Fast release of calcium from sarcoplasmic reticulum vesicles monitored by chlortetracycline fluorescence

Rapid Ca2+ release rate from sarcoplasmic reticulum vesicles was determined by the stopped flow method in terms of chlortetracycline fluorescence. Intensity of chlortetracycline fluorescence was proportional to the intravesicular free Ca2+ concentration. Ca2+ efflux was activated by extravesicular Ca2+ with an apparent dissociation constant of 25 microM and was inhibited with an inhibition constant of 120 microM in the absence of Mg2+. Caffeine enhanced the Ca2+ release rate by increasing only the affinity of Ca2+ for the activation site. Mg2+ reduced the Ca2+ release rate by competitive binding to the activation site. ATP increased the Ca2+ release rate very much without changing the affinities of Ca2+ for the activation and inhibition sites, i.e., ATP seems to increase the pore radius or number of the Ca2+ channels without affecting the gating mechanism of the channel. These results are consistent with those reported in skinned muscle sarcoplasmic reticulum. The maximum rate of Ca2+ release in the presence of ATP reached 80 s-1. This value is considered to be sufficient to cause muscular contraction.     

Sulfhydryl oxidation induces rapid calcium release from sarcoplasmic reticulum vesicles

Micromolar concentrations of cupric ion (Cu2+) and mercaptans such as cysteine, cysteamine, and homocysteine trigger large and rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles. At the concentrations used, Cu2+ alone does not induce Ca2+ release nor does cysteine alone; both are required to induce Ca2+ release from SR. Cu2+ is known to catalyze the autooxidation of cysteine to its disulfide form cystine; Cu2+/mercaptan-induced Ca2+ release appears to be caused by Cu2+-catalyzed formation of a mixed disulfide between the exogenous mercaptan and a critical sulfhydryl on a transmembrane protein. In the oxidized state the SR is highly permeable to Ca2+. Supporting evidence for this interpretation is as follows. The order of Ca2+-releasing reactivity of the mercaptans is the same as the order in which these compounds undergo oxidation to disulfide forms in the presence of Cu2+. Ca2+ efflux induced by cysteine and Cu2+ can be reversed by the addition of the disulfide reducing agent dithiothreitol. Hypochlorous acid and plumbagin, both potential sulfhydryl oxidants, induce rapid Ca2+ efflux from SR vesicles; in addition, Cu2+, which catalyzes H2O2 oxidation of cysteine, enhances H2O2-induced release. Oxidation-induced Ca2+ release from SR can be partially reversed or blocked by ruthenium red or the local anesthetics procaine and tetracaine. The Ca2+ efflux rates are strongly Mg2+ dependent and are significantly higher in heavy SR than in light SR. These data suggest that the Ca2+ efflux thus induced is via the "Ca2+ release channel" and that the oxidation state of a critical sulfhydryl group on this protein may be the principal means by which the Ca2+ permeability of the SR is regulated in vivo.     

The effects of sphingosine on sarcoplasmic reticulum membrane calcium release.

In this study, we report that sphingosine is a potent inhibitor of sarcoplasmic reticulum (SR) calcium release. Evidence is presented demonstrating a direct effect of sphingosine on the SR ryanodine receptor. Calcium release from "skinned" rabbit skeletal muscle fibers and isolated junctional SR derived from the terminal cisternae (TC) was measured in response to caffeine, doxorubicin, 5'-adenylyl-beta,gamma-imidodiphosphate or calcium. Sphingosine inhibited caffeine-induced release in a dose-dependent manner with an IC50 of 0.1 microM for the single muscle fibers and 0.5 microM for the isolated TC vesicles. Near complete blockage of TC calcium release rate was observed with 3 microM sphingosine. Neither sphingomyelin nor sphingosylphosphorylcholine had any effect at the 3 microM level, suggesting that the sphingosine effect was specific. Doxorubicin-induced calcium release and spontaneous calcium release were also blocked by sphingosine. Sphingosine was also capable of stimulating calcium transport in the isolated TC vesicles without an effect on Ca-ATPase activity. Ruthenium red was not capable of substantial additional stimulation of calcium transport nor inhibition of calcium release beyond the action of sphingosine. Sphingosine's blockage of calcium release was not reversed by the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2- methylpiperazine dihydrochloride, suggesting that the action of sphingosine on calcium release was not dependent on ryanodine receptor phosphorylation. Sphingosine significantly increased (8-fold) the Kd for specific [3H]ryanodine binding to TC membranes and decreased the Bmax with a dose dependence similar to the inhibition of calcium release, but sphingosine did not affect the pCa tension relationship of skinned skeletal muscle fibers. These data are consistent with a direct effect of submicromolar sphingosine on the ryanodine receptor. Substantially higher concentrations of sphingosine (30-50 microM) or sphingosylphosphorylcholine (10-20 microM) were capable of inducing calcium release by themselves. Preliminary data indicate that the transverse tubule and not the SR contain substantial sphingomyelinase activity consistent with a transverse tubule source of sphingosine production. Considering that sphingosine is found in micromolar concentrations in some cells, our data indicate that sphingosine generated by the transverse tubule membranes may be a physiologically relevant mechanism for modulating SR calcium release.     

