scholarly journals Cell membranes resist flow

2018 ◽  
Author(s):  
Zheng Shi ◽  
Zachary T. Graber ◽  
Tobias Baumgart ◽  
Howard A. Stone ◽  
Adam E. Cohen
Keyword(s):  
Endothelial Cells ◽  
Flow Resistance ◽  
Transmembrane Proteins ◽  
Vesicle Fusion ◽  
Membrane Tension ◽  
Membrane Flow ◽  
Intact Cells ◽  
Signaling Mechanism ◽  
Coordinate Changes ◽  
Fluid Dynamical Model

SUMMARYThe fluid-mosaic model posits a liquid-like plasma membrane, which can flow in response to tension gradients. It is widely assumed that membrane flow transmits local changes in membrane tension across the cell in milliseconds. This conjectured signaling mechanism has been invoked to explain how cells coordinate changes in shape, motility, and vesicle fusion, but the underlying propagation has never been observed. Here we show that propagation of membrane tension occurs quickly in cell-attached blebs, but is largely suppressed in intact cells. The failure of tension to propagate in cells is explained by a fluid dynamical model that incorporates the flow resistance from cytoskeleton-bound transmembrane proteins. In primary endothelial cells, local increases in membrane tension lead only to local activation of mechanosensitive ion channels and to local vesicle fusion. Thus membrane tension is not a mediator of long-range intra-cellular signaling, but local variations in tension mediate distinct processes in sub-cellular domains.

scholarly journals Posttranslational Modification of Bcl-2 Facilitates Its Proteasome-Dependent Degradation: Molecular Characterization of the Involved Signaling Pathway

2000 ◽  
Vol 20 (5) ◽  
pp. 1886-1896 ◽  
Author(s):  
Kristin Breitschopf ◽  
Judith Haendeler ◽  
Philipp Malchow ◽  
Andreas M. Zeiher ◽  
Stefanie Dimmeler
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Map Kinase ◽  
Specific Inhibitor ◽  
26S Proteasome ◽  
Apoptotic Pathway ◽  
Critical Determinant ◽  
Intact Cells ◽  
Antiapoptotic Protein ◽  
Tnf Α

ABSTRACT The ratio of proapoptotic versus antiapoptotic Bcl-2 members is a critical determinant that plays a significant role in altering susceptibility to apoptosis. Therefore, a reduction of antiapoptotic protein levels in response to proximal signal transduction events may switch on the apoptotic pathway. In endothelial cells, tumor necrosis factor alpha (TNF-α) induces dephosphorylation and subsequent ubiquitin-dependent degradation of the antiapoptotic protein Bcl-2. Here, we investigate the role of different putative phosphorylation sites to facilitate Bcl-2 degradation. Mutation of the consensus protein kinase B/Akt site or of potential protein kinase C or cyclic AMP-dependent protein kinase sites does not affect Bcl-2 stability. In contrast, inactivation of the three consensus mitogen-activated protein (MAP) kinase sites leads to a Bcl-2 protein that is ubiquitinated and subsequently degraded by the 26S proteasome. Inactivation of these sites within Bcl-2 revealed that dephosphorylation of Ser87 appears to play a major role. A Ser-to-Ala substitution at this position results in 50% degradation, whereas replacement of Thr74 with Ala leads to 25% degradation, as assessed by pulse-chase studies. We further demonstrated that incubation with TNF-α induces dephosphorylation of Ser87 of Bcl-2 in intact cells. Furthermore, MAP kinase triggers phosphorylation of Bcl-2, whereas a reduction in Bcl-2 phosphorylation was observed in the presence of MAP kinase-specific phosphatases or the MAP kinase-specific inhibitor PD98059. Moreover, we show that oxidative stress mediates TNF-α-stimulated proteolytic degradation of Bcl-2 by reducing MAP kinase activity. Taken together, these results demonstrate a direct protective role for Bcl-2 phosphorylation by MAP kinase against apoptotic challenges to endothelial cells and other cells.

Preliminary Study of Vascular Endothelial Ca2+ Response to Elevated Temperature

2000 ◽  
Author(s):  
Aaron Schen ◽  
Lisa X. Xu
Keyword(s):  
Endothelial Cells ◽  
Cardiac Output ◽  
Flow Resistance ◽  
Vascular Endothelial Cells ◽  
Cytosolic Calcium ◽  
Thermal Regulation ◽  
Vascular Endothelial ◽  
Local Flow ◽  
Preliminary Study ◽  
Diameter Change

Abstract The thermal regulation of tissue is controlled by the sympathetically mediated redistribution of cardiac output and change in local flow resistance of arterioles. The diameter change of the vessel from its resting level is governed by the state of the contractile proteins in vascular smooth muscle which can be influenced by the concentration of free cytosolic calcium ([Ca2+]i) in the vascular endothelial cells (Falcone, 1995).

