Arachidonic Acid and Leukotriene C4: Role in Transient Cerebral Ischemia of Gerbils

Accumulation of arachidonic acid (AA) is greatest in brain regions most sensitive to transient ischemia. Free AA released after ischemia is either: 1) reincorporated into the membrane phospholipids, or 2) oxidized during reperfusion by lipoxygenases and cyclooxygenases, producing leukotrienes (LT), prostaglandins, thromboxanes and oxygen radicals. AA, its metabolite LTC₄ and lipid peroxides (generated during AA metabolism) have been implicated in the blood-brain barrier (BBB) dysfunction, edema and neuronal death after ischemia/reperfusion. This report describes the time course of AA release, LTC₄ accumulation and association with the physiological outcome during transient cerebral ischemia of gerbils. Significant amount of AA was detected immediately after 10 min ischemia (0 min reperfusion) which returned to sham levels within 30 min reperfusion. A later release of AA occurred after 1 d. LTC₄ levels were elevated at 0–6 h and 1 d after ischemia. Increased lipid peroxidation due to AA metabolism was observed between 2–6 h. BBB dysfunction occurred at 6 h. Significant edema developed at 1 and 2 d after ischemia and reached maximum at 3 d. Ischemia resulted in ~80% neuronal death in the CA₁ hippocampal region. Pretreatment with a 5-lipoxygenase inhibitor, AA861 resulted in significant attenuation of LTC₄ levels (Baskaya et al. 1996. J. Neurosurg. 85:112–116) and CA₁ neuronal death. Accumulation of AA and LTC₄, together with highly reactive oxygen radicals and lipid peroxides, may alter membrane permeability, resulting in BBB dysfunction, edema and ultimately to neuronal death.     

Cyclooxygenase enzymes and prostaglandins in reproductive tract physiology and pathology

Prostaglandins, thromboxanes (TX) and leukotrienes, collectively referred to as eicosanoids, are cyclooxygenase (COX) metabolites of arachidonic acid (AA). Prostaglandins, have been recognised for many years as key molecules in regulating reproductive tract physiology and pathology. Numerous recent studies in in vitro model systems and knockout mouse models have demonstrated specific functional roles for the respective cyclooxygenase enzymes, prostaglandins and prostanoid receptors. Here we review the findings obtained in several of these studies with emphasis on the roles played by cyclooxygenase enzymes and prostaglandins, specifically prostaglandin E2 (PGE2) and F2alpha in reproductive tract physiology and pathology.     

Myocardial prostacyclin and thromboxane A2 synthetase activities during ischemia and reperfusion in isolated rabbit heart

The aim of the study was to determine the prostacyclin (PGI₂) and thromboxane A₂ (TXA₂) synthetase activities of myocardial tissue and their variation during ischemia and reperfusion. Regional ischemia was induced by 10 min occlusion of the left anterior descending coronary artery in isolated Langendorff rabbit hearts. Biosynthesis of PGI₂ and TXA₂ were carried out by using arachidonic acid as substrate and left ventricle microsomes (LVM) from ischemic and non-ischemic areas as sources of PGI₂ and TXA₂ synthetase. 6-keto-PGF_1α and TXB₂, stable metabolites of PGI₂ and TXA₂ respectively, were determined by radioimmunoassay. Experiments carried out under the adopted conditions showed that LVM were able to synthetise PGI₂ as well as TXA₂ from arachidonic acid. On the other hand, ischemia depressed both PGI₂ and TXA₂ synthetase activities of cardiac tissue: the depression was more pronounced on TXA₂ synthetase than on PGI₂ synthetase with no significant difference between ischemic and non-ischemic regions. Moreover, ischemia increased the ratio indicating therefore that it can facilitate the formation of PGI₂. The post ischemic reperfusion of the heart counteracted the decrease in PGI₂ synthetase induced by ischemia which returned to the normal level: reperfusion also slightly reversed the decrease in TXA₂ synthetase. However, the diminution in TXA₂ synthetase of non-ischemic myocardium was attenuated but it remained lower than the normal level. These results suggested that the whole left ventricle is affected by regional ischemia. Furthermore it appears that myocardial TXA₂ synthetase is more vulnerable than PGI₂ synthetase to a lack of oxygen and nutrients.     

