The functional coupling between Ca2+-ATPase and creatine phosphokinase in heart muscle sarcoplasmic reticulum]

The functional role of creatine phosphokinase (CPK) in the process of energy supply for the Ca2+-ATPase reaction and ion transport across the membrane of heart sarcoplasmic reticulum (SR) has been studied. It has been shown that isolated and purified preparations of heart SR contain significant activity of CPK. The localization of CPK on the membrane of SR has been revealed also by an electron microscopic histochemical method. Under conditions of the Ca+-ATPase reaction in the presence of creatine phosphate the release of creatine into the reaction medium is observed, the rate of the latter process being dependent upon the MgATP concentration in accordance with the kinetic parameters of the Ca2+-ATPase reaction. CPK localized on the SR membrane is able to maintain higher rate of calcium uptake by SR vesicles, as compared to that with added ATP-regenerating system. The results obtained demonstrate the close functional coupling between CPK and Ca2+-ATPase in the membrane of SR.     

Modeling of Nonlinear Chemical Reaction Systems and Two-Parameter Stochastic Resonance

The idea of stochastic resonance (SR) is extended to two-parameter dynamical systems based on the Oregonator model of the Belousov-Zhabotinsky (BZ) reaction. The first case presents the photosensitivity of the reaction, and light flux and a flow rate are the two control parameters. The second case presents the effect of temperature on the oscillatory behaviors, and temperature and a flow rate are the control parameters. Stochastic resonance is demonstrated in the first case in which a signal and noise are applied to the different inputs, respectively. The scenario and novel aspects of SR in two-parameter systems are discussed, and the possibility of the analogous SR in biological systems is also pointed out.     

Effects of ADP and AMPPNP on the hydrogen-deuterium exchange kinetics in Ca2+, Mg2+-ATpase of sarcoplasmic reticulum

The kinetics of the hydrogen-deuterium exchange reaction in sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle was followed by infrared absorption measurements. The exchange rate in SR was much lower than that in the soluble proteins reported so far. When adenylyl-imidophosphate (AMPPNP, an TP analog) was present, the exchange rate was lower than that in free SR and it was the lowest when ADP was present. The effect of the nucleotides on the exchange rate reflects the conformational change of the Ca2+, Mg2+-ATpase of SR membranes on binding the nucleotides. The structure of e Ca2+, Mg2+-ATPase is more restricted in the following order: SR + ADP greater than SR + AMPPNP greater than free SR.     

Self-sustained sequence replication (3SR): an alternative to PCR

 The amplification of target nucleic acids before hybridization is one of the most powerful approaches for the detection of low copy number RNA and DNA. The best known amplification reaction is PCR which has many applications. However, certain drawbacks of the PCR reaction provide a role for alternative amplification methods. One of these methods is the self-sustained sequence replication (3SR) reaction, which is an isothermal method for RNA amplification depending on the action of three enzymes. 3SR has been used in several in vitro applications and has also been modified for in situ use (IS-3SR). We have studied IS-3SR with the measles virus as a model and have found that it can significantly amplify the amount of intracellular RNA. Such a level of amplification could raise the amount of single copy RNA to the level of detection by conventional in situ hybridization. Although careful controls to insure its specificity must be carried out, IS-3SR has several advantages, including ease of use, preserved cell morphology, and specificity for RNA amplification, which make it an attractive alternative to the in situ PCR method. Accepted: 27 June 1997     

An assay for sarcoplasmic reticulum Ca2(+)-ATPase activity in muscle homogenates

A spectrophotometric method is described for the determination of sarcoplasmic reticulum (SR) Ca2(+)-ATPase activity (EC 3.1.6.38) in unfractionated muscle homogenates. Conditions were established that give maximal SR Ca2(+)-ATPase activity, while eliminating Ca2(+)-dependent myofibrillar ATPase activity and reducing Ca2(+)-independent or background ATPase activity. High [Ca2+] (20 mM) could be used to selectively inhibit the SR Ca2+ ATPase. Identification of the Ca2(+)-dependent ATPase activity in muscle homogenates as being SR Ca2+ ATPase was based on a comparison of several parameters using homogenate material and purified SR. The following parameters were compared and found to be the same in homogenate and SR: activation and inactivation between 0 and 20 mM Ca2+, temperature dependence, sensitivity toward Triton X-100, and the maximal level of inhibition of ATPase activity achieved by an antibody specific for SR Ca2+ ATPase. The method is illustrated with the analysis of homogenates prepared from freeze-dried muscle fibers and thin sections of muscles typically used in microscope analyses as well as an analysis of freshly prepared homogenates from various types of muscle, which shows a good correlation over a wide range between SR specific Ca2(+)-uptake and -ATPase activities. In addition, a simple, easily constructed cuvette is described which allows the analysis of less than 5 micrograms of tissue (wet weight) in a volume of 25 microliters.     

