Modulation of Cardiac Sarcoplasmic Reticulum Calcium Release by Aenosine: A protein Kinase C- Dependent Pathway

We have already reported that A₃ adenosine receptor stimulation reduces [³H]-ryanodine binding and sarcoplasmic reticulum Ca²⁺ release in rat heart. In the present work we have investigated the transduction pathway responsible for this effect. Isolated rat hearts were perfused for 20 min in the presence of the following substances: 100 nM N⁶-(iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA), an A₃ adenosine agonist; 10 μM U-73122, a phospholipase C inhibitor; 2 μM chelerythrine, a protein kinase C inhibitor. At the end of perfusion, the hearts were homogenized and [³H]-ryanodine binding was assayed. IB-MECA produced a significant decrease in ryanodine binding, which was abolished in the presence of chelerythrine but not in the presence of U-73122. RT-PCR experiments showed that ryanodine receptor gene expression was not affected by IB-MECA. In Western blot experiments, ryanodine receptor phosphorylation on serine 2809 was not modified after perfusion with IB-MECA. We conclude that modulation of SR Ca²⁺ release channel by IB-MECA is dependent on protein kinase C activation. However, in this model protein kinase C activation is not due to phospholipase C activation. In addition, changes in ryanodine receptor gene expression or direct phosphorylation of the ryanodine receptor on serine 2809 residue do not appear to occur.     

Transport of Ca2+ from Sarcoplasmic Reticulum to Mitochondria in Rat Ventricular Myocytes

Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) andmitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis thatthis proximity results in a transient exposure of mitochondrial Ca²⁺ uniporter (CaUP) to highconcentrations of Ca²⁺ following Ca²⁺ release from the SR and thus an influx of Ca²⁺into mitochondria. Single ventricular myocytes of rat were skinned by exposing them to aphysiological solution containing saponin (0.2 mg/ml). Cytosolic Ca²⁺ concentration ([Ca²⁺]_c)and mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) were measured with fura-2 and rhod2,respectively. Application of caffeine (10 mM) induced a concomitant increase in[Ca²⁺]_c and [Ca²⁺]_m.Ruthenium red, at concentrations that block CaUP but not SR release, diminished thecaffeine-induced increase in [Ca²⁺]_m but not[Ca²⁺]_c. In the presence of 1 mM BAPTA, a Ca²⁺ chelator,the caffeine-induced increase in [Ca²⁺]_m was reduced substantially less than [Ca²⁺]_c. Moreover,inhibition of SR Ca²⁺ pump with two different concentrations of thapsigargin caused anincrease in [Ca²⁺]_m, which was related to the rate of [Ca²⁺]_c increase. Finally, electronmicroscopy showed that sites of junctions between SR and T tubules from which Ca²⁺ is released,or Ca²⁺ release units, CRUs, are preferentially located in close proximity to mitochondria.The distance between individual SR Ca²⁺ release channels (feet or ryanodine receptors) isvery short, ranging between approximately 37 and 270 nm. These results are consistent withthe idea that there is a preferential coupling of Ca²⁺ transport from SR to mitochondria incardiac muscle cells, because of their structural proximity.     

大鼠心肌肌浆网的纯化及其性质研究

 用超声波破碎心肌细胞,差速离心法纯化大鼠心肌肌浆网(CSR)。SDS-聚丙烯酰胺凝胶电泳测得Ca~(2+)-ATPase分子量为98kD;电镜观察膜制备为完整的CSR微囊;标志酶哇巴因敏感型Na~(+),K~(+)-ATPase和叠氮化钠敏感型Mg~(2+)-ATPase活性表明膜制备中肌膜含量很低,但仍有线粒体污染。 用~(45)Ca~(2+)示踪微孔滤膜法研究Ca~(2+)跨膜转运,CSRCa~(2+)蓄集最大值为57nmol/mg蛋白。CSR Ca~(2+)-ATPase在4℃—21℃和21℃—49℃两区间反应活化能不同,前者大于后者。酶的最适pH为7.4。以ATP为底物,该酶有两个表观Km值:Km_1为3.7μmol/LKm_2为713μmol/L。     

The calcium uptake of the rat heart sarcoplasmic reticulum is altered by dietary lipid

