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[2] Measurement of Ca2+ release in skinned fibers from skeletal muscle
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C a 2+ F L U X E S A N D R E G U L A T I O N
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[2] M e a s u r e m e n t o f C a 2+ R e l e a s e in S k i n n e d F i b e r s from Skeletal Muscle
By M. ENDO and M. hNo Introduction C a 2+ release from the sarcoplasmic reticulum (SR) is one of the most important steps in excitation-contraction coupling of skeletal muscle. However, while various kinds of stimuli experimentally applied to the SR can be shown to cause Ca 2+ release, the mechanism of physiological Ca 2+ release from the SR is not yet known.l We have been trying to find out what kinds of stimuli directly applied to the SR of skinned muscle fibers cause Ca z+ release in the hope that, from the effective stimuli, we might be able to determine the mechanism utilized in physiological C a 2+ release. In this chapter, we will describe in detail the methods for measurement of Ca 2+ release in skinned fibers that are used in our laboratory.
General Considerations
In the experiments o n C a 2+ release from the SR, one must take the following facts into account. (1) There are several Ca 2+ release mechanisms in the SR, which are different at least in their activation mechanisms but probably also in their Ca 2+ channels. 1 Therefore, determinations regarding one of the release mechanisms can not be applied automatically to Ca 2+ release in general. (2) SR membrane also has a strong Ca 2+ uptake activity through the calcium pump ATPase. 2,3 Therefore, unless the Ca2+-pump is inhibited by the experimental condition, Ca 2+ released by any means tends to activate the pump and partly be taken up again. (3) Ca 2+ also activates one of the Ca 2+ release mechanisms present in the SR, the so-called Ca2+-induced Ca z+ release mechanism. 4,5 The secondary Ca z+ release caused by Ca z+ released by a primary process must be strictly distinguished from the primary release, if the properties of the latter are to be examined. For this reason, simple monitoring of M. Endo, in "Regulation of Calcium Transport across Muscle Membranes" (A. E. Shamoo, ed.), Current Topics in Membranes and Transport, Vol. 25, p. 181. Academic Press, New York, 1985. 2 S. Ebashi and F. Lipmann, J. Cell Biol. 14, 389 (1962). 3 W. Hasselbach and M. Makinose, Biochem. Z. 333, 518 (1961). 4 L. E. Ford and R. J. Podolsky, Science 167, 58 (1970). 5 M. Endo, M. Tanaka, and Y. Ogawa, Nature (London) 228, 34 (1970).
METHODS IN ENZYMOLOGY, VOL. 157
Copyright © 1988by Academic Press, Inc. All rights of reproduction in any form reserved.
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C a 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
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Ca 2+ concentrations outside the SR is not suitable for the measurement of the primary Ca 2+ release process. Instead Ca 2÷ concentrations outside the SR should be fixed as far as possible by the use of a Ca 2+ buffer. Under this condition, the Ca 2÷ release can be estimated by the time course of decrease in the amount of Ca 2÷ in the SR. Ca 2+ release from the SR can be studied in skinned fibers as described here or in vesicles of fragmented sarcoplasmic reticulum (FSR) as described in [32-34], this volume. Skinned fibers have a physiological advantage over the FSR and, probably for this reason, Ca 2÷ release is easier to evoke in skinned fibers than in FSR. In fact, many important modes of Ca 2+ release have been found in skinned fibers first and then confirmed with FSR. *-6 It is also easier to exchange the environmental solutions quickly in skinned fibers, whereas in FSR an exchange of solutions can only be made by filtration or centrifugation or, less precisely, by dilution. Another advantage (or possible disadvantage) of skinned fibers is the fact that the lumen of the whole SR is most probably continuous in skinned fibers, so that even if a single Ca 2+ channel throughout the whole SR is open, the Ca 2÷ release continues until the electrochemical potential gradient of Ca 2+ disappears between the inside and the outside of the SR lumen. In contrast, some FSR vesicles may not have a certain kind of Ca 2+ channel and thus may not respond to stimuli that activate that kind of channel. This heterogeneity of the vesicles must be kept in mind in interpreting results of FSR experiments. On the other hand, it is easier with FSR than with skinned fibers to follow Ca 2+ movements precisely and to conduct a large number of experiments by changing experimental conditions. Another disadvantage of skinned fibers is that while an exchange of solutions in the extrafiber space could be immediate, the change in composition in the real environment of the SR is rather slow due to a relatively long diffusion distance. Preparation of Skinned Fibers Preparing skinned fibers from muscles requires two steps: (1) isolation of a single fiber or a sufficiently thin bundle of fibers and (2) skinning or destruction of the semipermeability of the surface membrane. Skinning can be carried out before the isolation of single fibers or bundles if chemical skinning is used. Thicker bundles, of diameter larger than about 150 /xm, may be used for qualitative studies but they are not recommended for quantitative experiments because the diffusion time is too long. The best way to prepare a skinned fiber is first to isolate an intact 6 M. Endo and Y. Nakajima, Nature (London), New Biol. 246, 216 (1973).
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Ca2÷ FLUXESAND REGULATION
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single fiber from tendon to tendon in a physiological extracellular solution and then to skin it. This method assures that only damage will be due to skinning. Whenever possible, therefore, this procedure is recommended, but since isolating intact single fibers requires considerable skill and is time-consuming, an alternative is to isolate single fibers or a segment of fibers in a relaxing solution either before or after skinning. In this case, fibers should be examined under a microscope of a sufficient power to select parts with uniform striations for experimentation. The isolation of single fibers is done under a stereomicroscope of 40 × 80× magnification, with the aid of forceps and small scissors, knives, or needles. Tips of these instruments should be sharpened on an oil stone. The semitendinosus, iliofibularis, and tibialis anterior of amphibia are the muscles used most frequently for intact single fiber isolation. Slow tonic fibers can be obtained from amphibian iliofibularis muscles. 7 Mammalian fast twitch and slow twitch fibers can be obtained from extensor digitorum longus and soleus muscles, respectively. The strontium sensitivity of the contractile system is about one order of magnitude higher in both slow twitch 8,9 and slow tonic fibers 7 than in fast fibers and can be used to confirm fiber type. However, any kind of animal muscles can be used. Skinning can be carried out either mechanically or chemically.
Mechanical Skinning Mechanical skinning can be performed in either an oiP ° or a relaxing solution. 6 The entire surface membrane can be removed ~°or the fibers can be split into two (or more) longitudinal pieces to get partially skinned fibers. 6 The main difference between completely skinned fibers and partially skinned fibers is that, whereas in completely skinned fibers the disrupted T system membrane is likely to be sealed off and a potential difference across the T membrane may be reestablished with the aid of active sodium transport, in partially skinned fibers (if appropriately prepared) the mouths of the T tubules on the remaining sarcolemma may still be open to the outside and no ionic gradient across the T membrane can be established. 6 In the former, therefore, the real physiological stimulus to cause Ca 2÷ release, the depolarization of the T membrane, can still be effective, but in the latter, since the T membrane is completely depolarized from the beginning, it is extremely difficult, if not impossible, to 7 K. Horiuti, J. Physiol. (London) 373, 1 (1986). 8 S. K. B. Donaldson and W. G. L. Kerrick, J. Gen. Physiol. 66, 427 (1975). 9 A. Takagi and M. Endo, Exp. Neurol. 55, 95 (1977). to R. Natori, Jikeikai Med. J. 1, 119 (1954).
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C a 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
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cause Ca 2÷ release by manipulating the T membrane potential. To obtain well-resealed T tubules, skinning in an oil with a minimal quantity of extracellular fluid around the fiber is recommended, since Ca 2÷ seems to be required for the fusion of the disrupted membranes.
Chemical Skinning Chemical skinning for Ca 2÷ release experiments should be performed with an agent that destroys the semipermeability of the surface membrane but not that of the SR. Two techniques have been reported for skeletal muscles: (1) ethylenediaminetetraacetic acid (EDTA) or ethylene glycol bis(fl-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) treatment of human fibers and (2) treatment with saponin. EDTA-treated chemically skinned fibers were first described by Winegrad. H He showed that, when bundles of cardiac fibers are treated with a solution containing 3 mM EDTA for longer than 15 min at room temperature, the surface membrane became permeable to Ca 2÷, EGTA, ATP, and other small molecules but not to large molecules such as proteins. This method does not work for skeletal muscle fibers of amphibia, but human skeletal muscle fibers are reported to be skinned by a similar EGTA treatment, n However, this might not be purely chemical skinning but EGTA-assisted disruption of the surface membrane, unlike EDTAskinned cardiac fibers. 12 Treatment with 5-50/xg/ml saponin for 30 min specifically perforates the surface membrane in amphibian skeletal fast muscle fibers. ~3 A concentration of saponin higher than 150/xg/ml destroys the function of the SR as well. ~3Essentially the same applies to other kinds of fibers, but the concentration of saponin affecting the SR function differs in different kinds of fibers. For slow tonic fibers of amphibia and for mammalian skeletal muscle fibers, a lower concentration such as 20/zg/ml is recommended. In precise quantitative studies the possible effect of saponin on the SR should be checked under experimental conditions. The specificity of saponin comes from the fact that it acts on cholesterol molecules 14and that the cholesterol content of the surface membrane is much higher than that of the SR membraneJ 5,16 Therefore, besides saponin, other agents H S. Winegrad, J. Gen. Physiol. 58, 71 (1971). J2 D. S. Wood, J. Zollman, J. P. Reuben, and P. W. Brandt, Science 187, 1075 (1975). 13 M. Endo and M. Iino, J. Muscle Res. Cell Motil. 1, 89 (1980). 14 I. Ohtsuki, R. M. Manzi, G. E. Palade, and J. D. Jamieson, Biol. Cell. 31, 119 (1978). 15 A. Martonosi, Biochim. Biophys. Acta 150, 694 (1968). 16 K. Waku, Y. Uda, and Y. Nakazawa, J. Biochem. (Tokyo) 69, 483 (1971).
