Neutrophil Superoxide Generation Research Articles

Studies on the Superoxide Generation of Neutrophils in the Patients with Liver Disease

Studies on the Superoxide Generation of Neutrophils in the Patients with Liver Disease

Neutral Endopeptidase Modulates Substance P-Induced Activation of Human Neutrophils

Neutral endopeptidase (NEP; EC 3.4.24.11) is well recognized as a regulatory peptidase for substance P (SP)-induced responses in various tissues. To determine whether NEP regulates SP-induced activation of human neutrophils, we examined the effect of the NEP inhibitor phosphoramidon on SP-induced superoxide generation and chemotaxis in human blood neutrophils. SP (10(-6)-10(-4) M) induced superoxide generation and chemotaxis in the neutrophils dose dependently. The NEP inhibitor enhanced the SP-induced responses. Thus, phosphoramidon (10(-6) M) shifted the dose-response curves of SP-induced superoxide generation and chemotaxis of the neutrophils to the left by 0.5-0.6 log. Phosphoramidon prevented the hydrolysis of SP by the neutrophils, the NEP activity of the neutrophils being assessed as 125 +/- 13 pmol of SP/min/10(6) cells. The N-terminal peptide SP (up to 3 x 10(-4) M), which was a major degrading product by NEP of the neutrophils, did not activate the neutrophils. We conclude that NEP modulates SP-induced activation of human neutrophils.

Rheumatoid factor modulation of neutrophil superoxide generation enhancing activity of preformed immune complexes

Heat aggregated human (HAG) IgG pretreated with total rheumatoid factors isolated from the serum of rheumatoid arthritis patients showed decreased superoxide generation enhancing activity as compared with HAG pretreated with buffer alone. Similarly, monoclonal IgM rheumatoid factor isolated from the serum of a patient with macroglobulinemia complicated by rheumatoid arthritis inhibited superoxide generation enhancing activity of HAG. On the other hand, superoxide generation enhancing activity of BSA-antiBSA immune complexes was not affected by preincubation with rheumatoid factors isolated from the sera of either rheumatoid arthritis patients or the macroglobulinemia patient. Rheumatoid factors isolated from rheumatoid arthritis serum were fractionated by high performance liquid chromatography and IgM-class and IgG-class rheumatoid factors were obtained. IgG-class rheumatoid factor significantly enhanced the superoxide generation enhancing activity of HAG, whereas IgM rheumatoid factor inhibited it. Rheumatoid arthritis sera showed significantly higher superoxide generation enhancing activity than normal sera. HAG preincubated with rheumatoid arthritis sera showed significantly lower superoxide generation enhancing activity than HAG preincubated with normal sera. These results suggest that factors inhibiting superoxide generation enhancing activity of HAG are present in rheumatoid arthritis sera, and that the responsible is IgM rheumatoid factor, whereas IgG rheumatoid factor enhances it. The factors that express superoxide generation enhancing activity in rheumatoid arthritis sera are suggested to be intermediate size immune complexes.

Attenuated coronary relaxation after reperfusion: effects of superoxide dismutase and TxA2 inhibitor U 63557A

Previous studies demonstrate endothelium-dependent leukotriene D4 (LTD4)-induced relaxation of canine coronary arterial rings in vitro. We now show that coronary occlusion followed by reperfusion attenuates (P less than 0.01) the relaxation of canine coronary artery rings in response to LTD4 as well as acetylcholine (ACh), suggesting loss of endothelium-dependent coronary reactivity (n = 6 dogs). Since superoxide anions have been shown to cause breakdown of endothelium-derived relaxing factor (EDRF), we wondered whether treatment of dogs with superoxide anion scavenger superoxide dismutase (SOD) would modulate the effects of LTD4 and ACh on reperfused coronary artery rings. Indeed, treatment of dogs (n = 5) with SOD before coronary reperfusion resulted in preservation of LTD4- and ACh-induced relaxation of coronary rings. Treatment of another five dogs with selective thromboxane-synthetase blocker U 63557A before coronary reperfusion also resulted in preservation of coronary ring relaxation in response to LTD4 and ACh. To determine the mechanism of U 63557A-induced preservation of coronary reactivity, canine neutrophil superoxide anion generation in the presence of U 63557A was measured. Although U 63557A had no effect on superoxide anion generation in neutrophils alone, it markedly (P less than 0.02) inhibited superoxide anion generation in neutrophils in the presence of platelets, most likely via shunting of accumulated cyclic endoperoxide in platelets toward formation of prostacyclin, which inhibits neutrophil superoxide anion production. Thus SOD and U 63557A protect against loss of endothelium-mediated vascular relaxation by LTD4 and ACh after coronary occlusion and reperfusion.

