The biosynthesis of thromboxane (TX) B2 and immunoreactive prostaglandin (PG) F2 alpha in clotting whole blood ex vivo as well as collagen-induced platelet aggregation were determined before and up to 72 h after intravenous injection of 600 mg azapropazone 2H2O and intramuscular injection of 30 mg ketorolac tromethamine in six healthy subjects. The drug doses were selected on the basis of comparable analgesic activity (maximal recommended analgesic dose). Both platelet aggregation and prostanoid biosynthesis were inhibited by racketorolac to a significantly greater extent and for a longer period of time than by azapropazone. Correlations between serum concentrations and the inhibitory effects on TXB2 biosynthesis were observed for both drugs. Using the sigmoidal Emax model the mean serum concentration of azapropazone inhibiting platelet TXB2 generation by 50% (EC50) was found to be 98.1 +/- 41.9 (s.d.) micrograms ml-1, a value 1000 times higher than that for rac-ketorolac. The moderate inhibition of platelet function by azapropazone as compared with rac-ketorolac might be an advantage with regard to its use as a post-operative analgesic.

Arachidonic acid (AA), when cleaved from phospholipids by cytosolic phospholipase A2 alpha (cPLA2a), generates eicosanoids, with pro-hemostatic, pro-inflammatory, vasoactive and gastro-protective functions. We describe a patient (27-year-old man) and his twin-sister with early-onset bleeding diathesis and recurrent gastro-intestinal (GI) ulcers. Platelet aggregation/δ-granules secretion by collagen was impaired, but normal by AA; serum levels of thromboxane (Tx) B2 and 12-hydroxyeicosatetraenoic acid, and urinary levels of 11-dehydro-TxB2 were extremely low. Patients were homozygous for 1723G>C transition in PLA2G4A gene, which changed the codon for Asp575 to His. GI ulcers affected 5/14 heterozygous (< 40 years) and 1/16 wild-type homozygous >> 60 years) family members; none had bleeding diathesis. The proband, his sister and mother also had mildly reduced factor XI levels. Platelet messenger RNA expression did not differ among subjects with different PLA2G4A genotypes. Conversely, platelet cPLA2a was undetectable by Western Blotting in the proband and his sister, and decreased in 1723G>C heterozygous subjects, suggesting that the variant is transcribed, but not translated or translated into an unstable protein. We described a syndromic form of deficiency of cPLA2a , characterised by recurrent GI ulcers and bleeding diathesis, associated with mild inherited deficiency of factor XI. Unlike other reported patients with cPLA2a deficiency, these patients had extremely low levels of platelet TxA2 biosynthesis.

Nonsteroidal antiinflammatory drugs differ with respect to their effects on prostaglandin metabolism in various tissues, a property that may be partly responsible for some of the differences in the pharmacologic activities and side-effect profiles that are associated with their use. The effects of nabumetone on urinary prostaglandin excretion have not been reported. Fourteen healthy females, age 21-43 years, were treated with nabumetone (NAB) 1000 mg daily, sulindac (SUL) 200 mg every 12 hours, and indomethacin (IND) 50 mg every 12 hours for 7 days in a randomized period-balanced crossover study. The effects of drug treatment on urinary prostaglandin excretion (PGE2, 6-keto-PGF1 alpha, PGF2 alpha, thromboxane [TX] B2) and platelet function (collagen-induced whole blood platelet aggregation [CIPA] and template bleeding time) were determined on day 1 and day 7. For each treatment regimen, mean baseline urinary PG excretion values were comparable for each prostanoid, but the pattern of excretion differed in response to each drug. Treatment with NAB significantly increased the urinary excretion rates of PGE2 and PGF2 alpha, but 6-keto-PGF1 alpha and TXB2 excretion were unchanged. IND treatment did not result in a significant change in PGE2 excretion but did significantly reduce urinary 6-keto-PGF1 alpha and TXB2 excretion rates. Reduced excretion of PGF2 alpha was observed on both study days during treatment with IND and SUL. SUL treatment also resulted in increased urinary PGE2 excretion while significantly reducing 6-keto-PGF1 alpha excretion on day 7. Significant differences were observed between the NAB and SUL regimens with respect to PGF2 alpha excretion and between the NAB and SUL regimens for PGE2, PGF2 alpha, 6-keto-PGF alpha 1 (on day 1 only) and TXB2 (on day 1 only). Neither NAB nor SUL caused inhibition of CIPA or bleeding time although platelet aggregation was inhibited during IND treatment. That NAB treatment was neither associated with alterations in platelet function nor decreases in the urinary excretion of the vasodilatory prostaglandins, PGE2 and 6-keto-PGF1 alpha, suggests that NAB possesses renal sparing properties.

