Biosynthesis of polyunsaturated fatty acids by the australian field cricket,Teleogryllus commodus

Aspects of testicular fatty acid biochemistry from the Australian field cricket, Teleogryllus commodus, are reported. Over 10% of the phospholipid fatty acids were C20 polyunsaturated fatty acids (PUFAs), with nearly 6% arachidonic acid (20:4). The testes and ovaries accumulated a large proportion of label from radioactive arachidonic acid that was injected into the hemocoel (about 30%). Specificity in the uptake was shown by comparison to a similar study with labelled stearic acid, in which only 1.5% of the radioactivity was taken up by testes. Sixty percent of the radioactivity taken up by testes from [³H]20:4 was incorporated into phospholipids and 30% into triacylglycerols. Fat body of males and females incorporated 27% of the [³H]20:4 into phospholipids and 68% (males) or 55% (females) into triacylglcyerols. Radioactivity from [1-¹⁴C]acetate was incorporated into testicular linoleic acid and eicosatrienoic acid, but not eicosatetraenoic acid, suggesting the de novo biosynthesis of both 18:2 and a C20 PUFA by this species. Label from injected [U-¹⁴C]linoleic acid was recovered mostly as linoleic acid, with a small portion of the recovered radioactivity in eicosatrienoic acid, but not eicosatetraenoic acid. Very little label from injected linoleic acid occurred as monounsaturated or saturated fatty acids, indicating only slight, if any, β-oxidation of 18:2 to acetate and subsequent lipid synthesis.     

The effect of dietary supplementation with linoleic acid to late gestation ewes on the fatty acid composition of maternal and fetal plasma and tissues and the synthetic capacity of the placenta for 2-series prostaglandins

Linoleic acid (18:2n-6) is metabolised to arachidonic acid (20:4n-6), the precursor for 2-series prostaglandins (PGs). Increased consumption of 18:2n-6 during pregnancy may thus modify PG synthesis during labour. We have investigated whether increased 18:2n-6 composition during gestation altered the fatty acid consumption and PG synthesis of maternal and fetal tissues in the sheep. Ewes were fed a control diet or a diet providing 40% more 18:2n-6 from 96 days gestation. Half of each group received dexamethasone on day 136 to up-regulate the PG synthetic pathways promoting parturition. Maternal and fetal tissues were collected at 138 days. The 18:2n-6 diet significantly increased the 20:4n-6 content of maternal plasma, fetal plasma and allantochorion (51-81%) phosphatidylcholine, and fetal liver (40%) and maternal caruncular endometrium (57%) phosphatidylethanolamine. Increased 18:2n-6 intake increased production of PGF(2alpha) and PGE(2) in all placental tissues (maternal caruncular and intercaruncular endometrium and fetal allantochorion) by 23-98%, whereas dexamethasone increased it by 32-142%. This suggests that consumption of an 18:2n-6-enriched diet in late pregnancy enhanced placental PG production by increasing the supply of 20:4n-6. Variations in the extent to which the diet altered the polyunsaturated fatty acid (PUFA) content of the different tissues indicated complex interactions between nutrient availability and metabolic adaptation.     

Polyunsaturated fatty acids in the lipids from adult Galleria mellonella reared on diets to which only one unsaturated fatty acid had been added

Gas chromatographic analysis of tissue phospholipid and triacylglycerol fatty acids derived from adult moths, Galleria mellonella, reared on semi-synthetic basal medium supplemented with various single C18, C20 and C22 polyunsaturated fatty acids suggests that C20:2n6, C20:3n6 and C20:3n3 can be shortened to their C18 analogues. Only C18:2n6 and C18:3n3, or higher analogues convertible to these, can alleviate the pupal/adult ecdysis failure which results from essential fatty acid deficiency. C18, C20 and C22 polyunsaturates with double bonds on the carboxyl side of the n-9 carbon are all inadequate nutritionally. C18:3n6 is not saturated to the nutritionally active C18:2n6, and it appears that double bonds towards the carboxyl group interfere with the shortening of the higher polyunsaturated fatty acids. In addition to chain shortening, dietary C18:3n3 and C20:3n3 may be elongated/desaturated to C20:5n3, which is elevated in tissues of all moths reared to normal adult eclosion, and also is substantially accumulated in phospholipid of moths reared with dietary C20:5n3 even though their emergences are imperfect. Since the data, overall, indicate n3 and n6 fatty acids are not interconvertible, elevated C20:5n3 obtained with high dietary levels of the nutritionally adequate n6 acids, not so high as for dietary active n3 acids, probably derives from efficient sequestration of n3 fatty acids, possibly formed by symbiotic gut microorganisms. These results are discussed in terms of metabolic modifications of long-chain polyenoics by insects generally.     

