Reduced Ca2+-induced CaZ+release from skeletal muscle sarcoplasmic reticulum at low pH JAY H. WILLIAMS' AND C H R I S ~ P H E W.R WARD Muscukar finction hbomfory, Division of Health and Physical EBEeca~ton,Virginia Polytechnic Institute and state University, Blacksburg, VA 24061-0326pU.S.A.
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Received June 18, 1991 WLLIAMS, $. H., and WARD,C. W. 1992. Reduced &la2+-inducedCa2' release from skeletal muscle sarcoplasrnis reticulum at low pH. Can. J. Physiol. Pharmacol. 70: 926-938. The purpose of this investigation was to determine the effects of reduced pH on Ga"-induced Ca2' release (CIGR) from skeletal muscle sarcoplasmic reticulum (SW). Frog semitendinosus fiber bundles (1 -3lbkmcfle) were chemically skinned via saponin treatment (50 pg/mL, 20 min), which removes the sarcolema and leaves the SR functional. The SR was first depleted of Ca2' then loaded for 2 min at pea (log free Ca2+ concentration) 6.6. CIGR was then evoked by exposing the fibers to pCa 5-7 for 5-60 s. GICR was evoked both in the absence s f ATB and Mg2+ and in the presence sf fl,.y-methyleneadeno9ine-5'-triphosphate (AMPPCP, a nsnhydrolyzable form of ATP) and Mg2'. Ca" remaining in the SR was h e n assayed via caffeine (25 mM) contraemre. In all cases, GICR evoked at pH 6.5 resulted in larger caffeine contracmres than that evoked at 7.0, suggesting that more &la" was released during CICR at the higher pH. Accordingly, rate constants for CICR were significantly greater at pH 7.0 than at pH 6.5. These results indicate that reduced pH depresses CIGR from skeletal muscle SW. Key words : sarcoplasmic reticulum, skeletal muscle, calcium ions, hydrogen ions, fatigue. WILLIAMS, J. H., et WARD,C. W. 1992. Reduced @a2+-induced Ca2+ release from skeletal muscle sarcoplasrnis: reticulum at low pH. Can. J. Physiol. Bharmacol. 70 1 926-930. Le but de cette Chde a CtC de determiner les effets d'une diminution de pH sur Ba libbration de Ca" indutte par le &la2' (LCIC) du rCticulum sarcoplasmique (WS)du muscle squelettique. On a pel6 chimiquernent des faisceaux (1 -3lfaisceaux) de fibres musculaires demi-tendineuses de grenouille par un traitement ?I la sapwine (50 pg/mL, 20 min), qui extrait le sarcoleme et laisse le RS fonctionnel. Le RS a d'absrd Ctt appauvri en Ga2', puis chargC pendant 2 min B 6,6 pCa. La LCIC a CtC provoquCe en exposant les fibres i 5-7 pCn pendant 5 -60 s, ainsi qu'en l'absence d ' N P et de Mg2' et en prCsence de P,y-mCthyl&neadCnosine-5'-triphosphate(AMPPCP, une f o m e nsn hydrolysable Q'ATB) et de Mg2+. Le &la2' rCsiduel a ensuite CtC determind via une contraction induite par la cafCine (25 mM). Dans tsus les cas, la LClC obeeme B un pH 6,5 a provoqu6 de plus fortes contractions que celle obtenue ?I un pH 7,0, suggCrant un plus forte lib&ation de Ca2+ durant la LCIC obtenue au pH 7,0. De meme, les constantes de taux pour la LCIC one kt6 significativement plus fortes h un pH 7,0 qu'h un pH 6,5. Ces rCsultats indiquent qu'une rCduction de pH diminue la LCIC du RS du muscle squelettique. Moa clkms : r6ticulum sarcoplamique, muscle squelettique, ions calcium, ions hydrogkne, fatigue. [Traduit par la rkdaction]
Introduction Regulation of the intracellular milieu during sustained or repetitive skeletal muscle contractions involves a number of extremely complex processes. Continued activation of skeletal muscle typically leads to decreased levels of energy supplies and the accumulation of metabolic waste products such as lactic acid and hydrogen ions (H +). For example, during prolonged activity? intracelular pH (pHi) may decrease by 8.6 -0.8 pH units or from about 7.0 to 6.3 (Metzger and Fitts 1987; Westehlad and Liinnergren 1986). During sustained contractions, this change in pHi is temporally associated with a reduction in force output (i.e., fatigue). Unfortunately, whether these two phenomena are causally related is not present1y known. A number of skeletal muscle cellular processes are known to be altered by changes in intracellular H+ concentration. For example, in normal, intact muscle, C02-induced acidosis depresses maximal force output by about 30% (Renaud et dal. 1986). Similarly, increased H concentration depresses maximal cdcium (@a2+)activated force by approximately 15% and reduces the Ca2+ sensitivity of the contractile proteins of skinned fibers (Donaldson and Hermansen 1978; Fabiato and Fabiato 1978; Godt and Nosek 1989). This latter effect may +
ICorrespndence may be sent to the author at the following address: HPE Division, Virginia Tech, Blacksburg, V A 24061-0326, U.S.A. Printed in Canada / Imprim6 au Canada
be due to diminished Ca2+ binding to tropsnin and (or) decreased force production by individual crossbridges (Godt and Nosek 1989). However, such pH-induced changes in force output do not fully account for the rather large decline in force output that is typically observed during fatigue. The effects of changes in pHi on sarcoplasmic reticulum (SR) function are less well understood. Decreased pH is h o w n to reduce SR CaD-ATPase activity and to inhibit Ca2+ uptake by the SR (Fabiato and Fabiats 1978; Inesi and Hill 1983). The effects of changes in pHi on @a2+release, however, are equivocal. Increased pH is h o w n to stimulate the release of Ca2+ from the SR (Shoshan et al. 1981). Reduced pH, however, has been reported to decrease (Fabiato and Fabiato 1978; Meisner and Henderson 1987) and to increase (Allen et al. 1989; Lea and Ashley 1978) Ca2+ release. For example, Fabiato and Fabiats (1978) found increased SW Ca2+ loading of skinned skeletal and cardiac muscle at low pH and high free Ca2+ concentration. They attributed the increased loading to diminished @a2+-induced @a2+release (CHCW) at the low pH. However, this hypothesis was not rigorously tested in skeletal muscle. Conversely, Lea and Ashley (1978) and Allen et al. (1989) report that myoplasmic Ca2+ transients of whole muscle were elevated when P H ~was ~ d u c e dvia C02 exposure. Their results9 however, are difficult to interpret, since the increased Ca2+ transients were abolished in a zero CaD m d u m (Lea and Ashley
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1978), and since other organelles and proteins that bind and sequester @a2+might have contributed to the elevated myoplasmic Ca2+ concentration. Thus, there is some question as to the direct effects of reduced pHi on Caw release from skeletal muscle SR. The purpose of this investigation was to determine if reduced pHi influences SW Caw release by modifying the process of CICR.
