The Effect of Quinidine on Calcium Accumulation by Isolated Sarcoplasmic Reticulum of Skeletal and Cardiac Muscle

Quinidine potentiates twitch tension and (at higher concentrations) causes contracture of skeletal muscle whereas the same drug reduces tension development of cardiac muscle. To gain insight into the possible differences in the excitation-contraction coupling mechanism of the two types of muscle the effect of quinidine on calcium accumulation by isolated sarcoplasmic reticulum from skeletal and cardiac muscle was investigated. In a medium containing ATP, Mg⁺⁺, oxalate, and ⁴⁵Ca, pharmacologically active concentrations of the drug inhibited calcium accumulation by both skeletal and cardiac sarcoplasmic reticulum. The inhibition of the rates of calcium, uptake by the skeletal muscle preparation ranged from 11% with 10^-4 M quinidine to 90% with 10^-3 M quinidine. With the cardiac muscle preparation the inhibition ranged from 16% with 3 x 10^-6 M quinidine to 100% with 10^-3 M quinidine. With both preparations the inhibition of calcium transport was accompanied by an inhibition of the Ca⁺⁺-activated ATPase activity of the sarcoplasmic reticulum. The effect of quinidine on the skeletal sarcoplasmic reticulum supports the hypothesis that this compound produces twitch potentiation and contracture by interfering with intracellular calcium, sequestration. Its effect on cardiac sarcoplasmic reticulum. has been interpreted in terms of the hypothesis that cardiac contractility is a function of the amount of calcium released from the sarcoplasmic reticulum which is in turn dependent upon the absolute calcium content of the reticulum. Hence, following inhibition of calcium transport there would be less calcium available for coupling.     

Calcium-Binding Properties of Sarcoplasmic Reticulum As Influenced by ATP,Caffeine, Quinine,and Local Anesthetics

Calcium retained at binding sites of the sarcoplasmic reticulum membranes isolated from rabbit skeletal muscle requires 10^-5 - 10^-4 M ATP to exchange with ⁴⁵Ca added to the medium. The ATP requirement for Ca exchangeability was observed with respect to the "intrinsic" Ca of the reticulum membranes and the fraction of Ca that is "actively" bound in the presence of ATP. Furthermore, a concentration of free Ca in the medium higher than 10^-8 M is required for ATP to promote Ca exchangeability. This exchangeability is not influenced by caffeine, quinine, procaine, and tetracaine, and Ca that is either nonexchangeable (in the absence of ATP) or exchangeable (in the presence of ATP) is released by 1–5 mM quinine or tetracaine, but neither caffeine (6 mM) nor procaine (2–5 mM) has this effect. Quinine or tetracaine also releases Ca and Mg bound passively to the reticulum membranes. A possible role of ATP in maintaining the integrity of cellular membranes is discussed, and the effects of caffeine, quinine, and of local anesthetics on the binding of Ca by the isolated reticulum are related to the effects of these agents on ⁴⁵Ca fluxes and on the twitch output observed in whole muscles.     

The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum

At concentrations between 1 to 10 mM, caffeine reduced the Ca-accumulating capacity of fragmented reticulum obtained from frog and rabbit muscle. With 8 mM caffeine enough Ca was released from frog reticulum to account for the force of the contracture. Caffeine did not affect all reticulum membranes equally. The fraction which was spun down at 2000 g was more sensitive than the lighter fractions. The percentage of the total accumulated Ca released by caffeine decreased with decreasing Ca content of the reticulum. In parallel with their known effects on the caffeine contracture, a drop in temperature increased the caffeine-induced Ca release while procaine inhibited it. Caffeine also inhibited the rate of Ca uptake, which may in part account for the prolongation of the active state caused by caffeine.     

