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 myosin's 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.

Myoplasmic calcium transients monitored with purpurate indicator dyes injected into intact frog skeletal muscle fibers.

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after microinjection with tetramethylmurexide (TMX) or purpurate-3,3' diacetic acid (PDAA), two compounds from the purpurate family of absorbance Ca2+ indicators previously used in cut muscle fibers (Maylie, J., M. Irving, N. L. Sizto, G. Boyarsky, and W. K. Chandler. 1987. J. Gen. Physiol. 89:145-176; Hirota, A., W. K. Chandler, P. L. Southwick, and A. S. Waggoner. 1989. J. Gen. Physiol. 94:597-631.) The apparent longitudinal diffusion constant of PDAA (mol wt 380) in myoplasm was 0.99 (+/- 0.04, SEM) x 10(-6) cm2 s-1 (16-17 degrees C), a value which suggests that 24-43% of the PDAA molecules were bound to myoplasmic constituents of large molecular weight. The corresponding values for TMX (mol wt 322) were 0.98 (+/- 0.05) x 10(-6) cm2 s-1 and 44-50%, respectively. Muscle membranes (surface and/or transverse-tubular) appear to be permeable to TMX and, to a lesser extent, to PDAA, since the total amount of indicator contained within a fiber decreased with time after injection. The average time constants for disappearance of indicator were 46 (+/- 7, SEM) min for TMX and 338 (+/- 82) min for PDAA. The fraction of indicator in the Ca2(+)-bound state in resting fibers was significantly different from zero for TMX (0.070 +/- 0.008) but not for PDAA (0.026 +/- 0.009). In in vitro calibrations PDAA but not TMX appeared to react with Ca2+ with 1:1 stoichiometry. In agreement with Hirota et al. (Hirota, A., W. K. Chandler, P. L. Southwick, and A. S. Waggoner. 1989. J. Gen. Physiol. 94:597-631), we conclude that PDAA is probably a more reliable myoplasmic Ca2+ indicator than TMX. In fibers that contained PDAA and were stimulated by a single action potential, the calibrated peak value of the myoplasmic free [Ca2+] transient (delta[Ca2+]) averaged 9.4 (+/- 0.6) microM, a value about fivefold larger than that calibrated with antipyrylazo III under otherwise identical conditions (Baylor, S. M., and S. Hollingworth. 1988. J. Physiol. 403:151-192). The fivefold difference is similar to that previously reported in cut fibers with antipyrylazo III and PDAA. Since in both intact and cut fibers the percentage of PDAA bound to myoplasmic constituents is considerably smaller than that found for antipyrylazo III, the PDAA calibration of delta[Ca2+] is likely to be more accurate. Interestingly, in intact fibers the peak value of delta[Ca2+] calibrated with either PDAA or antipyrylazo III is about half that calibrated in cut fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Model of sarcomeric Ca2+ movements, including ATP Ca2+ binding and diffusion, during activation of frog skeletal muscle.

Cannell and Allen (1984. Biophys. J. 45:913-925) introduced the use of a multi-compartment model to estimate the time course of spread of calcium ions (Ca2+) within a half sarcomere of a frog skeletal muscle fiber activated by an action potential. Under the assumption that the sites of sarcoplasmic reticulum (SR) Ca2+ release are located radially around each myofibril at the Z line, their model calculated the spread of released Ca2+ both along and into the half sarcomere. During diffusion, Ca2+ was assumed to react with metal-binding sites on parvalbumin (a diffusible Ca2+- and Mg2+-binding protein) as well as with fixed sites on troponin. We have developed a similar model, but with several modifications that reflect current knowledge of the myoplasmic environment and SR Ca2+ release. We use a myoplasmic diffusion constant for free Ca2+ that is twofold smaller and an SR Ca2+ release function in response to an action potential that is threefold briefer than used previously. Additionally, our model includes the effects of Ca2+ and Mg2+ binding by adenosine 5'-triphosphate (ATP) and the diffusion of Ca2+-bound ATP (CaATP). Under the assumption that the total myoplasmic concentration of ATP is 8 mM and that the amplitude of SR Ca2+ release is sufficient to drive the peak change in free [Ca2+] (Delta[Ca2+]) to 18 microM (the approximate spatially averaged value that is observed experimentally), our model calculates that (a) the spatially averaged peak increase in [CaATP] is 64 microM; (b) the peak saturation of troponin with Ca2+ is high along the entire thin filament; and (c) the half-width of Delta[Ca2+] is consistent with that observed experimentally. Without ATP, the calculated half-width of spatially averaged Delta[Ca2+] is abnormally brief, and troponin saturation away from the release sites is markedly reduced. We conclude that Ca2+ binding by ATP and diffusion of CaATP make important contributions to the determination of the amplitude and the time course of Delta[Ca2+].

