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Stroke is the third leading cause of death in the United States, yet no neuroprotective agents for treatment are clinically available. There is a pressing need to understand the signaling molecules that mediate ischemic cell death and identify novel neuroprotective targets. Cyclopentenone isoprostanes (IsoPs), formed after free radical-mediated peroxidation of arachidonic acid, are used as markers of stress, but their bioactivity is poorly understood. We have recently shown that 15-A(2t)-IsoP is a potent neurotoxin in vitro and increases the free radical burden in neurons. In this work, we demonstrate that 15-A(2t)-IsoP is abundantly produced in stroke-infarcted human cortical tissue. Using primary neuronal cultures we found that minimally toxic exposure to 15-A(2t)-IsoP does not alter ATP content, but in combination with oxygen glucose deprivation resulted in a significant hyperpolarization of the mitochondrial membrane and dramatically increased neuronal cell death. In the presence of Ca(2+), 15-A(2t)-IsoP led to a rapid induction of the permeability transition pore and release of cytochrome c. Taken with our previous work, these data support a model in which ischemia causes generation of reactive oxygen species, calcium influx, lipid peroxidation, and 15-A(2t)-IsoP formation. These factors combine to enhance opening of the permeability transition pore leading to cell death subsequent to mitochondrial cytochrome c release. These data are the first documentation of significant 15-A(2t)-IsoP formation after acute ischemic stroke and suggest that the addition of 15-A(2t)-IsoP to in vitro models of ischemia may help to more fully recapitulate stroke injury.
Oxidative stress and subsequent lipid peroxidation are involved in the pathogenesis of numerous neurodegenerative conditions, including stroke. Cyclopentenone isoprostanes (IsoPs) are novel electrophilic lipid peroxidation products formed under conditions of oxidative stress via the isoprostane pathway. These cyclopentenone IsoPs are isomeric to highly bioactive cyclopentenone prostaglandins, yet it has not been determined if these products are biologically active or are formed in the brain. Here we demonstrate that the major cyclopentenone IsoP isomer 15-A2t-IsoP potently induces apoptosis in neuronal cultures at submicromolar concentrations. We present a model in which 15-A2t-IsoP induced neuronal apoptosis involves initial depletion of glutathione and enhanced production of reactive oxygen species, followed by 12-lipoxygenase activation and phosphorylation of extracellular signal-regulated kinase 1/2 and the redox sensitive adaptor protein p66shc, which results in caspase-3 cleavage. 15-A2t-IsoP application also dramatically potentiates oxidative glutamate toxicity at concentrations as low as 100 nm, demonstrating the functional importance of these molecules in neurodegeneration. Finally, we employ novel mass spectrometric methods to show that cyclopentenone IsoPs are formed abundantly in brain tissue under conditions of oxidative stress. Together these findings suggest that cyclopentenone IsoPs may contribute to neuronal death caused by oxidative insults, and that their activity should perhaps be addressed when designing neuroprotective therapies.
Cyclopentenone (A2/J2) isoprostanes (IsoPs) are a group of prostaglandin (PG)-like compounds generated in vivo from the free radical-induced peroxidation of arachidonic acid. Unlike other classes of IsoPs, cyclopentenone IsoPs contain highly reactive unsaturated carbonyl moieties on the prostane ring analogous to cyclooxygenase-derived PGA2 and PGJ2 that readily adduct relevant biomolecules such as thiols via Michael addition. The purpose of this review is to summarize our knowledge of the A2/J2-IsoPs. As a starting point, we will briefly discuss the formation and biological properties of PGA2 and PGJ2. Next, we will review studies definitively showing that cyclopentenone IsoPs are formed in large amounts in vivo. This is in marked contrast to cyclopentenone PGs, for which little evidence exists that they are endogenously produced. Subsequently, we will discuss studies related to the chemical syntheses of the 15-A2-IsoP series of cyclopentenone IsoPs. The successful synthesis of these compounds provides the recent impetus to explore the metabolism and biological properties of A-ring IsoPs, particularly as modulators of inflammation, and this work will be discussed. Finally, the formation of cyclopentenone IsoP-like compounds from other fatty acids such as linolenic acid and docosahexaenoic acid will be detailed.
