Time-resolved synchrotron X-ray diffraction studies of a single frog skeletal muscle fiber. Time courses of intensity changes of the equatorial reflections and intracellular Ca2+ transients.

Time-resolved X-ray equatorial diffraction studies on a single frog skeletal muscle fiber were performed with a 10 ms time resolution using synchrotron radiation in order to compare the time courses of the molecular changes of contractile proteins and the intracellular Ca2+ transient during an isometric twitch contraction at 2.7 degrees C. Measurements of the Ca2+ transient using aequorin as an intracellular Ca2+ indicator were conducted separately just before and after the X-ray experiments under very similar experimental conditions. The results, which showed a similar time course of tension to that observed in the X-ray experiment, were compared with the aequorin light signal, tension and the intensity changes of the 1,0 and 1,1 equatorial reflections. No appreciable change in both reflection spacings indicated that the effect of internal shortening of the muscle was minimized during contraction. The intensity change of the equatorial reflections generally occurred after the aequorin light signal. In the rising phase, the time course of increase in the 1,1 intensity paralleled that of the rise of the light signal and the intensity peak occurred 20-30 ms after the peak of the light signal. The decrease in the 1,0 intensity showed a time course similar to that of tension and the intensity minimum roughly coincided with the tension peak, coming at 80-90 ms and about 60 ms after the peaks of the light signal and the 1,1 intensity change, respectively. In the relaxation phase, the 1,1 intensity seemed to fall rapidly just before the tension peak and then returned to the original level in parallel with the decay of tension. The 1,0 intensity returned more slowly than the tension relaxation. Thus, the change of the 1,1 intensity was faster than that of the 1,0 intensity in both the rising and relaxation phases. When the measured aequorin light signal was corrected for the kinetic delay of the aequorin reaction with a first-order rate constant of either 50 or 17 s-1, the peak of the corrected light signal preceded that of the measured one by approx. 30 ms. Thus, the peak of the Ca2+ transient appeared earlier than the peaks of the 1,1 and 1,0 intensity changes by 50-60 and 110-120 ms, respectively. The time lag between the extent of structural change and the Ca2+ transient is discussed in relation to the double-headed attachment of a cross-bridge to actin.     

Kinetic investigations in single muscle fibres using luminescent and fluorescent Ca2+ probes

The Ca2+-sensitive photoprotein aequorin and the Ca2+-dependent fluorescent indicators quin 2 and TnCDANZ have been used to investigate contractile processes in single crustacean muscle fibres. The investigations with quin 2 indicate that the free Ca2+ rises to a maximum value before peak force as with aequorin light (approximately 200 msec delay at 12 degrees C) and subsequently decays more slowly, unlike the majority of the aequorin signal, although an aequorin 'tail' signal remains. The resting quin 2 fluorescence from the cell suggests an upper limit of 348 nM for the resting calcium concentration. Experiments with TnCDANZ indicate that this fluorescence response rises rapidly but then the rate of rise slows to reach a maximum value at a time when peak force is achieved and then the fluorescence signal decays more slowly than force. The latter result implies that Ca2+ is attached to the Ca2+-specific sites of TnC when externally recorded force is small.     

Contribution of sarcolemmal sodium-calcium exchange and intracellular calcium release to force development in isolated canine ventricular muscle

