HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (37) Citing Articles via Google Scholar Google Scholar Articles by Baylor, S. M. Articles by Hollingworth, S. Search for Related Content PubMed PubMed Citation Articles by Baylor, S. M. Articles by Hollingworth, S.

Measurement and Interpretation of Cytoplasmic [Ca²⁺] Signals From Calcium-Indicator Dyes

Stephen M. Baylor and Stephen Hollingworth

S. M. Baylor and S. Hollingworth are in the Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085.

	Abstract

 
Ca²⁺-indicator dyes are widely used in biology yet difficult to characterize inside cells. Studies in skeletal muscle fibers provide important information about indicator behavior and about Ca²⁺ signaling within the cytoplasm.

	Introduction

Top Introduction Properties of commonly used... Indicator properties are altered... Slow reaction kinetics in... Estimation of myoplasmic Ca2+... Use of a multicompartment... Spatially-resolved measurements... References

 
In most cells, the cytosolic free Ca²⁺ concentration ([Ca²⁺]) is low at rest (~0.1 µM) and difficult to measure precisely. However, [Ca²⁺] often rises manyfold during periods of cell activity (e.g., muscle contraction, neurosecretion, and cell division) and these changes in [Ca²⁺] ([Ca²⁺]), which serve as triggers for the underlying biological activity, can readily be monitored with the Ca²⁺-indicator dye technique. Typically, a fluorescent Ca²⁺ indicator such as fura 2, fluo 3, or furaptra is introduced into the cytosol, then the preparation is illuminated with ultraviolet or visible radiation and records are made of the indicator's resting fluorescence intensity (F) and changes in fluorescence intensity during activity (F). In some applications, the goal is simply to monitor a F signal that qualitatively reflects [Ca²⁺]. In others, calibration curves are applied to obtain quantitative estimates of [Ca²⁺], e.g., averaged over a substantial cytosolic region (spatially averaged [Ca²⁺]). With more recent technologies, such as confocal microscopy, the aim is to obtain "Ca²⁺ images" from the cell and thereby identify subcellular regions where [Ca²⁺] is large and localized (2, 3, 5, 8, 14). Such spatially-resolved measurements provide new information about the sites of Ca²⁺ entry into the cytosol, the diffusion of Ca²⁺ within the cytoplasm, and the complexation reactions between Ca²⁺ and key cytosolic constituents.

Here we consider some of the properties and organization of the intracellular environment that affect the interpretation of the optical measurements as well as some methods for quantitative assessment of intracellular Ca²⁺ movements. Most examples are drawn from experiments carried out by the authors on single-twitch fibers of the frog. These large accessible cells can be dissected "intact" (i.e., in essentially normal condition) and have highly developed mechanisms for generating large and brief [Ca²⁺] signals.

	Properties of commonly used Ca2+-indicator dyes

Top Introduction Properties of commonly used... Indicator properties are altered... Slow reaction kinetics in... Estimation of myoplasmic Ca2+... Use of a multicompartment... Spatially-resolved measurements... References

 
Many Ca²⁺ indicators in current use are derived from compounds first synthesized by R. Y. Tsien (12, 13), who chemically modified the Ca²⁺ buffer EGTA to obtain a key progenitor compound, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). BAPTA retains a high affinity for Ca²⁺ [dissociation constant (K_d) of ~0.1 µM in a simple salt solution], yet avoids the strong pH sensitivity characteristic of Ca²⁺ complexation by EGTA. Like EGTA, the reaction of BAPTA with Ca²⁺ has a 1:1 stoichiometry and a high selectivity over Mg²⁺, typically ~100,000-fold, which is appropriate physiologically, since free Mg²⁺ concentration in the cytosol is usually 1,000- to 10,000-fold higher than free [Ca²⁺]. To create a family of useful indicators, different chromophores have been attached to the BAPTA backbone, producing indicators with a range of Ca²⁺ affinities and optical properties (13). Frequently-used members of this family include fura 2, which is excitable with near-ultraviolet radiation and undergoes a substantial change in fluorescence intensity on binding Ca²⁺ (wavelength of maximum excitation sensitivity, ~380 nm), and fluo 3, which is excitable by visible wavelengths (e.g., 480 nm) and undergoes a remarkable increase in fluorescence, up to 200-fold (4), on binding Ca²⁺.