The Site of Calcium Binding in Relation to the Activation of Myofibrillar Contraction

Skeletal muscle myofibrils, in the presence of 2 mM MgCl₂ at pH 7.0, were found to have two classes of calcium-binding sites with apparent affinity constants of 2.1 x 10⁶ M ^-1 (class 1) and ∼3 x 10⁴ M ^-1 (class 2), respectively. At free calcium concentrations essential for the activation of myofibrillar contraction (∼10^-6 M) there would be significant calcium binding only to the class 1 sites. These sites could bind about 1.3 µmoles of calcium per g protein. Extraction of myosin from the myofibrils did not alter their calcium-binding parameters. Myosin A, under identical experimental conditions, had little affinity for calcium. The class 1 sites are, therefore, presumed to be located in the I filaments. The class 1 sites could only be detected in F actin and myosin B preparations which were contaminated with the tropomyosin-troponin complex. Tropomyosin bound very little calcium. Troponin, which in conjunction with tropomyosin confers calcium sensitivity on actomyosin systems, could bind 22 µmoles of calcium per g protein with an apparent affinity constant of 2.4 x 10⁶ M ^-1. In view of the identical affinity constants of the myofibrils and troponin and the much greater number of calcium-binding sites on troponin it is suggested that calcium activates myofibrillar contraction by binding to the troponin molecule.     

Studies on lysosomes. XI. Characterization of a hydrolase-rich fraction from human lymphocytes

Pure suspensions of human lymphocytes were separated from peripheral blood by means of nylon wool, homogenized in 0.34 M sucrose-0.01 M EDTA solution, and fractionated by differential centrifugation. The bulk of acid hydrolase activity was found to be concentrated in a 20,000 g x 20 min granular fraction, whereas nuclear, debris, and supernatant fractions contained lesser concentrations of hydrolases. Acid hydrolase activity present in the granular fraction showed appropriate "latency" as judged by its dose-dependent release into the 20,000 g x 20 min supernatant after exposure to membrane-disruptive agents such as streptolysin S, filipin, and lysolecithin. Heparin proved to be necessary in the suspending medium so that reproducible homogenization and cell fractionation could be obtained. Even excessive contamination of lymphocyte suspensions with platelets did not appreciably alter the acid hydrolase activity of lymphocyte homogenates or the distribution of enzymes in subcellular fractions. Discontinuous density-gradient centrifugation of a 500 g x 10 min supernatant, containing both acid hydrolase-rich organelles and mitochondria, resulted in partial resolution of hydrolase-rich organelles from mitochondria. Fine structural studies of the intact lymphocytes showed the presence of acid phosphatase-positive, membrane-bounded organelles. Electron microscopy of the "large granule" (20,000 g x 20 min) fraction of such lymphocytes demonstrated 80–90% mitochondria, 5–10% platelets, and 5–10% membrane-bounded acid phosphatase-positive structures. The data indicate the presence in human peripheral blood lymphocytes of acid hydrolase-rich granules which possess many of the biochemical and structural characteristics of lysosomes in other tissues.     

Adenosine triphosphate-induced rapid calcium release from fragmented sarcoplasmic reticulum

Calcium efflux from skeletal muscle fragmented sarcoplasmic reticulum was studied using a dilution technique and Millipore filtration. In the absence of Mg⁺⁺ and external Ca⁺⁺, addition of lmM adenosine triphosphate to the suspension resulted in an immediate loss of 26–55% of total vesicular calcium. The amount of calcium released was calculated to be sufficient to effect muscle contraction. After separation of the sarcoplasmic reticulum into light, intermediate and heavy vesicles, the light and heavy fractions were found to be only weakly responsive to adenosine triphosphate, whereas the intermediate fraction lost nearly half of its calcium. The significance of these results with respect to excitation-contraction coupling in muscle is discussed.     

