• The Korean Journal of Pharmacology
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• v.26 no.1
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• pp.55-66
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• 1990

In both resting and opsonized zymosan activated neutrophils, ATP stimulated superoxide generation, whereas adenosine inhibited it slightly. The superoxide generation in activated neutrophils to ATP was greater than that of resting neutrophils. In $Ca^{++}$ free medium, inhibitory effect of adenosine on superoxide generation was detectable, whereas ATP did not have any effect. The stimulatory effect of ATP on superoxide generation was inhibited by adenosine in a dose dependent manner. Neither ATP nor adenosine had any effect on NADPH oxidase acitivity. Effects of ATP or adenosine on superoxide generation were more prominent than that by other triphosphate nucleotides or nucleosides. ATP and ADP further stimulated $Ca^{++}$ uptake and increased cytosolic free $Ca^{++}$ level in neutrophils activated by opsonized zymosan, but adenosine inhibited a $Ca^{++}$ mobilization. Verapamil effectively and tetrodotoxin slightly inhibited an increase of cytosolic free $Ca^{++}$ level induced by ATP. Inhibitory effect of either verapamil or tetrodotoxin on superoxide generation in the ATP plus opsonized zymosan-activated neutrophils was greater than in the cells activated by opsonized zymosan alone. Tetraethylammonium chloride had no apparent effect on superoxide generation. CCCP, 2,4-dinitrophenol, diphenylhydantoin and procaine all inhibited superoxide generation in neutrophils activated by opsonized zymosan. Among these, CCCP only inhibited a stimulatory effect of ATP. ATP further stimulated a loss of sulfhydryl groups in activated neutrophils, whereas adenosine had no effect on it. These results suggest that functional responses of neutrophils may be regulated at least partly by purines. ATP and adenosine may further after functional responses of activated neutrophils through their effect on $Ca^{++}$ uptake, membrane phosphorylation and oxidation of soluble sulfhydryl groups.

• YAKHAK HOEJI
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• v.36 no.5
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• pp.460-468
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• 1992

Several bichemical parameters related to free radicals were estimated in senile-prone (P) and resistant(R) strains of male senescence-accelerated mice(SAM) at 2, 5 and 11 months of age. The superoxide generation was increased with age in SAM-R/1 and SAM-P/2. Compared to SAM-R/1, more generation of superoxide was significantly noted in the SAM-P/2 liver. The activities of Cu/Zn-superoxide dismutase and catalase were decreased during aging and these activities in SAM-P/2 were significantly lower than in SAM-R/l liver. The activities of glutathione S-transferase were varied with aging, whereas SAM-P/2 showed lower levels compared to SAM-R/l. The gradual decreases of glutathione, protein bound-SH and nonprotein bound-SH contents were noted with increasing age. SAM-P/2 liver contained lesser amounts of glutathione and nonprotein bound-SH compared to SAM-R. In conclusion, superoxide generation was increased whereas the antioxidant enzyme activities were decreased during aging in SAM-R/1. In addition, SAM-P/2 strain showed more superoxide generation and less antioxidant enzyme activities than SAM-R/1 in the liver, thus we assume that these factors might accelerate the senescence of SAM-P/2 strain.

• The Korean Journal of Pharmacology
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• v.25 no.1
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• pp.75-85
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• 1989

In opsonized zymosan activated PMN leukocytes, N-ethylamleiamide and $Hg^{++}$, penetrable sulfhydryl group inhibitors, inhibited superoxide generation, NADPH oxidase activity and lysosomal enzyme (lactic dehydrogenase and ${\beta}-glucuronidase$) secretion. P-Chloromercuribenzoic acid and p-chloromercuribenzenesulfonic acid, surface sulfhydryl group inhibitors did not affect superoxide generation but effectively inhibited both NADPH oxidase activity and lysosomal enzyme secretion. During phagocytosis, contents of surface and soluble sulfhydryl groups were gradually decreased with increasing incubation times. N-ethylmaleiamide and $Hg^{++}$ caused a loss of both surface and soluble sulfhydryl groups. P-Chloromercuribenzoic acid and p-chloromercuribenzenesulfonic acid significantly decreased the surface sulfhydryl content but did not after soluble sulfhydryl groups. Cysteine and mercaptopropionylglycine inhibited superoxide generation and lysosomal enzyme secretion. Glutathione had no effect on superoxide generation but remarkably inhibited lactic dehydrogenase release. Suppression of superoxide generation by N-ethylmaleiamide was reversed by cysteine and mercaptopropionyl-glycine but not by glutathione. Inactivation of NADPH oxidase by N-ethylmaleiamide was prevented by glutathione, cysteine or mercaptopropionylglycine. Stimulated superoxide generaion by carbachol was completely abolished by N-ethylrnaleiamide and antagonized by atropine. Thus, the expression of PMN leukocyte response to external stimuli may be associated with the change of sulfhydryl groups content. It is suggested that lysosomal enzyme secretion is influenced by both surface and soluble sulfhydryl groups, whereas superoxide generation by intracellular soluble sulfhydryl groups.