SR proteins are required for nematode trans-splicing in vitro.

SR (ser/arg) proteins have been shown to play roles in numerous aspects of pre-mRNA splicing, including modulation of alternative splicing, commitment of substrates to the splicing pathway, and splice site communication. The last of these, splice site communication, is particularly relevant to trans-splicing in which the 5' and 3' exons originate on separate molecules. The participation of SR proteins in naturally occurring, spliced leader RNA-dependent transsplicing has not been examined. Here, we have isolated SR proteins from an organism that performs both trans- and cis-splicing, the nematode Ascaris lumbricoides. To examine their activity in in vitro splicing reactions, we have also developed and characterized an SR protein-depleted whole-cell extract. When tested in this extract, the nematode SR proteins are required for both trans- and cis-splicing. In addition, the state of phosphorylation of the nematode SR proteins is critical to their activity in vitro. Interestingly, mammalian (HeLa) and A. lumbricoides SR proteins exhibit equivalent activities in cis-splicing, while the nematode SR proteins are much more active in trans-splicing. Thus, it appears that SR proteins purified from an organism that naturally trans-splices its pre-mRNAs promote this reaction to a greater extent than do their mammalian counterparts.     

The control of sarcoplasmic reticulum Ca content in cardiac muscle

Most of the calcium that activates contraction in the heart comes from the sarcoplasmic reticulum (SR) and it is therefore essential to control the SR Ca content. SR Ca content reflects the balance between uptake (via the SR Ca-ATPase, SERCA) and release, largely via the ryanodine receptor (RyR). Unwanted changes of SR Ca are prevented because, for example, an increase of SR Ca content increases the amplitude of the systolic Ca transient and this, in turn, results in increased loss of Ca from and decreased Ca entry into the cell thereby restoring cell and SR Ca towards control levels. We discuss the parameters that affect the steady level of SR Ca and how these may change in heart failure. Finally, we discuss disordered Ca regulation with particular emphasis on the condition of alternans where successive heartbeats alternate in amplitude. This behaviour can be explained by excessive feedback gain in the processes controlling SR Ca.     

Influence of signal filtering and sample rate on isometric torque – time parameters using a traditional isokinetic dynamometer

Isometric force- or torque-time parameters are commonly reported in the research literature. The processing methods of the electronic dynamometer-derived signal may influence the outcome measures. This study determined the influence of filtering and sample rate (SR) on isometric torque-time parameters and provides specific signal processing recommendations for future studies. Twenty-three subjects performed 49 isometric maximum voluntary contractions (MVCs) of the knee extensors on an isokinetic dynamometer. Outcome measures included peak torque (PT), and rate of torque development at peak (RTDPEAK), 50 (RTD50) and 200 (RTD200) ms for seven filter conditions including low-pass filter cutoffs at 5, 10, 20, 50, 100 and 150 Hz and a notch filter at 100 and 200 Hz. Comparisons were also made across four SR conditions at 100, 500, 1000 and 2000 Hz. The RTDPEAK variable was markedly changed (−5.4 to −37.9%) for all filter frequencies compared to the 150 Hz condition and the RTD50 variable was altered for all frequencies between 50 and 5 Hz. No differences were found for RTD200. For SR, compared to the 2000 Hz condition, differences were revealed for the 100 Hz condition for the RTDPEAK and RTD50 variables. The filtering or SR did not alter PT across any of the conditions. The filter and SR applied to the signal was capable of distorting the MVC signal and skewing the torque–time parameters, specifically for the early and maximum RTD variables of the MVC curve (RTD50 and RTDPEAK). For traditional isokinetic dynamometers, a low-pass filter cutoff of 150 Hz and a SR of at least 1000 Hz is recommended when assessing early isometric force- or torque-time MVC parameters.     

