Heavy Sarcoplasmic Reticulum Fractions Research Articles

Early over-expression of low-affinity [ 3H]ryanodine receptor sites in heavy sarcoplasmic reticulum fraction from dystrophic chicken pectoralis major

Heavy sacroplasmic reticulum (SR membranes enriched in [ 3H]ryanodine receptor have been isolated from pectoralis major (PM) of normal line 412 and dystrophic line 413 chickens paired at various stages during post-hatch development. Normal PM 2 days ex ovo yields 17% lower protein recovery in the heavy SR subfractions compared to preparations from paired dystophic PM (0.80 vs 0.96% mg/g PM, respectively). By 2 weeks ex ovo, protein recovery in normal SR subfractions decreases over 3-fold to 0.24 mg/g PM, whereas yields from dystrophic PM increase to 1.67 mg/g PM. Dystrophic preparations consistently give 7–9-fold higher recoveries of protein in heavy SR subfractions compared to normal PM when examined at 2, 4 and 5.5 weeks ex ovo. [ 3H]ryanodine binding to normal SR from PM 2 days ex novo exhibits nonlinear Scatchard plots which resolve into high-( K d,app = 18 nM; B max - 17 pmol /mg protein) and low-( K d,app = 532 nM; B max = 2.6 pmol/mg protein) affinity binding sites, whereas dystrophic preparations exhibit only high-affinity [ 3H]ryanodine binding sites ( K d,app = 31 nM; B max = 2.1 pmol/mg protein). Both normal and dystrophic PM have similar capacities to bind [ 3H]ryanodine (2.6 vs. 2.0 pmol/g PM, respectively) at 2 days ex ovo. However, at 2, 4, and 5.5 weeks ex ovo the density of high-affinity [ 3H]ryanodine binding sites in normal SR drops dramatically to 3.5, 1.2, and 0.4 pmol/mg protein, respetively, and corresponds with disappearance of the low-affinity binding sites. In marked contrast, heavy SR membranes from distrophic PM 2, 4, and 5.5 weeks ex ovo, maintain their high-affinity binding sites for [ 3H]ryanodine and exhibit high densities of low-affinity binding sites ( K d,app = 725−4500 nM; B max = 15.4–25.1 pmol/mg protein). Early developmental over-expression of [ 3H]ryanodine binding capacity in dystrophic PM ranges from 34-fold to 388-fold that of normal PM at 2 weeks and 5.5 weeks, respectively, and correspond to the intensity with which high molecular weight doublets of M r 340 000 and 320 000 stain on SDS-PAGE. Low-affinity [ 3H]ryanodine binding sites of dystrophic SR exhibit 4–6-fold higher sensitivity to activation by Ca 2+ and altered sensitivity to activation by caffein and adenine nucleotides. These results demonstrate that over-expression of junctional SR and [ 3H]ryanodine receptors having altered radioligand binding properties is a very early event in the post-hatch development of dystrophic PM. Since the [ 3H]ryanodine receptor is a specific marker for the SR Ca 2+ release channel at the muscle triad and a key component of excitation-contraction coupling, abnormal expression of [ 3H]ryanodine receptors may be of a fundamental importance to the etiology of muscular dystrophy in the chicken.

Uptake and release of calcium ions by heavy sarcoplasmic reticulum fraction of normal and malignant hyperthermia-susceptible human skeletal muscle

1. 1. Ca 2+ uptake, Ca 2+-dependent ATPase activity and halothane-induced Ca 2+ release from the heavy sarcoplasmic reticulum fraction of muscle from malignant hyperthermia susceptible individuals are similar to those of normal human muscle. 2. 2. Ca 2+-induced Ca 2+ release from the diseased muscle was increased by 13%.

