Ca2+ release from sarcoplasmic reticulum vesicles derived from longitudinal reticulum and terminal cisternae of frog skeletal muscle

Fragmented sarcoplasmic reticulum (FSR) of bullfrog skeletal muscle was fractionated into light and heavy sarcoplasmic reticulum (LSR and HSR) by sucrose density gradient centrifugation. Morphological and biochemical studies revealed that large parts of LSR and HSR were derived from longitudinal reticulum and terminal cisternae of SR, respectively. The Ca2+ uptake ability and ATPase activity of LSR were higher than those of HSR. Ca2+ release from Ca2+ preloaded SR vesicles by changing the medium from K-gluconate to KCl was suppressed by addition of 0.3 M sucrose or glucose; there was no correlation between Ca2+ release and membrane potential change either in LSR or HSR vesicles. Dantrolene sodium (DAN, 20 microM) had no effect on Ca2+ release. It is concluded that ion-induced Ca2+ release from SR (both HSR and LSR) in the isolated system is due to an osmotic effect.     

Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle

We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.     

Solubilization and separation of Ca2+-ATPase from the Ca2+-ryanodine receptor complex

Heavy sarcoplasmic reticulum (SR) preparations of rabbit skeletal muscle, which are enriched in Ca2+-release vesicles from the terminal cisternae (TC) and [3H]ryanodine receptor density, exhibit 60% of the Ca2+-ATPase activity, 58% of the EP level, and 30% of the steady state Ca2+ loading compared to membrane vesicles from the longitudinal SR. The Ca2+-ATPase of TC SR is solubilized and separated from the Ca2+-ryanodine receptor complex in the insoluble fraction on treatment with the detergent C12E9. However, a 50% decrease in receptor density is observed upon removal of the Ca2+-ATPase, suggesting a significant contribution of this protein to maintaining optimal receptor complex density.     

A study of passive Ca2+ flow through the sarcoplasmic reticulum of skeletal muscles. I. Passive Ca2+ efflux from vesicle membranes

The same level of passively loaded Ca2+ was observed both in the heavy (enriched in terminal cisternae) and light (enriched in longitudinal reticulum) sarcoplasmic reticulum (SR) fractions. The level of passively loaded Ca2+ of the both SR fractions decreased in the presence of 150 mM K+. However the rate and extent of Ca2+ release was greater from heavy SR fraction. The rate of Ca2+ release under conditions of antiport of K+, Na+, choline+ and gluconate-, Cl-, SCH- increased proportion with their permeability through the SR membrane. The initial rate of Ca2+ release also became higher under equal concentration of monovalent cation chloride both inside and outside the SR vesicles. Apparently, in this case Ca2+ release occurs through Ca-channels which are open at a membrane potential.     

Calcium release from two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium release from sarcoplasmic reticulum vesicles presumably derived from longitudinal tubules (LSR) and terminal cisternae (HSR) of rabbit skeletal muscle was investigated by dual wavelength spectrophotometry using the calcium-indicator antipyrylazo III. In 120 mM KCl, 5 mM MgCl2, 30 microM, CaCl2, 50 microM MgATP, 100 microM antipyrylazo III, 40 mM histidine (pH 6.8, 25 degrees C), LSR and HSR sequestered approx. 115 nmol calcium/mg, and then spontaneously released calcium. Analysis of ATP hydrolysis and phosphoenzyme level during LSR and HSR calcium sequestration indicated that this calcium release process was passive, occurring in the virtual absence of ATP and phosphoenzyme. Moreover, subsequent addition of ATP reinitiated the calcium sequestration-release sequence. Calcium release by HSR was more than 4-times faster than that by LSR. Analysis of the calcium release phase demonstrated a biexponential decay for both LSR (0.10 and 0.63 min-1) and HSR (0.26 and 1.65 min-1), suggestive of heterogeneity within each fraction. Replacement of 120 mM KCl with either 120 mM choline chloride, 240 mM sucrose, or H2O reduced maximal calcium sequestration by LSR, but had less effect on LSR calcium release rate constants. In the case of HSR, these changes in the ionic composition of the medium drastically reduced calcium release rate constants with little effect on calcium content. These marked differences between LSR and HSR are consistent with the hypothesis that the calcium permeability of the terminal cisternae is greater and more sensitive to the ionic environment than is that of the longitudinal tubules of sarcoplasmic reticulum.     

