Thrombin Stimulates Inositol Phosphate Accumulation and Prostacyclin Synthesis in Human Endothelial Cells from Umbilical Vein but Not from Omentum

1989 ◽  
Vol 61 (01) ◽  
pp. 122-126 ◽  
Author(s):  
A J Carter ◽  
W G Eisert ◽  
T H Müller
Keyword(s):  
Endothelial Cells ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Umbilical Vein ◽  
Microvascular Endothelial Cells ◽  
Human Endothelial Cells ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation ◽  
Microvascular Endothelial ◽  
Prostacyclin Synthesis

SummaryWe have compared the effects of thrombin on the accumulation of inositol phosphates and the synthesis of prostacyclin in cultured human endothelial cells from umbilical vein and the microvasculature of omentum. Active human thrombin induced a dose-dependent accumulation of inositol phosphates and a concomitant synthesis of prostacyclin in endothelial cells from human umbilical vein. However, thrombin at all concentrations tested was unable to stimulate inositol phosphate accumulation and prostacyclin synthesis in microvascular endothelial cells from human omentum. Bradykinin was able to stimulate these effects in both types of cell. These results demonstrate that although inositol phosphate turnover is an initial event associated with prostacyclin synthesis in endothelial cells, there are differences in the way microvascular endothelial cells respond to thrombin.

Histamine and inositol phosphate accumulation in endothelium: cAMP and a G protein

1989 ◽  
Vol 257 (4) ◽  
pp. L259-L264 ◽  
Author(s):  
M. R. Carson ◽  
S. S. Shasby ◽  
D. M. Shasby
Keyword(s):  
Endothelial Cells ◽  
Intracellular Calcium ◽  
G Protein ◽  
Inositol Phosphate ◽  
Umbilical Vein ◽  
Microvascular Permeability ◽  
Alpha Toxin ◽  
H1 Receptor ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation

Histamine increases microvascular permeability through a calcium-dependent process, and histamine occupancy of the H1-receptor increases calcium in cultured endothelial cells. Agents that increase adenosine 3',5'-cyclic monophosphate (cAMP) in endothelial cells prevent the in vivo increase in microvascular permeability that follows histamine exposure. In the current experiments, histamine occupancy of the H1-receptor increased the flux of albumin across monolayers of cultured human umbilical vein endothelial cells (HUVEC). This was prevented by pretreating the cells with theophylline, forskolin, and 8-bromo-cAMP (BrcAMP), which also decreased the flux of albumin across control monolayers. Exposing the cells to histamine increased inositol phosphate accumulation in the cells, and this was prevented by the H1-antagonist pyrilamine but not by theophylline, forskolin, and BrcAMP. Exposing the cells to histamine increased intracellular calcium measured with fura-2. The increase in cell calcium was prevented by pyrilamine but not by pretreatment with theophylline, forskolin, and BrcAMP. When endogenous cell GTP was depleted by permeabilizing the membranes of the endothelial cells with Staphylococcus aureus alpha-toxin, histamine-stimulated inositol phosphate accumulation was enhanced with addition of GTP but not with addition of GDP to the buffer. Addition of GTP alone to the buffer did not increase inositol phosphate accumulation in alpha-toxin-treated cells. Histamine stimulates inositol phosphate accumulation in HUVEC via a G protein. Inhibition of the edemagenic effects of histamine by cAMP does not occur by interrupting this signal transduction pathway between the binding of histamine to its receptor and the increase in intracellular calcium.

scholarly journals A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation

2004 ◽  
Vol 9 (4) ◽  
pp. 343-353 ◽  
Author(s):  
Elfrida R. Benjamin ◽  
Sarah L. Haftl ◽  
Dimitris N. Xanthos ◽  
Gregg Crumley ◽  
Mohamed Hachicha ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Column Chromatography ◽  
Large Volume ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Assay Method ◽  
Signal To Noise ◽  
Chromatography Method ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP3), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen™ assays, offer higher throughput. However, these techniques rely on measurement of IP3 itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP3. The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP3 and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.

scholarly journals Platelet-derived growth factor does not stimulate prostacyclin synthesis by cultured endothelial cells