Urinary levels of 2,3-dinorthromboxane B2 (Tx-M) and 2,3-dinor-6-ketoprostaglandin F1 alpha (PGI-M), measured by gas chromatography-negative ion-chemical ionization mass spectrometry, accurately reflect in vivo biosynthesis of thromboxane A2 (TxA2) and prostacyclin (PGI2), respectively. Although the basal excretion of Tx-M and PGI-M is adequately documented, no systemic data on the excretion during controlled exercise have been presented. We studied the effect of maximal tolerated exercise (2 h of bicycle ergometry) on the excretion of Tx-M and PGI-M in healthy humans. In addition, their urinary levels of epinephrine (E) and norepinephrine (NE) were analyzed. To address the impact of locally formed adenosine, all subjects were reinvestigated after administration of theophylline. Exercise did not affect the excretion of Tx-M (47 +/- 21 vs. 34 +/- 9 pg/mg creatinine) but increased the excretion of PGI-M (74 +/- 14 vs. 267 +/- 70 pg/mg creatinine; P less than 0.02). Theophylline augmented urinary NE and E but did not significantly change the PGI-M response to exercise. We suggest that the normal cardiovascular eicosanoid response to exercise is a platelet-independent increase in vascular PGI2 formation.

Slices of rat aorta were incubated in Krebs-Ringer bicarbonate buffer for measurements of immunoreactive 6-ketoprostaglandin F1 alpha, thromboxane (TX) B2, prostaglandin (PG)E2, and PGF2 alpha, and in Tris buffer (pH 9.3) for determination of prostacyclin (PGI2)-like activity. No significant generation of TXB2, PGE2, or PGF2 alpha by rat aortic tissue could be detected. The time-dependent release of 6-keto-PGF1 alpha Krebs-Ringer bicarbonate buffer closely correlated with PGI2 generation in alkaline Tris buffer. During a 30-min incubation period, 6-keto-PGF1 alpha, release was 79.8 +/- 3.3 pmol/mg at a buffer potassium concentration of 3.9 mmol/liter and significantly increased by 23% to 98.3 +/- 8.5 pmol/mg (P less than 0.025) in the absence of potassium in the incubation medium. A smaller decrease in buffer potassium concentration to 2.1 mmol/liter and an increase to 8.8 mmol/liter did not significantly alter aortic 6-keto-PGF1 alpha release. Changes in the incubation buffer sodium concentration from 144 mmol/liter to either 138 or 150 mmol/liter at a constant potassium concentration of 3.9 mmol/liter did not alter the recovery of 6-keto-PGF1 alpha. Our results support the concept that PGI2 is the predominant product of arachidonic acid metabolism in rat aorta. They further show that PGI2 can be recovered quantitatively as 6-keto-PGF1 alpha under the present in vitro conditions. In addition, this in vitro study points to the potassium ion as a modulator of vascular PGI2 synthesis with a stimulation at low potassium concentrations.

The measurement of antiphospholipid antibodies (aPL) has been an important aspect of antiphospholipid syndrome (APS) characterization since the disease was first described in the 1980s. Despite significant efforts geared toward the standardization of immunoassays that measure anticardiolipin antibodies and anti-β2-glycoprotein I spanning three decades, there are still reports of significant interassay and interlaboratory variation in the results of these assays. At the recent 13th International Congress on Antiphospholipid Antibodies (APLA 2010, April 13-16, 2010, Galveston, TX), a task force composed of internationally recognized experts in the field of APS was formed to address these issues. In this review, we discuss approaches that have been used in the past to achieve harmonization among aPL immunoassays as well as the ongoing efforts of the APLA task force. Our review also highlights the importance of cutoff determination in aPL assays and the clinical significance of positive aPL results of varying magnitudes.

