Simultaneous inhibition of TXA(2) and PGI(2) synthesis increases NO release in mesenteric resistance arteries from cirrhotic rats.

Our present study examines, in mesenteric resistance arteries, possible vasodilation alterations, and the role of NO and COX (cyclo-oxygenase) derivatives, in cirrhosis. The vasodilator response to acetylcholine was analysed in segments from control and cirrhotic rats. The effects of the non-specific COX inhibitor indomethacin, the specific COX-1 inhibitor SC-560 and the specific COX-2 inhibitor NS-398 were analysed in segments from both groups of rats. NO release was measured, and eNOS [endothelial NOS (NO synthase)], phospho-eNOS, iNOS (inducible NOS), COX-1 and COX-2 protein expression was also analysed. The effects of the TP receptor [TXA2 (thromboxane A(2)) receptor] antagonist SQ 29548, the TXA(2) synthesis inhibitor furegrelate, the PGI(2) (prostaglandin I(2)) synthesis inhibitor TCP (tranylcypromine) or TCP+furegrelate were only determined in segments from cirrhotic rats. The vasodilator response to acetylcholine was higher in segments from cirrhotic rats. Indomethacin, SC-560 and NS-398 did not modify the vasodilator response in control rats; however, indomethacin, NS-398 and TCP+furegrelate increased, whereas SC-560 did not modify and SQ 29548, furegrelate or TCP decreased, the vasodilator response to acetylcholine in cirrhotic rats. NO release was higher in cirrhotic rats. Furegrelate decreased, whereas TCP+furegrelate increased, the NO release in segments from cirrhotic rats. eNOS and COX-1 protein expression was not modified, whereas phosho-eNOS, iNOS and COX-2 protein expression was higher in cirrhotic rats. Therefore the increase in iNOS expression and eNOS activity may mediate increases in endothelial NO release. The COX-2 derivatives TXA(2) and PGI(2) may act simultaneously, producing a compensatory effect that reduces NO release and may limit the hyperdynamic circulation.

Production of poly-gama-glutamate (PGA) biopolymer by batch and semicontinuous cultures of immobilized Bacilluslicheniformis strain-R

Production of Polyglutamate (PGA) biopolymer by immobilized Bacilluslicheniformis strain-R was intensively investigated. Preliminary experiments were carried out to address the most suitable immobilization methodology. Entrapment of Bacillus cells in alginate-agar led optimal PGA production (36.75 g/l), with 1.32- and 2.18-fold increase in comparison with alginate- or K-carrageenan-immobilized cells, respectively. During semicontinuous cultivation of agar-alginate gel-cell mixture, production of PGA by 10 ml mixture was increased from 2nd to 3rd run whereas, increased till the 4th run using 15ml mixture. Adsorption was the most suitable immobilization technique for production of PGA and the sponge cubes was the preferred matrix recording 43.2 g/l of PGA with the highest cell adsorption. Furthermore, no PGA was detected when B. licheniformis cells were adsorbed on wood and pumice. Although luffa pulp-adsorbed cells recorded the highest PGA production (50.4 g/l), cell adsorption was the lowest. Semicontinuous cultivation of B. licheniformis cells adsorbed on sponge led to increase of PGA production till the 3rd run and reached 55.5 g/l then slightly decreased in the 4th run. The successful use of fixed-bed bioreactor for semicontinuous cultivation of B. licheniformis cells held on sponge cubes (3 runs, 96 hours/run) provides insight for the potential biotechnological production of PGA by immobilized cells.

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.

Evidence for oxylipin synthesis and induction of a new polyunsaturated fatty acid hydroxylase activity in Chondrus crispus in response to methyljasmonate.

