High salt diet enhances the physical coupling between TRPV4 and cPLA2 / 中国药理学通报

Aim To observe the physical coupling between transient receptor potential channel vanilloid type 4 (TRPV4 ) and cPLA2 in endothelial cells. Methods We investigated the physical association of TRPV4-cPLA2 coupling by immunofluorescence reso-nance energy transfer (immuno-FRET)to assess the spatial proximity between TRPV4 and cPLA2 in human microvascular endothelial cells (HMEC),primary cul-tured endothelial cells and in thoracic aortas rings from high salt-induced hypertension mice.Results At the cellular level,with high salt treatment,the physical in-teraction of TRPV4 and cPLA2 was significantly en-hanced in primary vascular endothelial cells and HMEC.Furthermore, in thoracic aortas rings from high salt-induced hypertension mice,we found an in-creases interaction between TRPV4 and cPLA2 in en-dothelial cells from arterial segments .Conclusion High-salt treatment increases the endothelial TRPV4-cPLA2 coupling,indicating that this coupling may pro-vide a new target for vascular endothelial dysfunction.

Characteristics of PLA2 activity and expression of PLA2 subtypes in rat pancreas after simple pancreatic trauma / 解放军医学杂志

Objective To study the characteristics of phospholipase A2 (PLA2) activity and expression of PLA2 subtypes in rat serum and pancreas tissue with an established simple pancreatic trauma model in rats. Methods One hundred and twenty Wistar rats were divided into two groups in a one-to-two ratio: control group and impact group. The two groups were subsequently divided into four subgroups at 6h, 24h, 72h, 7d time point. For the animals in the impact groups, the pancreas was injured by a BIM- III biotical impact machine, and the animals in control group were sham injured. The total catalytic activity of PLA2 in serum and pancreatic tissue, the expressions of mRNA and protein and immunohistochemical localization of secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2) and calcium independent PLA2 (iPLA2) in pancreatic tissue were determined after the corresponding pretreatments in each group. Results The total catalytic activity of PLA2 in serum and pancreatic tissue of impact groups increased at 6h after injury, and the PLA2 activity reached peak value at 24h in pancreatic tissue and at 72h in serum after trauma, and at 7d in serum and pancreatic tissue were still higher than that in control group at the same time (P<0.05). The results of RT-PCR, Western blotting and immunohistochemistry indicated that the expression of sPLA2 in pancreatic tissue was higher than that of cPLA2 and iPLA2. Conclusions PLA2 activity can be upregulated in serum and pancreas after pancreatic trauma. The expression of sPLA2 is increased most significantly in pancreatic tissue after pancreatic trauma. PLA2 activity may be detected in serum 6h-7d after pancreatic trauma, and is helpful for auxiliary diagnosis of simple pancreatic trauma.

Protective effect of PNS in spinal cord hemisection injury and its cPLA2 related mechanism / 中国医师杂志

Objective To investigate the protective effect and associated mechanism of PNS in spinal cord hemisection injury.Methods fifty-five adult SD rats were randomly divided into three groups,sham group(n =5),spinal cord injury group(n =25),PNS group(n =25).The rats were evaluated in behavioral test with BBB score,pathology and immunohistochemistry at 1 d,3d,7d,14d,21d after the procedures.Results Motor recovery was significantly better in PNS group than the spinal cord injury group at 3d,7d,14d and 21d.Nissl staining showed less neuron necrosis and more integrated neural cells in morphology.cPLA2 expression was inhabited in PNS group,and less number of positive cells were found in the group.Conclusion PNS can inhibit the expression of cPLA2 after spinal cord injury,which may be one of the mechanisms of its effect on promoting motor recovery.

