The occurrence of 15-keto-prostaglandins in the red alga Gracilaria verrucosa.

New 15-keto-prostaglandins (1-4) were isolated from the MeOH extract of the red alga, Gracilaria verrucosa. Their structures were determined to be prostaglandin B congeners (1-3) and a prostaglandin E congener (4) based on the NMR and MS data. Prostaglandins with a C-15 keto function are rare from natural sources. The presence of these metabolites in the alga is notable because 15-keto-prostaglandins (15-keto-PGs) are considered to be the metabolic products of regular prostaglandins in mammals. The occurrence of different prostaglandins in this alga might be due to the existence of different oxidative enzymes, as previously mentioned for oxygenated fatty acids of the red alga Gracilariopsis lemaneiformis. The antiinflammatory activity of these prostaglandins was examined by evaluating their inhibitory effects on nitric oxide production in lipopolysaccharide (LPS)-activated RAW264.7 murine macrophage cells. These prostaglandins showed weak activity on nitric oxide production.

Prostaglandin B(2) delivers a co-stimulatory signal leading to T cell activation.

Most of the data accumulated to date on the immunoregulatory effects of prostaglandins (PG) on T cell activation stem from the archetypal inhibitory effect of PGE(2). In this study we provide instead, the first evidence that exogenous PGB(2), a catabolic metabolite of PGE(2), synergizes with signals delivered by T cell receptor (TCR) engagement to induce interleukin-2 (IL-2) production and IL-2 receptor (IL-2R) alpha-expression in Jurkat cells. Accordingly, PGB(2) enhances the proliferation of anti-CD3-activated peripheral blood lymphocytes (PBL). In terms of cellular signaling, we present evidence that PGB(2) activates tyrosine kinase activities and efficiently increases c-fos mRNA expression and nuclear factor-kappa B (NF-kappa B) translocation to the nucleus. Owing to these features, PGB(2) appears as a new lipid mediator capable of delivering an ancillary signal leading to T lymphocyte activation.

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 µg/ml PGB2 inhibited virus yield by 60 percent, at the same dose PGA1 suppressed virus replication by more than 90 percent. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells.

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 micrograms/ml PGB2 inhibited virus yield by 60%, at the same dose PGA1 suppressed virus replication by more than 90%. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2.

Mitochondrial dysfunction during anoxia and acute cell injury.

Mitochondrial function is closely linked to the maintenance of mitochondrial integrity. During short-term anoxia, ion-transport systems in the inner membrane are inhibited to protect against loss of the promotive force and associated osmotic imbalance that can cause irreversible loss of mitochondrial integrity and function. In two models of chemically induced mitochondrial failure, a prostaglandin B1 derivative, di-calciphor, protected against mitochondrial failure and prevented cell death. Characteristics were similar to those observed in mitochondria during short-term anoxia. Thus, the results indicate that di-calciphor may represent a new type of mitochondrial protectant that inhibits ion transport and thus slows the loss of osmotic stability and delays mitochondrial dysfunction under traumatic and toxicologic conditions.

Effects of tri-Calciphor (trimer of 16, 16-dimethyl-15-dehydroprostaglandin B1) on glucose metabolism in liver cells.

Phosphorylase activity of isolated rat liver cells was increased about 2-fold on addition of tri-Calciphor (trimer of 16, 16-dimethyl-15-dehydroprostaglandin B1), epinephrine or the Ca2+ ionophore A23187, in all cases presumably due to an increase in cytosolic Ca2+. Extracellular Ca2+ was required with A23187, but not with either tri-Calciphor or epinephrine. Tri-Calciphor, however, did not stimulate a sustained release of glucose from hepatocytes as compared to the other Ca2+ mobilizing agents, even at concentrations 10-fold higher than that required to stimulate the phosphorylase activity. Tri-Calciphor did not alter the glucose release by epinephrine. It is concluded that tri-Calciphor can alter cytosolic Ca2+, but that its mechanism of action is more complex than that of a simple Ca2+ ionophore.

Protection of hepatocytes against death due to mitochondrial failure: effect of di-Calciphor on antimycin A-induced toxicity.