• BMB Reports
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• v.28 no.3
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• pp.210-215
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• 1995

Effects of staurosporine, genistein and pertussis toxin on PMA-induced superoxide generation and degranulation in neutrophils were investigated. Their effects were also examined in C5a-stimulated superoxide generation. PMA-induced superoxide generation was inhibited by staurosporine but was not affected by pertussis toxin. Genistein enhanced the stimulatory effect of PMA in a dose dependent fashion. C5a-induced superoxide generation was inhibited by staurosporine, genistein and pertussis toxin. An NADPH oxidase system of resting neutrophils was activated by PMA, and the stimulatory effect of PMA was inhibited by staurosporine but was not affected genistein and pertussis toxin. The activity of NADPH oxidase in the membrane fraction of PMA-activated neutrophils was not affected by staurosporine and genistein. PMA-induced acid phosphatase release was inhibited by staurosporine and genistein, whereas the effect of pertussis toxin was not detected. These results suggest' that the role of protein tyrosine kinase in neutrophil activation mediated by direct activation of protein kinase C may be different from receptor-mediated activation. The action of protein kinase C on the respiratory burst might be affected by the change of protein tyrosine kinase activity.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.1
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• pp.31-36
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• 2007

To elucidate a potential molecular link between diabetes and atherosclerosis, we investigated the role of Janus tyrosine kinase(JAK) for NAD(P)H oxidase-derived superoxide generation in the enhanced proliferative capacity of vascular smooth muscle cells(VSMC) of Otsuka Long-Evans Tokushima Fatty(OLETF) rat, an animal model of type 2 diabetes. An enhanced proliferative response to 10% fetal bovine serum(FBS) and superoxide generation with an increased NAD(P)H oxidase activity were observed in diabetic(OLETF) VSMC. Both the enhanced proliferation and superoxide generation in diabetic VSMC were significantly attenuated by AG490, JAK2 inhibitor, and PP2, Src kinase inhibitor. Tyrosine phosphorylation of proteins in diabetic VSMC, especially JAK2, was increased compared to control VSMC. Furthermore, the enhanced NAD(P)H oxidase activity in diabetic VSMC was significantly attenuated by AG490 in a dose-dependent manner. Together, these results indicate that the signal pathway which leads to diabetes-associated activation of Src kinase/JAK is critically involved in the diabetic VSMC proliferation through NAD(P)H oxidase activation and superoxide generation.

• Tuberculosis and Respiratory Diseases
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• v.41 no.1
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• pp.11-19
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• 1994

Background: Mycobacterium tuberculosis is a facultative intracellular pathogen which persists and multiplies within macrophage. Competent cell mediated immunity by cooperation of both T lymphocyte and macrophage of the host is required to kill the Mycobacterium tuberculosis. But a precise understanding of the pathogenesis of tuberculosis infection in pulmonary alveolar macrophage has not been achived. Research on the macrophage's basic microbicidal mechanism has elucidated the importance of oxygen-dependent or oxygen-independent components. Oxygen dependent processing begins with the reduction of oxygen by NADPH oxidase and generation of superoxide. In this study, the oxidative metabolic status of blood monocyte and pulmonary alveolar macrophage in patients with active pulmonary tuberculosis was accessed and compared with that of healthy control subjects to know whether there was a basic difference in superoxide generation by mononuclear cells between two groups. Methods: Pulmonary alveolar macrophage was purified after performing BAL(bronchoalveolar lavage) through the bronchi of infected lesion by plastic adhesion method. Blood monocyte was purified by Ficoll-Hypaque method. Superoxide generation by blood monocyte and pulmonary alveolar macrophage was measured by ferricytochrome-C reduction method after either stimulated with PMA(phorbol myristate acerate) or non-stimulated states. We also measured the effect of pulmonary tuberculosis patient's serum on superoxide generation by monocyte. Results: 1) Generation of superoxide by alveolar macrophage obtained from patients with pulmonary tuberculosis was little higher than those of controls, and PMA enhanced the generation of 2) Generation of superoxide by blood monocyte obtained from patients with pulmonary tuberculosis was little higher than those of control(p>0.05), and PMA more enhanced the generation of superoxide in patientswith pulmonary tuberculosis than those in controls(p<0.02). 3) Patient's serum enhanced the generation of superoxide by blood monocyte obtained from patients with pulmonary tuberculosis and controls, but not in the case of PMA stimulated blood monocyte. Conclusion: The present study suggest that the phenomenon of M.tuberculosis escape the microbicidal action of macrophage was not result of suppressed superoxide generation by blood monocyte and pulmonary alveolar macrophage, rather there might be a factor to stimulate the generation of superoxide by blood monocyte in pulmonary tuberculosis patient serum, but the comparision with effect of control's serum on superoxide generation needs further elucidation.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.5
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• pp.1494-1504
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• 2004

