Mechanisms underlying changes of tetanic [Ca2+]i and force in skeletal muscle

Force development in skeletal muscle is driven by an increase in myoplasmic free [Ca2+]i ([Ca2+]i) due to Ca2+ release from the sarcoplasmic reticulum (SR). The magnitude of [Ca2+]i elevation during stimulation depends on: (a) the rate of Ca2+ release from the SR; (b) the rate of Ca2+ uptake by the SR; and (c) the myoplasmic Ca2+ buffering. We have used fluorescent Ca2+ indicators to measure [Ca2+]i in intact, single fibres from mouse and Xenopus muscles under conditions where one or more of the above factors are changed. The following interventions resulted in increased tetanic [Ca2+]i: beta-adrenergic stimulation, which potentiates the SR Ca2+ release; application of 2.5-di(tert-butyl)-1,4-benzohydroquinone, which inhibits SR Ca2+ pumps; application of caffeine, which facilitates SR Ca2+ release and inhibits SR Ca2+ uptake; early fatigue, where the rate of SR Ca2+ uptake is reduced; acidosis, which reduces both the myoplasmic Ca2+ buffering and the rate of SR Ca2+ uptake. Reduced tetanic [Ca2+]i was observed in late fatigue, due to reduced SR Ca2+ release, and in alkalosis, due to increased myoplasmic Ca2+ buffering. Force is monotonically related to [Ca2+]i but depends also on the myofibrillar Ca2+ sensitivity and the maximum force cross-bridges can produce. This is clearly illustrated by changes of intracellular pH where, despite a lower tetanic [Ca2+]i, tetanic force is higher in alkalosis than acidosis due to increases of myofibrillar Ca2+ sensitivity and maximum cross-bridge force.     

Effect of intracellular and extracellular ion changes on E-C coupling and skeletal muscle fatigue

The causative factors in muscle fatigue are multiple, and vary depending on the intensity and duration of the exercise, the fibre type composition of the muscle, and the individual's degree of fitness. Regardless of the aetiology, fatigue is characterized by the inability to maintain the required power output and the decline in power can be attributed to a reduced force and velocity. Following high-intensity exercise, peak force has been shown to recover biphasically with an initial rapid (2 min) recovery followed by a slower (50 min) return to the pre-fatigued condition. The resting membrane potential depolarizes by 10-15 mV, while the action potential overshoot declines by a similar magnitude. Following high-frequency stimulation of the frog semitendinous muscle, we observed intracellular potassium [K+]1 decrease from 142 +/- 5 to 97 +/- 8 mM, while sodium [Na+]i rose from 16 +/- 1 to 49 +/- 6 mM. The [K+]i loss was similar to that observed in fatigued mouse and human skeletal muscle, which suggests that there may be a limit to which [K+]i can decrease before the associated depolarization begins to limit the action potential frequency. Fibre depolarization to- 60 mV (a value observed in some cells) caused a significant reduction in the t-tubular charge movement, and the extent of the decline was inversely related to the concentration of extracellular Ca2+. A decrease in intracellular pH (pHi) to 6.0 was observed, and it has been suggested by some that low pH may disrupt E-C coupling by directly inhibiting the SR Ca2+ release channel. However, Lamb at al. (1992) observed that low pH had no effect on Ca2+ release, and we found low pHi to have no effect on t-tubular charge movement (Q) or the Q vs. Vm relationship. The Ca2+ released from the SR plays three important roles in the regulation of E-C coupling. As Ca2+ rises, it binds to the inner surface of the t-tubular charge sensor to increase charge (Q gamma) and thus Ca2+ release, it opens SR Ca2+ channels that are not voltage-regulated, and as [Ca2+]i increases further it feeds back to close the same channels. The late stages of fatigue have been shown to be in part caused by a reduced SR Ca2+ release. The exact cause of the reduced release is unknown, but the mechanism appears to involve a direct inhibition of the SR Ca2+ channel.     

