Structural determinants of haloenol lactone-mediated suicide inhibition of canine myocardial calcium-independent phospholipase A2

Abstract

Haloenol lactones are potent mechanism-based inhibitors of a novel class of calcium-independent phospholipases A2 which have been implicated as the enzymic mediators of membrane dysfunction during myocardial ischemia (Hazen, S. L.; et al. J. Biol. Chem. 1991, 266, 7227-7232). Herein we demonstrate that the ring size, hydrophobic group, and cryptic electrophile in the haloenol lactone moiety are important and modifiable determinants of the inhibitory potency of haloenol lactone-mediated inhibition of calcium-independent phospholipase A2. Direct comparisons between haloenol lactone-mediated inhibition of calcium-independent phospholipase A2 and the absence of inhibition with calcium-dependent phospholipase A2 further underscore the marked differences in the catalytic strategy employed by these two classes of intracellular phospholipases A2.

Phospholipase A2-catalyzed hydrolysis of plasmalogen phospholipids in thrombin-stimulated human platelets

“…To determine whether the accelerated membrane phospholipid hydrolysis was mediated via iPLA 2 activity, platelets were incubated with1 μM BEL (a selective inhibitor of iPLA 2 ) [18] for 30 min prior to thrombin stimulation. Pretreatment with BEL significantly decreased basal PLA 2 activity measured in the cytosol using both plasmenylcholine and phosphatidylcholine substrates (Figure 1, dotted lines).…”

“…Although PLA 2 activity has been observed previously in platelets and there is evidence that PLA 2 -catalyzed plasmalogen hydrolysis occurs in response to thrombin [10,11], it was surprising to observe that platelet PLA 2 activity and arachidonic acid release was inhibited completely by BEL pretreatment since BEL does not significantly inhibit cPLA 2 [18]. Previous studies that have studied cPLA 2 activation in thrombin stimulated platelets have commonly used concentrations of thrombin at 1 to 2 IU/ml [3,24,28].…”

Long‐term potentiation requires activation of calcium‐independent phospholipase A2

“…IA) and over 95% of hippocampal phospholipase A2 activity was inhibited by the mechanismbased inhibitor BEL (10/,tM) which possesses over a 1000-fold selectivity for the inhibition of calcium-independent phospholipase A2 in comparison to calcium-dependent phospholipases A2 [18,19]. Remarkably, rat hippocampus was 7-fold enriched in calcium-independent phospholipase A 2 activity compared to phospholipase A 2 activity manifest in homogenates of whole brain (Fig.…”

“…Radiolabeled l-O-(Z)-hexadec-l'-enyl-2-[9,10-3H] octadec-9'-enoylsn-glycero-3-phosphocholine and the calcium-independent phospholipase A 2 inhibitor, BEL, were synthesized by established methods [18][19][20]. Recombinant 85 kDa cPLA2 (GenBank Accession #M68874) was purified from baculovirus infected Sf9 insect cells by FPLC Mono-Q chromatography.…”

Identification of Calcium-independent Phospholipase A2 (iPLA2) β, and Not iPLA2γ, as the Mediator of Arginine Vasopressin-induced Arachidonic Acid Release in A-10 Smooth Muscle Cells

“…Because rac-BEL inhibits both iPLA 2 ␤ and iPLA 2 ␥ at low micromolar concentrations (24,25,31,32), it is impossible to assign rac-BEL-mediated inhibition of AA release to iPLA 2 ␤ or iPLA 2 ␥ activities. Accordingly, it became necessary to develop pharmacologic approaches that could discriminate between iPLA 2 ␤ and iPLA 2 ␥ to facilitate identification of their biologic roles.…”