Two forms of Ca2+-dependent ATPase from sarcoplasmic reticulum]

Two highly purified sarcoplasmic reticulum membrane fractiones differing in their sensitivities to the uncoupling action of caffeine were isolated from white skeletal muscles of the rabbit. The main protein component of both fractions is a catalytical polypeptide of Ca2+-dependent ATPase. Treatment of the caffeine-sensitive reticular fraction by trypsin or DTNB completely removes the effect of caffeine. It was found that similar effects on the caffeine-sensitive reticular fraction are exerted by bemegride, camphor, ethymizole and cordiamine. Isolation of Ca2+-dependent ATPase from both reticular fractions and reconstruction of Ca2+-transporting vesicles were carried out. Ca2+ transport by the vesicles enriched by ATPase from the caffeine-sensitive reticular fraction is uncoupled under the effect of caffeine; however, caffeine has no effect on the vesicles enriched by caffeine-insensitive reticular ATPase. The molecular weight of caffeine-sensitive and caffeine-insensitive ATPases determined in the presence of sedium dodecyl sulfate are found to be identical. Electrophoresis in the presence of digitonin revealed different electrophoretic behaviour of the two forms of ATPase.     

The effect of total ischemia on the Ca2+-transporting activity of the sarcoplasmic reticulum of the rat myocardium

Different caffeine and calcium concentrations have been studied for their influence on Ca2(+)-pumping function of sarcoplasmic reticulum in a homogenate from control and ischemic rat myocardium. Ca2(+)-transporting system of sarcoplasmic reticulum terminal cisternae membranes from the ischemic myocardium was found to be more sensitive to Ca2+ and caffeine action, inhibiting Ca2+ uptake velocity, as compared to control. This may be one of causes leading to the contractibility disorder under myocardium ischemia.     

Intracellular localization of the caffeine-sensitive form of Ca-dependent ATPase in the sarcoplasmic reticulum

A Ca-selective electrode was used to study the effect of caffeine on different fractions of sarcoplasmic reticulum membranes of rabbit skeletal muscles. Caffeine was found to uncouple Ca2+ transport and ATP hydrolysis in a fraction, which is enriched with fragments of terminal cisterns according to the electron microscopy data. Caffeine does not produce any effect on the light fraction containing no fragments of terminal cisterns. It is concluded that caffeine-sensitive Ca2+-dependent ATPase is localized in terminal cisterns of the sarcoplasmic reticulum.     

Effect of caffeine on kinetics of accumulation and release of Ca2+ by vesicles of the sarcoplasmic reticulum of skeletal muscle

The action of caffeine was studied on the heavy sarcoplasmic reticulum fraction enriched by vesicles derived from terminal cisterns. Caffeine lowers the ATP-dependent accumulation of Ca2+ by vesicles and enhances the first rapid phase of the Ci2+ release from vesicles. The action of caffeine was transient, reversed, Ca2+-dependent. The data obtained suggest that the reduction of ATP-dependent calcium accumulation and enhancement of calcium release by caffeine are mediated by the mechanism of Ca2+-induced Ca2+ release and support the view that caffeine may regulate the equilibrium between open and closed states of Ca2+-channel by increasing the affinity of Ca2+-receptor site of the channel.     

The effect of carnosine on Ca-channels in rabbit skeletal muscle sarcoplasmic reticulum]

Carnosine (beta-alanyl-L-histidine), which is present in millimolar concentrations in skeletal muscles, induces Ca2+ release from the heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum by activation ruthenium red-sensitive Ca-release channels. The effect of carnosine is dose-dependent, which indicates the presence of saturable carnosine-binding sites in the Ca-release channel molecule. The half-maximal Ca2+ release is observed in the presence of 8.7 mM carnosine. At the same time, carnosine addition to the medium increases the affinity of sarcoplasmic reticulum Ca-channels for the Ca-release activators, caffeine and adenine nucleotides. It is concluded that carnosine is an endogenous regulator of skeletal muscle sarcoplasmic reticulum Ca-channels which modulates the affinity of these channels for different ligands.     

