Tritiated Arachidonic Acid Research Articles

Induction of apoptosis in circulating angiogenic cells by microparticles

Systemic sclerosis (SSc) is an autoimmune disease marked by aberrant activation and apoptosis of endothelial cells (ECs) and decreased numbers of circulating angiogenic cells (CACs). The aim of this study was to analyze whether microparticles might link pathologic activation and apoptosis of ECs with reduced numbers of CACs. Apoptosis was quantified by staining for annexin V and measurement of caspase 3 activity. The uptake of microparticles by CACs was determined by fluorescence-activated cell sorting and by fluorescence microscopy. Tritiated arachidonic acid and phosphatidylinositol 3,5-bisphosphate were used to demonstrate the transfer of arachidonic acid and highlight the role of the acid sphingomyelinase in microparticle-induced apoptosis of endothelial progenitor cells. Microparticles derived from activated or apoptotic ECs, the expression of which is strongly increased in the blood of patients with SSc, induce apoptosis in CACs in a dose-dependent manner. Microparticles, which are rich in arachidonic acid, are phagocytosed by CACs. Inhibition of phagocytosis prevents the induction of apoptosis in CACs by microparticles. Microparticles can transport arachidonic acid from ECs to CACs, and purified arachidonic acid mimics the proapoptotic effects of microparticles. Arachidonic acid activates the acid sphingomyelinase, and inhibition of acid sphingomyelinase prevents microparticle-induced apoptosis of CACs. Thus, phagocytosis of microparticles might stimulate the activity of acid sphingomyelinase and activate the apoptotic machinery. The induction of apoptosis in CACs by microparticles derived from ECs provides a novel link between aberrant activation or apoptosis of ECs, decreased numbers of CACs, and impaired formation of new vessels in SSc.

Role of platelet microparticles in the production of thromboxane by rabbit pulmonary artery.

This study examined the role of platelet microparticles in thromboxane A2 (TXA2) production. Incubation of microparticles with [14C]arachidonic acid and A23187 produced 14C-labeled TXB2, the stable metabolite of TXA2. To investigate the possibility that endothelial cells (ECs) transfer arachidonic acid to platelet microparticles and promote TXB2 synthesis, ECs with their cellular lipids prelabeled with tritiated arachidonic acid were incubated with microparticles. In the absence of microparticles, there was no production of tritiated TXB2 by the ECs. However, when microparticles were coincubated with prelabeled ECs, tritiated arachidonic acid was metabolized to tritiated TXB2. Aspirin was then used to inhibit cyclooxygenase. ECs coincubated with aspirin-treated platelet microparticles did not produce TXB2, as measured by radioimmunoassay. In contrast, aspirin-treated ECs coincubated with microparticles produced TXB2, and its production was enhanced by methacholine (10(-4) mol/L), indicating that endothelially derived arachidonic acid, and not endothelially derived prostaglandin endoperoxide, was transferred to the microparticle and further metabolized to TXA2. Additional studies with rabbit aorta and pulmonary artery investigated whether microparticles contributed to vascular contractions. Preincubation with microparticles enhanced arachidonic acid-induced contractions in the aorta and methacholine-induced contractions in the pulmonary artery. The thromboxane receptor antagonist SQ29548 and the thromboxane synthase inhibitor dazoxiben blocked these effects. Because TXA2 is an important mediator in various pathophysiologic states, including hypertension, the ability of platelet microparticles to act as a cellular source of TXA2 might provide new insight into the role of platelets and platelet microparticles in the control of vascular tone.

Olive Oil Isochromans Inhibit Human Platelet Reactivity

The effects of certain polyphenolic compounds in red wine, such as resveratrol and quercetin, have been widely investigated to determine the relationship between dietary phenolic compounds and the decreased risk of cardiovascular diseases. However, the effects of polyphenolic compounds contained in other foods, such as olive oil, have received less attention and little information exists regarding the biological activities of the phenol fraction in olive oil. The aim of this study was to evaluate the antiplatelet activity and antioxidant power of two isochromans [1-(3'-methoxy-4'-hydroxy-phenyl)-6,7-dihydroxy-isochroman (encoded L116) and 1-phenyl-6,7-dihydroxy-isochroman (encoded L137)] recently discovered in olive oil and synthesized in our laboratory from hydroxytyrosol. These compounds were effective free radical scavengers and inhibited platelet aggregation and thromboxane release evoked by agonists that induce reactive oxygen species-mediated platelet activation including sodium arachidonate and collagen, but not ADP. Release of tritiated arachidonic acid from platelets was also impaired by L116 and L137. These results indicate that other Mediterranean diet nutraceuticals also exhibit antioxidant activity that could be beneficial in the prevention of vascular diseases.

