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PubMed2004

Induction of endoplasmic reticulum stress by ellipticine plant alkaloids.

Anticancer drugs often show complex mechanisms of action, including effects on multiple cellular targets. Detailed understanding of these intricate effects is important for the understanding of cytotoxicity. In this study, we examined apoptosis induction by ellipticines, a class of cytotoxic plant alkaloids known to inhibit topoisomerase II. The potent ellipticine derivative 6-propanamine ellipticine (6-PA-ELL) induced rapid apoptosis in MDA-MB-231 breast cancer cells, preceded by a conformational change in Bak and cytochrome c release. Experiments using knock-out mouse embryo fibroblasts established that Bak was of particular importance for cytotoxicity. 6-PA-ELL increased the expression of the endoplasmic reticulum chaperones GRP78/BiP and GRP94, suggesting induction of endoplasmic reticulum stress. Induction of GRP78 expression was dependent on the endoplasmic reticulum stress response element (ERSE) of the GRP78 promoter. Examination of different ellipticine derivatives revealed a correlation between pro-apoptotic activity and the ability to induce GRP78 expression. Furthermore, 6-PA-ELL was found to induce splicing of the mRNA encoding the XBP1 transcription factor, characteristic of endoplasmic reticulum stress, and to induce activation of the endoplasmic reticulum-specific caspase-12 in mouse colon cancer cells. We finally demonstrate that 6-PA-ELL induces apoptotic signaling also in enucleated cells, consistent with the existence of a cytoplasmic target for this compound. Our data suggest that induction of endoplasmic reticulum stress may contribute to the cytotoxicity of ellipticines.

Animals↗
PubMed1995

Topology of prostaglandin H synthase-1 in the endoplasmic reticulum membrane.

Prostaglandin H synthase-1 is an integral endoplasmic reticulum membrane protein which catalyzes a key control step in prostaglandin biosynthesis. The overall arrangement of the prostaglandin H synthase-1 polypeptide with respect to the endoplasmic reticulum membrane was examined in transiently transfected COS-1 cells, using immunofluorescence microscopy. A bacterial toxin, streptolysin-O, was used for selective plasma membrane permeabilization and a detergent, saponin, for general membrane permeabilization. Treated cells were probed with six antibodies specific for particular prostaglandin H synthase-1 peptide segments and one antibody specific for an inserted viral reporter epitope. Control experiments established that actin, a cytoplasmic marker, was accessible to fluorescein-labeled phalloidin after streptolysin-O treatment, whereas antibodies against protein disulfide isomerase, an endoplasmic reticulum lumenal marker, bound only after saponin treatment, Using this approach to investigate prostaglandin H synthase-1, it was found that streptolysin-O treatment was sufficient to obtain staining of intracellular membranes by antibodies specific for the endogenous C-terminal segment, for the viral reporter inserted at the C-terminus, and for the protease-sensitive region near arg277. In contrast, saponin treatment was necessary for staining by antibodies specific for peptides spanning residues 51-66, 156-170, and 377-390. Antibodies targeted against residues 483-496 did not stain transfected cells even after saponin permeabilization, although they did bind to detergent-solubilized prostaglandin H synthase-1. These results indicate that the C-terminus and arg277 regions of the synthase can be exposed on the cytoplasmic side of the endoplasmic reticulum membrane, whereas regions near N-glycosylation sites are confined to the endoplasmic reticulum lumen and residues 483-496 are inaccessible from either side of the endoplasmic reticulum membrane.

Amino Acid Sequence↗
PubMed1998

Sarcoplasmic reticulum genes are selectively down-regulated in cardiomyopathy produced by doxorubicin in rabbits.