Concentration of components of the adenylate system in heavy and light fractions of the sarcoplasmic reticulum of skeletal muscles and sensitivity of these fractions to the effects of imidazole-containing compounds and caffeine]

Fragments of sarcoplasmic reticulum from rabbit sceletal muscles sedimented within the range from 2000 g to 8000 g (heavy fraction) and 8000 g to 40000 g (light fraction) and washed with 0.6 M KCl, were practically free of adenylatecyclase activity. Phosphodiesterase cAMP was not found in the light fraction, while its activity in the heavy fraction was 500 pmol of cAMP/min per mg of protein. Both fractions contain bound cAMP (1-2 pmol/mg of protein) and specific sites of cAMP binding, the binding constant being approximately 10(6)M-1. The number of binding sites is 60 pmol/mg of protein for the heavy and 30 pmol/mg of protein for the light fractions. The level of phosphodiesterase activity in the heavy fraction correlates with its sensitivity to imidazole, anserine and caffeine. Imidazole and anserine increase in 1.5-1.8 times the value of Ca2+/ATP in the heavy fraction and produce no effect on Ca2+ transport by the light fraction. Caffeine decreases almost twice the Ca2+/ATP value in the heavy fraction and has practically no effect on Ca2+ absorption by enzymes of the light reticulum fraction. Imidazole and anserine activate membrane-bound phosphodiesterase, while caffeine inhibits it. It is suggested that structural rearrangements of membrane-bound phosphodiesterase under the effect of caffeine, imidazole and anserine are responsible for changes in the efficiency of Ca2+ transport by fragments of the heavy reticulum fractions.     

Activation of the caffeine center of the sarcoplasmic reticulum at a reduced concentration of magnesium ions

The action of caffeine and Mg2+ on the efficacy of Ca2+ transport by terminal cisterns and longitudinal tubules of rabbit skeletal muscle sarcoplasmic reticulum (SR) was studied and compared. Addition of 5 to 10 mM caffeine to the incubation medium or a decrease in Mg2+ concentration from 4 to 0.1 mM led to a 3-fold diminution of the Ca/ATP ratio for the terminal cistern fraction. In longitudinal tubules, that effect was far less pronounced. The effects of caffeine and decreases in Mg2+ concentration were blocked by ruthenium red, tetracaine and dimethylsulfoxide. It is assumed that the decrease in Mg2+ concentration is accompanied by activation of the caffeine site of the SR, induced by the intravesicular caffeine-like factor.     

Cyclic AMP-induced enhancement of calcium accumulation by the sarcoplasmic reticulum with no modification of the sensitivity of the myofilaments to calcium in skinned fibres from a yeast skeletal muscle.

In the presence of low concentrations of total EGTA (5 . 10(-4) M) and free Mg2+ (3.16 . 10(-5) M) and in the presence of caffeine (8 . 10(-3) M), cyclic AMP (5 . 10(-6) M) produces a relaxation of the tension developed by skinned fibres from cat caudo-femoralis. The relaxation can be attributed to an enhancement of the Ca2+ accumulation by the sarcoplasmic reticulum, since cyclic AMP does not modify the sensitivity of the myofilaments of Ca2+. These results are similar to those previously reported for the effect of cyclic AMP on skinned cardiac cells in the presence of a higher free Mg2+ concentration and in the absence of caffeine. This similarity suggests that the mode of action of cyclic AMP on the sarcoplasmic reticulum is not fundamentally different in cardiac and fast skeletal muscles.     

