Phosphatidic acid and diacylglycerol directly activate NADPH oxidase by interacting with enzyme components.

The enzyme NADPH oxidase is regulated by phospholipase D in intact neutrophils and is activated by phosphatidic acid (PA) plus diacylglycerol (DG) in cell-free systems. We showed previously that cell-free NADPH oxidase activation by these lipids involves both protein kinase-dependent and -independent pathways. Here we demonstrate that only the protein kinase-independent pathway is operative in a cell-free system of purified and recombinant NADPH oxidase components. Activation by PA + DG was ATP-independent and unaffected by the protein kinase inhibitor staurosporine, indicating the lack of protein kinase involvement. Both PA and DG were required for optimal activation to occur. The drug reduced activation of NADPH oxidase by either arachidonic acid or PA + DG, with IC(50) values of 46 and 25 microm, respectively. The optimal concentration of arachidonic acid or PA + DG for oxidase activation was shifted to the right with, indicating interference of the drug with the interaction of lipid activators and enzyme components. inhibited the lipid-induced aggregation/sedimentation of oxidase components p47(phox) and p67(phox), suggesting a disruption of the lipid-mediated assembly process. The direct effects of on NADPH oxidase activation complicate its use as a "specific" inhibitor of DG kinase. We conclude that the protein kinase-independent pathway of NADPH oxidase activation by PA and DG involves direct interaction with NADPH oxidase components. Thus, NADPH oxidase proteins are functional targets for these lipid messengers in the neutrophil.

Phosphatidic acid regulates tyrosine phosphorylating activity in human neutrophils: enhancement of Fgr activity.

In human neutrophils, the activation of phospholipase D and the Tyr phosphorylation of proteins are early signaling events upon cell stimulation. We found that the pretreatment of neutrophils with ethanol (0.8%) or 1-butanol (0.3%), which results in the accumulation of phosphatidylalcohol at the expense of phosphatidic acid (PA), decreased the phorbol myristate acetate-stimulated Tyr phosphorylation of endogenous proteins (42, 115 kDa). When neutrophil cytosol was incubated in the presence or absence of PA, these and other endogenous proteins became Tyr-phosphorylated in a PA-dependent manner. In contrast, phosphatidylalcohols exhibited only 25% (phosphatidylethanol) or 5% (phosphatidylbutanol) of the ability of PA to stimulate Tyr phosphorylation in the cell-free assay. Similarly, other phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, polyphosphoinositides, and sphingosine 1-phosphate) showed little ability to stimulate Tyr phosphorylation. These data suggest that PA can function as an intracellular regulator of Tyr phosphorylating activity. Gel filtration chromatography of leukocyte cytosol revealed a peak of PA-dependent Tyr phosphorylating activity distinct from a previously described PA-dependent phosphorylating activity (Waite, K. A., Wallin, R., Qualliotine-Mann, D., and McPhail, L. C. (1997) J. Biol. Chem. 272, 15569-15578). Among the protein Tyr kinases expressed in neutrophils, only Fgr eluted exclusively in the peak of PA-dependent Tyr phosphorylating activity. Importantly, Fgr isolated from unstimulated neutrophil lysates showed increased activity in the presence of PA but not phosphatidylbutanol. Moreover, the pretreatment of neutrophils with 1-butanol decreased Fgr activity in cells stimulated with formyl-methionyl-leucyl phenylalanine plus dihydrocytochalasin B. Together, these results suggest a new second messenger role for PA in the regulation of Tyr phosphorylation.

Phosphorylation of p22phox is mediated by phospholipase D-dependent and -independent mechanisms. Correlation of NADPH oxidase activity and p22phox phosphorylation.

