PGH1, the precursor for the anti-inflammatory prostaglandins of the 1-series, is a potent activator of the pro-inflammatory receptor CRTH2/DP2.

Prostaglandin H(1) (PGH(1)) is the cyclo-oxygenase metabolite of dihomo-γ-linolenic acid (DGLA) and the precursor for the 1-series of prostaglandins which are often viewed as "anti-inflammatory". Herein we present evidence that PGH(1) is a potent activator of the pro-inflammatory PGD(2) receptor CRTH2, an attractive therapeutic target to treat allergic diseases such as asthma and atopic dermatitis. Non-invasive, real time dynamic mass redistribution analysis of living human CRTH2 transfectants and Ca(2+) flux studies reveal that PGH(1) activates CRTH2 as PGH(2), PGD(2) or PGD(1) do. The PGH(1) precursor DGLA and the other PGH(1) metabolites did not display such effect. PGH(1) specifically internalizes CRTH2 in stable CRTH2 transfectants as assessed by antibody feeding assays. Physiological relevance of CRTH2 ligation by PGH(1) is demonstrated in several primary human hematopoietic lineages, which endogenously express CRTH2: PGH(1) mediates migration of and Ca(2+) flux in Th2 lymphocytes, shape change of eosinophils, and their adhesion to human pulmonary microvascular endothelial cells under physiological flow conditions. All these effects are abrogated in the presence of the CRTH2 specific antagonist TM30089. Together, our results identify PGH(1) as an important lipid intermediate and novel CRTH2 agonist which may trigger CRTH2 activation in vivo in the absence of functional prostaglandin D synthase.

Cyclooxygenase-2, prostaglandin synthases, and prostaglandin H2 metabolism in traumatic brain injury in the rat.

Inflammatory mediators are important in traumatic brain injury (TBI). The objective of the present study was to investigate the expression of cyclooxygenase-2 (COX-2), prostaglandin E (PGE) and PGD synthases, and PGH2 metabolism in two rat models of TBI. Fluid percussion injury (FPI) resulted in bilateral induction of COX-2 mRNA in the dentate gyri and the cortex, whereas controlled cortical contusion injury (CCC) induced COX-2 mRNA in the ipsilateral dentate gyrus and intensely in the cortex as judged by in situ hybridization. The induction subsided within 24 h. COX-2 immunoreactivity was detectable in these areas and persisted in the ipsilateral cortex for at least 72 h after CCC. Regions with COX-2 induction co-localized with TUNEL staining, suggesting a link between COX-2 expression and cell damage. COX-2 forms PGH2, which can be isomerized to PGD2, PGE2, and PGF2alpha by enzymatic and non-enzymatic mechanisms. In situ hybridization showed that mRNA of PGD synthase and microsomal PGE synthase were present in the choroid plexus. The microsomal PGE synthase was induced bilaterally after FPI and unilaterally after CCC. Liquid chromatography-mass spectrometry showed that low speed supernatant of normal and traumatized cortex and hippocampus transformed PGH2 to PGD2 as main product. PGD2 was dehydrated in brain homogenates to biological active compounds, for example, 15-deoxy-delta12,14-PGJ2. Thus COX-2 increases in certain neurons following TBI without neuronal induction of PGD and microsomal PGE synthases, suggesting that PGH2 may decompose to PGD2 and its dehydration products by nonenzymatic mechanisms or to PGD2 by low constitutive levels of PGD synthase.

Rate of vasoconstrictor prostanoids released by endothelial cells depends on cyclooxygenase-2 expression and prostaglandin I synthase activity.