Cytochemistry of phosphatases of the sarcoplasmic reticulum. II. In situ localization of the Mg-dependent enzyme

The distribution of the Mg-dependent ATPase associated with a microsomal fraction of rabbit psoas muscle was studied histochemically and its localization in relation to the vesicles of the fraction and to the structure of intact fixed muscle was determined. Although enzyme activity was retained after fixation in hydroxyadipaldehyde and in glyoxal, it was lost after fixation in glutaraldehyde or after 4 hr fixation in formaldehyde. Activity was optimally demonstrated when incubations were conducted at 17°C, in media containing 125 mM Trismaleate buffer, pH 7.5, 5 mM ATP, 4 mM MgCl₂, and 1 mM Pb(NO₃)₂. After such incubations, activity was present throughout the sarcoplasmic reticulum, but was absent from the T system. Activation by Na or K could not be demonstrated histochemically. However, the other biochemical properties of the enzyme in the isolated vesicles and in intact muscle were similar with respect to Mg dependence, substrate specificity, inhibition by Ca, N-ethyl maleimide, p-hydroxymercuribenzoate, and lack of inhibition by ouabain.     

Further studies of the effect of calcium on the time course of action of carbamylcholine at the neuromuscular junction

The rate at which the postjunctional membrane of muscle fibers becomes desensitized to the action of carbamylcholine is increased after the muscle has been soaked in solutions containing increased concentrations of calcium. Some further aspects of this effect of calcium were investigated by measuring changes in the input resistance of single fibers of the frog sartorius during local perfusion of the neuromuscular junction with 2.73 x 10^-3 M carbamylcholine in isolated muscles immersed in 165 mM potassium acetate. It was found that (a) sudden changes in the local concentration of calcium brought about by perfusing fibers with carbamylcholine solutions containing 20 mM calcium, 40 mM oxalate, or 40 mM EDTA were followed within 20 sec by marked changes in the rate of desensitization; (b) prior to 13 sec after the introduction of carbamylcholine, however, no effect on the input resistance could be detected even though the muscle had been presoaked in 10 mM calcium; (c) the ability of high concentrations of calcium to bring about rapid desensitization disappears when a lower concentration of carbamylcholine (0.137 x 10^-3 M) is applied to the muscle fiber. These findings suggest that calcium present in the extracellular fluid can act directly on the postjunctional membrane to promote the desensitization process and that an increased permeability of the membrane to calcium brought about by the presence of carbamylcholine is a factor which contributes to this action.     

Kinetic analyses of calcium movements in cell cultures. 3. Effects of calcium and parathyroid hormone in kidney cells

Calcium compartments and fluxes were measured by kinetic analyses in kidney cell suspensions in a three-compartment closed system. The fast phase influx and compartment size increase linearly with the medium calcium and the half-time of exchange is only 1.3 min which suggests that the fast component is extracellular. The slow phase compartment rises linearly from 0.1 to 0.5 mmole calcium/kg cell water when the medium calcium is raised from 0.02 to 2.5 mM. The slow phase calcium influx exhibits the pattern of saturation kinetics with a V _max of 0.065 µµmole cm^-2 sec^-1 and a K_m of 0.3 mM indicating that it is a carrier-mediated transport process. PTH has no effect on the fast phase of calcium influx, but increases both calcium influx and the calcium pool size of the slow component. The maximum effect is obtained at medium calcium concentration of 1.3 mM. Below 0.3 mM extracellular calcium, the effects of the hormone cannot be demonstrated. PTH increases the V _max of calcium influx from 0.065 to 0.128 µµmole cm^-2 sec^-1 while the K_m rises from 0.3 to 1.15 mM. These findings suggest that PTH increases the translocation of the calcium-carrier complex across the membrane and not the carrier concentration or its binding affinity for calcium.     

ISOLATION OF AN INSULIN SECRETION GRANULE FRACTION

Goosefish islets were homogenized in 0.25 M sucrose and separated into nuclear, mitochondrial + secretion granule, microsomal, and supernatant fractions. Eighty per cent of the cytochrome oxidase activity and 75 per cent of the bioassayed insulin activity were found in the mitochondrial + secretion granule fraction (6000 g for 10 minutes). The mitochondrial + secretion granule fraction was further subfractionated by centrifugation (2 hours at 100,000 g and 0°C) using a continuous linear density gradient 1.0–2.0 M sucrose). Eighteen to 20 subfractions were collected by piercing the bottom of the tube and collecting drops. The total protein was distributed into a bimodal curve consisting of a high density component, which contained 90 per cent of the insulin (secretion granules), and a lower density component, which contained the cytochrome oxidase activity (mitochondria).     

