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1.
Mol Cell Biochem ; 225(1-): 1-6, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11716351

ABSTRACT

Our hypothesis is that sarcoplasmic and endoplasmic reticulum Ca2+ stores may be functionally distinct compartments in cardiomyocytes. Sarcoplasmic reticulum Ca2+ store is responsible for control of excitation-contraction coupling whereas endoplasmic reticulum compartment may provide Ca2+ for housekeeping and transcriptional functions.


Subject(s)
Endoplasmic Reticulum/metabolism , Myocardium/ultrastructure , Animals , Calcium/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calreticulin , Heart/growth & development , Humans , Intracellular Membranes/metabolism , Lectins/metabolism , Mice , Mice, Knockout , Myocardial Contraction , Myocardium/metabolism , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Sarcoplasmic Reticulum/metabolism , Transcription, Genetic
2.
J Cell Biol ; 154(3): 525-34, 2001 Aug 06.
Article in English | MEDLINE | ID: mdl-11489915

ABSTRACT

Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.


Subject(s)
Calsequestrin , Endoplasmic Reticulum/metabolism , Amino Acid Sequence , Animals , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calreticulin , Calsequestrin/chemistry , Calsequestrin/genetics , Calsequestrin/metabolism , HeLa Cells , Humans , Molecular Sequence Data , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/metabolism , Mutagenesis, Site-Directed/physiology , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Ribonucleoproteins/chemistry , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Transfection , Vacuoles/metabolism
3.
Cardiovasc Res ; 49(1): 118-26, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11121803

ABSTRACT

OBJECTIVE: Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P(2)) is not only a precursor to inositol 1,4,5-trisphosphate (Ins 1,4, 5-P(3)) and sn-1,2 diacylglycerol, but also essential for the function of several membrane proteins. The aim of this study was to evaluate the changes in the level of this phospholipid in the cell plasma membrane (sarcolemma, SL) of cardiomyopathic hamster (CMPH) heart. METHODS: We examined the cardiac SL PtdIns 4,5-P(2) mass and the activities of the enzymes responsible for its synthesis and hydrolysis in 250-day-old UM-X7.1 CMPH at a severe stage of congestive heart failure (CHF) and in age-matched controls (Syrian Golden hamsters). RESULTS: The SL PtdIns 4,5-P(2) mass in CMPH was reduced by 72% of the control value. The activities of PtdIns 4 kinase and PtdIns 4-P 5 kinase were depressed by 69 and 50% of control values, respectively. Although, the total phospholipase C (PLC) activity was moderately, although significantly, decreased (by 18% of control), PLCdelta(1) isoenzyme activity in the SL membrane was elevated, with a concomitant increase in its protein content, whereas PLCbeta(1) and gamma(1) isoenzyme activities were depressed despite the increase in their protein levels. A 2-fold increase in the Ins 1,4,5-P(3) concentration in the cytosol of the failing heart of CMPH was also observed. CONCLUSIONS: Reduced SL level of PtdIns 4, 5-P(2) may severely jeopardize cardiac cell function in this hamster model of CHF. In addition, the profound changes in the profile of heart SL PLC isoenzyme could alter the complex second messenger responses of these isoenzymes, and elevated Ins 1,4,5-P(3) levels may contribute to intracellular Ca(2+) overload in the failing cardiomyocyte.


Subject(s)
Heart Failure/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Sarcolemma/metabolism , 1-Phosphatidylinositol 4-Kinase/metabolism , Animals , Blotting, Western , Cricetinae , Cytosol/metabolism , Heart Failure/enzymology , Inositol 1,4,5-Trisphosphate/metabolism , Isoenzymes/metabolism , Male , Mesocricetus , Sarcolemma/enzymology , Second Messenger Systems/physiology , Type C Phospholipases/metabolism
4.
Arch Biochem Biophys ; 382(1): 48-56, 2000 Oct 01.
Article in English | MEDLINE | ID: mdl-11051096

