Intracellular production of reactive oxygen species in human neutrophils following activation by the soluble stimuli FMLP, dioctanoylglycerol and ionomycin.

The stimuli, sn-1, 2-dioctanoylglycerol; (DG8) the calcium specific ionophore, ionomycin, and the chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) can interact with normal human neutrophils and activate their superoxide/hydrogen peroxide generating NADPH-oxidase. In response to the peptide as well as DG8, the neutrophils produced both superoxide (O2-) and hydrogen peroxide (H2O2). Since interaction between the cells and ionomycin was not associated with any notable superoxide production and hydrogen peroxide was induced only in the presence of azide, a potent inhibitor of the hydrogen peroxide-consuming enzymes catalase and myeloperoxidase, we conclude that this stimulus can generate oxygen metabolites intracellularly. Since the DG8-induced production of hydrogen peroxide was increased in the presence of azide, whereas the FMLP-induced response was largely unaffected, we concluded that the three stimuli differ in their capacity to generate oxygen metabolites intracellularly. The use of sn-1,2-didecanoylglycerol (DG10) as stimulating agent did not result in any detectable activation of the NADPH-oxidase. However, preincubation caused an increased (primed) response during stimulation with the chemotactic peptide FMLP. The response of primed neutrophils to FMLP proceeds with a time-course different from that seen in normal cells. From the results presented on FMLP-induced activity in the presence of azide, we conclude that FMLP causes normal cells to produce oxygen radicals which are released from the cells. However, the primed cells are also capable of generating oxygen metabolites that are retained inside the cells. In fact, measurement of the intracellularly generated metabolites discloses this to be the predominant part of the response.     

Chemotactic factor-induced generation of superoxide radicals by human neutrophils: evidence for the role of sodium.

The role of sodium ion in superoxide (O2-) generation by human peripheral neutrophils was investigated. Cells were activated by exposure to the synthetic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), and O2- release was assessed by ferricytochrome c reduction after 5 min of incubation at 37 degrees C in the presence of FMLP 4 X 10(-8) M. In the absence of monovalent cations (isotonic glucose), negligible O2- generation occurred. There was a progressive increase in the magnitude of FMLP-induced O2- generation with increasing Na+ concentration up to 90 mM, where the response was noted to plateau. Varying the K+ concentration (1 to 10 mM) had no effect on the amount of O2- produced in the presence of Na+ 140 mM. FMLP also stimulated 22Na+ and 48Ca2+ uptake by the cells in a dose- and time-dependent fashion. FMLP-induced 22Na+ uptake appeared to be independent of the external Ca2+ concentration ( to 4 mM). In contrast, there was a progressive decrease in themagnitude of the FMLP-induced increase in 45Ca2+ uptake as the Na+ concentration was reduced by replacement with choline+ or glucose. These studies support a requirement for Na+ in FMLP-induced O2- generation and suggest that a Na+ influx may underlie the nature of this requirement. The data are also consistent with the hypothesis that a Na+ influx may precede the Ca2+ influx in the FMLP-induced activation sequence.     

Determination of serum glucose by horseradish peroxidase-catalysed imidazole chemiluminescence coupled to a micro-flow-injection system.

The reactivity of flow-injection (FI)-horseradish peroxidase (HRP)-catalysed imidazole chemiluminescence (CL) was studied for continuous determination of hydrogen peroxide (H(2)O(2)) and serum glucose with immobilized glucose oxidase. Light emission by the HRP-catalysed imidazole CL was obtained when immobilized HRP, alkaline imidazole (in Tricine solution, pH 9.3) and H(2)O(2) were reacted at room temperature. The optimal pH for the CL reaction was 9.3 and the optimal concentration of imidazole was 100 micromol/L. When no imidazole was added, the light intensity of the same H(2)O(2) specimen decreased to a level that could not be quantitatively determined. The spectrum of the light emitted by imidazole CL was in the range 400-600 nm with a peak at 500 nm. The calibration equation for determination of H(2)O(2) was y = 9860x(2) + 3830x + 11,700, where y = light intensity (RLU) and x = concentration of H(2)O(2) (micromol/L). The detection limit of H(2)O(2) was 5 pmol, and the reproducibility of the H(2)O(2) assay was 2.3% of the coefficient of variation (H(2)O(2) 48 micromol/L, n = 13). The CL method was successfully applied to assay glucose after on-line generation of H(2)O(2) with the immobilized glucose oxidase column, resulting in good reproducibility (CV = 3.3% and 1.0% for the standard glucose and the control serum, respectively).     

