31P NMR and freeze fracture studies of sarcoplasmic reticulum membranes from normal and malignant hyperthermic pigs: effect of halothane and dantrolene.

The effects of halothane and dantrolene on sarcoplasmic reticulum membranes isolated from normal and malignant hyperthermia pig muscle have been investigated using 31P NMR and freeze fracture electron microscopy. The dynamical and structural changes are estimated from the second moment, as calculated from 31P NMR spectra. For both membranes, addition of halothane induces a similar decrease in the spectral second moment. At high concentration of halothane, freeze fracture replicas show small unilamellar vesicles or mixed micelles, uniformly sprayed in the case of malignant hyperthermia membranes but mainly aggregated for the normal ones. The effect of halothane on both membranes is partially inhibited by adding dantrolene. These results suggest that (i) the malignant hyperthermia syndrome is not directly related to the polar heads of phospholipids and (ii) dantrolene counteracts unspecifically the disturbing effect of halothane at the lipid level.     

Abnormal membrane properties of the sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia: modes of action of halothane, caffeine, dantrolene, and two other drugs

The role of sarcoplasmic reticulum (SR) in malignant hyperthermia (MH) was studied using the heavy microsomal fraction prepared from semitendinosus muscles of both normal and genetically MH-susceptible pigs. In the presence of ATP, SR was loaded with 70 nmol Ca2+/mg SR protein. Under these conditions, MH-SR demonstrated Ca2+-induced Ca2+ release (Ca-ICaR) and halothane-induced Ca2+ release (halothane-ICaR; halothane concentrations as low as 10 microM). Normal SR did not demonstrate these release phenomena. Dantrolene inhibited the halothane-ICaR, but did not inhibit the Ca-ICaR. Ruthenium red and tetracaine inhibited both types of Ca2+ release. From the measurement of passive Ca2+ efflux, it was shown that dantrolene did not affect the Ca2+ permeability of the SR itself, but suppressed only the halothane-induced increment of the permeability. The membrane order parameter of the SR, as measured by the spin-probe EPR technique, indicated that halothane disordered the lipid bilayer of MH-SR to a greater extent than it did of normal SR. This halothane disordering effect on MH-SR was antagonized by dantrolene. Ruthenium red and tetracaine did not antagonize the halothane disordering effect. These results raise the possibility that halothane could disturb the structure of the lipoprotein complex in MH-SR in such a way that it could open the Ca2+-release channels. The Ca2+ thus released further opens the channel through the Ca-ICaR mechanism in a positive feedback fashion, thus triggering the MH syndrome. The efficacy of dantrolene in ameliorating the MH syndrome might be related to the inhibition of this halothane effect.     

Ethanol increases calcium permeability of heavy sarcoplasmic reticulum of skeletal muscle

The effects of ethanol on both Ca2+ pump activity and Ca2+-induced Ca2+ release in sarcoplasmic reticulum (SR) of rabbit skeletal muscle were studied. In concentrations of 0.1-1.0%, ethanol conspicuously enhanced Ca2+ release from the heavy fraction of SR, whereas a much smaller effect was noted in the light fraction. When Ca2+-induced Ca2+ release was inhibited by 10 mM Mg2+, the Ca2+ pump activities of both the heavy and light SR were the same; the activities were not significantly influenced by 1% ethanol. Ethanol itself did not release Ca2+ from the heavy SR. However, it appeared to render the heavy SR more permeable to Ca2+, thereby decreasing the amount of storable Ca2+. This action of ethanol may be related to the mechanism of its negative inotropic effect.     

Effect of tetracaine and sovcaine on the ATP-dependent calcium accumulation in sarcoplasmic reticulum vesicles of skeletal muscles

The tetracaine and cinchocaine in concentration less than 2 mM and 0.5 mM, respectively, stimulate ATP-dependent Ca-loading by enhancing the initial rate of Ca2+-accumulation, do not affect the Ca2+-binding and Ca-ATPase activity of sarcoplasmic reticulum vesicles. These data suggest blocking of Ca2+-efflux from vesicles which occurs during Ca-accumulation. Higher concentrations of the same compounds (above 2 mM and 0.5 mM for tetracaine and cinchocaine, respectively) caused inhibition of the Ca-ATPase activity and decreased the ability of SR vesicles to retain Ca2+, probably, due to their nonspecific lipophilic action.     