Preparation and characterization of longitudinal tubules of sarcoplasmic reticulum from fast skeletal muscle

Sarcoplasmic reticulum (SR) serves a central role in calcium uptake and release, thereby regulating muscle relaxation and contraction, respectively. Recently, we have isolated fractions referable to longitudinal tubules (R2) and terminal cisternae (R4), the two major types of sarcoplasmic reticulum (A. Saito et al. (1984) J. Cell Biol. 99, 875-885). The terminal cisternae contain two types of membranes, the calcium pump membrane and the junctional face membrane. The terminal cisternae are filled with electron-opaque contents which serve as a Ca2+ reservoir. The longitudinal tubules consist mainly of the calcium pump membrane. In this study, we describe a new longitudinal tubule fraction (F2) and characterize it together with the R2 and R4 SR fractions. The calcium pump membrane of the longitudinal tubules is a highly specialized membrane consisting of about 90% calcium pump protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive changes in morphology can be observed in the SR fractions referable to osmotic differences during the fixation conditions using either glutaraldehyde-tannic acid or osmium tetroxide fixatives. The changes include swelling or shrinkage and aggregation of the compartmental contents when the fixative contains calcium ions. The two types of SR have different osmotic permeability to the same medium, as indicated by differential swelling or shrinkage. Both longitudinal tubule and terminal cisternae vesicles of SR appear larger and are spherical vesicles when the glutaraldehyde-tannic acid fixative is isotonic as compared with the "standard" fixation method. We have previously reported that the ruthenium red-sensitive calcium release channels are localized to the terminal cisternae. The terminal cisternae as isolated are leaky to Ca2+ since these channels are in the "open state" (S. Fleischer et al. (1985) Proc. Natl. Acad. Sci USA 82, 7256-7259). Thus, the Ca2+, Mg2+-dependent ATPase (Ca2+ ATPase) rate is only slightly enhanced in the presence of a Ca2+ ionophore, which dissipates the Ca2+ gradient across the SR membrane. We now find that preincubation with ruthenium red restores the tight coupling of the Ca2+ ATPase activity to Ca2+ transport. That is to say, ATPase activity is reduced and the addition of ionophore stimulates the Ca2+ ATPase activity 4- to 7-fold. The Ca2+ ATPase activity in longitudinal tubules is already tightly coupled. It is minimal after a Ca2+ gradient has been generated, but can be stimulated 9- to 20-fold when the Ca2+ gradient is dissipated with ionophore. This finding suggests that the Ca2+ ATPase activity in SR is tightly coupled to Ca2+ transport in situ.     

Photolabeling of the integral proteins of skeletal muscle sarcoplasmic reticulum: comparison of junctional and nonjunctional membrane fractions

Rabbit skeletal muscle sarcoplasmic reticulum (SR) was fractionated by isopycnic density gradient centrifugation into longitudinal tubules (LSR) and terminal cisternae (TC). Junctional face membranes (JFM) were obtained by Triton X-100 treatment of the TC fraction (Costello, B., Chadwick, C., Saito, A., Chu, A., Maurer, A. and Fleischer, S. (1986) J. Cell Biol. 103, 741-753). Photoactivatable phospholipid analogs were introduced into LSR, TC, and JFM fractions to specifically label integral membrane proteins. Remarkably different labeling patterns were observed. Proteins of the following Mr were labeled and identified in the junctional sarcoplasmic reticulum (JFM): 350,000, 325,000, 80,000, 49,000, 37,000, 32,000, 30,000, and 6000. Polypeptides of Mr 105,000 (Ca2+-dependent ATPase), 77,000, 55,000, 41,000, 22,000, and 9000 (proteolipid) were labeled and found to be selectively localized in the nonjunctional sarcoplasmic reticulum (LSR). Calsequestrin, a key protein responsible for Ca2+ storage within the SR lumen, was never labeled, whether 1 mM CaCl2 was present or absent, and is termed a nonintegral membrane protein.     