Elevated calmodulin levels and reduced calmodulin‐stimulated calcium‐ATPase in Duchenne progressive muscular dystrophy

We determined the calmodulin concentration and Ca2+-ATPase activity in subcellular fractions recovered from samples of vastus lateralis muscle obtained from 18 patients with Duchenne muscular dystrophy, 10 patients with other primary myopathies, 5 with spinal muscular atrophy, and 16 age-matched controls. Calmodulin levels were increased in the cytosol, plasmalemma, and heavy sarcoplasmic reticulum fractions from Duchenne dystrophy patients; the greatest increases occurred at early stages of disease or in mildly progressive cases. The total Ca2+-ATPase activities were decreased in the Duchenne dystrophy muscles; calmodulin caused a minimal stimulation of the activity in calmodulin-depleted membranes from Duchenne dystrophy compared with control membranes. The changes in calmodulin concentration and Ca2+-ATPase activity complement previous observations of reduced calsequestrin and dystrophin concentrations in Duchenne dystrophy muscles and suggest that these muscles lose calcium regulatory functions at early stages of the disease process.

Ca-Release by Phosphoinositides from Sarcoplasmic Reticulum of Frog Skeletal Muscle1

To clarify the biological role of phosphoinositides including inositol trisphosphate (IP3) in the skeletal muscle, we examined the Ca-releasing action on the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle of IP3, phosphatidylinositol monophosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and glycerophosphoinositol 4,5-bisphosphate (GPIP2). Only PIP2 caused dose-dependent Ca release. IP3 (up to 55 microM), PIP (up to 37 microM), and GPIP2 (up to 33 microM) were ineffective. The PIP2-induced Ca release is due to the direct action of PIP2, but not its metabolite(s). The properties of the PIP2-induced Ca release are unique and cannot be accounted for by the Ca release mechanisms already reported, such as Ca2+-induced, ionic substitution-induced, or IP3-induced Ca release. The rate of the PIP2-induced Ca release, however, is so slow that it may have no physiological relevance unless stimulating factors or agents exist.

Polylysine induces a rapid Ca 2+ release from sarcoplasmic reticulum vesicles by mediation of its binding to the foot protein

The addition of polylysine to a heavy fraction of sarcoplasmic reticulum (SR) vesicles produces a rapid Ca 2+ release with no appreciable lag period. The polylysine concentration for half-maximal activation ( C 1 2 ) is approximately 0.99 μg/ml, or 0.3 μ m, the lowest C 1 2 for Ca 2+ release-inducing reagents reported in the literature. The time course and the [Ca 2+] dependence of polylysine-induced release are similar to those of caffeine-induced Ca 2+ release. At higher concentrations of polylysine (e.g., 10 μg/ml), however, little or no Ca 2+ release occurs. Upon photolysis of SR vesicles with the photocrosslinkable radiolabeled polylysine derivative, [ 3H]succinimidyl azido benzoate polylysine, 0.28 and 0.52–1.2 mol polylysine were bound to 1 mol of the 400-kDa foot protein at activating (3 μg/ml) and inhibitory (10 μg/ml) concentrations of polylysine, respectively. On the other hand, the amounts of polylysine bound to the other SR proteins (mol/mol) were negligible (e.g., ⩽0.0127 mol polylysine/mol calsequestrin). This suggests that the binding of polylysine to the foot protein is responsible not only for the induction of release but also for inactivation. These results provide direct evidence that the receptor for the chemical trigger of Ca 2+ release is localized within the foot protein. Ruthenium red, which inhibits polylysine-induced Ca 2+ release, does not inhibit polylysine binding to the foot protein, suggesting that the polylysine binding domain of the foot protein is different from the channel domain.

Caffeine treatment inhibits drug-induced calcium release from sarcoplasmic reticulum and caffeine contracture but not tetanus in frog skeletal muscle

Effects of pretreatment with caffeine on Ca2+ release induced by caffeine, thymol, quercetin, or p-chloromercuriphenylsulfonic acid (pCMPS) from the heavy fraction of sarcoplasmic reticulum (SR) were studied and compared with those effects on caffeine contracture and tetanus tension in single fibers of frog skeletal muscle. Caffeine (1-5 mM) did induce transient Ca2+ release from SR vesicles, but subsequent further addition of caffeine (10 mM, final concentration) induced little Ca2+ release. Ca2+ release induced by thymol, quercetin, or pCMPS was also inhibited by pretreatment with caffeine. In single muscle fibers, pretreatment with caffeine (1-5 mM) partially reduced the contracture induced by 10 mM caffeine. However, tetanus tension was almost maximally induced by electrical stimulus in caffeine-treated fibers. These results indicate that SR, which becomes less sensitive to caffeine, thymol, quercetin, or pCMPS by pretreatment with caffeine, can still respond to a physiological signal transmitted from transverse tubules.

