scholarly journals Spatial and temporal translocation of PKCα in single endothelial cell in response to focal mechanical stimulus

2017 ◽  
Author(s):  
Masataka Aarai ◽  
Toshihiro Sera ◽  
Takumi Hasegawa ◽  
Susumu Kudo
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Endothelial Cell ◽  
Mechanical Stress ◽  
Mechanical Stimulus ◽  
The Other ◽  
Protein Kinase Cα ◽  
Intracellular Ca ◽  
Summary Statement ◽  
Kinetics Of

AbstractWe observed the kinetics of protein kinase Cα (PKCα) and the intracellular Ca2+ wave in endothelial cells (ECs) in response to microscopic mechanical stress to investigate the effect of mechanical stress on PKCα translocation. The results show that a focal mechanical stimulus induced biphasic and directional PKCα translocation; PKCα initially translocated toward distinct spots near or at the membrane and then accumulated at the stimulus point. The low initial translocation occurred simultaneously in parallel with the increase in Ca2+. Initial translocation was inhibited in spite of Ca2+ increase when the diacylglycerol (DAG) binding domain of PKCα was inhibited, suggesting that translocation requires intracellular Ca2+ increase and DAG. On the other hand, high secondary translocation was delayed, occurring after the Ca2+ wave; however, this secondary translocation occurred even when Ca2+ release from the endoplasmic reticulum was inhibited, while it did not occur when the mechanosensitive (MS) channel was inhibited. These results indicated that at least Ca2+ influx through MS channels is required. Our results support the implication of PKCα in the Ca2+ signaling pathway in response to mechanical stress in ECs.Summary statementIn response to a focal mechanical stimulus, PKCα in an endothelial cell was initially translocated toward distinct spots near or at the membrane and then accumulated at the stimulus point.

Acetylcholinesterase in Human Erythroid Cells

1973 ◽  
Vol 12 (3) ◽  
pp. 911-923
Author(s):  
R. J. SKAER
Keyword(s):  
Endoplasmic Reticulum ◽  
Nervous System ◽  
Golgi Apparatus ◽  
Red Cells ◽  
The Other ◽  
Red Cell ◽  
Erythroid Cells ◽  
Cell Replacement ◽  
Kinetics Of ◽  
Human Red Cells

Acetylcholinesterase is present in human red cells but cannot be demonstrated by the copper thiocholine test. The enzyme is revealed, however, in the perinuclear cisterna, endoplasmic reticulum and Golgi apparatus of red cell precursors. It is suggested that 2 forms of the enzyme are present, one of which can be demonstrated by the copper thiocholine test, the other cannot; one form may be the precursor of the other. These observations may cast light on the kinetics of red cell replacement and on the interpretation of the results from the copper thiocholine test on other tissues such as the nervous system.

Homocysteine inhibits von Willebrand factor processing and secretion by preventing transport from the endoplasmic reticulum

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 683-689 ◽  
Author(s):  
SR Lentz ◽  
JE Sadler
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Protein Transport ◽  
Cell Protein ◽  
Carboxyl Terminal ◽  
Insulinoma Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Intracellular protein transport in endothelial cells is selectively inhibited by homocysteine, a thiol amino acid associated with both thrombosis and atherosclerosis. In a previous study, homocysteine decreased cell surface expression of the surface transmembrane glycoprotein thrombomodulin without decreasing secretion of another endothelial cell protein, plasminogen activator inhibitor-1. To define further the effects of homocysteine on protein transport, we examined the processing and secretion of the multimeric glycoprotein von Willebrand factor (vWF) in human umbilical vein endothelial cells. Incubation with 2 mmol/L homocysteine resulted in complete loss of vWF multimers and prevented asparagine-linked oligosaccharide maturation, propeptide cleavage, and secretion; these effects are consistent with impaired exit from the endoplasmic reticulum (ER). Dimerization was only partially inhibited, suggesting that homocysteine causes retention of provWF in the ER without preventing dimer formation. In pulse-chase incubations, intracellular provWF was degraded before exiting the ER in homocysteine-treated cells. Homocysteine also inhibited the processing and secretion of a carboxyl-terminal truncation mutant of human provWF expressed in rat insulinoma cells, indicating that retention in the endoplasmic reticulum can be mediated by regions of provWF apart from the carboxyl-terminal 20-Kd segment. These results suggest that retention of secretory proteins in the ER is regulated by redox mechanisms and imply that the intracellular transport of multiple endothelial cell proteins may be altered in patients with homocystinuria.