Di-Calciphor is a synthetic derivative of prostaglandin B1 that protects against cerebral and cardiac ischemia apparently by preserving mitochondrial function. To determine whether di-Calciphor specifically protects against mitochondrial failure, we studied its effects on mitochondrial functions in hepatocytes treated with the specific mitochondrial poison, antimycin A. The results show that 1 microM di-Calciphor protects against cell death at concentrations of antimycin A that inhibited mitochondrial respiration and caused cellular ATP depletion. Di-Calciphor did not protect against loss of ATP but did protect against the loss of mitochondrial delta psi and delta pH. In addition, di-Calciphor protected against antimycin A-induced loading of phosphate into mitochondria and an associated mitochondrial swelling. Thus, these results show that di-Calciphor protects against a specific mitochondrial poison and support the interpretation that di-Calciphor is a mitochondrial protective agent. In addition, the results suggest that the protection of the mitochondria involves preservation of mitochondrial ionic and osmotic stability and does not involve improved ATP supply.

Tri-Calciphor (16,16-dimethyl-15-dehydroprostaglandin B1 trimer)-mediated mitochondrial Ca2+ movements: modulation by phosphate.

The trimeric derivative of 16,16-dimethyl-15-dehydroprostaglandin B1 (termed tri-Calciphor), which protects tissues against ischemic damage, induced Ca2+ efflux and swelling in mitochondria in the absence of phosphate, Mg2+ and ATP. When glutamate/malate rather than succinate was the substrate, higher tri-Calciphor concentrations were required for the ionophoretic activity. Ca2+ efflux and mitochondrial swelling induced by tri-Calciphor were completely inhibited by ATP, phosphate and Mg2+ added together, and partially inhibited with phosphate plus either ATP or Mg2+. Between 0 and 7 microM added Ca2+ and in the presence of phosphate, ATP and Mg2+, tri-Calciphor stimulated the uptake of Ca2+ by mitochondria and increased the efficiency of buffering of extramitochondrial Ca2+. Thus, depending on the assay conditions, two different effects involving Ca2+ movements and mitochondria are observed with tri-Calciphor.

Reversal of chloroquine resistance in murine malaria parasites by prostaglandin derivatives.

An oligomeric ester of prostaglandin B2 (OC-5186) was found to reverse chloroquine resistance in the murine malarial parasite Plasmodium berghei. When mice were infected with either chloroquine-sensitive or -resistant P. berghei on day 0 (by intraperitoneal injection of 1 x 10(6) parasitized erythrocytes), they died before day 23. When treated with 15 mg/kg/day of chloroquine for the first four days of infection, all mice infected with the sensitive-strain survived, while all those infected with the resistant strain died before day 23. When OC-5186 (3-12 mg/kg/day) was administered in combination with chloroquine for the first four days, 60% of the animals infected with the resistant strain survived. The differences in the survival rate between the group treated with chloroquine only and the group treated with a combination of drugs (chloroquine plus 3-12 mg/kg/day of OC-5186) were significant. There was also a significant inhibition of parasitemia in the group treated with the combination of drugs. The combinations of chloroquine and a monomer ester of prostaglandin B2 (OC-5181) had some antimalarial activity, but the differences between the chloroquine-treated group and the combination treatment group were not significant in terms of both the parasitemia and the survival rate. Another oligomeric ester of prostaglandin E1 (MR-356) as well as unesterified monomer prostaglandins (PGA2 and PGB2) were ineffective by themselves and in combination with chloroquine.

1,3-Dioctanoylglycerol modulates arachidonate mobilization in human neutrophils and its inhibition by PGBx: evidence of a protein-kinase-C-independent role for diacylglycerols in signal transduction.

