J. Phy8iol. (1983), 341, pp. 579-593 579With 8 text-figuresPrinted in Great Britain

COMPARISON OF BIREFRINGENCE SIGNALS AND CALCIUMTRANSIENTS IN VOLTAGE-CLAMPED CUT SKELETAL MUSCLE

FIBRES OF THE FROG

BY L. KOVACS, R. A. SCHtMPERLI* AND G. SZtJCSFrom the Department of Physiology, University Medical School, Debrecen, Hungary4012 and * Institute of Physiology, University of Berne, 3012 Berne, Switzerland

(Received 10 September 1982)

SUMMARY

1. The characteristic features ofbirefringence and calcium transients were comparedin voltage-clamped cut skeletal muscle fibres.

2. Birefringence signals were measured by introducing crossed polarizers aboveand below the fibres (±450 to the fibre axis) and using light of 790 nm. Calciumtransients were monitored by the metallochromic indicator dye, Antipyrylazo IIIrecording the changes in fibre absorbance at 720 nm. The dye entered the myoplasmicspace by diffusion through the cut end.

3. The early large birefringence signals, related to excitation-contraction couplinghad a time course similar to that of calcium transients. The two signals hadsuperimposable onset but the change in optical retardation peaked later and declinedmore slowly than the calcium signal.

4. Using depolarizing pulses with increasing amplitudes both transients showed thesame voltage dependence in the rate of rise, the time-to-peak and the peak amplitude.

5. Birefringence signals recorded at different voltages along the strength-durationcurve for contraction threshold had the same amplitudes and similar time constantsfor the falling phase comparable to the properties of the calcium transients.

6. After applying dantrolene sodium both signals were reduced to the same extent.A shift in the contraction threshold was found towards more positive membranepotential values. The birefringence and calcium transients recorded at the newcontraction threshold during the dantrolene treatment showed nearly the same sizeand time course as threshold transients obtained before the treatment.

7. A subthreshold concentration of caffeine increased the peak amplitude ofbirefringence signals at a given voltage and decreased the latency of the signals.Birefringence transients at the new contraction threshold under caffeine were smallerthan controls. Both effects are very similar to the changes in calcium transients dueto caffeine treatment as previously reported.

8. Consequently the voltage-dependent properties of birefringence and calciumtransients and their responses to caffeine and dantrolene treatment are nearly thesame. These results support the view that the changes in optical retardation of thefibres reflect calcium bound to some sarcoplasmic binding site rather than a potentialchange of the sarcoplasmic reticulum.

19-2

L. Ko VACS, R. A. SCHUMPERLI AND G. SZUccs

INTRODUCTION

In recent years, optical techniques have become an important tool in the studyof excitation-contraction coupling. Apart from signals attributed to the surface andT-tubular action potential, several signals have been recorded during activation ofmuscle fibres which appear to reflect subsequent steps in excitation-contractioncoupling (birefringence, Baylor & Oetliker, 1975, 1977a, b; transparency changes,Kovacs & Schneider, 1977; signals from fibres stained with membrane-permeableso-called potentiometric dyes, Bezanilla & Horowicz, 1975; Oetliker, Baylor &Chandler, 1975; Vergara, Bezanilla & Salzberg, 1978; Baylor & Chandler, 1978). Animportant tool for the understanding of these signals of uncertain origin has beenprovided by the recent development of techniques to directly measure intracellularcalcium changes with metallochromic indicator dyes (Arsenazo III, Miledi, Parker& Schalow, 1977; Antipyrylazo III, Kovacs, Rios & Schneider, 1979). Comparisonsof the birefringence signal and a number of dye signals have been published (Baylor& Chandler, 1978; Baylor, Chandler & Marshall, 1981). While Suarez-Kurtz & Parker(1977) and Baylor, Chandler & Marshall (1979) agree that birefringence signals andArsenazo III transients start simultaneously, there is still no detailed comparison ofthe whole time course of birefringence signals and calcium transients.We now report measurements of calcium transients recorded with the metallo-

chromic calcium indicatorAntipyrylazo III and birefringence signals during activationof voltage-clamped cut skeletal muscle fibres under a variety of conditions. Whileboth signals have a very similar voltage dependence of onset, slope and peakamplitude and respond in the same way to dantrolene sodium and caffeine treatment,there is a consistent difference in the time course. After a simultaneous onset thebirefringence signal peaks later and declines more slowly than the Antipyrylazo IIIcalcium transient.