Skinned Bber preparation Bundles of muscle fibers (1 -3 fiberslbundle) were obtained from semitendinosus muscles of male grass frogs (Rana pipiens) and placed in cold relaxing solution. Bundles were then chemically skinned in the relaxing solution containing sapnin (50 pg/mL) for 20 min at room temperature. This treatment chemically removes the s a r c o l e m but leaves the SW intact and functional (Endo and Iino 1980). Skinned fibers were suspended horizontdly between a pair of self-closing forceps (Dumont No. 5), one of which was attached to a micromanipulator and the sther to an isometric force transducer (Grass FT-03), Fiber length was adjusted to and maintained at 130% of slack length. Experimental solutions were added to one of 12 wells ( = 500 pL) milled into a Plexiglas block. The volume of the solution in each well was such hat the surface of the solution protruded approximately 3 3above the surface of the block. The skinned fibers were placed just above the block and into the protruding p r tion of the solutions. The solutions to which the skinned fibers were exposed were changed by moving the block horizontally so that the fiber was transferred from one well to another. The output of the transducer was amplified (Grass 7P122, 030 Hz), displayed on a strip-chart recorder (Hanard), and, in some cases, sampled by microcomputer (MetraByte DAS-16 AID converter and Zenith PCIAT, 1898 Hz). In all cases, force output was computed from the strip-chart recordings. Sshtisns Sufficient potassium methanesulfonate (KMs) was added to all solutions to maintain an ionic strength of 0.2 M. All solutions also contained 20 mM irnidazole as a pH buffer, with find pH adjusted with KOH or KMs. In addition to the above constituents, the composition of the standard relaxing solution (R) was 5.0 rnkt MgS04, 4.6 mM Na,ATP, 10 mM ethylene glycol-bis(B-aminoehyl ether)N,N,N1,hi"tetraacetie acid (EGTA). A washing solution (W) was prepared as above except that the EGTA concentration was reduced to 2 mM. A rigor solution (&) was prepared as was R, except that the MgSO, was reduced to 1.5 mM and Na2ATB was omitted. The loading solution was prepared as was It, except hat CaSO, was added to obtain a log free Ca2+concentration (pCa) of 6.6. For these experiments, two sets of releasing solutions were prepared. One set was prepared as was R, except that MgSO, and Na2ATP were omitted. To more accurately simulate physiological conditions, a second set of releasing solutions was prepared as was R, except that Mg2+ was not removed but kept at f .5 d, and 1.0 mM 0,~-methyleneadenosine-5'-triphosphate (AMPPCP), a nonhydrolyzable analogue of ATB, was added (Ohta et al. 1989). For both sets of releasing solutions, CaSO, was added to obtain pCa 5, 6, and 7. For the loading and releasing solutions, the amount of CaSO, added to obtain each pCa was calculated using apparent stability constants (adjusted for ionic strength, pH 7.0 or 6.5, and 20°C) and the computer program published by Fabiato (1988). The apparent stability constants for AMPPCP were assumed to be the same as those of ATP (Ohta et al. 2989). Free Ca2+ concentrations in the Ca2+-containing solutions were also checked with a Ca2+-sensitive minielectrode N o r l d Precision %nstruments). Measurement sf &iaB -induced Ca" release The method of determining ClCR was based on that described by Endo (1977). The SR of the skinned fibers was first depleted of Ca2+ by exposure eo R containing 25 mM caffeine. The SR was then loaded by a 2-min exposure to the loading solution. Loading was ter-
FIG. 1. Typical effects of reducing pH from 7.0 to 6.5 on CICR. Both tracings were obtained from the same skinned fiber and were recorded after the standard 2-min loading period (pCa 6.6). Solid circles represent the transfer of h e fiber between solutions. See Methods for definition of solutions. CAHF, caffeine. minated and excess Ca2+removed by two rinses in R,, and the fiber was relaxed by two rinses in R. Following a rinse in R, to remove free ATP, CICR was evoked by exposing the fibers to one of the releasing solutions for 5, 15, 30, or 60 s. Because the releasing solutions contained no ATB, @a" was released but not taken up by the SR. Each fiber was then rinsed twice in R, to remove free Ca2+ and once in R to break the rigor bonds h a t were formed in the absence of AT$. The fibers were then rinsed twice in W to remove excess EGTA, and the amount of Ca" remaining in h e SR after CICR was determined by exposure to 25 mM caffeine (in W). The magnitude of this caffeine eontracture sewed as a measure of the amount of Ca2+ remaining in the SR. In each experiment, the rinsing perids were maintained at 10 s. Control experiments were performed as above but with the CICR step omitted. A set of CICR "runs" consisted of performing the above procedure four times, once each using CICR durations of 5, 15, 30, and 60 s. These runs were bracketed by both control measures and measures of maximal Ca2+-activated force (pea 4.5). Initial work indicated that at least two sets of runs could be performed on a single fiber bundle without causing significant 6'nm-dow~9'( > 10%). Thus, two sets of mns, one each at pH 7.0 and 6.5, were performed on a single fiber bundle. Bn half of the fibers tested, a set of runs with pH 7.0 was performed first and a set with pH 6.5 performed second. 'This protocol thus allowed a single fiber to serve as its own control. Statistical asaalysis Analysis of variance, adjusted for repeated measures performed on individual fibers, was used to determine differences in the extent of CICR between pH 7.0 and 6.5. Significance was established at p < 0.05.