Abstract 11427: Calcium Mobilization From Endoplasmic Reticulum Involves Calmodulin-mediated NADPH Oxidase-derived Reactive Oxygen Species Production in Porcine Aortic Endothelial Cells

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Keiichi Odagiri ◽  
Akio Hakamata ◽  
Hiroshi Watanabe
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Nadph Oxidase ◽  
Ros Production ◽  
Aortic Endothelial Cells ◽  
Intracellular Ca ◽  
Analysis System ◽  
Porcine Aortic Endothelial Cells ◽  
Dependent Pathway ◽  
Aortic Endothelial

Introduction: Previous studies indicate that calcium/calmodulin (Ca 2+ /CaM) mediates the phosphorylation and activation of NADPH oxidase (NOX). In endothelial cells, elevation of intracellular Ca 2+ concentration ([Ca 2+ ] i ) level consists of two components, mobilization of Ca 2+ from intracellular stores and subsequent store-operated Ca 2+ entry (SOCE). However, little is known which component is required to regulate NOX-derived ROS production via Ca 2+ /CaM dependent pathway in endothelial cells. Hypothesis: We hypothesized that Ca 2+ mobilization from endoplasmic reticulum (ER), but not SOCE, is required to regulate NOX-derived ROS via Ca 2+ /CaM dependent pathway in porcine aortic endothelial cells (PAECs). Methods: We evaluated the association between Ca 2+ /CaM mediated NOX-derived ROS production and Ca 2+ mobilization from ER. We measured [Ca 2+ ] i by fura-2/AM a production of ROS by C-DCDHF-DA and in primary cultured PAECs with a fluorescence imaging and analysis system. Results: (1) In the presence of 1mM extracellular Ca 2+ ([Ca 2+ ] o ), BK induced a rapid increase [Ca 2+ ] i and followed by a sustained increase. However, in the absence of [Ca 2+ ] o (0mM with EGTA 1mM), BK caused only a small and transient increase [Ca 2+ ] i which was cause by Ca 2+ mobilization from ER. (2) BK (1μM) rapidly increased fluorescence intensity of C-DCDHF-DA compared with control. (150.2±51.3% and 107.2±5.6% of the baseline, respectively, p<0.05). (3) BK-induced ROS production was inhibited by an inhibitor of NOX (VAS2870: 50μM) (125.5±9.9% of the baseline, respectively, p<0.05). (4) When cells were exposed to BK with or without [Ca 2+ ] o , there was no difference in BK-induced ROS production. (5) In the absence of [Ca 2+ ] o , BK-induced ROS production is inhibited by an inhibitor of calmodulin (W-7: 100μM) (121.3±13.1% of the baseline, p<0.05). Thapsigargin (an inhibitor of ER calcium ATPase: 1μM) and BAPTA/AM (100μM) eliminated BK-induced ROS production (110.0±5.1% and 115.7±9.5% of the baseline, respectively, p<0.05 vs 1mM [Ca 2+ ] o ). Conclusions: The NOX-derived ROS production by BK is mediated via Ca 2+ /CaM dependent pathway. This was strictly regulated by Ca 2+ mobilization from ER.

Increased expression of calreticulin is linked to ANG IV-mediated activation of lung endothelial NOS

1999 ◽  
Vol 277 (4) ◽  
pp. L794-L801 ◽  
Author(s):  
Jawaharlal M. Patel ◽  
Yong D. Li ◽  
Jianliang Zhang ◽  
Craig H. Gelband ◽  
Mohan K. Raizada ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Catalytic Activity ◽  
Endothelial Cell ◽  
Ang Ii ◽  
Tetraacetic Acid ◽  
Free Medium ◽  
Intracellular Ca ◽  
Ang Iv ◽  
Oxide Synthase

This study demonstrates that ANG IV-induced activation of lung endothelial cell nitric oxide synthase (ecNOS) is mediated through mobilization of Ca2+ concentration and by increased expression and release of the Ca2+ binding protein calreticulin in pulmonary artery endothelial cells (PAEC). In Ca2+-free medium and in the presence of the ANG II AT1 and AT2 receptor antagonists losartan and PD-123319 (1 μM each), respectively, ANG IV (5, 50, and 500 nM) significantly increased intracellular Ca2+ release in PAEC ( P < 0.05 for all concentrations). In contrast, ANG IV-mediated activation of ecNOS was abolished by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM. ANG IV stimulation resulted in significantly increased expression of calreticulin in cells as well as release of calreticulin into the medium of cells as early as 2 h after ANG IV stimulation ( P < 0.05). Catalytic activity of purified ecNOS in the absence of calmodulin was increased in a concentration-dependent fashion by calreticulin. Immunocoprecipitation studies revealed that ecNOS and calreticulin were coprecipitated in ANG IV-stimulated PAEC. These results demonstrate that ANG IV-mediated activation of ecNOS is regulated by intracellular Ca2+ mobilization and by increased expression of calreticulin, which appears to involve interaction of ecNOS and calreticulin proteins in PAEC.