Raju et al. (11) chemically modified the tetracarboxylate Ca²⁺-binding pocket common to EGTA, BAPTA, and related indicators, which use four carboxylate groups to coordinate Ca²⁺, to produce a new family of indicators. These tricarboxylate compounds use only three carboxylate groups to coordinate Ca²⁺ and react with Ca²⁺, with K_d generally >10 µM (classified here as lower-affinity indicators). For example, furaptra, which has the same chromophore group as fura 2, has a ~250-fold larger K_d, ~50 µM. The large increase in K_d appears to arise primarily from an increase in the dissociation rate constant (k_–1) rather than a decrease in the association rate constant (k₊₁) (K_d = k_–1/k₊₁; cf. Fig. 1A). Because, at a constant level of [Ca²⁺], the overall reaction rate of an indicator proceeds at k₊₁ [Ca²⁺] + k_–1, lower-affinity indicators (with large values of k_–1) trade off signal amplitude for a faster reaction speed. Tricarboxylate indicators generally have lower selectivity over Mg²⁺ and thus a greater potential for interference from Mg²⁺.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. A: scheme for a 1:1 complexation reaction of Ca2+ (Ca) with an indicator dye molecule (D). Association rate constant (k+1) has units of M–1•s–1, and dissociation rate constant (k–1) has units of s–1. B: in vitro fluorescence intensity (F) of fura 2 (ordinate) vs. –log10 Ca2+ concentration ([Ca2+]; pCa, abscissa). Circles indicate data obtained in a protein-free salt solution, whereas triangles indicate data obtained in a salt solution containing 55 mg/ml aldolase (predominant soluble protein by weight in skeletal muscle). Each data set was fitted with a steady-state theoretical curve that assumes reaction in A, with Kd (k–1/k+1) taken as an adjustable parameter. Displayed values of F were obtained as 100 times measured value divided by value of F in a Ca2+-free solution (Fmin) estimated for circle data. Dashed curve was obtained by scaling theoretical curve for triangle data to have same Fmin as circle data. For measurements, excitation band was 420 ± 15 nm and emission band was 550 ± 50 nm. C: recordings collected from different experiments on intact single-twitch fibers from frog muscle. Each fiber was bathed in normal Ringer solution at 16°C, stretched to long sarcomere length (3.5–4 µm), microinjected with a Ca2+ indicator at a nonbuffering concentration, and stimulated by an action potential at t = 0. Lowermost record is a representative twitch response, recorded with a tension transducer. Upper 4 records are optical signals [change () in absorbance (A), recorded with purpurate-3,3'-diacetic acid (PDAA); F, recorded with furaptra, fluo 3, and fura 2]. For display purposes, all signals have been scaled to have same peak amplitude. If calibrated in [Ca2+] units, peak of the PDAA signal is 22 µM (6) and furaptra signal is 19 µM (assumed intracellular Kd of furaptra, 98 µm; Ref. 15). No attempt was made to calibrate signals from slowly-responding indicators fluo 3 or fura 2. For fluorescence recording, bands of excitation and emission wavelengths were 410 ± 15 and 540 ± 60 nm for furaptra and fura 2, respectively, and 480 ± 15 and 560 ± 50 nm for fluo 3. Peak amplitudes of F/F were –0.154, 11.5, and –0.722 (furaptra, fluo 3, and fura 2). Adapted from Konishi et al. (9), with permission from the Biophysical Society, and unpublished experiments of the authors.

	Indicator properties are altered by the intracellular environment

Top Introduction Properties of commonly used... Indicator properties are altered... Slow reaction kinetics in... Estimation of myoplasmic Ca2+... Use of a multicompartment... Spatially-resolved measurements... References

An example is given in Fig. 1B, which shows fura 2 fluorescence measured in a simple salt solution without and with addition of 55 mg/ml soluble protein. Each data set considered individually is well fitted by a theoretical 1:1 binding curve after adjustment of three parameters: F_min (the value of fluorescence in a Ca²⁺-free solution), F_max (the value of fluorescence extrapolated to a saturating Ca²⁺ solution), and the apparent K_d. However, protein causes a large fractional change in at least two of the parameter values: F_min is increased by ~90% and apparent K_d is increased by 3.6-fold, from 0.19 to 0.69 µM. (A change in F_max may also be present but is difficult to resolve at the excitation wavelength used in Fig. 1B because F_max in both solutions is very close to zero.) If the calibrations of Fig. 1B were used to convert an intracellular fura 2 fluorescence level to [Ca²⁺], the inferred value would clearly depend on the choice of calibration curve.

