scholarly journals Oxidized glutathione decreases luminal Ca2+ content of the endothelial cell ins(1,4,5)P3-sensitive Ca2+ store

1995 ◽  
Vol 312 (2) ◽  
pp. 485-489 ◽  
Author(s):  
P N Henschke ◽  
S J Elliott
Keyword(s):  
Signal Transduction ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Vascular Tissue ◽  
Oxidant Stress ◽  
Inhibitory Effect ◽  
Oxidized Glutathione ◽  
Intact Cells

The model oxidant, t-butyl hydroperoxide (t-buOOH), inhibits Ins(1,4,5)P3-dependent Ca2+ signalling in calf pulmonary artery endothelial cells. Metabolism of t-buOOH within the cytosol is coupled to the oxidation of glutathione. In this study, we investigated whether oxidized glutathione (GSSG) is the intracellular moiety responsible for mediating the effects of t-buOOH on Ca2+ signalling. The increase in cytosolic [Ca2+] stimulated by application of 2,5-di-t-butylhydroquinone (BHQ) was used to estimate the luminal Ca2+ content of the Ins(1,4,5)P3-sensitive store in intact cells. Luminal Ca2+ content was unaffected by t-buOOH (0.4 mM, 0-3 h) unless intracellular GSSG content was concomitantly elevated. The effect was specific for increased GSSG and was not replicated by depletion of GSH. These results suggest that cytosolic GSSG, produced endogenously within the endothelial cell, decreases the luminal Ca2+ content of Ins(1,4,5)P3-sensitive Ca2+ stores. Depletion of internal Ca2+ stores by GSSG may represent a key mechanism by which some forms of oxidant stress inhibit signal transduction in vascular tissue. At the plasma membrane, t-buOOH is known to inhibit the capacitative Ca2+ influx pathway. Increased intracellular GSSG potentiated the inhibitory effect of t-buOOH on Ca2+ influx, thereby providing the first evidence that activity of the capacitative Ca2+ influx channel is sensitive to thiol reagents formed endogenously within the cell.

scholarly journals Cavin-1 regulates BMP/Smad signaling through the interaction of Caveolin-1 with BMPRII in pulmonary artery endothelial cells

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S Tomita ◽  
N Nakanishi ◽  
T Ogata ◽  
Y Tsuji ◽  
A Sakamoto ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Pulmonary Artery ◽  
Signaling Pathway ◽  
Knockout Mice ◽  
Bmp Signaling ◽  
Smad Signaling ◽  
Caveolin 1 ◽  
Smad Signaling Pathway

Abstract Background Pulmonary hypertension (PH) is a progressive disease associated with poor outcome. Caveolin-1 (Cav1) is a component of caveolae and classified as a related gene of pulmonary arterial hypertension (PAH). Gene mutations of bone morphogenetic protein type II receptor (BMPRII) is a most common cause of PAH. BMPRII is localized in caveolae and associates with Cav1. However, the role of the Caveolin-Cavin system on the BMP/Smad signaling and the PAH progression has not been well-known. Purpose The aim of our study is to investigate the relationship between Caveolin-Cavin system and BMP/Smad signaling pathway and explore the mechanism of downstream signal transduction of BMP signaling by the interaction between Caveolin and BMPRII. Methods Cav1 knockout mice were used to assess PH and caveolae in pulmonary artery endothelial cells were observed by electron microscope. Cav1 and Cavin-1, which is a component of caveolae and form a complex with Cav1, were knocked-down in human pulmonary artery endothelial cell (hPAEC) using siRNA and phosphorylation of Smad signal was evaluated. Apoptosis of these cells was explored by flow cytometry. We investigated the interaction between Cav1 and BMPRII, and evaluated whether Cavin-1 affects this interaction and signal transduction of BMP signaling. Results As previously described, deletion of Cav1 revealed disappearance of caveolae in pulmonary artery endothelial cells (PAECs), and Cav1 knockout mice exhibited PH with pulmonary vascular remodeling and right ventricular hypertrophy. We then examined roles of Cav1 in human PAECs (hPAECs). Cav1 knockdown in hPAECs reduced phosphorylation of Smad 1/5/9. In addition, Cav1 knockdown significantly increased hypoxia-induced apoptosis in hPAEC. Knockdown of Cavin-1 reversed phosphorylation of Smad 1/5/9 decreased by Cav1 knockdown in BMP9 stimulation. Cavin-1 reversed the expression of BMPRII decreased by overexpression of Cav1. Cav1 was associated with Cavin-1 at the plasma membrane in PAECs. Cav1 also associated with BMPRII at the membrane of hPAECs that was inhibited by Cavin-1, and Cavin-1 reduced the localization of BMPRII to the membrane of hPAECs. These results suggest that BMPRII interacts with Cav1 via Cavin-1-associated localization at the plasma membrane in hPAECs, resulting in regulating BMP/Smad signaling pathway and involving in the development of PAH. Conclusions Cavin-1 affects the interaction of Cav1 with BMPRII at the membrane of PAECs, and regulates BMP/Smad signaling. These results reveal a previously undescribed function of Cavin-Caveolin system in the development of PAH through regulation of BMP/Smad signaling. Funding Acknowledgement Type of funding source: None

