Evidence for a calcium-sensitive factor which alters the alkaline pH sensitivity of sarcoplasmic reticulum calcium transport

Oxalase-supported, ATP-dependent Ca2+ uptake by cardiac and skeletal muscle sarcoplasmic reticulum (SR) exhibits a pH profile with the maximal rate of Ca2+ uptake at pH 6.6-6.8 and marked inhibition (90-95%) at pH 7.4-7.6, a point at which Ca2+-dependent ATPase activity is optimal. These observations are noted when the SR is first preincubated in media containing no added Ca2+. This alkaline pH inhibition is not caused by an irreversible perturbation since the Ca2+ uptake rate is fully restored by changing the alkaline pH preincubation medium to pH 6.8. When SR is preincubated with added Ca2+, Ca2+ uptake at alkaline pH (7.4-7.6) is only inhibited by 10-30%. Ca2+ uptake at pH 6.8 is the same regardless of preincubation conditions. A depressed oxalate permeability is not a factor in the observed alkaline pH inhibition of Ca2+ uptake. At alkaline pH, the relationship between the preincubation Ca2+ concentration and the rate of Ca2+ uptake is hyperbolic; the half-maximal free Ca2+ concentration for stabilization of Ca2+ uptake is 8-15 microM with a Vmax equal to the velocity at the optimal pH. The Hill coefficient is 1.0, implying a single class of Ca2+-requiring sites for stabilization at alkaline pH. In contrast to its effect on Ca2+ uptake, the presence of Ca2+ during preincubation does not alter the pH sensitivity of Ca2+-dependent ATPase activity. Thus, the presence of Ca2+ during preincubation may stabilize a state of the CaATPase, conducive to the coupling of net Ca2+ translocation to Ca2+-dependent ATPase activity, which is ordinarily opposed by alkaline pH. The data suggest a single class of Ca2+-requiring sites which favors this coupled state.     

Myocardial stunning is associated with impaired calcium uptake by sarcoplasmic reticulum

Myocardial stunning (temporary post-ischaemic contractile dysfunction) may be caused by oxidative stress and/or impaired myocyte calcium homeostasis. Regional myocardial stunning was induced in open-chest pigs (segment shortening reduced to 68.3 ± 4.7% of baseline) by repetitive brief circumflex coronary occlusion (I/R). Reduced glutathione was depleted in stunned myocardium (1.34 ± 0.06 vs. 1.77 ± 0.11 nmol/mg, p = 0.02 vs. remote myocardium) indicating regional oxidant stress, but no regional differences were observed in protein-bound 3-nitrotyrosine or S-nitrosothiol content. Repetitive I/R did not affect myocardial quantities of the sarcolemmal sodium-calcium exchanger, L-type channel, SR calcium ATPase and phospholamban, or the kinetics of ligand binding to L-type channels and SR calcium release channels. However, initial rates of oxalate-supported ⁴⁵Ca uptake by SR were impaired in stunned myocardium (41.3 ± 13.5 vs. 73.0 ± 15.6 nmol/min/mg protein, p = 0.03). The ability of SR calcium ATPase to sequester cytosolic calcium is impaired in stunned myocardium. This is a potential mechanism underlying contractile dysfunction.     

Effects of alkaline pH on sarcoplasmic reticulum Ca2+ release and Ca2+ uptake.

Alkalinization-induced Ca2+ release from isolated frog or rabbit sarcoplasmic reticulum vesicles appears to consist of two distinct components: 1) a direct activation of ruthenium red-sensitive Ca2+ release channels in terminal cisternae and 2) an increased ruthenium red-insensitive Ca2+ efflux through some other efflux pathway distributed throughout the sarcoplasmic reticulum. The first of these releases exhibits an alkalinization-induced inactivation process and does not depend on the ruthenium red-insensitive form of Ca2+ release as a triggering agent for secondary Ca(2+)-induced Ca2+ release. Both releases are inhibited when the extravesicular (i.e. cytoplasmic) free [Ca2+] is reduced. This may reflect an increased sensitivity of the Ca2+ release channels to Ca2+ at alkaline pH. The pH sensitivity of the ruthenium red-sensitive Ca2+ release channels could be of significance during excitation-contraction coupling. The ruthenium red-insensitive form of Ca2+ release is less likely to be physiologically relevant, but it probably has contributed greatly to reports of alkalinization-induced decreases in net sarcoplasmic reticulum Ca2+ uptake, particularly under conditions where oxalate supported Ca2+ uptake is much less affected, as here.     

