• The Korean Journal of Physiology and Pharmacology
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• v.2 no.5
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• pp.617-628
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• 1998

In order to understand the pathogenetic mechanism of adult respiratory distress syndrome (ARDS), the role of phospholipase A2 (PLA2) in association with oxidative stress was investigated in rats. $Interleukin-1{\alpha}\;(IL-1,\;50\;{\mu}g/rat)$ was used to induce acute lung injury by neutrophilic respiratory burst. Five hours after IL-1 insufflation into trachea, microvascular integrity was disrupted, and protein leakage into the alveolar lumen was followed. An infiltration of neutrophils was clearly observed after IL-1 treatment. It was the origin of the generation of oxygen radicals causing oxidative stress in the lung. IL-1 increased tumor necrosis factor (TNF) and cytokine-induced neutrophil chemoattractant (CINC) in the bronchoalveolar lavage fluid, but mepacrine, a PLA2 inhibitor, did not change the levels of these cytokines. Although IL-1 increased PLA2 activity time-dependently, mepacrine inhibited the activity almost completely. Activation of PLA2 elevated leukotriene C4 and B4 (LTC4 and LTB4), and 6-keto-prostaglandin $F2{\alpha}\;(6-keto-PGF2{\alpha})$ was consumed completely by respiratory burst induced by IL-1. Mepacrine did not alter these changes in the contents of lipid mediators. To estimate the functional changes of alveolar barrier during the oxidative stress, quantitative changes of pulmonary surfactant, activity of gamma glutamyltransferase (GGT), and ultrastructural changes were examined. IL-1 increased the level of phospholipid in the bronchoalveolar lavage (BAL) fluid, which seemed to be caused by abnormal, pathological release of lamellar bodies into the alveolar lumen. Mepacrine recovered the amount of surfactant up to control level. IL-1 decreased GGT activity, while mepacrine restored it. In ultrastructural study, when treated with IL-1, marked necroses of endothelial cells and type II pneumocytes were observed, while mepacrine inhibited these pathological changes. In histochemical electron microscopy, increased generation of oxidants was identified around neutrophils and in the cytoplasm of type II pneumocytes. Mepacrine reduced the generation of oxidants in the tissue produced by neutrophilic respiratory burst. In immunoelectron microscopic study, PLA2 was identified in the cytoplasm of the type II pneumocytes after IL-1 treatment, but mepacrine diminished PLA2 particles in the cytoplasm of the type II pneumocyte. Based on these experimental results, it is suggested that PLA2 plays a pivotal role in inducing acute lung injury mediated by IL-1 through the oxidative stress by neutrophils. By causing endothelial damage, functional changes of pulmonary surfactant and alveolar type I pneumocyte, oxidative stress disrupts microvascular integrity and alveolar barrier.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.3
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• pp.219-226
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• 2000

In order to elucidate the role of platelet activating factor (PAF) in the acute lung injury induced by endotoxin (ETX), activities of phospholipase A2, lyso PAF acetyltransferase and oxidative stress by neutrophilic respiratory burst were probed in the present study. To induce acute lung injury, $100\;{\mu}g$ of E.coli ETX (type 0127; B8) was instilled directly into the tracheae of Sprague-Dawley rats. Five hours after the ETX instillation, induction of acute lung injury was confirmed by lung leak index and protein contents in the bronchoalveolar lavage (BAL) fluid. At the same time, lung phospholipase A2 (PLA2) activity and expression of group I and II secretory type PLA2 were examined. In these acutely injured rats, ketotifen fumarate, known as lyso PAF acetyltransferase inhibitor and mepacrine were administered to examine the role of PAF in the pathogenesis of the acute lung injury. To know the effect of the ETX in the synthesis of the PAF in the lungs, lyso PAF acetyltransferase activity and PAF content in the lungs were measured after treatments of ETX, ketotifen fumarate and mepacrine. In addition, the role of neutrophils causing the oxidative stress after ETX was examined by measuring lung myeloperoxidase (MPO) and enumerating neutrophils in the BAL fluid. To confirm the oxidative stress in the lungs, pulmonary contents of malondialdehyde (MDA) were measured. After instillation of the ETX in the lungs, lung leak index increased dramatically (p＜0.001), whereas mepacrine and ketotifen decreased the lung leak index significantly (p＜0.001). Lung PLA2 activity also increased (p＜0.001) after ETX treatment compared with control, which was reversed by mepacrine and ketotifen (p＜0.001). In the examination of expression of group I and II secretory PLA2, mRNA synthesis of the group II PLA2 was enhanced by ETX treatment, whereas ketotifen and WEB 2086, the PAF receptor antagonist, decreased the expression. The activity of the lysoPAF acetyltransferase increased (p＜0.001) after treatment of ETX, which implies the increased synthesis of PAF by the remodelling of lysoPAF in the lungs. Consequently, the contents of the PAF in the lungs were increased by ETX compared with control (p＜0.001), while mepacrine (p＜0.001) and ketotifen (p＜0.01) decreased the synthesis of the PAF in the lungs of ETX treated rats. The infiltration of the neutrophils was confirmed by measuring and enumerating lung MPO and the neutrophils in the BAL fluid respectively. Compared with control, ETX increased lung MPO and number of neutrophils in BAL significantly (p＜0.001) whereas mepacrine and ketotifen decrerased number of neutrophils (p＜0.001) and MPO (p＜0.05, p＜0.001, respectively). The lung MDA contents were also increased (p＜0.001) by ETX treatment, but treatment with mepacrine (p＜0.001) and ketotifen (p＜0.01) decreased the lung MDA contents. Collectively, we conclude that ETX increases PLA2 activity, and that the subsequently increased production of PAF was ensued by the remodelling of the lyso PAF resulting in tissue injury by means of oxidative stress in the lungs.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.1
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• pp.71-78
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• 1997

