Effect of 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin 1(1) (U-60,257), an inhibitor of leukotriene synthesis, on human neutrophil function

A23187, a calcium ionophore, stimulated a time-dependent generation of 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B₄), production of superoxide anion (O₂^?) and release of granule-associated β-glucuronidase and lysozyme by human neutrophils. Leukotriene B₄ also elicited the selective release of granule enzymes from cytochalasin B-treated neutrophils. U-60,257, a recently identified inhibitor of leukotriene (LT) C₄ and D₄ synthesis, caused a dose-related (1–10 μM) suppression of LTB₄ production by A23187-activated neutrophils. Degranulation and O₂^? generation by neutrophils exposed to A23187 and the chemotactic oligopeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), were also inhibited with U-60,257.     

Increase in glutathione peroxidase activity in erythrocytes from trisomy 21 subjects.

Glutathione peroxidase activity has been measured in erythrocytes from normal subjects and from trisomy 21 patients. The latter cases show about 50 % increase of this enzyme similar to the increase observed for superoxide dismutase (erythrocuprein) suggesting either localisation of the gene for glutathione peroxidase on chromosome 21 (as is the case for erythrocuprein) or regulation of this enzyme by intracellular levels of O₂^$\text{?}$, H₂O₂ or superoxide dismutase.     

Differential effects of 20-trifluoromethyl leukotriene B4 on human neutrophil functions

A leukotriene B₄ (LTB₄) analog, 20-trifluoromethyl LTB₄ (20CF₃−LTB₄), has been synthesized and evaluated with human neutrophils for effects on chemotaxis and degranulation. 20CF₃−LTB₄ was equipotent to LTB₄ as a chemoattractant (EC₅₀, 3 nM), produced 50% of maximal activity of LTB₄, and competed with [H] LTB₄ for binding to intact human neutrophil LTB₄ receptors. In contrast to chemotactic activity, 20CF₃−LTB₄ in nanomolar concentrations exhibited antagonist activity without agonist activity up to 10 μM on LTB₄-induced degranulation. The analog had no significant effect on degranulation induced by the chemoattractant peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP). Like LTB₄, 20CF₃−LTB₄ induced neutrophil desensitization to degranulation by LTB₄. The results indicate that hydrogen atoms at C-20 of LTB₄ are critical for its intrinsic chemotactic and degranulation activities. The fact that 20CF₃−LTB₄ is a partial agonist for chemotaxis and an antagonist for degranulation syggests that different LTB₄ receptor subtypes are coupled to these neutrophil functions. Desensitization of the neutrophil degranulation response to LTB₄ can result from receptor occupancy by an antagonist, and therefore, the desensitization is not specific for an agonist.     

Mycoplasma pneumoniae: A prokaryote which consumes oxygen and generates superoxide but which lacks superoxide dismutase

Superoxide dismutase and catalase were not detected in $\text{M}$. $\text{pneumoniae}$ and several other species of $\text{Mycoplasma}$ some of which consume oxygen and secrete H₂O₂. $\text{M}$. $\text{pneumoniae}$ in suspension formed O₂^? in the presence of NADH and flavins and extracts of $\text{M}$. $\text{pneumoniae}$ formed O₂^? in the presence of either NADH or NADPH. The lack of superoxide dismutase in $\text{M}$. $\text{pneumoniae}$ could not be attributed to superoxide dismutase in the complex medium in which the organisms were grown because organisms grown in medium in which the superoxide dismutase had been inactivated by heat still contained undetectable amounts. Mycoplasmas appear to be an exception to the rule that organisms which consume O₂ synthesize superoxide dismutase.     

A radioimmunoassay for leukotriene B4

A radioimmunoassay for leukotreine B₄ has been developed. The assay is sensitive; 5 pg LTB₄ caused significant inibition of binding of [³H]-LTB₄ and 50% displacement occurred with 30 pg. The specificity of the assay has been critically examined; prostaglandins, thromboxane B₂ and arachidonic acid do not exhibit detectable cross-reactions (< 0.03%). Flowever, some non-cyclic dihydroxy- and monohydroxy-eicosatetraeonic acids do cross-react slightly (e.g. diastereomers of 5,12-dihydroxy-6,8,10-$\text{trans}$-14-$\text{cis}$-elcosatetraenoic and 12-hydroxy-5,8,10,14-elcosatetraenoic acids cross-react 3.3% and 2.0% respectively). The assay has been used to monitor the release of LTB₄ from human neutrophils in response to the divalent cation ionophore, A23187. The immunoreactive material released during these incubations was confirmed as LTB₄ by reverse-phase high liquid chromatography follwing solvent extraction and silinic acid chromatography.     

