Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sarcoplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca²⁺ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca²⁺-ATPase activity in SR and changing of character of Ca²⁺ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca²⁺ transport of SR.     

质粒DNA增强大鼠缺血骨骼肌肌浆网钙转运功能

在大鼠肢体缺轿模型上观察质粒ｐｃＤＮＡ３对缺血骨骼肌肌浆网（ＳＲ）Ｃａ＾２＋转运的影响。结果显示,骨骼肌缺血时ＳＲＣａ＾２＋转运（Ｃａ＾２＋摄入与释放）较非缺血肌肉增强,而质粒ｐｃＤＮＡ３与ＳＲ上ＤＮＡ结合蛋白结合之后,可进一步增强缺备骨骼肌ＳＲＣａ＾２＋摄入（Ｐ＜０．０１）及释放速率（Ｐ＜０．０５）。提示质粒ＤＮＡ对正常及缺血大鼠骨骼肌的ＳＲＣａ＾２＋转运能力均有影响,其临床病理生理意义值得进一     

肾上腺髓质素对大鼠损伤性心肌肌浆网功能的改善

通过观察下述五个指标,评价肾上腺髓质素(adrenomedullin,Adm)对大鼠损伤性心肌肌浆网功能的改善程度左心室压力最大变化速率(±dp/dtmax)、肌浆网钙摄取和释放及钙泵活性.皮下注射异丙肾上腺素(isoproterenol,ISO,69μmol/kg体重)制备大鼠心肌损伤坏死模型.摘取心脏后用Adm灌流,观察左心室压力最大变化速率(±dp/dtmax);制备并提纯心肌肌浆网(sarcoplasmicreticulum,SR)膜,测定SRCa2+摄取和释放速率、SR钙泵活性和钙通道蛋白～3H-ryanodine受体的最大结合量.结果发现,5×10-5mol/LAdm灌流能使ISO损伤的大鼠心脏左室±dp/dtmax分别增加16.9%(2?135±281vs1?980±302)和29.2%(1?375±267vs1?064±355,均P<0.05);SRCa2+摄取和释放率分别增加23.0%(15.0±1.4vs12.2±1.2)和43.5%(6.6±1.0vs4.6±0.6,均P<0.01);SRCa2+-ATPase活性和～3H-ryanodine受体最大结合量(Bmax)分别增加24.2%(P<0.01)和42.2%(P<0.05).提示Adm对ISO诱导的大鼠心肌损伤具有保护作用,其机制可能与Adm增加SRCa2+-ATPase活性、增加～3H-ryanodine所致SRCa2+摄取和释放升高有关.外源性给予Adm对损伤心肌可能具有临床治疗作用.     

心肌细胞膜与肌质网膜的结构功能耦联及其病理重塑机制

心脏的收缩功能依赖心肌细胞膜（包括横管）与肌质网的结构耦联以及其中L型钙通道与肌质网钙释放通道之间的钙致钙释放过程。在一些病理条件下,细胞膜与肌质网的耦联结构发生重塑,钙致钙释放机制受损,心肌细胞收缩力下降。其中,junctophilin-2等蛋白分子表达量减少是心力衰竭疾病中心肌细胞收缩能力下降的关键因素。     

不同频率电刺激膈神经导致膈肌肌浆网Ca~(2 )-ATPase和Ca~(2 )释放-摄取动力学改变

研究不同频率慢性电刺激（ＣＥＳ）后兔膈肌肌浆网（ＳＲ）Ｃａ２＋－ＡＴＰａｓｅ活性以及ＳＲ Ｃａ２＋摄取－释放动力学对不同频率ＣＥＳ的适应性变化。建立不同频率ＣＥＳ组;用定磷法测定ＳＲ Ｃａ２＋－ＡＴＰａｓｅ活性;用Ｆｕｒａ－２荧光法测定ＳＲ　Ｃａ２＋摄取－释放动力学。与对照组比较,慢性低频电刺激１０ Ｈｚ和２０Ｈｚ组的ＳＲ　Ｃａ２＋－ＡＴＰａｓｅ活性明显降低（Ｐ＜０．０１）,Ｃａ２＋释放－摄取动力学也显著降低（Ｐ＜０．０１）;慢性高频电刺激５０ Ｈｚ和１００Ｈｚ组的ＳＲ Ｃａ２＋－ＡＴＰａｓｅ活性则显著升高（Ｐ＜０．０１）,Ｃａ２＋释放－摄取动力学亦明显升高（Ｐ＜０．０１）。实验提示,ＣＥＳ后不同频率ＣＥＳ导致膈肌ＳＲＣａ２＋－ＡＴＰａｓｅ、Ｃａ２＋摄取－释放动力学产生不同的适应性变化;对不同功能状态的膈肌应用不同频谱的慢性电刺激可能具有重要的临床意义。     

