scholarly journals Cytoprotection by glycine against ATP-depletion-induced injury is mediated by glycine receptor in renal cells1

2005 ◽  
Vol 390 (2) ◽  
pp. 447-453 ◽  
Author(s):  
Chao Pan ◽  
Xiaoming Bai ◽  
Leming Fan ◽  
Yong Ji ◽  
Xiaoyu Li ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Cell Injury ◽  
Glycine Receptor ◽  
Membrane Integrity ◽  
Cellular Membrane ◽  
Atp Depletion ◽  
Hek 293 ◽  
293 Cells ◽  
Marker Compounds

It is known that glycine protects mammalian cells against ischaemic cell injury by preventing cellular membrane leakage. However, the molecular mechanisms have not yet been clearly elucidated. The purpose of the present study was to clarify whether GlyR (glycine receptor) acts as a key mediator in cytoprotection of glycine. cDNA encoding human GlyRα1 (α1-subunit of glycine receptor) was transfected into HEK-293 cells. The membrane integrity of the cells with or without GlyRα1 was examined by the uptake of marker compounds, the release of LDH (lactate dehydrogenase) and the exclusion of Trypan Blue. Glycine prevented the permeability of 70 kDa dextrans and 140 kDa LDH in the cells in which GlyR was expressed under conditions of ATP depletion. The inhibition of endogenous GlyR expression by RNA interference attenuated the cytoprotection by glycine. Furthermore, the mutation of Tyr202 to phenylalanine in GlyRα1 blocked the glycine-mediated cytoprotection, while the mutation of Tyr202 to leucine abolished the cytoprotection by strychnine. Our results suggested that the cytoprotection of glycine against ATP-depletion-induced injury might be mediated by GlyR.

GFP Fusions for Fluorescence Detection of Ca2+ and Ca2+-Calmodulin in Living Cells

1998 ◽  
Vol 4 (S2) ◽  
pp. 1008-1009
Author(s):  
Anthony Persechini
Keyword(s):  
Mammalian Cells ◽  
Dissociation Constants ◽  
Hek 293 ◽  
Fluorescent Indicators ◽  
Calmodulin Binding ◽  
Binding Domains ◽  
293 Cells ◽  
20 Nm ◽  
Binding Sequence ◽  
Expression In Mammalian Cells

We have previously described fluorescent indicators for Ca2+ (FIP-CAs) and (Ca2+)4-calmodulin (FIP-CBs) whose responses are based on a ligand-dependent decrease in fluorescence energy transfer (FRET) between GFP variants. The indicators for (Ca2+)4-calmodulin contain calmodulin-binding domains, those for Ca2+ also contain an integral calmodulin (CaM) domain. We have developed new versions of these indicators constructed with enhanced blue- and red-shifted GFPs suitable for stable and transient expression in mammalian cells, and have begun to use them to investigate the relationships between the free intracellular concentrations of Ca2+ ([Ca2+]i) and (Ca2+)4-CaM ([(Ca2+)4-CaM]i). When the blue-shifted fluorophore is excited at 380 nm these constructs exhibit an emission peak at 505 nm due to FRET to the red-shifted fluorophore.We have made FIP-CBs with dissociation constants for (Ca2+)4-CaM of 0.5 nM, 20 nM, 300 nM and > 20 μM by introducing R →Q substitutions in the CaM-binding sequence, and have stablyexpressed them in HEK-293 cells (Fig. 1).

scholarly journals Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event

2004 ◽  
Vol 377 (1) ◽  
pp. 249-255 ◽  
Author(s):  
Adam COLE ◽  
Sheelagh FRAME ◽  
Philip COHEN
Keyword(s):  
Tyrosine Phosphorylation ◽  
Mammalian Cells ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Hek 293 ◽  
293 Cells ◽  
General Protein ◽  
The Stability

Phosphorylation of the endogenous GSK3α (glycogen synthase kinase-3α) at Tyr279 and GSK3β at Tyr216 was suppressed in HEK-293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein). GSK3β transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr216, but catalytically inactive mutants did not. GSK3β expressed in insect Sf 21 cells or E. coli was extensively phosphorylated at Tyr216, but the few molecules lacking phosphate at this position could autophosphorylate at Tyr216in vitro after incubation with MgATP. The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone. Wild-type GSK3β was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3β lacking phosphate at Tyr216. Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme.