Summary Small amounts of dietary n-3 fatty acids can have dramatic physiological effects, including the reduction of plasma triglycerides and an elevation of cellular eicosapentanoic (EPA) and docosahexanoic acids (DHA) at the expense of arachidonic acid (AA). We investigated the effects of alterations in the fatty acid compositions of cardiac sarcoplasmic reticulum (CSR) produced by dietary manipulation on the calcium pump protein that is required for energy dependent calcium transport. CSR was isolated from rats fed menhaden oil, which is rich in n-3 fatty acids, and from control animals that were given corn oil. Relative to control membranes, those isolated from rats fed menhaden oil, had a lower content of saturated phospholipids, an increased DHA/AA ratio, and an increased ratio of n-3 to n-6 fatty acids. These changes were associated with a 30% decrease in oxalate-facilitated, ATP-dependent calcium uptake and concomitant decreased Ca-ATPase activity in the membranes from the animals fed menhaden oil. In contrast, there was no alteration in active pump sites as measured by phosphoenzyme formation. Thus, the CSR Ca-ATPase function can be altered by dietary interventions that change the composition, and possibly structure, of the phospholipid membranes thereby affecting enzyme turnover.     

Mechanism of Inhibition of Calcium Uptake into Sarcoplasmic Reticulum by Notexin,a Neurotoxic and Myotoxic Polypeptide

Notexin belongs to a class of snake venom neurotoxins and myotoxins that have phospholipase A₂ activity. Previous studies have shown that these toxins affect target cells differently from phospholipases that are not neurotoxic or myotoxic. Notexin inhibited the Ca²⁺ uptake into fragmented sarcoplasmic reticulum from rabbit skeletal muscle, but it did not cause an efflux of previously accumulated Ca²⁺ or inhibit the Ca²⁺–ATPase activity. It is suggested that notexin specifically binds to and decreases the conductance for Ca²⁺ of the Ca²⁺ pump and/or the conductance of a channel for an ion that facilitates Ca²⁺ transport. The K⁺ ionophore valinomycin reversed the notexin-induced inhibition of Ca²⁺ uptake into sarcoplasmic reticulum, suggesting that the molecular target of notexin could be a K⁺ channel. Two types of reconstitution experiments make it unlikely that notexin acts by degrading a minor lipid that is resistant to hydrolysis by nontoxic phospholipases A₂. Notexininactivated sarcoplasmic reticulum vesicles were reactivated (with respect to Ca²⁺ uptake) by simple solubilization with detergent and subsequent reconstitution by detergent removal. Second, notexin was still active on sarcoplasmic reticulum vesicles after >94% of the lipids were replaced by soybean phosphoglycerides during the reconstitution procedure.     

Sarcoplasmic reticulum calsequestrins: Structural and functional properties

Calsequestrin is the major Ca²⁺-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its -helical content and the intrinsic fluorescence upon binding of Ca²⁺. Calsequestrin binds Ca²⁺ with high-capacity and with moderate affinity and it functions as a Ca²⁺ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca²⁺ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsquestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.     

Proton Countertransport and Coupled Gating in the Sarcoplasmic Reticulum Calcium Pump

The calcium pump of the sarcoplasmic reticulum (SERCA) is an ATP-driven active transporter of Ca²⁺ ions that functions via an “alternating-access” cycle mechanism. In each cycle, SERCA transports two Ca²⁺ ions toward the lumen of the sarcoplasmic reticulum and two to three protons to the cytoplasm. How the latter conformational transition is coupled to cytoplasmic release of protons remains poorly understood. The present computational study shows how the mechanism of proton countertransport is coupled to the alternating access gating process in SERCA. Molecular dynamics simulation trajectories are generated starting from a series of configurations taken along the E2 to E1 transition pathway determined by the string method with swarms-of-trajectories. Simulations of different protonation configurations at the binding sites reveal how deprotonation events affect the opening of the cytoplasmic gate. The results show that there is a strong coupling between the chronological order of deprotonation, the entry of water molecules into the TM region, and the opening of the cytoplasmic gate. Deprotonation of E309 and E771 is sequential with E309 being the first to lose the proton. The deprotonation promotes the opening of the cytoplasmic gate but leads to a productive gating transition only if it occurs after the transmembrane domain has reached an intermediate conformation. Deprotonation of E309 and E771 is unproductive when it occurs too early because it causes the re-opening of the luminal gate.     

