Phorbol 12-myristate 13-acetate inhibits binding of leukotriene B4 and platelet-activating factor and the responses they induce in neutrophils: site of action.

Abstract

The addition of the platelet-activating factor (PAF) to neutrophils causes an increase in cytoskeletal actin, a rise in the intracellular concentration of free calcium, release of arachidonic acid, and the synthesis of PAF. The PAF synthesis in human neutrophils stimulated by PAF is greatly potentiated by the human granulocyte-macrophage colonystimulating factor. Incubation of human neutrophils with the tumor copromoter phorbol 12-myristate 13-acetate (PMA) for 3 min prior to the addition of the stimulus inhibits all these responses produced by PAF. The inhibition is prevented when the cells are incubated with protein kinase C inhibitors such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine for 5 min prior to the addition of PMA. The rise in the intracellular concentration of free calcium in human neutrophils stimulated with leukotriene B4 is also inhibited by PMA, and this inhibition is prevented by protein kinase C inhibitors such as staurosporine. Unlike PMA, the inactive ester 4a-phorbol 12,13-didecanoate has no inhibitory effect on the stimulated rise in the intracellular concentration of free calcium. The binding of either PAF or leukotriene B4 to intact cells is inhibited by PMA. The most important rinding of the present studies is that PMA interferes with the binding of PAF and leukotriene B4 to their respective receptors. Whether PMA inhibits the binding of these lipid mediators by activating protein kinase C or by perturbing the membrane directly remains to be elucidated.Since its recent discovery, the role of protein kinase C (PKC) in initiating and modulating various cell functions has been under intense examination by several investigators using various cell types, including neutrophils. In neutrophils, PKC has been implicated in superoxide generation and possibly other cell functions (1-5). In addition to diacylglycerol, which is the natural stimulus, the tumor copromoter phorbol 12-myristate 13-acetate (PMA) binds to and activates PKC.Recently, it has been suggested that in several systems, including neutrophils, PKC activation plays both agonist and antagonist roles (6, 7). The wide occurrence of the PKC system in many cell types raises the possibility that one of the major physiological functions of the stimulation of PKC is to protect against cellular overstimulation. With respect to the agonist role of PKC, the site or sites of the excitationresponse sequence which may be affected are not known (7).The present studies were undertaken to examine three questions. First, does PMA inhibit human neutrophil responses produced by lipid mediators such as leukotriene B4 (LTB4) and platelet-activating factor (PAF)? Second, is the inhibition, if present, mediated by PKC activation? Third, what site in the excitation-response sequence is affected by PMA?MATERIALS AND METHODS Preparation of Cells. Human neutrophils were isolated from peripheral blood on Ficoll/Hypaque gradients as described by English and Andersen (8). Rabbit neutrophils (4-hr exudate) were collected and washed as described previou...

Protein Kinase C Inhibition Enhances Platelet-activating Factor-induced Eicosanoid Production in Human Eosinophils

“…Prior reports have implicated PKC in suppression of receptor-mediated cell activation. Responses to PAF in rat Kupffer cells (22), human platelets (23) and neutrophils (24,25), as well as guinea pig eosinophils (5) are downregulated by treatment of the cells with the PKC activator, PMA. Formyl peptide (fMLP)-induced arachidonic acid mobilization in human neutrophils is enhanced in the pres-ence of PKC inhibitors, which also increase the fMLPstimulated generation of LTB 4 and PAF while suppressing the PMA-induced production of O 2…”

Regulation of platelet-activating-factor receptors and the desensitization response in polymorphonuclear neutrophils

“…PAF induces PMN to mobilize PKC [23,24,[31][32][33]; PKC blockers inhibit PMN responses to PAF [23]; and PKC activators, e.g. PMA, share with PAF the ability to stimulate PMN to produce 02-, degranulate, and, of particular interest here, down-regulate high-affinity PAF receptors [33,34]. These data suggest that PKC may mediate not only the stimulating, but also the receptor-down-regulating, actions of PAF.…”

Platelet-activating factor: receptors and signal transduction

“…On the other hand, phorbol ester was found to suppress PAF-mediated signal transduction through modification of the GTP-binding proteins, as phorbol ester was found to abolish PAF-stimulated GTPase activity [51]. This observation suggests that protein kinase C selectively inhibits PAF effects by inactivating a GTP-binding protein coupled with PAF receptors, although activation of protein kinase C also down-regulates surface PAF receptors [57][58][59].…”

“…In neutrophils [57,58] and rat Kupffer cells [59] the PAF receptor is modulated by protein kinase C. In neutrophils, specific [3H]PAF receptors were modulated by the protein kinase C activator phorbol ester [57,58], as well as by LTB4 [58]. The regulatory effect of protein kinase C on specific PAF receptors was bidirectional, increasing specific [3H]PAF binding at low concentrations and decreasing binding at high concentrations.…”