Contribution of cyclooxygenase-1-dependent prostacyclin synthesis to bradykinin-induced dermal extravasation.

BACKGROUND: Non-allergic angioedema is a potentially life-threatening condition caused by accumulation of bradykinin and subsequent activation of bradykinin type 2 receptors (B2). Since COX activity plays a pivotal role in B2 signaling, the aim of this study was to determine which prostaglandins are the key mediators and which COX, COX-1 or COX-2, is predominantly involved. METHODS: We used Miles assays to assess the effects of inhibitors of COX, 5-lipoxygenase, epoxyeicosatrienoic acid generation, cytosolic phospholipase A2α and a variety of prostaglandin receptor antagonists on bradykinin-induced dermal extravasation in C57BL/6 and COX-1-deficient mice (COX-1-/-). In addition, the prostacyclin metabolite 6-keto-PGF1α was quantified by ELISA in subcutaneous tissue from C57BL/6 and human dermal microvascular endothelial cells. In the latter, 6-keto-PGF1α was also quantified and identified by LC-MS/MS. RESULTS: Unspecific COX inhibition by ibuprofen and diclofenac significantly reduced B2-mediated dermal extravasation in C57BL/6 but not COX-1-/-. Likewise, inhibition of cytosolic phospholipase A2α showed similar effects. Furthermore, extravasation in COX-1-/- was generally lower than in C57BL/6. Of the prostaglandin antagonists used, only the prostacyclin receptor antagonist RO1138452 showed a significant reduction of dermal extravasation. Moreover, 6-keto-PGF1α concentrations were increased after bradykinin treatment in subcutaneous tissue from C57BL/6 as well as in human dermal microvascular endothelial cells and this increase was abolished by diclofenac. CONCLUSION: Our findings suggest that COX-1-dependent prostacyclin production is critically involved in dermal extravasation after activation of B2 in small dermal blood vessels. Targeting prostacyclin production and/or signaling appears to be a suitable option for acute treatment of non-allergic angioedema.

Gastroprotective effect of Cochinchina momordica seed extract in nonsteroidal anti-inflammatory drug-induced acute gastric damage in a rat model.

BACKGROUND/AIMS: The major compounds of Cochinchina momordica seed extract (SK-MS10) include momordica saponins. We report that the gastroprotective effect of SK-MS10 in an ethanol-induced gastric damage rat model is mediated by suppressing proinflammatory cytokines and downregulating cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LOX), and the activation of calcitonin gene-related peptide. In this study, we evaluated the gastroprotective effects of SK-MS10 in the nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage rat model. METHODS: The pretreatment effect of SK-MS10 was evaluated in the NSAID-induced gastric damage rat model using aspirin, indomethacin, and diclofenac in 7-week-old rats. Gastric damage was evaluated based on the gross ulcer index by gastroenterologists, and the damage area (%) was measured using the MetaMorph 7.0 video image analysis system. Myeloperoxidase (MPO) was measured by enzyme-linked immunosorbent assay, and Western blotting was used to analyze the levels of cyclooxygenase (COX)-1, COX-2, cPLA2, and 5-LOX. RESULTS: All NSAIDs induced gastric damage based on the gross ulcer index and damage area (p<0.05). Gastric damage was significantly attenuated by SK-MS10 pretreatment compared with NSAID treatment alone (p<0.05). The SK-MS10 pretreatment group exhibited lower MPO levels than the diclofenac group. The expression of cPLA2 and 5-LOX was decreased by SK-MS10 pretreatment in each of the three NSAID treatment groups. CONCLUSIONS: SK-MS10 exhibited a gastroprotective effect against NSAID-induced acute gastric damage in rats. However, its protective mechanism may be different across the three types of NSAID-induced gastric damage models in rats.