In an attempt to investigate the role of phospholipase $A_2$($PLA_2$) in interleukin-l (IL-l) induced acute lung injury, mepacrine was tried to inhibit $PLA_2$ in IL-l induced ARDS rats. For confirmation of acute lung injury by IL-l, and to know the role of neutrophils in this injury, lung leak index, lung myeloperoxidase(MPO), number of neutrophils and protein content in the bronchoalveolar lavage (BAL) and wet lung weight were measured. At the same time lung $PLA_2$ was measured to know the effect of IL-l on $PLA_2$ activity. Pulmonary surfactant was also measured for an investigation of type II alveolar cell function. Neutrophil adhesion assay was performed to know the effect of $PLA_2$ inhibition in vitro with human umbilical vein endothelial cells (HUVEC). For precise location of injury by IL-l, morpholgical study was performed by electron microscopy. Five hours after instillation of IL-l (50 ng/rat), lung leak index, protein content, number of neutrophils, lung MPO and wet lung weight were increased significantly. Five hours after IL-l instillation lung $PLA_2$ activity was increased significantly, and increased surfactant release was observed in IL-l induced ARDS rats' BAL. In contrast, in rats given mepacrine and IL-l, there was decrease of acute lung injury i.e. decrease of lung leak index, wet lung weight, protein content, number of neutrophils in BAL and decreased lung MPO activity. Mepacrine decreased surfactant release also. Interestingly, inhibition of $PLA_2$ decreased adhesion of human neutrophils to HUVEC in vitro. Morphologically, IL-l caused diffuse necrosis of endothelial cells, type I and II epithelial cells and increased the infiltration of neutrophils in the interstitium of the lung but after mepacrine treatment these pathological findings were lessened. On the basis of these experimental results it is suggested that $PLA_2$ has a major role in the pathogenesis of acute lung injury mediated by neutrophil dependent manner in IL-l induced acute lung injury.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.3
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• pp.263-273
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• 1999

The role of phospholipase $A_2\;(PLA_2)$ in acute lung leak induced by intestinal ischemia was investigated in association with neutrophilic respiratory burst. To induce lung leak, we generated intestinal ischemia for 60 min prior to the 120 min reperfusion by clamping superior mesenteric artery in Sprague-Dawley rats. Acute lung leak was confirmed by the increased lung leak index and protein content in bronchoalveolar fluid. These changes were inhibited by mepacrine, the non-specific $PLA_2$ inhibitor. The lung myeloperoxidase (MPO) activity denoting the pulmonary recruitment of neutrophils was increased by intestinal I/R, but decreased by mepacrine. Simultaneously, the number of leukocytes in bronchoalveolar fluid was increased by intestinal ischemia/reperfusion (I/R) and decreased by mepacrine. Gamma glutamyl transferase activity, an index of oxidative stress in the lung, was increased after intestinal I/R but decreased by mepacrine, which implicates that $PLA_2$ increases oxidative stress caused by intestinal I/R. The $PLA_2$ activity was increased after intestinal I/R not only in the intestine but also in the lung. These changes were diminished by mepacrine. In the cytochemical electron microscopy to detect hydrogen peroxide, intestinal I/R increased the generation of the hydrogen peroxide in the lung as well as in the intestine. Expression of interleukin-1 (IL-1) in the lung was investigated through RT-PCR. The expression of IL-1 after intestinal I/R was enhanced, and again, the inhibition of $PLA_2$ suppressed the expression of IL-1 in the lung. Taken together, intestinal I/R seems to induce acute lung leak through the activation of $PLA_2$, the increase of IL-1 expression associated with increased oxidative stress by neutrophilic respiratory burst.