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic sarcoplasmic reticulum vesicles

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic smooth muscle sarcoplasmic reticulum vesicles was examined using the calcium indicator antipyrylazo III. Calcium release was initiated by addition of inositol 1,4,5-trisphosphate (IP₃) to aortic vesicles 7 min after initiation of ATP-supported calcium uptake. Half-maximal calcium release occurred at 1 μM IP₃, with maximal calcium release amounting to 25±2% of the intravesicular calcium (n=12, 9 preparations). Ruthenium red (10–20 μM), which has been reported to block IP₃-induced calcium release from skeletal muscle sarcoplasmic reticulum, did not inhibit aortic IP₃-induced calcium release. Elevation of Mg²⁺ concentration from 0.06 to 7.8 mM inhibited aortic IP₃-induced calcium release 75%, which contrasts with the Mg²⁺-insensitive IP₃-induced calcium release from platelet reticular membranes. The IP₃-dependence of aortic calcium release suggested that Mg²⁺ acted as a noncompetitive inhibitor. Thus, aortic sarcoplasmic reticulum vesicles contain an IP₃-sensitive calcium pathway which is inhibited by millimolar concentrations of Mg²⁺, but which is not inhibited by Ruthenium red and so differs from the previously described IP₃-sensitive calcium pathways in skeletal muscle and platelet reticular membranes.     

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic sarcoplasmic reticulum vesicles

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic smooth muscle sarcoplasmic reticulum vesicles was examined using the calcium indicator antipyrylazo III. Calcium release was initiated by addition of inositol 1,4,5-trisphosphate (IP3) to aortic vesicles 7 min after initiation of ATP-supported calcium uptake. Half-maximal calcium release occurred at 1 microM IP3, with maximal calcium release amounting to 25 +/- 2% of the intravesicular calcium (n = 12, 9 preparations). Ruthenium red (10-20 microM), which has been reported to block IP3-induced calcium release from skeletal muscle sarcoplasmic reticulum, did not inhibit aortic IP3-induced calcium release. Elevation of Mg2+ concentration from 0.06 to 7.8 mM inhibited aortic IP3-induced calcium release 75%, which contrasts with the Mg2+-insensitive IP3-induced calcium release from platelet reticular membranes. The IP3-dependence of aortic calcium release suggested that Mg2+ acted as a noncompetitive inhibitor. Thus, aortic sarcoplasmic reticulum vesicles contain an IP3-sensitive calcium pathway which is inhibited by millimolar concentrations of Mg2+, but which is not inhibited by Ruthenium red and so differs from the previously described IP3-sensitive calcium pathways in skeletal muscle and platelet reticular membranes.     

Oxidation induced by phthalocyanine dyes causes rapid calcium release from sarcoplasmic reticulum vesicles