Hypochlorous acid modifies calcium release channel function from skeletal muscle sarcoplasmic reticulum.

We have previously demonstrated that H2O2 at millimolar concentrations induces Ca(2+) release from actively loaded sarcoplasmic reticulum (SR) vesicles and induces biphasic [(3)H]ryanodine binding behavior. Considering that hypochlorous acid (HOCl) is a related free radical and has been demonstrated to be a more effective oxidant of proteins, we evaluated the effects of HOCl on sarcoplasmic reticulum Ca(2+)-channel release mechanism. In a concentration-dependent manner, HOCl activates the SR Ca(2+) release channel and induces rapid release of Ca from actively loaded vesicles. HOCl-induced Ca(2+) release is inhibited in the presence of millimolar concentrations of DMSO. High-affinity [(3)H]ryanodine binding is also enhanced at concentrations from 10 to 100 microM. At HOCl concentrations of >100 microM, equilibrium binding is inhibited. HOCl stimulation of binding is inhibited by the addition of dithiothreitol. The direct interaction between HOCl and the Ca(2+) release mechanism was further demonstrated in single-channel reconstitution experiments. HOCl, at 20 microM, activated the Ca(2+) release channel after fusion of a SR vesicle to a bilayer lipid membrane. At 40 microM, Ca(2+)-channel activity was inhibited. Pretreatment of SR vesicles with HOCl inhibited the fluorescence development of a fluorogenic probe specific to thiol groups critical to channel function. These results suggest that HOCl at micromolar concentrations can modify SR Ca(2+) handling.     

Spectroscopic determination of sarcoplasmic reticulum Ca2+ uptake and Ca2+ release

In this report we describe the application of spectroscopic methods to the study of Ca2+ release by isolated native sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle. To date, dual-wavelength spectroscopy of arsenazo III and antipyrylazo III difference absorbance have been the most common spectroscopic methods for the assay of SR Ca2+ transport. The utility of these methods is the ability to manipulate intraluminal Ca2+ loading of SR vesicles. These methods have also been useful for studying the effect of both agonists and antagonists upon SR Ca2+ release and Ca2+ uptake. In this study, we have developed the application of Calcium Green-2, a long-wavelength excitable fluorescent indicator, for the study of SR Ca2+ uptake and release. With this method we demonstrate how ryanodine receptor Ca2+ channel opening and closing is regulated in a complex manner by the relative distribution of Ca2+ between extraluminal and intraluminal Ca2+ compartments. Intraluminal Ca2+ is shown to be a key regulator of Ca2+ channel opening. However, these methods also reveal that the intraluminal Ca2+ threshold for Ca2+-induced Ca2+ release varies as a function of extraluminal Ca2+ concentration. The ability to study how the relative distribution of a finite pool of Ca2+ across the SR membrane influences Ca2+ uptake and Ca2+ release may be useful for understanding how the ryanodine receptor is regulated, in vivo.     

Calcium binding proteins of junctional sarcoplasmic reticulum: detection by 45Ca ligand overlay

