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Smooth-muscle endoplasmic reticulum contains a cardiac-like form of calsequestrin.

It is proposed that smooth-muscle endoplasmic reticulum contains calsequestrin and that this protein in smooth muscle resembles cardiac calsequestrin more than the skeletal-muscle form. This proposal is based on seven similarities between the smooth-muscle protein and cardiac calsequestrin. Proteins with an Mr of 55,000 can be extracted from the membranes of smooth muscle and of cardiac muscle using 100 mM Na2CO3. The protein from smooth muscle binds to phenyl-Sepharose in the absence of Ca2+ and is released by 10 mM CaCl2, as has been observed for cardiac calsequestrin. The protein from smooth muscle comigrates with the cardiac calsequestrin on Laemmli-type SDS-polyacrylamide gel electrophoresis. The protein of Mr 55,000 from smooth muscle and cardiac calsequestrin both strain blue with the carbocyanine dye Stains-all. Both proteins present similar one-dimensional Cleveland peptide maps although minor differences might exist. From an analysis of subcellular membranes separated by sucrose gradient centrifugation it is concluded that the protein with Mr 55,000 from the smooth muscle is confined to the endoplasmic reticulum, the same subcellular structure from which, in heart muscle, calsequestrin can be isolated. Antibodies raised against canine cardiac calsequestrin bind to a protein of similar Mr in smooth-muscle endoplasmic reticulum. In addition to the calsequestrin, three other extrinsic proteins with an Mr of 130,000, 100,000 and 63,000, stain blue with Stains-all and occur in the endoplasmic reticulum of smooth muscle.

Animals↗

Drug-induced calcium release from heavy sarcoplasmic reticulum of skeletal muscle.

Calcium release from isolated heavy sarcoplasmic reticulum of rabbit skeletal muscle by several calmodulin antagonistic drugs was measured spectrophotometrically with arsenazo III and compared with the properties of the caffeine-induced calcium release. Trifluoperazine and W7 (about 500 microM) released all actively accumulated calcium (half-maximum release at 129 microM and 98 microM, respectively) in the presence 0.5 mM MgCl2 and 1 mg/ml sarcoplasmic reticulum protein; calmidazolium (100 microM) and compound 48/80 (70 micrograms/ml) released maximally 30-40% calcium, whilst bepridil (100 microM) and felodipin (50 microM) with calmodulin antagonistic strength similar to trifluoperazine (determined by inhibition of the calcium, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum) did not cause a detectable calcium release, indicating that this drug-induced calcium release is not due to the calmodulin antagonistic properties of the tested drugs. Calcium release of trifluoperazine, W7 and compound 48/80 and that of caffeine was inhibited by similar concentrations of magnesium (half-inhibition 1.4-4.2 mM compared with 0.97 mM for caffeine) and ruthenium red (half-inhibition for trifluoperazine, W7 and compound 48/80 was 0.22 microM, 0.08 microM and 0.63 micrograms/ml, respectively, compared with 0.13 microM for caffeine), suggesting that this drug-induced calcium release occurs via the calcium-gated calcium channel of sarcoplasmic reticulum stimulated by caffeine or channels with similar properties.

Animals↗

Characterization of anion binding sites of sarcoplasmic reticulum vesicles by 35Cl-NMR.

Sarcoplasmic reticulum vesicles were prepared from rabbit skeletal muscle. It could be demonstrated that the anion binding sites on this membrane can be studied by 35Cl-NMR spectroscopy. Titration of sarcoplasmic reticulum vesicles with the sulfate exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) revealed specific binding of this compound to the sarcoplasmic reticulum membrane. A new inhibitor, pyridoxalphosphate-6-azophenyl-2'-sulfonic acid (PPAPS) was introduced and proved to displace chloride equally well from its binding sites. Two binding sites could be distinguished by titration with inorganic phosphate in the presence and absence of the inhibitors. Because of the insensitivity of 35Cl-NMR spectroscopy these anion binding sites have to be located on a protein being present in considerable amount in the sarcoplasmic reticulum membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

Oxytocin pretreatment of pregnant rat uterus inhibits Ca2+ uptake in plasma membrane and sarcoplasmic reticulum.