The copper containing phthalocyanine dyes, alcian blue, copper phthalocyanine tetrasulfonic acid, and Luxol fast blue MBSN are found to induce rapid calcium efflux from actively loaded sarcoplasmic reticulum (SR) vesicles. Alcian blue (5 microM), with 1 mM free Mg2+ triggered Ca2+ efflux at rates greater than 20 nmol/mg of SR/s. As in the case of Ca2+ efflux induced by calcium, heavy metals, or SH oxidation with Cu2+/cysteine, efflux induced by phthalocyanines is also stimulated by adenine containing nucleotides and inhibited by millimolar Mg2+ and submicromolar ruthenium red (RR). In addition, analogs of RR, such as hexamminecobalt(III) chloride or hexammineruthenium(III) chloride also inhibit Ca2+ efflux but are effective at somewhat higher concentrations (approximately 50 microM). Calcium release stimulated by phthalocyanines is specific for SR derived from the terminal cisternae region rather than longitudinal SR. Preincubation of alcian blue with the reducing agents, sodium dithionite, dithiothreitol, or cysteine causes complete loss of Ca2+ release activity from SR vesicles. Reoxidation of the alcian blue leads to return of the Ca2+ release activity of the phthalocyanine dye. The copper containing phthalocyanine dyes appear to cause rapid Ca2+ release from SR vesicles by oxidizing sulfhydryl groups associated with the calcium release channel. Moreover, phthalocyanines appear to act by oxidizing a pair of neighboring sulfhydryls to a disulfide because subsequent additions of the reducing agent dithiothreitol promote the closure of the Ca2+ channel and calcium re-uptake.     

A spin-label study of the effects of drugs on calcium release from isolated sarcoplasmic reticulum vesicles

The effects of caffeine, thymol, and procaine on calcium release from fragmented sarcoplasmic reticulum (FSR) from rabbit skeletal white muscle were investigated by the spin label method at the organellar level. Two thiol-directed spin labels, 4-maleimide-2,2,6,6-tetramethylpiperidinooxyl and 4-(2-iodoacetamide)-2,2,6,6-tetramethylpiperidinooxyl, were used for the labeling of SR proteins. The ratio (W/S) of the weakly (W) and strongly (S) immobilized ESR signals was measured for the maleimide and iodoacetamide labeled FSR. The two labels gave different W/S values, which means that the two labels report conformational changes at different loci of SR proteins. The dependences of the W/S ratios on the concentration of the drugs showed that conformational changes of SR proteins induced by these drugs are not the same. From measurements of the distribution of 5-doxyldecanoic acid methylester between the lipid and water phases, it was found that the conformational changes of the SR proteins caused by thymol or procaine induced a disorder in local regions of the phospholipid bilayers of FSR, while such disordering was not induced by caffeine. On the other hand, caffeine and thymol showed definite effects on calcium release from FSR, while procaine did not. These results indicate that the effects of the drugs on the protein conformations can be well characterized at the organellar level by means of the spin label technique and that some specific changes in the conformations of SR proteins are necessary for calcium release from FSR.     

Chloride-induced release of actively loaded calcium from light and heavy sarcoplasmic reticulum vesicles

Summary Light and heavy sarcoplasmic reticulum vesicles (LSR, HSR) isolated from rabbit leg muscle have been used in a study of chloride-induced Ca²⁺ release. The biochemical and morphological data indicate that LSR is derived from the longitudinal reticulum and HSR is derived from the terminal cisternae of the sarcoplasmic reticulum. LSR and HSR were both able to accumulate Ca²⁺ in the presence of ATP to amounts greater than 100 nmol Ca²⁺/mg of protein in less than 1 min. LSR and HSR each had a biphasic time course of Ca²⁺ uptake. The initial uptake was followed by a rapid release, after approximately 1 min, of 30–40% of the accumulated Ca²⁺, which was then followed by a slower phase of Ca²⁺ accumulation. Ca²⁺ taken up by the SR vesicles could be released from both the LSR and HSR by changing the anion outside the vesicles from methanesulfonate to chloride. Due to the difference in permeability between methanesulfonate and chloride, this change should result in a decreased positivity inside the vesicles with respect to the exterior. It could also result in osmotic swelling of the vesicles. Changing the ionic medium from chloride to methanesulfonate caused no release of Ca²⁺. The amount of accumulated Ca²⁺ released in 6 sec by changing the anion outside the vesicles from methanesulfonate to chloride was 30–35 nmol/mg membrane protein for LSR and HSR, respectively. Osmotic buffering with 200mm sucrose caused a slight inhibition of chloride-induced Ca²⁺ release from HSR (17%15%) but it greatly reduced the release of Ca²⁺ from LSR (32%15%). The specificity of Ca²⁺ release was measured using SR vesicles which were passively loaded with 10mm ²²Na⁺. LSR released five times more²²Na⁺ than HSR under same conditions as chloride-induced Ca²⁺ release occurred. Na dantrolene (20 m) had no effect on the release of Ca²⁺ from LSR but it inhibited the chloride-induced Ca²⁺ release from HSR by more than 50%. Na dantrolene also increased the Ca²⁺ uptake in the HSR by 20% while not affecting LSR Ca²⁺ uptake. Our results indicate the presence of a chloride-induced, Na dantrolene inhibited, Ca²⁺ release from HSR, which is not due to osmotic swelling.     

Calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles. General characteristics

Isolated canine cardiac sarcoplasmic reticulum exhibits Ca2+-induced Ca2+ release from both actively and passively loaded vesicles. The rate and extent of Ca2+ release depend on the extravesicular ionized Ca2+ concentration ( [Ca2+]o) at the onset of release. Maximal release following ATP-dependent, phosphate-facilitated Ca2+ loading (up to 360 nmol of Ca2+/mg of protein/min at 37 degrees C) occurs at 1.5-2 microM [Ca2+]o, with reduced release at both lower and higher Ca2+ concentrations (half-maximal Ca2+ release at approximately 0.8 and 5.5 microM [Ca2+]o). Only a portion of the accumulated Ca2+ is released and the release is followed by reuptake of Ca2+. A similar Ca2+ dependence is obtained in the absence of ATP and Pi by measuring unidirectional Ca2+ efflux from passively loaded vesicles (maximal Ca2+ efflux at 1 microM [Ca2+]o; half-maximal Ca2+-dependent efflux at approximately 0.15 and 13 microM [Ca2+]o). Although the Ca2+ release rates observed in this study are several orders of magnitude lower than the rate of Ca2+ release which occurs in muscle cells in vivo, this Ca2+ release phenomenon may be related to the Ca2+-induced Ca2+ release which has been described for skinned cardiac cells ( Fabiato , A. (1983) Am. J. Physiol. 245, C1-C14). Ca2+ release occurs in the presence of an ATP-regenerating system and is not accompanied by a reduction in ATP hydrolysis. Also, since unidirectional Ca2+ efflux (as high as 860 nmol of Ca2+/mg of protein/min at 37 degrees C) exceeds net Ca2+ release under similar conditions, Ca2+ influx proceeds during the period of net Ca2+ release. Therefore, Ca2+ release does not involve reversal or cessation of inward Ca2+ pumping. Other data indicate that Ca2+ release is not mediated through the Ca2+ pump protein, but occurs through a separate Ca2+-dependent efflux pathway, possibly a channel.     

Rate of calcium release and ATP synthesis in sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum vesicles can catalyze the synthesis of ATP coupled to the efflux of calcium. The rate of this reaction is much faster when the vesicles are loaded in a medium containing phosphate than when oxalate is the precipitating agent. Two components of ATP synthesis can be observed when vesicles loaded with calcium phosphate are used. In the millisecond range and when the loaded vesicles are phosphorylated by Pi, the addition of ADP leads to an initial burst of ATP synthesis and after 50 ms approximately 3.0 nmol of ATP/mg protein are synthesized. This burst is not inhibited by ATP and is enhanced by physiological concentrations of KCl. The slow component of ATP synthesis is inhibited by both ATP and 100 mM KCl. In the physiological pH range, betaine, a trimethylamine present in different tissues, increases the level of phosphoenzyme formed by Pi and enhances the amount of ATP synthesized during the first turn of the reversal of the calcium pump.     

Ca2+ uptake and membrane potential in sarcoplasmic reticulum vesicles

The rate of calcium uptake by sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was stimulated by inside-negative membrane potential generated by K+ gradients in the presence of valinomycin. The increase in the calcium transport rate was accompanied by a proportional increase in the rate of calcium-dependent ATP hydrolysis, without significant change in the steady state level of the phosphorylated enzyme intermediate. Changes in the sarcoplasmic reticulum membrane potential during calcium transport were monitored with the optical probe, 3,3'-diethylthiadicarbocyanine. The decrease in the absorbance of 3,3'-diethylthiadicarbocyanine at 660 nm following generation of inside-negative membrane potential was reversed during ATP-induced calcium uptake. These observations support an electrogenic mechanism for the transport of calcium by the sarcoplasmic reticulum.     

