scholarly journals Phospholipid metabolism, calcium flux, and the receptor-mediated induction of chemotaxis in rabbit neutrophils.

1982 ◽  
Vol 93 (3) ◽  
pp. 690-697 ◽  
Author(s):  
D L Bareis ◽  
F Hirata ◽  
E Schiffmann ◽  
J Axelrod
Keyword(s):  
Arachidonic Acid ◽  
Calcium Influx ◽  
Calcium Ionophore ◽  
Receptor Activation ◽  
Calcium Flux ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Arachidonic Acid Release ◽  
Chemotactic Peptide ◽  
Acid Release

Rabbit neutrophils were stimulated with the chemotactic peptide fMet-Leu-Phe in the presence of the methyltransferase inhibitors homocysteine (HCYS) and 3-deazaadenosine (3-DZA). HCYS and 3-DZA inhibited chemotaxis, phospholipid methylation, and protein carboxymethylation in a dose-dependent manner. The chemotactic peptide-stimulated release of [14C]arachidonic acid previously incorporated into phospholipid was also partially blocked by the methyltransferase inhibitors. Stimulation by fMet-Leu-Phe or the calcium ionophore A23187 caused release of arachidonic acid but not of previously incorporated [14C]-labeled linoleic, oleic, or stearic acids. Unlike the arachidonic acid release caused by fMet-Leu-Phe, release stimulated by the ionophore could not be inhibited by HCYS and 3-DZA, suggesting that the release was caused by a different mechanism or by stimulating a step after methylation in the pathway from receptor activation to arachidonic acid release. Extracellular calcium was required for arachidonic acid release, and methyltransferase inhibitors were found to partially inhibit chemotactic peptide-stimulated calcium influx. These results suggest that methylation pathways may be associated with the chemotactic peptide receptor stimulation of calcium influx and activation of a phospholipase A2 specific for cleaving arachidonic acid from phospholipids.

Reduced prostacyclin formation after reoxygenation of anoxic endothelium

1990 ◽  
Vol 259 (5) ◽  
pp. C738-C745 ◽  
Author(s):  
S. L. Hempel ◽  
D. L. Haycraft ◽  
J. C. Hoak ◽  
A. A. Spector
Keyword(s):  
Arachidonic Acid ◽  
Umbilical Vein ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
High Rate ◽  
Ionophore A23187 ◽  
Arachidonic Acid Release ◽  
Cyclooxygenase Activity ◽  
Platelet Adherence ◽  
Acid Release

Human umbilical vein endothelial cells subjected to 24 h of anoxia followed by reoxygenation released less prostacyclin (PGI2) in response to thrombin, calcium ionophore A23187, or arachidonic acid. This was associated with a substantial increase in stimulated platelet adherence. Increased lactate dehydrogenase and 51Cr release occurred after 1 h of reoxygenation, but the high rate of release did not persist during the subsequent 23 h of reoxygenation. The changes in platelet adherence and PGI2 release partially resolved over 24 h. PGI2 formation from prostaglandin H2 was not reduced, suggesting that cyclooxygenase activity, but not prostacyclin synthase, is affected by reoxygenation. A decrease in arachidonic acid release from cellular lipids also occurred. The reduction in cyclooxygenase activity, but not arachidonic acid release, was prevented by the presence of ibuprofen during reoxygenation. Addition of catalase or superoxide dismutase during reoxygenation increased PGI2 release but did not completely overcome the reduction relative to control cultures. These findings suggest that the increase in platelet adherence during reoxygenation may be mediated in part by a change in cyclooxygenase activity. This is only partly overcome by extracellular oxygen species scavengers but is prevented by the presence of a reversible cyclooxygenase inhibitor during reoxygenation.

Influence of growth oxygen level on eicosanoid release from lung endothelial cells during hypoxia

1992 ◽  
Vol 263 (4) ◽  
pp. L454-L459
Author(s):  
W. E. Holden ◽  
E. M. Burnham ◽  
M. A. Lee ◽  
S. P. Bagby
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Acute Hypoxia ◽  
Calcium Ionophore ◽  
Pulmonary Vasculature ◽  
Ionophore A23187 ◽  
Oxygen Level ◽  
Arachidonic Acid Release ◽  
Eicosanoid Release ◽  
Acid Release