Preincubation of human neutrophils with 1-oleoyl-2-acetylglycerol (OAG) enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization. We have recently demonstrated that preincubation of neutrophils with OAG also reverses inhibition of A23187 stimulated [3H]arachidonate mobilization by the phospholipase A2 inhibitors, PGBx and aristolochic acid. The present study has compared the effects of 1,2-sn-dioctanoylglycerol (1,2-diC8) and 1,3-dioctanoylglycerol (1,3-diC8) on these cellular events. Dose-dependent priming (ED50 < 2.5 microM) of fMLP-stimulated [3H]arachidonate mobilization is obtained with both 1,2-diC8 and 1,3-diC8. Both diC8s also enhance fMLP-stimulated synthesis of leukotriene B4, 5-hydroxyeicosatetraenoic acid and platelet-activating factor, and generation of superoxide. Furthermore, both 1,2-diC8 and 1,3-diC8 reverse the effects of PGBx on A23187-stimulated [3H]arachidonate mobilization and platelet-activating factor synthesis. By contrast, higher concentrations (5-10 microM) of 1,2-diC8, but not 1,3-diC8, directly stimulate both [3H] arachidonate mobilization and superoxide generation. Since 1,3-diC8 does not activate protein kinase C (PKC), these results suggest that PKC is involved in direct activation of neutrophils by diacylglycerols but not in priming. Furthermore, reversal of the inhibitory effects of PGBx by diacylglycerols also appears to involve a PKC-independent mechanism.

Neuroprotective activity of dimer of 16,16'-dimethyl-15-dehydroprostaglandin B1 (di-Calciphor) in cerebral ischemia.

Post-ischemic treatment of di-Calciphor (16,16'-dimethyl-15- dehydroprostaglandin B1) significantly improves animal survival and prevents ischemia-induced neurodegeneration of vulnerable forebrain regions assessed with histochemical and biochemical techniques in gerbils. Neuronal degeneration seen by Cresyl violet staining and silver impregnation in the CA1 sector of the hippocampus and the dorso-lateral sector of the striatum was significantly reduced in animals treated with di-Calciphor. In addition, the early onset of selective degradation of calpain I substrates spectrin and microtubule-associated protein (MAP2) in these same vulnerable regions was prevented. The lack of adverse side effects may facilitate the potential therapeutic use of this drug in preventing neuronal damage caused by stroke.

Role of human sperm phospholipase A2 in fertilization: effects of a novel inhibitor of phospholipase A2 activity on membrane perturbations and oocyte penetration.

Phospholipase A2 was isolated from human sperm and its potential role in the membrane fusion events of fertilization was examined. Highly purified enzyme hydrolyzed the phospholipids of [1-14C]oleate-labeled Escherichia coli optimally at neutral to alkaline pH with 5 mM CaCl2 and 150 mM NaCl (specific activity = 20 mumol/min/mg). Activity was inhibited in a dose-dependent manner by an oligomer of prostaglandin B1 (IC50 = 1.5 microM) reported to inhibit human phospholipases A2 in vitro and in situ. Sperm phospholipase A2 injected into mouse foot pad induced a dose-dependent edema that was inhibited by oral administration of prostaglandin Bx (IC50 < or = 10 mg/kg) or by pretreatment of the enzyme with 4-bromophenacyl bromide. Human sperm phospholipase A2 (10 micrograms) induced fusion of phosphatidylserine vesicles in the presence of 1 mM calcium chloride by approximately 80% (+/- 10%) as determined by monitoring turbidity (O.D.400) and efficiency of fluorescence resonance energy transfer. This enzyme-induced fusion was accompanied by phospholipid hydrolysis, and both fusion and phospholipid degradation were inhibited by more than 60% when enzyme was preincubated with 5 microM prostaglandin Bx. Sperm penetration of zona pellucida-free hamster oocytes was inhibited in a dose-dependent fashion when sperm were incubated with prostaglandin Bx (IC50 approximately 15 microM) during capacitation; sperm motility was not affected by this treatment. Capacitation in the presence of prostaglandin Bx had little to no effect on the in vitro acrosome reaction. These results suggest that sperm phospholipase A2 and its modulators may contribute to membrane fusion events in mammalian fertilization.

Protective effect of a prostaglandin oligomer on liver mitochondria in situ: time-shared measurements of fluorescence and reflectance in the cold-preserved rat liver.