The aim of this work was to determine the relationship between peak twitch amplitude and sarcoplasmic reticulum (SR) Ca2+ content during changes of stimulation frequency in isolated canine ventricle, and to estimate the extent to which these changes were dependent upon sarcolemmal Na(+)-Ca2+ exchange. In physiological [Na+]o, increased stimulation frequency in the 0.2-2-Hz range resulted in a positive inotropic effect characterized by an increase in peak twitch amplitude and a decrease in the duration of contraction, measured as changes in isometric force development or unloaded cell shortening in intact muscle and isolated single cells, respectively. Action potentials recorded from single cells indicated that the inotropic effect was associated with a progressive decrease of action potential duration and a marked reduction in average time spent by the cell near the resting potential during the stimulus train. The frequency-dependent increase of peak twitch force was correlated with an increase of Ca2+ uptake into and release from the SR. This was estimated indirectly using the phasic contractile response to rapid (less than 1 s) lowering of perfusate temperature from 37 degrees C to 0-2 degrees C and changes of twitch amplitude resulting from perturbations in the pattern of electrical stimulation. Lowering [Na+]o from 140 to 70 mM resulted in an increase of contractile strength, which was accompanied by a similar increase of apparent SR Ca2+ content, both of which could be abolished by exposure to ryanodine (1 x 10(-8) M), caffeine (3 x 10(-3) M), or nifedipine (2 x 10(-6) M). Increased stimulation frequency in 70 mM [Na+]o resulted in a negative contractile staircase, characterized by a graded decrease of peak isometric force development or unloaded cell shortening. SR Ca2+ content estimated under identical conditions remained unaltered. Rate constants derived from mechanical restitution studies implied that the depressant effect of increased stimulation frequency in 70 mM [Na+]o was not a consequence of a decreased rate of refilling of a releasable pool of Ca2+ within the cell. These results demonstrate that frequency-dependent changes of contractile strength and intracellular Ca2+ loading in 140 mM [Na+]o require the presence of a functional sarcolemmal Na(+)-Ca2+ exchange process. The possibility that the negative staircase in 70 mM [Na+]o is related to inhibition of Ca(2+)-induced release of Ca2+ from the SR by various cellular mechanisms is discussed.     

Radial spread of aequorin Ca2+ signal in single frog skeletal muscle fibers

The Ca²⁺-sensitive photoprotein aequorin was injected into single frog skeletal muscle fibers, and the intracellular aequorin light intensity during muscle activation with different maneuvers was mapped with digital imaging microscopy. During 50 Hz electrical activation (tetanus), the aequorin light intensity from different locations in the muscle fiber rose with very similar time course. Caffeine (10 mM) application, on the other hand, caused aequorin light signals to show significantly different time courses, with an earlier increase in Ca²⁺ concentration near the surface of the fiber than near the core. The non-uniform rise of intracellular Ca²⁺ concentration with caffeine treatment is consistent with the slow inward diffusion of caffeine and subsequent Ca²⁺ release from sarcoplasmic reticulum.     

心肌α1—肾上腺素受体激动对豚鼠心室乳头肌的影响

The alpha-adrenoceptor agonist phenylephrine (5.0 x 10(-6) mol/L) was used to stimulate myocardial alpha-adrenoceptors of the guinea-pig ventricular papillary muscle, and changes of transmembrane action potential and contractile force of the muscle were observed. The alpha 1-adrenoceptor blocker prazosin (5.0 x 10(-7) mol/L) and the alpha 2-adrenoceptor blocker yohimbine (5.0 x 10(-7) mol/L) were used to determine which subtype of alpha-adrenoceptor is responsible for the effects. The beta-adrenoceptor blocker propranolol (1.0 x 10(-6) mol/L) was used throughout the experiment. The results show that the myocardial alpha 1-adrenoceptor stimulation (1) increases the contractile force of the guinea-pig ventricular papillary muscle, (2) prolongs the time to peak contractile force while the duration of relaxation is not altered, (3) prolongs the fast response action potential duration, and (4) increases the maximal rate of depolarization during the phase 0 of the slow response action potential. It is suggested that the electrophysiological and positive inotropic effects of myocardial alpha 1-adrenoceptor stimulation might be due to the activation of the slow inward current and an increase in Ca2+ influx.     

Differential effects of tetracaine on charge movements and Ca2+ signals in frog skeletal muscle

The effects of tetracaine on charge movements and on antipyrylazo III signals monitoring intracellular delta [Ca2+] were compared in cut frog semitendinosus muscle fibers in a single vaseline gap-voltage clamp. Low tetracaine concentrations (25-40 microM) markedly reduced delta [Ca2+] signals and shifted the rheobase. However, they neither influenced charge movement nor that peak delta [Ca2+] value associated with the contractile threshold. Higher tetracaine concentrations (100-200 microM) partly inhibited charge movements in cut fibers. They separated a steeply voltage-sensitive charge, some of whose features resembled 'q gamma' reported in intact fibers, and whose movement preceded delta [Ca2+] signals at threshold. These findings: (a) directly confirm an earlier suggestion that tetracaine acts on steps in excitation-contraction coupling rather than myofilament activation; (b) show that tetracaine at low concentrations can directly interfere with sarcoplasmic reticular calcium release without modifying charge movement; (c) show that the tetracaine-sensitive charge, first found in intact fibers, also exists in cut fibers; and (d) make it unlikely that tetracaine-sensitive charge transfer is a consequence of Ca2+ release as suggested on earlier occasions.     