Typical effects of reducing pH from 7.8 to 6.5 on Ca2+ release from the SR are shown in Fig, 1, Both tracings were recorded from the same fiber, in which CICR was evoked for 15 s with a pCa of 6.8 and in the absence of Mg2+ and ATP. As can be seen, the caffeine contracture recorded after evoking CICR at pH 7.8 is approximately 30% smaller than that induced after CICR was evoked at pH 6.5. This suggests that more Ca2+ remained in the SR after CICW at the low pH. Figure 2 shows the relative magnitude of caffeine contracfure c corded after evoking CICR9 in the absence of N P and Mg2'. for 5 -60 s at P C 5~-7- In this figure, 100% refers to the magnitude of the caffeine contractwe recorded without prior CICR (i.e., control). FOPall durations of CICR and each
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CAN. 5. BHYSIOL. PHARMACOL. VOL. 70, 1992
01
6
I
I
I
I
%B
96
46
60
CICR DUUTION (s) FIG.2. Relative magnitude of the caffeine contractures after evoking CICR at pH 7.0 and 6.5. Experiments were performed in the absence of both ATP and I&IIgZf.100% refers to the control contraca r e (no CICR). Values are given as means i SE (in some cases, the error bars are obscured by the symbol). *p < 0.05, n = 8.
pCa, caffeine contractures following CICR at pH 6.5 were slgnificantly greater than those evoked after CICR at pH 7.0 ( p < 0.05). The effects of reduced pH on CICR in the presence of Mg2' and AMPPCP are shown in Fig. 3. At both pH '7.0 and 6.5, larger caffeine contracture forces were obtained after CICR in the presence of these compounds than in their absence. However, the effects of reduced pH were qualitatively similar in that at each pCa level and duration of CICR, significantly larger contractures were obtained after CICR at pH 6.5. Because the magnitude of these assay caffeine contractuses indicates the amount of Ca2+ that remained in the SR after CICR, the results of Pigs. 2 and 3 suggest that less Ca2+ was released from the SR whew CICR was evoked at low pH. The decline in contracture force as a function of increasing
PIG.3. Relative magnitude of the caffeine contractures afier evoking CICR at pH 7.0 and 6.5. Experiments were performed in the presence of 1.5 mM MgZf rand 1 mM AMPPCP. *p < 0.05, n = 6.
CICR durations follows first-order kinetics (Ohta et al . 1989). Thus, the fractional rate (k) of CICR per unit time (i. e., rate constant of CICR) was computed for each fiber and pH condition as k = -On B)lt, where B is the relative force of caffeine contracture (P/Bo) and t is the duration of CICR (Ohta et d. 1989). Fitting of each fiber's data to this exponential function yielded r2 values between 0.96 and 0.99. Figure 4 shows rate constants computed for experiments performed in the absence of ATP and Mg2+ (top) and in the presence of Rag2+ and AMPPCP (bottom). En both cases, for each pCa the rate constants of CICR were significantly greater when CICR was evoked at pH 7.0 than at pH 6.5. To determine if reduced pH affected passive CaD efflux from the SR, fibers were exposed to R, (at pH 7.0 or 6.5) for 10 min. No significant differences in the size of the subsequent caffeine contracture between pH 4.5 and 7.0 were noted.
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WILLIAMS AND WARD
to point out that during this procedure @a2+ loading was always performed at pH 7.0 for 2 min and only after the SR had been depleted of Ca". Thus, any influence sf low pH on the SR Ca2+ ATPase and Ca2+ uptake would not have affected subsequent caffeine contractures, since loading occurred under identical conditions. Also, free Ca2 concentration and ionic strength of the releasing solutions were held constant between pH levels. Since pH affects the binding of Ca" to EGTA and free Ca" concentration, apparent stability constants that were adjusted for pH were used when preparing low and high pH releasing solutions (Fabiato 1988). Finally, assay caffeine contractures were always evoked at pH 7 -0so that any pH effects on crossbridge formation and activation would not have influenced force production. Thus, differences in the magnitude of caffeine contractures observed in this study were due to the effects of pH on CICR and support the notion that decreased pH inhibits Ca2+ release from skeletal muscle SR. One advantage of using a skinned fiber preparation to assay Ca2+ release from the SR is the ability to expose the same fiber to different conditions. However, the skinned fiber technique does not allow for quantitative measurements of Ca2+ exchange by the SR. In addition, this protocol becomes less sensitive at high Ca2+ concentrations as saturation of the Ca2+ binding sites on troponin begins to occur. To overcome this, we selected a loading condition that does not allow for full loading of the SR. Bur preliminary experiments showed that increasing the free Ca2+ concentration from pCa 6.6 to 6.0 during the 2-min loading period resulted in slightly, but significantly, larger subsequent caffeine contractures. Nevertheless, our data indicate that on a qualitative basis less Ca2+ is released during CICR at pH 6.5 than at pH 7.0 and do not represent a quantitative (either absolute or relative) amount of Ca2+ released from the SR. Although, the exact stimulus for Ca2+ release by the SR during excitation-contraction coupling is not known, most agree that CECR is not the major mode of Ca2+ release under normal conditions. However, recent evidence suggests that this component of Ca2+ release may ampli$l SR @a2+evoked by activation of the transverse-tubule voltage sensor (Lamb and Stephenson 1991). Under some circumstances, CICR may be more important. For example, it has been suggested that CICR plays a role in Ca2' release during prolonged, repetitive, and chemical activation of intact fibers (Palade et al. 1989; Volpe and Stephenson 1986; Williams 1990). In addition, it seems to participate heavily in pathological conditions such as malignant hyperthermia (Ohta et al. 1989). The extent to which CICR affects total SR Ca2+ release under various activation conditions and the changes in the CICR process that affect subsequent muscle performance await hrther exploration. At present, it is unclear how low pHi might inhibit SR CICR. It is evident from our studies that the effects of pH are not permanent but are hlly reversible. It is possible that low pH inhibits @a2+binding to and activation of the SW Ca2+release channel. Alternatively, low pH might modi$l Ca2+ movement from the SR by interacting directly with the release channel. Accordingly, Rousseau and Pinkos (1990) have shown that acidification of the cytoplasmic side of the SR alters the gating behavior of the channel and reduces its probability of opening whereas reduced pH of the luminal side of the SR reduces ion conductance through the channel. En either case, the flux of Ca2+ into the myoglasm, Ca2+ binding to troponin, and subsequent force production would be diminished by lowered pH. Clearly additional studies are needed to
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+
PCP 1 EnM
FIG.4. Rate constants for CICR as a function of pCa. Experiments were performed in the absence of both ATP and Mg2+ (top, n = 8) and in the presence of 1.5 mM Mg2+ and 1 mM A M P K P (bottom, n = 6). *p < 0.05.