Comparative biological activities of synthetic leukotriene b4 and its ω-oxidation products

Synthetic leukotriene B₄ (LTB₄) and its ω-oxidation products, 20 OH-LT₄ and 20 COOH-LTB₄, were tested for their ability to induce the aggregation of rat neutrophils $\text{in}$$\text{vitro}$, to contract the guinea pig parenchymal strip $\text{in}$$\text{vitro}$ and to cause vascular permeability changes in rabbit skin $\text{in}$$\text{vivo}$. 20 OH-LTB₄ had 10, 100 and 20% of the activity of LTB₄ in the neutrophil aggregation, parenchymal strip and vascular permeability assays respectively. 20 C00H-LTB₄ was inactive $\text{in}$$\text{vivo}$ and showed <1% of the activity of LTB₄$\text{in}$$\text{vitro}$. These results show that while ω-oxidation is a route for biological inactivation of LTB₄, 20 OH-LTB₄ still retains significant biological activity.     

Leukotriene B4 is a complete secretagogue in human neutrophils: a kinetic analysis

The interactions have been studied of leukotriene B₄ (LTB₄) and 20-COOH-LTB₄ with human neutrophils (PMN). Kinetic studies, utilizing continuous recording techniques, showed that LTB₄ activates PMN with respect to aggregation, mobilization of membrane-associated Ca²⁺, $\underset{\dot{}}{?}$ generation, and degranulation within seconds of exposure. Dose-response studies indicate 1) that LTB₄ is much more potent than its dicar?ylic acid derivative (20-COOH-LTB₄) or its all trans-isomer, and 2) that PMN responses to these agents are largely dependent upon pretreatment of the cells with cytochalasin B. These properties were similar to those of the microbial ionophores, ionomycin and A23187. Results demonstrate that LTB₄ rapidly activates PMN and indicate that LTB₄ serves as a complete secretagogue. Moreover, they provide additional evidence that oxidized fatty acids activate human PMN.     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.     

Kinetic study of O-2 DISMUTATION BY BOVINE SUPEROXIDE DISMUTASE. Evidence for saturation of the catalytic sites by O-2.

Dismutation of O₂^? by bovine copper-zinc superoxide $\text{di}$ smutase has been studied at different O₂^? concentrations with a polarographic method. Saturation of the enzyme by the substrate was observed and K_m and Vmax values were calculated. Inhibition by OH^? and CN^? was shown to be of the competitive type. The data support on inner sphere mechanism for the reaction between O₂^? and copper.     

Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system.

Ascorbic acid was found to be oxidized by O₂^$\text{?}$ which was generated by the xanthine-xanthine oxidase system. From a kinetic analysis of the inhibition of this reaction by superoxide dismutase, the second-order rate constant for the reaction between ascorbic acid and O₂^? at pH 7.4 was estimated to be 2.7 × 10⁵ M^?1 sec^?1. A function of ascorbic acid as a defense against O₂^? is presented.     

The accumulation of superoxide radical during the aerobic action of xanthine oxidase A requiem for H2O4−

The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O₂^? during the first few minutes of the reaction. H₂O₂ decreases this accumulation of O₂^? presumably because of the Haber-Weiss reaction ($\text{H}{}_2\text{O}{}_2\text{+}\text{O}{}_2{}^{\mathrm{?}}\text{→}\text{OH}{}^{\mathrm{?}}\text{+}\text{OH}\text{+}\text{O}{}_2$) and very small amounts of superoxide dismutase eliminate it. This accumulation of O₂^? was demonstrated in terms of a burst of reduction of cytochrome $\text{c}$, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H₂O₄^?, can now be satisfactorily explained entirely on the basis of known radical intermediates.     

Activation of the human neutrophil respiratory burst with zymosan-activated serum

Zymosan-activated serum (ZAS) stimulated a time- and concentration-dependent generation of superoxide anion (O₂^?) by human neutrophils. O₂^? production was rapid with maximum generation occurring 2 minutes after cell exposure to ZAS. O₂^? generation is markedly reduced if cells are not preincubated with cytochalasin B prior to contact with ZAS. The amount of O₂^? produced by ZAS stimulated neutrophils was enhanced in the presence of extracellular calcium. However, the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy) benzoate hydrochloride (TMB-8), caused a dose-related inhibition of ZAS-elicited O₂^? production. Neutrophils pretreated with ZAS were desensitized to the subsequent exposure to this stimulus. The fact that pretreatment of neutrophils with ZAS did not diminish the capacity of these cells to generate O₂^? in response to 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), N-formyl-methionyl-leucyl-phenylalanine (FMLP) or 5(5),12(R)-dihydroxy-6,14-cis-8,10-transeicosatetraenoic acid (LTB₄), demonstrates the stimulus specific nature of ZAS-induced desensitization. Thus, ZAS, which contains the complement-derived neutrophil activator, C5a, a naturally occurring phlogistic mediator, represénts a relevent probe for investigating neutrophil function.     

