共查询到20条相似文献,搜索用时 15 毫秒
1.
Inflammatory bowel diseases (IBD) such as Crohn’s disease and ulcerative colitis are inflammatory disorders associated with
decreased colonic contractility. Here we show that, in experimental colitis in rat induced by trinitrobenzenesulfonic acid,
there is a decrease in contraction in response to carbamoylcholine and the sarco/endoplasmic reticulum Ca+2 (SERCA) pump inhibitor thapsigargin. However, the decrease in contractility may occur due to decrease in the SERCA pump levels
or their inactivation. Therefore, we examined the protein and mRNA levels for SERCA2 isoform, which is predominant isoform
in colonic smooth muscle. There was a decrease in the levels of SERCA2 protein and mRNA levels in inflamed colonic muscle.
These findings suggest that decreased SERCA pump levels is responsible for a decrease in the Ca+2 stores in the sarco/endoplasmic reticulum that causes a decrease in the contractility in colonic smooth muscle leading to
poor bowel movements. 相似文献
2.
Liu XH Zhang ZY Andersson KB Husberg C Enger UH Ræder MG Christensen G Louch WE 《Cell calcium》2011,49(4):201-207
Reduced sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA2) contributes to the impaired cardiomyocyte Ca(2+) homeostasis observed in heart failure. We hypothesized that a reduction in SERCA2 also elicits myocardial ER/SR stress responses, including unfolded protein responses (UPR) and cardiomyocyte apoptosis, which may additionally contribute to the pathophysiology of this condition. Left ventricular myocardium from mice with cardiomyocyte-specific tamoxifen-inducible disruption of Serca2 (SERCA2 KO) was compared with aged-matched controls. In SERCA2 KO hearts, SERCA2 protein levels were markedly reduced to 2% of control values at 7 weeks following tamoxifen treatment. Serca2 disruption caused increased abundance of the ER stress-associated proteins CRT, GRP78, PERK, and eIF2α and increased phosphorylation of PERK and eIF2α, indicating UPR induction. Pro-apoptotic signaling was also activated in SERCA2 KO, as the abundance of CHOP, caspase 12, and Bax was increased. Indeed, TUNEL staining revealed an increased fraction of cardiomyocytes undergoing apoptosis in SERCA2 KO. ER-Tracker staining additionally revealed altered ER structure. These findings indicate that reduction in SERCA2 protein abundance is associated with marked ER/SR stress in cardiomyocytes, which induces UPR, apoptosis, and ER/SR structural alterations. This suggests that reduced SERCA2 abundance or function may contribute to the phenotype of heart failure also through induction of ER/SR stress responses. 相似文献
3.
Status epilepticus is associated with sustained and elevated levels of cytosolic Ca(2+). To elucidate the mechanisms associated with changes of cytosolic Ca(2+) after status epilepticus, this study was initiated to evaluate the effect of pilocarpine-induced status epilepticus on Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in microsomes isolated from rat cortex, because the Ca(2+) uptake mechanism plays a major role in regulating intracellular Ca(2+) levels. The data demonstrated that the initial rate and overall Ca(2+) uptake in microsomes from pilocarpine treated animals were significantly inhibited compared with those in microsomes from saline-treated control animals. It was also shown that the inhibition of Ca(2+) uptake caused by status epilepticus was not an artifact of increased Ca(2+) release from microsomes, selective isolation of damaged microsomes from the homogenate, or decreased Mg(2+)/Ca(2+) ATPase protein in the microsomes. Pretreatment with the NMDA antagonist dizocilpine maleate blocked status epilepticus-induced inhibition of the initial rate and overall Ca(2+) uptake. The data suggest that inhibition of microsomal Mg(2+)/Ca(2+) ATPase Ca(2+) uptake is involved in NMDA-dependent deregulation of cytosolic Ca(2+) homeostasis associated with status epilepticus. 相似文献
4.
5.