Signaling cascades involving oxygenated derivatives (oxylipins) of polyunsaturated fatty acids (PUFAs) are known to operate in response to external stimuli. The marine red alga Chondrus crispus uses both oxygenated derivatives of C18 (octadecanoids) and C20 (eicosanoids) PUFAs as developmental or defense hormones. The present study demonstrates that methyljasmonate (MeJA) triggers a cascade of oxidation of PUFAs leading to the synthesis of prostaglandins and other oxygenated fatty acids. As a result of a lipoxygenase-like activation, MeJA induces a concomitant accumulation of 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) and 13-oxo-9Z,11E-octadecadienoic acid (13-oxo-ODE) in a dose-dependent manner in C. crispus. Furthermore, MeJA increases the level of mRNA encoding a gluthatione S-transferase and induces the activity of a new enzyme catalyzing the regio- and stereoselective bisallylic hydroxylation of polyunsaturated fatty acids from C(18) to C(22). The enzyme selectively oxidized the omega minus 7 carbon position (omega-7) and generated the stereoselective (R)-hydroxylated metabolites with a large enantiomeric excess. The enzyme specificity for the fatty acid recognition was not dependent of the position of double bonds but at least requires a methylene interrupted double bond 1,4-pentadiene motif involving the omega-7 carbon.

Isolation of three marine prostanoids, possible biosynthetic intermediates for clavulones, from the Okinawan soft coral Clavularia viridis.

Three marine prostanoids, 1, 2, and 3, were isolated from the extract of the Okinawan soft coral Clavularia viridis. The structures of these compounds were assigned based on the results of spectroscopic analysis. Compound 1 was shown to be preclavulone-A methyl ester, and this is the first isolation of the ester of preclavulone-A as a natural product. Preclavulone-A is proposed to be the key intermediate in the biosynthesis of marine prostanoids exemplified by clavulones in C. viridis. The new prostanoid 3 was suggested to be a biosynthetic intermediate from preclavulone-A to clavulones, and a possible biogenetic pathway via 3 is proposed.

Prostaglandin biosynthesis by midgut tissue isolated from the tobacco hornworm, Manduca sexta.

We describe prostaglandin (PG) biosynthesis by isolated midgut preparations from tobacco hornworms, Manduca sexta. Microsomal-enriched midgut preparations yielded four PGs, PGA/B(2), PGD(2), PGE(2) and PGF(2alpha), all of which were confirmed by analysis on gas chromatography--mass spectrometry (GC--MS). PGA and PGB are double bond isomers which do not resolve on TLC but do resolve by GC; for convenience, we use the single term PGA(2) for this product. PGA(2) was the major product under most conditions. The midgut preparations were sensitive to reaction conditions, including radioactive substrate, protein concentration (optimal at 1mg/reaction), reaction time (optimal at 0.5 min), temperature (optimal at 22 degrees C), buffer pH (highest at pH 6), and the presence of a co-factor cocktail composed of reduced glutathione, hydroquinine and hemoglobin. In vitro PG biosynthesis was inhibited by two cyclooxygenase inhibitors, indomethacin and naproxen. Subcellular localization of PG biosynthetic activity in midgut preparations, determined by ultracentrifugation, revealed the presence of PG biosynthetic activity in the cytosolic and microsomal fractions, although most activity was found in the cytosolic fractions. This is similar to other invertebrates, and different from mammalian preparations, in which the activity is exclusively associated with the microsomal fractions. Midgut preparations from M. sexta pupae, adult cockroach, Periplaneta americana, and corn ear worms, Helicoverpa zea, also produced the same four major PG products. We infer that insect midguts are competent to biosynthesize PGs, and speculate they exert important, albeit unrevealed, actions in midgut physiology.

Prostaglandin biosynthesis by fat body from true armyworms, Pseudaletia unipuncta.

We describe prostaglandin (PG) biosynthesis by microsomal-enriched fractions of fat body prepared from true armyworms, Pseudaletia unipuncta. PG biosynthesis was sensitive to experimental conditions, including incubation time, temperature, pH, substrate and protein concentration. Optimal PG biosynthesis conditions included 1 mg of microsomal-enriched protein, incubated at 28 degrees C for 7.5 min at pH 8. These preparations yielded four major PGs: PGA(2), PGE(2), PGD(2) and PGF(2alpha). PGA(2) and PGE(2) were the predominant eicosanoids produced under these conditions. Two non-steroidal anti-inflammatory drugs, indomethacin and naproxen, effectively inhibited PG biosynthesis. Unlike other invertebrate PG biosynthetic systems studied so far, the true armyworm system appeared to be independent of the usual exogenous co-factors required by mammalian and other invertebrate systems. These findings are discussed with respect to PG biosynthesis in other invertebrate and vertebrate systems.

