No description available.

Most embryonal mortality occurs during early embryonic development. Two experiments were designed to study aspects of early embryonic development. Experiment 1 was to determine if early ovine embryos were capable of metabolizing arachidonic acid. Cyclic ewes were estrous synchronized with 6⍺-methyl-17β-hydroxy progesterone acetate (MPA) pessaries, superovulated with follicle stimulating hormone (FSH) and bred artificially. Embryos were collected on d 4, 8, 10, 12 or 14 of pregnancy and incubated with 1 μCi of [¹⁴C] arachidonic acid in an atmosphere of 5% CO₂, 45% O₂ and 50% N at 37°C for 24 h. Embryos from all days of pregnancy metabolized arachidonic acid to a number of compounds. Embryos produced primarily an unidentified polar compound, 6-keto-prostaglandin F₁⍺ (6-keto-PGF₁⍺), prostaglandin F₂⍺ (PGF₂⍺), prostaglandin E₂ (PGE₂), 13,14-dihydro-15-keto prostaglandin F₂⍺ (PGFM), prostaglandin B₂ (PGB₂) and 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Experiment 2 was to determine whether prostaglandins have a role in embryo hatching from the zona pellucida. Ewes were superovulated and bred artificially, and embryos were collected on d 7 of pregnancy. Embryos were incubated with ethanol (control), indomethacin, PGE₂ or indomethacin and PGE₂ in an atmosphere of 5% CO₂ and 95% air at 37°C for 24 h. Indomethacin appeared to decrease embryo hatching rate (indomethacin, 34.5% vs control, 46.4%). Prostaglandin E₂ appeared to increase embryo hatching rate (PGE₂, 60.0% vs. control, 46.4%). However, hatching rates for indomethacin and PGE₂ treatment groups were not different from control (P > .05). When compared to any group with indomethacin treatment, PGE₂ increased (P < .05) embryo hatching rate. The results of this study indicated that early ovine embryos can convert arachidonic acid to various compounds in vitro. Although not conclusive, indomethacin may decrease and PGE₂ may increase embryo hatching rate. Therefore, embryo-produced prostaglandins may be involved in hatching of sheep embryos from the zona pellucida. / Master of Science

[Truncated abstract] Arachidonic acid is oxidised in vivo by non-enzymatic (free radical) or enzymatic pathways (cyclooxygenase, lipoxygenase, and cytochrome P450) to form a range of biologically active eicosanoids. Specifically, arachidonic acid is metabolised by cytochrome P450 -hydroxylase to produce vasoactive 20-hydroxyeicosatetraenoic acid (20-HETE), and by 5-lipoxygenase to produce proinflammatory leukotriene B4 (LTB4), which can further be metabolised by -hydroxylase to from 20-OH-LTB4 and 20-COOH-LTB4. F2-Isoprostanes (F2-IsoPs) are produced through free radical attack on arachidonic acid and have been recognised as the most reliable markers of lipid peroxidation in vivo. The metabolic syndrome (MetS) is characterised by abdominal obesity, hypertension, insulin resistance, glucose intolerance, and dyslipidemia. It is associated with low-grade inflammation and oxidative stress and an increased risk of developing cardiovascular diseases. Dietary weight loss is strongly recommended for the management of the MetS and can potentially minimise the risk of cardiovascular diseases and diabetes in individuals with the MetS. Little is known regarding the role of these arachidonic acid metabolites in the MetS and the effect of weight loss on their metabolism. Chapter three comprised of three in vitro studies aimed to examine 20-HETE synthesis in human blood cells. 20-HETE acts as a second messenger for vasoconstrictor actions of angiotensin II (Ang II) and endothelin-1 (ET-1) in renal and mesenteric beds. Human neutrophils and platelets are integral to the inflammatory process. ... Production of LTB4 and 20-OH-LTB4 was significantly lower compared with controls (P<0.005) and remained so after adjustment for neutrophil count (P<0.05).The weight loss intervention resulted in a 4.6kg reduction in body weight and a 6.6cm decrease in waist circumference and a significant increase in LTB4 and 20-OH- LTB4 in the weight loss group. Chapter Five continued to investigate the role of other arachidonic acid metabolites, 20-HETE and F2-IsoPs in the MetS and the effect of weight loss. In the case-control study (Human study 1), plasma and urinary 20-HETE and F2-IsoPs were significantly elevated in the MetS group, but no significant difference was found in stimulated-neutrophil 20-HETE. A significant gender x group interaction was observed in that women with the MetS had higher urinary 20-HETE and F2-IsoPs compared to controls (P<0.0001). In a randomised controlled trial (Human study 2), relative to the weight- maintenance group, a 4.6 kg loss in weight resulted in a 2 mmHg fall in blood pressure but did not alter the production of 20-HETE or F2-IsoPs. No significant differences were shown in 20-HETE released from stimulated-neutrophils before and after weight loss. 20-HETE and oxidative stress may be important mediators of cardiovascular disease risk in the MetS. Although a 4% reduction in body weight reduced BP, there were no changes in plasma or urinary 20-HETE or F2-IsoPs. In summary, in vitro studies show that human neutrophils and platelets can produce 20-HETE in response to Ang II and ET-1, and human studies demonstrate that the presence of MetS has a significant impact on arachidonic acid metabolism and effective weight loss can restore leukocyte synthesis of LTB4.