ABSTRACT

Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) is the substrate for phosphoinositide-phospholipase C (PLC) and is required for the function of several cardiac cell plasma membrane (sarcolemma, SL) proteins. PtdIns 4,5-P2 is synthesized in the SL membrane by coordinated and successive actions of PtdIns 4-kinase and PtdIns 4-phosphate 5-kinase. These kinases and the generation of PtdIns 4,5-P2 may be a factor in the cardiac dysfunction during pathophysiological conditions of oxidative stress. Therefore, we examined the effects of different reactive oxygen species (ROS) on the kinases' activities and subsequent generation of PtdIns 4,5-P2. Exposure to the xanthine-xanthine oxidase-ROS generating system significantly reduced both SL kinase activities. Superoxide dismutase did not prevent this inhibition; however, catalase significantly prevented the xanthine-xanthine oxidase induced inhibition. Treatment of SL with hydrogen peroxide (H2O2) resulted in inhibition of both the kinases, which was prevented by catalase and dithiothreitol (DTT). Hypochlorous acid also inhibited both the kinases, which was prevented by DTT. Deferoxamine (an iron chelator) and mannitol (an *OH scavenger) did not modify the H2O2-induced depression of the kinases, eliminating any role of *OH. Furthermore, the IC50 of H2O2 on PtdIns 4-kinase and PtdIns 4-P 5-kinase was 27 and 81 microM, respectively. In addition, inclusion of reduced glutathione in the assay of the kinases in the absence of H2O2 did not affect the activities of the kinases; however, oxidized glutathione induced a significant depression. Also, a significant decline of the PtdIns 4-kinase and PtdIns 4-P 5-kinase activities due to changing of the redox ratio was observed. Thiol modifiers (N-ethylmaleimide, methyl methanethiosulfonate, or p-chloromercuriphenylsulfonic acid) were detected to depress the kinases' activities, which were substantially prevented by DTT. The results suggest that functionally critical thiol groups may be associated with PtdIns 4-kinase and PtdIns 4-P 5-kinase and that changes of their redox state by ROS can impair their activities, which may be an important factor in the oxidant-induced cardiac dysfunction.


Subject(s)
Myocardium/metabolism , Oxidants/metabolism , Phosphatidylinositol 4,5-Diphosphate/biosynthesis , Reactive Oxygen Species/metabolism , Sarcolemma/metabolism , 1-Phosphatidylinositol 4-Kinase/metabolism , Animals , Dose-Response Relationship, Drug , Glutathione/pharmacology , Glutathione Disulfide/pharmacology , Heart/drug effects , Hydrogen Peroxide/pharmacology , Hypochlorous Acid/pharmacology , Inhibitory Concentration 50 , Kinetics , Oxidation-Reduction , Oxidative Stress , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Rats , Sarcolemma/drug effects , Time Factors
5.
Biochem J ; 344 Pt 2: 281-92, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-10567207

ABSTRACT

The endoplasmic reticulum (ER) plays a critical role in the synthesis and chaperoning of membrane-associated and secreted proteins. The membrane is also an important site of Ca(2+) storage and release. Calreticulin is a unique ER luminal resident protein. The protein affects many cellular functions, both in the ER lumen and outside of the ER environment. In the ER lumen, calreticulin performs two major functions: chaperoning and regulation of Ca(2+) homoeostasis. Calreticulin is a highly versatile lectin-like chaperone, and it participates during the synthesis of a variety of molecules, including ion channels, surface receptors, integrins and transporters. The protein also affects intracellular Ca(2+) homoeostasis by modulation of ER Ca(2+) storage and transport. Studies on the cell biology of calreticulin revealed that the ER membrane is a very dynamic intracellular compartment affecting many aspects of cell physiology.


Subject(s)
Calcium-Binding Proteins/metabolism , Calcium/metabolism , Endoplasmic Reticulum/metabolism , Lectins/metabolism , Molecular Chaperones/metabolism , Ribonucleoproteins/metabolism , Amino Acid Sequence , Animals , Calcium-Binding Proteins/genetics , Calreticulin , Homeostasis , Lectins/genetics , Mice , Molecular Chaperones/genetics , Molecular Sequence Data , Ribonucleoproteins/genetics
6.
J Biol Chem ; 274(21): 15085-94, 1999 May 21.
Article in English | MEDLINE | ID: mdl-10329714

ABSTRACT

We used two cell lines expressing fast (RPEfast) and slow (RPEslow) attachment kinetics to investigate mechanisms of cell-substratum adhesion. We show that the abundance of a cytoskeletal protein, vinculin, is dramatically decreased in RPEfast cells. This coincides with the diminished expression level of an endoplasmic reticulum chaperone, calreticulin. Both protein and mRNA levels for calreticulin and vinculin were decreased in RPEfast cells. After RPEfast cells were transfected with cDNA encoding calreticulin, both the expression of endoplasmic reticulum-resident calreticulin and cytoplasmic vinculin increased. The abundance of other adhesion-related proteins was not affected. RPEfast cells underexpressing calreticulin displayed a dramatic increase in the abundance of total cellular phosphotyrosine suggesting that the effects of calreticulin on cell adhesiveness may involve modulation of the activities of protein tyrosine kinases or phosphatases which may affect the stability of focal contacts. The calreticulin and vinculin underexpressing RPEfast cells lacked extensive focal contacts and adhered weakly but attached fast to the substratum. In contrast, the RPEslow cells that expressed calreticulin and vinculin abundantly developed numerous and prominent focal contacts slowly, but adhered strongly. Thus, while the calreticulin overexpressing RPEslow cells "grip" the substratum with focal contacts, calreticulin underexpressing RPEfast cells use close contacts to "stick" to it.