Cytoplasmic sodium, calcium and free energy change of the Na+/Ca2+-exchanger in rat ventricular myocytes

The relationship between changing driving force of the Na+/Ca2+-exchanger (deltaG(exch)) and associated cytosolic calcium fluxes was studied in rat ventricular myocytes. DeltaG(exch) was abruptly reversed by the reduction of extracellular sodium ([Na+]o) with or without sustained depolarization by the elevation of potassium ([K+]o). Cytosolic sodium ([Na+]i) and calcium ([Ca2+]i) were measured with SBFI and indo-1 respectively and the time course of recovery of deltaG(exch) was calculated. Following abrupt reversal of deltaG(exch) from +4.1 to -9.2 kJ/mol [Na+]i exponentially decreased from 9.6-2.5 mmol/l (t(1/2) about 30 s) and [Ca2+]i transiently increased to a peak value after about 30 s. Negative values of deltaG(exch) were associated with an increase and positive values with a decrease of [Ca2+]i. Equilibrium (deltaG(exch) = 0) was reached after about 30 s coinciding with the time to peak [Ca2+]i. After 180 s deltaG(exch) reached a new steady state at +3.5 kJ/mol. Inhibition of SR with ryanodine or thapsigargin reduced the amplitude of the [Ca2+]i transient and shifted its peak to 80 s, but did not affect the time course of [Na+]i changes. In the presence of ryanodine or thapsigargin the time required for deltaG(exch) to recover to equilibrium was also shifted to 80 s. When we changed the deltaG(exch) to the same extent by the reduction of [Na+]o in combination with a sustained depolarization, [Na+]i decreased less and the amplitude of [Ca2+]i transient was much enhanced. This increase of [Ca2+]i was completely abolished by verapamil. DeltaG(exch) only recovered to a little above equilibrium (+1 kJ/mol). Inhibition of the Na+/K+-ATPase with ouabain entirely prevented the decrease of [Na+]i and caused a much larger increase of [Ca2+]i, which remained elevated; deltaG(exch) recovered to equilibrium and never returned to positive values. The rate of change of total cytosolic calcium was related to deltaG(exch), despite the fact that the calcium flux associated with the exchanger itself contributed only about 10%; SR related flux contributed by about 90% to the rate of change of total cytosolic calcium. In summary, reduction of [Na+]o causes reversal of the Na+/Ca2+-exchanger and its driving force deltaG(exch), a transient increase of [Ca2+]i and a decrease of [Na+]i. The influx of calcium associated with reversed deltaG(exch) triggers the release of calcium from SR. Both the decrease of [Na+]i and the increase of [Ca2+]i contribute to the recovery of deltaG(exch) to equilibrium. The time at which deltaG(exch) reaches equilibrium always coincides with the time to peak of [Ca2+]i transient. Activation of the Na+/K+-ATPase is required to reduce [Na+]i and recover deltaG(exch) to positive values in order to reduce [Ca2+]i. We conclude that deltaG(exch) is a major regulator of cytosolic calcium by interaction with SR.     

Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world

We measured [Ca2+]i and [Na+]i in isolated transgenic (TG) mouse myocytes overexpressing the Na+-Ca2+ exchanger and in wild-type (WT) myocytes. In TG myocytes, the peak systolic level and amplitude of electrically stimulated (ES) [Ca2+]i transients (0.25 Hz) were not significantly different from those in WT myocytes, but the time to peak [Ca2+]i was significantly prolonged. The decline of ES [Ca2+]i transients was significantly accelerated in TG myocytes. The decline of a long-duration (4-s) caffeine-induced [Ca2+]i transient was markedly faster in TG myocytes, and [Na+]i was identical in TG and WT myocytes, indicating that the overexpressed Na+-Ca2+ exchanger is functionally active. The decline of a short-duration (100-ms) caffeine-induced [Ca2+]i transient in 0 Na+/0 Ca2+ solution did not differ between the two groups, suggesting that the sarcoplasmic reticulum (SR) Ca2+-ATPase function is not altered by overexpression of the Na+-Ca2+ exchanger. There was no difference in L-type Ca2+ current density in WT and TG myocytes. However, the sensitivity of ES [Ca2+]i transients to nifedipine was reduced in TG myocytes. This maintenance of [Ca2+]i transients in nifedipine was inhibited by Ni2+ and required SR Ca2+ content, consistent with enhanced Ca2+ influx by reverse Na+-Ca2+ exchange, and the resulting Ca2+-induced Ca2+ release from SR. The rate of rise of [Ca2+]i transients in nifedipine in TG myocytes was much slower than when both the L-type Ca2+ current and the Na+-Ca2+ exchange current function together. In TG myocytes, action potential amplitude and action potential duration at 50% repolarization were reduced, and action potential duration at 90% repolarization was increased, relative to WT myocytes. These data suggest that under these conditions, overexpression of the Na+-Ca2+ exchanger in TG myocytes accelerates the decline of [Ca2+]i during relaxation, indicating enhanced forward Na+-Ca2+ exchanger function. Increased Ca2+ influx also appears to occur, consistent with enhanced reverse function. These findings provide support for the physiological importance of both these modes of Na+-Ca2+ exchange.     