Myometrium from rats in varying stages of pregnancy (from 17 to 22 days) was treated with oxytocin (0.1-10 microM) and plasma membranes and sarcoplasmic reticulum were isolated using a Percoll gradient. When the myometrium had been treated with oxytocin, Ca2+ uptake was reduced by 29.4% in the plasma membrane and by 32.6% in the sarcoplasmic reticulum. The inhibitory action of oxytocin was highly dependent upon the stage of gestation: Only membranes from rats close to term (21-22 days) exhibited reduced Ca2+ transport activity after hormone treatment. This effect correlated highly with a significant decrease in the serum progesterone level of these animals. In plasma membrane vesicles, oxytocin reduced the maximal velocity of the Ca2+ pump without significantly affecting the affinity for Ca2+. Oxytocin did not affect the passive permeability of the plasma membranes, nor their proportion of sealed inside-out vesicles nor the amount of Ca2(+)-pump protein in these membranes. In addition, oxytocin caused no change in the passive permeability of the membrane nor in the rate of inositol trisphosphate-induced Ca2+ release from the sarcoplasmic reticulum. These results suggest that there is a specific action of oxytocin on the activity of the myometrial plasma membrane and sarcoplasmic reticulum Ca2+ pumps which may contribute to the maintenance of an elevated intracellular calcium level during parturition.

Animals↗

Evidence for the lumenal location of the 53 kDa glycoprotein of sarcoplasmic reticulum.

The 53 kDa glycoprotein from sarcoplasmic reticulum was shown to be protected from proteolysis by trypsin, V8 proteinase and proteinase K in intact vesicles yet readily digested in the presence of the non-denaturing detergent C12E8. Competitive ELISAs with a library of seven monoclonal antibodies raised against the 53 kDa glycoprotein showed that the epitopes for these antibodies were only accessible in C12E8 solubilised and not intact sarcoplasmic reticulum. When the monoclonal antibodies against the 53 kDa glycoprotein were assessed for their effect on the uptake of Ca2+ by sarcoplasmic reticulum no effect was detected; neither were these antibodies able to augment the inhibitory influences of anti-(Ca(2+)-Mg2+)-ATPase monoclonal antibodies on Ca2+ uptake. These data indicate that the 53 kDa glycoprotein is located in the lumen of the sarcoplasmic reticulum.

Amino Acid Sequence↗

The relationship between phospholipid content and Ca2+-ATPase activity in the sarcoplasmic reticulum.

The relationship between the phospholipid composition of sarcoplasmic reticulum and the activity of the Ca2+, Mg2+-stimulated ATPase was analyzed by digestion of membrane phospholipids with phospholipase C and A2 enzymes of diverse specificity and by detergent extraction. Phospholipase C of Clostridium perfringens and Clostridium welchii, that hydrolyze preferentially phosphatidylcholine (PC), inhibited the Ca2+-ATPase activity parallel with the depletion of phosphatidylcholine from the membrane. Phospholipase C of Bacillus cereus hydrolyzed in addition to PC, phosphatidylethanolamine (PE) and phosphatidylserine (PS), causing complete inhibition of Ca2+-stimulated ATPase activity. Digestion of sarcoplasmic reticulum with the phospholipase A2 of snake or bee venom produced similar effects. The phosphatidylinositol (PI)-specific phospholipases of B. cereus and Bacillus thuringiensis caused less than 10% inhibition of the Ca2+-ATPase, accompanied by the hydrolysis of more than 70% of the phosphatidylinositol content of the membrane, without significant change in PC, PE and PS content. The inhibition of ATPase activity by the C type phospholipases was nearly completely reversed by octaethyleneglycol dodecyl ether (C12E8). These experiments suggest that the full phospholipid content of native sarcoplasmic reticulum (congruent to 100 mol phospholipid per mol Ca2+-ATPase), is required for ATPase activity and there is no indication that PE, PS, and PI play a specific role in ATP hydrolysis. Extraction of sarcoplasmic reticulum phospholipids by detergents such as deoxycholate, cholate and C12E8 also caused proportional inhibition of ATPase activity with the decrease in phospholipid content; the parallel extraction of PC, PE and PI left the phospholipid composition largely unchanged during delipidation. These observations do not support the requirement for a 'lipid annulus' of congruent to 30 phospholipid molecules/Ca2+-ATPase as proposed by Hesketh et al. ((1976) Biochemistry 15, 4145-4151) or the specific interaction of phosphatidylethanolamine with the ATPase molecule proposed by Bick et al. ((1991) Arch. Biochem. Biophys. 286, 346-352).