The effects of calcium ions on the activities of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from various muscles

1. The effects of Ca²⁺ on the activities and regulatory properties of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from vertebrate red and white muscle and insect fibrillar and non-fibrillar muscle have been investigated. These muscles were selected because of the possible difference in the role of glycolysis in energy production in the vertebrate muscles, and the possible difference in the role of Ca²⁺ in the control of contraction in the two types of insect muscle. An increase in Ca²⁺ concentration from 0.001μm to 10μm did not modify the activities nor did it modify the regulatory properties of these enzymes from these various muscles. 2. Concentrations of Ca²⁺ above 0.1mm inhibited the activities of hexokinase and phosphofructokinase from the different muscles. It has been suggested that this inhibition may provide the basis for a theory of regulation of glycolysis (Margreth et al., 1967). If phosphofructokinase is located within the sarcoplasmic reticulum, its activity will be inhibited when the muscle is at rest, but the release of Ca²⁺ from the reticulum during contraction will lead to a stimulation of its activity and hence an increase in glycolytic flux. The distribution of hexokinase and phosphofructokinase in the various cell fractions of these muscles was very variable. In particular, both enzymes were present almost exclusively in the 100000g supernatant fraction in the extracts of insect flight muscles. Thus there is no correlation between the properties of the enzymes and their distribution in muscle. 3. It is concluded that Ca²⁺ does not control the activities of the important regulatory enzymes of glycolysis in muscle. It is suggested that in some muscles the sensitivity of the control mechanism at the level of phosphofructokinase to changes in the concentration of AMP may be increased by a process known as `substrate-cycling'.     

Characterization of cell membrane and sarcoplasmic reticulum from bovine uterine smooth muscle

Subcellular fractions, enriched in sarcoplasmic reticulum or in cell membrane, were separated from one another. Starting material was a microsomal pellet (15–40 × 1000g) obtained by differential centrifugation from the uteri of close-to-term pregnant cows. A microsomal fraction enriched in ATP-dependent calcium accumulation was shown to contain sarcoplasmic reticulum and cell membrane. Only 8% or less of the protein in this fraction could be recovered, using affinity chromatography on Sepharose 6MB wheat germ agglutinin. The small yields did not allow extensive characterization. A method was developed to separate sarcoplasmic reticulum from cell membrane using discontinuous sucrose density gradient centrifugation. Protein was collected at the 24–28, the 28–33, and the 33–45% sucrose interfaces. Characterization was by enzyme assays and by specific receptor assay. ATP-dependent calcium accumulation was fourfold greater in the 24–28% sucrose layer than in the 33–45% layer. In contrast, 5′-nucleotidase was more than threefold as high in the 33–45% sucrose layer as in the 24–28% layer. Ouabain-inhibited p-nitrophenylphosphatase doubled and ouabain-inhibited Na,K-ATPase tripled in the 28–33% layer, compared with the 24–28% layer, specific ouabain binding was also doubled in the 28–33% sucrose layer. ¹²⁵I-Labeled wheat germ agglutinin binding was greatest in the 33–45% sucrose layer. It is concluded that the 24–28% layer consists primarily of sarcoplasmic reticulum, whereas the 28–33 and the 33–45% layers are concentrated in the cell membrane. Specific prostaglandin (PGE₂) binding was found to be a property of the cell membrane.     