Ovarian follicular fluid eicosanoid concentrations during the pre-ovulatory period in humans

Prostaglandins are involved in ovulation and in every mammal studied so far, ovulation has been inhibited by prostaglandin inhibition. Information regarding the role of leukotrienes and thromboxanes in ovulation is more limited. In order to study the production of eicosanoids in human pre-ovulatory follicular fluid, follicular aspiration was timed by means of serial ultrasound scans and human chorionic gonadotrophin (hCG) to be immediately pre-ovulatory. 11 women were studied and the eicosanoids measured by radioimmunoassay (RIA). The follicular fluid was found to contain leukotrienes (LT) B4, LTC4 (D4, E4), prostaglandin (PG) E2, PGF2 alpha 6 keto PGF1 alpha k and thromboxane (TX) B2. This is the first published report of leukotrienes in human follicular fluid in spontaneous cycles, and is one of the few reports showing prostaglandins and thromboxanes. The significance of demonstrating leukotrienes in human follicular fluid is discussed as is the correlation between individual eicosanoids in the human ovary.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α₂-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α₂-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α₂-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α₂-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α₂-adrenergic receptor stimulation.     

Eicosanoids in human ventricular cerebrospinal fluid following severe brain injury

Recent evidence has shown that a variety of prostaglandins and leukotrienes can be produced in brain tissue after injury in animals. It has also been speculated that increases in brain prostaglandins occur in humans following injury. Ventricular cerebrospinal fluid (CSF) samples have been obtained from children with static lesions (controls) as well as children with acute brain injury and eicosanoids measured by immunologic techniques. Metabolites of prostacyclin (6-keto-PGF1 a) and thromboxane A2 (thromboxane B2) were the major eicosanoids found in CSF, and levels of these compounds were increased 3-10 times in acutely injured patients. Prostaglandin E2 was also found in lower amounts, although in one case its level was very high. Prostaglandin D2 was also present, but in low amounts. No leukotrienes were found in CSF samples that were purified by HPLC prior to immunoassay. Elevated levels of hydroxyeicosatetraenoic acids (HETEs) were observed in those samples stored frozen, but these metabolites were most probably due to autooxidation of arachidonic acid in CSF. Arachidonic acid concentration in CSF was typically found to be in the range of 10-200 ng/ml, but was found to be 5-10 fold higher in one severely injured patient. Thus, elevated free arachidonic acid and various oxygenated metabolites were observed in CSF following brain injury.     

Platelet-activating factor and cardiac diseases: therapeutic potential for PAF inhibitors

Platelet-activating factor (PAF) is a potent phospholipid mediator released from inflammatory cells in response to diverse immunologic and non-immunologic stimuli. Animal studies have implicated PAF as a major mediator involved in coronary artery constriction, modulation of myocardial contractility and the generation of arrhythmias which may bear on cardiac disorders such as ischemia, infarction and sudden cardiac death. PAF effects are induced by direct actions of PAF on cardiac tissue to modify chronotropic and inotropic activity, or indirectly via the release of eicosanoids such as thromboxane A₂ (TXA,), leukotrienes (LT) or cytokines (TNFx). The development of selective, high affinity PAF receptor antagonists has permitted investigations on the role of PAF in experimental animal models of cardiac injury. In vivo and in vitro studies strongly suggest that PAF receptor antagonists might convey therapeutic benefits in ischemic conditions and certain arrhythmias. In addition, PAF antagonists might have a cardiac allograft-preservation effect. Although clinical studies with PAF receptor antagonists in patients with cardiac diseases have not yet been reported, the experimental results to date suggest that PAF receptor antagonist might be useful in some specific cardiac disorders in humans.     

