manoalogue and 4-bromophenacyl-bromide

Contractile activities of nPLA2, pPLA2 and hPLA2 were characterized on pleural strips of guinea pig lung. The rank order of potency for these PLA2s was nPLA2 > pPLA2 > hPLA2. The concentration-related contractions induced by nPLA2 (0.0002-0.67 micrograms/ml), pPLA2 (0.006-20 micrograms/ml) and hPLA2 (0.1-30 micrograms/ml) appear to be mediated primarily by the formation of cyclooxygenase products and to a lesser extent by 5-lipoxygenase products, as revealed by experiments using indomethacin and BW A4C. To further support a PLA2-related mechanism, the selectivity and inhibitory effects of two irreversible PLA2 inhibitors, parabromophenacyl bromide (pBPB) and manoalogue, were evaluated against the contractile responses induced by each PLA2. Various concentrations of manoalogue and pBPB were incubated with individual PLA2s for 24 hr before initiating experiments. Both agents suppressed each PLA2-induced contractile activity in a concentration-related manner. The inhibitory effects of pBPB were similar at the highest concentration, whereas manoalogue was more effective in blocking contractions induced by pPLA2 and hPLA2. Conversely, methylated manoalogue, an inactive analog, failed to reduce the PLA2-induced contractions. These results demonstrate that hPLA2 has the ability to catalytically induce the release of arachidonic acid and the formation of proinflammatory eicosanoids that contract the pleural strips. This tissue bath preparation may be a useful model for the evaluation of novel PLA2 inhibitors as potentially useful therapeutic agents.

Topics: Acetophenones; Animals; Arachidonic Acid; Elapid Venoms; Guinea Pigs; Humans; In Vitro Techniques; Lung; Male; Methylation; Muscle Contraction; Muscle, Smooth; Pancreas; Phospholipases A; Phospholipases A2; Pleura; Recombinant Proteins; Swine; Terpenes

In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostagl

Topics: Acetophenones; Calcimycin; Cell Line; Dinoprostone; Fatty Acids, Unsaturated; Kinetics; Macrophages; Melitten; Phospholipases; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Substrate Specificity; Terpenes