Local control of excitation-contraction coupling in rat heart cells

1. Cytosolic free calcium ion concentration ([Ca2+]i) and whole-cell L-type Ca2+ channel currents were measured during excitation-contraction (E-C) coupling in single voltage-clamped rat cardiac ventricular cells. The measurements were used to compute the total cellular efflux of calcium ions through sarcoplasmic reticulum (SR) Ca2+ release channels (FSR,rel) and the influx of Ca2+ via L-type Ca2+ channels (FICa). 2. FSR,rel was elicited by depolarizing voltage-clamp pulses 200 ms in duration to membrane potentials from -30 to +80 mV. Over this range, peak FSR,rel had a bell-shaped dependence on clamp pulse potential. In all cells, the 'gain' of the system, measured as the ratio, FSR,rel(max)/FICa(max), declined from about 16, at 0 mV, to much lower values as clamp pulse voltage was made progressively more positive. We named this phenomenon of change in gain as a function of membrane potential, 'variable gain'. At clamp pulse potentials in the range -30 to 0 mV, the gain differed from cell to cell, being constant at about 16 in some cells, but decreasing from high values (approximately 65) at -20 mV in others. 3. At clamp pulse potentials at which Ca2+ influx (FICa) was maintained, FSR,rel also had a small maintained component. When macroscopic Ca2+ influx was brief (1-2 ms, during 'tails' of FICa), FSR,rel rose rapidly to a peak after repolarization and then declined with a half-time of about 9 ms (typically). 4. The rising phase of [Ca2+]i transients could be interrupted by stopping Ca2+ influx rapidly (by voltage clamp). We therefore termed this phenomenon 'interrupted SR Ca2+ release'.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possible role of altered extracellular Ca2+ concentration ([Ca2+]o) in skeletal muscle fatigue was tested on isolated slow-twitch soleus and fast-twitch extensor digitorum longus muscles of the mouse. The following findings were made. 1) A change from the control solution (1.3 mM [Ca2+]o) to 10 mM [Ca2+]o, or to nominally Ca2+-free solutions, had little effect on tetanic force in nonfatigued muscle. 2) Almost complete restoration of tetanic force was induced by 10 mM [Ca2+]o in severely K+-depressed muscle (extracellular K+ concentration of 10-12 mM). This effect was attributed to a 5-mV reversal of the K+-induced depolarization and subsequent restoration of ability to generate action potentials (inferred by using the twitch force-stimulation strength relationship). 3) Tetanic force depressed by lowered extracellular Na+ concentration (40 mM) was further reduced with 10 mM [Ca2+]o. 4) Tetanic force loss at elevated extracellular K+ concentration (8 mM) and lowered extracellular Na+ concentration (100 mM) was partially reversed with 10 mM [Ca2+]o or markedly exacerbated with low [Ca2+]o. 5) Fatigue induced by using repeated tetani in soleus was attenuated at 10 mM [Ca2+]o (due to increased resting and evoked forces) and exacerbated at low [Ca2+]o. These combined results suggest, first, that raised [Ca2+]o protects against fatigue rather than inducing it and, second, that a considerable depletion of [Ca2+]o in the transverse tubules may contribute to fatigue.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg2+ (0.05-10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 microM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 microM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 microM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 microM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 microM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve activity but not intracellular calcium determines the time course of endocytosis at the frog neuromuscular junction

