1.Roles of intracellular calcium and monomeric G-proteins in regulating exocytosis of human neutrophils.
Ying ZHU ; Jun-Han WANG ; Jian-Min WU ; Tao XU ; Chun-Guang ZHANG
Acta Physiologica Sinica 2003;55(6):699-704
Neutrophils play a major role in host defense against microbial infection. There are some clues indicate that neutrophils may also play a role in the pathophysiology of the airway obstruction in chronic asthma. We studied the roles of intracellular calcium and GTP gamma S in the regulation of neutrophils exocytosis using pipette perfusion and membrane capacitance measurement technique in whole cell patch clamp configuration. The results showed that the membrane capacitance increase induced by calcium revealed a biphasic process. The first phase occurred when the calcium level was between 0.2-14 micromol/L with a plateau amplitude of 1.23 pF and a calcium EC50 of 1.1 micromol/L. This phase might correspond to the release of the tertiary granules. The second phase occurred when the calcium concentration was between 20-70 micromol/L with a plateau increment of 6.36 pF, the calcium EC50 being about 33 micromol/L. This phase might represent the release of the primary and secondary granules. Intracellular calcium also simultaneously increased the exocytotic rate and the eventual extent in neutrophils. On the other hand, GTP gamma S can increase the exocytotic rate in a dose-dependent manner but had no effect on the eventual extent of membrane capacitance increment (>6 pF) if the cell was stimulated for a long period (>20 min). GTP gamma S (ranging from 20 to 100 micromol/L) induced the neutrophils to release all four types of the granules at very low intracellular calcium level.
Calcium
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metabolism
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Cell Degranulation
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drug effects
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Exocytosis
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drug effects
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GTP-Binding Proteins
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metabolism
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physiology
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Guanosine Triphosphate
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analogs & derivatives
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pharmacology
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Humans
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Neutrophils
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metabolism
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physiology
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Patch-Clamp Techniques
2.Effect of spearmint oil on lipopolysaccharide induced emphysema-like changes and expression of matrix metalloproteinase-9.
Junbo LIU ; Yan WANG ; Fadi TANG ; Chenxi YU ; Mengshan HUANG ; Xiaojing ZHAO ; Youfa ZHU
China Journal of Chinese Materia Medica 2011;36(8):1054-1059
OBJECTIVETo investigate the effect of spearmint oil on emphysema-like changes and the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta(IL-1beta), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-9) in lipopolysaccharide (LPS) treated rats.
METHODEmphysematous changes model was induced by intratracheal instillation of LPS once a week for up to 8 weeks in rats. Rats were divided into control, dexamethasone (0.3 mg x kg(-1)), and spearmint oil (10, 30,100 mg x kg(-1)) groups. Each group was treated with saline, dexamethasone, and spearmint of oil respectively for 4 weeks. Then total and different white blood cell counts in bronchoalveolar lavage fluid(BALF) were carried out. The pathologic changes of lung tissue such as alveolar structure, airway inflammation, and goblet cell metaplasia were observed by HE and AB-PAS staining. Expression of TNF-alpha, IL-1beta, TIMP-1 and MMP-9 were measured.
RESULTBoth spearmint and dexamethasone decreased the destruction of pulmonary alveolus. The total and different white blood cell counts in BALF including neutrophile and lymphocyte of spearmint oil 100 mg x kg(-1) and dexamethasone group were significantly reduced, and the goblet cell metaplasia was also inhibited. Dexamethasone had inhibitory effect on the expression of TNF-alpha, IL-1beta, TIMP-1 and MMP-9. Spearmint oil 30, 100 mg x kg(-1) significantly reduced TNF-alpha and IL-1beta respectively. Spearmint oil 10, 30 and 100 mg x kg(-1) had no effect on the expression of TIMP-1, but could decrease the expression of MMP-9 significantly in lung tissues.
CONCLUSIONSpearmint oil has protective effect on rats with emphysematous changes, since it improves alveolar destruction, pulmonary inflammation, and goblet cell metaplasia. The mechanism may include reducing TNF-alpha, IL-1beta content and inhibiting overexpression of matrix metalloproteinase-9 in lung tissues.
