William C. Hope

Influence of dietary vitamin E on prostaglandin biosynthesis in rat blood.

A vitamin E (alpha-tocopherol) deficient diet stimulated prostaglandin biosynthesis in coagulating rat blood. Prostaglandins were extracted from serum, purified and bioassayed. The identity of prostaglandin E2 was confirmed by gas chromatography-mass spectrometry. Withholding vitamin E from the diet caused a marked increase in PGE2 and a lesser increase in PGF2alpha production in serum. In rats maintained on diets containing different concentrations of vitamin E, serum concentrations of PGE2 and PGF2alpha were inversely related to serum concentrations of alpha-tocopherol. These data suggest that in vitro alpha-tocopherol inhibits the endogenous conversion of arachidonic acid into PGE2 and PGF2alpha. The possibility that alpha-tocopherol may inhibit the formation of endoperoxide intermediates of PGE2 and PGF2alpha biosynthesis and subsequent induction of platelet aggregation is discussed.

Cyclic AMP regulation of prostaglandin biosynthesis in fat cells

Abstract A prostaglandin generating system has been demonstrated in rat isolated fat cells using both a sensitive bioassay and gas chromatography combined with mass fragmentography. Accumulation of PGE 2 , but not PGE 1 or PGF 2α was stimulated by addition of norepinephrine or theophylline, agents which cause elevation of intracellular cyclic AMP in fat cells, and by addition of dibutyryl cyclic AMP. Biosynthesis of PG was blocked by indomethacin. Arachidonic acid, the precursor for PGE biosynthesis, was found in high concentrations in fat cell phospholipids. The significance of these findings with reference to a negative feedback regulation of hormone activity is discussed.

Interleukin-1β stimulates cytosolic phospholipase A2 in rheumatoid synovial fibroblasts

Phospholipase A2 (PLA2) activities in rheumatoid synovial fibroblasts (RSF) stimulated with interleukin-1 beta (IL-1 beta) were investigated. RSF incubated in the presence of IL-1 beta (120 pg/ml) for 18 h secreted 35 fold more PGE2 than did those incubated without IL-1 beta. IL-1 beta treatment did not increase the level of secretory PLA2 (sPLA2) activity or sPLA2 protein in the conditioned medium or subcellular fractions of lysed RSF. In contrast, the cell-associated PLA2 activity increased 3 to 4 fold in IL-1 beta stimulated RSF when compared with the control. The IL-1 beta stimulated, cell-associated PLA2 required submicromolar concentrations of calcium for activity, a characteristic consistent with the calcium sensitivity of cytosolic PLA2 (cPLA2) activity reported in other cell types, such as U937 cells. These findings demonstrate that an elevation in a cytosolic PLA2, rather than a sPLA2, is associated with increased PGE2 production in IL-1 beta stimulated RSF.

Retinoids inhibit phospholipase A2 in human synovial fluid and arachidonic acid release from rat peritoneal macrophages

Retinoids have demonstrated antiinflammatory activity in certain animal models and human disease states. The mechanism by which retinoids elicit this activity is unknown. Some retinoids are known to inhibit arachidonic acid (AA) release and metabolism in intact cells in vitro. Retinoids may exert their antiinflammatory effects by inhibiting phospholipase A2 (PLA2) and the resultant production of inflammatory AA metabolites. Retinoids were evaluated in vitro as inhibitors of the PLA2 activity in human synovial fluid (HSF-PLA2). Of the naturally occurring, nonaromatic retinoids tested, all-trans-retinal, all-trans-retinoic acid (all-trans-RA) and 13-cis-RA were the most potent inhibitors (IC50 s 6–15ΜM), whereas all-trans-retinol was much less potent. Of the synthetic aromatic retinoids and arotinoids examined, the free carboxylic, sulfonic, and sulfinic acid forms were more than 15-fold more potent inhibitors of HSF-PLA2 than their corresponding ethyl esters. These retinoids also were evaluated as inhibitors of calcium ionophore A23187-induced AA release from rat peritoneal macrophages. All-trans-RA and 13-cis-RA were potent inhibitors of AA release from these cells (IC50 s 4ΜM), while the other natural retinoids were inactive. Of the aromatic retinoids and arotinoids tested, the free acid forms (IC50 s 2–6ΜM) were 5- to 21-fold more potent inhibitors of AA release from the macrophages than their corresponding ethyl esters. The potencies of the arotinoids as inhibitors of HSF-PLA2 appeared to correlate with their potencies as inhibitors of AA release from A23187-stimulated rat peritoneal macrophages. These data support the hypothesis that one possible mechanism for the known antiinflammatory activity of some retinoids may be by inhibition of phospholipase A2.