Plasma from patients with severe Lassa fever profoundly modulates f‐met‐leu‐phe induced superoxide generation in neutrophils

A recurrent theme in studies of the pathology of fatal Lassa fever in man is the lack of histological lesions to explain disordered cell function and death. Recently, we demonstrated the existence of a factor in the plasma of patients with Lassa fever which markedly inhibits the aggregation responses of normal platelets in vitro. To assess whether this factor could mediate more global cellular dysfunction, we studied the effects of Lassa plasma on the respiratory burst of neutrophils. Thirteen of 15 samples from patients in the acute phase of Lassa fever profoundly inhibited the amount of superoxide generated by normal neutrophils in response to the chemotactic peptide, f-met-leu-phe (FMLP) (mean superoxide generated = 54.7 +/- 6.1% of control). In contrast, eight of nine samples from patients who had infections other than Lassa fever enhanced the neutrophil response to the peptide. All Lassa samples which inhibited the ADP-induced aggregation responses of normal platelets inhibited the neutrophil response to FMLP. Unlike the effect on platelets, however, the inhibition of neutrophils was only apparent when the cells were stimulated within 5 min of exposure to the plasma. The inhibition of neutrophils is not due to either interference with FMLP-neutrophil binding or an effect on the NADPH-oxidase, suggesting a suppression of signal transduction. Our data suggest the inhibitory factor in Lassa plasma has global effects on cellular function, and may play a central role in the pathogenesis of this often fatal illness.

In vitro modulation of human neutrophil superoxide anion generation by various calcium channel antagonists used in ischemia-reperfusion resuscitation

Generation of toxic oxygen species by activated polymorphonuclear leukocytes (PMNs) may represent an important mechanism of ischemia-reperfusion injury. Concentration-response data concerning inhibition of Superoxide anion (O 2 −) generation by NADPH oxidoreductase (NADPH OR) from isolated human PMN were generated for five calcium channel antagonists commonly utilized in ischemia-reperfusion investigational therapeutics. Regression analysis derived IC 50 values for verapamil, nimodipine, nicardipine and lidoflazine were 45, 20, 12 and 7 μM respectively. Inhibition of the extent of reaction at 5 min paralleled inhibition of initial velocity. No inhibition by flunarizine was noted at concentrations ≤ 25 μM (where it did not alter reaction mixture composition). Only nicardipine demonstrated a significant concentration-response effect relative to prolonging lag time preceding O 2 − synthesis. Inhibition appeared at least partially reversible for all five agents. Neither PMN activation/ desensitization, free-radical scavenging, nor PMN cytotoxicity appeared to be involved in the inhibition of PMN O 2 − synthesis by these agents. Ca 2+ antagonist inhibition of PMN NADPH OR appears to involve more than simple inhibition of Ca 2+ flux across the PMN plasma membrane. Direct inhibition of the intracellular events involved in the activation and/or activity of NADPH OR may be operative.