Animals ; Azo Compounds ; pharmacology ; Bronchoalveolar Lavage Fluid ; cytology ; Goblet Cells ; drug effects ; Interleukin-1beta ; drug effects ; metabolism ; Leukocytes ; drug effects ; metabolism ; Lipopolysaccharides ; Lymphocytes ; drug effects ; metabolism ; Matrix Metalloproteinase 9 ; drug effects ; metabolism ; Mentha spicata ; chemistry ; Metaplasia ; Monocytes ; drug effects ; metabolism ; Neutrophils ; drug effects ; metabolism ; Phytotherapy ; Plant Oils ; therapeutic use ; Pulmonary Emphysema ; chemically induced ; drug therapy ; enzymology ; pathology ; Rats ; Respiratory System ; drug effects ; pathology ; Tissue Inhibitor of Metalloproteinase-1 ; drug effects ; metabolism ; Tumor Necrosis Factor-alpha ; drug effects ; metabolism
3.Modulation of lianbizi injection (andrographolide) on some immune functions.
Guang-yong PENG ; Feng ZHOU ; Ru-ling DING ; Huan-di LI ; Kun YAO
China Journal of Chinese Materia Medica 2002;27(2):147-150
OBJECTIVETo study the effects of andrographolide on immune functions and the immune mechanism of its clinical application.
METHODThe amounts of IFN-alpha, IFN-gamma, TNF-alpha, IL-8 in the peripheral blood mononuclear cells(PBMCs) culture supernatants deal with by different concentrations of LianBiZhi (LBZ) injection made of andrographolide were detected by biological activity test or ELISA in vitro. The effects of LBZ injection on macrophage phagocytotic function and natural killer cells cytotoxicity were examined by means of macrophage to phagocytize cock erythrocyte and measurement of lactate dehydrogenase(LDH) activity released from the damaged cells, respectively.
RESULTThe LBZ injection could promote IFN-alpha, IFN-gamma, TNF-alpha inductions of PBMCs, but had no effect on IL-8. At the same time, the LBZ injection could not only enhance the phagocytosis activity of peritoneal macrophage from guinea pig to phagocytosis cock erythrocyte, but also augment the cytotoxicity mediated by natural killer cells from PBMCs to damage the K562 cell lines.
CONCLUSIONAndrographolide is an immunostimulant agent which can modulate both antigen specific and nonspecific immune function by means of its natural killer cells and macrophage and cytokines induction.
Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Chickens ; Diterpenes ; pharmacology ; Guinea Pigs ; Humans ; Injections ; Interferon-alpha ; metabolism ; Interferon-gamma ; metabolism ; Killer Cells, Natural ; drug effects ; Macrophages, Peritoneal ; drug effects ; Neutrophils ; metabolism ; Phagocytosis ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism
4.Effect of haw leaf extract and its preparation on polymorphonuclear leucocyte adhesion during HUVEC anoxia/reoxygenation injury.
Peng LI ; Jian-hua FU ; Xin-zhi LI
Chinese Journal of Integrated Traditional and Western Medicine 2008;28(8):716-720
OBJECTIVETo study the effect and molecular mechanism of two haw leaf extracts, Vitexin-rhamnoside (VR) and Vitexin-glucoside (VG), and their preparation, Aoshaen injection (AI), on the polymorphonuclear leucocyte (PMN) adhesion during human umbilical vein endothelial cell (HUVEC) anoxia/reoxygenation (A/R) injury.
METHODSThe cell model of A/R injury duplicated by breaking off the oxygen supplying of HUVEC for 60 min followed with reoxygenating for 30 min (phase 1) or 240 min (phase 2) was taken as the experimental objective. The effects of testing drugs (VR, VG and AI) on PMN adhesion in the model cells were measured by enzyme immunoassay, and their effects on PMN superficial adhesion molecule CD11/CD18 expression were measured by flow cytometer respectively.
RESULTSAfter 60 min of anoxia, HUVEC was shrunk and deformed. The adhesion between PMN and HUVEC significantly revealed at phase 1 in the model group, but it was fewer in the normal cell group, and also lesser in the groups treated with various drugs. The condition of cell adhesion revealed at phase 2 was the similar to that at phase 1. All testing drugs, VR, VG and AI, showed inhibitory effect on the cell adhesion at either phase 1 or phase 2, showing a certain dose-effect relationship. The expression of CD11/ CD18 was also inhibited by the testing drugs, and a good dose-effect relation was shown by VG and AI.
CONCLUSIONAt the resting condition, there are almost no expression of CD11/CD18 molecule, but it could be enhanced by incubating PMN with supernate of A/R injured HUVEC culture, and more marked at phase 1. Adding the test drugs into the supernate could inhibit the enhancing of CD11/CD18 molecule expression and reduce the PMN-HUVEC adhesion, which may be one of the molecular mechanisms of haw leaf extracts and their preparation in protecting heart against A/R injury.