Localization of carbonic anhydrase on pulmonary artery endothelial cells in culture

1982 ◽  
Vol 53 (4) ◽  
pp. 914-919 ◽  
Author(s):  
U. S. Ryan ◽  
P. L. Whitney ◽  
J. W. Ryan
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Carbonic Anhydrase ◽  
Pulmonary Artery ◽  
Venous Blood ◽  
Carbonic Anhydrase Activity ◽  
Intact Cells ◽  
Cells In Culture ◽  
Carbonic Anhydrase B ◽  
Arterial Blood

Bovine pulmonary artery endothelial cells in culture possess carbonic anhydrase activity and immunoreactivity. The intact cells and cell homogenates lower the pH of 25 mM triethanolamine sulfate buffer saturated with CO2 (starting pH 8.1). The intact cells are more reactive than the cell homogenates, and the enzymic activity is enriched in association with the plasma membrane fraction. Specific immunofluorescence is obtained when the cells are incubated with rabbit antibovine erythrocyte carbonic anhydrase B and then with goat antirabbit immunoglobulin G coupled to fluorescein. At the level of electron microscopy, antibodies to carbonic anhydrase B are reactive with sites along the plasma membrane and associated caveolae. Multivesicular bodies are the only intracellular sites labeled and appear to correspond to the globular sites of intracellular immunofluorescence. Cells maintained and propagated in culture in the absence of an exogenous source of carbonic anhydrase nonetheless possess carbonic anhydrase activity, suggesting that the cells are capable of synthesizing the enzyme. Taken together, our results indicate that pulmonary artery endothelial cells possess carbonic anhydrase situated so that the enzyme could readily catalyze the dehydration of plasma HCO-3 to facilitate CO2 excretion and participate in the regulation of blood pH as central venous blood is converted into systemic arterial blood.

scholarly journals Role of thromboxane in retinal microvascular degeneration in oxygen-induced retinopathy

2001 ◽  
Vol 90 (6) ◽  
pp. 2279-2288 ◽  
Author(s):  
Martin H. Beauchamp ◽  
Ana Katherine Martinez-Bermudez ◽  
Fernand Gobeil ◽  
Anne Marilise Marrache ◽  
Xin Hou ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Endothelial Cell ◽  
Oxidant Stress ◽  
Microvascular Endothelial Cell ◽  
Oxygen Induced Retinopathy ◽  
Rat Pups ◽  
Endothelial Cell Death ◽  
Peroxidation Product ◽  
Synthase Inhibitor

Microvascular degeneration is an important event in oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity. Because oxidant stress abundantly generates thromboxane A2(TxA2), we tested whether TxA2plays a role in retinal vasoobliteration of OIR and contributes to such vascular degeneration by direct endothelial cytotoxicity. Hyperoxia-induced retinal vasoobliteration in rat pups (80% O2exposure from postnatal days 5–14) was associated with increased TxB2generation and was significantly prevented by TxA2synthase inhibitor CGS-12970 (10 mg · kg−1· day−1) or TxA2-receptor antagonist CGS-22652 (10 mg · kg−1· day−1). TxA2mimetics U-46619 (EC5050 nM) and I-BOP (EC505 nM) caused a time- and concentration-dependent cell death of neuroretinovascular endothelial cells from rats as well as newborn pigs but not of smooth muscle and astroglial cells; other prostanoids did not cause cell death. The peroxidation product 8-iso-PGF2, which is generated in OIR, stimulated TxA2formation by endothelial cells and triggered cell death; these effects were markedly diminished by CGS-12970. TxA2-dependent neuroretinovascular endothelial cell death was mostly by necrosis and to a lesser extent by apoptosis. The data identify an important role for TxA2in vasoobliteration of OIR and unveil a so far unknown function for TxA2in directly triggering neuroretinal microvascular endothelial cell death. These effects of TxA2might participate in other ischemic neurovascular injuries.

scholarly journals Galectin-3- Mediated Transdifferentiation of Pulmonary Artery Endothelial Cells Contributes to Hypoxic Pulmonary Vascular Remodeling