scholarly journals An Association between Mitochondria and the Endoplasmic Reticulum in Cells of the Pseudobranch Gland of a Teleost

1959 ◽  
Vol 5 (3) ◽  
pp. 393-396 ◽  
Author(s):  
D. E. Copeland ◽  
A. J. Dalton
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Carbonic Anhydrase ◽  
Basement Membrane ◽  
The Other ◽  
Secretory Cells ◽  
Specific Cell ◽  
Primary Products ◽  
In Cells

An elaborate and apparently unique specialization of the endoplasmic reticulum having the form of tubules and a precise orientation with respect to the mitochondria has been described for the specific cell of the pseudobranch gland. The tubules also are concentrated near the vascular border of the cell where they show continuity with the plasma membrane and open directly against the basement membrane. On the other side of the basement membrane, the endothelial cells of the sinusoid show openings or discontinuities characteristically associated with secretory cells. The pseudobranch gland is presumed to have carbonic anhydrase as one of its primary products, if not its only one, and the elaborate ultrastructure is thought to be associated with the special problems of secreting this enzyme.

scholarly journals Multifunctional Liposomes Modulate Purinergic Receptor-Induced Calcium Wave in Cerebral Microvascular Endothelial Cells and Astrocytes: New Insights for Alzheimer’s disease

2021 ◽  
Author(s):  
Greta Forcaia ◽  
Beatrice Formicola ◽  
Giulia Terribile ◽  
Sharon Negri ◽  
Dmitry Lim ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Purinergic Receptor ◽  
P2y Receptors ◽  
Microvascular Endothelial Cells ◽  
Cultured Astrocytes ◽  
Intracellular Ca ◽  
Microvascular Endothelial

AbstractIn light of previous results, we assessed whether liposomes functionalized with ApoE-derived peptide (mApoE) and phosphatidic acid (PA) (mApoE-PA-LIP) impacted on intracellular calcium (Ca2+) dynamics in cultured human cerebral microvascular endothelial cells (hCMEC/D3), as an in vitro human blood-brain barrier (BBB) model, and in cultured astrocytes. mApoE-PA-LIP pre-treatment actively increased both the duration and the area under the curve (A.U.C) of the ATP-evoked Ca2+ waves in cultured hCMEC/D3 cells as well as in cultured astrocytes. mApoE-PA-LIP increased the ATP-evoked intracellular Ca2+ waves even under 0 [Ca2+]e conditions, thus indicating that the increased intracellular Ca2+ response to ATP is mainly due to endogenous Ca2+ release. Indeed, when Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) activity was blocked by cyclopiazonic acid (CPA), the extracellular application of ATP failed to trigger any intracellular Ca2+ waves, indicating that metabotropic purinergic receptors (P2Y) are mainly involved in the mApoE-PA-LIP-induced increase of the Ca2+ wave triggered by ATP. In conclusion, mApoE-PA-LIP modulate intracellular Ca2+ dynamics evoked by ATP when SERCA is active through inositol-1,4,5-trisphosphate-dependent (InsP3) endoplasmic reticulum Ca2+ release. Considering that P2Y receptors represent important pharmacological targets to treat cognitive dysfunctions, and that P2Y receptors have neuroprotective effects in neuroinflammatory processes, the enhancement of purinergic signaling provided by mApoE-PA-LIP could counteract Aβ-induced vasoconstriction and reduction in cerebral blood flow (CBF). Our obtained results could give an additional support to promote mApoE-PA-LIP as effective therapeutic tool for Alzheimer’s disease (AD).

scholarly journals Homocysteine inhibits von Willebrand factor processing and secretion by preventing transport from the endoplasmic reticulum

