Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: Ca2+-dependent passive Ca2+ efflux

Characterization of the putative Ca2+-gated Ca2+ channel of sarcoplasmic reticulum, which is thought to mediate Ca2+-induced Ca2+ release, was carried out in order to elucidate the mechanism of Ca2+-induced Ca2+ release. Heavy and light fractions of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle were loaded passively with Ca2+, and then passive Ca2+ efflux was measured under various conditions. The fast phase of the Ca2+ efflux depended on the extravesicular free Ca2+ concentration and was assigned to the Ca2+ efflux through the Ca2+-gated Ca2+ channel. Vesicles with the Ca2+-gated Ca2+ channels comprised about 85% of the heavy fraction and about 40% of the light fraction. The amount of Ca2+ loaded in FSR was found to be much larger than that estimated on the basis of vesicle inner volume and the equilibration of intravesicular with extravesicular Ca2+, indicating Ca2+ binding inside FSR. Taking this fact into account, the Ca2+ efflux curve was quantitatively analyzed and the dependence of the Ca2+ efflux rate constant on the extravesicular free Ca2+ concentration was determined. The Ca2+ efflux was maximal, with the rate constant of 0.75 s-1, when the extravesicular free Ca2+ was at 3 microM. Caffeine increased the affinity for Ca2+ of Ca2+-binding sites for opening the channel with only a slight change in the maximum rate of Ca2+ efflux. Mg2+ inhibited the Ca2+ binding to the sites for opening the channel while procaine seemed to inhibit the Ca2+ efflux by blocking the ionophore moiety of the channel.     

The effect of phenothiazines on Ca2+ fluxes in skeletal muscle sarcoplasmic reticulum

The effect of phenothiazines (trifluoperazine, chlorpromazine, methochlorpromazine, and imipramine) on Ca2+ fluxes in light and heavy sarcoplasmic reticulum (SR) isolated from rabbit fast-twitch skeletal muscle was investigated. These drugs inhibited Ca2+ loading and (Ca2+,Mg2+)-ATPase activity, but had no effect on unidirectional Ca2+ efflux from vesicles loaded either actively or passively with Ca2+. Chlorpromazine, which is membrane permeable, and its quaternary analog, methochlorpromazine, which is membrane impermeable, gave identical results. It is concluded that (a) the enhancement of net Ca2+ release by phenothiazines is due to inhibition of Ca2+ influx mediated by the Ca2+ pump rather than to the opening of a Ca2+ channel; and (b) phenothiazines act at the outer (myoplasmic) face of the SR membrane.     

Silver ions trigger Ca2+ release by acting at the apparent physiological release site in sarcoplasmic reticulum

Rapid Ca2+ release from Ca2+ -loaded sarcoplasmic reticulum vesicles (SR) was previously shown to occur upon the addition of micromolar concentrations of heavy metals, and the extent of Ca2+ release was dependent on the binding affinity of the metal to sulfhydryl group(s) on an SR protein (Abramson, J.J., Weden, L., Trimm, J.L., and Salama, G. (1982) Biophys. J. 37, 134a; Abramson, J.J., Trimm, J.L., Weden, L., and Salama, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1526). The nature of this Ca2+ release site was examined further and found to be predominantly distributed in heavy SR (HSR) rather than light SR fractions. Ag+ -induced Ca2+ release from heavy SR was blocked by local anesthetics and ruthenium red which are known to inhibit Ca2+ release in skeletal fibers and in heavy SR, respectively. The rate of Ca2+ efflux from SR triggered by Ag+ was dependent on pH, Mg2+, and ionic strength of the medium. Efflux rates increased by a factor of 4 from pH 6.0 to 7.0 and then decreased in more alkaline reaction mixtures. Efflux rates from actively or passively loaded SR increased by a factor of 2.5 with increasing Mg2+ from 0 to 1 mM and then decreased in the range of 1 to 10 mM Mg2+. ATP-dependent Ca2+ uptake by SR was similar in 100 mM KCl and in 200 mM sucrose solutions, but the extent and rate of Ca2+ efflux induced by Ag+ were dramatically reduced with decreasing ionic strength of the medium. In solutions containing 5 mM Mg2+, the rate of Ca2+ efflux from heavy SR averaged over the first 1.5 s after the addition of Ag+ was 58 nmol of Ca2+/mg of SR/s, a value comparable to the fast initial rate of ATP-dependent Ca2+ uptake. The maximum initial rate of Ag+ -induced Ca2+ efflux from heavy SR in 1 mM Mg2+ may be comparable to the rate of Ca2+ release and tension development in muscle fibers. Our data indicate that Ag+ reacts with a protein or proteins in the SR, probably not the (Ca2+, Mg2+)-ATPase, to induce a rapid release of Ca2+, possibly from the physiological Ca2+ release site.     

Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides

A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.     

Rapid filtration studies of Ca2+-induced Ca2+ release from skeletal sarcoplasmic reticulum. Role of monovalent ions

We have developed a rapid filtration technique for the measurement of Ca2+ release from isolated sarcoplasmic reticulum vesicles. Using this technique, we have studied the Ca2+-induced Ca2+ release of sarcoplasmic reticulum vesicles from rabbit skeletal muscle passively loaded with 5 mM Ca2+. The effect of known effectors (adenine nucleotides and caffeine) and inhibitors (Mg2+ and ruthenium red) of this release were investigated. In a medium composed of 100 mM KCl buffered at pH 6.8 with 20 mM K/3-(N-morpholino)propanesulfonic acid the Ca2+ release rate was maximal (500 nmol of Ca2+ released.(mg of protein)-1.s-1) at 1 micron external Ca2+ and 5 mM ATP. We also observed a rapid Ca2+ release induced by micromolar Ag+ in the presence of ATP (at 1 nM Ca2+). The Ag+-induced Ca2+ release was totally inhibited by 5 micron ruthenium red. We have also investigated the effect of monovalent ions on the Ca2+ release elicited by Ca2+ or Ag+. We show that the Ca2+ release rate: 1) was dependent upon the presence of K+ or Na+ in the release medium and 2) was influenced by a K+ gradient created across the sarcoplasmic reticulum membrane. These results directly support the idea of the involvement of an influx of K+ (through K+ channels) during the Ca2+ release and allow to reconsider a possible influence of the membrane potential of the sarcoplasmic reticulum on the Ca2+ release.     

Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs.

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.     

A possible role of protein phosphorylation in the inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum

The Ca2+-induced Ca2+ release channel in the heavy fraction of the sarcoplasmic reticulum (SR) from rabbit skeletal muscle is inactivated during ATP-dependent Ca2+ uptake (Morii, H., Takisawa, H., & Yamamoto, T. (1985) J. Biol. Chem. 260, 11536-11541). AMP, one of the adenine nucleotides which activate the Ca2+ release, delayed the onset of the channel inactivation when added early during the course of the Ca2+ uptake. However, AMP could no longer activate the channel but accelerated the inactivation when added during the later phase of the Ca2+ uptake. In SR passively loaded with Ca2+, the Ca2+ channel which had been activated by AMP and Ca2+ was not spontaneously inactivated. Similarly, during GTP-dependent Ca2+ uptake, the channel activated by AMP was not inactivated. In addition acid phosphatase markedly delayed the onset of the inactivation during ATP-dependent Ca2+ uptake, without affecting Ca2+ ATPase activity or GTP-dependent Ca2+ uptake by heavy SR. The effect of the phosphatase was completely blocked by ruthenium red, a potent inhibitor of the channel. These results suggest that the channel is inactivated through an ATP-dependent process, presumably phosphorylation of proteins in the SR membrane. This was supported by the findings that the reactivation of the inactivated channel by added Ca2+ was markedly accelerated by the addition of acid phosphatase and that several proteins of heavy SR were phosphorylated during ATP-dependent Ca2+ uptake.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