Absorbance signals from resting frog skeletal muscle fibers injected with the pH indicator dye, phenol red.

Singly dissected twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with the pH indicator dye, phenol red. Dye-related absorbances in myoplasm, denoted by A0(lambda) and A90(lambda), were estimated as a function of wavelength lambda (450 nm less than or equal to lambda less than or equal to 640 nm) with light polarized parallel (0 degrees) and perpendicular (90 degrees) to the fiber axis respectively. At all lambda, A0(lambda) was slightly greater than A90(lambda), indicating that some of the phenol red molecules were bound to oriented structures accessible to myoplasm. The phenol red "isotropic" signal, [A0(lambda) + 2A90(lambda)]/3, a quantity equal to the average absorbance of all the dye molecules independent of their orientation, had a spectral shape that was red-shifted by approximately 10 nm in comparison with in vitro dye calibration curves measured in 140 mM KCl. The red-shifted spectrum also indicates that some phenol red molecules were bound in myoplasm. A quantitative estimate of indicator binding was obtained from measurements of the dye's apparent diffusion constant in myoplasm, denoted by Dapp. The small value of Dapp, 0.37 x 10(-6) cm2 s-1 (at 16 degrees C), can be explained if approximately 80% of the dye was bound to myoplasmic sites of low mobility. To estimate the apparent myoplasmic pH, denoted by pHapp, the isotropic absorbance of phenol red was fitted by in vitro calibration spectra. pHapp was found to be independent of dye concentration (0.2-2 mM), but varied widely (range, 6.8-7.5; mean value, 7.17) among fibers judged from functional characteristics to be normal. When fibers were subjected to acid or alkaline loads by exposure to Ringer's solution containing, respectively, dissolved CO2 or NH3, the changes in pHapp were in agreement with those expected from pH micro-electrode studies. It is concluded that in spite of the several indications for the presence of bound phenol red inside muscle cells, the pHapp signal from the indicator is useful for monitoring changes in myoplasmic pH in response to physiological and pharmacological manipulations.

Changes in phenol red absorbance in response to electrical stimulation of frog skeletal muscle fibers.

Intact single twitch fibers from frog muscle were stretched to long sarcomere length, micro-injected with the pH indicator dye phenol red, and activated by action potential stimulation. Indicator-related absorbance changes (denoted by delta A0 and delta A90) were measured with 0 degree and 90 degrees polarized light (oriented, respectively, parallel and perpendicular to the fiber axis). Two components of delta A were detected that had generally similar time courses. The "isotropic" component, calculated as the weighted average (delta A0 + 2 delta A90)/3, had the wavelength dependence expected for a change in myoplasmic pH. If calibrated in pH units, this signal's peak amplitude, which occurred 15-20 ms after stimulation, corresponded to a myoplasmic alkalization of average value 0.0025 +/- 0.0002 (+/- SEM; n = 9). The time course of this change, as judged from a comparison with that of the fibers' intrinsic birefringence signal, was delayed slightly with respect to that of the myoplasmic free [Ca2+] transient. On average, the times to half-peak and peak of the phenol red isotropic signal lagged those of the birefringence signal by 2.4 +/- 0.2 ms (+/- SEM; n = 8) and 8.4 +/- 0.5 ms (+/- SEM; n = 4), respectively. The other component of the phenol red signal was "dichroic," i.e., detected as a difference (delta A0-delta A90 greater than 0) between the two polarized absorbance changes. The wavelength dependence of this signal was similar to that of the phenol red resting dichroic signal (Baylor and Hollingworth. 1990. J. Gen. Physiol. 96:449-471). Because of the presence of the active dichroic signal, and because approximately 80% of the phenol red molecules appear to be bound in the resting state to either soluble or structural sites, the possibility exists that myoplasmic events other than a change in pH underlie the phenol red isotropic signal.