Similar effects of protein on in vitro calibrations have been observed with other indicator dyes. For example, fura red, analyzed by the "ratio" technique, which provides a normalization method that adjusts for variations in indicator concentration (13), revealed the following protein-related effects on the four key parameters of the calibration (10): 1) R_min (the ratio of fluorescence excited with 420-nm light divided by that with 480-nm light, measured in a Ca²⁺-free solution) was decreased by 32%; 2) R_max (the ratio of fluorescence excited with 420-nm light divided by that with 480-nm light, extrapolated to a saturating Ca²⁺ solution) was decreased by 60%; 3) ß (the ratio of 480-nm fluorescence measured in a Ca²⁺-free solution divided by that extrapolated for a saturating Ca²⁺ solution) was decreased by 2.6-fold; and 4) apparent K_d was increased 4.4-fold, from 0.36 to 1.59 µM. As expected, these substantial changes strongly affect the estimation of cytosolic [Ca²⁺]. In frog muscle fibers injected with fura red, the estimated resting level of [Ca²⁺] was 0.04 µM if based on an in vitro calibration without protein but was 0.46 µM if based on a protein-containing solution (10).

A third example concerns fluo 3. In in vitro calibrations, addition of protein increased apparent K_d by 2.1-fold, from 0.5 µM to 1.1 µm, and increased F_min by 1.5-fold. In contrast, F_max remained essentially unchanged (4). Again, estimation of resting [Ca²⁺] in skeletal muscle varied with the choice of in vitro calibration, 0.05 µM without protein or 0.10 µM with protein. [Note, to calibrate the myoplasmic signal from fluo 3, Harkins et al. (4) relied on both absorbance and fluorescence signals from the indicator.]

In summary, steady-state indicator behavior is strongly affected by protein, and, at the concentration of soluble proteins found in myoplasm, a major fraction of the indicators (0.5–0.9) appears to be protein-bound (4, 9, 10). Most indicators are also largely bound within muscle fibers (15). Although it is difficult to pinpoint the exact nature of the in vivo binding sites, both soluble and structural proteins likely participate. In consequence, in vitro calibrations, even with added protein, may be unreliable predictors of indicator behavior in the intracellular environment.

	Slow reaction kinetics in the cytosol can significantly delay an indicator's response to [Ca2+]

Top Introduction Properties of commonly used... Indicator properties are altered... Slow reaction kinetics in... Estimation of myoplasmic Ca2+... Use of a multicompartment... Spatially-resolved measurements... References

 
Because the apparent value of K_d is altered by the binding of indicator to protein, the apparent value of k₊₁, k_–1, or both must be altered (cf. Fig. 1, A and B). In fact, for many indicators the evidence suggests that the apparent values of both k₊₁ and k_–1 are reduced by the cytosol, with k₊₁ being reduced fractionally more than k_–1 (1, 15). For example, in muscle fibers at 16°C, the apparent values of k₊₁ and k_–1 for fluo 3 are estimated to be ~3.5 x 10⁷ M^–1•s^–1 and ~55 s^–1, respectively (K_d = 1.6 µM) (1). These values are approximately ninefold and threefold smaller, respectively, than expected for fluo 3 at the same temperature in a solution with the viscosity of myoplasm. Reductions of this magnitude decrease significantly the ability of fluo 3 and related high-affinity indicators (e.g., indo 1, fura 2, fura red, calcium orange, and so forth) to track rapidly changing [Ca²⁺] signals (1, 4, 9, 10, 15).