Pathways of calcium release from heavy sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle

The active uptake and efflux of Ca2+ from suspensions of vesicles from heavy rabbit muscle sarcoplasmic reticulum have been examined using the antipyrylazo III dye method in the presence of various nucleotide triphosphate substrates to support active Ca2+ accumulation. On addition of ATP, Ca2+ is rapidly accumulated and maintained at high internal concentrations until the substrate for pump protein is exhausted. Ca2+-induced Ca2+ release which is inhibited by ruthenium red can be demonstrated. The kinetics of Ca2+ release via these channels is different from the Ca2+ efflux observed after substrate exhaustion. This rate was found to be dependent on the type of nucleotide triphosphate, decreasing in the order ATP greater than GTP greater than CTP greater than ITP UTP. It is suggested that different conformations of the Ca2+ pump protein induced by the different substrates may result in the creation of pathways for the facilitated diffusion of Ca2+.     

Pharmacology of calcium release from sarcoplasmic reticulum

Calcium release from sarcoplasmic reticulum (SR) has been elicited in response to additions of many different agents. Activators of Ca²⁺ release are here tentatively classified as activators of a Ca²⁺-induced Ca²⁺ release channel preferentially localized in SR terminal or as likely activators of other Ca²⁺ efflux pathways. Some of these pathways may be associated with several different mechanisms for SR Ca²⁺ release that have been postulated previously. Studies of various inhibitors of excitation-contraction coupling and of certain forms of SR Ca²⁺ release are summarized. The sensitivity of isolated SR to certain agents is unusually affected by experimental conditions. These effects can seriously undermine attempts to anticipate effects of the same pharmacological agentsin situ. Finally, mention is made of a new preparation (sarcoballs) designed to make the pharmacological study of SR Ca²⁺ release more accessible to electrophysiologists, and some concluding speculations on the future of SR pharmacology are offered.     

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.     

Kinetic studies of calcium-induced calcium release in cardiac sarcoplasmic reticulum vesicles

Fast Ca(2+) release kinetics were measured in cardiac sarcoplasmic reticulum vesicles actively loaded with Ca(2+). Release was induced in solutions containing 1.2 mM free ATP and variable free [Ca(2+)] and [Mg(2+)]. Release rate constants (k) were 10-fold higher at pCa 6 than at pCa 5 whereas Ryanodine binding was highest at pCa < or =5. These results suggest that channels respond differently when exposed to sudden [Ca(2+)] changes than when exposed to Ca(2+) for longer periods. Vesicles with severalfold different luminal calcium contents exhibited double exponential release kinetics at pCa 6, suggesting that channels undergo time-dependent activity changes. Addition of Mg(2+) produced a marked inhibition of release kinetics at pCa 6 (K(0.5) = 63 microM) but not at pCa 5. Coexistence of calcium activation and inhibition sites with equally fast binding kinetics is proposed to explain this behavior. Thimerosal activated release kinetics at pCa 5 at all [Mg(2+)] tested and increased at pCa 6 the K(0.5) for Mg(2+) inhibition, from 63 microM to 136 microM. We discuss the possible relevance of these results, which suggest release through RyR2 channels is subject to fast regulation by Ca(2+) and Mg(2+) followed by time-dependent regulation, to the physiological mechanisms of cardiac channel opening and closing.     

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.     

Luminal calcium regulation of calcium release from sarcoplasmic reticulum

This article discusses how changes in luminal calcium concentration affect calcium release rates from triad-enriched sarcoplasmic reticulum vesicles, as well as single channel opening probability of the ryanodine receptor/calcium release channels incorporated in bilayers. The possible participation of calsequestrin, or of other luminal proteins of sarcoplasmic reticulum in this regulation is addressed. A comparison with the regulation by luminal calcium of calcium release mediated by the inositol 1,4,5-trisphosphate receptor/calcium channel is presented as well.     

Quinacrine inhibits the calcium-induced calcium release in heavy sarcoplasmic reticulum vesicles

Quinacrine is a fluorescence probe useful for studying the effect of local anesthetics. The interaction of quinacrine and sarcoplasmic reticulum membranes measured by fluorescence spectroscopy indicates the presence of a saturable binding site. Typical local anesthetics are able to displace quinacrine bound to heavy sarcoplasmic reticulum membranes. The effectiveness of that displacement decreases in the order dibucaine greater than tetracaine greater than benzocaine greater than lidocaine greater than procaine greater than procainamide, indicating that the size and hydrophobicity of quinacrine are major determinants in the binding process. The use of radioactive tracer and a rapid filtration technique reveals that quinacrine interacts, at lower concentrations, with sarcoplasmic reticulum membranes by blocking the Ca2+-induced Ca2+ release. Higher quinacrine concentrations also affect the Ca2+-pump activity.