Polyunsaturated fatty acids differentially alter PGF(2alpha) and PGE(2) release from bovine trophoblast and endometrial tissues during short-term culture

Two studies tested the hypothesis that eicosapentaenoic (20:5omega3; EPA), docosahexaenoic acids (22:6omega3; DHA) or linoleic acid (C18:2omega6; LIN) reduced bovine endometrial and trophoblast prostaglandin F(2alpha) (PGF(2alpha)) and prostaglandin E(2) (PGE(2)) release during short-term culture. In Study 1, endometrial tissues were collected from non-lactating, non-pregnant cows and endometrial plus trophoblast tissues from pregnant cows 16 days post-insemination. In Study 2, endometrial and trophoblast tissues were collected on day 17 of pregnancy, from cows synchronised using a double prostaglandin (PG) or Ovagentrade mark synchronisation. Tissues were incubated in medium only (M) or media supplemented with fatty acids: eicosapentaenoic (20:5omega3; EPA), docosahexaenoic acids (22:6omega3; DHA) or linoleic acid (C18:2omega6; LIN). In Study 1, PGE(2) release from 'pregnant' endometria was higher (P=0.094) than from 'non-pregnant' endometria, while PGF(2alpha) concentrations were similar. Fatty acids treatment had no effect on PGF(2alpha) or PGE(2) release from either pregnant or non-pregnant endometria. Individual fatty acid treatments had no effect on the ratio of PGF(2alpha) to PGE(2) from trophoblast tissues, but when the data from the 3 fatty acid treatments were combined (EPA, DHA and LIN treatment groups) the ratio of PGF(2alpha) to PGE(2) was reduced (P=0.026) when compared to medium only. In Study 2, PGE(2) concentrations were higher (P=0.013) from the trophoblast collected from Ovagentrade mark cows as compared to that of the PG synchrony group. When the data from the 3-omega fatty acids were combined (DHA and EPA treatment groups), the 3-omega treatments decreased (P<0.05) PGE(2) biosynthesis from both endometrial and trophoblast tissues from animals synchronised following PG synchrony but not Ovagentrade mark synchrony. Short-term culture with low concentrations of 3-omega fatty acids tended to reduce prostaglandin release from trophoblast collected 16 days after insemination, with the type of synchrony modifying PGE(2) production from the trophoblast tissues collected 17 days after insemination. The ability of exogenous fatty acids to modify embryonic prostaglandin release needs to be examined in the context of supplementing dairy cows with different sources of fats. Synchronisation method altered trophoblast PGE(2) release, highlighting the importance of the hormonal environment in modifying embryonic prostaglandin synthesis and release.     

Formation of prostaglandins by ovarian carcinomas

Tissue contents of prostaglandins (PG) PGE2, PGE2a and 6-keto-PGF1a (degradation product of PGI2) were determined in specimens of advanced human ovarian cancer (n = 11). The PG levels (ng/mg tissue protein) varied widley: PGE2 17-515; PGF2a 2-43 and 6-keto-PGF1a 5-105. Tumors of patients without response to chemotherapy contained more PGE2, PGF2a and 6-keto-PGF1a than did tumors responding to chemotherapy. PG production was investigated in two ovarian carcinoma-derived cell lines. The ability of these cells to synthesize PG varied depending on the cell density. An increase of cell number was associated with a decrease of PG yield. PG formation was inhibited by indomethacin in a concentration-dependent manner. The present study suggests that ovarian carcinoma cells form PG in vivo and vitro.     