Animals↗

Preferential heme transport through endoplasmic reticulum associated with mitochondria in rat liver.

The transport of de novo synthesized protoheme into the conventional microsomal fraction and endoplasmic reticulum associated with mitochondria (MAER) was studied by injecting amino[14C]levulinic acid (ALA) into phenobarbital-treated rats to evaluate the role of MAER in the trafficking of heme between mitochondria and endoplasmic reticulum. In mitochondria, the specific radioactivity of the radiolabeled heme reached a maximum level at 4 min after the injection of 14C-ALA. The specific radioactivity in cytosol was about 2-fold lower than that in microsomes, suggesting that the cytosolic pathway of the heme transport from mitochondria to endoplasmic reticulum is not predominant, because the specific radioactivity of heme in cytosol should be higher than that in microsomes if heme is transported mainly through cytosol. MAER showed higher specific radioactivity than the conventional microsomal fraction up to 4 min and thereafter the specific radioactivities in MAER and the conventional microsomal fraction became nearly the same. The extents of decrease in cytochrome P-450 and the radioactivity in microsomes by the treatment with allylisopropylacetamide which destroyed cytochrome P-450 but not cytochrome b5, were essentially the same, suggesting that most of the radiolabeled heme in microsomes was incorporated into cytochrome P-450. These results suggest that MAER is a preferential site for the protoheme transport from mitochondria to endoplasmic reticulum.

Animals↗

Cell-free transfer of phospholipids between the endoplasmic reticulum and the Golgi apparatus of leek seedlings.

The transfer of lipids between the endoplasmic reticulum and the Golgi apparatus was investigated in vitro using a cell-free system from leek seedlings. Lipids of the donor membranes (endoplasmic reticulum) were radiolabeled either by incubating leek seedlings with [1-14C]acetate or [3H]acetate. Acceptor membranes (Golgi apparatus) were unlabeled and immobilized on nitrocellulose strips. The assay measured the lipid transfer resulting from both an ATP-independent process and an ATP- and cytosol-dependent process. A significant ATP- and cytosol-dependent lipid transfer was observed only in the case of the endoplasmic reticulum as donor and the Golgi apparatus as acceptor. Lipids transferred in an ATP-dependent manner were chiefly phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The stimulation of lipid transfer by ATP as compared to the ATP-independent process was +79% (PC), +123% (PS) and +69% (PE). On the other hand, PI was not transferred in an ATP-dependent manner (the stimulation by ATP was only 20%). This supports the theory that a sorting of phospholipids takes place in the donor membrane. Moreover, a formation of lysoPC was observed only in the presence of ATP (+330%). The ATP-dependent lipid transfer was inhibited by N-ethylmaleimide, indicating the involvement of cytosolic (but no phospholipid transfer proteins) or membrane proteins in the transfer process. The ATP-dependent transfer of lipids was also diminished at 12 degrees C showing the sensitivity to low temperatures of the transfer of lipids between the endoplasmic reticulum and the Golgi apparatus.

Adenosine Triphosphate↗

The phosphoprotein intermediate of a Ca2+ transport ATPase in rat liver endoplasmic reticulum.