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which affect cholesterol, such as digitonin, may have a similar effect. Such agents could also be used for chemical skinning for Ca 2÷ release measurement, if their effects on the SR are negligible. In chemical skinning, the T tubule membrane is also permeabilized along with the surface membrane, so that no potential gradient can be established across the T membrane, as in the case of partially skinned fibers. C a 2+ M e a s u r e m e n t
As described under General Considerations, measurement of Ca 2+ release from the SR should be made by determining the time course of decrease in the amount of Ca 2÷ in the SR at a fixed Ca 2÷ concentration, to avoid the secondary Ca2÷-induced Ca 2÷ release. Measurement of the amount of Ca 2÷ in the SR can be made directly by using 45Ca or by discharging all of Ca 2÷ and measuring the amount discharged. A more-or-less continuoUs monitoring of Ca 2÷ content of the SR in skinned fibers with 45Ca is probably possible, since such experiments in intact single fibers have been reported. 17,1aHowever, it requires 45Ca of a very high specific activity and very careful experimental apparatus and design because counts of/3-emission of 45Ca that is of low energy are sharply diminished by small increases in distance between fiber and counter. Alternatively, if 45Ca is extracted from the fiber for counting, 19,2° the measurement is reliable. However, a single time course cannot be determined with a single fiber, but requires a large number of fibers, which makes the experiment cumbersome. On the other hand, under appropriate conditions a high concentration of Caffeine causes an almost complete release of Ca 2+ from the SR without any damage to the SR, so that many time courses can be determined in one skinned fiber. Since this kind of experiment can be more conveniently conducted than the 45Ca experiments mentioned above, this is the method of choice in general Ca 2+ release measurements. The amount of Ca 2÷ discharged from the SR can be determined by using (1) the size of resulting contracture of the skinned fiber (bioassay), (2) Ca 2+ indicator dyes, (3) a luminescent protein, aequorin, 21 and (4) probably 45Ca. Only the first two methods will be described. 17 B. A. Curtis, J. Gen. Physiol. 50, 255 (1966). 18 B. A. Curtis and R. S. Eisenberg, J. Gen. Physiol. 85, 383 (1982). 19 L I E . Ford and R. J. Podolsky, J. Physiol. (London) 223, 1 (1972). 20 E. W. Stephenson, J. Gen. Physiol. 71, 411 (1978). 21 j. R. Blinks, W. G. Wiet, P. Hess, and F. G. Prendergast, Prog. Biophys. Mol. Biol. 40, 1 (1982).
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C a 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
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Cae+ Measurement by Utilizing Contraction of the Skinned Fiber Under appropriate conditions, application of a high concentration of caffeine causes contraction of skinned fibers in a relaxing solution due to the release of Ca 2÷ from the SR. zz Caffeine causes this by stimulating the Ca2+-induced Ca 2+ release. 23 The contractions are transient because the Ca 2÷ released in the fiber space quickly diffuses into the relatively large volume of medium bathing the fiber. Free EGTA in the relaxing solution in turn diffuses into the fiber space. In order to obtain a good estimate of Ca 2+ release from the size of contraction, the time course of Ca 2+ released by caffeine must be rapid compared with that of diffusion. After a single caffeine treatment, the SR becomes practically empty of Ca 2+. Reapplication of caffeine (after the drug is washed out once) no longer causes any response until the SR is allowed to reaccumulate Ca 2+. The completeness of the Ca 2÷ release by caffeine can be checked either directly by determining the amount of 45Ca present in the SR before and after the caffeine treatment or indirectly and less precisely by showing that incubation with a low concentration of Ca 2+, e.g., 0.03 /zM, can cause a recovery of detectable caffeine contracture. This indicates that caffeine reduces the level of Ca 2+ in the SR below the equilibrium level with the low Ca 2+ concentration. 2z To obtain rapid and complete Ca 2+ release, the effect of caffeine should be strong. To achieve this, (1) a high concentration of caffeine must be used (25-50 mM). (2) EGTA concentration in a relaxing solution during caffeine application should be appropriate. If the EGTA concentration is too high, the Ca 2+ concentration will not be raised sufficiently as a result of the Ca 2+ release, which makes (a) Ca z+ release rather slow, z4 and (b) contractile response too small. If the concentration of EGTA is too low, distortion of the linearity of the contractile response with the amount of Ca 2+ released becomes greater due to saturation of contraction and fibers run down rapidly because of long intensive contractions. (3) Free Mg 2+ ion concentration must also be adjusted. CaZ+-induced Ca 2+ release and hence caffeine-induced Ca 2+ release are inhibited by raising Mg 2+ concentration in the medium. 25 Therefore, lower Mg 2÷ concentrations are preferable for rapid and complete Ca 2÷ release by caffeine. On the other hand, since Ca 2+ is assayed by contraction, a Mg 2+ concentra22 M. Endo, Physiol. Rev. 57, 71 (1977). 23 M. E n d o , Proc. Jpn. Acad. 51, 479 (1975). 24 B e c a u s e caffeine-induced Ca 2+ release is stronger at higher Ca 2+ concentrations, a very rapid Ca 2÷ release is obtained w h e n the Ca 2+ ion released in turn accelerates a further release o f C a 2÷. 25 M. Endo, Proc. Jpn. Acad. 51, 467 (1975).
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Ca 2+ FLUXES AND REGULATION
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TABLE I MAIN COMPOSITIONS OF SOLUTIONS USED FOR MEASUREMENTOF Ca2+-INDUCED Ca 2+ RELEASE BY UTILIZING CONTRACTION OF SKINNED FIBERSa
Solutions For Step 1 G2 relaxing solution G10 relaxing solution Loading solution For Step 2 Rigor solution Prereleasing solution c Releasing solution c Stopping solution For Step 3 Preassay solution A a Preassay solution B e Assay solution A d Assay solution B e
[Mg2+] b (mM)
[MgATP2-] b (mM)
[EGTA] total (mM)
[Ca:+] b (M)
[Caffeine] (mM)
[Procaine +] (mM)
1 1 1
4 4 4
2 10 10
0 0 1-5 × 10-7
0 0 0
0 0 0
1 0 0 10
0 0 0 0
2 2 10 10
0 0 Variable 0
0 0 0 0
0 0 0 10
0.5-2 0.1 0.5-2 0.1
0 0 0 0
0 0 25-50 25-50
0 0-5 0 0
1 1 1 0.02
4 4 4 l J"
All solutions contain total 20 mM piperazine-N,N'-bis(2-ethanesulfonicacid) (PIPES), pH 7.0, at 0-5 ° for amphibian fibers and 20-25 ° for mammalian fibers. Ionic strength of all solutions is adjusted to 0.17 M and 0.2 M for amphibian and mammalian fibers, respectively, by adding an appropriate amount of potassium methanesulfonate. b To calculate free Ca2~" and Mg2÷ concentrations etc., refer to some original papers, e.g., M. Iino, J. Physiol. (London) 320, 513 (198!) or K. Horiuti, J. Physiol. (London) 373, 1 (1986). c Depending on the purpose of the experiment, necessary modifications must of course be made. For example, if the effect of Mg2÷ ion is to be examined, an appropriate concentration of Mg2÷ ions should be added. d A is for amphibian fast fibers. e B is for amphibian slow fibers or mammalian fibers. s Total ATP is nearly 5 mM.
tion is required to obtain a sufficient concentration of MgATP. Values for these three concentration factors should be chosen according to fiber types. The Ca2÷-accumulating capacity of the SR per unit fiber volume is dependent on fiber type and the time of diffusion is strongly dependent on the diameter of the skinned fiber preparation. Typical figures are given in Table I. The reproducibility of the assay system can be demonstrated by the fact that repeated caffeine tests after the same procedure always give the same magnitude of contracture if slow run-down of the fiber is taken into a c c o u n t . 26 26 At the beginning of experiments in each fiber, contractile responses may not decrease but may increase slightly for the first two or three trials.