Chronic alcohol ingestion alters the calcium permeability of sarcoplasmic reticulum of rat skeletal muscle

Heavy sarcoplasmic reticulum (SR) was prepared from skeletal muscle of control and chronic alcoholic rats, and the effect of in vitro addition of ethanol on the passive Ca2+ permeability was studied. The SR was loaded with Ca2+ in the absence of ATP. Then efflux was initiated by adding an EGTA solution to decrease the extravesicular Ca2+ concentration. The decrease of Ca2+ content of the SR was measured by an optical method using an encapsulated metallochromic indicator (calcein). The Ca2+ permeability of alcoholic rat SR was higher than that of control rats, especially at low external Ca2+ concentrations (below 1 microM). An in vitro (acute) exposure of SR to ethanol increased the Ca2+ permeability of the SR. However, the degree of increase in alcoholic rat SR was smaller than that in control rat SR.     

Abnormal human sarcoplasmic reticulum Ca2+ release channels in malignant hyperthermic skeletal muscle.

Single sarcoplasmic reticulum (SR) Ca2+ release channels were reconstituted from normal and malignant hyperthermic (MH) human skeletal muscle biopsies (2-5 g samples). Conduction, gating properties, and myoplasmic Ca2+ dependence of human SR Ca2+ release channels were similar to those in other species (rabbit, pig). The MH diagnostic procedure distinguishes three phenotypes (normal, MH-equivocal, and MH-susceptible) on the basis of muscle contracture sensitivity to caffeine and/or halothane. Single channel studies reveal that human MH muscles (both MH phenotypes) contain SR Ca2+ release channels with abnormally greater caffeine sensitivity. Muscles from MH-equivocal and MH-susceptible patients appear to contain channels with the same abnormality. Further, our data (n = 115, 21 channels, 11 patients) reveals that human MH muscles (both phenotypes) may contain two populations of SR Ca2+ release channels, possibly corresponding to normal and abnormal isoforms. Thus, whole cell phenotypic variation (MH-equivocal vs. MH-susceptible) arises in muscles containing channels with similar caffeine sensitivity suggesting that human MH does not arise from a single defect. These results have important ramifications concerning (a) correlation of functional and genetic MH studies, (b) identification of other, yet to be determined, factors which may influence MH expression, and (c) characterization of normal SR Ca2+ release channel function by exploring genetic channel defects.     

The effect of calcium load on the calcium permeability of sarcoplasmic reticulum

The effect of intravesicular and extravesicular calcium concentration on the passive efflux from sarcoplasmic reticulum (SR) vesicles isolated from cardiac and skeletal muscle was determined by measuring net efflux of calcium after stopping pump-mediated fluxes. The apparent permeability, calculated as the passive efflux divided by the total intravesicular calcium, depended on calcium load. This dependence of the apparent permeability on calcium load could be explained by the presence of intravesicular calcium-binding sites with a dissociation constant less than 10(-3) M. When the intravesicular bound calcium was taken into account, passive calcium efflux was found to be linearly related to the difference in calcium concentration across the SR membrane. Thus the permeability of the SR membrane is independent of intravesicular and extravesicular calcium concentration in the ranges investigated. The average first order rate constant for passive calcium efflux for six preparations was 0.8 +/- 0.2 min-1 for skeletal and 0.7 +/- 0.1 min-1 for cardiac SR. The amount of intravesicular bound calcium for the same preparations was 33 +/- 6 nmol mg-1 for skeletal and 13 +/- 2 nmol mg-1 for cardiac SR. The first order rate constants were unaffected by Mg concentration between 0.1 +/- 15.1 mM and by the presence of an ATP-regenerating system. The results suggest that some minimal calcium load may be required in order to observe a substantial passive calcium efflux, the passive calcium efflux is not carrier mediated, and passive calcium efflux is not a likely route of calcium release during excitation-contraction coupling.     