The kinetics relating calcium and force in skeletal muscle.

The kinetics relating Ca2+ transients and muscle force were examined using data obtained with the photoprotein aequorin in skeletal muscles of the rat, barnacle, and frog. These data were fitted by various models using nonlinear methods for minimizing the least mean square errors. Models in which Ca2+ binding to troponin was rate limiting for force production did not produce good agreement with the observed data, except for a small twitch of the barnacle muscle. Models in which cross-bridge kinetics were rate limiting also did not produce good agreement with the observed data, unless the detachment rate constant was allowed to increase sharply on the falling phase of tension production. Increasing the number of cross-bridge states did not dramatically improve the agreement between predicted and observed force. We conclude that the dynamic relationship between Ca2+ transients and force production in intact muscle fibers under physiological conditions can be approximated by a model in which (a) two Ca2+ ions bind rapidly to each troponin molecule, (b) force production is limited by the rate of formation of tightly bound cross-bridges, and (c) the rate of cross-bridge detachment increases rapidly once tension begins to decline and free Ca2+ levels have fallen to low values after the last stimulus. Such a model can account not only for the pattern of force production during a twitch and tetanus, but also the complex, nonlinear pattern of summation which is observed during an unfused tetanus at intermediate rates of stimulation.     

Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3.

Fluo-3 is an unusual tetracarboxylate Ca2+ indicator. For recent lots supplied by Molecular Probes Inc. (Eugene, OR), FMAX, the fluorescence intensity of the indicator in its Ca(2+)-bound form, is approximately 200 times that of FMIN, the fluorescence intensity of the indicator in its Ca(2+)-free form. (For earlier lots, impurities may account for the smaller reported values of FMAX/FMIN, 36-40). We have injected fluo-3 from a high-purity lot into intact single fibers from frog muscle and measured the indicator's absorbance and fluorescence signals at rest (A and F, respectively) and changes in absorbance and fluorescence following action potential stimulation (delta A and delta F signals substantially lagged behind that of the myoplasmic free Ca2+ transient. Our analysis of fluo-3's signals from myoplasm therefore focused on information about the level of resting myoplasmic free [Ca2+] ([Ca2+]r). From A, delta A, and in vitro estimates of fluo-3's molar extinction coefficients, the change in the fraction of fluo-3 in the Ca(2+)-bound form during activity (delta f) was estimated. From delta f, delta F, and F, the fraction of the indicator in the Ca(2+)-bound form in the resting fiber (fr) was estimated by fr = (delta f x F/delta F) + (1-FMAX/FMIN)-1. Since FMAX/FMIN is large, the contribution of the second term to the estimate of fr is small. At 16 degrees C, the mean value (mean +/- S.E.) of fr was 0.086 +/- 0.004 (N = 15). From two estimates of the apparent dissociation constant of fluo-3 for Ca2+ in the myoplasm, 1.09 and 2.57 microM, the average value of [Ca2+]r is calculated to be 0.10 and 0.24 microM, respectively. The smaller of these estimates lies near the upper end of the range of values for [Ca2+]r in frog fibers (0.02-0.12 microM) estimated by others with aequorin and Ca(2+)-selective electrodes. The larger of the estimates lies within the range of values (0.2-0.3 microM) previously estimated in this laboratory with fura red. We conclude that [Ca2+]r in frog fibers is at least 0.1 microM and possibly as large as 0.3 microM.     