Synthesis of tritium labelled arachidonic acid amide and ester derivatives with dopamine, serotonin, vanillylamine, and ethyleneglycol moieties

AbstractTritium labelled arachidonic acid amides with dopamine, serotonin, vanillyl‐amine and the ethyleneglycol ester moieties with high specific activity (120 Ci/mmol) and yield (70–90%) were prepared from tritiated arachidonic acid by condensation with the corresponding amines and alcohol via mixed anhydrides or acyl fluorides. The labelled compounds were used for studying their uptake by mouse spleen lymphocytes. From the data obtained it was suggested that arachidonic acid amides permeate the membrane by means of passive diffusion, while transmembrane transport of arachidonoylethyleneglycol seems to be driven by the concentration gradient, maintained by hydrolytic enzymes. The compounds synthesized by the reported methods can also be used in receptor binding studies and in the oxidative metabolism of fatty acids amides and esters. Copyright © 2002 John Wiley & Sons, Ltd.

Effects of topical corticosteroids on inflammatory mediator–induced eicosanoid release by human airway epithelial cells

Background: Airway epithelial cells are among the first cells to come in contact with aerosolized corticosteroids. However, the relative potencies and time course of action of the several commonly used aerosolized corticosteroids on eicosanoid production by airway epithelial cells are unknown. Objectives: This study compared the effects of fluticasone, budesonide, and triamcinolone on eicosanoid output by human airway epithelial cells in vitro. We also determined the spectrum of eicosanoids affected and the mechanism for corticosteroid action. Methods: Cultured BEAS-2B airway epithelial cells (a transformed cell line) were exposed to corticosteroids (1 nmol/L to 1 μmol/L) for 2 to 48 hours and then assayed for basal- and bradykinin (BK)-stimulated eicosanoid output. The eicosanoid profile was identified by HPLC in tritiated arachidonic acid prelabelled cells, and PGE 2, the major eicosanoid product, was quantitated by RIA. The effect of corticosteroids on the immunoreactivity of key proteins involved in eicosanoid metabolism (ie, cyclooxygenase [COX], phospholipase A2 [PLA 2], and Clara cell protein, a PLA 2 inhibitor) was determined by Western blotting. Results: Eicosanoid output was largely confined to prostaglandins with values of 5 ± 2 and 82 ± 35 ng PGE 2/10 6 cells for basal- and BK stimulation, respectively (n = 8). All 3 corticosteroids inhibited basal- and BK-induced PGE 2 output in a dose- and time-dependent manner. Fluticasone and budesonide completely eliminated PGE 2 output in nanomolar concentrations in contrast to triamcinolone, which required micromolar concentration. The rank order of potency was: fluticasone = budesonide > triamcinolone. The time course of action for PGE 2 inhibition also differed, with budesonide acting more slowly than the other 2 corticosteroids ( P = .04). All 3 corticosteroids markedly reduced COX2 with little effect on COX1, cPLA 2 (Type IV), or iPLA 2 (Type VI) immunoreactivity or their relative distribution in cytosol versus membrane fractions. Clara cell protein immunoreactivity was undetectable in control and corticosteroid-treated cell lysates. Conclusion: These results show that in a human airway epithelial cell line, the 3 inhaled corticosteroids commonly used to treat asthma differ in onsets of action as inhibitors of prostaglandin synthesis and vary considerably in potency. All 3 corticosteroids act mechanistically in similar fashion by inhibiting COX2 synthesis. (J Allergy Clin Immunol 1999;103:1081-91.)