Preliminary reports of these findings have been presented as abstracts (Kovacs,Schiimperli & Szuics, 1981; Schfimperli, Kovacs & Szuics, 1982).

METHODS

Single fibres dissected from the semitendinosus muscle of the frog (Rana esculenta) were cut inrelaxing solution and mounted in a single-gap chamber. A voltage-clamp circuit was used torepolarize the fibres and to apply depolarizing pulses. The cut-fibre preparation and the single-gapvoltage-clamp technique have been described by Kovacs & Schneider (1978).For further details of the methods not described below, see the previous paper (Kovacs & Szucs,

1983).

SolutionsThe dissection was carried out in Ringer solution containing the following constituents

(mmol 1-1): NaCl, 115; KCl, 2-5; CaCI2, 1-8; Tris buffer, 2. The composition of the external solutionapplied to the closed end pool was (mmol l'-): tetraethyl ammonium sulphate ((TEA)2504), 76;CS2S04, 5; Tris buffer 5; CaSO4 8; and 3-1 x 10- mol 1-1 tetrodotoxin (TTX). The internal (relaxing)solution for the mounting procedure contained (mmol 1-'): K glutamate, 120; MgCl2, 2; Tris buffer,5; ethyleneglycol-bis(fl-aminoethylether)N,N'-tetraaceticacid (EGTA), 0.1. After completing theseparation, internal solution containing EGTA 1 mmol 1-1, and adenosine 5'-triphosphate (ATP),0 5 mmol 1-1 was applied to the open end pool. The pH of all solutions was adjusted to 7-0. Theexperiments were carried out at low temperature (2-4 °C).

580

BIREFRINGENCE AND Ca2+ SIGNALS IN MUSCLE

Drug effectsDantrolene sodium and caffeine were dissolved in the external solution and were applied to the

closed end pool. The measurements were started 5 min after drug application. No additional changein the drug effect was found during the experiments. Usually a recovery period was observed afterchanging the solution at the closed end pool for drug-free external solution.

Measurement of birefringence signalsTo measure birefringence transients, linear polarizers (Polarex P-W 40) were placed in the path

of monochromatic light of 790 nm both before the fibre (polarizer) and between the fibre and thephotodiode analyzerr) oriented in a crossed position, i.e. ±450 with respect to the fibre axis (Fig.1). The resting retardation of the fibres was determined with a Babinet compensator which allowedthe introduction of a variable amount of retardation.

In all Figures the upwards deflexion of the birefringence records represents a decrease in lightintensity, to facilitate the comparison with the calcium transients. Fibre contraction usuallyresulted in a delayed downward deflexion due to transmission and birefringence changes.The signal-to-noise ratio for birefringence transients was usually worse than for calcium signals,

therefore digital filtering was used to improve the birefringence records. In the smoothing procedurethe nth sample point was replaced by the sum: n/2 + (n-i1)/4 + (n + 1)/4 (Armstrong & Gilly, 1979).Kinetic parameters of the transients were determined before filtering.

RESULTS

Validity of the methodThe validity of a detailed comparison of Antipyrylazo III and birefringence

transients is limited by the degree of contamination of the signals by artifacts. Themain source of artifacts in optical measurements using muscle fibres, is mechanicalmovement. Therefore, we avoided fibre contraction whenever possible by usingsubthreshold or just-threshold depolarizing pulses. Calcium-dependent absorptionchanges of Antipyrylazo III were recorded at 720 nm. If movement was present, itstime course could be detected by measuring fibre transmission at 790 nm, where thefree Antipyrylazo III as well as the Ca-dye complex have negligible absorption (seeScarpa, Brinley & Dubyak, 1978, Kovacs et al. 1979).