Human neutrophils participate in the host innate immune response, partly mediated by the multicomponent superoxide-generating enzyme NADPH oxidase. A correlation between phosphorylation of cytosolic NADPH oxidase components and enzyme activation has been identified but is not well understood. We previously showed that p22(phox), the small subunit of the membrane-bound oxidase component flavocytochrome b(558), is an in vitro substrate for both a phosphatidic acid-activated kinase and conventional protein kinase C isoforms (Regier, D. S., Waite, K. A., Wallin, R., and McPhail, L. C. (1999) J. Biol. Chem. 274, 36601-36608). Here we show that several neutrophil agonists (phorbol myristate acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine) induce p22(phox) phosphorylation in intact neutrophils. To determine if phospholipase D (PLD) is needed for p22(phox) phosphorylation, cells were pretreated with ethanol, which reduces phosphatidic acid production by PLD in stimulated cells. Phorbol myristate acetate-induced phosphorylation of p22(phox) and NADPH oxidase activity were not reduced by ethanol. In contrast, ethanol reduced both activities when cells were stimulated by N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan. Varying the time of stimulation with opsonized zymosan showed that the phosphorylation of p22(phox) coincides with NADPH oxidase activation. GF109203X, an inhibitor of protein kinase C and the phosphatidic acid-activated protein kinase, decreased both p22(phox) phosphorylation and NADPH oxidase activity in parallel in opsonized zymosan-stimulated cells. Stimulus-induced phosphorylation of p22(phox) was on Thr residue(s), in agreement with in vitro results. Overall, these data show that NADPH oxidase activity and p22(phox) phosphorylation are correlated and suggest two mechanisms (PLD-dependent and -independent) by which p22(phox) phosphorylation occurs.

Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase.

The neutrophil NADPH-oxidase can be activated by protein kinase C (PKC) agonists such as phorbol myristate acetate (PMA), resulting in superoxide anion release. This superoxide release is independent of phosphatidylinositol 3-kinase (PI 3-kinase) because the inhibitor wortmannin does not affect the response. In this study, PMA is shown to also induce a wortmannin-sensitive NADPH-oxidase activation, however, not resulting in release of superoxide but in intracellular production of the radical. This indicates that two pools of NADPH-oxidase, one localized in the plasma membrane and the other in the granule membranes, are separately regulated and the signal transduction pathways leading to activation of these pools differ regarding involvement of PI 3-kinase. Activation of both pools was dependent on ERK/MAPK kinase (MEK) activity and protein phosphatase 1 and/or 2A. As the two oxidase responses were differently affected by the inhibitor Gö-6850, different PKC isozymes are suggested to take part in the two signal transduction pathways.

A phosphatidic acid-activated protein kinase and conventional protein kinase C isoforms phosphorylate p22(phox), an NADPH oxidase component.

Using a phosphorylation-dependent cell-free system to study NADPH oxidase activation (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931-7935), we previously showed that p47(phox), a cytosolic NADPH oxidase component, is phosphorylated. Now, we show that p22(phox), a subunit of the NADPH oxidase component flavocytochrome b(558), also is phosphorylated. Phosphorylation is selectively activated by phosphatidic acid (PA) versus other lipids and occurs on a threonine residue in p22(phox). We identified two protein kinase families capable of phosphorylating p22(phox): 1) a potentially novel, partially purified PA-activated protein kinase(s) known to phosphorylate p47(phox) and postulated to mediate the phosphorylation-dependent activation of NADPH oxidase by PA and 2) conventional, but not novel or atypical, isoforms of protein kinase C (PKC). In contrast, all classes of PKC isoforms could phosphorylate p47(phox). In a gel retardation assay both the phosphatidic acid-dependent kinase and conventional PKC isoforms phosphorylated all molecules of p22(phox). These findings suggest that phosphorylation of p22(phox) by conventional PKC and/or a novel PA-activated protein kinase regulates the activation/assembly of NADPH oxidase.

Protein kinase C (PKC) isoforms translocate to Triton-insoluble fractions in stimulated human neutrophils: correlation of conventional PKC with activation of NADPH oxidase.