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 683-689 ◽  
Author(s):  
SR Lentz ◽  
JE Sadler
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Protein Transport ◽  
Cell Protein ◽  
Carboxyl Terminal ◽  
Insulinoma Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Intracellular protein transport in endothelial cells is selectively inhibited by homocysteine, a thiol amino acid associated with both thrombosis and atherosclerosis. In a previous study, homocysteine decreased cell surface expression of the surface transmembrane glycoprotein thrombomodulin without decreasing secretion of another endothelial cell protein, plasminogen activator inhibitor-1. To define further the effects of homocysteine on protein transport, we examined the processing and secretion of the multimeric glycoprotein von Willebrand factor (vWF) in human umbilical vein endothelial cells. Incubation with 2 mmol/L homocysteine resulted in complete loss of vWF multimers and prevented asparagine-linked oligosaccharide maturation, propeptide cleavage, and secretion; these effects are consistent with impaired exit from the endoplasmic reticulum (ER). Dimerization was only partially inhibited, suggesting that homocysteine causes retention of provWF in the ER without preventing dimer formation. In pulse-chase incubations, intracellular provWF was degraded before exiting the ER in homocysteine-treated cells. Homocysteine also inhibited the processing and secretion of a carboxyl-terminal truncation mutant of human provWF expressed in rat insulinoma cells, indicating that retention in the endoplasmic reticulum can be mediated by regions of provWF apart from the carboxyl-terminal 20-Kd segment. These results suggest that retention of secretory proteins in the ER is regulated by redox mechanisms and imply that the intracellular transport of multiple endothelial cell proteins may be altered in patients with homocystinuria.

Endothelial Cell Thiol Isomerases: Potential Role in Thrombus Formation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3709-3709 ◽  
Author(s):  
Reema Jasuja ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Disulfide Bonds ◽  
Cellular Localization ◽  
Cell Activation ◽  
Potential Role ◽  
Ionophore A23187 ◽  
Platelet Surface

Abstract Thiol isomerases are multifunctional enzymes that contain a variable number of thioredoxin-like domains and catalyze the formation and isomerization of disulfide bonds. Members of the thiol isomerase family, including PDI, ERp5, ERp46, ERp57 and ERp72, are found in the endoplasmic reticulum (ER) where they play important roles during protein synthesis. Despite having an ER retention sequence some members of this family have been identified at cellular locations outside the ER with the potential to regulate the activity of proteins with labile disulfide bonds. Some of these proteins are involved in hemostasis, and include tissue factor and platelet surface receptors. Here, we describe the cellular localization and secretion of PDI and the first identification of two additional thiol isomerases in human umbilical vein endothelial cells (HUVEC). Our studies show a rapid secretion of PDI from HUVECs upon activation with thrombin or the calcium ionophore A23187 as detected by immunoblotting of proteins in the culture medium. We have estimated approximately 48000 ± 3600 molecules of PDI per cell. Approximately 8% of HUVEC PDI is secreted over 5 minutes upon cell activation. This secreted PDI is functionally active as measured by the reduction of disulfide bonds in the insulin transhydrogenase assay. Subcellular fractionation studies demonstrated PDI localized in different cellular compartments in activated and quiescent HUVECs. PDI was detected in the ER colocalized with sarcoplasmic and endoplasmic reticulum calcium ATPase (SERCA2) in both resting and activated cells. However PDI was detected in plasma membrane-containing fractions colocalized with alkaline phosphatase in thrombin-activated HUVECs while it was predominantly microsomal in buffer-treated control cells. PDI does not appear colocalized with the Weibel-Palade body marker von Willebrand factor (vWF) in subcellular fractions of activated or resting HUVECs nor is it detected in preparations of isolated Weibel-Palade bodies. Immunocytochemistry confirms localization of PDI in the ER but also shows punctate localization in the cytoplasm. Immunostaining for PDI in human aortic endothelial cells (HAEC) showed a similar staining pattern as HUVEC. Since PDI is secreted following cell activation, it is likely that the punctate PDI staining observed in the cytoplasm indicates PDI localization in secretory granules distinct from Weibel Palade bodies. We have also demonstrated the presence of two thiol isomerases, ERp57 and Erp72, in endothelial cells. Both ERp57 and ERp72 are constitutively secreted from HUVECs with no difference in secretory pattern pre and post activation. In contrast to PDI, ERp57 and ERp72 are detected in the microsomal as well as the plasma membrane fractions regardless of the activation state of the HUVECs. ERp57 was also detected in nuclear fractions consistent with previous immunocytochemical observations for ERp57. The regulated secretion of active PDI from endothelial cells and its rapid kinetics of release along with its subcellular localization suggest a potential role for endothelial cell PDI in modulation of protein structure and function in proteins regulated by endothelial cell injury, such as inflammation and thrombosis.

scholarly journals Endoplasmic Reticulum (ER) Stress-Generated Extracellular Vesicles (Microparticles) Self-Perpetuate ER Stress and Mediate Endothelial Cell Dysfunction Independently of Cell Survival