It has been reported that the prostaglandin (PG) precursor, arachidonic acid, produces divergent hemodynamic responses in the feline pulmonary vascular bed. However, the pattern of arachidonic acid products formed in the lung of this species is unknown. In order to determine the type and activity of terminal enzymes in the lung, prostaglandin biosynthesis by microsomes from cat lung was studied using the prostaglandin endoperoxide, PGH2, as a substrate. The major products of incubations of PGH2 with microsomes were thromboxane (TX) B2 (the major metabolite of TXA2), 6-keto-PGF1 alpha (the breakdown product of PGI2) and 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Formation of TXB2 was markedly reduced by imidazole. Tranylcypromine decreased the formation of TXB2 and HHT and inhibited the formation of 6-keto-PGF1 alpha. At low PGH2 concentrations, equal production of TXB2 and 6-keto-PGF1 alpha was observed. However, as PGH2 concentration increased, 6-keto-PGF1 alpha production approached early saturation while TXB2 production increased in a linear fashion. These results suggest that enzymatic formation of TXA2 and PGI2 is a function of substrate availability in the lung. These findings provide a possible explanation for the divergent hemodynamic responses to arachidonic acid infusions at high and low concentrations in the feline pulmonary vascular bed.

Endothelin-1 (ET-1), a potent mediator released by airway epithelial cells, often exerts its effects in the lung through stimulation of arachidonic acid (AA) metabolism. To investigate its range of influence, we studied the action of ET-1 on the synthesis and release of thromboxane (TX)B2, prostaglandin (PG)D2, and histamine from canine airway cells obtained by bronchoalveolar lavage (BAL). ET-1 (10(-10), 10(-9) and 10(-8)M) stimulated production of TXB2 and PGD2 by BAL cell preparations in a dose-related manner in the absence of measurable histamine release. Release of TXB2 was 10-fold higher than that of PGD2. The effect of ET-1 on AA metabolism in alveolar macrophages was evaluated in preparations of purified (greater than 99%) cells labelled for 20-22 hrs with 3H-AA prior to stimulation. ET-1 (10(-8), 10(-7), 10(-6)M) induced significant, dose-related release of 3H-AA and its metabolites from alveolar macrophages, to levels 350% above control. These studies indicate that low levels of ET-1 can stimulate AA metabolism in resident luminal airway cells, including alveolar macrophages, and suggest that the function of these luminal cells may be modulated by the epithelium, in vivo, through the release of this peptide into the airways.

The pulmonary vascular effects of thromboxane (TX) B2 were investigated in the intact chest dog and cat. In both species, TXB2 increased lobar arterial pressure in a dose-related fashion without affecting left atrial pressure. Since blood flow to the lobe was maintained constant, the increases in lobar arterial pressure reflect increases in pulmonary lobar vascular resistance. In the dog, the increases in lobar arterial pressure were associated with increases in small vein pressure suggesting that TXB2 increases lobar vascular resistance by constricting pulmonary veins and upstream segments. In both the cat and dog, TXB2 and prostaglandin E2 had comparable pressor activity, whereas both substances were less potent than prostaglandin F2 chi. Responses to TXB2 were similar in experiments in which blood flow was controlled or when pulmonary flow varied naturally. Pulmonary vasoconstrictor responses to TXB2 were similar when the lung was ventilated, during bronchial occlusion in the dog and during an apneic interval in the cat. The increases in vascular resistance in both species were not altered by doses of indomethacin that blocked responses to arachidonic acid. The present studies demonstrate that TXB2 has significant vasoconstrictor activity in the feline and canine pulmonary vascular bed and suggest that responses to TXB2 are not dependent on alterations in bronchomotor tone or enhanced prostaglandin synthesis in the lung.