Caffeine ~ontracture forces in the absence of Mg2+ and AMPPCP were 97.2 f 2-6 and 96-1 f 3.3% of control bH 3 * 1 and 6 - 5 and 7.0, respectively p < 0.05) and 92-8 91.0 f 2.9% ( p < 0.05). This indicates that the low pH probably does not have a major influence On the passive leak sf Ca2+ from the SR. 9
Discussion The results of this investigation suggest that a reduction of pH from 7.0 to 6.5 depresses CICR in skinned skeletal muscle fibers. In addition, this pH effect is not influenced by the presence of Mg2+ and ATP, both of which are known to affect the CICW process (Endo 1975; Endo and Kitmawa 1976). Evidence that pH reduces SR Ca2+ release has been reported for cardiac muscle (Fabiato and Fabiato 1978; Meissner and Henderson 1987). However, as noted above, a rigorous test of the mtion that low pH inhibits Ca2+ release from skeletal muscle SW has not been previously conducted. In this investigation, the magnitude of CICW was assessed indirectly by the caffeine contracture method. It is important
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CAN. J. PHYSIOE. PHARMACOL. VOL. 70, 1992
further clarify the precise mechanism(s) by which low pH reduces SR @as release. Based on the present results, it is possible that reduced CICR by low pH may account, in part, for the decline in force prsduction observed during fatigue. Recent reports indicate that during fatigue of normal skeletal muscle, a 50% decline in tetanic force is accomgmied by a 15 - 30% reduction in the peak rnyoplasmic Ca2+ transient (Allen et al. 1989; Lee et al. 1991). Application of high concentrations of Caffeine (e. g ., 10 mM) returned both tension and peak Ca2+ transient to near normal levels (Allen et al. 1989). These changes in force and myoplasmic Ca2+ concentration have furthered the speculation that fatigue may be due to an inability of the SR to release stored Ca2+. This could occur as the result of a reduced magnitude of the stimulus for Ca2+ release and (or) a reduced sensitivity of the release mechanism to the stimulus. Our data show that when a "fatigued" condition is simulated ( i s . , low pH), the sensitivity of the release mechanism to @a2+is reduced. Unfortunately, the exact stimulus for SW Ca" release during normal excitation -contraction coupling is not known. What is more significant than the specific releasing agent used in the present investigation is our finding that acidosis depresses a component of Ca2+ release from skeletal muscle SR. A pH-mediated depression s f CICW might result in a reduction in myoplasmic Ca2+ levels and subsequent force production during fatiguing contractions. Although this notion is speculative at present, it is entirely compatible with recent studies that suggest that skeletal muscle fatigue is due, in part, to a reduction in the ability of the SR to release Ca2+ (Allen et al. 1989; Lee et al. 8991). It remains to be seen if reduced pH depresses other components of Ca2+ release. Finally, it is tempting to speculate that pH-induced depression sf SR Ca2+ release might serve as a protective ~ s e h a n ism against muscle damage caused by Ca2+. Elevations in resting @aD +odd adversely affect further functioning of the muscle by stimulating @a2+-activated proteases. These proteases are h o w n to degrade a number of muscle proteins, including the contractile proteins (Kasuga and Umazume 1990) and the Ca2+-release channel of the SR (Seiler et al. 8984). It has been shown that during fatigue resting myopfasmic Ca2+ levels are elevated by more than 2-fold (Lee et d. 1991). If SR @aD release were unchecked during sustained contractions, further accumulation of myoplasmic Ca2+ could occur, which would lead to severe damage s f the active muscle fibers. Thus, it is plausible that Bow pH-induced depression of CICR prevents large elevations of resting myoplasmic Ca2+ and subsequent protein degradation. Such a mechanism would compromise force prsduction but lessen the chance of irreversible muscle damage. In summary, the present results show that CICR from the SR of skinned skeletal muscle fibers is depressed by reducing pH from 7.0 to 6.5. It is possible that such an effect accounts, in part, for the diminished SW Ca2+ release and reduced force output that is observed during fatigue. Allen, D. G., Lee, J. A., and Westerblad, H. 1989. Intracellular cakcium and tension during fatigue in isolated muscle fibers from %enspus Inevis. J. Physiol. (London), 415: 433 -458. Donaldson, S. K. B., and Hemansem, L. 1978. Differential, direct effects of H ' on Ca2+-activated force of skinned fibers from the soleus, cardiac and adduetor magnus muscles of rabbits. Pfluegers Arch. 376: 55-65. Ends, M e 1975. Conditions required for calcium-induced release of calcium from the sarcoplasmic reticulum. Proc. Jpn. Acad. 51: 467 -472.