Smooth endoplasmic reticulum vesicles from rat liver display an ATP-supported Ca2+ transport which is mediated by a (Ca2+ + Mg2+)-ATPase. During the catalytic cycle the terminal phosphate from ATP is incorporated to form an acid-precipitable reaction product(118 000-Mr in SDS-gel electrophoresis) with stability characteristics of an acylphosphate. Comparative studies with sarcoplasmic reticulum vesicles from fast-twitch skeletal muscle suggest that the 118 000-Mr phosphopeptide may be identified with the phosphorylated reaction intermediate of a Ca2+ transport ATPase in endoplasmic reticulum, similar to that in sarcoplasmic reticulum of muscle.

Adenosine Triphosphatases↗

Inhibition of liver endoplasmic reticulum calcium pump by CCl4 and release of a sequestered calcium pool.

One of the earliest effects observed in rat liver after CCl4 administration is inhibition of an ATP-dependent calcium pump found at the endoplasmic reticulum. This report confirms that the amount of calcium associated with the microsomal fraction is reduced after CCl4 administration and, for the first time, demonstrates time-, dose-, and metabolism-dependent relationships between inhibition of the liver microsomal calcium pump and the amount of calcium found in the microsomal fraction. Furthermore, release of calcium from the endoplasmic reticulum is shown to cause activation of a cytoplasmic enzyme that responds to increases of ionized calcium, glycogen phosphorylase. This suggests that the endoplasmic reticulum calcium pump sequesters an intracellular pool of calcium within the endoplasmic reticulum. This pool of calcium may be released into the cytoplasm as a consequence of inhibition of the calcium pump by CCl4.

Animals↗

Propranolol inhibition of the neutral phospholipases A of rat heart mitochondria, sarcoplasmic reticulum and cytosol.

Membrane damage caused by phospholipase A action is thought to be an important factor in ischemic myocardial injury. Propranolol has been shown previously to have beneficial effects in both animal experiments and clinical trials, and it has membrane-stabilizing properties in vitro. To investigate the possibility that these effects might be due, in part, to effects on phospholipases, we determined the effects of propranolol on rat heart phospholipases A at physiological pH using small unilamellar liposomes of di[1-14C]oleoylphosphatidylcholine as substrate. Propranolol inhibited heart phospholipases A in vitro. The concentration required to give 50% inhibition was 0.2 mM for the mitochondrial and cytosolic phospholipases A and 2.9 mM for sarcoplasmic reticulum phospholipase A. The binding of [4-3H]propranolol to fresh membrane preparations was studied using an ultracentrifugation method. Propranolol bound readily to both membrane fractions in vitro with no significant difference in the saturation number (0.20 to 0.28 mol drug per mol phospholipid) but the association constant, KA, was higher for mitochondrial membranes (3760 +/- 350) than for the sarcoplasmic reticulum (2190 +/- 390). Our results show that propranolol inhibited heart phospholipases A in vitro at physiological pH. The mitochondrial and cytosolic phospholipases A were more susceptible to inhibition than the phospholipase A of sarcoplasmic reticulum. Propranolol bound to mitochondria and sarcoplasmic reticulum in vitro, suggesting the possibility that propranolol binding to heart membranes in vivo could result in drug concentrations in these membranes high enough to inhibit phospholipase A. This could represent an additional mechanism by which propranolol exerts beneficial effects in myocardial ischemia.

Animals↗

Alterations in the enzyme activity and polypeptide composition of rat hepatic endoplasmic reticulum during acute exposure to 2-acetylaminofluorene.

Studies have been made of the morphology, enzyme activity and protein composition of liver endoplasmic reticulum in rats exposed to acute doses of the carcinogen, 2-acetylaminofluorene (2-AAF). Electron microscopic examination revealed numerous ultrastructural changes in the hepatocyte; most consistent alterations were the disorganisation of endoplasmic reticulum system with apparent increase of smooth endoplasmic reticulum. Administration of 2-AAF to rats immediately depressed microsomal glucose-6-phosphatase activity and eventually induced epoxide hydratase activity 6--7-fold over control activity. The induction was time-dependent and maximal rates of induction were observed at dosages greater than 40 mg/kg body wt. The treatment also induced cytochrome b5 content, NADH and NADPH cytochrome c reductase activities (1.0--1.5-fold). Only very small changes in the total content of cytochrome P-45- were noted. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of microsomal proteins from 2-AAF pretreated animals showed time-dependent induction of two polypeptides which differed slightly in migration, in the region of Mr = 48000; the fast-migrating induced polypeptide has been identified as epoxide hydratase. Two-dimensional PAGE analysis of microsomal proteins from 2-AAF exposed rats showed a reproducible deletion of a protein with molecular weight in the region of 67000. The basis for the alterations in the protein composition of endoplasmic reticulum in response to 2-AAF treatment is discussed.

