scholarly journals Ecto-phosphorylation on aortic endothelial cells. Exquisite sensitivity to staurosporine

1992 ◽  
Vol 285 (2) ◽  
pp. 585-591 ◽  
Author(s):  
S Pirotton ◽  
O Boutherin-Falson ◽  
B Robaye ◽  
J M Boeynaems
Keyword(s):  
Endothelial Cells ◽  
Kinase Inhibitor ◽  
Serum Proteins ◽  
Inositol Phosphates ◽  
Calf Serum ◽  
P2y Receptors ◽  
Two Dimensional Gel Electrophoresis ◽  
Aortic Endothelial Cells ◽  
Cell Functions ◽  
Aortic Endothelial

One- and two-dimensional gel electrophoresis of proteins from bovine aortic endothelial cells (BAEC) incubated with [gamma-32P]ATP revealed the preferential labelling of a cell-associated 21 kDa substrate. The labelling of this band was detectable within 30 s, increased up to 30 min and was stable for at least 3 h following the wash-out of the ATP. This protein was also labelled after incubation of the cells with [gamma-35S]ATP. Incorporation of radioactivity into the 21 kDa band did not occur if the endothelial cells were treated with low concentrations of trypsin (0.01%) before or after the labelling period. The pattern of BAEC protein phosphorylation by [gamma-32P]ATP was completely different from that of the fetal calf serum used for the cell culture. The presence of serum during the incubation of BAEC with [gamma-32P]ATP did not modify qualitatively the labelling pattern and, in particular, did not enhance the phosphorylation of the 21 kDa substrate; this suggests that neither the kinase nor the 21 kDa substrate are adsorbed serum proteins. Staurosporine, a protein kinase inhibitor with low specificity, decreased the labelling of the 21 kDa protein with an IC50 of 2 nM. In contrast, at 100 nM, staurosporine did not decrease the accumulation of inositol phosphates induced by ATP via the activation of P2y receptors. These data indicate the presence of aortic endothelial cells of an ecto-kinase which uses extracellular ATP to produce the selective and long-lived phosphorylation of a 21 kDa endothelial substrate. Ecto-phosphorylation of this protein might play a role in the modulation of endothelial cell functions by ATP, in addition to the P2y receptors [Boeynaems & Pearson (1990) Trends Pharmacol. Sci. 11, 34-37]. The exquisite sensitivity of ecto-phosphorylation to inhibition by staurosporine and its specific inhibition by some isoquinolinesulphonamide compounds provide potential pharmacological tools to investigate this hypothesis.

scholarly journals Metabolism of inositol phosphates in ATP-stimulated vascular endothelial cells

1991 ◽  
Vol 277 (1) ◽  
pp. 103-110 ◽  
Author(s):  
S Pirotton ◽  
B Verjans ◽  
J M Boeynaems ◽  
C Erneux
Keyword(s):  
Endothelial Cells ◽  
Inositol Phosphates ◽  
Vascular Endothelial Cells ◽  
P2y Receptors ◽  
Vascular Endothelial ◽  
Aortic Endothelial Cells ◽  
Control Value ◽  
Time Courses ◽  
Fold Stimulation ◽  
Aortic Endothelial

The accumulation of InsP1, InsP2, InsP3 and InsP4 isomers was investigated in bovine aortic endothelial cells labelled with [3H]inositol and stimulated with ATP. The separation of these isomers was performed by ion-pairing reverse-phase h.p.l.c. on a mu Bondapack C18 column for the InsP3 and InsP4 isomers and by ion-exchange h.p.l.c. on a Partisil SAX column for the InsP1 and InsP2 isomers. In unstimulated endothelial cells, a large amount of material was co-eluted with InsP5 and InsP6, whereas amounts of InsP3 and InsP4 were small. The addition of ATP (100 microM) induced a striking (35-fold stimulation) and transient increase of Ins(1,4,5)P3 that was maximal around 15 s. This peak was followed by a more sustained accumulation of Ins(1,3,4,5)P4 and Ins(1,3,4)P3, but the amounts of these two metabolites accumulated in response to ATP were much smaller than that of Ins(1,4,5)P3. The increase in InsP2 isomers in response to ATP had similar characteristics: a rapid and transient accumulation of Ins(1,4)P2, followed by an increase of Ins(3,4)P2 and Ins(1,3)P2, which was more sustained but had a smaller magnitude. ATP also induced the accumulation of both Ins1P and Ins4P, but with different time courses: the level of Ins4P was maximal at 1 min (60 times the control value) and returned to baseline after 5 min, whereas the increase in Ins1P was undetectable at 1 min and reached a maximum after 5 min, which represented 240% of the basal level. These data indicate that Ins(1,4,5)P3, which is rapidly formed in aortic endothelial cells as a result of activation of P2Y receptors, is preferentially metabolized at early times (less than 1 min) by a 5-phosphatase, with the sequential formation of Ins(1,4)P2 and Ins4P. Afterwards, a small but sustained increase in the content of Ins(1,3,4)P3, Ins(1,3)P2, Ins(3,4)P2 and Ins1P was observed, reflecting the activation of the Ins(1,4,5)P3 3-kinase.