Figure 1C compares the spatially averaged responses of two lower-affinity and two high-affinity indicators in skeletal muscle fibers activated by an action potential. The trace-labeled purpurate-3,3'-diacetic acid (PDAA), which has a time-to-peak after stimulation of 4.5 ms, is the A signal from PDAA, an absorbance indicator with a very large K_d (~0.9 mM). Three features of PDAA support the conclusion that its A tracks spatially averaged [Ca²⁺] in a rapid and linear fashion (6). Firstly, because PDAA has a large value of K_d and thus k_–1, its effective reaction rate with Ca²⁺ (k₊₁ [Ca²⁺] + k_–1) is extremely fast at all levels of [Ca²⁺]. Secondly, the large value of K_d implies that the change in concentration of Ca²⁺-indicator complex ([CaD], proportional to A) is far from saturation in all regions of the cytosol (see below). Thirdly, PDAA is the least bound of any indicator yet studied in skeletal muscle; thus alterations in indicator properties due to intracellular binding are likely to be less serious with PDAA than with other indicators. (Note, PDAA also has some significant disadvantages; for example, because its optical change is small and because it reports a A, not a F, it is subject to greater interference from movement artifacts.) The upper three traces in Fig. 1C show the time course of the F signals measured with furaptra, fluo 3, and fura 2; these traces have times-to-peak of F of 4.5, 10.5, and 16 ms, respectively. A comparison among these indicators reveals a clear correlation between the time-to-peak of F and the value of K_d and hence k_–1. For example, with furaptra (in vitro K_d, 50 µM; estimated myoplasmic value of k_–1 >5,000 s^–1 at 16°C), the time-to-peak of F is essentially identical to that of A from PDAA; in contrast, with fura 2 and fluo 3 (in vitro K_d, 0.2 and 0.5 µM, respectively; estimated myoplasmic values of k_–1, ~35 and ~55 s^–1, respectively), the time-to-peak of F is substantially greater than that of A. This general kinetic pattern is confirmed in experiments with a number of other indicators (15).

If the delay between [Ca²⁺] and the F of a high-affinity indicator reflected only limitations inherent in the effective values of k₊₁ and k_–1, it should be possible to use the reaction scheme of Fig. 1A to kinetically correct F and thus obtain an accurate estimate of the time course of spatially averaged [Ca²⁺]. Unfortunately, a second complication–-the existence of significant myoplasmic gradients in [Ca²⁺]–-renders this approach approximate at best for high-affinity indicators like fura 2 and fluo 3. In skeletal muscle, [Ca²⁺] results from the release of stored Ca²⁺ through specific channels [ryanodine receptors (RYRs)] in the membranes of the sarcoplasmic reticulum (SR). These channels are found at the "triadic junctions," the special anatomical sites within the fiber volume where the transverse tubular and SR membranes come into close apposition (cf. Fig. 2A). During release, [Ca²⁺] near the junctions rises rapidly to high levels. Then, as a result of diffusion, [Ca²⁺] rises at other cytosolic locations, although to less elevated levels (cf. Figs. 3 and 4, described below). The existence of large gradients in [Ca²⁺] implies the existence of large gradients in [CaD]. With high-affinity indicators like fura 2 and fluo 3, [CaD] in response to an action potential rises quickly to saturated levels near the triadic junctions, whereas away from the junctions [CaD] is still in the linear range. Consequently, the full time course of spatially averaged [Ca²⁺] cannot be estimated accurately with a simple kinetic correction applied to the spatially-averaged F signal (1).

