Báo cáo khoa học: Mechanisms of accumulation of arachidonate in phosphatidylinositol in yellowtail A comparative study of acylation systems of phospholipids in rat and the fish species Seriola quinqueradiata pot

Mechanisms of accumulation of arachidonate in phosphatidylinositol in yellowtail A comparative study of acylation systems of phospholipids in rat and the fish species Seriola quinqueradiata Tamotsu Tanaka, Dai Iwawaki, Masahiro Sakamoto, Yoshimichi Takai, Jun-ichi Morishige, Kaoru Murakami and Kiyoshi Satouchi Department of Applied Biological Science, Fukuyama University, Japan It is known that phosphatidylinositol (PtdIns) contains abundant arachidonate and is composed mainly of 1-stea- royl-2-arachidonoyl species in mammals. We investigated if this characteristic of PtdIns applies to the PtdIns from yellowtail (Seriola quinqueradiata), a marine fish. In common with phosphatidylcholine (PtdCho), phosphatidylethanol- amine (PtdEtn) and phosphatidylserine (PtdSer) from brain, heart, liver, spleen, kidney and ovary, the predominant polyunsaturated fatty acid was docosahexaenoic acid, and levels of arachidonic acid were less than 4.5% (PtdCho), 7.5% (PtdEtn) and 3.0% (PtdSer) in these tissues. In striking contrast, arachidonic acid made up 17.6%, 31.8%, 27.8%, 26.1%, 25.4% and 33.5% of the fatty acid composition of PtdIns from brain, heart, liver, spleen, kidney and ovary, respectively. The most abundant molecular species of PtdIns in all these tissues was 1-stearoyl-2-arachidonoyl. Assay of acyltransferase in liver microsomes of yellowtail showed that arachidonic acid was incorporated into PtdIns more effect- ively than docosahexaenoic acid and that the latter inhibited incorporation of arachidonic acid into PtdCho without inhibiting the utilization of arachidonic acid for PtdIns. This effect of docosahexaenoic acid was not observed in similar experiments using rat liver microsomes and is thought to contribute to the exclusive utilization of arachidonic acid for acylation to PtdIns in yellowtail. Inositolphospholipids and their hydrolysates are known to act as signaling molecules in cells. The conserved hydrophobic structure of PtdIns (the 1-stearoyl-2-arachidonoyl moiety) may have physio- logical significance not only in mammals but also in fish. Keywords: acyltransferase; arachidonic acid; fish; phospha- tidylinositol; yellowtail. Biological membranes are composed of several phospho- lipid classes, and glycerophospholipid classes are further separated into molecular species based on the combination of acyl (alkyl, alkenyl) residues at positions sn-1 and sn-2. One well-known characteristic of phosphatidylinositol (PtdIns) is an abundance of arachidonate. This has been demonstrated in several mammalian tissues [1–12] and confirmed in this study in most tissues of the rat. At the molecular level, PtdIns has been reported to be composed mainly of 1-stearoyl-2-arachidonoyl species in guinea pig brain [6], bovine brain [7], rat liver [8], human platelets [9,10], human endothelial cells [11] and rabbit macrophages [12]. We confirm here that this molecular conservation of PtdIns is a feature distinct from other phospholipids in most tissues of the rat. The molecular conservation of PtdIns is thought to have physiological importance for (a) eicosanoid precursor storage, (b) donation of potent activators of protein kinase C (PKC), such as 1-stearoyl-2-arachidonoyl- glycerol [13], and (c) donation of arachidonate-containing biologically active molecules, such as 2-arachidonoylglycerol [14]. However, the exact physiological meaning of the conservation of PtdIns molecular species has not been fully resolved. Several enzymatic systems are involved in the accumu- lation of arachidonate in PtdIns. CoA)1-acyl-2-lyso-PtdIns acyltransferase activity, operating in the remodeling path- ways of phospholipid biosynthesis, is known to utilize arachidonoyl-CoA as substrate [15,16]. Both diacylglycerol kinase [17–20] and CDP-sn-1,2-diacylglycerol synthase [21], enzymes involved in the PtdIns cycle, have been reported to contribute to the enrichment of arachidonate in PtdIns. With respect to the biosynthesis of PtdIns, we and others [22,23] have demonstrated that sciadonic acid (20:3, D-5c,11c,14c), an n)6 series trienoic acid that lacks the D8 double bond of arachidonic acid, is metabolized in a similar manner to arachidonic acid in the biosynthesis of PtdIns [24,25]. We have also presented evidence suggesting that the nonarachidonic acid and utilizable polyunsaturated fatty Correspondence to T. Tanaka, Department of Applied Biological Science, Fukuyama University, Fukuyama, 729-0292, Japan. Fax: + 81 84 936 2459, Tel.: + 81 84 936 2111, Ext. 4056, E-mail: tamot@fubac.fukuyama-u.ac.jp Abbreviations: PKC, protein kinase C; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; PUFA, polyunsaturated fatty acid. Enzymes: acylCoA:lysophospholipid acyltransferase (EC 2.3.1.23); CDP-diacylglycerol synthase (CTP-phosphatidate:cytidylyltrans- ferase; EC 2.7.7.41); diacylglycerol kinase (EC 2.7.1.107); phospho- lipase A 2 (EC 3.1.1.4); phospholipase C (EC 3.1.4.3); protein kinase C (EC 2.7.1.37). (Received 26 September 2002, revised 13 December 2002, accepted 10 February 2003) Eur. J. Biochem. 270, 1466–1473 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03512.x acid (PUFA) for PtdIns is a potential tool with which to clarify the significance of the arachidonic acid residue of bioactive lipids of PtdIns origin [25]. In general, lipids from terrestrial mammals are rich in n)6 series PUFAs, such as linoleic acid and arachidonic acid. In contrast, the predominant PUFAs in lipids from marine fish are n)3 series fatty acids such as docosahexaenoic acid and eicosapentaenoic acid. Does the molecular conservation in which PtdIns is composed mainly of arachidonate-contain- ing molecular species apply to marine fish? Studies have shown that 1-stearoyl-2-arachidonoyl-PtdIns is the pre- dominant molecular species even in codfish roe [26] and salmon sperm [27], salmon liver [28] and rainbow trout retina [29]. If molecular conservation of PtdIns is found in marine fish, what mechanisms operate to accumulate arachidonic acid into PtdIns in an environment in which arachidonic acid is limited? In this study, we investigated the composition of PtdIns from brain, heart, liver, spleen, kidney and ovary in Seriola quinqueradiata, the marine fish known as yellowtail. The results show that PtdIns is rich in arachidonic acid despite the predominance of n)3 series PUFAs in these tissues. We also investigated mechanisms for the accumulation of arachidonic acid into PtdIns through experiments with microsomes from the liver of yellowtail, and found that the one-sided accumulation of arachidonic acid into PtdIns is attained in the presence of large amounts of docosahexa- enoic acid and that several acyltransferase activities are involved in the process in yellowtail. Materials and methods Materials Yellowtails (S. quinqueradiata) were obtained from a local market. Standard fatty acids were purchased from Serdary Research Laboratories (London, ON, Canada). [1- 14 C]Arachidonic acid (55 mCiÆmmol )1 )and[1-1 4 C]doco- sahexaenoic acid (55 mCiÆmmol )1 ) were from NEM Life Sciences Products, Inc. (Boston, MA, USA). Essentially fatty acid-free BSA, ATP, CoA, 1-palmitoyl-2-lyso-phos- phatidylcholine (lysoPtdCho), phospholipase C (from Bacillus cereus) and phospholipase A 2 (from Crotalus adamanteus venom) were from Sigma Chemical Co. (St Louis, MO, USA). By treatment with phospholipase A 2 [30], 1-acyl-2-lyso-PtdIns (lysoPtdIns) was prepared from bovine liver PtdIns (Sigma). The resulting lysoPtdIns was purified by TLC using chloroform/acetone/methanol/ acetic acid/water (50 : 20 : 10 : 13 : 5, v/v/v/v/v). Lyso- PtdIns was extracted from the silica gel by the method of Bligh & Dyer [31] under slightly acidic (HCl) conditions. All other reagents were of reagent grade. Fatty acid composition of phospholipids and molecular species composition of PtdIns Brain, heart, liver, spleen, kidney and ovary of yellowtails were isolated, and total lipids were extracted by the method of Bligh & Dyer [31]. After separation of the phospholipid fraction by silicic acid column chromatography, PtdCho, phosphatidylethanolamine (PtdEtn) and the mixture of phosphatidylserine (PtdSer), PtdIns and sphingomyelin were separated by TLC with the solvent system chloro- form/methanol/28% ammonia (65 : 35 : 5, v/v/v). The