Secretory phospholipase A2 inhibitors and calmodulin antagonists as inhibitors of cytosolic phospholipase A2

Human cytosolic phospholipase A2 (cPLA2, 85 kDa) appears to be pharmacologically distinct from human secretory phospholipase A2 (sPLA2, 14 kDa). Marine natural products and PLA2 substrate and product analogs were potent inhibitors of human recombinant sPLA2 (r-sPLA2), whereas these compounds stimulated, weakly inhibited, or had no effect on cPLA2 activity from the human monocytic cell line U937. In contrast, within a series of seven reported calmodulin (CaM) antagonists tested, significant correlations among the rank order of potencies of these compounds as inhibitors of cPLA2, r-sPLA2, and a CaM-dependent phosphodiesterase were observed. The correlated inhibitory effects of the hydrophobic CaM antagonists on cPLA2 and sPLA2 may reflect a common feature (possibly a hydrophobic domain) shared by these two types of enzymes.

Antiflammin-2 (HDMNKVLDL) does not inhibit phospholipase A2 activities

A basic nonapeptide P2 (antiflammin-2, HDMNKVLDL) which is identical to a portion of the amino acid sequence (residues 246–254) of lipocortin I, has been described to have antiinflammatory activity in a rat paw edema model (Nature 335: 726–730 [1988]). P2 (0.05 μM) was also reported to inhibit porcine pancreatic phospholipase A2 (PLA2). The effect of synthetic P2 (98% pure) on PLA2 was evaluated in two assay systems. Using porcine pancreatic PLA2 and phosphatidylcholine/deoxycholate mixed micellar substrate, P2 (0.005–50 μM) had no effect on PLA2 activity, even in the presence of 2-mercaptoethanol to prevent peptide oxidation. In another assay, using human synovial fluid PLA2 as the enzyme and [14C]-oleate-labelledE. coli substrate, P2 (0.005–50 μM) had no significant effect on PLA2 activity. A reported PLA2 inhibitor, manoalide, was a potent inhibitor of PLA2 in both assay systems. On the basis of these results, we conclude that P2 is devoid of PLA2 inhibitory activity.

In vitro studies on the mechanism of action of a new antiallergic, Ro 21-7634.

The effects of Ro 21-7634 and disodium cromoglycate (cromoglycate) on the in vitro release of mediators of anaphylaxis from rat peritoneal cells and guinea pig lung tissue were compared. Ro 21-7634 was 25 fold more potent than cromoglycate as an inhibitor of antigen-induced histamine release from passively sensitized (IgE) rat peritoneal cells. Ro 21-7634 was also the more potent inhibitor of both compound 48/80- and concanavalin A-induced histamine release from rat peritoneal cells. The two drugs shared the common properties of producing the same maximal level of inhibition in each of the above releasing systems and exhibiting a time and concentration dependent loss of inhibitory activity when added to the cells prior to the releasing agent. Neither drug inhibited ionophore A23187- or ionophore X537A-induced histamine release from these cells. Ro 21-7634 inhibited antigen-induced (IgG1) histamine and SRS-A release from actively sensitized guinea pig lung fragments, whereas cromoglycate did not. The results indicate that Ro 21-7634 and cromoglycate act through a common mechanism to inhibit allergic mediator release and that Ro 21-7634 is the more potent inhibitor.