In skeletal muscle, the junctional sarcoplasmic reticulum (JFM) plays a crucial role in excitation-contraction coupling and Ca2+ release. In the present report, the sarcoplasmic reticulum (SR) was fractionated into longitudinal SR (LSR), terminal cisternae (TC), and JFM. Each fraction had a unique protein profile as detected by SDS-polyacrylamide gel electrophoresis as well as specific Ca2+ binding proteins as judged by 45Ca ligand overlay of nitrocellulose blots. Ca2+ binding proteins of LSR were the Ca2+ ATPase (Mr of 115K), an 80K polypeptide, and the intrinsic glycoprotein (Mr of 160K); Ca2+ binding proteins of JFM were polypeptides with the following Mr values: 350K and 325K (feet components), 200K, 170K, a doublet of 140K, 118K, 65K (calsequestrin), and 52K. Measurements of Ca2+ binding to SR fractions by equilibrium dialysis indicated that 8-17 nmol Ca2+/mg of protein was specifically bound. After EDTA extraction of calsequestrin, JFM still bound Ca2+ (5-6 nmol/mg of protein), suggesting the existence of specific Ca2+ binding sites. The Ca2+ binding sites of Ca2+-gated Ca2+ release channels might be on two JFM polypeptides (Mr's of 350K and 170K) which are putative channel constituents (F. Zorzato, A. Margreth, and P. Volpe (1986) J. Biol. Chem. 261, 13252-13257).     

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).     

Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle

[3H]Ryanodine binding to skeletal muscle and cardiac sarcoplasmic reticulum (SR) vesicles was compared under experimental conditions known to inhibit or stimulate Ca2+ release. In the skeletal muscle SR, ryanodine binds to a single class of high-affinity sites (Kd of 11.3 nM). In cardiac SR vesicles, more than one class of binding sites is observed (Kd values of 3.6 and 28.1 nM). Ryanodine binding to skeletal muscle SR vesicles requires high concentrations of NaCl, whereas binding of the drug to cardiac SR is only slightly influenced by ionic strength. In the presence of 5'-adenylyl imidodiphosphate (p[NH]ppA), increased pH, and micromolar concentration of Ca2+ (which all induce Ca2+ release from SR) binding of ryanodine to SR is significantly increased in skeletal muscle, while being unchanged in cardiac muscle. Ryanodine binding to skeletal but not to cardiac muscle SR is inhibited in the presence of high Ca2+ or Mg2+ concentrations (all known to inhibit Ca2+ release from skeletal muscle SR). Ruthenium red or dicyclohexylcarbodiimide modification of cardiac and skeletal muscle SR inhibit Ca2+ release and ryanodine binding in both skeletal and cardiac membranes. These results indicate that significant differences exist in the properties of ryanodine binding to skeletal or cardiac muscle SR. Our data suggest that ryanodine binds preferably to site(s) which are accessible only when the Ca2+ release channel is in the open state.     

Doxorubicin directly binds to the cardiac-type ryanodine receptor

The clinical use of doxorubicin, an antineoplasmic agent, is limited by its extensive cardiotoxicity which is mediated by the mobilization of intracellular Ca2+ from SR. In order to elucidate the mechanism of Ca2+ release, we analyzed the binding sites of doxorubicin on rabbit cardiac SR (sarcoplasmic reticulum). One of the binding sites was identified as cardiac-type ryanodine receptor (RyR2) which was purified by immunoprecipitation from solubilized cardiac SR in the presence of DTT. Ligand blot analysis revealed the direct binding of doxorubicin to RyR2. The binding of doxorubicin to RyR2 was specific and displaced by caffeine. Both doxorubicin and caffeine enhanced [3H]-ryanodine binding to RyR2 in a Ca2+ dependent manner. These results suggest that there is a doxorubicin binding site on RyR2.     

Increased Ca2+ storage capacity in the sarcoplasmic reticulum by overexpression of HRC (histidine-rich Ca2+ binding protein)

The histidine-rich Ca(2+) binding protein (HRC) is a high capacity Ca(2+) binding protein in the sarcoplasmic reticulum (SR). Because HRC appears to interact directly with triadin, HRC may play a role in the regulation of Ca(2+) release during excitation-contraction coupling. In this study, we examined the physiological effects of HRC overexpression in rat neonatal cardiomyocytes. Both caffeine-induced and depolarization-induced Ca(2+) release from the SR were increased significantly in the HRC overexpressing cardiomyocytes. Consistently, the Ca(2+) content, normally depleted from the SR in the presence of cyclopiazonic acid (CPA), remained elevated in these cells. In contrast, the density and the ryanodine-binding kinetics of the ryanodine receptor (RyR)/Ca(2+) release channel were slightly reduced or not significantly altered in the HRC overexpressing cardiomyocytes. We suggest that HRC is involved in the regulation of releasable Ca(2+) content into the SR.     