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Single-compartment estimates of myoplasmic Ca2+ binding and sarcoplasmic reticulum (SR) Ca2+ release in response to an action potential. A: schematic of a portion of a sarcomere of one myofibril. Arrows illustrate flow of Ca2+ to and from SR and onto and off major Ca2+-binding sites of myoplasm. [CaD], concentration of Ca2+ indicator complex; [CaTrop], [CaATP], and [CaParv], Ca2+ binding to Ca2+ buffer sites of myoplasm of troponin C, ATP, and parvalbumin, respectively. B: furaptra F and F measurements from a frog single fiber at 16°C provided direct estimates of [CaD] and [Ca2+]. [Ca2+] was then used to drive the single-compartment model to calculate the remaining traces. Uppermost trace (Release) estimates flux of Ca2+ across membranes of SR. On ordinate, all concentrations have been referred to myoplasmic water volume. [CaT], change in total myoplasmic [Ca2+]. Adapted from Baylor and Hollingworth (1), with permission from the Rockefeller University Press.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Top: geometry of a multicompartment model of a half-sarcomere (z- to m-line). Myofibrillar axis extends horizontally, and radial symmetry is assumed (note different vertical and horizontal scales). Troponin is restricted to 9 compartments located within 1 µm of the z-line, whereas diffusible Ca2+ buffers (ATP and parvalbumin) have access to all 18 compartments. Bottom: model calculations are shown as follows. A: spatially-averaged [Ca2+]. B: occupancy of troponin regulatory sites with Ca2+. A value of 446 µM on the ordinate corresponds to 100% occupancy. Three largest [CaTrop] traces report changes from 3 compartments adjacent to z-line; among these, peak rate of rise decreases from outer edge to middle to center of myofibril. A similar progression applies to 3 traces of intermediate size, which are from middle troponin-containing compartments, and to 3 smallest traces, which are from the troponin-containing compartments most distant from the z-line. C: [CaATP] responses from all 18 compartments. Amplitudes decrease progressively in manner described for B. Only 3 largest changes (from 3 compartments adjacent to the z-line) are well resolved radially; for other changes, radial gradient is almost negligible. As a set, 18 [CaATP] traces have essentially same relative shapes as do 18 [Ca2+] traces (not shown); scaling factor relating them ([CaATP]/[Ca2+]) is ~3.6. D: [CaParv] responses in 18 compartments; amplitudes decrease as described in B and C. Adapted from Baylor and Hollingworth (1), with permission from the Rockefeller University Press.

View larger version (73K): [in this window] [in a new window]   FIGURE 4. Confocal measurements of fluo 3 fluorescence obtained on a Nikon TE300 inverted microscope equipped with an "Oz" laser scanning system (Noran Instruments, Middleton, WI) and a 60x, 1.2 numerical aperture water-immersion objective (17°C). A: spatial (x-y) image (64.4 µm x 60.4 µm) of a region of a resting frog muscle fiber injected with fluo 3; 64 images, taken with low laser power, were averaged. Vertical dimension (x) runs parallel to fiber axis (sarcomere length, ~2.9 µm). B: single spatiotemporal (x-t) image (64.4 µm x 485 ms) from another frog fiber, also injected with fluo 3. Bright comet-like object is a fluo 3 "calcium spark." Seven minutes before measurement, caffeine (at 1 mM) was added to normal Ringer solution bathing fiber (sarcomere length, ~3.4 µm). C: average time course of F/F obtained from 3 adjacent x locations in x-t image where spark amplitude was maximum. Raw data of both images (512 x 480 pixels) were filtered by a 9-point (3 x 3) smoothing algorithm. Unpublished results of the authors.

	Estimation of myoplasmic Ca2+ movements from spatially averaged [Ca2+] signals

Top Introduction Properties of commonly used... Indicator properties are altered... Slow reaction kinetics in... Estimation of myoplasmic Ca2+... Use of a multicompartment... Spatially-resolved measurements... References

	Use of a multicompartment model allows inclusion of effects due to diffusion

Top Introduction Properties of commonly used... Indicator properties are altered... Slow reaction kinetics in... Estimation of myoplasmic Ca2+... Use of a multicompartment... Spatially-resolved measurements... References