Bromo-eudistomin D, a novel inducer of calcium release from fragmented sarcoplasmic reticulum that causes contractions of skinned muscle fibers

Bromo-eudistomin D induced a contraction of the chemically skinned fibers from skeletal muscle at concentrations of 10 microM or more. This contractile response to bromo-eudistomin D was completely blocked by 10 mM procaine. The extravascular Ca2+ concentrations of the heavy fractions of the fragmented sarcoplasmic reticulum (HSR) were measured directly by a Ca2+ electrode to examine the effect of bromo-eudistomin D on the sarcoplasmic reticulum. After the HSR was loaded with Ca2+ by the ATP-dependent Ca2+ pump, the addition of 10 microM bromo-eudistomin D caused Ca2+ release that was followed by spontaneous Ca2+ reuptake. In the presence of 2 microM ruthenium red or 4 mM MgCl2, no Ca2+ release was induced by 20 microM bromo-eudistomin D. The rate of 45Ca2+ efflux from HSR, which had been passively preloaded with 45Ca2+, was accelerated 7 times by 10 microM bromo-eudistomin D. The concentration of bromo-eudistomin D for half-maximum effect on the apparent efflux rate was 1.5 microM, while that of caffeine was 0.6 mM. The bromo-eudistomin D-evoked efflux of 45Ca2+ was abolished by 2 microM ruthenium red or 0.5 mM MgCl2. Bromo-eudistomin D was found to be 400 times more potent than caffeine in its Ca2+-releasing action but was similar in its action in other respects. These results indicate that bromo-eudistomin D may induce Ca2+ release from the sarcoplasmic reticulum through physiologically relevant Ca2+ channels.     

Effect of caffeine on the Ca2+-transport function of sarcoplasmic reticulum vesicles in the rat myocardium

The effects of caffeine on active transport of Ca2 by heavy and light fractions of rat myocardial microsomes were investigated with the use of a Ca2+-selective electrode and nephelometry. It was found that under the effect of caffeine (5 mM) the rate of Ca2 transport in the presence of oxalate decreased by 30 to 40%. The caffeine-induced inhibition was prevented by ruthenium and tetracaine, thus suggesting the inhibitor specificity. Since caffeine is a specific blocker of Ca2 transport to the terminal cisterns of the skeletal muscle sarcoplasmic reticulum, it is assumed that the microsomal fraction of rat myocardium contains terminal cistern fragments.     

Modification of calcium fluxes by dimethyl sulfoxide and 2-butoxyethanol in sarcoplasmic reticulum vesicles: a possible mechanism for skeletal muscle relaxation induced by dimethyl sulfoxide

In order to investigate the mechanism of skeletal muscle relaxation induced by dimethyl sulfoxide, 2-butoxyethanol and dimethyl sulfoxide were examined for their effects on 1) Ca2+ uptake into and efflux from sarcoplasmic reticulum vesicles prepared from rabbit fast skeletal muscle and crayfish tail muscle by the murexide method, 2) ATPase activities of rabbit reticulum vesicles, 3) the isolated phrenic nerve-diaphragm preparation of the rat and 4) crayfish opener muscle preparation. Ca2+ efflux rate from rabbit reticulum vesicles was markedly decreased with increasing concentrations (5-20% v/v) of dimethyl sulfoxide without affecting the maximum Ca2+ uptake by the reticulum. 2-Butoxyethanol showed quite contrary effects. Dimethyl sulfoxide strongly inhibited the activity of basal ATPase rather than of Ca2+-dependent ATPase. 2-Butoxyethanol did not significantly inhibit the activity of basal ATPase, but markedly increased Ca2+-dependent ATPase activity. Antagonisms between dimethyl sulfoxide and caffeine were demonstrated either in contractions of crayfish opener muscles or in the Ca2+ release from crayfish sarcoplasmic reticulum vesicles. These results indicate a possibility that dimethyl sulfoxide reversibly induces skeletal muscle relaxation mainly in the sarcoplasmic reticulum by means of decreasing the rate and the amount of Ca2+ release from the reticulum.     

Enhanced Ca2+-induced calcium release by isolated sarcoplasmic reticulum vesicles from malignant hyperthermia susceptible pig muscle

To further define the possible involvement of sarcoplasmic reticulum calcium accumulation and release in the skeletal muscle disorder malignant hyperthermia (MH), we have examined various properties of sarcoplasmic reticulum fractions isolated from normal and MH-susceptible pig muscle. A sarcoplasmic reticulum preparation enriched in vesicles derived from the terminal cisternae, was further fractionated on discontinuous sucrose density gradients (Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374). The resultant MH-susceptible and normal sarcoplasmic reticulum fractions, designated F0-F4, did not differ in yield, cholesterol and phospholipid content, or nitrendipine binding capacity. Calcium accumulation (0.27 mumol Ca/mg per min at 22 degrees C), Ca2+-ATPase activity (0.98 mumol Pi/mg per min at 22 degrees C), and calsequestrin content were also similar for MH-susceptible and normal sarcoplasmic reticulum fraction F3. To examine sarcoplasmic reticulum calcium release, fraction F3 vesicles were passively loaded with 45Ca (approx. 40 nmol Ca/mg), and rapidly diluted into a medium of defined Ca2+ concentration. Upon dilution into 1 microM Ca2+, the extent of Ca2+-dependent calcium release measured after 5 s was significantly greater for MH-susceptible than for normal sarcoplasmic reticulum, 65.9 +/- 2.8% vs. 47.7 +/- 3.9% of the loaded calcium, respectively. The C1/2 for Ca2+ stimulation of this calcium release (5 s value) from MH-susceptible sarcoplasmic reticulum also appeared to be shifted towards a higher Ca2+-sensitivity when compared to normal sarcoplasmic reticulum. Dantrolene had no effect on calcium release from fraction F3, however, halothane (0.1-0.5 mM) increased the extent of calcium release (5 s) similarly in both MH-susceptible and normal sarcoplasmic reticulum. Furthermore, Mg2+ was less effective at inhibiting, while ATP and caffeine were more effective in stimulating, this Ca2+-dependent release of calcium from MH-susceptible, when compared to normal sarcoplasmic reticulum. Our results demonstrate that while sarcoplasmic reticulum calcium-accumulation appears unaffected in MH, aspect(s) of the sarcoplasmic reticulum Ca2+-induced calcium release mechanism are altered. Although the role of the Ca2+-induced calcium release mechanism of sarcoplasmic reticulum in situ is not yet clear, our results suggest that an abnormality in the regulation of sarcoplasmic reticulum calcium release may play an important role in the MH syndrome.     

Caffeine interaction with the Ca-release channels of heavy sarcoplasmic reticulum. Evidence that 170 kD Ca-binding protein is a caffeine receptor of the Ca-channels

The study of Ca2+- and caffeine-induced Ca2+ release from heavy sarcoplasmic reticulum vesicles under the different conditions suggests that Ca2+ and caffeine can interact with the common receptor of the Ca-release channels. The reticulum membranes were solubilized using nonionic detergent polyoxyethylene 9-lauryl ether, and affinity chromatography on reactive red 120-agarose was carried out. The 170 kD Ca-binding protein which is eluted by caffeine is the most probable candidate for the caffeine receptor of the Ca-channels.     

Release of Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle induced by heparin. Relation between the Ca2+ release caused by Ca2+ ions and caffeine

Using a Ca2+-selective electrode and Quin 2 and chlortetracycline fluorescence, a Ca2+ release from terminal cysterns of skeletal muscle sarcoplasmic reticulum under effects of heparin, caffeine and Ca2+ has been studied. It was shown that Ca2+ release induced by heparin is insensitive to the blockers of Mg2+-dependent system of Ca2+-induced Ca2+ release, i.e., Mg2+, tetracaine and dimethylsulfoxide. Preliminary release of Ca2+ in the presence of caffeine, which activates Mg2+-dependent Ca2+ release, does not prevent the heparin-induced Ca2+ release. At the same time, after Ca2+ release caused by Ca2+ in a Mg2+-independent system, heparin cannot cause additional efflux of Ca2+. It has been shown that the heparin-induced release of Ca2+ diminishes with a decrease in a decrease in Ca2+ concentration. This effect is less pronounced in the presence of Na+ than with K+. The data obtained suggest that sarcoplasmic reticulum terminal cysterns contain two systems of Ca2+-induced release of Ca2+, i.e., a Mg2+-dependent, caffeine-sensitive and a Mg2+-independent heparin-sensitive ones. The mechanism of activation of both systems by caffeine and heparin consists, in all probability, in their increased affinity for Ca2+.     

Effect of caffeine on active Ca2+ ion transport in a homogenate of skeletal muscles and myocardium

A Ca2-selective electrode was used to study active transport of Ca2+ by sarcoplasmic reticulum fragments of rabbit skeletal muscle and myocardium homogenates. The specific Ca2+ transport activities (mumol Ca2+/min/mg tissue) are 40 = 60 and 3 = 5 units for fast and slow muscles and the myocardium, respectively. Caffeine (5 mM) exerts a powerful inhibitory influence on Ca2+ transport in skeletal muscle homogenates. For fast muscles, the degree of inhibition exceeds 50%. The rate of Ca2+ transport in the myocardium homogenate increases in the presence of creatine phosphate. The latter produces no effect on Ca2+ transport in skeletal muscle homogenates. The high sensitivity of Ca2 transport to caffeine, a specific blocker of Ca2+ transport to the terminal cisterns of the sarcoplasmic reticulum, suggests that the terminal cisterns, apart from being a reservoir for Ca2+ needed for contraction trigger, may play an essential role in muscle relaxation.     

Effect of temperature on [3H]ryanodine binding to sarcoplasmic reticulum from bullfrog skeletal muscle

It has been clarified that ryanodine binds to Ca2(+)-induced Ca release channels in the open state in sarcoplasmic reticulum. While the pharmacological action of ryanodine is known to be retarded at a low temperature, the Ca-releasing action of caffeine is potentiated at a low temperature. In order to obtain deeper insight into the molecular mechanism underlying Ca-release, the effect of temperature on ryanodine binding to the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle was examined. Although Ca2+ is indispensable for ryanodine binding, Ca2+ alone cannot cause ryanodine binding in a reaction medium of a salt concentration similar to that of the sarcoplasm. In addition to Ca2+, caffeine and/or beta,gamma-methylene adenosine triphosphate (AMPOPCP) are necessary. [3H]Ryanodine binding at 25 degrees C closely paralleled the Ca release activity in respect of the Ca2(+)-dependence in the presence of caffeine and/or AMPOPCP, and the effects of inhibitors. A Scatchard plot for ryanodine binding gave a straight linear line, indicating a single class of homogeneous binding sites. At 0 degrees C, the rate of ryanodine binding decreased. Q10 being about 3 on average. The affinity for ryanodine was reduced to about half that at 25 degrees C, with no change in the maximum number of binding sites. The temperature-dependent change in apparent affinity for Ca2+ on ryanodine binding is not always consistent with that in the case of Ca-release activity. The bound ryanodine may be in an occluded state because it did not dissociate for up to 90 h at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium releasing action of quercetin on sarcoplasmic reticulum from frog skeletal muscle