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 131-134
Author(s):  
KS Callahan ◽  
A Schorer ◽  
JM Harlan
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Umbilical Vein ◽  
Platelet Derived Growth Factor ◽  
Human Endothelial Cells ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Prostacyclin Synthesis ◽  
Release Product ◽  
Stimulation Of

We examined the effect of highly purified platelet-derived growth factor (PDGF) on prostacyclin (PGI2) release by cultured human umbilical vein and bovine aortic endothelial cells. PDGF tested at concentrations equal to or exceeding those observed in serum did not increase endothelial cell PGI2 synthesis as measured by radioimmunoassay of its metabolite, 6-keto-PGF1 alpha. In contrast, cells incubated with 20% human whole blood serum (WBS) demonstrated significantly increased PGI2 production (fivefold stimulation). Addition of anti-PDGF antibody to the 20% WBS did not attenuate the increased synthesis of PGI2. Incubation with 20% plasma-derived serum (PDS) that was deficient in PDGF produced stimulation of PGI2 release similar to 20% WBS. These results demonstrate that PDGF does not cause increased PGI2 synthesis in cultured human endothelial cells of human or bovine origin, and further suggest that the stimulation observed with serum is not due to a platelet-release product.

Adrenergic stimulation of inositol phosphate accumulation in tracheal epithelium

1989 ◽  
Vol 66 (1) ◽  
pp. 504-508 ◽  
Author(s):  
T. Bainbridge ◽  
R. D. Feldman ◽  
M. J. Welsh
Keyword(s):  
Adrenergic Receptor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Adrenergic Stimulation ◽  
Cl Secretion ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation ◽  
Stimulation Of

To determine whether inositol phosphates are important second messengers in the regulation of Cl- secretion by airway epithelia, we examined the relationship between inositol phosphate accumulation and Cl- secretion in response to adrenergic agonists. We found that epinephrine stimulated Cl- secretion and inositol phosphate accumulation with similar concentration dependence. Although isoproterenol stimulated Cl- secretion, there was no effect of beta-adrenergic receptor activation on inositol phosphate accumulation. In contrast, alpha 1-adrenergic receptor activation stimulated inositol phosphate accumulation but failed to induce Cl- secretion. Another Cl- secretagogue, prostaglandin E1, also failed to stimulate inositol phosphate accumulation. These data suggest that inositol phosphate accumulation is neither sufficient nor required for stimulation of Cl- secretion in cultured canine tracheal epithelial cells.

scholarly journals Streptococcus suis Serotype 2 Interactions with Human Brain Microvascular Endothelial Cells

2000 ◽  
Vol 68 (2) ◽  
pp. 637-643 ◽  
Author(s):  
Nathalie Charland ◽  
Victor Nizet ◽  
Craig E. Rubens ◽  
Kwang Sik Kim ◽  
Sonia Lacouture ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Damage ◽  
Umbilical Vein ◽  
Bacterial Species ◽  
Streptococcus Suis ◽  
Neonatal Meningitis ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Group B Streptococci ◽  
Microvascular Endothelial

ABSTRACT Streptococcus suis serotype 2 is a worldwide causative agent of many forms of swine infection and is also recognized as a zoonotic agent causing human disease, including meningitis. The pathogenesis of S. suis infections is poorly understood. Bacteria circulate in the bloodstream in the nonimmune host until they come in contact with brain microvascular endothelial cells (BMEC) forming the blood-brain barrier. The bacterial polysaccharide capsule confers antiphagocytic properties. It is known that group B streptococci (GBS) invade and damage BMEC, which may be a primary step in the pathogenesis of neonatal meningitis. Interactions betweenS. suis and human endothelial cells were studied to determine if they differ from those between GBS and endothelial cells. Invasion assays performed with BMEC and human umbilical vein endothelial cells demonstrated that unlike GBS, S. suisserotype 2 could not invade either type of cell. Adherence assays showed that S. suis adhered only to BMEC, whereas GBS adhered to both types of cell. These interactions were not affected by the presence of a capsule, since acapsular mutants from both bacterial species adhered similarly compared to the wild-type strains. Lactate dehydrogenase release measurements indicated that some S. suis strains were highly cytotoxic for BMEC, even more than GBS, whereas others were not toxic at all. Cell damage was related to suilysin (S. suis hemolysin) production, since only suilysin-producing strains were cytotoxic and cytotoxicity could be inhibited by cholesterol and antisuilysin antibodies. It is possible that hemolysin-positive S. suis strains use adherence and suilysin-induced BMEC injury, as opposed to direct cellular invasion, to proceed from the circulation to the central nervous system.