KCNQ4 channels expressed in mammalian cells: functional characteristics and pharmacology

2001 ◽  
Vol 280 (4) ◽  
pp. C859-C866 ◽  
Author(s):  
Rikke Søgaard ◽  
Trine Ljungstrøm ◽  
Kamilla Angelo Pedersen ◽  
Søren-Peter Olesen ◽  
Bo Skaaning Jensen
Keyword(s):  
Mammalian Cells ◽  
Inward Rectification ◽  
Excitable Cells ◽  
Independent Manner ◽  
Hek 293 ◽  
M Current ◽  
Gating Charge ◽  
293 Cells ◽  
Boltzmann Function ◽  
Instantaneous Current

Human cloned KCNQ4 channels were stably expressed in HEK-293 cells and characterized with respect to function and pharmacology. Patch-clamp measurements showed that the KCNQ4 channels conducted slowly activating currents at potentials more positive than −60 mV. From the Boltzmann function fitted to the activation curve, a half-activation potential of −32 mV and an equivalent gating charge of 1.4 elementary charges was determined. The instantaneous current-voltage relationship revealed strong inward rectification. The KCNQ4 channels were blocked in a voltage-independent manner by the memory-enhancing M current blockers XE-991 and linopirdine with IC50 values of 5.5 and 14 μM, respectively. The antiarrhythmic KCNQ1 channel blocker bepridil inhibited KCNQ4 with an IC50 value of 9.4 μM, whereas clofilium was without significant effect at 100 μM. The KCNQ4-expressing cells exhibited average resting membrane potentials of −56 mV in contrast to −12 mV recorded in the nontransfected cells. In conclusion, the activation and pharmacology of KCNQ4 channels resemble those of M currents, and it is likely that the function of the KCNQ4 channel is to regulate the subthreshold electrical activity of excitable cells.

scholarly journals IRP1 Ser-711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase activities

2005 ◽  
Vol 388 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Carine FILLEBEEN ◽  
Annie CALTAGIRONE ◽  
Alain MARTELLI ◽  
Jean-Marc MOULIS ◽  
Kostas PANTOPOULOS
Keyword(s):  
Protein Misfolding ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Phosphorylation Site ◽  
Hek 293 ◽  
293 Cells ◽  
Critical Residue ◽  
Flanking Sequences ◽  
Iron Responsive Elements

In iron-starved cells, IRP1 (iron regulatory protein 1) binds to mRNA iron-responsive elements and controls their translation or stability. In response to increased iron levels, RNA-binding is inhibited on assembly of a cubane [4Fe-4S] cluster, which renders IRP1 to a cytosolic aconitase. Phosphorylation at conserved serine residues may also regulate the activities of IRP1. We demonstrate that Ser-711 is a phosphorylation site in HEK-293 cells (human embryonic kidney 293 cells) treated with PMA, and we study the effects of the S711E (Ser-711→Glu) mutation on IRP1 functions. A highly purified preparation of recombinant IRP1S711E displays negligible IRE-binding and aconitase activities. It appears that the first step in the aconitase reaction (conversion of citrate into the intermediate cis-aconitate) is more severely affected, as recombinant IRP1S711E retains approx. 45% of its capacity to catalyse the conversion of cis-aconitate into the end-product isocitrate. When expressed in mammalian cells, IRP1S711E completely fails to bind to RNA and to generate isocitrate from citrate. We demonstrate that the apparent inactivation of IRP1S711E is not related to mutation-associated protein misfolding or to alterations in its stability. Sequence analysis of IRP1 from all species currently deposited in protein databases shows that Ser-711 and flanking sequences are highly conserved in the evolutionary scale. Our results suggest that Ser-711 is a critical residue for the control of IRP1 activities.