Effect of auranofin on eicosanoids and protein kinase C in human neutrophils

Auranofin (AF), a lipophilic chrysotherapeutic agent, was investigated for its effect on the formation of lipoxygenase products and the activity of protein kinase C in human neutrophils. We have previously shown that inhibition of LTB4 formation by 5-lipoxygenase (5-LO) inhibitors is intimately associated with a marked increased in 15-HETE in excess of arachidonic acid. The calcium- and phospholipid-dependent protein kinase, protein kinase C, is activated in FMLP- and A23187-stimulated neutrophils, is hypothesized to stimulate superoxide generation, and plays an essential role in eicosanoid production. AF dose-dependently inhibited the generation of leukotriene B4(LTB4) in FMLP-stimulated neutrophils, the ID50 was approximately 4.5 micrograms/ml. Unlike known 5-LO inhibitors, AF did not enhance the production of 15-HETE. In neutrophils stimulated with the calcium ionophore, A23187, AF did not inhibit the generation of LTB4 nor did AF change the 15-HETE levels. AF inhibited superoxide generation in FMLP-stimulated neutrophils dose-dependently, but did not change the activation of protein kinase C in the cells. We therefore conclude, that AF inhibition of LTB4 production in neutrophils is different from 5-lipoxygenase inhibitors and is elicited at a step distal to protein kinase C activation.

Effect of fluoride on movement of concanavalin A-acceptor molecules of human neutrophils.

The effects of fluoride (F) on neutrophil protuberance formation and induced Con A acceptor molecule migration were assessed microscopically. Below 5 mM, F had little effect on acceptor migration, while it markedly inhibited formation of colchicine-induced protuberances. The anion also increased the rate at which preformed protuberances regressed. Since protuberance formation is enhanced by disassembly of microtubules, these data suggest that F promotes and/or stabilizes microtubule assembly. Microtubule assembly is favored by binding of GTP to tubulin subunits, while GDP binding favors disassembly of microtubules. Since F binds with GDP, forming a new complex that mimics GTP, the anion would be expected to enhance microtubule assembly. Over the same F concentration range, the anion failed to inhibit acceptor polarization, but did inhibit cytochalasin B-enhanced dispersion of prepolarized Con A acceptors, implying that, at low concentrations, F also affected microfilament cycling. Concentrations of F in excess of 5 mM inhibited acceptor migration as well as protuberance formation. At 20 mM, the anion abolished both events, yet at this same concentration F induced neutrophil superoxide generation and degranulation, suggesting that acceptor migration is not a prerequisite for these two neutrophil effector activities.

Three new and bioactive icosanoids from the temperate red marine algaFarlowia mollis

Three new dihydroxyicosanoids, 12(R),13(R)-dihydroxyicosa-5(Z),8(Z),10(E),14(Z)-tetraenoic acid, 12(R),13(R)-dihydroxyicosa-5(Z),8(Z),10(E),14(Z),17(Z)-pentaeno ic acid and 10(R*),11(R*)-dihydroxyoctadeca-6(Z),8(E),12(Z)-trienoic acid, have been isolated from a previously unstudied temperate red marine alga, Farlowia mollis (Cryptonemiales, Rhodophyta). The structures of these new metabolites have been deduced from detailed nuclear magnetic resonance and mass spectrometry analyses on stabilized diacetate-methyl esters and stereochemistry deduced by 1H NMR couplings and CD analysis of a dibenzoate derivative. Collectively, these new natural products modulate fMLP-induced superoxide anion generation in human neutrophils, inhibit the conversion of arachidonic acid to lipoxygenase products by human neutrophils, and inhibit the functioning of the dog kidney Na+/K+ ATPase.

Regulation of human neutrophil functions by adenine nucleotides.