Mechanotransduction by integrin is essential for IL-6 secretion from endothelial cells in response to uniaxial continuous stretch

2005 ◽  
Vol 288 (5) ◽  
pp. C1012-C1022 ◽  
Author(s):  
Akitoshi Sasamoto ◽  
Masato Nagino ◽  
Satoshi Kobayashi ◽  
Keiji Naruse ◽  
Yuji Nimura ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Kinase Inhibitor ◽  
Umbilical Vein ◽  
Nuclear Factor Κb ◽  
Inhibitory Peptide ◽  
Signaling Mechanism ◽  
Iκb Kinase ◽  
Intracellular Ca ◽  
Protein Kinase C Inhibitor ◽  
Umbilical Vein Endothelial Cells

We previously reported that uniaxial continuous stretch in human umbilical vein endothelial cells (HUVECs) induced interleukin-6 (IL-6) secretion via IκB kinase (IKK)/nuclear factor-κB (NF-κB) activation. The aim of the present study was to clarify the upstream signaling mechanism responsible for this phenomenon. Stretch-induced IKK activation and IL-6 secretion were inhibited by application of α5β1 integrin-inhibitory peptide (GRGDNP), phosphatidylinositol 3-kinase inhibitor (LY-294002), phospholipase C-γ inhibitor (U-73122), or protein kinase C inhibitor (H7). Although depletion of intra- or extracellular Ca2 + pool using thapsigargin (TG) or EGTA, respectively, showed little effect, a TG-EGTA mixture significantly inhibited stretch-induced IKK activation and IL-6 secretion. An increase in the intracellular Ca2 + concentration ([Ca2 +]i) upon continuous stretch was observed even in the presence of TG, EGTA, or GRGDNP, but not in a solution containing the TG-EGTA mixture, indicating that both integrin activation and [Ca2 +]i rise are crucial factors for stretch-induced IKK activation and after IL-6 secretion in HUVECs. Furthermore, while PKC activity was inhibited by the TG-EGTA mixture, GRGDNP, LY-294002, or U-73122, PLC-γ activity was retarded by GRGDNP or LY-294002. These results indicate that continuous stretch-induced IL-6 secretion in HUVECs depends on outside-in signaling via integrins followed by a PI3-K-PLC-γ-PKC-IKK-NF-κB signaling cascade. Another crucial factor, [Ca2 +]i increase, may at least be required to activate PKC needed for NF-κB activation.

Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function

1997 ◽  
Vol 273 (4) ◽  
pp. C1250-C1258 ◽  
Author(s):  
Ashok K. Grover ◽  
Sue E. Samson
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Contractile Function ◽  
Ischemia Reperfusion ◽  
Muscle Cells ◽  
Intact Cells ◽  
Serca Pump

We examined the effects of peroxide on the sarco(endo)plasmic reticulum Ca2+ (SERCA) pump in pig coronary artery endothelium and smooth muscle at three organizational levels: Ca2+ transport in permeabilized cells, cytosolic Ca2+ concentration in intact cells, and contractile function of artery rings. We monitored the ATP-dependent, azide-insensitive, oxalate-stimulated45Ca2+uptake by saponin-permeabilized cultured cells. Low concentrations of peroxide inhibited the uptake less effectively in endothelium than in smooth muscle whether we added the peroxide directly to the Ca2+ uptake solution or treated intact cells with peroxide and washed them before the permeabilization. An acylphosphate formation assay confirmed the greater resistance of the SERCA pump in endothelial cells than in smooth muscle cells. Pretreating smooth muscle cells with 300 μM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolic Ca2+ concentration in a Ca2+-free solution, but it did not affect the endothelial cells. Peroxide pretreatment inhibited the CPA-induced contraction in deendothelialized arteries with a 50% inhibitory concentration of 97 ± 13 μM, but up to 500 μM peroxide did not affect the endothelium-dependent, CPA-induced relaxation. Similarly, 500 μM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced, endothelium-dependent relaxation by only 40 ± 13%. The greater resistance of the endothelium to reactive oxygen may be important during ischemia-reperfusion or in the postinfection immune response.

Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury

1997 ◽  
Vol 273 (4) ◽  
pp. L856-L865 ◽  
Author(s):  
Bruce R. Pitt ◽  
Margaret Schwarz ◽  
Elizabeth S. Woo ◽  
Emily Yee ◽  
Karla Wasserloos ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Metal Binding ◽  
High Performance ◽  
Binding Capacity ◽  
Peroxyl Radicals ◽  
Intact Cells ◽  
Tert Butyl Hydroperoxide ◽  
Liquid Chromatography Separation

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein with extensive metal binding capacity and potential nonenzymatic antioxidant activity. Despite the sensitivity of vascular endothelium to either heavy metal toxicity or oxidative stress, little is known regarding the role of MT in endothelial cells. Accordingly, we determined the sensitivity of cultured sheep pulmonary artery endothelial cells (SPAEC) that overexpressed MT to tert-butyl hydroperoxide ( t-BOOH), hyperoxia, or 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN; peroxyl radical generator). Nontoxic doses of 10 μM Cd increased MT levels from 0.21 ± 0.03 to 2.07 ± 0.24 μg/mg and resulted in resistance to t-BOOH and hyperoxia as determined by reduction of Alamar blue or [3H]serotonin transport, respectively. SPAEC stably transfected with plasmids containing either mouse or human cDNA for MT were resistant to both t-BOOH and hyperoxia. In addition, we examined transition metal-independent, noncytotoxic AMVN-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid into phospholipids and high-performance liquid chromatography separation. SPAEC that overexpressed MT after gene transfer completely inhibited peroxyl oxidation of phosphatidylserine, phosphatidylcholine, and sphingomyelin (but not phosphatidylethanolamine) noted in wild-type SPAEC. These data show for the first time that MT can 1) protect pulmonary artery endothelium against a diverse array of prooxidant stimuli and 2) directly intercept peroxyl radicals in a metal-independent fashion, thereby preventing lipid peroxidation in intact cells.

Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism

2003 ◽  
Vol 284 (6) ◽  
pp. H1978-H1984 ◽  
Author(s):  
M. Ursula Norman ◽  
Shane B. Reeve ◽  
Vincent Dive ◽  
A. Ian Smith ◽  
Rebecca A. Lew
Keyword(s):  
Endothelial Cells ◽  
High Performance ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Hybrid Cell ◽  
Fluorescent Substrate ◽  
Intact Cells ◽  
Aortic Endothelial Cells

The closely related metalloendopeptidases EC 3.4.24.15 (EP24.15; thimet oligopeptidase) and 24.16 (EP24.16; neurolysin) cleave a number of vasoactive peptides such as bradykinin and neurotensin in vitro. We have previously shown that hypotensive responses to bradykinin are potentiated by an inhibitor of EP24.15 and EP24.16 (26), suggesting a role for one or both enzymes in bradykinin metabolism in vivo. In this study, we have used selective inhibitors that can distinguish between EP24.15 and EP24.16 to determine their activity in cultured endothelial cells (the transformed human umbilical vein endothelial hybrid cell line EA.hy926 or ovine aortic endothelial cells). Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-d-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation.

Regulation of Ca(2+)-dependent nitric oxide synthase in bovine aortic endothelial cells

1995 ◽  
Vol 269 (3) ◽  
pp. C757-C765 ◽  
Author(s):  
B. J. Buckley ◽  
Z. Mirza ◽  
A. R. Whorton
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
No Synthase ◽  
No Production ◽  
Intact Cells ◽  
Aortic Endothelial Cells ◽  
A 23187 ◽  
Transient Rise ◽  
Synthase Inhibitor ◽  
Sustained Increase

Vascular endothelium responds to Ca(2+)-mobilizing agonists by producing nitric oxide (NO), a potent vasodilator and inhibitor of platelet aggregation. Regulation of constitutively expressed endothelial NO synthase (eNOS) in intact cells is not well understood. We investigated the kinetics of NO formation in response to Ca(2+)-mobilizing agonists, the requirement for extracellular L-arginine, and the role of NO in regulating eNOS activity. When endothelial cells were stimulated with bradykinin and ATP in the presence of 100 microM L-arginine, we observed a rapid and transient rise in intracellular Ca2+ concentration ([Ca2+]i) from 50 +/- 8 nM to 698 +/- 74 and 637 +/- 53 nM, respectively, and a rapid and transient rise in NO production from a basal level of 37 pmol.min-1.mg protein-1 to 256 and 275 pmol.min-1.mg protein-1, respectively. When cells were stimulated with A-23187 or thapsigargin in the presence of 100 microM L-arginine, we observed a sustained increase in [Ca2+]i and a sustained increase in NO production. The rate of NO synthesis was linear over 30 min, rising above control levels of 7 pmol/min to 53 pmol/min for A-23187 and 62 pmol/min for thapsigargin. Thapsigargin stimulated NO production and [Ca2+]i with 50% effective concentration values of 0.01 and 0.05 microM, respectively. Ca(2+)-stimulated NO production was attenuated by the NO synthase inhibitor NG-monomethyl-L-arginine, the removal of extracellular L-arginine, and the Ca(2+)-chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. When we exposed cells to NO gas (3.1 mM for 15 min) and S-nitrosoglutathione (10 mM for 1 h) thapsigargin-stimulated NO production was decreased by 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine stimulation of prostaglandin and HETE synthesis in human endothelial cells