The clinical utility of doxorubicin, an antineoplastic agent, is limited by its cardiotoxicity. Our objective was to determine whether expression of genes encoding proteins that affect Ca2+ homeostasis were altered in the hearts of rabbits chronically treated with doxorubicin. Twelve male New Zealand white rabbits received an injection of doxorubicin (2.5 mg/kg i.v.) once a week for 8 weeks. Eight rabbits were similarly injected with saline as controls. The cardiac function of both groups was evaluated 8 weeks after the final injection, as were the levels of expression of mRNA for Ca2+ transport proteins in the sarcoplasmic reticulum and plasma membrane. The amount of the sarcoplasmic reticulum Ca2+-ATPase and the Ca2+ uptake capacity of the protein were also quantitated. Cardiac output was significantly decreased in the doxorubicin-treated group (71+/-21 ml/min, P<0.05) compared with the control group (118+/-15 ml/min). The mRNA levels for the sarcoplasmic reticulum proteins were significantly diminished in the doxorubicin-treated hearts: ryanodine receptor-2 (relative expression level compared with controls, 0.35+/-0.13, P<0.01), sarcoplasmic reticulum Ca2+-ATPase (0.56+/-0.13, P<0.01), phospholamban (0.62+/-0.20, P<0.01) and cardiac calsequestrin (0. 57+/-0.26, P<0.01). In addition, both relative amount of sarcoplasmic reticulum Ca2+-ATPase protein (doxorubicin-treated group, 69+/-17% of control, P<0.01) and the Ca2+ uptake capacity (46. 9+/-9.8 nmol Ca2+/mg protein-5 min in doxorubicin group v 63.2+/-10. 4 in the control group, P<0.01) were concomitantly decreased with its mRNA expression level. Conversely, the mRNA levels for the plasma membrane proteins did not differ from those of control rabbits: the dihydropyridine receptor (relative expression level, 1. 03+/-0.30, N.S.), plasma membrane Ca2+-ATPase (0.93+/-0.33, N.S.) and the Na+/Ca2+ exchanger (0.87+/-0.34, N.S.). These findings suggest that a selective decrease in mRNA expression for sarcoplasmic reticulum Ca2+ transport proteins is responsible for the impaired Ca2+ handling, and thus, for the reduced cardiac function seen in the cardiomyopathy induced in rabbits by the long-term treatment with doxorubicin.

Animals↗
PubMed2002

Sequential NO production by mitochondria and endoplasmic reticulum during induced apoptosis.

Early stages of rat thymocyte apoptosis measured as annexin-V positive events and induced by methylprednisolone (MPS), etoposide, and thapsigargin, showed a sequential increase in nitric oxide (NO) production by mitochondrial and endoplasmic reticulum membranes. Thapsigargin induced the highest NO production, a sevenfold increase as compared with untreated thymocytes, in mitochondrial and microsomal membranes. MPS and etoposide were equally effective in increasing NO production by mitochondrial membranes by a factor of 4-5, with only a slight increase in NO production by endoplasmic reticulum membranes. Western blot analysis of both types of membrane indicated that a nitric oxide synthase (NOS) isoenzyme is present in mitochondrial membranes and reacts with antibodies to i-NOS (type II), while reactivity to antibodies to e-NOS (type III) was restricted to endoplasmic reticulum. The participation of endoplasmic reticulum during apoptosis was further determined by alterations in UDP-Glucosyltransferase (UDP-GT) and NADPH cytochrome P450 reductase. Increased UDP-GT activity was observed after thapsigargin treatment, and no changes were found after treatment with etoposide or MPS. NADPH cytochrome P450 reductase activity markedly decreased during apoptosis, being stronger after thapsigargin treatment. The latest stage of the apoptotic process was measured by caspase activities. Caspase 3 activity was markedly increased by the three apoptosis inducers; caspase 6 was only activated by MPS and etoposide, while caspase 8 was not activated by any of these inducers. It is clear that mitochondria and endoplasmic reticulum are involved in thapsigargin induced thymocyte apoptosis. Meanwhile, other thymocyte apoptotic pathways, such as those induced by MPS or etoposide, seem to centrally involve mitochondria but not endoplasmic reticulum.

Animals↗
PubMed1990

A non-specific Ca2+ (or Mg2+)-stimulated ATPase in rat heart sarcoplasmic reticulum.