A study of passive Ca2+ flow through the sarcoplasmic reticulum of skeletal muscles. II. Stimulation of passive Ca2+ influx with monovalent cations and anions

The initial rate of passive Ca2+ influx into "heavy" and "light" fractions of sarcoplasmic reticulum (SR) vesicles increases in the presence of univalent cation chlorides. Stimulation of passive Ca2+ influx decreases in the following order: KCl + valinomycin-KSCN- + valinomycin greater than KSI = NaCl greater than choline chloride. K-gluconate + valinomycin and K-gluconate have no effect on the passive Ca2+ influx into SR vesicles. It is supposed that KCl-stimulation of passive Ca2+ influx into SR vesicles under conditions used may be caused by depolarization of the SR membrane.     

A model for the uptake and release of Ca2+ by sarcoplasmic reticulum.

On addition of ATP to vesicles derived from the sarcoplasmic reticulum (SR) of skeletal muscle, Ca2+ is accumulated from the external medium. Following uptake, spontaneous release of Ca2+ occurs in the presence or in the absence of ATP. These processes of Ca2+ uptake and release were simulated by using the models derived for ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227; Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730] and for Ca2+ release from passively loaded vesicles [McWhirter, Gould, East & Lee (1987) Biochem. J. 245, 713-722]. The simulations are consistent with measurements of the effects of pH, K+, Ca2+ and Mg2+ on uptake and release of Ca2+. The increase in maximal Ca2+ accumulation observed in the presence of maleate is explained in terms of complexing of Ca2+ and maleate within the SR. The calculated concentration of ADP generated by hydrolysis of ATP has a large effect on the simulations. The effects of an ATP-regenerating system on the measured Ca2+ uptake is explained in terms of both removal of ADP and precipitation of Ca3(PO4)2 within the vesicles. It is concluded that both the process of Ca2+ uptake and the process of Ca2+ release seen with SR vesicles can be interpreted quantitatively in terms solely of the properties of the Ca2+ + Mg2+-activated ATPase.     

Adenine nucleotide stimulation of Ca2+-induced Ca2+ release in sarcoplasmic reticulum

Rabbit skeletal muscle sarcoplasmic reticulum was fractionated into a "Ca2+-release" and "control" fraction by differential and sucrose gradient centrifugation. External Ca2+ (2-20 microM) caused the release of 40 nmol of 45Ca2+/mg of protein/s from Ca2+-release vesicles passively loaded at pH 6.8 with an internal half-saturation Ca2+ concentration of 10-20 mM. Ca2+-induced Ca2+ release had an approximate pK value of 6.6 and was half-maximally inhibited at an external Ca2+ concentration of 2 X 10(-4) M and Mg2+ concentration of 7 X 10(-5) M. 45Ca2+ efflux from control vesicles was slightly inhibited at external Ca2+ concentrations that stimulated the rapid release of Ca2+ from Ca2+-release vesicles. Adenine, adenosine, and derived nucleotides caused stimulation of Ca2+-induced Ca2+ release in media containing a "physiological" free Mg2+ concentration of 0.6 mM. At a concentration of 1 mM, the order of effectiveness was AMP-PCP greater than cAMP approximately AMP approximately ADP greater than adenine greater than adenosine. Other nucleoside triphosphates and caffeine were minimally effective in increasing 45Ca2+ efflux from passively loaded Ca2+-release vesicles. La3+, ruthenium red, and procaine inhibited Ca2+-induced Ca2+ release. Ca2+ flux studies with actively loaded vesicles also indicated that a subpopulation of sarcoplasmic reticulum vesicles contains a Ca2+ permeation system that is activated by adenine nucleotides.     

Mechanism of chloride-dependent release of Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle.