Evaluation of the degree of medical radiation damage with a highly sensitive chemiluminescence method.

A highly sensitive chemiluminescence (CL) method for evaluation of medical radiation damage degree is presented. According to the principle of cell stress response to ionizing radiation, lymphocytes will produce reactive oxygen species (ROS) after irradiation. The ROS produced can react with 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha] pyrazin-3-one (MCLA), a specific CL probe for superoxide anion (O(.-) (2)) and singlet oxygen ((1)O(2)), to emit light at 465 nm. The CL intensity is positively related to the amount of generated ROS detected 30 min after irradiation. Cell viability, which is inversely related to cell mortality, was determined by MTT assay after 3 days' culture. The results show that both CL intensity and cell mortality of lymphocytes increase with the increase of the radiation dose when the dosage is no more than 3 Gy, suggesting a positive relationship between the degree of lymphocyte cell damage and the amount of ROS generated. In addition, the effects of catalase, Cu-Zn superoxide dismutase (SOD), mannitol, sodium azide (NaN(3)), and D(2)O on MCLA-dependent CL of lymphocytes are discussed. We believe that the MCLA-dependent CL method would potentially provide an easy way for evaluating the degree of lymphocyte damage induced by radiation.     

Noncytotoxic activation of neutrophils by eosinophil granule major basic protein. Effect on superoxide anion generation and lysosomal enzyme release

Eosinophil granule major basic protein (MBP) and neutrophils have each been implicated in the inflammatory late phase events of allergic disease. Based on this association and flow cytometric evidence presented in this report for MBP binding to neutrophils, we examined the ability of MBP to activate human neutrophils. Incubation of neutrophils with 0.5 to 3.0 microM MBP at room temperature produced a concentration-dependent chemiluminescence (CL) response that peaked after 50 to 70 min. Reduced-and-alkylated MBP, eosinophil cationic protein, and eosinophil-derived neurotoxin did not induce CL. MBP-induced CL was abrogated in the absence of Ca2+ and was absent in neutrophils isolated from two individuals with chronic granulomatous disease. MBP also stimulated release of superoxide anion (O2-) and lysozyme but not beta-glucuronidase or lactate dehydrogenase. Additionally, 1.5 microM MBP in combination with FMLP or platelet-activating factor stimulated a synergistic increase in O2- release from cytochalasin B-treated neutrophils. The degree of synergism with FMLP or platelet-activating factor was inversely related (p less than 0.005) to the level of MBP-induced O2- release. These results indicate that MBP activates neutrophils in a noncytolytic fashion and provide evidence that eosinophil-neutrophil collaboration may contribute to the pathogenesis observed in allergic late phase reactions.     

Chemiluminescence flow biosensor for glucose based on gold nanoparticle-enhanced activities of glucose oxidase and horseradish peroxidase

In this work, a novel chemiluminescence (CL) flow biosensor for glucose was proposed. Glucose oxidase (GOD), horseradish peroxidase (HRP) and gold nanoparticles were immobilized with sol-gel method on the inside surface of the CL flow cell. The CL detection involved enzymatic oxidation of glucose to d-gluconic acid and H(2)O(2), and then the generated H(2)O(2) oxidizing luminol to produce CL emission in the presence of HRP. It was found that gold nanoparticles could remarkably enhance the CL respond of the glucose biosensor. The enhanced effect was closely related to the sizes of gold colloids, and the smaller the size of gold colloids had the higher CL respond. The immobilization condition and the CL condition were studied in detail. The CL emission intensity was linear with glucose concentration in the range of 1.0 x 10(-5)molL(-1) to 1.0 x 10(-3)molL(-1), and the detection limit was 5 x 10(-6)molL(-1) (3sigma). The apparent Michaelis-Menten constant of GOD in gold nanoparticles/sol-gel matrix was evaluated to be 0.3mmolL(-1), which was smaller than that of GOD immobilized in sol-gel matrix without gold nanoparticles. The proposed biosensor exhibited short response time, easy operation, low cost and simple assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.     

