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At least 19 recordsLinked to original sources
PubMed1992

The endoplasmic reticulum-sarcoplasmic reticulum connection: distribution of endoplasmic reticulum markers in the sarcoplasmic reticulum of skeletal muscle fibers.

The skeletal muscle sarcoplasmic reticulum (SR) was investigated for the presence of well-known endoplasmic reticulum (ER) markers: the lumenal protein BiP and a group of membrane proteins recognized by an antibody raised against ER membrane vesicles. Western blots of SR fractions revealed the presence of BiP in fast- and slow-twitch muscles of the rabbit as well as in rat and chicken muscles. Analyses of purified SR subfractions, together with cryosection immunofluorescence and immunogold labeling, revealed BiP evenly distributed within the longitudinal SR and the terminal cisternae. Within the terminal cisternae BiP appeared not to be mixed with calsequestrin but to be distributed around the aggregates of the latter Ca2+ binding protein. Of the various membrane markers only calnexin (91 kDa) was found to be distributed within both SR subfractions, whereas the other markers (apparent molecular masses of 64 kDa and 58 kDa and a doublet around 28 kDa) were concentrated in the terminal cisternae. These results suggest that the SR is a specialized ER subcompartment in which general markers, such as the ones we have investigated, coexist with the major SR proteins specifically responsible for Ca2+ uptake, storage, and release. The differential distribution of the ER markers reveals new aspects of the SR molecular structure that might be of importance for the functioning of the endomembrane system.

Animals↗
PubMed1993

The endoplasmic reticulum-sarcoplasmic reticulum connection. II. Postnatal differentiation of the sarcoplasmic reticulum in skeletal muscle fibers.

The postnatal differentiation of sarcoplasmic reticulum (SR) of rabbit skeletal muscles (the slow-twitch soleus and the fast-twitch adductor muscles) was monitored between Days 1 and 12 by following on Western blots the expression and accumulation of molecular markers specific not only for the muscle endomembrane system, i.e., calsequestrin (CS) and the ryanodine-sensitive Ca2+ release channel, but also for the endoplasmic reticulum (ER) at large, i.e., BiP, calnexin (CN) and calreticulin. Our results demonstrate that SR development, documented by the increase of the SR fractional volume, terminal cisternae proliferation, and reorientation of triads, is accompanied by the accumulation of the SR-specific proteins and also of CN, with no change of the other ER general markers. Moreover, the distribution of two of the markers, BiP and CS, was investigated by immunocytochemistry at both the light and the electron microscope level. At Day 1 CS was found to be concentrated both within the few recognizable triad terminal cisternae and within the lumen of numerous, apparently discrete cisternae and tubules, widely scattered throughout both the contractile and the subplasmalemmal areas of the cytoplasm. These structures remain evident until Day 12, when most triad junctions have acquired proper configuration, composition and orientation. BiP, on the other hand, appears widely distributed within the ER/SR of the fibers. From the early stages of postnatal development it does colocalize with the Ca2+ binding protein in the lumen of the CS-rich structures and appears also within the longitudinal SR and the conventional ER cisternae.

Animals↗
PubMed1996

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus.

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca2+-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca2+-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca2+. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca2+-activated K+ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca2+ influx was triggered through the Na+/Ca2+ exchanger and the other Ca2+ entry pathways were blocked. Thapsigargin, another specific SR Ca2+-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca2+-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca2+ influx presumably via nifedipine-sensitive Ca2+ channels. This study suggests the important role of SR Ca2+-ATPase in the modulation of the muscle tone and the function of SR as a "buffer barrier" to Ca2+ entry in the cat gastric fundus smooth muscle.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1976

Association of gylcogenolysis with cardiac sarcoplasmic reticulum.