Birefringence signals were tested for artifacts by inserting half a wave-length ofoptical retardation between polarizer and analyzer. Under these conditions, bire-fringence signals are expected to have equal time course and absolute amplitude(the same absolute change in light intensity, Al, independent of the background lightintensity, I) but opposite polarity (Baylor & Oetliker, 1977 b). Trace b in Fig. 1 B showsthat the average of traces a (birefringence signal without additional retardation) andtrace c (birefringence signal after the addition ofA/2 of additional retardation) is closeto a straight line, indicating negligible contribution from fibre transmission changes.A late, movement related component of opposite polarity (compared to the early partof the signal) sometimes seen in the birefringence signals (e.g. Fig. 7 A, trace e; Fig.8A, trace c) was also partially inverted by A/2 of additional retardation, indicatingthat this movement artifact involves changes in birefringence.

Birefringence signals are optimally recorded if the fibre's resting retardation is closeto A/4 (Baylor & Oetliker, 1977b). The resting retardation in the middle of the fibrewas usually in the range of A/5 to A/3 (160 nm-280nm). In a few cases ofexceptionally thick fibres, the resting retardation approached or even exceeded A/2.Signals from such fibres are difficult to interpret and were excluded from analysis.

581

582 L. Ko vics, R. A. SCHCMPERLI AND G. SZ CS

To prevent any slow changes in fibre properties from influencing the comparison,birefringence and Antipyrylazo III signals under identical conditions were alwaysmeasured in rapid succession.

A B

|IZ D .:7I.n, | P2 'a1~P

F

Do C

|L _l l

Fig. 1. A, schematic diagram of the optical set-up. L, monochromatic light; P,, polarizerfilter; B, Babinet compensator; C, objective; F, fibre; W, water immersion objective; P2,analyzer filter; D, photodiode. B, birefringence signals evoked by 60-ms-long depolarizingpulses (indicated by vertical bars). The membrane potential was -52-9 mV. Trace a wasmeasured before, while trace c after the addition of half a wave-length of opticalretardation. Trace b represents the average of a and c. Amplitude calibration representsthe absolute change in light intensity, AI, expressed as a voltage change 0 5 mV. Restinglight intensity (I) was 1112 mV (a) and 1278 mV (c). Fibre 00912, d = 84 #sm, 8 = 2-7 /sm.Eight sweeps averaged.

Time course of calcium and birefringence signalsFig. 2 shows a calcium transient and a birefringence signal recorded from a typical

fibre in response to a depolarizing pulse just at the threshold for visible movement(threshold pulse, 20 ms; from -100 mV to -20-6 mV). The scale in Fig. 2A has beenadjusted to the same peak amplitude for both signals. Following a sigmoidal foot,both signals have a nearly linear rising phase and decline to base line with a timecourse following a single exponential. However, after an apparently simultaneousonset, the birefringence signal rises more slowly, has a longer time to peak (20-5 msV8. 17 ms) and an increased time constant of the falling phase (31-9 ms vs. 14-7 ms).

Fig. 2B indicates that, after appropriate scaling, most of the rising phase of thebirefringence signal superimposes the rising phase of the calcium transients. Thedifference appears to develop at the peak of the calcium transient.The signal onsets, though indistinguishable, are not all well resolved in Fig. 2A, B.

Several separate, high resolution records have been obtained of this critical signal

BIREFRINGENCE AND Ca2+ SIGNALS IN MUSCLE

part. Fig. 2C shows two pairs ofsignals in response to two different depolarizing pulsesrecorded at high gain and temporal resolution (02 ms per point, sixteen sweepsaveraged). After scaling, there is no detectable difference between the birefringenceand the calcium traces; the signals are superimposable.

A

C

b

CI

Fig. 2. A, B, comparison of the time course of calcium transients (continuous lines) andbirefringence signals (dotted lines) evoked by the same depolarizing pulses indicated byvertical bars (10 ms to -20-6 mY). A, superimposed traces with equal peak amplitudes.B, superimposed traces having the same rate of rise after appropriate scaling. Amplitudecalibration for the birefringence signal, AI/I, is 1-5 x 10-4 in both A and B. Al/I representsthe fractional change in light intensity. Amplitude calibration for the calcium transients,AA/A560, 0.5 X 10-4 in A and 0-6 x 10-4 in B.AA/A6,50 represents the fractional changeof fibre absorbance at 720 nm relative to the resting absorbance at 550 nm. Fibre 00924,d = 98 m, 8 = 2-8,Xm. Eight sweeps averaged. C, comparison of the onset of birefringencesignals (a, c) and calcium transients (b, d). The membrane potential during the pulse was-20g8mY for 15 ms (a, b) and -e39k4m for 30 ms (c, d). The beginning of the pulsesis indicated as a vertical bar below the individual traces. The time calibration represents10 is. Amplitude calibration (Af/I) for a,4A 5 x 10-; 5 X10-4 for c and AA/AI/ 0 for b1o2 x 102; for d1b 4 xa10 . Fibre 10205 (a, b) data not recorded. Fibre 11020, d = 91 4m,d = 2948um (c, d). Eight (a, b) and sixteen (c, d) sweeps averaged.