We used FM1-43 imaging and intracellular recordings of synaptic potentials to measure the time course of endocytosis in frog motor nerve terminals following tetanic nerve stimulation, and we used fura-2 imaging of intraterminal Ca2+ concentration to compare endocytic rate and [Ca2+]i. Following a 30 Hz tetanus, endocytosis declined exponentially with a time constant that depended on the duration of stimulation. The level of [Ca2+]i rose from a resting value of about 100 nM to more than 500 nM during 30 Hz stimulation, and rapidly declined to 200-250 nM after stimulation. [Ca2+]i returned to resting level with a time course that, like endocytosis, depended on the duration of tetanic stimulation. However, the rate of [Ca2+]i recovery was much slower than the rate of endocytosis, leading to the conclusion that endocytic rate is not determined solely by the instantaneous level of [Ca2+]i.     

MgADP promotes a catch-like state developed through force-calcium hysteresis in tonic smooth muscle

Tonic rabbit femoral artery and phasic rabbit ileum smooth muscles permeabilized with Triton X-100 were activated either by increasing [Ca2+] from pCa > 8.0 to pCa 6.0 (calcium-ascending protocol) or contracted at pCa 6.0 before lowering [Ca2+] (calcium-descending protocol). The effects of, respectively, high [MgATP]/low [MgADP] [10 mM MgATP + creatine phosphate (CP) + creatine kinase (CK)] or low [MgATP]/[MgADP] (2 mM MgATP, 0 CP, 0 CK) on the "force-[Ca]" relationships were determined. In femoral artery at low, but not at high, [MgATP]/[MgADP] the force and the ratio of stiffness/force at pCa 7.2 were significantly higher under the calcium-descending than calcium-ascending protocols (54% vs. 3% of Po, the force at pCa 6.0) (force hysteresis); the levels of regulatory myosin light chain (MLC20) phosphorylation (9 +/- 2% vs. 10 +/- 2%) and the velocities of unloaded shortening V0 (0.02 +/- 0.004 l/s with both protocols) were not significantly different. No significant force hysteresis was detected in rabbit ileum under either of these experimental conditions. [MgADP], measured in extracts of permeabilized femoral artery strips by two methods, was 130-140 microM during maintained force under the calcium-descending protocol. Exogenous CP (10 mM) applied during the descending protocol reduced endogenous [MgADP] to 46 +/- 10 microM and abolished force hysteresis: residual force at low [Ca2+] was 17 +/- 5% of maximal force. We conclude that the proportion of force-generating nonphosphorylated (AMdp) relative to phosphorylated cross-bridges is higher on the Ca2+-descending than on the Ca2+-ascending force curve in tonic smooth muscle, that this population of positively strained dephosphorylated cross-bridges has a high affinity for MgADP, and that the dephosphorylated AMdp . MgADP state makes a significant contribution to force maintenance at low levels of MLC20 phosphorylation.     

Effects of angiotensin II on inotropy and intracellular Ca2+ handling in normal and hypertrophied rat myocardium