Sarcoplasmic reticulum fragments isolated from dog cardiac muscle possess a calcium-accumulating system associated with a series of enzymes linked to glycogenolysis. These enzymes include: adenylate cyclase, cyclic AMP-dependent protein kinase, phosphorylase b kinase, phosphorylase (b/a, 30/1),"debrancher" enzyme, and glycogen (0.3 to 0.7 mg/mg of protein). The sarcoplasmic reticulum preparation produced glucose 1-phosphate and glucose from either endogenous or exogenous glycogen. Both the calcium-accumulating and glycogenolytic enzymes sediment in a single peak at 33% sucrose on a linear continous sucrose density gradient, and the complex remains intact throughout repeated washing. Glycogen particles appear to be associated with the sarcoplasmic reticulum in situ as well as in the isolated microsomal fraction. The sarcoplasmic reticulum-glycogenolytic complex, monitored by a linked enzyme spectrophotometric assay, shows several features: (a) activation of phosphorylase activity to peak rate occurs over a very rapid time course which cannot be duplicated using combinations of purified enzymes; (b) activation is inhibited by protein kinase inhibitor; (c) phosphorylase b functions as in the purified form with respect to AMP (Km, 0.3 mM); (d) in the presence of limiting amounts of glycogen, optimal phosphorylase b activity in the sarcoplasmic reticulum requires the presence of debrancher, and the activity is sensitive to inhibitors of that enzyme such as Tris, which suggests the possiblity that the enzymes bear a specific structual relationship to the glycogen present. Phosphorylase b leads to a activation in the sarcoplasmic reticulum was completely resistant to ethylene glycol bis(beta-aminoethyl either)-N,N'-tetraacetic acid (EGTA). Inhibition of calcium accumulation by or release of bound calcium from sarcoplasmic reticulum by X537A (RO 2-2985) did not alter the EGTA resistance. These results suggest that cardiac sarcoplasmic reticulum is a complex organelle containing functions that may be related to excitation-contraction coupling and intermediary metabolism.

Adenosine Monophosphate↗
PubMed1981

A proton gradient controls a calcium-release channel in sarcoplasmic reticulum.

Sarcoplasmic reticulum vesicles from mammalian skeletal muscle have previously been shown to develop a proton gradient (alkaline inside) of 0.15-0.5 pH units during active Ca2+ uptake. We found that dissipation of this gradient by the proton ionophores gramicidin, nigericin, and carbonyl cyanide p-trichloromethoxyphenylhydrazone caused a rapid transient tension in skinned rabbit psoas muscle fibers. Increases, but not decreases, in medium pH of approximately 0.2 units over the range from pH 6.5 to pH 7.5 also elicited transient tensions. In isolated vesicles, physiological levels of Ca2+ (3.3 microM), inhibited pH-induced Ca2+ release. Dicyclohexylcarbodiimide blocked pH- and ionophore-induced Ca2+ release under conditions in which it could bind to sarcoplasmic reticulum proteins but did not inhibit Ca2+ uptake. We propose that a proton gradient generated across sarcoplasmic reticulum membranes during Ca2+ uptake maintains a Ca2+ release channel in a closed conformation and that dissipation of this gradient permits the Ca2+ release channel to open. We further propose that elevated myoplasmic Ca2+ also causes the Ca2+ channel to close, permitting Ca2+ uptake through Ca2+/Mg2+-ATPase to function effectively. As the proteolipids of sarcoplasmic reticulum bind dicyclohexylcarbodiimide under conditions in which Ca2+ release is blocked and as they have previously been shown to have Ca2+ ionophoric activity, we propose that the Ca2+-release channel either resides in the proteolipids or is controlled by H+ fluxes through the proteolipids.

Animals↗
PubMed1966

Studies on the in vitro interaction of electrical stimulation and Ca++ movement in sarcoplasmic reticulum.

Sarcoplasmic reticulum fragments (S.R.F.) were isolated from skeletal and heart muscles. These fragments were found to take up Ca(++) very actively from media. When monophasic square waves were passed through the S.R.F. suspension, the Ca(++) uptake by S.R.F. was decreased. When the suspension was stimulated electrically after the Ca(++) was taken up by S.R.F., the initiation and the cessation of the stimulation were followed by the release and re-uptake of Ca(++) by S.R.F., respectively. The degree of inhibition of the Ca(++) uptake as well as of the Ca(++) release by electrical stimulation was dependent on the voltage and the frequency of stimulation. The presence of inorganic phosphate or oxalate modified the influence of electrical stimulation on the release and the uptake of Ca(++) by S.R.F. Attempts were made to observe the release of Ca(++) by electrical stimulation from unfractionated sarcoplasmic reticulum remaining in myofibers, and the interaction of the released Ca(++) with myofibrils in vitro. For this purpose, the glycerol-extracted fiber was selected as a muscle model, since it contains both sarcoplasmic reticulum and myofibrils. It was found that electrical stimulation of skeletal and heart glycerol-extracted fibers resulted in the contraction of fibers. It appeared that the contraction of glycerol fibers by electrical stimulation was caused by the Ca(++) release from sarcoplasmic reticulum by stimulation.

Adenosine Triphosphate↗
PubMed1986

Fluorescence conformational probe study of calcium release from sarcoplasmic reticulum.