Simulation of calcium sparks in cut skeletal muscle fibers of the frog.

Spark mass, the volume integral of Delta F/F, was investigated theoretically and with simulations. These studies show that the amount of Ca2+ bound to fluo-3 is proportional to mass times the total concentration of fluo-3 ([fluo-3T]); the proportionality constant depends on resting Ca2+ concentration ([Ca2+]R). In the simulation of a Ca2+ spark in an intact frog fiber with [fluo-3T] = 100 microM, fluo-3 captures approximately one-fourth of the Ca2+ released from the sarcoplasmic reticulum (SR). Since mass in cut fibers is several times that in intact fibers, both with similar values of [fluo-3T] and [Ca2+]R, it seems likely that SR Ca2+ release is larger in cut fiber sparks or that fluo-3 is able to capture a larger fraction of the released Ca2+ in cut fibers, perhaps because of reduced intrinsic Ca2+ buffering. Computer simulations were used to identify these and other factors that may underlie the differences in mass and other properties of sparks in intact and cut fibers. Our spark model, which successfully simulates calcium sparks in intact fibers, was modified to reflect the conditions of cut fiber measurements. The results show that, if the protein Ca2+-buffering power of myoplasm is the same as that in intact fibers, the Ca2+ source flux underlying a spark in cut fibers is 5-10 times that in intact fibers. Smaller source fluxes are required for less buffer. In the extreme case in which Ca2+ binding to troponin is zero, the source flux needs to be 3-5 times that in intact fibers. An increased Ca2+ source flux could arise from an increase in Ca2+ flux through one ryanodine receptor (RYR) or an increase in the number of active RYRs per spark, or both. These results indicate that the gating of RYRs, or their apparent single channel Ca2+ flux, is different in frog cut fibers--and, perhaps, in other disrupted preparations--than in intact fibers.

The amplitude and time course of the myoplasmic free [Ca2+] transient in fast-twitch fibers of mouse muscle.

Bundles of 10-100 fibers were dissected from the extensor digitorum longus muscle of mouse, mounted in an apparatus for optical recording, and stretched to long sarcomere length (> or = 3.6 microns). One fiber within the bundle was microinjected with furaptra, a fluorescent indicator that responds rapidly to changes in myoplasmic free [Ca2+] (delta [Ca2+]). Twitches and brief tetani were initiated by external stimulation. At myoplasmic furaptra concentrations of approximately 0.1 mM, the indicator's fluorescence signal during fiber activity (delta F/F) was well resolved. delta F/F was converted to delta [Ca2+] under the assumption that furaptra's myoplasmic dissociation constant for Ca2+ is 98 microM at 16 degrees C and 109 microM at 28 degrees C. At 16 degrees C, the peak amplitude of delta [Ca2+] during a twitch was 17.8 +/- 0.4 microM (+/-SEM; n = 8) and the half-width of delta [Ca2+] was 4.6 +/- 0.3 ms. At 28 degrees C, the peak and half-width values were 22.1 +/- 1.8 microM and 2.0 +/- 0.1 ms, respectively (n = 4). During a brief high-frequency tetanus, individual peaks of delta [Ca2+] were also well resolved and reached approximately the same amplitude that resulted from a single shock; the initial decays of delta [Ca2+] from peak slowed substantially during the tetanus. For a single twitch at 16 degrees C, the amplitude of delta [Ca2+] in fast-twitch fibers of mouse is not significantly different from that recently measured in fast-twitch fibers of frog (16.5 +/- 0.9 microM; Zhao, M., S. Hollingworth, and S.M. Baylor. 1996. Biophys. J. 70:896-916); in contrast, the half-width of delta [Ca2+] is surprisingly brief in mouse fibers, only about half that measured in frog (9.6 +/- 0.6 ms). The estimated peak rate at which Ca2+ is released from the sarcoplasmic reticulum in response to an action potential is also similar in mouse and frog, 140-150 microM/ms (16 degrees C).