2018 ◽  
Vol 51 (2) ◽  
pp. 763-777 ◽  
Author(s):  
Li Zhang ◽  
Yu-mei Li ◽  
Xi-xi Zeng ◽  
Xiao-yan Wang ◽  
Shao-kun Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Ultrasound Biomicroscopy ◽  
Pulmonary Artery Hypertension ◽  
Carbohydrate Binding ◽  
Vessel Remodeling ◽  
Galectin 3

Background/Aims: Vascular muscularity is a key event in vessel remodeling during pulmonary artery hypertension (PAH). Endothelial-mesenchymal transdifferentiation (EndMT) has been increasingly reported to play a role in disease occurrence. Galectin-3, a carbohydrate-binding protein regulates cell proliferation, differentiation, migration and neovascularization. However, whether galectin-3 controls endothelial cell transdifferentiation during the development of PAH is unknown. Methods: Rats were exposed to normoxic or hypoxic conditions (fraction of inspired O2 0.10) for 21 d to establish PAH models. Hemodynamic changes were evaluated through surgery of the right jugular vein and ultrasound biomicroscopy inviVue. And vessel pathological alterations were detected by H&E staining. Galectin-3 (Gal-3)-induced pulmonary artery endothelium cell (PAEC) dynamic alterations were measured by MTT assays, Cell immunofluorescence, Flow cytometry, Real-time PCR and Western blot. Results: Our study demonstrated that Gal-3 was expressed in hypoxic pulmonary vascular adventitia and intima. The increased Gal-3 expression was responsible for hypoxic vessel remodeling and PAH development in vivo. Gal-3 was found to inhibit cell proliferation and apoptosis in cultured endothelial cells. Meanwhile endothelial cell morphology was altered and exhibited smooth muscle-like cell features as demonstrated by the expression of α-SMA after Gal-3 treatment. Gal-3 activated Jagged1/Notch1 pathways and induced MyoD and SRF. When MyoD or SRF were silenced with siRNAs, Gal-3-initiated transdifferentiation in endothelial cells was blocked as indicated by a lack of α-SMA. Conclusion: These results suggest that Gal-3 induces PAECs to acquire an α-SMA phenotype via a transdifferentiation process which depends on the activation of Jagged1/Notch1 pathways that mediate MyoD and SRF expression.

scholarly journals Fluorescent, short-chain C6-NBD-sphingomyelin, but not C6-NBD-glucosylceramide, is subject to extensive degradation in the plasma membrane: implications for signal transduction related to cell differentiation

1995 ◽  
Vol 309 (3) ◽  
pp. 905-912 ◽  
Author(s):  
J W Kok ◽  
T Babia ◽  
K Klappe ◽  
D Hoekstra
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Cell Differentiation ◽  
Plasma Membranes ◽  
Cell Types ◽  
Short Chain ◽  
Synthetic Activity ◽  
Intact Cells ◽  
Ht29 Cells ◽  
Extensive Degradation

The involvement of the plasma membrane in the metabolism of the sphingolipids sphingomyelin (SM) and glucosylceramide (GlcCer) was studied, employing fluorescent short-chain analogues of these lipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoylsphingosylphosphorylcholine (C6-NBD-SM), C6-NBD-GlcCer and their common biosynthetic precursor C6-NBD-ceramide (C6-NBD-Cer). Although these fluorescent short-chain analogues are metabolically active, some caution is to be taken in view of potential changes in biophysical/biochemical properties of the lipid compared with its natural counterpart. However, these short-chain analogues offer the advantage of studying the lipid metabolic enzymes in their natural environment, since detergent solubilization is not necessary for measuring their activity. These studies were carried out with several cell types, including two phenotypes (differing in state of differentiation) of HT29 cells. Degradation and biosynthesis of C6-NBD-SM and C6-NBD-GlcCer were determined in intact cells, in their isolated plasma membranes, and in plasma membranes isolated from rat liver tissue. C6-NBD-SM was found to be subject to extensive degradation in the plasma membrane, due to neutral sphingomyelinase (N-SMase) activity. The extent of C6-NBD-SM hydrolysis showed a general cell-type dependence and turned out to be dependent on the state of cell differentiation, as revealed for HT29 cells. In undifferentiated HT29 cells N-SMase activity was at least threefold higher than in its differentiated counterpart. In contrast, in all cell types studied, very little if any biosynthesis of C6-NBD-SM from the precursor C6-NBD-Cer occurred. Moreover, in the case of C6-NBD-GlcCer, neither hydrolytic nor synthetic activity was found to be associated with the plasma membrane. These results are discussed in the context of the involvement of the sphingolipids SM and GlcCer in signal transduction pathways in the plasma membrane.