Changes in extracellular pH affect glycine receptor channels expressed in HEK 293 cells

2010 ◽  
Vol 636 (1-3) ◽  
pp. 59-64 ◽  
Author(s):  
Yun-Ping Song ◽  
Friedrich Schlesinger ◽  
Daniela Ragancokova ◽  
Roger Calixto ◽  
Reinhard Dengler ◽  
...  
Keyword(s):  
Glycine Receptor ◽  
Extracellular Ph ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Human haem oxygenase-1 induction by nitro-linoleic acid is mediated by cAMP, AP-1 and E-box response element interactions

2009 ◽  
Vol 422 (2) ◽  
pp. 353-361 ◽  
Author(s):  
Marcienne M. WRIGHT ◽  
Junghyun KIM ◽  
Thomas D. HOCK ◽  
Norbert LEITINGER ◽  
Bruce A. FREEMAN ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Site Directed Mutagenesis ◽  
Related Factor ◽  
Intact Cells ◽  
Response Element ◽  
Hek 293 ◽  
293 Cells ◽  
E Box ◽  
Haem Oxygenase ◽  
Anti Inflammatory

Nitro-fatty acid products of oxidative inflammatory reactions mediate anti-inflammatory cell signalling responses. LNO2 (nitrolinoleic acid) induces expression of HO-1 (haem oxygenase-1), an enzyme that catabolizes haem into products exhibiting potent anti-inflammatory properties. In the present manuscript, the molecular mechanisms underlying HO-1 induction by LNO2 were examined in HAEC (human aortic endothelial cells), HEK-293 (human embryonic kidney 293) cells, and in transcription factor-deficient MEF (mouse embryonic fibroblasts). LNO2 induced HO-1 expression in Nrf2 [NF-E2 (nuclear factor-erythroid 2)-related factor 2]-deficient MEF and in HEK-293 cells transfected with Nrf2-specific shRNA (small-hairpin RNA), supporting the fact that LNO2-mediated HO-1 induction can be regulated by Nrf2-independent mechanisms. LNO2 activated expression of a −4.5 kb human HO-1 promoter construct, whereas a −4.0 kb construct with deletion of 500 bp from the 5′ region was unresponsive. Site-directed mutagenesis of a CRE (cAMP-response element) or of a downstream NF-E2/AP-1 (activating protein-1) element, individually, within this 500 bp region modestly reduced activation of the HO-1 promoter by LNO2. Mutations of both the CRE and the NF-E2/AP-1 site also attenuated LNO2-mediated HO-1 promoter expression, whereas the addition of a third mutation in the proximal E-box sequence completely abolished LNO2-induced HO-1 expression. Chromatin immunoprecipitation assays confirmed CREB (CRE-binding protein)-1 binding to the CRE (located at −4.0 kb) and E-box regions (located at −44 bp) of the human HO-1 promoter. A 3C (Chromosome Conformation Capture) assay of intact cells showed LNO2-induced interactions between the CRE- and E-box- containing regions. These observations indicate that regulation of human HO-1 expression by LNO2 requires synergy between CRE, AP-1 and E-box sequences and involves the participation of CREB-1.

scholarly journals Human geneSLC41A1encodes for the Na+/Mg2+exchanger

2012 ◽  
Vol 302 (1) ◽  
pp. C318-C326 ◽  
Author(s):  
Martin Kolisek ◽  
Axel Nestler ◽  
Jürgen Vormann ◽  
Monika Schweigel-Röntgen
Keyword(s):  
Mammalian Cells ◽  
Molecular Level ◽  
Transport Activity ◽  
Human Embryonic Kidney ◽  
Efflux System ◽  
Dependent Protein Kinase ◽  
Complete Replacement ◽  
Hek 293 ◽  
293 Cells ◽  
Dependent Protein