• Journal of Life Science
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• v.19 no.6
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• pp.818-824
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• 2009

The mechanisms responsible for ischemia/reperfusion (I/R) injury have direct or indirect relevance to clinical lung injury after severe shock, cardiopulmonary bypass, and transplantation. This study investigated the effects of aspirin on intestinal I/R-induced acute lung injury (ALI) in rats. Lipopolysaccharide (LPS) induced cyclooxygenase-2 (COX-2) expression in A549 and RAW264.7 cells. RAW264.7 macrophages had shown greater expression of COX-2 than A549 cells. In addition, the NADPH oxidase inhibitor apocynin and p38 MAPK inhibitor SB203580 attenuated LPS-stimulated COX-2 expression. To induce ALI, intestinal ischemia was performed for 60 min prior to the 4 hr reperfusion by clamping the superior mesenteric artery in Sprague-Dawley rats. In order to test and compare the effect of non-specific COX inhibitor aspirin with the effect of mepacrine, a well known phospholipase$A_{2}$ inhibitor, rats were divided into 4 groups: Sham, I/R, Mepa+I/R (mepacrine, 60 mg/kg, i.p.), ASA+I/R (aspirin, 10 mg/kg, i.p.). In the present investigation, myeloperoxidase activities in the lung and intestinal tissues were increased by I/R. These changes were reduced by single pretreatment of mepacrine (60 mg/kg, i.p.) or aspirin (10 mg/kg, i.p.) 30 min before I/R. Structural studies demonstrated that the tissue injuries in the lung and intestine after I/R were also attenuated by the pretreatment of mepacrine or aspirin. These results suggest that I/R-induced ALI is mediated, in part, by the activation of COX. In addition, pretreatment of aspirin might be helpful for the prevention of ALI in ARDS-prone patients. In addition, the p38 MAPK inhibitor and apocynin also might be helpful to ALI through the inhibition of COX-2 expression.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.3
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• pp.181-187
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• 2009

Intestinal ischemia/reperfusion (I/R) is one of common causes of acute lung injury (ALI). Early and accurate diagnosis of patients who are like to develop serious acute respiratory distress syndrome (ARDS) would give a therapeutic advantage. Ferritin and heme oxygenase-1 (HO-1) are increased by oxidative stress and are potential candidates as a predictive biomarker of ARDS. However, the mechanisms responsible for the increases of ferritin and HO-1, and their relationship to ALI, are unclear. In order to elucidate the interactions between ferritin and HO-1, we studied the changes in ferritin and HO-1 levels in serum and bronchoalveolar lavage (BAL) fluid after intestinal I/R injury in rats. Leukocyte number and protein contents in BAL fluid were elevated following I/R, and the increases were attenuated by mepacrine pretreatment. Both serum ferritin and HO-1 concentrations were progressively elevated throughout the 3 h observation period. Mepacrine pretreatment attenuated the increase of serum and BAL fluid ferritin concentrations, but did not suppress the increase of serum HO-1. Moreover, BAL fluid HO-1 levels did not change after I/R or after mepacrine pretreated I/R compared with sham rats. Unlike ferritin, HO-1 levels are not exactly matched with the ALI. Therefore, there might be a different mechanism between the changes of ferritin and HO-1 in intestinal I/R-induced ALI model.

