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.

Inhibition of arachidonic acid release and cytosolic phospholipase A2α activity by d-erythro-sphingosine

2004 ◽  
Vol 484 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Hiroyuki Nakamura ◽  
Testuya Hirabayashi ◽  
Akiyoshi Someya ◽  
Masaya Shimizu ◽  
Toshihiko Murayama
Keyword(s):  
Arachidonic Acid ◽  
Arachidonic Acid Release ◽  
Cytosolic Phospholipase ◽  
Acid Release ◽  
Cytosolic Phospholipase A2α

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.

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.

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