It is known that thromboxane (TX)B2, the metabolite of the potent vasoconstrictor TXA2, is elevated markedly in the serum of the patients immediately postburn. We had shown that extensive thermal injury causes a reduction in mesenteric blood flow that can lead to bacterial translocation from the intestine. In this study, we tested the hypothesis that the TX synthetase inhibitor, OKY-046, prevents increased mesenteric vascular resistance (MVR) and decreases the rate of translocation of bacteria seen after extensive thermal injury. Pigs in groups 1 (n = 6) and 2 (n = 6) had third degree burns of 40 per cent total body surface area under general anesthesia and were resuscitated according to the Parkland formula. Pigs in group 2 received 10 milligrams per kilogram of OKY-046 as a bolus just before the burn and 10 grams per kilogram per minute for 16 hours as a continuous infusion. Pigs in group 3 (control, n = 6) underwent general anesthesia only and received daily maintenance fluids of lactated Ringer's solution, 2 milliliters per kilogram per hour. OKY-046 prevented the significant increase in MVR seen during the first eight hours after burn. The total peripheral resistance (TPR) showed an early increase and a late decrease in the burn group, while the cardiac index (CI) and temperature (T) significantly increased after 24 hours. Administration of OKY-046 kept TPR, Cl, and T remarkably stable. OKY-046 reduced the rate of translocation of bacteria seen in the burn group from 67 to 17 per cent. Our results show that the blockade of thromboxane synthesis by OKY-046 prevented the early mesenteric vasoconstriction and the late hyperdynamic response seen after thermal injury and was useful in reducing the incidence of postburn translocation of bacteria.

We wanted to evaluate whether the kidney tissue can metabolize thromboxane (Tx) B2 and, specifically, whether the 2,3-dinor metabolite might be formed in the kidney and excreted in the urine. For this purpose, we used an isolated perfused kidney preparation exposed to vehicle or TxB2 at different infusion rates. Approximately 96% of the total TxB2 infused was recovered in the venous effluent, whereas approximately 1% was found in urine. Isolated perfused kidneys exposed to [3H]TxB2 eliminated in the urine 1.2% of the [3H]TxB2 infused, measured by thin-layer chromatographic analysis, and actively metabolized [3H]TxB2 to 2,3-dinor-TxB2, 11-dehydro-TxB2, and possibly 2,3,4,5-tetranor-TxB1. No metabolites of TxB2 were recovered in the venous effluent. As a marker of renal TxB2 metabolic activity, urinary 2,3-dinor-TxB2 was quantified by high-resolution gas chromatography-negative-ion chemical ionization mass spectrometry. The 2,3-dinor-TxB2 was detected both before and during TxB2 infusion in urinary samples but not in the venous effluent. The ratio of 2,3-dinor-TxB2-TxB2 increased during the infusion reaching a peak value immediately after stopping the TxB2 infusion. These results indicate that, in the rat, the kidney tissue metabolizes TxB2 to 2,3-dinor-TxB2, and both TxB2 and 2,3-dinor-TxB2 are excreted in the urine.

The mechanisms whereby bacterial endotoxins stimulate arachidonic acid metabolism in macrophages are uncertain. Both protein kinase C activation and de novo protein synthesis occur in macrophages in response to endotoxin. In this study we evaluated the time course and role of protein kinase C and de novo protein synthesis in endotoxin stimulated arachidonic acid metabolism in resident rat peritoneal macrophages. Thromboxane (TX) B2 was measured as the representative arachidonic acid metabolite synthesized in response to Salmonella enteritidis endotoxin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate (PMA). The effect of inhibition of protein kinase C by 1-(5-isoquinolinsulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine on endotoxin- and A23187-induced TXB2 synthesis was examined. The potential roles of transcriptional and translational events in endotoxin- and A23187-stimulated TXB2 synthesis were determined by utilizing the transcriptional inhibitors camptothecin (10 microM) or actinomycin D (0.08 microM), and the translational inhibitor cycloheximide (0.1 microM). Whereas, A23187 stimulated maximal TXB2 synthesis within 15 min, endotoxin showed a more prolonged time course with a 12-fold increase in TXB2 synthesis above basal levels after 3 h (P less than 0.05). PMA induced an approx. 8-fold increase above basal TXB2 levels that was blocked by inhibition of transcription with actinomycin D. H-7 (10 microM to 50 microM) inhibited endotoxin- and A23187-stimulated eicosanoid synthesis. Staurosporine (0.2 microM) produced a selective 66% inhibition of endotoxin, but not A23187-stimulated TXB2 synthesis. Endotoxin-induced TXB2 production was significantly (P less than 0.05) inhibited by staurosporine, camptothecin, actinomycin D or cycloheximide at intervals from 30 min prior to, through 60 min after endotoxin stimulation. These studies suggest a role for protein kinase C activation and de novo protein synthesis in endotoxin signal transduction events leading to increased macrophage arachidonic acid metabolism. These intracellular events are essential in sustaining the prolonged inflammatory response to endotoxin.