DIFFERENTIAL STIMULATION OF INOSITOL TRISPHOSPHATE ACCUMULATION IN CULTURED HUMAN ENDOTHELIAL CELLS BY THROMBIN

1987 ◽  
Author(s):  
A J Carter ◽  
W G Eisert ◽  
T H Mμller
Keyword(s):  
Endothelial Cells ◽  
Time Course ◽  
Inositol Phosphate ◽  
Umbilical Vein ◽  
Inositol Trisphosphate ◽  
Specific Antigen ◽  
Enzymic Activity ◽  
Small Vessel ◽  
Human Endothelial Cells ◽  
Large Vessels

Vascular endothelial cells possess specific receptors for thrombin, and thrombin can interact with these receptors to activate the endothelial cells. However, the signal transduction mechanisms which mediate the cellular responses are not yet characterised. The aim of this study therefore, was to determine whether thrombin influenced the inositol phosphate transduction pathway in cultured human endothelial cells. Endothelial cells were isolated from both large and small vessels; these were human umbilical vein and the microvasculature of human omentum respectively. The endothelial cells stained positively with antibodies against Factor VIII antigen and another endothelial cell specific antigen (BMA 120). Pure human thrombin (0.1 - 10 units/ml) induced a dose-dependent formation of inositol phosphate, inositol biphosphate and inositol trisphosphate (IP3) in endothelial cells from large vessels prelabelled with tritiated inositol. The formation of IP3 was significantly increased after 15 sec., maximal after 1 min. and had returned almost to baseline levels after 4 min. This time course is consistent with its role as a second messenger. When the enzymic activity of thrombin was removed with phenylalanyl-prolyl-arginine chloromethyl ketone or d i i sopr opyIfluorophosphate, thrombin lost its ability to stimulate the accumulation of IP3. Thrombin at all concentrations tested was unable to stimulate the formation of IP3 in small vessel endothelial cells. However, IP3 formation could be stimulated by bradykinin (0.1-10 μM) in cells from both small and large vessels. The results demonstrate that active thrombin can induce the formation of IP3 in large vessel endothelium. But that there are differences in the way small vessel endothelium responds to thrombin.

CULTURED HUMAN MICROVASCULAR ENDOTHELIAL CELLS SYNTHESIZE CYCLOOXYGENASE METABOLITES FROM ARACHIDONIC ACID IN RESPONSE TO LEUKOTRIENES C4 AND D4

1987 ◽  
Author(s):  
L O Carreras ◽  
J Maclouf ◽  
G Tobelem ◽  
J P Caen
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Time Course ◽  
Umbilical Vein ◽  
Calcium Ionophore ◽  
Microvascular Endothelial Cells ◽  
Ionophore A23187 ◽  
Total Production ◽  
Dependent Manner ◽  
Microvascular Endothelial

Several investigators have demonstrated that endothelial cells have heterogeneous intrinsic properties depending on their vascular origin. In this respect, very limited knowledge exists concerning the production of eicosanoids by human microvascular endothelial cells (HMEC). The aim of this study was to determine: 1) the pattern of the production of cyclooxygenase metabolites by cultured HMEC from omental adipose tissue as compared to the classical study of human umbilical vein endothelial cells (HUVEC); 2) the modification of this metabolism upon leukotrienes (LTs) stimulation. Cultured HMEC produced prostaglandin (PG) E2, PGF2 , 6-keto-PGF1 , and PGD2 (measured by enzymoimmunoassay). In basal conditions, PGD2 was the main product released in the supernatant. Upon stimulation with thrombin, arachidonic acid and calcium ionophore A23187, a marked increase in the production of PGE2, PGF2 , and 6-keto-PGFj , was observed; these results were quite different from HUVEC. In contrast, PGD2 remained unchanged under our experimental conditions and thromboxane B2 was always undetectable. In all cases, the release of PGE2 and PGF2 , was higher than that of 6-keto-PGFj . A considerable amount of the metabolites produced remained cell-associated. The total production (release + cell bound) of cyclooxygenase products was stimulated by LTC4 and LTD4 in a dose-dependent manner (10-9 to 10-6 M). The production of PGD2 was unchanged. LTC4 and LTD4 were almost equally potent, but LTB4 was unable to stimulate PG synthesis (n=4). The production of metabolites induced by 1 uM LTC4 or LTD4 was even higher than that obtained in the presence of high concentrations of thrombin (5 U/ml). This contrasted with the more pronounced stimulation of thrombin on HUVEC as compared to LTs. In the kinetic studies (n=2) we have observed a slow time-course of release of PGE2 and 6-keto-PGF1 into the supernatant of LTs-stimulated HMEC (half-maximal formation at 14-15 min). The stimulatory activity of LTC4 and LTD4 on the production of vasoactive cyclooxygenase metabolites by HMEC could be relevant in inflammatory processes.