Comparison of simulated and measured calcium sparks in intact skeletal muscle fibers of the frog.

Calcium sparks in frog intact skeletal muscle fibers were modeled as stereotypical events that arise from a constant efflux of Ca(2+) from a point source for a fixed period of time (e.g., 2.5 pA of Ca(2+) current for 4.6 ms; 18 degrees C). The model calculates the local changes in the concentrations of free Ca(2+) and of Ca(2+) bound to the major intrinsic myoplasmic Ca(2+) buffers (troponin, ATP, parvalbumin, and the SR Ca(2+) pump) and to the Ca(2+) indicator (fluo-3). A distinctive feature of the model is the inclusion of a binding reaction between fluo-3 and myoplasmic proteins, a process that strongly affects fluo-3's Ca(2+)-reaction kinetics, its apparent diffusion constant, and hence the morphology of sparks. DeltaF/F (the change in fluo-3's fluorescence divided by its resting fluorescence) was estimated from the calculated changes in fluo-3 convolved with the microscope point-spread function. To facilitate comparisons with measured sparks, noise and other sources of variability were included in a random repetitive fashion to generate a large number of simulated sparks that could be analyzed in the same way as the measured sparks. In the initial simulations, the binding of Ca(2+) to the two regulatory sites on troponin was assumed to follow identical and independent binding reactions. These simulations failed to accurately predict the falling phase of the measured sparks. A second set of simulations, which incorporated the idea of positive cooperativity in the binding of Ca(2+) to troponin, produced reasonable agreement with the measurements. Under the assumption that the single channel Ca(2+) current of a ryanodine receptor (RYR) is 0.5-2 pA, the results suggest that 1-5 active RYRs generate an average Ca(2+) spark in a frog intact muscle fiber.

Calcium inactivation of calcium release in frog cut muscle fibers that contain millimolar EGTA or Fura-2.

Cut muscle fibers from Rana temporaria (sarcomere length, 3.4-4.2 microns) were mounted in a double Vaseline-gap chamber (14-15 degrees C) and equilibrated with end-pool solutions that contained 20 mM EGTA and 1.76 mM Ca. Sarcoplasmic reticulum (SR) Ca release was estimated from changes in pH (Pape, P. C., D.-S. Jong, and W.K. Chandler. 1995. Journal of General Physiology. 106:000-000). Although the amplitude and duration of the [Ca] transient, as well as its spatial spread from the release sites, are reduced by EGTA, SR Ca release elicited by either depolarizing voltage-clamp pulses or action potentials behaved in a manner consistent with Ca inactivation of Ca release. After a step depolarization to -20 or 10 mV, the rate of SR Ca release, corrected for SR Ca depletion, reached a peak value within 5-15 ms and then rapidly decreased to a quasi-steady level that was about half the peak value; the time constant of the last half of the decrease was usually 2-4 ms. Immediately after an action potential or a 10-15 ms prepulse to -20 mV, the peak rate of SR Ca release elicited by a second stimulation, as well as the fractional amount of release, were substantially decreased. The rising phase of the rate of release was also reduced, suggesting that at least 0.9 of the ability of the SR to release Ca had been inactivated by the first stimulation. There was little change in intramembranous charge movement, suggesting that the changes in SR Ca release were not caused by changes in its voltage activation. These effects of a first stimulation on the rate of SR Ca release elicited by a second stimulation recovered during repolarization to -90 mV; the time constant of recovery was approximately 25 ms in the action-potential experiments and approximately 50 ms in the voltage-clamp experiments. Fura-2, which is able to bind Ca more rapidly than EGTA and hence reduce the amplitude of the [Ca] transient and its spatial spread from release sites by a greater amount, did not prevent Ca inactivation of Ca release, even at concentrations as large as 6-8 mM. These effects of Ca inactivation of Ca release can be simulated by the three-state, two-step model proposed by Schneider, M. F., and B. J. Simon (1988, Journal of Physiology. 405:727-745), in which SR Ca channels function as a single uniform population of channels. (ABSTRACT TRUNCATED AT 400 WORDS)

Effect of fura-2 on action potential-stimulated calcium release in cut twitch fibers from frog muscle.

