Caffeine potentiation of calcium release in frog skeletal muscle fibres

J. Physiol. (1986), 375, pp. 535-559 535With 11 text-figuresPrinted in Great Britain

CAFFEINE POTENTIATION OF CALCIUM RELEASEIN FROG SKELETAL MUSCLE FIBRES

BY M. DELAY, B. RIBALET AND J. VERGARAFrom the Department of Physiology, Jerry Lewis Neuromuscular Research Center,Ahmanson Laboratory of Neurobiology, University of California at Los Angeles,

Los Angeles, CA U.S.A.

(Received 2 April 1985)

SUMMARY

1. The effects of caffeine at concentrations up to 3 mm were studied on Ca signalsobtained using the metallochromic Ca indicator dyes Arsenazo III and Antipyryl-azo III in cut frog skeletal ntiscle fibres mounted in a triple Vaseline-gap chamberand stimulated by voltage clamp or action potential.

2. The peak amplitude of the transient absorbance change due to Ca2+ releasefollowing action potential stimulation is potentiated by an amount dependent oncaffeine concentration up to 0-5 mm, and by a concentration-independent amountbetween 0 5 and 2 mm. At 3 mM-caffeine, the potentiation is reduced, and the Ca signalcan have a smaller amplitude than under the control condition. The time course ofthe rising phase of the Ca signal is preserved by the potentiating effect of caffeine;however, the decay rate of the Ca signal is increasingly slowed at caffeine concentra-tions greater than 0-5 mM.

3. No substantial change was found in the resting myoplasmic Ca2+ level at caffeineconcentrations near 0-5 mm. Even if the free Ca2+ concentration in the presence ofthis level of caffeine were to increase by 0-04 /M (the threshold of delectability), thecalculated potentiation of the Ca signal due to increased partial saturation ofintracellular Ca2+ buffers would amount to only about 7 %. This value is significantlyless than the amount ofpotentiation observed (up to 40 %) following action potentialsat caffeine levels of 0 5 mm and above.

4. Experiments made with the impermeant potentiometric dye NK2367 show noalteration by caffeine of the electrical properties of the tubular system.

5. Caffeine at up to moderate concentrations causes a substantial increase in themaximal Ca2+ release obtainedfollowing large depolarizations. The voltage dependenceof the Ca2+ release is characterized by a caffeine concentration-dependent shifttowards more negative membrane potentials.

6. The potentiation of Ca2+ release by caffeine was found to be independent of theexternal free Ca2+ level.

7. The potentiation of the Ca2+ release process by caffeine is likely to occur at astep subsequent to the depolarization of the transverse tubule membrane, andsuggests the presence ofan intermediate step, or second messenger, in the excitation-contraction coupling process.

M. DELA Y, B. RIBALET AND J. VERGARA

INTRODUCTION

Caffeine potentiates twitch tension in skeletal muscle at concentrations near 1 mM(Sandow, Taylor, Isaacson & Seguin, 1964). Several factors may in principlecontribute to this effect, but can be eliminated on the basis of previous work. Forexample, this level of caffeine does not cause substantial change to surface membraneelectrical properties (Axelsson & Thesleff, 1958; Sandow et al. 1964; Lfittgau &Oetlicker, 1968). The hypothesis that twitch potentiation is an effect of a caffeine-induced increase in the resting myoplasmic-free Ca concentration (Sandow et al. 1964)was addressed by Lopez, Alamo, Caputo, DiPolo & Vergara (1983), who usedCa2+-sensitive micro-electrodes to show that 1-2 mM-caffeine does not detectablyincrease the resting free Ca2+ level in single muscle fibres, within a resolution of0-01 /M. Caffeine has also been shown to increase the force-generating ability of thecontractile filaments (Wendt & Stephenson, 1983); however, this effect is small near1 mM-caffeine, and cannot account for the observed magnitude of twitch potentiation.These results therefore raise the possibility that low levels of caffeine directlypotentiate Ca2+ release in skeletal muscle.Kovacs & Szucs (1983) studied the effects of caffeine upon the transient increase

in myoplasmic Ca2+ following stimulation (the 'calcium transient'), and found that0 5 mM-caffeine shifts the voltage dependence of the Ca signal amplitude. However,a simple shift in the voltage dependence of the Ca transient cannot account for themagnitude oftwitch tension increase, since the voltage dependence of tension has beenshown to saturate at membrane potentials lower than that reached by an actionpotential (Hodgkin & Horowicz, 1960). The further observation by Kovacs & Szucs(1983) that caffeine does not alter the voltage dependence of the non-linear chargemovement suggests that caffeine may not modify electrical properties of thetransverse (T-) tubular membrane. It may be proposed, therefore, that low concen-trations of caffeine potentiate electrically stimulated Ca2+ release, and that this takesplace at a step subsequent to the electrical excitation of the T-tubule.

This paper gives the results of a direct test of this hypothesis on the basis ofcomparative measurements ofCa signals, using Ca-sensitive dyes, in the presence andabsence of caffeine. We report results using two dyes with different Ca2+-bindingcharacteristics, Arsenazo III and Antipyrylazo III, in order to eliminate possiblebiases due to specific properties ofeither dye. The experiments were made on cut singleskeletal muscle fibres mounted in a triple Vaseline-gap chamber, with the myoplasmdiffused from the ends with a solution containing a Ca-sensitive indicator dye andup to 3 mM-EGTA, used in order to reduce developed tension and lessen the effectof the consequent fibre motion upon the Ca signals.The effect of caffeine upon the magnitude and voltage dependence of Ca signals

was studied in voltage-clamped fibres. Luittgau & Oetlicker (1968) proposed thatcaffeine may affect the electrical conductances present in the transverse tubularsystem; T-tubule potential was therefore studied on fibres stained with an impermeantpotentiometric dye. Since caffeine may increase the flux of external Ca2+ into themyoplasm (Bianchi, 1961), the effect of caffeine on Ca signals was studied usingexternal solutions containing Ni2+ or low free Ca2+ concentrations. Preliminaryresults have been presented elsewhere (Delay, Ribalet & Vergara, 1983).

536

CAFFEINE POTENTIATION OF Ca RELEASE

METHODS

The apparatus and general methods used in these experiments are fully described in previouspublications (Vergara, Bezanilla & Salzberg, 1978; Palade & Vergara, 1982). Compositions of thestandard internal and external solutions are given in Table 1; the legend for each Figure gives any

TABLE 1. Compositions of solutions used in the experiments. The levels ofK EGTA and Ca indicatordye varied with the experiment, and the values given here are representative. The values for eachexperiment presented in a Figure, if different from these, are given in the Figure legend

1. K- or Ca-based internal solution (mM)K aspartate 100MgCl2 (3 mm was used in some experiments) 2Na ATP 3Na creatine phosphate 5K MOPS 20K EGTA 1.5Up to 1-5 mM-Arsenazo III or 2-5 mM-Antipyrylazo III.

The Cs-based solutions contained Cs substituted for K.All solutions used in these experiments were adjusted to pH 7x1.2. Normal Ringer solution

NaCl 115KCl 2x5CaCl2 1-8K MOPS 5Dextrose 10

3. 1 Na-, I TMA-based external solutionNaCl 57.5TMA Cl 57.5

in addition to other unchanged components of normal Ringer solution.4. TTX-containing external solution

0-2 ,uM-TTX in normal Ringer solution.5. Low Ca2+ external solution

CaCl2 0 4K EGTA 1MgCl2 3

and other unchanged components of normal Ringer solution.The Ca2+ concentration of this solution measured 0-2 /M using Ca2+-sensitivemicro-electrodes. Further low external Ca experiments were performedwith a solution of the following composition, the Ca2+ levelof which was estimated to be less than 0-01 #m:

NaCl 115KCI 1.5K EGTA 1MgCl2 5Na MOPS 5Dextrose 10

6. Ni-containing external solution1 mM-NiCl2 in normal Ringer solution.

differences with respect to these. Normal Ringer solution was applied externally (Solution 2 ofTable 1), unless otherwise stated. The internal solution which diffused into the myoplasm throughthe cut ends of the fibre contained ethyleneglycol-bis(/1-aminoethylether)N-N'-tetracetic acid(EGTA) and one of the metallochromic Ca2+ indicators Arsenazo III or Antipyrylazo III as listed

537

538 M. DELA Y, B. RIBALET AND J. VERGARA

in Table 1. It was found that both Arsenazo III and Antipyrylazo III diffuse readily into the fibre;typically 25-35 min were required to admit enough dye to permit the measurement of Ca signalswith a high signal-to-noise ratio. Experiments were performed starting at this time. Diffusion ofthe dye into the fibre has considerable advantage over other techniques reported previously(ionophoresis for Arsenazo III; ionophoresis and pressure injection for Antipyrylazo III) (Miledi,Parker & Schalow, 1977a; Palade & Vergara, 1982; Baylor, Chandler &.Marshall, 1982) in that