Eicosanoid products of arachidonic acid are suspected modulators of hypoxic vasoconstriction in the pulmonary vasculature. Vascular endothelial cells (EC) release several eicosanoids, but there is disagreement regarding the effect of hypoxia on EC eicosanoid release. We postulated that the oxygen level of growth in culture might influence the release of eicosanoids during acute hypoxia. We studied EC cultured from the main pulmonary arteries of pigs and grown at either 5% or near 20% oxygen, representing the normal limits of oxygen exposure to endothelium in normal lungs. Although cultures grown in 5% oxygen grew slightly faster by 4 days, the confluent cell number, protein content, and baseline eicosanoid release were no different compared with paired cultures grown in 20% oxygen. However, with an acute decrease in oxygen level, cultures grown in 5% oxygen released less prostaglandin E2, F2 alpha, and 6-ketoprostaglandin F1 alpha compared with amounts released at the growth oxygen level. In contrast, cultures grown in 20% oxygen released increased amounts of these eicosanoids compared with release at the growth oxygen level. Release of thromboxane B2 was not significantly different during hypoxia between cultures grown at 5% vs. 20% oxygen. In other experiments, cyclooxygenase activity, stimulated arachidonic acid release by calcium ionophore A23187, and uptake of arachidonic acid were no different in cultures grown at 5% vs. 20% oxygen. However, arachidonic acid release during hypoxia was reduced in 5% cultures and increased in 20% cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a novel inhibitor of cytosolic phospholipase A2α, pyrrophenone

2002 ◽  
Vol 363 (3) ◽  
pp. 727-735 ◽  
Author(s):  
Takashi ONO ◽  
Katsutoshi YAMADA ◽  
Yukiko CHIKAZAWA ◽  
Masahiko UENO ◽  
Shozo NAKAMOTO ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Arachidonic Acid Release ◽  
Eicosanoid Synthesis ◽  
Cytosolic Phospholipase ◽  
Ic50 Value ◽  
Acid Release ◽  
Cytosolic Phospholipase A2α ◽  
Dose Dependent

Cytosolic phospholipase A2α (cPLA2α), one of the three subtypes of cPLA2 (α, β and γ), is thought to be a rate-limiting enzyme in eicosanoid biosynthesis. We developed a novel and potent cPLA2α inhibitor with an optically active pyrrolidine, termed pyrrophenone, and characterized this compound in detail using enzyme and cellular assay systems. Pyrrophenone, which shows strong inhibition of cPLA2α activity, is one of the most potent cPLA2α inhibitors reported to date. Similar inhibitory potencies for cPLA2α were obtained from three different assays. The inhibitory activity of pyrrophenone is two or three orders of magnitude more potent than arachidonyl trifluoromethyl ketone (AACOCF3) under the same assay conditions. Pyrrophenone shows reversible inhibition of cPLA2α and displays no characteristics of the slow-binding inhibition observed for AACOCF3. Pyrrophenone also inhibited the esterase and lysophospholipase activities of cPLA2α. However, the inhibition by pyrrophenone of 14kDa secretory PLA2s, types IB and IIA, was over two orders of magnitude less potent than that for cPLA2α. Pyrrophenone strongly inhibited arachidonic acid release in calcium ionophore (A23187)-stimulated human monocytic cells (THP-1 cells) in a dose-dependent manner with an IC50 value of 0.024μM, followed by suppression of eicosanoid synthesis, and also showed dose-dependent inhibition for interleukin-1-induced prostaglandin E2 synthesis in human renal mesangial cells with an IC50 value of 0.0081μM. The mechanism of inhibition of eicosanoid synthesis in these cell-based assays was due to inhibition of only one step of arachidonic acid release without any effect on cyclo-oxygenase or lipoxygenase pathways. These results suggest that pyrrophenone could be a potential therapeutic agent for inflammatory diseases.

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.

Calcium-dependent release of arachidonic acid in response to purinergic receptor activation in airway epithelium

1994 ◽  
Vol 266 (2) ◽  
pp. C406-C415 ◽  
Author(s):  
E. R. Lazarowski ◽  
R. C. Boucher ◽  
T. K. Harden
Keyword(s):  
Arachidonic Acid ◽  
Inositol Phosphate ◽  
Purinergic Receptor ◽  
Receptor Activation ◽  
Normal Donor ◽  
Maximal Effect ◽  
Airway Epithelial ◽  
Arachidonic Acid Release ◽  
Receptor Agonists ◽  
Acid Release