Cut fibers (striation spacing, 3.6-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14-15 degrees C. One or both of the Ca indicators fura-2 and purpurate-3,3' diacetic acid (PDAA) were introduced into the optical recording site by diffusion from the end pools. Sarcoplasmic reticulum (SR) Ca release was elicited by action potential stimulation. With resting [fura-2] = 0 mM at the optical site, the [Ca] transient measured with PDAA was used to estimate SR Ca release (Baylor, S.M., W.K. Chandler, and M.W. Marshall. 1983. Journal of Physiology. 344:625-666). With resting [fura-2] > 0 mM, the contribution from Ca complexation by fura-2 was added to the estimate. When resting [fura-2] was increased from 0 to 0.5-2 mM, both the amount of SR Ca release and the maximal rate of release were increased by approximately 20%. These results are qualitatively similar to those obtained in intact fibers (Baylor, S.M., and S. Hollingworth. 1988. Journal of Physiology. 403:151-192; Hollingworth, S., A. B. Harkins, N. Kurebayashi, M. Konishi, and S. M. Baylor. 1992. Biophysical Journal. 63:224-234) and are consistent with a reduction of Ca inactivation of SR Ca release produced by 0.5-2 mM fura-2. With resting [fura-2] > or = 2 mM, the PDAA [Ca] transient was reduced to nearly zero and SR Ca release could be estimated from delta [Cafura-2] alone. When resting [fura-2] was increased from 2-4 to 5-6 mM, both the amount of SR Ca release and the maximal rate of release were decreased by approximately half, consistent with a possible reduction of Ca-induced Ca release (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophysical Journal. 60:867-873) or a possible pharmacological effect of fura-2.

Reduction of calcium inactivation of sarcoplasmic reticulum calcium release by fura-2 in voltage-clamped cut twitch fibers from frog muscle.

Cut fibers from Rana temporaria and Rana pipiens (striation spacing, 3.9-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14 degrees C. The Ca indicator purpurate-3,3' diacetic acid (PDAA) was introduced into the end pools and allowed to diffuse into the optical recording site. When the concentration at the site exceeded 2 mM, step depolarizations to 10 mV were applied and the [Ca] transient measured with PDAA was used to estimate Ca release from the sarcoplasmic reticulum (SR) (Baylor, S. M., W. K. Chandler, and M. W. Marshall. 1983. Journal of Physiology. 344:625-666). With depolarization, the rate of SR Ca release increased to an early peak and then rapidly decreased several-fold to a quasi-steady level. The total amount of Ca released from the SR at the time of peak rate of release appeared to be independent of SR Ca content, consistent with the idea that a single activated channel might pass, on average, a fixed number of ions, independent of the magnitude of the single channel flux. A possible explanation of this property is given in terms of locally induced Ca inactivation of Ca release. The solution in the end pools was then changed to one with PDAA plus fura-2. SR Ca release was estimated from the [Ca] transient, as before, and from the delta [Cafura-2] signal. On average, 2-3 mM fura-2 increased the quasi-steady level of the rate of SR Ca release by factors of 6.6 and 3.8, respectively, in three fibers from Rana temporaria and three fibers from Rana pipiens. The peak rate of release was increased in five of the six fibers but to a lesser extent than the quasi-steady level. In all fibers, the amplitude of the free [Ca] transient was markedly reduced. These increases in the rate of SR Ca release are consistent with the idea that Ca inactivation of Ca release develops during a step depolarization to 10 mV and that 2-3 mM fura-2 is able to reduce this inactivation by complexing Ca and thereby reducing free [Ca]. Once the concentration of fura-2 becomes sufficiently large, a further increase reduces the rate of SR Ca release. On average, 5-6 mM fura-2 increased the quasi-steady rate of release, compared with 0 mM fura-2, by 6.5 and 2.9, respectively, in four fibers from Rana temporaria and three from Rana pipiens.(ABSTRACT TRUNCATED AT 400 WORDS)

Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra.

Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with a chromophore group similar to that of fura-2 (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. J. Biol. Chem. 260:3440-3450). In vitro calibrations indicate that furaptra reacts with Ca2+ and Mg2+ with 1:1 stoichiometry, with dissociation constants of 44 microM and 5.3 mM, respectively (16-17 degrees C; ionic strength, 0.15 M; pH, 7.0). Thus, in a frog skeletal muscle fiber stimulated electrically, the indicator is expected to respond to the change in myoplasmic free [Ca2+] (delta[Ca2+]) with little interference from changes in myoplasmic free [Mg2+]. The apparent longitudinal diffusion constant of furaptra in myoplasm was found to be 0.68 (+/- 0.02, SEM) x 10(-6) cm2 s-1 (16-16.5 degrees C), a value which suggests that about half of the indicator was bound to myoplasmic constituents of large molecular weight. Muscle membranes (surface and/or transverse-tubular) appear to have some permeability to furaptra, as the total quantity of indicator contained within a fiber decreased after injection; the average time constant of the loss was 302 (+/- 145, SEM) min. In fibers containing less than 0.5 mM furaptra and stimulated by a single action potential, the calibrated peak value of delta[Ca2+] averaged 5.1 (+/- 0.3, SEM) microM. This value is about half that reported in the preceding paper (9.4 microM; Konishi, M., and S. M. Baylor. 1991. J. Gen. Physiol. 97:245-270) for fibers injected with purpurate-diacetic acid (PDAA). The latter difference may be explained, at least in part, by the likelihood that the effective dissociation constant of furaptra for Ca2+ is larger in vivo than in vitro, owing to the binding of the indicator to myoplasmic constituents. The time course of furaptra's delta[Ca2+], with average values (+/- SEM) for time to peak and half-width of 6.3 (+/- 0.1) and 9.5 (+/- 0.4) ms, respectively, is very similar to that of delta[Ca2+] recorded with PDAA. Since furaptra's delta[Ca2+] can be recorded at a single excitation wavelength (e.g., 420 nm) with little interference from fiber intrinsic changes, movement artifacts, or delta[Mg2+], furaptra represents a useful myoplasmic Ca2+ indicator, with properties complementary to those of other available indicators.

Perturbation of sarcoplasmic reticulum calcium release and phenol red absorbance transients by large concentrations of fura-2 injected into frog skeletal muscle fibers.

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with two indicator dyes: phenol red, to monitor a previously described signal (denoted delta pHapp; Hollingworth and Baylor. 1990. J. Gen. Physiol. 96:473-491) possibly reflective of a myoplasmic pH change following action potential stimulation; and fura-2, to monitor the associated change in the myoplasmic free calcium concentration (delta[Ca2+]). Additionally, it was expected that large myoplasmic concentrations of fura-2 (0.5-1.5 mM) might alter delta pHapp, since it was previously found (Baylor and Hollingworth. 1988. J. Physiol. 403:151-192) that the Ca2(+)-buffering effects of large fura-2 concentrations: (a) increase the estimated total concentration of Ca2+ (denoted by delta[CaT]) released from the sarcoplasmic reticulum (SR), but (b) reduce and abbreviate delta[Ca2+]. The experiments show that delta pHapp was increased at the larger fura-2 concentrations; moreover, the increase in delta pHapp was approximately in proportion to the increase in delta[CaT]. At all fura-2 concentrations, the time course of delta pHapp, through time to peak, was closely similar to, although probably slightly slower than, that of delta[CaT]. These properties of delta pHapp are consistent with an hypothesis proposed by Meissner and Young (1980. J. Biol. Chem. 255:6814-6819) and Somlyo et al. (1981. J. Cell Biol. 90:577-594) that a proton flux from the myoplasm into the SR supplies a portion of the electrical charge balance required as Ca2+ is released from the SR into the myoplasm. A comparison of the amplitude of delta pHapp with that of delta[CaT] indicates that, in response to a single action potential, 10-15% of the charge balance required for Ca2+ release may be carried by protons.

