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.     

白细胞介素-2对大鼠心肌Ca2+ATPase和Na+ /K+ATPase的影响

为了探讨IL－2对心肌细胞内钙影响的可能机制,用光学法检测心肌肌浆网Ca^2 ATPase的活性,以及细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性。结果：（1）用IL－2（10、40、200、800U/ml）灌流心脏后,其肌浆网Ca^2 ATPase的活性随IL－2浓度的升高而增强;（2）在ATP浓度为0.1-4mmol/L时,Ca^2 ATPase的活性随ATP浓度的升庙则增强,由IL－2（200U／ml）灌流后的心脏获得肌浆网（SR）,其Ca^2 ATPase的活性对ATP的反应强于对照组;（3）在[Ca^2 ]为1－40μmol/L时,心脏SR Ca^2 ATPase的活性随[Ca^2 ]增加而增强,而IL－2灌流心脏后分离的SR,其Ca^2 ATPase活性在[Ca^2 ]升高时没有明显改变;（4）用nor-BNI(10nmol/L）预处理5min后,IL－2（200U／ml）灌流后不再使SR Ca^2 ATPase的活性增强;（5）用PTX（5mg/L）预处理后,IL－2对SR Ca^2 ATPase的影响减弱;（6）用磷脂酶C（PLC）抑制剂U73122（5μmol/L）处理后,IL－2不再使SR Ca^2 ATPase活性增高;（7）用IL－2直接处理从正常大鼠分离的SR后,对SR Ca^2 ATPase活性无明显影响;（8）IL－2灌流后,对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase活性没有显著。上述结果表明,IL－2灌流心脏后使心肌肌浆网Ca^2 ATPase的活性增加,心肌细胞膜上的κ－阿片受体及其下游的G蛋白和PLC介导了IL－2的作用。尽管IL－2提高SR Ca^2 ATPase对ATP的反应性,但却抑制SR Ca^2 ATPase对钙离子的敏感性。IL－2对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性无明显影响。     

Fura-2 fluorescent technique for the assessment of Ca2+ homeostasis in cardiomyocytes

Ca2+ homeostasis plays a pivotal role in maintaining cell growth and function. Many heart diseases are related to the abnormalities in Ca2+ mobilization and extrusion. Ca2+-sensitive fluorescent dyes have been used successfully to estimate intracellular free Ca2+ ([Ca2+]i) level and the mechanisms of Ca2+ movements in living cells. This article is focused on the methodology involving the use of Fura-2/AM or free Fura-2 to measure agonist-induced Ca2+ mobilization as well as the mechanisms of changes in [Ca2+]i in cardiomyocytes. Methods involving Fura-2 technique for the measurement of Ca2+ extrusion from the cells and Ca2+ reuptake by sarcoplasmic reticulum (SR) are also described. The prevention of KCl-induced increase in the intracellular Ca2+ is shown by chelating the extracellular Ca2+ with EGTA or by the presence of Ca2+-channel inhibitors such as verapamil and diltiazem. The involvement of SR in the ATP-induced increase in intracellular Ca2+ is illustrated by the use of Ca2+-pump inhibitors, thapsigargin and cyclopiazonic acid as well as ryanodine which deplete the SR Ca2+ storage. The use of 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate (NCDC), an inhibitor of inositol 1,4,5-trisphosphate (IP3) production, is described for the attenuation of phosphatidic acid (PA) induced increase in Ca2+-mobilization. The increase in intracellular Ca2+ in cardiomyocytes by PA, unlike that by KCl or ATP, was observed in diabetic myocardium. Thus, it appears that the Fura-2 method for the measurement of Ca2+ homeostasis in cardiomyocytes is useful in studying the pathophysiology and pharmacology of Ca2+ movements.     

Chemical modification of an arginine residue in the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by phenylglyoxal

Phenylglyoxal (PGO) was used as a reagent for chemical modification of the ATP-binding site of Ca2+-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum (SR-ATPase). When 1 mM PGO was reacted with SR-ATPase at 30°C at pH 8.5, PGO was bound to the ATPase molecule in two-to-one stoichiometry with concomitant loss of activity of the ATPase to form the phosphorylated intermediate (E-P). ATP and ADP prevented the binding of PGO and thereby protected the enzyme from inactivation. The SR membranes were labeled with [14C]PGO and then digested with pepsin to identify the attachment site of PGO. A 14C-labeled peptide (402lle-Arg*-Ser-Gly-Gln406) was purified to homogeneity by C18-reversed phase HPLC (Arg* denotes the binding site of [14C]PGO). These results indicate that Arg403 is located in the ATP binding site of the SR-ATPase.     