BACKGROUND

omega-3 Fatty acids may have a major impact on immune responses involved in heart transplant rejection. We compared the effects of posttransplant intravenous supplementation with omega-3-rich versus omega-6-rich lipid emulsions on graft survival, plasma fatty acid profiles, and levels of arachidonic acid versus eicosapentaenoic acid-derived lipid mediators.

RESULTS

Inbred PVG and Wistar-Kyoto rats were used as donors and recipients, respectively, in a model of heterotopic heart transplantation. Animals received 9 g/kg body wt per day of either fish oil-derived (n = 8) or soybean oil-derived fat (n = 7) in the form of a continuously infused lipid emulsion; controls were sham-infused with saline (n = 8). Graft rejection was assessed by loss of activity of the transplant. The fish oil-derived preparation but not that originating from soybean oil caused an increase in total and free plasma fatty acids. Substantial quantities of eicosapentaenoic acid and docosahexaenoic acid appeared in the free fatty acid fraction, surpassing those of arachidonic acid. Ex vivo stimulation of neutrophils with the Ca2+ ionophore A23187 demonstrated an increase in 5-series leukotriene (LT) generation in animals undergoing omega-3 lipid infusion (LTB5, omega-oxidation products of LTB5, LTA5 secretion), with 5-series/4-series LT ratios ranging between 0.08 and 0.36. Ratios of TX B3/B2 liberated from ex vivo stimulated platelets even approached 1:1 in omega-3 supplemented rats. Graft survival was 7.6 +/- 0.3 (mean +/- SEM) days in saline-infused, 10.4 +/- 0.7 in omega-6 lipid-infused, and 12.9 +/- 0.4 in omega-3 lipid-infused animals.

CONCLUSIONS

Posttransplant intravenous alimentation with fish oil-derived lipid emulsions prolongs heart transplant survival in excess to omega-6 lipids. Profound changes in fatty acid profiles and lipid mediator generation may underlie this finding.

The syntheses of prostaglandin (PG) F2 alpha, E2 and D2, and thromboxane (TX) B2 from [14C]arachidonic acid were studied in frontal cortex of human control and Alzheimer's disease (AD) brains using the microsomal fractions. Under the assay conditions employed, it was found that the major metabolite of [14C]arachidonic acid was PGE2 accounting for 63% of total prostanoid production; PGF2 alpha accounted for 21.5%, TXB2 for 9%, and PGD2 for 6.5%. When AD samples were compared to control samples, microsomal PG synthesis was significantly decreased, with reduced production of PGE2, PGF2 alpha and PGD2. Such decreases in AD brain seem unrelated to age, sex, postmortem delay and, as far as could be determined, antemortem state. In both control and Alzheimer groups, a history of anti-inflammatory therapy seemed to correlate with increased PG synthesis.

Renal glomeruli were isolated from rat kidneys using a passive mechanical sieving technique. Glomerular microsomal fraction, glomerular homogenate, or intact glomeruli were incubated with [1-14C]arachidonic acid, and the profile of prostaglandin (PG) synthesis was determined by thin-layer chromatography. The three incubation systems produced 15.3, 20.8, and 40.4% 6-keto-PGF1 alpha; 19.1, 23.5, and 15.3 PGF2 alpha; 5.7, 9.1, and 3.9% thromboxane (TX) B2; 36.0, 35.1, and 37.0% PGE2; and 23.9, 11.3, and 3.4% PGD2, respectively. Glomeruli were placed in suspension within glass chambers and superfused with Krebs solution. Superfusion with 1.6 x 10(-4) M arachidonic acid stimulated a significant release of renin from glomeruli, whereas 2.7 x 10(-6) M PGE1, PGE2, PGF2 alpha, TXB2, PGD2, or a stable analog of PGH2 had no effect on renin. When the rapid breakdown of PGI2 was counteracted by either increasing the concentration to 1.7 x 10(-4) M or stabilizing in Krebs at pH 9.4, it stimulated a significant increase in renin release. Reducing the arachidonic acid concentration to 1.6 x 10(-5) M eliminated both renin release and PGI2 synthesis, while increased PGE2 synthesis persisted. Finally, using an inhibitor of PGI2 synthesis, azo analog 1 (2.8 x 10(-6) M), 6-keto-PGF1 alpha produced in response to arachidonic acid was eliminated, as was the concurrent release of renin, but PGE2 synthesis was not affected. These results suggest that the mechanism of direct interaction between renal PG and renin in isolated glomeruli is selectively due to the action of PGI2.