Evidence for the formation of a novel cyclopentenone isoprostane, 15-A2t-isoprostane (8-iso-prostaglandin A2) in vivo.

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.

Clavulones and related tert-hydroxycyclopentenone fatty acids: occurrence, physiological activity and problem of biogenetic origin.

The present review is concerned with a group of prostanoids from soft corals, namely, clavulones and their congeners, as well as with related octadecanoids found in plants of the genus Chromolaena (Compositae). Known physiological properties of these prostaglandin-like fatty acids are discussed. Special attention is paid to published hypotheses on the biogenetic origin of clavulones and related fatty acids. According to a newly proposed biosynthetic route, acyclic alpha-ketols are metabolic precursors of cyclopentenolones.

Prostaglandin biosynthesis by fat body from the tobacco hornworm, Manduca sexta.

We describe prostaglandin (PG) biosynthesis by microsomal-enriched preparations of fat body from larvae of the tobacco hornworm Manduca sexta. Four major PGs were synthesized under most experimental conditions, PGA2, PGE2, PGD2 and PGF2 alpha. PGA2, was the predominant product under most conditions. Unlike mammals, in which PGA2, is generally thought to arise from non-enzymatic rearrangements of PGE2, the fat body preparations did not convert exogenous PGE2 into PGA2. These findings suggest that PGA2 is an important fat body product that is synthesized by a route that does not involve PGE2. The PG synthase activity and the overall profile of PG synthesis were sensitive to experimental conditions, including incubation time, temperature, and protein concentration. Optimal PG biosynthesis was observed with 1 mg of microsomal-rich protein, incubated at 30 degrees C for 1-2 min. The fat body preparations is sensitive to two non-steroidal anti-inflammatory drugs, indomethacin and naproxen, both of which inhibited PG synthesis at low dosages.

Investigation of the allene oxide pathway in the coral Plexaura homomalla: formation of novel ketols and isomers of prostaglandin A2 from 15-hydroxyeicosatetraenoic acid.

Prostaglandin A2 is a major constituent of the gorgonian Plexaura homomalla, and there is evidence that its biosynthesis involves a noncyclooxygenase pathway. The coral contains an 8(R)-lipoxygenase and an allene oxide synthase; from arachidonic acid, the sequential action of these enzymes gives an allene epoxide, the cyclization of which forms an analogue of prostaglandin A2 (PGA2) with no 15-hydroxyl group. In this study we examined the metabolic fate of 15-hydroxyeicosatetraenoic acid (15-HETE), which via analogous reactions could lead to PGA2. The 8(R)-lipoxygenase metabolized preferentially the 15(R) enantiomer of 15-HETE, and this reaction was stimulated fivefold by including 1 M NaCl in the incubation. Further enzymic steps were detected by comparing the metabolic profiles of the 8(R)-hydroperoxy-15(R)-hydroxy intermediate with that of its 8(S),15(S) enantiomer. Two main products were formed exclusively from the 8(R),15(R) enantiomer: an allene epoxide and the comparatively stable epoxide, 8,9-epoxy-10,15-dihydroxyeicosa-5,11,14-trienoic acid. Formation of the allene oxide was inferred from detection of its hydrolysis and cyclization products. It cyclized to give two isomers of PGA2 which have a "cis" arrangement of the side chains. The main hydrolysis product (8,15-dihydroxy-9-ketoeicosa-5,11,13-trienoic acid) was unstable and prone to oxygenation, giving 8,14,15-trihydroxy-9-ketoeicosa-5,10,12-trienoic acids after reduction of the 14-hydroperoxide. We conclude that metabolism of a 15-hydroxy eicosanoid is a potential route to the A series prostaglandins, although the low yield and lack of stereochemical control suggest that this is not the natural pathway of biosynthesis in P. homomalla. Unexpectedly, the major end products of the pathway are trihydroxy ketols and the single diastereomer of a stable epoxyalcohol.