The startle reaction to an acoustic stimulus in the rat: the effect of noradrenaline administered by microinjection into the pontomedullary reticular formation

The effect of the local administration of noradrenaline (NA) by microinjection into the pontomedullary reticular formation on the startle reaction (SR) evoked by an acoustic stimulus was studied in waking, non-immobilized rats. NA (10(-6) mol.l-1 in 2.5 microliters artificial cerebrospinal fluid) was administered 21 times to five animals in 13 experiments. In nine cases (43%), a significant change in the mean amplitude of the SR to a series of 30 stimuli (recorded in the third minute after administering NA) was observed; in eight cases (38%) it rose and in only one (5%) it fell. Binomial statistics confirmed the preponderance of results with an increase in SR amplitude. The results indicated that the potentiating effect of NA could actively help to regulate the function of the pontomedullary part of the neuronal circuit for the acoustic SR.     

On the specificity of the histochemical technique for sarcoplasmic reticular adenosine triphosphatase: a light and electron microscopic study.

The specificity of the histochemical localization of the calcium activated adenosine triphosphatase (ATPase) activity of the sarcoplasmic reticulum (SR) at pH 7.4 was studied using a calcium-citro-phosphate technique. The latter involves the splitting of ATP by ATPase producing phosphate ions which then react with calcium and citrate to form an insoluble reaction product. This reaction product was detected by both light and electron microscopy. Light microscopic examination showed a darkly stained continuous reticular pattern of reaction product which surrounded individual myofibrils. This reticular pattern of reaction product was distinctly dissimilar to that found when the histochemical reactions for mitochondrial or myofibrillar ATPase were performed. Ultrastructural investigations demonstrated the presence of discrete foci of electron dense reaction product in close association with the membranes of the SR in striated muscle fibres. Only occasional flecks were seen in the vicinity of mitochondria or myofilaments. The possibility is considered that the reticular pattern of staining achieved by the calcium-citro-phosphate technique may reflect the distribution of the "extra ATPase" of the SR, an enzyme implicated in the process of calcium uptake and muscle relaxation.     

Sarcoplasmic reticulum Ca-ATPase: distinction of phosphoenzymes formed from MgATP and CaATP as substrates and interconversion of the phosphoenzymes by Mg2+ and Ca2+

In order to characterize the phosphoenzymes (EPs) formed from MgATP and CaATP as substrates, the effects of Mg2+ and Ca2+ outside SR vesicles on the hydrolysis rates of EPs were examined by using purified and unpurified Ca-ATPases of sarcoplasmic reticulum (SR) at low [gamma-32P]ATP (4-10 microM), 0.1 M KCl, pH 7.0, and 0 degrees C. When the phosphorylation reaction was stopped by adding an excess of EDTA over Ca and Mg, two components of EP, EPfast (rate constant, kfast = 15-20 min-1), and EPslow (kslow = 0.3-0.4 min-1), were recognized in the time course of EP decomposition. These two rates did not depend on the Ca2+ or Mg2+ concentration in the medium during the phosphorylation reaction, although the proportions of EPfast and EPslow essentially depended on the concentrations of MgATP and CaATP in the phosphorylation reaction medium. The proportion of EPfast increased with increasing [MgATP]/[CaATP] in the medium, whereas that of EPslow decreased. The rate of EPslow hydrolysis in the presence of excess EDTA was basically the same as that of EP formed from CaATP. These results suggest that EPfast and EPslow are derived from MgATP and CaATP, respectively, and EPfast is a reaction intermediate with Mg bound at the substrate site (MgEP), while the main EPslow is a reaction intermediate with Ca bound at the substrate site (CaEP) which is readily converted to metal-free EP by EDTA addition (Shigekawa et al., (1983) J. Biol. Chem. 258, 8698-8707). Mg2+ added outside SR vesicles stimulated the conversion of CaEP to MgEP and inhibited the hydrolysis of MgEP in the relatively high concentration range (K(Mg) = 7.9 mM). Ca2+ added outside SR vesicles stimulated the conversion of MgEP to CaEP and inhibited the conversion of CaEP to MgEP by Mg2+ addition. The Ca2+ outside SR vesicles did not essentially affect the hydrolysis of MgEP. These results suggest that the interconversion between MgEP and CaEP takes place during the reaction by exchange of the divalent cation on the substrate site. The following scheme is proposed. (formula: see text)     

Mechanism of the stimulation of calcium ion dependent adenosine triphosphatase of cardiac sarcoplasmic reticulum by adenosine 3',5'-monophosphate dependent protein kinase