Toxicity of sulfur mustard in primary neuron culture

The toxicity of sulfur mustard (HD) was assessed in primary cultures of chick embryo forebrain neurons using several different endpoints. Mature neurons were found to be very sensitive to the toxic effects of this agent and tritiated arachidonic acid release, as well as the MTT, neutral red and alamarBlue cytotoxicity assays all gave LC 50 values in the low μ m range. Maximal toxicity was initiated within minutes of culture exposure to HD and was not found to be associated with toxic mediator release into the medium. The characteristics of toxicity were quite different when comparing immature cultures to mature ones. Mature cultures were more sensitive to the toxicity of HD than were immature cultures, and maximal toxicity in mature cultures took longer to be expressed. In addition, the toxicity was found to be dependent on the initial seeding density, as well as on the age of the cultures at the time of chemical treatment. Although the reasons for these observations are unclear, the apparent dependence of HD toxicity on the differentiative maturity of the cultures may eventually provide some clues as to the mechanism of action of this chemical agent. Furthermore, the extreme sensitivity of these cells to the toxic effects of HD makes them a useful model system with which to screen for potential protective drug regimens against this chemical warfare agent.

Preterm labor and bacterial intraamniotic infection: Arachidonic acid liberation by phospholipase A 2 of Fusobacterium nucleatum

Objective: The studies presented in this report were undertaken to evaluate whether Fusobacterium nucleatum , a common anaerobic isolate in intrauterine infection, stimulates arachidonic acid metabolism, a rate-limiting step for prostaglandin synthesis, in the human uterine endometrium. Study Design: Effects of F nucleatum on arachidonic acid liberation from human uterine endometrial cells and of F nucleatum extract on lysophosphatidylcholine production in human uterine endometrial cells were investigated. Results: When human uterine endometrial cells labeled with tritiated arachidonic acid to an isotopically steady state were exposed to an extract of F nucleatum , arachidonic acid liberation was stimulated, accompanied by lysophospholipid formation. Similar stimulatory effects on phospholipid degradation were also observed in the experiment with bacterially conditioned media. Conclusions: These results suggest that F nucleatum stimulates endometrial phospholipid metabolism, related to activity of phospholipase A 2 , which might induce the onset of labor associated with intraamniotic infection. (Am J Obstet Gynecol 1998;179:1579-82.)

Inhibitory effect of peptides derived from the N-terminus of lipocortin 1 on arachidonic acid release and proliferation in the A549 cell line: identification of E-Q-E-Y-V as a crucial component.

1. The ability of the glucocorticoid-induced protein lipocortin 1 (LC1) to inhibit arachidonic acid release and cell proliferation in A549 cells may be mimicked by a sequence taken from the N-terminal, LC1(13-25) (FIENEEQEYVQTV). We have now synthesized and tested for biological activity a library of 25 smaller peptides derived from this sequence. 2. Peptides were tested in two assays: A549 cells were prelabelled with tritiated arachidonic acid and thapsigargin (50 nM) and EGF (10 nM) used to stimulate the release of this fatty acid. Cell proliferation was determined by counting cell numbers following 3 day incubation with these peptides, or controls. 3. Many of the peptides were highly insoluble but could be more readily dissolved in aqueous solution in the presence of commercial liposomes or phosphatidyl serine (5 microM). Since neither of these agents alone had any effect on arachidonic acid release or cell proliferation, all peptides were tested in the presence of 5 microM phosphatidyl serine. Under these conditions LC1(13-25) was active in both assay systems with an IC40 of 40.7 and 57.0 microM respectively. 4. Deletion of amino acids from the C-terminus of the peptide progressively diminished (2-3 fold) the molar potency of LC1(13-25) in both assays: after the removal of Val22 biological activity was virtually undetectable or very weak (< 30% of LC1[13-25]). 5. Removal of amino acids from the N-terminus also lead to a progressive reduction (3-5 fold) in the molar potency of the peptides and biological activity became undetectable, or very weak, after the removal of Glu18. 6. All active peptides contained the core sequence EQEYV(Glu-Gln-Glu-Tyr-Val) which seems to represent a crucial component of the pharmacophore, although this sequence on its own was inactive and the shortest peptide with significant activity was LC1(18-25) (EQEYVQTV). 7. Methoxylation of Tyr21 abolished the ability of LC1(18-25) to inhibit cell proliferation and arachidonic acid release. A cyclized version of LC1(18-25) was also tested and found to be inactive. 8. LC1(18-25) (178 microM) inhibits cPLA2 activation in A549 cells as judged by a band-shift assay, whereas equimolar concentrations of an inactive peptide LC1(19-25) were without effect in this assay system. 9. Several possible mechanisms whereby these peptides act are discussed in the light of LC1 biology and of the effect of glucocorticoids on cell function.