 
The calculations of Fig. 2B do not take into account the existence of [Ca²⁺] gradients within the sarcomere and the inhomogeneities in Ca²⁺ binding that result from these gradients. These effects can be estimated with a multicompartment model. Figure 3 (top) illustrates one such model, which considers the subdivision of a half sarcomere of one myofibril into 18 compartments of equal volume (1). Ca²⁺ entry is assumed to take place only into the outer compartment immediately adjacent to the z-line (appropriate to the location of the triadic junctions in frog fibers). Ca²⁺ entry into the remaining compartments occurs by diffusion, which depends on the gradients in free [Ca²⁺], [CaATP], and [CaParv]. ([CaTrop] does not diffuse, since troponin is fixed to the thin filament; [CaD] has been ignored since it is generally small.) Calculations for an action potential stimulus are shown in Fig. 3, A–D. (Note, Fig. 3 does not show the individual [Ca²⁺] waveforms for the 18 compartments, but these are proportional to the [CaATP] waveforms of Fig. 3C; see legend.) To initiate the calculation, a Ca²⁺-release waveform with a time course similar to that of the Release trace of Fig. 2B was assumed. Time was advanced in the calculation by simultaneous integration of ~100 first-order differential equations. To obtain reasonable agreement between the calculated and measured versions of spatially averaged [Ca²⁺], it was necessary to reduce the amplitude of SR Ca²⁺ release by ~20% in comparison with that shown in Fig. 2B. The need for this adjustment reflects some inaccuracy in the single-compartment calculation of Fig. 2B, which is not designed to account for the effects of Ca²⁺ gradients. The multicompartment model thus improves the accuracy of the estimation of SR Ca²⁺ release and provides explicit estimates of the subsarcomeric Ca²⁺ gradients. It also establishes the importance of including ATP as a significant myoplasmic Ca²⁺ buffer. Without ATP, the multicompartment calculation did not produce acceptable agreement between the measured and modeled versions of spatially averaged [Ca²⁺] (1).

	Spatially-resolved measurements of intracellular Ca2+ movements

Top Introduction Properties of commonly used... Indicator properties are altered... Slow reaction kinetics in... Estimation of myoplasmic Ca2+... Use of a multicompartment... Spatially-resolved measurements... References

 
An exciting recent advance in the field of excitation-contraction coupling is the use of laser confocal microscopy to monitor Ca²⁺ signals at different locations within the sarcomere (2, 3, 8, 14). This advance is possible because a confocal microscope equipped with a high-numerical aperture objective is able to measure fluorescence from a small volume, e.g., 0.4 x 0.4 x 0.8 µm³. With a scanning system to move the focal point of the objective, spatial images of fluorescence (termed x-y images; Fig. 4A) or spatiotemporal images (termed x-t images; Fig. 4B) can be obtained.

The x-y image in Fig. 4A was acquired from a resting frog fiber that contained a relatively large concentration of fluo 3 (0.2–0.3 mM). A banding pattern in fluo 3 fluorescence is apparent that reflects the sarcomeric organization of the fiber. On average, the value of F detected from the middle of each sarcomere (m-line location) is ~15% larger than that from the ends of the sarcomere (z-line and triadic junction locations). This pattern probably reflects a differential binding of fluo 3 to myofilament proteins (e.g., more fluo 3 may be bound to the thick than to the thin filaments).

The x-t image in Fig. 4B (from a different fiber from that of the x-y image) was obtained by a left-to-right alignment of 480 vertical line scans, which were taken repetitively along a single linear position oriented parallel to the fiber axis. The acquisition period was 1.01 ms per line scan, so that the horizontal axis in the x-t image corresponds to 485 ms. During the first 350 ms of the image, no Ca²⁺-related activity is apparent. At 350 ms, a brief but substantial F occurred, indicative of an SR Ca²⁺-release event. This event probably lasted about 10 ms, which is the rise time of the signal in the spatial location of maximal F/F (Fig. 4C). As expected for frog muscle, the location of this Ca²⁺ "source" is near a z-line (8, 14). Diffusion of Ca²⁺ and Ca²⁺-fluo 3 away from the source (toward the nearest m-lines) is reflected in the x-t image as a V-shape that expands to the right. Isolated events of this type are termed "calcium sparks" (2, 8) and are thought to reflect the brief opening of one or a few RYRs. The spark in Fig. 4B was elicited by exposure of the fiber to 1 mM caffeine, an agonist of the RYRs. The frequency of caffeine-activated sparks increases with exposure time in caffeine and, after ~10 minutes, the occurrence of one or more sparks per x-t image is commonplace. This contrasts with the frequency of sparks in an intact fiber at rest in a standard Ringer solution, in which events are rare.

The x-t image in Fig. 4B illustrates one example of the use of confocal microscopy to examine the spatial location of Ca²⁺ entry and the time course of Ca²⁺ spread within the cytosol. Many laboratories are now using this and related technologies to measure both local and global Ca²⁺ signals in a wide variety of cells. A full analysis of the recorded information requires consideration of complex issues, such as those addressed in Figs. 1–3. Accordingly, these and related issues are active research topics in many laboratories. Their elucidation is bringing an expanding horizon to studies of Ca²⁺ signaling in living cells.