Sarcoplasmic reticulum isolated from rabbit skeletal muscle was labeled with a limited (0.625 nmol/mg sarcoplasmic reticulum protein) amount of the fluorescent thiol reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The fluorescence intensity of the membrane-attached DACM decreased concurrently with (Ca2+ and caffeine)-induced Ca2+ release, depolarization-induced Ca2+ release and Ca2+-dependent dependent passive efflux of Ca2+. The decreased DACM fluorescence level initiated by a Ca2+ jump was subsequently reversed under passive efflux conditions when there was no ATP-dependent Ca2+ uptake, suggesting spontaneous closing of the channels. Therefore, the higher fluorescence level corresponds to a larger population of closed channels, whereas the lower level represents a larger population of opened channels. Under conditions when the Ca2+ release-coupled fluorescence change was maximal, a stoichiometric incorporation of DACM took place only into a 32-kDa protein. Furthermore, reconstituted vesicles, in which purified DACM-labeled 32-kDa protein was incorporated into unlabeled sarcoplasmic reticulum vesicles, were capable of both (Ca2+ and caffeine)-induced Ca2+ release and the release-coupled DACM fluorescence change. These results suggest that the 32-kDa protein is a constituent of the Ca2+ release channel or a protein which is in close contact with the channel.

Adenosine Triphosphate↗
PubMed1976

Synthesis of adenosine triphosphate during release of intravesicular and membrane-bound calcium ions from passively loaded sarcoplasmic reticulum.

Sarcoplasmic reticulum isolated from rabbit skeletal muscle and incubated in a medium containing Ca2+ in the absence of ATP retains intravesicular and/or membrane-bound Ca2+. The synthesis of ATP coupled with the release of intravesicular Ca2+ is totally inhibited by the ionophore X-537A. Release of the membrane-bound Ca2+, retained after short periods of incubation (10min) or after release of the intravesicular Ca2+ by ionophore X-537A, still supports some synthesis of ATP. The ratios of Ca2+ released to ATP synthesized are 2.5-3.2, when bound and intravesicular Ca2+ are released simultaneously, and 3.1-4.0, when only bound Ca2+ is released. The results show that the synthesis of ATP by sarcoplasmic reticulum during release of passively accumulated Ca2+ by EGTA [ethanedioxybis(ethylamine)tetra-acetic acid] is accompanied by a loss of membrane-bound Ca2+.

Adenosine Diphosphate↗
PubMed1988

Tryptic cleavage inhibits but does not uncouple Ca2+ATPase of sarcoplasmic reticulum.

Sarcoplasmic reticulum Ca2+ATPase is cleaved by trypsin at two sites, T1 and T2. Cleavage at T1 is complete, whereas only about 50% of the Ca2+ATPase is digested at the T2 site. In the absence of Ca2+ ionophor, Ca2+-ATPase activity of the digested enzyme remains virtually unchanged. In the presence of Ca2+ ionophor, however, the calculated specific activity of the doubly cleaved Ca2+ATPase is decreased by about 40%. The decrease in Ca2+ transport activity is much more rapid than cleavage of the T2 site, and could be correlated with an increased leak of Ca2+ from the digested vesicles. We obtained evidence that this leakiness is independent of the digestion of the Ca2+ATPase itself and is presumably due to the digestion of some other components of the sarcoplasmic reticulum vesicles. Examination of steady-state phosphoenzyme levels resulting from phosphorylation by ATP and Pi, or dephosphorylation by ADP or ADP/EGTA revealed no difference between the digested and the undigested Ca2+ATPase indicating no change in the equilibria caused by the T2 cleavage. Analysis of the substrate concentration dependence of the Ca2+ATPase activity also led to the conclusion that the digestion at T2 reduced the Vmax of ATP hydrolysis but leaves the Km unchanged. The above results are consistent with the model that cleavage at the T2 site reduces the turnover rate of the Ca2+ATPase reaction cycle by about 40% by slowing down or altering the rate-limiting step without affecting the equilibrium constants of the examined steps. We found no evidence of true uncoupling of Ca2+ transport from ATP hydrolysis correlated with cleavage at the T2 site.

Adenosine Triphosphate↗
PubMed1981

31P-NMR studies of oriented multilayers formed from isolated sarcoplasmic reticulum and reconstituted sarcoplasmic reticulum.