Validation of different chemilumigenic substrates for detecting extracellular generation of reactive oxygen species by phagocytes and endothelial cells

Chemiluminescence is a widely used tool to detect extracellular generation of reactive oxygen species (ROS). In the present study we tested four different chemilumigenic substrates (CLS)—luminol, isoluminol, lucigenin and pholasin—to detect extracellular CL in different cell types: polymorphonuclear leukocytes (PMN); DMSO‐differentiated HL‐60 cells; murine macrophages (RAW 264.7); and TNFα‐stimulated human endothelial cells (HUVEC). Extracellular ROS production was calculated by subtracting intracellular CL response in the presence of superoxide dismutase and catalase from the overall CL response in the absence of enzymes. CL varied considerably in dependence on the CLS and the stimulus used to evoke ROS generation. Luminol (oxidized LDL and zymosan stimulation) and isoluminol (FMLP and PMA stimulation) were the most effective CLS for PMN. Using 5 µmol/L lucigenin as CLS, small but consistent CL responses could be obtained in macrophages stimulated with PMA, zymosan or oxidized LDL. FMLP‐stimulated extracellular CL in H‐60 cells, HUVEC and macrophages was detected with the greatest sensitivity by pholasin. Our results demonstrate that none of the investigated CLS consistently yielded the highest CL quantum, either in different cell types with one stimulating agent or by different stimulating agents in one cell type. To get the highest CL quantum in experimental studies, we recommend optimizing the CLS depending on the cell type and the ROS‐generating stimulus used. Copyright © 2003 John Wiley & Sons, Ltd.     

Ca2+-independent synergistic augmentation of O2- production by FMLP and PMA in HL-60 cells

N-Formyl-Met-Leu-Phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) caused a synergistic augmentation of superoxide anion (O2-) production in neutrophil-like HL-60 cells differentiated with dibutyryl cAMP. The present study was undertaken to investigate the mechanism of the synergistic augmentation of O2- production. FMLP increased intracellular free Ca2+ concentration ([Ca2+]i), which was slightly suppressed by PMA and completely inhibited by an intracellular Ca2+ chelating agent, O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM). Although FMLP-induced O2- production was inhibited by BAPTA-AM, a major part of the synergistic augmentation of O2- production by FMLP and PMA remained after BAPTA-AM treatment, suggesting that a Ca2+-independent mechanism might be involved in the augmentation. FMLP and PMA caused an activation of phospholipase D (PLD) almost additively in a Ca2+-sensitive manner. The synergistic activation of mitogen-activated protein kinase (MAPK) was evoked by combined addition of PMA and FMLP in a BAPTA-AM resistant manner. However, PD98059, a MAPK kinase inhibitor, did not affect the synergistic augmentation of O2- production, although it potently inhibited the synergistic augmentation of tyrosine phosphorylation of MAPK. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FMLP-induced O2- production, but it did not inhibit the synergistic augmentation of O2- production by PMA and FMLP. In contrast, staurosporine and GF109203X, protein kinase C inhibitors, reduced the synergistic augmentation induced by PMA and FMLP. In addition, pertussis toxin (PT) abolished the synergistic augmentation of O2- production. It is concluded that the synergistic augmentation of O2- production induced by PMA and FMLP is mediated through a PT-sensitive G protein and a protein kinase C in a Ca2+-independent manner.     

Chemiluminescence study of active oxygen species produced by TiO2 photocatalytic reaction.

Two chemiluminescence approaches have been used for study of active oxygen species produced by the TiO2 photocatalytic reaction. One is based on flow injection analysis (FIA)-luminol chemiluminescence (CL); another is a time-resolved CL method. In the FIA-CL experiment, an UV-illuminated TiO2 suspension and water were passed into a mixing cell by two separate flow lines. Luminol solution was injected into the water flow line at different times. The injected luminol reacted with active oxygen species generated by the TiO2 photocatalytic reaction in a mixing coil and produced CL. It was found that the maximum CL was detected at the first injection of luminol. CL intensity decreased with time of injection. When the luminol was injected after 5 min, the CL intensity was almost unchanged. Addition of scavengers of active oxygen species indicated that the CL produced early in the 5 min was caused by O2- and H2O2, while CL after 5 min was only from H2O2. In the time-resolved CL, the third harmonic wavelength of Nd:YAG laser (355 nm) was used as a UV light source, and CL was detected by a PMT and recorded in a millisecond time scale using a digital oscilloscope. It was found that CL induced by the photocatalytic reaction increased with concentration of the TiO2 suspension. Scavengers of active oxygen species of *OH, O2- and H2O2 were added to study the involvement of the active oxygen species.     