	Acknowledgments

 
This work was supported by a grant from the National Institutes of Health (NS-17620). Helpful comments on the manuscript were provided by Dr. W. K. Chandler and Mr. Jon Peet.

	References

Top Introduction Properties of commonly used... Indicator properties are altered... Slow reaction kinetics in... Estimation of myoplasmic Ca2+... Use of a multicompartment... Spatially-resolved measurements... References

 

1. Baylor, S. M., and S. Hollingworth. Model of sarcomeric Ca²⁺ movements, including ATP Ca²⁺ binding and diffusion, during activation of frog skeletal muscle. J. Gen. Physiol. 112: 297–316, 1998.[Abstract/Free Full Text]
2. Cheng, H., W. J. Lederer, and M. B. Cannell. Calcium sparks: elementary events underlying excitation-contraciton coupling in heart muscle. Science 262: 740–745, 1993.[Abstract/Free Full Text]
3. Escobar, A. L., J. R. Monk, J. M. Fernandez, and J. L. Vergara. Localisation of the site of Ca²⁺release at the level of a single sarcomere in skeletal muscle fibers. Nature 367: 739–774, 1994.[Medline]
4. Harkins, A. B., N. Kurebayashi, and S. M. Baylor. Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3. Biophys. J. 65: 865–881, 1993.[Abstract/Free Full Text]
5. Hernandez-Cruz, A., F. Sala, and P. R. Adams. Subcellular calcium transients visualized by confocal microscopy in a voltage-clamped vertebrate neuron. Science 247: 858–862, 1990.[Abstract/Free Full Text]
6. Hirota, A., W. K. Chandler, P. L. Southwick, and A. S. Waggoner. Calcium signals recorded from two new purpurate indicators inside frog cut twitch fibers. J. Gen. Physiol. 94: 597–631, 1989.[Abstract/Free Full Text]
7. Jong, D.-S., P. Pape, S. M. Baylor, and W. K. Chandler. Calcium inactivation of calcium release in frog cut muscle fibers that contain millimolar EGTA or Fura-2. J. Gen. Physiol. 106: 337–388, 1995.[Abstract/Free Full Text]
8. Klein, M. G., H. Cheng, L. F. Santana, Y.-H. Jiang, W. J. Lederer, and M. F. Schneider. Two mechanisms of quantized calcium release in skeletal muscle. Nature 379: 455–458, 1996.[Medline]
9. Konishi, M., A. Olson, S. Hollingworth, and S. M. Baylor. Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements. Biophys. J. 54: 1089–1104, 1988.[Abstract/Free Full Text]
10. Kurebayashi, N., A. B. Harkins, and S. M. Baylor. Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers. Biophys. J. 64: 1934–1960, 1993.[Abstract/Free Full Text]
11. Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. A fluorescent indicator for measuring cytosolic free magnesium. Am. J. Physiol. Cell Physiol. 256: C540–C548, 1989.[Abstract/Free Full Text]
12. Tsien, R. Y. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19: 2396–2404, 1980.[Medline]
13. Tsien, R. Y. Intracellular signal transduction in four dimensions: from molecular design to physiology. Am. J. Physiol. Cell. Physiol. 263: C723–C728, 1992.[Abstract/Free Full Text]
14. Tsugorka, A., E. Rios, and L. A. Blatter. Imaging elementary events of calcium release in skeletal muscle cells. Science 269: 1723–1726, 1995.[Abstract/Free Full Text]
15. Zhao, M., S. Hollingworth, and S. M. Baylor. Properties of tri- and tetra-carboxylate Ca²⁺ indicators in frog skeletal muscle fibers. Biophys. J. 70: 896–916, 1996.[Abstract/Free Full Text]

This article has been cited by other articles:

				C. Hille and B. Walz Characterisation of neurotransmitter-induced electrolyte transport in cockroach salivary glands by intracellular Ca2+, Na+ and pH measurements in duct cells J. Exp. Biol., February 15, 2008; 211(4): 568 - 576. [Abstract] [Full Text] [PDF]

