Sarcoplasmic Reticulum System Research Articles

Lipid oxidation in a chicken muscle model system: oxidative response of lipid classes to iron ascorbate or methemoglobin catalysis.

Catalysis by iron ascorbate and activated methemoglobin generated different oxidative responses in chicken muscle model systems. In iron ascorbate systems, large increases in hydroperoxides and thiobarbituric acid-reactive substances (TBARS) occurred during the initial stage of incubation. Thereafter, iron ascorbate catalysis led to a slow increase in the oxidation of triacylglcyerol (TG) and sarcoplasmic reticulum (SR) membrane lipids. By the end of incubation, 24, 36, and 32% of the initial content of n-3 fatty acids in free fatty acids, TG, and SR single-lipid model systems catalyzed by iron ascorbate had been lost. Reduced losses of n-3 fatty acids were observed in the SR and TG fractions (0 and 24%, respectively) when iron ascorbate model systems contained all three lipid fractions (mix). Hydroperoxides and TBARS in model systems catalyzed by activated methemoglobin were characterized by a lag phase during most of the incubation. Consistent with their role as antioxidants, losses of alpha-tocopherol (42-49%), gamma-tocopherol (36-42%), and protein sulfhydryls (41-52%) were observed in model systems catalyzed by activated methemoglobin. SR and mix model systems were 30-50% slower to oxidize than TG model systems when activated methemoglobin served as the catalytic agent.

Ultra‐high‐resolution scanning electron microscopy of the sarcoplasmic reticulum of the rat atrial myocardial cells

Background The sarcoplasmic reticulum (SR) of mammalian ventricular and atrial muscles share common features but also differ because T-tubules are rare and extended junctional SR is exclusively seen in the atrium. This scanning electron microscope (SEM) study was undertaken to clarify the three-dimensional organization of the rat atrial SR system. Specific preparations were examined with an ultra-high-resolution SEM. Methods Fixed right rat atria were frozen, fractured, and macerated by the aldehyde-osmium-DMSO-osmium method to remove myofibrils and cytoplasmic matrix. Left exposed were mitochondria, SR, and sarcolemma. Dried specimens were then impregnated by osmium-hydrazine and examined without metal coating. Results In place of conventional T-tubules, a prominent type of sarcotubules, Z-tubules, were found at the Z-line level. Branches from these tubules joined the cisternal SR, which was 100–300 nm in diameter and localized near the Z-line, and formed extensive SR meshworks and polygonal patches. Bulbous swellings, the corbular SR, were also evident. Sarcotubular reticulum completely surrounded each myofibril. The inter-myofibrillar SR, especially Z-tubules, joined the peripheral subsarcolemmal SR, which was also arranged as a meshwork and was closely apposed to the sarcolemma. Conclusions These SEM observations confirm the organization of the rat atrial SR system and present new, detailed, three-dimensional images of Z-tubules, cisternal SR, extended junctional SR, and peripheral SR, which provide further structural insight. Anat Rec 248:70–75, 1997. © 1997 Wiley-Liss, Inc.

Ultra-high-resolution scanning electron microscopy of the sarcoplasmic reticulum of the rat atrial myocardial cells

The sarcoplasmic reticulum (SR) of mammalian ventricular and atrial muscles share common features but also differ because T-tubules are rare and extended junctional SR is exclusively seen in the atrium. This scanning electron microscope (SEM) study was undertaken to clarify the three-dimensional organization of the rat atrial SR system. Specific preparations were examined with an ultra-high-resolution SEM. Fixed right rat atria were frozen, fractured, and macerated by the aldehyde-osmium-DMSO-osmium method to remove myofibrils and cytoplasmic matrix. Left exposed were mitochondria, SR, and sarcolemma. Dried specimens were then impregnated by osmium-hydrazine and examined without metal coating. In place of conventional T-tubules, a prominent type of sarcotubules, Z-tubules, were found at the Z-line level. Branches from these tubules joined the cisternal SR, which was 100-300 nm in diameter and localized near the Z-line, and formed extensive SR meshworks and polygonal patches. Bulbous swellings, the corbular SR, were also evident. Sarcotubular reticulum completely surrounded each myofibril. The intermyofibrillar SR, especially Z-tubules, joined the peripheral subsarcolemmal SR, which was also arranged as a meshwork and was closely apposed to the sarcolemma. These SEM observations confirm the organization of the rat atrial SR system and present new, detailed, three-dimensional images of Z-tubules, cisternal SR, extended junctional SR, and peripheral SR, which provide further structural insight.

Characterization of the sarcoplasmic reticulum proteins in the thermogenic muscles of fish.