ATPase activity in rat heart sarcoplasmic reticulum was stimulated in a concentration-dependent manner by both Ca2+ and Mg2+ in the complete absence of the other cation. Increasing concentrations of Mg2+ produced an apparent inhibition of the Ca2(+)-dependent ATP hydrolysis. CDTA (trans-1,2-diaminocyclo-hexane-N,N,N',N'-tetraacetate) had no effect on these responses. The results indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase in rat heart sarcoplasmic reticulum. However, sarcoplasmic reticulum vesicles transported Ca2+ with a high affinity (K0.5 Ca2+ = 0.41 microM) suggesting the presence of a high affinity Ca2(+)-transporting ATPase. Calmodulin did not stimulate rat heart sarcoplasmic reticulum ATPase activity over a range of Ca2+ and Mg2+ concentrations and failed to stimulate membrane phosphorylation and Ca2+ transport into sarcoplasmic reticulum vesicles. Calmodulin antagonists trifluoperazine and compound 48/80 did not affect the ATPase activity. Catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating the ATPase activity. These results suggest the presence of an ATPase activity in rat heart sarcoplasmic reticulum with different properties from the high affinity Ca2(+)-pumping ATPase previously characterized in dog heart and other species.

Animals↗
PubMed1989

Effects of dietary n-3 polyunsaturated fatty acids on phospholipid composition and calcium transport in mouse cardiac sarcoplasmic reticulum.

The effects of dietary n-3 and n-6 polyunsaturated fatty acids on the fatty acid composition of phospholipid, Ca++. Mg++ ATPase and Ca++ transport activities of mouse sarcoplasmic reticulum were investigated. Mice were fed a 2 weight percent fat diet containing either 0.5 weight percent ethyl esters of 18:3n-3, 20:5n-3 or 22:6n-3 as a source of n-3 polyunsaturated fatty acid or 0.5 weight percent safflower oil as a source of n-6 polyunsaturated fatty acid for 10 days. Olive oil (2 weight percent) was used as a control diet. Although feeding n-6 polyunsaturated fatty acid induced very little modifications of the phospholipid sarcoplasmic reticulum fatty acid composition, feeding n-3 polyunsaturated fatty acid altered it markedly. Inclusion of 18:3n-3, 20:5n-3 or 22:6n-3 in the diet caused an accumulation of 22:6n-3, which replaced 20:4n-6 and 18:2n-6 in phospholipid sarcoplasmic reticulum. The saturated fatty acids were significantly increased with a concurrent reduction of 18:1n-9. These changes in the fatty acid composition resulted in a decrease in the values of the n-6/n-3 polyunsaturated fatty acid ratio and a decrease in the ratio of 20 carbon to 22 carbon fatty acids esterified in the phospholipid sarcoplasmic reticulum. This was associated with a decrease in Ca++ uptake by n-3 polyunsaturated fatty acid enriched sarcoplasmic reticulum vesicles as compared with n-6 fatty acid and control diet sarcoplasmic reticulum vesicles. However, neither the affinity for Ca++ nor the maximal velocity of ATP hydrolysis activity of Ca++.MG++ATPase were altered by the different diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

The content of glycogen phosphorylase and glycogen in preparations of sarcoplasmic reticulum-glycogenolytic complex is enhanced in diabetic rat skeletal muscle.