The direct inotropic effect of angiotensin II on the myocardium is still controversial and little information exists as to its potential modification by heart disorders. Therefore, this study performed simultaneous measurements of isometric force and intracellular Ca2+ concentrations ([Ca2+]i) in left ventricular papillary muscles from sham-operated and aortic-banded rats at 10 weeks post-surgery. Angiotensin II (10(-6) M) induced a reduction of peak systolic [Ca2+]i (0.56 +/- 0.03 to 0.48 +/- 0.04 microM; P<0.05) and a parallel but insignificant diminution of developed tension (10.5 +/- 1.3 to 9.6 +/- 0.8 mN/mm2) in normal papillary muscles from sham-operated animals. Hypertrophied papillary muscles from aortic-banded rats demonstrated a significant decline in both peak systolic [Ca2+]i (0.51 +/- 0.02 to 0.44 +/- 0.01 microM; P<0.05) and developed tension (8.4 +/- 1.1 to 6.8 +/- 1.7 mN/mm2; P<0.05) after addition of angiotensin II. The time courses of the mechanical contraction and the intracellular Ca2+ signal were prolonged by angiotension II in both groups. Isoproterenol dose-dependently increased developed tension and peak systolic [Ca2+]i in papillary muscles from sham-operated rats. In contrast, the positive inotropic response to isoproterenol was markedly reduced in hypertrophied muscles despite a seemingly unimpaired increase in peak systolic [Ca2+]i. Pretreatment with angiotensin II (10(-6) M) resulted in a significant attenuation of the systolic [Ca2+]i response to isoproterenol stimulation in both normal and hypertrophied papillary muscles. Neither the bradykinin B2 antagonist icatibent (10(-6) M) nor the nitric oxide (NO) inhibitor L-NMMA (10(-6) M) abolished the depressant effects of angiotension II. Thus, ANG II induces a parallel decline of the mechanical performance and Ca2+ availability in rat myocardium. These effects are more distinct in hypertrophied than in normal muscle and become accentuated during beta-adrenergic stimulation. The underlying mechanism is not associated with the NO pathway but might involve a negative functional coupling between the angiotensin and beta-adrenergic-receptor complex.     

Intracellular free calcium dynamics in stretch-injured astrocytes

We have previously developed an in vitro model for traumatic brain injury that simulates a major component of in vivo trauma, that being tissue strain or stretch. We have validated our model by demonstrating that it produces many of the posttraumatic responses observed in vivo. Sustained elevation of the intracellular free calcium concentration ([Ca2+]i) has been hypothesized to be a primary biochemical mechanism inducing cell dysfunction after trauma. In the present report, we have examined this hypothesis in astrocytes using our in vitro injury model and fura-2 microphotometry. Our results indicate that astrocyte [Ca2+]i is rapidly elevated after stretch injury, the magnitude of which is proportional to the degree of injury. However, the injury-induced [Ca2+]i elevation is not sustained and returns to near-basal levels by 15 min postinjury and to basal levels between 3 and 24 h after injury. Although basal [Ca2+]i returns to normal after injury, we have identified persistent injury-induced alterations in calcium-mediated signal transduction pathways. We report here, for the first time, that traumatic stretch injury causes release of calcium from inositol trisphosphate-sensitive intracellular calcium stores and may uncouple the stores from participation in metabotropic glutamate receptor-mediated signal transduction events. We found that for a prolonged period after trauma astrocytes no longer respond to thapsigargin, glutamate, or the inositol trisphosphate-linked metabotropic glutamate receptor agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid with an elevation in [Ca2+]i. We hypothesize that changes in calcium-mediated signaling pathways, rather than an absolute elevation in [Ca2+]i, is responsible for some of the pathological consequences of traumatic brain injury.     

Spontaneous and repetitive calcium transients in C2C12 mouse myotubes during in vitro myogenesis

Fluorescence videomicroscopy was used to monitor changes in the cytosolic free Ca2+ concentration ([Ca2+]i) in the mouse muscle cell line C2Cl2 during in vitro myogenesis. Three different patterns of changes in [Ca2+]i were observed: (i) [Ca2+]i oscillations; (ii) faster Ca2+ events confined to subcellular regions (localized [Ca2+]i spikes) and (iii) [Ca2+]i spikes detectable in the entire myotube (global [Ca2+]i spikes). [Ca2+]i oscillations and localized [Ca2+]i spikes were detectable following the appearance of caffeine-sensitivity in differentiating C2Cl2 cells. Global [Ca2+]i spikes appeared later in the process of myogenesis in cells exhibiting coupling between voltage-operated Ca2+ channels and ryanodine receptors. In contrast to [Ca2+]i oscillations and localized [Ca2+]i spikes, the global events immediately stopped when cells were perfused either with a Ca2+-free solution, or a solution with TTX, TEA and verapamil. To explore further the mechanism of the global [Ca2+]i spikes, membrane currents and fluorescence signals were measured simultaneously. These experiments revealed that global [Ca2+]i spikes were correlated with an inward current. Moreover, while the depletion of the Ca2+ stores blocked [Ca2+]i oscillations and localized [Ca2+]i spikes, it only reduced the amplitude of global [Ca2+]i spikes. It is suggested that, during the earlier stages of the myogenesis, spontaneous and repetitive [Ca2+]i changes may be based on cytosolic oscillatory mechanisms. The coupling between voltage-operated Ca2+ channels and ryanodine receptors seems to be the prerequisite for the appearance of global [Ca2+]i spikes triggered by a membrane oscillatory mechanism, which characterizes the later phases of the myogenic process.     