For the purpose of characterizing changes, the time to detectable beginning of thesignals can be calculated. This 'latency time' (defined as time from pulse onset tothe first point beyond two standard deviations from the mean base line) is longer forthe birefringence signals (Fig. 2C, trace a,842 ins; trace c, 12c0 ins) than for thecorresponding calcium transients (trace b, 7t4ns; trace d, 10i8 is), due to the bettersignal-to-noise ratio for the latter signals. Thus, 'latency' can be different from thetime to signal onset. However, it is useful to compare signals of the same kind under

583

L. KOVACS, R. A. SCHUMPERLI AND G. SZUCS

different conditions. In Fig. 2 C, for example, the 'latencies' are shorter for bothsignals in response to the larger depolarization (traces a and b compared to traces cand d). The minimum latency time calculated in our experiments was 6 ms. Thisindicates a voltage dependence of the signals which is discussed in moredetail below.

AA/ 5 X10-4

B AA 4X10-3A550

.1

8.1

_,. 4.*-,-- -28 6

-33.3

-38 2

Aid.A. . -:IVY-429

Fig. 3. Birefringence (A) and calcium (B) transients evoked by 100 ms depolarizing pulses(indicated by vertical bars). Membrane potential values are shown at each pair of traces.The total dye concentration in the myoplasmic space of intact terminated segment (DT)was 0-26 mmol 1-1. Vertical calibration for B corresponds to the change in free myoplasmiccalcium concentration (ACa) of 1-21 /zmol 1-1. The ACa was determinated using thecalibration procedure of Rios & Schneider (1981). Fibre 11022, d = 95 jUm, s = 2 7 Itm.Four or eight sweeps averaged.

Voltage dependence of calcium and birefringence signalsIn order to prevent movement artifacts from aifecting the time course of the

optical signals, experiments to study the voltage dependence of birefringence andcalcium transients were limited to pulse amplitudes below contraction threshold. Insome fibres, however, a threshold less negative than usual (possibly due to warmadaptation, see Kovacs & Schneider, 1977) permitted the use of relatively large andlong depolarizing pulses. In Fig. 3, records from a fibre are shown, which had acontraction threshold more positive than-20 mV for 100 ms rheobase pulses. Theuse of long pulses has the advantage that the whole rising phase and the peak of thesignals occur during the depolarization.

It is clearly visible in these traces that in both the birefringence (Fig. 3A) and thecalcium transients (Fig. 3B), the take off occurs earlier, the slope of the rising phaseand the peak amplitude increase and the time-to-peak shortens with increasing pulseamplitude.

584

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--l-.11,

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I.,.: I-

: il ,..% ^

f

BIREFRINGENCE AND Ca2+ SIGNALS IN MUSCLE

The voltage dependence of rate of rise, time-to-peak and peak amplitude of thesesignals is analysed in Fig. 4. Due to the rather noisy base lines of the birefringencesignals, 'latencies' were not calculated.

Fig. 4A compares the peak amplitudes of birefringence and calcium signals afterscaling to the same amplitude of the largest pair of signals. There is no detectabledifference in the voltage dependence of the signal amplitudes. Fig. 4B shows that

12 go 8

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MembrodstoAan potential (mV) Tesclswr Membranedtspoentialos (mVeFmpig.d4.Voltaebige-dpnetpigaraBtmeters mtepleoseotepaof birefringencesinl(cre)adcliusigame experaiumen tansientFi.3.C, peake ampitue of birefringence signals((A/I)t, left

ordinate) and calcium transients ((AA/A~c0)/At, right ordinate). The scales were adjustedto show the similarity in the voltage dependence of the rate of rise of both signals. D,relative rate of rise, rate of rise divided by peak amplitude of each signal.

