Prostaglandins, Leukotrienes and Essential Fatty Acids
Profiling of prostanoids in zebrafish embryonic development
Introduction
Prostanoids (PGs) possess potent biological activities in vascular, pulmonary, reproductive and renal physiology as well as in pathophysiology of inflammation, thrombosis and cancer [1]. Their biosynthesis is initiated by the action of phospholipase to release arachidonic acid from membrane phospholipids, followed by the action of prostaglandin H synthase (also known as cyclooxygenase, COX). Two different COX isozymes, COX-1 and COX-2, encoded by separate genes, have been identified [2]. Their common product, prostaglandin H2 (PGH2), is an unstable compound with a half-life of ∼10 min in aqueous solution [3]. PGH2 is used by the specific enzymes for the formation of each PG. The type and quantity of the terminal PGs generated appear to be governed by the specific PG-synthesizing enzyme present in a given cell. PGs then exert their actions through specific receptors on plasma membranes [4].
In mammals, the major terminal PGs are prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2α (PGF2α), prostaglandin I2 (PGI2, also known as prostacyclin (PGI2) and thromboxane A2 (TXA2) [5]. PGD2 is a major PG synthesized in the central nervous system and is involved in the regulation of sleep and pain responses [6]. It is also actively produced by mast cells, basophils and Th2 cells, acting as an allergic and inflammatory mediator [7]. Two distinctive types of PGD synthase have been identified: one is the lipocalin-type enzyme and the other, the hematopoietic enzyme. PGE2 is the most common PG and is ubiquitously produced in the body. It has four subtypes of PGE receptors to carry out its diversified functions including pain, fever, inflammation, ovulation, fertilization and bone metabolism [8]. There are also two forms of PGE synthases derived from separate genes: cytosolic form and membrane-bound form. PGF2α is an inducer of leuteolysis and has been implicated in parturition [9]. PGI2 is a potent vasodilator and inhibits platelet aggregation [10]. In contrast, TXA2 is a potent inducer of vasoconstriction and platelet aggregation. The balance of PGI2 and TXA2 is therefore crucial for hemostasis. Both PGI2 and TXA2 are very unstable in water and are non-enzymatically converted to 6-keto PGF1α and TXB2, respectively. Their synthesizing enzymes, PGI synthase and TXA synthase, are members of cytochrome P450 [11]. PGI synthase is mainly present in endothelial and smooth muscle cells, whereas TXA synthase is present in platelets, monocytes and macrophages.
The physiological roles of PGs were also explored from the studies of mice lacking the specific PG-synthesizing enzyme and/or specific PG receptor. In general, these knockout genes are non-lethal and the knockout mice reveal minor phenotypes [12]. For example, both TXA synthase- and TXA2 receptor-knockout mice revealed mild bleeding disorders and altered vascular responses to arachidonic acid [13], [14]. PGI2 receptor-knockout mice suffered obstructive thrombi upon FeCl3-induced endothelial injury [15], and 6-month-old PGI synthase-knockout mice showed vascular disorders with the thickening of vascular walls and interstitial fibrosis [16]. Lipocalin-type PGD synthase-knockout mice failed to respond to a touch-evoked pain, but had a normal hyperalgesia response [17]. Interestingly, the knockout mice were also found to be glucose-intolerant and insulin-resistant on a low fat diet [18]. In addition, the lipocalin-type PGD synthase-knockout mice developed nephropathy and an aortic thickening on a high-fat diet, suggesting that PGD2 plays an important role in the vasculature, especially in atherosclerosis and diabetes.
Zebrafish has been used as a model to study embryonic development of COX-1 and COX-2 [19], [20]. COX-1 serves as a house-keeping function and is a constitutive enzyme, whereas COX-2 is highly regulated by growth factor, cytokine and mitogen. COX-1 is found ubiquitously early in the zebrafish embryos during blastula and gastrula stages. During somitogenesis, COX-1 is enriched in the posterior intermediate mesoderm, and at 24 h post-fertilization (hpf) it is confined to the distal end of nephric duct and developing vasculature. Knockdown of COX-1 by a morphonino oligonucleotide resulted in gastrulation arrest or defect in vascular tube formation. Conversely, knockdown of COX-2 caused no noticeable phenotype. These findings are in sharp contrast to those obtained with knockout mice. COX-1 knockout mice survived well and were fertile [21]. However, COX-2 knockout mice suffered higher mortality due to gastric peritonitis or kidney failure [22], [23]. The COX-2 knockout females also revealed multiple reproductive defects including ovulation, fertilization, implantation and decidualization. These controversial results can be explained by the possibility that PG knockout mice acquire maternal PG to allow normal development of embryos in uterus. Therefore, the physiological roles of PGs may not reflect the developmental roles of PGs in the knockout mice. In this regard, zebrafish as a vertebrate is well suited for studying the embryonic roles of PGs because the embryos develop externally from maternal body.
In order to fully exploit the advantages of zebrafish for studying the developmental roles of PGs, it needs information of the specific PG-synthesizing enzymes and PG receptors in this species to enable detection of gene expression and examination of gene-perturbed phenotypes. Zebrafish PGE synthase, both cytosol and membrane-bound forms, is the only terminal PG-synthesizing enzyme that was reported [24], [25]. As an onset to further understand the developmental roles of PGs, we report here the biosynthetic capacity of PGs in the zebrafish embryonic stages. PGE2 is the major PG throughout the embryonic stages. However, at the early developmental stages, PGI2 is synthesized at a higher level than TXA2, suggesting an important role of PGI2 besides hemostasis. While most PGs remain at the fairly constant levels during embryogenesis, levels of PGD2 biosynthesis decreases as embryo matures, suggesting an important role of PGD2 in the early stage of embryo development.
Section snippets
Sample preparation and product analysis
[1-14C]-PGH2 was synthesized by ovine COX-1 using 5 μl of [1-14C] arachidonic acid (Amersham Pharmacia; 55 mCi/mmol, 10 μCi/200 μl) mixed with 1 mg non-radiolabelled arachidonic acid (NuCheck) as the substrate. PGH2 was purified by a normal phase silica HPLC column as described previously [26].
About 50 zebrafish embryos were collected at different developmental stages. Each batch of embryos was washed three times with 0.2 ml PBS and resuspended in 0.2 ml of PBS. The embryos were lysed by sonication
Results and discussion
Zebrafish embryos at different embryonic stages were collected and subjected to sonication. Cell debris was removed by low-speed centrifugation, and supernatant was used as the embryo homogenate. Because arachidonic acid can also be used by lipoxygenase to generate leukotrienes, we thus used [1-14C]-PGH2 as the substrate and incubated it with embryo homogenates to obtain PGs. The PGs were extracted with ether and resolved by HPLC over a reverse phase C18 column. Extraction efficiency for each
Acknowledgments
We thank Dr. Xinping Zhao at the Department of Ophthalmology for providing zebrafish embryos. This work is supported by Grant HL60625 from the National Institutes of Health
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