Cell Adhesion ; drug effects ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Crataegus ; chemistry ; Female ; Humans ; Hypoxia ; drug therapy ; physiopathology ; Neutrophils ; drug effects ; physiology ; Oxygen ; metabolism ; Plant Extracts ; pharmacology ; Plant Leaves ; chemistry ; Pregnancy ; Umbilical Veins ; cytology ; drug effects
5.Effects of hydrocortisone on oxygen free radicals released by polymorphonuclear neutrophils in lipopolysaccharide-induced acute lung injury in mice.
Li ZHANG ; Kun-ling SHEN ; Tao ZHOU ; Yue-qiang XUE ; Peng YANG
Chinese Journal of Pediatrics 2004;42(9):659-662
BACKGROUNDCorticosteroid treatment of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) has been one of the most controversial clinical issues in critical care. Although the administration of high-dose corticosteroids does not benefit patients with early septic shock and ARDS, recent clinical trials have indicated that treatment with relatively low-dose corticosteroids (2 to 3 mg/kg/day of methylprednisolone or equivalent) may improve outcome when used for late ARDS or persistent septic shock. The underlying mechanism was not fully clarified. Whether the administration of corticosteroids can arrest neutrophil-driven organ injury once started remains to be elucidated.
OBJECTIVETo observe the effects of hydrocortisone (HC, 6 mg/kg) on oxygen free radicals (OFR) released by PMN and pulmonary pathological changes in rat ALI model induced by lipopolysaccharide (LPS), to investigate the possible mechanism through which corticosteroids exert protective effect on ALI.
METHODSA rat model of ALI was induced by peritoneal injection of 2 x 10(12) Escherichia coli/kg. Fifty-six rats were randomly divided into three groups: normal control group, LPS group and HC group (6 mg/kg). Samples were collected 2 h, 4 h and 6 h after giving LPS to LPS and HC group (6 h after giving normal saline in normal control group) to measure the level of OFR released by PMN using chemiluminescence method based on lumino, and to compae of pulmonary pathological changes among the three groups.
RESULTSPathological examination with light microscope in LPS group showed thickened pulmonary interstitia, inflammatory cell infiltration, edema and hemorrhage, which were in accordance with the features of ALI. There were significant differences in the release of OFR by PMN among the three groups (P < 0.01). The level of OFR released by PMN in LPS group was significantly higher than that of the control group, and continued to increase during the observation period (2 - 6 h after LPS). The release of OFR by PMN in HC group was significantly suppressed as compared with LPS group, which was peaked at 4 h after LPS injection (to 98.2%); there were also significant differences in the grades of ALI pathologic changes among the three groups (P < 0.01). The grades of ALI pathologic changes in LPS group were significantly increased when compared with the normal control group (P < 0.05) while significantly decreased in HC group as compared with LPS group (P < 0.05).
CONCLUSIONIt was demonstrated in the LPS induced ALI model that OFR might play an important role in onset of ALI. Intervening with HC (6 mg/kg) treatment could ameliorate the lung injury and exert significant and sustained suppression on the release of OFR by PMN, showing that HC has a protective effect on LPS induced ALI and its theraputic effect occurs possibly through suppression on the release of OFR by PMN.
Acute Lung Injury ; etiology ; immunology ; Animals ; Disease Models, Animal ; Free Radicals ; metabolism ; Glucocorticoids ; pharmacology ; Hydrocortisone ; pharmacology ; Lipopolysaccharides ; adverse effects ; Lung ; immunology ; pathology ; Mice ; Neutrophils ; drug effects ; metabolism
6.Prolongation of functional life-span of neutrophils by recombinant verotoxin 2.
Jiajia LIU ; Tao HE ; Yanzheng HE ; Zhongjie ZHANG ; Tohru AKAHOSHI ; Hirobumi KONDO ; Sen ZHONG
Chinese Medical Journal 2002;115(6):900-903
OBJECTIVEVerotoxin-producing Escherichia coli (VTEC) strains of serotype O157 : H7 have been implicated in a wide spectrum of diseases, including blood diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). To further explore the pathological role of verotoxin (VT) in HUS and other VTEC associated diseases, we investigated the effects of recombinant verotoxin 2 (rVT2) on the biological activity of neutrophils.
METHODSThe technique of flow cytometry, a fluorescent probe 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM), and the assay of reduced cytochrome c to detect superoxide production were used in this study.