Protein-facilitated Export of Arachidonic Acid from Pig Neutrophils

Activated neutrophils release a variety of eicosanoids into the extracellular medium including arachidonic acid, 5-hydroxyicosatetraenoic acid, and leukotriene A4 and B4. In this study, the mechanism of arachidonic acid export has been examined using inside-out plasma membrane vesicles from pig polymorphonuclear leukocytes. Tritiated arachidonic acid associated rapidly with the membrane vesicles and crossed the membrane into the intravesicular space in a time-dependent and saturable manner. Half the maximal influx rate was measured at an arachidonate concentration of 5.7 microM, and a maximal influx velocity of 3.0 nmol/mg x min was determined at pH 6.8. Influx into vesicles was sensitive to a number of common anion transport inhibitors including pentachlorophenol, phloretin, diiodosalicylic acid, and quercetin as well as to the proteases trypsin and Pronase, suggesting a protein-dependent process. Furthermore, influx was temperature-sensitive with an energy of activation of 11.6 kcal/mol. Varying extravesicular concentration of ATP, Na+, or K+ had no impact on arachidonate influx, whereas changes in pH had a profound effect; optimum transport activity was observed at an extravesicular pH of 6, whereas raising the pH to 9.5 essentially abolished uptake. These results indicate and initially characterize a novel protein-facilitated arachidonate export mechanism in pig neutrophils.

Leukotriene C 4 from vascular endothelium enhances neutrophil adhesiveness

We have examined the synthesis of leukotriene C 4 from bovine aortic and pulmonary artery endothelium. Under basal conditions, neither aortic nor pulmonary artery endothelium revealed significant amounts of hydroxy fatty acids. Following incubation with ionophore A23187, several peaks including one which co-migrated with authentic LTC 4 could be demonstrated from both aortic and pulmonary endothelium. LTC 4 production was maximal after 30 min incubation, was inhibitable by the lipoxygenase inhibitor nordihydroguairetic acid, and was synthesized by bovine endothelium from tritiated arachidonic acid substrate. The putative LTC 4 from endothelium was shown to be identical to authentic LTC 4 by chromatography and scanning UV spectroscopy. Endothelial-derived LTC 4 increased the adherence of bovine aortic endothelium for neutrophils in a concentration dependent pattern similar to authentic LTC 4. These data suggest that vascular endothelium may influence leukocyte-endothelial interactions through synthesis of biologically active arachidonic acid metabolites such as LTC 4.

Preferential esterification of arachidonic acid into ethanolamine phospholipids in epithelial cells from ovine endometrium.

In sheep, the pulsatile release of prostaglandin F2 alpha by the endometrium is necessary to achieve luteolysis which occurs at the end of the oestrous cycle. The production of prostaglandins is known to depend upon the availability of arachidonic acid, the fatty acid precursor of prostaglandin biosynthesis. Consequently, the mechanisms controlling intracellular amounts of arachidonate may be involved in the regulation of prostaglandin synthesis. Since arachidonic acid is mostly found in phospholipids and the endometrial epithelium is the primary source of prostaglandin F2 alpha during luteolysis, the fate of arachidonic acid when incorporated into epithelial cells from the ovine uterus was investigated. Endometrial epithelial cells isolated from cyclic ewes at day 15 after oestrus were cultured in the presence of [3H]arachidonic acid. Incorporation and distribution of the radiolabelled arachidonic acid into the various phospholipid classes were examined using HPLC. We observed that ethanolamine glycerophospholipids contained 61% of the total tritiated arachidonic acid incorporated into cellular lipids, whereas phosphatidylinositols, phosphatidylcholines and phosphatidylserines contained 17%, 13% and 4.7%, respectively. In addition, the radioactivity measured within phosphatidylethanolamines was preferentially detected in the 1-alkenyl-2-acyl (44%) forms of ethanolamine phospholipids, also called plasmalogens. The kinetic study of arachidonic acid uptake into ethanolamine phospholipids showed that arachidonic acid was rapidly esterified into the diacyl forms and then uptake decreased, whereas the incorporation increased continuously into the plasmalogen forms for at least 24 h. These results demonstrate that the primary pool of esterified arachidonic acid is found in ethanolamine plasmalogens of epithelial cells from the ovine endometrium. The high arachidonate content of ethanolamine plasmalogens suggests that these phospholipids play a crucial role in the control of arachidonic acid availability and ultimately in the regulation of prostaglandin synthesis.