The responses of human neutrophils (PMN) involve reorganization and phosphorylation of cytoskeletal components. We investigated the translocation of protein kinase C (PKC) isoforms to PMN cytoskeletal (Triton-insoluble) fractions, in conjunction with activation of the respiratory burst enzyme NADPH oxidase. In resting PMN, PKC-delta (29%) and small amounts of PKC-alpha (0.6%), but not PKC-betaII, were present in cytoskeletal fractions. Upon stimulation with the PKC agonist PMA, the levels of PKC-alpha, PKC-betaII, and PKC-delta increased in the cytoskeletal fraction, concomitant with a decrease in the noncytoskeletal (Triton-soluble) fractions. PKC-delta maximally associated with cytoskeletal fractions at 160 nM PMA and then declined, while PKC-alpha and PKC-betaII plateaued at 300 nM PMA. Translocation of PKC-delta was maximal by 2 min and sustained for at least 10 min. Translocation of PKC-alpha and PKC-betaII was biphasic, plateauing at 2-3 min and then increasing up to 10 min. Under maximal stimulation conditions, PKC isoforms were entirely cytoskeletal associated. Translocation of the NADPH oxidase component p47phox to the cytoskeletal fraction correlated with translocation of PKC-alpha and PKC-betaII, but not with translocation of PKC-delta. Oxidase activity in cytoskeletal fractions paralleled translocation of PKC-alpha, PKC-betaII, and p47phox. Stimulation with 1,2-dioctanoylglycerol resulted in little translocation of PKC isoforms or p47phox, and in minimal oxidase activity. We conclude that conventional PKC isoforms (PKC-alpha and/or PKC-betaII) may regulate PMA-stimulated cytoskeletal association and activation of NADPH oxidase. PKC-delta may modulate other PMN responses that involve cytoskeletal components.

A novel protein kinase target for the lipid second messenger phosphatidic acid.

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.

Opsonized zymosan stimulates the redistribution of protein kinase C isoforms in human neutrophils.

We examined the ability of opsonized zymosan (OPZ) to stimulate translocation of protein kinase C (PKC) isoforms in human neutrophils. Neutrophils express five PKC isoforms (alpha, betaI, betaII, delta, and zeta), but little is known of their individual roles in neutrophil activation. As determined by immunoblotting, OPZ caused a time-dependent translocation of the predominant PKC isoforms (betaII, delta, and zeta) to neutrophil membranes, with a concomitant loss from the cytosol. Maximal translocation of all three isoforms occurred by 3 min. No PKC immunoreactivity was observed in a crude nuclear fraction, but PKC-delta and -zeta were found in the granule fraction after degranulation (10 min). PKC activity (Ca2+-dependent and -independent) increased 50- and 19-fold, respectively, by 10 min in the granules from OPZ-stimulated cells. Curiously, no immunoreactive cPKC (alpha and beta(I/II)) could be localized in the granule fraction to account for the Ca2+-dependent PKC activity. Localization of PKC isoforms in the neutrophil membranes and granules suggests their involvement in the regulation of functional responses triggered by OPZ. PKC isoform translocation to membranes from OPZ-stimulated cells preceded both p47phox (a cytosolic component of the NADPH oxidase) translocation and NADPH oxidase assembly. The presence of both PKC isoforms and p47phox in the membrane was transient, with the loss of p47phox occurring sooner than either the loss of membrane-associated PKC or that of NADPH oxidase activity. The apparent EC50 values for PKC translocation and NADPH oxidase assembly were similar. These data suggest that PKC isoforms regulate the assembly and activation of NADPH oxidase induced by OPZ.

Phosphatidic acid-mediated phosphorylation of the NADPH oxidase component p47-phox. Evidence that phosphatidic acid may activate a novel protein kinase.