mixed fraction of PtdIns, PtdSer and sphingomyelin was further separated by TLC with chloroform/acetone/meth- anol/acetic acid/water (50 : 20 : 10 : 13 : 5, v/v/v/v/v) to obtain PtdIns and PtdSer. Detection was with 0.01% primuline (in acetone/water, 4 : 1, v/v) under UV light. The fatty acid composition of each phospholipid was analyzed by GC after transmethylesterification. A portion of the PtdIns was subjected to phospholipase C treatment, and the resulting diacylglycerol was converted into dinitrobenzoyl derivatives as described by Kito et al. [32]. The diacyl- glyceroldinitrobenzoyl derivative was analyzed by HPLC with a 0.45 · 25 cm Inertsil ODS-2 column (GL Science Inc., Tokyo, Japan) using acetonitrile/propan-2-ol (80 : 20, v/v) as eluent. The major peaks were assigned by the direct analysis with GC after transmethylesterification. Lipids of male Sprague–Dawley rats (250–300 g) were analysed by the same method as those of yellowtail. Acyltransferase assay The isolated liver of yellowtail was homogenized in 50 m M potassium phosphate buffer (pH 7.0) containing 1.5 m M glutathione, 0.15 M KCl, 1 m M EDTA and 0.25 M sucrose (homogenizing buffer) with a Potter–Elvehjem glass/Teflon homogenizer. The microsome fraction was prepared by sequential centrifugation [25]. Microsomes from the liver of male Sprague–Dawley rats (250–300 g) were prepared by the same method. The final microsomal pellet was suspen- ded in the homogenizing buffer (omitting EDTA), and the protein content was determined by the method of Lowry et al. [33]. Acyltransferase was assayed as described previ- ously [25]. Each incubation contained 32 nmol lysoPtdCho (1-acyl) or lysoPtdIns (1-acyl), 0.5 m M nicotinamide, 1.5 m M glutathione, 0.15 M KCl, 5 m M MgCl 2 , 0.25 M sucrose, 3.0 m M ATP, 0.1 m M CoA, 50 m M potassium phosphate buffer (pH 7.0), 0.1 mg protein of the micro- somal fraction, and radiolabeled fatty acid (0.05 lCi per 25 nmol) in a total volume of 1.0 mL. After incubation at 37 °C for 10 min, lipids were extracted, the resulting PtdCho and PtdIns were isolated by 2D TLC as described previously [25], and radioactivities were determined. The inhibitory effect of unlabeled docosahexaenoic acid on the incorporation of labeled arachidonic acid into lysoPtdCho and lysoPtdIns was determined by experiments with 0.1 mg liver microsomal protein, 10 nmol labeled arachidonic acid, and the indicated amount of unlabeled docosahexaenoic acid in the presence of 6.4 nmol lysoPtdIns and 6.4 nmol lysoPtdCho. Results Fatty acid composition of phospholipids from tissues of yellowtail and rat The fatty acid compositions of PtdCho, PtdEtn, PtdSer and PtdIns of brain, heart, spleen, kidney and ovary of yellowtail were investigated (Tables 1–4). In all the tissues, the most abundant PUFA in the PtdCho fraction was docosahexa- enoic acid. The proportion of eicosapentaenoic acid in PtdCho was relatively high compared with that in PtdEtn Ó FEBS 2003 Arachidonate in phosphatidylinositol of yellowtail (Eur. J. Biochem. 270) 1467 and PtdSer, except in brain where oleic acid was abundant. In both the PtdEtn and PtdSer fractions, docosahexaenoic acid was the predominant PUFA in all the tissues. The presence of dimethylacetals in PtdEtn suggested the exist- ence of a substantial amount of an alkenylacyl subclass in these tissues. In common with PtdCho, PtdEtn and PtdSer, levels of arachidonic acid were very low compared with those of docosahexaenoic acid in these tissues. In striking contrast, larger amounts of arachidonic acid existed in PtdIns from all tissues investigated (Fig. 1). The propor- tion of it in PtdIns was highest in ovary (33.5%) and lowest in brain (17.6%). In all tissues, the proportion of Table 3. Fatty acid composition of PtdSer from various tissues of yellowtail. Values are weight percentages, given as the mean ± SD. Tissues were obtained from three different yellowtails. Fatty acid Brain Heart Liver Spleen Kidney Ovary 14:0 0.7 ± 0.3 0.9 ± 1.0 0.5 ± 0.2 0.8 ± 0.2 0.4 ± 0 0.4 ± 0.7 16:0 2.7 ± 1.6 7.2 ± 2.0 16.7 ± 3.2 6.3 ± 0.9 11.1 ± 2.0 8.5 ± 2.4 16:1 1.8 ± 1.0 1.3 ± 1.3 0.4 ± 0.2 1.6 ± 0.7 0.5 ± 0.3 0.4 ± 0.2 18:0 26.4 ± 2.4 31.3 ± 4.9 25.7 ± 3.4 32.0 ± 1.3 28.8 ± 1.1 31.7 ± 3.0 18:1(n)9) 19.6 ± 2.2 4.2 ± 0.7 3.3 ± 1.1 4.4 ± 1.3 4.2 ± 1.5 7.6 ± 3.1 18:1(n)7) – 2.5 ± 0.5 1.6 ± 0.2 3.2 ± 0.3 2.4 ± 0.4 2.8 ± 0.6 18:2(n)6) 2.1 ± 2.3 0.7 ± 0.4 – 2.1 ± 2.2 0.6 ± 0.3 0.7 ± 0.2 20:4(n)6) 0.5 ± 0.2 0.9 ± 0.4 1.4 ± 1.0 1.1 ± 0.3 2.0 ± 0.6 3.0 ± 2.0 20:5(n)3) 0.9 ± 0.1 1.5 ± 0.6 1.0 ± 0.2 1.5 ± 0.7 2.6 ± 2.0 1.5 ± 1.0 22:5(n)3) 4.3 ± 1.5 4.1 ± 0.8 2.1 ± 0.5 2.2 ± 0.7 2.8 ± 0.6 2.8 ± 1.2 22:6(n)3) 36.1 ± 1.4 33.7 ± 5.6 41.0 ± 0.9 36.4 ± 2.6 34.3 ± 2.4 26.0 ± 5.9 Table 1. Fatty acid composition of PtdCho from various tissues of yellowtail. Values are weight percentages, given as the mean ± SD. Tissues were obtained from three different yellowtails. Fatty acid Brain Heart Liver Spleen Kidney Ovary 14:0 0.7 ± 0.3 0.8 ± 0.3 1.4 ± 0.2 1.8 ± 0.8 2.0 ± 1.2 1.2 ± 0.9 16:0 21.4 ± 1.4 33.0 ± 1.6 32.4 ± 3.3 35.0 ± 1.8 32.9 ± 3.5 34.8 ± 1.0 16:1 5.5 ± 1.4 1.3 ± 0.5 1.8 ± 0.7 3.8 ± 0.6 3.7 ± 0.7 2.5 ± 1.0 18:0 8.6 ± 0.3 3.9 ± 0.5 3.9 ± 0.8 3.6 ± 0.3 3.7 ± 0.5 3.8 ± 1.0 18:1(n)9) 29.2 ± 1.2 7.2 ± 1.3 7.4 ± 0.6 13.1 ± 1.8 12.2 ± 1.9 12.5 ± 2.2 18:1(n)7) 0.9 ± 0.8 2.6 ± 0.3 2.1 ± 0.3 4.0 ± 0.3 3.3 ± 0.6 2.5 ± 1.3 18:2(n)6) 1.4 ± 1.2 1.0 ± 0.4 1.3 ± 0.1 2.6 ± 1.0 2.4 ± 0.5 2.1 ± 0.7 20:4(n)6) 1.2 ± 0.4 4.5 ± 0.3 2.1 ± 0.2 3.7 ± 0.8 3.6 ± 0.9 3.6 ± 0.8 20:5(n)3) 1.8 ± 0.2 10.6 ± 1.5 8.4 ± 0.5 10.7 ± 1.1 11.1 ± 2.6 11.1 ± 1.2 22:5(n)3) 6.5 ± 0.4 1.7 ± 0.4 2.1 ± 0.6 1.7 ± 0.7 1.6 ± 0.4 1.6 ± 0.2 22:6(n)3) 15.3 ± 0.2 28.3 ± 3.1 31.5 ± 3.4 14.3 ± 0.4 19.7 ± 3.1 19.6 ± 5.5 Table 2. Fatty acid composition of PtdEtn from various tissues of yellowtail. Values are weight percentages, given as the mean ± SD. Tissues were obtained from three different yellowtails. DMA, Dimethylacetal. Fatty acid Brain Heart Liver Spleen Kidney Ovary 14:0 – – 0.4 ± 0.2 0.4 ± 0.1 – 0.8 ± 0.2 16:0DMA 1.2 ± 0.2 6.0 ± 0.8 0.5 ± 0.1 7.9 ± 2.0 6.3 ± 0.7 8.3 ± 1.7 16:0 4.6 ± 0.4 6.5 ± 1.3 22.2 ± 3.2 8.0 ± 1.0 12.5 ± 4.3 11.8 ± 1.8 16:1 1.0 ± 0.1 0.4 ± 0.3 0.8 ± 0.4 0.5 ± 0.1 0.7 ± 0.4 1.0 ± 0.4 18:0DMA 19.3 ± 2.0 1.5 ± 0.9 – 4.1 ± 0.4 3.5 ± 2.1 4.9 ± 1.5 18:1DMA 4.0 ± 1.1 1.0 ± 0.4 – 5.1 ± 0.5 3.4 ± 0.8 3.2 ± 0.8 18:0 10.2 ± 0.6 21.0 ± 0.3 13.6 ± 2.0 8.8 ± 0.3 9.0 ± 1.2 7.4 ± 1.2 18:1(n)9) 24.3 ± 2.3 3.3 ± 1.3 5.2 ± 0.9 3.5 ± 0.6 3.9 ± 0.7 4.7 ± 1.0 18:1(n)7) – 3.0 ± 0.6 3.1 ± 0.2 3.2 ± 0.4 3.0 ± 0.4 2.6 ± 0.3 18:2(n)6) – 0.9 ± 0 0.7 ± 0.4 0.7 ± 0.3 0.8 ± 0.3 0.8 ± 0.2 20:4(n)6) 2.9 ± 0.7 2.6 ± 0.3 1.6 ± 0.2 4.4 ± 1.3 5.5 ± 0.5 7.5 ± 1.4 20:5(n)3) 3.6 ± 0.6 3.8 ± 0.6 3.6 ± 0.3 6.1 ± 0.3 8.7 ± 3.0 6.7 ± 2.1 22:5(n)3) 1.3 ± 0.2 4.0 ± 0.3 1.8 ± 0.7 2.1 ± 0.8 1.7 ± 0.6 1.5 ± 0.7 22:6(n)3) 19.2 ± 4.9 41.5 ± 5.0 39.1 ± 4.5 37.7 ± 1.9 32.9 ± 3.7 27.4 ± 4.3 1468 T. Tanaka et al.(Eur. J. Biochem. 270) Ó FEBS 2003 docosahexaenoic acid in PtdIns was lower than in other phospholipids in corresponding tissues. Fatty acid compositions of PtdCho, PtdEtn, PtdSer and PtdIns from brain, heart, lung, liver, pancreas, kidney and testis of rat were investigated. Only the proportions of arachidonic acid in each phospholipid are presented in Fig. 1. The proportions of arachidonic acid in PtdCho, PtdEtn and PtdSer from these rat tissues varied from 4.7% to 21.4%, from 10.6% to 39.1% and from 4.8% to 32.6%, respectively. In contrast, 38.5%, 33.6%, 38.7%, 32.9%, 36.2% and 34.7% of total fatty acids in the PtdIns of rat brain, heart, lung, liver, pancreas, kidney and testis, respectively, were arachidonic acid. These results confirm that PtdIns is rich in arachidonic acid in rat tissues. Furthermore, it is evident that this characteri- stic of PtdIns also applies to yellowtail, a fish species living in seawater. Molecular species composition of PtdIns from tissues of yellowtail and rat The molecular species compositions of PtdIns from various tissues of yellowtail were analyzed by HPLC as diacyl- glyceroldinitrobenzoyl derivatives. To assign major peaks of these derivatives, we collected the eluate corresponding to each molecular species peak, and directly analysed the fatty acids of each fraction by GC. Under our analytical conditions, one pair of molecular species, 18:1/22:6 and 16:0/20:5, could not be resolved. Therefore, the amounts of these molecular species are shown as mixed components. As expected from the fatty acid analyses, the most abundant molecular