Endo, M. 1977. Calcium release from the sarcoplasmic reticuliun~. Physiol. Rev. 57: 479 -484. Emdo, M . , and Iino, M. 1980. Specific perforation of muscle cell membranes with preserved SR functions by sapanin treatment. J. Muscle Wes. Cell Motil. 1: 89 - 100. Endo, M., and Kitazawa, T. 1976. The effect of ATP on calcium release mechanisms in the sarcoplasmlc reticulum of skinned muscle fibers. Proc. Jpn. Acad. 52: 599 -602. Fabiato, A. 1988. Computer programs for calculating total and specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 157: 378-417. Fabiato, A., and Fabiato, F. 1978. Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. 3. Physiol. (London), 276: 233 -255. Godt, R. E., and Nosek, T. M. 1989. Changes in intracellular milieu with fatigue or hypoxia depresses contraction of skinned rabbit skeletal and cardiac muscle. J. Physisl. (London), 412: 155- 180. Inesi, G., and Hill, T. E. 1983. Calcium and proton dependence of sarcoplasmic reticulum ATPase. Biophys. J. 44: 27 1 -280. Kasuga, N., and Umazume, Y. 1990. Deterioration induced by physiological concentrations sf calcium ions in skinned muscle fibers. J. Muscle Res. Cell Motil. 11: 41 -47. Lamb, G . D., and Stephenson, D. G. 1991. Effect of Mg2' on the control of Ca2+ release in skeletal muscle fibers sf the toad. J. Physiol. (London), 434: 507 -538. Lea, T. J., and Ashley, C. C. 1978. Increase in free Ca2+in muscle after exposure to Co2. Nature (London), 275: 236 -238. Lee, J. A., Westerblad, H., and Allen, D. 6. 1991. Changes in tetanic and resting [Ca2'Ii during fatigue and recovery of single muscle fibers frsm %enopus kaevis. J. Physiol. (London), 433: 30'7 - 324. Meissner, G . , and Henderson, J. S. 198%. Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca" and is modulated by Mg", adenine muclestide, and calmsdulin. J. Biol. Chem. 262: 3065 - 3073. Metzger, J. M., and Fitts, R. H. 1387. Role of intracellular pH in muscle fatigue. 9. Appl. Physiol. 62: 1392- 1397. Ohta, T., Ewdo, M., Nakano, T., Morohoshi, Y., Wanikawa, K., and Ohga, A. 1989. Ca-induced Ca release in malignant hyperthemia-susceptible pig skeletal muscle, Am. J. Physiol. 256: C358 -C367. Palade, P., Dettbarn. C., Bmnder, B., Stein, B., and Hals, 6 . 1989. Pharmacology of calcium release frsm sarcoplasmic reticulum. J. Bioenerg. Biornembr. 21: 295 -320. Renaud, J. M., Allard, Y., and Maimwood, G. W. 1986. Hs the change in intracellular pH during fatigue large enough to be the main cause of fatigue? Can. J. Physiol. Pharmacol. 64:764 -767. Rousseau, E., and Pinkos, J. 1990. pH modulates conducting and gating behavior of single calcium release channels. Pfluegers Arch. 415: M5 -644. Seiler, S., Wegener, A. D., Whang, D. D., Hathaway, D. W., and Jones, L. R. 1984. High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phospho~ylatedand are degraded by Ca2+ activated proteases. J. Biol. Chem. 259: 8550-8557. Shoshan, V., MacLennan, D. H., and Wood, D. S. 1981. A proton gradient controls a calcium-release channel in sarcoplasmic reticulum. Proc. Natl. Acad. Sci. U.S.A. 98: 4828 -4832. Volpe, P., and Stephenson, E. W. 1986. Ca2+dependence of transvirse tubule-mediated calcium release in skinnd skeletal muscle fibers. J. @en. Bhysiol. 87: 271 -288. Westerblad, H., and Lannergrem, J. 1986. The relation between force and intracellular pH in fatigued, single %emopus muscle fibers. Acta Physiol . Scand. 133: $3 - 89. Williams, J. H. 1990. Effects of low calcium and calcium antagonists on skeletal muscle staircase and htigue. Muscle Nerve, 13: 1118-1124.
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Received June 18, 1991 WLLIAMS, $. H., and WARD,C. W. 1992. Reduced &la2+-inducedCa2' release from skeletal muscle sarcoplasrnis reticulum at low pH. Can. J. Physiol. Pharmacol. 70: 926-938. The purpose of this investigation was to determine the effects of reduced pH on Ga"-induced Ca2' release (CIGR) from skeletal muscle sarcoplasmic reticulum (SW). Frog semitendinosus fiber bundles (1 -3lbkmcfle) were chemically skinned via saponin treatment (50 pg/mL, 20 min), which removes the sarcolema and leaves the SR functional. The SR was first depleted of Ca2' then loaded for 2 min at pea (log free Ca2+ concentration) 6.6. CIGR was then evoked by exposing the fibers to pCa 5-7 for 5-60 s. GICR was evoked both in the absence s f ATB and Mg2+ and in the presence sf fl,.y-methyleneadeno9ine-5'-triphosphate (AMPPCP, a nsnhydrolyzable form of ATP) and Mg2'. Ca" remaining in the SR was h e n assayed via caffeine (25 mM) contraemre. In all cases, GICR evoked at pH 6.5 resulted in larger caffeine contracmres than that evoked at 7.0, suggesting that more &la" was released during CICR at the higher pH. Accordingly, rate constants for CICR were significantly greater at pH 7.0 than at pH 6.5. These results indicate that reduced pH depresses CIGR from skeletal muscle SW. Key words : sarcoplasmic reticulum, skeletal muscle, calcium ions, hydrogen ions, fatigue. WILLIAMS, J. H., et WARD,C. W. 1992. Reduced @a2+-induced Ca2+ release from skeletal muscle sarcoplasrnis: reticulum at low pH. Can. J. Physiol. Bharmacol. 70 1 926-930. Le but de cette Chde a CtC de determiner les effets d'une diminution de pH sur Ba libbration de Ca" indutte par le &la2' (LCIC) du rCticulum sarcoplasmique (WS)du muscle squelettique. On a pel6 chimiquernent des faisceaux (1 -3lfaisceaux) de fibres musculaires demi-tendineuses de grenouille par un traitement ?I la sapwine (50 pg/mL, 20 min), qui extrait le sarcoleme et laisse le RS fonctionnel. Le RS a d'absrd Ctt appauvri en Ga2', puis chargC pendant 2 min B 6,6 pCa. La LCIC a CtC provoquCe en exposant les fibres i 5-7 pCn pendant 5 -60 s, ainsi qu'en l'absence d ' N P et de Mg2' et en prCsence de P,y-mCthyl&neadCnosine-5'-triphosphate(AMPPCP, une f o m e nsn hydrolysable Q'ATB) et de Mg2+. Le &la2' rCsiduel a ensuite CtC determind via une contraction induite par la cafCine (25 mM). Dans tsus les cas, la LClC obeeme B un pH 6,5 a provoqu6 de plus fortes contractions que celle obtenue ?I un pH 7,0, suggCrant un plus forte lib&ation de Ca2+ durant la LCIC obtenue au pH 7,0. De meme, les constantes de taux pour la LCIC one kt6 significativement plus fortes h un pH 7,0 qu'h un pH 6,5. Ces rCsultats indiquent qu'une rCduction de pH diminue la LCIC du RS du muscle squelettique. Moa clkms : r6ticulum sarcoplamique, muscle squelettique, ions calcium, ions hydrogkne, fatigue. [Traduit par la rkdaction]
Introduction Regulation of the intracellular milieu during sustained or repetitive skeletal muscle contractions involves a number of extremely complex processes. Continued activation of skeletal muscle typically leads to decreased levels of energy supplies and the accumulation of metabolic waste products such as lactic acid and hydrogen ions (H +). For example, during prolonged activity? intracelular pH (pHi) may decrease by 8.6 -0.8 pH units or from about 7.0 to 6.3 (Metzger and Fitts 1987; Westehlad and Liinnergren 1986). During sustained contractions, this change in pHi is temporally associated with a reduction in force output (i.e., fatigue). Unfortunately, whether these two phenomena are causally related is not present1y known. A number of skeletal muscle cellular processes are known to be altered by changes in intracellular H+ concentration. For example, in normal, intact muscle, C02-induced acidosis depresses maximal force output by about 30% (Renaud et dal. 1986). Similarly, increased H concentration depresses maximal cdcium (@a2+)activated force by approximately 15% and reduces the Ca2+ sensitivity of the contractile proteins of skinned fibers (Donaldson and Hermansen 1978; Fabiato and Fabiato 1978; Godt and Nosek 1989). This latter effect may +
