously for caffeine. Potentiated Ca2+ release could be terminated by repolarization as fast as under control conditions both with 15 mM and 0.1 mM internal EGTA. 8. The effects of 4-CmC may result from a direct opening of the release channel combined with an enhancement of the transduction mechanism that couples channel opening to displacement of voltage sensor charges.

Voltage-controlled Ca2+ release in normal and ryanodine receptor type 3 (RyR3)-deficient mouse myotubes.

1. Primary cultured myotubes were derived from satellite cells of the diaphragm obtained from both normal mice (RyR3+/+) and mice with a targeted mutation eliminating expression of the type 3 isoform of the ryanodine receptor (RyR3-/-). Using the whole-cell patch clamp technique, L-type Ca2+ currents were measured during step depolarizations. Simultaneously, intracellular Ca2+ transients were recorded with the fluorescent indicator dye fura-2. 2. After correction for non-instantaneous binding of Ca2+ to the indicator dye and taking into account the dynamics of Ca2+ binding to intracellular constituents, an estimate of the time course of the Ca2+ release rate from the sarcoplasmic reticulum (SR) was obtained. 3. The calculated SR Ca2+ release flux exhibited a marked peak within less than 12 ms after the onset of the voltage-clamp depolarization and fell rapidly thereafter to a five times lower, almost steady level. It declined rapidly after termination of the depolarization. 4. Signals in normal and RyR3-deficient myotubes showed no significant difference in the activation of Ca2+ conductance and in amplitude, time course and voltage dependence of the Ca2+ efflux from the SR. 5. In conclusion, the characteristics of voltage-controlled Ca2+ release reported here are similar to those of mature mammalian muscle fibres. In contrast to differences observed in the contractile properties of RyR3-deficient muscle fibres, a contribution of RyR3 to excitation-contraction coupling could not be detected in myotubes.

Voltage-dependent calcium release in human malignant hyperthermia muscle fibers.

Malignant hyperthermia (MH) results from a defect of calcium release control in skeletal muscle that is often caused by point mutations in the ryanodine receptor gene (RYR1). In malignant hyperthermia-susceptible (MHS) muscle, calcium release responds more sensitively to drugs such as halothane and caffeine. In addition, experiments on the porcine homolog of malignant hyperthermia (mutation Arg615Cys in RYR1) indicated a higher sensitivity to membrane depolarization. Here, we investigated depolarization-dependent calcium release under voltage clamp conditions in human MHS muscle. Segments of muscle fibers dissected from biopsies of the vastus lateralis muscle of MHN (malignant hyperthermia negative) and MHS subjects were voltage-clamped in a double vaseline gap system. Free calcium was determined with the fluorescent indicator fura-2 and converted to an estimate of the rate of SR calcium release. Both MHN and MHS fibers showed an initial peak of the release rate, a subsequent decline, and rapid turn-off after repolarization. Neither the kinetics nor the voltage dependence of calcium release showed significant deviations from controls, but the average maximal peak rate of release was about threefold larger in MHS fibers.

Fura-2 calcium signals in skeletal muscle fibres loaded with high concentrations of EGTA.

Fura-2 is one of the most frequently used fluorescent Ca indicator dyes; yet it has limitations in tracking large intracellular Ca transients due to its high affinity for Ca. Since high affinity is of advantage when small Ca changes are to be detected, we tried the application of Fura-2 in skeletal muscle fibres which had been loaded with 15 mM internal EGTA to eliminate contractile artifacts. Under these conditions, the free Ca transients are considerably reduced in amplitude and strong saturation of Fura-2 is avoided. Cut segments of isolated muscle fibres were voltage-clamped in a double vaseline gap set-up. In the presence of high internal EGTA, free Ca (as measured with the rapid metallochromic indicator antipyrylazo III) drops rapidly from one value to a lower quasi steady-state value at the end of a depolarizing voltage pulse. This property allowed inspection of the dissociation kinetics of Ca from Fura-2 in the myoplasmic environment. The dissociation rate constant koff in the fibre was determined from the time constant of the exponential decay of the Fura-2 signal as a function of the final level of free Ca. We obtained a value of 26 s-1 at the experimental temperature of 12 degrees C. Knowledge of koff in the cell is essential for reconstructing the time course of free Ca from indicator bound Ca and for estimating the time course of the rate of release from the sarcoplasmic reticulum. The described combination of high EGTA buffering with Fura-2 fluorescence recording may be particularly useful for the determination of Ca release in small muscle cells.

Numerical methods to determine calcium release flux from calcium transients in muscle cells.

Several methods are currently in use to estimate the rate of depolarization-induced calcium release in muscle cells from measured calcium transients. One approach first characterizes calcium removal of the cell. This is done by determining parameters of a reaction scheme from a fit to the decay of elevated calcium after the depolarizing stimulus. In a second step, the release rate during depolarization is estimated based on the fitted model. Using simulated calcium transients with known underlying release rates, we tested the fidelity of this analysis in determining the time course of calcium release under different conditions. The analysis reproduced in a satisfactory way the characteristics of the input release rate, even when the assumption that release had ended before the start of the fitting interval was severely violated. Equally good reconstructions of the release rate time course could be obtained when the model used for the analysis differed in structure from the one used for simulating the data. We tested the application of a new strategy (multiple shooting) for fitting parameters in nonlinear differential equation systems. This procedure rendered the analysis less sensitive to ill-chosen initial guesses of the parameters and to noise. A locally adaptive kernel estimator for calculating numerical derivatives allowed good reconstructions of the original release rate time course from noisy calcium transients when other methods failed.