Comparative Study of Natural and Synthetic Retinoids as Inhibitors of Arachidonic Acid Release and Metabolism in Rat Peritoneal Macrophages

The effect of several natural and synthetic retinoids on the release and metabolism of arachidonic acid (20:4) in rat peritoneal macrophages (M phi), stimulated in vitro by either Ca2+ ionophore A23187 (A23187), opsonized zymosan (OZ) or 12-O-tetradecanoylphorbol-13-acetate (TPA), was investigated. With the exception of Ro 10-1670, the retinoids containing a free carboxylic acid group [i.e., all-trans-retinoic acid (all-trans-RA), 13-cis-RA, Ro 13-7652, Ro 12-7310 and Ro 13-7410] inhibited 20:4 metabolite formation in A23187- and OZ-stimulated Mø at 1-33 microM. However, only all-trans-RA, Ro 12-7310 and Ro 13-7410 inhibited the formation of 20:4 metabolites in TPA-stimulated Mø. These data suggest that part of the therapeutic effect of retinoids in inflammatory, hyperproliferative dermatologic conditions might be attributed to reduced 20:4 metabolite production.

Analogs of arachidonic acid methylated at C-7 and C-10 as inhibitors of leukotriene biosynthesis

The syntheses and biological activity of (all Z)-7,7-dimethyl-5,8,11,14- eicosatetraenoic acid, (all Z)-7,7,-dimethyl-5,8,11-eicosatrienoic acid, (Z,Z)-7,7-dimethyl-5,8-eicosadienoic acid, (all Z)-10,10-dimethyl-5,8,11,14-eicosatetraenoic acid, (all Z)-10,10-dimethyl-5,8,11-eicosatrienoic acid, and rac.-(Z,Z)-15-hydroxy-7,7-dimethyl-5,8-eicosadienoic acid are described. These arachidonic acid analogs are all inhibitors of ionophore-induced SRS-A biosynthesis in rat peritoneal cells. Their mode of action may involve inhibition of phospholipase A2 rather than delta 5-lipoxygenase. These compounds failed to exhibit significant activity in an in vivo model designed to detect inhibitors of antigen-induced, leukotriene-mediated bronchoconstriction in sensitized guinea pigs.

Relationship of serum trigkyceride lowering to changes in hepatic composition induced by different classes of drugs

Abstract Dose-related serum trigylceride lowering has been demonstrated in the rat with the microsomal enzyme inducers, phenobarbital, 5,5′-diphenyl-2-thiohydantoin and chlorcyclizine and with the clinically tested anti-hyperlipidemic agents, clofibrate, 1-methyl-4-piperidyl bis(p-chlorophenoxy) acetate (SaH42-348) and 2-methyl-2-[p-(1,2,3,4-tetrahydro-1-naphthyl)-phenxoy] propionic acid (Su-13437). With both classes of drugs, serum lipid lowering was accompanied by hepatomegaly due to an increase in liver protein and phospholipid. Hepatomegaly was reversible upon cessation of drug treatment. The anti-hyperlipidemic drugs caused an increase in microsomal N-demethylase activity but this was slight when compared with the induction produced by known microsomal enzyme inducers. Examination of the subcellular compartments indicated that, even though both mitochondrial protein and phospholipid was increased with all the agents studied, the dominant change in the case of the microsomal enzyme inducers was in the microsomal compartment, whereas the anti-hyperlipidemic agents induced a larger increase in the mitochondrial fraction. The total accumulation of new membrane material, however, was similar for both classes of drugs. These data demonstrate that two classes of drugs which produce hepatomegaly through a proliferation of intracellular membranes also lower serum trigylceride levels.