Analysis of the Ca2+ response of mycelial fungi to external effects by the recombinant aequorin method

Using the mutant strain Aspergillus awamori 66A producing a recombinant Ca2+-dependent photosensitive protein aequorin, the dynamics of Ca2+ was studied for the first time in the cytosol of the micromycetes exposed to stressful factors, such as an increase in extracellular Ca2+ to 50 mM, hypoosmotic shock, and mechanical shock. Cell response to stress proved to involve an increase in the Ca2+ concentration in the cytosol, which was determined from the amplitude of aequorin luminescence and the time of the amplitude enhancement and relaxation. The level of Ca2+ response depended on the physiological stimulus. Inhibitory analysis with various agents that block Ca2+ channels and with agonists that specifically enhance the activity of the channels suggested that (1) the level of Ca2+ in the cytosol of micromycetes increases in response to stress because of the ion influx from both the growth medium and intracellular reservoirs and (2) the potential-dependent transport systems play the major role in the Ca2+ influx into the cytosol of the micromycete cells.     

Properties of chloride-stimulated 45Ca flux in skinned muscle fibers

Isometric force and 45Ca loss from fiber to bath were measured simultaneously in skinned fibers from frog muscle at 19 degrees C. In unstimulated fibers, 45Ca efflux from the sarcoplasmic reticulum (SR) was very slow, with little or no dependence on EGTA (0.1-5 mM) or Mg++ (20 micrometer-1.3 mM). Stimulation by high [Cl] at 0.11 mM Mg++ caused rapid force transients (duration approximately 10 s) and 45Ca release. This response was followed for 55 s, with 5 mM EGTA added to chelate myofilament space (MFS) Ca either (a) after relaxation, (b) near the peak of the force spike, or (c) before or with the stimulus. When EGTA was present during Cl application, stimulation of 45Ca release was undetectable. Analysis of the time-course of tracer loss during the three protocols showed that when EGTA was absent, 16% of the fiber tracer was released from the SR within approximately 3 s, and 70% of the tracer still in the MFS near the peak of the force spike was subsequently reaccumulated. The results suggest that (a) the Cl response is highly Ca-dependent; (b) stimulation increases 45Ca efflux from the SR at least 100-200-fold; and (c) the rate of reaccumulation is much slower than the influx predicted from published data on resting fibers, raising the possibility that depolarization inhibits active Ca transport by the SR.     

Effects of palmatine on isometric force and intracellular calcium levels of arterial smooth muscle

The effects of palmatine on isometric force and intracellular free calcium levels ([Ca2+]i) were determined in isolated rat arterial strips. Palmatine dose-dependently relaxed the contractile responses stimulated by phenylephrine (PE) in aortic strips. In contrast, it only partially relaxed aortic strips contracted by 51 mM KCl. Pretreatment with palmatine shifted the dose-response curves of PE both rightwards and downwards in a dose-dependent manner. When Ca2+-free solution and re-addition of Ca2+ were applied to assess PE-induced phasic and tonic contractions, palmatine was found to be effective in inhibiting both contractions. The effects of palmatine on intracellular calcium levels were measured with the bioluminescent calcium indicator aequorin in rat tail artery strips. Palmatine caused a concomitant, dose-dependent decrease in PE-activated isometric force and [Ca2+]i, resulting in small changes in the [Ca2+]i-force relationship. These results suggest that vasodilatory effect of palmatine was mediated by reducing [Ca2+]i as well as affecting [Ca2+]i sensitivity of the contractile apparatus. Palmatine-induced [Ca2+]i decreases appeared to involve decreases in both Ca2+ release from intracellular stores and Ca2+ influx through calcium channels.     