A

A E

A-OlAA 0-002

660nm * 710 n

-J

0.0011B 0-0021 F _

790nm 790nm

C, G

0-01 0-002

-J J - * \~%b-D t H\ 50omVL

50mV 20 ms

20 msFig. 1. An illustration ofthe procedure used to subtract motion-related contributions fromCa-dye dignals in skeletal muscle fibres. The traces on the left show the procedure forArsenazo III, and those on the right, for Antipyrylazo III. Traces D and H show themembrane action potential used to stimulate the fibre. Traces A and E show theabsorbance changes, AA, following stimulation at wave-lengths where both Ca signals andmotion contributions are present. Traces B and F show pure motion signals recorded atlong wave-length, and traces C and a show the result of the subtraction procedure. Seetext for details. The internal solutions used in these experiments were those of Table 1,except for 1-25 mM-Arsenazo III and 3 mM-MgCl, for the left-hand traces, in which theintracellular dye concentration was 0-4 mm, and 2-5 mM-Arsenazo III, 3 mm-EGTA and3 mM-MgCl2 for the right-hand traces, in which the intracellular dye level was 1 mm. Notethat the intracellular dye concentration was measured over the region of the fibreilluminated by incident light, where fibre absorbance was monitored. Fibre diameters:Arsenazo III fibre, 100 /m; Antipyrylazo III fibre, 120,um.

the diffusion method allows the introduction of both dyes in an identical manner and avoids themechanical and electrical damage to the preparation and the spatial non-homogeneity of dyeconcentration that may result from the use of micro-electrode impalement. The myoplasmic dyeconcentration usually increased over the course of each experiment. Fibre movement consequentto stimulation diminished steadily with time, probably due to an increase in internal levels of dyeand EGTA. Dye concentration was estimated approximately using the relation C = Af/ed, whereC is the dye concentration, Af is the absorbance of the fibre measured at 530 nm for Arsenazo IIIand 550 nm for Antipyrylazo III, e is the extinction coefficient at that wave-length (given in Palade& Vergara (1983) for Arsenazo III and Palade & Vergara (1981) for Antipyrylazo III), and d theeffective path length through the fibre. The experiments were performed at near 12 °C; comparativemeasurements ofCa signals under different conditions were performed within approximately 0-2 °C


of each other. The sarcomere lengths of the fibres used were typically near 2-2 ,m. Following achange in the external solution, such as the addition of caffeine, the temperature returned to nearthe set-point value after 2 min, at which time measurements could be resumed. Successivestimulations were made at 3 min intervals in order to allow complete recovery of the Ca2+ releaseprocess.Two methodological problems were encountered in these experiments, which are discussed here:

(i) the removal of contributions to the optical signals due to stimulated motion of the fibre; and(ii) the scaling of Ca signals obtained at different times in the presence of monotonically increasingmyoplasmic dye concentrations. The motion subtraction procedure relied upon the lack of anyabsorbance by either dye at sufficiently long wave-lengths, and upon the absence ofany dependenceof the motion signal time course upon the wave-length (Delay, Ribalet & Vergara, 1982). Fig. 1shows the procedure used for motion subtraction in the presence of Arsenazo III (traces A-C) andAntipyrylazo III (traces E-G) following fibre stimulation by action potential (traces D and H).Traces A and E show the changes in absorbance, AA (defined below), measured at 660 nm and710 nm respectively, where both the early Ca signal and the later movement signal contribute.Traces B and F show the pure motion signal recorded at 790 nm. The signal in trace B is then scaledto that in trace A over the time period following the cessation of the Ca signal, and subtractedfrom trace A over the entire sample period. This procedure is also followed for trace F with respectto trace E. The results are shown in traces C and G. This can be done independently of the amountof dye and EGTA used (Delay et al. 1982), though in the presence of sufficiently high dye levelsmost of the motion is abolished. A comparison of traces C and G shows that the Arsenazo III signalhas a slower falling phase than does the Antipyrylazo III signal. This may be becauseAntipyrylazo III has a lower affinity for Ca2+ than Arsenazo III (Palade & Vergara, 1982), perhapsresulting in a faster off rate constant for the Antipyrylazo III-Ca reaction; for a review of thissubject see Vergara & Delay (1985).

In order to allow comparisons of relative Ca signals, we calculated the magnitude of the changein optical absorbance, AA, at the peak of the Ca signal; here AA is defined as logl0 (II(I+ AI)), whereI is the resting and AI is the peak change in light intensity measured at 660 nm for Arsenazo IIIand 710 nm for Antipyrylazo III. In many previous reports, including ones from this laboratory,AA has often been scaled by Af at 530 nm for Arsenazo III or 550 nm for Antipyrylazo III, in orderto approximately compensate for variations in dye concentration (Delay et al. 1983). We have found(unpublished observations) that the quantity AA/Af may introduce excessive dependence on theresting dye concentration and therefore does not always allow unbiased comparison of opticalsignals due to Ca2+ release. An empirical and more accurate method of scaling in the presence ofa steadily increasing dye concentration, based on repeated observations made under controlconditions, is briefly illustrated in Fig. 2 for a particular experiment. Absorbance changes AA weremeasured in a fibre diffused with Arsenazo III following stimulation by action potential. TracesA, B and C show the signals obtained at steadily increasing times, corresponding to intracellulardye concentrations of 0-11 mm, 0-26 mm and 0-38 mm respectively. The signal amplitude increasesmonotonically, but not linearly, with dye concentration. Traces D and F are the same as B andC, respectively; trace E was recorded, in the presence of 0 5 mM-caffeine, at a time between B andC when the dye concentration was 0-32 mm. A small amount of remaining motion contribution(enhanced by caffeine) is apparent after the end of the Ca signal. An increase of the Ca signalamplitude is seen in E relative to both D and F; however, part of the increase is due purely tothe increase in the level of the dye. A smooth curve is drawn through the points on a graph ofAAV8. Af obtained in the absence of caffeine. On the assumption that the size of the Ca2+ release inabsence of caffeine is invariant with time, this curve describes the actual dependence ofAA on dyeconcentration. Finally, traces G, H and I of Fig. 2 correspond to D, E and F, respectively, afterthe peak amplitudes are arbitrarily scaled (using the calibration curve) to a dye concentration of0-26 mm. Traces G and I show equal values of peak AA, but trace H shows a clear increase in AArelative to these. The reversibility of the effects of caffeine, upon which this method depends, isdemonstrated below in Results. This scaling procedure allows consistently unbiased comparisonsbetween Ca signals, and has been used in this report in obtaining all Figures and results whichinvolve comparisons of relative Ca signal amplitude.Some experiments were performed using the membrane-impermeant potentiometric dye

NK2367. The procedures followed in this regard are essentially identical to Heiny & Vergara(1982).

539

540 M. DELA Y, B. RIBALET AND J. VERGARA

High concentrations of caffeine (3 mM) have been directly shown to increase the myoplasmic freeCa2+ level (Lopez, Alamo, Caputo, DiPolo & Vergara, 1983). Measurements of resting lightabsorbance levels in cut fibres diffused with Arsenazo III showed (Appendix A) that no substantialchanges in the resting free Ca2+ concentration are likely in presence of 0-75 mM-caffeine. In orderto assess the effects of a possibly undetected increase in free myoplasmic Ca2+ level upon thesensitivity of the dye to transiently released Ca2+, a computer program was developed toapproximately calculate the partition of this Ca2+ between the Ca buffers Arsenazo III, troponinC and parvalbumin, in presence of Mg2+, ATP, and a given level of free Ca2+. The conclusions ofthis model are presented in Results, with details reserved for Appendix B.

A B CAA 0 003

40 ms

D E F

G H /

Fig. 2. An illustration of the procedure used to scale Ca signals recorded over a periodof time when the concentration of dye is gradually increasing. Traces A, B and C showCa signals recorded following action potential stimulation, with intracellular dyeconcentrations of 0.10, 0-26 and 0-38 mm respectively. D is identical to A, and F to C; Ewas recorded in the presence of0-5 mM-caffeine with a dye concentration of0-32 mm. TraceO is identical to D, and H and I correspond to those of E and F respectively followingtheir scaling to trace D using the procedure given in the text. The internal solution useddiffered from that in Table 1 by the presence of 0-6 mm-Arsenazo III and 3 mM-MgCl2.Fibre diameter 120 ,um.

