Stephen M. Baylor

Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements.

Binding of the fluorescent Ca2+ indicator dye fura-2 by intracellular constituents has been investigated by steady-state optical measurements. Fura-2s (a) fluorescence intensity, (b) fluorescence emission anisotropy, (c) fluorescence emission spectrum, and (d) absorbance spectra were measured in glass capillary tubes containing solutions of purified myoplasmic proteins; properties b and c were also measured in frog skeletal muscle fibers microinjected with fura-2. The results indicate that more than half, and possibly as much as 85%, of fura-2 molecules in myoplasm are in a protein-bound form, and that the binding changes many properties of the dye. For example, in vitro characterization of the Ca2+-dye reaction indicates that when fura-2 is bound to aldolase (a large and abundant myoplasmic protein), the dissociation constant of the dye for Ca2+ is three- to fourfold larger than that measured in the absence of protein. The problems raised by intracellular binding of fura-2 to cytoplasmic proteins may well apply to cells other than skeletal muscle fibers.

A small-molecule inhibitor of skeletal muscle myosin II

We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosins interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.

Sarcoplasmic reticulum calcium release in frog skeletal muscle fibres estimated from Arsenazo III calcium transients.

Single twitch fibres, dissected from frog muscle, were injected with the metallochromic dye Arsenazo III. Changes in dye‐related absorbance measured at 650 or 660 nm were used to estimate the time course of myoplasmic free [Ca2+] following either action potential stimulation or voltage‐clamp depolarization (temperature, 15‐17 degrees C). The amplitude of the Ca2+ transient decreased when fibres were stretched to sarcomere spacings approaching 4 microns. The effect appeared to be less marked in H2O Ringer than in D2O Ringer, where a reduction of about 40% was observed in going from 3.0 microns to 3.7‐3.9 microns. In fibres heavily injected with dye (1.5‐2.2 mM‐dye) at least 0.1 mM‐Ca2+ was complexed with Arsenazo III following a single action potential, implying that at least 0.1 mM‐Ca2+ was released from the sarcoplasmic reticulum (s.r.) into the myoplasm. Computer simulations were carried out to estimate the flux of Ca2+ between the s.r. and myoplasm (in fibres containing no more that 0.8 mM‐dye). The amounts and time courses of Ca2+ bound to the Ca2+‐regulatory sites on troponin and to the Ca2+, Mg2+ sites on parvalbumin were estimated from the free [Ca2+] wave form and the law of mass action. In the computations the total myoplasmic [Ca2+] was taken as the total amount of Ca2+ existing either as free ion or as ion complexed with dye, troponin or parvalbumin. The time derivative of total myoplasmic [Ca2+] was used as an estimate of net Ca2+ flux (release minus uptake) from the s.r. into myoplasm. Rate constants for formation of cation: receptor complex were taken from published values. For the Ca2+‐regulatory sites on troponin, three sets of rate constants, corresponding to two values of dissociation constant (0.2 and 2 microM) were used. Each set of three simulations was carried out both with and without parvalbumin. The simulations show that following action potential stimulation, 0.2‐0.3 mM‐Ca2+ enters the myoplasm from the s.r. The wave form of s.r. Ca2+ release is early and brief compared with the wave form of free [Ca2+]. Neither the selection of troponin rate constants nor the inclusion of parvalbumin has much effect on the shape of the release wave form; the main effect of varying these parameters is to change the magnitude. After the initial, rapid phase of Ca2+ release from the s.r. there is a longer, maintained period of Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

Fura-2 calcium transients in frog skeletal muscle fibres.

1. Intact single twitch fibres from frog muscle were mounted at long sarcomere spacing (3.5‐4.2 microns) on an optical bench apparatus for the measurement of absorbance and fluorescence signals following the myoplasmic injection of either or both of the Ca2+ indicator dyes Fura‐2 and Antipyrylazo III. Dye‐related signals were measured at 16‐17 degrees C in fibres at rest and stimulated electrically to give a single action potential or brief train of action potentials. 2. The apparent diffusion constant of Fura‐2 in myoplasm, Dapp, was estimated from Fura‐2 fluorescence measured as a function of time and distance from the site of dye injection. On average (N = 7), Dapp was 0.36 x 10(‐6) cm2 s‐1, a value nearly 3‐fold smaller than expected if all the Fura‐2 was freely dissolved in the myoplasmic solution. The small value of Dapp is explained if approximately 60‐65% of the Fura‐2 molecules were bound to relatively immobile sites in myoplasm. 3. In resting fibres the fraction of Fura‐2 in the Ca2+‐bound form was estimated to be small, on average (N = 11) 0.06 of total dye. However, because of the large fraction of Fura‐2 not freely dissolved in myoplasm, and the indirect method employed for estimating Ca2+‐bound dye, calibration of the resting level of myoplasmic free Ca2+ ([Ca2+]) from the fraction of Ca2+‐bound dye was not considered reliable. 4. In response to a single action potential, large changes in Fura‐2 fluorescence (delta F) and absorbance (delta A) were detected, which had identical time courses. As expected, the directions of these transients corresponded to an increase in Ca2+‐dye complex. For wavelengths, lambda, between 380 and 460 nm, peak delta A(lambda) was closely similar to the Ca2+‐dye difference spectrum for Fura‐2 determined in in vitro calibrations. Beers law was used to calibrate the concentration of Ca2+‐dye complex formed during activity (delta[CaFura‐2]) from the delta A(lambda) signal. Peak delta[CaFura‐2] was found to vary between 0.01 and 0.4 mM, depending on the total concentration of injected Fura‐2 ([Fura‐2T]), which ranged as high as 0.9 mM. 5. In fibres in which peak delta[CaFura‐2] was less than 0.06 mM, delta[CaFura‐2] had a limiting minimal half‐width of 50‐60 ms. However, as peak delta[CaFura‐2] increased (up to 0.3‐0.4 mM), delta[CaFura‐2] half‐width became markedly prolonged (up to 150‐200 ms), indicative of a strong buffering action of large concentrations of Fura‐2 on the underlying [Ca2+] transient (delta[Ca2+]).(ABSTRACT TRUNCATED AT 400 WORDS)

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 indicators 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-3s 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-3s 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.

Sarcoplasmic reticulum calcium release compared in slow- twitch and fast-twitch fibres of mouse muscle

Experiments were carried out to compare the amplitude and time course of Ca2+ release from the sarcoplasmic reticulum (SR) in intact slow‐twitch and fast‐twitch mouse fibres. Individual fibres within small bundles were injected with furaptra, a low‐affinity, rapidly responding Ca2+ indicator. In response to a single action potential at 16 °C, the peak amplitude and half‐duration of the change in myoplasmic free [Ca2+] (Δ[Ca2+]) differed significantly between fibre types (slow‐twitch: peak amplitude, 9.4 ± 1.0 μM (mean ± S.E.M.); half‐duration, 7.7 ± 0.6 ms; fast‐twitch: peak amplitude 18.5 ± 0.5 μM; half‐duration, 4.9 ± 0.3 ms). SR Ca2+ release was estimated from Δ[Ca2+] with a computational model that calculated Ca2+ binding to the major myoplasmic Ca2+ buffers (troponin, ATP and parvalbumin); buffer concentrations and reaction rate constants were adjusted to reflect fibre‐type differences. In response to an action potential, the total concentration of released Ca2+ (Δ[CaT]) and the peak rate of Ca2+ release ((d/dt)Δ[CaT]) differed about 3‐fold between the fibre types (slow‐twitch: Δ[CaT], 127 ± 7 μM; (d/dt)Δ[CaT], 70 ± 6 μM ms−1; fast‐twitch: Δ[CaT], 346 ± 6 μM; (d/dt)Δ[CaT], 212 ± 4 μM ms−1). In contrast, the half‐duration of (d/dt)Δ[CaT] was very similar in the two fibre types (slow‐twitch, 1.8 ± 0.1 ms; fast‐twitch, 1.6 ± 0.0 ms). When fibres were stimulated with a 5‐shock train at 67 Hz, the peaks of (d/dt)Δ[CaT] in response to the second and subsequent shocks were much smaller than that due to the first shock; the later peaks, expressed as a fraction of the amplitude of the first peak, were similar in the two fibre types (slow‐twitch, 0.2–0.3; fast‐twitch, 0.1–0.3). The results support the conclusion that individual SR Ca2+ release units function similarly in slow‐twitch and fast‐twitch mammalian fibres.

Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers

Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicators dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.

Use of metallochromic dyes to measure changes in myoplasmic calcium during activity in frog skeletal muscle fibres

1. Changes in transmission of quasi‐monochromatic light were measured in singly dissected, dye‐injected twitch fibres following a single propagated action potential. The records, after correction for the intrinsic transmission signal, indicate changes in dye‐related absorbance, ΔA. This paper describes the different components of dye‐related signals in fibres injected with either Arsenazo III, Antipyrylazo III or Dichlorophosphonazo III.

Intracellular calcium movements during excitation–contraction coupling in mammalian slow-twitch and fast-twitch muscle fibers

In skeletal muscle fibers, action potentials elicit contractions by releasing calcium ions (Ca2+) from the sarcoplasmic reticulum. Experiments on individual mouse muscle fibers micro-injected with a rapidly responding fluorescent Ca2+ indicator dye reveal that the amount of Ca2+ released is three- to fourfold larger in fast-twitch fibers than in slow-twitch fibers, and the proportion of the released Ca2+ that binds to troponin to activate contraction is substantially smaller.

Sarcomeric Ca2+ gradients during activation of frog skeletal muscle fibres imaged with confocal and two-photon microscopy.

1 Intra‐sarcomeric gradients of [Ca2+] during activation of action potential stimulated frog single fibres were investigated with the Ca2+ indicator fluo‐3 and confocal and two‐photon microscopy. The object of these experiments was to look for evidence of extra‐junctional Ca2+ release and examine the microscopic diffusion of Ca2+ within the sarcomere. 2 By exploiting the spatial periodicity of sarcomeres within the fibre, we could achieve a high effective line‐scanning rate (≈8000 lines s−1), although the laser scanning microscope was limited to < 1000 lines s−1. At this high time resolution, the time course of fluorescence changes was very different at the z‐ and m‐lines, with a significant delay (≈1 ms; 22 °C) between the rise of fluorescence at the z‐line and the m‐line. 3 To calculate the expected fluorescence changes, we used a multi‐compartment model of Ca2+ movements in the half‐sarcomere in which Ca2+ release was restricted to triadic junctions (located at z‐lines). Optical blurring by the microscope was simulated to generate fluorescence signals which could be compared directly to experimental data. The model which reproduced our experimental findings most accurately included Ca2+ binding by ATP, as well as indicator binding to immobile sarcomeric proteins. After taking sarcomeric misregistration within the fibre into account, there was very good agreement between the model and experimental results. 4 We conclude that there is no experimental evidence for Ca2+ release at locations other than at z‐lines. In addition, our calculations support the conclusion that rapidly diffusing Ca2+ buffers (such as ATP) are important in shaping the Ca2+ transient and that the details of intracellular indicator binding need to be considered to explain correctly the time course of fluorescence change in the fibre.