RESULTSgammaVT2 significantly inhibited spontaneous apoptosis in neutrophils. Neutrophils with prolonged survival due to gammaVT2 maintained various biological functions, such as the expression of adhesion molecules (shading CD62L and raising CD11b/CD18), adherence to human umbilical vein endothelial cells (HUVECs), and generation of superoxide (O(2)(-)).
CONCLUSIONProlongation of the functional life-span of neutrophils by gammaVT2 may accelerate inflammatory responses at sites of inflammation. This may play a crucial role in neutrophil-mediated tissue injury in HUS and other VTEC-associated diseases.
Apoptosis ; drug effects ; Cell Adhesion ; Endothelium, Vascular ; cytology ; Humans ; Neutrophils ; drug effects ; physiology ; Recombinant Proteins ; toxicity ; Shiga Toxin 2 ; toxicity ; Superoxides ; metabolism
7.Inhibition of Janus activated kinase-3 protects against myocardial ischemia and reperfusion injury in mice.
Young Bin OH ; Min AHN ; Sang Myeong LEE ; Hyoung Won KOH ; Sun Hwa LEE ; Suhn Hee KIM ; Byung Hyun PARK
Experimental & Molecular Medicine 2013;45(5):e23-
Recent studies have documented that Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) pathway can modulate the apoptotic program in a myocardial ischemia/reperfusion (I/R) model. To date, however, limited studies have examined the role of JAK3 on myocardial I/R injury. Here, we investigated the potential effects of pharmacological JAK3 inhibition with JANEX-1 in a myocardial I/R model. Mice were subjected to 45 min of ischemia followed by varying periods of reperfusion. JANEX-1 was injected 1 h before ischemia by intraperitoneal injection. Treatment with JANEX-1 significantly decreased plasma creatine kinase and lactate dehydrogenase activities, reduced infarct size, reversed I/R-induced functional deterioration of the myocardium and reduced myocardial apoptosis. Histological analysis revealed an increase in neutrophil and macrophage infiltration within the infarcted area, which was markedly reduced by JANEX-1 treatment. In parallel, in in vitro studies where neutrophils and macrophages were treated with JANEX-1 or isolated from JAK3 knockout mice, there was an impairment in the migration potential toward interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), respectively. Of note, however, JANEX-1 did not affect the expression of IL-8 and MCP-1 in the myocardium. The pharmacological inhibition of JAK3 might represent an effective approach to reduce inflammation-mediated apoptotic damage initiated by myocardial I/R injury.
Animals
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Apoptosis/drug effects
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Cell Movement/drug effects
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Chemokines/pharmacology
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Heart Function Tests/drug effects
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Inflammation/pathology
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Janus Kinase 3/*antagonists & inhibitors/metabolism
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Macrophages/drug effects/metabolism/pathology
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Male
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Mice
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Mice, Inbred C57BL
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Myocardial Reperfusion Injury/drug therapy/*enzymology/physiopathology/*prevention & control
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Myocardium/enzymology/pathology
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Myocytes, Cardiac/drug effects/metabolism/pathology
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Neutrophils/drug effects/metabolism/pathology
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Quinazolines/pharmacology/therapeutic use
8.A preliminary in vitro study on the activation of polymorphonuclear cells and vascular endothelial cells co-cultured with and stimulated by lipopolysaccharide.
Jianming LI ; Qian CAI ; Hong ZHOU ; Guangxia XIAO
Chinese Journal of Burns 2002;18(2):78-80
OBJECTIVETo explore the activation of polymorphonuclear cells (PMNs) and vascular endothelial cells co-cultured with and stimulated by lipopolysaccharide.
METHODSPMNs in concentration of 2 x 10(6)/ml isolated from healthy volunteers by Percoll gradient were added to monolayer of ECV-304 cells grown to confluency, then different groups were prepared according to final concentration of lipopolysaccharide. The morphological change was observed under invert microscope. The changes in TNFalpha and IL-6 levels of the supernatant of the cultured cells were determined at 4, 8, 12 and 24 hours after culturation.