Effect of intravenous eicosapentaenoic acid on cerebral blood flow, edema and brain prostaglandins in ischemic gerbils

Eicosapentaenoic acid is converted by cyclo-oxygenase to the prostacyclin, PGI₃. Consequently eicosapentaenoic acid might protect the brain from the impairment in cerebral blood flow that follows temporary cerebral artirial occlusion. We studied the effect of 90% pure eicosapentaenoic acid, given intravenously, on cerebral blood flow, brain water and prostaglandins after ischemia in gerbils. Ischemia was produced by bilateral carotid occlusion for 15 min followed by reperfusion for 2 h. In experimental gerbils, 0.833 mg or 0.167 mg of eicosapentaenoic acid (Na salt) was given intravenously followed by a continuous infusion of 1 mg h⁻¹. Control gerbils were given 0.167 mg of linoleic acid (Na salt) intravenously followed by a continuous infusion of 1 mg h⁻¹ or a saline infusion. Regional cerebral blood flow was measured by the hydrogen clearance method and brain water by the specific gravity technique. Brain diene prostaglandins were measured by radioimmunoassay. In control gerbils cerebral blood flow decreased significantly during reperfusion and remained depressed after 2 h of reperfusion. In eicosapentaenoic acid treated gerbils blood flow decreased initially but after 2 h of reperfusion blood flow was significantly higher than in control gerbils. Brain edema and brain diene prostaglandins were not significantly different between control and experimental groups.Our study indicates that eicosapentaenoic acid, given intravenously, improves cerebral blood flow after ischemia and reperfusion. We speculate that this effect may be due to the formation of the prostacyclin, PGI₃.     

Advances in eicosanoid research, novel therapeutic implications

Eicosanoids are a family of oxygenated metabolites of arachidonic acid, including the prostaglandins, thromboxanes, leukotrienes and lipoxins. These lipid mediators play essential roles in normal cellular homeostasis as well as in a number of disease states. This review will focus on recent advances in the field of eicosanoids and highlight specific discoveries and achievements. Emphasis will be placed on structure and receptor biology, which are of significant pharmacological and clinical relevance.     

Myocardial prostacyclin and thromboxane A2 synthetase activities during ischemia and reperfusion in isolated rabbit heart

The aim of the study was to determine the prostacyclin (PGI2) and thromboxane A2 (TXA2) synthetase activities of myocardial tissue and their variation during ischemia and reperfusion. Regional ischemia was induced by 10 min occlusion of the left anterior descending coronary artery in isolated Langendorff rabbit hearts. Biosynthesis of PGI2 and TXA2 were carried out by using arachidonic acid as substrate and left ventricle microsomes (LVM) from ischemic and non-ischemic areas as sources of PGI2 and TXA2 synthetase. 6-keto-PGF1 alpha and TXB2, stable metabolites of PGI2 and TXA2 respectively, were determined by radioimmunoassay. Experiments carried out under the adopted conditions showed that LVM were able to synthetise PGI2 as well as TXA2 from arachidonic acid. On the other hand, ischemia depressed both PGI2 and TXA2 synthetase activities of cardiac tissue: the depression was more pronounced on TXA2 synthetase than on PGI2 synthetase with no significant difference between ischemic and non-ischemic regions. Moreover, ischemia increased the ratio 6-keto-PGF1 alpha/TXB2 indicating therefore that it can facilitate the formation of PGI2. The post ischemic reperfusion of the heart counteracted the decrease in PGI2 synthetase induced by ischemia which returned to the normal level: reperfusion also slightly reversed the decrease in TXA2 the decrease in TXA2 synthetase. However, the diminution in TXA2 synthetase of non-ischemic myocardium was attenuated but it remained lower than the normal level. These results suggested that the whole left ventricle is affected by regional ischemia. Furthermore it appears that myocardial TXA2 synthetase is more vulnerable than PGI2 synthetase to a lack of oxygen and nutrients.     

Human salivary eicosanoids: circadian variation

A circadian rhythm in the concentrations of prostaglandins (PG) E2, PGF2, PGI2 (measured as 6-keto-PGF1 alpha), immunoreactive h hydroxyeicosatetraenoic acids and immunoreactive 6-sulfidopeptide containing leukotrienes in human mixed saliva was observed. The rhythm reflected changes in the absolute amounts of these compounds in saliva. Under usual sleep-wake cycles a single peak occurred during sleep with maximal levels at 5:00 AM; the amplitude of the peak varied for each product. The rhythm was sleep-dependent and a shift occurred when the sleep-wake cycles were displaced. Basal levels of these eicosanoids were maintained even without sleep.     

