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Synthesis of 8-epi-prostaglandin F2alpha by human endothelial cells: role of prostaglandin H2 synthase.

ABSTRACT: The experiments described in this paper were designed to determine the mechanism underlying the increase in 8-isoprostaglandin F(2alpha) (8-epi-PGF(2alpha)) production by cultured human endothelial cells during reoxygenation following hypoxia. Human umbilical artery endothelial cells were grown on microcarrier beads and exposed to sequential periods of normoxia, hypoxia, and reoxygenation. The amount of 8-epi-PGF(2alpha) in the medium was determined by ELISA. The production of 8-epi-PGF(2alpha) decreased by greater than 90% during hypoxia. Upon reoxygenation 8-epi-PGF(2alpha) production increased linearly for 90 min reaching nearly 3 times normoxic levels. When added to the medium during reoxygenation, neither superoxide dismutase nor Tiron, a cell-permeable superoxide scavenger, inhibited 8-epi-PGF(2alpha) production. However, 8-epi-PGF(2alpha) production was inhibited by catalase. The production of 8-epi-PGF(2alpha) was also inhibited by indomethacin and aspirin. Exogenous hydrogen peroxide stimulated 8-epi-PGF(2alpha) production by normoxic cells, and aspirin inhibited the hydrogen peroxide-mediated increase in 8-epi-PGF(2alpha) production. These results indicate that the reactive oxygen species responsible for 8-epi-PGF(2alpha) synthesis during reoxygenation is hydrogen peroxide and that in endothelial cells 8-epi-PGF(2alpha) synthesis is mediated by prostaglandin H(2) synthase (PGHS). To verify the role of PGHS in 8-epi-PGF(2alpha) synthesis, human PGHS-1 was expressed in COS-7 cells, a PGHS negative cell line that does not synthesize 8-epi-PGF(2alpha). In the presence of exogenous arachidonic acid the COS-7 cells expressing human PGHS-1 produced substantial amounts of PGE(2) and 8-epi-PGF(2alpha). These data indicate that human PGHS-1 can support the synthesis of 8-epi-PGF(2alpha) and that 8-epi-PGF(2alpha) synthesis by cultured human endothelial cells during reoxygenation is dependent on the activity of PGHS-1.

SUBMITTER: Watkins MT

PROVIDER: S-EPMC1220696 | biostudies-other | 1999 Dec

REPOSITORIES: biostudies-other

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Project description:For decades, the binding of prostaglandin H(2) (PGH(2)) to multiple target proteins of unrelated protein structures which mediate diverse biological functions has remained a real mystery in the field of eicosanoid biology. Here, we report that the structure of a PGH(2) mimic, U46619, bound to the purified human TP, was determined and compared with that of its conformation bound to the COX-downstream synthases, prostacyclin synthase (PGIS) and thromboxane A(2) synthase (TXAS). Active human TP protein, glycosylated and in full length, was expressed in Sf-9 cells using a baculovirus (BV) expression system and then purified to near homogeneity. The binding of U46619 to the purified receptor in a nonionic detergent-mimicked lipid environment was characterized by high-resolution NMR spectroscopy. The conformational change of U46619, upon binding to the active TP, was evidenced by the significant perturbation of the chemical shifts of its protons at H3 and H4 in a concentration-dependent manner. The detailed conformational changes and 3D structure of U46619 from the free form to the TP-bound form were further solved by 2D (1)H NMR experiments using a transferred NOE (trNOE) technique. The distances between the protons of H11 and H18, H11 and H19, H15 and H18, and H15 and H19 in U46619 were shorter following their binding to the TP in solution, down to within 5A, which were different than that of the U46619 bound to PGIS and U44069 (another PGH(2) mimic) bound to TXAS. These shorter distances led to further separation of the U46619 alpha and omega chains, forming a unique "rectangular" shape. This enabled the molecule to fit into the ligand-binding site pocket of a TP model, in which homology modeling was used for the transmembrane (TM) domain, and NMR structures were used for the extramembrane loops. The proton perturbations and 3D conformations in the TP-bound U46619 were different with that of the PGH(2) mimics bound to PGIS and TXAS. The studies indicated that PGH(2) can adopt multiple conformations in solution to satisfy the specific and unique shapes to fit the different binding pockets in the TP receptor and COX-downstream enzymes. The results also provided sufficient information for speculating the molecular basis of how PGH(2) binds to multiple target proteins even though unrelated in their protein sequences.

Project description:Background/objectivesObesity has been associated with both changes in adipose tissue lipid metabolism and inflammation. A key class of lipid-derived signalling molecules involved in inflammation are the prostaglandins. In this study, we aimed to determine how obesity affects the levels of prostaglandins within white adipose tissue (WAT) and determine which cells within adipose tissue produce them. To avoid the effects of cellular stress on prostaglandin levels, we developed a multivariate statistical approach in which metabolite concentrations and transcriptomic data were integrated, allowing the assignment of metabolites to cell types.Subjects/methodsEicosanoids were measured by liquid chromatography-tandem mass spectrometry and mRNA levels using real-time PCR. Eicosanoid levels and transcriptomic data were combined using principal component analysis and hierarchical clustering in order to associate metabolites with cell types. Samples were obtained from C57Bl/6 mice aged 16 weeks. We studied the ob/ob genetically obese mouse model and diet-induced obesity model. We extended our results in mice to a cohort of morbidly obese humans undergoing bariatric surgery.ResultsUsing our modelling approach, we determined that prostglandin D₂ (PGD₂) in adipose tissue was predominantly produced in macrophages by the haematopoietic isoform of prostaglandin D synthase (H-Pgds). Analysis of sub-fractionated WAT confirmed that H-Pgds was expressed in adipose tissue macrophages (ATMs). Furthermore, H-Pgds expression in ATMs isolated from lean and obese mice was consistent with it affecting macrophage polarisation. Functionally, we demonstrated that H-PGDS-produced PGD₂ polarised macrophages toward an M2, anti-inflammatory state. In line with a potential anti-inflammatory role, we found that H-PGDS expression in ATMs was positively correlated with both peripheral insulin and adipose tissue insulin sensitivity in humans.ConclusionsIn this study, we have developed a method to determine the cellular source of metabolites within an organ and used it to identify a new role for PGD₂ in the control of ATM polarisation.

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Synthesis of 8-epi-prostaglandin F2alpha by human endothelial cells: role of prostaglandin H2 synthase.

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