Magnesium (Mg2+), the second most abundant divalent intracellular cation, is involved in the vast majority of intracellular processes, including the synthesis of nucleic acids, proteins, and energy metabolism. The concentration of intracellular free Mg2+([Mg2+]i) in mammalian cells is therefore tightly regulated to its optimum, mainly by an exchange of intracellular Mg2+for extracellular Na+. Despite the importance of this process for cellular Mg2+homeostasis, the gene(s) encoding for the functional Na+/Mg2+exchanger is (are) still unknown. Here, using the fluorescent probe mag-fura 2 to measure [Mg2+]ichanges, we examine Mg2+extrusion from hSLC41A1-overexpressing human embryonic kidney (HEK)-293 cells. A three- to fourfold elevation of [Mg2+]iwas accompanied by a five- to ninefold increase of Mg2+efflux. The latter was strictly dependent on extracellular Na+and reduced by 91% after complete replacement of Na+with N-methyl-d-glucamine. Imipramine and quinidine, known unspecific Na+/Mg2+exchanger inhibitors, led to a strong 88% to 100% inhibition of hSLC41A1-related Mg2+extrusion. In addition, our data show regulation of the transport activity via phosphorylation by cAMP-dependent protein kinase A. As these are the typical characteristics of a Na+/Mg2+exchanger, we conclude that the human SLC41A1 gene encodes for the Na+/Mg2+exchanger, the predominant Mg2+efflux system. Based on this finding, the analysis of Na+/Mg2+exchanger regulation and its involvement in the pathogenesis of diseases such as Parkinson's disease and hypertension at the molecular level should now be possible.

Effects of chloride channel inhibitors on H2O2-induced renal epithelial cell injury

2000 ◽  
Vol 278 (1) ◽  
pp. F83-F90 ◽  
Author(s):  
Xianmin Meng ◽  
W. Brian Reeves
Keyword(s):  
Lipid Peroxidation ◽  
Dna Damage ◽  
Cell Death ◽  
Epithelial Cell ◽  
Small Molecules ◽  
Cell Injury ◽  
Membrane Integrity ◽  
Atp Depletion ◽  
Renal Epithelial Cell ◽  
Epithelial Cell Injury

Oxidative stress contributes to renal epithelial cell injury in certain settings. Chloride influx has also been proposed as an important component of acute renal epithelial cell injury. The present studies examined the role of Cl− in H2O2-induced injury to LLC-PK1 renal epithelial cells. Exposure of LLC-PK1 cells to 1 mM H2O2 resulted in the following: depletion of intracellular ATP content; DNA damage; lipid peroxidation; and a loss of membrane integrity to both small molecules, e.g., trypan blue, and macromolecules, e.g., lactate dehydrogenase (LDH), and cell death. Substitution of Cl− by isethionate or the inclusion of certain Cl− channel blockers, e.g., diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino)· benzoate (NPPB), and niflumic acid, prevented the H2O2-induced loss of membrane integrity to LDH. In addition, the H2O2-induced loss of membrane integrity was prevented by raising the osmolality of the extracellular solutions, by depletion of cell ATP, and by inhibitors of volume-sensitive Cl− channels. However, these maneuvers did not prevent the H2O2-induced permeability to small molecules or H2O2-induced ATP depletion, DNA damage, lipid peroxidation, or cell death. These results support the view that volume-sensitive Cl− channels play a role in the progressive loss of cell membrane integrity during injury.

Tension and electrolyte changes with Na+-K+ pump inhibition in rat papillary muscle

1989 ◽  
Vol 257 (3) ◽  
pp. H942-H953
Author(s):  
D. Parsons ◽  
K. P. Burton ◽  
H. K. Hagler ◽  
J. T. Willerson ◽  
L. M. Buja
Keyword(s):  
Electron Probe ◽  
Cell Injury ◽  
Membrane Integrity ◽  
Bimodal Distribution ◽  
High Energy ◽  
Atp Depletion ◽  
X Ray ◽  
Elemental Concentrations ◽  
Intracellular Ca ◽  
The Mean