We investigated the effect of Cl- on the Ca2+ permeability of rabbit skeletal muscle junctional sarcoplasmic reticulum (SR) using 45Ca2+ fluxes and single channel recordings. In 45Ca2+ efflux experiments, the lumen of the SR was passively loaded with solutions of 150 mM univalent salt containing 5 mM 45Ca2+. Release of 45Ca2+ was measured by rapid filtration in the presence of extravesicular 0.4-0.8 microM free Ca2+ and 150 mM of the same univalent salt loaded into the SR lumen. The rate of release was 5-10 times higher when the univalent salt equilibrated across the SR-contained Cl- (Tris-Cl, choline-Cl, KCl) instead of an organic anion or other halides (gluconate-, methanesulfonate-, acetate-, HEPES-, Br-, I-). Cations (K+, Tris+) could be interchanged without a significant effect on the release rate. To determine whether Cl- stimulated ryanodine receptors, we measured the stimulation of release by ATP (5 mM total) and caffeine (20 mM total) and the inhibition by Mg2+ (0.8 mM estimated free) in Cl(-)-free and Cl(-)-containing solutions. The effects of ATP, caffeine, and Mg2+ were the largest in K-gluconate and Tris-gluconate, intermediate in KCl, and notably poor or absent in choline-Cl and Tris-Cl. Procaine (10 mM) inhibited the caffeine-stimulated release measured in K-gluconate, whereas the Cl- channel blocker clofibric acid (10 mM) but not procaine inhibited the caffeine-insensitive release measured in choline-Cl. Ruthenium red (20 microM) inhibited release in all solutions. In SR fused to planar bilayers we identified a nonselective Cl- channel (PCl: PTris: PCa = 1:0.5:0.3) blocked by ruthenium red and clofibric acid but not by procaine. These conductive and pharmacological properties suggested the channel was likely to mediate Cl(-)-dependent SR Ca2+ release. The absence of a contribution of ryanodine receptors to the Cl(-)-dependent release were indicated by the lack of an effect of Cl- on the open probability of this channel, a complete block by procaine, and a stimulation rather than inhibition by clofibric acid. A plug model of Cl(-)-dependent release, whereby Cl- removed the inhibition of the nonselective channel by large anions, was formulated under the assumption that nonselective channels and ryanodine receptor channels operated separately from each other in the terminal cisternae. The remarkably large contribution of Cl- to the SR Ca2+ permeability suggested that nonselective Cl- channels may control the Ca2+ permeability of the SR in the resting muscle cell.     

Reactive disulfide compounds induce Ca2+ release from cardiac sarcoplasmic reticulum

Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to a disulfide bond, 2,2'dithiodipyridine (2,2' DTDP) and 4,4' dithiodipyridine (4,4' DTDP), induce Ca2+ release from isolated canine cardiac sarcoplasmic reticulum (SR) vesicles. RDSs are absolutely specific to free sulfhydryl (SH) groups and oxidize SH sites of low pKa via a thiol-disulfide exchange reaction, with the stoichiometric production of thiopyridone in the medium. As in skeletal SR, this reaction caused large increases in the Ca2+ permeability of cardiac SR and the number of SH sites oxidized by RDSs was kinetically and quantitatively measured through the absorption of thiopyridone. RDS-induced Ca2+ release from cardiac SR was characterized and compared to the action of RDSs on skeletal SR and to Ca2(+)-induced Ca2+ release. (i) RDS-induced Ca2+ release from cardiac SR was dependent on ionized Mg2+, with maximum rates of release occurring at 0.5 and 1 mM Mg2+free for 2,2' DTDP and 4,4' DTDP, respectively. (ii) In the presence of adenine nucleotides (0.1-1 mM), the oxidation of SH sites in cardiac SR by exogenously added RDS was inhibited, which, in turn, inhibited Ca2+ release induced by RDSs. (iii) Conversely, when the oxidation reaction between RDSs and cardiac SR was completed and Ca2+ release pathways were opened, subsequent additions of adenine nucleotides stimulated Ca2+ efflux induced by RDSs. (iv) Sulfhydryl reducing agents (e.g., dithiothreitol, DTT, 1-5 mM) inhibited RDS-induced Ca2+ efflux in a concentration-dependent manner. (v) RDSs elicited Ca2+ efflux from passively loaded cardiac SR vesicles (i.e., with nonfunctional Ca2+ pumps in the absence of Mg-ATP) and stimulated Ca2(+)-dependent ATPase activity, which indicated that RDS uncoupled Ca2+ uptake and did not act at the Ca2+, Mg2(+)-ATPase. These results indicate that RDSs selectively oxidize critical sulfhydryl site(s) on or adjacent to a Ca2+ release channel protein channel and thereby trigger Ca2+ release. Conversely, reduction of these sites reverses the effects of RDSs by closing Ca2+ release channels, which results in active Ca2+ reuptake by Ca2+, Mg2(+)-ATPase. These compounds can thus provide a method to covalently label and identify the protein involved in Ca2+ release from cardiac SR.     