No description available.

<p>Prostaglandin (PG) E₂ is an eicosanoid derived from the polyunsaturated twenty carbon fatty acid arachidonic acid (AA). PGE₂ has physiological as well as pathophysiological functions and is known to be a key mediator of inflammatory responses. Formation of PGE₂ is dependent upon the activities of three specific enzymes involved in the AA cascade; phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGEs). Although the research within this field has been intense for decades, the regulatory mechanisms concerning the PGE₂ synthesising enzymes are not completely established.</p><p>PGE₂ was investigated in human monocytes with or without lipopolysaccharide (LPS) pre-treatment followed by stimulation with calcium ionophore, opsonised zymosan or phorbol myristate acetate (PMA). Cytosolic PLA₂a (cPLA₂a) was shown to be pivotal for the mobilization of AA and subsequent formation of PGE₂. Although COX-1 was constitutively expressed, monocytes required expression of COX-2 protein in order to convert the mobilized AA into PGH₂. The conversion of PGH₂ to the final product PGE₂ was to a large extent due to the action of microsomal PGEs-1 (mPGEs-1). In addition, experiments with inhibitors of extracellular signal regulated kinase and p38 activation, indicated that phosphorylation of cPLA₂α was markedly advantageous for the formation of PGE₂.</p><p>Ellagic acid, a natural polyphenolic compound found in fruits and nuts, was shown to inhibit stimuli induced release of PGE₂ in human monocytes. The effect of ellagic acid was not due to a direct effect on the activities of the enzymes but rather to inhibition of the LPS-induced protein expression of COX-2, mPGEs-1 and cPLA₂a.</p>

Linoleic acid, with a DRI of 12-17g/d, is the most highly consumed polyunsaturated fatty acid in the Western diet and is found in virtually all commonly consumed foods. The concern with dietary linoleic acid, being the metabolic precursor of arachidonic acid, is its consumption may enrich tissues with arachidonic acid and contribute to chronic and overproduction of bioactive eicosanoids. However, no systematic review of human trials regarding linoleic acid consumption and subsequent changes in tissue levels of arachidonic acid has been undertaken. In this study, we reviewed the human literature that reported changes in dietary linoleic acid and its subsequent impact on changing tissue arachidonic acid in erythrocytes and plasma/serum phospholipids. We identified, reviewed, and evaluated all peer-reviewed published literature presenting data outlining changes in dietary linoleic acid in adult human clinical trials that reported changes in phospholipid fatty acid composition (specifically arachidonic acid) in plasma/serum and erythrocytes within the parameters of our inclusion/exclusion criteria. Decreasing dietary linoleic acid up to 90% was not significantly correlated with changes in tissue arachidonic acid levels (p=0.39). Similarly, when dietary linoleic acid levels were increased six fold, no significant correlations with tissue arachidonic acid levels were observed (p=0.72). However, there was a positive relationship between dietary gamma-linolenic acid and arachidonic acid on changes in tissue arachidonic levels. Our results do not support the concept that modifying current intakes of dietary linoleic acid has an effect on changing tissue levels of arachidonic acid in adults consuming Western-type diets.