Comparison between strontium and calcium uptake by the fragmented sarcoplasmic reticulum.

The ATP-supported uptake of strontium by the fragmented sarcoplasmic reticulum is monophasic and proceeds more rapidly than the fast uptake of calcium. Strontium uptake is not activated by Pi. The accumulation of strontium is nearly proportional to the external strontium concentration even in the millimolar range. Internal and external strontium quickly equilibrate. One mole of strontium is stored for every mole of ATP split by the Sr2+-activated ATPase. In the absence of oxalate most of the strontium is taken up with a transport ratio of one. On the opposite, the transport ratio of calcium decreases immediately, especially when ADP is not instantaneously phosphorylated to ATP. In this case, energy conversion is uncoupled more effectively by the simultaneous action of ADP and free internal calcium, resulting in the interruption of the fast uptake. After depletion of ATP most of the stored strontium is released and the remaining fraction appears to be not exchangeable. Strontium activates the slow uptake of calcium, but reduces the amplitude of the fast uptake. The calcium induced release of strontium, and vice versa, is partial and transient. The strontium activated ATPase does not transport calcium at low ionic calcium concentrations.     

Electron cytochemistry of calcium uptake in the fragmented sarcoplasmic reticulum

Summary Ultrastructural aspects and cytochemical localisation of Ca-uptake in the fragmented sarcoplasmic reticulum (FSR) have been studied with positive and various negative staining techniques.Effect of fixation, staining and other preparative procedures for electron microscopy on the result of the cytochemical reaction have been investigated.Calcium outflow from loaded vesicles occurs in various degree depending on the method used so that no quantitative estimation of Ca-uptake appears possible by ultrastructural studies alone.Ultrastructure and proportion of calcium containing vesicles also vary considerably and no certain correlations can be made between their size and shape and localisation of Ca-uptake. Certain informations can be obtained only by comparison of results with different methods, taking into account also the limits of each method applied.     

Effects of beta-bungarotoxin on calcium uptake by sarcoplasmic reticulum from rabbit skeletal muscle

A fluorescent chelate probe and a Millipore filtration technique have been used to study the effects of β-bungarotoxin (β-toxin) on passive and active Ca⁺⁺ uptake and ATPase in fragmented sarcoplasmic reticulum (SR) of rabbit skeletal muscle. β-Toxin at 3 × 10^?6 M did not affect ATPase activity. In the absence of ATP, β-Toxin increased the passive uptake of Ca⁺⁺; in the presence of ATP, active Ca⁺⁺ uptake was inhibited. The effect of β-toxin in SR can be detected at concentrations as low as 10^?9 M. The results suggest that β-toxin induces Ca⁺⁺ leakage in SR membranes.     

Observation of calcium uptake by isolated sarcoplasmic reticulum employing a fluorescent chelate probe

The fluorescent chelate probe technique is employed to observe the accumulation and binding of Ca⁺⁺ to isolated sarcoplasmic reticulum from skeletal and cardiac muscle. Chlorotetracycline serves as a fluorescent chelate probe which chelates to membrane bound Ca⁺⁺ giving rise to an intensely fluorescence adduct. An increase in fluorescence of chlorotetracycline is caused by ATP induced Ca⁺⁺ transport in both skeletal and cardiac muscle microsomes. The fluorescence spectra indicate that Ca⁺⁺ lies on the membrane surface in a relatively polar environment.     

Changes in luminal pH caused by calcium release in sarcoplasmic reticulum vesicles.

Fast (milliseconds) Ca2+ release from sarcoplasmic reticulum is an essential step in muscle contraction. To electrically compensate the charge deficit generated by calcium release, concomitant fluxes of other ions are required. In this study we investigated the possible participation of protons as counterions during calcium release. Triad-enriched sarcoplasmic reticulum vesicles, isolated from rabbit fast skeletal muscle, were passively loaded with 1 mM CaCl2 and release was induced at pCa = 5.0 and pH = 7.0 in a stopped-flow fluorimeter. Accompanying changes in vesicular lumen pH were measured with a trapped fluorescent pH indicator (pyranin). Significant acidification (approximately 0.2 pH units) of the lumen occurred within the same time scale (t(1/2) = 0.75 s) as calcium release. Enhancing calcium release with ATP or the ATP analog 5'-adenylylimidodiphosphate (AMPPNP) produced >20-fold faster acidification rates. In contrast, when calcium release induced with calcium with or without AMPPNP was blocked by Mg2+, no acidification of the lumen was observed. In all cases, rate constants of luminal acidification corresponded with reported values of calcium release rate constants. We conclude that proton fluxes account for part (5-10%) of the necessary charge compensation during calcium release. The possible relevance of these findings to the physiology of muscle cells is discussed.     