1988 ◽  
Vol 255 (2) ◽  
pp. C214-C225 ◽  
Author(s):  
G. E. Revtyak ◽  
M. J. Hughes ◽  
A. R. Johnson ◽  
W. B. Campbell
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Umbilical Artery ◽  
Epoxyeicosatrienoic Acids ◽  
Human Endothelial Cells ◽  
Intact Cells ◽  
Histamine Stimulation ◽  
Human Umbilical Artery ◽  
A Minor ◽  
Stimulation Of

Endothelial cells (EC) cultured from human umbilical artery (UA) and vein (UV) metabolized [14C]arachidonic acid to prostaglandins (PGs), monohydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs). Major radioactive products were identified as 6-keto-PGF1 alpha, PGE2, PGF2 alpha, 12-hydroxy heptadecatrienoic acid, 15-HETE, and 11-HETE. In addition, extracts from UV ECs contained 12-HETE, 5-HETE, 14,15-EET, and 5,6-EET as minor products, whereas extracts from UA ECs contained only 12-HETE as a minor product. UA ECs also produced metabolites comigrating with 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET. Histamine increased the release of [14C]PGs and [14C]HETEs from [14C]arachidonic acid-labeled ECs. Indomethacin, aspirin, and nordihydroguauretic acid completely inhibited synthesis of both [14C]PGs and [14C]HETEs from exogenous [14C]arachidonic acid in these cells. Microsomes metabolized [14C]arachidonic acid to the same [14C]PGs and [14C]HETEs as intact cells. Pretreatment of microsomes with indomethacin completely inhibited formation of these products. These data indicate that UA ECs and UV ECs metabolize endogenous and exogenous arachidonic acid to both PGs and HETEs. Also 15-HETE and 11-HETE appear to be synthesized by a microsomal enzyme with the properties of cyclooxygenase.

Dimerization of P-selectin in platelets and endothelial cells

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3070-3077 ◽  
Author(s):  
Fern J. Barkalow ◽  
Kurt L. Barkalow ◽  
Tanya N. Mayadas
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Platelet Activation ◽  
Cell Activation ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Monomeric Form ◽  
Intact Cells ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

P-selectin is a leukocyte adhesion receptor stored in platelets and endothelial cells and is translocated to the surface upon cell activation. Purified P-selectin is oligomeric and has increased avidity for its ligand relative to the monomeric form, but whether P-selectin self-associates in the membrane of intact cells is not known. A chemical cross-linking approach was used to show that P-selectin is present as noncovalent dimers in resting platelets, human umbilical vein endothelial cells, and heterologous RIN5F cells expressing P-selectin. The results of 2-dimensional isoelectric focusing are consistent in showing P-selectin dimers as homodimers, but they are composed of a more basic subset of P-selectin than the monomers. This suggests that the dimers are a biochemically distinct subset of P-selectin. P-selectin dimers form in the endoplasmic reticulum and Golgi compartments of human umbilical vein endothelial cells only after synthesis of the mature P-selectin subunit, and are not preferentially stored in Weibel-Palade bodies as compared with the monomeric form. Platelet activation with thrombin receptor–activating peptide leads to the presence of P-selectin monomers and homodimers on the cell surface as well as P-selectin heterodimers, which are composed of P-selectin and an unidentified protein of approximately 81 kd molecular weight. In summary, these studies demonstrate that P-selectin is homodimeric in situ and that platelet activation leads to the formation of an additional activation-specific heterodimeric species. In addition, the homodimer has unique biochemical characteristics compared with the monomeric form, and dimerization occurs in the endoplasmic reticulum and Golgi compartments of endothelial cells.

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