Canine cardiac sarcoplasmic reticulum (SR) is known to be phosphorylated by adenosine 3',5'-monophosphate (cAMP) dependent protein kinase on a 22 000-dalton protein. Phosphorylation enhances the initial rate of Ca2+ uptake and Ca2+-ATPase activity. To determine the molecular mechanism by which phosphorylation regulates the calcium pump in SR, we examined the effect of cAMP-dependent protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Cardiac sarcoplasmic reticulum was preincubated with cAMP and cAMP-dependent protein kinse in the presence (phosphorylated SR) and absence (control) of adenosine 5'-triphosphate (ATP). Control and phosphorylated SR were subsequently assayed for formation (4-200 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase in media containing 100 microM [ATP] and various free [Ca2+]. cAMP-dependent phosphorylation of SR resulted in pronounced stimulation of initial rates and levels of E approximately P formed at low free [Ca2+] (less than or equal to 7 microM), but the effect was less at high free Ca2+ (greater than or equal to 10 microM). This stimulation was associated with a decrease in the dissociation constant for Ca2+ binding and a possible increase in Ca2+ sites. The observed rate constant for E approximately P formation of calcium-preincubated SR was not significantly altered by phosphorylation. Phosphorylation also increased the initial rate of E approximately P decomposition. These findings indicate that phosphorylation of cardiac SR by cAMP-dependent protein kinase regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the calcium pump observed at steady state.     

Calcium alternans in a couplon network model of ventricular myocytes: role of sarcoplasmic reticulum load

Intracellular calcium (Ca) alternans in cardiac myocytes have been shown in many experimental studies, and the mechanisms remain incompletely understood. We recently developed a "3R theory" that links Ca sparks to whole cell Ca alternans through three critical properties: randomness of Ca sparks; recruitment of a Ca spark by neighboring Ca sparks; and refractoriness of Ca release units. In this study, we used computer simulation of a physiologically detailed mathematical model of a ventricular myocyte couplon network to study how sarcoplasmic reticulum (SR) Ca load and other physiological parameters, such as ryanodine receptor sensitivity, SR uptake rate, Na-Ca exchange strength, and Ca buffer levels affect Ca alternans in the context of 3R theory. We developed a method to calculate the parameters used in the 3R theory (i.e., the primary spark rate and the recruitment rate) from the physiologically detailed Ca cycling model and paced the model periodically to elicit Ca alternans. We show that alternans only occurs for an intermediate range of the SR Ca load, and the underlying mechanism can be explained via its effects on the 3Rs. Furthermore, we show that altering the physiological parameters not only directly changes the 3Rs but also alters the SR Ca load, having an indirect effect on the 3Rs as well. Therefore, our present study links the SR Ca load and other physiological parameters to whole cell Ca alternans through the framework of the 3R theory, providing a general mechanistic understanding of Ca alternans in ventricular myocytes.     

Monoclonal antibodies to dog heart sarcoplasmic reticulum. Antibodies that inhibit Ca2+-pump systems of cardiac and skeletal muscles

Purified sarcoplasmic reticulum (SR) vesicles from dog heart were used as an antigen to produce monoclonal antibodies (mAbs) to the Ca2+-ATPase. Nine of twelve clones of hybridoma cells produce mAbs which cross-react with seven SR preparation isolated from cardiac and skeletal muscles of various species. Three mAbs of IgM type interact with the 45-kDa tryptic fragment of rabbit skeletal muscle Ca2+-ATPase and markedly inhibit Ca2+ uptake (by 95%) and ATPase activity (by 80%) and decrease (by 30-50%) the steady-state level of the Ca2+-ATPase phosphoenzyme. The ATPase activity could be completely blocked by one of these mAbs if the incubation medium was supplemented with 2 microM orthovanadate. On the other hand, when SR vesicles were treated with increasing concentrations of a nonionic detergent C12E8, the inhibiting effect of mAb 4B4 is diminished. It is concluded that the mAbs inhibit the Ca2+-ATPase only if the enzyme exists in an oligomeric form. The inhibition of the SR activities is due to an effect of the mAbs on the whole active center of the enzyme, rather than on a single partial reaction.     

Reverse mode of the sarcoplasmic reticulum calcium pump and load-dependent cytosolic calcium decline in voltage-clamped cardiac ventricular myocytes

We have characterized [Ca](i) decline in voltage-clamped rabbit ventricular myocytes with progressive increases in sarcoplasmic reticulum (SR) calcium load. "Backflux" through the SR calcium pump is a critical feature which allows realistically small values for SR calcium leak fluxes to be used. Total cytosolic calcium was calculated from the latter part of [Ca](i) decline using rate constants for cellular calcium buffers. Intra-SR calcium buffering characteristics were also deduced. We found that the net SR calcium pump flux and rate of [Ca](i) decline decreased as the SR free [Ca] rose, with pump parameters held constant. We have therefore characterized for the first time in intact myocytes both forward and reverse SR calcium pump kinetics as well as intra-SR calcium buffering and SR calcium leak. We conclude that the reverse flux through the SR calcium pump is an important factor in comprehensive understanding of dynamic SR calcium fluxes.     