Phosphatidic acid (PA), generated by phospholipase D activation, has been linked to the activation of the neutrophil respiratory burst enzyme, NADPH oxidase; however, the intracellular enzyme targets for PA remain unclear. We have recently shown (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931-7935) that a PA-activated protein kinase is involved in the activation of NADPH oxidase in a cell-free system. This protein kinase phosphorylates numerous endogenous proteins, including p47-phox, a component of the NADPH oxidase complex. Phospholipids other than PA were less effective at inducing endogenous protein phosphorylation. Several of these endogenous substrates were also phosphorylated during stimulation of intact cells by opsonized zymosan, an agonist that induces phospholipase D activation. We sought to identify the PA-activated protein kinase that phosphorylates p47-phox. The PA-dependent protein kinase was shown to be cytosolic. cis-Unsaturated fatty acids were poor inducers of protein kinase activity, suggesting that the PA-activated protein kinase is not a fatty acid-regulated protein kinase (e.g. protein kinase N). Chromatographic techniques separated the PA-activated protein kinase from a number of other protein kinases known to be activated by PA or to phosphorylate p47-phox. These included isoforms of protein kinase C, p21 (Cdc42/Rac)-activated protein kinase, and mitogen-activated protein kinase. Gel filtration chromatography indicated that the protein kinase has an apparent molecular size of 125 kDa. Screening of cytosolic fractions from several cell types and rat brain suggested the enzyme has widespread cell and tissue distribution. The partially purified protein kinase was sensitive to the same protein kinase inhibitors that diminished NADPH oxidase activation and was independent of guanosine 5'-3-O-(thio)triphosphate and Ca2+. Phosphoamino acid analysis showed that serine and tyrosine residues were phosphorylated on p47-phox by this kinase(s). These data indicate that one or more potentially novel protein kinases are targets for PA in neutrophils and other cell types. Furthermore, a PA-activated protein kinase is likely to be an important regulator of the neutrophil respiratory burst by phosphorylation of the NADPH oxidase component p47-phox.

Identification and regulation of protein kinase C-delta in human neutrophils.

The intracellular mechanisms that regulate the function of human neutrophils are not well understood. Receptor-initiated signaling events result in the production of several second messengers (e.g., Ca2+, diacylglycerol, phosphatidic acid, and arachidonic acid) with the potential to activate members of the protein kinase C (PKC) family of signaling enzymes. The mixture of second messenger signaling molecules produced usually varies, depending on the particular receptor engaged. Previous work suggests that PKC has complex regulatory effects on neutrophil function. This may be due to the presence of multiple isoforms of the enzyme family, responding differentially to the second messengers produced. In studies to identify the PKC isoforms present in human neutrophils, we discovered the presence of the PKC isoform delta in these cells. Like other previously identified isoforms (alpha, beta I, beta II, and zeta), delta is a cytosolic enzyme in unstimulated neutrophils and partially translocates to membrane-containing fractions in cells stimulated by either the PKC activator PMA or the chemoattractant FMLP. Partial purification of cytosolic PKC gave two peaks of activity. The beta isoforms predominated in peak I, while the delta isoform predominated in peak II. The identification of delta indicates that neutrophils contain at least one member of the Ca(2+)-independent, diacylglycerol-dependent subfamily of PKC isoforms. Thus, this isoform may participate in Ca(2+)-independent, but diacylglycerol-dependent signaling events in these cells.

Cell-free activation of neutrophil NADPH oxidase by a phosphatidic acid-regulated protein kinase.