Biochemical characterization of the Ca2+ release channel of skeletal and cardiac sarcoplasmic reticulum

Summary Rapid mixing-vesicle ion flux and planar lipid bilayer-single channel measurements have shown that a high-conductance, ligand-gated Ca²⁺ release channel is present in heavy, junctional-derived membrane fractions of skeletal and cardiac muscle sarcoplasmic reticulum. Using the release channel-specific probe, ryanodine, a 30S protein complex composed of polypeptides of M_r 400 000 has been isolated from cardiac and skeletal muscle. Reconstitution of the complex into planar lipid bilayers has revealed a Ca²⁺ conductance with properties characteristic of the native Ca²⁺ release channel.     

Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.     

Methylglyoxal induces cell death through endoplasmic reticulum stress‐associated ROS production and mitochondrial dysfunction

Diabetic retinopathy (DR) and age‐related macular degeneration (AMD) are two important leading causes of acquired blindness in developed countries. As accumulation of advanced glycation end products (AGEs) in retinal pigment epithelial (RPE) cells plays an important role in both DR and AMD, and the methylglyoxal (MGO) within the AGEs exerts irreversible effects on protein structure and function, it is crucial to understand the underlying mechanism of MGO‐induced RPE cell death. Using ARPE‐19 as the cell model, this study revealed that MGO induces RPE cell death through a caspase‐independent manner, which relying on reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) loss, intracellular calcium elevation and endoplasmic reticulum (ER) stress response. Suppression of ROS generation can reverse the MGO‐induced ROS production, MMP loss, intracellular calcium increase and cell death. Moreover, store‐operated calcium channel inhibitors MRS1845 and YM‐58483, but not the inositol 1,4,5‐trisphosphate (IP3) receptor inhibitor xestospongin C, can block MGO‐induced ROS production, MMP loss and sustained intracellular calcium increase in ARPE‐19 cells. Lastly, inhibition of ER stress by salubrinal and 4‐PBA can reduce the MGO‐induced intracellular events and cell death. Therefore, our data indicate that MGO can decrease RPE cell viability, resulting from the ER stress‐dependent intracellular ROS production, MMP loss and increased intracellular calcium increase. As MGO is one of the components of drusen in AMD and is the AGEs adduct in DR, this study could provide a valuable insight into the molecular pathogenesis and therapeutic intervention of AMD and DR.     

Hierarchical domain‐motion analysis of conformational changes in sarcoplasmic reticulum Ca2+‐ATPase

Sarco(endo)plasmic reticulum Ca²⁺‐ATPase transports two Ca²⁺ per ATP‐hydrolyzed across biological membranes against a large concentration gradient by undergoing large conformational changes. Structural studies with X‐ray crystallography revealed functional roles of coupled motions between the cytoplasmic domains and the transmembrane helices in individual reaction steps. Here, we employed “Motion Tree (MT),” a tree diagram that describes a conformational change between two structures, and applied it to representative Ca²⁺‐ATPase structures. MT provides information of coupled rigid‐body motions of the ATPase in individual reaction steps. Fourteen rigid structural units, “common rigid domains (CRDs)” are identified from seven MTs throughout the whole enzymatic reaction cycle. CRDs likely act as not only the structural units, but also the functional units. Some of the functional importance has been newly revealed by the analysis. Stability of each CRD is examined on the morphing trajectories that cover seven conformational transitions. We confirmed that the large conformational changes are realized by the motions only in the flexible regions that connect CRDs. The Ca²⁺‐ATPase efficiently utilizes its intrinsic flexibility and rigidity to response different switches like ligand binding/dissociation or ATP hydrolysis. The analysis detects functional motions without extensive biological knowledge of experts, suggesting its general applicability to domain movements in other membrane proteins to deepen the understanding of protein structure and function. Proteins 2015; 83:746–756. © 2015 Wiley Periodicals, Inc.     

Ca2+ homeostasis and cytotoxicity in isolated hepatocytes: Studies with extracellular adenosine 5′-triphosphate

The incubation of isolated rat hepatocytes with extracellular adenosine 5′-trihosphate (ATP) resulted in an inhibition of Ca²⁺ efflux. The ATP-induced Ca²⁺ accumulation as determined by the increase in phosphorylase a activity and the Ca²⁺ -sensitive fluorescent indicator (2-[(2-bis-[carboxymethyl]-amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis-[carboxymethyl] aminoquinoline-tetrakis-[acetoxymethyl]ester) (Quin 2-AM) was associated with both the hydrolysis of ATP and the phosphorylation of a 110 kDa protein. No significant alteration in the intracellular ATP level was observed. The appearance of surface blebs and cytotoxicity followed the rise in cytosolic Ca²⁺, suggesting that the increased free Ca²⁺ may be responsible for the loss of viability. When a calmodulin inhibitor, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy] ethyl]-1H-imidazolium chloride (calmidazolium), was included in the medium prior to ATP addition, bleb formation was reduced and the loss of viability was completely prevented, indicating that a Ca²⁺ -calmodulin process may be involved in the initiation of cytotoxicity.     