Cyclopentenone isoprostanes (IsoPs), A(2)/J(2)-IsoPs, are one class of IsoPs formed via the free radical-initiated peroxidation of arachidonic acid. These compounds, which are structurally similar to cyclooxygenase-derived PGA(2) and PGJ(2), contain highly reactive alpha,beta-unsaturated carbonyl moieties. A(2)/J(2)-IsoPs are generated in vivo in humans esterified in glycerophospholipids. Unlike other classes of IsoPs, however, cyclopentenone IsoPs cannot be detected in the free form; we postulated that this might be due to their rapid adduction to various thiol-containing biomolecules via Michael addition. Recently, we reported that the A-ring IsoP, 15-A(2t)-IsoP, is efficiently conjugated with glutathione in vitro by certain human and rat glutathione transferases (GSTs), with the isozyme GSTA4-4 displaying the highest activity. Herein, we examined the metabolic disposition of 15-A(2t)-IsoP in HepG2 cells. We report that 15-A(2t)-IsoP is primarily metabolized by these cells via conjugation to glutathione. Within 6 h, approximately 60% of 15-A(2t)-IsoP added to HepG2 cells was present in the form of a water soluble conjugate(s). Structural characterization of the adduct(s) by liquid chromatography-tandem mass spectrometry revealed four major conjugates. These include the intact 15-A(2t)-IsoP-GSH conjugate, the GSH conjugate in which the carbonyl at C-9 of 15-A(2t)-IsoP is reduced, and the corresponding cysteine conjugates. These studies thus show that the primary pathway of metabolic disposition of endogenously derived cyclopentenone IsoPs occurs via conjugation with thiols.
Glutathione transferases (GSTs) are a large family of enzymes that can be divided into different classes based on structure. There has been considerable interest in the ability of GSTs to conjugate and inactivate endogenously derived reactive lipid peroxidation products that contain alpha,beta-unsaturated carbonyl moieties such as 4-hydroxyalkenals. One enzyme with prominent activity toward these substrates is human GST A4-4. Recently, we described a novel series of compounds termed A(2)/J(2)-isoprostanes (IsoPs) that are formed endogenously in humans from the free radical-initiated peroxidation of arachidonic acid. These compounds contain alpha,beta-unsaturated carbonyl groups and have structures similar to cyclooxygenase-derived PGA(2) and PGJ(2). Because of their chemical reactivity, these compounds may mediate tissue injury associated with oxidant stress. Herein, we report that the A-ring IsoP 15-A(2t)-IsoP (8-iso-PGA(2)) is efficiently conjugated to glutathione (GSH) by human GST A4-4 with a k(cat)/K(m) value of >200 s(-)(1) mM(-)(1). The k(cat)/K(m) value for conjugation of 15-A(2t)-IsoP by the homologous rat GST A4-4 is >2000 s(-)(1) mM(-)(1). Similar high enzyme activities were observed when PGA(2) was used as a substrate. In contrast, the human GSTs A1-1, M1-1, M2-2, P1-1, and T1-1 and rat GST T2-2 did not significantly metabolize 15-A(2t)-IsoP. These studies have therefore defined a potentially important route by which cyclopentenone IsoPs are metabolized that may serve as a mechanism for the inactivation of these highly reactive compounds.
The highest concentrations of prostaglandins in nature are found in the Caribbean gorgonian Plexaura homomalla. Depending on its geographical location, this coral contains prostaglandins with typical mammalian stereochemistry (15S-hydroxy) or the unusual 15R-prostaglandins. Their metabolic origin has remained the subject of mechanistic speculations for three decades. Here, we report the structure of a type of cyclooxygenase (COX) that catalyzes transformation of arachidonic acid into 15R-prostaglandins. Using a homology-based reverse transcriptase--PCR strategy, we cloned a cDNA corresponding to a COX protein from the R variety of P. homomalla. The deduced peptide sequence shows 80% identity with the 15S-specific coral COX from the Arctic soft coral Gersemia fruticosa and approximately 50% identity to mammalian COX-1 and COX-2. The predicted tertiary structure shows high homology with mammalian COX isozymes having all of the characteristic structural units and the amino acid residues important in catalysis. Some structural differences are apparent around the peroxidase active site, in the membrane-binding domain, and in the pattern of glycosylation. When expressed in Sf9 cells, the P. homomalla enzyme forms a 15R-prostaglandin endoperoxide together with 11R-hydroxyeicosatetraenoic acid and 15R-hydroxyeicosatetraenoic acid as by-products. The endoperoxide gives rise to 15R-prostaglandins and 12R-hydroxyheptadecatrienoic acid, identified by comparison to authentic standards. Evaluation of the structural differences of this 15R-COX isozyme should provide new insights into the substrate binding and stereospecificity of the dioxygenation reaction of arachidonic acid in the cyclooxygenase active site.