Elisa Bianchini Stefania Testoni Arcangelo Gentile Tito Calì Denis Ottolini Antonello Villa Marisa Brini Romeo Betto Francesco Mascarello Poul Nissen Dorianna Sandonà Roberta Sacchetto 《The Journal of biological chemistry》2014,289(48):33073-33082
A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. Skeletal muscles of affected cattle are characterized by a selective reduction of SERCA1 in sarcoplasmic reticulum membranes. In this study, we provide evidence that the ubiquitin proteasome system is involved in the reduced density of mutated SERCA1. The treatment with MG132, an inhibitor of ubiquitin proteasome system, rescues the expression level and membrane localization of the SERCA1 mutant in a heterologous cellular model. Cells co-transfected with the Ca2+-sensitive probe aequorin show that the rescued SERCA1 mutant exhibits the same ability of wild type to maintain Ca2+ homeostasis within cells. These data have been confirmed by those obtained ex vivo on adult skeletal muscle fibers from a biopsy from a pseudomyotonia-affected subject. Our data show that the mutation generates a protein most likely corrupted in proper folding but not in catalytic activity. Rescue of mutated SERCA1 to sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca2+ concentration and prevent the appearance of pathological signs of cattle pseudomyotonia. 相似文献
6.
Trópia MJ Cardoso AS Tisi R Fietto LG Fietto JL Martegani E Castro IM Brandão RL 《Biochemical and biophysical research communications》2006,343(4):1234-1243
In this work, we show that glucose-induced activation of plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is strongly dependent on calcium metabolism and that the glucose sensor Snf3p works in a parallel way with the G protein Gpa2p in the control of the pathway. The role of Snf3p is played by the Snf3p C-terminal tail, since in a strain with the deletion of the SNF3 gene, but also expressing a chimera protein formed by Hxt1p (a glucose transporter) and the Snf3p C-terminal tail, a normal glucose-activation process can be observed. We present evidences indicating that Snf3p would be the sensor for the internal signal (phosphorylated sugars) of this pathway that would connect calcium signaling and activation of the plasma membrane ATPase. We also show that Snf3p could be involved in the control of Pmc1p activity that would regulate the calcium availability in the cytosol. 相似文献
7.
Takashi Daiho Kazuo Yamasaki Stefania Danko Hiroshi Suzuki 《The Journal of biological chemistry》2014,289(45):31241-31252
The actuator (A) domain of sarco(endo)plasmic reticulum Ca2+-ATPase not only plays a catalytic role but also undergoes large rotational movements that influence the distant transport sites through connections with transmembrane helices M1 and M2. Here we explore the importance of long helix M2 and its junction with the A domain by disrupting the helix structure and elongating with insertions of five glycine residues. Insertions into the membrane region of M2 and the top junctional segment impair Ca2+ transport despite reasonable ATPase activity, indicating that they are uncoupled. These mutants fail to occlude Ca2+. Those at the top segment also exhibited accelerated phosphoenzyme isomerization E1P → E2P. Insertions into the middle of M2 markedly accelerate E2P hydrolysis and cause strong resistance to inhibition by luminal Ca2+. Insertions along almost the entire M2 region inhibit the dephosphorylated enzyme transition E2 → E1. The results pinpoint which parts of M2 control cytoplasm gating and which are critical for luminal gating at each stage in the transport cycle and suggest that proper gate function requires appropriate interactions, tension, and/or rigidity in the M2 region at appropriate times for coupling with A domain movements and catalysis. 相似文献
8.
Nathaniel J. Traaseth 《生物化学与生物物理学报:生物膜》2010,1798(2):77-15414
Phospholamban (PLN) is a dynamic single-pass membrane protein that inhibits the flow of Ca2+ ions into the sarcoplasmic reticulum (SR) of heart muscle by directly binding to and inhibiting the SR Ca2+ATPase (SERCA). The PLN monomer is the functionally active form that exists in equilibrium between ordered (T state) and disordered (R state) states. While the T state has been fully characterized using a hybrid solution/solid-state NMR approach, the R state structure has not been fully portrayed. It has, however, been detected by both NMR and EPR experiments in detergent micelles and lipid bilayers. In this work, we quantitatively probed the μs to ms dynamics of the PLN excited states by observing the T state in DPC micelles using CPMG relaxation dispersion NMR spectroscopy under functional conditions for SERCA. The 15N backbone and 13Cδ1 Ile-methyl dispersion curves were fit using a two-state equilibrium model, and indicate that residues within domain Ia (residues 1-16), the loop (17-22), and domain Ib (23-30) of PLN undergo μs-ms dynamics (kex = 6100 ±800 s- 1 at 17 °C). We measured kex at additional temperatures, which allowed for a calculation of activation energy equal to ∼ 5 kcal/mol. This energy barrier probably does not correspond to the detachment of the amphipathic domain Ia, but rather the energy needed to unwind domain Ib on the membrane surface, likely an important mechanism by which PLN converts between high and low affinity states for its binding partners. 相似文献
9.