Reduction in the Ca2(+)-induced Ca2+ release from canine cardiac sarcoplasmic reticulum following endotoxin administration

Effects of endotoxin administration on the Ca2(+)-induced Ca2+ release from canine cardiac sarcoplasmic reticulum (SR) were studied. Results show that the Ca2(+)-induced Ca2+ release from either passively or actively loaded SR vesicles was decreased by 28 to 46% (p less than 0.05) 4 h after endotoxin administration. Kinetic analysis reveals that the Vmax for Ca2+ was decreased significantly without changing the S0.5 and the Hill coefficient values. The binding of [3H]ryanodine to cardiac SR was reduced by 25.3% (p less than 0.01) following endotoxin administration. These data demonstrate that the Ca2(+)-induced Ca2+ release via the ryanodine-sensitive Ca2+ channel in canine cardiac SR was reduced during endotoxin shock. A reduction in the SR Ca2(+)-induced Ca2+ release may have a pathophysiological significance in contributing to the development of myocardial depression during endotoxin shock.     

Fast release of 45Ca2+ induced by inositol 1,4,5-trisphosphate and Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle: evidence for two types of Ca2+ release channels.

The kinetics of Ca2+ release induced by the second messenger D-myoinositol 1,4,5 trisphosphate (IP3), by the hydrolysis-resistant analogue D-myoinositol 1,4,5 trisphosphorothioate (IPS3), and by micromolar Ca2+ were resolved on a millisecond time scale in the junctional sarcoplasmic reticulum (SR) of rabbit skeletal muscle. The total Ca2+ mobilized by IP3 and IPS3 varied with concentration and with time of exposure. Approximately 5% of the 45Ca2+ passively loaded into the SR was released by 2 microM IPS3 in 150 ms, 10% was released by 10 microM IPS3 in 100 ms, and 20% was released by 50 microM IPS3 in 20 ms. Released 45Ca2+ reached a limiting value of approximately 30% of the original load at a concentration of 10 microM IP3 or 25-50 microM IPS3. Ca(2+)-induced Ca2+ release (CICR) was studied by elevating the extravesicular Ca2+ while maintaining a constant 5-mM intravesicular 45Ca2+. An increase in extravesicular Ca2+ from 7 nM to 10 microM resulted in a release of 55 +/- 7% of the passively loaded 45Ca2+ in 150 ms. CICR was blocked by 5 mM Mg2+ or by 10 microM ruthenium red, but was not blocked by heparin at concentrations as high as 2.5 mg/ml. In contrast, the release produced by IPS3 was not affected by Mg2+ or ruthenium red but was totally inhibited by heparin at concentrations of 2.5 mg/ml or lower. The release produced by 10 microM Ca2+ plus 25 microM IPS3 was similar to that produced by 10 microM Ca2+ alone and suggested that IP3-sensitive channels were present in SR vesicles also containing ruthenium red-sensitive Ca2+ release channels. The junctional SR of rabbit skeletal muscle may thus have two types of intracellular Ca2+ releasing channels displaying fast activation kinetics, namely, IP3-sensitive and Ca(2+)-sensitive channels.     