Marlins, sailfish, spearfishes, and swordfish have extraocular muscles that are modified into thermogenic organs beneath the brain. The modified muscle cells, called heater cells, lack organized myofibrils and are densely packed with sarcoplasmic reticulum (SR), transverse (T) tubules, and mitochondria. Thermogenesis in the modified extraocular muscle fibers is hypothesized to be associated with increased energy turnover due to Ca2+ cycling at the SR. In this study, the proteins associated with sequestering and releasing Ca2+ from the SR (ryanodine receptor, Ca2+ ATPase, calsequestrin) of striated muscle cells were characterized in the heater SR using immunoblot and immunofluorescent techniques. Immunoblot analysis with a monoclonal antibody that recognizes both isoforms of nonmammalian RYRs indicates that the fish heater cells express only the alpha RYR isoform. The calcium dependency of [3H]ryanodine binding to the RYR isoform expressed in heater indicates functional identity with the non-mammalian alpha RYR isoform. Fluorescent labeling demonstrates that the RYR is localized in an anastomosing network throughout the heater cell cytoplasm. Measurements of oxalate supported 45Ca2+ uptake, Ca2+ ATPase activity, and [32P]phosphoenzyme formation demonstrate that the SR contains a high capacity for Ca2+ uptake via an ATP dependent enzyme. Immunoblot analysis of calsequestrin revealed a significant amount of the Ca2+ binding protein in the heater cell SR. The present study provides the first direct evidence that the heater SR system contains the proteins necessary for Ca2+ release, re-uptake and sequestration, thus supporting the hypothesis that thermogenesis in the modified muscle cells is achieved via an ATP-dependent cycling of Ca2+ between the SR and cytosolic compartments.

Calcium transport activity of rat cardiac sarcoplasmic reticulum isolated in the presence of dithiothreitol

We examined the effect of dithiothreitol (DTT) on the isolation and calcium transport properties of rat cardiac sarcoplasmic reticulum (SR). DTT at a concentration of 4 mM was included in the homogenization/initial isolation and the high-salt (KCI) buffers used in the preparation of the SR fraction. The inclusion of DTT in the SR isolation media did not affect the total amount of SR protein isolated from ventricular tissue. However, SR isolated in the presence of DTT exhibited a significantly increased ( p < 0.05) rate of calcium transport at free calcium concentrations ranging from 0.1 μm up to 2.0 μm, and a 26% increase in the apparent affinity of the SR calcium transport system for Ca 2+. DTT included in the calcium-transport reaction medium did not significantly alter the rate of SR calcium transport in SR preparations isolated in the absence or presence of DTT. These results demonstrate that functionally important sulfhydryl groups of the SR calcium transport pump may be oxidized during the isolation of the membrane fraction and that such oxidation can be reduced by including DTT in the isolation buffers.

Effect of continuous vasopressin infusion on circadian rhythms of food and water intake, diuresis, and electrolyte excretion in brattleboro rats

In the experiments on rats a significant decrease of resistance to the contracture and arrhythmogenic effect of increased Ca2+ on the isolated hearts of animals after short term immobilization stress has been shown. After a long term stress (6 hours) the changes of the studied physiological parameters of the isolated hearts were not observed. Transport in the homogenates of the rat hearts was measured by the Ca--selective electrode on the ionometer ORION EA 940. After a 1-hour stress the rate of Ca--transport in sarcoplasmic reticulum (SR) substantially decreased as compared with the control (3.5 fold at 20 mcM), but at the higher concentration the Ca-transport was inhibited. The long term stress lead to a slower damage of functional Ca-transport system of SR--at 20 mcM the rate decreased by 25% as compared with control and at the higher concentration of Ca2+ depression of transport was not observed.

A comparative study of th Ca 2+-Mg 2+ dependent ATPase from skeletal muscles of young, adult and old rats

Sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle of Sprague—Dawley rats ranging in age from 4 months to 28 months were studied and comapred. A marked decline, with age, was observed in the amount of (total) SR proteins isolated per gram of muscle tisue used. This decline is in line with the known loss of muscle fiber mass and size with advancing age; however, whether the magnitudes of these two effects are indeed identical, remains to be studied. In contrast, no analogous age-related change was detected in the amount of SR protein per unit mass of rat cardiac muscle. The calcium contents, per mg protein, in SR vesicles isolated from rats of all age groups studied did not differ significantly, and represented only a small fraction of the total capacity of the vesicles for this cation. This capacity was found to decline at old age and this effect, combined with the age-related decrease in the concentration of SR proteins in the tissue, indicate a significant decline in calcium sequestration ability in old muscle. Both basal (Ca 2+ independent) and calcium stimulated ATPase activities were found not to be affected by age. In contrast, the efficiency of Ca 2+ transport across the SR membrane, as reflected by the number of calcium ions pumped into the vesicles per ATP cleaved, declined from a value of 0.37 at 3–4 months to 0.15 at 24 months. This change may represent an age-related reduction in the fraction of coupled SR vesicles, possibly due to alterations in the membrane. SR vesicle preparations from both young and old rats displayed strongly biphasic inactivation kinetics when incubated at 37°C. This may reflect the heterogeneity of muscles in the tissue used, or be due to the presence of a mixture of coupled and uncoupled vesicles in the SR preparations. The rate of the first step in the ATPase inactivation, in which about 75% of the activity is lost, was found to be affected by age, the old SR vesicles being markedly more labile than their young counterparts. In contrast, no difference was detected between the inactivation kinetics of young and old ATPase proteins dissolved in Triton X 100 and the inactivation was monophasic down to less than 6% of the original activity. These results indicate that the age-related modifications in the stability of the SR calcium pump system involve the membrane but not the ATPase protein. The inactivation of the SR ATPase is believed to proceed via dissociation of the dimeric enzyme to (unstable) subunits. Its is therefore likely that changes in the SR membrane in old muscle render the ATPase more dissociable.