J. -P. Hildebrandt 《The Journal of membrane biology》1995,144(1):49-58
We tested lysophosphatidic acid (LPA), known to induce inositol phosphate generation and calcium signals as well as rearrangements of the cytoskeleton and mitogenic responses in fibroblasts, for its ability to activate phospholipase C in an exocrine cell system, the salt-secreting cells from the avian nasal salt gland. LPA (>10 nmol/l) caused the generation of inositol phosphates from membrane-bound phosphatidylinositides. The resulting calcium signals resembled those generated upon activation of muscarinic receptors, the physiological stimulus triggering salt secretion in these cells. However, close examination of the LPA-mediated calcium signals revealed that the initial calcium spike induced by high concentrations of LPA (>10 μmol/l) may contain a component that is not dependent upon generation of inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) and may result from calcium influx from the extracellular medium induced by LPA in a direct manner. Low concentrations of LPA (<10 μmol/l), however, induce inositol phosphate generation, Ins(1,4,5)P3-mediated release of calcium from intracellular pools and calcium entry. These effects seem to be mediated by a specific plasma membrane receptor and a G protein transducing the signal to phospholipase C in a pertussis-toxin-insensitive manner. Signaling pathways of the muscarinic receptor and the putative LPA-receptor seem to merge at the G-protein level as indicated by the fact that carbachol and LPA trigger hydrolysis of the same pool of phosphatidylinositol (4,5)-bisphosphate (PIP2) and mobilize calcium from the same intracellular stores. 相似文献
10.
The functional expression and distribution of intracellular ATPase (sarco(endo)plasmic reticulum Ca(2+)-ATPase: SERCA) and plasma membrane Ca(2+)-ATPase (PMCA) was analyzed in the developing chick cerebellum. The activity and Ca(2+) uptake increase with development for both ATPases. However, the protein content increases with the stage of development only for SERCA, remaining constant for PMCA. Immunohistochemical assays showed that the ontogenesis of these ATPases goes along with definite stages of cerebellum histogenesis, and is complete at hatching. The SERCA is mainly distributed in Purkinje neurons, whereas the PMCA seems to be expressed initially in climbing fibers, shifting to soma and spiny branchlets of Purkinje cells at late embryonic stages. Granule cells express both ATPases according to their degree of maturity, whereas only PMCA is present in cerebellar glomeruli. These pumps are present in deep nuclei and the choroid plexus, although in this latter tissue their expression declines with development. The spatio-temporal distribution of SERCA and PMCA must be closely related to their association with the development of specific cells and processes of the chick cerebellum. 相似文献
11.
Stefanie Eichstaedt Karoline Gäbler Sabine Below Jan-Peter Hildebrand 《Journal of receptor and signal transduction research》2013,33(6):591-612
Mechanical clearance of inhaled dust particles and microorganisms is an important part of the innate defense mechanisms of mammalian airways. Airway epithelia are composed of various cell types with different degrees of cell polarity. Serous cells regulate composition and volume of luminal periciliary fluid and mucus. Autocrine, paracrine, or neuronal messengers determine the secretory and reabsorptive rates of electrolytes and water via cAMP-or inositol triphosphate/calcium-mediated intracellular signals. Comparison of the expression of calcium-mobilizing receptor types (G protein-coupled-, growth factor-, and cytokine receptors) in two types of human immortalized airway epithelial cells (S9, 16HBE14o-) revealed that receptor populations were qualitatively and quantitatively different in the two cell types. Sustained calcium signals were elicited by activation of purinergic receptors in 16HBE14o-cells or muscarinic acetylcholine or histamine receptors in S9 cells. These G protein-coupled receptors mobilized calcium from intracellular stores and activated capacitative calcium influx. The experimental cells may represent different types of original airway epithelial cells and seem to be suited as model cells to study cell signaling and protein expression during interaction with pathogens or their secretory products (e.g., virulence factors). 相似文献
12.