species in all the tissues was 1-stearoyl-2-arachi- donoyl-PtdIns (Table 5). Although the proportion of this molecular species was relatively low in brain, about half of the total molecular species of PtdIns were 1-stearoyl-2- arachidonoyl species in liver, heart, spleen and ovary (Table 5). The next most abundant molecular species was 18:0/20:5 in all tissues. We also analyzed the molecular species composition of PtdIns obtained from tissues of rat: 65.6 ± 4.2%, 63.0 ± 7.8%, 54.4 ± 5.9%, 65.3 ± 4.8%, 65.7 ± 2.2%, 60.2 ± 2.7% and 53.5 ± 7.8% of the total molecular species of PtdIns from rat brain, heart, lung, liver, pancreas, kidney and testis, respectively, were 1-stearoyl- 2-arachidonoyl species. The molecular conservation observed in mammalian tissues that PtdIns is composed mainly of 1-stearoyl-2-arachidonoyl species also applies to tissues of yellowtail. Accumulation of arachidonic acid in PtdIns in the presence of the large amounts of docosahexaenoic acid in yellowtail Lipids from yellowtail have a preponderance of docosa- hexaenoic acid over arachidonic acid. In fact, docosahexa- enoic acid and arachidonic acid made up 28.4% and 3.5%, respectively, of the fatty acid composition of the total lipid fraction of yellowtail liver (data not shown). Despite such a one-sided PUFA composition, arachidonic acid is exclu- sively accumulated in PtdIns. Therefore, there must be a mechanism that selects arachidonic acid from the large amounts of docosahexaenoic acid for acylation to lyso- PtdIns in fish cells. To investigate this, we assessed the efficacy of the acylation of [ 14 C]arachidonic acid or [ 14 C]docosahexaenoic acid into sn-2 of lysoPtdIns (1-acyl) or lysoPtdCho (1-acyl) in fish liver microsomes. In prelimi- nary experiments, the optimum temperature for acylation of arachidonic acid to lysophospholipids was found to be 37 °C, so the assay was conducted at this temperature. When lysoPtdIns was used as an acyl acceptor, arachidonic acid was incorporated into sn-2 of PtdIns more effectively than docosahexaenoic acid (Fig. 2A). The saturation levels of acylation for arachidonic acid and docosahexaenoic acid were  70 and 7 nmol per 10 min per mg protein, respect- ively. When lysoPtdCho was used as an acyl acceptor, the acyltransferase activity of the fish liver microsomes acylated docosahexaenoic acid more effectively than arachidonic acid (Fig. 2B). At a fatty acid concentration of 50 l M , the amounts of docosahexaenoic acid and arachidonic acid incorporated into PtdCho were 129 and 94 nmol per 10 min per mg protein, respectively. The same experiments were conducted with rat liver microsomes: docosahexaenoic acid was found to be a poor acyl donor not only for lysoPtdIns but also for lysoPtdCho compared with arachi- donic acid (Fig. 2C,D). At a fatty acid concentration of 50 l M , the level of acylation of docosahexaenoic acid to lysoPtdIns was 19.4 nmol per 10 min per mg protein, which was about one-fifth of that obtained with the same con- centration of arachidonic acid (90.4 nmol per 10 min per mg Table 4. Fatty acid composition of PtdIns from various tissues of yellowtail. Values are weight percentages, given as the mean ± SD. Tissues were obtained from three different yellowtails. Fatty acid Brain Heart Liver Spleen Kidney Ovary 14:0 0.8 ± 0.5 0.4 ± 0.1 0.3 ± 0 0.4 ± 0 0.3 ± 0.2 0.5 ± 0.4 16:0 11.1 ± 2.4 4.6 ± 0.6 5.3 ± 0.9 6.2 ± 0.9 5.7 ± 0.7 6.1 ± 1.0 16:1 0.7 ± 0.1 0.6 ± 0.2 0.3 ± 0.1 0.5 ± 0 0.3 ± 0 0.6 ± 0 18:0 29.7 ± 1.3 33.7 ± 1.6 37.7 ± 3.2 34.0 ± 0.7 36.4 ± 3.2 32.8 ± 1.0 18:1(n)9) 8.0 ± 1.0 6.6 ± 0.6 7.3 ± 0.8 7.7 ± 0.7 6.0 ± 2.4 7.0 ± 0.9 18:1(n)7) 1.8 ± 0.1 2.0 ± 0.1 1.4 ± 0.2 2.1 ± 0.3 1.6 ± 0.5 1.9 ± 0.5 18:2(n)6) 0.5 ± 0.2 0.6 ± 0.2 0.3 ± 0.1 0.7 ± 0.2 0.4 ± 0.2 0.4 ± 0.1 20:4(n)6) 17.6 ± 1.7 31.8 ± 1.1 27.8 ± 6.5 26.1 ± 4.2 25.4 ± 3.5 33.5 ± 3.3 20:5(n)3) 11.5 ± 1.4 9.1 ± 1.6 12.5 ± 0.6 8.8 ± 0.4 13.0 ± 1.3 2.0 ± 0.1 22:5(n)3) 1.4 ± 0.4 0.7 ± 0.2 0.6 ± 0.3 1.7 ± 0.9 1.2 ± 0.3 0.9 ± 0.4 22:6(n)3) 13.1 ± 1.3 3.1 ± 0.8 3.5 ± 2.1 7.4 ± 1.3 7.0 ± 1.7 10.0 ± 1.1 Ó FEBS 2003 Arachidonate in phosphatidylinositol of yellowtail (Eur. J. Biochem. 