Effects of pyrrophenone, an inhibitor of group IVA phospholipase A₂, on eicosanoid and PAF biosynthesis in human neutrophils

Abstract

Background and Purpose: The biosynthesis of leukotrienes (LT) and platelet-activating factor (PAF) involves the release of their respective precursors, arachidonic acid (AA) and lyso-PAF by the group IVA PLA 2 (cPLA 2 a). This paper aims at characterizing the inhibitory properties of the cPLA 2 a inhibitor pyrrophenone on eicosanoids and PAF in human neutrophils (PMN). Experimental Approach: Freshly isolated human PMN were activated with physiological and pharmacological agents (fMLP, PAF, exogenous AA, A23187 and thapsigargin) in presence and absence of the cPLA 2 a inhibitor pyrrophenone and biosynthesis of LT, PAF, and PGE 2 was measured. Key Results: Pyrrophenone potently inhibited LT, PGE 2 and PAF biosynthesis in PMN with IC 50 s in the range of 1-20 nM. These inhibitory effects of pyrrophenone were specific (the consequence of substrate deprivation), as shown by the reversal of inhibition by exogenous AA and lyso-PAF. Comparative assessment of pyrrophenone, methyl-arachidonoyl-fluorophosphonate (MAFP) and arachidonoyl-trifluoromethylketone (AACOCF 3 ) demonstrated that pyrrophenone was more specific and 100-fold more potent than MAFP and AACOCF 3 for the inhibition of LT biosynthesis in A23187-activated PMN. The inhibitory effect of pyrrophenone on LT biosynthesis was reversible as LT biosynthesis was recovered when pyrrophenonetreated PMN were washed with autologous plasma. No alteration of phospholipase D (PLD) activity in fMLP-activated PMN was observed with up to 10 mM pyrrophenone, suggesting that the cPLA 2 a inhibitor does not directly inhibit PLD. Conclusions and Implications: Pyrrophenone is a more potent and specific cPLA 2 a inhibitor than MAFP and AACOCF 3 and represents an excellent pharmacological tool to investigate the biosynthesis and the biological roles of eicosanoids and PAF.

The Endocannabinoid 2-Arachidonoyl-Glycerol Activates Human Neutrophils: Critical Role of Its Hydrolysis and De Novo Leukotriene B4 Biosynthesis

“…5), indicating that NAM and URB-602 were also inhibiting an enzyme involved in LT biosynthesis. Importantly, the effect of 100 nM MAFP on A23187-induced LT biosynthesis was minimal and in line with our previous observations, showing its inhibitory effect on cytosolic phospholipase A 2 a (cPLA 2 a) occurred in the low micromolar range (45).…”

Phospholipase A2 Enzymes: Physical Structure, Biological Function, Disease Implication, Chemical Inhibition, and Therapeutic Intervention

“…Pyrrophenone potently inhibited LT, PGE 2 and PAF biosynthesis in human neutrophils with IC 50 s in the range of 1–20 nM. 297 A structurally related inhibitor, pyrroxyphene ( 57 ), displayed anti-arthritic and anti-bone destructive action in a murine arthritis model. 298 …”

Osteoblast retraction induced by adherent neutrophils promotes osteoclast bone resorption: implication for altered bone remodeling in chronic gout

“…After 48 h of coincubation and two vigorous washes, cells were analyzed by microscopy. OBs or neutrophils were preincubated 30 min with 5 mg/ml blocking anti-CD49d Ab or control rat IgG2b, 10 mg/ml of anti-VCAM-1 or anti-MadCAM-1 mAbs or control mouse IgG1, 150 mg/ml of CS1 peptide, or 5 min with vehicle or 300 nM pyrrophenone (cPLA 2a inhibitor), 20 1 mM BN52021 (PAF receptor antagonist), 1 mM CP105696 (LTB 4 receptor antagonist), 500 nM NS-398 (COX-2 inhibitor), 1 mM indomethacin (COX-1/2 inhibitor), 5 mM SB203580 (p38 inhibitor) or 8 mM PD98069 (ERK inhibitor). All inhibitors and antagonists were used at concentrations optimal for their specific effects in incubation medium containing 10% FBS, and no toxicity (PI exclusion test) on cells was observed as assessed by cytofluorometry.…”