The effect of purinergic receptor agonists on arachidonic acid release was investigated in [3H]arachidonic acid-prelabeled human airway epithelial cells. Exposure of bronchial epithelial BEAS39 cells to extracellular ATP resulted in a marked release of unesterified [3H]arachidonic acid with maximal effect observed within 60-90 s. [3H]diacylglycerol and [3H]phosphatidic acid accumulated in parallel with [3H]arachidonic acid. ATP-stimulated [3H]arachidonic acid release with a K0.5 of 9 +/- 2 microM and UTP was equipotent; no effect was observed with P2Y- or P2X-purinergic receptor agonists or with adenosine. Similar results were obtained with primary cultures of normal human nasal epithelium, CF/T43 and HBE1 airway epithelial cell lines derived from a cystic fibrosis patient and from a normal donor, respectively, and HT-29 human colon carcinoma cells. ATP stimulated inositol phosphate formation in BEAS39 cells with a concentration dependence identical to that for [3H]arachidonic acid release. The effect of ATP on both [3H]arachidonic acid release and inositol phosphate formation was equally inhibited by pertussis toxin. The Ca2+ ionophore A-23187 mimicked the effects of ATP or UTP on arachidonic acid release, and a marked inhibitory effect was observed with thapsigargin. The protein kinase C inhibitor staurosporine partially inhibited ATP-stimulated [3H]arachidonic acid release. These data are consistent with the hypothesis that phospholipase A2 activation is secondary to P2U-purinergic receptor stimulation of D-myoinositol 1,4,5-trisphosphate production and calcium mobilization from intracellular stores.

Regulation of Arachidonic Acid Release by Calcium Influx in Human Endothelial Cells

1999 ◽  
Vol 36 (3) ◽  
pp. 235-244 ◽  
Author(s):  
Elisabeth Millanvoye-Van Brussel ◽  
Monique David-Dufilho ◽  
Thuc Do Pham ◽  
Lahcen Iouzalen ◽  
Marie Aude Devynck
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Calcium Influx ◽  
Arachidonic Acid Release ◽  
Human Endothelial Cells ◽  
Acid Release

scholarly journals Stress stimuli increase calcium-induced arachidonic acid release through phosphorylation of cytosolic phospholipase A2

1999 ◽  
Vol 344 (2) ◽  
pp. 359-366 ◽  
Author(s):  
Marcus BUSCHBECK ◽  
Farideh GHOMASHCHI ◽  
Michael H. GELB ◽  
Steve P. WATSON ◽  
Angelika G. BÖRSCH-HAUBOLD
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Cytosolic Phospholipase A2 ◽  
Ionophore A23187 ◽  
Arachidonic Acid Release ◽  
High Osmolarity ◽  
Cytosolic Phospholipase ◽  
Acid Release ◽  
Sb 203580

Stress stimuli such as free radicals, high osmolarity or arsenite activate stress-activated protein kinases (SAPKs) in a wide variety of cells. In the present study, we have investigated the ability of several stress stimuli to activate SAPKs in platelets and to induce phosphorylation of their substrates. Treatment of human platelets with H2O2 stimulated SAPK2a and its downstream target mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2). Kinase activity reached a maximum after 2-5 min and declined towards basal levels after 15 min. Arsenite caused a steady increase of MAPKAP-K2 activity up to 15 min. The level of maximal kinase activation by H2O2 and arsenite was comparable with the effect caused by the physiological platelet stimulus thrombin. A high osmolarity solution of sorbitol induced comparatively small activation of SAPK2a and MAPKAP-K2. The 42-kDa extracellular signal-regulated kinase (ERK) 2 was not activated by H2O2, sorbitol or arsenite. None of these stimuli triggered significant arachidonic acid release on their own. However, H2O2 and sorbitol enhanced the release of arachidonic acid induced by the calcium ionophore A23187. This effect was reversed by the inhibitor of SAPK2a, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl) imidazole (SB 203580), but not by the inhibitor of the ERK2-activating pathway, 2-(2-amino-3-methoxyphenyl)-oxanaphthalen-4-one (PD 98059). Both H2O2 and sorbitol increased phosphorylation of cytosolic phospholipase A2 (cPLA2) and its intrinsic activity; both responses were blocked by SB 203580. Phosphorylation of cPLA2 by H2O2 occurred on Ser-505, a reaction that is known to increase the intrinsic lipase activity of the enzyme. Our results demonstrate that activation of SAPKs by stress stimuli primes cPLA2 activation through phosphorylation. In vivo, this mechanism would lead to the sensitization of platelet activation and may be an important risk factor in thrombotic disease.

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