The heavy metal ions Ag+ and Hg2+ trigger calcium release from cardiac sarcoplasmic reticulum

Heavy metal ions have been shown to induce Ca2+ release from skeletal sarcoplasmic reticulum (SR) by binding to free sulfhydryl groups on a Ca2+ channel protein and are now examined in cardiac SR. Ag+ and Hg2+ (at 10-25 microM) induced Ca2+ release from isolated canine cardiac SR vesicles whereas Ni2+, Cd2+, and Cu2+ had no effect at up to 200 microM. Ag(+)-induced Ca2+ release was measured in the presence of modulators of SR Ca2+ release was compared to Ca2(+)-induced Ca2+ release and was found to have the following characteristics. (i) Ag(+)-induced Ca2+ release was dependent on free [Mg2+], such that rates of efflux from actively loaded SR vesicles increased by 40% in 0.2 to 1.0 mM Mg2+ and decreased by 50% from 1.0 to 10.0 mM Mg2+. (ii) Ruthenium red (2-20 microM) and tetracaine (0.2-1.0 mM), known inhibitors of SR Ca2+ release, inhibited Ag(+)-induced Ca2+ release. (iii) Adenine nucleotides such as cAMP (0.25-2.0 mM) enhanced Ca2(+)-induced Ca2+ release, and stimulated Ag(+)-induced Ca2+ release. (iv) Low Ag+ to SR protein ratios (5-50 nmol Ag+/mg protein) stimulated Ca2(+)-dependent ATPase activity in Triton X-100-uncoupled SR vesicles. (v) At higher ratios of Ag+ to SR proteins (50-250 nmol Ag+/mg protein), the rate of Ca2+ efflux declined and Ca2(+)-dependent ATPase activity decreased gradually, up to a maximum of 50% inhibition. (vi) Ag+ stimulated Ca2+ efflux from passively loaded SR vesicles (i.e., in the absence of ATP and functional Ca2+ pumps), indicating a site of action distinct from the SR Ca2+ pump. Thus, at low Ag+ to SR protein ratios, Ag+ is very selective for the Ca2+ release channel. At higher ratios, this selectivity declines as Ag+ also inhibits the activity of Ca2+,Mg2(+)-ATPase pumps. Ag+ most likely binds to one or more sulfhydryl sites "on" or "adjacent" to the physiological Ca2+ release channel in cardiac SR to induce Ca2+ release.     

Ca2+ movements in sarcoplasmic reticulum of rat soleus fibers after hindlimb suspension.

The functional capacity of skeletal muscle sarcoplasmic reticulum (SR) was examined in the slow soleus of rats submitted to 15 days of disuse produced by hindlimb suspension (HS). By using caffeine-induced contractions of single skinned fibers, Ca2+ uptake, Ca2+ release, and passive Ca2+ leakage through the SR membrane were investigated. In the SR of atrophied muscles, the amounts of Ca2+ uptake and Ca2+ release were significantly higher than in the control muscles and were close to those found for a fast muscle, the plantaris. Moreover, the study of the Ca2+ leakage showed that the time required to empty the SR previously loaded with Ca2+ was reduced by a factor of two after HS. Such disturbances of the Ca2+ movements in the SR suggested that alterations of the SR membrane occurred after HS. The results supported the idea that after hindlimb unweighting the slow soleus muscle acquired SR properties that were very much like those of a faster muscle.     