Calcium sparks in intact skeletal muscle fibers of the frog.

Calcium sparks were studied in frog intact skeletal muscle fibers using a home-built confocal scanner whose point-spread function was estimated to be approximately 0.21 microm in x and y and approximately 0.51 microm in z. Observations were made at 17-20 degrees C on fibers from Rana pipiens and Rana temporaria. Fibers were studied in two external solutions: normal Ringer's ([K(+)] = 2.5 mM; estimated membrane potential, -80 to -90 mV) and elevated [K(+)] Ringer's (most frequently, [K(+)] = 13 mM; estimated membrane potential, -60 to -65 mV). The frequency of sparks was 0.04-0.05 sarcomere(-1) s(-1) in normal Ringer's; the frequency increased approximately tenfold in 13 mM [K(+)] Ringer's. Spark properties in each solution were similar for the two species; they were also similar when scanned in the x and the y directions. From fits of standard functional forms to the temporal and spatial profiles of the sparks, the following mean values were estimated for the morphological parameters: rise time, approximately 4 ms; peak amplitude, approximately 1 DeltaF/F (change in fluorescence divided by resting fluorescence); decay time constant, approximately 5 ms; full duration at half maximum (FDHM), approximately 6 ms; late offset, approximately 0.01 DeltaF/F; full width at half maximum (FWHM), approximately 1.0 microm; mass (calculated as amplitude x 1.206 x FWHM(3)), 1.3-1.9 microm(3). Although the rise time is similar to that measured previously in frog cut fibers (5-6 ms; 17-23 degrees C), cut fiber sparks have a longer duration (FDHM, 9-15 ms), a wider extent (FWHM, 1.3-2.3 microm), and a strikingly larger mass (by 3-10-fold). Possible explanations for the increase in mass in cut fibers are a reduction in the Ca(2+) buffering power of myoplasm in cut fibers and an increase in the flux of Ca(2+) during release.

Excitation-contraction coupling in skeletal muscle fibers injected with the InsP3 blocker, heparin.

Heparin, an inhibitor of inositol trisphosphate (InsP3)-induced Ca2+ release in smooth muscle and non-muscle cells, was injected into intact frog skeletal muscle fibres. Ca2+ release from the sarcoplasmic reticulum was elicited by the normal action potential mechanism and monitored by both fura-2 fluorescence and an intrinsic birefringence signal. Both optical signals, and hence Ca2+ release, were unaffected by high concentrations of heparin. This result argues against a major physiological role of InsP3 as a chemical messenger of excitation-contraction coupling in skeletal muscle.

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. Beer's 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)

Measurement of myoglobin diffusivity in the myoplasm of frog skeletal muscle fibres.