Mechanism of calcium release from skeletal sarcoplasmic reticulum

Summary Ca²⁺-induced Ca²⁺ release at the terminal cisternae of skeletal sarcoplasmic reticulum was demonstrated using heavy sarcoplasmic reticulum vesicles. Ca²⁺ release was observed at 10 m Ca²⁺ in the presence of 1.25mm free Mg²⁺ and was sensitive to low concentrations of ruthenium red and was partially inhibited by valinomycin. These results suggest that the Ca²⁺-induced Ca²⁺ release is electrogenic and that an inside negative membrane potential created by the Ca²⁺ flux opens a second channel that releases Ca²⁺. Results in support of this formulation were obtained by applying a Cl^– gradient or K⁺ gradient to sarcoplasmic reticulum vesicles to initiate Ca²⁺ release. Based on experiments the following hypothesis for the excitation-contraction coupling of skeletal muscle was formulated. On excitation, small amounts of Ca²⁺ enter from the transverse tubule and interact with a Ca²⁺ receptor at the terminal cisternae and cause Ca²⁺ release (Ca²⁺-induced Ca²⁺ release). This Ca²⁺ flux generates an inside negative membrane potential which opens voltage-gated Ca²⁺ channels (membrane potential-dependent Ca²⁺ release) in amounts sufficient for contraction.     

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.     

Characterization of the tetraphenylboron-induced calcium release from skeletal sarcoplasmic reticulum

Release of Ca2+ from skeletal sarcoplasmic reticulum vesicles was studied by the spectrophotometric stopped-flow technique using tetraphenylboron as a releasing agent. The extent of Ca2+ release shows a sigmoidal response, with respect to the tetraphenylboron concentration, being dependent on Ca2+ preloading and Ca2+-ATPase activity, since these experiments were performed on actively loaded vesicles. The release process has a rapid component with an apparent rate constant of 6-8 s-1, showing a linear relationship between the rapid rate of Ca2+ release and the Ca2+ content of the vesicles. The release is not mediated by the reversal of the Ca2+ pump. Since the amphipathic anion tetraphenylboron was unable to elicit a Ca2+-release response when added to a preparation of sarcoplasmic reticulum phospholipid vesicles, it is suggested that there may be an interaction with some membrane protein(s) at the hydrophobic/hydrophilic interface leading to the opening of some specific Ca2+-release pathway.     

Thiophosphorylation of skeletal muscle sarcoplasmic reticulum in porcine malignant hyperpyrexia

1. The basal, Ca2+-dependent and Mg2+-dependent thiophosphorylation of malignant hyperpyrexia-susceptible (MHS) porcine skeletal muscle was investigated. 2. Seven major proteins of Mr 100,000-11,000 were substrates for thiophosphorylation. 3. Sodium molybdate significantly elevated all levels of thiophosphorylation in control sarcoplasmic reticulum, but did not effect the Ca2+-dependent thiophosphorylation of MHS samples. 4. These results suggest that MHS sarcoplasmic reticulum may have altered sensitivity to protein phosphatase inhibition.     

Solubilization and reconstitution of the calcium release channel of skeletal sarcoplasmic reticulum

Fragmented heavy sarcoplasmic reticulum was solubilized by cholate, and proteins were subsequently fractionated by using diethylaminoethyl-cellulose (DEAE-cellulose) column chromatography. A fraction was collected in which proteins with molecular weights between 31,000 and 45,000 u were major components. This fraction, when incorporated into Ca2+ -loaded liposomes, facilitated the Ca2+ efflux. The rate of efflux was regulated by the external Ca2+ concentration, reaching a maximum at 3 microM Ca2+. The Ca2+ efflux was suppressed by Mg2+.     