Delta9-tetrahydrocannabinol activates [Ca2+]i increases partly sensitive to capacitative store refilling

Delta9-tetrahydrocannabinol induces [Ca2+]i increases in DDT1MF-2 smooth muscle cells. Both Ca2+ entry and release from intracellular Ca2+ stores were concentration dependently activated. The Ca2+ entry component contributed most to the increases in [Ca2+]i. Stimulation with delta9-tetrahydrocannabinol after functional downregulation of intracellular Ca2+ stores by longterm thapsigargin treatment, still induced a major Ca2+ entry and a minor Ca2+ release component. Thapsigargin sensitive influx and release were selectively inhibited by the cannabinoid CB1 receptor antagonist SR141716A. No effects on [Ca2+]i were obtained after stimulation with the CB2 receptor agonist palmitoylethanolamide. This study is the first demonstration of (1) Ca2+ release from thapsigargin sensitive intracellular stores and capacitative Ca2+ entry via CB1 receptor stimulation and of (2) an additional delta9-tetrahydrocannabinol induced thapsigargin insensitive component, mainly representing Ca2+ influx which is neither mediated by CB1 nor CB2 receptor stimulation.     

Steady state relation between cytoplasmic free Ca2+ concentration and force in intact frog skeletal muscle fibers

The steady state relation between cytoplasmic Ca2+ concentration ([Ca2+]i) and force was studied in intact skeletal muscle fibers of frogs. Intact twitch fibers were injected with the dextran-conjugated Ca2+ indicator, fura dextran, and the fluorescence signals of fura dextran were converted to [Ca2+]i using calibration parameters previously estimated in permeabilized muscle fibers (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123-1150). In the first series of experiments, [Ca2+]i and isometric force were simultaneously measured during high K+ depolarization. Slow changes in [Ca2+]i and force induced by 15-30 mM K+ appeared to be in equilibrium, as instantaneous [Ca2+]i versus force plot tracked the common path in the rising and relaxation phases of K+ contractures. In the second series of experiments, 2,5-di-tert-butylhydroquinone (TBQ), an inhibitor of the sarcoplasmic reticulum Ca2+ pump, was used to decrease the rate of decline of [Ca2+]i after tetanic stimulation. The decay time courses of both [Ca2+]i and force were dose-dependently slowed by TBQ up to 5 micro M; the instantaneous [Ca2+]i- force relations were nearly identical at >/=1 micro M TBQ, suggesting that the change in [Ca2+]i was slow enough to reach equilibrium with force. The [Ca2+]i-force data obtained from the two types of experiments were consistent with the Hill curve using a Hill coefficient of 3.2-3.9 and [Ca2+]i for half activation (Ca50) of 1.5-1.7 micro M. However, if fura dextran reacts with Ca2+ with a 2.5-fold greater Kd as previously estimated from the kinetic fitting (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123-1150), Ca50 would be 3.7-4.2 micro M. We also studied the [Ca2+]-force relation in skinned fibers under similar experimental conditions. The average Hill coefficient and Ca50 were estimated to be 3.3 and 1.8 microM, respectively. Although uncertainties remain about the precise levels of [Ca2+]i, we conclude that the steady state force is a 3rd to 4th power function of [Ca2+]i, and Ca50 is in the low micromolar range in intact frog muscle fibers, which is in reasonable agreement with results obtained from skinned fibers.     