Subject(s)
Calcium-Binding Proteins/physiology , Cell Adhesion/physiology , Molecular Chaperones/physiology , Receptors, G-Protein-Coupled , Ribonucleoproteins/physiology , Calcium-Binding Proteins/biosynthesis , Calreticulin , Cells, Cultured , Eye Proteins/genetics , Eye Proteins/physiology , GTP-Binding Proteins/physiology , Molecular Chaperones/biosynthesis , Phenotype , Phosphotyrosine/biosynthesis , Receptors, Cell Surface/genetics , Receptors, Cell Surface/physiology , Ribonucleoproteins/biosynthesis , Vinculin/physiology
7.
J Cell Biol ; 144(5): 857-68, 1999 Mar 08.
Article in English | MEDLINE | ID: mdl-10085286

ABSTRACT

Calreticulin is a ubiquitous Ca2+ binding protein, located in the endoplasmic reticulum lumen, which has been implicated in many diverse functions including: regulation of intracellular Ca2+ homeostasis, chaperone activity, steroid-mediated gene regulation, and cell adhesion. To understand the physiological function of calreticulin we used gene targeting to create a knockout mouse for calreticulin. Mice homozygous for the calreticulin gene disruption developed omphalocele (failure of absorption of the umbilical hernia) and showed a marked decrease in ventricular wall thickness and deep intertrabecular recesses in the ventricular walls. Transgenic mice expressing a green fluorescent protein reporter gene under the control of the calreticulin promoter were used to show that the calreticulin gene is highly activated in the cardiovascular system during the early stages of cardiac development. Calreticulin protein is also highly expressed in the developing heart, but it is only a minor component of the mature heart. Bradykinin-induced Ca2+ release by the InsP3-dependent pathway was inhibited in crt-/- cells, suggesting that calreticulin plays a role in Ca2+ homeostasis. Calreticulin-deficient cells also exhibited impaired nuclear import of nuclear factor of activated T cell (NF-AT3) transcription factor indicating that calreticulin plays a role in cardiac development as a component of the Ca2+/calcineurin/NF-AT/GATA-4 transcription pathway.


Subject(s)
Calcium-Binding Proteins/physiology , Heart/embryology , Nuclear Proteins , Ribonucleoproteins/physiology , Animals , Base Sequence , Biological Transport , Calcium/metabolism , Calcium-Binding Proteins/genetics , Calreticulin , Cell Nucleus/metabolism , DNA Primers , DNA-Binding Proteins/metabolism , Embryonic and Fetal Development/physiology , Inositol 1,4,5-Trisphosphate/metabolism , Mice , Mice, Knockout , Mice, Transgenic , NFATC Transcription Factors , Phenotype , Promoter Regions, Genetic , Ribonucleoproteins/genetics , Transcription Factors/metabolism
8.
Arterioscler Thromb Vasc Biol ; 17(11): 2359-68, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9409202

ABSTRACT

Both thrombotic and inflammatory responses to arterial injury have been implicated in atherosclerotic plaque growth. Calreticulin is a ubiquitous calcium-binding protein with antithrombotic activity and, in addition, is associated with leukocyte activation. We are investigating calreticulin as a potential vascular regulatory protein. The development of intimal hyperplasia was studied at sites of balloon injury in iliofemoral arteries from 91 rats. Calreticulin was infused directly into the artery immediately before balloon injury, and plaque growth was then assessed at 4 weeks' follow-up. Parallel studies of the effects of each calreticulin domain as well as a related calcium-binding protein, calsequestrin, were examined. The effects of calreticulin on platelet activation, clot formation, and mononuclear cell migration were also studied. When infused before balloon injury in rat iliofemoral arteries, calreticulin, or its high-capacity Ca(2+)-binding C domain, significantly reduces plaque development, whereas calsequestrin, a related calcium-binding protein that lacks the multifunctional nature of calreticulin, does not decrease plaque area (saline: 0.037 +/- 0.007 mm2, calsequestrin: 0.042 +/- 0.021 mm2, calreticulin: 0.003 +/- 0.002 mm2, n = 46, P < .04). The N domain and more specifically the P domain, a low-capacity, high-affinity calcium-binding domain in calreticulin, do not reduce intimal hyperplasia (N + P domain: 0.038 +/- 0.012 mm2, C domain: 0.003 +/- 0.002 mm2, n = 45 rats, P < .0001). Calreticulin reduces macrophage and T cell staining in the arterial wall after injury but has no direct effect on monocyte migration in vitro (percent medial area staining positive for macrophage 24 hours after injury (N + P: 4.06 +/- 1.42, calreticulin: 0.273 +/- 0.02; n = 26, P < .009). Calreticulin does, however, reduce platelet-dependent whole blood clotting time, in vitro (baseline: 78.23 +/- 2.04 seconds, calreticulin: 113.5 +/- 1.95 seconds; n = 5, P < .002). We conclude that calreticulin significantly reduces intimal hyperplasia after arterial injury, potentially acting as a vascular regulatory protein.


