A comparison of vesicles derived from terminal cisternae and longitudinal tubules of sarcoplasmic reticulum isolated from rabbit skeletal muscle

Sarcoplasmic reticulum vesicles were separated into heavy (derived from terminal cisternae) and light (derived from longitudinal tubules) fractions, according to Meissner [Biochim. Biophys. Acta, 389, 51-68 (1975)]. The similar Ca2+ sensitivities of phosphoprotein formation, ATPase activity and calcium uptake, and the similar phosphoprotein turnover rates (ATPase/phosphoprotein formation) of both fractions indicate that the same ATPase enzyme is present in the terminal cisternae and longitudinal sarcoplaxmic reticulum. The higher V for Ca2+-activated ATPase activity and calcium uptake in the light fraction correlated with the higher concentration of ATPase enzyme per mg of membrane protein in this fraction. In both the presence and absence of calcium-precipitating anions, the light fraction stored more calcium than the heavy. The Ca2+ dependence of calcium release after addition of EGTA appeared similar in both fractions, but the rate of calcium release was more rapid in the light fraction. These findings suggest that calcium release may occur more rapidly from longitudinal than terminal cisternae portions of the sarcoplasmic reticulum and that calcium release, like calcium uptake, may be mediated by the ATPase enzyme in the sarcoplasmic reticulum membrane. Although the activation energies for Ca2+-activated ATPase activity above and below the transition temperature were significantly different for the heavy and light fractions, their transition temperatures were similar. Partial purification of the ATpase enzyme by deoxycholate treatment modified the activation energies of the light but not the heavy fraction and caused the activation energies to become similar. The phosphoprotein levels of heavy and light vesicles did not become similar after deoxycholate treatment, although gel electrophoretograms indicated both samples contained > 90% ATPase protein. These results indicate the protein-lipid associations in these two fractions may be different.     

Heparin induces Ca2+ release from the terminal cisterns of skeletal muscle sarcoplasmic reticulum

Using a Ca2+-selective electrode and the chlorotetracycline fluorescence technique, the effects of heparin on Ca2+ transport in the sarcoplasmic reticulum (SR) of skeletal muscles in the absence of oxalate were investigated. It was shown that heparin (0.5-10 micrograms/ml) causes a rapid release of 40-50 nmol Ca2+/mg protein from the terminal cistern SR vesicles bound to 130-150 nmol/mg protein of Ca2+ in the presence of ATP. However, heparin has practically no effect on the longitudinal cistern fraction of SR. The effects of heparin can be prevented by ruthenium red. No influence of heparin is observed in the case of the Ca2+-induced release of Ca2+ from the terminal cisterns. When the Ca2+ release is induced by heparin, no Ca2+-induced release of Ca2+ takes place.     

Effect of halothane on Ca2+-induced Ca2+ release from sarcoplasmic reticulum vesicles isolated from rat skeletal muscle

Halothane induces the release of Ca2+ from a subpopulation of sarcoplasmic reticulum vesicles that are derived from the terminal cisternae of rat skeletal muscle. Halothane-induced Ca2+ release appears to be an enhancement of Ca2+-induced Ca2+ release. The low-density sarcoplasmic reticulum vesicles which are believed to be derived from nonjunctional sarcoplasmic reticulum lack the capability of both Ca2+-induced and halothane-induced Ca2+ release. Ca2+ release from terminal cisternae vesicles induced by halothane is inhibited by Ruthenium red and Mg2+, and require ATP (or an ATP analogue), KCl (or similar salt) and extravesicular Ca2+. Ca2+-induced Ca2+ release has similar characteristics.     

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.     

Ca transport and heat production in vesicles derived from the sarcoplasmic reticulum terminal cisternae: Regulation by K

In this work, we compared the effect of K⁺ on vesicles derived from the longitudinal (LSR) and terminal cisternae (HSR) of rabbit white muscle. In HSR, K⁺ was found to inhibit both the Ca²⁺ accumulation and the heat released during ATP hydrolysis by the Ca²⁺-ATPase (SERCA1). This was not observed in LSR. Valinomycin abolished the HSR Ca²⁺-uptake inhibition promoted by physiological K⁺ concentrations, but it did not modify the thermogenic activity of the Ca²⁺ pump. The results with HSR are difficult to interpret, assuming that a single K⁺ is binding to either the ryanodine channel or to the Ca²⁺-ATPase. It is suggested that an increase of K⁺ in the assay medium alters the interactions among the various proteins found in HSR, thus modifying the properties of both the ryanodine channel and SERCA1.     

Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum

Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Characterization of the terminal cisternae and longitudinal tubules of sarcoplasmic reticulum from malignant hyperpyrexia susceptible porcine skeletal muscle

1. The sarcoplasmic reticulum (SR) from malignant hyperpyrexia susceptible (MHS) and control porcine skeletal muscle was separated into vesicular fractions enriched in the membrane elements of the terminal cisternae and longitudinal tubules. 2. The two membrane preparations were highly purified and had distinctive features which were associated with their origins in the SR membraneous network. 3. Calsequestrin and calcium were enriched in the terminal cisternae fraction (HSR), in comparison to longitudinal tubule preparations (LSR). 4. The HSR membrane also had a greater total capacity to store Ca2+ and Ca2+ release was more rapid than from LSR preparations. 5. No distinction could be made between the membrane morphology, Ca2+ -fluxes or Ca2+ -dependent ATPase activities, associated with these functionally distinct regions of MHS and control preparations.     

Ca2+ release from the sarcoplasmic reticulum compared in amphibian and mammalian skeletal muscle

Puzzled by recent reports of differences in specific ligand binding to muscle Ca2+ channels, we quantitatively compared the flux of Ca2+ release from the sarcoplasmic reticulum (SR) in skeletal muscle fibers of an amphibian (frog) and a mammal (rat), voltage clamped in a double Vaseline gap chamber. The determinations of release flux were carried out by the "removal" method and by measuring the rate of Ca2+ binding to dyes in large excess over other Ca2+ buffers. To have a more meaningful comparison, the effects of stretching the fibers, of rapid changes in temperature, and of changes in the Ca2+ content of the SR were studied in both species. In both frogs and rats, the release flux had an early peak followed by fast relaxation to a lower sustained release. The peak and steady values of release flux, Rp and Rs, were influenced little by stretching. Rp in frogs was 31 mM/s (SEM = 4, n = 24) and in rats 7 +/- 2 mM/s (n = 12). Rs was 9 +/- 1 and 3 +/- 0.7 mM/s in frogs and rats, respectively. Transverse (T) tubule area, estimated from capacitance measurements and normalized to fiber volume, was greater in rats (0.61 +/- 0.04 microns-1) than in frogs (0.48 +/- 0.04 micron-1), as expected from the greater density of T tubuli. Total Ca in the SR was estimated as 3.4 +/- 0.6 and 1.9 +/- 0.3 mmol/liter myoplasmic water in frogs and rats. With the above figures, the steady release flux per unit area of T tubule was found to be fourfold greater in the frog, and the steady permeability of the junctional SR was about threefold greater. The ratio Rp/Rs was approximately 2 in rats at all voltages, whereas it was greater and steeply voltage dependent in frogs, going through a maximum of 6 at -40 mV, then decaying to approximately 3.5 at high voltage. Both Rp and Rs depended strongly on the temperature, but their ratio, and its voltage dependence, did not. Assuming that the peak of Ca2+ release is contributed by release channels not in contact with voltage sensors, or not under their direct control, the greater ratio in frogs may correspond to the relative excess of Ca2+ release channels over voltage sensors apparent in binding measurements. From the marked differences in voltage dependence of the ratio, as well as consideration of Ca(2+)-induced release models, we derive indications of fundamental differences in control mechanisms between mammalian and amphibian muscle.     