Superoxide-dependent formation of hydroxyl radicals: Detection of hydroxyl radicals by the hydroxylation of aromatic compounds

A mixture of xanthine or hypoxanthine and xanthine oxidase generates the superoxide radical, O₂^$\text{?}$, and H₂O₂. In the presence of iron salts, O₂^$\text{?}$ and H₂O₂ can interact to produce the hydroxyl radical, OH·. Superoxide-dependent formation of OH· can be measured by its ability to hydroxylate salicylate as followed by an improved colorimetric assay described in this paper. A more accurate analysis of OH· can be obtained using its ability to hydroxylate phenol, the hydroxylated products being separated and measured after derivatization using gas-liquid chromatography and electron-capture detection. The derivatization and separation techniques are described.     

The relative effectiveness of .OH,H2O2,O2−, and reducing free radicals in causing damage to biomembranes: A study of radiation damage to erythrocyte ghosts using selective free radical scavengers

The relative effectiveness of oxidizing (^.OH, H₂O₂), ambivalent (O₂^?) and reducing free radicals (e^? and CO₂^?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ($\text{R}$(enz)) were measured and the rates of damage to membranes ($\text{R}$(mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. ^.OH, O₂^? and H₂ O₂ were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e^? and CO₂^? were the least effective. $\text{R}$(enz) values of O₂^? and H₂O₂ were 10-times and of ^.OH 15-times that of e^?. $\text{R}$(mb) values were quite similar for e^? and H₂O₂ (about twice that of O₂^?), while that of ^.OH was 3-times that of O₂^?. Hence, with respect to $\text{R}$(mb): ${}^{\mathrm{.}}\text{OH}\text{>}\text{e}{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{O}{}_2{}^{\mathrm{?}}$ , and with respect to $\text{R}$(enz): ${}^{\mathrm{.}}\text{OH}\text{>}\text{O}{}_2{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{e}{}^{\mathrm{?}}$. The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H₂O₂ added as a chemical reagent and H₂O₂ formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H₂O₂ but markedly susceptible to the latter.     

Inactivation of the human CuZn superoxide dismutase during exposure to O-2 and H2O2

Human copper-zinc superoxide dismutase undergoes inactivation when exposed to O₂^? and H₂O₂ generated during the oxidation of acetaldehyde by xanthine oxidase at pH 7.4 and 37° C. In contrast, human manganese superoxide dismutase is not inactivated under the same conditions. Catalase and Mn-superoxide dismutase protect CuZn superoxide dismutase from inactivation. Similar protection is observed with hydroxyl radical (OH.) scavengers, such as formate and mannitol. In contrast, other OH. scavengers such as ethanol and tert-butyl alcohol, have no protective action. The latter results indicate that “free OH.” is not responsible for the inactivation. Furthermore, H₂O₂ generated during the oxidation of glucose by glucose oxidase, i.e., without production of O₂^?, does not induce CuZn superoxide dismutase inactivation. A mechanism accounting for this $\text{O}{}_2{}^{\mathrm{?}}\text{H}{}_2\text{O}{}_2$-dependent inactivation of CuZn superoxide dismutase is proposed.     

Cannabinoid induced degranulation of rabbit neutrophils

We have examined the effects of various cannabinoids on the degranulation of rabbit peritoneal neutrophils. Several cannabinoids were found to cause a dose-dependent and noncytotoxic release of lysosomal enzymes from the neutrophils. The degranulation induced by cannabidiol is rapid ($\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.3 min}$), and enhanced by extracellular calcium and cytochalasin B. In addition to their intrinsic activity, cannabinoids also modulate the neutrophils' responses to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine. This investigation represents the initial step toward the characterization of the effect of cannabinoids on the excitation-activation coupling sequence of hormonally responsive cells.     