Differentiation of human peripheral blood monocytes to macrophages is associated with changes in the cellular respiratory burst activity.

When phagocytic leukocytes, e.g. neutrophils, monocytes and macrophages, interact with soluble or particulate stimuli, the cells respond with an increased production of reactive oxygen metabolites. This production can be measured with the luminol-amplified chemiluminescence (CL) technique. In the present study, the CL reaction induced in monocyte-derived macrophages was investigated and compared to the responses of neutrophils and monocytes. In systems without additives the CL response of macrophages to soluble stimuli (FMLP, PMA and ionomycin) was very low. Addition of a peroxidase (HRP) to the reaction mixtures resulted in a pronounced increase in CL activity. The cellular CL response in macrophages is thus limited by the amount of peroxidase available. The macrophage response differs qualitatively from the responses of neutrophils and monocytes, in that the intracellular phase of the response is missing.     

Degranulation in human neutrophils primes the cells for subsequent responsiveness to the chemoattractant N-formylmethionylleucylphenylalanine but does not increase the sensitivity of the NADPH-oxidase to an intracellular calcium rise

Both the chemotactic peptide formylmethionylleucylphenylalanine (FMLP) and the calcium-specific ionophore ionomycin can activate the NADPH-oxidase in human neutrophils. However, since ionomycin and FMLP activity differ in their requirement for azide, a potent inhibitor of the hydrogen peroxide consuming enzymes catalase and myeloperoxidase, we propose that the two stimuli can activate different pools of the oxidase. Degranulation, induced in vitro by sn-1,2-dedecaoylglycerol or in vivo by an exudation process, resulted in a priming of the cells using FMLP as stimulating agent as well as in a reduced capacity to generate H2O2 in response to ionomycin. The sensitivity of the plasma membrane-bound NADPH-oxidase to an intracellular [Ca2+] rise, induced by the ionophore was, however, not changed by the degranulation. From these results we propose that FMLP activates the plasma membrane-bound oxidase, whereas the ionophore is capable of activating a granule-bound pool of the oxidase.     

Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using nanoCuS tags

A novel and sensitive biosensor for the determination of short sequence of DNA based on flow injection (FI)-chemiluminescence (CL) system of luminol-H2O2-Cu2+ was developed in the present work. The DNA probe labeled with copper sulfide nanoparticles (CuS NPs) could hybridize with target DNA immobilized on glass-carbon electrode (GCE). The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu2+ after the cupric ions was dissolved from the hybrids. A preconcentration process of cupric ions was performed by anodic stripping voltammetry (ASV) technology to improve the sensitivity of the biosensor. Under the optimum conditions, the CL intensity was proportional to the concentration of target DNA in the range of 2.0 x 10(-12)-1.0 x 10(-10)M. A detection limit of 5.5 x 10(-13)M of target DNA was achieved. The CL intensity of two-base mismatched sequences and noncomplementary sequences were also detected. The experiments indicated that two-base mismatched sequences showed weaker CL intensity and noncomplementary sequences gave no response at all.     

Superoxide responses to formyl-methionyl-leucyl-phenylalanine in primed neutrophils. Role of intracellular and extracellular calcium.

For superoxide (O2-) responses of human neutrophils stimulated by FMLP, experiments were designed to assess the requirement of extracellular calcium [( Ca2+]o) for priming of O2- responses by platelet-activating factor (PAF), PMA, or ionomycin. Although priming by PMA did not require [Ca2+]o, there was, as expected, a requirement for [Ca2+]o for the optimal priming effects of PAF and ionomycin. The ED50 value for [Ca2+]o in the priming function of PAF was 105 microM. The [Ca2+]o-dependent priming with ionomycin was bimodal with two ED50 values for [Ca2+]o of 90 microM and 3.2 mM. Optimal priming by PAF required at least 4-min exposure of cells to [Ca2+]o. Cells primed by PAF exhibited faster initial rates of O2-production after addition of FMLP, but the duration of O2- production was not prolonged. Whereas PAF-primed responses to FMLP are usually associated with increases in intracellular calcium [( Ca2+]i) after addition of FMLP, two conditions were found in which O2- responses to FMLP in PAF-primed cells occurred in the absence of any detectable increase in [Ca2+]i. When cells were loaded with the calcium chelator, bis-(O-aminophenoxy)-ethane-H,N,N',N'-tetraacetic acid, and then primed with PAF, normal amounts of inositol 1,4,5-trisphosphate were formed, but no increase in [Ca2+]i occurred after addition of FMLP even though the cells exhibited a fully primed O2- response; in Ca2(+)-depleted and ionomycin-permeabilized cells that were primed with PAF and then stimulated with FMLP, O2- was generated in amounts comparable to reference control (primed) cells, but there was suppressed production of inositol 1,4,5-trisphosphate and no increase in [Ca2+]i after addition of FMLP to PAF-primed cells. These data confirm the requirement of [Ca2+]o for optimal priming of neutrophils by PAF and ionomycin (but not cells primed by PMA) and indicate that, under certain conditions, generation of O2- in response to FMLP in PAF-primed neutrophils can occur independent of any increase in [Ca2+]i.     