Gastroprotective Effect of Cochinchina momordica Seed Extract in Nonsteroidal Anti-Inflammatory Drug-Induced Acute Gastric Damage in a Rat Model

BACKGROUND/AIMS: The major compounds of Cochinchina momordica seed extract (SK-MS10) include momordica saponins. We report that the gastroprotective effect of SK-MS10 in an ethanol-induced gastric damage rat model is mediated by suppressing proinflammatory cytokines and downregulating cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LOX), and the activation of calcitonin gene-related peptide. In this study, we evaluated the gastroprotective effects of SK-MS10 in the nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage rat model. METHODS: The pretreatment effect of SK-MS10 was evaluated in the NSAID-induced gastric damage rat model using aspirin, indomethacin, and diclofenac in 7-week-old rats. Gastric damage was evaluated based on the gross ulcer index by gastroenterologists, and the damage area (%) was measured using the MetaMorph 7.0 video image analysis system. Myeloperoxidase (MPO) was measured by enzyme-linked immunosorbent assay, and Western blotting was used to analyze the levels of cyclooxygenase (COX)-1, COX-2, cPLA2, and 5-LOX. RESULTS: All NSAIDs induced gastric damage based on the gross ulcer index and damage area (p<0.05). Gastric damage was significantly attenuated by SK-MS10 pretreatment compared with NSAID treatment alone (p<0.05). The SK-MS10 pretreatment group exhibited lower MPO levels than the diclofenac group. The expression of cPLA2 and 5-LOX was decreased by SK-MS10 pretreatment in each of the three NSAID treatment groups. CONCLUSIONS: SK-MS10 exhibited a gastroprotective effect against NSAID-induced acute gastric damage in rats. However, its protective mechanism may be different across the three types of NSAID-induced gastric damage models in rats.

Effects of synthetic sphingosine-1-phosphate analogs on cytosolic phospholipase A2alpha-independent release of arachidonic acid and cell toxicity in L929 fibrosarcoma cells: the structure-activity relationship.

Sphingolipid metabolites including ceramide, sphingosine, and their phosphorylated products [sphingosine-1-phosphate (S1P) and ceramide-1-phosphate] regulate cell functions including arachidonic acid (AA) metabolism and cell death. The development of analogs of S1P may be useful for regulating these mediator-induced cellular responses. We synthesized new analogs of S1P and examined their effects on the release of AA and cell death in L929 mouse fibrosarcoma cells. Among the analogs tested, several compounds including DMB-mC11S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMB-mC9S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-nonyl)phenylpropyl phosphate] released AA within 1 h and caused cell death 6 h after treatment. The release of AA was observed in C12 cells [a L929 variant lacking a type alpha cytosolic phospholipase A(2) (cPLA(2)alpha)] and L929-cPLAalpha-siRNA cells (L929 cells treated with small interference RNA for cPLA(2)alpha). Treatment with pharmacological inhibitors of secretory and Ca(2+)-independent PLA(2)s decreased the DMB-mC11S-induced release of AA. The effect of the S1P analogs tested on the release of AA was comparable to that on cell death in L929 cells, and a high correlation coefficient was observed. Two analogs lacking a butoxycarbonyl moiety [DMAc-mC11S (dimethyl (2S,3R)-2-acetamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMAm-mC11S [dimethyl (2S,3R)-2-amino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate)] had inhibitory effects on the release of AA and cell toxicity induced by DMB-mC11S. Synthetic phosphorylated lipid analogs may be useful for studying PLA(2) activity and its toxicity in cells. [Supplementary Fig. 1: available only at http://dx.doi.org/10.1254/jphs.08284FP].

Effects of ceramide, ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2alpha; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling.

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A(2) (PLA(2)) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca(2+) binding regions in the C2 domain of alpha type cytosolic PLA(2) (cPLA(2)alpha), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA(2)alpha has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4beta-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca(2+) ionophore (A23187) -induced release of AA via cPLA(2)alpha's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca(2+) nor the phosphorylation of cPLA(2)alpha in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA(2)alpha that is independent of the Ca(2+) signal and the PKC-ERK-mediated phosphorylation signal.