Simultaneous measurement of Ca2+ in muscle with Ca electrodes and aequorin. Diffusible cytoplasmic constituent reduces Ca(2+)-independent luminescence of aequorin

Estimates of cytoplasmic Ca2+ concentration ([Ca2+]i) were made essentially simultaneously in the same intact frog skeletal muscle fibers with aequorin and with Ca-selective microelectrodes. In healthy fibers under truly resting conditions [Ca2+]i was too low to be measured reliably with either technique. The calibration curves for both indicators were essentially flat in this range of [Ca2+], and the aequorin light signal was uniformly below the level to be expected in the total absence of Ca2+. When [Ca2+]i had been raised to a stable level below the threshold for contracture by increasing [K+]o to 12.5 mM, [Ca2+]i was 38 nM according to aequorin and 59 nM according to the Ca-selective microelectrodes. These values are not significantly different. Our estimates of [Ca2+]i are lower than most others obtained with microelectrodes, probably because the presence of aequorin in the cells allowed us to detect damaging microelectrode impalements that otherwise we would have had no reason to reject. The observation that the light emission from aequorin-injected fibers in normal Ringer solution was below the level expected from the Ca(2+)-independent luminescence of aequorin in vitro was investigated further, with the conclusion that the myoplasm contains a diffusible macromolecule (between 10 and 30 kD) that interacts with aequorin to reduce light emission in the absence of Ca2+.     

Calcium in excitation--contraction coupling of frog skeletal muscle

A principal step in the process leading to muscle contraction is the intracellular release of Ca2+. We have detected and compared some physical and chemical events that reflect Ca2+ release in contracting frog skeletal muscle cells, described the effects of some agents that are believed to alter intracellular Ca2+ release during contraction, and speculated about the role of Ca2+ release in influencing some of the mechanical properties of frog muscle. The specific physical features recorded were changes in striation spacing, myofibrillar orientation, and force development. The chemical feature was the relative change in intracellular [Ca2+] recorded as light emission from cells microinjected with the Ca2+-sensitive protein aequorin. The presence or absence of a correlation among these variables has been used (i) to evaluate the action of some agents thought to change intracellular Ca2+ release in excitation--contraction (E--C) coupling, (ii) to further substantiate the effects of cell length on Ca2+ release, and (iii) to examine some details of models for E--C coupling. The results showed that potentiating agents enhance and prolong intracellular Ca2+ release without changing the rate of Ca2+ removal during E--C coupling. This extra Ca2+ does not produce the same effect on contractions at all lengths. Contractility is inversely related to cell length, and Ca2+-induced activation is normally less than maximum not only at short lengths but also at optimal striation spacings.     

Effects of ryanodine on intracellular Ca2+ transients in mammalian cardiac muscle

We observed the effects of ryanodine on the aequorin luminescence, membrane potential, and contraction of canine cardiac Purkinje fibers and ferret ventricular muscle. In canine Purkinje fibers, ryanodine (10 nM to 1 microM) abolished the spontaneous spatiotemporal fluctuations in [Ca2+] that occur as a result of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR) during exposure to low-Na+ solutions. Ryanodine strongly reduced the twitch and both components of the intracellular aequorin luminescence signal (L1 and L2), which normally accompanies contraction. The small luminescence signals that remained in ryanodine could be abolished by a Ca2+ channel blocker (nitrendipine, 10 microM). The plateau phase of the action potential was reduced by nitrendipine in the presence of ryanodine, which suggests that Ca2+ current was not blocked by ryanodine. In ferret ventricular tissue, ryanodine (1 microM) prolonged the action potential and reduced the peak amplitudes of both the aequorin transient and the twitch, while greatly prolonging the time-to-peak of both signals. Increases in extracellular [Ca2+] restored the peak amplitudes of the twitch and the aequorin luminescence, but did not restore the normal time-to-peak. The results show that in both tissues, the negative inotropic effect of ryanodine is due to the reduction of the intracellular [Ca2+] transient. Inasmuch as neither Ca2+ entry via surface membrane Ca2+ channels nor Na+-Ca2+ exchange appears to be blocked by ryanodine, the most probable cause of reduction of the [Ca2+] transient is an inhibition of Ca2+ release by the SR.     

Subcellular analysis of Ca2+ homeostasis in primary cultures of skeletal muscle myotubes.

Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca2+ concentration in different subcellular compartments of differentiated skeletal muscle myotubes. For the cytosol, mitochondria, and nucleus, the previously described chimeric aequorins were utilized; for the sarcoplasmic reticulum (SR), a new chimera (srAEQ) was developed by fusing an aequorin mutant with low Ca2+ affinity to the resident protein calsequestrin. By using an appropriate transfection procedure, the expression of the recombinant proteins was restricted, within the culture, to the differentiated myotubes, and the correct sorting of the various chimeras was verified with immunocytochemical techniques. Single-cell analysis of cytosolic Ca2+ concentration ([Ca2+]c) with fura-2 showed that the myotubes responded, as predicted, to stimuli known to be characteristic of skeletal muscle fibers, i.e., KCl-induced depolarization, caffeine, and carbamylcholine. Using these stimuli in cultures transfected with the various aequorin chimeras, we show that: 1) the nucleoplasmic Ca2+ concentration ([Ca2+]n) closely mimics the [Ca2+]c, at rest and after stimulation, indicating a rapid equilibration of the two compartments also in this cell type; 2) on the contrary, mitochondria amplify 4-6-fold the [Ca2+]c increases; and 3) the lumenal concentration of Ca2+ within the SR ([Ca2+]sr) is much higher than in the other compartments (> 100 microM), too high to be accurately measured also with the aequorin mutant with low Ca2+ affinity. An indirect estimate of the resting value (approximately 1-2 mM) was obtained using Sr2+, a surrogate of Ca2+ which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr2+, the kinetics and amplitudes of the changes in [cation2+]sr evoked by the various stimuli could also be directly analyzed.     

Characterization of the effects of Mg2+ on Ca2+- and Sr2+-activated tension generation of skinned rat cardiac fibers

Submaximum and maximum forces of the cardiac muscle contractile apparatus, activated by Ca2+ or Sr2+, were determined as a function of Mg2+ concentration. Apical left ventricular tissue from Sprague-Dawley rats was broken by homogenization into small bundles of fibers with disrupted sarcolemmas (skinned). Tension generation was activated by and graded according to the concentration of Ca2+ or Sr2+ in solutions bathing the skinned fibers and measured with a photodiode force transducer. Steady-state tensions for various levels of activation at each of four concentrations of Mg2+ (5 x 10(-5), 1 x 10(-3), 5 x 10(-3), and 10 x 10(-3) M) in the bathing solutions were analyzed. Other bathing solution constituents and parameters mimicked significant normal intracellular conditions while providing adequate buffering of [H+], [Ca2+], and [MgATP2-] (magnesium adenosine triphosphate). To assess changes in sensitivity of the mechanical system to activation by Ca2+ (or Sr2+), each submaximum tension was expressed as a percentage of the given fiber bundle's maximum force generated at saturating [Ca2+] (or [Sr2+]) at the same [Mg2+]. When plotted as saturation curves these data demonstrate that increasing [Mg2+] depresses Ca2+ sensitivity of the force-generating mechanism. The Ca2+ and Sr2+ sensitivity of the cardiac force-generating apparatus is similar at every [Mg2+], indicating that the magnitude of Mg2+ effect is similar for both types of activation. However, absolute maximum tensions at saturating activating cation concentration increased as [Mg2+] increased; the effect of Mg2+ on maximum force was proportionately the same for Ca2+ and Sr2+ activation. But because saturating [Ca2+] always resulted in a lower maximum force than saturating [Sr2+], this site of Ca2+-Mg2+ interaction appears distinct from the one influencing Ca2+ sensitivity.     

Ionized calcium concentrations in squid axons

Values for ionized [Ca] in squid axons were obtained by measuring the light emission from a 0.1-mul drop of aequorin confined to a plastic dialysis tube of 140-mum diameter located axially. Ionized Ca had a mean value of 20 x 10(-9) M as judged by the subsequent introduction of CaEGTA/EGTA buffer (ratio ca. 0.1) into the axoplasm, and light measurement on a second aequorin drop. Ionized Ca in axoplasma was also measured by introducing arsenazo dye into an axon by injection and measuring the Ca complex of such a dye by multichannel spectrophotometry. Values so obtained were ca. 50 x 10(-9) M as calibrated against CaEGTA/EGTA buffer mixtures. Wth a freshly isolated axon in 10 mM Ca seawater, the aequorin glow invariably increased with time; a seawater [Ca] of 2-3 mM allowed a steady state with respect to [Ca]. Replacement of Na+ in seawater with choline led to a large increase in light emission from aequorin. Li seawater partially reversed this change and the reintroduction of Na+ brought light levels back to their initial value. Stimulation at 60/s for 2-5 min produced an increase in aequorin glow about 0.1% of that represented by the known Ca influx, suggesting operationally the presence of substantial Ca buffering. Treatment of an axon with CN produced a very large increase in aequorin glow and in Ca arsenazo formation only if the external seawater contained Ca.     