RESULTS

We find that caffeine has a concentration-dependent effect on Ca signals. At lowconcentrations (between the lowest caffeine concentrations used, 0-125 mm, and0-5 mM), a monotonically increasing potentiation of the Ca signal is seen with caffeinelevel, where potentiation refers to the increase in the absorbance change measuredat the peak of the Ca signal following action potential stimulation. For moderatecaffeine levels, between 0 5 and 2 mm, the potentiation is independent of caffeine level,and a substantial slowing is seen in the decay phase of the Ca signal. Finally, for highlevels of caffeine (from 2 to 3 mM) the Ca signal amplitude is often smaller than thepotentiated signal at moderate caffeine levels, and may even be smaller than a signalobtained under control conditions. The results presented below are grouped according


to the appropriate caffeine level, though this is not meant to imply three distinctmechanisms of action of caffeine.

Ca signal potentiation at low levels of caffeineLow concentrations of caffeine, below 05 mM, potentiate the Ca signal following

electrical stimulation. Fig. 3A andBshows, fortwo fibres, the effects ofO05 mM-caffeineupon the Ca signals elicited by an action potential. The Ca signals are shown in the

A B

AA 0 1 AA 5 X10-4

20Oms l0ins

100mV _ 100mv

Fig. 3. The upper traces show Ca signals recorded in control solution (smaller amplitudetraces) and in 0-5 mM-caffeine (larger amplitude traces). Panel A was recorded inArsenazo III and B in Antipyrylazo III. The lower traces show the membrane actionpotentials which elicited Ca release; control is shown in the upper trace, and caffeine inthe lower. Internal solutions were standard (Table 1) except for the presence of1-25 mM-Arsenazo III (0-58 mm in pool A of the Vaseline-gap chamber) and 3 mM-MgCl2for A, and 2 mM-Antipyrylazo III (0 35 mm in pool A) and 2 mM-EGTA for B. Fibrediameters: Arsenazo III fibre, 100 /m; Antipyrylazo III fibre, 140 /sm.

upper traces; Fig. 3A was obtained using Arsenazo III, and B using Antipyrylazo III.It is apparent that both the Ca signals obtained using Arsenazo III and Antipyryl-azo III are potentiated by caffeine, andfurthermore thatthe magnitude ofpotentiationis comparable for both dyes. The lower traces show the membrane action potentials,which are clearly not modified by this level of caffeine. The amounts of potentiationof Ca signals by low levels of caffeine, following action potential stimulation, werefoundtobe1 0O atO-125 mM(n = 2fibres), 16% atO-25 mM (n = 4) and22% atO 5 mM(n = 3); thus, the potentiation is monotonic in caffeine concentration. Herepotentiation is defined as the ratio of peak AA values between caffeine and controlCa signals following action potential stimulation.

541


Caffeine has separate effects on the time courses of the rising and falling phasesofCa signals. At these low levels ofcaffeine, the rising phase ofthe caffeine-potentiatedCa signal is almost always identical to that of the control signal when the signals arescaled to the same peak height. This can be seen by examination of the Ca signalsin Fig. 3. The falling phase of the Ca signal, however, is affected to a variable extentby caffeine. In order to demonstrate this quantitatively, a single exponential wasfitted to the falling phase of the Ca signals using a least-squares procedure, similarto Miledi, Parker & Zhu (1983). For the traces in Fig. 3A and B, the time constantsare not substantially affected by caffeine (28 ms vs. 29 ms for Arsenazo III and 2-9 msvs. 2-0 ms for Antipyrylazo III, for control and caffeine respectively); however, inother fibres under identical conditions, the time constant was noticeably increasedin the presence of caffeine (e.g. see Fig. 5).

0-002 AA

20 ms

a b

Fig. 4. Ca signals recorded with Arsenazo III following stimulation by action potential inpresence of caffeine at the following concentrations: trace a, control; trace b, 005 mM;trace c, I 0 mm. The internal solution applied at the cut ends contained 05 mm-Antipyrylazo III, and 0-36 mm was present intracellularly. Fibre diameter was 150 jsm,and interstimulus interval was 3 min.

Effects of moderate levels of caffeine upon Ca signal amplitudeAt moderate concentrations ofcaffeine (from 0 5 to 2 mM), the extent ofpotentiation

of Ca signals by caffeine is approximately independent of the caffeine concentration.Fig. 4 shows the Ca signals obtained over several concentrations of caffeine from onefibre diffused with Arsenazo III and stimulated by a brief voltage-clamp pulse of80 mV for 5 ms. The Ca signal in 05 mM-caffeine (trace b) is increased relative tocontrol conditions (trace a), but is not increased further upon addition oft mM-caffeine(trace c). For these caffeine levels, the amount of potentiation was observed to varyfrom fibre to fibre, ranging from 10 to 40 %, with most values between 20 and 30 %.However, for any particular fibre, the amount of potentiation did not vary with thecaffeine concentration over this range.

It should be noted that this effect is not due to saturation of the dye, since astimulation in absence of caffeine by a longer pulse (80 mV, 20 ms), which would beexpected to increase Ca2+ release, was observed to produce a 68% increase in peakAA relative to control conditions, an increase greater than that due to caffeine.

542


The potentiation of Ca signal amplitude by low and moderate concentrations ofcaffeine is reversible. This was found by applying depolarizing pulses of 70 mV for20 ms to a voltage-clamped fibre during successive alternations between the controlsolution and one containing 0 75 mM-caffeine over a time period of 75 min. Theamount of caffeine potentiation was repeatedly found to be near 29% and, followingreplacement of caffeine-containing solution with control, the Ca signal amplitudeconsistently returned to the pre-caffeine level. This point is further supported by theresults shown in Fig. 6. The reversibility of the caffeine effect is required by theprocedure used to scale Ca signals with different dye concentrations, as is describedin Methods.

2-

VV

0~~~~~~~~~~

0 1 2 3

[Caffeine] (mM)Fig. 5. A comparison of the falling phase time constant and the amount of potentiationofCa signals in a fibre diffused with Arsenazo III and stimulated by action potential. Thetriangles show the potentiation of the peak Ca signal and the circles the inverse timeconstant, both relative to their respective control values. Internal solutions were as givenfor Fig. 4. Fibre diameter 150 /sm.

Effects of moderate levels of caffeine on the falling phase of the Ca signalFig. 4 shows that, as has previously been pointed out for low levels of caffeine, the

rising phase of the Ca signal is unchanged in time course by application of moderatelevels of caffeine when scaled to the control signal. There is, however, a significantslowing of the falling phase of these signals, as was found by Kovacs & Szucs (1983)in recordings made following long depolarizations. We find that the time constant,which characterizes the single exponential fit to the falling phase of the Ca signal ina particular fibre, often increases monotonically with increasing caffeine concentrationover the range 0-5-2 mm. The results from one such fibre are shown in Fig. 5, whereeach filled circle represents the inverse ratio of the time constant measured in caffeinerelative to the control value. The monotonic increase is found in fibres diffused witheither Arsenazo III or Antipyrylazo III. In contrast to the increased slowing of thefalling phase with increasing caffeine level, the amount of potentiation (defined asthe ratio of the peak Ca signals in the presence and absence of caffeine, and shown

543


as triangles in Fig. 5) measured immediately after change of external solution isessentially constant over the range of caffeine concentrations 0'5 to above 2 mM.

A-

0005 &A

30 ms

B

C

Fig. 6. The effect of moderate to high levels of caffeine upon Ca signals. The smalleramplitude trace in A was recorded using Arsenazo III following an action potential undercontrol conditions. This trace is repeated as the smaller amplitude transient in B and thelarger amplitude transient in B. The larger amplitude trace in A and the smaller amplitudetrace inB were recorded in 0 5 and 3 mM-caffeine, respectively. The trace in C was recordedafter return to control solution. The intracellular Arsenazo III level was 0-5 mm, the fibrediameter 140 /sm, and interstimulus interval 3 min.

The effects of high levels of caffeine upon Ca signalsIn contrast to results obtained at lower concentrations, levels of caffeine above

2 mm are associated with smaller potentiation; in extreme cases the Ca signal evenfalls below the control value. Fig. 6A shows superimposed records of the Ca transientin the presence of 0-5 mM-caffeine (larger amplitude trace), and in control solution(smaller amplitude trace), illustrating that potentiation is present. PanelB shows thatthe Ca signal in presence of3 mM-caffeine (smaller amplitude trace) is ofmuch smalleramplitude than the control trace (larger amplitude trace). The decline of the signalat these high levels of caffeine is not due to progressive deterioration of fibreexcitability, since many fibres survived long-lasting experiments (from 3 to 5 h) inwhich removal of a high caffeine concentration resulted in quick recovery of the Ca

544


signal to normal amplitude. This is illustrated in Fig. 6C, which was recordedfollowing Fig. 6B after restoration of the control external solution. The amplitudeis almost completely recovered; the falling phase time constant has returned to 40 s,compared to 43 s for the control. Note also that the time-to-peak of the Ca signal isunchanged by caffeine over the course of the experiment shown in Fig. 6, suggestingthat the rising phase is not affected by these levels of caffeine.