scholarly journals Phorbol ester inhibition of the hormone-stimulated phosphoinositide cycle in WRK-1 cells

1986 ◽  
Vol 236 (1) ◽  
pp. 171-175 ◽  
Author(s):  
M E Monaco ◽  
R A Mufson
Keyword(s):  
Binding Affinity ◽  
Phorbol Ester ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Tumour Cells ◽  
Mammary Tumour ◽  
Phosphate Accumulation ◽  
Phosphoinositide Cycle ◽  
Inositol Phosphate Accumulation

WRK-1 rat mammary tumour cells respond to vasopressin with increased accumulation of inositol phosphates as well as increased precursor incorporation into phosphatidylinositol. The phorbol ester, phorbol 13-myristate 12-acetate (PMA) inhibits by 80% both inositol phosphate accumulation and increased precursor incorporation. This inhibition is much less evident at early times (2 min) than at later times (25 min). The vasopressin-induced rise in cytosolic free Ca2+ is inhibited in a similar manner. Oleoylacetylglycerol is inactive with respect to inhibition of vasopressin-induced increases in incorporation of 32P into phosphoinositides. PMA has no effect on vasopressin binding at saturating concentrations of the hormone and does not affect the binding affinity.

scholarly journals Thrombin Modulates the Expression of a Set of Genes Including Thrombospondin-1 in Human Microvascular Endothelial Cells

2005 ◽  
Vol 280 (23) ◽  
pp. 22172-22180 ◽  
Author(s):  
Joseph N. McLaughlin ◽  
Maria R. Mazzoni ◽  
John H. Cleator ◽  
Laurie Earls ◽  
Ana Luisa Perdigoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelium ◽  
Disease States ◽  
Similar Expression Profile ◽  
Thrombospondin 1 ◽  
Microvascular Endothelial

Thrombospondin-1 (THBS1) is a large extracellular matrix glycoprotein that affects vasculature systems such as platelet activation, angiogenesis, and wound healing. Increases in THBS1 expression have been liked to disease states including tumor progression, atherosclerosis, and arthritis. The present study focuses on the effects of thrombin activation of the G-protein-coupled, protease-activated receptor-1 (PAR-1) on THBS1 gene expression in the microvascular endothelium. Thrombin-induced changes in gene expression were characterized by microarray analysis of ∼11,000 different human genes in human microvascular endothelial cells (HMEC-1). Thrombin induced the expression of a set of at least 65 genes including THBS1. Changes in THBS1 mRNA correlated with an increase in the extracellular THBS1 protein concentration. The PAR-1-specific agonist peptide (TFLLRNK-PDK) mimicked thrombin stimulation of THBS1 expression, suggesting that thrombin signaling is through PAR-1. Further studies showed THBS1 expression was sensitive to pertussis toxin and protein kinase C inhibition indicating Gi/o- and Gq-mediated pathways. THBS1 up-regulation was also confirmed in human umbilical vein endothelial cells stimulated with thrombin. Analysis of the promoter region of THBS1 and other genes of similar expression profile identified from the microarray predicted an EBOX/EGRF transcription model. Expression of members of each family, MYC and EGR1, respectively, correlated with THBS1 expression. These results suggest thrombin formed at sites of vascular injury increases THBS1 expression into the extracellular matrix via activation of a PAR-1, Gi/o, Gq, EBOX/EGRF-signaling cascade, elucidating regulatory points that may play a role in increased THBS1 expression in disease states.

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