Subject(s)
Calcium-Binding Proteins/pharmacology , Femoral Artery/injuries , Iliac Artery/injuries , Ribonucleoproteins/pharmacology , Tunica Intima/drug effects , Angioplasty, Balloon/adverse effects , Animals , Blood Coagulation/drug effects , Calcium-Binding Proteins/chemistry , Calreticulin , Calsequestrin/analysis , Chemotaxis, Leukocyte/drug effects , Femoral Artery/drug effects , Femoral Artery/pathology , Humans , Hyperplasia , Iliac Artery/drug effects , Iliac Artery/pathology , Macrophages/drug effects , Macrophages/pathology , Monocytes/drug effects , Monocytes/pathology , Peptide Fragments/pharmacology , Platelet Activation/drug effects , Rabbits , Rats , Rats, Sprague-Dawley , Ribonucleoproteins/chemistry , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/pathology , Thrombosis/prevention & control , Tunica Intima/injuries , Tunica Intima/pathology
9.
J Cell Biol ; 138(3): 547-57, 1997 Aug 11.
Article in English | MEDLINE | ID: mdl-9245785

ABSTRACT

We have isolated and characterized a 12-kb mouse genomic DNA fragment containing the entire calreticulin gene and 2.14 kb of the promoter region. The mouse calreticulin gene consists of nine exons and eight introns, and it spans 4.2 kb of genomic DNA. A 1.8-kb fragment of the calreticulin promoter was subcloned into a reporter gene plasmid containing chloramphenicol acetyltransferase. This construct was then used in transient and stable transfection of NIH/ 3T3 cells. Treatment of transfected cells either with the Ca2+ ionophore A23187, or with the ER Ca2+-ATPase inhibitor thapsigargin, resulted in a five- to sevenfold increase of the expression of chloramphenicol acetyltransferase protein. Transactivation of the calreticulin promoter was also increased by fourfold in NIH/3T3 cells treated with bradykinin, a hormone that induces Ca2+ release from the intracellular Ca2+ stores. Analysis of the promoter deletion constructs revealed that A23187- and thapsigargin-responsive regions are confined to two regions (-115 to -260 and -685 to -1,763) in the calreticulin promoter that contain the CCAAT nucleotide sequences. Northern blot analysis of cells treated with A23187, or with thapsigargin, revealed a fivefold increase in calreticulin mRNA levels. Thapsigargin also induced a fourfold increase in calreticulun protein levels. Importantly, we show by nuclear run-on transcription analysis that calreticulin gene transcription is increased in NIH/3T3 cells treated with A23187 and thapsigargin in vivo. This increase in gene expression required over 4 h of continuous incubation with the drugs and was also sensitive to treatment with cycloheximide, suggesting that it is dependent on protein synthesis. Changes in the concentration of extracellular and cytoplasmic Ca2+ did not affect the increased expression of the calreticulin gene. These studies suggest that stress response to the depletion of intracellular Ca2+ stores induces expression of the calreticulin gene in vitro and in vivo.


Subject(s)
Calcium-Binding Proteins/genetics , Calcium/metabolism , Gene Expression Regulation , Promoter Regions, Genetic , Ribonucleoproteins/genetics , 3T3 Cells , Animals , Base Sequence , Bradykinin/pharmacology , Calcimycin/pharmacology , Calreticulin , Cell Line , Chelating Agents/pharmacology , Cloning, Molecular , Cytoplasm/metabolism , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Endoplasmic Reticulum/metabolism , Gene Expression Regulation/drug effects , Humans , Ionophores , Kinetics , Mice , Molecular Sequence Data , Promoter Regions, Genetic/drug effects , Thapsigargin/pharmacology
10.
J Mol Cell Cardiol ; 29(1): 237-46, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9040038