Potentiation of Ca signal by caffeine was observed using either of the two dyes,suggesting that the effect is not dependent upon a fortuitous combination of dyecharacteristics. Cuvette calibrations performed in a manner similar to Palade &Vergara (1983) show no change in the dye spectra with caffeine concentrations upto 2-5 mm and free Ca2+ levels of either trace amounts or 0 5 mM.

A B C

AAf2X1O0 A4;~~>0.1 V

Fig. 7. The upper three traces show changes in optical absorbance recorded at 670 nm ina fibre stained with the non-permeant potentiometric dye NK2367 and stimulated byaction potential. The records of electrically recorded membrane potential are shown inthe lower traces. A, records made in the absence of caffeine; B, records made with0-5 mM-caffeine; and C, a comparison of the two optical traces after scaling to the samepeak heights to compensate for bleaching of the dye. The external solution containedNK2367 0-5 mg ml-; the internal, 3 mM-MgCl2 and 20 mM-EGTA.

Electrical properties of surface and tubular membranes are unchanged by caffeineThe T-system possesses activatable conductances responsible for the regenerative

radial propagation of potential (Costantin, 1970; Adrian & Peachey, 1973; Vergara& Bezanilla, 1981; Heiny & Vergara, 1982; Vergara, Delay, Heiny & Ribalet, 1983).The possibility that caffeine could alter these conductances justifies examination ofthe effect of caffeine upon the transient changes in tubular membrane potential(Luttgau & Oetlicker, 1968).

Optical signals were recorded at 670 nm from fibres stained with the non-penetratingpotentiometric dye NK2367; at this wave-length the dye response derives primarilyfrom the tubular system (Heiny & Vergara, 1982; Heiny & Vergara, 1984). Theabsorbance change, AA, monitoring the tubular potential change is shown in theupper trace of Fig. 7A for a fibre stimulated to elicit an action potential, shown inthe lower trace. A comparison of these two traces shows, as was previously describedby Heiny & Vergara (1982), that the time course of the optical signal is somewhatslower than that of the surface action potential. The upper trace of Fig. 7B shows

545

18 PHY 375


the optical signal obtained in the presence of 0 5 mM-caffeine. The scaled comparisonof the upper traces of Fig. 7A and B are shown superimposed in Fig. 7 C,demonstrating that there is no significant effect of caffeine upon the tubular actionpotential. Furthermore, a comparison of the time courses of the electrical records(lower traces) in Fig. 7A and B shows that caffeine has no effect upon the surfacemembrane action potential. Identical conclusions were drawn from experimentsperformed using voltage-clamp stimulation, and further experiments at caffeine levelsup to 2 mm confirmed that caffeine has no effect upon either surface or tubularpotential signals.

0 005

tAA

0'-80 -60 -40 -20

mVFig. 8. The dependence ofpeak absorbance changes ofCa signals upon membrane potentialmeasured using Antipyrylazo III. Triangles represent data taken in the absence ofcaffeine, and circles, with O-5 mM-caffeine. The line shows the least-squares fit to aBoltzmann function, y = pil/(1 +exp((x-P2)/P3)), where Pi, P2 and p3 are respectively4*50, 61 1 and 6-0 for control and 5-69, 56-9 and 6-4 for caffeine. The internal solutioncontained 1-5 mM-Antipyrylazo III, 3 mm-EGTA and 3 mM-MgCl2; the external solutioncontained 0-2 ,sM-TTX (Solution 4 of Table 1) with 2 mM-MgCl2; and the pulse durationwas 20 ms or longer. Fibre diameter 120 ,sm.

Effects of caffeine upon the voltage dependence of Ca2+ releaseA voltage-sensitive coupling mechanism between the tubular system and the

sarcoplasmic reticulum would account for the dependence of Ca2+ release upon theapplied surface depolarization (Hodgkin & Horowicz, 1960; Schneider & Chandler,1973). It was of interest to examine the effect of caffeine upon the voltage dependenceof the Ca2+ release over the full range of depolarizing voltages, in order to test fora potentiation of the maximal Ca signal. This was done under voltage-clampconditions, which allowed the imposition of a steady-state T-tubular potential, asopposed to the inherently strong time dependence of the potential during an actionpotential.

546


Experiments were performed on fibres treated with either tetrodotoxin (TTX) orzero Na+ external solutions in order to eliminate the surface and tubular membraneNa+ conductance. Ca signals recorded under this condition are shown in Palade &Vergara (1983). The results for a representative fibre in presence of TTX (Solution4 of Table 1) are given in Fig. 8, where the dots represent control conditions. In thepresence of 0'5 mM-caffeine (triangles), however, there is an apparent shift of thevoltage dependence towards more negative potentials. Note that the shift results ina very large increase in the Ca signal amplitude (ca. 300 %) at potentials close to thethreshold for discernible Ca signal. The linear portion of the dependence is increased

A B1 &~~~~~~~~~~~~~~~005-

00

X 0E x

E

0- 0-80 -60 -40 -20 0 1 2 3

mV [Caffeine] (mM)Fig. 9. A, the same fibre and conditions as in Fig. 8; the symbols correspond to thefollowing concentrations of caffeine: circles, 0 mM; crosses, 0 5 mM; triangles, 2 mM; andsquares, 3 mm. The absorbance changes are normalized to their respective values recordedat depolarizations to -20 mV. The data for 0 and 0-5 mM-caffeine are repeated fromFig. 8. B, the dependence upon caffeine level of the peak absorbance change of Ca signalsrecorded at depolarizations to -20 mV for the data of Fig. IOA.

in slope by about 1-3 compared to the control curve. This is consistent with thepotentiation of the maximal Ca signal obtained at large depolarizations, which alsoamounts to a factor of about 1-3. Similar amounts of potentiation were obtained atlarge depolarizating voltages when using an external solution based on 1 Na, 12 '2tetramethylammonium (TMA) (Solution 3 of Table 1). It may be concluded, sincecaffeine potentiates both the maximal Ca signal obtained via large depolarizationsand the slope of the linear part of the voltage dependence, that caffeine has apotentiating effect upon the releasing mechanism rather than causing only a simpleshift in its sensitivity to voltage.The voltage dependence of Ca2+ release was studied using a range of caffeine

concentrations up to 3 mm. Fig. 9A shows the results of such an experiment on thesame fibre as in Fig. 8, but with the peak absorbance changes scaled to their respectivemaximal values obtained via depolarizations to -20 mV. The shift in the voltagedependence is clearly monotonic with caffeine levels up to 2 mm, beyond which no

18-2

547


further shift is apparent. The peak absorbance change of the Ca signals obtained atdepolarizations to -20 mV for the data of Fig. 9A are shown in Fig. 9B.

Caffeine potentiation of Ca2+ release occurs both in the absence of external Ca2+ or in thepresence of Ni2+The dependence of caffeine potentiation on external Ca2+ was tested in experiments

using highly buffered solutions with low free Ca2+ and containing Mg2+ (Solution 5of Table 1). The results of one of these experiments are shown in Fig. 10. The trace

AAA0005

20ms

JFig. 10. Ca signals recorded following action potential stimulation in the presence ofexternal solutions containing a low Ca2+ level (Solution 5 of Table 1); see text for details.The smaller amplitude trace corresponds to control solution, and the larger amplitudetrace to 05 mM-caffeine. The measured Ca2+ concentration was 02 /SM. The internalsolutions bathing the cut ends contained 1-25 mM-Arsenazo III, with 0-8 mm presentintracellularly. Fibre diameter 120 ,um.

with smaller peak height shows AA in control conditions; the larger amplitude trace,in presence of0 5 mM-caffeine. The fact that Ca signals can be elicited in zero externalCa2+ was previously shown by Miledi, Parker & Schalow (1977 b). The caffeine-inducedpotentiation of Ca2+ release is seen despite the absence ofexternal Ca2+; furthermore,the amplitude ofthe Ca signal and its potentiation under this condition are consistentwith those obtained in external solutions with normal levels of external Ca2+. Theseresults were observed in experiments on three fibres, all of which showed potentiatedCa signals in presence of caffeine. Following replacement with a caffeine-free solutioncontaining normal external Ca2+, the Ca signal amplitude returned to the controllevel.

Externally applied Ni2+ has been reported to be an effective blocker of Ca2+movement into the myoplasm (Almers, Fink & Palade, 1981). If an influx of Ca2+were required for Ca2+ release and its potentiation by caffeine, the presence of Ni2+would abolish or sharply reduce this potentiation. Fig. 11 shows the results of anexperiment to test the effect of Ni2+; the upper traces show AA due to Ca2+ releasefollowing stimulation by the action potential shown in the lower traces. Fig. 11 A wasobtained under control conditions, and Fig. 11 B, following the replacement of theexternal solution with one containing 1 mM-Ni2+ (Solution 6 ofTable 1). The additionofNi2+ is seen to slightly alter the time course ofthe repolarization phase ofthe actionpotential without any change to its rising phase (lower trace in Fig. 11 B) in agreement

548


with Sandow & Isaacson (1966). The falling phase ofthe Ca signal is slowed, and thereis a small decrease in the rate of rise, though there is no significant change in theamplitude of the Ca signal. Fig. 11C was observed in the presence of a solutioncontaining both Ni2+ and 0-5 mM-caffeine. This causes a characteristic potentiationof the Ca signal, with no further effects on the time course of the action potentialor the Ca signal. Externally applied Co2+ shows identical results, and the effects ofboth Co2+ and Ni2+ are fully reversible.