Ca2+ channel agonist BAY-k 8644 does not elicit Ca2+ release from skeletal muscle sarcoplasmic reticulum

BAY-k 8644, a nifedipine analogue, promotes Ca2+ influx into excitable cells via plasma membrane voltage-sensitive Ca2+ channels. We report here that sarcoplasmic reticulum (SR) Ca2+ release channels are insensitive to BAY-k 8644, as studied in highly purified isolated fractions and in chemically skinned fibers of rabbit skeletal muscle. This result suggests that a subcellular heterogeneity exists among Ca2+ channels, at least with respect to drug-receptor sites. In the course of this study, however we found that BAY-k 8644 reversibly inhibits the SR Ca2+ pump, i.e., it decreases Ca2+ influx into the SR lumen, although at concentrations (IC50 = 3-5 X 10(-5) M) much higher than those effective on voltage-sensitive Ca2+ channels.     

Rose bengal activates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

The photooxidizing xanthene dye rose bengal (10 nM to 1 microM) stimulates rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles. Following fusion of sarcoplasmic reticulum (SR) vesicles to an artificial bilayer, reconstituted Ca2+ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. Rose bengal does not appear to affect K+ channels present in the SR. Following reconstitution of the sulfhydryl-activated 106-kDa Ca2+ channel protein into a bilayer, rose bengal activates the isolated protein in a light-dependent manner. Ryanodine at a concentration of 10 nM is shown to lock the 106-kDa channel protein in a subconductance state which can be reversed by subsequent addition of 500 nM rose bengal. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is also directly observed upon measurement of [3H]ryanodine binding to JSR vesicles. These observations indicate that photooxidation of rose bengal causes a stimulation of the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum by interacting with the ryanodine binding site. Furthermore, similar effects of rose bengal on isolated SR vesicles, on single channel measurements following fusion of SR vesicles, and following incorporation of the isolated 106-kDa protein strongly implicates the 106-kDa sulfhydryl-activated Ca2+ channel protein in the Ca2+ release process.     

Phosphatidylinositol 4,5-bisphosphate-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum terminal cisternal membranes. Ca2+ flux and single channel studies.

We report here that the inositol 1,4,5-trisphosphate (IP3) precursor, L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) is a potent molecule (1 microM) which activates the ryanodine-sensitive Ca2+ release channel from rabbit skeletal muscle terminal cisternae incorporated into a phospholipid bilayer. It also stimulates Ca2+ release from these membrane vesicles. Therefore, it may play a modulating role in excitation-contraction coupling. In the bilayer, PIP2 added on the cytoplasmic side increased the mean channel opening probability 2-12-fold in the presence and absence of physiological Mg2+ and ATP. From flux studies, PIP2-induced Ca2+ release, occurring through the ryanodine-sensitive Ca2+ release channel, displayed saturation kinetics. The rate of Ca2+ release induced by PIP2 was approximately greater than 50% slower than the rates induced by other agents (e.g. caffeine, Ca2+, ATP). PIP2, and not IP3, effectively elicited Ca2+ release from terminal cisternae. On the contrary, IP3, and not PIP2, specifically mediated Ca2+ release from dog brain cerebellum microsomes, where IP3 receptors are known to be found. The PIP2-induced Ca2+ release from muscle membranes was not dependent on medium [Ca2+] (from less than 10(-9) to approximately 10(-4) M). However, IP3 could activate the terminal cisternae Ca2+ channel in the bilayer when there was low Ca2+ (less than 10(-7) M). The data suggest that the ionic microenvironment around the Ca2+ channel may be different for observing the two phosphoinositide actions.     

Effects of azumolene on doxorubicin-induced Ca2+ release from skeletal and cardiac muscle sarcoplasmic reticulum

The mechanism of doxorubicin-induced Ca2+ release from skeletal and cardiac muscle sarcoplasmic reticulum (SR) was studied by examining the effects of azumolene (a water soluble dantrolene analog) on doxorubicin-mediated Ca2+ release and ryanodine binding. Doxorubicin induced a rapid Ca2+ release from both skeletal and cardiac SR in a similar concentration range (EC50 = 5-10 microM). Maximal doxorubicin-induced Ca2+ release was seen at 2 and 0.2 microM Ca2+ for skeletal and cardiac SR, respectively. Addition of 400 microM azumolene caused approx. 30% inhibition of doxorubicin-induced Ca2+ release from both skeletal and cardiac SR; skeletal SR had significantly higher sensitivity to azumolene than cardiac SR. In the presence of Ca2+, doxorubicin increased [3H]ryanodine binding to both skeletal and cardiac SR; whereas in the absence of Ca2+, doxorubicin led to significant ryanodine binding to skeletal SR, but not to cardiac SR. In both types of SR, doxorubicin-activated, but not Ca2+ activated ryanodine binding was inhibited by azumolene. Azumolene sensitivity for inhibition of doxorubicin-activated ryanodine binding was much higher in skeletal SR than cardiac SR, consistent with the results for effects of azumolene on Ca2+ release. Our results are consistent with the possibility that azumolene inhibits doxorubicin binding by direct competition for the drug receptor(s).     