AIMS/HYPOTHESIS: We have examined the effect of diabetes and pharmacological insulin treatment on the content of glycogen phosphorylase and glycogen associated with the sarcoplasmic reticulum-glycogenolytic complex from rat skeletal muscle. METHODS: Diabetes was induced in rats by streptozotocin injection. Enzymatic activities were measured using spectrophotometric methods. Glycogen phosphorylase was determined measuring the pyridoxal-5' -phosphate content and using polyacrylamide gel electrophoresis. Glycogen content was measured by enzymatic and the phenol sulfuric methods. RESULTS: The content of glycogen phosphorylase associated with the sarcoplasmic reticulum glycogenolytic complex gradually arises after diabetes induction. The content of glycogen phosphorylase was restored to a control value by pharmacological insulin treatment. In addition, the content of glycogen in preparations of sarcoplasmic reticulum-glycogenolytic complex of diabetic animals was also increased, whereas the content of glycogen in total muscle of diabetic rats was similar to that of the control rats. The absolute and relative amount of glycogen associated with sarcoplasmic reticulum seemed to increase in diabetic animals. These effects on the compartmentalisation of glycogen were suppressed by insulin treatment. Additionally, the rate of conversion of glycogen phosphorylase b to a, an index of the phosphorylase kinase activity, was 50 % lower in diabetic rats, increasing the dephosphorylated form of glycogen phosphorylase and, as a consequence, its association with sarcoplasmic reticulum membranes. CONCLUSION/INTERPRETATION: These results suggest that under diabetic conditions, both glycogen phosphorylase and a small percentage of muscle glycogen are relocalized in the sarcoplasmic reticulum-glycogenolytic complex.

Animals↗
PubMed1983

Induced Ca2+ release in skeletal muscle sarcoplasmic reticulum by sulfhydryl reagents and chlorpromazine.

Sarcoplasmic reticulum is a specialized membrane system in muscle involved in the energized uptake, storage, and release of Ca2+. The sulfhydryl content of normal and reconstituted sarcoplasmic reticulum was measured using Ellman reagent. For both preparations, we find 17 and 26 mol sulfhydryls per mole calcium pump protein assayed in the absence and presence of sodium dodecyl sulfate. The release of Ca2+ from sarcoplasmic reticulum, which triggers muscle contraction, likely involves the regulation of a channel. This report deals with an experimental approach to studying the Ca2+ release in isolated sarcoplasmic reticulum. We find that sulfhydryl agents of which water-soluble mercurials were most effective induce Ca2+ release. Chlorpromazine acts synergistically with the sulfhydryl reagents. Ca2+ release under optimal conditions is very rapid compared with calcium leakage from preloaded but untreated sarcoplasmic reticulum. The imposed rapid release of Ca2+ is suggestive of the opening of a channel. Ca2+ release by mercurials is retained in reconstituted sarcoplasmic reticulum membrane vesicles.

Animals↗
PubMed1981

Phosphoenzyme decomposition in sarcoplasmic reticulum isolated from cat caudofemoralis, tibialis and soleus.

Decomposition of phosphoenzyme (E approximately P) in sarcoplasmic reticulum isolated from caudofemoralis, tibialis and soleus of cat hind leg skeletal muscles was studied under various conditions of monovalent cations. In the presence of Li+, Na+, and K+ chosen for E approximately P formation and decomposition after quenching of E approximately P with EGTA, E approximately P in the caudofemoralis and tibialis sarcoplasmic reticulum decomposed faster than that in the soleus sarcoplasmic reticulum. Quenching the E approximately P formation with EGTA and ADP revealed that 30-40% of the total E approximately P formed in all types of sarcoplasmic reticulum was 'ADP sensitive'. Decomposition of the remaining E approximately P in caudofemoralis and tibialis sarcoplasmic reticulum was enhanced by ADP, which resulted in a multiphasic decomposition pattern. A larger portion of the remaining E approximately P in the soleus sarcoplasmic reticulum, on the other hand decomposed in a monophasic manner and was not significantly influenced by ADP. The data on E approximately P decomposition clearly differentiate between the fast and slow muscle types.

Animals↗
PubMed1984

Calcium release from sarcoplasmic reticulum of normal and dystrophic mice.