Involvement of the 60 kDa phosphoprotein in the regulation of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles

Junctional sarcoplasmic reticulum (SR) vesicles isolated from back muscles of normal and malignant hyperthermia susceptible (MHS) pigs were phosphorylated by addition of MgATP in the presence of 5 mM Ca2+ and 1 microM calmodulin (CaM). The major site of phosphorylation was a 60 kDa protein both in normal and MHS SR. The maximal amount of phosphorylation in MHS SR (5 pmol P/mg SR) was significantly lower than that in the normal SR (12 pmol P/mg SR). The phosphorylated 60 kDa protein was spontaneously dephosphorylated both in normal and MHS SR. Ca2+ release from the passively loaded SR was induced by a Ca2+-jump, and monitored by stopped-flow fluorometry using chlorotetracycline. In the absence of preincubation with MgATP, no significant difference was found in any of the kinetic parameters of Ca2+ release between normal and MHS SR. Upon addition of 20 microM MgATP to the passively loaded SR to phosphorylate the 60 kDa protein, the initial rate of Ca2+ release in normal SR significantly decreased from 659 +/- 102 to 361 +/- 105 nmol Ca2+/mg SR per s, whereas in MHS SR the rate decreased from 749 +/- 124 to 652 +/- 179 nmol Ca2+/mg SR per s. Addition of 20 microM adenosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppA) did not significantly alter the initial rate of Ca2+ release both in normal and MHS SR. These results suggest that the previously reported higher Ca2+ release rate in MHS SR (Kim et al. (1984) Biochim. Biophys. Acta 775, 320-327) is at least partly due to the reduced extent of the Ca2+/CaM-dependent phosphorylation of the 60 kDa protein. Two-dimensional gel electrophoresis study showed that amount of a protein with Mr = 55,000 was significantly lower in MHS SR than in normal SR suggesting that the abnormally lower amount of 55 kDa protein would cause the lower amount of phosphorylation of the 60 kDa protein in MHS SR.     

Effect of luminal calcium on Ca2+ release channel activity of sarcoplasmic reticulum in situ.

Ca2+ influx into empty SR in the absence of Ca2+ pump activity was determined in skinned frog skeletal muscle fibers and compared with Ca2+ efflux from loaded SR (i.e., Ca2+ release) to deepen our understanding of the properties of the Ca2+ release channel (CRC). Calcium content in SR increased approximately in a first-order kinetics and finally reached the equilibrium level determined by cytoplasmic Ca2+ ([Ca2+]c). Because AMP caused an increase in the rate of Ca2+ influx, and procaine, Mg2+, and high concentrations of Ca2+ caused a characteristic decrease, the major Ca2+ influx pathway was concluded to be the CRC, as is true of Ca2+ release. The apparent rate constant (k(app)) of Ca2+ efflux did not significantly change when the loading level was decreased to one-third. At a given [Ca2+]c, the same equilibrium level of calcium in SR was attained with a similar k(app) by both Ca2+ influx and Ca2+ efflux. The relationship between [Ca2+]c and calcium in SR indicated the Ca2+ binding sites in SR. These results, together with the anticipated effects of these Ca2+ buffer sites on kinetics, are consistent with the idea that luminal Ca2+ inhibits the CRC.     

Intra- and extraluminal sarcoplasmic reticulum membrane regulatory sites for Ca2(+)-induced Ca2+ release.

A heavy skeletal muscle sarcoplasmic reticulum (SR) fraction was actively loaded stepwise with calcium until Ca2(+)-induced Ca2+ release occurred. The total Ca2+ load, T1, at which release occurred is postulated to be regulated by an intraluminal, low-affinity receptor. After obtaining T1, the critical concentration of Ca2+ required extraluminally (T2) was determined. T1 averaged 58.6 +/- S.D., 6.9 nmol Ca2+/mg SR and T2 averaged 2.14 +/- S.D., 0.24 microM. Both T1 and T2 were increased by Mg2+ and decreased by caffeine. Ruthenium red increased T2 more than T1 while ryanodine had no effect on T1 but markedly increased T2. The results suggest that two Ca2+ regulatory sites may be functional for Ca2(+)-induced Ca2+ release from SR.