Effects of conjugated linoleic acid on prostaglandins produced by cells isolated from maternal intercotyledonary endometrium, fetal allantochorion and amnion in late pregnant ewes

The anticarcinogenic properties of conjugated linoleic acid (CLA) are, at least partially, attributed to its ability to interrupt the n-6 polyunsaturated fatty acid (PUFA) metabolic pathway for the biosynthesis of eicosanoids, including prostaglandins (PG). Both PGE(2) and PGF(2alpha) play key roles in parturition. In the present study, we compared the effects of CLA (a mixture of cis- and trans-9, 11- and -10, 12-octadecadienoic acid) and linoleic acid (LA) on PG production by cells isolated from maternal intercotyledonary endometrium, fetal allantochorion and amnion from late pregnant ewes. The results demonstrated that supplementation of LA and CLA significantly affected both the proportions and the amounts of PGs produced by all three tissue types. The ability of the uterus and placenta to respond to oxytocin (OT, endometrium only) and lipopolysaccharide (LPS) was also affected. LA inhibited PGE(2) and PGF(2alpha) production in the absence or presence of either oxytocin or LPS. In endometrial cells with or without oxytocin or LPS, CLA dose-dependently suppressed PGF(2alpha) generation, whereas low doses of CLA (20 microM) increased PGE(2) generation. Supplementation with CLA therefore increased the PGE(2)/PGF(2alpha) ratio in the endometrial cells. These results suggest that dietary supplementation of LA or CLA may affect both the initiation and progression of parturition.     

The biosynthesis of the 3-series prostaglandins in rat uterus after alpha-linolenic acid feeding: Mass spectroscopy of prostaglandins E and F produced by rat uteri in tissue culture

The abnormal uterine activity associated with dietary n-3 fatty acids may result from competitive inhibition of PG2 production. Uterine synthesis of 2- and 3-series prostaglandins F(PGF) and E(PGE) was studied using mass spectrophotometry in rats fed diets containing predominantly n-3 fatty acid, n-6 fatty acid, or control pelleted diet. Mass spectra of PGF (Me, TMS and Me, TBDMS derivatives) synthesised by uteri of n-3 fed rats were characterised by 8 ions containing the n-3 double bond, and m.i.d. of the 651/653 ions of PGF-Me, TBDMS indicated PGF3 alpha synthesis (44 +/- 8% and 13 +/- 2% of PGF release by uteri incubated + or -5 micrograms/ul calcium ionophore A23187 respectively). In uteri from the control diet group incubated with ionophore, PGF3 alpha ions were detected and PGF 3 alpha represented 9.5 +/- 1.0% of PGF alpha release. Similarly, analysis of PGE from uteri of n-3 fed rats indicated that PGE3 (16 +/- 6% of PGE) was released in the presence of ionophore A23187. Synthesis of 3-series PG by rat uteri was detected after only 3 weeks of n-3 diet. The capacity to synthesise 3-series PG increased at intracellular calcium concentrations which mimicked cell calcium during decidual autolysis at parturition. These experiments suggest that uterine synthesis of 3-series PG is regulated by the specifity of enzymes incorporating fatty acids, rather than by the cyclooxygenase enzyme.     

De novo biosynthesis of arachidonic acid and 5,11,14-eicosatrienoic acid in the cricket Teleogryllus commodus

The de novo biosynthesis of 5,11,14-eicosatrienoic acid (5,11,14-20:3), arachidonic acid (20:4(n - 6] and eicosadienoic acid (20:2(n - 6] and the elongation/desaturation of linoleic acid (18:2(n - 6] to 20:4(n - 6) and alpha-linolenic acid (18:3(n - 3] to eicosapentaenoic acid (20:5(n - 3] were demonstrated in adult males of the field cricket Teleogryllus commodus. Sodium [1-14C]acetate, [1-14C]18:2(n - 6) and [1-14C]18:3(n - 3) were injected into adult male crickets and after an incubation period, the testes and remaining tissues were extracted and the methyl esters obtained from the phospholipid and triacylglycerol fractions were analyzed. After 5 days of daily injections of [1-14C]acetate, the methyl esters of the triene and tetraene fatty acids from the testicular phospholipid fraction were purified by AgNO3-TLC and HPLC and analyzed by GLC, radio-HPLC, and radio-GLC of ozonolysis products. The results demonstrate the de novo biosynthesis of 20:2(n - 6), 20:4(n - 6) and an isomer of 20:3(n - 6) with double bonds in the 5,11,14 positions. the elongation/desaturation of 18:2(n - 6) to 20:4(n - 6) and 18:3(n - 3) to 20:5(n - 3) was demonstrated by analysis of the methyl esters derived from the testicular phospholipid fraction by radio-HPLC after injecting crickets with radiolabeled substrates.     

Phospholipid fatty acids from exocrine and reproductive tissues of male american cockroaches,Periplaneta americana (L.)