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19
Ca 2÷ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS 1 0
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Loading time (min) Flo. 1. Time course of Ca2. uptake by the SR of the skinned slow tonic fiber of Xenopus at various Ca 2+ concentrations, pCa values are identified on each curve. The Ca 2+ ion in the SR is discharged by caffeine, and the area under the resulting contracture tension curve (tension-time integral) is plotted after normalization to that at pCa 6.6 and 3 rain. The maximum level of loading at pCa 5.0 is lower than at higher pCa values, probably because of the operation of the Ca2÷-induced Ca 2+ release mechanism. Results for three slow fibers are combined. [Reproduced from K. Horiuti, J. Physiol. (London) 373, 1 (1986).]
Areas under the contracture curve (tension-time integrals) but not peak tensions are recommended as the index of the magnitude of contracture and hence of the amount of Ca 2÷ released. With an increase in the amount of Ca 2+ released peak tension does reach saturation at some point, but the tension-time integral does not because the duration of the contracture can still increase. For this reason the distortion of linearity is less if the tension-time integral is used. The approximate linearity of the assay system using tension-time integrals of caffeine contracture is supported by the fact that the time course of Ca 2÷ uptake is approximately exponential (Fig. 1). 27 As shown later, the time course of Ca 2÷ release is also approximately exponential. Both ends of a skinned fiber segment, 5 mm long, are tied with a single silk thread to hooks, one of which is connected to a tension transducer. The output from the strain-gauge transducer (UL-2, UL-20 of NMB, Ja27 A closer examination of Fig. 1 reveals that the initial parts of Ca 2+ uptake curves have a concave form. This is due to the fact that when the amount of Ca 2+ released is small enough, it only binds to EGTA in the solution and cannot evoke any contraction at all. With lower EGTA concentrations, this distortion of linearity is smaller.
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C a 2+ FLUXES AND REGULATION
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A
2 rain
1
2
3 4
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FIG. 2. (A) Tension record of a single run during a Ca 2+ release experiment on a skinned guinea pig skeletal muscle fiber. The procedure consists of three steps: (1) Ca 2+ loading to a fixed level, (2) Ca 2+ release, and (3) assay. In each step several solution exchanges are made at the time indicated by the artifacts on the tension record. The composition of the solutions is given in Table I. Solution 1, G2 relaxing solution; 2, loading solution; 3, GI0 relaxing solution; 4, rigor solution; 5, prereleasing solution; 6, releasing solution; 7, stopping solution; 8, 9, preassay solution; 10, assay solution. Note that the time scale is different for
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C a 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
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pan or AE801, AME, Norway) is amplified by a strain meter (DSA-601-B, NMB, Japan) and recorded on a pen recorder (Recticorder, NihonKohden, Japan). The length of the skinned fiber is set, using a microscope or laser diffraction, at a longer-than-optimum length (around 2.8/zm per sarcomere) because rundown of the fiber is slower at longer lengths. By exchanging solutions in a logical order, Ca 2+ release can be measured. To exchange solutions, a volume of desired solution, which is several times that of the experimental trough, is rapidly injected from a syringe through a thin short tubing and the overflow is aspirated. The following method may also be used. Solutions are placed in small wells (about 0.5 ml in volume), which are drilled in aluminum, brass, or plastic plate and resin-coated, so that the surface of the solution is about 2-3 mm above plate surface. The skinned fiber is set horizontally in the convex solution. By moving the plate horizontally, the solution exchange can easily be made. The temperature of the solution is kept constant by circulating water just underneath the trough or the plate. In the latter case a small magnetic bar is put in the solution and a magnetic stirrer is placed under the stage holding the plate. Temperatures of 00-5 ° for amphibian muscles and 200-25 ° for mammalian and avian muscles are recommended. Higher temperatures can be used, but they tend to cause a very rapid rundown of skinned fibers. Protocols for the measurement of Ca2? release in skinned fibers essentially consist of three steps. Starting from the empty SR, (I) load the SR with Ca 2÷ to a fixed level by immersing the fiber in a medium containing Ca 2÷ and MgATP for a period of time, and allow the SR to accumulate Ca z÷ by Ca2+-pump ATPase. (2) Stimulate Ca 2+ release for various periods of time by suppressing the CaZ+-pump, and by preventing secondary Ca2+-induced Ca 2÷ release. (3) Totally discharge Ca z+ remaining in the SR and measure the amount. An example of the detailed protocol for Ca 2÷induced Ca z÷ release is given in Fig. 2A. Here the run starts with the empty SR since Step 3 immediately preceding the run should have discharged all the Ca 2+ in the SR. The assay solution used in Step 3 is replaced by a relaxing solution with 2 mM EGTA. If. the EGTA concen-
solutions 9 and 10. Peak tension is 20 mg. Room temperature. (B) Time course of decrease in the amount of Ca 2÷ in the SR of a Xenopus fast fiber during Ca 2÷ release stimulated by various Ca 2+ concentrations. Experiments were performed as in (A) and described in the text. The tension-time integral during the application of solution 10 was plotted after normalization to that of the control run, which was done in exactly the same way except that the application of the releasing solution was omitted. [Reproduced from M. Endo, in "Regulation of Calcium Transport across Muscle Membranes" (A. E. Shamoo, ed.), Current Topics in Membranes and Transport, Vol. 25,' p. 181. Academic Press, New York, 1985.]
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C a 2+ FLUXES AND REGULATION
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tration in this relaxing solution is too high, the start of Ca 2+ loading is delayed by the diffusion time. If it is too low, Ca 2÷ loading may already start in the relaxing solution because of contaminating Ca 2+. Ca 2÷ loading (Step 1) starts by applying the loading solution (typical composition is given in Table I). A Ca 2+ concentration that does not directly activate the contractile system is usually preferred. The Ca 2+ concentration should be buffered with a high concentration of a Ca 2÷buffer. After an appropriate period of time, the loading solution is replaced by a relaxing solution having a high concentration of EGTA to stop the loading immediately. The loading period is chosen so as to obtain a 30-70% maximum level of loading. With excessive loading Ca 2÷ tends to be released very easily or even spontaneously. 28 On the other hand, the time course of Ca 2+ decrease in the SR due to a stimulus may be difficult to determine accurately with too small a level of initial loading. Before the Ca2÷-releasing stimulus is applied in Step 2, the solution must be changed appropriately. First, ATP is removed to stop the Ca 2÷ pump. The removal of ATP takes a relatively long time because of its slow diffusion due to the presence of concentrated ATP-binding sites in the fiber. In the example given in Fig. 2A, several exchanges of ATP-free rigor solution (Table I) are made. Removal of MgATP is indicated by rigor tension development. If a sufficient amount of Mg 2÷ ion is present as in the rigor solution of this example, this can approximately be equated with the removal of total ATP. Prereleasing solution (Table I), which is the same as releasing solution except that it does not contain Ca2÷-releasing stimulus, was then applied. Sufficient time must be allowed so that each constituent in the prereleasing solution can reach equilibrium. Then, the Ca2÷-releasing stimulus, in this example, the Ca 2+ ion, (releasing solution, Table I), is applied for a predetermined period of time. Care should be taken to initiate and to terminate the releasing stimulus as quickly as possible, especially if the period of stimulation is short. In the case of Ca2÷-induced Ca 2÷ release, a Ca 2÷ solution with a high concentration of Ca 2+ buffer is used to initiate Ca 2+ stimulus quickly. A special stopping solution, which is not only devoid of Ca 2+, but also contains inhibitors of Ca2+-induced Ca 2÷ release, Mg 2÷ ion, and procaine 19,29(Table I), is used to shut off the Ca2+-induced Ca 2÷ release as quickly as possible. In Step 3, before the assay solution is applied, EGTA concentration must be reduced to the level of that in the preassay solution by applying preassay solution (Table I). However, if the skinned fiber is kept in a 28 This is probably ascribable to s e c o n d a r y Ca 2÷ release due to activation o f Ca2+-induced Ca 2+ release by Ca 2÷ leakage from the SR. T h e rate of Ca 2+ leakage should be nearly proportional to the level o f Ca 2+ loaded. 29 S. T h o r e n s and M. Endo, Proc. Jpn. Acad. 51, 473 (1975).