The protective effect of a new oligomeric derivative of prostaglandin B2, known as OC-5186, was evaluated using time-sharing spectrofluorometry in the cold-preserved rat liver. Experiments were divided into three groups: in group A, a 5000 ng dose of OC-5186 was administered via the peripheral vein, 1000 ng via the portal vein, and 200 ng/ml in University of Wisconsin (UW) solution; in group B, the OC-5186 dosage was ten times greater than that in group A; in group C (control group), liver procurement and storage were performed without OC-5186. At 0, 12, and 24 h after cold preservation at 4 degrees C, the liver was perfused for 30 min at 12 degrees C with oxygenized Krebs-Henseleit solution, after which the perfusate was switched to deoxygenized Krebs-Henseleit solution. Time sharing spectrofluorometry was used to follow NADH fluorescence at 450 nm with a 360-nm excitation wavelength, as well as the reflectance of cytochrome aa3 with 605 minus 620 nm from oxidation to reduction. Rate constants of NADH fluorescence and cytochrome aa3 reflectance were used as indices of integrity of the mitochondrial respiratory chain. In group C, the rate constant of NADH fluorescence decreased significantly (P < 0.05) from the control value of 8.31 +/- 0.21 x 10(-3) (sec-1) to 4.97 +/- 0.15 x 10(-3) and 5.58 +/- 0.16 x 10(-3) (mean +/- SEM) at 12 and 24 h after cold preservation, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Protective effect of the dimer of 16,16-diMePGB1 against KCN-induced mitochondrial failure in hepatocytes.

The dimer and trimer of 16,16-dimethyl-15-dehydroprostaglandin B1 (16,16-diMePGB1) previously have been shown to have protective effects on mitochondrial function. To examine the potential mechanisms involved in protection against mitochondrial failure, we have studied the effects of the dimer of 16,16-diMe-PGB1 (dicalciphor) on mitochondrial function in hepatocytes exposed to KCN. Addition of micromolar concentrations of dicalciphor provided substantial protection against KCN-induced toxicity in a concentration- and time-dependent manner. Dicalciphor, however, had no effect on total or mitochondrial ATP losses in KCN-treated cells. The dimer prevented the marked loss of mitochondrial membrane potential (delta psi) and delta pH that occurs as a result of KCN treatment and prevented KCN-induced loading of phosphate in mitochondria. Furthermore, the dimer of 16,16-diMePGB1 also prevented KCN-induced mitochondrial and cellular swelling. These results demonstrate that dicalciphor protects against KCN-induced damage and that this protection is associated with regulation of specific mitochondrial ion transport functions.

The effects of the phospholipase A2 inhibitors aristolochic acid and PGBx on A23187-stimulated mobilization of arachidonate in human neutrophils are overcome by diacylglycerol or phorbol ester.

Aristolochic acid and PGBx, two structurally unrelated, protein-targeted inhibitors of isolated phospholipases A2, are effective antagonists of calcium ionophore A23187-stimulated mobilization of [3H]arachidonate from human neutrophils. We now report that preincubation of neutrophils with oleoylacetylglycerol (OAG, 15 microM) substantially reverses the inhibitory effect of 200 microM aristolochic acid (from 70 to 24% inhibition). Similarly, OAG increases the IC50 for PGBx from 2.5 to greater than 20 microM. The effects of OAG on inhibition by either aristolochic acid or PGBx are dose-dependent, with an ED50 of 2.5 microM. Protection against inhibition by either aristolochic acid or PGBx is also observed with phorbol myristate acetate (PMA, ED50 3 nM), but not 4-alpha-phorbol didecanoate. Aristolochic acid and PGBx do not inhibit PMA-stimulated superoxide generation, and are thus not protein kinase C inhibitors. Furthermore, neither aristolochic acid nor PGBx inhibit diglyceride generation through the phospholipase D/phosphatidate phosphohydrolase pathway. A23187-stimulated [3H]arachidonate mobilization is increased by 20-50% when neutrophils are preincubated with OAG or PMA. The present results indicate that OAG and PMA also modulate the A23187-stimulated [3H]arachidonate mobilization so as to render it less sensitive to inhibitors of phospholipase A2.

Treatment of severe brain ischemia with di- and tri-Calciphor (dimer and trimer of 16,16'-dimethyl prostaglandin B1).