A retrograde signal from calsequestrin for the regulation of store-operated Ca2+ entry in skeletal muscle

Calsequestrin (CSQ) is a high capacity Ca(2+)-binding protein present in the lumen of sarcoplasmic reticulum (SR) in striated muscle cells and has been shown to regulate the ryanodine receptor Ca(2+) release channel activity through interaction with other proteins present in the SR. Here we show that overexpression of wild-type CSQ or a CSQ mutant lacking the junction binding region (amino acids 86-191; Delta junc-CSQ) in mouse skeletal C2C12 myotube enhanced caffeine- and voltage-induced Ca(2+) release by increasing the Ca(2+) load in SR, whereas overexpression of a mutant CSQ lacking a Ca(2+) binding, aspartate-rich domain (amino acids 352-367; Delta asp-CSQ) showed the opposite effects. Depletion of SR Ca(2+) by thapsigargin initiated store-operated Ca(2+) entry (SOCE) in C2C12 myotubes. A large component of SOCE was inhibited by overexpression of wild-type CSQ or Delta junc-CSQ, whereas myotubes transfected with Delta asp-CSQ exhibited normal function of SOCE. These results indicate that the aspartate-rich segment of CSQ, under conditions of overexpression, can sustain structural interactions that interfere with the SOCE mechanism. Such retrograde activation mechanisms are possibly taking place at the junctional site of the SR.     

硒对心肌质膜(Ca~(2+)·Mg~(2+))-ATPase和肌浆网Ca~(2+)-ATPase活力的影响

本文以豚鼠和大白鼠心肌肌浆网膜(SR)Ca~(2+)-ATPase的活力,心肌质膜(SL)(Ca~(2+)Mg~(2+))-ATPase的活力和电子显微镜的方法探索克山病病区粮中低硒与心肌细胞钙转运调控的共系,实验结果为硒对克山病有预防作用的观点提供了新的理论依据,并进一步支持了“克山病是一种心肌线粒体病”的观点。     

Charge changes in sarcoplasmic reticulum and Ca2+-ATPase induced by calcium binding and release: a study using lipophilic ions

Changes in the charge of sarcoplasmic reticulum (SR) vesicles are studied using lipophilic ions, which are adsorbed by the membrane phase. Upon addition of MgATP, phenyldicarbaundecaborane (PCB-) and tetraphenylboron (TPB-) are taken up by the SR vesicles, while tetraphenylphosphonium (TPP+) is released into the water phase. The PCB- uptake occurs as well under conditions when SR membrane is shunted by high Cl- concentration. MgATP induces minor additional binding of PCB- in the presence of oxalate and it is followed by release of the lipophilic anion from the vesicles. EGTA partly reverses the ATP effect, and calcium ionophore A23187 plus EGTA reverses it completely. Vesicles that were preliminarily loaded by Ca2+ demonstrated higher passive and lower ATP-dependent PCB- binding. Activation of isolated Ca2+-ATPase in the presence of 0.1 mM EGTA results in PCB- release into the medium and additional TPP+ binding to the enzyme. We suggest that the redistribution of the lipophilic ions between the water phase and SR membrane reflects charge changes in Ca2+-binding sites inside both SR vesicles and Ca2+-ATPase molecules in the course of Ca2+ translocation.     

Regulation of cardiac sarcoplasmic reticulum Ca release by luminal [Ca] and altered gating assessed with a mathematical model

Cardiac excitation-contraction coupling is initialized by the release of Ca from the sarcoplasmic reticulum (SR) in response to a sudden increase in local cytosolic [Ca] ([Ca]i) within the junctional cleft. We have tested the hypothesis that functional ryanodine receptor (RyR) regulation plays a major role in the regulation of myocyte Ca. A mathematical model with unique characteristics was used to simulate Ca homeostasis. Specifically, the model was designed to accurately represent the SR [Ca]-dependence of release from a variety of experimentally produced data sets. The simulated data for altered RyR Ca sensitivity demonstrated a regulatory feedback loop that resulted in the same release at lower [Ca]SR. This suggests that the primary role of myocyte RyR regulation may be to decrease SR [Ca] without decreasing the size of the [Ca]i transient. The model results suggest that this action moderates the increased SR [Ca] observed with adrenergic stimulation and may keep the [Ca]SR below the threshold for delayed afterdepolarizations and arrhythmia. However, increased Ca affinity of the RyR increased the probability of delayed afterdepolarizations when heart failure was simulated. We conclude that RyR regulation may play a role in preventing arrhythmias in healthy myocytes but that the same regulation may have the opposite effect in chronic heart failure.     