We present the phospholipid fatty acid compositions, determined by GLC,* of four individual tissues (testes, mushroom glands, conglobate glands, and salivary glands) from adult male cockroaches, P. americana. The testes phospholipids contained higher proportions of palmitic acid than did the exocrine tissues (16% vs about 8%). This was also true for palmitoleic (2.3% vs 0.4–1.3%), oleic (49% vs 30–35%), and linolenic acids (7% vs 0.5–2.8%). Testicular extracts were lower in linoleic acid (16%) than were the exocrine gland extracts (at 40–45%). All four tissues also contained low proportions of several long-chain polyunsaturated fatty acids of both omega-3 and omega-6 metabolic families, including C20:3n6, C20:4n6, and C20:5n3. The data suggest that the phospholipid fatty acid profiles of specific tissues differ from each other and from those obtained from whole-insect extracts. The presence of several polyenoics from the n3 and n6 metabolic families is interpreted in the context of complex fatty acid requirements at the tissue level.     

The effect of partial outlet obstruction on prostaglandin generation in the rabbit urinary bladder

Partial outlet obstruction of the urinary bladder has been demonstrated to induce specific dysfunctions in cellular and sub-cellular membrane structures within the bladder's smooth muscle and mucosal compartments. Recent studies have linked these membrane dysfunctions to alterations in phospholipid metabolism leading to mobilization of free arachidonic acid, the precursor for synthesis of prostaglandins (PG). The purpose of this study was to determine if partial outlet obstruction of the urinary bladder induces changes in the capacity of bladder smooth muscle and mucosa to generate PG. PG were isolated from control and partially obstructed urinary bladder smooth muscle and mucosa of male New Zealand White (NZW) rabbits. PG concentrations (PGE2, PGF2alpha and PGI2, as its stable metabolite 6-keto-PGF1alpha) were determined after 30 minute incubations using enzyme-linked immunoassay (ELISA) kits. In both control and obstructed rabbit urinary bladders, PG generation was significantly higher in isolated mucosa than muscle tissues. A significantly higher concentration of PGF2alpha, and 6-keto-PGF1alpha was measured in obstructed muscle tissue relative to controls. The concentration of 6-keto-PGF1alpha was also significantly higher than the concentrations measured for PGE2 and PGF2alpha in both control and obstructed smooth muscle samples. The generation of PGE2 was significantly higher in rabbit urinary bladder mucosa than either PGF2alpha or 6-keto-PGF1alpha in both control and obstructed samples. The capacity of obstructed mucosal tissue to generate 6-keto-PGF1alpha was significantly higher than control tissue, while no significant differences in PGE or PGF2alpha generation were noted. These data suggest obstruction of the urinary bladder induce specific elevations in PG in both smooth muscle and mucosal tissues.     

Release of prostaglandins and monohydroxy and trihydroxy metabolites of linoleic and arachidonic acids by adult and fetal aortae and ductus arteriosus

The conversion of arachidonic acid (20:4) to prostaglandins by vascular tissue is important in the adult because of the antithrombotic effect of prostacyclin and in the fetus because of the vasodilatory effect of prostaglandin (PG) E2 on the ductus arteriosus. We have shown that vascular tissue converts various polyunsaturated fatty acids to monohydroxy and trihydroxy metabolites derived from hydroperoxides, which may be involved in regulating prostaglandin synthesis. We have now measured the amounts of these hydroperoxide metabolites, as well as those of prostaglandins, released from slices of rat, rabbit and bovine aortae, as well as from fetal calf aorta and ductus arteriosus. The major oxygenated polyunsaturated fatty acid metabolite formed by rat and bovine blood vessels was 6-oxo-PGF1 alpha. Fetal calf aorta and ductus arteriosus produced about five times as much 6-oxo-PGF1 alpha as adult bovine aorta. Much smaller amounts of the cyclooxygenase products, PGE2, 12-hydroxy-5,8,10-heptadecatrienoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid (11-hydroxy-20:4), and 15-hydroxy-20:4, were released by aortae. Small amounts of the lipoxygenase product, 12-hydroxy-20:4, were also detected. Substantial amounts of free and esterified monohydroxy and trihydroxy metabolites of linoleic acid (18:2) were detected, especially in rat and rabbit aortae. Rabbit aorta, which had low cyclooxygenase activity, formed more oxygenated 18:2 metabolites than 20:4 metabolites. Indomethacin did not inhibit the formation of the 18:2 metabolites, indicating that cyclooxygenase was not involved. Neither exogenous 13-hydroxy-18:2 nor trihydroxyoctadecenoic acid was incorporated to a large extent into lipids from vascular endothelial or smooth muscle cells, suggesting that the esterified 18:2 oxygenation products had arisen mainly via direct oxygenation of lipids.     