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.     

The effects of calmodulin antagonist drugs on isolated sarcoplasmic reticulum from malignant hyperpyrexia susceptible swine

1. Because calcium antagonist drugs increase contracture in both control and malignant hyperpyrexia susceptible (MHS) skeletal muscle, the effect of these drugs on the sarcoplasmic reticulum (SR) was investigated. 2. The calmodulin antagonist drugs inhibited the Ca2+ dependent ATPase activity and the ATP-dependent Ca2+ uptake, and accelerated the efflux of Ca2+ from isolated SR preparations from both control and MHS skeletal muscle. These effects of calmodulin antagonist drugs on SR Ca2+ transport functions were consistent with their in vitro pharmacological effects on control and MHS muscle.     

Thermogenic activity of Ca2+-ATPase from skeletal muscle heavy sarcoplasmic reticulum: the role of ryanodine Ca2+ channel

The sarcoplasmic reticulum Ca(2+) ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca(2+) transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca(2+) channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (DeltaH(cal)) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg(2+) or ruthenium red, conditions that close the ryanodine Ca(2+) channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the DeltaH(cal) values of ATP hydrolysis increased significantly. Neither Mg(2+) nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the DeltaH(cal) of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca(2+) channel is opened or closed.     

Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle

Longitudinal tubules and junctional sarcoplasmic reticulum (SR) were prepared from heart muscle microsomes by Ca2+-phosphate loading followed by sucrose density gradient centrifugation. The longitudinal SR had a high Ca2+ loading rate (0.93 +/- 0.08 mumol.mg-1.min) which was unchanged by addition of ruthenium red. Junctional SR had a low Ca2+ loading rate (0.16 +/- 0.02 mumol.mg-1.min) which was enhanced about 5-fold by ruthenium red. Junctional SR had feet structures observed by electron microscopy and a high molecular weight protein with Mr of 340,000, whereas longitudinal SR was essentially devoid of both. Thus, these subfractions have similar characteristics to longitudinal and junctional terminal cisternae of SR from fast twitch skeletal muscle. Ryanodine binding was localized to junctional cardiac SR as determined by [3H]ryanodine binding. Scatchard analysis of the binding data showed two types of binding (high affinity, Kd approximately 7.9 nM; low affinity, Kd approximately 1 microM), contrasting with skeletal junctional terminal cisternae where only one site with Kd of approximately 50 nM was observed. The ruthenium red enhancement of Ca2+ loading rate in junctional cardiac SR was blocked by pretreatment with low concentrations of ryanodine as reported for junctional terminal cisternae of skeletal muscle SR. The Ca2+ loading rate of junctional cardiac SR was enhanced by preincubation with high concentrations of ryanodine. The apparent inhibition constant (Ki approximately 7 nM) and stimulation constant (Km approximately 1.1 microM) for ryanodine on junctional SR corresponded to the Kd for high affinity binding (Kd approximately 7.9 nM) and low affinity binding (Kd approximately 1.1 microM), respectively. These results suggest that high affinity ryanodine binding locks the Ca2+ release channels in the open state and that low affinity binding closes the Ca2+ release channels of the junctional cardiac SR. The characteristics of the Ca2+ release channels of junctional cardiac SR appear to be similar to that of skeletal muscle SR, but the Ca2+ release channels of cardiac SR are more sensitive to ryanodine.     