ICorrespndence may be sent to the author at the following address: HPE Division, Virginia Tech, Blacksburg, V A 24061-0326, U.S.A. Printed in Canada / Imprim6 au Canada
be due to diminished Ca2+ binding to tropsnin and (or) decreased force production by individual crossbridges (Godt and Nosek 1989). However, such pH-induced changes in force output do not fully account for the rather large decline in force output that is typically observed during fatigue. The effects of changes in pHi on sarcoplasmic reticulum (SR) function are less well understood. Decreased pH is h o w n to reduce SR CaD-ATPase activity and to inhibit Ca2+ uptake by the SR (Fabiato and Fabiats 1978; Inesi and Hill 1983). The effects of changes in pHi on @a2+release, however, are equivocal. Increased pH is h o w n to stimulate the release of Ca2+ from the SR (Shoshan et al. 1981). Reduced pH, however, has been reported to decrease (Fabiato and Fabiato 1978; Meisner and Henderson 1987) and to increase (Allen et al. 1989; Lea and Ashley 1978) Ca2+ release. For example, Fabiato and Fabiats (1978) found increased SW Ca2+ loading of skinned skeletal and cardiac muscle at low pH and high free Ca2+ concentration. They attributed the increased loading to diminished @a2+-induced @a2+release (CHCW) at the low pH. However, this hypothesis was not rigorously tested in skeletal muscle. Conversely, Lea and Ashley (1978) and Allen et al. (1989) report that myoplasmic Ca2+ transients of whole muscle were elevated when P H ~was ~ d u c e dvia C02 exposure. Their results9 however, are difficult to interpret, since the increased Ca2+ transients were abolished in a zero CaD m d u m (Lea and Ashley
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1978), and since other organelles and proteins that bind and sequester @a2+might have contributed to the elevated myoplasmic Ca2+ concentration. Thus, there is some question as to the direct effects of reduced pHi on Caw release from skeletal muscle SR. The purpose of this investigation was to determine if reduced pHi influences SW Caw release by modifying the process of CICR.
Skinned Bber preparation Bundles of muscle fibers (1 -3 fiberslbundle) were obtained from semitendinosus muscles of male grass frogs (Rana pipiens) and placed in cold relaxing solution. Bundles were then chemically skinned in the relaxing solution containing sapnin (50 pg/mL) for 20 min at room temperature. This treatment chemically removes the s a r c o l e m but leaves the SW intact and functional (Endo and Iino 1980). Skinned fibers were suspended horizontdly between a pair of self-closing forceps (Dumont No. 5), one of which was attached to a micromanipulator and the sther to an isometric force transducer (Grass FT-03), Fiber length was adjusted to and maintained at 130% of slack length. Experimental solutions were added to one of 12 wells ( = 500 pL) milled into a Plexiglas block. The volume of the solution in each well was such hat the surface of the solution protruded approximately 3 3above the surface of the block. The skinned fibers were placed just above the block and into the protruding p r tion of the solutions. The solutions to which the skinned fibers were exposed were changed by moving the block horizontally so that the fiber was transferred from one well to another. The output of the transducer was amplified (Grass 7P122, 030 Hz), displayed on a strip-chart recorder (Hanard), and, in some cases, sampled by microcomputer (MetraByte DAS-16 AID converter and Zenith PCIAT, 1898 Hz). In all cases, force output was computed from the strip-chart recordings. Sshtisns Sufficient potassium methanesulfonate (KMs) was added to all solutions to maintain an ionic strength of 0.2 M. All solutions also contained 20 mM irnidazole as a pH buffer, with find pH adjusted with KOH or KMs. In addition to the above constituents, the composition of the standard relaxing solution (R) was 5.0 rnkt MgS04, 4.6 mM Na,ATP, 10 mM ethylene glycol-bis(B-aminoehyl ether)N,N,N1,hi"tetraacetie acid (EGTA). A washing solution (W) was prepared as above except that the EGTA concentration was reduced to 2 mM. A rigor solution (&) was prepared as was R, except that the MgSO, was reduced to 1.5 mM and Na2ATB was omitted. The loading solution was prepared as was It, except hat CaSO, was added to obtain a log free Ca2+concentration (pCa) of 6.6. For these experiments, two sets of releasing solutions were prepared. One set was prepared as was R, except that MgSO, and Na2ATP were omitted. To more accurately simulate physiological conditions, a second set of releasing solutions was prepared as was R, except that Mg2+ was not removed but kept at f .5 d, and 1.0 mM 0,~-methyleneadenosine-5'-triphosphate (AMPPCP), a nonhydrolyzable analogue of ATB, was added (Ohta et al. 1989). For both sets of releasing solutions, CaSO, was added to obtain pCa 5, 6, and 7. For the loading and releasing solutions, the amount of CaSO, added to obtain each pCa was calculated using apparent stability constants (adjusted for ionic strength, pH 7.0 or 6.5, and 20°C) and the computer program published by Fabiato (1988). The apparent stability constants for AMPPCP were assumed to be the same as those of ATP (Ohta et al. 2989). Free Ca2+ concentrations in the Ca2+-containing solutions were also checked with a Ca2+-sensitive minielectrode N o r l d Precision %nstruments). Measurement sf &iaB -induced Ca" release The method of determining ClCR was based on that described by Endo (1977). The SR of the skinned fibers was first depleted of Ca2+ by exposure eo R containing 25 mM caffeine. The SR was then loaded by a 2-min exposure to the loading solution. Loading was ter-
FIG. 1. Typical effects of reducing pH from 7.0 to 6.5 on CICR. Both tracings were obtained from the same skinned fiber and were recorded after the standard 2-min loading period (pCa 6.6). Solid circles represent the transfer of h e fiber between solutions. See Methods for definition of solutions. CAHF, caffeine. minated and excess Ca2+removed by two rinses in R,, and the fiber was relaxed by two rinses in R. Following a rinse in R, to remove free ATP, CICR was evoked by exposing the fibers to one of the releasing solutions for 5, 15, 30, or 60 s. Because the releasing solutions contained no ATB, @a" was released but not taken up by the SR. Each fiber was then rinsed twice in R, to remove free Ca2+ and once in R to break the rigor bonds h a t were formed in the absence of AT$. The fibers were then rinsed twice in W to remove excess EGTA, and the amount of Ca" remaining in h e SR after CICR was determined by exposure to 25 mM caffeine (in W). The magnitude of this caffeine eontracture sewed as a measure of the amount of Ca2+ remaining in the SR. In each experiment, the rinsing perids were maintained at 10 s. Control experiments were performed as above but with the CICR step omitted. A set of CICR "runs" consisted of performing the above procedure four times, once each using CICR durations of 5, 15, 30, and 60 s. These runs were bracketed by both control measures and measures of maximal Ca2+-activated force (pea 4.5). Initial work indicated that at least two sets of runs could be performed on a single fiber bundle without causing significant 6'nm-dow~9'( > 10%). Thus, two sets of mns, one each at pH 7.0 and 6.5, were performed on a single fiber bundle. Bn half of the fibers tested, a set of runs with pH 7.0 was performed first and a set with pH 6.5 performed second. 'This protocol thus allowed a single fiber to serve as its own control. Statistical asaalysis Analysis of variance, adjusted for repeated measures performed on individual fibers, was used to determine differences in the extent of CICR between pH 7.0 and 6.5. Significance was established at p < 0.05.