A

j

0m02

20 ms

i

Jo' J''100 mV

Fig. 11. The upper traces show records of Ca signals recorded in the presence of normaland Ni2+-containing solutions; see text for details. A, normal external solution; B, externalsolution contained Ni+ (Solution 6 of Table 1); and C, Ni2+ and 0-5 mM-caffeine. Theinternal solution at the cut ends contained 1-25 mM-Arsenazo III (0 5 mM-intracellularly),2-5 mm-EGTA and 3 mm-MgCl,. Fibre diameter 120 /sm.

Observed levels ofCa signal potentiation by caffeine cannot be due to increased myoplasmicCa2+ levels

It has been proposed that caffeine increases the net Ca2+ influx into muscle fibres(Bianchi, 1961), and may increase the steady rate ofCa2+ release from the sarcoplasmicreticulum (s.r.) (Weber & Herz, 1968). Either of these effects could increase theamount of Ca2+ bound to the myoplasmic Ca2+ buffers under steady-state conditions.In the experiments reported here, the myoplasm was diffused with EGTA and eitherArsenazo III or Antipyrylazo III, which compete with the myoplasmic Ca2+ buffersfor available Ca2+ (Baylor, Chandler & Marshall, 1983; Kovacs, Rios & Schneider,1983). A proportionally greater increase in loading ofthe intracellular buffers, relativeto the dye, may grant the dye a greater ability to compete for Ca2+ during anelectrically stimulated release than it has under control -conditions. This could inprinciple result in an increased Ca signal amplitude independent of any increase inreleased Ca2+. Two approaches were used to show that this possibility could notaccount for our results. First, resting Arsenazo III dye absorbance measurementswere used to show that the resting myoplasmic Ca2+ level did not change within a

549


resolution of 0-04 /M in the presence of up to 0 75 mM-caffeine. Secondly, we showedwith the aid of a model that a plausible increase in the free Ca2+ level would resultin a substantially smaller amount of Ca signal potentiation than was foundexperimentally. Further details of these methods are presented in Appendices A andB, respectively; the results suggest that elevated myoplasmic Ca2+ levels cannotaccount for the observed potentiation of Ca signal.

DISCUSSION

Potentiation of Ca signals by caffeineThe results described in this paper show that, in cut fibres the myoplasm of which

is diffused with a Ca2+ indicator dye, caffeine causes a consistent increase of between10 % and 40 % in the peak Ca signals. The amount of increase is concentrationdependent up to 0'5 mM-caffeine and is concentration dependent from 0'S to 2 mm. Thispotentiating effect of caffeine is reproducible, having been observed in a large numberof fibres (n - 100) under a number of different internal and external solutions. Thepotentiation was present in fibres using either Arsenazo III or Antipyrylazo III, thepeak Ca signals showing very similar amounts of potentiation for the two dyes.

Does an increased Ca signal imply an increased Ca2+ release?The Ca signal observed using a Ca2+ indicator dye represents the ability of that

dye to bind Ca2+ in competition with other myoplasmic Ca2+ buffers. The interpretationof the Ca signal therefore bears on the characteristics of this competition. Inparticular, the hypothesis may be made that a potentiated Ca signal is due to alteredbehaviour of the other Ca2+ buffers, without any increase in the amount of Ca2+released by the s.r. With this in mind, we have considered three general effects ofcaffeine upon physiological processes which could give rise to an increase in the Casignal: first, an increased steady-state loading of intracellular Ca2+ buffers with nochange in the Ca2+ release; secondly, a diminished rate of Ca2+ removal from the dyeagain with an unaltered release of Ca2+; and finally, an increased Ca2+ release. Thefollowing section discusses these hypotheses.

(1) The possibility that the Ca signal potentiation results from increased loadingof intracellular Ca2+ buffers is unlikely, since any such increase in loading must beaccompanied by increases in the resting free myoplasmic Ca2+ level, and nosubstantial changes in Ca2+ concentration were found (Lopez et al. 1983, and Results).Any change in Ca2+ concentration which might have occurred within the resolutionof the method can account for only a small portion of the potentiation observed. Itis to be expected that the presence of EGTA and Ca2+-sensitive dyes diffused intothe cut fibre would result in even less change in Ca2+ concentration than in the intactfibres used by Lopez et al. (1983). In support of this, we find that no contracture isinduced in the cut fibre in presence of 3 mM-caffeine; this level does inducecontracture in uncut fibres (Caputo, 1966), presumably due to the increased free Ca2+level of 0-31 #M (Lopez et al. 1983), which is close to the threshold for generation oftension (Fabiato & Fabiato, 1978).The results cited above do not eliminate the possibility that a small increase in

resting Ca2+ concentration, below the limits of resolution of the respective methods

550


used, may take place in the presence of low levels of caffeine. If this increase wereto cause an increase in the level of Ca2+ stored in the s.r., a larger Ca transient mightresult. However, our results show clear potentiation in the absence of external Ca2+.Since no Ca2+ entry can occur from the external medium, any increase in myoplasmicCa2+ concentration must deplete the s.r. stores, resulting if anything in a smaller Catransient. Furthermore, Endo (1975) has found in skinned skeletal muscle that0 5 mM-caffeine has no significant effect on the amount of Ca2+ taken up by the s.r.It is therefore unlikely that the observed potentiation is due to an increase in restingmyoplasmic free Ca2+ level.

(2) The Ca signal may be considered to originate as the net effect of twoindependent processes: the Ca2+ release, which could be responsible for the risingphase of the signal; and the removal of Ca2+ from the myoplasm, which results inthe falling phase. Since moderate levels of caffeine can slow the rate of decline of thefalling phase of the Ca signal, it is conceivable that caffeine, by inhibiting the removalprocess and without changing the Ca2+ release, could effect a larger amplitude Casignal with a higher rate of rise and a slower falling phase. However, as can be seenfor example in Fig. 3, at 0'5 mM-caffeine the slowing of the falling phase time courseis almost negligible, and it is extremely unlikely in this case that this slowing canaccount for the large potentiation. In any case, we have shown (Fig. 5) that theamount of potentiation remains constant despite the monotonically increasingprolongation of the falling phase with caffeine concentration.

(3) By elimination of the two possibilities enumerated above, we infer that theincrease in the peak Ca absorbance signals due to caffeine very likely implies anincrease in amount of Ca2+ released from the s.r.

Effect of caffeine upon voltage dependence of Ca szgnaleWe have shown in Fig. 8 that 0-5 mM-caffeine causes both a potentiation of

maximal Ca signal and a shift of the voltage dependence by about 6 mV towards morenegative potentials. The shift in the potential dependence is consistent with theresults ofKovacs & Szucs (1983). As a consequence of this shift, the apparent Ca signalpotentiation for membrane potentials just above -60 mV is extremely large,amounting to severalfold. This strongly suggests that the caffeine potentiation ofCa signals cannot result solely from an increase in myoplasmic free Ca2+ level, sincean effect of this magnitude is not likely to arise from any reasonable change in Ca2+concentration.

Several hypotheses may account for the shift, and are discussed here. First, it maybe hypothesized that the dye might not be able to bind released Ca2+ until substantialsaturation of a buffer with fast Ca-binding kinetics, such as the Ca-specific site oftroponin C (TnCc). Under this hypothesis, the voltage shift would result if a slightrise in myoplasmic free Ca2+ level were to increase the resting level of Ca2+ boundto TnCc, and allow saturation by a smaller amount of released Ca2+. We estimatedthe change in resting Ca2+ concentration required to obtain the Ca signal amplitudesfound at different caffeine levels for 60 mV depolarization in Fig. 9A. Assuming that150 sM-Ca2+ is released following an action potential (Endo, 1977), and that all theTnCc must bind Ca2+ before the dye can do so, the free Ca2+ level must rise to 0-9and 2-9 UM at 05 and 2'0 mM-caffeine, respectively. This is very unlikely, givenprevious arguments on changes in myoplasmic Ca2+ level.

551


Secondly, the shift in the voltage dependence could be due to non-linearity inresponse of the Ca-sensitive dye. Since peak Ca signals have the same threshold andvoltage dependence whether recorded from fibres diffused with either Arsenazo IIIor Antipyrylazo III, which have quite different affinities and extinction coefficients(Palade & Vergara, 1981; Palade & Vergara, 1982), this alternative is unlikely.

