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.

Enhanced incorporation of exogenous arachidonic acid into phosphatidylinositol and other phospholipids during the early stages of thrombin-induced aggregation in gerbil platelets.

The degradation of platelet phospholipids via phospholipase activity is known to occur during thrombin-induced platelet aggregation. Both phosphatidylinositol and phosphatidylcholine are considered to be sources of the released arachidonic acid which becomes a substrate for prostaglandin and thromboxane A2 formation. In this work, the effect of thrombin on the incorporation of exogenous arachidonic acid into platelet membrane phospholipids was studied. Suspensions of gerbil platelets were incubated in aggregometer cuvettes with [14C] arachidonic acid in the absence or presence of thrombin, and product formation was monitored by thin layer chromatography and scintillation counting. Within 30 sec, the entry of arachidonic acid into phosphatidylinositol was increased by 165% in thrombin-stimulated platelets over controls. Under identical conditions, the incorporation into phosphatidylcholine was increased by only 57%. These results suggest that the incorporation of exogenous arachidonic acid via lysophosphatidylinositol and lysophosphatidylcholine acyltransferase activities may be intimately associated with thrombin-induced platelet aggregation in the gerbil.

Evidence for 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) as a proaggregating agent in rat and human platelets.

In view of the established biological role for 1,25-dihydroxycholecalciferol (DHCC) in calcium mobilization and the significance of calcium availability in platelet activation, it was of interest to investigate the potential for DHCC to act as a proaggregating agent. The present results provide evidence that DHCC at a concentration of 0.01 micrograms/microL can both aggregate human platelets and potentiate ADP-induced platelet aggregation. DHCC was also capable of aggregating rat platelets.

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.

Platelet aggregation in platelet suspensions from Mongolian gerbils (Meriones unguiculatus).

Blood obtained from Mongolian gerbils (Meriones unguiculatus) by cardiac puncture was used to develop a method for preparing platelet suspensions suitable for biochemical and aggregometric studies. The aggregation responses of gerbil platelet suspensions to 10 microM adenosine diphosphate and to 0.2 U/mL thrombin were immediate and irreversible. In addition, thrombin produced a short lag period. Bovine acid-soluble collagen (1:800) produced a long lag period coupled with an irreversible aggregation. Indomethacin (10 microM) significantly depressed adenosine diphosphate--induced aggregation but had no effect on thrombin-induced aggregation. Indomethacin significantly prolonged the lag period and reduced the rate of collagen-induced aggregation. Our results strongly indicate that platelet suspensions from the Mongolian gerbil, an animal which is responsive to hypercholesterolemic diets, may be useful in studying dietary lipid factors which influence platelet aggregation.

Self-regulation of phosphate intake in the rat: the influence of age, vitamin D and parathyroid hormone.

Growing rats offered a choice of four pairs of diets, one low in P (0.1%) and the others containing 0.3, 0.6, 1.2 or 1.8% P, selected food mixtures in each case with nearly identical P contents (0.23-0.24%) (Ca:P = 2.2:1). Mature rats offered the same dietary choices exhibited less rigid diet selection but clearly preferred a diet higher in P (0.64-0.69%) (Ca:P = 0.9:1). Vitamin D-deficient animals selected less P than controls and parathyroidectomized rats severely limited their P intake. The increase in self-determined P consumption relative to Ca in mature rats is consonant with the greater decrease in the requirement for Ca associated with maturation and cessation of bone growth. Susceptibility to hypocalcemia in vitamin D deficiency and parathyroidectomy is a probable factor in the increased sensitivity to excess dietary P, which further depresses plasma Ca. These experiments confirm the existence of a feedback mechanism that regulates the voluntary consumption of P in accordance with physiological needs.

Binding and germination of Aspergillus fumigatus conidia on cultured A549 pneumocytes.

Invasive pulmonary aspergillosis is acquired through inhalation of conidia. Little is known about early interactions between Aspergillus fumigatus conidia and alveolar epithelial cells, so an in vitro model was developed to study binding between conidia and A549 cells, a line of type II pneumocytes. Conidia rapidly became attached to confluent monolayers of A549 cells in serum-free medium, reaching a plateau within 40 min. Scanning electron microscopy (EM) showed a random pattern of early adherence; viable conidia subsequently became clustered on pneumocyte surfaces. Following germination of pneumocyte-adherent conidia for 12 h, direct penetration of epithelial cells by hyphae could be demonstrated by scanning and transmission EM. These data suggest that an early event following inhalation of A. fumigatus conidia may be binding of conidia to pneumocytes, followed by hyphal penetration of the epithelial cell layer.

Mass determination of receptor-mediated accumulation of phosphatidate and diglycerides in human neutrophils measured by Coomassie blue staining and densitometry.