2-Acetylaminofluorene↗

Membrane properties of the sarcolemma and sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia. Action of halothane.

The role of sarcolemma and sarcoplasmic reticulum in malignant hyperthermia was studied by the esr technique using the trapezius muscle membrane of both normal and genetically susceptible pigs. Normal and malignant hyperthermia membranes from sarcolemma as well as from sarcoplasmic reticulum did not show significant differences near the polar heads of the phospholipidic bilayer. In contrast, the fluidity and activation energy of normal membranes differed from those in malignant hyperthermia; in both sarcolemma and sarcoplasmic reticulum the mobility of the label was greater than the controls. The presence of halothane was examined, by inducing this disease anesthetically. The drug effect confirmed the above results, i.e. the disease affects mainly the hydrophobic core of the lipid bilayer of both sarcolemma and sarcoplasmic reticulum membranes.

Animals↗

Evidence of endoplasmic reticulum-related Ca2+ ATPase in human microvascular endothelial cells.

We have demonstrated by immunological and molecular methods the presence of a reticulum endoplasmic-related Ca2+-ATPase in human omental microvascular endothelial cells (HOME cells). HOME cells reacted positively with a previously characterized sarcoplasmic reticulum Ca2+-ATPase antibody as demonstrated by indirect immunofluorescence. Western blotting revealed that the antibody recognized a 95-100 kDa protein. 35S-Metabolic labeling led to the detection of a similar protein with which the purified sarcoplasmic reticulum Ca2+-ATPase competed. Dot-blotting experiments indicated that a substantial amount of Ca2+-ATPase was present in HOME cell membranes. In addition, Northern blot analysis using a cDNA probe from cardiac sarcoplasmic reticulum showed the presence of mRNA species of 4-kb. As these experiments were conducted in comparison with cell types with well-defined Ca2+-ATPase in HOME cells.

Animals↗

Effects of phospholamban phosphorylation catalyzed by adenosine 3':5'-monophosphate- and calmodulin-dependent protein kinases on calcium transport ATPase of cardiac sarcoplasmic reticulum.

To elucidate the role of 22000-dalton protein phospholamban, a putative regulator of Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum, we examined the relationship between cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban and their effects on ATPase activity and calcium transport of cardiac sarcoplasmic reticulum. Cardiac microsomes were incubated with [gamma-32P]ATP or unlabeled ATP, catalytic subunit of cyclic AMP-dependent protein kinase and/or exogenous calmodulin, and subsequently assayed for ATPase activity and calcium uptake by cardiac sarcoplasmic reticulum. Cyclic AMP-dependent phosphorylation of phospholamban was independent of Ca2+, whereas calmodulin-dependent phosphorylation of phospholamban was dependent on Ca2+ within a range between 0.2 and 50 microM. Cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban occurred independently; when both kinases were operative, the amounts of phosphorylation were additive. Under these conditions, the phosphoproteins formed by cyclic AMP- and calmodulin-dependent protein kinases electrophoretically migrated as 11000-dalton components when sodium dodecyl sulfate-solubilized phosphoproteins were boiled prior to polyacrylamide gel electrophoresis. The ATPase activity was stimulated by either cyclic AMP- or calmodulin-dependent phosphorylation of phospholamban at Ca2+ concentrations up to 2 microM. The extents of stimulation of ATPase activity were additive when both types of phosphorylation were functional. Calcium uptake was similarly augmented by cyclic AMP- and/or calmodulin-dependent phosphorylation of phospholamban. These results indicate that Ca2+-dependent ATPase and calcium transport of cardiac sarcoplasmic reticulum are regulated by phospholamban phosphorylation catalyzed by cyclic AMP- and calmodulin-dependent protein kinases, thus suggesting a dual role of phospholamban in active calcium transport.