				D. Szczesna-Cordary, M. Jones, J. R. Moore, J. Watt, W. G. L. Kerrick, Y. Xu, Y. Wang, C. Wagg, and G. D. Lopaschuk Myosin regulatory light chain E22K mutation results in decreased cardiac intracellular calcium and force transients FASEB J, December 1, 2007; 21(14): 3974 - 3985. [Abstract] [Full Text] [PDF]

				O. Ostrovskaya, R. Goyal, N. Osman, C. E. McAllister, I. N. Pessah, J. R. Hume, and S. M. Wilson Inhibition of Ryanodine Receptors by 4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) in Canine Pulmonary Arterial Smooth Muscle Cells J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 381 - 390. [Abstract] [Full Text] [PDF]

				N. Yagi A Structural Origin of Latency Relaxation in Frog Skeletal Muscle Biophys. J., January 1, 2007; 92(1): 162 - 171. [Abstract] [Full Text] [PDF]

				J. D. Rojas, S. R. Sennoune, D. Maiti, K. Bakunts, M. Reuveni, S. C. Sanka, G. M. Martinez, E. A. Seftor, C. J. Meininger, G. Wu, et al. Vacuolar-type H+-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1147 - H1157. [Abstract] [Full Text] [PDF]

				S. Sanchez-Armass, S. R. Sennoune, D. Maiti, F. Ortega, and R. Martinez-Zaguilan Spectral imaging microscopy demonstrates cytoplasmic pH oscillations in glial cells Am J Physiol Cell Physiol, February 1, 2006; 290(2): C524 - C538. [Abstract] [Full Text] [PDF]

				D. F. Reiff, A. Ihring, G. Guerrero, E. Y. Isacoff, M. Joesch, J. Nakai, and A. Borst In Vivo Performance of Genetically Encoded Indicators of Neural Activity in Flies J. Neurosci., May 11, 2005; 25(19): 4766 - 4778. [Abstract] [Full Text] [PDF]

				J. R. Lopez, N. Linares, I. N. Pessah, and P. D. Allen Enhanced response to caffeine and 4-chloro-m-cresol in malignant hyperthermia-susceptible muscle is related in part to chronically elevated resting [Ca2+]i Am J Physiol Cell Physiol, March 1, 2005; 288(3): C606 - C612. [Abstract] [Full Text] [PDF]

				K.H.S. Arun, C.L. Kaul, and P. Ramarao AT1 receptors and L-type calcium channels: functional coupling in supersensitivity to angiotensin II in diabetic rats Cardiovasc Res, February 1, 2005; 65(2): 374 - 386. [Abstract] [Full Text] [PDF]

				G. Ji, M. E. Feldman, K.-Y. Deng, K. S. Greene, J. Wilson, J. C. Lee, R. C. Johnston, M. Rishniw, Y. Tallini, J. Zhang, et al. Ca2+-sensing Transgenic Mice: POSTSYNAPTIC SIGNALING IN SMOOTH MUSCLE J. Biol. Chem., May 14, 2004; 279(20): 21461 - 21468. [Abstract] [Full Text] [PDF]

				D. L. Wokosin, C. M. Loughrey, and G. L. Smith Characterization of a Range of Fura Dyes with Two-Photon Excitation Biophys. J., March 1, 2004; 86(3): 1726 - 1738. [Abstract] [Full Text] [PDF]

				R. P. Schuhmeier, B. Dietze, D. Ursu, F. Lehmann-Horn, and W. Melzer Voltage-Activated Calcium Signals in Myotubes Loaded with High Concentrations of EGTA Biophys. J., February 1, 2003; 84(2): 1065 - 1078. [Abstract] [Full Text] [PDF]

				N. Yagi An X-Ray Diffraction Study on Early Structural Changes in Skeletal Muscle Contraction Biophys. J., February 1, 2003; 84(2): 1093 - 1102. [Abstract] [Full Text] [PDF]

				Y. Wang and W. G. L. Kerrick The off rate of Ca2+ from troponin C is regulated by force-generating cross bridges in skeletal muscle J Appl Physiol, June 1, 2002; 92(6): 2409 - 2418. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (37) Citing Articles via Google Scholar Google Scholar Articles by Baylor, S. M. Articles by Hollingworth, S. Search for Related Content PubMed PubMed Citation Articles by Baylor, S. M. Articles by Hollingworth, S.