Comparison of the effects of the membraneassociated Ca2+/calmodulin-dependent protein kinase on Ca2+-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum

In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca²⁺-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca²⁺-ATPase. Studies comparing, the effects of PKA and CaM kinase on cardiac Ca²⁺-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca²⁺-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca²⁺-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 M) directly to the Ca²⁺ transport assay medium results in minimal ( 112–130% of control) stimulation of Ca²⁺ uptake activity when the Ca²⁺ uptake reaction is initiated by the addition of either ATP or Ca²⁺/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca²⁺ transport assay medium results in stimulation of Ca²⁺ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca²⁺ uptake markedly (215% of control) when the Ca²⁺ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca²⁺/EGTA but minimally (130% of control) when the Ca²⁺ uptake reaction is initiated by the addition of ATP. These findings imply that (a) phospholamban is coupled to the Ca²⁺-ATPase in slow-twitch skeletal muscle SR (as in cardiac SR), and (b) the amount of Ca²⁺ uptake stimulation seen upon the addition of calmodulin or PKA depends strongly on the assay conditions employed. Our observations help to explain the wide range of effects of calmodulin or PKA addition reported in previous studies. It should be noted that, since CaM kinase is now known to phosphorylate the Ca²⁺-ATPase in addition to phospholamban, further studies are required to determine the relative contributions of phospholambanversus Ca²⁺-ATPase phosphorylation in the stimulation of Ca²⁺-ATPase function by CaM kinase. Also, earlier studies attributing all of the effects of CaM kinase stimulation of Ca²⁺ uptake and Ca²⁺-ATPase activity to phospholamban phosphorylation need to be re-examined.     

Asperpyrone A attenuates RANKL‐induced osteoclast formation through inhibiting NFATc1, Ca2+ signalling and oxidative stress

Imbalance of osteoblast and osteoclast in adult leads to a variety of bone‐related diseases, including osteoporosis. Thus, suppressing the activity of osteoclastic bone resorption becomes the main therapeutic strategy for osteoporosis. Asperpyrone A is a natural compound isolated from Aspergillus niger with various biological activities of antitumour, antimicrobial and antioxidant. The present study was designed to investigate the effects of Asperpyrone A on osteoclastogenesis and to explore its underlining mechanism. We found that Asperpyrone A inhibited RANKL‐induced osteoclastogenesis in a dose‐dependent manner when the concentration reached 1 µm, and with no cytotoxicity until the concentration reached to 10 µm. In addition, Asperpyrone A down‐regulated the mRNA and protein expression of NFATc1, c‐fos and V‐ATPase‐d2, as well as the mRNA expression of TRAcP and Ctsk. Furthermore, Asperpyrone A strongly attenuated the RNAKL‐induced intracellular Ca²⁺ oscillations and ROS (reactive oxygen species) production in the process of osteoclastogenesis and suppressed the activation of MAPK and NF‐κB signalling pathways. Collectively, Asperpyrone A attenuates RANKL‐induced osteoclast formation via suppressing NFATc1, Ca²⁺ signalling and oxidative stress, as well as MAPK and NF‐κB signalling pathways, indicating that this compound may become a potential candidate drug for the prevention or treatment of osteoporosis.     

Putative roles of Ca2+‐independent phospholipase A2 in respiratory chain‐associated ROS production in brain mitochondria: influence of docosahexaenoic acid and bromoenol lactone

Ca²⁺‐independent phospholipase A₂ (iPLA₂) is hypothesized to control mitochondrial reactive oxygen species (ROS) generation. Here, we modulated the influence of iPLA₂‐induced liberation of non‐esterified free fatty acids on ROS generation associated with the electron transport chain. We demonstrate enzymatic activity of membrane‐associated iPLA₂ in native, energized rat brain mitochondria (RBM). Theoretically, enhanced liberation of free fatty acids by iPLA₂ modulates mitochondrial ROS generation, either attenuating the reversed electron transport (RET) or deregulating the forward electron transport of electron transport chain. For mimicking such conditions, we probed the effect of docosahexaenoic acid (DHA), a major iPLA₂ product on ROS generation. We demonstrate that the adenine nucleotide translocase partly mediates DHA‐induced uncoupling, and that low micromolar DHA concentrations diminish RET‐dependent ROS generation. Uncoupling proteins have no effect, but the adenine nucleotide translocase inhibitor carboxyatractyloside attenuates DHA‐linked uncoupling effect on RET‐dependent ROS generation. Under physiological conditions of forward electron transport, low micromolar DHA stimulates ROS generation. Finally, exposure of RBM to the iPLA₂ inhibitor bromoenol lactone (BEL) enhanced ROS generation. BEL diminished RBM glutathione content. BEL‐treated RBM exhibits reduced Ca²⁺ retention capacity and partial depolarization. Thus, we rebut the view that iPLA₂ attenuates oxidative stress in brain mitochondria. However, the iPLA₂ inhibitor BEL has detrimental activities on energy‐dependent mitochondrial functions.