Quantitation of 1,2-diacylglycerol (AAG), 1-0-alkyl-2-acylglycerol (EAG) and phosphatidic acid (PA) was conducted in polymorphonuclear leukocytes (PMN) labeled with 1-0-[3H]alkyl-2-acyl-GPC following stimulation with 1 microM fMLP using Coomassie blue staining and densitometry. At 5s AAG and PA increased by 80% and 107%, respectively, over controls. The accumulation of PA, which reached a maximum by 30s, was higher than AAG by 302% at 5s, and 550% at 30s. EAG accumulation was delayed by 15s following stimulation of PMN. These results show that AAG accumulates before EAG and support the role of AAG in cellular activation, perhaps, via the stimulation of protein kinase C (PKC). EAG may serve to counter the effects of AAG or may itself elicit responses. The high concentrations of PA which accumulate early suggest that PA may be generated by the activation of phospholipase D in PMN stimulated with fMLP.

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.

Aspergillus fumigatus complement inhibitor: production, characterization, and purification by hydrophobic interaction and thin-layer chromatography.

Aspergillus fumigatus has previously been shown to produce a soluble extracellular inhibitor of the alternative complement pathway, called Aspergillus complement inhibitor, or CI. We now report an efficient method for production of CI which relies on the fact that poorly conidiating cultures yielded CI activity with approximately sevenfold-higher potency than CI produced by conidiating cultures. CI from poorly conidiating cultures provided 50% inhibition of alternative pathway-mediated binding of 125I-labeled complement component C3 to cryptococcal blastoconidia at a mean concentration of 60 micrograms/ml. The ability of crude CI to inhibit the alternative complement pathway seemed to be independent of intact protein or polysaccharide structure, as evidenced by resistance of inhibitory activity to digestion by proteases, including subtilisin, alpha-chymotrypsin, papain, and pepsin as well as endoglycosidases F and H. Separation of the active inhibitory component of CI from contaminating materials contained in crude CI preparations was achieved by using Phenylsuperose hydrophobic interaction chromatography in a fast protein liquid chromatography system. The active material proved to be extremely hydrophobic, desorbing from the column only during elution with ethanol; it contained only 15% protein and 5% polysaccharide. Furthermore, results from preparative thin-layer chromatography indicated that lipids which comigrated with phosphatidylserine/phosphatidylinositol and phosphatidylethanolamine possessed significant complement-inhibitory activity. Taken together, these data suggested that phospholipids from A. fumigatus contributed to the functional activity of CI.

Choline-linked phosphoglycerides. A source of phosphatidic acid and diglycerides in stimulated neutrophils.

Stimulation of human polymorphonuclear leukocytes (PMN) may result in the metabolism of phospholipids other than phosphoinositides to generate second-messenger intermediary metabolites. We investigated agonist-induced breakdown of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC), which constitutes almost half the diradyl-GPC fraction in human PMN (Mueller, H. W., O'Flaherty, J. T., Green, D. G., Samuel, M. P., and Wykle, R. L. (1984) J. Lipid Res. 25: 383-388), in cells prelabeled with 1-O-[3H] alkyl-2-acyl-GPC. We also utilized normal-phase high pressure liquid chromatography to quantitate the accumulation of diradylglycerols (1-O-alkyl-2-acylglycerols and diacylglycerols) in stimulated PMN. Phorbol-12-myristate-13-acetate (PMA), 1-oleoyl-2-acetyl-sn-glycerol-, calcium ionophore A23187-, and f-methionyl-leucyl-phenylalanine (fMLP) stimulation of PMN resulted in a time- and concentration-dependent hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA) and 1-O-[3H]alkyl-2-acylglycerol. In all cases formation of 1-O-[3H]alkyl-2-acyl-PA preceded that of 1-O-[3H]alkyl-2-acylglycerol. The times between addition of stimulus and appearance of 1-O-[3H] alkyl-2-acylglycerol varied for PMA (40 s at 1.6 microM), A23187 (5 min at 5 microM), and fMLP (30 sec at 1 microM). Preincubation of cells with 1 microgram/ml pertussis toxin (PT) inhibited the breakdown of 1-O-[3H]alkyl-2-acyl-GPC in cells stimulated with 1 microM fMLP, indicating a role for a PT-sensitive G protein with this stimulus. Quantitation of diglycerides as diradylglycerobenzoates in PMN stimulated with PMA (10 min), A23187 (10 min), or fMLP demonstrated marked accumulation of both 1-O-alkyl-2-acylglycerols and diacylglycerols. The highest increases over controls were observed for fMLP (33-fold for 1-O-alkyl-2-acylglycerols and 17-fold for diacylglycerols). In stimulated PMN prelabeled with 1-O-[3H]hexadecyl-2-acyl-GPC and 1-O-alkyl-2-acyl-sn-glycero-3-[32P]phosphocholine, the ratio of 3H to 32P in 1-O-alkyl-2-acyl-PA compared to 1-O-alkyl-2-acyl-GPC suggested the involvement of a phospholipase D in the hydrolysis of 1-O-[3H]-alkyl-2-acyl-GPC. Thus, stimulation of human PMN results in the hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC to yield 1-O-[3H] alkyl-2-acyl-PA and 1-O-[3H]alkyl-2-acylglycerol possibly initiated by activation of a phospholipase D.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.