Alterations of intrauterine eicosanoid production in pregnancy-induced hypertension: Decreased production of 12-hydroxyeicosatetraenoic acid in the placenta

The important role of eicosanoids in pregnancy-induced hypertension is generally accepted. Because of the lack of innervation of the uteroplacental vessels, humoral vasoactive factors are important for the regulation of vascular tone. Until now, mainly the balance of vasodilatative and vasoconstrictive prostaglandins has been studied. We were able to confirm their intrauterine imbalance in hypertensive pregnancies. In addition, the placental production of less known lipoxygenase metabolites has been analyzed in this study. Intrauterine tissues (30–100mg wet weight) were examined for their release of eicosanoids. Short term tissue cultures were performed in Hanks balanced salts solution (HBSS) at 37°C in an atmosphere of 95% air/5% CO 2 with and without incorporation of tritiated arachidonic acid. The arachidonate metabolites in culture media were analyzed by High Performance Liquid Chromatography (HPLC) with radioactivity detection or by enzymeimmunoassays or radioimmunoassays, respectively. All intrauterine tissues released more lipoxygenase metabolites than cyclooxygenase metabolites with 12-hydroxyeicosatetraenic acid (12-HETE) as their main metabolite. The placental release of 12-HETE was significantly decreased in hypertensive pregnancies. In hypertensive pregnancies the ratio TXB 2/6-keto-PGF 1α synthesis was increased. Lipoxygenase metabolites, especially 12-HETE, seem to have impotant physiological and pathophysiological functions in the intrauterine compartment. Their biological role in this context needs further investigation.

Lindane-Induced Elimination of Gap Junctional Communication in Rat Uterine Myocytes Is Mediated by an Arachidonic Acid-Sensitive cAMP-Independent Mechanism

Previous studies by this laboratory showed that the pesticide lindane rapidly and potently inhibits gap junctional communication in myometrial smooth muscle cells. This study examined the possible role of cAMP or arachidonic acid in lindane′s elimination of myometrial gap junctional communication. Lindane produced concentration-dependent increases in cAMP of 1.21, 2.94, 6.06, and 8.69 pmol/mg protein with 0.1, 1, 30, and 100 μM lindane, respectively, compared to solvent-treated controls (1.27 pmol/mg protein). Lindane also increased release of tritiated arachidonic acid to 342, 509, 852, 1236, 1639, and 4454 dpm/μg protein with 0.01, 0.1, 1, 10, and 100 μM lindane, respectively, compared to solvent controls (342 dpm/mu g protein). Transfer of Lucifer Yellow dye was used as a measure of gap junctional communication. Both 8-br-cAMP (98, 97, 54, and 4% transfer seen with 0, 1, 10, and 100 μM cAMP) and arachidonic acid (98, 73, 54, 31, and 0% dye transfer for 0.1, 1, 10, 100, and 1000 nM arachidonic acid) depressed dye transfer in cultured myocytes. Although the adenylate cyclase inhibitor 2′,3′-dideoxyadenosine completely reversed forskolin-induced depression of dye transfer (1 μM forskolin, 22% transfer), it had no effect with lindane, indicating that lindane′s depression of dye transfer was independent of adenylate cyclase activation. Lindane′s inhibition of dye transfer was effectively reversed by growing myometrial cells under arachidonic acid-free conditions in the presence of eicosapentaenoic acid, a fatty acid that competes with arachidonic acid for the sn-1,2 position of membrane phospholipids: 0, 15, 40, and 88% dye transfer occurred in the presence of 0.01, 0.1, 1, and 10 μM eicosapentaenoic acid with 30 μM lindane. This implies that arachidonic acid release may be a critical event associated with lindane′s inhibition of gap junctional communication in uterine myocytes.

Interleukin-1 and tumor necrosis factor stimulate arachidonic acid release and phospholipid metabolism in human myometrial cells