time-to-peak decreases for both signals in a very similar way but the birefringencesignals are consistently slower. The voltage dependence of the rate of rise of the twosignals is again indistinguishable (Fig. 40). The scales have been adjusted to facilitatethe comparison. If the rate of rise is divided by the peak amplitude of each signal(Fig. 4D) this relative rate of rise still increases with pulse amplitude for both signals.Apart from the smallest pair of signals (membrane potential -42*9 mV) where thedetermination of slope and signal amplitude are most uncertain, the relative rate ofrise of the calcium signals is consistently larger. This confirms the conclusions drawnfrom Fig. 2A.

585

586 L. KovAcs, R. A. SCHCMPERLI AND G. SZLTs

Calcium and birefringence signals at the contraction thresholdDepolarizing pulses along a strength-duration curve for just-visible movement

allow the observation of signals over a wide range ofmembrane potentials in normallycontracting fibres without the complications introduced by fibre movement. Since allthese pulses bring the fibre just to the contraction threshold, it can be expected that

..~.* .-

-31 -6 - -2 5-.A*~~ ~~~~~-, '% ..

5~ ~ ~ ~ 2 *.:-22 1 ::,>J ~~~~~~~6 32~~~2

4 X.

-127

Fig. 5. Birefringence signals at the contraction threshold. After determination of thestrength-duration curve for the threshold, depolarizing pulses (parameters calculated)were applied to evoke the optical signals (see Methods for further details). Pulse durationsare indicated by vertical bars. Time calibration represents 20 ms. Membrane potentialvalues are shown next to the corresponding traces. Fibre 01124, d = 112 jtm, 8 = 2-4 /m.Eight sweeps averaged.

they activate excitation-contraction coupling to the same extent. In agreement withthis prediction, it has been shown that pulses along a strength-duration curveproduce constant amounts of charge movement (Horowicz & Schneider, 1981)andthat threshold calcium transients have constant amplitude (Kovacs & Szuics, 1981,1983). In addition, the time constant of the falling phase of the threshold calciumtransients does not depend on the previous pulse voltage (Kovacs & Szufcs, 1983).

Fig. 5 demonstrates that threshold birefringence signals share these properties.They have equal amplitude and a voltage-independent falling phase along a strength-duration curve. It is important to note that the major part of these signals occursafter the membrane has been repolarized, in contrast to the situation in Fig. 3.Table 1 presents a quantitative analysis of amplitude and time constant of the

falling phase of the birefringence (Fig. 5) and calcium signals obtained on the samefibre and confirms the voltage independence of both parameters in birefringence and

BIREFRINGENCE AND Ca2+ SIGNALS IN MUSCLE

calcium signals. The time constant of the falling phase of the birefringence signalsis about three times longer, confirming the differences in signal time course alreadynoted.

In Fig. 6, threshold signals in response to rheobase (100 ms, - 36-5 mV) and short(5 ms, + 14-3 mV) pulses are compared in another fibre. While the signal amplitudeagain is not voltage-dependent, there is a striking similarity in the influence of the

TABLE 1. Comparison of birefringence and calcium transients at contraction thresholdBirefringence Calcium

Depolarization(AI/I) max ACa

Vm (mV) tth (MS) (x 10-4) T (Ms) (Smol 1-1) T (Ms)-31-6 12-2 6-04 16-1 1.51 5-7-22-1 9-2 6-28 14-4 1P55 5-3- 12-7 7-4 6-32 15-0 1-58 5-4-2-5 6-1 540 18-3 1-49 5-6+6-3 5-3 5-92 15.1 1-40 5-2

A -, B, *. *.

*.. *. s %v s

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. r ; >o . 36-2 Gore-ssors>

A/ 5x10-4

11

M 2x10-3A550

It

.I

+14-3 i, NAs11v~

Fig. 6. Threshold birefringence (A) and calcium (B) transients evoked by long (100 ms)and short (5 ms) depolarizing pulses (indicated by vertical bars below the individualtraces). The membrane potential values during the pulses are shown between thecorresponding traces. Fibre 11025, d = 91 um, 8 = 2-6 em. Eight sweeps averaged.

pulse duration on the shape of both signals. Birefringence (Fig. 6A) and calcium(Fig. 6B) transients in response to the rheobase pulse develop more slowly anddecline to a plateau during the depolarization. The kinetics of the calcium signalsare again faster than those of the birefringence transients, which is particularlynoticeable in the rheobase signals (Fig. 6, upper traces).

