Sarcoplasmic Reticulum Vesicle Preparations Research Articles

Greater Susceptibility of the Sarcoplasmic Reticulum to H2O2 Injuries in Diaphragm Muscle from mdx Mice

The aim of the present study was to investigate the direct effects of a reactive oxygen species, H(2)O(2), on the contractile function and sarcoplasmic reticulum properties of dystrophin-deficient diaphragm using chemically skinned fibers and sarcoplasmic reticulum vesicle preparations. The results obtained using Triton X-100-skinned fibers demonstrate that exposure to 1 mM H(2)O(2) had similar effects on the maximal Ca(2+)-activated tension and on the Ca(2+) sensitivity of the contractile apparatus of diaphragm fibers in Bl10 and mdx mice. The effects of H(2)O(2) were also assessed on sarcoplasmic reticulum function using saponin-skinned fibers and sarcoplasmic reticulum vesicle preparations. We found that H(2)O(2) induced changes in sarcoplasmic reticulum properties, particularly in the Ca(2+) pump function. The most important finding was that diaphragm muscle from mdx mice displayed increased sensitivity to the oxidant. Furthermore, in isolated superfused diaphragm muscle from mdx mice, the data demonstrate that the amount of superoxide anion produced under fatiguing conditions was increased. Our study shows that the sarcoplasmic reticulum, and the Ca(2+) pump in particular, in dystrophin-deficient muscles display increased susceptibility to H(2)O(2) injuries. This suggests that free radicals might, therefore, be involved in the pathophysiological pathway and dysregulation of Ca(2+) homeostasis of muscular dystrophy.

Impaired Ca2+ Store Functions in Skeletal and Cardiac Muscle Cells from Sarcalumenin-deficient Mice

Sarcalumenin (SAR), specifically expressed in striated muscle cells, is a Ca2+-binding protein localized in the sarcoplasmic reticulum (SR) of the intracellular Ca2+ store. By generating SAR-deficient mice, we herein examined its physiological role. The mutant mice were apparently normal in growth, health, and reproduction, indicating that SAR is not essential for fundamental muscle functions. SAR-deficient skeletal muscle carrying irregular SR ultrastructures retained normal force generation but showed slow relaxation phases after contractions. A weakened Ca2+ uptake activity was detected in the SR prepared from mutant muscle, indicating that SAR contributes to Ca2+ buffering in the SR lumen and also to the maintenance of Ca2+ pump proteins. Cardiac myocytes from SAR-deficient mice showed slow contraction and relaxation accompanied by impaired Ca2+ transients, and the mutant mice exhibited a number of impairments in cardiac performance as determined in electrocardiography, ventricular catheterization, and echocardiography. The results obtained demonstrate that SAR plays important roles in improving the Ca2+ handling functions of the SR in striated muscle.

Peptide and protein modulation of local Ca2+ release events in permeabilized skeletal muscle fibers

Local discrete elevations in myoplasmic Ca2+ (Ca2+ sparks) arise from the opening of a small group of RyRs. Summation of a large number of Ca2+ sparks gives rise to the whole cell Ca2+ transient necessary for muscle contraction, Unlike sarcoplasmic reticulum vesicle preparations and isolated single channels in artificial membranes, the study of Ca2+ sparks provides a means to understand the regulation of a small group of RyRs in the environment of a functionally intact triad and in the presence of endogenous regulatory proteins. To gain insight into the mechanisms that regulate the gating of RyRs we have utilized laser scanning confocal microscopy to measure Ca2+ sparks in permeabilized frog skeletal muscle fibers. This review summarizes our recent studies using both exogenous (ImperatoxinA and domain peptides) and endogenous (calmodulin) modulators of RyR to gain insight into the number of RyR Ca2+ release channels underlying a Ca2+ spark, how domain-domain interactions within RyR regulate the functional state of the channel as well as gating mechanisms of RyR in living muscle fibers.