Identification of a Ca 2+-ATPase in cerebellar Purkinje cells

The expression of a sarcoplasmic reticulum (SR)-like Ca 2+-ATPase was studied in the adult chicken cerebellum. A monoclonal antibody, CaS/Cl-IgG, specific for the cardiac/slow-twitch skeletal muscle SR Ca 2+-ATPase, was used as a probe of protein expression. An immunoblot analysis showed that CaS/Cl-IgG recognized similar size polypeptides in adult chicken heart and cerebellum. CaS/Cl-IgG recognized fragments of similar size after limited tryptic digestion of cardiac and cerebellar membranes. A two-dimensional α-chymotryptic peptide map analysis demonstrated that the cardiac and cerebellar Ca 2+-ATPases were structurally very similar. Immunofluorescence microscopy localized the cerebellar Ca 2+-ATPase to Purkinje cell bodies and dendritic trees. These results suggest that the well-known Ca 2+ uptake system of skeletal and cardiac muscle SR has a remarkably similar counterpart in some neurons.

Cardiac sarcoplasmic reticulum characteristics in hypertrophic hearts from spontaneously hypertensive rats.

Sarcoplasmic reticulum (SR) from left ventricles of rats that developed spontaneous hypertension was studied in vitro. Similar increases of left ventricular mass were found when grouping the animals into mild and severe hypertensives (average systolic arterial pressure of 168 +/- 4 and 202 +/- 6 mmHg, respectively). The amount of SR protein (mg/g of left ventricle) was higher when obtained from hypertrophic ventricles of both hypertensive groups than from ventricles of the control group. The result agreed with the enhanced Ca2+ uptake exhibited by left ventricular homogenates of the hypertensive groups. Consequently, Ca2+ uptake in SR microsomes isolated per gram of left ventricle (nmol Ca2+/g muscle) was 51.62 +/- 10.06 and 64.99 +/- 12.84 in mildly and severely hypertensive groups vs. 17.37 +/- 5.79 in the control group (P less than 0.05). The SR microsomes obtained from ventricles of hypertensive rats showed an enhanced Ca2+ activated ATPase activity that was not accompanied by increased Ca2+ uptake at saturating calcium concentrations, but by increased affinity for calcium (K'app. of 1.09 +/- 0.28 and 2.67 +/- 0.16 microM in SR microsomes of hypertrophic and control ventricles respectively; P less than 0.05). The rates of calcium loss, measured in SR vesicles passively loaded with 45Ca, were similar when assayed in SR obtained from ventricles of both hypertensive and normal rats. These results enable us to suggest that in hearts of rats presenting spontaneous hypertension, the function of the SR system could account for a normal handling of cytosolic calcium. They might support the absence of mechanical alterations described in hearts of young rats of the SHR strain.

Biochemical alterations in heart after exhaustive swimming in rats.

This study investigated alterations in glycogen, catecholamines, and the function of various subcellular membranes of the heart after exhaustive swimming in rats. The rats were exhausted by daily exercise over 1, 3, or 7 consecutive days. Glycogen content of the heart and three selected skeletal muscles was depleted after a single bout of exhaustive exercise. Repeated bouts of exhaustive swimming elicited a depletion of glycogen in only the plantaris and gastrocnemius skeletal muscles. Plasma norepinephrine and epinephrine levels were highly elevated, and cardiac concentrations of these hormones were significantly depleted immediately after all exercise sessions. Cardiac sarcoplasmic reticulum (SR) Ca2+ transport was depressed after a single exhaustive exercise period. After three exercise bouts SR Ca2+ accumulation remained depressed; however, mitochondrial Ca2+ transport was found to be augmented. If the exhaustive exercise protocol was continued up to seven days, only mitochondrial Ca2+ accumulation was depressed. Various parameters of sarcolemmal membrane function were observed to be unaltered after exhaustive exercise. These findings demonstrate that exhaustive swimming exercise in rats is capable of producing significant alterations in the Ca2+ transport capacity of the SR and mitochondrial membrane systems of the heart but is without apparent effect on the sarcolemmal membrane.

Resolution of Ca++-ATPase of sarcoplasmic reticulum into subunits.

The Ca++-ATPase system of sarcoplasmic reticulum (SR) was resolved into several subunits by isoelectric focusing and isotachophoresis in acrylamide gels. The results obtained support the concept that the ATPase system of SR is oligomeric forming a tetramer of 100,000 mol.wt subunits.