Contraction of skeletal muscle is triggered by release of calcium from the sarcoplasmic reticulum. In this study, highly purified normal and dystrophic mouse sarcoplasmic reticulum vesicles were compared with respect to calcium release characteristics. Sarcoplasmic reticulum vesicles were actively loaded with calcium in the presence of an ATP-regenerating system. Calcium fluxes were followed by dual wavelength spectrophotometry using the metallochromic indicators antipyrylazo III and arsenazo III, and by isotopic techniques. Calcium release from sarcoplasmic reticulum vesicle was elicited by (a) changing the free calcium concentration of the assay medium (calcium-induced calcium release); (b) addition of a permeant anion to the assay medium, following calcium loading in the presence of a relatively impermeant anion (depolarization-induced calcium release); (c) addition of the lipophilic anion tetraphenylboron (TPB-) to the assay medium and (d) using specific experimental conditions, i.e. high phosphate levels and low magnesium (spontaneous calcium release). Drugs known to influence Ca2+ release were shown to differentially affect the various types of calcium release. Caffeine (10 mM) was found to enhance calcium-induced calcium release from isolated sarcoplasmic reticulum. Ruthenium red (20 microM) inhibited both calcium-induced calcium release and tetraphenylboron-induced calcium release, and partially inhibited spontaneous calcium release and depolarization-induced calcium release. Local anesthetics inhibited spontaneous calcium release in a time-dependent manner, and inhibited calcium-induced calcium release instantaneously, but did not inhibit depolarization-induced calcium release. Use of pharmacological agents indicates that several types of calcium release operate in vitro. No significant differences were found between normal and dystrophic sarcoplasmic reticulum in calcium release kinetics or drug sensitivities.

Animals↗
PubMed1987

Lack of effects of calcium X calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum.

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium X calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca2+ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium X calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca2+ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium X calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca2+ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent 45Ca2+-40Ca2+ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium X calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca2+ release and 45Ca2+-40Ca2+ exchange.

Animals↗
PubMed1990

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital.

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.

Adenosine Triphosphate↗
PubMed1993

Effects of thapsigargin and ryanodine on vascular contractility: cross-talk between sarcoplasmic reticulum and plasmalemma.

Thapsigargin and ryanodine are proposed to interfere with Ca2+ storage in sarcoplasmic reticulum by different mechanisms. Thapsigargin inhibits Ca2+ transport into and ryanodine enhances Ca2+ out of the sarcoplasmic reticulum. Contractility studies were performed in the rat aorta and dog mesenteric artery. Ryanodine was found to reduce phenylephrine-induced (10 microM) contraction in Ca(2+)-free medium of rat aorta and dog mesenteric artery in a concentration-dependent manner. Each agent alone caused a slow contraction in the rat aorta. In this tissue, the tension caused by ryanodine (30 microM) but not that by thapsigargin (1 microM) was found to be dependent on the status of the sarcoplasmic reticulum: prior stimulation with K+ (60 mM) enhanced the rate of development of ryanodine-induced tension compared with when the sarcoplasmic reticulum was previously depleted with phenylephrine stimulation in Ca(2+)-free medium. Sodium nitroprusside (1 microM) or isoproterenol (1 microM) fully antagonized the contraction induced by ryanodine or phenylephrine. However, thapsigargin-induced contraction was antagonized fully by sodium nitroprusside and only partially by isoproterenol. This result suggests that cAMP elevation by isoproterenol required a functioning sarcoplasmic reticulum Ca2+ pump for its relaxant effect while cGMP elevation by sodium nitroprusside did not. These findings are consistent with the view that ryanodine promotes Ca2+ release from the sarcoplasmic reticulum and that thapsigargin inhibits the ability of cAMP to stimulate Ca2+ uptake into the store by blocking its Ca2+ pump. In the dog mesenteric artery, when the phenylephrine-sensitive Ca2+ pool was emptied and thapsigargin was added to block Ca2+ uptake into the store, restoration of Ca2+ in the Ca(2+)-free medium caused a transient contraction (absent in controls). This contraction was replaced by a significantly larger amplitude and more sustained contraction in low Na+ medium indicating the involvement of the Na+/Ca2+ exchanger in the homeostasis of cytosolic [Ca2+]. In the presence of nifedipine (2 microM), repletion of the phenylephrine-sensitive store was inhibited. It is possible that refilling occurs in part through L-type Ca2+ channels.