270) 1469 protein) in rat liver microsomes. When experiments were conducted with lysoPtdCho and 50 l M fatty acid, the amount of docosahexaenoic acid incorporated into PtdCho (28.4 nmol per 10 min per mg protein) was about one- seventh of that of arachidonic acid (209.5 nmol per 10 min per mg protein) in rat liver microsomes. In an extended study, the inhibitory effect of docosa- hexaenoic acid on the incorporation of [ 14 C]arachidonic acid into lysophospholipid was investigated. This experi- ment was conducted in the presence of both lysoPtdIns and lysoPtdCho to elucidate the distribution of [ 14 C]arachidonic acid incorporated into these phospholipids. We also modi- fied the experimental conditions so that the addition of an equimolar quantity of unlabeled arachidonic acid achieved 50% inhibition of the acylation of [ 14 C]arachidonic acid to lysophospholipids. In experiments using microsomes from yellowtail liver (Fig. 3A), the distribution of [ 14 C]arachi- donic acid between PtdCho and PtdIns was approximately 1 : 1 in the absence of docosahexaenoic acid. In contrast, the addition of docosahexaenoic acid at equimolar, two and fourtimesmolarexcessover[ 14 C]arachidonic acid modified the distribution of [ 14 C]arachidonic acid between PtdCho and PtdIns from 1 : 1 to 1 : 3, 1 : 4 and 1 : 5, respectively. These results obtained in the presence of large amounts of docosahexaenoic acid are in good agreement with the distribution patterns of arachidonic acid between PtdCho and PtdIns observed in the fatty acid analysis of tissues of yellowtail. Furthermore, they indicate that the one-sided incorporation of arachidonic acid into lysoPtdIns can be accomplished in the presence of large amounts of docosa- hexaenoic acid. A similar experiment was conducted with rat liver microsomes (Fig. 3B). Unlike the results obtained with liver microsomes from yellowtail, the ratios of distri- bution of [ 14 C]arachidonic acid between PtdCho and PtdIns were not much changed by the addition of docosahexaenoic acid at equimolar, two and four times molar excess over [ 14 C]arachidonic acid, remaining about 1 : 0.6–0.7. This ratio was similar to that obtained in the absence of docosahexaenoic acid (1 : 0.6). Because of the difference in the phospholipid acylation systems of rat and yellowtail in the preference for docosahexaenoic acid over arachidonic acid for acylation to lysoPtdCho (Fig. 2), the docosahexa- enoic acid preference of the enzymatic activity of yellowtail contributes to the one-sided distribution of arachidonic acid between PtdCho and PtdIns in yellowtail. Discussion Unlike terrestrial animals, lipids from marine fish have a preponderance of n)3 series PUFAs over n)6series PUFAs. Despite this PUFA composition, phospholipid acylation systems operating in yellowtail utilize arachidonic acid exclusively for acylation to lysoPtdIns. In this study, we have clarified several mechanisms concerning this point. The key enzymatic activity for construction of the final molecular species of PtdIns is considered to be acylCoA– lysoPtdIns acyltransferase activity operating in the remode- ling pathway of phospholipid biosynthesis. This enzymatic activity in liver microsomes of yellowtail strictly recognized arachidonic acid and hardly utilized docosahexaenoic acid at all. This one-sided efficacy was remarkable compared with that observed in rat liver microsomes. This strict recognition must contribute to the accumulation of arachi- donic acid in PtdIns in yellowtail. Docosahexaenoic acid is predominantly acylated to PtdCho in tissues of yellowtail like other marine fish species [26–29,34]. Consistent with these observations, lysoPtdCho acyltransferase activity in liver microsomes of the yellowtail preferred docosahexaenoic acid. This enzymatic activity also utilized arachidonic acid with significant efficacy. Therefore, in the absence of docosahexaenoic acid, arachi- donic acid was acylated into both lysoPtdCho and lyso- PtdIns at similar levels (Fig. 3A). The result indicates that, in yellowtail, there is no selectivity for incorporation of Fig. 1. Arachidonic acid contents of PtdCho, PtdEtn, PtdSer and PtdIns obtained from several tissues of yellowtail and rat. 1470 T. Tanaka et al.(Eur. J. Biochem. 