Regulation of Ca2+ release from sarcoplasmic reticulum in skeletal muscles

Ca²⁺ release from skeletal sarcoplasmic reticulum (SR) could be regulated by at least three mechanisms: 1) Ca²⁺, 2) calmodulin, and 3) Ca²⁺/calmodulin-dependent phosphorylation. Bell-shaped Ca²⁺-dependence, of Ca²⁺ release from both actively- and passively-loaded SR vesicles suggest that opening and closing of the Ca²⁺ release channel could be regulated by [Ca²⁺ _o] . The time- and concentration-dependent inhibition of Ca ²⁺ release from skeletal SR by calmodulin was also studied using passively-Ca²⁺ loaded SR vesicles. Up to 50% of Ca ²⁺ release was inhibited by calmodulin (0.01–0.5 µM); this inhibition required 5–15 min preincubation time. The hypothesis that Ca²⁺/calmodulin-dependent phosphorylation of a 60 kDa protein regulates Ca²⁺ release from skeletal SR was tested by stopped-flow fluorometry using passively-Ca²⁺-loaded SR vesicles. Approximately 80% of the initial rates of Ca²⁺-induced Ca²⁺ release was inhibited by the phosphorylation within 2 min of incubation of the SR with Mg·ATP and calmodulin. We identified two types of 60 kDa phosphoproteins in the rabbit skeletal SR, which was distinguished by solubility of the protein in CHAPS. The CHAPS-soluble 60 kDa phosphoprotein was purified by column chromatography on DEAE-Sephacel, heparin-agarose, and hydroxylapatite. Analyses of the purified protein indicate that the CHAPS-soluble 60 kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding isoforms of PGM were cloned and sequenced using synthetic oligonucleotides. Two types of PGM isoforms (Type I and Type 11) were identified. The translated amino acid sequences show that Type II isoform is SR-form. Our results are significant in terms of understanding evidence of an association of glycolytic and glycogenolytic enzymes with SR and a role in the regulation of SR functions. (Mol Cell Biochem 114: 105-108, 1992)     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Rapid filtration measurements of Ca2+ release from cisternal sarcoplasmic reticulum vesicles

A radioactive tracer and rapid filtration method was applied to the study of Ca2+ release from sarcoplasmic reticulum (SR) vesicles which were preloaded passively (equilibration with millimolar Ca2+) or actively (in the presence of ATP or acetyl phosphate). The method allows complete substitution of the loading mixture with release medium in constant flow, and time resolution between 0.01 and 10.0 s. Net release can be clearly distinguished from isotope exchange. The latter is prominent in longitudinal SR vesicles. Net Ca2+ release is observed only from cisternal SR vesicles, is Ca2+ (micromolar) dependent, and is accelerated by inactive ATP analogues, or ATP itself, even in the presence of Mg2+. Net release has a strong pH dependence (between 6 and 7), and very little temperature dependence (consistent with a passive channel). In media of physiological significance (1 mM ATP, 1 mM magnesium, and free Ca2+ in the micromolar range), net Ca2+ release proceeds with a rate constant of approx. 100 s-1.     

Oligomerisation of sarcoplasmic reticulum Ca2+-ATPase monomers from skeletal muscle

The fast-twitch SERCA1 isoform of the sarcoplasmic reticulum Ca(2+)-ATPase was purified to homogeneity and conjugated to peroxidase. The SERCA1 probe showed high affinity binding to the immobilized monomeric enzyme, but not crosslinker-stabilized oligomers. This suggests a preferential complex formation via homo-dimerization, rather than interactions with established oligomeric structures.