Animals↗
PubMed1987

Distribution and calcium-sequestering ability of smooth endoplasmic reticulum in olfactory axon terminals of frog brain.

In the present study, the structural and functional role of smooth endoplasmic reticulum was investigated in bullfrog olfactory axon terminals. Structural evidence obtained from this study indicated that this vesiculotubular organelle becomes a more elaborate network of anastomosing tubules near the nerve terminal, located in the olfactory lobe of frog brain. Further structural evidence suggested that membranes of the smooth endoplasmic reticulum pinch off to give rise to some electron-lucent vesicles of approximately 50 nm diameter (microvesicles). Ultrastructural cytochemistry was employed in the present study to demonstrate that olfactory axon terminal smooth endoplasmic reticulum actively sequesters Ca2+. However, a variable amount of electron-dense product (calcium oxalate) was associated with microvesicles located at a distance from the synapse, in contrast to those clustered near the synapse which usually did not contain this reaction product. Results from Ca2+-Mg2+-adenosine-5'-triphosphatase (ATPase) cytochemistry showed a similar pattern of distribution, with smooth endoplasmic reticulum being densely labeled with ATPase reaction product (lead phosphate), but aggregated microvesicles in the nerve terminal generally lacking this electron-dense product. Therefore, it is concluded that olfactory axonal smooth endoplasmic reticulum plays a role in the regulation of intraneuronal Ca2+ levels, and that the Ca2+-sequestering activity of this membranous organelle is dependent upon enzymatic hydrolysis of ATP. Conversely, the microvesicles, particularly those accumulated near the synapse, lack this Ca2+-pumping capacity. Thus, if some of the microvesicles originate from smooth endoplasmic reticulum membranes which are capable of pumping Ca2+, but these vesicles themselves lack this capacity, one can postulate that the Ca2+ pumps are either removed from the newly formed microvesicle membranes or are somehow incapacitated in situ in the membrane.

Adenosine Triphosphate↗
PubMed2005

Interaction of PTPB with the insulin receptor precursor during its biosynthesis in the endoplasmic reticulum.

PTP1B is a protein tyrosine-phosphatase predominantly located on the cystosolic surface of the endoplasmic reticulum. This tyrosine-phosphatase plays a major role in the regulation of the activity of the insulin receptor (IR). We have studied the interaction of the IR with PTP1B in living cells using bioluminescence resonance energy transfer (BRET). The IR was fused to Renilla luciferase and a substrate-trapping mutant of PTP1B was fused to the yellow variant of the green fluorescent protein (YFP). When the two partners interacted, an energy transfer occurred between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, could be detected. The interaction of the IR with PTP1B could be monitored in real time for more than 30 min. Insulin rapidly and dose-dependently stimulated this interaction. The basal (insulin-independent) interaction of IR with PTP1B was much lower with a soluble form than with the endoplasmic reticulum-targeted form of PTP1B, indicating that this basal interaction mainly occurred in the endoplasmic reticulum. In the basal state, PTP1B and the IR indeed co-localized in the endoplasmic reticulum, as demonstrated by confocal microscopy and cell fractionation experiments. Moreover, inhibition of IR processing with tunicamycin indicated that the basal interaction of PTP1B with IR occurred during biosynthesis of the IR precursor in the endoplasmic reticulum. These results strongly suggest that PTP1B not only dephosphorylates the insulin receptor that has been activated by insulin, but also regulates the insulin receptor precursor during its biosynthesis. Localisation of PTP1B to the endoplasmic reticulum may be important to prevent insulin-independent autonomous activity of the immature insulin receptor precursor.

Animals↗
PubMed2004

Tubular aggregates are from whole sarcoplasmic reticulum origin: alterations in calcium binding protein expression in mouse skeletal muscle during aging.