The phosphorylation-dependent mechanisms regulating activation of the human neutrophil respiratory-burst enzyme, NADPH oxidase, have not been elucidated. We have shown that phosphatidic acid (PA) and diacylglycerol (DG), products of phospholipase activation, synergize to activate NADPH oxidase in a cell-free system. We now report that activation by PA plus DG involves protein kinase activity, unlike other cell-free system activators. NADPH oxidase activation by PA plus DG is reduced approximately 70% by several protein kinase inhibitors [1-(5-isoquinolinesulfonyl)piperazine, staurosporine, GF-109203X]. Similarly, depletion of ATP by dialysis reduces PA plus DG-mediated NADPH oxidase activation by approximately 70%. Addition of ATP, but not a nonhydrolyzable ATP analog, to the dialyzed system restores activation levels to normal. In contrast, these treatments have little effect on NADPH oxidase activation by arachidonic acid or SDS plus DG. PA plus DG induces the phosphorylation of a number of endogenous proteins. Phosphorylation is largely mediated by PA, not DG. A predominant substrate is p47-phox, a phosphoprotein component of NADPH oxidase. Phosphorylation of p47-phox precedes activation of NADPH oxidase and is markedly reduced by the protein kinase inhibitors. In contrast, arachidonic acid alone or SDS plus DG is a poor activator of protein phosphorylation in the cell-free system. Thus, PA induces activation of one or more protein kinases that regulate NADPH oxidase activation in a cell-free system. This cell-free system will be useful for identifying a functionally important PA-activated protein kinase(s) and for dissecting the phosphorylation-dependent mechanisms responsible for NADPH oxidase activation.

Activation of phospholipase D by interleukin-8 in human neutrophils.

Interleukin 8 (IL-8), a member of the C-X-C branch of the chemokine superfamily, stimulated the breakdown of 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine ([3H]EAPC) and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid ([3H]-EAPA) in human polymorphonuclear leukocytes (PMN) in the presence of cytochalasin B. In addition, the mass of diradyl-PA was increased with similar kinetics. In the presence of ethanol, 1-O-[3H]alkyl-2-acyl-phosphatidylethanol ([3H]EAPEt) was formed at the expense of [3H]EAPA formation, indicating the activation of phospholipase D by the cytokine. The effect was time- and concentration-dependent, reaching a plateau at 30 seconds with the maximally activating concentration of 120 nmol/L IL-8. Preincubation of cells with 1 microgram/mL Bordetella pertussis toxin inhibited the breakdown of [3H]EAPC and [3H]EAPA formation, indicating a role for a pertussis toxin-sensitive guanosine triphosphate-binding protein. Formation of phosphatidic acid (PA) correlated with activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the oxidative burst enzyme, with both events occurring in the same concentration range. Inhibition of PA formation, by the presence of ethanol, also inhibited the oxidative burst stimulation by IL-8. Pretreatment of PMN with 10 nmol/L platelet-activating factor potentiated both [3H]EAPA accumulation and activation of NADPD oxidase by IL-8. Collectively, these data show that IL-8 stimulates the metabolism of choline-containing phosphoglycerides in human PMN and support a role for PA in the signaling mechanisms used by IL-8 to stimulate PMN function.

Phosphatidic acid and diacylglycerol synergize in a cell-free system for activation of NADPH oxidase from human neutrophils.

NADPH oxidase, the respiratory burst enzyme of human neutrophils, is a multi-component complex that is assembled and activated during stimulation of the cells by inflammatory or phagocytic stimuli. The signal mechanisms leading to activation of the enzyme are unclear, but it is likely that phospholipases are involved. Recent work has shown that phosphatidic acid, the initial product of phospholipase D activation, is a weak activator of NADPH oxidase in a cell-free system. We now show that diacylglycerol enhances the cell-free activation of NADPH oxidase activation by phosphatidic acid. 1,2-Didecanoyl phosphatidic acid (10:0-PA) and 1,2-dioctanoylglycerol (8:0-DG) each increased levels of NADPH oxidase activity in mixtures of membrane and cytosolic fractions about 2-fold. The combination of both lipids increased NADPH oxidase activity approximately 12-fold, indicative of a synergistic response. Fatty acid and neutral lipid metabolites of 10:0-PA or 8:0-DG were ineffective, suggesting activation is directly mediated by phosphatidic acid and diacylglycerol. Activation was time- and concentration-dependent with maximum activation at 30-60 min and a sharp peak of maximal activity at 10 microM 10:0-PA and 30 microM 8:0-DG. In lipid specificity studies, activity of PA or DG decreased with increasing acyl chain length but was restored by introducing unsaturation in the acyl chain. Natural forms of PA stimulated levels of activity comparable to that seen with 10:0-PA. Synthetic and natural phosphatidylserines, but not other phospholipids, could replace phosphatidic acid in the synergistic response. These studies provide direct evidence for a synergistic interaction between phosphatidic acid and diacylglycerol in mediating a cellular function: the assembly and activation of NADPH oxidase. Our results support the concept that the generation of second messenger lipids by phospholipase D is a key step in activation of the respiratory burst enzyme.