Our aim was to evaluate the effects of the cytokines interleukin-1 and tumor necrosis factor on arachidonic acid release in human myometrial cells. Primary monolayer cultures of human myometrial cells prelabeled with tritiated arachidonic acid were exposed to interleukin-1 or tumor necrosis factor for varying periods and the release of tritiated arachidonic acid and its loss from phospholipids were measured by radiochromatography. To gain some information on the biologic action of interleukin-1 the contractile response to oxytocin was measured in myometrial strips preincubated with this cytokine. Data were statistically evaluated with analysis of variance or Student's test. Both cytokines caused a dose-dependent increase in tritiated arachidonate release that was suppressed by the protein synthesis inhibitor cycloheximide. Tritiated arachidonic acid release was maximal after 24 hours of stimulation with interleukin-1. Both interleukin-1 and tumor necrosis factor stimulated the release of the isotopically labeled fatty acid from phosphatidylcholine. In addition, interleukin-1 also increased the loss of arachidonic acid from phosphatidic acid and significantly potentiated the oxytocin-evoked myometrial contractility. Both interleukin-1 and tumor necrosis factor enhance arachidonic acid release, probably by inducing the synthesis of phospholipase A2 and possibly other enzymes involved in the metabolism of phospholipids. In turn, arachidonic acid itself may act as a second messenger, synergizing with other uterotonic agents, as well as serving as the precursor for prostaglandins and various other bioactive eicosanoids.

Biosynthesis, structure and biological activity of hydroxyeicosatetraenoic acids in Hydra vulgaris.

Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the control of body pattern, head and tentacle regeneration and bud formation in Hydra spp. Here we describe for the first time the biosynthesis of hydroxyeicosatetraenoic acids (HETEs) in vitro by hydroid cytosolic extracts. Incubation of both unlabelled and tritiated AA with homogenates of Hydra vulgaris led to the conversion of up to 11% of the exogenous fatty acid into mainly two metabolites. These were characterized as 11-hydroperoxyeicosatetraenoic acid (11-HPETE) and 11-HETE by means of a combination of chromatographic, chemical, 1H-n.m.r. and electron-impact m.s. techniques. Trace amounts of 9-HETE and 12-HETE were also found. Analysis of 11-HETE by chiral-phase h.p.l.c. revealed that this metabolite was composed mainly of the R enantiomer. The production of 11-HPETE and 11-HETE was found to be: (1) associated with the cytosolic fraction of Hydra homogenates; (2) dependent on AA concentration, incubation time and protein amount in the homogenates; (3) unaffected by co-incubation with the 5- and 12-lipoxygenase inhibitors, 5,8,11-eicosatriynoic acid and nordihydroguaiaretic acid, the cyclo-oxygenase inhibitor, indomethacin, or the cytochrome P-450 inhibitors, proadifen and methoxalen. These results strongly suggest the presence of a very active (R)-11-lipoxygenase in H. vulgaris. The activity of both R and S enantiomers of synthetic 9-, 11- and 12-HETE and of 'endogenous' 11-HETE was studied on tentacle regeneration and bud formation in decapitated Hydra. Although almost all compounds tested inhibited budding, only endogenous 11-HETE and synthetic (R)-11-HETE significantly enhanced the average number of tentacles, thus suggesting that this eicosanoid might be one of the cellular regulators of regeneration in H. vulgaris.

Metabolism of arachidonic acid by caruncular and allantochorionic tissues in cows with retained fetal membranes (RFM)

The metabolism of arachidonic acid (AA) by caruncular and allantochorionic tissues and its regulation was studied in normal cows (n=13) and those with retained fetal membranes (RFM; n=9). Tissues were taken via the vagina about 6 hours postpartum and incubated for 6 hours in minimum essential medium containing tritiated AA alone or in the presence of oxytocin, platelet activating factor (PAF), epidermal growth factor (EGF) or ionophore calcium (A23187). The metabolites of AA were separated by reverse phase-high pressure-liquid chromatography. Tissue concentrations of prostaglandin F 2α (PGF 2α) and prostaglandin E 2 (PGE 2) and plasma 13,14-dihydro-15-keto-PGF 2α (PGFM) concentration were also measured by radioimmunoassay. For caruncular tissue, less thromboxane B 2 (TXB 2) and more 6-keto prostaglandin F 1α (PGIM) was synthesized in tissue from the animals with RFM than in the controls. Oxytocin, PAF, EGF and A23187 increased only PGIM production in the control animals; A23187 also decreased TBX 2 synthesis. For the allantochorion, more PGE 2, leukotriene B 4 (LTB 4) and PGIM and less TXB 2, PGF 2α and hydroxyecosatetranoic acids (HETE) was synthesized in tissue from cows with RFM than from animals that delivered normally. All of the substances used in this study increased PGIM, PGF 2α and LTB 4 and decreased TXB 2 production by the allantochorionic tissue in control animals. The metabolism of AA by the allantochorionic tissue seems quantitatively under hormonal control. The metabolism of AA at the level of both maternal and fetal components of the placenta in cows with RFM differed from that seen in animals that expelled the membranes normally.