A Biochemical and Morphological Study of Rat Liver Microsomes

Microsomes isolated by differential centrifugation from a rat liver homogenate in 0.88 M sucrose solution have been studied from the biochemical and morphological point of view. 1. Under these experimental conditions, the "total microsome" fraction was obtained by centrifuging the cytoplasmic extract free of nuclei and mitochondria, for 3 hours at 145,000 g. Morphologically, the total microsomes consist mainly of "rough-surfaced membranes" and "smooth" ones. 2. The total microsomes have been divided into 2 subfractions so that the 1st microsomal fraction contains the "rough" vesicles (2 hours centrifugation at 40,000 g) while the 2nd microsomal fraction consists essentially of smooth vesicles, free particles, and ferritin (centrifugation of the supernatant at 145,000 g for 3 hours). 3. By the action of 0.4 per cent sodium deoxycholate in 0.88 M sucrose, it was possible to obtain a pellet for each of the 2 fractions which consisted of dense particles, rich in RNA, poor in lipids, and which represented about 50 to 60 percent of the RNA and 10 to 15 per cent of the proteins. The results have been discussed taking into consideration the hypothesis of the presence of RNA in the membranes of microsomal vesicles.     

NOTES ON ULTRASTRUCTURE AND SOME PROPERTIES OF TRANSPORT WITHIN THE LIVING MITOTIC SPINDLE

The sites of lead phosphate precipitation in mouse bladder smooth muscle incubated with adenosine triphosphate and lead nitrate were studied by electron microscopy. The media constituents and incubating conditions were independently varied so that we could determine optimal conditions for histochemical demonstration of ATPase activity in agranular endoplasmic reticulum. Specimens of glutaraldehyde-fixed bladder muscle, frozen, cut into 10–40-µ sections, and incubated for 1 hr at 25°C in medium containing 0.025 M ATP, 0.0025 M lead nitrate, 0.05 M magnesium chloride, and 0.09 M sodium acetate buffer at pH 6.2, exhibited microcrystalline deposits in agranular endoplasmic reticulum and pinocytotic vesicles. Lead salt deposition was also noted in terminal cisternae of sarcoplasmic reticulum in skeletal muscle similarly treated, suggesting that the organelle systems in the two types of muscle cells subserve a common function.     

Nature of the Schwann cell electrical potential. Effects of the external ionic concentrations and a cardiac glycoside

The effects on the Schwann cell electrical potential of external ionic concentrations and of K-strophanthoside were investigated. Increasing (K)_o depolarized the cell. The potential is related to the logarithm of (K)_o in a quasi-linear fashion. The linear portion of the curve has a slope of 45 mv/ten-fold change in (K)_o. Diminutions of (Na)_o and (Cl)_o produced only small variations in the potential. Calcium and magnesium can be replaced by 44 mM calcium without altering the potential. Increase of (Ca)_o to 88 mM produced about 10 mv hyperpolarization. The cell was hyperpolarized by 11 mv and 4 mv within 1 min after applying K-strophanthoside at concentrations of 10^-3 and 10^-5 M, respectively. No variations of cellular potassium, sodium, or chloride were observed 3 min after applying the glycoside. The hyperpolarization caused by 10^-3 M K-strophanthoside was not observed when (K)_o was diminished to 1 or 0.1 mM or was increased to 30 mM. At a (K)_o of 30 mM, 10^-2 M strophanthoside was required to produce the hyperpolarizing effect. In high calcium, the cell was further hyperpolarized by the glycoside. The initial hyperpolarization caused by the glycoside was followed by a gradual depolarization and a decrease of the cellular potassium concentration. The results indicate that the Schwann cell potential of about -40 mv is due to ionic diffusion, mainly of potassium, and to a cardiac glycoside-sensitive ion transport process.     