Stimulation Of Prostacyclin Production In Aortic Endothelial Cells By A Non-Dialysable Serum Factor

1981 ◽  
Author(s):  
E R Hall ◽  
M Rafelson ◽  
K Wu
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Calf Serum ◽  
Dependent Manner ◽  
Freezing And Thawing ◽  
Concentration Dependent Manner ◽  
Aortic Endothelial Cells ◽  
Prostacyclin Production ◽  
Aortic Endothelial

The production of prostacyclin (PGI2) by vascular endothelial cells is thought to be of primary importance in maintaining normal hemostasis. We have investigated the production of prostacyclin in bovine arterial endothelial cells maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 30% fetal calf serum. Intact, confluent monolayers of endothelial cells (3x106 cells) in passages 2 through 6 were used. The growth medium was removed and the cells were washed in DMEM that did not contain serum. 3 mls of medium alone or containing normal plasma or serum was then added and incubated at 37°C for 15 min. Then, 1 mg of arachidonic acid was added and the cells incubated for an additional hour. The test medium was removed, centrifuged to remove any loose cells and stored at -70°C. To determine the production of PGI2 by the endothelial cells, the medium was assayed for 6-keto-PGF1α, the stable metabolite of PGI2, by radioimmunoassay. The synthesis of prostacyclin by bovine aortic endothelial cells was significantly increased in a concentration dependent manner by both normal platelet poor plasma and normal serum. This increase in prostacyclin production was inhibited by both aspirin and indomethacin, indicating an increase in synthesis rather than the release of PGI2. Furthermore, this increase could be demonstrated in the presence or absence of added arachidonic acid. The active component in plasma and serum was non-dialysable, eliminating the possibility of a small compound such as bradykinin or angiotensin II. This active factor was present after freezing and thawing the plasma and serum and was heat stable (60°C, 5 min). The presence of an endogenous prostacyclin stimulating factor may be significant in the in vivo regulation of prostacyclin production.

H2 receptor-mediated responses of aortic endothelial cells to histamine

1992 ◽  
Vol 262 (1) ◽  
pp. H220-H224 ◽  
Author(s):  
G. Hekimian ◽  
S. Cote ◽  
J. Van Sande ◽  
J. M. Boeynaems
Keyword(s):  
Endothelial Cells ◽  
Inositol Phosphates ◽  
Umbilical Vein ◽  
Bovine Aortic Endothelial Cell ◽  
Camp Content ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
H2 Antagonist ◽  
Umbilical Vein Endothelial Cells ◽  
Aortic Endothelial

It is well known that umbilical vein endothelial cells express H1 receptors that mediate the various responses of these cells to histamine, including accumulation of inositol phosphates, rise of cytosolic Ca2+, increased permeability to macromolecules, and release of prostacyclin. In bovine aortic endothelial cells, histamine did not increase the level of inositol phosphates nor the release of prostacyclin. In contrast, it increased the adenosine 3',5'-cyclic monophosphate (cAMP) content of these cells. That response was obtained in the 1 to 100 microM range of concentrations and reached a maximum within 2 min of histamine addition. It was mimicked by the H2-specific agonist dimaprit, inhibited by the H2 antagonist ranitidine, and insensitive to the H1 antagonist mepyramine. Histamine reduced the permeability to albumin of bovine aortic endothelial cell monolayers; this paradoxical effect is likely to be mediated by the rise in cAMP, which is known to enhance the barrier property of the endothelium. In conclusion, bovine aortic endothelial cells are responsive to histamine, and this response is mediated by H2 and not H1 receptors.

Tunicamycin increases intracellular calcium levels in bovine aortic endothelial cells

1997 ◽  
Vol 273 (4) ◽  
pp. C1298-C1305 ◽  
Author(s):  
Barbara J. Buckley ◽  
A. R. Whorton
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Intracellular Calcium ◽  
Inositol Phosphates ◽  
Protein Glycosylation ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Intracellular Ca ◽  
Oxide Synthase ◽  
Aortic Endothelial