1. Experiments were carried out on intact, single skeletal muscle fibres from frog in order to estimate the apparent diffusion constant of myoglobin (denoted DAPP) in the myoplasm of living muscle cells. An optical technique was employed to measure myoglobin concentration along the fibre axis following injection of metmyoglobin (denoted metMb) at a point source. The concentration profiles were fitted by the one-dimensional diffusion equation to give estimates of DAPP. The method relied on the fact that myoglobin is normally absent from these frog fibres, thus permitting resolution of the myoglobin-related absorbance above the intrinsic absorbance of the fibre. 2. One complication in the method was that metMb became significantly reduced to oxymyoglobin (denoted MbO2) during the elapsed time before measurement of the concentration profile. The rate of reduction was evaluated by fitting myoglobin-related absorbance spectra, measured at different times following injection of metMb, with in vitro absorbance spectra of metMb and MbO2. Results from four experiments indicated that reduction could be described by a first-order, irreversible reaction having an average rate constant of 0.0164 min-1 (22 degrees C). The effect of reduction on the fitting of DAPP was taken into account. 3. DAPP was determined under three fibre conditions: (1) long sarcomere spacing (3.6-3.8 microns) at 16 degrees C, (2) long sarcomere spacing at 22 degrees C, and (3) normal sarcomere spacing (2.4-2.7 microns) at 22 degrees C. The average values for DAPP under these conditions were: (1) 0.12 (n = 5); (2) 0.17 (n = 5); and (3) 0.15 (n = 7) x 10(-6) cm2 s-1. The average value at 22 degrees C, 0.16 x 10(-6) cm2 s-1, is about 4 times smaller than values for myoglobin diffusivity at 20 degrees C commonly assumed in models of facilitated transport of oxygen by myoglobin. 4. In order to test the possibility that the unexpectedly low value of DAPP found in intact fibres might be due to the binding of myoglobin to relatively immobile sites in myoplasm, experiments were carried out in a cut-fibre preparation using a technique described by Maylie, Irving, Sizto & Chandler (1987 b) for determining the diffusion constants and degree of myoplasmic binding of absorbance dyes. Values for DAPP and the factor (denoted 1 + beta) by which the total myoglobin concentration exceeded the free myoglobin concentration were obtained by fitting the absorbance data by solutions of the one-dimensional diffusion equation.(ABSTRACT TRUNCATED AT 400 WORDS)

A large birefringence signal preceding contraction in single twitch fibres of the frog.

1. When a single muscle fibre was externally stimulated to give a propagated action potential, a large decrease in light intensity was measured with the fibre positioned between crossed-polarizers oriented at +/- 45 degrees with respect to the fibre axis. This large optical signal begins just after stimulation and its man phase precedes the development of positive tension. 2. The peak (or plateau) amplitude of the signal, expressed as the peak (plateau) change in light intensity, delta I, divided by the resting light intensity, I, was typically (minus) 1 to 3x10-3 and the time-to-peak (plateau) was 4 to 6 ms (20 degrees C). 3. When mechanical activity was minimized by stretch or Ringer replacement with D2O or hypertonic solution, the peak tension response was reduced in far greater proportion than the peak optical response, suggesting that the early optical signal is not due to changes in tension or gross fibre movement. 4. The magnitude of the optical response was increased by nitrate and double-shock stimulatin, procedures which potentiate the twitch response. 5. The optical signal was shown to propagate along the fibre length with a conduction velocity appropriate to the surface action potential. 6. The above results suggest that the large, early birefringence signal reflects a step or steps in the sequence of events leading to contractile activation.

The optical properties of birefringence signals from single muscle fibres.

1. The optical retardation of single muscle fibres at rest and the optical properties of the large, early birefringence signal detectable during a twitch (Baylor & Oetliker, 1975, 1977a) were investigated. 2. The resting birefringence, B, which is the factor relating resting retardation, R, to the light path length through the fibre, L, was found to be 2.25 x 10-3 (i.e. R = 2.25 X 10-3 X L) and to be independent of wavelength ( lambda = 480-660 nm). 3. When the angle of incidence, psi, of the crossed polarizers with respect to the fibre axis was varied, the resting light intensity and large, early change in light intensity were related by the function sin2 psi-cos2psi. When the net phase shift, phi lambda, of a narrow longitudinal strip of fibre plus compensator was varied, the resting light intensity was described by the function (1-cos phi lambda), whereas the early change in light intensity followed sin phi lambda. These results make it likely that the optical mechanism underlying the early birefringence signal is a change in retardation. 4. When a narrow longitudinal strip of fibre was illuminated by monochromatic light in the range 480-690 nm, the magnitude of the signal varied approximately as expected if the retardation change is independent of wave-length. 5. The spatial characteristics of the signal were examined by moving a small slit of light across the fibre width as well as by measuring the signal collected from the entire fibre width as a function of wave-length. The results from both experiments support the idea that the large, early change in retardation is due to a volume-related rather than surface-related structure. 6. Under the assumption that the retardation change is distributed as fibre volume, its average magnitude was calculated. For fibres in normal Ringer the peak of the early retardation change compared with resting is about 1.7 x 10-3, and for fibres in D2O Ringer about 0.7 x 10-3.