Subcellular electrolyte alterations during progressive hypoxia and following reoxygenation in isolated neonatal rat ventricular myocytes.

This study characterizes the sequential alterations of, and relations between, multiple electrolytes in cytoplasm, mitochondria, and whole cells during hypoxia and on reoxygenation in isolated neonatal rat ventricular myocytes. Subcellular electrolyte content and distribution were measured by electron probe x-ray microanalysis, membrane phospholipid degradation by tritiated arachidonic acid release, and cell morphology by electron microscopy. At 1-2 hours of hypoxia, the myocyte population showed a loss of cytoplasmic potassium, magnesium, and chlorine without alteration of cytoplasmic sodium or calcium. Mitochondria showed increased potassium with unchanged magnesium content. There was no morphological evidence of cell injury or tritiated arachidonic acid release. At 3-5 hours of hypoxia, the myocyte population showed a further loss of cytoplasmic potassium and magnesium and an increase in cytoplasmic sodium, chlorine, and calcium. At a single-cell level, the increase in cytoplasmic sodium preceded the increase in cytoplasmic calcium. Mitochondria showed increased sodium and chlorine and decreased magnesium before increased calcium content; potassium loss was manifest only at 5 hours of hypoxia. At 3-5 hours of hypoxia, there was also tritiated arachidonic acid release and morphological evidence of cell injury. Reoxygenation for 1 hour after 5 hours of hypoxia partially reversed the mean alterations of all electrolytes, except calcium, in the cytoplasm of the myocyte population, whereas analysis was required at a single-cell level to show a partial reversal in calcium levels in cytoplasm of reoxygenated cells. Reoxygenation for 1 hour after 5 hours of hypoxia partially reversed the mean alterations of all electrolytes, including calcium, in the mitochondria of the myocyte population. Recovery of potassium in the cytoplasm correlated with reduction of mitochondrial calcium content on reoxygenation and best predicted recovery of cellular homeostasis of sodium, chlorine, magnesium, and calcium. This study demonstrates that in this experimental model of hypoxia 1) initial losses of cytoplasmic potassium and magnesium occur in the absence of cell injury; 2) increases of sodium, chlorine, and calcium occur in association with cell injury, with sodium increasing before calcium; 3) membrane phospholipid degradation and electrolyte derangement, including increased calcium, may contribute to reversible and irreversible phases of cell injury; 4) analysis of calcium at a subcompartmental level and at a single-cell level is required to correlate reduction of calcium on reoxygenation with recovery of cell homeostasis; 5) reduction of calcium content in mitochondria may predict recovery of cell homeostasis; and 6) recovery of potassium on reoxygenation best predicts recovery of cell membrane function and cell homeostasis.

Differences in arachidonic acid metabolism by human myelomonocytic cell lines.

The production of arachidonic acid metabolites by the HL60, ML3, and U937 human phagocyte cell lines was determined after incubation with interferon-gamma (IFN-gamma, 500 U/ml) or vehicle for 4 days. Cells were prelabeled with tritiated arachidonic acid, [3H]AA, for 4 h, and media supernatants were analyzed by high-performance liquid chromatography. None of the cell lines produced [3H]AA metabolites in large amounts during an unstimulated, basal release period (30 or 60 min). In response to 10 microM calcium ionophore A23187 incubation (30 min), undifferentiated and IFN-gamma-differentiated HL60 cells formed both cyclooxygenase products (thromboxane and prostaglandins) and lipoxygenase products (leukotrienes and hydroxyeicosatetraenoic acids). In contrast to the HL60 cells, IFN-gamma-differentiated U937 cells formed primarily cyclooxygenase products and undifferentiated and IFN-gamma-differentiated ML3 cells did not form any [3H]AA metabolites in response to A23187. These results indicate the need to be careful in selecting a cell line for use in a phagocyte assay system when cyclooxygenase and/or lipoxygenase products could influence the assay results.

