Pergamon Press
Life Sciences Vol. 18, pp . 773-780 Printed in the U .S .A .
MINIREVIEVP MECHANISMS OF CALCIUM RELEASE IN SARCOPLASMIC RETICULUM G . Ineai and N . Malen Laboratory of Physiology and Biophysics University of the Pacific San Francisco, California 94115 The involvement of Sarcoplasmic Reticulum (SR) in relaxation of skeletal muscle has been studied extensively since vesicular fragments of SR membrane were found in the microsomal frnction of muscle homogenates (1,2) . It was shown that the isolated SR vesicles ezhibit ATP dependent calcium transport in vitro, reducing the Cat+ concentration in the medium to levels (3) and at rates (4,5) compatible with relaxation of myofibrils in physiological conditions (6) . The question of calcium release, however, has been elusive for a long time . In this regard it ie known that skeletal muscle SR is able to atnre an amount of calcium which is sufficient for activation of myofibrils . Therefore, it is simply assumed that upon membrane excitation calcium is released from SR, thereby raising the Cat+ concentration in the myoplaem and initiating contraction . Recently various experiments were performed demonstrating that calcium release from SR can occur by different mechanisms of great interest and possibly of physiological relevance . These mechanisms will be discussed here . Reversal of the Pump The release of calcium from SR vesicles loaded by active transport is very slow if the activity of the pump is stopped by procedures reducing Cat+ or ATP concentrations in the outside me dium . However, in the presence of ADP and inorganic phosphate, calcium release from loaded vesicles occurs at rates comparable to those of inward active transport (7,8) . The calcium release ie accompanied by the formation of ATP, indicatiaq that potential energy derived from the transmembrane calcium gradient can be utilised for phosphorylation of ADP (9) . it was also shows that a phosphorylnted form of the SR ATPase is an intnrmediate step during both outward translocation of calcium in these conditions and inward translocation during active transport (10,11) . Tha occurrence of calcium release in the presence of ADP and inorganic phosphate clearly demoaatrates that the calcium puap of sarcoplasmic reticulum can be reversed . However, such a mechanism of release is unlikely to play a physiological role in excitationcontraction coupling since (a) the rates of efflux are too slow to provide sufficient oalcium duriaq the short time of mechanical coupling, and (b) it is not apparent how the myoplaematic concentration 773
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of ADP would significantly increase as an immediate consequence of membrane depolarization . Io nophore induced Release Several antibiotics have been found to increase the permeability of natural and artificial membranes to cations, due to selective formation of cation-antibiotic complexes which are lipid solu ble (12,13) . Some of these antibiotics facilitate diffusion of ions across memb canes by functioning ae carriers of ions, and were descriptively names "ionophorea" (14) . Two ionophorea of particular relevance to the question of calcium release from SR are the antibiotics X-537 A (15) and A 23187 (16) . The two antibiotics, isolated from different strains of atreptomyces, possess the ability to function as divalent cation ionophorea . Addition of either of the two to loaded SR vesicles produces immediate calcium release (17,18), while neither ATPase inhibition nor structural damage to the vesicles is apparent (19) . Other antibiotics which are known to facilitate tranamembrane diffusion of monovalent, but not divalent ca ons, have no effect on Cat+ release by SR (19) .
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The ionophore induced release definitely demonstrates that a tranamembrane gradient of calcium ion is formed in SR vesicles as a consequence of ATP dependent calcium uptake . It also shows that the accumulated calcium can be released at a very rapid rate, if the 3R membrane permeability to calcium ie suitably increased . However, the involvement of ionophorea in the physiological mechanism of excitation-contraction coupling is as open question since no low molecular weight muscle component, meeting the definition of calcium ionophore, has been as yet isolated from muscle . Calcium-triggered Release A group of very interesting experiments related to the question of Ca t} release was performed with skinned muscle fibers (20) . In these preparations earcolemma is peeled off the fiber, while SR remains with the myofibrils . The remaining structure then exhibits phenomena which are dependent on the relation between SR and myofibrils . In the skinned fibers release of Ca t+ from SR can be inferred from the occurrence of contractile responses, and the actual exit of calcium from SR can be directly demonstrated by use of a radioactive calcium isotope (21,22) or by measuring the bioluminescence of the calcium sensitive protein Aquorin (23) . In this ezperimental system it was shown that the addition of calcium ion, itself, can trigger the release of calcium previously sequestered by the SR (24,25,26) . In our laboratory we have found that calcium-triggered release may be demonstrated even with isolated SR vesicles (Fig . 1) . Therefore this mechanism involves directly the SR membrane . Thnae observations offer a very attractive hypothesis viewing excitation-contraotion coupling as a calcium trigqered regenerative process involving calcium release from SR . However, the required concentrations of triggering calcium era too high to be consistent with the physiological mechanism of excitation-contraction coupling in skeletal muscle (27) . On the other hand the calcium trigqered
