Inhibition of neutrophil activation by p‐bromophenacyl bromide and its effects on phospholipase A₂

Abstract

1 In an effort to elucidate the nature ofthe inhibitory effects ofp-bromophenacyl bromide (pBPB) on neutrophil stimulation, we have examined its effects on several stages of stimulus-response coupling. 2 Pretreatment of rat neutrophils with pBPB resulted in a dose-and time-dependent irreversible inhibition of both N-formylmethionyl-leucylphenylalanine (f Met-Leu-Phe)-induced lysosomal enzyme release and change in transmembrane potential. 3 Inhibition of the biological responses to the chemotactic peptide fMet-Leu-Phe was not due to receptor inactivation since fMet-Leu-[3H]-Phe binding to the formyl peptide receptor was not significantly altered by pBPB pretreatment. 4 Inhibition by pBPB of phorbol myristate acetate (PMA)-induced changes in transmembrane potential and the generation of superoxide (02) was also observed. 5 pBPB treatment appeared to inhibit activation of the NADPH oxidase without a direct effect on the oxidase itself. 6 This inhibitory effect was not accompanied by cell death or decrease in cellular titratable sulphydryl groups (at least at doses <20 pM).7 There was, however, significant inhibition of a membranous fraction of fMet-Leu-Phe-induced phospholipase A2 activity by pretreatment with 10 pM pBPB, although total cellular phospholipase A2 was only minimally (<20% inhibition) affected. 8 These data would indicate that pBPB inhibits an early event associated with stimulus-response coupling in rat polymorphonuclear. leukocytes (i.e. change in transmembrane potential). The inhibitory effects of pBPB may be secondary to the inhibition of a critical membranous fraction of cell bound phospholipase A2 activity or its activation, necessary for the initiation of cell activation.

Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans

“…In contrast, both compounds inhibited PLB of P. notatum [39] and phospholipase A # (PLA # ) from secreted venoms [40]. p-Bromophenacyl bromide, an effective inhibitor of mammalian secretory PLA # and of P. notatum PLB, did not inhibit the activity of cryptococcal phospholipase [41,42]. Most major membrane phospholipids (including DPPC and PG, the most abundant components of lung surfactant) and lysophospholipids were hydrolysed by the pure enzyme, whereas the less accessible, acidic inner-leaflet phospholipids, PS, PI and PA were preferred substrates for PLBs from non-pathogenic fungi [11,12,16].…”

Biochemical and Functional Characterisation of Secreted Phospholipase Activities from Cryptococcus Neoformans in their Naturally Occurring State

“…The PLA2 inhibitor, p-bromophenacyl bromide [20], appeared to increase the LPL/LPTA activities (n = 2) [20]. Mild enhancement (18% and 10%) of PLB activity was noted in the presence of phenylglyoxal or tripolyphosphate (n = 2).…”

“…The tryptophan-modifjring agent, N-bromosuccinimide, and p-bromophenacyl bromide (reportedly an effective inhibitor of mammalian phopholipase A2 [20] and f?…”

“…Strain BL-1 was grown to confluence on SDA for 72 h at 30"C, washed and resuspended in harvesting buffer (10 mM imidazole, 2 M CaC12, 2 mM MgC12, 56mM D-glucose, in NaCl 0.9% w/v, pH 5.5) for incubation (20)(21)(22)(23)(24) h at 37°C). The cell-free supernate was assayed for total protein content and stored at -70°C until required.…”

Human polymorphonuclear neutrophil activation with arachidonic acid

“…PMNs are capable of generating metabolites of arachidonic acid (AA) lipoxygenation such as 5(S), 12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4) (Payan et al, 1984), which stimulates these cells to express various functions (Payan et al, 1984;Smith et al, 1984a). To the extent that lipoxygenase inhibitors suppress neutrophil activation (Naccache et al, 1979;Smolen & Weissmann, 1980;Duque et al, 1986;Smith et al, 1982a;1986a,b), products of AA lipoxygenation have also been implicated in the PMN activation pathway. However, AA itself has been demonstrated to induce PMN aggregation (O'Flaherty et al, 1979) and oxygen radical production (Badwey et al, 1981;.…”