Adaptive cytoprotection against alcohol injury in the rat stomach is not due to increased prostanoid synthesis.

This study evaluated the effects of 25% ethanol, a mild irritant, on endogenous prostanoid synthesis in the rat stomach before and after exposure to oral 100% ethanol. Rats received water or 25% ethanol orally. After 15 min, a portion of each group was sacrificed and the remaining animals treated with 100% ethanol prior to sacrifice one minute later. Microsomal membrane fractions were prepared from the glandular gastric mucosa in all groups and incubated with 14C arachidonic acid in the presence of cofactors. Endogenous mucosal prostanoid synthesis was analyzed by radiochromatography and results correlated with the presence or absence of gastric injury macroscopically. Prostanoids measured included PGI2, PGF2 alpha, PGE2, PGD2, PGA2, and thromboxane A2. Additional experiments were performed in like manner to those just described with the exception that indomethacin (5 mg/kg intraperitoneally) pretreatment was rendered. Stomachs exposed to water or 25% ethanol alone demonstrated a modest and equivalent level of synthesis of all prostanoids measured. Exposure to 100% ethanol (with and without mild irritant pretreatment) significantly increased prostanoid synthesis (especially PGI2, PGF2 alpha, and PGE2) compared with stomachs exposed to water or 25% ethanol alone; only mild irritant treated mucosa was protected from injury by 100% ethanol. Indomethacin pretreatment reversed the increased prostanoid synthesis in mucosa exposed to 100% ethanol, with or without mild irritant pretreatment, and partially reversed the protective effect of 25% ethanol. Other experiments using tissue slices in which perturbations in mucosal levels of prostanoids were measured by radioimmunoassay under identical experimental conditions exhibited similar results. These data dispute the notion that adaptive cytoprotection is mediated by increased endogenous prostanoid synthesis. The partial reversal of this process by indomethacin was most likely secondary to some other action of this agent, such as a reduction in gastric blood flow, rather than direct effects on prostanoid synthesis.

Role of antidiuretic hormone in the attenuated furosemide response observed during indomethacin administration.

Recently we demonstrated that increased chloride reabsorption in Henle's loop is a major contributor to the blunted furosemide response observed during prostaglandin synthesis inhibition. Because antidiuretic hormone (ADH) modulates chloride reabsorption in the loop and because prostaglandin synthesis inhibition potentiates ADH-mediated water reabsorption, ADH may be necessary for the attenuated furosemide response observed during prostaglandin synthesis inhibition. If such were the case, then prostaglandin synthesis inhibition should have no effect on furosemide's chloruretic response in the absence of ADH. To test this hypothesis, the effect of indomethacin on furosemide chloruresis was determined in homozygous (ADH-deficient) Brattleboro rats and in homozygous Brattleboro rats receiving ADH (2.4 mU/hr) over a short period of time. Furosemide-induced chloruresis was not different (P was not significant) between indomethacin-treated homozygous Brattleboro rats and homozygous Brattleboro rats receiving the indomethacin vehicle (fractional excretion of chloride: 6.28% +/- 1.08% vs. 6.24% +/- 0.98%). However, in ADH-infused Brattleboro rats, furosemide chloruresis was lower in indomethacin-treated rat groups than in vehicle-treated rat groups (fractional excretion of chloride: 3.09% +/- 0.62% vs. 6.61% +/- 0.88%; P less than 0.02) and lower than in indomethacin-treated ADH-deficient Brattleboro rats as well (P less than 0.05). Mean arterial pressure, inulin clearance, and renal blood flow were not different between any groups. Urinary prostaglandin excretion rates were not different between ADH-deficient Brattleboro rats and ADH-treated Brattleboro rats during furosemide administration and were markedly reduced by indomethacin in both circumstances. Thus, ADH is necessary for the blunted furosemide response observed during prostaglandin synthesis inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum-induced arachidonic acid release and prostaglandin biosynthesis are potentiated by oxygenated sterols in NRK 49F cells.