RESULTSThe TNFalpha production of cultured pure ECV-304 exhibited no remarkable change when stimulated by different concentrations of LPS. But the TNFalpha production of the ECV-304 increased significantly when co-cultured with PMNs at 4 hr and stimulated by LPS in concentration of 10 micro g/ml, and increased at 8 hours and lasted up to 24 hours of culturation in higher levels (P < 0.05). The IL-6 production of cultured pure ECV-304 increased obviously along with the increase of LPS concentration, and it showed no change when PMNs co-cultured with ECV-304. While the IL-6 level in the supernatant of co-cultured ECV-304 with PMNs increased sharply when stimulated by both low (100 ng/ml) and high (1 micro g/ml) concentrations of LPS and maintained at high levels up to 24 hours of culturation. The higher the concentration of LPS was, the quicker the IL-6 level increased.
CONCLUSIONCo-cultured PMNs and endothelial cells could be activated and activation state could be maitained by low concentrations of LPS.
Adult ; Cell Line ; Cells, Cultured ; Coculture Techniques ; Dose-Response Relationship, Drug ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Humans ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; pharmacology ; Neutrophils ; cytology ; drug effects ; metabolism ; Time Factors ; Tumor Necrosis Factor-alpha ; metabolism
9.Antagonistic effect of myricetin on platelet activing factor.
Bao-xia ZANG ; Ming JIN ; Wei WU ; Wen-mei CHEN ; Yong-zhe PIAO ; Jin-rong LI
Acta Pharmaceutica Sinica 2003;38(11):831-833
AIMTo study the antagonistic effect of myricetin on platelet activing factor (PAF).
METHODSThe specific binding of [3H] PAF to rabbit platelet receptor was investigated using radio ligand binding assay (RLBA). Platelet adhesion induced by PAF was measured with spectrophotometry. The elevation of inner free calcium concentration in rabbit polymorphonuclear leukocytes (PMNs) induced by PAF was assayed by Fura-2 fluorescent technique.
RESULTSThe specific binding inhibition potency of Myr was found to be concentration-dependent. The IC50 of Myr in [3H] PAF 1, 2 and 4 nmol.L-1 were 34.8, 85.7 and 118.6 mumol.L-1, respectively. The PAF induced reactions of rabbit platelet adhesion and PMNs inner free calcium concentration increase were inhibited by Myr in a dose-dependent manner. The IC50 of Myr to inhibit platelet adhesion was 13.1 mumol.L-1.
CONCLUSIONThe specific receptor binding of PAF can be antagonized by myricetin.
Animals ; Calcium ; metabolism ; Flavonoids ; pharmacology ; Male ; Neutrophils ; metabolism ; Platelet Activating Factor ; antagonists & inhibitors ; metabolism ; Platelet Activation ; drug effects ; Platelet Adhesiveness ; drug effects ; Platelet Aggregation Inhibitors ; pharmacology ; Platelet Membrane Glycoproteins ; metabolism ; Rabbits ; Receptors, G-Protein-Coupled ; metabolism
10.Inhibitory effect of kaempferol against binding of platelet activating factor to its receptor.
Bao-Xia ZANG ; Ming JIN ; Wei WU ; Wen-Mei CHEN ; Yong-Zhe PIAO ; Jin-Rong LI
China Journal of Chinese Materia Medica 2004;29(8):789-791
OBJECTIVETo observe the platelet activating factor (PAF) antagonistic effect of kaempferol.
METHODThe specific binding of [3H] PAF to rabbit platelet receptor was investigatedwith radio ligand binding assay (RLBA). Platelet adhesion induced by PAF was measured with spectrophotometry. The elevation of inner free calcium concentration in rabbit polymorphonuclear leukocytes (PMNs) induced by PAF was determined with Fura-2 fluorescent technique.
RESULTThe 1, 2 or 4 nmol x L(-1) [3H]PAF specific binding to rabbit platelet receptor was inhibited by Kae dosage dependently and the IC50 were 30.8, 74.6 and 92.0 micro mol x L(-1), respectively. The PAF induced reactions of rabbit platelet adhesion and PMNs inner free calcium concentration elevation were inhibited by Kae in a dose-dependent manner. The IC50 of Kae to inhibit platelet adhesion was 65 micromol x L(-1).
CONCLUSIONKae is effective in inhibiting the action of PAF and it is a new PAF receptor antagonist.
Animals ; Blood Platelets ; drug effects ; physiology ; Calcium ; metabolism ; Kaempferols ; pharmacology ; Male ; Neutrophils ; metabolism ; Platelet Activating Factor ; metabolism ; Platelet Adhesiveness ; drug effects ; Platelet Membrane Glycoproteins ; antagonists & inhibitors ; metabolism ; Rabbits ; Radioligand Assay ; Receptors, G-Protein-Coupled ; antagonists & inhibitors ; metabolism