Defective Ca(2+) handling proteins regulation during heart failure

Abnormal release of Ca(2+) from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction in heart failure (HF). We previously demonstrated that RyR2 macromolecular complexes from HF rat were significantly more depleted of FK506 binding protein (FKBP12.6). Here we assessed expression of key Ca(2+) handling proteins and measured SR Ca(2+) content in control and HF rat myocytes. Direct measurements of SR Ca(2+) content in permeabilized cardiac myocytes demonstrated that SR luminal [Ca(2+)] is markedly lowered in HF (HF: DeltaF/F(0) = 26.4+/-1.8, n=12; control: DeltaF/F(0) = 49.2+/-2.9, n=10; P<0.01). Furthermore, we demonstrated that the expression of RyR2 associated proteins (including calmodulin, sorcin, calsequestrin, protein phosphatase 1, protein phosphatase 2A), Ca(2+) ATPase (SERCA2a), PLB phosphorylation at Ser16 (PLB-S16), PLB phosphorylation at Thr17 (PLB-T17), L-type Ca(2+) channel (Cav1.2) and Na(+)- Ca(2+) exchanger (NCX) were significantly reduced in rat HF. Our results suggest that systolic SR reduced Ca(2+) release and diastolic SR Ca(2+) leak (due to defective protein-protein interaction between RyR2 and its associated proteins) along with reduced SR Ca(2+) uptake (due to down-regulation of SERCA2a, PLB-S16 and PLB-T17), abnormal Ca(2+) extrusion (due to down-regulation of NCX) and defective Ca(2+) -induced Ca(2+) release (due to down-regulation of Cav1.2) could contribute to HF.     

Polylysine induces a rapid Ca2+ 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 Ca2+ release with no appreciable lag period. The polylysine concentration for half-maximal activation (C1/2) is approximately 0.99 micrograms/ml, or 0.3 microM, the lowest C 1/2 for Ca2+ release-inducing reagents reported in the literature. The time course and the [Ca2+] dependence of polylysine-induced release are similar to those of caffeine-induced Ca2+ release. At higher concentrations of polylysine (e.g., 10 micrograms/ml), however, little or no Ca2+ 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 micrograms/ml) and inhibitory (10 micrograms/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., less than or equal to 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 Ca2+ release is localized within the foot protein. Ruthenium red, which inhibits polylysine-induced Ca2+ 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.     

Ryanodine-affinity chromatography purifies 106 kD Ca release channels from skeletal and cardiac sarcoplasmic reticulum

A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the 'physiological' Ca2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca2+ release channels which are a minor component (0.1-0.3%) of SR proteins.     

Calcium release from isolated sarcoplasmic reticulum due to 4,4'-dithiodipyridine

The effects of SH reagents on Ca2+ release from sarcoplasmic reticulum (SR) vesicles were examined by the tracer method using 45Ca2+. Among the various SH reagents tested, 4,4'-dithiodipyridine (PDS) was found to induce Ca2+ release most specifically from the heavy fraction of SR vesicles. Further, the following results were obtained. (i) PDS bound covalently to proteins in the SR membrane and induced Ca2+ release. (ii) The Ca2+ release was further enhanced by ATP and caffeine, but inhibited by procaine, ruthenium red and various divalent cations. (iii) PDS enhanced the Ca2+ release in the whole range of Ca2+ concentrations tested. (iv) Choline permeability was also enhanced by PDS. Further, the electrical conductance of the Ca2+-induced Ca2+ release channels was studied by incorporating them into lipid bilayers and it was found that PDS increased the probability of opening of the channels. These results suggest that PDS binds to certain SH groups of the Ca2+-induced Ca2+ release channels in the SR membrane and thus induces Ca2+ release.