Cyclopentenone prostaglandins A2 and J2 are reactive compounds that possess unique biological activities. However, the extent to which they are formed in vivo remains unclear. In this study, we explored whether D2/E2-isoprostanes undergo dehydration in vivo to form A2/J2-isoprostanes. Oxidation of arachidonic acid in vitro generated a series of compounds that were confirmed to be A2/J2-isoprostanes by mass spectrometric analyses. A2/J2-isoprostanes were detected in vivo esterified to lipids in livers from normal rats at a level of 5. 1 +/- 2.3 ng/g, and levels increased dramatically by a mean of 24-fold following administration of CCl4. An A2-isoprostane, 15-A2t-isoprostane, was obtained and found to readily undergo Michael addition with glutathione and to adduct covalently to protein. A2/J2-isoprostanes could not be detected in the circulation, even following CCl4 administration, which we hypothesized might be explained by rapid formation of adducts. This was supported by finding that essentially all the radioactivity excreted into the urine following infusion of radiolabeled 15-A2t-isoprostane into a human volunteer was in the form of a polar conjugate(s). These data identify a new class of reactive compounds that are produced in vivo as products of the isoprostane pathway that can exert biological effects relevant to the pathobiology of oxidant injury.
A2/J2-Isoprostanes (IsoPs) are prostaglandin (PG) A2/J2-like compounds that are produced in vivo as dehydration products of D2/E2-IsoPs. One A2-IsoP that should be formed is 15-A2t-IsoP (8-iso-PGA2). Analogous to cyclopentenone PGs, 15-A2t-IsoP readily undergoes nucleophilic addition to various biomolecules suggesting the compound is capable of exerting potent bioactivity. However, proof that it is definitively formed in vivo is lacking. Evidence is now presented that 15-A2t-IsoP, in fact, is generated in vivo by demonstrating that an endogenous A2-IsoP with a retention time on capillary GC identical with that 15-A2t-IsoP co-chromatographs through four high resolving HPLC purification procedures with authentic radiolabeled 15-A2t-IsoP.
Prostaglandin A2 (PGA2) reversibly blocked the cell cycle progression of NIH 3T3 cells at G1 and G2/M phase. When it was applied to cells synchronized in G0 or S phase, cells were blocked at G1 and G2/M, respectively. The G2/M blockage was transient. Microinjected oncogenic leucine 61 Ras protein could not override the PGA2 induced G1 blockage, nor could previous transformation with the v-raf oncogene. The serum-induced activation of mitogen-activated protein kinase was not inhibited by PGA2 treatment. These data suggest that PGA2 blocks cell cycle progression without interfering with the cytosolic proliferative signaling pathway. Combined microinjection of E2F-1 and DP-1 proteins or microinjected adenovirus E1A protein, however, could induce S phase in cells arrested in G1 by PGA2, indicating that PGA2 does not directly inhibit the process of DNA synthesis. In quiescent cells, PGA2 blocked the normal hyperphosphorylation of the retinoblastoma susceptible gene product and the activation of cyclin-dependent kinase (CDK) 2 and CDK4, in response to serum stimulation. PGA2 treatment elevated the p21Waf1/Cip1/Sdi1 protein expression level. These data indicate that PGA2 may arrest the cell cycle in G1 by interfering with the activation of G1 phase CDKs.
We investigated prostacyclin (PGI2) biosynthesis during pregnancy by measuring urinary excretion of 2,3-dinor-6-keto-prostaglandin F1 alpha (dinor) and 15-keto-13,14-dihydro-2,3-dinor-6-keto-prostaglandin F1 alpha (15 kd dinor) with the use of specific gas chromatography-mass spectrometry assays. Nine normotensive nonpregnant women, five normotensive women in the mid-trimester of pregnancy, eight normotensive women in the third trimester of pregnancy, and six women who developed hypertension during the third trimester provided 24-hour samples of urine. Normal pregnant women had a fivefold increase in urinary excretion of dinor in comparison to nonpregnant women (253 +/- 21 ng dinor/gm creatinine for controls vs. 1,224 +/- 110 and 1,127 +/- 152 for second and third trimesters) (mean +/- SEM). Pregnant subjects with hypertension had a significant (50%) reduction in urinary dinor excretion in comparison to normotensive pregnant subjects (561 +/- 105 ng dinor/gm creatinine). In subjects selected from each group, the ratio of dinor to 15 kd dinor remained constant. We conclude that PGI2 biosynthesis is increased during normal pregnancy, and that this increase is less in pregnancy-induced hypertension. This raises the possibility that PGI2 helps mediate hemodynamic changes during normal pregnancy, and that a relative decrease in production might be related to the pathogenesis of pregnancy-induced hypertension.