The effects of prostaglandins (PGs) D2, E2, F2 alpha, an epoxymethano analogue of PGH2 (U-46619), prostacyclin (PGI2), 6-keto-PGF1 alpha and thromboxane (TX) B2 were tested on spirally-cut strips of guinea-pig isolated ileum or colon. In the ileum no prostanoid exerted a marked effect on the resting tissue, but PGD2, PGE2 or PGI2 1 ug ml-1 inhibited submaximal contraction to KC1. U-46619 1 ug ml-1 either inhibited or increased contractions in KC1, but PGF2 alpha, 6-keto-PGF1 alpha or TXB2 1 ug ml-1 had no significant effect. PGE2 relaxed colonic strips whereas the other prostanoids caused contraction, except for TXB2 which had no effect. The PG antagonist SC-19220 blocked colonic contractions to the prostanoids, and the residual inhibitory effect of PGD2, U-46619 or PGI2 was demonstrated by the reduction of submaximal contractions to acetylcholine. Our results suggest that prostanoid receptors mediating inhibitory responses of circular muscle predominate in the ileum, whereas in the colon both excitatory and inhibitory prostanoid receptors occur.

Pieces of rat brain incubated in oxygenated Tyrode solution were stimulated with the divalent cation ionophore A 23187. Incubation media were assayed for leukotriene (LT) C4-like immunoreactivity as well as for prostaglandin (PG) D2, 6-keto-PGF1 alpha and thromboxane (TX) B2 using specific and sensitive radioimmunoassays. Spontaneous release of the cyclooxygenase products but only of trace amounts of LTC4-like immunoreactivity was detected. Stimulation with ionophore A 23187 increased the level of cyclooxygenase products and to a smaller extent the level of LTC4-like immunoreactivity. Inhibition of cyclooxygenase by indomethacin was paralleled by a significantly increased release of LTC4-like material under basal conditions as well as in the presence of ionophore A 23187. On the other hand, the synthesis of LTC4-like material was significantly decreased by the lipoxygenase inhibitors quercetin and nordihydroguaiaretic acid. The presence of LT-like material in rat brain incubates was confirmed by bioassay. The capacity of brain tissue to synthesize LT-like material may be important for cerebral blood flow as well as cellular functions in the central nervous system.

We have developed a method for the simultaneous estimation of the levels of the prostanoids 6-keto prostaglandin (PG) Flalpha, PGB2, PGD2, PGE2, PGF2(alpha), PGJ2, and thromboxane (TX) B2 in blood- or serum-containing medium using liquid chromatography-tandem mass spectrometry. These prostanoids and their deuterium derivatives, which were used as internal standards, were subjected to solid-phase extraction using Empore C18 HD disk cartridges and analyzed in the selected reaction-monitoring mode. A linear response curve starting at 10 pg of prostanoid/tube was observed for each prostanoid. The accuracy of the method was demonstrated with samples containing known amounts of the prostanoids. Furthermore, we used this method to analyze the prostanoids produced in mouse bone marrow-derived mast cells stimulated with arachidonic acid, which resulted in the production of PGD2, PGE2, PGF2alpha, and TXB2. The results suggest that this simultaneous quantification method is useful for the analysis of the production of biomedically important prostanoids.