Purification from brain of an intrinsic membrane protein fraction enriched in (Na+ + K+)-ATPase

A microsomal fraction from canine brain gray matter has been extracted with the detergent sodium dodecyl sulfate to partially purify the membrane bound $\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ adenosine triphosphatase. Phospholipid, glycolipid, and a family of other glycoproteins are also enriched by the procedure; it is proposed that the product is an intrinsic membrane protein fraction. 6–8-fold purification of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ is obtained without solubilizing the enzyme and without irreversibly altering its turnover number. Final specific activities are 350–400 μmol of ATP hydrolyzed/h per mg protein. The stimulation and reversible inactivation of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ by dodecyl sulfate were examined for information relevant to the mechanism of action of the detergent.     

Clomipramine and Related Structures as Inhibitors of the Skeletal Sarcoplasmic Reticulum Ca2+ Pump

The Ca²⁺-pumping activity of skeletal sarcoplasmic reticulum vesicles is half-maximallyinhibited by 120 M clomipramine, 250 M desipramine, and 500 M imipramine or trimipramine.The inhibition is attributed to the dihydrodibenzazepine moiety, since3-(dimethylamino)propionitrile, reproducing the aliphatic amine chain, has no inhibitory action. The inhibitionis shown as a marked decrease of Ca²⁺ binding at equilibrium in theabsence of ATP and asa reduction of phosphorylation of the Ca²⁺-free conformation byinorganic phosphate. Therefore,the drug effect is consistent with preferential interaction of tricyclic antidepressants withthe Ca²⁺-free conformation of the nonphosphorylated enzyme. An additional decrease in theapparent rate constant of enzyme dephosphorylation, i.e., in the release of phosphate fromATP during enzyme cycling was also noticed.     

亲和层析纯化肌质网Ca2+－ATP酶

建立了一种亲和层析纯化肌质网Ca²⁺-ATP酶的方法．用非离子型去污剂C₁₂E₈ 溶解肌质网,再通过反应红－120琼脂糖亲和层析柱使肌质网Ca²⁺-ATP酶纯度从粗品中的65％提高到99％,并具有较高ATP水解活性．经SDS－聚丙烯酰胺凝胶电泳检测,为电泳纯．     

GM3掺入肌质网膜及其对Ca2+-ATP酶的正向调节

用微量提取和高效薄板层析方法研究了外源性神经节苷脂GM3掺入兔肌质网膜的动力学过程.将掺入量分别相对于掺入浓度、时间和温度作图,显示掺入曲线均呈抛物线形式.当掺入体系中GM3浓度为8 μmol／L、掺入时间为90 min、掺入温度为35℃时,其掺入量达到最大值,约为掺入体系中GM3量的50%.上述结果表明外源性GM3对肌质网膜的作用不仅仅是一种简单的水相反应,而是一个依赖于掺入浓度、时间和温度,并具有一定的饱和度的掺入到肌质网膜脂双层中的动力学过程.进一步的实验表明外源性GM3的掺入能明显增加肌质网Ca^2＋-ATP酶的活力.这为从分子水平上研究糖脂对细胞内膜系统的结构与功能的调节作用提供了重要的基础.     

Role of the Sarcoplasmic Reticulum Ca2+-ATPase on Heat Production and Thermogenesis

The sarcoplasmic reticulum of skeletal muscle retains a membrane bound Ca²⁺-ATPase which is able to interconvert different forms of energy. A part of the chemical energy released during ATP hydrolysis is converted into heat and in the bibliography it is assumed that the amount of heat produced during the hydrolysis of an ATP molecule is always the same, as if the energy released during ATP cleavage were divided in two non-interchangeable parts: one would be converted into heat, and the other used for Ca²⁺ transport. Data obtained in our laboratory during the past three years indicate that the amount of heat released during the hydrolysis of ATP may vary between 7 and 32 kcal/mol depending on whether or not a transmembrane Ca²⁺ gradient is formed across the sarcoplasmic reticulum membrane. Drugs such as heparin and dimethyl sulfoxide are able to modify the fraction of the chemical energy released during ATP hydrolysis which is used for Ca²⁺ transport and the fraction which is dissipated in the surrounding medium as heat.     