Thirdly, the shift in the voltage dependence could take place in the T-tubulemembrane due to charge screening (Chandler, Hodgkin & Meves, 1965). However,since caffeine is not an ion and can readily permeate the cell membrane, presumablyexplaining its reversibility of effect, this possibility is not likely. Furthermore, theT-tubule membrane is apparently not affected by these concentrations of caffeine,since experiments we have performed on the voltage-dependent characteristics of theT-system showed no altered behaviour at up to 2 mM-caffeine.

In the absence of other plausible arguments, we conclude that the shift of thevoltage dependence of Ca2+ release is a genuine effect of caffeine.

Possible effects of caffeine upon the s.r. Ca(2+ levelThe absence of concentration-dependent potentiation at moderate concentrations

of caffeine and the marked decrease ofthe peak Ca signal amplitude at concentrationshigher than 2 mM suggest that these levels of caffeine may reduce the ability of thes.r. to release Ca2+ in a concentration-dependent manner. This may be related to acaffeine-induced depletion of Ca2+ in the s.r. There are several points of experimentalsupport from other studies for a depletion hypothesis: (i) terminal cisternae of frogsemitendinosus muscle exposed to 5 mM-caffeine show a substantial drop in total Cacontent (Yoshioka & Somlyo, 1984); (ii) caffeine concentrations between 1 and 10 mMreduce the Ca accumulating ability of the s.r. in a concentration-dependent manner(Herz & Weber, 1965); and (iii) 3 mM-caffeine causes an increased resting freemyoplasmic Ca2+ level of 0-31 /M (Lopez et al. 1983).An alternative hypothesis may be proposed, that a concentration of caffeine near

3 mm might increase the binding ofCa2+ by the myofibrils, consistent with the shiftingofthe pCa-tension curve found at even higher levels ofcaffeine (Wendt & Stephenson,1983). Since the dye and the myofibrils presumably compete for released Ca2 , thisincreased myofibrillar affinity for Ca2+ would result in a correspondingly smalleramount of Ca bound to dye and hence to a smaller Ca signal. This effect is unlikelyto have occurred, since the increase in tension would be expected to in turn increasethe optical signal associated with the fibre movement. No such increase was foundin our experiments, as the smaller Ca signal at caffeine levels over 2 mm was associatedwith smaller motion signals.

If the reduced ability of the s.r. to release Ca2+ were present and graded withcaffeine level, it would tend to counteract the potentiation of Ca signal. In this case,the true amounts of caffeine potentiation of Ca signal would be expected to exceedthose measured at higher caffeine levels.

Potentiation and shift in voltage dependence may be related effects of caffeineFig. 9A shows that increasing the caffeine level causes an increasing shift in the

voltage dependence of Ca signals, but suggests that the incremental amount of shiftis greatest for low caffeine levels, decreases as caffeine level is increased, and is almost

552


absent above about 2 mm. The amount of potentiation has a parallel dependence oncaffeine concentration, showing the greatest increases at low caffeine levels, andtending to stabilize at higher caffeine levels. The parallelism may even extend to3 mM-caffeine, considering that depletion of s.r. Ca2+ may tend to reduce the Ca signalamplitude at this level. This suggests that caffeine acts on excitation-contractioncoupling by a mechanism that includes both potentiation and a shift.

The effect of caffeine may involve an intermediate step in excitation-contraction couplingIt is appropriate to pose some conjectures on excitation-contraction coupling that

would readily account for the phenomena described in this report. Caffeine maypotentiate Ca2+ release by augmenting the effect of a voltage-dependent process,which takes place at the T-tubule membrane, but at a later step than the membranedepolarization itself. This requires an intermediate step, or second messenger, whichis released in an earlier voltage-dependent process, and whose ability to cause s.r. Ca2+release can be modulated by caffeine. The existence of this intermediate step isnecessary to explain the simultaneous potentiation and shift in the voltage dependenceof Ca2+ release, since caffeine does not appear to modify the electrical properties ofthe T-tubule membrane. A single step mechanism in which, for example, caffeine wereto act only by increasing the conductance of the s.r. Ca2+ channel would predict apotentiation of Ca2+ release, but could not account for the shift. On the other hand,a simultaneous shift and potentiation can be accounted for in an intermediate stepmodel if for example the basal level or efficiency of second messenger is increased bycaffeine. An implication of this conjecture is that higher concentrations of caffeinemay result in greater basal levels of the second messenger, perhaps causing sustainedrelease of Ca2+ and ultimately leading to contracture. In this picture, the action ofcaffeine may take place via a single mechanism over a wide concentration range,though we do not rule out the possibility of direct caffeine-induced release of s.r. Ca2+or inhibition of net s.r. Ca2+ uptake, particularly at very high caffeine concentrations.

APPENDIX A

Measurement of changes in resting free Ca2+ concentration using Arsenazo IIIChanges in the ratio of absorbance R = A(530 nm)/A(660 nm) for Arsenazo III

were related to approximate changes in the free Ca2+ level at the steady state,essentially independent of dye concentration, given the formation of several dye-Cacomplexes with known association constants and extinction coefficients (Palade &Vergara, 1983). Briefly, the ratio R was calculated as a function of free Ca2+concentration, considering the total Mg2+ and ATP concentrations which are diffusedinto the muscle; the levels of Mg2+ and ATP are important because Mg2+ affects theresting absorbance spectrum of Arsenazo III (Palade & Vergara, 1983). Experimentswere performed to measure the effects upon R of external solutions containing eithercaffeine or altered free Ca2+ levels; the results are given in Table 2. When the totalexternal Ca level was raised to 4-8 mm, R decreased over about 10 min by an amountcorresponding to a calculated increase in internal free Ca2+ concentration of about008 um relative to control (first fibre). When the external solution was changed fromcontrol to one with a low external Ca level, the change in R corresponded to a decrease

553


in internal free Ca2+ concentration of about 004 /M (second fibre). Finally, upontransitions between control solution and ones containing 0 5 and 0 75 mM-caffeine,measurements ofR made at 1 min intervals between 2 and 4 min following solutionchange were consistent with no changes in resting Ca2+ concentration within aresolution of about 004 /SM (third and fourth fibres).The presence ofEGTA within the myoplasm raises the possibility that the changes

in Ca2+ found here might not be characteristic of a physiologically normal fibre, dueto the extra Ca2+ buffering. However, the purpose of the measurement was to definechanges in resting Ca2+ concentration under our experimental conditions. Within theresolution of the method (about 004 gM), therefore, no changes in resting Ca2+concentration take place in the presence of up to 075 mM-caffeine.

TABLE 2. Measurements ofR = A(530 nm)/A(660 nm) on fibres diffused with Arsenazo III, underdifferent external solutions

Fibre R S.D. n Conditions

M0304A 7 30 0-16 3 4-8 mM-total external Ca2+8-77 0104 4 Control

M0205 9*54 0-13 9 3 mm-external EGTA8-94 0-14 5 Control

M0301 9-93 0-25 3 0-5 mM-caffeine9-82 0 19 5 Control

M0825 9-63 0-57 5 0-75 mM-caffeine9-58 0-17 3 Control

The mean value ofn observations is shown for each case with the standard deviation of the mean(S.D.). The external solution consisted ofRinger solution, modified as shown under Conditions. Usingthe model described in Appendix A, a change in R of 0-7 was calculated to correspond to a changein resting free Ca2+ concentration of about 0104 /M for the intracellular conditions assumed there.

APPENDIX B

The effect of increased renting myopla8mic Caa2+ level upon Arsenazo III sensitivityThe measurements ofresting myoplasmic Ca2+ concentration cited here cannot rule

out a small increase below the level of resolution. This might be argued to cause aproportionally greater loading of myoplasmic Ca2+ buffers than of the Ca2+-sensitivedye, and to result in an effectively increased ability of the dye to bind released Ca2 .The increase in the amplitude of the Ca signal might therefore be observedindependent of any increased Ca2+ release.