Identification of protein coding regions of rice genes using alternative spectral rotation measure and linear discriminant analysis

An improved method, called Alternative Spectral Rotation (ASR) measure, for predicting protein coding regions in rice DNA has been developed. The method is based on the Spectral Rotation (SR) measure proposed by Kotlar and Lavner, and its accuracy is higher than that of the SR measure and the Spectral Content (SC) measure proposed by Tiwari et al. In order to increase the identifying accuracy,we chose three different coding characters, namely the asymmetric, purine, and stop-codon variables as parameters, and an approving result was presented by the method of Linear Discriminant Analysis (LDA).     

Nitric oxide-dependent modification of the sarcoplasmic reticulum Ca-ATPase: localization of cysteine target sites

Skeletal muscle contraction and relaxation is modulated through the reaction of sarcoplasmic reticulum (SR) protein thiols with reactive oxygen and nitrogen species. Here, we have utilized high-performance liquid chromatography-electrospray mass spectrometry and a specific thiol-labeling procedure to identify and quantify cysteine residues of the SR Ca-ATPase that are modified by exposure to nitric oxide (NO). NO and/or NO-derived species inactivate the SR Ca-ATPase and modify a broad spectrum of cysteine residues with highest reactivities towards Cys364, Cys670, and Cys471. The selectivity of NO and NO-derived species towards the SR Ca-ATPase thiols is different from that of peroxynitrite. The efficiency of NO at thiol modification is significantly higher compared with that of peroxynitrite. Hence, NO has the potential to modulate muscle contraction through chemical reaction with the SR Ca-ATPase in vivo.     

Change in the passive influx of Ca2+ into sarcoplasmic reticulum vesicles as a result of chemical modification of surface amino groups

Treatment of sarcoplasmic reticulum (SR) vesicles with trinitrobenzene (TNBS) and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) stimulates the initial rate of passive influx of Ca2+ into SR vesicles, but does not affect either the binding or the maximal passive loading of SR vesicles with Ca2+. The changes in the kinetics of KCl-stimulated passive influx of Ca2+ depend on the reagent used. It is supposed that stimulation of passive influx of Ca2+ into SR vesicles and the changes in the reaction kinetics may be caused by modification of the Ca2+ channel gating behaviour as a result of binding of surface amino groups.     

Post-infarct sleep disruption and its relation to cardiac remodeling in a rat model of myocardial infarction

Sleep disruption after myocardial infarction (MI) by affecting ubiquitin–proteasome system (UPS) is thought to contribute to myocardial remodeling and progressive worsening of cardiac function. The aim of current study was to test the hypothesis about the increased risk of developing heart failure due to experience of sleep restriction (SR) after MI. Male Wistar rats (n = 40) were randomly assigned to four experimental groups: (1) Sham, (2) MI, (3) MI and SR (MI + SR) (4) Sham and SR (Sham + SR). MI was induced by permanent ligation of left anterior descending coronary artery. Twenty-four hours after surgery, animals were subjected to chronic SR paradigm. Blood sampling was performed at days 1, 8 and 21 after MI for determination of serum levels of creatine kinase-MB (CK-MB), corticosterone, malondialdehyde (MDA) and nitric oxide (NO). Finally, at 21?days after MI, echocardiographic parameters and expression of MuRF1, MaFBx, A20, eNOS, iNOS and NF-kB in the heart were evaluated. We used H&;E staining to detect myocardial hypertrophy. We found out that post infarct SR increased corticosterone levels. Our results highlighted deteriorating effects of post-MI SR on NO production, oxidative stress, and echocardiographic indexes (p < 0.05). Moreover, its detrimental effects on myocardial damage were confirmed by overexpression of MuRF1, MaFBx, iNOS and NF-kB (p < 0.001) in left ventricle and downregulation of A20 and eNOS (p < 0.05). Furthermore, histological examination revealed that experience of SR after MI increased myocardial diameter as compared to Sham subjects (p < 0.05). Our data suggest that SR after MI leads to an enlargement of the heart within 21?days, marked by an increase in oxidative stress and NO production as well as an imbalance in UPS that ultimately results in cardiac dysfunction and heart failure.