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FIG . 1 Calcium-triggered release in SR vesicles . Calcium loading was initiated by the addition of ATP (2 .5 mM) to a reaction miature containing SR vesicles (0 .25 mq protein/ml), MOPS buffer pH 6 .8 (20 mM), &CL (125 mM), MgC1 2 (2 .5 mM), 45 Ca " CaC1 2 ( .1 mM) and EGTA ( .1 mM) . After 60 seconds .5 mM (~ 45 Ca " CaCl 2 (0 ) , or . 5 mM 45 Ca " CaC1 2 + 5 mM MgCl Z ) , or .5 mM 45 Ca " CaC1 2 + .5 mM EGTA (~) were added . The specific radioactivity of all CaC1 2 èolutions was identical . Temp . : 37~ C. release may play a role in special circumstances such ae in the presence of caffeine and in heart muscle preparations (28,29) . The Effect of Caffeine The contractile effects of caffeine on skeletal muscle are mediated through calcium release from SR (30) . In fact, such a release can be induced by caffeine on isolated SR vesicles (31, 32) . One interesting aspect of the caffeine effect is its relation to the calcium triggered release . Certain analogies between these two phenomena were clearly pointed out by Endo (33) : a) In both cases calcium release is facilitated by lowering the concentration of free magnesium or raising the concentration of free calcium in the mediums b) The two phenomena potentiate each other ; c) Hoth mechanisms require that the SR be preloaded with calcium at near maximal levels and are inhibited by procaine . It is therefore apparent that, in physiological conditions, caffeine readers the
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calcium triggered release operative even at low sarcoplasmic concentrations of calcium. "Depolarization" of Sarcoplasmic Reticulum Experimentation with skinned fibers has permitted the observation of yet another mechanism of calcium release, which is induced by changes in the electrolyte composition of the medium . In these experiments the fibers are first incubated in media containing ATP, magnesium, proprionate (or methaneaulfonate) and a Ca-EGTA buffer to allow filling of sarcoplaemic reticulum with calcium and (presumably) diffusion of large anions (proprionate or methaneaulfonate) into the SR, while the fibers are relaxed . After suitable incubation, replacement of large anions with chloride in the bathing medium is followed by transient contractions (34,25,35) . The interpretation of these experiments is based on the assumption that the SR membrane is more permeable to chloride than to other large anions (e .g ., methaneaulfonate) . Following aubati tution of the bathing medium, chloride diffuses into SR faster than large anions diffuse out . This produces net positive charge outside the SR, a condition that may be compared to that occurring during membrane depolarization in intact muscle . Similar results are detected by loading SR with calcium in the presence of a small cation such as potassium, and then adding a medium containing a large cation such as TRIS ( (hydroximethyl) aminomethane) (35) . Thin produces net positive charge on the outside (cytoplasmic aide) of the SR, if the SR membrane is not permeable to TRIS . Opposite maneuvers designed to produce net negative charge on Therefore, the calthe outside of the SR are not affective (35) . cium release and contractile responses obtained by electrolyte changes in the media appear to be an experimental model reflecting a physiological mechanism and suggest that electrical phenomena affect SR membranes even in the absence of myolamma . A doubt, however, remains as to whether the intervention of membranes other than SR is completely eliminated in the skinned fibers . For instance, remaining T tubules could reseal to form close compartments retaining Na+ pump and electrical properties similar to those oP the outer membrane (34) . Depolarization of the resealed tubules would then generate a stimulus for the SR . Attempts to eliminate this possibility were made by the use of partially skinned fibers, on which remnants oP interrupted myolemma prevent T tubules from resealing (35) . Nevertheless, the primary involvement of the SR membrane by manipulations of the electrolyte composition of the medium would be beat demonstrated if Cat+ release could be obtained from isolated SR vesicles . In our laboratory, we have investigated this possibility by first loading SR vesicles with calcium in the presence of ATP, and then adding to the reaction mixture small volumes of concentrated solutions of different electrolytes in order to change the composition of the medium . The effects of these experimental maneuvers on the steady state levels of SR filling are determined by following the distribution of radioactive calcium tracer .
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FIG . 2 Effect of various nations on the steady state levels of accumulated calcium in SR vesicles . Calcium loading was initiated by the addition of ATP (2 .5 mM) to a reaction mizture containing 3R vesicles ( .25 mg protein/ml), MOPS buffer pH 6 .8 (20 mM), 1CC1 (50 mM), MgC12 (2 .5 mM), CaCaC12 ( .1 mM), EGTA ( .1 mM) and sucrose (200 mM) . After 60 seconds 250 mM RC1 (~), or (CH3)4 NC1 (~) or TRIS C1 (4) were added . Temp . : 37 °C In Fiq . 2 it is shown that addition of chloride and various monovalent catione to vesicles loaded in the presence of potassium chloride produces sudden calcium release . in these experiments osmotic effects are minimised by the presence of 200 mM sucrose, but cannot be definitely ezcluded . A more pronounced release is obtained when larger ca nons are added, probably reflecting slower transmembrane diffusion of the various aatione as compared to that of chloride . Therefore these effects, in analogy to those obtained with skinned fibers (35), may be related to asymmetric charge distribution across the SR membrane. If such an interpretation is correct, the experiments suggest that the 3R membrane is sensitive to electrical phenomena and that calcium release may be induced directly by changes in the electrical potential of the SR membrane .
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Conclusion The ATP dependent calcium uptake by SR results in a transmemThe accumulated calcium can be brane gradient of calcium ion . released by a reversal of the pump or by other procedures increas ing the SR permeability to calcium . It is apparent that the physiological mechanism of calcium release is one involving direct electrical effects on the 3R membrane . Açknowledgementa This work was partially supported by grants from the NIH (HL 16607), the Muscular Dystrophy Association of America, Potchefatroom University for CHE (South Africa) and the South African Medical Research Institute .
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