Calcium binding proteins of junctional sarcoplasmic reticulum: detection by 45Ca ligand overlay

In skeletal muscle, the junctional sarcoplasmic reticulum (JFM) plays a crucial role in excitation-contraction coupling and Ca2+ release. In the present report, the sarcoplasmic reticulum (SR) was fractionated into longitudinal SR (LSR), terminal cisternae (TC), and JFM. Each fraction had a unique protein profile as detected by SDS-polyacrylamide gel electrophoresis as well as specific Ca2+ binding proteins as judged by 45Ca ligand overlay of nitrocellulose blots. Ca2+ binding proteins of LSR were the Ca2+ ATPase (Mr of 115K), an 80K polypeptide, and the intrinsic glycoprotein (Mr of 160K); Ca2+ binding proteins of JFM were polypeptides with the following Mr values: 350K and 325K (feet components), 200K, 170K, a doublet of 140K, 118K, 65K (calsequestrin), and 52K. Measurements of Ca2+ binding to SR fractions by equilibrium dialysis indicated that 8-17 nmol Ca2+/mg of protein was specifically bound. After EDTA extraction of calsequestrin, JFM still bound Ca2+ (5-6 nmol/mg of protein), suggesting the existence of specific Ca2+ binding sites. The Ca2+ binding sites of Ca2+-gated Ca2+ release channels might be on two JFM polypeptides (Mr's of 350K and 170K) which are putative channel constituents (F. Zorzato, A. Margreth, and P. Volpe (1986) J. Biol. Chem. 261, 13252-13257).     

Functional characteristics of reconstituted sarcoplasmic reticulum membranes as a function of the lipid-to-protein ratio

The ATP-induced Ca2+ accumulation efficiency and rates of Ca2+ uptake of the reconstituted sarcoplasmic reticulum (RSR) model membrane system were measured over an extended range of lipid-to-protein (L/P) molar ratios and were compared to those of isolated light sarcoplasmic reticulum (LSR). Highly purified sarcoplasmic reticulum (SR), dissociated in the presence of deoxycholate, was reconstituted for several L/P ratios, according to the same procedure, forming closed membranes vesicles composed of greater than 95% Ca2+ pump protein and SR lipids which were capable of ATP-induced Ca2+ accumulation in the absence of oxalate or other Ca2+ precipitating agents. This suggests that dissociation of SR and reconstitution to form RSR does not significantly affect the ability of the Ca2+ pump protein incorporated into the SR lipid bilayer to establish Ca2+ gradients. Electron micrographs of fixed and stained dispersions of RSR revealed a structural organization of the membrane that was dependent upon the L/P molar ratio. RSR with L/P greater than 88 were composed of closed vesicles whose membranes stained asymmetrically, similar to that observed for LSR. Closed vesicles of RSR with L/P less than 88 were composed of membrane that stained symmetrically. In addition, reconstituted SR preparations with well-defined L/P molar ratios greater than 88 possess a functional behavior similar to that of LSR (in the absence of oxalate, energy efficiencies are 60-70% and apparent initial uptake rates are 80% that of isolated LSR controls); RSR preparations with L/P less than 88 are characterized by significantly depressed values of the energy efficiencies and apparent initial uptake rates especially at low L/P ratios. Thus, we are the first to report a reconstituted SR model membrane system capable of attaining rates of Ca2+ uptake comparable to isolated LSR controls at comparable L/P ratios in the absence of oxalate or other Ca2+ precipitating agents.     

Comparison of the catalytic and structural properties of Ca2+-ATPase in longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum

The catalytic behavior and structural features of Ca2+-ATPase in the vesicles of longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum isolated from rabbit skeletal muscles was analysed. pH measurements have shown under optimal conditions Ca2+-ATPase has similar catalytic behavior both in the fractions of longitudinal tubules and terminal cisternae. Under non-optimal conditions, the behavior similarity was not observed. The specific activity of the ATPase enzyme under optimal conditions was shown to be much higher in the fraction of longitudinal tubules than in the fraction of terminal cisternae. Caffeine added to both fractions had no effect on the catalytic behavior of Ca2+-ATPase. As judged from fluorescence analysis, the structure of Ca2+-ATPase of longitudinal tubules differs from that structure of terminal cisternae. In sarcoplasmic reticulum membrane, at least half of the tryptophan residues of Ca2+-ATPase was shown to be buried in the lipid bilayer. Our findings suggest that in terminal cisternae some of the Ca2+-ATPase molecules exist as an oligomeric protein and do not participate in ATP hydrolysis (named "silent" Ca2+-ATPase).     