31P-NMR spectra were obtained from oriented multilayer preparations of normal sarcoplasmic reticulum and reconstituted sarcoplasmic reticulum with lipid to protein ratios varying between 41 : 1 and 110 : 1. The dependence of the 31P-NMr spectra on the alignment of the membranes with respect to the magnetic field was used to draw two conclusions about the motion of the phospholipid molecules that contribute to the observed spectra. First, the phosphate group and the two adjacent methylene groups are able to rapidly rotate (i.e., tau R much less than 10-5 S) around the normal to the plane of the membrane. Second, the restricted internal motion of the phosphate group and the glycerol CH2OP group is very similar to that found in liposomes formed from sarcoplasmic reticulum phospholipids. Calibration experiments showed that all (100 +/- 7%) of the phospholipid molecules in the membrane can be accounted for in the observed spectra. Thus, essentially all the phospholipid molecules in the sarcoplasmic reticulum and the reconstituted sarcoplasmic reticulum membranes have the same motion in the polar headgroup region as found in model bilayer membranes. Since a large fraction of the phospholipid molecules (between one-quarter and one-half, depending on ;the lipid to protein ratio) are immediately surrounding the calcium-pump protein, we conclude that the calcium-pump protein does not perturb the motion of these 'boundary-layer' lipids.

Animals↗
PubMed1977

Subunits of the calcium ion-pump system of sarcoplasmic reticulum.

Sarcoplasmic reticulum membranes with high content of Ca2+ -ATPase (80% of total protein) were dissolved in a non ionic medium and were submitted to isoelectric focusing in polyacrylamide gels. The membrane protein was resolved into six main bands whose isoelectric points range from 6 to 5. The mol. wt. of these peptides is about 100 000 as estimated by second dimension electrophoresis in sodium dodecyl sulfate-polyacrylamide system. The electrophoretic behaviour of the purified ATPase enzyme is similar to that of crude membranes.

Adenosine Triphosphatases↗
PubMed1994

Transmembrane Ca2+ gradient-mediated change of fluidity in the inner layer of phospholipids modulates Ca(2+)-ATPase of sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) vesicles with (1000 folds) or without transmembrane Ca2+ gradient have been prepared. Different fluorescence probes (DPH, TMA-DPH and n-AS), were used to determine the effect of transmembrane Ca2+ gradient on the lipid fluidity both in outer and inner layer of Ca(2+)-ATPase-containing SR vesicles. The results showed that transmembrane Ca2+ gradient could significantly decrease the fluidity of the inner layer of SR membrane, while no obvious change was monitored in the outer layer. This may be deduced that Ca(2+)-ATPase might be modulated mainly by the transmembrane Ca2+ gradient-mediated alteration of physical state of phospholipid in the inner layer of SR membrane.

Animals↗
PubMed1976

Comparative study of the lipid composition of rabbit and lobster sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) membranes isolated from rabbit and lobster muscles have similar phospholipid classes, but they differ in plasmalogen content. The plasmalogenic species are mostly distributed among phosphatidylethanolamines (PE's) and make up about 62% of the total in rabbit SR and about 46% in lobster membranes. Lobster SR phospholipids contain large amounts of polyunsaturated fatty acids which are present in low amounts in rabbit membranes. The total unsaturated fatty acids of phosphatidylcholines (PC's) represent about 53% and 73% of the total fatty chains for rabbit and lobster SR, respectively. The values found for PE's were about 56% and 64%, respectively. Furthermore, lobster membranes contain significant amounts of PC and PE molecular species with unsaturated fatty acids in positions 1 and 2, whereas rabbit SR contain low amounts.

Adenosine Triphosphatases↗
PubMed1997

Lactate inhibits Ca(2+) -activated Ca(2+)-channel activity from skeletal muscle sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) Ca(2+)-release channel function is modified by ligands that are generated during about of exercise. We have examined the effects of lactate on Ca(2+)- and caffeine-stimulated Ca2+ release, [3H]ryanodine binding, and single Ca(2+)-release channel activity of SR isolated from rabbit white skeletal muscle. Lactate, at concentrations from 10 to 30 mM, inhibited Ca(2+)- and caffeine-stimulated nodine binding to and inhibited Ca(2+)- and caffeine-stimulated [3H]ryanodine binding to and inhibited Ca(2+)- and caffeine-stimulated Ca2+ release from SR vesicles. Lactate also inhibited caffeine activation of single-channel activity in bilayer reconstitution experiments. These findings suggest that intense muscle activity, which generates high concentrations of lactate, will disrupt excitation-contraction coupling. This may lead to decreases in Ca2+ transients promoting a decline in tension development and contribute to muscle fatigue.