Following 20 min occlusion of both carotid arteries, female gerbils were subjected to treatment with di- or tri-Calciphor (dimer or trimer of 16,16'-dimethyl prostaglandin B1). Dimer was injected i.p. at 5 and 10 mg/kg at 5 min and again at 24 h, 30 min and 24 h, 60 min and 24 h or 180 min and 24 h postischemia (N = 25/group). Trimer was given i.p. at 5, 10 or 15 mg/kg at 5 min and 24 h postischemia (N = 25/group.) The controls (N = 25) were injected with the vehicle. Neurological status and postischemic survival of the animals were monitored for 14 days postischemia. Survival of the treated gerbils was significantly improved following the treatment with either di- or tri-Calciphor administered at 10 mg/kg at 5 min and 24 h postischemia (36 vs. 68% di- and 64% tri-Calciphor, P less than 0.05), and with di-Calciphor at 5 mg/kg at 180 min and 24 h postischemia (64%). All other treatment regimens with either drug resulted in a numerical, statistically insignificant improvement. In addition, treatment with either drug reduced the intensity of postischemic neurological impairment. Treatment with di-Calciphor injected at 10 mg/kg at 5 min and 24 h post 20 min ischemia substantially reduced the period of postischemic locomotor hyperactivity. The drug had no impact on either body temperature or blood pressure. There is evidence that the effects of Calciphor may be mediated via calcium regulatory mechanisms. The results of the present study are discussed in the light of such possibility.

Inhibition of lipid peroxidation by prostaglandin oligomeric derivatives.

The inhibition of lipid peroxidation by oligomeric derivatives synthesized from prostaglandin E1 (PGE1) and PGB2 was studied using two rat models. In an in vitro model, the brain was exposed to decapitation-ischemia, the cortex was removed and homogenized, and the formation of thiobarbituric acid reactive substances (TBAR) was measured after exposing the homogenate to in vitro reoxygenation either in the presence or absence of oligomers. It was found that these oligomers could inhibit lipid peroxidation, and that their activities were higher than that of superoxide dismutase (SOD). In an in vivo administration model, either the oligomer or the vehicle was injected i.p. 30 min before decapitation. The brain was exposed to decapitation-ischemia, the cortex was homogenized and exposed to 'in vitro' reoxygenation, after which TBAR value was determined. Ester-type compounds had a greater activity than free-acid type compounds in inhibiting lipid peroxidation. A possible mechanism of the protective effect of these oligomers in ischemia/reperfusion injury may be to scavenge oxygen free radicals.

Inhibitory effect of prostaglandin oligomeric derivatives on 9,10-dimethyl-1,2-benzanthracene-induced hamster lingual carcinomas.

The inhibitory effects of prostaglandin oligomeric derivatives OC-3186 and OC-5186 were examined in hamster lingual carcinoma induced by 9,10-dimethyl-1,2-benzanthracene (DMBA). These compounds caused a regression of 40-90% in the size of lingual carcinomas in the hamster within several days after systemic or local administration.

Mechanism(s) of cytoprotective and anti-inflammatory activity of PGB1 oligomers: PGBx has potent anti-phospholipase A2 and anti-oxidant activity.

We postulate that the anti-PLA2 and anti-oxidant activities may account for the broad spectrum protective effects of PGBx that were previously described. These dual properties are demonstrable in vitro, in situ and in vivo, and would have profound effects on stabilization of membrane structure and function, which in turn, would protect organelles, cells, tissues, and organs from inflammation and injury, and possibly alter patterns of aging involving senescence and cell death.

[31P]MRS study of the protective effects of prostaglandin oligomers on forebrain ischemia in rats.

Two ester-type prostaglandin oligomeric compounds were synthesized, one from prostaglandin E1 (termed MR-356) and the other from prostaglandin B2 (termed OC-5186). Using in vivo [31P]MRS, the protective effects of these oligomers on forebrain ischemia (15 min) were evaluated in a rat model. Forebrain ischemia caused a decrease in intracellular high energy phosphates and intracellular pH (pHi) in the control and compounds-treated groups, but changes of these values in the OC-5186-treated group were significantly smaller than those in the control group. Moreover, the cerebral energy metabolism of the OC-5186-treated group returned to the preischemia level more rapidly than in the control group after forebrain ischemia. MR-356 had some effects, but the differences were not significant.