Phospholipases and activation of the NADPH oxidase.

The signal transductional mechanisms regulating the activation of NADPH oxidase, the respiratory burst enzyme in phagocytic cells, are not completely understood. Receptors for most physiologic stimuli trigger the activation of various phospholipases, including phospholipases A2, C, and D. The lipid mediators formed (arachidonic acid, 1,2-diacylglycerol, and phosphatidic acid) have been implicated as second messengers in the induction of the respiratory burst. In intact cells, we have correlated phospholipase D activation and the production of phosphatidic acid with the activation of NADPH oxidase, using the drug propranolol. Phosphatidic acid activated NADPH oxidase in a cell-free system, but the level of activation was low. 1,2-Diacylglycerol markedly enhanced NADPH oxidase activation by phosphatidic acid. The synergistic effect required the diacyl species, since mono- or tri-acylglycerols were ineffective. Phosphatidic acid could be replaced by either lysophosphatidic acid or phosphatidylserine, but not by phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol, suggesting specificity for an anionic phospholipid. Since other cell-free activators of NADPH oxidase (arachidonic acid, sodium dodecyl sulfate) are also anionic amphiphiles, phosphatidic acid may directly interact with an enzyme component(s). The targets for phosphatidic acid and diacylglycerol in the cell-free system are currently under investigation. These results emphasize the critical importance of phospholipases, particularly phospholipase D, in the regulation of the respiratory burst.

Propranolol, a phosphatidate phosphohydrolase inhibitor, also inhibits protein kinase C.

Propranolol, a beta-adrenergic receptor antagonist, also inhibits phosphatidate phosphohydrolase, the enzyme that converts phosphatidic acid into diacylglycerol. This latter effect has prompted recent use of propranolol in studies examining the importance of diacylglycerol and phosphatidic acid in cellular signalling events. Here, we show that propranolol is also an inhibitor of protein kinase C. At concentrations greater than or equal to 20 microM, propranolol reduced [3H]phorbol dibutyrate binding (IC50 = 200 microM) and phorbol myristate acetate-stimulated superoxide anion release (IC50 = 130 microM) in human neutrophils. Scatchard analysis showed that propranolol lowers the number of phorbol diester binding sites without significantly affecting their affinity. In vitro kinetic analysis, performed in a mixed micellar assay with protein kinase C purified from human neutrophils, suggested a competitive inhibition of propranolol with the cofactor phosphatidylserine. Complex kinetic patterns were observed with respect to diacylglycerol and ATP, approximating competitive and noncompetitive inhibition, respectively. Taken together, these results suggest that the drug interacts at the level of the regulatory domain of the enzyme. Fifty % inhibition occurred at approximately 150 microM propranolol. Similar levels of inhibition were obtained using exogenous (histone) and endogenous (p47-phox, a NADPH oxidase component) substrates. Protein kinase C-alpha and protein kinase C-beta, two protein kinase C isozymes present in human neutrophils, were inhibited by propranolol in a comparable manner. In the range of concentrations tested (30-1000 microM), neither cAMP-dependent protein kinase nor neutrophil protein tyrosine kinases were affected. The racemic form of propranolol and the (+) and the (-) stereoisomers were equally active, and other beta-adrenergic receptor antagonists (pindolol) and agonists (isoproterenol) were inactive. This suggests that the inhibitory action of propranolol on protein kinase C is related to the amphipathic nature of the drug rather than to its beta-adrenergic receptor blocking ability. Analogs of propranolol were synthesized and found to be more potent protein kinase C inhibitors, with IC50 values in the 10-20 microM range. We conclude that the ability of propranolol to inhibit both protein kinase C and PA phosphohydrolase complicates interpretation of results when this drug is used in signal transduction studies. In addition, propranolol may be a useful prototype for the synthesis of new protein kinase C inhibitors.

