Neurotoxic lipid peroxidation species formed by ischemic stroke increase injury.

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.

Cyclopentenone isoprostanes are novel bioactive products of lipid oxidation which enhance neurodegeneration.

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.

Overexpression of amyloid precursor protein is associated with degeneration, decreased viability, and increased damage caused by neurotoxins (prostaglandins A1 and E2, hydrogen peroxide, and nitric oxide) in differentiated neuroblastoma cells.

Inflammatory reactions are considered one of the important etiologic factors in the pathogenesis of Alzheimer's disease (AD). Prostaglandins such as PGE2 and PGA1 and free radicals are some of the agents released during inflammatory reactions, and they are neurotoxic. The mechanisms of their action are not well understood. Increased levels of beta-amyloid fragments (Abeta40 and Abeta42), generated through cleavage of amyloid precursor protein (APP), oxidative stress, and proteasome inhibition, are also associated with neurodegeneration in AD brains. Therefore, we investigated the effect of PGs and oxidative stress on the degeneration and viability of cyclic AMP-induced differentiated NB cells overexpressing wild-type APP (NBP2-PN46) under the control of the CMV promotor in comparison with differentiated vector (NBP2-PN1) or parent (NBP2) control cells. Results showed that differentiated NBP2-PN46 cells exhibited enhanced spontaneous degeneration and decreased viability in comparison with differentiated control cells, without changing the level of Abeta40 and Abeta42. PGA1 or PGE2 treatment of differentiated cells caused increased degeneration and reduced viability in all three cell lines. These effects of PGs are not due to alterations in the levels of vector-derived APP mRNA or human APP holoprotein, secreted levels of Abeta40 and Abeta42, or proteasome activity. H2O2 or SIN-1 (an NO donor) treatment did not change vector-derived APP mRNA levels, but H2O2 reduced the level of human APP protein more than SIN-1. Furthermore, SIN-1 increased the secreted level of Abeta40, but not of Abeta42, whereas H2O2 had no effect on the level of secreted Abeta fragments. Both H2O2 and SIN-1 inhibited proteasome activity in the intact cells. The failure of neurotoxins to alter APP mRNA levels could be due to the fact that they do not affect CMV promoter activity. These results suggest that the mechanisms of action of PGs on neurodegeneration are different from those of H2O2 and SIN-1 and that the mechanisms of neurotoxicity of H2O2 and SIN-1 are, at least in part, different from each other.

Regulation of prostaglandin A1-induced heat shock protein expression in isolated cardiomyocytes.

Prostaglandins of the A-type (PGAs) induce heat shock protein (HSP) synthesis in a wide variety of mammalian cells resulting in protection against cellular stresses. The effect of PGAs on HSP-induction in cardiac myocytes is unknown. Therefore, we investigated the effect of PGA1 on HSP synthesis in adult rat cardiac myocytes. After 24 h of treatment, HSP72 was significantly increased 2.9-, 5.6- and 5.0-fold by PGA1 used at concentrations of 10, 20 or 40 microg/ml, respectively (P<0.05). However, the PGA1-concentration of 40 microg/ml, was found to be cytotoxic as evidenced by the release of LDH. In addition to HSP72, HSP32 was significantly increased by PGA1. The HSP32 induction was more vigorous with a marked increase with only 4 microg/ml of PGA1. No differences in the levels of HSP27, HSP60 or HSP90 were detected. When isolated cardiac myocytes were treated with PGA1, clear activation of heat shock factor (HSF) 1, one of the transcription factors for HSPs, was observed. In addition, another stress-induced transcription factor NFkappaB was also activated by PGA exposure. Despite the significant upregulation of both HSP72 and HSP32 cytoprotective properties against hypoxia and reoxygenation were absent. In conclusion, these experiments show for the first time that PGA1 induces differential expression of heat shock proteins in cardiac myocytes probably mediated through the activation of both HSF1 and NFkappaB.

Induction of the stress response with prostaglandin A1 increases I-kappaBalpha gene expression.

I-kappaBalpha is an intracellular protein that functions as a primary inhibitor of the proinflammatory transcription factor NF-kappaB. Induction of the stress response with heat shock was previously demonstrated to induce I-kappaBalpha gene expression. Because the stress response can also be induced by nonthermal stimuli, we determined whether induction of the stress response with prostaglandin A1 (PGA1) would induce I-kappaBalpha gene expression. Treatment of human bronchial epithelium (BEAS-2B cells) with PGA1 induced nuclear translocation of heat shock factor 1, thus confirming that PGA1 induces the stress response in BEAS-2B cells. Induction of the stress response with PGA1 increased I-kappaBalpha mRNA expression in a time-dependent manner and increased I-kappaBalpha peptide expression. Transient transfection assays involving a human I-kappaBalpha promoter-luciferase reporter construct demonstrated that induction of the stress response with PGA1 activated the I-kappaBalpha promoter. Induction of the stress response with PGA1 and concomitant induction of I-kappaBalpha were associated with inhibition of TNF-alpha-mediated secretion of interleukin 8 and with inhibition of TNF-alpha-mediated nuclear translocation and activation of NF-kappaB. These data demonstrate that induction of the stress response, by a nonthermal stimulus, increases I-kappaBalpha gene expression by a mechanism involving activation of the I-kappaBalpha promoter. Coupled with previous data demonstrating heat shock-mediated induction of I-kappaBalpha gene expression, these data suggest that I-kappaBalpha may be considered to be one of the stress proteins. The functional consequences of stress response-mediated I-kappaBalpha gene expression may involve attenuation of cellular proinflammatory responses.