The present paper reports the effects of Hwaotang an atherosclerosis using a spontaneous experimental model, We have also investigated the pharmacological effect of Hwaotang on collagen- and ADP-induced blood platelet aggregation, thrombin-induced conversion of fibrinogen and fibrinolysis in in vitro experiments, and various effects on stimuli-induced superoxide generation in human neutrophils. Hwao-tang was shown to have inhibitory effect on collagen- and ADP-induced blood platelet aggregation, on thrombin-induced conversion of fibrinogen to fibrin and on the activity of plasminogen or plasmin. Hwao-tang also significantly inhibited fMLP-induced superoxide generation in a concentration-dependent manner, but not that induced by arachidonic acid. Hwao-tang inhibited neutrophil functions, including degranulation, superoxide generation, and leukotriene B4 production, without any effect on 5-lipoxygenase activity. In conclusion, the protection of extracts of Hwao-tang on the ischemic infarction induced artificially might be involved to their inhibition of thrombotic action. The results also indicate that Hwao-tang exerts the effects on superoxide generation related to the inhibition of neutrophil functions.

• The Korean Journal of Pharmacology
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• v.23 no.1
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• pp.33-44
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• 1987

NADPH oxidase dependent superoxide generation and phagocytosis in neutrophils stimulated with opsonized zymosan or heat aggregated IgG were coincided with the process of calcium uptake. The responses in activated neutrophils were enhanced with increasing concentrations of extracellular calcium and these effects were significantly inhibited by calcium chelators, EGTA and EDTA. The superoxide generation in activated neutrophils was reduced by dantrolene and chlorpromazine. Calcium antagonists, bepredil, diltiazem, verapamil, nifedipine and nimodipine effectively inhibited the calcium uptake, superoxide generation and phagocytosis in activated neutrophils, and NADPH oxidase activity was also inhibited. The results suggest that calcium antagonists may inhibit the superoxide generation and phagocytosis in activted neurtophils by the inhibition of calcium influx and by the action on intracellular redistribution of calcium and NADPH oxidase system.

• Environmental Mutagens and Carcinogens
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• v.22 no.4
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• pp.229-235
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• 2002

Thimerosal, a widely used preservative, has been well known to induce intracellular calcium mobilization in various cell types. However, the mechanism of its calcium mobilization is not clearly understood yet. For studying the mechanism of thimerosal-mediated calcium release, we have used HL60 cells in calcium-free Lockes solution that has no extracellular calcium. Thimerosal significantly reduced the lag period of initial calcium release whereas it enhanced the rate and magnitude of the calcium release in a dose-dependent manner. At the same time, we found that thimerosal generated superoxide anion by activating NADPH oxidase in dose- and time-dependent manner. Interestingly, the kinetics and the dosedependency of superoxide anion generation were very similar to those of intracellular calcium mobilization. In inhibitors study, the thimerosal-induced superoxide anion generation was significantly suppressed by DMSO as well as superoxide dismutase but not by genistein or EGTA. Surprisingly, the pretreatment with N-Acetyl-$_{L}$-Cysteine blocked almost completely the thimerosal-induced calcium increase, indicating that ROS playa key role in the calcium mobilization. The present results suggest that thimerosal-induced calcium mobilization is possibly mediated by the activation of NADPH oxidase and subsequent ROS generation.n.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.2
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• pp.477-481
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• 2006

A clinical report indicates that 'Geijibokryunghwan(GBH) is very effective in treating thrombosis in those patients who have difficulties with more conventional antithrombotic drugs. The isolation and identification of various compounds from this plant and the same genus have been reported by several groups. However, the pharmaceutical effect of the GBH on superoxide generation in human neutrophils has not been studied. In the present report, we investigated the possibility of using herbal medicine as an alternative therapy. In particular, we studied tremor in antiatheroscleosis. In this report, we shows the GBH extract can be used as a potential atherosclerosis preventive agent in human. The effect of GBH on stimulus-induced superoxide generation and phosphorylation of tyrosine residues of protein in human neutrophils was investigated. In a conclusion, GBH suppressed tyrosine phosphorylase in a dose-dependent manner, and may have pharmacoceutical applications. These data suggest that GBH extracts merits investigation as a potential anti-atherosclerogenic agent in humans.