Cholinesterase activity per unit surface area of conducting membranes

According to theory, the action of acetylcholine (ACh) and ACh-esterase is essential for the permeability changes of excitable membranes during activity. It is, therefore, pertinent to know the activity of ACh-esterase per unit axonal surface area instead of per gram nerve, as it has been measured in the past. Such information has now been obtained with the newly developed microgasometric technique using a magnetic diver. (1) The cholinesterase (Ch-esterase) activity per mm² surface of sensory axons of the walking leg of lobster is 1.2 x 10^-3 µM/hr. (σ = ± 0.3 x 10^-3; SE = 0.17 x 10^-3); the corresponding value for the motor axons isslightly higher: 1.93 x 10^-3 µM/hr. (σ = ± 0.41 x 10^-3; SE = ± 0.14 x 10^-3). Referred to gram nerve, the Ch-esterase activity of the sensory axons is much higher than that of the motor axons: 741 µM/hr. (σ = ± 73.5; SE = ± 32.6) versus 111.6 µM/hr. (σ = ± 28.3; SE = ± 10). (2) The enzyme activity in the small fibers of the stellar nerve of squid is 3.2 x 10^-4 µM/mm²/hr. (σ = ± 0.96 x 10^-4; SE = ± 0.4 x 10^-4). (3) The Ch-esterase activity per mm² surface of squid giant axon is 9.5 x 10^-5 µM/hr. (σ = ± 1.55 x 10^-5; SE = ± 0.38 x 10^-5). The value was obtained with small pieces of carefully cleaned axons after removal of the axoplasm and exposure to sonic disintegration. Without the latter treatment the figurewas 3.85 x 10^-5 µM/mm²/hr. (σ = ± 3.24 x 10^-5; SE = ± 0.93 x 10^-5). The experiments indicate the existence of permeability barriers in the cell wall surrounding part of the enzyme, since the substrate cannot reach all the enzyme even when small fragments of the cell wall are used without disintegration. (4) On the basis of the data obtained, some tentative approximations are made of the ratio of ACh released to Na ions entering the squid giant axon per cm² per impulse.     

Effects of the Intracellular Ca Ion Concentration upon the Excitability of the Muscle Fiber Membrane of a Barnacle

The membrane excitability and contraction were examined in single barnacle muscle fibers with different internal Ca⁺⁺ concentrations by using buffer solutions made up with EGTA and Ca-gluconate in various proportions. During the passage of dc currents the membrane shows all-or-none spike potentials for internal Ca⁺⁺ concentrations below about 8 x 10^-8 M, oscillatory potential changes in the range between 8 x 10^-8 to 5 x 10^-7 M, but neither oscillatory nor spike potentials were seen for concentrations above 5 x 10^-7 M. All-or-none spike potentials were suppressed when the internal Mg⁺⁺ concentration exceeded 5 mM. The suppression threshold of the internal Ca⁺⁺ concentration for the Sr spike is much higher than that for the Ca spike. The threshold concentration of internal Ca⁺⁺ for contraction was about 8 x 10^-7 M.     

The Action of Serotonin on Calcium-45 Efflux from the Anterior Byssal Retractor Muscle of Mytilus edulis

⁴⁵Ca efflux was studied in resting anterior byssal retractor muscle. The data are described by a three-compartment system. The most rapidly exchanging compartment, with an average time constant of 7 min, contains about 0.9 mM Ca/liter muscle, and probably represents extracellular space. A second compartment, with a time constant of 83 ± 5 min, contains 1.2 mM Ca/liter, and may represent a membrane calcium store. The presence of a third, or more, compartments, probably representing sarcoplasmic reticulum and contractile proteins, is indicated by the fact that the final time constant is 10 times the 83 min time constant of the second compartment. Serotonin (5HT), on initial application, increases ⁴⁵Ca efflux from this third compartment(s). This effect has a typical dose-response relationship with a maximum response appearing at 10^-7 M5HT. In addition, removal of 5HT causes a secondary increase in ⁴⁵Ca efflux which has a maximum at a 5HT concentration of 10^-7 M and declines at both higher and lower doses.     

Highly purified sarcoplasmic reticulum vesicles are devoid of Ca2+-independent (‘basal’) ATPase activity

On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca²⁺-ATPase is selectively extracted while approximately half of the initial Mg²⁺-, or ‘basal’, ATPase remains in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca²⁺-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg²⁺-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the ‘basal’ ATPase separated from the Ca²⁺-ATPase by detergent extraction originates from mitochondrial contaminants.To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90–95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg²⁺-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca²⁺-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures.     

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

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