We aimed to examine the prospective association between plasma FAs, oxylipins, and risk of acute myocardial infarction (AMI) in a Singapore Chinese population. A nested case-control study with 744 incident AMI cases and 744 matched controls aged 47-83 years was conducted within the Singapore Chinese Health Study. Nineteen plasma FAs and 12 oxylipins were quantified using MS. These were grouped into 12 FA clusters and 5 oxylipin clusters using hierarchical clustering, and their associations with AMI risk were assessed. Long-chain n-3 FAs [odds ratio (OR) = 0.67 per SD increase, 95% confidence interval (CI): 0.53-0.84, P < 0.001] and stearic acid (OR = 0.65, 95% CI: 0.44-0.97, P = 0.03) were inversely associated with AMI risk, whereas arachidonic acid (AA) was positively associated with AMI risk (OR = 1.25, 95% CI: 1.03-1.52, P = 0.02) in the multivariable model with adjustment for other FAs. Further adjustment for oxylipins did not substantially change these associations. An inverse association was observed between AA-derived oxylipin, thromboxane (TX)B2, and AMI risk (OR = 0.81, 95% CI: 0.71-0.93, P = 0.003). Circulating long-chain n-3 FAs and stearic acid were associated with a lower and AA was associated with a higher AMI risk in this Chinese population. The association between the oxylipin TXB2 and AMI requires further research.

The formation and composition of cysteinyl-leukotrienes (LT) in primary astroglial cell cultures prepared from newborn rat brain has been studied. Small amounts of cysteinyl-LT determined in terms of LTC4-like material in the supernatants of the cultures, became detectable after stimulation of the cells with 10(-5) M ionophore A23187. Cysteinyl-LT formation increased with time, reaching about 600 pg (mg protein)-1 after 60 min incubation. In contrast, considerable thromboxane (TX) B2 synthesis was found at 5 min following A23187-stimulation (about 30 ng TXB2 (mg protein)-1). The synthesis of cysteinyl-LT was abolished by 5 x 10(-5) M nordihydroguaiaretic acid (NDGA). Irrespective of the duration of incubation, blockage of prostanoid synthesis by 10(-6) M indomethacin did not result in increased cysteinyl-LT production. Reversed phase HPLC combined with radioimmunological detection showed that, after 60 min incubation in the presence of A23187, LTC4 and LTD4 accounted for practically all the LTC4-like immunoreactive material in the supernatants of cell cultures. No significant amounts of LTE4 could be detected. The results show that astrocytes may contribute to brain LTC4 and LTD4 synthesis. However, the cellular site of cerebral LTE4 formation seems to be other than the astroglia.

The effect of short-term beta blockade with the nonselective antagonist propranolol and with the cardioselective antagonist metoprolol on arterial blood pressure, heart rate, serum renin activity, plasma thromboxane (Tx) B2 (the stable metabolite of TxA2). and plasma and urinary prostaglandin (PG) E2 and F2 alpha levels were examined in 11 normal subjects. After propranolol (160 mg by mouth), supine and standing mean arterial pressure (MAP) fell from 82 +/- 2 and 88 +/- 4 mm Hg to 71 +/- 3 (P less than 0.001) and 78 +/- 3 mm Hg (P less than 0.01) within 13 hr. MAP fell from 78 +/- 2 and 88 +/- 3 mm Hg to 72 +/- 2 (P less than 0.025) and 80 +/- 2 mm Hg (P less than 0.01) after metoprolol (200 mg by mouth). These blood pressure effects were associated with beta blockade; both drugs induced reduction in heart rate and serum renin activity and the reductions were of the same order. Propranolol and metoprolol also reduced plasma TxB2 levels by 33% (P less than 0.005) and 46% (P less than 0.05), but plasma and urinary PGE2 and PGF2 alpha levels were not changed by either drug. These findings suggest that changes in PGE2 or PGF2 alpha levels are unlikely to contribute to the short-term hypotensive effects of propranolol or metoprolol in normal subjects. Alterations in the synthesis or metabolism of the potent vasoconstrictor and proaggregatory drug TxA2, however, may be involved in the hypotensive, cardioprotective, and antiplatelet effects of beta-adrenergic antagonists.