ABSTRACT

Although phosphoinositide-specific phospholipase C (PLC) is involved in signal transduction mechanisms of the myocardial cell. very little is known about its status in congestive heart failure (CHF). We have examined the PLC activity in sarcolemmal and cytosolic fractions isolated from the viable left ventricle of rats at 8 weeks (moderate stage of CHF) and 16 weeks (severe stage of CHF) after occlusion of the left anterior descending coronary artery; the hypertrophied right ventricle was used for comparison. At 8 weeks, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by sarcolemmal PLC was reduced by 37% of sham control values only in the left ventricle, whereas at 16 weeks, PLC-mediated hydrolysis was depressed in both left and right ventricles by 25% and 30%, respectively. The hydrolysis of phosphatidylinositol 4-monophosphate (PIP) was reduced by 25% of control value only in the severely failing left ventricle, while the phosphatidylinositol (PI) hydrolysis remained unaltered. Kinetic studies of PLC activity in the left ventricle showed a depression of V(max) at moderate and severe failure stages, whereas the affinity for the substrate was increased in the left ventricle at 8 weeks and decreased in the right ventricle at 16 weeks. The only difference observed between experimental and control groups at the cytosolic level, was a significant enhancement of PLC activity in the severely failing left ventricle when PIP was given as a substrate, and in the corresponding right ventricle when PI was the substrate. The results of this study identify time-related defects in sarcolemmal PLC in right and left ventricles during the development of CHF due to myocardial infarction.


Subject(s)
Cytosol/enzymology , Heart Failure/enzymology , Heart Ventricles/enzymology , Sarcolemma/enzymology , Type C Phospholipases/metabolism , Analysis of Variance , Animals , Heart Failure/pathology , Hemodynamics/physiology , Male , Rats , Rats, Sprague-Dawley
11.
J Cell Biol ; 135(6 Pt 2): 1913-23, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8991101

ABSTRACT

Calreticulin is an ubiquitous and highly conserved high capacity Ca(2+)-binding protein that plays a major role in Ca2+ storage within the lumen of the ER. Here, using L fibroblast cell lines expressing different levels of calreticulin, we show that calreticulin plays a role in the control of cell adhesiveness via regulation of expression of vinculin, a cytoskeletal protein essential for cell-substratum and cell-cell attachments. Both vinculin protein and mRNA levels are increased in cells overexpressing calreticulin and are downregulated in cells expressing reduced level of calreticulin. Abundance of actin, talin, alpha 5 and beta 1 integrins, pp125 focal adhesion kinase, and alpha-catenin is not affected by the differential calreticulin expression. Overexpression of calreticulin increases both cell-substratum and cell-cell adhesiveness of L fibroblasts that, most surprisingly, establish vinculin-rich cell-cell junctions. Upregulation of calreticulin also affects adhesion-dependent phenomena such as cell motility (which decreases) and cell spreading (which increases). Downregulation of calreticulin brings about inverse effects. Cell adhesiveness is Ca2+ regulated. The level of calreticulin expression, however, has no effect on either the resting cytoplasmic Ca2+ concentration or the magnitude of FGF-induced Ca2+ transients. Calreticulin, however, participates in Ca2+ homeostasis as its level of expression affects cell viability at low concentrations of extracellular Ca2+. Consequently, we infer that it is not the Ca2+ storage function of calreticulin that affects cell adhesiveness. Neither endogenous calreticulin nor overexpressed green fluorescent protein-calreticulin construct can be detected outside of the ER. Since all of the adhesion-related effects of differential calreticulin expression can be explained by its regulation of vinculin expression, we conclude that it is the ER-resident calreticulin that affects cellular adhesiveness.


Subject(s)
Calcium-Binding Proteins/genetics , Fibroblasts/cytology , Ribonucleoproteins/genetics , Vinculin/genetics , Animals , Blotting, Northern , Blotting, Western , Calcium/metabolism , Calreticulin , Cell Adhesion/physiology , Cell Line/chemistry , Cell Line/cytology , Cell Line/physiology , Cell Movement/physiology , Cell Size , Cytoplasm/chemistry , Cytoskeleton/physiology , Endoplasmic Reticulum/chemistry , Fibroblasts/chemistry , Fibroblasts/physiology , Fluorescent Antibody Technique , Gene Expression/physiology , Integrins/analysis , Intercellular Junctions/chemistry , Mice , Molecular Chaperones/genetics , RNA, Messenger/analysis , Transfection
12.
J Biol Chem ; 271(46): 29436-45, 1996 Nov 15.
Article in English | MEDLINE | ID: mdl-8910610