This hypothesis was tested quantitatively using a model for the partition of Ca2+among known myoplasmic buffers including Arsenazo III; the methods and resultsof the calculation are presented here. In principle, such a model should take intoaccount the kinetic formation of several Arsenazo III-:Ca2+ complexes, since the dyeforms multiple complexes with Ca2+ at steady state (Palade & Vergara, 1981; Palade& Vergara, 1983). However, due to the lack of complete data on the rate constantsof formation and decay for these complexes, the analysis cannot predict the actualtime course and absolute magnitude of Ca2+ release. However, the goal of this reportis to estimate relative amounts ofCa2+ released in the presence and absence of caffeine

554


and, under these circumstances, it is reasonable to make estimates using theapproximations given here.The purpose of the model was to estimate the partitioning of released Ca2+ at

the time of the peak Ca signal, about 5 ms after signal onset, since the potentiationwas measured at that time. It was therefore sufficient to assume that the onlymyoplasmic buffers which compete with Arsenazo III for released Ca2+ over the firstfew milliseconds are the uncomplexed Ca2+-specific sites on troponin C (TnC), theCa2+, Mg2+-specific TnC, and parvalbumin, for the following reasons. (i) TheCa2+-specific TnC sites are only diffusion-limited in their binding to Ca2+ (for a reviewsee Gillis, Thomason, Lefevre & Kretsinger, 1982). (ii) Of the other fast buffers, mostof the parvalbumin and Ca2+, Mg2+_specific sites of TnC are bound to either Mg2+ orCa2+ at rest, since the cut fibres are diffused with 2-3 mM-Mg. Those buffer sites boundto Mg2+-require prior dissociation before they can bind Ca2+. Since this is a slow step(see Gillis et al. 1982), the fraction of these buffers bound to Mg2+ at rest are thereforeunable to effectively compete with the dye for Ca2+ during the short time of interest.(iii) However, a small fraction of these sites is free in the resting state and cantherefore bind Ca2+. Finally, (iv) the on rate constant for Ca2+ binding to EGTA canbe estimated by extrapolation to 12 0C from the results of Smith, Liesegang, Berger,Czerlinski & Podolsky (1984) to be about 0-5 x 106 M-1 s-1. Since the on rate constantfor binding of free TnC or parvalbumin to Ca2+ is diffusion-limited, and that forArsenazo III and Ca2+ has been found under particular experimental conditions toexceed 4 x 106 M-1 s- (Ogawa, Harafuji & Kuregayashi, 1980), it is likely that freeArsenazo III, TnC and parvalbumin will bind all but a few percent of the releasedCa2+ in the first few milliseconds. The presence ofEGTA will therefore have relativelylittle effect on the peak Ca signal amplitude. This has been observed by Palade &Vergara (1982), who found in cut fibres that high concentrations ofEGTA markedlyreduce the optical motion signal associated with generated tension, while causing onlyminor changes in the Ca signals. We have therefore neglected EGTA in calculatingthe rapid partition of released Ca2 .

It was assumed that the rate of arrival of Ca2+ to proximity with either dye or TnCis substantially slower than the rate at which these buffers may bind Ca2+. This islikely true for TnC and parvalbumin, which have fast forward rate constants; forArsenazo III, however, in the absence of published values for rate constants forinteraction with Ca2+, the arguments must necessarily be indirect. It is known thatthe two dyes Arsenazo III and Antipyrylazo III have markedly different affinitiesfor Ca2+; for example, the one-to-one dye-Ca2+ complex has association constants7830 M-1 for Arsenazo III (Palade & Vergara, 1983) and about 460 M-1 for Antipyryl-azo III (Palade & Vergara, 1981). It is therefore unlikely that the formation of thiscomplex would have equal rate constants for both dyes. Since the rising phases ofboth Arsenazo III and Antipyrylazo III Ca signals are quite similar (e.g. Fig. 4), itis unlikely that the dye limits the rate of formation of dye-Ca2+ complex in ourexperiments.

If Ca2+ release is the rate-limiting step of binding to free Arsenazo III, TnC andparvalbumin, we may solve the partition problem at steady state. It was furtherassumed that all buffers are located in a point volume, i.e. that there is no spatialsegregation between them. This is in principle not the case, since Ca2+ presumably

555


has to diffuse through the dye before reaching the myofilaments. However, this isa conservative assumption, since it would tend to exaggerate the buffer loading effect.The following myoplasmic constituents were considered in the model: Ca2+

(abbreviated C), Arsenazo III (abbreviated A), Mg2+ (abbreviated M, complexingwith Arsenazo III as A.M and with ATP as M.ATP), ATP, the Ca2+-specific siteson TnC (abbreviated TnCc or Bi), parvalbumin and the Ca2+, Mg2+-specific sites onTnC (treated identically and abbreviated TnCCM or B2) and EGTA. It was assumedthat Arsenazo III binds Ca2+ as AC, A2C and A22 and Mg2+ as A.M (Palade &Vergara, 1983). The concentrations of the constituents and the association constantsused are given in Table 3. A FORTRAN computer program was written to solve the

TABLE 3. Constants used in calculating the partition of Ca2+Constituents (mM)

Ca-specific binding sites on TnC 0-14Ca, Mg binding sites on TnC 0X14Parvalbumin 0-7Mg2+ 3ATP 3Arsenazo III 1EGTA 1

Association constants (see Appendix B for key to abbreviations) (M-,)Fabiato & Fabiato (1979) Mg ATP 1-24 x 10'Potter & Zot (1982) TnCc Ca 5 x 105Gillis et al. (1982) TnCcM Ca 10 x 10'Gillis et al. (1982) TnCcM Mg 1-3 x 10'

Parameters of Arsenazo III-Ca and Arsenazo III-Mg interactions were taken from Palade &Vergara (1983).

systems ofnon-linear equations corresponding to the steady-state partition ofthe ionsamong the buffers. The total concentrations of Arsenazo III, Ca2+, Mg2+, ATP, andEGTA were entered as input parameters, from which the program calculated theconcentrations of all complexes and free species.The problem was solved in two steps. First, the program was used to find the

concentrations of all species in the presence of a free Ca2+ level of 0412 FM, found byLopez et al. (1983) under control conditions. The calculated concentrations are givenin line 1 of Table 4. In the second step, an additional 150 FM of Ca2+ was partitionedbetween the remaining free concentrations ofthe buffers A, BI and B2, correspondingto an action-potential-stimulated Ca2+ release (Endo, 1977) into the myoplasm withthe above resting free Ca2+ concentration. The absorbance change in the dye wascalculated at 660 nm, assuming 100 ,um path length, from the changes in concen-trations of the Ca2+-Arsenazo III complexes, and are shown on line 2 of Table 4.The problem was then solved again assuming a reasonable increase in the resting

free Ca2+ concentration, hypothetically due to the presence of 1 mM-caffeine. Lopezet al. (1983) estimated no such change in 1 mm-caffeine within a standard deviationof 0-01 FM. For the purpose of this calculation, an increase of 2 S.D. was assumed, toCa2+ concentration = 0-14 FM. Line 3 of Table 4 shows the resulting concentrationsof species, and line 4 shows the effect of the partition of an additional 150 FM-Ca2+

556


amongst the free concentrations of buffers. The increase in the absorbance changeat 660 nm due to buffer loading is near 4%.For the sake of argument, the above calculations were repeated assuming that the

myoplasmic Ca2+ was to rise linearly with caffeine concentration between 0 and 3 mm,at which level Lopez et al. (1983) measured a significantly higher free Ca2+ level of0-31 /M, and where the Ca2+ micro-electrode performance is improved. In this case,

TABLE 4. Results of Ca partition calculations[A.M] [M.ATP] [B1.C] [B1.M] [B2.C] [B2.M] [AC] [E.C]102 2380 7-5 7-8 595 195 16 321

-- 26-5 41 82 - AA = 0-0218104 2390 8-6 7-9 619 177 19 356

- 29-2 45 87 - A = 0-0227All concentrations are in jM. Abbreviations: A, Arsenazo III; M, Mg; B1, Ca-specific site on TnC;

B2, Ca, Mg site on TnC, including parvalbumin; E, EGTA. By AC is meant all Arsenazo III-Cacomplexes. The first line of figures gives the concentrations of all species in the presence of0-12/M-free Ca. The second line shows the additional Ca-buffer concentrations formed by free A,B1 and B2 given an additional 150,u/M-total Ca; AA gives the resulting absorbance change at660 nm. The third line shows the results of a calculation as in the first line, but for 0-14 /sM-freeCa, and the last line, the results corresponding to the procedure in line 2 for the free buffers of line3. See Appendix B for details.

a Ca2+ level of 0-16 /M would be expected at 0 5 mm-caffeine, or about 4 S.D. awayfrom the results of Lopez et al. (1983), and at the limit of resolution of theArsenazo III-based method used above to measure changes in resting Ca2+. Even inthis case, the expected potentiation of the Ca signal amounts to only 7 %. Increasedloading of myoplasmic Ca2+ buffers due to plausible increases in resting myoplasmicCa2+ concentration can therefore not account for the much larger potentiation (upto 40 %) of Ca signal seen in these experiments.

The authors thank Dr D. Hilgemann for many helpful discussions, Mr V. Cordova for help withelectronic equipment, and Mr D. Curtis for mechanical designs and construction. This work wassupported by Post-doctoral Fellowships to Dr Ribalet from the American Heart Association(GLAA), and grants from the Crump Foundation, the Laubisch Fund, USPHS AM-25201,USPHS EY-041 10 and Project No. 2-JLNRC of the Muscular Dystrophy Association.