Alkalinization within sarcoplasmic reticulum during the uptake of calcium ions.

The pH indicator, bromothymol blue, was incorporated into sarcoplasmic reticulum vesicles which bind more than 90% of the total added dye. The sequestered dye does not respond to changes in external pH upon addition of acid to the medium, since the decrease of absorbance at 616 nm is very slow. The absorbance of sequestered dye at 616 nm increases suddenly after triggering the transport of Ca²⁺ by ATP at a rate much higher than that of Ca²⁺ uptake, and declines when Ca²⁺ has been accumulated. When the uptake of Ca²⁺ is followed in the presence of oxalate, the absorbance of the indicator declines after the first phase of Ca²⁺ uptake. The results suggest that a transient alkalinization occurs rapidly inside the vesicles and reflects the formation of a transmembrane proton gradient responsible for sustaining the Ca²⁺ transport.     

Time-dependent changes of calcium influx and efflux rates in rabbit skeletal muscle sarcoplasmic reticulum

Unfractionated and low buoyant density sarcoplasmic reticulum vesicles released calcium spontaneously after ATP- or acetyl phosphate-supported calcium uptake when internal Ca²⁺ was stabilized by the use of 50 mM phosphate as calcium-precipitating anion. This spontaneous calcium release could not be attributed to falling Ca²⁺ concentration outside the vesicles (Ca₀²⁺), substrate depletion, ADP accumulation, nonspecific membrane deterioration or the attainment of a high vesicular calcium content. Instead, spontaneous calcium release was directly proportional to Ca₀²⁺ at the time that calcium content was maximal. A causal relationship between high Ca₀²⁺ and spontaneous calcium release was suggested by the finding that elevation of Ca₀²⁺ from less than 1 μM to 3–5 μM increased the rate and extent of calcium release.The spontaneous calcium release was due both to acceleration of calcium efflux and slowing of calcium influx that was not accompanied by a significant change in the rate of ATP hydrolysis. Neither reversal of the transmembrane KCl gradient nor incubation with cation and proton ionophores abolished the spontaneous calcium release. The persistence of calcium release under conditions where the membrane was permeable to both anions and cations makes it unlikely that this phenomenon is due to a changing transmembrane potential.     

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.     

Calmodulin participation in oxygen radical-induced cardiac sarcoplasmic reticulum calcium uptake reduction

The effect of scavengers of oxygen radicals on canine cardiac sarcoplasmic reticulum (SR) Ca2+ uptake velocity was investigated at pH 6.4, the intracellular pH of the ischemic myocardium. With the generation of oxygen radicals from a xanthine-xanthine oxidase reaction, there was a significant depression of SR Ca2+ uptake velocity. Xanthine alone or xanthine plus denatured xanthine oxidase had no effect on this system. Superoxide dismutase (SOD), a scavenger of .O2-, or denatured SOD had no effect on the depression of Ca2+ uptake velocity induced by the xanthine-xanthine oxidase reaction. However, catalase, which can impair hydroxyl radical (.OH) formation by destroying the precursor H2O2, significantly inhibited the effect of the xanthine-xanthine oxidase reaction. This effect of catalase was enhanced by SOD, but not by denatured SOD. Dimethyl sulfoxide (Me2SO), a known .OH scavenger, completely inhibited the effect of the xanthine-xanthine oxidase reaction. The observed effect of oxygen radicals and radical scavengers was not seen in the calmodulin-depleted SR vesicles. Addition of exogenous calmodulin, however, reproduced the effect of oxygen radicals and the scavengers. The effect of oxygen radicals was enhanced by the calmodulin antagonists (compounds 48/80 and W-7) at concentrations which showed no effect alone on Ca2+ uptake velocity. Taken together, these findings strongly suggest that .OH, but not .O2-, is involved in a mechanism that may cause SR dysfunction, and that the effect of oxygen radicals is calmodulin dependent.     