587

A

I

t

Nlw%

I-

-e-,.

L. KOVACS, R. A. SCHUMPERLI AND G. SZUCS

Effect of dantrolene sodium on calcium and birefringence signalsDantrolene sodium is a muscle relaxant known to interfere directly with calcium

release (e.g. Lopez, Helland, Wanek, Rildel & Taylor, 1979). Therefore, the effectsof this substance on birefringence and calcium signals were compared. By observingthreshold signals, we expected to obtain some information about a possible additional

AB

5 X10-4

a .-Amp

A.r_ ACa 1-5 mu11~ ~ Z~ 15

D:

Dantrolerart_-~~~~~_-36 2 .:'

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b*'

: I.. .Dantrolene sodium

-

d ..&~- --=a=-f -

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ds-s at_ -

'-Se -2

36-2

36 2-7

62

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S.

Fig. 7. The effect of dantrolene sodium at a concentration of one-tenth saturated on thebirefringence (A) and calcium (B) transients evoked by 100-ms-long depolarizing pulses(indicated by vertical bars). Membrane potential values are shown as in the previousFigures. Records a, d, and e were obtained in control solution; b and c in external solutioncontaining dantrolene sodium. See text for further details. DT = 0-31-0O50 mmol 1-1. Dueto the definite increase in dye concentration traces represent ACa in B instead of AA/A550.Fibre 11025, d = 91 ,um, 8 = 2-6 Gem. Eight sweeps averaged.

influence ofdantrolene on the contractile proteins. A dantrolene concentration ofone-tenth saturated was chosen which produced a moderate and reversible shift of thecontraction threshold to more positive membrane potentials (about 6-10 mV forlonger pulses). One-twentieth saturated solution had almost no effect and halfsaturated and saturated solution appeared to lead to irreversible fibre damage.

In Fig. 7, the effect of dantrolene sodium (one-tenth saturated) is compared forcalcium transients and birefringence signals in response to 100 ms depolarizations.Traces a represent control signals at the threshold for just-visible movement. Afterthe application of dantrolene, the signals b were recorded at the same membranepotential and pulse duration (now subthreshold). The amplitude decreased by 50%.Increasing the pulse amplitude to the new threshold potential essentially restored the

588

-11-7o-

"I.

I

BIREFRINGENCE AND Ca2+ SIGNALS IN MUSCLE

control signal amplitude, as shown in traces c. After washout of dantrolene, tracesd and e show the signals produced by the control threshold pulse and by the thresholdpulse under dantrolene, respectively. The last signals with an amplitude of 1200 arenow clearly above the threshold for just-visible movement and contaminated bymovement artifacts. Dantrolene did not significantly affect the time course of thesignals.

A B

.^<ark / ~~~~10o-3'9-

,fl,~~ -....--b

a

b

CaffeineCaffeine.

- ~ ~ ~ ~ ~ ~ ~ ~ ~~ C

Fig. 8. Effect of caffeine 0 5 mmol 1-l on the birefringence signals evoked by long (100 ms;A) and short (10 ms; B) depolarizing pulses (indicated by vertical bars at the bottom ofthe Figure). Traces a, b and d were recorded in control solution while traces denoted byc were obtained in external solution containing caffeine. Membrane potential values duringthe pulses were in A -48-6 mV (a) and -52-4 mV (b-d); in B-33-3 mV (a) and 38-1 mV(b-d) respectively. See text for further details. Fibre 11027, d = 91 min, s = 2-7 #mm. Fouror eight sweeps averaged.

Effect of caffeine on birefringence signalsCaffeine is a well studied twitch potentiator in skeletal muscle (LUttgau & Oetliker,

1968) and its effect on intracellular calcium transients has been described recently(Kovacs & Szuics, 1981, 1983). At a concentration below the threshold for activatinga contracture (0-5 mmol 1-1 at 3 0C), the amplitude of calcium transients is increasedand their latency reduced by caffeine. The threshold for just-visible movement isshifted to more negative membrane potentials. At the new threshold, calciumtransients are smaller and have a shorter latency than control threshold signals. Wetested whether birefringence signals are affected in a similar way by caffeine.

In Fig. 8, birefringence signals are shown under control conditions at threshold(traces a) and well below threshold (traces b) in response to 100 ms (Fig. 8A) and 10 ms(Fig. 8B) pulses. After the application of caffeine (0-5 mmol 1-1) the birefringencesignal amplitude increased by a factor of 6 for the 100 ms pulse (trace c, Fig. 8A)

589

L. Kovics, R. A. SCHCMPERLI AND G. SZUCS

and by a factor ofabout 2-5 for the short pulse (trace c, Fig. 8B). 'Latency' decreasedfrom 30 ms to 17 ms in A and from 13 ms to 11-5 ms in B. These effects werecompletely reversibly (traces d). Due to the shift in threshold to more negativepotentials, the long pulse under caffeine (trace c, Fig. 8A) is now above thresholdand the signal shows a movement component.Due to the caffeine treatment, the threshold strength-duration curve is shifted to

more negative potentials at given pulse durations or to shorter pulses at givenmembrane potentials. To investigate the effects of caffeine on birefringence signalsat contraction threshold, the pulse duration was reduced at given pulse amplitudesto eliminate voltage-dependent changes in the signals.As already shown for calcium transients, the birefringence signals at threshold were

smaller in the presence of caffeine than in control solution by an average value of35% (ranging from 15 to 64% in seven determinations) and the 'latency' was reducedalso.

DISCUSSION

The purpose of our study was a detailed comparison of birefringence signalssupposedly related to excitation-contraction coupling of skeletal muscle and thechanges in myoplasmic calcium concentration accompanying the depolarization ofmuscle fibres. The birefringence transients recorded in our system are assumed to bethe equivalent of the early large birefringence signal described by Baylor & Oetliker(1977 a, b) in intact fibres based on the following reasons: both signals have the samepolarity and are almost pure birefringence changes (see Fig. 1 B). Birefringencetransients following action potentials at 3 OC have an estimated delay of 4-5 ms(Schfimperli & Oetliker, 1981), in good agreement with the calculated minimal'latency' of 6 ms observed in our experiments using short and large voltage-clamppulses. Both signals can be separated from the optical changes related to fibrecontraction.The Antipyrylazo III transients measured at 720 nm can be taken, within certain

limits, as a linear measure of intracellular calcium concentration changes with a timecourse only minimally affected by changes in other intracellular ions (Scarpa et al.1978; Kovacs et al. 1979; Rios & Schneider, 1981; Baylor, Chandler & Marshall,1982 a). Therefore, the comparison ofthe birefringence andAntipyrylazo III transientscan give information about the relation between the birefringence and myoplasmiccalcium concentration changes.The main result of our study is that both calcium and birefringence signals are

affected in a very similar way by all parameters and interventions tested. At the sametime we find that after a simultaneous onset, the birefringence signal consistentlypeaks later and declines more slowly than the calcium transient.The voltage dependence of the optical signals was investigated with pulses lasting

longer than the time-to-peak of the transients (Figs. 3 and 4). The time-to-peakdecreased, while the rate of rise and the peak amplitude increased with increasingpulse amplitude in both signals in the same way. The voltage dependence of thebirefringence signal in our study is similar to that reported by Baylor & Chandler(1978) using a 2 micro-electrode voltage clamp.Along a strength-duration curve for just-visible movement the rising phase and

590

BIREFRINGENCE AND Ca2+ SIGNALS IN MUSCLE

time-to-peak of the signals again were voltage-dependent, but the peak amplitudeand the time constant of the falling phase were constant (Figs. 5 and 6; Table 1) inagreement with findings already reported for threshold calcium transients (Kovacs& Szucs, 1983).