Impacts of Cytosolic Phospholipase A2, 15-Prostaglandin Dehydrogenase, and Cyclooxygenase-2 Expressions on Tumor Progression in Colorectal Cancer

PURPOSE: In addition to cyclooxygenase-2 (COX-2) which is related to prostaglandin E2 synthesis, other enzymes such as cytosolic phospholipase A2 (cPLA2), microsomal prostaglandin E2 synthase-1 (mPGES-1), and 15-prostaglandin dehydrogenase (15-PGDH) have been suggested to be related to carcinogenesis of colorectal cancer (CRC). The aim of this study was to investigate the roles of cPLA2, COX-2, mPGES-1, and 15-PGDH in tumor progression. MATERIALS AND METHODS: cPLA2, COX-2, mPGES-1, 15-PGDH, and vascular endothelial growth factor (VEGF) expressions were immunohistochemically examined in 89 CRC, and their expressions were compared with each other or clinicopathologic parameters as well as VEGF as tumor progression parameters. RESULTS: cPLA2 was expressed in 54.5%, COX-2 in 80.5%, mPGES-1 in 96.4%, 15-PGDH in 46.1%, and VEGF in 65.9%. The expression of cPLA2 correlated with VEGF expression. COX-2 expression was correlated with the depth of invasion, tumor stage, cPLA2, and VEGF expressions. Moreover, VEGF revealed the highest expression in the tissues positive for both cPLA2 and COX-2. Furthermore, 15-PGDH expression was inversely correlated with VEGF expression. CONCLUSION: The present study demonstrates that cPLA2 and mPGES-1, in addition to COX-2, are constitutively overexpressed, and that 15-PGDH might be attenuated in colorectal cancer. Furthermore, cPLA2 and 15-PGDH as well as COX-2 could have an important role in tumor progression.

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na+-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na+-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na+-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na+ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na+-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A2 (cPLA2). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA2 specific inhibitor AACOCF3. ATP depletion-induced alterations in Na+-dependent phosphate uptake and cell viability were prevented by AACOCF3. Inhibition of Na+-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA2 activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

Cytosolic Phospholipase A2 Activity in Neutrophilic Oxidative Stress of Platelet-activating Factor-induced Acute Lung Injury / 결핵및호흡기질환

BACKGROUND: The present investigation was performed in rats and isolated human neutrophils in order to confirm the presumptive role of the positive feedback loop of cytosolic phospholipase A2 (cPLA2) activation by platelet- activating factor (PAF). METHODS: The possible formation of the positive feedback loop of the cPLA2 activation and neutrophilic respiratory burst was investigated in vivo and in vitro by measurement of the parameters denoting acute lung injury. In addition, morphological examinations and electron microscopic cytochemistry were performed for the detection of free radicals in the lung. RESULTS: Five hours after intratracheal instillation of PAF (5 microgram/rat), the lung leak index, lung myeloperoxidase (MPO) activity, the number of neutrophils and the concentration of cytokine-induced neutrophil chemoattractant (CINC) in bronchoalveolar lavage fluid were increased by PAF as compared with those of control rats. The NBT assay and cytochrome-c reduction assay revealed an increased neutrophilic respiratory burst in isolated human neutrophils following exposure to PAF. Lung and neutrophilic cPLA2 activity were increased following PAF exposure and exposure to hydrogen peroxide increased cPLA2 activity in the lung. Histologically, inflammatory findings of the lung were observed after PAF treatment. Remarkably, as determined by CeCl3 cytochemical electron microscopy, increased production of hydrogen peroxide was identified in the lung after PAF treatment. CONCLUSION: PAF mediates acute oxidative lung injury by the activation of cPLA2, which may provoke the generation of free radicals in neutrophils.