Flumethason-induced calving is preceded by a period of myometrial inhibition during luteolysis

The temporal relationship among changes of the concentrations of the 13,14-dihydro-15-keto metabolite of prostaglandin F2 alpha (PGFM), estrone (E1) and estrone sulphate (E1S) in maternal arterial plasma (MP) and amniotic fluid (AF), the prepartum progesterone (P4) decline in MP, and the evolution of uterine electromyographic (EMG) activity was investigated in 6 cows. Calving was induced by a single i.m. injection of 5 mg flumethason on Day 270 of gestation. The period under investigation was subdivided into four consecutive periods: Period 1 covered the last 2 days before flumethason treatment; Period 2 (mean +/- SEM duration: 16.1 +/- 2.5 h), Period 3 (8.8 +/- 1.1 h), and Period 4 (13.0 +/- 1.5 h) together included the interval between injection and the onset of the expulsive stage of induced parturition. Each was defined by its pattern of uterine EMG activity. During Periods 1 and 2, this activity occurred in long episodes (2-20 min; contractures) at a similar mean (+/- SEM) frequency (0.51 +/- 0.14/h and 0.42 +/- 0.07/h, respectively). No significant differences in hormonal concentrations in MP and AF between these two periods were detected. During Period 3, contractures nearly disappeared (freq: 0.09 +/- 0.05/h), and in MP mean P4 levels were significantly lower and PGFM levels were significantly higher than before. Mean PGFM concentrations in AF were not significantly changed during Period 3. Finally, during Period 4, EMG activity reappeared and a parturient EMG pattern gradually evolved in the presence of a further significant decline of P4 levels and significant increase of PGFM concentrations in MP.(ABSTRACT TRUNCATED AT 250 WORDS)     

不同活性氧胁迫下Bacillus sp. F26以抗氧化物酶合成为特征的应激响应

采用不同的活性氧发生源, 研究了· 、H2O2和OH·胁迫下Bacillus sp. F26以抗氧化物酶合成为特征的应激响应。结果表明, 细胞对氧胁迫的应激响应程度取决于活性氧种类、胁迫程度和形式(瞬时和持续)。Bacillus sp. F26对H2O2胁迫的响应程度最高, 过氧化氢酶的快速合成对细胞抵抗H2O2胁迫至关重要, 当细胞及时分解进入胞内的H2O2, 胁迫对细胞的氧化损伤程度并不高, 相反会刺激细胞的生长和底物消耗, 当胁迫超过过氧化氢酶的分解能力时, H2O2会迅速抑制细胞生长和过氧化氢酶合成; 由于 ·与细胞作用的方式和效果与H2O2不同, 超氧化物歧化酶和过氧化氢酶的快速合成并不能保证细胞及时有效地清除胞内的活性氧, 因此, 细胞对 ·胁迫的响应程度要低于H2O2胁迫; 在所考察的3种活性氧中, OH·胁迫(Fenton反应体系)对细胞的氧化损伤程度最大, 胁迫强烈地抑制了细胞生长和抗氧化物酶的合成。由此表明, 由于不同活性氧的化学性质有所不同, 细胞对不同种类、程度和形式的活性氧胁迫会表现出不同的生物学效应, 为了提高自身对氧胁迫的抵抗能力, 微生物会通过自身的代谢调节适应新的环境, 包括调整抗氧化物酶合成水平、改变生长速度以及底物消耗速率等。     