ABSTRACT

Calreticulin is a ubiquitously expressed Ca2+-binding protein of the endoplasmic reticulum (ER), which inhibits DNA binding in vitro and transcriptional activation in vivo by steroid hormone receptors. Transient transfection assays were carried out to investigate the effects of different intracellular targeting of calreticulin on transactivation mediated by glucocorticoid receptor. BSC40 cells were transfected with either calreticulin expression vector (ER form of calreticulin) or calreticulin expression vector encoding calreticulin minus leader peptide, resulting in cytoplasmic localization of the recombinant protein. Transfection of BSC40 cells with calreticulin expression vector encoding the ER form of the protein led to 40-50% inhibition of the dexamethasone-sensitive stimulation of luciferase expression. However, in a similar experiment, but using the calreticulin expression vector encoding cytoplasmic calreticulin, dexamethasone-stimulated activation of the luciferase reporter gene was inhibited by only 10%. We conclude that the ER, but not cytosolic, form of calreticulin is responsible for inhibition of glucocorticoid receptor-mediated gene expression. These effects are specific to calreticulin, since overexpression of the ER lumenal proteins (BiP, ERp72, or calsequestrin) has no effect on glucocorticoid-sensitive gene expression. The N domain of calreticulin binds to the DNA binding domain of the glucocorticoid receptor in vitro; however, we show that the N+P domain of calreticulin, when synthesized without the ER signal sequence, does not inhibit glucocorticoid receptor function in vivo. Furthermore, expression of the N domain of calreticulin and the DNA binding domain of glucocorticoid receptor as fusion proteins with GAL4 in the yeast two-hybrid system revealed that calreticulin does not interact with glucocorticoid receptor under these conditions. We conclude that calreticulin and glucocorticoid receptor may not interact in vivo and that the calreticulin-dependent modulation of the glucocorticoid receptor function may therefore be due to a calreticulin-dependent signaling from the ER.


Subject(s)
Calcium-Binding Proteins/metabolism , Endoplasmic Reticulum/metabolism , Gene Expression Regulation/drug effects , Glucocorticoids/pharmacology , Ribonucleoproteins/metabolism , 3T3 Cells , Animals , Calreticulin , Mice , Nuclear Proteins/metabolism , Rats , Receptors, Glucocorticoid/metabolism , Signal Transduction , Transcriptional Activation
13.
J Biol Chem ; 271(16): 9332-9, 1996 Apr 19.
Article in English | MEDLINE | ID: mdl-8621597

ABSTRACT

The widely distributed and highly conserved Ca(2+)-binding protein calreticulin has been suggested to play a role as a Ca2+ storage protein of intracellular Ca+ stores. To test this hypothesis, we have generated a mouse L fibroblast cell line stably transfected with a calreticulin expression vector. The calreticulin content of the overexpressers was increased by 1.6 +/- 0.2-fold compared with mock-transfected cells. The total cellular Ca2+ content of calreticulin-overexpressing and control cells, as assessed by equilibrium 45Ca+2 uptake, was 141 +/- 8 and 67 +/- 6 pmol of Ca2+/10(6) cells, respectively (i.e. a 2.1 +/- 0.2-fold increase in the Ca2+ content of calreticulin-overexpressing cells). Over 80% of the increased Ca2+ content was found within thapsigargin-sensitive Ca2+ stores. The pattern of calreticulin distribution, revealed by immunofluorescence microscopy, showed an endoplasmic reticulum-like pattern and was identical in overexpressers and control cells. In overexpressers, cytosolic free [Ca2+] elevations due to Ca2+ release were enhanced when either ATP or a combination of ionomycin and thapsigargin was used as a stimulus. In contrast, thapsigargin-induced Ca2+ and Mn2+ influxes from the extracellular space were markedly diminished in calreticulin-overexpressing cells, suggesting an active involvement of calreticulin in the regulation of store-operated Ca2+ influx.


Subject(s)
Calcium-Binding Proteins/metabolism , Calcium/metabolism , Ribonucleoproteins/metabolism , Adenosine Triphosphate/pharmacology , Animals , Calcium-Binding Proteins/analysis , Calcium-Binding Proteins/biosynthesis , Calcium-Transporting ATPases/antagonists & inhibitors , Calreticulin , Dimethyl Sulfoxide/pharmacology , Enzyme Inhibitors/pharmacology , Fluorescent Antibody Technique , Homeostasis , Ionomycin/pharmacology , Kinetics , L Cells , Manganese/metabolism , Mice , Molecular Sequence Data , Monensin/pharmacology , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Restriction Mapping , Ribonucleoproteins/analysis , Ribonucleoproteins/biosynthesis , Terpenes/pharmacology , Thapsigargin , Transfection
15.
Biochem Biophys Res Commun ; 191(2): 487-94, 1993 Mar 15.
Article in English | MEDLINE | ID: mdl-8384843

ABSTRACT

It is generally assumed that the fatty acid compositions of the phosphoinositides are identical. To investigate this in myocardium, inositol lipids extracted from rat and pig ventricular homogenates were absorbed to neomycin-coated glass beads, eluted and quantitated by fatty acid analysis after thin-layer chromatography. The percentages of stearic, oleic, linoleic and arachidonic acid (20:4n-6) in the rat were, respectively, 49, 4, 7 and 26 for phosphatidylinositol, 62, 1, 4 and 18 for phosphatidylinositol-4-monophosphate and 63, 2, 4, 18 for phosphatidylinositol-4,5-bisphosphate. Equal distribution patterns of fatty acids were found in homogenate and sarcoplasmic reticulum of pig myocardium. Cultured rat ventricular myocytes were utilized to study the incorporation (25 h) of [14C]20:4n-6 relative to that of myo-[3H]inositol into phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate which were, respectively, 1.61 and 1.22. The data indicate that in myocardium phosphatidylinositol-4,5-bisphosphate represents a relatively modest source of 20:4n-6.