Prostaglandins act as neurotoxin for differentiated neuroblastoma cells in culture and increase levels of ubiquitin and beta-amyloid.

Although chronic inflammatory reactions have been proposed to cause neuronal degeneration associated with Alzheimer's disease (AD), the role of prostaglandins (PGs), one of the secretory products of inflammatory reactions, in degeneration of nerve cells has not been studied. Our initial observation that PGE1-induced differentiated neuroblastoma (NB) cells degenerate in vitro more rapidly than those induced by RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, has led us to postulate that PGs act as a neurotoxin. This study has further investigated the effects of PGs on differentiated NB cells in culture. Results showed that PGA1 was more effective than PGE1 in causing degeneration of differentiated NB cells as shown by the cytoplasmic vacuolation and fragmentation of soma, nuclei, and neurites. Because increased levels of ubiquitin and beta-amyloid have been implicated in causing neuronal degeneration, we studied the effects of PGs on the levels of these proteins during degeneration of NB cells in vitro by an immunostaining technique, using primary antibodies to ubiquitin and beta-amyloid. Results showed that PGs increased the intracellular levels of ubiquitin and beta-amyloid prior to degeneration, whereas the degenerated NB cells had negligible levels of these proteins. These data suggest that PGs act as external neurotoxic signals which increase levels of ubiquitin and beta-amyloid that represent one of the intracellular signals for initiating degeneration of nerve cells.

Assessment of antiviral activity, efficacy, and toxicity of prostaglandin A2 in a rabbit model of herpetic keratitis.

Prostaglandin A2 (PGA2) inhibited the replication of herpes simplex virus type 1 in rabbit and human cornea stromal cells at concentrations of 1 to 5 microM while causing significant toxicity at 55 to 150 microM. Despite favorable therapeutic indices in cultured cells, PGA2 was not effective as a therapeutic agent in the treatment of herpetic keratitis in a rabbit model. The sequelae of disease appeared more severe in animals receiving PGA2 than in untreated or placebo-treated controls. The recovery of virus from tissues of latently infected rabbits was not affected by therapy. PGA2 therapy alone induced breakdown of the blood-aqueous barrier, indicating that pharmacologically active concentrations of drug were achieved in the eye. Thus, PGA2 had antiviral activity, but its proinflammatory effects appeared to be more detrimental than beneficial in the treatment of herpetic keratitis.

Effects of prostaglandins F2 alpha, A2, and their esters in glaucomatous monkey eyes.

The effect of prostaglandin (PG) F2 alpha-isopropyl ester (IE), PGA2, or PGA2-IE on intraocular pressure (IOP) was tested in eight cynomolgus monkey eyes with argon laser-induced glaucoma. Dose-response testing and baseline IOP measurements were done. For multiple dose testing, 5 micrograms in 25 microliters (0.02%) of each PG was topically applied twice daily for 5 days. The IOP was measured at 30- or 60-minute intervals for 6 hours after the morning dose each day. A significant (P less than 0.05) reduction of IOP peaked at 5-9 mm Hg below baseline values on the 5th day of treatment for each PG. The ocular hypotensive effect of these PGs progressively became more pronounced during the course of twice-daily dosing, with a significant reduction maintained at least 17 hours after some doses. No more than trace aqueous flare and no cells were observed in any eye during the course of treatment. These findings demonstrate that PGs other than F2 alpha are potent ocular hypotensive agents in primates.

Toxicity of prostaglandins A1 and A2 for cells in culture.

Prostaglandins A1 and A2, in concentrations near 3 x 10(-5) M, produced striking toxicity to muscle, skin and liver cells in culture. Prostaglandins E and F2alpha were much less active in this regard. Toxicity could be measured by reduction in viable cell number, protein and DNA synthesis in the cultures. The sensitivity of cultured cells was related to their age and population density. Dense cultures were sensitive early, in the first 2 days, and resistant after they manifested confluent growth. Sparse cultures remained sensitive later while they continued DNA synthesis and active cell division. It is hypothesized that the prostaglandin A effect is related to active cell division and DNA synthesis.

A comparison of the central actions of prostaglandins A1, E1, E2, F1alpha, and F2alpha in the rat. I. Behavioral, antinociceptive and anticonvulsant actions of intraventricular prostaglandins in the rat.

The effect of prostaglandins (PGs) A1, E1, E2, F1alpha, and F2alpha administered intraventricularly at doses of 0.02--4.0 mug/rat were studied in some behavioral, antinociceptive and anticonvulsant tests in rats. Exploratory and locomotor activity were decreased by all PGs except A1 and F2alpha which had no effect on locomotor activity. All PGs studied, except A1, induced hyperthermia and afforded protection in the 'hot-plate' analgesic test and against maximal electro-shock seizures.

Pregnancy and progeny in rats treated with prostaglandin A.

The effects of long-acting prostaglandin A1 upon pregnancy and fetal development were investigated in Long-Evans rats. The animals were treated by the intrauterine route either on gestational day 14 with 25 mug PGA1 or with 50 mug on day 17 of gestation. The incidence of fetal resorptions was significantly increased compared to the controls following treatment on gestational day 17. A significantly high incidence of nonspecific malformations was also induced in the offspring as a result of the treatment. There were no significant differences between experimental and control fetal and placental weights. The total protein content of fetal liver following treatment with PGA1 was not significantly reduced. Microscopic examination of placentas showed a consistent decrease in the thickness of the decidua basalis following treatment with 25 mug PGA1 on day 14 of gestation. The ovaries and umbilical cords showed no changes. In addition, treatment of rats daily with 200 mug PGA1, administered subcutaneously, on gestational days 9-12 or 12-15 did not affect the development of the conceptuses.