In the presence of dantrolene sodium and caffeine, the changes ofcalcium transientsand birefringence signals were proportional. The muscle relaxant dantrolene sodium,by suppressing calcium release, decreased the amplitude ofboth signals in comparablemanner. The finding that both calcium and birefringence signals are virtually restoredto control amplitude at the new elevated threshold under dantrolene is in a goodagreement with the results of other type of experiments that this substance does notaffect the contractile proteins directly (Hainaut & Desmedt, 1974; Takauji, Takahashi& Nagai, 1975; Takauji, Takahashi, Suzuki & Nagai, 1977; Lopez et al. 1979). Onthe other hand, caffeine facilitates the release of calcium. The birefringence signalsare increased and accelerated at a given depolarization and the threshold signals aresmaller and earlier in the presence of caffeine than in control circumstances. Botheffects have already been described for the calcium transients by Kovacs & Szucs(1983).

Careful studies of the early parts of calcium and birefringence signals indicateindistinguishable onsets (Fig. 2C), in agreement with reports by Suarez-Kurtz &Parker (1977), Baylor et al. (1979) and Baylor, Chandler & Marshall (1982 b). However,under all experimental conditions tested, the birefringence signal peaked later anddeclined more slowly. There is some fibre-to-fibre variation in the extent of thisdifference but we never recorded fully superimposable signals. These differencescannot be due to the recording system, since both signals were measured with thesame apparatus in the same range of light intensities and at similar wave-lengths.They are not due to disturbance of the birefringence signals by a high restingretardation which usually was between A/5 and A/3 (i.e. 160 nm-280 nm). They alsocannot be attributed to small changes in fibre transparency, since contributions fromtransmission changes are much smaller and, if present, are visible in both signals (seeFig. lB and Kovacs et al. 1979).The most important question concerning the present study is what kind of

processes are reflected in the birefringence transients? Originally it has beensuggested that the birefringence and the potentiometric dye signals of similar timecourse might reflect sarcoplasmic reticulum (s.r.) membrane potential (Baylor &Oetliker, 1975, 1977a; Bezanilla & Horowicz, 1975).Recent evaluation ofthis hypothesis indicated that a large and sustained membrane

potential change of the s.r. during calcium release is highly unlikely (Oetliker, 1982).Therefore, our data rather suggest that the changes in birefringence are related tothe changes in myoplasmic calcium concentration. This view is supported by thestrong similarity in the voltage dependence and time course of the two signals andby the comparable influences ofpharmacological agents observed in our experiments.The mechanism by which the calcium ions can modify the optical retardation of

the fibres is uncertain at the moment.Although the contraction of the fibres was avoided as far as possible by our

experimental conditions, the subthreshold activity of the contractile proteins cannotbe excluded. For example the calcium ions can bind to different binding sites (e.g.

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592 L. KO VACS, R. A. SCHCMPERLI AND G. SZ(CS

troponin or the regulatory light chains of myosin) and change the properties of thesehighly oriented structures.Another possible calcium-binding site is the s.r. membrane. Recent experiments

by Oetliker (1980, 1981 a, b) suggest that the Ca-ATPase of the s.r. may be involvedin optical signals during excitation-contraction coupling. He showed that changes inextravesicular calcium concentration evoke an indodicarbocyanine fluorescencesignal in intact, as well as leaky, s.r. vesicles and even in a delipidated Ca-ATPasepreparation. Since the indodicarbocyanine fluorescence and the early large bi-refringence signal in intact fibres are remarkably similar (Oetliker et at. 1975; Baylor& Chandler, 1978) a common mechanism could underlie both signals. In agreementwith this interpretation, measurements of birefringence in partially fused andflattened s.r. vesicles indicate highly ordered protein domains in the calciumtransport molecule contributing to the intrinisic birefringence of the s.r. membrane(Stromer & Hasselbach, 1976).Our results do not permit us to exclude completely the possibility that the

birefringence signal represents a potential change ofthe s.r. membrane, but it appearsmuch more plausible that it reflects calcium bound to certain sarcoplasmic bindingsites.

We thank Professor E. Varga for continuous support, Dr M. F. Schneider for the critical readingof the manuscript and for valuable suggestions, Dr H. Oetliker for helpful comments on the results,M. Fuxreiter for helpful assistance. The experiments were carried out in Debrecen, Hungary andwere sponsored by the Hungarian Ministry of Health (grant no. 17/2.06/072). Dr Schfimperli's staywas supported by University of Bern, Switzerland.

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