270) Ó FEBS 2003 arachidonic acid itself, whether into PtdCho or PtdIns. However, in the presence of a large amount of docosahexa- enoic acid, docosahexaenoic acid effectively inhibits the incorporation of arachidonic acid into PtdCho without inhibiting the utilization of arachidonic acid for PtdIns (Fig. 3A). A possible explanation of this phenomenon is that docosahexaenoic acid competes with arachidonic acid effectively only in the case of incorporation into PtdCho. This docosahexaenoic acid effect would explain the relat- ively low content of arachidonic acid in PtdCho, and may contribute to the exclusive utilization of arachidonic acid for acylation to PtdIns in living fish cells. In the experiment with rat liver microsomes, a large amount of docosahexaenoic acid did not affect the distribution of arachidonic acid incorporated into PtdCho and PtdIns (Fig. 3B). This observation is in good agreement with the results showing that docosahexaenoic acid is a poor acyl donor compared with arachidonic acid, not only for lysoPtdIns but also for lysoPtdCho (Fig. 2C,D). The preference for arachidonic acid over docosahexaenoic acid for acylation to these lysophospholipids has been reported in microsomes of porcine platelets, porcine liver and rat liver [35]. The arachidonic acid-specific acyltransferase and acylCoA syn- thase present in mammals could be involved in these processes [36]. Besides the acylation systems in the remode- ling of phospholipids, both diacylglycerol kinase [17–20] and CDP-sn-1,2-diacylglycerol synthase [21], enzymes operating in the PtdIns cycle, have been reported to contribute to the enrichment of arachidonate in PtdIns in mammals. Further experiments are needed to clarify the involvement of the PtdIns cycle in the accumulation of arachidonic acid in PtdIns of fish. Fig. 3. Effects of docosahexaenoic acid (DHA) on the incorporation of [ 14 C]arachidonic acid (*AA) into exogenously added lysoPtdCho and lysoPtdIns in microsomes from liver of yellowtail and liver of rat. Microsomes (0.1 mg protein ) from liver of yellowtail (A) or rat (B) were incubated at 37 °C for 10 min with 10 nmol labeled arachidonic acid (*AA) and the indicated amount of unlabeled DHA in the pre- sence of both 6.4 nmol 1-acyl-2-lyso-PtdIns and 6.4 nmol 1-acyl- 2-lyso-PtdCho. After the incubation, phospholipids were separated by 2D TLC, and radioactivity was measured. Therefore, only the amount of arachidonic acid incorporated into each lysophospholipid could be determined. Similar results were obtained in three independent experiments with microsomes from different yellowtails or rats. Table 5. Molecular species composition of PtdIns from various tissues of yellowtail. The isolated PtdIns was converted to dinitrobenzoyl derivative as described in materials and methods and analyzed by HPLC. Values are mol percentages, given as the mean ± SD. Tissues were obtained from three different yellowtails. Molecular species Brain Heart Liver Spleen Kidney Ovary 18:1/20:5(n)3) 5.8 ± 1.9 2.5 ± 0.3 3.6 ± 1.0 2.5 ± 1.1 1.6 ± 1.5 1.5 ± 0.4 18:1/22:6(n)3)+16:0/20:5(n)3) 11.5 ± 2.5 3.1 ± 1.0 4.1 ± 1.0 4.6 ± 2.3 4.7 ± 4.4 4.6 ± 1.1 16:0/22:6(n)3) 6.2 ± 1.2 1.0 ± 0.5 1.7 ± 1.5 2.3 ± 1.2 1.4 ± 1.3 2.7 ± 1.4 18:1/20:4(n)6) 9.1 ± 1.6 9.9 ± 0.2 9.4 ± 2.5 6.9 ± 1.3 6.8 ± 0.8 8.8 ± 2.7 16:0/20:4(n)6) 7.3 ± 2.2 4.7 ± 0.6 4.7 ± 1.8 4.9 ± 3.0 3.3 ± 1.6 6.7 ± 2.7 18:0/20:5(n)3) 16.7 ± 0.1 17.6 ± 3.4 18.9 ± 4.8 15.0 ± 6.3 15.5 ± 6.3 9.0 ± 4.6 18:0/22:6(n)3) 11.8 ± 1.8 1.9 ± 0.5 3.0 ± 3.2 9.4 ± 4.8 5.3 ± 1.3 8.6 ± 4.2 18:0/20:4(n)6) 19.8 ± 2.6 54.8 ± 1.2 44.4 ± 5.5 45.4 ± 5.9 31.9 ± 4.1 47.4 ± 6.4 Fig. 2. Incorporation of [ 14 C]arachidonic acid or [ 14 C]docosahexaenoic acid (DHA) into exogenously added lysoPtdCho or lysoPtdIns in microsomes from liver of yellowtail or liver of rat. The incubation was conducted at 37 °Cfor10minwith0.1mgproteinfrommicrosomes of yellowtail liver in the presence of 32 nmol 1-acyl-2-lyso-PtdIns (A) or 32 nmol 1-acyl-2-lyso-PtdCho (B). The same experiments were conducted with rat liver microsomes in the presence of lysoPtdIns (C) or lysoPtdCho (D). After the incubation, phospholipids were separ- ated by 2D TLC, and radioactivity was measured. Values are means ± SD (three microsomal preparations from different yellow- tailsorrats). Ó FEBS 2003 Arachidonate in phosphatidylinositol of yellowtail (Eur. J. Biochem. 