Molecular Mechanisms of Regulation of the Activity of Sarcoplasmic Reticulum Ca-Release Channels (Ryanodine Receptors), Muscle Fatigue, and Severin''s Phenomenon

In this short review of the literature and our own data the characteristics of structural organization of sarcoplasmic reticulum Ca-release channels (ryanodine receptors) in different types of muscles, the participation of other sarcoplasmic reticulum proteins in excitation–contraction coupling and Ca-release channel operation, and the regulation of the channel activity by endogenous low molecular weight compounds are analyzed. Special attention is given to changes that occur in muscle cells during exhausting work and to the role of sarcoplasmic reticulum Ca-release channels in the loss of muscle contractile activity during the development of fatigue. It is concluded that the protection of muscle fibers against fatigue in the presence of the histidine-containing dipeptide carnosine, called in the literature Severin's phenomenon, is primarily connected with modulation of sarcoplasmic reticulum Ca-release channel activity by carnosine.     

Calcium binding proteins in the sarcoplasmic/endoplasmic reticulum of muscle and nonmuscle cells

In this paper we review some of the large quantities of information currently available concerning the identification, structure and function of Ca²⁺-binding proteins of endoplasmic and sarcoplasmic reticulum membranes. The review places particular emphasis on identification and discussion of Ca²⁺ storage proteins in these membranes. We believe that the evidence reviewed here supports the contention that the Ca²⁺-binding capacity of both calsequestrin and calreticulin favor their contribution as the major Ca²⁺-binding proteins of muscle and nonmuscle cells, respectively. Other Ca²⁺-binding proteins discovered in both endoplasmic reticulum and sarcoplasmic reticulum membranes probably contribute to the overall Ca²⁺ storage capacity of these membrane organelles, and they also play other important functional role such as posttranslational modification of newly synthesized proteins, a cytoskeletal (structural) function, or movement of Ca²⁺ within the lumen of the sarcoplasmic/endoplasmic reticulum towards the storage sites.Abbreviations SR Sarcoplasmic Reticulum - ER Endoplasmic Reticulum - InsP₃ Inositol 1,4,5-trisphosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - PDI Protein Disulphide Isomerase - T₃BP Thyroid Hormone Binding Protein - Grp Glucose regulated proteins - HCP Histidine-rich Ca²⁺ binding Protein - LDL Low Density Lipoprotein     

Trypsin-induced calcium efflux from sarcoplasmic reticulum: Evidence for the involvement of the (Ca2++Mg2+)-ATPase

Summary Trypsin digestion of the sarcoplasmic reticulum membrane at 35 to 43°C leads to an increased calcium permeability, the temperature dependence of which suggests tryptic exposure or creation of a channel rather than tryptic release of a mobile carrier (K.C. Toogood et al.,Membr. Biochem. 5:49–75, 1983). Here we show that: (1) the digested vesicles both pump and leak calcium, demonstrating that the vesicles remain intact; (2) an increased rate of efflux is not observed for membranes digested and kept at 15°C, but a temperature shift to 35°C following arrested digestion leads to the development of increased calcium permeability, indicating that a digestion step at the lower temperature potentiates increased permeability which develops rapidly as a result of a trypsin-facilitated protein conformational change at the higher temperature; (3) two inhibitors of the ATPase, adenyl-5-yl imidodiphosphate and dicyclohexyl-carbodiimide, both measurably retard the development of increased permeability at the higher temperature following arrested digestion, suggesting that these inhibitors bind to the target protein and prevent the conformational change responsible for the permeability increase, and further suggesting that the ATPase is the target for the trypsin; (4) digestion of the ATPase at 15°C follows the same initial cleavage pattern as at 35°C, but the cleavage stops or drastically slows down after the second digestion step at the lower temperature, whereas the digestion continues beyond the second step at the higher temperature, showing that an early digestion step may be responsible for potentiating increased permeability; (5) the permeability increase following digestion at 15°C and incubation at 35°C correlates (r>0.98) with the second tryptic cleavage step of the calcium ATPase, providing more support for the ATPase as the trypsin-sensitive efflux site; and (6) the rate of efflux depends on the concentration of the doubly cleaved ATPase molecules to the first power; the null hypothesis that the efflux actually depends on the cleaved ATPase concentration to the second or higher power was examined using the F test and can be rejected (confidence>0.90 to 0.98), suggesting that the efflux pathway is through a single ATPase molecule. We speculate that the pathway for increased calcium permeability is the one employed during calcium uptake and that there is a functional separation of the ATPase and calcium channel activities by trypsin digestion at 15°C followed by incubation at 35°C.     

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: Functional and structural considerations

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca²⁺ ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca²⁺ binding site are thereby changed, permitting vectorial dissociation of bound Ca²⁺ against a concentration gradient. A long range linkage between phosphorylation and Ca²⁺ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.     