2020 ◽  
Vol 7 ◽  
Author(s):  
Aisha Osman ◽  
Heba El-Gamal ◽  
Mazhar Pasha ◽  
Asad Zeidan ◽  
Hesham M. Korashy ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Endothelial Cell ◽  
Endothelial Dysfunction ◽  
Endothelial Function ◽  
Er Stress ◽  
Extracellular Vesicles ◽  
Chemical Chaperone ◽  
No Release ◽  
The Impact

Circulating extracellular vesicles (EVs) are recognized as biomarkers and effectors of endothelial dysfunction, the initiating step of cardiovascular abnormalities. Among these EVs, microparticles (MPs) are vesicles directly released from the cytoplasmic membrane of activated cells. MPs were shown to induce endothelial dysfunction through the activation of endoplasmic reticulum (ER) stress. However, it is not known whether ER stress can lead to MPs release from endothelial cells and what biological messages are carried by these MPs. Therefore, we aimed to assess the impact of ER stress on MPs shedding from endothelial cells, and to investigate their effects on endothelial cell function. EA.hy926 endothelial cells or human umbilical vein endothelial cells (HUVECs) were treated for 24 h with ER stress inducers, thapsigargin or dithiothreitol (DTT), in the presence or absence of 4-Phenylbutyric acid (PBA), a chemical chaperone to inhibit ER stress. Then, MPs were isolated and used to treat cells (10–20 μg/mL) for 24–48 h before assessing ER stress response, angiogenic capacity, nitric oxide (NO) release, autophagy and apoptosis. ER stress (thapsigargin or DDT)-generated MPs did not differ quantitatively from controls; however, they carried deleterious messages for endothelial function. Exposure of endothelial cells to ER stress-generated MPs increased mRNA and protein expression of key ER stress markers, indicating a vicious circle activation of ER stress. ER stress (thapsigargin)-generated MPs impaired the angiogenic capacity of HUVECs and reduced NO release, indicating an impaired endothelial function. While ER stress (thapsigargin)-generated MPs altered the release of inflammatory cytokines, they did not, however, affect autophagy or apoptosis in HUVECs. This work enhances the general understanding of the deleterious effects carried out by MPs in medical conditions where ER stress is sustainably activated such as diabetes and metabolic syndrome.

scholarly journals Intracellular organelles and calcium homeostasis in rods and cones

2007 ◽  
Vol 24 (5) ◽  
pp. 733-743 ◽  
Author(s):  
TAMAS SZIKRA ◽  
DAVID KRIŽAJ
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Body ◽  
Cyclopiazonic Acid ◽  
Rods And Cones ◽  
Fold Reduction ◽  
Intracellular Ca ◽  
Intracellular Organelles ◽  
Spatial Buffering ◽  
Kinetics Of

The role of intracellular organelles in Ca2+homeostasis was studied in salamander rod and cone photoreceptors under conditions that simulate photoreceptor activation by darkness and light. Sustained depolarization evoked a Ca2+gradient between the cell body and ellipsoid regions of the inner segment (IS). The standing pattern of calcium fluxes was created by interactions between the plasma membrane, endoplasmic reticulum (ER), and mitochondria. Pharmacological experiments suggested that mitochondria modulate both baseline [Ca2+]i in hyperpolarized cells as well as kinetics of Ca2+entry via L type Ca2+channels in cell bodies and ellipsoids of depolarized rods and cones. Inhibition of mitochondrial Ca2+sequestration by antimycin/oligomycin caused a three-fold reduction in the amount of Ca2+accumulated into intracellular organelles in both cell bodies and ellipsoids. A further 50% decrease in intracellular Ca2+content within cell bodies, but not ellipsoids, was observed after suppression of SERCA-mediated Ca2+uptake into the ER. Inhibition of Ca2+sequestration into the endoplasmic reticulum by thapsigargin or cyclopiazonic acid decreased the magnitude and kinetics of depolarization-evoked Ca2+signals in cell bodies of rods and cones and decreased the amount of Ca2+accumulated into internal stores. These results suggest that steady-state [Ca2+]i in photoreceptors is regulated in a region-specific manner, with the ER contribution predominant in the cell body and mitochondrial buffering [Ca2+] the ellipsoid. Local [Ca2+]i levels are set by interactions between the plasma membrane Ca2+channels and transporters, ER and mitochondria. Mitochondria are likely to play an essential role in temporal and spatial buffering of photoreceptor Ca2+.

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