Dissociation between aggregation and superoxide production in human granulocytes

Aggregation and the activation of the granulocyte (PMN) superoxide (O2-) generating system occur when certain stimuli are added to resting cells. It had previously been postulated that PMN aggregation is essential for maximal O2- production. This study was undertaken to test the hypothesis that PMN aggregation is required for full expression of PMN O2- production. We examined aggregation and O2- production induced by four stimuli; concanavalin A (Con A), phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (FMLP), and ionophore A23187. Cytochalasin B enhanced aggregation by all four stimuli but only enhanced the rate of O2- production by Con A; 2-deoxyglucose inhibited aggregation by all stimuli. Dissociation of PMN aggregation and O2- production was achieved by using NEM, TPCK, and divalent cations. NEM and TPCK prevent Con A-induced O2- production but have no effect on Con A-induced aggregation. PMA-stimulated PMN generate O2- in the presence or absence of Ca++ and Mg++. In contrast, PMA stimulated maximum PMN aggregation only in the presence of both Ca++ and Mg++. Thus PMN can generate O2- without aggregating, and PMN can aggregate without producing O2-. PMN from patients with chronic granulomatous disease do not generate O2- or undergo membrane potential depolarization in response to PMA. These PMN aggregated when stimulated with PMA, providing evidence that depolarization is not required for PMN aggregation. We conclude that aggregation and the activation of the O2- generating system, though temporally related, are not necessarily causally related.     

Effect of psoralens on Fenton-like reaction generating reactive oxygen species

Psoralens (psoralen, 5-methoxypsoralen, 8-methoxypsoralen, khellin, and visnagin) in 1 mM doses were shown to enhance the generation of reactive oxygen species, such as the hydroxyl radical (HO*), the superoxide anion radical (O2(-)), and singlet oxygen ((1)O(2)), from the system generating chemiluminescence (CL), as well as free radicals in the absence of light. The system that generated CL was made up of CoCl(2) and H(2)O(2). Incubation of psoralens in 0.2 mM doses with the generating system showed that only 8-methoxypsoralen and khellin have antioxidative effects. Antioxidative effects were also observed in the case of visnagin but in low concentration (0.05 mM). High doses of psoralens (1 mM) showed prooxidative effects. Measurements were done using a deoxyribose assay, the CL method, and spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide and 2,2,6,6-tetramethylpiperidine combined with electron spin resonance spectroscopy and spectrophotometry methods.     

The mechanism of action of lymphokines. IX. The enzymatic basis of hydrogen peroxide production by lymphokine-activated macrophages

The purpose of this study was to elucidate the biochemical basis of the enhanced hydrogen peroxide (H2O2) production by guinea pig peritoneal macrophages (MP) cultured in lymphokine (LK)-containing medium. The markedly augmented H2O2 generation by these cells, demonstrable by the horseradish peroxidase (HRP)-catalyzed oxidation of phenol red, is distinguished by its lack of dependence on a second stimulus. We demonstrate that H2O2 production is truly spontaneous and is not caused by a stimulant present among the H2O2 assay reagents. The principal candidate for such a role was HRP type II (a mixture of five isoenzymes) that was reported to be capable of eliciting an oxidative burst in MP. Four distinct HRP isoenzymes that were found incapable of provoking an oxidative response were nevertheless adequate for demonstrating H2O2 production by LK-activated MP. Blocking the MP receptor for mannose by the addition of mannan to the assay system resulted in enhanced detection of H2O2 by low concentrations of HRP type II and by three out of four HRP isoenzymes. Treatment of MP with LK-containing medium for 72 hr did not result in a significant change in the activity of cellular superoxide dismutase (SOD) compared with MP cultured for the same length of time in control medium. By using the specific inhibitor of copper, zinc-containing SOD, sodium diethyldithiocarbamate (DDC), and the universal SOD inhibitor, sodium nitroprusside, we found that the predominant enzyme in guinea pig peritoneal MP is probably manganese-containing SOD. Incubation of LK-activated MP with nitroprusside resulted in almost total inhibition of H2O2 production and a simultaneous switch to superoxide (O2-) liberation. Similar exposure to DDC had no effect. These data indicate that H2O2 produced by LK-activated MP is derived exclusively by enzymatic dismutation of O2- mediated by a manganese-containing SOD. The increase in spontaneous H2O2 production induced by LK is therefore secondary to augmented O2- production that occurs at a cellular location where O2- is accessible to SOD. The enzymatic basis of the enhanced oxygen radical production was investigated by determining the kinetic parameters of the O2- -forming NADPH oxidase of resting LK-treated MP in a cellfree system in which O-2 production was induced by sodium dodecyl sulfate. The Km for NADPH and the Vmax of the enzyme of LK-treated MP were not different from those of the enzyme of MP incubated in control medium. We conclude that LK treatment of MP does not modulate the NADPH oxidase itself but, most likely, a process related to activation of the enzyme.     