Valeriy Lukyanenko Aristide Chikando W.J. Lederer 《The international journal of biochemistry & cell biology》2009,41(10):1957-1971
Ca2+ signaling is of vital importance to cardiac cell function and plays an important role in heart failure. It is based on sarcolemmal, sarcoplasmic reticulum and mitochondrial Ca2+ cycling. While the first two are well characterized, the latter remains unclear, controversial and technically challenging.In mammalian cardiac myocytes, Ca2+ influx through L-type calcium channels in the sarcolemmal membrane triggers Ca2+ release from the nearby junctional sarcoplasmic reticulum to produce Ca2+ sparks. When this triggering is synchronized by the cardiac action potential, a global [Ca2+]i transient arises from coordinated Ca2+ release events. The ends of intermyofibrillar mitochondria are located within 20 nm of the junctional sarcoplasmic reticulum and thereby experience a high local [Ca2+] during the Ca2+ release process. Both local and global Ca2+ signals may thus influence calcium signaling in mitochondria and, reciprocally, mitochondria may contribute to the local control of calcium signaling. In addition to the intermyofibrillar mitochondria, morphologically distinct mitochondria are also located in the perinuclear and subsarcolemmal regions of the cardiomyocyte and thus experience a different local [Ca2+].Here we review the literature in regard to several issues of broad interest: (1) the ultrastructural basis for mitochondrion – sarcoplasmic reticulum cross-signaling; (2) mechanisms of sarcoplasmic reticulum signaling; (3) mitochondrial calcium signaling; and (4) the possible interplay of calcium signaling between the sarcoplasmic reticulum and adjacent mitochondria.Finally, this review discusses experimental findings and mathematical models of cardiac calcium signaling between the sarcoplasmic reticulum and mitochondria, identifies weaknesses in these models, and suggests strategies and approaches for future investigations. 相似文献
13.
《Molecular membrane biology》2013,30(3):75-87
AbstractLike other integral membrane proteins, the activity of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) is regulated by the membrane environment. Cholesterol is present in the endoplasmic reticulum membrane at low levels, and it has the potential to affect SERCA activity both through direct, specific interaction with the protein or through indirect interaction through changes of the overall membrane properties. There are experimental data arguing for both modes of action for a cholesterol-mediated regulation of SERCA. In the current study, coarse-grained molecular dynamics simulations are used to address how a mixed lipid-cholesterol membrane interacts with SERCA. Candidates for direct regulatory sites with specific cholesterol binding modes are extracted from the simulations. The binding pocket for thapsigargin, a nanomolar inhibitor of SERCA, has been suggested as a cholesterol binding site. However, the thapsigargin binding pocket displayed very little cholesterol occupation in the simulations. Neither did atomistic simulations of cholesterol in the thapsigargin binding pocket support any specific interaction. The current study points to a non-specific effect of cholesterol on SERCA activity, and offers an alternative interpretation of the experimental results used to argue for a specific effect. 相似文献
14.
15.