     

Phaeomoniella chlamydospora‐induced Oxidative Burst in Vitis vinifera Cell Suspensions: Role of NADPH Oxidase and Ca2+

The biphasic oxidative burst induced by Phaeomoniella chlamydospora extract (Pce) in Vitis vinifera (Vv) cell suspensions was investigated. Treatment of cell suspensions with diphenyleneiodonium chloride, an inhibitor of NADPH oxidase, prevented the Pce‐induced biphasic reactive oxygen species (ROS) accumulation, suggesting that NADPH oxidase is the primary ROS source in the oxidative burst induced by Pce elicitation of Vv cells. The role of Ca²⁺ in the oxidative burst was also investigated using a Ca²⁺ chelator and several Ca²⁺ channel blockers. The treatment of Vv cell suspensions with the Ca²⁺ chelator ethylene glycol‐bis(2‐aminoethylether)‐N, N, N’; N’‐tetraacetic acid (EGTA) completely inhibited Pce‐induced ROS accumulation, suggesting that Ca²⁺ availability is necessary for occurrence of the induced oxidative burst. However, only the Ca²⁺ channel blocker ruthenium red strongly inhibited the Pce‐induced ROS accumulation, suggesting that the specific Ca²⁺ channel types from which Ca²⁺ influx is originated also play an important role in the Pce‐induced oxidative burst. Furthermore, Ca²⁺ availability seems to be necessary for the Pce‐induced activity of NADPH oxidase.     

Ca2+ and mitochondria as substrates for deficits in synaptic plasticity in normal brain ageing

Normal brain ageing is associated with a degree of functional impairment of neuronal activity that results in a reduction in memory and cognitive functions. One mechanism proposed to explain the age-dependent changes was the "Ca(2+) hypothesis of ageing" but data accumulated in the last decade revealed a number of inconsistencies. Two important questions were raised: (a) which are, if any, the most reliable age-associated change in neuronal Ca(2+) homeostasis and (b) are these changes primary, and thus determinant of the ageing phenotype, or are they secondary to other changes in the physiology of the aged neurones. After a brief review of the evidence accumulated for the age-induced changes in synaptic plasticity, we assess the proposal that these changes are, ultimately, determined by changes in the metabolic state of the aged neurones, that are manifest particularly after neuronal stimulation. In this context, it appears that the changes in mitochondrial status and function are of primary importance.     

Ca2+‐regulated and diurnal rhythm‐regulated Na+/Ca2+ exchanger AtNCL affects flowering time and auxin signalling in Arabidopsis

Calcium (Ca²⁺) is vital for plant growth, development, hormone response and adaptation to environmental stresses, yet the mechanisms regulating plant cytosolic Ca²⁺ homeostasis are not fully understood. Here, we characterize an Arabidopsis Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger AtNCL that regulates Ca²⁺ and multiple physiological processes. AtNCL was localized to the tonoplast in yeast and plant cells. AtNCL appeared to mediate sodium (Na⁺) vacuolar sequestration and meanwhile Ca²⁺ release. The EF‐hand domains within AtNCL regulated Ca²⁺ binding and transport of Ca²⁺ and Na⁺. Plants with diminished AtNCL expression were more tolerant to high CaCl₂ but more sensitive to both NaCl and auxin; heightened expression of AtNCL rendered plants more sensitive to CaCl₂ but tolerant to NaCl. AtNCL expression appeared to be regulated by the diurnal rhythm and suppressed by auxin. DR5::GUS expression and root responses to auxin were altered in AtNCL mutants. The auxin‐induced suppression of AtNCL was attenuated in SLR/IAA14 and ARF6/8 mutants. The mutants with altered AtNCL expression also altered flowering time and FT and CO expression; FT may mediate AtNCL‐regulated flowering time change. Therefore, AtNCL is a vacuolar Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger that regulates auxin responses and flowering time.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles

The Ca²⁺-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca²⁺-uptake and Ca²⁺-ATPase activities were significantly increased (2–3 fold). The highest activation was obtained at a 50:50 molar ratio of PSYC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca²⁺-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca²⁺-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca²⁺-ATPase were similar in either PC or PC: PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca²⁺-ATPase. Regulation of the Ca²⁺-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca²⁺-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.