Regulation of Ca 2+ transport by sarcoplasmic reticulum Ca 2+-ATPase at limiting [Ca 2+

The factors regulating Ca 2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca 2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca 2+] at approximately 10 μM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca 2+] of 100 to 200 nM, and has been defined as [Ca 2+] lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca 2+ fluophores, where it is presumed that sarcoplasmic free [Ca 2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca 2+] lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K 0.5 value for histidine was 3 mM, suggesting that residual [Ca 2+] lim fluorescence is due to Zn 2+. The level of Zn 2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47±0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254–263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca 2+] lim of 49±1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible ‘pump/leak’ mechanism was tested by the effects of varying active Ca 2+ transport 1 to 2 orders with temperature and pH. [Ca 2+] lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca 2+] lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca 2+] lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca 2+] lim was estimated by the unidirectional flux of 45Ca 2+, and directly, following depletion of ATP, by measuring release of 40Ca 2+, and was 0.02% of V max. Constant infusion of CaCl 2 at [Ca 2+] lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca 2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were V max=853 nmol/min per mg, K 0.5(Ca)=279 nM, and n H(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca 2+ transients, and in the relaxed state, will minimise contribution of Zn 2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca 2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca 2+] lim in the resting state is a characteristic feature of Ca 2+ pump function, rather than a balance between active transport and passive leakage pathways.

Isolation and characterization of the Mg 2+-ATPase from rabbit skeletal muscle sarcoplasmic reticulum membrane preparations

Preparations of sarcoplasmic reticulum vesicles, obtained according to the method of Eletr and Inesi (Biochim. Biophys. Acta (1972) 282, 174), contained both Mg 2-ATPase and Ca 2+,Mg 2+-ATPase activity. The two enzymes were solubilized by a mixture of digitonin and lysophosphatidylcholine and separated on a DEAE-cellulose column eluted with a discontinuous gradient of NaCl. The Mg 2+-ATPase activity was eluted with 0.43 M NaCl. The Ca 2+,Mg 2+-ATPase was obtained by increasing the NaCl concentration of the elution medium to 0.40 M. The fraction eluted with 0.043 M NaCl was insensitive to micromolar concentrations of calcium, resistant to oligomycin, ouabain, orthovanadate and thiocyanate, and was inhibited by low concentrations of Triton X-100. The enzyme showed a single apparent K m for MgATP in the range of 0.2 mM and a V m of 2.9 μmol P l • min −1 • mg −1 protein. Activity was maximal over a broad peak between pH 6.0–8.0. Hydrolysis of ATP was unaffected by dimethylsulfoxide concentrations up to 20% ( v v ) and was inhibited at higher concentrations. The enzyme was not phosphorylated by either 32P i or [γ- 32P]ATP at significant levels when compared with the Ca 2+,Mg 2+-ATPase in an EGTA-containing medium. The kinetic pattern of the Mg 2+-ATPase was distinctly different from that of the Ca 2+,Mg 2+-ATPase under the same conditions. The fraction eluted from the DEAE-cellulose column was subjected to electrophoresis under non-denaturing conditions. Only one band with Mg 2+-ATPase activity was detected. The Mg 2+-ATPase migrated much slower than the Ca 2+,Mg 2+-ATPase under non-denaturing conditions, whereas both enzymes had a molecular mass of 105 kDa on SDS gel electrophoresis.

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.

Type B monoamine oxidase and functions of Ca2+, Mg2+-dependent adenosinetriphosphatase in preparations from sarcoplasmic reticulum vesicles

Oxidative deamination of β-phenylethylamine or benzylamine by type B monoamine oxidases (MAO) in preparations of sarcoplasmic reticulum vesicles from rabbit skeletal muscles is accompanied by inhibition both of active Ca2+ transport into the vesicles and of the activity of Ca2+, Mg2+-dependent ATPase, which is preventable by deprenil, a specific inhibitor of type B MAO. Aldehydes formed during enzymatic deamination of substrates of type B MAO may perhaps participate in the regulation of Ca2+, Mg2+-dependent ATPase, activity.