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Ca 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
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relaxing solution containing a low concentration of EGTA with minimal buffering capacity, a small amount of Ca 2÷ leaking out of the SR may not be effectively buffered and may stimulate the Ca2÷-induced Ca :÷ release before the assay is made. To avoid this, the Mg 2÷ concentration in the preassay solution should be increased and, if necessary, procaine should be added. After equilibration with the preassay solution, the assay solution is applied and the resulting contraction is recorded. In this assay, the decrease in Mg 2÷ and procaine concentrations to the level of the assay solution is obviously not immediate. However, unlike Ca 2÷ release by the releasing solution (the main purpose of the experiment), what is important in the assay is to apply the assay solution under exactly the same conditions and to obtain a rapid enough Ca 2÷ release. An accurate time course of Ca 2÷ release by the assay solution is not necessary. Experiments as shown in Fig. 2A are repeated by changing the Ca 2÷ concentration and the application time of each releasing solution. To account for a slow run down of the skinned fiber, controls are run after every 4-6 experiments. The same series of solution exchanges is made without any releasing solution for the controls. The result of experiments is presented as a value relative to that of the controls in Fig. 2B. The amount of Ca 2÷ in the control runs is taken as 100%. Figure 2B shows that the time courses are roughly exponential, and, therefore, the rate constant of the decline can be taken as the index of magnitude of Ca 2÷ release under these conditions. Essentially the same procedures can be followed for amphibian and mammalian (or avian) muscles. The main differences in procedures for these classes occur in experimental temperature, ionic strength of solutions, and composition of the assay solution. (For amphibian slow tonic fibers, assay solution similar to that for mammalian muscle is suitable, probably due to the smaller Ca2÷-accumulating capacity of the SR of these fibers than amphibian fast fibers.) For other kinds of Ca2÷-releasing stimuli, essentially the same procedures can be used with appropriate modifications. C a 2+ M e a s u r e m e n t by a Fluorescent Ca 2+ Indicator
Amount of Ca 2+ discharged from the SR of skinned fiber can also be measured by the change in the optical properties of a Ca 2+ indicator dye. Fura-2, a fluorescent Ca 2+ indicator, 3° is an EGTA analog which has a dissociation constant for Ca 2+ as low as 200 nM. Therefore fura-2 can bind most of the Ca 2+ discharged from the SR, when it is the major Ca 2+ 3o G. Grynkiewicz, M. Poenie, and R. Y. Tsien, J. Biol. Chem. 2609 3440 (1985).
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C a 2+ FLUXES AND REGULATION
[2]
buffer in the assay solution. When this dye is excited by 340 nm ultraviolet light, the fluorescence intensity increases 3-fold upon binding of Ca 2+. For our experiments, we use a glass capillary as a cuvette and a fluorescence microscope equipped with a photomultiplier tube as a fluorometer. Conventional cuvettes and spectrofluorometers should not be used for the following reasons: (1) The maximum amount of Ca 2÷ released from a skinned skeletal muscle fiber with a diameter of 100/zm is of the order of I0 pmol/mm of fiber length. If the amount of Ca 2÷ is then diluted with 1 ml of the assay solution, it will result in only a 0.1/zM increase in Ca 2÷ concentration even if a 10-mm-long skinned fiber is used. On the other hand, in a capillary with an internal diameter of 400/zm, several tens of a micromolar change in Ca :+ concentration can be produced. (2) Solution changes cannot be carried out rapidly using a conventional cuvette and large quantities of solutions containing fura-2 (which is rather expensive) are discarded. This problem can be circumvented if a capillary cuvette is used. Using silk filaments, skinned fiber, 5 mm in length, is tied to a resincoated tungsten wire with a diameter of 100/xm. It is then inserted into and fixed in a glass capillary (internal diameter 400 ~m, length 32 mm) and mounted on the stage of a microscope. Both ends of the capillary are linked to silicone tubing. One of the tubings is connected to a peristaltic pump, so that, by placing the free end of the silicone tubing in solution and running the pump, it is possible to rapidly change and to perfuse the solution in the capillary cuvette. A fluorescence microscope is used for the detection of fura-2 fluorescence. Excitation light (340 nm) is provided by a Xenon lamp via a narrow bandwidth interference filter. A 0.8-mm length of the cuvette is epiilluminated through a 20x objective. Emitted light is collected by the same objective, and focused on the photomultiplier tube through 500 nm band-pass filter. Calibration of the system using 40-100 p,M of fura-2 gives a linear relationship between the total Ca 2÷ concentration and the change in fluorescence intensity up to one-half of the maximum change as theoretically expected from one-to-one binding between Ca 2÷ and the dye. Since ultraviolet light is known to be harmful to the SR, 31 it is desirable to have a shutter in the excitation light path and to keep the exposure time as short as possible. In our system, the shutter is pneumatically opened for 50 msec every 5 sec. The shutter operation, as well as the peristaltic pump operation and data collection, is controlled by a microcomputer. The procedure for the Ca 2+ assay is essentially the same as that de31 T. Nagai, M. Makinose, and W. Hasselbach, Biochim. Biophys. Acta 43, 223 (1960).
[2]
C a 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
25
IO-lmax
30s Time FIG. 3. Fluorescence change of fura-2 during Ca 2+ assay. Caffeine-containing assay solution is injected into the capillary cuvette at the time marked by the arrow. Upper trace is obtained after Ca 2+ loading at pCa 6.7 for 60 sec. No Ca 2÷ loading is carried out for the lower trace. Fura-2 concentration is 40/xM, and, therefore, the vertical bar corresponds to 4/zM of Ca 2+. Guinea pig EDL; fiber width, 40/zm.
scribed in the previous section, and 50 mM caffeine is used to discharge Ca 2+ from the SR. There are, however, a few modifications. ATP is withdrawn prior to the assay and replaced by 25 mM AMP during the assay. This will ensure that the fiber remains unmoved on increasing Ca 2÷ concentration. AMP is introduced because adenine nucleotides are known to enhance Ca2+-induced Ca 2÷ release) 2,33 Both Mg2+ and EGTA are also removed prior to the assay in order to enhance the Ca 2+ release by caffeine and to allow fura-2 to bind all the Ca 2+ discharged. The change in fluorescence intensity takes place in two phases after the application of the caffeine solution (Fig. 3). First, it increases rapidly within I0 to 15 sec (step), then there is a slow, more-or-less linear increase (creep). A part of the step change is due to the effect of caffeine on fura-2 fluorescence and is Ca 2+ independent. The slope of the creep is dependent on the duration and Ca 2+ concentration of the Ca 2+ treatment before the assay, but is present even if ATP is withdrawn during the Ca 2+ treatment. Therefore, the creep seems to be due to very slow release of Ca 2+ passively trapped or bound by the skinned fiber. Step amplitude minus Ca2+independent step amplitude is both Ca 2+- and ATP-dependent, and should 32 M. Endo and T. Kitazawa, Proc. Jpn. Acad. 52, 595 (1976). 33 y . Kakuta, Pfluegers Arch. 400, 72 (1984).
26
C a 2+ F L U X E S
AND REGULATION
[3]
represent the amount of Ca :+ which has been stored in the SR and is discharged by caffeine. Since the Ca 2+ signal is obtained from the center of a 5-mm-long skinned fiber, any distortion of the fluorescence change due to diffusion of Ca 2÷ along the fiber length seems minimal. The concentration of fura-2 chosen is such that the size of the step change in fluorescence intensity does not exceed one-half of the maximum response. The time course of both Ca 2+ uptake by and Ca2+-induced Ca 2+ release from the SR of skinned fibers appears to be exponential. This provides another test for the linearity of the Ca2+-measuring system. This Ca2+-measuring system has also been successfully applied to cardiac and smooth muscle chemically skinned fibers in our laboratory. 34 34 M. Iino, Biochem. Biophys. Res. Commun. 142, 47 (1987).
[3] I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f S a r c o l e m m a l Vesicles from Rabbit Fast Skeletal Muscle
By STEVEN SEILER and SIDNEY FLEISCHER The study of sarcolemmal ion transport using whole muscle cells is complicated by contributions of the internal membrane systems and their respective transport activities. The availability of purified sarcolemmal membrane vesicles simplifies the study of ion transport referable to sarcolemma. This report describes the preparation of skeletal muscle sarcolemma in the form of sealed, predominantly inside-out vesicles, which are suitable for transport studies. Overview of the procedure--the sarcolemmal vesicle isolation procedure includes subjecting ground fast skeletal muscle to several limited blendings and washings in 0.6 M KC1. A low-speed sediment is rehomogenized in buffered sucrose and a microsomal fraction is obtained by differential centrifugation. The microsomes are then fractionated using sequential isopycnic sucrose and dextran T-10 density gradient centrifugations. ~,2 t These studies were supported by a grant from the National Institutes of Health DK 14632 and the Muscular Dystrophy Association of America, and a Biomedical Research Support Grant from the National Institutes of Health administered by Vanderbilt University (SF). 2 S. Seiler and S. Fleischer, J. Biol. Chem. 257, 13862 (1982).