Arachidonate-derived dihomoprostaglandin production observed in endotoxin-stimulated macrophage-like cells

Eicosanoids, including the prostaglandins, leukotrienes, hydroxyeicosatetraenoic acids, epoxyeicosatetraenoic acids, and related compounds, are biosynthetic, bioactive mediators derived from arachidonic acid (AA), a 20:4(n-6) fatty acid. We have developed a comprehensive and sensitive mass spectral analysis to survey eicosanoid release from endotoxin-stimulated RAW 264.7 macrophage-like cells that is capable of detecting over 70 diverse eicosanoids and eicosanoid metabolites, should they be present. We now address the question: Are biologically significant eicosanoids being overlooked? Herein, we illustrate a general approach to diverse isotope metabolic profiling of labeled exogenous substrates using mass spectrometry (DIMPLES/MS), demonstrated for one substrate (AA) and its resultant products (eicosanoids). RAW cells were incubated in medium supplemented with deuterium-labeled AA. When the cells are stimulated, two sets of eicosanoids are produced, one from endogenous AA and the other from the supplemented (exogenous) deuterium-labeled form. This produces a signature mass spectral "doublet" pattern, allowing for a comprehensive and diverse eicosanoid search requiring no previous knowledge or assumptions as to what these species may be, in contrast to traditional methods. We report herein observing unexpected AA metabolites generated by the cells, some of which may constitute novel bioactive eicosanoids or eicosanoid inactivation metabolites, as well as demonstrating differing metabolic pathways for the generation of isomeric prostaglandins and potential peroxisome proliferator-activated receptor activators. Unexpectedly, we report observing a series of 1a, 1b-dihomologue prostaglandins, products of adrenic acid (22:4(n-6)), resulting from the two-carbon elongation of AA by the RAW cells.     

Redox-mediated programed death of myocardial cells after cardiac arrest and cardiopulmonary resuscitation

Abstract

Besides the fact that prolonged whole-body ischemia causes tissue and organ injury during cardiac arrest, additional damage occurs after the restoration of spontaneous circulation, during which the reperfusion activates a host of intracellular responses. These responses may lead to an increased threshold of oxidant-mediated injury and redox-mediated programed cell death in the stunned myocardium. The aim of this article is to summarize the major intracellular responses occurring from the onset of cardiac arrest until the post-resuscitation period that may lead to redox-mediated programed death of myocardial cells.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.     

Molecular evolution of enzymes involved in the arachidonic acid cascade

The metabolic pathway called the arachidonic acid cascade produces a wide range of eicosanoids, such as prostaglandins, thromboxanes and leukotrienes with potent biological activities. Recombinant DNA techniques have made it possible to determine the nucleotide sequences of cDNAs and/or genomic structures for the enzymes involved in the pathway. Sequence comparison analyses of the accumulated sequence data have brought great insights into the structure, function and molecular evolution of the enzymes. This paper reviews the sequence comparison analyses of the enzymes involved in the arachidonic acid cascade.     

Eicosanoids: Prostaglandins, Thromboxanes, Leukotrienes, and Other Derivatives of Carbon-20 Unsaturated Fatty Acids

In recent years, knowledge of the biochemistry of oxygenated metabolites of arachidonic acid has greatly increased. Their biological functions in acceleration and prevention of platelet aggregation and in inflammatory and immune reactions are becoming much clearer. The therapeutic value, particularly of PGI2 as well as selective inhibitors of synthesis, is also rapidly advancing. Despite much effort, the functional importance of prostaglandins and thromboxanes in the cNS in normal ongoing physiological processes is still quite uncertain. However, when parenchymal or vascular elements are damaged or invaded by extraneural cells, the synthesis of one or the other member of the eicosanoids is greatly increased and contributes significantly to pathophysiological reactions. Thus, prevention of synthesis is likely to have increasing importance in clinical neurology, particularly in cerebrovascular diseases.     