The signal transduction events occurring in monocytes in response to endotoxin (LPS) stimulation are incompletely delineated, although pertussis toxin (PT)-sensitive G proteins and the mitogen-activated protein kinase (MAPK) cascade have been implicated. Cellular desensitization in response to 18-h pre-exposure to 1 microgram/mL LPS alters signal transduction pathways of cellular activation and decreases production of certain inflammatory mediators such as thromboxane (Tx)B2, the stable metabolite of TxA2. We hypothesized that LPS stimulation of the human monocyte cell line THP-1 occurs via MAPK activation, and that LPS desensitization, induced by pre-exposure to LPS, is associated with altered signaling through the MAPK cascade. Involvement of a specific MAPK, ERK, in LPS-stimulated TxB2 production was further tested using a specific MAPK cascade inhibitor, PD98059 (PD). PD inhibited LPS and phorbol myristate acetate (PMA)-stimulated ERK activation as demonstrated by immunoblots using anti-activated ERK antibodies. PD significantly inhibited LPS and PMA-stimulated TxB2 synthesis to non-detectable levels, suggesting an involvement of MAPK in LPS-stimulated activation. Because PT-sensitive G proteins mediate LPS-stimulated signal transduction, their role in MAPK activation was tested. Pretreatment with PT inhibited basal and LPS-stimulated, but not PMA-stimulated ERK activation. Activation of ERK after LPS desensitization was also assessed. LPS pre-exposure resulted in a profound decrease in LPS-stimulated activation of ERK, but did not affect PMA activation of ERK. These data implicate the involvement of the MAPK cascade in LPS-stimulated activation of THP-1 cells and suggest coupling of Gi proteins and MAPKs in LPS-stimulated events. LPS desensitization is associated with decreased MAPK activation, but does not impair MAPK activation by PMA. Thus, LPS desensitization appears to selectively alter signal transduction upstream of ERK.

In previous studies, it had been shown that human gray and white matter tissue slices have the capacity to synthesize large amounts of cysteinyl-leukotrienes (cysteinyl-LT) in vitro. This study was initiated to investigate cysteinyl-LT formation by tissue slices from intracranial tumors in comparison with cyclooxygenase products such as prostaglandin (PG) F2 alpha and thromboxane (TX) B2. Tissue slices from meningiomas and astrocytomas were found to release large amounts of cysteinyl-LT spontaneously and even higher amounts after ionophore A 23187 stimulation, which could not be accounted for by blood possibly remaining in the tissue slices. Cysteinyl-LT were identified by their immunoreactive characteristics, their biological activity in the guinea pig ileum bioassay, and their retention time on reversed-phase HPLC. With increasing malignancy, astrocytomas were shown to have an increasing biosynthetic capacity for cysteinyl-LT and TXB2 in vitro. In comparison with meningioma patients, malignant astrocytoma patients had an enhanced urinary excretion of LTE4, the major urinary metabolite of cysteinyl-LT in humans, which dropped in level within 7 days after operation by 79%. A correlation exists between the in vivo cysteinyl-LT production in patients with malignant astrocytomas and that observed under basal conditions in the tissue slices in vitro. The results provide evidence that in malignant astrocytoma patients, the tumor tissue produces large amounts of cysteinyl-LT, which may be detected in the patients' urine.

The adequacy, selectivity and long-term persistence of inhibition in cyclooxygenase-dependent platelet function by a daily low-dose (0.45 mg kg-1 day-1) aspirin treatment have been evaluated in 15 patients after a recent (less than 17 days) acute myocardial infarction. Serum thromboxane (TX) B2, an index of platelet TXA2 production, was decreased by 94-98% (P less than 0.001) by aspirin, while urinary excretion of 6-keto-prostaglandin F1 alpha, as an index of extraplatelet cyclooxygenase activity, remained unchanged. Compared to placebo, aspirin induced a persistent increase in bleeding time (% difference 45.6 +/- 21.4, mean +/- SD) and a decrease in platelet aggregation by ADP, epinephrine, collagen and arachidonic acid. No tendency towards an attenuation of the effects was apparent for the period of aspirin administration (4 weeks). Aspirin 0.45 mg kg-1 day-1 is adequate and selective in the long-term inhibition of TX-related platelet function in patients after acute myocardial infarction. The clinical effectiveness of such a regimen remains to be proven in clinical trials.