METHODS IN ENZYMOLOGY, VOL. 1.57
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
C a 2+ F L U X E S A N D R E G U L A T I O N
[2]
[2] M e a s u r e m e n t o f C a 2+ R e l e a s e in S k i n n e d F i b e r s from Skeletal Muscle
By M. ENDO and M. hNo Introduction C a 2+ release from the sarcoplasmic reticulum (SR) is one of the most important steps in excitation-contraction coupling of skeletal muscle. However, while various kinds of stimuli experimentally applied to the SR can be shown to cause Ca 2+ release, the mechanism of physiological Ca 2+ release from the SR is not yet known.l We have been trying to find out what kinds of stimuli directly applied to the SR of skinned muscle fibers cause Ca z+ release in the hope that, from the effective stimuli, we might be able to determine the mechanism utilized in physiological C a 2+ release. In this chapter, we will describe in detail the methods for measurement of Ca 2+ release in skinned fibers that are used in our laboratory.
General Considerations
In the experiments o n C a 2+ release from the SR, one must take the following facts into account. (1) There are several Ca 2+ release mechanisms in the SR, which are different at least in their activation mechanisms but probably also in their Ca 2+ channels. 1 Therefore, determinations regarding one of the release mechanisms can not be applied automatically to Ca 2+ release in general. (2) SR membrane also has a strong Ca 2+ uptake activity through the calcium pump ATPase. 2,3 Therefore, unless the Ca2+-pump is inhibited by the experimental condition, Ca 2+ released by any means tends to activate the pump and partly be taken up again. (3) Ca 2+ also activates one of the Ca 2+ release mechanisms present in the SR, the so-called Ca2+-induced Ca z+ release mechanism. 4,5 The secondary Ca z+ release caused by Ca z+ released by a primary process must be strictly distinguished from the primary release, if the properties of the latter are to be examined. For this reason, simple monitoring of M. Endo, in "Regulation of Calcium Transport across Muscle Membranes" (A. E. Shamoo, ed.), Current Topics in Membranes and Transport, Vol. 25, p. 181. Academic Press, New York, 1985. 2 S. Ebashi and F. Lipmann, J. Cell Biol. 14, 389 (1962). 3 W. Hasselbach and M. Makinose, Biochem. Z. 333, 518 (1961). 4 L. E. Ford and R. J. Podolsky, Science 167, 58 (1970). 5 M. Endo, M. Tanaka, and Y. Ogawa, Nature (London) 228, 34 (1970).
METHODS IN ENZYMOLOGY, VOL. 157
Copyright © 1988by Academic Press, Inc. All rights of reproduction in any form reserved.
[2]
C a 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
13
Ca 2+ concentrations outside the SR is not suitable for the measurement of the primary Ca 2+ release process. Instead Ca 2÷ concentrations outside the SR should be fixed as far as possible by the use of a Ca 2+ buffer. Under this condition, the Ca 2÷ release can be estimated by the time course of decrease in the amount of Ca 2÷ in the SR. Ca 2+ release from the SR can be studied in skinned fibers as described here or in vesicles of fragmented sarcoplasmic reticulum (FSR) as described in [32-34], this volume. Skinned fibers have a physiological advantage over the FSR and, probably for this reason, Ca 2÷ release is easier to evoke in skinned fibers than in FSR. In fact, many important modes of Ca 2+ release have been found in skinned fibers first and then confirmed with FSR. *-6 It is also easier to exchange the environmental solutions quickly in skinned fibers, whereas in FSR an exchange of solutions can only be made by filtration or centrifugation or, less precisely, by dilution. Another advantage (or possible disadvantage) of skinned fibers is the fact that the lumen of the whole SR is most probably continuous in skinned fibers, so that even if a single Ca 2+ channel throughout the whole SR is open, the Ca 2÷ release continues until the electrochemical potential gradient of Ca 2+ disappears between the inside and the outside of the SR lumen. In contrast, some FSR vesicles may not have a certain kind of Ca 2+ channel and thus may not respond to stimuli that activate that kind of channel. This heterogeneity of the vesicles must be kept in mind in interpreting results of FSR experiments. On the other hand, it is easier with FSR than with skinned fibers to follow Ca 2+ movements precisely and to conduct a large number of experiments by changing experimental conditions. Another disadvantage of skinned fibers is that while an exchange of solutions in the extrafiber space could be immediate, the change in composition in the real environment of the SR is rather slow due to a relatively long diffusion distance. Preparation of Skinned Fibers Preparing skinned fibers from muscles requires two steps: (1) isolation of a single fiber or a sufficiently thin bundle of fibers and (2) skinning or destruction of the semipermeability of the surface membrane. Skinning can be carried out before the isolation of single fibers or bundles if chemical skinning is used. Thicker bundles, of diameter larger than about 150 /xm, may be used for qualitative studies but they are not recommended for quantitative experiments because the diffusion time is too long. The best way to prepare a skinned fiber is first to isolate an intact 6 M. Endo and Y. Nakajima, Nature (London), New Biol. 246, 216 (1973).
14
Ca2÷ FLUXESAND REGULATION
[2]
single fiber from tendon to tendon in a physiological extracellular solution and then to skin it. This method assures that only damage will be due to skinning. Whenever possible, therefore, this procedure is recommended, but since isolating intact single fibers requires considerable skill and is time-consuming, an alternative is to isolate single fibers or a segment of fibers in a relaxing solution either before or after skinning. In this case, fibers should be examined under a microscope of a sufficient power to select parts with uniform striations for experimentation. The isolation of single fibers is done under a stereomicroscope of 40 × 80× magnification, with the aid of forceps and small scissors, knives, or needles. Tips of these instruments should be sharpened on an oil stone. The semitendinosus, iliofibularis, and tibialis anterior of amphibia are the muscles used most frequently for intact single fiber isolation. Slow tonic fibers can be obtained from amphibian iliofibularis muscles. 7 Mammalian fast twitch and slow twitch fibers can be obtained from extensor digitorum longus and soleus muscles, respectively. The strontium sensitivity of the contractile system is about one order of magnitude higher in both slow twitch 8,9 and slow tonic fibers 7 than in fast fibers and can be used to confirm fiber type. However, any kind of animal muscles can be used. Skinning can be carried out either mechanically or chemically.
Mechanical Skinning Mechanical skinning can be performed in either an oiP ° or a relaxing solution. 6 The entire surface membrane can be removed ~°or the fibers can be split into two (or more) longitudinal pieces to get partially skinned fibers. 6 The main difference between completely skinned fibers and partially skinned fibers is that, whereas in completely skinned fibers the disrupted T system membrane is likely to be sealed off and a potential difference across the T membrane may be reestablished with the aid of active sodium transport, in partially skinned fibers (if appropriately prepared) the mouths of the T tubules on the remaining sarcolemma may still be open to the outside and no ionic gradient across the T membrane can be established. 6 In the former, therefore, the real physiological stimulus to cause Ca 2÷ release, the depolarization of the T membrane, can still be effective, but in the latter, since the T membrane is completely depolarized from the beginning, it is extremely difficult, if not impossible, to 7 K. Horiuti, J. Physiol. (London) 373, 1 (1986). 8 S. K. B. Donaldson and W. G. L. Kerrick, J. Gen. Physiol. 66, 427 (1975). 9 A. Takagi and M. Endo, Exp. Neurol. 55, 95 (1977). to R. Natori, Jikeikai Med. J. 1, 119 (1954).
[2]
C a 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
15
cause Ca 2÷ release by manipulating the T membrane potential. To obtain well-resealed T tubules, skinning in an oil with a minimal quantity of extracellular fluid around the fiber is recommended, since Ca 2÷ seems to be required for the fusion of the disrupted membranes.
Chemical Skinning Chemical skinning for Ca 2÷ release experiments should be performed with an agent that destroys the semipermeability of the surface membrane but not that of the SR. Two techniques have been reported for skeletal muscles: (1) ethylenediaminetetraacetic acid (EDTA) or ethylene glycol bis(fl-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) treatment of human fibers and (2) treatment with saponin. EDTA-treated chemically skinned fibers were first described by Winegrad. H He showed that, when bundles of cardiac fibers are treated with a solution containing 3 mM EDTA for longer than 15 min at room temperature, the surface membrane became permeable to Ca 2÷, EGTA, ATP, and other small molecules but not to large molecules such as proteins. This method does not work for skeletal muscle fibers of amphibia, but human skeletal muscle fibers are reported to be skinned by a similar EGTA treatment, n However, this might not be purely chemical skinning but EGTA-assisted disruption of the surface membrane, unlike EDTAskinned cardiac fibers. 12 Treatment with 5-50/xg/ml saponin for 30 min specifically perforates the surface membrane in amphibian skeletal fast muscle fibers. ~3 A concentration of saponin higher than 150/xg/ml destroys the function of the SR as well. ~3Essentially the same applies to other kinds of fibers, but the concentration of saponin affecting the SR function differs in different kinds of fibers. For slow tonic fibers of amphibia and for mammalian skeletal muscle fibers, a lower concentration such as 20/zg/ml is recommended. In precise quantitative studies the possible effect of saponin on the SR should be checked under experimental conditions. The specificity of saponin comes from the fact that it acts on cholesterol molecules 14and that the cholesterol content of the surface membrane is much higher than that of the SR membraneJ 5,16 Therefore, besides saponin, other agents H S. Winegrad, J. Gen. Physiol. 58, 71 (1971). J2 D. S. Wood, J. Zollman, J. P. Reuben, and P. W. Brandt, Science 187, 1075 (1975). 13 M. Endo and M. Iino, J. Muscle Res. Cell Motil. 1, 89 (1980). 14 I. Ohtsuki, R. M. Manzi, G. E. Palade, and J. D. Jamieson, Biol. Cell. 31, 119 (1978). 15 A. Martonosi, Biochim. Biophys. Acta 150, 694 (1968). 16 K. Waku, Y. Uda, and Y. Nakazawa, J. Biochem. (Tokyo) 69, 483 (1971).