Flooding-induced membrane damage, lipid oxidation and activated oxygen generation in corn leaves

Flooding effects on membrane permeability, lipid peroxidation and activated oxygen metabolism in corn (Zea mays L.) leaves were investigated to determine if activated oxygens are involved in corn flooding-injury. Potted corn plants were flooded at the 4-leaf stage in a controlled environment. A 7-day flooding treatment resulted in a significant increase in chlorophyll breakdown, lipid peroxidation (malondialdehye content), membrane permeability, and the production of superoxide (O ₂ ^- ) and hydrogen peroxide (H₂O₂) in corn leaves. The effects were much greater in older leaves than in younger ones. Spraying leaves with 8-hydroxyquinoline (an O ₂ ^- scavenger) and sodium benzoate (an .OH scavenger) reduced the oxidative damage and enhanced superoxide dismutase (SOD) activity. A short duration flooding treatment elevated the activities of SOD, catalase, ascorbate peroxidase (AP), and glutathione reductase (GR), while further flooding significantly reduced the enzyme activities but enhanced the concentrations of ascorbic acid and reduced form glutathione (GSH). It was noted that the decline in SOD activity was greater than that in H₂O₂ scavengers (AP and GR). The results suggested that O ₂ ^- induced lipid peroxidation and membrane damage, and that excessive accumulation of O ₂ ^- is due to the reduced activity of SOD under flooding stress.     

Kinetic studies of 18O exchange from inorganic phosphate catalyzed by Mg2+-activated unadenylylated glutamine synthetase (E. coli w).

Oxygen-18 exchange out of [¹⁸O]Pi catalyzed by Mg²⁺-activated unadenylated glutamine synthetase from $\text{E.}$$\text{coli}$ was followed by ³¹P-NMR in the presence of the other substrates, ADP and L-glutamine. The pattern of the ${}^{16}\text{O}{}^{18}\text{O}$ in the species P¹⁸O₄, P¹⁸O₃¹⁶O₁, P¹⁸O₂¹⁶O₂, P¹⁸O₁¹⁶O₃, P¹⁶O₄ during the exchange followed a binomial distribution consistent with indiscriminate removal of any of the four oxygens of P_i. The rate constant for ${}^{16}\text{O}{}^{18}\text{O}$ exchange was 410±40 min^?1 while the rate constant for net reaction (ATP formation) was 62±4 min^?1. Thus exchange proceeds ～7 times faster than net reaction, a finding in accord with that of Stokes and Boyer ($\text{J.}$$\text{Biol.}$$\text{Chem.}$ (1976) $\text{251}$, 5558) for the Mn²⁺-activated adenylylated glutamine synthetase. A model for the overall catalytic events first derived from rapid kinetic fluorescence experiments (Rhee and Chock, Proc. Natl. Acad. Sci. USA, (1976) $\text{73}$, 476) was successfully used to fit the oxygen exchange data in this paper.     

Inhibition of leukotriene B4-induced neutrophil degranulation by leukotriene B4-dimethylamide

Leukotriene B₄ (LTB₄) is a potent mediator of pro-inflammatory responses including neutrophil degranulation. Leukotriene B₄ dimethylamide has been synthesized and shown to inhibit neutrophil degranulation induced by LTB₄. The inhibition required time to develop (～60 secs), and had a K_D of circa 2 × 10^?7M, and occurred at concentrations where LTB₄ dimethylamide had negligible agonist activity.     

Structural requirements for chemotactic activity of leukotriene B4 (LTB4)

LTB₄ (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB₄ and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo- LTB₄. Dose response curves for neutrophil chemotaxis to these compounds were obtained using a modified Boyden chamber. The mean distance cells moved into the filter was determined after 30 minutes. Peak chemotactic activity of LTB₄ was at 10⁻⁷M. At higher concentrations, chemotactic activity was decreased. The shape of the dose response curve was similar to that of FMLP except that maximum chemotaxis to LTB₄ was consistently greater than chemotaxis to FMLP. A mixture of the two epimers at C-5 and c-12 shifted the response curve to the right but did not lower maximum activity. Increasing or decreasing the chain by one carbon between the first hydroxyl group and the carboxyl group also shifted the response curve to the right without lowering maximal activity. Changing the 6 double bond from cis to trans has a greater effect. Activity was only detectable at high concentrations and maximum activity achieved was less than 50% that of LTB₄. Thus the chain length between the carboxyl and C-5 hydroxyl groups, the c-5 and c-12 absolute stereochemistry and the stereochemistry of the delta⁶ double bond are all important structural features for chemotactic activity with delta⁶ stereochemistry apparently having the greatest contribution. The relative potencies of these compounds in inducing aggregation were comparable to their chemotactic potencies. The data suggested that they acted at the same receptor since even the less active isomers were able to desensitive the neutrophils to LTB₄.