The release of Ca by quercetin from the sarcoplasmic reticulum has been claimed to be a result of the well-known inhibition of Ca2+-ATPase activity, or to be due to an intrinsic property of quercetin. To get a clearer understanding of the effect of quercetin, we examined it using fragmented sarcoplasmic reticulum (FSR) from bullfrog skeletal muscle. The rapid phase of Ca release (hereafter simply referred to as "Ca release") from loaded FSR was almost completed within 5 s after addition of quercetin in the presence of ATP. It cannot be ascribed to the inhibition of Ca2+-ATPase activity on the basis of following findings. First, when Ca uptake was driven by carbamylphosphate, no or little Ca release was observed in marked contrast to a stronger reduction in the rate of Ca uptake. Secondly, procaine reverses the Ca releasing action of quercetin, whereas it show a synergistic action in the inhibition of Ca2+-ATPase activity. Thirdly, HFSR released more Ca than LFSR, while the Ca2+-ATPase activities of both fractions were inhibited to a similar extent. The Ca release by quercetin is enhanced by ATP or beta, gamma-methylene adenosine triphosphate, and decreased by procaine or a high concentration of Mg2+. In the presence of 2.5 mM caffeine, the amount of Ca2+ released by quercetin was decreased, and the dose-effect relationship was shifted to higher doses of quercetin. This indicates that quercetin and caffeine probably overlap in the site(s) of the action, but that quercetin is dissimilar from halothane in the mode of its Ca-releasing action.     

The use of a model water-lipid system for the study of the electric function of Ca2+, Mg2+-ATPase of the sarcoplasmic reticulum

The method of dynamic capacity in the model organic phase-water system was used to investigate a possibility of studying the electrical function of Ca2+,Mg2+-ATPase from sarcoplasmic reticulum of the rabbit hind limb skeletal muscles. Decane and decane solution of azolectin were used as an organic phase. It is stated that in the model systems the sarcoplasmic reticulum Ca2+,Mg2+-ATPase did not cause ATP-dependent changes in the boundary Volta potential (delta phi) irrespective of the presence of polyvalent cation chelates in the organic phase. The fragmented sarcoplasmic reticulum is able of realizing Mg-ATP, Ca2+-dependent generation of delta phi only with phospholipids present in the organic phase. It is supposed that generation of delta phi of the fragmented sarcoplasmic reticulum is due to the active transport of calcium ions by the reticulum Ca2+,Mg2+-ATPase.     

Effects of rapid application of caffeine on intracellular calcium concentration in ferret papillary muscles

In this paper we investigate the effects of caffeine (5-20 mM) on ferret papillary muscle. The intracellular Ca2+ concentration ( [Ca2+]i) was measured from the light emitted by the photoprotein aequorin, which had previously been microinjected into superficial cells. Isometric tension was measured simultaneously. The rapid application of caffeine produced a transient increase of [Ca2+]i, which decayed spontaneously within 2-3 s and was accompanied by a transient contracture. The removal of extracellular Na+ or an increase in the concentration of intracellular Na+ (produced by strophanthidin) increased the magnitude of the caffeine response. Cessation of stimulation for several minutes or stimulation at low rates decreased the magnitude of the stimulated twitch and Ca2+ transient. These maneuvers also decreased the size of the caffeine response. These results are consistent with the hypothesis that the caffeine-releasable pool of Ca2+ (sarcoplasmic reticulum) is modulated by maneuvers that affect contraction. Ryanodine (10 microM) decreased the magnitude of the caffeine response as well as that of the stimulated twitch. In contrast, the rapid removal of external Ca2+ abolished the systolic Ca2+ transient within 5 s, but had no effect on the caffeine response. From this we conclude that the abolition of twitch by Ca2+-free solutions is not due to depletion of the sarcoplasmic reticulum of Ca2+, but may be due to a requirement of Ca2+ entry into the cell to trigger Ca2+ release from the sarcoplasmic reticulum.     

Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum.

The Ca2+ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca2+ and surface membrane-bound Ca2+ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca2+ in the absence of ATP, 10-12 h are necessary for measurable amount of Ca2+ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca2+ from 'loaded' vesicles only after this period of incubation. A fraction of Ca2+ of 50-60 nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg2+ and K+ in the medium and is readily released by ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca2+ (30-40 nmol/mg protein) is rapidly released X-537A. The results indicate that most of the Ca2+ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca2+ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.