This article describes a new apparatus for making simultaneous optical measurements on single muscle fibers at three different wavelengths and two planes of linear polarization . There are two modes of operation : mode 1 measures the individual absorbances of light linearly polarized along and perpendicular to the fiber axis, and mode 2 measures retardation (or birefringence) and the average of the two absorbance components . 89 :41-143), was routinely employed so that all our cut fiber results would be comparable. A simple method, which does not require microelectrodes, allowed continual estimation of a fiber's membrane (r ,) and internal longitudinal (r) resistances as well as the external resistance (re) under the Vaseline seals . The values of r , and r; obtained from cut fibers with this method agree reasonably well with values obtained from intact fibers using microelectrode techniques . Optical measurements were made on resting and action potential-stimulated fibers . The intrinsic fiber absorbance, defined operationally as logo of the ratio of incident light to transmitted light intensity, was similar in intact and cut preparations, as were the changes that accompanied stimulation . On the other hand, the resting birefringence and the peak of the active change in cut fibers were, respectively, only 0.8 and 0.7 times the corresponding values in intact fibers . Both the amplitude and the half-width of the active retardation signal increased considerably during the time course of cut fiber experiments ; a

The Ca indicator antipyrylazo III was introduced into cut frog twitch fibers by diffusion (Maylie, J ., M . Irving, N . L. Sizto, and W . K. Chandler . 1987 . Journa l of General Physiology . 89 :41-81) . Like arsenazo 111, antipyrylazo III was largely bound to or sequestered by intracellular constituents ; on average, a fraction 0.68 was so immobilized . After action potential stimulation, there was an early change in absorbance, with a wavelength dependence that nearly matched a cuvette Ca-difference spectrum. As with arsenazo III, this signal became prolonged as experiments progressed. In a freshly prepared cut fiber containing 0.3 mM indicator, the absorbance change had an average half-width of 10 ms at 18°C . The peak amplitude of this Ca signal depended on the indicator concentration in a roughly parabolic manner, which is consistent with a 1 :2 stoichiometry for Ca :indicator complexation and, for indicator,concentrations 50.4 mM, constant peak free [Ca] . If all the antipyrylazo III inside a fiber can react normally with Ca, peak free [Ca] is 3 jM at 18°C. If only freely diffusible indicator can react, the estimate is 42 pM . The true amplitude probably lies somewhere in between . The time course of Ca binding to intracellular buffers and of Ca release from the sarcoplasmic reticulum is estimated from the 3-and 42-AM myoplasmic [Ca] transients . After action potential stimulation, the release waveform is rapid and brief, its latency after the surface action potential is 2-3 ms and its half-width is 2-4 ms . This requires rapid coupling between the action potential in the transverse tubular system and Ca release from the sarcoplasmic reticulum . The peak fractional occupancy calculated for Ca-regulatory sites on troponin is 0.46 for the 3-AM transient and 0.93 for the 42-EtM transient. During a I00-ms tetanus at 100 Hz, the corresponding fractional occupancies are 0.56 and 0 .94 . The low value of occupancy associated with the low-amplitude [Ca] calibration seems inconsistent with a brief tetanus being able to produce near-maximal activation (Blinks, J . R., R.

The Ca indicator tetramethylmurexide was introduced into cut fibers, mounted in a double-Vaseline-gap chamber, by diffusion from the endpool solutions . The indicator diffused rapidly to the central region of a fiber where optical recording was done and, if removed, diffused away equally fast. The time course of concentration suggests that, on average, a fraction 0.27 of indicator was reversibly bound to myoplasmic constituents and the free diffusion constant was 1 .75 X 10-' cm2/s at 18°C . The shape of the resting absorbance spectrum suggests that a fraction 0 .11-0.15 of tetramethylmurexide inside a fiber was complexed with Ca . After action potential stimulation, there was a rapid transient change in indicator absorbance followed by a maintained change of opposite sign . The wavelength dependence of both changes matched a cuvette Ca-difference spectrum. The amplitude of the early peak varied linearly with indicator concentration and corresponded to an average rise in free [Ca] of 17 uM . These rather diverse findings can be explained if the sarcoplasmic reticulum membranes are permeable to Ca-free indicator . Both Ca-free and Ca-complexed indicator inside the sarcoplasmic reticulum would appear to be bound by diffusion analysis and the Ca-complexed form would be detected by the resting absorbance spectrum . The transient change in indicator absorbance would be produced by myoplasmic Ca reacting with indicator molecules that freely diffuse in myoplasmic solution . The maintained signal, which reports Ca dissociating from indicator complexed at rest, would come from changes within the sarcoplasmic reticulum . A method, based on these ideas, is described for separating the two components of the tetramethylmurexide signal . The estimated myoplasmic free [Ca] transient has an average peak value of 26 yM at 18°C . Its time course is similar to, but possibly faster than, that recorded with antipyrylazo III (Mayliej, M . Irving, N .

Junge and McLaughlin (1987) derived an expression for the apparent diffusion constant of protons in the presence of both mobile and immobile buffers. Their derivation applies only to cases in which the values of pH are considerably greater than the largest pK of the individual buffers, a condition that is not expected to hold in skeletal muscle or many other cell types. Here we show that, if the pH gradients are small, the same expression for the apparent diffusion constant of protons can be derived without such constraints on the values of the pK's. The derivation is general and can be used to estimate the apparent diffusion constant of any substance that diffuses in the presence of both mobile and immobile buffers. The apparent diffusion constant of protons is estimated to be 1-2 x 10(-6) cm2/s at 18 degrees C inside intact frog twitch muscle fibers. It may be smaller inside cut fibers, owing to a reduction in the concentration of mobile myoplasmic buffers, so that in this preparation a pH gradient, if established within a sarcomere following action potential stimulation, could last 10 ms or longer after stimulation ceased.

Antipyrylazo III was introduced into frog cut twitch fibers (17-19~ by diffusion. After action potential stimulation, the change in indicator absorbance could be resolved into two components that had different time courses and wavelength dependences. The first component was early and transient and due to an increase in myoplasmic free [Ca] (Maylie, J., M. Irving, N.L. Sizto, and W. [C Chandler, 1987,Journal of General Physiology, 89:83-143). The second component, usually measured at 590 nm (near the isosbestic wavelength for Ca), developed later than the Ca transient and returned towards baseline about 100 times more slowly. Although the wavelength dependence of this component is consistent with an increase in either free [Mg] or pH, its time course is clearly different from that of the signals obtained with the pH indicators phenol red and 4',5'-dimethyl-5-(and -6-) carboxyfluorescein, suggesting that it is mainly due to an increase in free [Mg]. After a single action potential in freshly prepared cut fibers that contained 0.3 mM antipyrylazo III, the mean peak amplitude of &A(590) would correspond to an increase in free [Mg] of 47 #M if all the signal were due to a change in [Mg] and all the intracellular indicator reacted with Mg as in cuvette calibrations. With either repetitive action potential stimulation or voltage-clamp depolarization, the AA(590) signal continued to develop throughout the period when free [Ca] was elevated and then recovered to within 40-90% of the prestimulus baseline with an average rate constant between 0.5 and 1.0 s -1. With prolonged voltage-clamp depolarization, both the amplitude and rate of development of the &A(590) signal increased with the amplitude of the depolarization and appeared to saturate at levels corresponding to an increase in free [Mg] of 0.8-1.4 mM and a maximum rate constant of 3-4 s -1, respectively. These results are consistent with the idea Address reprint requests to Dr.