Myocardial ischemic injury results in altered membrane integrity, energy depletion, and electrolyte shifts leading to accumulation of intracellular Ca. However, analysis of the direct effects of Ca accumulation is complicated by other concomitant cellular changes produced by ischemia. The purpose of this study was to examine the effects of Ca loading in rat papillary muscles produced by Na+-K+ pump inhibition in oxygenated K+-free buffer. Changes in contractile characteristics, high energy phosphate, and elemental concentrations of subcellular compartments were measured. Electron probe X-ray microanalysis was used to assess elemental concentrations in cryosections. After 3 h of Na+-K+ pump inhibition, resting tension (RT) increased to 164% and developed tension (DT) fell to 16.8% of control values. One hour after return to complete buffer, RT and DT partially recovered but remained significantly different from the 180 to 240-min values for the control muscles. Electron probe X-ray microanalysis showed increases in cytoplasmic and mitochondrial Na and Ca and a decrease in K during Na+-K+ pump inhibition. Mitochondrial Ca was greater than 100-fold greater than Ca in control mitochondria. Morphologically, the majority of cells showed ultrastructural damage. The mean ATP level was 20% of control. After 1 h of recovery, the cells appeared more heterogeneous, and the mean mitochondrial Ca decreased, whereas mean cytoplasmic Ca increased. Further statistical analysis showed a bimodal distribution for Na, Ca, K, Mg, and Cl, which coincided with the morphologically mixed population of cells. This suggests that replacement of extracellular K+ was associated with restored electrolyte gradients in some cells and the persistent or further alteration of electrolytes in others. These results suggest that variable Ca accumulation and associated ATP depletion without the compounding effects of ischemia lead to cell injury similar to reperfusion injury reported in ischemic myocardium.

scholarly journals Regulation of GPR54 Signaling by GRK2 and β-Arrestin

2009 ◽  
Vol 23 (12) ◽  
pp. 2060-2074 ◽  
Author(s):  
Macarena Pampillo ◽  
Natasha Camuso ◽  
Jay E. Taylor ◽  
Jacob M. Szereszewski ◽  
Maryse R. Ahow ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Molecular Mechanisms ◽  
Control Cell ◽  
Cytoplasmic Tail ◽  
Intracellular Loop ◽  
Membrane Expression ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Abstract Kisspeptin and its receptor, GPR54, are major regulators of the hypothalamic-pituitary-gonadal axis as well as regulators of human placentation and tumor metastases. GPR54 is a Gq/11-coupled G protein-coupled receptor (GPCR), and activation by kisspeptin stimulates phosphatidy linositol 4, 5-biphosphate hydrolysis, Ca2+ mobilization, arachidonic acid release, and ERK1/2 MAPK phosphorylation. Physiological evidence suggests that GPR54 undergoes agonist-dependent desensitization, but underlying molecular mechanisms are unknown. Furthermore, very little has been reported on the early events that regulate GPR54 signaling. The lack of information in these important areas led to this study. Here we report for the first time on the role of GPCR serine/threonine kinase (GRK)2 and β-arrestin in regulating GPR54 signaling in human embryonic kidney (HEK) 293 cells, a model cell system for studying the molecular regulation of GPCRs, and genetically modified MDA MB-231 cells, an invasive breast cancer cell line expressing about 75% less β-arrestin-2 than the control cell line. Our study reveals that in HEK 293 cells, GPR54 is expressed both at the plasma membrane and intracellularly and also that plasma membrane expression is regulated by cytoplasmic tail sequences. We also demonstrate that GPR54 exhibits constitutive activity, internalization, and association with GRK2 and β- arrestins-1 and 2 through sequences in the second intracellular loop and cytoplasmic tail of the receptor. We also show that GRK2 stimulates the desensitization of GPR54 in HEK 293 cells and that β-arrestin-2 mediates GPR54 activation of ERK1/2 in MDA-MB-231 cells. The significance of these findings in developing molecular-based therapies for treating certain endocrine-related disorders is discussed.

Sign in / Sign up

Export Citation Format

Share Document