Typical effects of reducing pH from 7.8 to 6.5 on Ca2+ release from the SR are shown in Fig, 1, Both tracings were recorded from the same fiber, in which CICR was evoked for 15 s with a pCa of 6.8 and in the absence of Mg2+ and ATP. As can be seen, the caffeine contracture recorded after evoking CICR at pH 7.8 is approximately 30% smaller than that induced after CICR was evoked at pH 6.5. This suggests that more Ca2+ remained in the SR after CICW at the low pH. Figure 2 shows the relative magnitude of caffeine contracfure c corded after evoking CICR9 in the absence of N P and Mg2'. for 5 -60 s at P C 5~-7- In this figure, 100% refers to the magnitude of the caffeine contractwe recorded without prior CICR (i.e., control). FOPall durations of CICR and each
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CAN. 5. BHYSIOL. PHARMACOL. VOL. 70, 1992
01
6
I
I
I
I
%B
96
46
60
CICR DUUTION (s) FIG.2. Relative magnitude of the caffeine contractures after evoking CICR at pH 7.0 and 6.5. Experiments were performed in the absence of both ATP and I&IIgZf.100% refers to the control contraca r e (no CICR). Values are given as means i SE (in some cases, the error bars are obscured by the symbol). *p < 0.05, n = 8.
pCa, caffeine contractures following CICR at pH 6.5 were slgnificantly greater than those evoked after CICR at pH 7.0 ( p < 0.05). The effects of reduced pH on CICR in the presence of Mg2' and AMPPCP are shown in Fig. 3. At both pH '7.0 and 6.5, larger caffeine contracture forces were obtained after CICR in the presence of these compounds than in their absence. However, the effects of reduced pH were qualitatively similar in that at each pCa level and duration of CICR, significantly larger contractures were obtained after CICR at pH 6.5. Because the magnitude of these assay caffeine contractuses indicates the amount of Ca2+ that remained in the SR after CICR, the results of Pigs. 2 and 3 suggest that less Ca2+ was released from the SR whew CICR was evoked at low pH. The decline in contracture force as a function of increasing
PIG.3. Relative magnitude of the caffeine contractures afier evoking CICR at pH 7.0 and 6.5. Experiments were performed in the presence of 1.5 mM MgZf rand 1 mM AMPPCP. *p < 0.05, n = 6.
CICR durations follows first-order kinetics (Ohta et al . 1989). Thus, the fractional rate (k) of CICR per unit time (i. e., rate constant of CICR) was computed for each fiber and pH condition as k = -On B)lt, where B is the relative force of caffeine contracture (P/Bo) and t is the duration of CICR (Ohta et d. 1989). Fitting of each fiber's data to this exponential function yielded r2 values between 0.96 and 0.99. Figure 4 shows rate constants computed for experiments performed in the absence of ATP and Mg2+ (top) and in the presence of Rag2+ and AMPPCP (bottom). En both cases, for each pCa the rate constants of CICR were significantly greater when CICR was evoked at pH 7.0 than at pH 6.5. To determine if reduced pH affected passive CaD efflux from the SR, fibers were exposed to R, (at pH 7.0 or 6.5) for 10 min. No significant differences in the size of the subsequent caffeine contracture between pH 4.5 and 7.0 were noted.