This study was undertaken to investigate the enzymatic regulation of the biosynthesis of vasoconstrictor prostanoids by resting and interleukin (IL)-1(beta)stimulated human umbilical vein endothelial cells (HUVECs). Biosynthesis of eicosanoids in response to IL-1beta, exogenous labeled arachidonic acid (AA), or histamine, as well as their spontaneous release, was evaluated by means of HPLC and RIA. HUVECs exposed to IL-1beta produced prostaglandin (PG) I2 for no longer than 30 seconds after the substrate was added irrespective of the cyclooxygenase (COX) activity, whereas the time course of PGE2 and PGD2 formation was parallel to the COX activity. The ratio of PGE2 to PGD2 produced by HUVECs was similar to that obtained by purified COX-1 and COX-2. Production of PGF2alpha from exogenous AA was limited and similar in both resting and IL-1beta-treated cells. PGF2alpha was the main prostanoid released into the medium during exposure to IL-1beta, whereas when HUVECs treated with IL-1beta were stimulated with histamine or exogenous AA, PGE2 was released in a higher quantity than PGF2alpha. PGF2alpha released into the medium during treatment with IL-1beta and the biosynthesis of PGE2 and PGD2 in response to exogenous AA or histamine increased with COX-2 expression, whereas this did not occur in the case of PGI2. We observed that PGI synthase (PGIS) mRNA levels were not modified by the exposure to IL-1beta, but the enzyme was partially inactivated. When SnCl2 was added to the incubation medium, the transformation of exogenous AA-derived PGH2 into PGE2 and PGD2 was totally diverted toward PGF2alpha. Overall, these results support the conclusions that PGE2 and PGD2 (and also probably PGF2alpha) were nonenzymatically derived from PGH2 in HUVECs. The concept that a high ratio of PGH2 was released by the IL-1beta-treated HUVECs and isomerized outside the cell into PGE2 and PGD2 was supported by the biosynthesis of thromboxane B2 by COX-inactivated platelets, indicating the uptake by platelets of HUVEC-derived PGH2. The IL-1beta-induced increase in the release of PGH2 by HUVECs was suppressed by the COX-2-selective inhibitor SC-58125 and correlated with both COX-2 expression and PGIS inactivation. An approach to the mechanism of inactivation of PGIS by the exposure to IL-1beta was performed by using labeled endoperoxides as substrate. The involvement of HO. in the PGIS inactivation was supported by the fact that deferoxamine, pyrrolidinedithiocarbamate, DMSO, mannitol, and captopril antagonized the effect of IL-1beta on PGIS to different degrees. The NO synthase inhibitor NG-monomethyl-L-arginine also antagonized the PGIS inhibitory effect of IL-1beta, indicating that NO. was also involved. NO. reacts with O2-. to form peroxynitrite, which has been reported to inactivate PGIS. Homolytic fission of the O-O bond of peroxynitrite yields NO2. and HO.. The fact that 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which reacts with NO. to form NO2., dramatically potentiated the IL-1beta effect suggests that NO2. could be a species implicated in the inactivation of PGIS. Cooperation of HO. was supported by the fact that DMSO partially antagonized the effect of carboxy-PTIO. Although our results on the exact mechanism of the inactivation of PGIS caused by IL-1beta were not conclusive, they strongly suggest that both NO. and HO. were involved.

Lipopolysaccharide induces time-dependent increases in prostaglandin H synthase-2 and cytosolic phospholipase A2 mRNA in cultured human microvessel-derived endothelial cells.

The effect of lipopolysaccharide (LPS) upon the cellular content of mRNA for several enzymes associated with the arachidonic acid cascade was determined in human microvessel-derived endothelial cells. Cells were treated with either vehicle or 10 ng/mL LPS for up to 24 h. Reverse transcription followed by DNA amplification and Southern blotting were used to quantify mRNA for prostaglandin H synthase-1, prostaglandin H synthase-2, cytoplasmic PLA2, and a group II secretory PLA2. LPS treatment resulted in an increase in prostaglandin H synthase-2 mRNA after 4 h with a second peak occurring at 12 h of incubation. The expression of prostaglandin H synthase-1 mRNA was not altered by LPS treatment. An increase in cytoplasmic PLA2 mRNA but not group II PLA2 mRNA was seen after 12 h. These data demonstrate that LPS can increase mRNA production for the inducible form of prostaglandin H synthase and for cytoplasmic PLA2 in a time-dependent manner in human microvessel-derived endothelial cells. These multiple, specific increases in the prostaglandin H synthase-2 and phospholipase A2 mRNA values suggest a complex interaction between bacterial LPS and the endothelial cell eicosanoid system.