The characterization of differential calcium signalling in tobacco guard cells

Two novel approaches for the study of Ca2+-mediated signal transduction in stomatal guard cells are described. Stimulus-induced changes in guard-cell cytosolic Ca2+ ([Ca2+]cyt) were monitored using viable stomata in epidermal strips of a transgenic line of Nicotiana plumbaginifolia expressing aequorin (the proteinous luminescent reporter of Ca2+) and in a new transgenic line in which aequorin expression was targeted specifically to the guard cells. The results indicated that abscisic acid (ABA)-induced stomatal closure was accompanied by increases in [Ca2+]cyt in epidermal strips. In addition to ABA, mechanical and low-temperature signals directly affected stomatal behaviour, promoting rapid closure. Elevations of guard-cell [Ca2+]cyt play a key role in the transduction of all three stimuli. However, there were striking differences in the magnitude and kinetics of the three responses. Studies using Ca2+ channel blockers and the Ca2+ chelator EGTA further suggested that mechanical and ABA signals primarily mobilize Ca2+ from intracellular store(s), whereas the influx of extracellular Ca2+ is a key component in the transduction of low-temperature signals. These results illustrate an aspect of Ca2+ signalling whereby the specificity of the response is encoded by different spatial or kinetic Ca2+ elevations.     

Fluorescence changes from single striated muscle fibres injected with labelled troponin C (TnCDANZ)

A fluorescently labelled derivative of the calcium binding subunit of troponin, TnC, has been injected into isolated striated muscle fibres from the barnacle Balanus nubilus. The Ca2+ affinity of isolated TnC is close to that of intact troponin when located in the thin filament. Excitation of the TnCDANZ within the muscle cell (325nm) revealed a marked fluorescence at 510 nm and was similar to that observed in vitro, which was absent at 400 or 600 nm after subtraction of the fibre autofluorescence. High Ca2+ salines increased the fluorescence at 510 nm by roughly 2 times. Single voltage clamp pulses produced a rapid rise in fluorescence at 510 nm after allowing for any non-specific changes at 400 nm, and this signal preceded force development by approx. 55 ms at 22 degrees C. It reached a maximum at the same time as force and subsequently decayed more slowly. The fluorescence signal increased in magnitude with increase in stimulus intensity. These results suggest that Ca2+ attaches rapidly to the contractile filament, but is lost relatively slowly and imply a slow decay of the activation process.     

The effect of calcium and Ca antagonists upon excitation-contraction coupling

The effect of a Ca2+-free tetraethylammonium sulfate solution on force development in short skeletal muscle fibres of the frog was investigated under voltage clamp control. Maximum force could still be reached under this condition. The removal of external Ca2+, however, caused an acceleration of force inactivation leading to a shift of the steady-state potential dependence of force inactivation to more negative potentials. With reference to the "modulated-receptor hypothesis" this result was explained by assuming a potential-dependent binding of Ca2+ to a force-controlling system in the T-tubular membrane, with a low affinity in the depolarized-inactivated state. A dissociation of Ca2+ is assumed to turn the system into a secondary inactivated state (paralysis) from which it only slowly recovers after repolarization. Ca antagonists like D600 and diltiazem accelerated the shift into paralysis, probably by an allosteric displacement of Ca2+ from its binding site. The application of 1-2 microM of the Ca antagonist nifedipine blocked the inward Ca2+ current and caused a prolongation of the transient force development following a depolarization. A similar retardation of force inactivation and a threshold shift to more negative potentials occurred when the Ca2+ chelator ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) was injected into the fibre and when in Ca2+-free solutions sodium ions entered the cell through Ca2+ channels.