Previous work has shown that platelet-derived adenine nucleotides modulate neutrophil superoxide anion (O2-) generation. Additional studies were undertaken to characterize the effects of authentic adenosine (ADO) and its nucleotide derivatives on the inflammatory functions of human neutrophils. Stimulus-specific inhibition of neutrophil O2- generation by ADO in response to FMLP was verified. In addition, the ability of ATP, ADP, and AMP to limit neutrophil O2- generation induced by FMLP (0.2 to 0.5 microM) was demonstrated. The concentration producing 50% inhibition for nucleotide inhibition of neutrophil O2- generation was in the rank order of ADO (0.1 microM) less than AMP (0.5 microM) less than ADP less than or equal to ATP (5 microM). Guanine and inosine nucleotides (0.01 to 100 microM) did not inhibit FMLP-stimulated neutrophil O2- generation. Neutrophil degranulation in response to FMLP was only modestly inhibited by adenine nucleotides and ADO. Adenosine and ADP failed to affect chemotaxis of neutrophils stimulated with FMLP. The inability of non-metabolizable analogs to mimic the inhibitory effects of authentic ATP or ADP on the neutrophil O2- response suggested that metabolism of added nucleotides is necessary for their effectiveness. Both TLC and HPLC confirmed that ATP and ADP were converted to AMP and ADO after their incubation with unstimulated or FMLP-activated neutrophils. The addition of adenosine deaminase to neutrophil reaction mixtures in which conversion of added nucleotides was apparent removed detectable ADO but failed to completely abrogate the inhibition of neutrophil O2- generation by accumulated AMP. The kinetics of inhibition of FMLP-induced neutrophil O2- generation by ATP and ADP also indicated that conversion of these nucleotides to ADO and/or AMP may be essential for their ability to reduce neutrophil responses.

The effect of azelastine on neutrophil and eosinophil generation of superoxide

Azelastine is a new investigational drug used to treat rhinitis and asthma. In addition to described actions that include inhibition of immunologic release of histamine from mast cells and basophils, blockade of smooth muscle contraction to various spasmogenic mediators, and relaxation of airway smooth muscle, azelastine has been ascribed anti-inflammatory properties. To understand further the mechanisms by which azelastine may regulate inflammation, its effect on neutrophil and eosinophil superoxide (O 2 -) generation was evaluated. Purified suspension of neutrophils (>95% pure) and eosinophils (>85% pure) were isolated from human peripheral blood samples by continuous density gradients of Percoll. The isolated granulocyte suspensions (1 × 10 5 cells) were added to 96-well microtiter plates in the presence of cytochrome c, activated by either N-formyl-methionyl-leucyl-phenylalanine, phorphol myristate actetate, calcium ionophore A23187, or opsonized zymosan particles; O 2 - generation was measured by the reduction of cytochrome c. We found that azelastine significantly inhibited both neutrophil and eosinophil generation of O 2 - in a dose-dependent fashion (10 −7 to 10 −5 mol/L) with each activator except zymosan. Furthermore, the degree to which azelastine suppressed O 2 - was similar in neutrophils and eosinophils. Thus, azelastine, in concentrations achieved therapeutically, inhibited granulocyte generation of O 2 -. This anti-inflammatory effect may also be beneficial in the treatment of asthma.

Halothane, an inhalation anesthetic, activates protein kinase C and superoxide generation by neutrophils

The rate of superoxide generation of guinea pig intraperitoneal neutrophils by a chemotactic peptide or 12- O-tetradecanoylphorbol-13-acetate (TPA) was increased by 2-bromo-2-chloro-1,1,1,-trifluoroethane (halothane), an inhalation anesthetic. This increase was inhibited by 1-(5-isoquinolinesulfonyl)methylpiperazine dihydrochloride (H-7), a specific inhibitor of Ca 2+- and phospholipid-dependent protein kinase C (PKC). Halothane was found to significantly activate partially purified PKC. The activation required phosphatidylserine (PS) and Ca 2+. Dioleoylglycerol- or TPA-activated PKC activity was further increased by halothane. The cytoplasmic proteins of guinea pig neutrophils phosphorylated by halothane-activated PKC were similar to those phosphorylated by PMA-activated PKC. The phosphorylation of a 48 kDa protein, a phosphorylated protein required for NADPH oxidase activation, was also increased by halothane. These data suggest that the increase of superoxide production by halothane is correlated with its activation of PKC.

Enhancement of phorbol ester-induced protein kinase activity in human neutrophils by platelet-activating factor.

We have shown that platelet-activating factor (PAF), a weak primary stimulus for neutrophil superoxide generation, synergistically enhances neutrophil oxidative responses to the tumor promoter phorbol myristate acetate (PMA). Since PMA is known to cause cytosol-to-membrane shift of calcium-activated, phospholipid-dependent protein kinase (protein kinase c, PKC) in human neutrophils, we investigated the role of PAF in modifying PMA-induced PKC activation/translocation. Protein kinase activity was measured as the incorporation of 32P from gamma-32P-ATP into histone H1 induced by enzyme in cytosolic and particulate fractions from sonicated human neutrophils. PAF did not alter the sharp decrease in cytosolic PKC activity induced by PMA. However, in the presence of PAF and PMA, total particulate protein kinase activity increased markedly over that detected in the presence of PMA alone (144 +/- 9 pmoles 32P/10(7)PMN/minute in cells treated with 20 ng/ml PMA compared to 267 +/- 24 pmoles 32P in cells exposed to PMA and 10(-6)M PAF). The increase in total particulate protein kinase activity was synergistic for the two stimuli, required the presence of cytochalasin B during stimulation, and occurred at PAF concentrations of 10(-7) M and above. Both PKC and calcium-, phospholipid-independent protein kinase activities in whole particulate fractions were augmented by PAF as were both activities in detergent-extractable particulate subfractions. PAF did not directly activate PKC obtained from control or PMA-treated neutrophils. However, the PKC-enhancing effect of PAF was inhibited in the absence of calcium during cellular stimulation. PAF also increased particulate protein kinase activity in cells simultaneously exposed to FMLP but the effect was additive for these stimuli. These results suggest that PAF enhances PMA-induced particulate PKC activity by a calcium-dependent mechanism. The enhancing effect of PAF may be directly involved in the mechanism whereby the phospholipid "primes" neutrophils for augmented oxidative responses to PMA.

Inhibition of Neutrophil Superoxide Anion Generation by Platelet Products: Role of Adenine Nucleotides

Previously, we demonstrated the stimulus-specific ability of platelet lysate or release products to inhibit neutrophil functions in response to f-met-leu-phe (fMLP) or phorbol myristate acetate (PMA). The present study further characterizes these activities and identifies one of the platelet inhibitors of neutrophil superoxide anion (O2-) generation. Sephadex G-200 chromatography revealed two fMLP-specific inhibitory activities in platelet lysate. One component eluted with materials less than 13 kilodaltons (kD) molecular weight, whereas the second fractionated at the void volume (Mr greater than 200 kD). PMA-specific inhibitor(s) cofractionated at the void volume with the lysate's fMLP-inhibitory activity. Release products of activated platelets contained low-molecular-weight (less than 13 kD), fMLP-specific inhibitor activity but failed to demonstrate PMA-specific inhibition of neutrophil O2- generation. Biochemical characterization identified two distinct high-molecular-weight inhibitors in platelet lysate. Pretreatment with adenosine deaminase (ADA) completely abrogated the maximum inhibitory effect (43.8 +/- 7.5%) of the high-molecular-weight, fMLP-specific lysate component but failed to reduce maximum PMA-specific inhibition (93.0 +/- 2.0%) of neutrophil O2- generation. Likewise, while the lysate's fMLP-specific inhibitor was heat stable, heating reduced the PMA-specific inhibition 56.5 +/- 10.4%. Equal sensitivity to ADA, resistance to trypsin, and heat stability were demonstrated by the low-molecular-weight inhibitor(s) in platelet lysate and release products. Neither high- nor low-molecular-weight platelet inhibitors scavenged O2-. High-performance liquid chromatography (HPLC) of the low-molecular-weight inhibitory activity from both platelet lysate and release products revealed physiological levels of adenine nucleotides. Addition of pure adenine nucleotides at physiological levels (1-10 microM) mimicked the effectiveness of the platelet inhibitor. We conclude that stimulus-specific limitation of neutrophil O2- generation by platelet adenine nucleotides may represent a mechanism for reducing inadvertent tissue damage during inflammation.

Temperature-dependent inhibition of fmet-leu-phe-stimulated superoxide generation by C-I and H-7 in human neutrophils

Contrary to previous reports by other investigators, protein kinase inhibitors C-I and H-7 (at 10(-6)M) caused a significant inhibition of fmet-leu-phe-stimulated superoxide (O2-) generation in human neutrophils. The observed inhibition of O2- production was a highly temperature-sensitive event and occurred only when C-I or H-7 was added to neutrophils at 37 degrees C. When the temperature at which C-I or H-7 added to neutrophils was varied between 16 degrees C to 37 degrees C, no significant inhibition of fmet-leu-phe-stimulated O2- production by C-I or H-7 was observed even at 35 degrees C. However, when added at 37 degrees C, both the maximal rate and the final extent of fmet-leu-phe induced O2- production were significantly inhibited (greater than 50%) by 10(-6)M C-I or H-7. A relatively weaker protein kinase C antagonist, HA1004, was not inhibitory under identical experimental conditions. In contrast, inhibition of the PMA-induced O2- generation by C-I or H-7 was not found to be similarly temperature-dependent. These results indicate that some temperature-dependent cellular event(s) is critically involved in the observed inhibition of fmet-leu-phe-induced O2- generation by C-I or H-7, and suggest a role for protein kinase C in the signal transduction process.

Inhibition of neutrophil functions by platelets and platelet-derived products: description of multiple inhibitory properties.

The effect of platelets and their products on in vitro responses of human neutrophils to f-met-leu-phe (fMLP) and phorbol myristate acetate (PMA) was studied. Platelets produced a concentration-dependent inhibition of neutrophil superoxide anion (O2-) generation with maximum inhibition of the response to fMLP approaching 42.2 +/- 5.4% and that to PMA approaching 85.9 +/- 3.4%. Supernates of thrombin-activated platelets produced maximal inhibition of neutrophil O2- generation in response to fMLP (66.3 +/- 4.2%) at a ratio of 10 platelet equivalents (PEQ) to 1 neutrophil but failed to affect the cells' response to PMA. In contrast, lysate of sonicated platelets inhibited neutrophil O2- generation in response to both fMLP (59.6 +/- 2.0%) and PMA (90.1 +/- 1.3%). Biochemical characterization of platelet lysate identified at least two stimulus-specific inhibitory activities which differ in size, thermal stability and enzyme sensitivity. Platelet lysate also maximally inhibited neutrophil degranulation and chemotaxis at a ratio of 20 PEQ to 1 neutrophil. Our findings indicate that physiologically achievable levels of platelets or their products modulate the response of neutrophils in a stimulus-specific manner and are thereby capable of potentially limiting tissue damage which accompanies neutrophil activation during inflammation.

Superoxide generation and cytotactic response of irradiated neutrophils.

Irradiation of blood components has been used to prevent transfusion-related graft-versus-host disease (GVHD) in immunocompromised patients. This study was designed to determine the effect of irradiation on neutrophil aggregation, chemotaxis, and superoxide generation. Purified neutrophils were irradiated with a Cesium source at four doses ranging from 0 to 17,500 rads. Formyl-methionyl-leucyl-phenylalanine (FMLP) and zymosan-treated serum (ZTS) cytotaxin-induced chemotaxis and migration were determined in the agarose assay. Neutrophil aggregation to FMLP was determined by aggregometry. Superoxide generation and random migration were not affected by irradiation at doses up to 17,500 rads. When compared to nonirradiated controls, the chemotactic response to ZTS remained normal, with an insignificant decline from 174 +/- 31.0 to 150 +/- 42.3 (mean +/- SD) units. The chemotactic response to FMLP declined insignificantly, from 228 +/- 31.3 at 0 rad to 207 +/- 26.4 at 17,500 rads. The aggregation response to FMLP remained within the normal range but declined from 0.78 +/- 0.11 to 0.61 +/- 0.18. At the radiation doses currently used to reduce the risk of transfusion-related GVHD, neutrophil superoxide generation and chemotactic response remain essentially normal.