Comparison of diglyceride production from choline-containing phosphoglycerides in human neutrophils stimulated with N-formylmethionyl-leucylphenylalanine, ionophore A23187 or phorbol 12-myristate 13-acetate.

The turnover of choline-containing phosphoglycerides (PC) in response to agonist stimulation is well documented in human neutrophils. We have now compared the enzymic pathways of N-formylmethionyl-leucylphenylalanine (fMLP)-, A23187- and phorbol-12-myristate 13-acetate (PMA)-induced diglyceride (DG) and phosphatidic acid (PA) generation in these cells. In order to distinguish between phospholipase C- and D-mediated PC breakdown, human neutrophils were radiolabelled with 1-O-[3H]alkyl-2-acyl-glycero-3-phosphocholine and stimulated in the presence of ethanol or propranolol. The addition of 0.5% ethanol to the incubation mixture resulted in the production of phosphatidylethanol, indicative of phospholipase D activation, in response to all three stimuli. Concomitant with phosphatidylethanol formation was a partial block of PA production. The production of DG was also partially blocked by addition of ethanol. Propranolol (200 microM) was also used to assess the contributions of phospholipases C and D toward DG generation. Inhibition of PA phosphohydrolase by propranolol resulted in the complete abolition of DG generation when neutrophils were stimulated with fMLP. In contrast, propranolol only partially inhibited DG generation in response to A23187 and PMA. These results suggested that DG production in response to fMLP stimulation is mediated via the activation of phospholipase D, whereas A23187- or PMA-induced DG generation may involve more than one pathway. However, examination of the water-soluble choline metabolites produced indicated that phospholipase D was responsible for the production of PA and DG in response to all three stimuli.

Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase.

Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway.

Regulation of phospholipase D-induced hydrolysis of choline-containing phosphoglycerides by cyclic AMP in human neutrophils.

In human neutrophils, the chemotactic tripeptide FMLP and the protein kinase C activator PMA stimulate the breakdown of 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine ([3H]EAPC) and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid ([3H]-EAPA) and 1-O-[3H]alkyl-2-acylglycerol ([3H]EAG) via mechanism(s) that may involve the activation of phospholipase D. We have investigated the regulation of phospholipase D by determining the effects of elevation of intracellular levels of cAMP on receptor-mediated and PMA-induced breakdown of [3H]-EAPC and formation of products. Pretreatment of neutrophils with either 10(-3) M dibutyryl-cAMP or 10(-5) M PGE2, in the presence of 4 x 10(-4) M isobutylmethylxanthine (IBMX), inhibited FMLP- and leukotriene B4-induced breakdown of [3H]EAPC and formation of [3H]EAPA and [3H]EAG. Inhibition was apparent at all time points of stimulation examined (15-120 s). In addition, the mass of diradyl-phosphatidic acid was decreased in FMLP-stimulated neutrophils pretreated with PGE2 and IBMX. In contrast, pretreatment of cells with PGE2 and IBMX did not inhibit PMA-induced breakdown of [3H]EAPC and the formation of products at 3 and 10 min after stimulation. Furthermore, formation of 1-O-[3H]alkyl-2-acyl-phosphatidylethanol, produced by phospholipase D in the presence of ethanol by a transphosphatidylation reaction, was significantly inhibited by pretreatment of cells with PGE2 and IBMX in FMLP- but not PMA-stimulated neutrophils. This differential modulation by cAMP of receptor-mediated and PMA-induced activation of phospholipase D suggests agonist-dependent activation of separate pathways and/or activation of separate phospholipase D enzymes. Thus, cAMP elevation may exert inhibitory effects directly on the phospholipase D activated by FMLP and/or on sites proximal to the enzyme that are involved in signal transmission.