Cytosolic Phospholipase A2 Activity in Neutrophilic Oxidative Stress of Platelet-activating Factor-induced Acute Lung Injury / 결핵및호흡기질환

BACKGROUND: The present investigation was performed in rats and isolated human neutrophils in order to confirm the presumptive role of the positive feedback loop of cytosolic phospholipase A2 (cPLA2) activation by platelet- activating factor (PAF). METHODS: The possible formation of the positive feedback loop of the cPLA2 activation and neutrophilic respiratory burst was investigated in vivo and in vitro by measurement of the parameters denoting acute lung injury. In addition, morphological examinations and electron microscopic cytochemistry were performed for the detection of free radicals in the lung. RESULTS: Five hours after intratracheal instillation of PAF (5 microgram/rat), the lung leak index, lung myeloperoxidase (MPO) activity, the number of neutrophils and the concentration of cytokine-induced neutrophil chemoattractant (CINC) in bronchoalveolar lavage fluid were increased by PAF as compared with those of control rats. The NBT assay and cytochrome-c reduction assay revealed an increased neutrophilic respiratory burst in isolated human neutrophils following exposure to PAF. Lung and neutrophilic cPLA2 activity were increased following PAF exposure and exposure to hydrogen peroxide increased cPLA2 activity in the lung. Histologically, inflammatory findings of the lung were observed after PAF treatment. Remarkably, as determined by CeCl3 cytochemical electron microscopy, increased production of hydrogen peroxide was identified in the lung after PAF treatment. CONCLUSION: PAF mediates acute oxidative lung injury by the activation of cPLA2, which may provoke the generation of free radicals in neutrophils.

Effect of Uteroglobin on cPLA2, COX-2 Expression and ERK Activation in NSCLC Cells / 결핵

BACKGROUND: Uteroglobin is a protein produced by the normal bronchial epithelium and its expression level is lower in non-small cell lung cancer tissues and cell lines. It mainly functions as an anti-inflammatory, and when it is overexpressed in cancer cells, the neoplastic phenotype is antagonized. cPLA2 and COX-2, which are also associated with inflammation, were reported to be related to cancer. The relationship between cPLA2, COX-2 and uteroglobin is unclear. The relationship between uteroglobin and ERK, which is related to cell growth, is also not unclear. This study investigated the changes in the cPLA2 and COX-2 expression levels and the ERK activities after the overexpression of uteroglobin in non-small cell lung cancer cell lines. METHODS: The A549 and NCI-H460 cell lines were infected by adenovirus-null and adenovirus- uteroglobin. The cChange in the cPLA2, COX-2 expression level and ERK activity after uteroglobin overexpression was measured by Western blot. The change in MMP activity was measured by zymography. RESULTS: Western blot revealed decreased expression levels of cPLA2, and COX-2, and increased pERK levels in nonsmall cell lung cancer cells after uteroglobin overexpression. Zymography revealed no changes in the MMP-2 activity and lower MMP-9 activity. U0126, which is a specific inhibitor of ERK-activating kinase MEK-1/-2, prevented the decrease in the MMP-9 activity CONCLUSIONS: A decrease in cPLA2 expression, COX-2 expression, MMP-9 activity and a increase in ERK activity may be related to the anticancer effects of uteroglobin in nonsmall cell lung cancer cells.

The inhibition of IL-1? and IL-6 release in LPS induced Hela cells by cPLA_2 antisense oligonucleotide / 中国免疫学杂志

Objective:To investigate the effect of cPLA 2(cytosolic PLA 2, 85-kD PLA 2) on the signaling pathways of IL-1?、IL-6 release in LPS induced Hela cells.Methods:①LPS stimulated Hela cells were exposed to the complex of cPLA 2 initiation site-directed antisense oligonucleotide and liposome in the indicated doses and time, and then,analyzed the changes of cPLA 2 both in mRNA and in protein by RT-PCR and Western blot.②At the same time, the supernatants were collected to detect the level of IL-1? and IL-6 under radioimmunoassay(RIA).Results:①The transfection led to a dose-dependent decrease in cPLA 2 protein, while the change of mRNA is not notable.②The levels of IL-1?、IL-6 in the transfected cells were reduced following the repression of cPLA 2 compared with that of LPS added only.Conclusion:cPLA 2 probably plays an important role in the signaling mechanisms on IL-1?、IL-6 release in LPS-induced Hela cell. [