A quantitative measurement of the dependence of short-term synaptic enhancement on presynaptic residual calcium

We simultaneously measured presynaptic free calcium ion concentration ([Ca2+]i) and synaptic strength at the crayfish claw opener neuromuscular junction (nmj) under a variety of experimental conditions. Our experiments were designed both to test the hypothesis that elevated [Ca2+]i is necessary and sufficient for the induction of a form of synaptic enhancement that persists for several seconds after tetanic stimulation--augmentation--and to determine the quantitative relationship between elevated [Ca2+]i and this enhancement. Action potential trains increased [Ca2+]i and enhanced transmission. During the decay phase of synaptic enhancement known as augmentation (time constant of decay approximately 7 sec at 20 degrees C with < 200 microM fura-2 in terminals), [Ca2+]i was elevated 700 nM or less above rest and an essentially linear relationship between [Ca2+]i and enhancement was observed. Introduction of exogenous Ca2+ buffers into the presynaptic terminal slowed the buildup and recovery kinetics of both [Ca2+]i and the component of synaptic enhancement corresponding to augmentation. The slope of the relationship relating delta [Ca2+]i to augmentation was not changed. The time course of augmentation and recovery of [Ca2+]i remained correlated as the temperature of the preparation was changed from about 10 degrees C to 20 degrees C, but the quantitative relationship of enhancement to [Ca2+]i was increased more than two- to threefold. During moderate frequency trains of action potentials, a slowly developing component of the total synaptic enhancement was approximately linearly related to residual [Ca2+]i measured with fura-2. The quantitative relationship between [Ca2+]i and this component of synaptic enhancement during trains was the same as that during synaptic augmentation after trains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ryanodine binding sites measured in small skeletal muscle biopsies

A method allowing measurement of the concentration of [3H]ryanodine binding sites in small skeletal muscle specimens (> 10-20 mg) was developed. A membrane fraction containing 87% of the [3H]ryanodine binding sites of the tissue and exhibiting one single KD of 18-27 nmol l-1 in rat and 8 nmol l-1 in human muscles (p < 0.05) was obtained. Maximum binding to rat EDL and soleus muscles equalled 59.1 and 16.2 pmol g-1 wet wt, whereas in human gluteus muscles binding was 12.3 pmol g-1 wet wt. The [3H]ryanodine binding showed a dependency on Mg2+ and pH similar to previously published results. As measured by Ca2+ selective mini-electrodes, the [Ca2+] causing 50% of maximum [3H]ryanodine binding (K0.5) was 200-400 nmol l-1 for different muscles. [Ca2+] higher than 1 mmol l-1 caused strong inhibition of the [3H]ryanodine binding, and both high and low [Ca2+] caused rapid dissociation of the complex. At ionic strength lower than 100 mmol l-1, more than 50% of the [3H]ryanodine was bound to particles with size less than 1.2 microns which were not retained by GF/C filters. Thus, we have obtained an almost complete quantitative recovery of functional RyRs from small muscle specimens exhibiting high affinity for Ca2+, which stimulated ligand binding.     

Sarcoplasmic reticulum-sarcolemma interactions and vascular smooth muscle tone

A characteristic of vascular smooth muscle cell morphology is a close apposition of its peripheral sarcoplasmic reticulum (SR) with the sarcolomma; this arrangement gives rise to important functional interactions whereby the peripheral SR regulates Ca2+ influx and vascular tone. We review here the key evidence supporting the following aspects of SR-sarcolemma interactions while establishing a conceptual framework encompassing (i) the SR ultrastructure and functions, (ii) the integration of the sarcolemmal Na+-Ca2+ exchanger and the peripheral SR in the mediation of a bidirectional Ca2+ exchange between the peripheral SR and the extracellular space, (iii) the existence of a higher myoplasmic free Ca2+ concentration [Ca2+]myo in the subsarcolemmal space formed between the sarcolemma and the peripheral SR relative to the [Ca2+]myo of the inner myoplasm in the resting smooth muscle cell, (iv) the division of the subsarcolemmal space into functional microdomains, (v) the existence of spontaneous localized bursts of Ca2+ release from the peripheral SR (Ca2+ sparks) towards the sarcolemma, (vi) the physiological triggering of nonlocalized Ca2+ release from the peripheral SR by Ca2+ influx (Ca2+-induced Ca2+ release), and (vii) capacitative Ca2+ entry in vascular smooth muscle. We present an overview of the physiological and pathological implications of these interactions.     