Drug-induced calcium release from heavy sarcoplasmic reticulum of skeletal muscle

Calcium release from isolated heavy sarcoplasmic reticulum of rabbit skeletal muscle by several calmodulin antagonistic drugs was measured spectrophotometrically with arsenazo III and compared with the properties of the caffeine-induced calcium release. Trifluoperazine and W7 (about 500 microM) released all actively accumulated calcium (half-maximum release at 129 microM and 98 microM, respectively) in the presence 0.5 mM MgCl2 and 1 mg/ml sarcoplasmic reticulum protein; calmidazolium (100 microM) and compound 48/80 (70 micrograms/ml) released maximally 30-40% calcium, whilst bepridil (100 microM) and felodipin (50 microM) with calmodulin antagonistic strength similar to trifluoperazine (determined by inhibition of the calcium, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum) did not cause a detectable calcium release, indicating that this drug-induced calcium release is not due to the calmodulin antagonistic properties of the tested drugs. Calcium release of trifluoperazine, W7 and compound 48/80 and that of caffeine was inhibited by similar concentrations of magnesium (half-inhibition 1.4-4.2 mM compared with 0.97 mM for caffeine) and ruthenium red (half-inhibition for trifluoperazine, W7 and compound 48/80 was 0.22 microM, 0.08 microM and 0.63 micrograms/ml, respectively, compared with 0.13 microM for caffeine), suggesting that this drug-induced calcium release occurs via the calcium-gated calcium channel of sarcoplasmic reticulum stimulated by caffeine or channels with similar properties.     

Mechanism of anthraquinone-induced calcium release from skeletal muscle sarcoplasmic reticulum

The anthraquinones, doxorubicin, mitoxantrone, daunorubicin and rubidazone are shown to be potent stimulators of Ca2+ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles and to trigger transient contractions in chemically skinned psoas muscle fibers. These effects of anthraquinones are the direct consequence of their specific interaction with the [3H] ryanodine receptor complex, which constitutes the Ca2+ release channel from the triadic junction. In the presence of adenine nucleotides and physiological Mg2+ concentrations (approximately 1.0 mM), channel activation by doxorubicin and daunorubicin exhibits a sharp dependence on submicromolar Ca2+ which is accompanied by a selective, dose-dependent increase in the apparent affinity of the ryanodine binding sites for Ca2+, in a manner similar to that previously reported with caffeine. Unlike caffeine, however, anthraquinones increase [3H]ryanodine receptor occupancy to the level observed in the presence of adenine nucleotides. A strong interaction between the anthraquinone and the caffeine binding sites on the Ca2+ release channel is also observed when monitoring Ca2+ fluxes across the SR. Millimolar caffeine both inhibits anthraquinone-stimulated Ca2+ release, and reduces anthraquinone-stimulated [3H]ryanodine receptor occupancy, without changing the effective binding constant of the anthraquinone for its binding site. The degree of cooperativity for daunorubicin activation of Ca2+ release from SR also increases in the presence of caffeine. These results demonstrate that the mechanism by which anthraquinones stimulate Ca2+ release is caused by a direct interaction with the [3H]ryanodine receptor complex, and by sensitization of the Ca2+ activator site for Ca2+.     

Effects of diltiazem or verapamil on calcium uptake and release from chicken skeletal muscle sarcoplasmic reticulum