• Journal of Life Science
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• v.19 no.7
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• pp.878-885
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• 2009

Serum ferritin levels are elevated in subjects with acute lung injury (ALI), and abnormalities in plasma and lung iron chemistry have also been demonstrated in ALI and acute respiratory distress syndrome (ARDS). Stress-inducible heme oxygenase-1 (HO-1), as well as ferritin, had shown anti-inflammatory actions. Biomarkers for early detection in patients who are likely to develop ARDS would give several therapeutic chances to the patients. In order to verify the predictability in severe hemorrhage-induced ALI in rats, we measured serum ferritin and HO-1 concentrations before and after hemorrhage. Severe hemorrhages significantly increased the number of leukocytes in bronchoalveolar lavage (BAL) fluid and lung tissue myeloperoxidase activity. Both serum ferritin and HO-1 levels increased following hemorrhage, but ferritin levels were elevated earlier than HO-1. In BAL cell immunohistochemical studies, ferritin and HO-1 expressions increased after hemorrhage and localized in the cytoplasm of leukocytes. These findings suggest that inflammatory leukocytes in BAL fluid can secrete ferritin and HO-1, and serum ferritin levels might be more valid factor in predicting ARDS than HO-1 levels in hemorrhage-induced ALI.

• YAKHAK HOEJI
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• v.44 no.1
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• pp.80-86
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• 2000

TEA, glibenclamide, L-NAME and SKF 525A-induced contraction were investigated using acetylcholine, sodium nitroprusside (SNP, NO donor) and pinacidil (ATP sensitive $K^{+}$ channel opener) in rat abdominal and thoracic aorta. The relaxant effects of acetylcholine, SNP and pinacidil were not different in the abdominal aorta and in the thoracic aorta. Acetylcholine-induced relaxation was dependent on endothelial cell, but pinacidil was independent endothelia cell. In the presence of TEA, glibenclamide, L-NAME, mepacrine and SKF 525A, acetylcholine and SNP did not change, but pinacidil-induced relaxation was significantly reduced in presence of glibenclamide, which is ATP sensitive $K^{+}$ channel blocker. SKF 525A, which is inhibitor of cytochrome P$_{450}$ dependent epoxygenase, partially inhibited the pinacidil-induced relaxation. These results indicate that the pinacidil-induced relaxation may be mediated by ATP sensitive $K^{+}$ channel and partially by EETs, which is produced by cytochrome P$_{450}$ dependent epoxygenase.enase.

• Proceedings of the KOSOMBE Conference
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• v.1997 no.11
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• pp.333-336
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• 1997

The adhesion of activated and normal platelets to fibrinogen requires the receptor binding site of GPIIb/IIIa. These recognition sites exists in the A ${\alpha}$ chain(RGDS at 572-575 and RGDF at 95-98) and the carboxy-terminal of ${\gamma}$ chain (HHLGGAKQAGDV at 400-411) of fibrinogen. In this study, we developed RGDF-immobilized surface to detect the unctional state of platelet. RGDF-immobilized surface was prepared on the glass using photolithographic technology. Platelet adhesion to RGDF-immobilized surface was observed by staining platelets with mepacrine using a fluorescence microscope using mepacrine. Using the RGDF peptide of fragment E, we observed that the platelets pretreated with PGE1 interacted incompletely with RGDF-immobilized surface, whereas ADP activated platelets interacted with the surface extensively. These results show that the distinct selectivity of RGDF-immobilized micro-patterned surface can be used to detect the unctional state of platelets.

• Tuberculosis and Respiratory Diseases
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• v.51 no.6
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• pp.503-516
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• 2001

Background : The present study was carried out in association with neutrophilic respiratory burst in the lung in order to clarify the pathogenesis of acute respiratory distress syndrome(ARDS) following acute severe hemorrhage. Because oxidative stress has been suggested as one of the principal factors causing tissue injury, the role of free radicals from neutrophils was assessed in acute hemorrhage-induced lung injury. Method : In Sprague-Dawley rats, hemorrhagic shock was induced by withdrawing blood(20 ml/kg of B.W) for 5 min and the hypotensive state was sustained for 60 min. To determine the mechanism and role of oxidative stress associated with phospholipase A2(PLA2) by neutrophils, the level of lung leakage, pulmonary myeloperoxidase(MPO), and the pulmonary PLA2 were measured. In addition, the production of free radicals was assessed in isolated neutrophils by cytochemical electron microscopy in the lung. Results : In hypotensive shock-induced acute lung injury, the pulmonary MPO, the level of lung leakage and the production of free radicals were higher. The inhibition of PLA2 with mepacrine decreased the pulmonary MPO, level of lung leakage and the production of free radicals from neutrophils. Conclusion : A. neutrophilic respiratory burst is responsible for the oxidative stress causing acute lung injury followed by acute, severe hemorrhage. PLA2 activation is the principal cause of this oxidative stress.