16
C a 2+ FLUXES AND REGULATION
[2]
which affect cholesterol, such as digitonin, may have a similar effect. Such agents could also be used for chemical skinning for Ca 2÷ release measurement, if their effects on the SR are negligible. In chemical skinning, the T tubule membrane is also permeabilized along with the surface membrane, so that no potential gradient can be established across the T membrane, as in the case of partially skinned fibers. C a 2+ M e a s u r e m e n t
As described under General Considerations, measurement of Ca 2+ release from the SR should be made by determining the time course of decrease in the amount of Ca 2÷ in the SR at a fixed Ca 2÷ concentration, to avoid the secondary Ca2÷-induced Ca 2÷ release. Measurement of the amount of Ca 2÷ in the SR can be made directly by using 45Ca or by discharging all of Ca 2÷ and measuring the amount discharged. A more-or-less continuoUs monitoring of Ca 2÷ content of the SR in skinned fibers with 45Ca is probably possible, since such experiments in intact single fibers have been reported. 17,1aHowever, it requires 45Ca of a very high specific activity and very careful experimental apparatus and design because counts of/3-emission of 45Ca that is of low energy are sharply diminished by small increases in distance between fiber and counter. Alternatively, if 45Ca is extracted from the fiber for counting, 19,2° the measurement is reliable. However, a single time course cannot be determined with a single fiber, but requires a large number of fibers, which makes the experiment cumbersome. On the other hand, under appropriate conditions a high concentration of Caffeine causes an almost complete release of Ca 2+ from the SR without any damage to the SR, so that many time courses can be determined in one skinned fiber. Since this kind of experiment can be more conveniently conducted than the 45Ca experiments mentioned above, this is the method of choice in general Ca 2+ release measurements. The amount of Ca 2÷ discharged from the SR can be determined by using (1) the size of resulting contracture of the skinned fiber (bioassay), (2) Ca 2+ indicator dyes, (3) a luminescent protein, aequorin, 21 and (4) probably 45Ca. Only the first two methods will be described. 17 B. A. Curtis, J. Gen. Physiol. 50, 255 (1966). 18 B. A. Curtis and R. S. Eisenberg, J. Gen. Physiol. 85, 383 (1982). 19 L I E . Ford and R. J. Podolsky, J. Physiol. (London) 223, 1 (1972). 20 E. W. Stephenson, J. Gen. Physiol. 71, 411 (1978). 21 j. R. Blinks, W. G. Wiet, P. Hess, and F. G. Prendergast, Prog. Biophys. Mol. Biol. 40, 1 (1982).
[9_]
C a 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
17
Cae+ Measurement by Utilizing Contraction of the Skinned Fiber Under appropriate conditions, application of a high concentration of caffeine causes contraction of skinned fibers in a relaxing solution due to the release of Ca 2÷ from the SR. zz Caffeine causes this by stimulating the Ca2+-induced Ca 2+ release. 23 The contractions are transient because the Ca 2÷ released in the fiber space quickly diffuses into the relatively large volume of medium bathing the fiber. Free EGTA in the relaxing solution in turn diffuses into the fiber space. In order to obtain a good estimate of Ca 2+ release from the size of contraction, the time course of Ca 2+ released by caffeine must be rapid compared with that of diffusion. After a single caffeine treatment, the SR becomes practically empty of Ca 2+. Reapplication of caffeine (after the drug is washed out once) no longer causes any response until the SR is allowed to reaccumulate Ca 2+. The completeness of the Ca 2÷ release by caffeine can be checked either directly by determining the amount of 45Ca present in the SR before and after the caffeine treatment or indirectly and less precisely by showing that incubation with a low concentration of Ca 2+, e.g., 0.03 /zM, can cause a recovery of detectable caffeine contracture. This indicates that caffeine reduces the level of Ca 2+ in the SR below the equilibrium level with the low Ca 2+ concentration. 2z To obtain rapid and complete Ca 2+ release, the effect of caffeine should be strong. To achieve this, (1) a high concentration of caffeine must be used (25-50 mM). (2) EGTA concentration in a relaxing solution during caffeine application should be appropriate. If the EGTA concentration is too high, the Ca 2+ concentration will not be raised sufficiently as a result of the Ca 2+ release, which makes (a) Ca z+ release rather slow, z4 and (b) contractile response too small. If the concentration of EGTA is too low, distortion of the linearity of the contractile response with the amount of Ca 2+ released becomes greater due to saturation of contraction and fibers run down rapidly because of long intensive contractions. (3) Free Mg 2+ ion concentration must also be adjusted. CaZ+-induced Ca 2+ release and hence caffeine-induced Ca 2+ release are inhibited by raising Mg 2+ concentration in the medium. 25 Therefore, lower Mg 2÷ concentrations are preferable for rapid and complete Ca 2÷ release by caffeine. On the other hand, since Ca 2+ is assayed by contraction, a Mg 2+ concentra22 M. Endo, Physiol. Rev. 57, 71 (1977). 23 M. E n d o , Proc. Jpn. Acad. 51, 479 (1975). 24 B e c a u s e caffeine-induced Ca 2+ release is stronger at higher Ca 2+ concentrations, a very rapid Ca 2÷ release is obtained w h e n the Ca 2+ ion released in turn accelerates a further release o f C a 2÷. 25 M. Endo, Proc. Jpn. Acad. 51, 467 (1975).
18
Ca 2+ FLUXES AND REGULATION
[2]
TABLE I MAIN COMPOSITIONS OF SOLUTIONS USED FOR MEASUREMENTOF Ca2+-INDUCED Ca 2+ RELEASE BY UTILIZING CONTRACTION OF SKINNED FIBERSa
Solutions For Step 1 G2 relaxing solution G10 relaxing solution Loading solution For Step 2 Rigor solution Prereleasing solution c Releasing solution c Stopping solution For Step 3 Preassay solution A a Preassay solution B e Assay solution A d Assay solution B e
[Mg2+] b (mM)
[MgATP2-] b (mM)
[EGTA] total (mM)
[Ca:+] b (M)
[Caffeine] (mM)
[Procaine +] (mM)
1 1 1
4 4 4
2 10 10
0 0 1-5 × 10-7
0 0 0
0 0 0
1 0 0 10
0 0 0 0
2 2 10 10
0 0 Variable 0
0 0 0 0
0 0 0 10
0.5-2 0.1 0.5-2 0.1
0 0 0 0
0 0 25-50 25-50
0 0-5 0 0
1 1 1 0.02
4 4 4 l J"
All solutions contain total 20 mM piperazine-N,N'-bis(2-ethanesulfonicacid) (PIPES), pH 7.0, at 0-5 ° for amphibian fibers and 20-25 ° for mammalian fibers. Ionic strength of all solutions is adjusted to 0.17 M and 0.2 M for amphibian and mammalian fibers, respectively, by adding an appropriate amount of potassium methanesulfonate. b To calculate free Ca2~" and Mg2÷ concentrations etc., refer to some original papers, e.g., M. Iino, J. Physiol. (London) 320, 513 (198!) or K. Horiuti, J. Physiol. (London) 373, 1 (1986). c Depending on the purpose of the experiment, necessary modifications must of course be made. For example, if the effect of Mg2÷ ion is to be examined, an appropriate concentration of Mg2÷ ions should be added. d A is for amphibian fast fibers. e B is for amphibian slow fibers or mammalian fibers. s Total ATP is nearly 5 mM.
tion is required to obtain a sufficient concentration of MgATP. Values for these three concentration factors should be chosen according to fiber types. The Ca2÷-accumulating capacity of the SR per unit fiber volume is dependent on fiber type and the time of diffusion is strongly dependent on the diameter of the skinned fiber preparation. Typical figures are given in Table I. The reproducibility of the assay system can be demonstrated by the fact that repeated caffeine tests after the same procedure always give the same magnitude of contracture if slow run-down of the fiber is taken into a c c o u n t . 26 26 At the beginning of experiments in each fiber, contractile responses may not decrease but may increase slightly for the first two or three trials.