Postnatal development of Ca2+-sequestration by the sarcoplasmic reticulum of fast and slow muscles in normal and dystrophic mice.

Ca2+-uptake activities of the sarcoplasmic reticulum (SR) were determined with a Ca2+-sensitive electrode in homogenates from fast- and slow-twitch muscles from both normal and dystrophic mice (C57BL/6J strain) of different ages. Immunochemical quantification of tissue Ca2+-ATPase content allowed determination of the specific Ca2+-transport activity of the enzyme. In 3-week-old mice of the dystrophic strain specific Ca2+ transport was already significantly lower than in the normal strain. It progressively decreased with maturation and reached only 40-50% and 30-50% of the normal values in fast- and slow-twitch muscles of adult dystrophic animals, respectively. Tissue contents of calsequestrin were reduced in both types of muscle leading to an increased Ca2+-ATPase to calsequestrin protein ratio. Equal amounts of the Ca2+-ATPase protein (detected by Coomassie blue staining of polyacrylamide gels) were present in SR vesicles isolated by Ca2+-oxalate loading from adult normal and dystrophic fast-twitch muscles. However, the specific ATP-hydrolysing activity of the enzyme was approximately 50% lower in dystrophic than in normal SR. The reduced ATP-hydrolysing activity was correlated with decreased Ca2+-transport activity, phosphoprotein formation and fluorescein isothiocyanate labeling as determined in total microsomal and heavy SR fractions. Although the Ca2+ and ATP affinities of the enzyme were unaltered, its ATPase activity was reduced at all levels of ATP in the dystrophic SR. Taken together, these findings point to a markedly impaired function of the SR and an increase in the population of inactive SR Ca2+-ATPase molecules in murine muscular dystrophy.

Ethanol increases calcium permeability of heavy sarcoplasmic reticulum of skeletal muscle

The effects of ethanol on both Ca 2+ pump activity and Ca 2+-induced Ca 2+ release in sarcoplasmic reticulum (SR) of rabbit skeletal muscle were studied. In concentrations of 0.1–1.0%, ethanol conspicuously enhanced Ca 2+ release from the heavy fraction of SR, whereas a much smaller effect was noted in the light fraction. When Ca 2+-induced Ca 2+ release was inhibited by 10 m m Mg 2+, the Ca 2+ pump activities of both the heavy and light SR were the same; the activities were not significantly influenced by 1% ethanol. Ethanol itself did not release Ca 2+ from the heavy SR. However, it appeared to render the heavy SR more permeable to Ca 2+, thereby decreasing the amount of storable Ca 2+. This action of ethanol may be related to the mechanism of its negative inotropic effect.

Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle.

We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.

Post‐Mortem Changes in Subcelllular Fractions from Normal and Pale, Soft, Exudative Porcine Muscle. 2. Electron Microscopy.

SUMMARY— Myofibrillar, mitochondrial, heavy sarcoplasmic reticulum, and light sarcoplasmic reticulum fractions were isolated from homogenates of normal and pale, soft, exudative (PSE) porcine muscle at 0 and 24 hr post‐mortem and examined by electron microscopy. No differences were observed between normal and PSE myofibrils obtained at death. PSE myofibrils prepared at 24 hr post‐mortem had more granular appearing filaments and wider Z lines than normal myofibrils at 24 hr. The PSE heavy sarcoplasmic reticulum fraction obtained at death had a higher proportion of granular material than the same fraction from normal muscle. Several structural differences between the other PSE and normal fractions were also observed, especially at 24 hr postmortem. This study indicated that the composition of the subcellular fractions changed with time post‐mortem and that this change should be considered when analyzing biochemical data from these fractions. However, the differences observed could not explain the large changes in calcium accumulating ability that have been shown to occur post‐mortem.