Animals↗
PubMed1986

Effect of age of winter flounder on some properties of the sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) from winter flounder (Pseudopleuronectes americanus) of various ages was investigated with regard to fluorescent age pigment (FAP), potential of enzymic lipid peroxidation, fatty acid composition, protein pattern and Ca2+-sequestering ability. FAP accumulation, protein pattern and Ca2+-sequestering ability were also evaluated after the SR were incubated in vitro with the peroxidizing cofactors: NADH, ADP and Fe3+. No difference in FAP content was observed in SR isolated from fish of different ages. However, a higher accumulation of FAP in muscle tissue from old fish was observed. A slow but significant increase in FAP in SR was observed when the SR from both young and old fish were incubated with peroxidizing cofactors. It was demonstrated that 4-year-old fish had higher levels of NADH-dependent lipid peroxidation in their SR than 2-year-old fish. No differences in fatty acid composition were observed in the SR of fish aged from 2 to 8 years old. Two species of high-molecular-weight protein were found to increase in SR isolated from aging fish. These species resembled the polymerized proteins derived after incubation of the SR with the peroxidizing cofactors. Ca2+-uptake decreased when the SR was incubated with peroxidizing cofactors, but the results are difficult to interpret since Ca2+-uptake of SR was affected by captive stress.

Aging↗
PubMed1988

Presence of a Ca2+-sensitive CDPdiglyceride-inositol transferase in canine cardiac sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) and plasma membranes from canine left ventricle were used to evaluate the presence of the enzyme CDPdiglyceride-inositol transferase in these membranes. (K+,-Ca2+)-ATPase activity, a marker for SR, was 79.2 +/- 5.0 (SE) and 11.2 +/- 2.0 mumol.mg-1.h-1 in SR and plasma membrane preparations, respectively, and (Na+,K+)-ATPase activity, a marker for plasma membranes, was 5.6 +/- 1.2 and 99.2 +/- 8.0 mumol.mg-1.h-1, respectively. Contamination of SR and plasma membrane preparations by mitochondria was estimated to be 2% and 8%, respectively, and by Golgi membranes, 0.9% and 1.8%, respectively. Transferase activity, measured at pH 6.8, was 1.32 +/- 0.04 (SE) and 0.28 +/- 0.04 nmol of [3H]phosphatidylinositol ([3H]PtdIns).mg-1.min-1 in three SR and plasma membrane preparations, respectively. The transferase activity detected in the plasma membrane preparation could be accounted for largely, but not entirely, by contaminating SR membranes. The pH optimum for the SR transferase activity was between 8.0 and 9.0; little or no activity was detectable at pH 6.3 and 5.5, the lowest pH tested. Ca2+ inhibited the enzyme, half-maximal inhibition occurring at about 10 microM Ca2+; removal of the Ca2+ by addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid restored activity. No loss of [3H]PtdIns could be detected when membranes were incubated in the presence or absence of Ca2+. The Ca2+ inhibition of the transferase was noncompetitive with respect to CDP-dipalmitin while that with respect to myo-inositol was slightly noncompetitive at low [Ca2+] and became uncompetitive at higher [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1983

Energy transfer between fluorescent dyes attached to Ca2+,Mg2+-ATPase in the sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle was solubilized with a nonionic detergent, dodecyl octaethyleneglycol monoether (C12E8), at a weight ratio of detergent to protein of greater than 10, so that the Ca2+, Mg2+ dependent ATPase existed mainly in a monomeric form (7). The solubilized ATPase was reacted with 10 microM N-1-P or 5 microM DACM in the presence of 5 mM CaCl2, 0.4 M KCl, 20% glycerol and 50 mM TES at pH 7.5 and 20 degrees C. Under these conditions, about 1 mol of N-1-P was incorporated into 10(5) g SR protein on 10 min incubation and 1 mol of DACM was incorporated into the same amount of SR on 5 min incubation. Analysis of the tryptic digest of the N-1-P- or DACM-labeled. ATPase on SDS polyacrylamide gel revealed that almost all the fluorescence was associated with the 30K m.w. subfragment of the ATPase protein. Even when the amount of the probe incorporated into SR-ATPase was increased from 1 to 3 mol per 10(5) g SR protein, all was incorporated into the 30K subfragment. Both the activities of formation and decomposition of the phosphorylated intermediate (EP) were unaffected by these modifications. When the separately labeled ATPases were mixed together in the presence of C12E8 and the detergent was removed by incubation with Bio-Beads SM-2, a significant amount of fluorescence energy transfer was observed between N-1-P and DACM. However, energy transfer did not occur when the labeled ATPases were mixed after removal of C12E8.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