Thyroid hormone downregulates the expression and function of sarcoplasmic reticulum-associated CaM kinase II in the rabbit heart

Phosphorylation of sarcoplasmic reticulum (SR) Ca2+-cycling proteins by a membrane-associated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is a well-documented physiological mechanism for regulation of transmembrane Ca2+ fluxes and the cardiomyocyte contraction-relaxation cycle. The present study investigated the effects of L-thyroxine-induced hyperthyroidism on protein expression of SR CaM kinase II and its substrates, endogenous CaM kinase II-mediated SR protein phosphorylation, and SR Ca2+ pump function in the rabbit heart. Membrane vesicles enriched in junctional SR (JSR) or longitudinal SR (LSR) isolated from euthyroid and hyperthyroid rabbit hearts were utilized. Endogenous CaM kinase II-mediated phosphorylation of ryanodine receptor-Ca2+ release channel (RyR-CRC), Ca2+-ATPase, and phospholamban (PLN) was significantly lower (30-70%) in JSR and LSR vesicles from hyperthyroid than from euthyroid rabbit heart. Western immunoblotting analysis revealed significantly higher (approximately 40%) levels of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) in JSR, but not in LSR, from hyperthyroid than from euthyroid rabbit heart. Maximal velocity of Ca2+ uptake was significantly increased in JSR (130%) and LSR (50%) from hyperthyroid compared with euthyroid rabbit hearts. Apparent affinity of the Ca2+-ATPase for Ca2+ did not differ between the two groups. Protein levels of PLN and CaM kinase II were significantly lower (30-40%) in JSR, LSR, and ventricular tissue homogenates from hyperthyroid rabbit heart. These findings demonstrate selective downregulation of expression and function of CaM kinase II in hyperthyroid rabbit heart in the face of upregulated expression and function of SERCA2 predominantly in the JSR compartment.     

Ca transport and heat production in vesicles derived from the sarcoplasmic reticulum terminal cisternae: Regulation by K

In this work, we compared the effect of K⁺ on vesicles derived from the longitudinal (LSR) and terminal cisternae (HSR) of rabbit white muscle. In HSR, K⁺ was found to inhibit both the Ca²⁺ accumulation and the heat released during ATP hydrolysis by the Ca²⁺-ATPase (SERCA1). This was not observed in LSR. Valinomycin abolished the HSR Ca²⁺-uptake inhibition promoted by physiological K⁺ concentrations, but it did not modify the thermogenic activity of the Ca²⁺ pump. The results with HSR are difficult to interpret, assuming that a single K⁺ is binding to either the ryanodine channel or to the Ca²⁺-ATPase. It is suggested that an increase of K⁺ in the assay medium alters the interactions among the various proteins found in HSR, thus modifying the properties of both the ryanodine channel and SERCA1.     

Thermogenic activity of Ca-ATPase from skeletal muscle heavy sarcoplasmic reticulum: The role of ryanodine Ca channel

The sarcoplasmic reticulum Ca²⁺ ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca²⁺ transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca²⁺ channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (ΔH^cal) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg²⁺ or ruthenium red, conditions that close the ryanodine Ca²⁺ channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the ΔH^cal values of ATP hydrolysis increased significantly. Neither Mg²⁺ nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the ΔH^cal of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca²⁺ channel is opened or closed.     

Abnormal ryanodine receptor channels in malignant hyperthermia.