270) 1471 It has been widely reported that PtdIns contains abun- dant arachidonate and is composed mainly of 1-stearoyl- 2-arachidonoyl species in mammals [1–12]. We have confirmed this characteristic in rat tissues in this study. This was also the case for tissues of chicken: the arachidonic acid contents of PtdIns from chicken brain, heart, liver and kidney were 40.6%, 30.9%, 33.7% and 30.3%, respectively (T. Tanaka, T. Hiyama & K. Satouchi, unpublished results). In this study, we have demonstrated that this characteristic of PtdIns also applies to tissues of yellowtail, a marine fish. We do not know if all fish species have this feature, but it has been reported that 1-stearoyl-2-arachidonoyl-PtdIns is the predominant molecular species in codfish roe [26] and several tissues of salmon [27–29]. In plants and some insects [37], arachidonic acid is not a lipid constituent, therefore the molecular conservation of PtdIns is not a characteristic of all multicellular organisms. However, in preliminary experi- ments, PtdIns from liver of Xenopus laevis, a frog, was found to contain abundant arachidonate compared with other phospholipids. Inositolphospholipids are known to be a source of diacylglycerol, which activates PKC. There is evidence that PKC activation correlates with the transient accumulation of diacylglycerol derived from inositolphospholipid [38]. PKC isoforms that can be activated by diacylglycerol have been reported to exist even in fish cells [39,40]. The molecular conservation of PtdIns gives rise to the unifica- tion of diacylglycerol molecular species produced in response to agonistic stimulation. It is still unclear whether PKC discriminates the structural difference between 1-stearoyl-2-arachidonoylglycerol and other PUFA- containing diacylglycerol molecular species. Bell & Sargent [40] have reported that n)3-rich diacylglycerols prepared from cod roe have a similar potency to 1-stearoyl- 2-arachidonoylglycerol for increasing PKC activity in vitro. Similar results have been reported with synthetic 1-stearoyl- 2-docosahexaenoylglycerol [41]. On the other hand, evi- dence has emerged that activation of PKC is dependent on the composition of diacylglycerol molecular species [38,42,43] and that diacylglycerols containing PUFAs, such as arachidonic acid and mead acid (20:3, D-5c,8c,11c), are more potent activators of PKC [44]. In addition, 1-stearoyl- 2-arachidonoylglycerol has been reported to be a more potent activator of PKC than diacylglycerols rich in n)3 series PUFA under certain conditions [45]. It has been reported that 1-stearoyl-2-arachidonoylglycerol attains a V-shaped conformation in biological membranes that facilitates anchoring of PtdSer-requiring proteins [46]. Furthermore, some Ca 2+ channels that mediate the influx of Ca 2+ across the plasma membrane are directly activated by 1-stearoyl-2-arachidonoylglycerol [47]. The physiological significance of the enrichment of arachidonate in PtdIns can be clarified by investigating the functions of cells in which the arachidonic acid residue of PtdIns has been replaced with another fatty acid. We have demonstrated that polymethylene-interrupted fatty acids, such as sciadonic acid (20:3, D-5c,11c,14c), mimic arachidonic acid in the biosynthesis of PtdIns in cells [25]. We are now conducting experiments to clarify whether such an acyl residue modi- fication of PtdIns affects the cell response to agonistic stimulation using Swiss 3T3 cells. In conclusion, the characteristic that PtdIns con- tains abundant arachidonate and is composed mainly of 1-stearoyl-2-arachidonoyl species also applies to tissues of yellowtail. Lysophospholipid acyltransferase systems of the yellowtail enable PtdIns to accumulate arachidonate in the presence of large amounts of docosahexaenoic acid and a limited supply of arachidonic acid. 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Mechanisms of accumulation of arachidonate in phosphatidylinositol in yellowtail A comparative study of acylation systems of phospholipids in rat and the. the fish species Seriola quinqueradiata Tamotsu Tanaka, Dai Iwawaki, Masahiro Sakamoto, Yoshimichi Takai, Jun-ichi Morishige, Kaoru Murakami and Kiyoshi Satouchi Department