Arachidonic acid metabolism by bovine placental tissue during the last month of pregnancy

Conversion of tritiated arachidonic acid (AA) into metabolites of the cyclo- and lipoxygenase pathways by bovine fetal placental tissue (200 mg) and fetal plus maternal placental tissue (400 mg) of Days 255, 265, 275 of gestation and at parturition (n = 5) during a 30 min incubation was measured using reverse-phase high pressure liquid chromatography. Fetal placental tissue produced 13,14-dihydro-15-keto-prostaglandin E 2 (PGEM) as the major metabolite, the synthesis of which increased from Day 265 to Day 275 and parturition by 150 % and 475 %, respectively. In tissues collected at parturition, PGE 2 synthesis was also detected. On Day 275 and at parturition fetal placental tissue synthesized the metabolite 12-hydroxyheptadecatrienoic acid (HHT), and throughout the experimental period the lipoxygenase product 15-HETE was detected with synthesis rates increasing over time of gestation. In addition, an unidentified metabolite was regularly found in the radiochromatograms which eluted at 1 h and 1 min (U101), between HHT and 15-HETE. The synthesis of this metabolite decreased as pregnancy progressed. Furthermore, various other polar and nonpolar metabolites pooled under the heading UNID were eluted, the production of which increased over time of gestation. The presence of maternal placental tissue did not influence the synthesis of PGEM, 15-HETE and U101, but the production of HHT was decreased when maternal tissue was present. Also, as pregnancy progressed, maternal placental tissue seemed to contribute to the pool of unidentified metabolites. In conclusion, fetal placental tissue seems to be the major source of the AA metabolites when compared with maternal placental tissue, and AA metabolism by bovine placental tissue is markedly increased throughout the last month of pregnancy, suggesting a role for AA metabolites in mechanisms controlling parturition.

Blockade by lipoxygenase inhibitors of Ca 2+-dependent insulin secretion from permeabilized rat islets : A molecular mechanism distinct from that of α2-adrenergic agonists

To evaluate the regulation and effects of pancreatic islet lipoxygenase, adult rat islets were permeabilized, using digitonin or staphylococcal α-toxin, and then were studied in a medium simulating an intracellular milieu at fixed ambient concentrations of Ca 2+. Permeabilized islets retained 12-lipoxygenase activity, as indicated by conversion of tritiated arachidonic acid to a predominant peak of [ 3H]12-hydroxy-eicosatetraenoic acid (12-HETE); this activity was inhibited (89–98%) by the lipoxygenase blockers nor-dihydroguaiaretic acid (35 μM), BW755c (250 μM) or ETYA (35 μM). Lesser amounts of compounds coeluting with 15- and 11-HETE (but little or no 5-HETE) were formed; however, 11-HETE (and possibly some 15-HETE) was probably synthesized (at least in part) via cyclooxygenase, as suggested by the partial synthesis blockade induced by 50 μM ibuprofen. The production of 12-HETE did not require the presence of Ca 2+, Mg 2+ or ATP; it also was not stimulated by addition of cyclic AMP, a phorbol ester, or calmodulin. However, it was augmented modestly by provision of a basal cytosolic free Ca 2+ concentration of 60–80 nM, with no further increase at physiologically elevated levels of 260–530 nM. Elevations in cytosolic free Ca 2+ concentrations induced insulin release which was inhibited by cooling, epinephrine or protein kinase inhibitors and, therefore, was exocytotic in nature. Lipoxygenase inhibitors blocked this insulinotropic effect of calcium at submaximal or saturating Ca 2+ concentrations (with or without its potentiation by 12- O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C) by 53–82%. However, they did not reduce the Ca 2+-independent secretory effects (at subnanomolar Ca 2+ concentrations) of the phorbol ester alone. Similar results were seen using dibutyryl cyclic AMP to activate protein kinase A. The α 2-adrenergic agonists epinephrine or clonidine inhibited Ca 2+-, TPA- or cyclic AMP-induced insulin release without reducing HETE formation. We conclude that (1) islet lipoxygenase is constitutively expressed and is not physiologically regulated by α 2-adrenergic agonism, Ca 2+ or protein kinases; (2) lipoxygenase modulates insulin release; HETE production is not merely an epiphenomenon reflecting the activation (or inhibition) of exocytotic secretion; (3) islet lipoxygenase inhibitors reduce insulin secretion, at least in part, by blocking the direct effects of Ca 2+ on exocytosis and/or its synergism with Ca 2+-binding proteins such as protein kinase C; and (4) these same inhibitors do not directly poison protein kinase C or A, or the exocytotic apparatus. Furthermore, the retention of significant activity and biological effects of lipoxygenase in leaky islets suggests that this enzyme may be associated with, and act upon, cell membranes in islets.