Previous studies have demonstrated a defect associated with the calcium release mechanism of sarcoplasmic reticulum (SR) from individuals susceptible to malignant hyperthermia (MH). To examine whether SR calcium release channels were indeed altered in MH, SR vesicles were purified from normal and MH susceptible (MHS) porcine muscle. The Ca2+ dependence of calcium efflux rates from 45Ca2(+)-filled SR vesicles was then compared with the Ca2+ dependence of single-channel recordings of SR vesicles incorporated into planar lipid bilayers. The rate constants of 45Ca2+ efflux from MHS SR were two to threefold larger than from normal SR over a wide range of myoplasmic Ca2+. Normal and MHS single channels were progressively activated in a similar fashion by cis Ca2+ from pCa 7 to 4. However, below pCa 4, normal channels were inactivated by cis Ca2+, whereas MHS channels remained open for significantly longer times. The altered Ca2+ dependence of channel inactivation in MHS SR was also evident when Ca2+ was increased on the trans side while cis Ca2+ was held constant. We propose that a defect in a low-affinity Ca2+ binding site is responsible for the altered gating of MHS SR channels. Such a defect could logically result from a mutation in the gene encoding the calcium release channel, providing a testable hypothesis for the molecular basis of this inherited disorder.     

Regulation of the apolipoprotein B in heterozygous hypobeta-lipoproteinemic knock-out mice expressing truncated apoB,B81. Low production and enhanced clearance of apoB cause low levels of apoB

Meat quality of pigs is dependent on biochemical and biophysical processes in the time course post mortem (p.m.) and is associated with the intracellular Ca2+ homeostasis. However, there is little known about changes in the Ca2+ transporting proteins controlling the Ca2+ uptake of sarcoplasmic reticulum (SR) in the time course p.m. In this study changes in the Ca2+ transporting proteins were investigated in homogenates of longissimus muscles of 4 malignant hyperthermia susceptible (MHS) and 6 malignant hyperthermia resistant (MHR) Pietrain pigs. Muscle samples were obtained at different time intervals: biopsy 2 h prior slaughtering and from the carcass immediately after exsanguination (0 h), 45 min, 4 h, and 22 h p.m. The SR Ca2+ uptake rate was measured immediately after homogenization with closed calcium release channel (CRC), with opened CRC and without manipulation of CRC. Additionally the SR Ca2+ ATPase activity was determined.The results show: (i) The ability of SR to sequester Ca2+ declined to about 60% in the first 45 min p.m. in MHS samples irrespective of CRC state, whereas in MHR samples this decline was about 5%; (ii) Ca2+ uptake and Ca2+ ATPase activity were not different between the biopsy and 0 h samples, i.e. the stress of slaughter was of no immediate influence; (iii) The Ca2+ ATPase activity of the SR declined at about the same rate as the Ca2+ uptake in both MHS and MHR pig samples in the course of time p.m.; (iv) In samples, taken immediately after exsanguination, the Ca2+ ATPase activity of MHS pigs was higher than that of MHR pigs. However, in samples taken 4 h p.m. Ca2+ ATPase activity of MHS pigs has declined to about 30% of the value at 0 h; (v) The CRC can be closed and opened in all samples up to 22 h p.m. and seems to be fully functional at all sampling times; (vi) The CRC of MHS pigs is almost fully open, whereas the CRC of MHR pigs is only partially open at all sampling times; (vii) The permeability of the SR membrane to Ca2+ (determined as the ratio of SR Ca2+ ATPase with and without ionophore A23187) is the same in both MHS and MHR and did not change with ongoing time; (viii) No uncoupling of uptake from ATP hydrolysis occurred up to 4 h p.m., but the coupling differed between MHS and MHR for all time intervals with lower values for MHS pigs. The results suggest that the decreasing Ca2+ uptake rate of homogenates, sampled at different times p.m., is essentially caused by changes in the Ca2+ pump and not by changes in the CRC or an increased phospholipid membrane permeability to Ca2+.     

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.