Eicosanoid production and transfer in the placenta of the diabetic pregnancy

The metabolism of arachidonic acid (AA) and the transfer of its metabolites was determined in in vitro perfused placental tissue from normal pregnancies and those complicated by maternal insulin-dependent diabetes mellitus (IDDM). 14C-labelled AA was recirculated in the fetal circulation for 60 min while 3H-AA was recirculated in the maternal circulation. Placental effluent was subjected to high performance liquid chromatography (HPLC) and analysis of dual-label scintillation counts. Placentae from IDDM pregnancies converted 3-6 times more radiolabelled AA to eicosanoids than did normal placentae. In addition, the transfer of eicosanoids into the opposing circulation was doubled in placentae from IDDM pregnancies compared to normal placentae. The predominant direction of eicosanoid transfer in both groups of placentae was in the fetal-to-maternal direction. The relative amounts of eicosanoids produced was also altered in placentae from IDDM pregnancies. Increased amounts of thromboxane (Tx) B2 and hydroxyeicosatetraenoic acids (HETEs) were present in both circulations of placentae from IDDM pregnancies. Levels of 6-keto prostaglandin F1a (6KPGF1a) were significantly reduced in both circulations in placentae from IDDM pregnancies. Thus, the ratio of TxA2 to PGI2 and the ratio of HETEs to PGI2 were both significantly increased in placentae from IDDM pregnancies. These results suggest an imbalance in eicosanoid production which may be relevant to abnormal placental structure and function in IDDM pregnancies.     

Alterations of vascular prostacyclin and thromboxane A2 in Dahl genetical strain susceptible to salt-induced hypertension

To assess the implications of vascular eicosanoids system in the hypertension of Dahl salt-sensitive (Dahl S) strain, we investigated the production of vascular vasodepressor and vasoconstrictor eicosanoids in Dahl S rats. 14-week-old Dahl S rats on a 0.11% NaCl diet (normotension) or a 0.3% NaCl diet (borderline hypertension) had a significantly lowered generation of vascular prostacyclin (PGI₂), compared with Dahl salt-resistant (Dahl R) rats. The impairment of vascular PGI₂ in Dahl S rats was restored to the normal level of Dahl R rats with the elevation of blood pressure induced by a high salt diet (4% NaCl). The production of vascular PGI₂ was closely related to the height of blood pressure. The deterioration of vascular PGI₂ was also found in 4-week-old Dahl S rats with normotension. Conversely, vascular thromboxane A₂ (TXA₂) was significantly enhanced in 14-week-old Dahl S rats in all of the feeding groups. Thus, it seems possible that the proved alterations of the vasodepressor and vasoconstrictor eicosanoids partially contribute to the genesis of salt hypertension. Although the exact mechanisms remain obscure, the adaptation of vascular PGI₂ on a high salt diet may be suitable to compete with the high blood pressure and to protect against the vascular damage.     

三七总皂甙和灯盏花素复方注射剂对结扎大鼠冠脉致心肌缺血的保护作用

采用结扎大鼠冠脉造成急性心肌缺血模型 ,观察了三七总皂甙和灯盏花素复方注射剂 (简称复方注射剂 )抗实验性心肌缺血作用。结果表明 :大鼠静脉注射复方注射剂 5 0mg/kg和 10 0mg/kg对急性心肌缺血均有一定程度的保护作用 ,在改善心电图S T段的上移、降低血清CPK、LDH方面两个剂量组均有显著作用 (P <0 .0 5 ,P <0 .0 1)以 10 0mg/kg组缩小心肌梗塞范围为显著 (P <0 .0 5 )。复方注射剂与丹参注射剂均能保护超氧化物歧化酶活性 (P <0 .0 5 ,P <0 .0 1) ,显著降低丙二醛含量 (P <0 .0 1)。复方注射剂两个剂量组均能显著增加前列环素合成 (P <0 .0 5 ,P <0 .0 1)。提示三七总皂甙和灯盏花素复方注射剂对心肌缺血的保护作用与抗脂质过氧化作用有关 ,其促进前列环素合成作用可能与其抗心肌缺血有关。