[2]
19
Ca 2÷ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS 1 0
j
IO
T/° ; 6 2
e~ e-
1
IU~
J
S
50 ~
=
j O
O
j
I
J
~(9 j O
+
%
pCaoT~~ 0
I
0
I
t
I
I
I
I
1
2
3
6
9
~ , ~ 1
18
Loading time (min) Flo. 1. Time course of Ca2. uptake by the SR of the skinned slow tonic fiber of Xenopus at various Ca 2+ concentrations, pCa values are identified on each curve. The Ca 2+ ion in the SR is discharged by caffeine, and the area under the resulting contracture tension curve (tension-time integral) is plotted after normalization to that at pCa 6.6 and 3 rain. The maximum level of loading at pCa 5.0 is lower than at higher pCa values, probably because of the operation of the Ca2÷-induced Ca 2+ release mechanism. Results for three slow fibers are combined. [Reproduced from K. Horiuti, J. Physiol. (London) 373, 1 (1986).]
Areas under the contracture curve (tension-time integrals) but not peak tensions are recommended as the index of the magnitude of contracture and hence of the amount of Ca 2÷ released. With an increase in the amount of Ca 2+ released peak tension does reach saturation at some point, but the tension-time integral does not because the duration of the contracture can still increase. For this reason the distortion of linearity is less if the tension-time integral is used. The approximate linearity of the assay system using tension-time integrals of caffeine contracture is supported by the fact that the time course of Ca 2÷ uptake is approximately exponential (Fig. 1). 27 As shown later, the time course of Ca 2÷ release is also approximately exponential. Both ends of a skinned fiber segment, 5 mm long, are tied with a single silk thread to hooks, one of which is connected to a tension transducer. The output from the strain-gauge transducer (UL-2, UL-20 of NMB, Ja27 A closer examination of Fig. 1 reveals that the initial parts of Ca 2+ uptake curves have a concave form. This is due to the fact that when the amount of Ca 2+ released is small enough, it only binds to EGTA in the solution and cannot evoke any contraction at all. With lower EGTA concentrations, this distortion of linearity is smaller.
20
C a 2+ FLUXES AND REGULATION
[2]
A
2 rain
1
2
3 4
=Step 1 = =
5
,
6
Step 2
78
9
• 4
,
~
)
10 Step 3
B
'f.O~
c~2.' ~,5×~o-G.M__
I
C~-~.
fft~ .c
0.5
OO@,~
E E
~ ° ~ 0.0 0
i
L
I 2 Duration of Ca2*Treatrnent
i
3
rnin
FIG. 2. (A) Tension record of a single run during a Ca 2+ release experiment on a skinned guinea pig skeletal muscle fiber. The procedure consists of three steps: (1) Ca 2+ loading to a fixed level, (2) Ca 2+ release, and (3) assay. In each step several solution exchanges are made at the time indicated by the artifacts on the tension record. The composition of the solutions is given in Table I. Solution 1, G2 relaxing solution; 2, loading solution; 3, GI0 relaxing solution; 4, rigor solution; 5, prereleasing solution; 6, releasing solution; 7, stopping solution; 8, 9, preassay solution; 10, assay solution. Note that the time scale is different for
[2]
C a 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
21
pan or AE801, AME, Norway) is amplified by a strain meter (DSA-601-B, NMB, Japan) and recorded on a pen recorder (Recticorder, NihonKohden, Japan). The length of the skinned fiber is set, using a microscope or laser diffraction, at a longer-than-optimum length (around 2.8/zm per sarcomere) because rundown of the fiber is slower at longer lengths. By exchanging solutions in a logical order, Ca 2+ release can be measured. To exchange solutions, a volume of desired solution, which is several times that of the experimental trough, is rapidly injected from a syringe through a thin short tubing and the overflow is aspirated. The following method may also be used. Solutions are placed in small wells (about 0.5 ml in volume), which are drilled in aluminum, brass, or plastic plate and resin-coated, so that the surface of the solution is about 2-3 mm above plate surface. The skinned fiber is set horizontally in the convex solution. By moving the plate horizontally, the solution exchange can easily be made. The temperature of the solution is kept constant by circulating water just underneath the trough or the plate. In the latter case a small magnetic bar is put in the solution and a magnetic stirrer is placed under the stage holding the plate. Temperatures of 00-5 ° for amphibian muscles and 200-25 ° for mammalian and avian muscles are recommended. Higher temperatures can be used, but they tend to cause a very rapid rundown of skinned fibers. Protocols for the measurement of Ca2? release in skinned fibers essentially consist of three steps. Starting from the empty SR, (I) load the SR with Ca 2÷ to a fixed level by immersing the fiber in a medium containing Ca 2÷ and MgATP for a period of time, and allow the SR to accumulate Ca z÷ by Ca2+-pump ATPase. (2) Stimulate Ca 2+ release for various periods of time by suppressing the CaZ+-pump, and by preventing secondary Ca2+-induced Ca 2÷ release. (3) Totally discharge Ca z+ remaining in the SR and measure the amount. An example of the detailed protocol for Ca 2÷induced Ca z÷ release is given in Fig. 2A. Here the run starts with the empty SR since Step 3 immediately preceding the run should have discharged all the Ca 2+ in the SR. The assay solution used in Step 3 is replaced by a relaxing solution with 2 mM EGTA. If. the EGTA concen-
solutions 9 and 10. Peak tension is 20 mg. Room temperature. (B) Time course of decrease in the amount of Ca 2÷ in the SR of a Xenopus fast fiber during Ca 2÷ release stimulated by various Ca 2+ concentrations. Experiments were performed as in (A) and described in the text. The tension-time integral during the application of solution 10 was plotted after normalization to that of the control run, which was done in exactly the same way except that the application of the releasing solution was omitted. [Reproduced from M. Endo, in "Regulation of Calcium Transport across Muscle Membranes" (A. E. Shamoo, ed.), Current Topics in Membranes and Transport, Vol. 25,' p. 181. Academic Press, New York, 1985.]
22
C a 2+ FLUXES AND REGULATION
[2]
tration in this relaxing solution is too high, the start of Ca 2+ loading is delayed by the diffusion time. If it is too low, Ca 2÷ loading may already start in the relaxing solution because of contaminating Ca 2+. Ca 2÷ loading (Step 1) starts by applying the loading solution (typical composition is given in Table I). A Ca 2+ concentration that does not directly activate the contractile system is usually preferred. The Ca 2+ concentration should be buffered with a high concentration of a Ca 2÷buffer. After an appropriate period of time, the loading solution is replaced by a relaxing solution having a high concentration of EGTA to stop the loading immediately. The loading period is chosen so as to obtain a 30-70% maximum level of loading. With excessive loading Ca 2÷ tends to be released very easily or even spontaneously. 28 On the other hand, the time course of Ca 2+ decrease in the SR due to a stimulus may be difficult to determine accurately with too small a level of initial loading. Before the Ca2÷-releasing stimulus is applied in Step 2, the solution must be changed appropriately. First, ATP is removed to stop the Ca 2÷ pump. The removal of ATP takes a relatively long time because of its slow diffusion due to the presence of concentrated ATP-binding sites in the fiber. In the example given in Fig. 2A, several exchanges of ATP-free rigor solution (Table I) are made. Removal of MgATP is indicated by rigor tension development. If a sufficient amount of Mg 2÷ ion is present as in the rigor solution of this example, this can approximately be equated with the removal of total ATP. Prereleasing solution (Table I), which is the same as releasing solution except that it does not contain Ca2÷-releasing stimulus, was then applied. Sufficient time must be allowed so that each constituent in the prereleasing solution can reach equilibrium. Then, the Ca2÷-releasing stimulus, in this example, the Ca 2+ ion, (releasing solution, Table I), is applied for a predetermined period of time. Care should be taken to initiate and to terminate the releasing stimulus as quickly as possible, especially if the period of stimulation is short. In the case of Ca2÷-induced Ca 2÷ release, a Ca 2÷ solution with a high concentration of Ca 2+ buffer is used to initiate Ca 2+ stimulus quickly. A special stopping solution, which is not only devoid of Ca 2+, but also contains inhibitors of Ca2+-induced Ca 2÷ release, Mg 2÷ ion, and procaine 19,29(Table I), is used to shut off the Ca2+-induced Ca 2÷ release as quickly as possible. In Step 3, before the assay solution is applied, EGTA concentration must be reduced to the level of that in the preassay solution by applying preassay solution (Table I). However, if the skinned fiber is kept in a 28 This is probably ascribable to s e c o n d a r y Ca 2÷ release due to activation o f Ca2+-induced Ca 2+ release by Ca 2÷ leakage from the SR. T h e rate of Ca 2+ leakage should be nearly proportional to the level o f Ca 2+ loaded. 29 S. T h o r e n s and M. Endo, Proc. Jpn. Acad. 51, 473 (1975).
