SUMMARY1. 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 0C).2. The amplitude of the Ca2+ transient decreased when fibres were stretched to sarcomere spacings approaching 4 /sm. The effect appeared to be less marked in H20 Ringer than in D20 Ringer, where a reduction of about 40 % was observed in going from 3 0 psm to 3 7-3 9 ,sm.3. 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 041 mM-Ca2+ was released from the sarcoplasmic reticulum (s.r.) into the myoplasm.4. 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 /IM) were used. Each set of three simulations was carried out both with and without parvalbumin.5. The simulations show that following action potential stimulation, 0-2-0-3 mMCa2+ 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 ofparvalbumin has much effect on the shape 626 S. M. BA YLOR, W. K. CHANDLER AND M. W. MARSHALL of the release wave form; the main effect of varying these parameters is to change the magnitude.6. After the initial, rapid phase of Ca2+ release from the s.r. there is a longer, maintained period of Ca2+ uptake. The peak magnitude of this uptake divided by the concentration of s.r. Ca2+ pump sites is used to estimate the turnover rate of the sites. The numbers so obtained, 2-4-6-3 s-1, are in good agreement with previously published values based on biochemical experiments. Thus, the known biochemical properties of the s.r. Ca2+ pump can explain the final phase of the computed s.r. Ca2+ flux and therefore, within the framework of the model, the falling phase of the free [Ca2+] transient.7. Simulations, based on Ca2+ transients obtained during a train of action potentials spaced 10 ms apart, indicate that s.r. Ca2+ release associ...

SUMMARY1. 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, AA. This paper describes the different components of dye-related signals in fibres injected with either Arsenazo III, Antipyrylazo III or Dichlorophosphonazo III.2. Fibres injected with Arsenazo III can show two kinds of changes in dyerelated absorbance, an early isotropic change and a later dichroic change. The isotropic signal, which is the main subject of this paper, is transient in nature; it starts to develop before tension, reaches a peak in about 10 msec and is nearly over by 0-1 see (16 0C). This signal is largest at 650-660 nm and measurements in this range indicate that the peak AA varies approximately linearly with dye concentration between 0-2 and 0 7 mm. The wavelength dependence of the peak amplitude can be qualitatively fitted by the Ca2+-difference spectrum determined from cuvette calibration measurements. There may be a small maintained (0A4-0 5 see) absorbance change of a few percent of the peak value at 650-660 nm, possibly reflecting a maintained increase in myoplasmic pH or free [Mg2+].3. In a fibre injected with approximately 0 5 mM-Antipyrylazo III, there were two kinds of dye-related absorbance signals, both of which were isotropic. There was no signal that was obviously dichroic. The earlier signal was similar in time course to the early isotropic Ca2+ signal which was measured with Arsenazo III, and its magnitude followed the wavelength dependence of the Ca2+-difference spectrum determined from cuvette calibration measurements. By contrast, the wavelength dependence of the later absorbance change was similar to either the H+ or Mg2+_ difference spectrum. The direction of this late signal (0-2 see after stimulus) would correspond to an increase in either myoplasmic pH or free [Mg2+]. Records of the absorbance change at all wavelengths can be fitted by a linear combination of the Ca2+ waveform and the H+/Mg2+ waveform.4. Fibres injected with Dichlorophosphonazo III showed three dye-related absorbance changes. There was an early isotropic signal, a later dichroic signal and a second 140 S. M. BA YLOR, W. K. CHANDLER AND M. W. MARSHALL isotropic signal. The wavelength dependence of the first part of the early signal is similar to the Ca2+-difference spectrum whereas the wavelength dependence of the second isotropic signal is similar to the H+-or Mg2+-difference spectrum. As was the case with Arsenazo III and Antipyrylazo III, the direction of the second signal at late times would correspond to an increase in either pH or free [Mg2+]. Replacing H20 with D20 resulted in a marked diminution of the dichroic signal. In D2O, linear combinations of two basic isotropic waveforms were sufficient to account for the absorbance changes measured at all wavelengths.5. With all three metallochromic dyes, the time course of t...

SUMMARY1. Single twitch fibres were isolated from frog muscle, then mounted in a chamber which was positioned on an optical bench. The fibres were immobilized by high stretch (sarcomere spacing 3-9-4-3 jsm) and by placement on a pedestal. Their 3. The earliest change in absorbance following an action potential was a small, transient increase which was followed by a larger decrease. The decrease in fibre absorbance varied from 0.5 x 10-4 to 3 x 10-4 units.4. Resting myoplasmic pH was determined by comparing the absorbance spectrum from fibres injected with Phenol Red with that obtained from calibrating solutions in cuvettes. The muscle measurements were corrected for the intrinsic absorbance of fibre without dye. The average value of pH in two fibres was 6-9. The change in absorbance following an action potential in these highly stretched fibres was small. In one experiment the change, if due to pH alone, corresponded to an increase in pH of 0 004 peak and 0 002 maintained (relative to a resting level of 6 9). The maintained signal can be satisfactorily explained by the known amount of phosphocreatine hydrolysis. This variability probably means that at least two, and possibly all three dyes behave differently inside muscle fibres than they do in calibrating solutions. The most likely explanation is that dye, once injected, can bind to cellular contents and that this alters its properties. 6. Changes in absorbance of fibres injected with Arsenazo I, a dye three times more sensitive to Mg2+ than to Ca2+, were used to determine whether changes in free [Mg2+] occur following an action potential. The observed changes were small and could be due to a small increase in pH, of the magnitude measured with Phenol Red, and/or free [Mg2+]. In terms of a change in free [Mg2+], the results set an upper limit of 2 %.7. The conclusion from the action potential experiments is that neither intracellular pH nor free [Mg2+] changes appreciably in highly stretched fibres. Changes in these two quantities can therefore be neglected in analysing the relatively large 650-660 nm Ca2+ signal in fibres injected with the Ca2+ (but also pH and Mg2+) sensitive indicator dye Arsenazo III.

SUMMARY1. A method is described which allows the approximate computation of membrane current from measurements with three electrodes in the midregion of a muscle fibre.2. Measurements of inward sodium current in frog muscle are compared with the results of previous clamping studies to test the validity of the new method.3. Sodium current in rat muscle (extensor digitorum longus) is in general similar to sodium current in frog muscle. Two differences in detail have been found between sodium current in rat and frog muscle:(a) at the same temperature (in the range 0-20°C) inactivation is slower in the rat than in the frog; (b) in rat the steady-state activation is shifted negatively on the voltage axis by some 10-15 mV.4. Delayed outward current and charge movement (Schneider & Chandler, 1973) are present in rat muscle.5. Rat muscle fibres are more resistant than frog muscle fibres to the action of tetrodotoxin. Inward current is still detectable in rat muscle at 100 nM tetrodotoxin. We found no evidence to suggest the existence in rat muscle of two kinds of sodium channel, one sensitive and one less sensitive to tetrodotoxin.