Identification of a Cytoskeleton-bound Form of Phospholemman with Unique C-Terminal Immunoreactivity 总被引:1,自引:0,他引:1
Phospholemman (PLM) is a 72-amino acid transmembrane protein thought to function in Na,K-ATPase regulation or assembly, similar to other members of the FXYD family of proteins. Unique to PLM among these regulatory proteins are sites for C-terminal phosphorylation by PKA and PKC, although a role for phosphorylation in PLM function remains unclear. To study PLM phosphorylation, we used PLM phosphopeptides to generate antibodies to specifically detect phosphorylated PLM. Peptide affinity chromatography isolated two populations of antibodies: one reacting with standard PLM, a collection of closely-spaced 15-kDa protein bands by SDS-PAGE. About 20% of PLM antibodies reacted specifically with a single distinct form of PLM. Levels of this second immunological form (PLM-b) were increased with overexpression of PLM cDNA, and also reacted with a monoclonal antibody against the PLM N-terminus. In complete contrast to standard PLM, however, PLM-b was quantitatively insoluble in nonionic detergents and was released from tight binding by colchicine. Antibodies to PLM-b were present in two different antisera raised to the phosphorylated C-terminal peptide (residues 57-70), but not in antiserum raised to the non-phosphorylated C-terminal peptide. Despite an apparent relationship between PLM-b and phosphorylated PLM, PLM-b levels were not affected by treatment of heart cells with isoproterenol. PLM-b appears to represent a cytoskeleton-attached detergent-insoluble form of PLM with distinctive C-terminal immunoreactivity that might have implications for PLM structure and function. 相似文献
16.
A plasma membrane-enriched fraction (fraction 1B) has been obtained from rat aortic myocytes grown in primary culture. Plasma membrane markers, 5′-nucleotidase and ouabain-sensitive (Na+ + K+)-ATPase, are enriched 4.1- and 8.7-fold, respectively, in this fraction. Although endoplasmic reticulum marker NADPH-cytochrome c reductase is the most enriched in mitochondrial and heavy sucrose density gradient fractions, substantial enrichment of this marker is also observed in membrane fraction 1. This membrane preparation therefore contains a certain quantity of endoplasmic reticulum. Cytochrome c oxidase is de-enriched by a factor of 0.04 in fraction 1, indicating that it is essentially clear of mitochondrial contamination. Homogenization of aortic media-intima layers using a whole-tissue technique induces greater disruption of mitochondria and subsequent contamination of membrane fractions than does the procedure for cell disruption. Analysis of electrophoretic gels, vesicle density distribution and electron micrographs of enriched membrane fractions provide evidence that plasma membrane enriched from cultured myocytes is less traumatized than comparable fractions obtained from intact tissue. The potential value of such a highly enriched, minimally disrupted plasma membrane preparation is discussed. 相似文献
17.
Tsunoda Y Song I Taylor LP Owyang C 《Biochemical and biophysical research communications》2004,314(3):861-869
Key amino acids of the cholecystokinin (CCK) peptide for receptor binding are sulfated Y27, W30, D32, and F33-NH(2). Three-dimensional modeling showed that the CCK-A receptor (CCK-AR) antagonist devazepide penetrated into the transmembrane (TM) domains, whereas CCK was placed on the surface of the CCK-AR. Four types of rat CCK-AR cDNAs were transfected into CHO-K1 and COS-7 cells: normal CCK-AR cDNA transfected cells (wild type, WT); K120 substituted with V; K130V; and R352V. Binding of [3H]CCK-8 was observed in WT and K130V, but not in K120V and R352V. CCK caused Ca(2+) spiking in WT and K130V, whereas K120V and R352V had no effect. Three chimeras including the CCK-AR/3ibeta2 adrenergic receptor (beta2AR), 3Nibeta2AR, and 3Cibeta2AR were constructed. Two groups of point mutations in the CCK-AR3i were also made: Y252V, S274V, S281V, and S289V (non-phospho-acceptor Y or S); S260V, S264V, S271V, and S275V (phospho-acceptor S). WT and CCK-AR/3Cibeta2AR increased [Ca(2+)](i) in response to CCK; 3Nibeta2AR was vice versa. CCK failed to increase [IP(3)] in phospho-acceptor S to V without affecting binding. Non-phospho-acceptor S or Y to V showed normal response. Thus, Lys120 outside the TM2 and Arg352 outside the TM6 of the CCK-AR are amino acids interacting with Tyr[SO(3)H]27 and Asp32 of the CCK peptide for binding. Phospho-acceptor Ser groups in the CCK-AR 3Ni are amino acids for initiating cell signaling. 相似文献
18.