Fetal calf serum is able to activate arachidonic acid release from phospholipids in NRK 49F cells. We showed that this phenomenon can be potentiated by adding oxysterols to the culture medium. The oxysterol effect was dose-dependent and was not observed in the absence of fetal calf serum. Greater amounts of prostaglandin E2 and prostaglandin F2 alpha were released into the medium in the presence of oxysterols without apparent modification of the cyclooxygenase activity. The most effective oxysterols, in descending order, were the following: calcitriol greater than 7 alpha-hydroxycholesterol greater than 7 beta-hydroxycholesterol greater than 25-hydroxycholesterol. Cholesterol and 7-ketocholesterol were unable to activate phospholipase activity. The mechanism of this activation by oxysterols is still unknown.

Biosynthesis of prostaglandins in gingiva of patients with chronic periodontitis.

This study was undertaken to determine the ability of inflamed and normal gingival tissues to synthesize prostaglandins (PGs) from the precursor arachidonic acid. Thirteen samples of inflamed human gingival tissue and six samples of normal human gingival tissue were studied. The inflammation was characterized histologically. After incubation of the tissue with [14C]arachidonate, PG metabolites were separated by thin-layer chromatography and identified by comparison with co-chromatographed standards. Inflamed gingival tissue synthesized significantly larger amounts, compared to normal tissue, of 6-keto-PGF1 alpha (P less than 0.05), thromboxane B2 (P less than 0.01), PGD2 (P less than 0.05), and PGA2 (P less than 0.001). Some unidentified metabolites, possibly lipoxygenase products were detected in significantly (P less than 0.001) larger amounts in inflamed than in normal tissue.

[Demonstration of the biosynthesis of prostaglandins from arachidonic acid by rat adrenal cortex cells in culture]. / Mise en évidence de la biosynthèse de prostaglandines à partir d'acide arachidonique par les cellules corticosurrénaliennes de rat en culture.

The biosynthesis of prostaglandins of the E pathway was shown in new-born rat adrenocortical cells in long-term primary culture, by direct incubation of labelled arachidonic acid or by incubation with prelabelled cells. A rapid method of extraction was developed using silanised silica cartridges.

The relationship between the antiviral action of interferon and prostaglandins in virus-infected murine cells.

The relationship between prostaglandins (PG) and interferon (IFN) was investigated. IFN induced the synthesis of immunoreactive PGE and PGA at early and late stages, respectively, of vaccinia virus infection in mouse L fibroblasts. Only species-specific IFN possessed this activity and PG synthesis was stimulated in virus-infected cells, while normal L cells were not affected. The vaccinia virus infection did not significantly alter PG synthesis in the absence of IFN. Indomethacin increased the rate of vaccinia virus replication and partially inhibited the IFN-induced protection of L cells. The addition of exogenous PGA1 only partially reversed this effect. Finally, short-term PGA treatment induced the synthesis of two enzymes (protein kinase and 2,5A synthetase) thought to be partially responsible for the antiviral action of interferon. These findings suggest that a prostaglandin or PG-related compound seems to mediate at least one aspect of IFN action.

[Kinetic study of prostaglandin biosynthesis. A mathematical model]. / Kineticheskoe issledovanie biosinteza prostaglandinov. Matematicheskaia model'.

A mathematical model has been suggested to describe the kinetics of the prostanoid biosynthesis in the Plexaura homomalla coral. It allows to predict the changes in prostaglandin A2 concentration at various pH and concentration of sodium ions citrate when the latter is not too high. The degradation of prostaglandin biosynthesis intermediates is shown to proceed as two consecutive first-order reactions. For the second step the reaction rate grows into the raise of prostaglandin A2 concentration, apparently due to autocatalytic character of the process.

Aspectos fisiologicos y farmacologia de las prostaglandinas (PGs) E2 y F2 / Physiologic and pharmacologic aspects of the E2 and F2 prostaglandins (PGs)

Las prostaglandinas (PG) E-2 y F-2 alfa tienen variadas acciones fisiologicas y farmacologicas. El presente trabajo es una revision bibliografica de estas acciones, profundizando en los efectos uterinos, farmacocinetica, las indicaciones, precauciones y contraindicaciones en su uso