Abstract 327: Mitochondrial Functional Variation Contributes to Endothelial Cell Activation

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
David M Krzywanski ◽  
Bing Cheng ◽  
Xinggui Shen ◽  
Christopher Kevil
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Ethnic Groups ◽  
Mitochondrial Function ◽  
Cell Activation ◽  
Oxidant Stress ◽  
Oxygen Utilization ◽  
Endothelial Cell Activation ◽  
Inflammatory Conditions ◽  
Oxidant Production

Vascular oxidant stress contributes to endothelial dysfunction and plays a critical role in early stage cardiovascular disease (CVD) development. Changes in endothelial function due to oxidant stress may contribute to CVD initiation and progression through the development of a pro-inflammatory environment. Differences in mitochondrial function may contribute to this process and provide insight into why age of onset and clinical outcomes differ amongst individuals form distinct ethnic groups; but no reports demonstrate distinct mitochondrial functional parameters between normal cells. Consequently, we hypothesized that significant variations in normal mitochondrial function and oxidant production exist between endothelial cells from donors representing different ethnic groups. Aspects of mitochondrial oxygen utilization and oxidant production were assessed under basal and inflammatory conditions in human aortic endothelial cells (HAECs) isolated from African Americans (AA) and Caucasians (CA). Bioenergetic analysis indicates that compared to CA, AA HAEC utilized significantly less oxygen for ATP production, possess a lower maximal respiratory capacity, and have reduced electron leak. Significant differences in mitochondrial membrane potential, decreased expression of endothelial nitric oxide synthase, and increased levels of superoxide were also observed and AA HAEC supporting a pro-inflammatory phenotype. As a marker of endothelial cell activation, AA HAEC expressed increased levels of intercellular cell adhesion molecule-1 under both basal and inflammatory conditions that could be partially mitigated but treatment with the mitochondrially targeted antioxidant MitoTEMPO. These data demonstrate that fundamental differences exist in mitochondrial oxygen utilization and oxidant production between CA and AA HAEC and that these changes may affect endothelial cell activation. These findings are consistent with the hypothesis that differences in “normal” mitochondrial function amongst ethnic groups could influence individual susceptibility by contributing to vascular inflammation, providing important insights into the mechanisms that contribute human CVD development.

scholarly journals Sickle erythrocytes inhibit human endothelial cell DNA synthesis

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2146-2152 ◽  
Author(s):  
R Weinstein ◽  
MA Zhou ◽  
A Bartlett-Pandite ◽  
K Wenc
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Function ◽  
Dna Synthesis ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Inhibitory Effect ◽  
Human Endothelial Cell ◽  
Endothelial Monolayers ◽  
Sickle Erythrocytes

Abstract Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling. These results indicate that interaction between sickle erythrocytes and the endothelium may result in altered endothelial function. This altered endothelial function may contribute to the development of vascular occlusive phenomena in patients with sickle cell anemia.

Serotonin transport and fluidity in plasma membrane vesicles: effect of hyperoxia

1988 ◽  
Vol 254 (6) ◽  
pp. C781-C787 ◽  
Author(s):  
N. P. Sheridan ◽  
E. R. Block
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Pulmonary Artery ◽  
Membrane Fluidity ◽  
Fluorescence Probe ◽  
Membrane Vesicles ◽  
Phase System ◽  
Maximal Velocity ◽  
Plasma Membrane Vesicles ◽  
Plasma Membrane Fluidity

Plasma membrane vesicles were prepared from porcine pulmonary artery endothelial cells by a dextran-polyethylene glycol two-phase system. Specific carrier-mediated transport of 5-hydroxytryptamine (5-HT) into the vesicles was examined. Transport required a Na+ gradient (out greater than in) across the membrane, and accumulated 5-HT rapidly effluxed out of the vesicles when the ionophore gramicidin was added. Transport was inhibited by the antidepressant imipramine. 5-HT transport into plasma membrane vesicles appeared saturable and exhibited Michaelis-Menten kinetics (Km 7.4 microM, maximal velocity 217 pmol.min-1.mg membrane protein-1). A 24-h exposure to 95% O2 at 1 atmosphere absolute resulted in a 21% decrease (P less than 0.05) in specific 5-HT transport by plasma membrane vesicles. Hyperoxia also caused a significant (P less than 0.01) decrease in plasma membrane fluidity, as measured with the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. These results indicate that pulmonary artery endothelial cell plasma membrane vesicles provide a good model for studying 5-HT transport activity in vitro. Hyperoxia affects plasma membrane fluidity and 5-HT transport in pulmonary artery endothelial cells, suggesting a possible cause-and-effect relationship between the two.

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