REFERENCES

ADRIAN, R. H. & PEACHEY, L. D. (1973). Reconstruction of the action potential of frog sartoriusmuscle. Journal of Phy8iology 235, 103-131.

ALMERS, W., FINK, R. & PALADE, P. T. (1981). Calcium depletion in frog muscle tubules: the declineof calcium current under maintained depolarization. Journal of Phy8iology 312, 177-207.

AxEmssoN, J. & THESLEFF, S. (1958). Activation of the contractile mechanism in striated muscle.Ada phygiologica 8candinavica 44, 55-66.

BAYLOR, S. M., CHANDLER, W. K. & MARSHALL, M. W. (1982). Optical measurements ofintracellularpH and magnesium in frog skeletal muscle fibres. Journal of Phy8iology 331, 105-137.

BAYLOR, S. M., CHANDLER, W. K. & MARSHATLL, M. W. (1983). Sarcoplasmic reticulum calciumrelease in frog skeletal muscle fibres estimated from arsenazo III calcium transients. Journal ofPhy8iology 544, 625-666.

557


BIANCHI, C. P. (1961). The effect of caffeine on radiocalcium movement in frog sartorius. Journalof General Physiology 44, 845-858.

CAPUTO, C. (1966). Caffeine- and potassium-induced con ractures of frog striated muscle fibers inhypertonic solutions. Journal of General Physiology 50, 129-139.

CHANDLER, W. K., HODGKIN, A. L. & MEVES, H. (1965). The effect of changing the internal solutionon sodium inactivation and related phenomena in giant axon. Journal ofPhysiology 180, 821-836.

COSTANTIN, L. L. (1970). The role ofsodium current in the radial spread ofcontraction in frog musclefibers. Journal of General Physiology 55, 703-715.

DELAY, M. J., RIBALET, B. & VERGARA, J. (1982). Correlation of movement with calcium signalsin muscle using calcium-sensitive dyes. Biophysical Journal 37, 22a.

DELAY, M. J., RIBALET, B. & VERGARA, J. (1983). Caffeine potentiation ofcalcium release in skeletalmuscle observed using calcium- and voltage-sensitive dyes. Biophysical Journal 41, 396 a.

ENDO, M. (1975). Mechanism of action of caffeine on the sarcoplasmic reticulum of skeletal muscle.Proceedings of the Japan Academy 51, 479-484.

ENDO, M. (1977). Calcium release from the sarcoplasmic reticulum. Physiological Reviews 57, 71-108.FABIATO, A. & FABIATO, F. (1978). Cyclic AMP-induced enhancement of calcium accumulation by

the sarcoplasmic reticulum with no modification of the sensitivity of the myofilaments to calciumin skinned fibres from a fast skeletal muscle. Biochimica et biophysics acta 39, 253-260.

FABIATO, A. & FABIATO, F. (1979). Calculator programs for computing the composition of solutionscontaining multiple metals and ligands used for experiments in skinned muscle cells. Journal dephysiologie 75, 463-505.

GILLIS, J. M., THOMASON, D., LEFEVRE, J. & KRETSINGER, R. H. (1982). Parvalbumins and musclerelaxation: a computer simulation study. Journal ofMuscle Research and Cell Motility 3, 377-398.

HEINY, J. A. & VERGARA, J. (1982). Optical signals from surface and T system membranes inskeletal muscle fibers. Journal of General Physiology 80, 203-230.

HEINY, J. A. & VERGARA, J. (1984). Dichroic behavior of the absorbance signals from dyes NK2367and WW375 in skeletal muscle fibers. Journal of General Physiology 84, 805-837.

HERZ, R. & WEBER, A. (1965). Caffeine inhibition of Ca uptake by muscle reticulum. FederationProceedings 24, 208.

HODGKIN, A. L. & HOROWICZ, P. (1960). Potassium contractures in single muscle fibres. Journalof Physiology 153, 386-403.

KOVACS, L., Rios, E. & SCHNEIDER, M. F. (1983). Measurement and modification of free calciumtransients in frog skeletal muscle fibres by a metallochromic indicator dye. Journal of Physiology343, 161-196.

KOVACS, L. & Szucs, G. (1983). Effect of caffeine on intramembrane charge movement and calciumtransients in cut skeletal muscle fibres of the frog. Journal of Physiology 341, 559-578.

LOPEZ, J. R., ALAMO, L., CAPUTO, C., DIPOLO, R. & VERGARA, J. (1983). Determination offree ionicCa2+ in frog skeletal muscle fibers. Biophysical Journal 43, 1-4.

LUTTGAU, H. C. & OETLICKER, H. (1968). The action of caffeine on the activation of the contractilemechanism in striated muscle fibres. Journal of Physiology 194, 51-74.

MILEDI, R., PARKER, I. & SCHALOW, G. (1977 a). Measurement of changes in intracellular calciumin frog skeletal muscle fibres using arsenazo III. Journal of Physiology 269, 11-13 P.

MILEDI, R., PARKER, I. & SCHALOW, G. (1977 b). Measurement of calcium transients in frog muscleby the use of arsenazo III. Proceedings of the Royal Society B 198, 201-210.

MILEDI, R., PARKER, I. & ZHU, P. H. (1983). Calcium transients in frog skeletal muscle fibresfollowing conditioning stimuli. Journal of Physiology 339, 223-242.

OGAWA, Y., HARAFUJI, H. & KUREGAYASHI, N. (1980). Comparison of the characteristics of fourmetallochromic dyes as potential calcium indicators for biological experiments. Journal ofBiochemistry 87, 1293-1303.

PALADE, P. & VERGARA, J. (1981). Detection of Ca2+ with optical methods. In The Regulation ofMuscle Contraction: Excitation-Contraction Coupling, ed. GRINNELL, A. D. & BRAZIER, M. A. B.,pp. 143-158. New York: Academic Press.

PALADE, P. & VERGARA, J. (1982). Arsenazo III and antipyrylazo III calcium transients in singleskeletal muscle fibers. Journal of General Physiology 79, 679-707.

PALADE, P. & VERGARA, J. (1983). Stoichiometries of arsenazo III-Ca complexes. BiophysicalJournal 43, 355-369.

POTTER, J. D. & ZOT, H. G. (1982). The role of actin in modulating Ca2+ binding to troponin.BiophysiCal Journal 37, 43 a.

558


SANDOW, A. & ISAACSON, A. (1966). Topochemical factors in potentiation of contraction by heavymetal cations. Journal of General Physiology 49, 937-961.

SANDOW, A., TAYLOR, S. R., ISAACSON, A. & SEGUIN, J. J. (1964). Electromechanical coupling inpotentiation of muscular contractions. Science 143, 577-579.

SCHNEIDER, M. F. & CHANDLER, W. K. (1973). Voltage dependent charge movement in skeletalmuscle: a possible step in excitation-contraction coupling. Nature 242, 244-248.

SMITH, P. D., LIESEGANG, G. W., BERGER, R. L., CZERLINSKI, G. & PODOLSKY, R. J. (1984). Astopped-flow investigation of the rate of calcium ion binding by ethyleneglycol bis(f-aminoethyl ether)-N,N'-tetraacetic acid. Analytic Biochemistry 143, 188-195.

VERGARA, J. & BEZANILLA, F. (1981). Optical studies of E-C coupling with potentiometric dyes.In The Regulation of Muscde Contraction: Excitation-Contraction Coupling, ed. GRINNELL, A. D.& BRAZIER, M. A. B., pp. 67-76. New York: Academic Press.

VERGARA, J., BEZANILLA, F. & SALZBERG, B. M. (1978). Nile blue fluorescence signals from cut singlemuscle fibers under voltage or current clamp conditions. Journal of General Physiology 72,775-800.

VERGARA, J. & DELAY, M. (1985). The use of metallochromic Ca indicators in skeletal muscle. CellCalcium 6, 119-132.

VERGARA, J., DELAY, M., HEINY, J. & RIBALET, B. (1983). Optical studies of T-system potentialand calcium release in skeletal muscle fibers. In The Physiology of Excitable Cells, ed. GRINNELL,A. & MOODY, W., pp. 343-355. New York: Alan R. Liss.

WEBER, A. & HERZ, R. (1968). The relationship between caffeine contracture of intact muscle andthe effect of caffeine on reticulum. Journal of General Physiology 52, 750-759.

WENDT, I. R. & STEPHENSON, D. G. (1983). Effects of caffeine on Ca-activated force productionin skinned cardiac and skeletal muscle fibres of the rat. Pfluigers Archiv 398, 210-216.

YOSHIOKA, T. & SOMLYO, A. P. (1984). Composition of sarcoplasmic reticulum and mitochondriaduring caffeine contracture. Biophysical Journal 45, 319a.

559

Documents

Caffeine potentiation of calcium release in frog skeletal muscle fibres