Biological Transport, Active↗

Analysis of sarcoplasmic reticulum proteins in patients susceptible to malignant hyperthermia.

It has been suspected that the cause of malignant hyperthermia (MH) is an abnormality in the sarcoplasmic reticulum of skeletal muscle. We isolated the sarcoplasmic reticulum from malignant hyperthermia-susceptible (MHS) patients and controls and analysed the protein composition with sodium dodecyl sulfate polyacrylamide gel electrophoresis. There were no remarkable changes in the sarcoplasmic reticulum protein composition profile of the scanned gel of the patients. Quantitative measurement of the relative proportion of each band in the gel, however, revealed a slight decrease in calsequestrin and a slight increase in protein of molecular weight 23,000. (Ca2+ -Mg2+)ATPase had no altered subfragments in MHS patients. Crude mitochondrial proteins and myoplasmic proteins showed minor alterations in composition in some patients. The data supported the thesis that malignant hyperthermia is due to defects in several different cell membranes including the sarcoplasmic reticulum and the mitochondria.

Disease Susceptibility↗

Comparison of three tetramic acids and their ability to alter membrane function in cultured skeletal muscle cells and sarcoplasmic reticulum vesicles.

Cyclopiazonic acid is a potent inhibitor of calcium uptake and Ca(2+)-ATPase activity in sarcoplasmic and endoplasmic reticulum. In L6 muscle myoblasts, cyclopiazonic acid stimulates the uptake of tetraphenylphosphonium, a lipophilic membrane potential probe, and has antioxidant properties. The purpose of the present study was to investigate the structural requirements necessary for causing the surface charge alterations, and the antioxidant activity in L6 skeletal muscle myoblasts, and for inhibition of calcium transport by rat skeletal muscle sarcoplasmic reticulum vesicles. This was accomplished by comparing the effects of two structurally related tetramic acids, cyclopiazonic acid imine and tenuazonic acid, with cyclopiazonic acid. Cyclopiazonic acid imine inhibited oxalate-assisted 45Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles and stimulated tetraphenylphosphonium accumulation by L6 muscle myoblasts. However, these effects required an approximately fourfold higher concentration than that of cyclopiazonic acid. Tenuazonic acid, up to 1 mM, had no effect on oxalate-assisted 45Ca2+ uptake or Ca(2+)-ATPase activity in sarcoplasmic reticulum vesicles and did not stimulate tetraphenylphosphonium accumulation by L6 muscle myoblasts. Cyclopiazonic acid was only slightly more effective than cyclopiazonic acid imine at preventing the patulin-induced increase in thiobarbituric acid positive substance (used to estimate lipid peroxidation); tenuazonic acid was totally ineffective. Previously, it was shown that cyclopiazonic acid was twice as effective as cyclopiazonic acid imine at preventing increases in thiobarbituric acid positive substance in cultured renal cells, LLC-PK1. Thus, the indole nucleus of cyclopiazonic acid is essential for the membrane-associated biological activity; however, modification of the acetyl group reduces the potency of the activity.

Animals↗

Effects of a myotoxic phospholipase A2 isolated from Bothrops asper venom on skeletal muscle sarcoplasmic reticulum.

The myotoxin from B. asper snake venom inhibited Ca-ATPase activity of rabbit sarcoplasmic reticulum after incubation in vitro. Inhibition was non-competitive and albumin enhanced the effect of the toxin. Furthermore, B. asper myotoxin hydrolyzed sarcoplasmic reticulum phospholipids and induced a dose-dependent release of horseradish peroxidase that had been trapped in sarcoplasmic reticulum vesicles. Binding studies indicated that myotoxin does not bind to any particular protein of this membrane, suggesting that the toxin might interact with phospholipids. Inhibition of Ca-ATPase is probably a consequence of an alteration in sarcoplasmic reticulum phospholipids.

Animals↗