Tubular aggregates are observed in various muscle disorders and appear as densely packed tubules believed to arise from sarcoplasmic reticulum of striated muscle. They are found both in human skeletal muscle, especially from patients suffering from 'tubular aggregate myopathy' and in fast twitch skeletal muscle of the male inbred mouse during aging. In this work, we studied tubular aggregates present in inbred male mouse skeletal muscle using electron microscopy as well as histochemistry and Western blotting with the main markers of the sarcoplasmic reticulum. We show that mouse tubular aggregates include the proteins SERCA 1, sarcalumenin (longitudinal sarcoplasmic reticulum), calsequestrin (terminal cisternae) and RyR1 (junctional sarcoplasmic reticulum). We demonstrate also that 95 and 51 kDa triadin isoforms are present in mouse skeletal muscle and are both components of tubular aggregates. These results support the hypothesis that tubular aggregates form a tubular arrangement of a complete sarcoplasmic reticulum containing the junctional, cisternae and longitudinal components of sarcoplasmic reticulum implicated in calcium homeostasis. During mouse skeletal muscle aging, however, densitometry of Western blots reveals a persistent decrease in the expression of the calcium binding protein calreticulin as well as a continuous increase in calsequestrin-like protein expression which both appear unrelated to the tubular aggregate formation.

Age Factors↗
PubMed2000

Ca(2+) uptake function of sarcoplasmic reticulum during contraction of rat arterial smooth muscles.

To determine the Ca(2+) uptake function of the sarcoplasmic reticulum during contraction, the effects of cyclopiazonic acid or thapsigargin, agents that inhibit sarcoplasmic reticulum Ca(2+)-ATPase, on the contractile responses to K(+) or norepinephrine were compared in endothelium-denuded strips of femoral, mesenteric and carotid arteries of the rat. The addition of K(+) (3-20 mM) to the strips caused a concentration-dependent contraction, and the sensitivity to K(+) was much higher in the carotid artery than in the other arteries. The preincubation of strips with cyclopiazonic acid (10 microM) or thapsigargin (100 nM) caused a leftward shift of the concentration-response curve for K(+), and this effect was smaller in the carotid artery than in the other arteries. Inhibition of sarcoplasmic reticulum Ca(2+) uptake caused the sensitivity to K(+) to be similar in the three arteries. Similar results were obtained when the contractile responses to norepinephrine were determined. Cyclopiazonic acid itself induced similar transient contractions in the three arteries. The addition of caffeine (20 mM) caused a transient contraction that was smaller in the carotid artery than in the other arteries. We conclude that (1) the Ca(2+) influx during stimulation with K(+) or norepinephrine is buffered by the sarcoplasmic reticulum in femoral and mesenteric arteries, (2) this function is weak in the carotid artery, probably because the sarcoplasmic reticulum of this artery is almost filled with Ca(2+) in the resting state, and (3) the Ca(2+) uptake function of the sarcoplasmic reticulum during contraction is reflected by the contractile sensitivity in these arteries.

Animals↗
PubMed2000

Attributes and dynamics of the endoplasmic reticulum in mammalian eggs.

The endoplasmic reticulum is a multifunctional continuous network of membrane-enclosed sacs and tubules that extends throughout the cell. The endoplasmic reticulum is the site of protein synthesis and assembly, as well as lipid and membrane synthesis. Additionally, the endoplasmic reticulum contains calcium pumps, intraluminal calcium storage proteins, and specific calcium-releasing channels. Thus, this membrane system plays a central role in intracellular signaling through the storage and release of calcium. At fertilization, the sperm triggers a large and dramatic release of calcium from the endoplasmic reticulum, which activates the egg to begin development. The ability of the egg to fully elevate calcium depends on biochemical and structural changes during oocyte maturation. The sensitivity of the calcium-releasing system increases and the endoplasmic reticulum is reorganized during maturation of the oocyte; together, these dynamic changes place a substantial calcium storage compartment just beneath the membrane, near the site of sperm-egg fusion. Localization of the calcium store may also contribute to the long-lasting calcium oscillations that are characteristic of mammalian fertilization. Examination of the endoplasmic reticulum in living eggs is leading to a better understanding of calcium release at fertilization.

Animals↗