Tunicamycin is a nucleoside antibiotic that inhibits protein glycosylation and palmitoylation. The therapeutic use of tunicamycin is limited in animals because of its toxic effects, particularly in cerebral vasculature. Tunicamycin decreases palmitoylation of the endothelial isoform of nitric oxide synthase, stimulates nitric oxide synthesis, and increases the concentration of intracellular calcium ([Ca2+]i) in bovine aortic endothelial cells (B. J. Buckley and A. R. Whorton. FASEB J. 11: A110, 1997). In the present study, we investigated the mechanism by which tunicamycin alters [Ca2+]iusing the Ca2+-sensitive dye fura 2. We found that tunicamycin increased [Ca2+]iwithout increasing levels of inositol phosphates. When cells were incubated in the absence of extracellular Ca2+, [Ca2+]irapidly rose in response to tunicamycin, although a full response was not achieved. The pool of intracellular Ca2+ mobilized by tunicamycin overlapped with that mobilized by thapsigargin. Extracellular nickel blocked a full response to tunicamycin when cells were incubated in the presence of extracellular Ca2+. The effects of tunicamycin on [Ca2+]iwere partially reversed by washing out the drug, and the remainder of the response was inhibited by removing extracellular Ca2+. These results indicate that tunicamycin mobilizes Ca2+ from intracellular stores in a manner independent of phospholipase C activation and increases the influx of Ca2+ across the plasma membrane.

Ghrelin has novel vascular actions that mimic PI 3-kinase-dependent actions of insulin to stimulate production of NO from endothelial cells

2007 ◽  
Vol 292 (3) ◽  
pp. E756-E764 ◽  
Author(s):  
Micaela Iantorno ◽  
Hui Chen ◽  
Jeong-a Kim ◽  
Manfredi Tesauro ◽  
Davide Lauro ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
Nitric Oxide Synthase ◽  
Map Kinase ◽  
Kinase Inhibitor ◽  
Dependent Manner ◽  
Aortic Endothelial Cells ◽  
Oxide Synthase ◽  
Aortic Endothelial

Ghrelin is an orexigenic peptide hormone secreted by the stomach. In patients with metabolic syndrome and low ghrelin levels, intra-arterial ghrelin administration acutely improves their endothelial dysfunction. Therefore, we hypothesized that ghrelin activates endothelial nitric oxide synthase (eNOS) in vascular endothelium, resulting in increased production of nitric oxide (NO) using signaling pathways shared in common with the insulin receptor. Similar to insulin, ghrelin acutely stimulated increased production of NO in bovine aortic endothelial cells (BAEC) in primary culture (assessed using NO-specific fluorescent dye 4,5-diaminofluorescein) in a time- and dose-dependent manner. Production of NO in response to ghrelin (100 nM, 10 min) in human aortic endothelial cells was blocked by pretreatment of cells with NG-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor), wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor], or (d-Lys3)-GHRP-6 (selective antagonist of ghrelin receptor GHSR-1a), as well as by knockdown of GHSR-1a using small-interfering (si) RNA (but not by mitogen/extracellular signal-regulated kinase inhibitor PD-98059). Moreover, ghrelin stimulated increased phosphorylation of Akt (Ser473) and eNOS (Akt phosphorylation site Ser1179) that was inhibitable by knockdown of GHSR-1a using siRNA or by pretreatment of cells with wortmannin but not with PD-98059. Ghrelin also stimulated phosphorylation of mitogen-activated protein (MAP) kinase in BAEC. However, unlike insulin, ghrelin did not stimulate MAP kinase-dependent secretion of the vasoconstrictor endothelin-1 from BAEC. We conclude that ghrelin has novel vascular actions to acutely stimulate production of NO in endothelium using a signaling pathway that involves GHSR-1a, PI 3-kinase, Akt, and eNOS. Our findings may be relevant to developing novel therapeutic strategies to treat diabetes and related diseases characterized by reciprocal relationships between endothelial dysfunction and insulin resistance.

Effects of Dipyrone on Prostaglandin Production by Human Platelets and Cultured Bovine Aortic Endothelial Cells

1983 ◽  
Vol 49 (02) ◽  
pp. 132-137 ◽  
Author(s):  
A Eldor ◽  
G Polliack ◽  
I Vlodavsky ◽  
M Levy
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Competitive Inhibition ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Aortic Endothelial

SummaryDipyrone and its metabolites 4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoan- tipyrine inhibited the formation of thromboxane A2 (TXA2) during in vitro platelet aggregation induced by ADP, epinephrine, collagen, ionophore A23187 and arachidonic acid. Inhibition occurred after a short incubation (30–40 sec) and depended on the concentration of the drug or its metabolites and the aggregating agents. The minimal inhibitory concentration of dipyrone needed to completely block aggregation varied between individual donors, and related directly to the inherent capacity of their platelets to synthesize TXA2.Incubation of dipyrone with cultured bovine aortic endothelial cells resulted in a time and dose dependent inhibition of the release of prostacyclin (PGI2) into the culture medium. However, inhibition was abolished when the drug was removed from the culture, or when the cells were stimulated to produce PGI2 with either arachidonic acid or ionophore A23187.These results indicate that dipyrone exerts its inhibitory effect on prostaglandins synthesis by platelets or endothelial cells through a competitive inhibition of the cyclooxygenase system.

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