Impaired calcium uptake by cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock

Effects of endotoxin administration on the ATP-dependent Ca²⁺ uptake by canine cardiac sarcoplasmic reticulum (SR) were investigated. Results obtained 4 h after endotoxin administration show that ATP-dependent Ca²⁺ uptake by cardiac SR was decreased by 27–43% (p < 0.05). Kinetic analysis indicates that the Vmax values for Ca²⁺ and for ATP were significantly decreased while the S_0.5 and the Hill coefficient values were not affected during endotoxin shock. Magnesium (1–5 mM) stimulated while vanadate (25–50 M) inhibited the ATP-dependent Ca²⁺ uptake, but the Mg²⁺-stimulated and the vanadate-inhibited activities remained significantly lower in the endotoxin-treated animals. Phosphorylation of SR by the exogenously added catalytic subunit of the cAMP-dependent protein kinase or by the addition of calmodulin stimulated the ATP-dependent Ca²⁺ uptake activities both in the control and endotoxin-injected dogs. However, the phosphorylation-stimulated activities remained significantly lower in the endotoxin-injected dogs. Dephosphorylation of SR decreased the ATP-dependent Ca²⁺ uptake, but the half-time required for the maximal dephosphorylation was reduced by 31% (p < 0.05) 4 h post-endotoxin. These data indicate that endotoxin administration impairs the ATP-dependent Ca²⁺ uptake in canine cardiac SR and the endotoxininduced impairment in the SR calcium transport is associated with a mechanism involving a defective phosphorylation and an accelerated dephosphorylation of SR membrane protein. Since ATP-dependent Ca²⁺ uptake by cardiac SR plays an important role in the regulation of the homeostatic levels of the contractile calcium, our findings may provide a biochemical explanation for myocardial dysfunction that occurs during endotoxin shock.     

Oxalate, calcium uptake and ATPase activity of sarcoplasmic reticulum vesicles.

Ca++-uptake and Mg++-Ca++-dependent ATPase activity of skeletal muscle sarcoplasmic reticulum vesicles were reciprocally affected by increasing the oxalate concentration from 0 to 4 mM. At 0-0.1 mM oxalate approximately 17% of the calcium was removed by the vesicles from the medium while the ATPase activity was maximal (approximately 0.66 mumoles Pi mg-1 protein min-1). Between 0.1 to 0.2 mM oxalate the ATPase activity was reduced to one-fifth but the uptake rose sharply and 100% of the 45Ca++ was removed from the medium. The uptake was maintained at this level at oxalate concentrations greater than 0.4 mM but the ATPase activity remained inhibited. The kinetics of Ca++-uptake and ATPase activity were also differentially affected by oxalate. In the presence of oxalate, ruthenium red had only a very slight inhibitory effect on the calcium uptake. Addition of 0.1 mM EGTA removed 80% of the Ca++ from preloaded vesicles within 10 min. The formation of insoluble Ca-oxalate salt on the surface of the vesicle is suggested by these results. Calculations based on the Ksp of the calcium oxalate salt are presented to show its formation and the possible speciation of a Ca-oxalate complex which may affect the Ca++-uptake and ATPase activity.     

Possible effects of membrane polarization on calcium uptake by and release from sarcoplasmic reticulum vesicles

Rates of calcium uptake by and calcium release from sarcoplasmic reticulum vesicles isolated from skeletal muscle of the crab seem to depend on membrane potential generated by potassium (K) and chloride (Cl) gradients. This does not appear to be due to an effect of the ions themselves since media of different ionic compositions leading to the same membrane potential, also lead to the same ATP hydrolysis and the same Ca uptake by SR vesicles. From a large positive intravesicular potential (conditions termed "normal" in this paper), membrane depolarization of passively Ca loaded vesicles, produced by changes in K and Cl concentrations in the media, resulted in: i) decrease in rate of calcium uptake; ii) decrease in calcium loading; iii) increase in rate of calcium release despite a decrease in the driving force for calcium ions. Moreover, the addition of caffeine (5 mmol/l) to the different polarization media resulted in a increase in calcium release.     

3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity

3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA.