Phospholipase A2 inhibitors from marine algae

Twelve out of twenty-nine compounds isolated from benthic marine algae from the phyla Chlorophyta, Phaeophyta and Rhodophyta have been found to be potent inhibitors of bee venom derived phospholipase A₂ (PLA₂) (> 50%) in the M range. The compounds investigated were from: Bryopsis pennata, Rhipocephalus phoenix, Caulerpa prolifera, C. racemosa, C. bikinensis, Cymopolia barbata, Laurencia cf. palisada, Laurencia sp., Ochtodes crockeri, Liagora farinosa, Sphaerococcus coronipifolius, Phacelocarpus labillardieri, Dictyota sp., B furcaria galapagensis, Stypopodium zonale, Dictyopteris undulata, Stoechospermum marginatum, Dictyopteris divaricata, Dilophus fasciola and Dilophus sp. This is the first report of bee venom PLA₂ inhibition in vitro by pure compounds isolated from marine algae.     

Phospholipase A2 inhibitors: monoacyl, monoacylamino-glycero-phosphocholines

In this study the relative affinities of natural lecithins and slightly modified lecithin analogues to the active site of porcine pancreatic phospholipase A2 were determined. It was found that the replacement of the phospholipase-fissile fatty acid ester bond in lecithins by an acylamino function results in the formation of potent competitive inhibitors. Substitution of the non-phospholipase-susceptible ester bond by the acylamino linkage does not result in increased affinity of the lecithin analogue to the enzyme. Most probably only the former lecithin analogues partially mimic the structure of the transition state and bind more tightly to the enzyme than the equivalent substrate molecule.     

Antagonists of embryo-derived platelet-activating factor prevent implantation of mouse embryos

Inhibitors of platelet activation, alprazolam, iloprost and SRI 63-441, were used to demonstrate the necessity of embryo-derived platelet-activating factor (PAF) activity for the establishment of pregnancy in mice. In a splenectomized mouse bioassay 6 micrograms alprazolam inhibited, for 3 h, the thrombocytopenia induced by 0.1 micrograms PAF; 4 micrograms iloprost and 0.5 microgram SRI 63-441 were effective for 6 and 12h respectively. The administration of 2 micrograms iloprost/30 g body weight on Days 1 and 4 of pregnancy and twice daily on Days 2 and 3 caused a 50% reduction (P less than 0.0005) in the number of implantation sites in the uterus at Day 8 of pregnancy, without affecting (P greater than 0.05) the number of corpora lutea. A similar reduction in the number of implantation sites was achieved with 20 micrograms SRI 63-441/30 g body weight/day. The reduction in implantation rate was evident on Day 5 of pregnancy by visualizing the implantation sites with pontamine sky blue. SRI 63-441 had no effect on peripheral blood progesterone concentrations from Day 1 to Day 9 of pregnancy, and did not appear to inhibit implantation by blocking the preimplantation surge of oestradiol. The number and morphology of blastocysts flushed from the uterus of Day 4 inhibitor-treated mice was not different (P greater than 0.05) from the controls. The cleavage rate and morphology of embryos cultured from the 2-cell to blastocyst stage in media containing SRI 63-441 or iloprost (10 micrograms/ml) were normal, precluding a gross toxic effect. Simultaneous administration of 1 microgram PAF-acether to treated animals re-established pregnancy rates to levels not significantly different (P greater than 0.05) from the controls.     

Adenosine A 2A receptor antagonists prevent the increase in striatal glutamate levels induced by glutamate uptake inhibitors

Active uptake by neurons and glial cells is the main mechanism for maintaining extracellular glutamate at low, non-toxic concentrations. Activation of adenosine A(2A) receptors increases extracellular glutamate levels, while A(2A) receptor antagonists reduce stimulated glutamate outflow. Whether a modulation of the glutamate uptake system is involved in the effects elicited by A(2A) receptor blockers has never been investigated. This study examined the ability of adenosine A(2A) receptor antagonists to prevent the increase in glutamate levels induced by blockade of the glutamate uptake. In rats implanted with a microdialysis probe in the dorsal striatum, perfusion with 4 mm l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a transportable competitive inhibitor of glutamate uptake), or 10 mm dihydrokainic acid (DHK, a non-transportable competitive inhibitor that mainly blocks the glial glutamate transporter GLT-1), significantly increased extracellular glutamate levels. The effects of PDC and DHK were completely prevented by the adenosine A(2A) receptor antagonists SCH 58261 (0.01 mg/kg i.p.) and/or ZM 241385 (5 nm via probe). Since an impairment in glutamate transporter function is thought to play a major role in neurodegenerative disorders, the regulation of glutamate uptake may be one of the mechanisms of the neuroprotective effects of A(2A) receptor antagonists.     

Radioligand binding of antagonists of platelet-activating factor to intact human platelets

Two new antagonists of platelet-activating factor (PAF), the pyrrolothiazole derivative 52770 RP and the triazolodiazepine WEB 2086, have been studied as radioligands in intact human platelets. [3H]52770 RP and [3H]WEB 2086 bound specifically to high-affinity sites with dissociation constants (Kd) of 14.8 and 6.1 nM, respectively. The maximal number of sites for [3H]52770 RP binding was approx. 15-fold higher than for [3H]PAF and [3H]WEB 2086. In addition, C16-PAF, lyso-PAF, WEB 2086 and 52770 RP had Ki values which were nearly identical for both [3H]PAF and [3H]WEB 2086, whereas only 52770 RP competed for [3H]52770 RP-binding sites. These results demonstrate that in human platelets the sites of [3H]WEB 2086 binding are identical to [3H]PAF-binding sites, whereas those of [3H]52770 RP are not. [3H]WEB 2086 appears, therefore, to be a suitable antagonist radioligand for labelling PAF receptors.     

Calmodulin antagonists inhibit formation of platelet-activating factor in stimulated human neutrophils

Conversion of native, 97-100 kDa rat liver microsomal HMG CoA reductase to membrane-bound 62 kDa and soluble 52-56 kDa catalytically active forms was catalyzed in vitro by the calcium-dependent, leupeptin- and calpastatin-sensitive protease calpain-II purified from rat liver cytosol. Cleavage of the native 97-100 kDa reductase was enhanced by pretreatment (inactivation) of microsomes with ATP(Mg2+) and liver reductase kinase (compared to protein phosphatase-pretreated controls). This was reflected in a loss of the 97-100 kDa species and an increase in the soluble 52-56 kDa species (total enzyme activity and specific immunoblot recovery).     

Inhibition of immune complex-induced enteropathy by three different platelet-activating factor receptor antagonists

We previously showed that intravenous injection of rat anti-BSA-BSA complexes (IC) prepared in 5x antigen excess rapidly induced a striate pattern of serosal (to mucosal) hemorrhage and vascular congestion throughout the small intestine of the Sprague-Dawley rat. In this study, we tested the effect of three different platelet-activating factor (PAF) receptor antagonists on the development of lesions. L-652, 731, a synthetic derivative of kadsurenone (at doses of 1.3–2.7 mg/kg), SRI 63–675, a substituted quinolinium compound (6.7–15 mg/kg), and WEB 2086, a thienotriazolodiazepine (5–25 mg/kg) were each capable of completely or partially inhibiting IC-induced enteropathy in the majority of animals tested. Pretreatment with WEB 2086 prevented IC-induced hemoconcentration but not neutropenia. The antagonists did not lower the level of blood complement nor interfere with the fall in complement induced by administration of IC. The ability of PAF receptor antagonists to completely or partially inhibit IC-induced small intestinal lesions suggests that endogenous PAF is a major mediator of IC-induced enteropathy.     

PrPSc binding antibodies are potent inhibitors of prion replication in cell lines

Conversion of the cellular alpha-helical prion protein (PrP(C)) into a disease-associated isoform (PrP(Sc)) is central to the pathogenesis of prion diseases. Molecules targeting either normal or disease-associated isoforms may be of therapeutic interest, and the antibodies binding PrP(C) have been shown to inhibit prion accumulation in vitro. Here we investigate whether antibodies that additionally target disease-associated isoforms such as PrP(Sc) inhibit prion replication in ovine PrP-inducible scrapie-infected Rov cells. We conclude from these experiments that antibodies exclusively binding PrP(C) were relatively inefficient inhibitors of ScRov cell PrP(Sc) accumulation compared with antibodies that additionally targeted disease-associated PrP isoforms. Although the mechanism by which these monoclonal antibodies inhibit prion replication is unclear, some of the data suggest that antibodies might actively increase PrP(Sc) turnover. Thus antibodies that bind to both normal and disease-associated isoforms represent very promising anti-prion agents.     

Glyceroglycolipids,a novel class of platelet-activating factor antagonists from Kalimeris indica

The bioassay-guided purification of chloroform extracts of Kalimeris indica (Linn) Schultz-Bip led to the isolation of three diacylglycerols: 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoylglycerol (1), 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoyl-3-O-α-(6-sulfoquinovopyranosyl)glycerol (2), 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoyl-3-O-[α-d-galactopyranosyl(1 → 6)-O-β-d-galactopyranosyl]glycerol (3). Their structures were established on the basis of spectral and chemical methods. The glyceroglycolipids 2 and 3 exhibited significant PAF receptor binding inhibitory activities. Our studies have identified diacylglycolipids as a novel class of potent PAF antagonist.     

Regulation of platelet-activating factor (PAF) activity in human diseases by phospholipase A2 inhibitors, PAF acetylhydrolases, PAF receptor antagonists and free radical scavengers.

The aim of this review is to present recent findings indicating the likely involvement of platelet-activating factor (PAF) in human diseases, and possible ways of alleviating its harmful effects. PAF is a potent proinflammatory mediator and promotes adhesive interactions between leukocytes and endothelial cells, leading to transendothelial migration of leukocytes, by a process of juxtacrine intercellular signalling. This process leads to activation of leukocytes and the release of reactive oxygen radicals, lipid mediators, cytokines and enzymes. These reaction products subsequently contribute to the pathological features of various inflammatory diseases. The reactive oxygen radicals cause low density lipoprotein (LDL) oxidation which mediates the development of atherosclerosis. Oxidized LDL may damage cellular and subcellular membranes, leading to tissue injury and cell death. Among the therapeutic approaches considered are agents that inhibit/degrade proinflammatory mediators and thereby have anti-inflammatory and/or anti-atherogenic potential. These include inhibitors of phospholipase A2 activity, PAF-acetylhydrolases, PAF antagonists and free radical scavengers/antioxidants, the latter protecting against oxidized LDL-induced cytotoxicity.     

Phospholipase A2 antagonists inhibit constitutive retrograde membrane traffic to the endoplasmic reticulum

Eukaryotic cells contain a variety of cytoplasmic Ca²⁺-dependent and Ca²⁺-independent phospholipase A₂s (PLA₂s; EC 2.3.1.2.3). However, the physiological roles for many of these ubiquitously-expressed enzymes is unclear or not known. Recently, pharmacological studies have suggested a role for Ca²⁺-independent PLA₂ (iPLA₂) enzymes in governing intracellular membrane trafficking events in general and regulating brefeldin A (BFA)-stimulated membrane tubulation and Golgi-to-endoplasmic reticulum (ER) retrograde membrane trafficking, in particular. Here, we extend these studies to show that membrane-permeant iPLA₂ antagonists potently inhibit the normal, constitutive retrograde membrane trafficking from the trans -Golgi network (TGN), Golgi complex, and the ERGIC-53-positive ER-Golgi-intermediate compartment (ERGIC), which occurs in the absence of BFA. Taken together, these results suggest that iPLA₂ enzymes play a general role in regulating, or directly mediating, multiple mammalian membrane trafficking events.     

Phospholipase A2

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate arachidonic acid (AA), a precursor of eicosanoids including prostaglandins (PGs) and leukotrienes (LTs). The same reaction also produces lysophosholipids, which represent another class of lipid mediators. So far, at least 19 enzymes that possess PLA2 activity have been identified in mammals. The secretory PLA2 (sPLA2) family, in which 10 isozymes have been identified, consists of low-molecular-weight, Ca2+-requiring, secretory enzymes that have been implicated in a number of biological processes, such as modification of eicosanoid generation, inflammation, host defense, and atherosclerosis. The cytosolic PLA2 (cPLA2) family consists of 3 enzymes, among which cPLA2alpha plays an essential role in the initiation of AA metabolism. Intracellular activation of cPLA2alpha is tightly regulated by Ca2+ and phosphorylation. The Ca2+-independent PLA2 (iPLA2) family contains 2 enzymes and may play a major role in membrane phospholipid remodeling. The platelet-activating factor (PAF) acetylhydrolase (PAF-AH) family represents a unique group of PLA2 that contains 4 enzymes exhibiting unusual substrate specificity toward PAF and/or oxidized phospholipids. In this review, we will overview current understanding of the properties and functions of each enzyme belonging to the sPLA2, cPLA2, and iPLA2 families, which have been implicated in signal transduction.     

Inhibition of immune complex-induced enteropathy by three different platelet-activating factor receptor antagonists.

We previously showed that intravenous injection of rat anti-BSA-BSA complexes (IC) prepared in 5x antigen excess rapidly induced a striate pattern of serosal (to mucosal) hemorrhage and vascular congestion throughout the small intestine of the Sprague-Dawley rat. In this study, we tested the effect of three different platelet-activating factor (PAF) receptor antagonists on the development of lesions. L-652,731, a synthetic derivative of kadsurenone (at doses of 1.3-2.7 mg/kg), SRI 63-675, a substituted quinolinium compound (6.7-15 mg/kg), and WEB 2086, a thienotriazolodiazepine (5-25 mg/kg) were each capable of completely or partially inhibiting IC-induced enteropathy in the majority of animals tested. Pretreatment with WEB 2086 prevented IC-induced hemoconcentration but not neutropenia. The antagonists did not lower the level of blood complement nor interfere with the fall in complement induced by administration of IC. The ability of PAF receptor antagonists to completely or partially inhibit IC-induced small intestinal lesions suggests that endogenous PAF is a major mediator of IC-induced enteropathy.     

A superfusion bioassay for platelet-activating factor

A superfusion bioassay for platelet-activating factor is described using various types of tissues. By washing the tissue with 0.1-0.5% bovine serum albumin for 2-3 min after each addition of platelet-activating factor, desensitization did not develop in most tissues studied. Because of the ability to apply a sample directly onto an assay tissue with negligible dilution, this bioassay can detect smaller amounts of platelet-activating factor than those previously reported in which an organ bath was utilized. The ascending colon of the rat and dog appeared to be the most sensitive of the tissues tested, with a limited of detectability in the range of 100-500 fg. Repeated additions of platelet-activating factor could be made for up to 4 h without desensitization. Release of platelet-activating factor from samples of rat stomach was measured using the superfusion bioassay and a platelet aggregation bioassay. There was a significant correlation (r = 0.96; p less than 0.01) between the values obtained using the two assay systems. Thus, the sensitivity, the reproducibility, and the inexpensive nature of this bioassay make it an attractive alternative to existing bioassays for platelet-activating factor.     

Cyclic platelet-activating factor analogues derived from 2-deoxy-D-erythro-pentose

A method for the synthesis of chiral cyclic analogues of platelet-activating factor (PAF) is reported. Treatment of suitably substituted derivatives of 2-deoxy-D-erythro-pentose with phosphorus oxychloride, followed by choline p-toluenesulfonate generates cyclic phospholipids in good yield. Further chemical modification produces other compounds including optically active gamma-butyrolactones such as 2-deoxy-5-O-hexadecyl-3-O-phosphocholyl-D-erythro-pentono-1, 4-lactone and 2-deoxy-3-O-hexadecyl-5-O-phosphocholyl-D-erythro-pentono-1, 4-lactone. All phospholipids were poor antagonists of PAF-induced aggregation of human platelets, and two analogs were poor agonists. The chemistry presented should be useful for the syntheses of other conformationally restricted analogues of PAF.     

Lipoprotein-associated phospholipase A2 (platelet-activating factor acetylhydrolase) and cardiovascular disease

PURPOSE OF REVIEW: Plasma lipoproteins carry a number of highly active enzymes in the circulation. One of these is lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), also known as platelet-activating factor acetylhydrolase. This review addresses the molecular properties of Lp-PLA(2), the controversy surrounding its role in atherosclerosis and the regulation of its plasma levels in humans. RECENT FINDINGS: Recent reports indicate that the enzyme Lp-PLA(2) found in both LDL and HDL may be independently regulated in these lipoprotein subclasses and have distinct roles in atherogenesis. Seminal findings establishing the response-to-retention hypothesis of atherosclerosis support further the potentially damaging role that in-situ release of LDL-associated oxidative products by Lp-PLA(2) may have in the formation of arterial wall lesions. In the mouse, where Lp-PLA(2) circulates mainly bound to HDL, overexpression leads to reduced atherosclerosis, raising the possibility that the enzyme in HDL may have a protective role. Further evidence for a potential protective role is seen in studies of partial or complete deficiency of the enzyme. In the more general setting of population studies, however, it is clear that Lp-PLA(2) is a positive risk factor for coronary disease and measurements of its mass may contribute to the prediction of coronary heart disease risk, especially in individuals with low LDL cholesterol levels. SUMMARY: Lp-PLA(2) is an enzyme with potentially multiple risks in atherosclerosis. In humans the weight of evidence suggests that it is a positive risk factor for coronary heart disease - an observation commensurate with its position in the direct pathological sequence leading from formation of oxidized LDL in the artery wall to cellular dysfunction and formation of lesions.     

Phospholipase A2 in cnidaria

Phospholipase A2 (PLA2) is an enzyme present in snake and other venoms and body fluids. We measured PLA2 catalytic activity in tissue homogenates of 22 species representing the classes Anthozoa, Hydrozoa, Scyphozoa and Cubozoa of the phylum Cnidaria. High PLA2 levels were found in the hydrozoan fire coral Millepora sp. (median 735 U/g protein) and the stony coral Pocillopora damicornis (693 U/g) that cause skin irritation upon contact. High levels of PLA2 activity were also found in the acontia of the sea anemone Adamsia carciniopados (293 U/g). Acontia are long threads containing nematocysts and are used in defense and aggression by the animal. Tentacles of scyphozoan and cubozoan species had high PLA2 activity levels: those of the multitentacled box jellyfish Chironex fleckeri contained 184 U/g PLA2 activity. The functions of cnidarian PLA2 may include roles in the capture and digestion of prey and defense of the animal. The current observations support the idea that cnidarian PLA2 may participate in the sting site irritation and systemic envenomation syndrome resulting from contact with cnidarians.