Lysophospholipid-mediated alterations in the calcium transport systems of skeletal and cardiac muscle sarcoplasmic reticulum

Summary The effects of various lysophospholipids on the calcium transport activity of sarcoplasmic reticulum (SR) from rabbit skeletal and canine cardiac muscles were examined. The lipids decreased calcium transport activity in both membrane types; the effectiveness being in the order lysoPC > lsyoPS, lysoPG > lysoPE. The maximum inhibition induced by lysoPC, lysoPG and lysoPS was greater than 85% of the normal Ca²⁺-transport rate. In cardiac SR lysoPE had a maximal inhibition of about 50%. Half maximal inhibition of calcium transport by lysoPC was achieved at 110 nmoles lysoPC/mg SR. At this concentration of lysoPC, the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺-uptake activities were inhibited to the same extent (about 60%) in skeletal sarcoplasmic reticulum, while in cardiac sarcoplasmic reticulum, there was less than 20% inhibition of the Ca²⁺ + Mg²⁺-ATPase activity. Studies with EGTA-induced passive calcium efflux showed that up to 200 nmoles lysoPC/mg SR did not alter calcium permeability significantly in cardiac sarcoplasmic reticulum. In skeletal muscle membranes the lysophospholipid mediated decrease in calcium uptake correlated well with the increase in passive calcium efflux due to lysophosphatidylcholine. The difference in the lysophospholipid-induced effects on the sarcoplasmic reticulum from the two muscle types probably reflects variations in protein and other membrane components related to the respective calcium transport systems.     

Appraisal of the physiological relevance of two hypotheses for the mechanism of calcium release from the mammalian cardiac sarcoplasmic reticulum: calcium-induced release versus charge-coupled release

Recent studies correlating the calcium current with, respectively, the clamp-imposed voltage and the calcium current in intact isolated mammalian cardiac myocytes are reviewed. The major findings are the following: [1] With the exception of one group, all investigators agree that a calcium transient is never observed in the absence of a calcium current. In addition, there is a good correlation between voltage dependence of the calcium current and that of the calcium transient, although this correlation may vary among the cardiac tissues from different animal species. [2] Repolarization clamp pulses from highly positive potentials produce a tail current which is associated with a tail calcium transient. [3] The calcium transient is inhibited when the calcium current is blocked by calcium deprivation or substitution, or by the addition of calcium current antagonists, despite the fact that sarcoplasmic reticulum still contains calcium that can be released by caffeine (with inhibition of this release by ryanodine). These three findings are strongly in favor of a calcium-induced release of calcium and against the hypothesis of charge-movement-coupled release of calcium from the sarcoplasmic reticulum. [4] The only finding that would be more in favor of the latter hypothesis (although till reconciliable with the former) is that repolarization occurring before the rapid rise of calcium transient is complete curtails the calcium transient. Thus, the possibility that charge movement might somehow regulate calcium-induced release of calcium cannot be excluded.     

离体缺血再灌注鼠心肌钙离子的变化

用液体闪烁计数法测定离体再灌注鼠心肌肌质网(SR)和线粒体(Mit)内 ⁴⁵Ca²⁺放射性强度(cpm),比较能量制剂ATP-MgCl₂,活性氧自由基清除剂SOD和钙阻滞剂Verapamil对离体缺血再灌注鼠心肌细胞SR和Mit钙离子浓度的影响.结果表明,SR内ATp-MgCl₂,SOD和Verapamil组 ⁴⁵Ca²⁺的cpm均高于对照组(P<0.0l或P<0.05),而Mit内均低于对照组(P<0.01).此三种药均能提高离体大鼠心肌细胞内SR ⁴⁵Ca²⁺和降低Mit ⁴⁵Ca²⁺积聚,从而保护了心肌细胞,防止缺血再灌注损伤.     

Model of the c-subunit oligomer in the membrane domain of F-ATPases

A model is described of a dodecameric complex consisting of the integral membrane component subunit c of the H⁺-transporting F_o domain of Escherichia coli F-ATPase. A high-resolution partial structure of monomeric subunit c resulting from ¹H-NMR studies [1] was used for constructing the model. The validity of the proposed arrangement of protomers in the dodecameric complex was tested by amino acid substitution analysis and chemical, biochemical and genetic data on subunit c.