Prostaglandins, released from Kupffer cells, have been shown to mediate the increase in hepatic glycogenolysis by various stimuli such as zymosan, endotoxin, immune complexes, and anaphylotoxin C3a involving prostaglandin (PG) receptors coupled to phospholipase C via a G(0) protein. PGs also decreased glucagon-stimulated glycogenolysis in hepatocytes by a different signal chain involving PGE(2) receptors coupled to adenylate cyclase via a G(i) protein (EP(3) receptors). The source of the prostaglandins for this latter glucagon-antagonistic action is so far unknown. This study provides evidence that Kupffer cells may be one source: in Kupffer cells, maintained in primary culture for 72 hours, glucagon (0.1 to 10 nmol/ L) increased PGE(2), PGF(2 alpha), and PGD(2) synthesis rapidly and transiently. Maximal prostaglandin concentrations were reached after 5 minutes. Glucagon (1 nmol/L) elevated the cyclic adenosine monophosphate (cAMP) and inositol triphosphate (InsP(3)) levels in Kupffer cells about fivefold and twofold, respectively. The increase in glyco gen phosphorylase activity elicited by 1 nmol/L glucagon was about twice as large in monocultures of hepatocytes than in cocultures of hepatocytes and Kupffer cells with the same hepatocyte density. Treatment of cocultures with 500 mu mol/L acetylsalicylic acid (ASA) to irreversibly inhibit cyclooxygenase (PGH-synthase) 30 minutes before addition of glucagon abolished this difference. These data support the hypothesis that PGs produced by Kupffer cells in response to glucagon might participate in a feedback loop inhibiting glucagon-stimulated glycogenolysis in hepatocytes.

The neuropeptides orexin-A and orexin-B were discovered in 1998 and were first described as regulators of feeding behaviour. Later research has shown that they have an important role in the regulation of sleep. Two G protein-coupled receptors, OX1 and OX2 orexin receptors, mediate the cellular responses to orexins. The overall aim of this thesis was to investigate the OX1 orexin receptors signalling to phospholipases. Previous investigations have determined that orexin receptors induce Ca2+ elevations through both receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs). In this thesis we investigated the importance of these influxpathways on orexin-mediated phospholipase (PLC) activation. The results demonstrate that ROC influx is enough to fully support orexin-stimulated PLC activation but that SOC influx has a further amplifying role. We also investigated the metabolites generated after PLC activation, inositolphosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities one of them leading to DAG production without co-occurring IP3 production at low orexin receptor stimulation. The results also suggest that at even lower orexin receptor stimulation DAG is produced via the activation of phospholipase D. In this thesis we also investigated if the ubiquitous phospholipase A2 (PLA2) signalling system is involved in orexin receptor signalling. The results demonstrate that stimulation of the OX1 orexin receptors leads to arachidonic acid (AA) release. This release is fully dependent on Ca2+ influx, probably through ROC, and at the same time the studies demonstrate that ROC influx is partly dependent on PLA2 activation. At low orexin receptor activation the AA release seemed to in part rely on extracellular signal-regulated kinase. We also devised two methods to aid in these investigations. The first method enabled studies of the receptor-operated Ca2+ influx without interference of the co-occurring store-operated Ca2+ influx. This was done by the expression of IP3-metabolising enzymes IP3-3-kinase-A and IP3-5-phosphatase-I. The second method enables quantification of DAG and IP3 signalling in fixed cells using GFP-fused indicators, leading to a semi-quantitative but easily applicable pharmacological assay.

Intrauterine growth restriction (IUGR) reduces bone mass by 10-30% and impairs arachidonic (AA) and docosahexaenoic (DHA) acid status in infants. Because AA and DHA enhance neonatal bone mass, the aim of this study was to determine the effects of dietary 0.5% AA and 0.2% DHA (w/w) prior to weaning on bone and growth. 40 guinea pigs were randomized to either a control (C) or low-protein diet (LP) during pregnancy and the C diet or the C diet with AA+DHA during lactation. Measurements included bone mass, metabolism, and strength, and erythrocyte lipid of sows and offspring from birth to 16 wk post-partum. The LP diet induced IUGR, while the AA+DHA increased bone mass by 5-20% in sows and offspring and corrected growth and bone mass in IUGR pups. Thus, AA+DHA provided in lactation rescues the growth trajectory in an IUGR state and is beneficial to maternal and neonatal bone mass.

Although the etiology of diabetic nephropathy is still uncertain, proteinuria due to podocyte injury and loss (podocytopenia) are early features of the disease. Significant increases in thromboxane A2 (TXA2) production as well as expression of its receptor in animal models of diabetic nephropathy led to the hypothesis that TXA2 acting via its thromboxane-prostanoid (TP) receptor induces podocytopenia resulting in proteinuria. Systemic infusion of a TP antagonist demonstrated an important role of TXA2/TP signalling in our model of streptozotocin induced type-1 diabetic nephropathy by reducing kidney damage including proteinuria. Podocyte specific TP overexpressing mice did not demonstrate more pathologic or dynamic kidney damage than non-transgenic mice in STZ-induced diabetic nephropathy. Further assessment of the TP transgene functionality in this mice line is necessary to validate those results. Whereas the importance of TXA2/TP signalling is undeniable in diabetic nephropathy, it appears that podocyte TP receptors might not be directly targeted.