Prostaglandin E1 inhibits N-formyl-methionyl-leucyl-phenylalanine-mediated depolarization responses by decreasing the proportion of responsive cells without affecting chemotaxin-induced forward light scatter changes

Hypaque-Ficoll-purified human polymorphonuclear neutrophils (PMN) equilibrated with the membrane potential-sensitive probe 3,3'dipentyloxacarbocyanine [di-O-C(5)(3)] were incubated with buffer or cytochalasin B (cyto B) followed by incubation with prostaglandin E1 (PGE1) (0 to 10(-5) M) for 5 min at 37 degrees C. The cells were then stimulated with N-formyl-methionyl-leucyl-phenylalanine (FMLP) (0 to 10(-5) M). Changes in forward light scatter (FWD-SC), 90 degrees scatter (90 degrees -SC), and fluorescence intensity were measured by flow cytometry to determine the effects of PGE1 on FMLP-induced shape change, secretion, and membrane potential responses, respectively. In other experiments, the effects of PGE1 preincubation on FMLP +/- cyto B and phorbol myristate acetate-induced (O2) production were measured by superoxide dismutase-inhibitable cyto c reduction. PGE1 had no direct effects on the FWD-SC, 90 degrees-SC, or resting potential fluorescence of unstimulated or cyto B-pretreated PMN. PGE1 produced a dose-dependent inhibition of the proportion of depolarizing PMN in response to FMLP, which was maximal at 10(-6) M (42.1 +/- 6.9% inhibition, p less than 0.005), but was apparent at 10(-8) M. The PGE1-induced inhibition was maximal after 30 sec of incubation at 37 degrees C and was caused by a decrease in the maximal percentage of depolarizing PMN without a significant change in the FMLP dose-response curve (Km = 2.43 vs 3.62 X 10(-8) M, control vs PGE1-treated) or an inhibition in the degree of depolarization by the responding subpopulation. PGE1 also inhibited the loss of 90 degrees-SC induced by FMLP in cyto B-pretreated cells (secretion response) (46.2 +/- 16.5% inhibition of the maximal 90 degrees-SC loss, n = 5, p less than 0.005), but did not affect the increase in FWD-SC seen with FMLP-induced PMN activation or the ability of cyto B to recruit more PMN to depolarize. PGE1 also inhibited FMLP +/- cyto B-induced O2 production in a dose-dependent fashion; phorbol myristate acetate-induced O2 production was also slightly inhibited, but only at high PGE1 concentrations. The data indicate that PGE1 inhibits FMLP-induced cell activation by a mechanism that involves a step distal to the recruitment of unresponsive PMN by cyto B, and that PGE1 is capable of inhibiting depolarization responses without affecting FMLP-induced shape change, providing more support for a dissociation between the two activation pathways.     

Study on immunocapture-chemiluminescence assay of lipase activity in a biological sample.

A new approach for the determination of lipase (triacylglycerol lipase, EC.3.1.1.3) activity in a biological sample was investigated by combining an immunocapture technique with a chemiluminescence (CL) assay method in order to eliminate interference with CL detection. The proposed method consists of an immunocapture step to trap lipase and a subsequent step for CL detection of the activity of the captured lipase. The CL detection is based on the luminol-hydrogen peroxide (H(2)O(2))-horseradish peroxidase (HRP) reaction and utilizes a proenhancer substrate [a lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI)] which liberates an active enhancer, HDI, by enzymatic hydrolysis. A polyclonal antibody prepared with porcine pancreas lipase was used for the immunocapture. The proposed immunocapture-CL method effectively eliminated the interference with the CL reaction from biological components and enabled the determination of spiked porcine pancreas lipase activity in serum samples in the range 0.41-1.1 U(HDI) (1 U(HDI) corresponds to the amount which liberates 1 pmol HDI/min at 37 degrees C from the substrate). The method was further applied to the assay of the activity for human pancreas lipase in serum and the results showed good correlation (r = 0.871) with those by the conventional colorimetric method.