New advances in sex preselection

The effects of peroxynitrite (ONOO-) on cultured cardiac myocytes were examined by simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and contractile function. On exposure to 0.2 mM ONOO-, [Ca2+]i increased to beyond the systolic level within 5 min with a concomitant decrease in spontaneous contraction of myocytes followed by complete arrest. Addition of a L-type Ca2+ channel blocker or removal of extracellular Ca2+ prevented the ONOO(-)-induced increase in [Ca2+]i, indicating that the increase in [Ca2+]i was caused by the enhanced influx of Ca2+ through the plasma membrane and not by the enhanced release from sarcoplasmic reticulum (SR). Plasma membrane fluidity and concentration of the thiobarbiturate acid-reactive substance (TBARS) in the cells remained unchanged by the ONOO- treatment. The complete cessation of contraction of myocytes persisted even under the massive increase in [Ca2+]i, which was induced by an additional saponin (5 microM) treatment. In conclusion, ONOO- increases [Ca2+]i in myocytes through disturbance of Ca2+ transport systems in the plasma membrane and impairs contractile protein.     

Fluorescence probe study of the lumenal Ca2+ of the sarcoplasmic reticulum vesicles during Ca2+ uptake and Ca2+ release

A limited amount of information is available about the lumenal Ca2+ kinetics of the sarcoplasmic reticulum (SR). Incubation of mag-fura-2AM permitted to incorporate a sufficient amount of the probe into the SR vesicles, as determined by Mn2+ quenching. Rapid changes in the lumenal [Ca2+] ([Ca2+]lum) during Ca2+ uptake and release could be monitored by following the signal derived from the lumenal probe while clamping the extra-vesicular Ca2+ ([Ca2+]ex) at various desired levels with a BAPTA/Ca buffer. Changes in the [Ca2+]lum during uptake and release show the characteristics intrinsic to the SR Ca2+ pump (the [Ca2+]ex-dependence of the activation and inhibition by thapsigargin) and the Ca2+ release channel (blocking by ruthenium red), respectively. A new feature revealed by the [Ca2+]lum measurement is that during the uptake reaction the free [Ca2+]lum showed a significant oscillation. Several pieces of evidence suggest that this is due to some interactions between the Ca2+ pump and lumenal proteins.     

Selectivity of felodipine for depolarized ventricular myocytes: a study at the single-cell level

We studied the effects of felodipine (a second-generation dihydropyridine Ca2+ channel blocker) on excitation-contraction coupling (E-C coupling) in single isolated guinea-pig ventricular myocytes, using the whole-cell perforated patch-clamp technique or the Ca indicator, indo-1. Felodipine inhibited both L-type Ca2+ channel currents (ICa) and cell contractions in a concentration-dependent manner (10 pM to 100 nM) when we used a holding potential of -80 mV or -40 mV. The potency of felodipine was sharply dependent on a holding potential. Namely, use of a more depolarized holding potential markedly increased the potency of felodipine for inhibition of ICa and cell contraction. Next we current-clamped cells and obtained the resting membrane potential of -82 +/- 8 mV. When cells were current-injected at 0.1 Hz, exposure to 10 nM felodipine slightly but significantly diminished the amplitude of cell contractions (7.2 +/- 1.6 to 6.7 +/- 1.7 microns, P < 0.05) within 10 min. When cells were field stimulated, exposure of cells to 10 nM felodipine also slightly diminished the amplitude of cell shortening (5.1 +/- 2.0 to 4.6 +/- 1.9 microns, P < 0.05) and [Ca2+]i transients. We observed clear voltage-dependent blockade of E-C coupling by felodipine in ventricular myocytes. Thus, therapeutic concentrations (1-10 nM) of felodipine could inhibit E-C coupling in depolarized ventricular myocytes, which might simulate an ischemic or failing heart.