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WILLIAMS AND WARD
to point out that during this procedure @a2+ loading was always performed at pH 7.0 for 2 min and only after the SR had been depleted of Ca". Thus, any influence sf low pH on the SR Ca2+ ATPase and Ca2+ uptake would not have affected subsequent caffeine contractures, since loading occurred under identical conditions. Also, free Ca2 concentration and ionic strength of the releasing solutions were held constant between pH levels. Since pH affects the binding of Ca" to EGTA and free Ca" concentration, apparent stability constants that were adjusted for pH were used when preparing low and high pH releasing solutions (Fabiato 1988). Finally, assay caffeine contractures were always evoked at pH 7 -0so that any pH effects on crossbridge formation and activation would not have influenced force production. Thus, differences in the magnitude of caffeine contractures observed in this study were due to the effects of pH on CICR and support the notion that decreased pH inhibits Ca2+ release from skeletal muscle SR. One advantage of using a skinned fiber preparation to assay Ca2+ release from the SR is the ability to expose the same fiber to different conditions. However, the skinned fiber technique does not allow for quantitative measurements of Ca2+ exchange by the SR. In addition, this protocol becomes less sensitive at high Ca2+ concentrations as saturation of the Ca2+ binding sites on troponin begins to occur. To overcome this, we selected a loading condition that does not allow for full loading of the SR. Bur preliminary experiments showed that increasing the free Ca2+ concentration from pCa 6.6 to 6.0 during the 2-min loading period resulted in slightly, but significantly, larger subsequent caffeine contractures. Nevertheless, our data indicate that on a qualitative basis less Ca2+ is released during CICR at pH 6.5 than at pH 7.0 and do not represent a quantitative (either absolute or relative) amount of Ca2+ released from the SR. Although, the exact stimulus for Ca2+ release by the SR during excitation-contraction coupling is not known, most agree that CECR is not the major mode of Ca2+ release under normal conditions. However, recent evidence suggests that this component of Ca2+ release may ampli$l SR @a2+evoked by activation of the transverse-tubule voltage sensor (Lamb and Stephenson 1991). Under some circumstances, CICR may be more important. For example, it has been suggested that CICR plays a role in Ca2' release during prolonged, repetitive, and chemical activation of intact fibers (Palade et al. 1989; Volpe and Stephenson 1986; Williams 1990). In addition, it seems to participate heavily in pathological conditions such as malignant hyperthermia (Ohta et al. 1989). The extent to which CICR affects total SR Ca2+ release under various activation conditions and the changes in the CICR process that affect subsequent muscle performance await hrther exploration. At present, it is unclear how low pHi might inhibit SR CICR. It is evident from our studies that the effects of pH are not permanent but are hlly reversible. It is possible that low pH inhibits @a2+binding to and activation of the SW Ca2+release channel. Alternatively, low pH might modi$l Ca2+ movement from the SR by interacting directly with the release channel. Accordingly, Rousseau and Pinkos (1990) have shown that acidification of the cytoplasmic side of the SR alters the gating behavior of the channel and reduces its probability of opening whereas reduced pH of the luminal side of the SR reduces ion conductance through the channel. En either case, the flux of Ca2+ into the myoglasm, Ca2+ binding to troponin, and subsequent force production would be diminished by lowered pH. Clearly additional studies are needed to
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+
PCP 1 EnM
FIG.4. Rate constants for CICR as a function of pCa. Experiments were performed in the absence of both ATP and Mg2+ (top, n = 8) and in the presence of 1.5 mM Mg2+ and 1 mM A M P K P (bottom, n = 6). *p < 0.05.
Caffeine ~ontracture forces in the absence of Mg2+ and AMPPCP were 97.2 f 2-6 and 96-1 f 3.3% of control bH 3 * 1 and 6 - 5 and 7.0, respectively p < 0.05) and 92-8 91.0 f 2.9% ( p < 0.05). This indicates that the low pH probably does not have a major influence On the passive leak sf Ca2+ from the SR. 9
Discussion The results of this investigation suggest that a reduction of pH from 7.0 to 6.5 depresses CICR in skinned skeletal muscle fibers. In addition, this pH effect is not influenced by the presence of Mg2+ and ATP, both of which are known to affect the CICW process (Endo 1975; Endo and Kitmawa 1976). Evidence that pH reduces SR Ca2+ release has been reported for cardiac muscle (Fabiato and Fabiato 1978; Meissner and Henderson 1987). However, as noted above, a rigorous test of the mtion that low pH inhibits Ca2+ release from skeletal muscle SW has not been previously conducted. In this investigation, the magnitude of CICW was assessed indirectly by the caffeine contracture method. It is important
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CAN. J. PHYSIOE. PHARMACOL. VOL. 70, 1992
further clarify the precise mechanism(s) by which low pH reduces SR @as release. Based on the present results, it is possible that reduced CICR by low pH may account, in part, for the decline in force prsduction observed during fatigue. Recent reports indicate that during fatigue of normal skeletal muscle, a 50% decline in tetanic force is accomgmied by a 15 - 30% reduction in the peak rnyoplasmic Ca2+ transient (Allen et al. 1989; Lee et al. 1991). Application of high concentrations of Caffeine (e. g ., 10 mM) returned both tension and peak Ca2+ transient to near normal levels (Allen et al. 1989). These changes in force and myoplasmic Ca2+ concentration have furthered the speculation that fatigue may be due to an inability of the SR to release stored Ca2+. This could occur as the result of a reduced magnitude of the stimulus for Ca2+ release and (or) a reduced sensitivity of the release mechanism to the stimulus. Our data show that when a "fatigued" condition is simulated ( i s . , low pH), the sensitivity of the release mechanism to @a2+is reduced. Unfortunately, the exact stimulus for SW Ca" release during normal excitation -contraction coupling is not known. What is more significant than the specific releasing agent used in the present investigation is our finding that acidosis depresses a component of Ca2+ release from skeletal muscle SR. A pH-mediated depression s f CICW might result in a reduction in myoplasmic Ca2+ levels and subsequent force production during fatiguing contractions. Although this notion is speculative at present, it is entirely compatible with recent studies that suggest that skeletal muscle fatigue is due, in part, to a reduction in the ability of the SR to release Ca2+ (Allen et al. 1989; Lee et al. 8991). It remains to be seen if reduced pH depresses other components of Ca2+ release. Finally, it is tempting to speculate that pH-induced depression sf SR Ca2+ release might serve as a protective ~ s e h a n ism against muscle damage caused by Ca2+. Elevations in resting @aD +odd adversely affect further functioning of the muscle by stimulating @a2+-activated proteases. These proteases are h o w n to degrade a number of muscle proteins, including the contractile proteins (Kasuga and Umazume 1990) and the Ca2+-release channel of the SR (Seiler et al. 8984). It has been shown that during fatigue resting myopfasmic Ca2+ levels are elevated by more than 2-fold (Lee et d. 1991). If SR @aD release were unchecked during sustained contractions, further accumulation of myoplasmic Ca2+ could occur, which would lead to severe damage s f the active muscle fibers. Thus, it is plausible that Bow pH-induced depression of CICR prevents large elevations of resting myoplasmic Ca2+ and subsequent protein degradation. Such a mechanism would compromise force prsduction but lessen the chance of irreversible muscle damage. In summary, the present results show that CICR from the SR of skinned skeletal muscle fibers is depressed by reducing pH from 7.0 to 6.5. It is possible that such an effect accounts, in part, for the diminished SW Ca2+ release and reduced force output that is observed during fatigue. Allen, D. G., Lee, J. A., and Westerblad, H. 1989. Intracellular cakcium and tension during fatigue in isolated muscle fibers from %enspus Inevis. J. Physiol. (London), 415: 433 -458. Donaldson, S. K. B., and Hemansem, L. 1978. Differential, direct effects of H ' on Ca2+-activated force of skinned fibers from the soleus, cardiac and adduetor magnus muscles of rabbits. Pfluegers Arch. 376: 55-65. Ends, M e 1975. Conditions required for calcium-induced release of calcium from the sarcoplasmic reticulum. Proc. Jpn. Acad. 51: 467 -472.
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