Granulocyte-macrophage colony-stimulating factor enhances neutrophil function in acquired immunodeficiency syndrome patients.

We conducted a clinical trial of human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) in leukopenic patients with acquired immunodeficiency syndrome (AIDS) and analyzed neutrophil function before, during, and after in vivo administration of rGM-CSF. Prior to GM-CSF infusion, AIDS patients' neutrophil superoxide generation and neutrophil antibody-dependent cell-mediated cytotoxicity were enhanced normally by in vitro exposure to GM-CSF. Neutrophil phagocytosis and intracellular killing of Staphylococcus aureus were also normal in the majority of these patients. Two patients, however, had discrete neutrophil functional defects: one in phagocytosis and one in intracellular killing. During the period of GM-CSF infusion, these abnormalities were corrected. The number of circulating neutrophils increased in all patients treated with GM-CSF in a dose-dependent manner. Neutrophils produced in vivo in response to GM-CSF administration functioned normally and there was evidence for neutrophil priming and activation in vivo. We conclude that GM-CSF treatment of AIDS patients leads to the production of functionally active neutrophils, suggesting therapeutic potential for GM-CSF in the treatment of patients with impaired host defense.

Stimulation of cAMP Accumulation and Superoxide Production in Human Neutrophils and Monocytes

The effect of sodium fluoride (NaF) on superoxide generation and cyclic adenosine monophosphate (cAMP) levels in human neutrophils and monocytes was investigated. NaF (greater than 10 mM) stimulated superoxide (O2-) production in both cell types in a time dependent manner. NaF (0.5 to 20 mM) increased cAMP levels by 1.5- to 3.-fold in both neutrophils and monocytes. Increases in cAMP levels were time-dependent; the maximal level was attained within 5 minutes after the addition of NaF, and cAMP levels remained elevated for up to 10 minutes. Only high concentrations of NaF (10 and 20 mM) increased both cAMP levels and O2- production. Therefore, a direct role of cAMP in O2- generation is not likely. It is speculated that since NaF (greater than 10 mM) can complex with extracellular Ca++, and thus reduce free Ca++ concentration required for O2- generation, a NaF-dependent increase in cAMP may restore cytosolic free Ca++ by mobilizing intracellular stores of Ca++. Further, in view of the proposed involvement of a phosphorylation-dephosphorylation mechanism in the regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, we speculate that NaF, by inhibiting phosphoprotein phosphatase activity, may indirectly activate the NADPH oxidase system and thus superoxide generation.

Biosynthesis and biological activities of lipoxin A5 and B5 from eicosapentaenoic acid.

[1-14C]-Eicosapentaenoic acid (EPA) was incubated with porcine leukocytes. Three polar metabolites were isolated after RP-HPLC separation in addition to pentaene leukotrienes and mono-hydroxy fatty acids. These compounds display U.V. absorbance with U.V. lambda max at 302 nm with shoulders at 289 and 317 nm which were typical of a conjugated tetraenes. Using an alkaline RP-HPLC solvent system, it was found that these three compounds co-eluted with synthetic standards of lipoxin A5, lipoxin B5 and 5S, 6S, 15S-lipoxin A5 [6-S-LXA5] and were identified accordingly. Their structures were further confirmed by GC/MS analysis. When tested for biological activities, it was found that both lipoxin A5 and lipoxin B5 induce superoxide anion generation in canine neutrophils. Furthermore, LXA5 caused a dose-related contraction of isolated rat tail artery. The biological potency of 5-series lipoxins were similar to those of 4-series.