Subject(s)
Fatty Acids/analysis , Myocardium/chemistry , Phosphatidylinositols/chemistry , Animals , Cells, Cultured , Chromatography, Thin Layer , Myocardium/cytology , Rats , Rats, Wistar , Swine
16.
Mol Cell Biochem ; 117(2): 181-9, 1992 Nov 18.
Article in English | MEDLINE | ID: mdl-1336811

ABSTRACT

Phosphatidylinositol (PtdIns) kinase and phosphatidylinositol 4-phosphate (PtdIns4P) kinase have been studied in a purified sarcolemmal fraction isolated from rat heart. Both enzymes were Mg(2+)-dependent and their activities were maximal at 2.5 mM Mg2+ and pH 7.5. Kinetic analysis of endogenous substrate phosphorylation by ATP showed that the apparent Km and Vmax values for PtdIns kinase were 292 +/- 17 microM and 1390 +/- 80 pmol.mg-1.min-1, respectively, while the apparent Km and Vmax values for PtdIns4P kinase were 398 +/- 25 microM and 382 +/- 24 pmol.mg-1.min-1. Under normal conditions, the activity of PtdIns4P kinase was lower than that of PtdIns kinase; however, the former activity increased several fold in the presence of PtdIns4P as an exogenous substrate. The enzymatic synthesis of intramembranal PtdIns4P and phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P2) was maximally enhanced by 0.1% Triton X-100 and inhibited by micromolar concentrations of Ca2+. Inhibition of PtdIns and PtdIns4P kinase showed IC50 values for Ca2+ of 20 and 6 microM, respectively, and was independent of either Ca(2+)-induced activation of phospholipase C and polyphosphoinositide monophosphoesterases or low ATP concentrations. The results indicate that purified rat heart sarcolemmal membranes contain a very active phosphoinositide phosphorylation system which is regulated by micromolar levels of Ca2+. The Ca2+ effect may contribute to the feedback inhibition of the receptor-activated formation of inositol 1,4,5-trisphosphate.


Subject(s)
Calcium/pharmacology , Myocardium/enzymology , Phosphotransferases/metabolism , Sarcolemma/enzymology , 1-Phosphatidylinositol 4-Kinase , Adenosine Triphosphate/metabolism , Animals , Hydrogen-Ion Concentration , Kinetics , Magnesium Chloride/pharmacology , Male , Neomycin/pharmacology , Phosphatidylinositol 3-Kinases , Rats , Rats, Sprague-Dawley
17.
Can J Cardiol ; 8(4): 387-95, 1992 May.
Article in English | MEDLINE | ID: mdl-1377592

ABSTRACT

The effects of plain ischemia (34 degrees C) and the protective role of hypothermia (20 degrees C) alone or in combination with cardioplegia (St Thomas' Hospital [STH] or glucose-potassium-nifedipine [GPN]) on the intracellular kinetics of the activator calcium of cardiac muscle were quantified and compared from the interval-force behaviour (mechanical restitution) of right and left ventricles of the perfused rat heart. Plain ischemia caused a major depression in the restitution of force of contraction of both ventricles, deranged the mixed linear-exponential functions by significantly increasing the time constants of the fitted mechanical restitution curves (MRC) and altered the control right/left ventricle interval-force relationship. The right ventricle was found to be more susceptible to ischemic damage than the left ventricle, and its inotropic reserve was virtually abolished by 1 h of plain ischemia. Hypothermic preservation during ischemia improved the mechanical restitution, salvaged the inotropic reserve and optimized right/left ventricle interval-force relationship, but the time constants of the fitted MRCs were still prolonged. However, both the cardioplegic formulations were equally effective in normalizing the time constants of the fitted curves. In general, right ventricle functions were better preserved by STH cardioplegia and left ventricle functions were better preserved by GPN cardioplegia. Cardioplegic interventions did not further improve the ventricular inotropic reserve compared with hypothermic preservation. Additional beneficial effects of cardioplegic formulations were directed towards stabilizing the linear-exponential functions and hence restitution of force of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Calcium/physiology , Cardioplegic Solutions/pharmacology , Ischemia/physiopathology , Myocardial Contraction/physiology , Ventricular Function/physiology , Animals , Bicarbonates/pharmacology , Calcium Chloride/pharmacology , Cardiac Complexes, Premature/physiopathology , Heart Arrest, Induced , Heart Ventricles/drug effects , Homeostasis , Hypothermia, Induced , In Vitro Techniques , Kinetics , Magnesium/pharmacology , Male , Myocardial Contraction/drug effects , Potassium Chloride/pharmacology , Rats , Rats, Inbred Strains , Sodium Chloride/pharmacology , Ventricular Function/drug effects , Ventricular Function, Left/drug effects , Ventricular Function, Left/physiology , Ventricular Function, Right/drug effects , Ventricular Function, Right/physiology
18.
Can J Cardiol ; 8(2): 164-72, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1373104