[2]
Ca 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
23
relaxing solution containing a low concentration of EGTA with minimal buffering capacity, a small amount of Ca 2÷ leaking out of the SR may not be effectively buffered and may stimulate the Ca2÷-induced Ca :÷ release before the assay is made. To avoid this, the Mg 2÷ concentration in the preassay solution should be increased and, if necessary, procaine should be added. After equilibration with the preassay solution, the assay solution is applied and the resulting contraction is recorded. In this assay, the decrease in Mg 2÷ and procaine concentrations to the level of the assay solution is obviously not immediate. However, unlike Ca 2÷ release by the releasing solution (the main purpose of the experiment), what is important in the assay is to apply the assay solution under exactly the same conditions and to obtain a rapid enough Ca 2÷ release. An accurate time course of Ca 2÷ release by the assay solution is not necessary. Experiments as shown in Fig. 2A are repeated by changing the Ca 2÷ concentration and the application time of each releasing solution. To account for a slow run down of the skinned fiber, controls are run after every 4-6 experiments. The same series of solution exchanges is made without any releasing solution for the controls. The result of experiments is presented as a value relative to that of the controls in Fig. 2B. The amount of Ca 2÷ in the control runs is taken as 100%. Figure 2B shows that the time courses are roughly exponential, and, therefore, the rate constant of the decline can be taken as the index of magnitude of Ca 2÷ release under these conditions. Essentially the same procedures can be followed for amphibian and mammalian (or avian) muscles. The main differences in procedures for these classes occur in experimental temperature, ionic strength of solutions, and composition of the assay solution. (For amphibian slow tonic fibers, assay solution similar to that for mammalian muscle is suitable, probably due to the smaller Ca2÷-accumulating capacity of the SR of these fibers than amphibian fast fibers.) For other kinds of Ca2÷-releasing stimuli, essentially the same procedures can be used with appropriate modifications. C a 2+ M e a s u r e m e n t by a Fluorescent Ca 2+ Indicator
Amount of Ca 2+ discharged from the SR of skinned fiber can also be measured by the change in the optical properties of a Ca 2+ indicator dye. Fura-2, a fluorescent Ca 2+ indicator, 3° is an EGTA analog which has a dissociation constant for Ca 2+ as low as 200 nM. Therefore fura-2 can bind most of the Ca 2+ discharged from the SR, when it is the major Ca 2+ 3o G. Grynkiewicz, M. Poenie, and R. Y. Tsien, J. Biol. Chem. 2609 3440 (1985).
24
C a 2+ FLUXES AND REGULATION
[2]
buffer in the assay solution. When this dye is excited by 340 nm ultraviolet light, the fluorescence intensity increases 3-fold upon binding of Ca 2+. For our experiments, we use a glass capillary as a cuvette and a fluorescence microscope equipped with a photomultiplier tube as a fluorometer. Conventional cuvettes and spectrofluorometers should not be used for the following reasons: (1) The maximum amount of Ca 2÷ released from a skinned skeletal muscle fiber with a diameter of 100/zm is of the order of I0 pmol/mm of fiber length. If the amount of Ca 2÷ is then diluted with 1 ml of the assay solution, it will result in only a 0.1/zM increase in Ca 2÷ concentration even if a 10-mm-long skinned fiber is used. On the other hand, in a capillary with an internal diameter of 400/zm, several tens of a micromolar change in Ca :+ concentration can be produced. (2) Solution changes cannot be carried out rapidly using a conventional cuvette and large quantities of solutions containing fura-2 (which is rather expensive) are discarded. This problem can be circumvented if a capillary cuvette is used. Using silk filaments, skinned fiber, 5 mm in length, is tied to a resincoated tungsten wire with a diameter of 100/xm. It is then inserted into and fixed in a glass capillary (internal diameter 400 ~m, length 32 mm) and mounted on the stage of a microscope. Both ends of the capillary are linked to silicone tubing. One of the tubings is connected to a peristaltic pump, so that, by placing the free end of the silicone tubing in solution and running the pump, it is possible to rapidly change and to perfuse the solution in the capillary cuvette. A fluorescence microscope is used for the detection of fura-2 fluorescence. Excitation light (340 nm) is provided by a Xenon lamp via a narrow bandwidth interference filter. A 0.8-mm length of the cuvette is epiilluminated through a 20x objective. Emitted light is collected by the same objective, and focused on the photomultiplier tube through 500 nm band-pass filter. Calibration of the system using 40-100 p,M of fura-2 gives a linear relationship between the total Ca 2÷ concentration and the change in fluorescence intensity up to one-half of the maximum change as theoretically expected from one-to-one binding between Ca 2÷ and the dye. Since ultraviolet light is known to be harmful to the SR, 31 it is desirable to have a shutter in the excitation light path and to keep the exposure time as short as possible. In our system, the shutter is pneumatically opened for 50 msec every 5 sec. The shutter operation, as well as the peristaltic pump operation and data collection, is controlled by a microcomputer. The procedure for the Ca 2+ assay is essentially the same as that de31 T. Nagai, M. Makinose, and W. Hasselbach, Biochim. Biophys. Acta 43, 223 (1960).
[2]
C a 2+ RELEASE IN SKINNED SKELETAL MUSCLE FIBERS
25
IO-lmax
30s Time FIG. 3. Fluorescence change of fura-2 during Ca 2+ assay. Caffeine-containing assay solution is injected into the capillary cuvette at the time marked by the arrow. Upper trace is obtained after Ca 2+ loading at pCa 6.7 for 60 sec. No Ca 2÷ loading is carried out for the lower trace. Fura-2 concentration is 40/xM, and, therefore, the vertical bar corresponds to 4/zM of Ca 2+. Guinea pig EDL; fiber width, 40/zm.
scribed in the previous section, and 50 mM caffeine is used to discharge Ca 2+ from the SR. There are, however, a few modifications. ATP is withdrawn prior to the assay and replaced by 25 mM AMP during the assay. This will ensure that the fiber remains unmoved on increasing Ca 2÷ concentration. AMP is introduced because adenine nucleotides are known to enhance Ca2+-induced Ca 2÷ release) 2,33 Both Mg2+ and EGTA are also removed prior to the assay in order to enhance the Ca 2+ release by caffeine and to allow fura-2 to bind all the Ca 2+ discharged. The change in fluorescence intensity takes place in two phases after the application of the caffeine solution (Fig. 3). First, it increases rapidly within I0 to 15 sec (step), then there is a slow, more-or-less linear increase (creep). A part of the step change is due to the effect of caffeine on fura-2 fluorescence and is Ca 2+ independent. The slope of the creep is dependent on the duration and Ca 2+ concentration of the Ca 2+ treatment before the assay, but is present even if ATP is withdrawn during the Ca 2+ treatment. Therefore, the creep seems to be due to very slow release of Ca 2+ passively trapped or bound by the skinned fiber. Step amplitude minus Ca2+independent step amplitude is both Ca 2+- and ATP-dependent, and should 32 M. Endo and T. Kitazawa, Proc. Jpn. Acad. 52, 595 (1976). 33 y . Kakuta, Pfluegers Arch. 400, 72 (1984).
26
C a 2+ F L U X E S
AND REGULATION
[3]
represent the amount of Ca :+ which has been stored in the SR and is discharged by caffeine. Since the Ca 2+ signal is obtained from the center of a 5-mm-long skinned fiber, any distortion of the fluorescence change due to diffusion of Ca 2÷ along the fiber length seems minimal. The concentration of fura-2 chosen is such that the size of the step change in fluorescence intensity does not exceed one-half of the maximum response. The time course of both Ca 2+ uptake by and Ca2+-induced Ca 2+ release from the SR of skinned fibers appears to be exponential. This provides another test for the linearity of the Ca2+-measuring system. This Ca2+-measuring system has also been successfully applied to cardiac and smooth muscle chemically skinned fibers in our laboratory. 34 34 M. Iino, Biochem. Biophys. Res. Commun. 142, 47 (1987).
[3] I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f S a r c o l e m m a l Vesicles from Rabbit Fast Skeletal Muscle
By STEVEN SEILER and SIDNEY FLEISCHER The study of sarcolemmal ion transport using whole muscle cells is complicated by contributions of the internal membrane systems and their respective transport activities. The availability of purified sarcolemmal membrane vesicles simplifies the study of ion transport referable to sarcolemma. This report describes the preparation of skeletal muscle sarcolemma in the form of sealed, predominantly inside-out vesicles, which are suitable for transport studies. Overview of the procedure--the sarcolemmal vesicle isolation procedure includes subjecting ground fast skeletal muscle to several limited blendings and washings in 0.6 M KC1. A low-speed sediment is rehomogenized in buffered sucrose and a microsomal fraction is obtained by differential centrifugation. The microsomes are then fractionated using sequential isopycnic sucrose and dextran T-10 density gradient centrifugations. ~,2 t These studies were supported by a grant from the National Institutes of Health DK 14632 and the Muscular Dystrophy Association of America, and a Biomedical Research Support Grant from the National Institutes of Health administered by Vanderbilt University (SF). 2 S. Seiler and S. Fleischer, J. Biol. Chem. 257, 13862 (1982).
METHODS IN ENZYMOLOGY, VOL. 1.57
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.