The purpose of this study was to determine the effects of 2 Ca2+ channel blockers, verapamil and diltiazem, on calcium loading (active Ca2+ uptake) and the following Ca2+ release induced by silver ion (Ag+) and Ca2+ from the membrane of heavy sarcoplasmic reticulum (SR) of chicken skeletal muscle. A fluorescent probe technique was employed to determine the calcium movement through the SR. Pretreatment of the medium with diltiazem and verapamil resulted in a significant decrease in the active Ca2+ uptake, with IC50 of about 290 micromol/L for verapamil and 260 micromol/L for diltiazem. Inhibition of Ca2+ uptake was not due to the development of a substantial drug-dependent leak of Ca2+ from the SR. It might, in part, have been mediated by a direct inhibitory effect of these drugs on the Ca2+ ATPase activity of the SR Ca2+ pump. We confirmed that Ca2+ channel blockers, administered after SR Ca2+ loading and before induction of Ca2+ release, caused a dose-dependent inhibition of both Ca2+- and Ag+-induced Ca2+ release rate. Moreover, if Ca2+ channel blockers were administered prior to SR Ca2+ loading, in spite of Ca2+ uptake inhibition the same reduction in Ca2+- and Ag+-induced Ca2+ release rate was seen. We showed that the inhibition of Ag+-induced Ca2+ release by L-channel blockers is more sensitive than Ca2+-induced Ca2+ release inhibition, so the IC50 for Ag+- and Ca2+-induced Ca2+ release was about 100 and 310 micromol/L for verapamil and 79 and 330 micromol/L for diltiazem, respectively. Our results support the evidence that Ca2+ channel blockers affect muscle microsome of chicken skeletal muscle by 2 independent mechanisms: first, reduction of Ca2+ uptake rate and Ca2+-ATPase activity inhibition, and second, inhibition of both Ag+- and Ca2+-induced Ca2+ release by Ca2+ release channels. These findings confirm the direct effect of Ca2+ channel blockers on calcium release channels. Our results suggest that even if the SR is incompletely preloaded with Ca2+ because of inhibition of Ca2+ uptake by verapamil and diltiazem, no impairment in Ca2+ release occurs.     

Caffeine treatment inhibits drug-induced calcium release from sarcoplasmic reticulum and caffeine contracture but not tetanus in frog skeletal muscle

Effects of pretreatment with caffeine on Ca2+ release induced by caffeine, thymol, quercetin, or p-chloromercuriphenylsulfonic acid (pCMPS) from the heavy fraction of sarcoplasmic reticulum (SR) were studied and compared with those effects on caffeine contracture and tetanus tension in single fibers of frog skeletal muscle. Caffeine (1-5 mM) did induce transient Ca2+ release from SR vesicles, but subsequent further addition of caffeine (10 mM, final concentration) induced little Ca2+ release. Ca2+ release induced by thymol, quercetin, or pCMPS was also inhibited by pretreatment with caffeine. In single muscle fibers, pretreatment with caffeine (1-5 mM) partially reduced the contracture induced by 10 mM caffeine. However, tetanus tension was almost maximally induced by electrical stimulus in caffeine-treated fibers. These results indicate that SR, which becomes less sensitive to caffeine, thymol, quercetin, or pCMPS by pretreatment with caffeine, can still respond to a physiological signal transmitted from transverse tubules.     

Dependence of calcium permeability of sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations.

The ability of sarcoplasmic reticulum vesicles to retain calcium following ATP-supported calcium uptake in the presence of the calcium-precipitating anions oxalate and phosphate depends on Cao (calcium ion concentration outside the vesicles) and Cai (calcium ion concentration within the vesicles). Calcium efflux rates at any level of Cai are accelerated when Cao is increased. Higher Cao at the time that calcium uptake reactions reach steady state is associated with a spontaneous calcium release that reflects this effect of increased Cao. Increasing Cai at any level of Cao causes little or no acceleration of calcium efflux rate so that calcium permeability coefficients, estimated by dividing calcium efflux rates by Cai, the "driving force", are inversely proportional to Cai. Calcium permability coefficients thus correlate, as a first approximation, with the ratio Cai/Cao, decreasing 1000-fold as this ratio increases over a 3000-fold range (Cao = 0.1 to 3.3 muM, Cai =4 to 750 muM). Oscillations in both the calcium content of the vesicles and Cao are seen as calcium uptake reactions approach steady state, suggesting that calcium permeability undergoes time-dependent variations. Sudden reduction of Cao to levels that markedly inhibit calcium influx via the calcium pump unmasks a calcium efflux that decreases slowly over 60 to 90 s.The maximal calcium permeability observed in the present study would allow the calcium efflux rate from the sarcoplasmic reticulum at a Cai of 100 muM to be approximately 10(-10) mol/cm2/s, which is about 1 order of magnitude less than that estimated for the sarcoplasmic reticulum of activated skeletal muscle in vivo. The release of most of the stored calcium in some experiments indicates that the observed permeability changes can occur over a large portion of the surface of the sarcoplasmic reticulum.