ABSTRACT

Interval-force relationship of right and left ventricles of the isolated perfused rat heart was quantified by fitting polynomial, linear and mixed linear-exponential functions to the mechanical restitution (MRC) and post extrasystolic potentiation (PESPC) curves. Ventricular maximum developed pressure (Pmax) and its first derivative (dP/dtmax) were used as indices of contractility. MRCs and PESPCs could be separated into two distinct phases: phase A and phase B of MRCs; phase I and phase II of PESPCs. These phases for the right and left ventricle of the rat heart could be explained on the same model of cellular kinetics of the activator calcium, but showed distinct differences from other species. Right and left ventricle inotropic reserve (CRmax), as quantified from the centre of mass of the phase B of MRCs (from normalized Pmax), was (mean +/- SE): 132.4 +/- 2.05% and 132.1 +/- 1.7% at 1 Hz, which increased significantly (P less than 0.001) to 181.0 +/- 5.8% and 182.3 +/- 5.2% at 3.3 Hz, respectively. Linear regression of normalized right ventricle extrasystolic responses on the left ventricle responses gave a high correlation coefficient (typically r2 = 0.97). Time constants of the fitted mechanical restitution (TMRC) and post extrasystolic potentiation (TPESPC) curves were at 1 Hz, TMRC and TPESPC (from normalized dP/dtmax) were (mean +/- SE): 161.6 +/- 10.8 and 159.0 +/- 13.2 ms for right ventricle, and 196.1 +/- 14.5 and 188.3 +/- 10.7 ms for left ventricle, respectively. The results of this study indicate that interval-force relationship of the rat heart, as exemplified by CRmax and time constants of the fitted curves, could provide a useful index for quantifying and comparing right and left ventricular functions.


Subject(s)
Myocardial Contraction/physiology , Ventricular Function/physiology , Action Potentials/physiology , Animals , Cardiac Complexes, Premature/physiopathology , Cardiac Pacing, Artificial , In Vitro Techniques , Male , Perfusion , Rats , Rats, Inbred Strains , Ventricular Function, Left , Ventricular Function, Right
19.
Mol Cell Biochem ; 103(1): 85-96, 1991 Apr 24.
Article in English | MEDLINE | ID: mdl-1857347

ABSTRACT

The effect of reagents that modify sulfur-containing amino acid residues in the phosphatidylethanolamine N-methyltransferase was studied in the isolated rat cardiac sarcolemma by employing S-adenosyl-L-[methyl-3H]methionine as a methyl donor. Dithiothreitol protected the sulfhydryl groups in the membrane and caused a concentration- and time-dependent increase of phospholipid N-methylation at three different catalytic sites. This stimulation was highest (9-fold) in the presence of 1 mM MgCl2 and 0.1 microM S-adenosyl-L-[methyl-3H]methionine at pH 8.0 (catalytic site I), and was associated with an enhancement of Vmax without changes in Km for the methyl donor. Thiol glutathione was less stimulatory than dithiothreitol; glutathione disulfide inhibited the phosphatidylethanolamine N-methylation by 50%. The alkylating reagents, N-ethylmaleimide and methylmethanethiosulfonate, inhibited the N-methylation with IC50 of 6.9 and 14.1 microM, respectively; this inhibition was prevented by 1 mM dithiothreitol. These results indicate a critical role of sulfhydryl groups for the activity of the cardiac sarcolemmal phosphatidylethanolamine N-methyltransferase and suggest that this enzyme system in cardiac sarcolemma may be controlled by the glutathione/glutathione disulfide redox state in the cell.


Subject(s)
Dithiothreitol/pharmacology , Membrane Lipids/metabolism , Methyltransferases/metabolism , Myocardium/metabolism , Phospholipids/metabolism , Sarcolemma/metabolism , Animals , Cell Fractionation , Heart Ventricles , Kinetics , Male , Methylation , Myocardium/ultrastructure , Rats , Rats, Inbred Strains , S-Adenosylmethionine/metabolism , Sarcolemma/ultrastructure , Sulfhydryl Compounds/metabolism
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