Summary Growing pollen tubes of tobacco germinated in suspension culture, were labelled with [3H]leucine and after varying times of chase with unlabelled leucine at 23, 16, or 4°C, were separated into plasma membrane-enriched and plasma membrane-depleted fractions by aqueous two-phase partition. At 23°C, the specific radioactivity of the plasma membrane increased with time to a maximum at 60 min. At 16°C and 4°C, labelling of the plasma membrane was respectively 40% and 10% that at 23°C. However, if labelling was at 23°C and subsequent transfer was at 4°C, plasma membrane labelling was much less affected and labelling of the plasma membrane was 60% that at 23°C. Additionally, quantitation of various morphological parameters revealed no accumulations of 50–70 nm transition vesicles in the space between endoplasmic reticulum and cis Golgi apparatus that might suggest formation of a low temperature compartment similar to those described for mammalian cells and tissues. Similarly, growth of pollen tubes was reduced but not blocked even at temperatures of 12°C. The results suggest that tube elongation is accompanied by a steady state flow of membranes to the cell surface that is relatively insensitive to interruption by low temperatures. Whereas leucine incorporation is reduced by low temperature even at 16°C, the flow pathway to the cell surface, including the endoplasmic reticulum to Golgi apparatus transfer step, as well as elongation growth does not exhibit a pronounced low temperature block in this tip growing system. 相似文献
19.
Summary Tobacco (Nicotiana tabacum L.) pollen, germinated 4 hours in suspension culture, was labeled with radioactive leucine and fractionated into constituent membranes by the technique of preparative free-flow electrophoresis. Tubes were ruptured by sonication directly into the electrophoresis buffer. Unfortunately, the Golgi apparatus of the rapidly elongating pollen tubes did not survive the sonication step. However, it was possible to obtain useful fractions of endoplasmic reticulum and mitochondria. To obtain Golgi apparatus, glutaraldehyde was added to the homogenization buffer during sonication. Plasma membrane, which accounted for only about 3% of the total membrane of the homogenates as determined by staining with phosphotungstate at low pH, was obtained in insufficient quantity and fraction purity to permit analysis. Results show rapid incorporation of [3H]leucine into endoplasmic reticulum followed by rapid chase out. The half-time for loss of radioactivity from the pollen tube endoplasmic reticulum was about 10 minutes. Concomitant with the loss of radioactivity from endoplasmic reticulum, the Golgi apparatus fraction was labeled reaching a maximum 20 minutes post chase. The findings suggest flow of membranes from endoplasmic reticulum to the Golgi apparatus during pollen tube growth. 相似文献
20.
Classic phenylketonuria (PKU) is characterized by brain lesions. However, its underlying neurotoxic mechanisms remain unknown.
Based on our previous studies, we hypothesized that calcium might participate in PKU-associated neuropathy. In cultured cortical
neurons, cytoplasmic free calcium concentration ([Ca2+]i) decreased dramatically when treatment with phenylalanine (Phe) and phenyllactic acid, while phenylacetic acid treatment
immediately increased [Ca2+]i, which began to decrease after 3 min. Moreover, [Ca2+]i decreased dramatically after Phe treatment in the presence of EGTA suggesting that Phe might increase [Ca2+]i efflux. Phe-induced [Ca2+]i decrease was strongly inhibited by vanadate, a non-specific plasma membrane Ca2+-ATPase (PMCA) antagonist, suggesting that Phe might increase [Ca2+]i efflux throught modulating PMCA. These findings were further supported by the facts that Phe could increase membrance 45Ca-uptake capability and PMCA activity. In contrast, treatment of KBR7943 or thapsigargin, antagonists to Na/Ca Exchanger
(NCX) and Sarco/Endoplasmic reticulum Ca2+-ATPase (SERCA), respectively, did not elicit any changes in [Ca2+]i. Specific siRNA against PMCA had an effect similar to vanadate. Since the brain injury induced by phenylalaninemia was thought
to be a chronic process, we cultured cortical neurons in the presence of Phe for 2 weeks and measured [Ca2+]i, PMCA activity and 45Ca-uptake capability at days 3, 7, 9 and 14, respectively. PMCA activity and 45Ca-uptake capability decreased from day 9, at the same time [Ca2+]i increase was observed. In conclusion, PMCA participate in regulating Phe-induced initial rapid decrease in [Ca2+]i and subsequent long-term increase in [Ca2+]i. 相似文献