Maturation-related variations in prostaglandin and fatty acid content of ovary in the kuruma prawn (Marsupenaeus japonicus)

Total lipid, fatty acids and prostaglandins (PGF(2 alpha) and PGE(2)) in the ovary of kuruma prawns (Marsupenaeus japonicus) were measured during ovarian development. The level of ovarian total lipid increased with an increase in the gonad-somatic index (GSI). No significant difference was found in fatty acid composition among different stages of ovarian development. However, the content of arachidonic acid (precursor of PG(2)), but not eicosapentanoic acid (precursor of PG(3)), was significantly lower at stages I and II than at stage V (P<0.01). When ovarian PGF(2 alpha) and PGE(2) levels were plotted against GSI, no correlation was found in either PG. However, in terms of ovarian developmental stages, the level of ovarian PGs was high (approx. 20 pg/mg) at stage I, followed by marked decreases at stages IV and V (PGF(2 alpha), P<0.01) and stage IV (PGE(2), P<0.01). These results suggest that ovarian PGs and arachidonic acid are deeply involved in ovarian maturation in kuruma prawns.     

Utilization of gammalinolenic acid by mouse peritoneal macrophages.

To delineate the metabolism of gammalinolenic acid (18:3(n-6] by macrophages, primary cultures of resident mouse peritoneal macrophages were incubated with [14C]18:3(n-6). At 3, 6 or 20 h, the majority (greater than 85%) of the radiolabel was recovered in cell phospholipids. With increasing time of incubation, a relative reduction of 14C in glycerophosphocholine (ChoGpl, 58.1% to 46.2%) was noted. This was offset by a corresponding increase in glycerophosphoethanolamine (EtnGpl) labeling (from 8.8% to 18.9%). There was also a time-dependent redistribution of 14C from diacyl to ether-containing phospholipid subclasses in ChoGpl and EtnGpl. Analysis of cell extracts by reverse-phae HPLC following transmethylation demonstrated that 18:3(n-6) was extensively elongated (greater than 80%) to dihomogammalinolenic acid (20:3(n-6] by 3 h. The major radiolabeled phospholipid molecular species in the diacyl (PtdCho) and alkylacylglycerophosphocholine (PakCho) subclasses was 16:0-20:3(n-6). In contrast, diacyl (PtdEtn) and alkenylacylglycerophosphoethanolamine (PlsEtn) subclasses contained primarily [14C]18:0-20:3(n-6) and 16:0-20:3(n-6), respectively. Macrophages prelabeled with [14C]18:3(n-6) for 20 h and stimulated with calcium ionophore A23187 or zymosan synthesized [14C]prostaglandin E1 (PGE1). These data demonstrate that macrophages possess an active long chain polyunsaturated fatty acid elongase capable of converting 18:3(n-6) to 20:3(n-6) which can, upon stimulation, be converted to PGE1.     

Formation of cyclic adenosine monophosphate (cAMP) in the preovulatory rabbit follicle: role of prostaglandins and steroids

The preovulatory increase in follicular prostaglandins (PG) stimulated by luteinizing hormone (LH) is dependent upon 3'-5'-cyclic adenosine monophosphate (cAMP) and is essential for ovulation. It has been proposed that follicular PG stimulate a second rise in cAMP, independent of LH. This study examined the temporal relationships among PGE2, PGF2 alpha 6-keto-PGF1 alpha, estradiol-17 beta, progesterone, testosterone, androstenedione and the biphasic increases of cAMP in follicles of rabbits. Does received indomethacin (IN, 20 mg/kg, i.v.; n = 30) or phosphate buffer (C; n = 30), 0.5 h before 50 ug of LH. At laparotomy at 0, 0.5, 1, 2, 4 or 8 h after LH, blood was collected from each ovarian vein and two follicles per ovary were aspirated of fluid and excised. Plasma and follicular tissue and fluid were assayed for PG and steroids. Tissue and fluid were assayed for cAMP. In C does, cAMP (pmol/follicle) in tissue increased from 11.3 at 0 h to 14.2 at 0.5 h, decreased at 1 h (5.4) and increased linearly through 8 h to 14.5. In IN-treated does, cAMP remained high from 0.5 (13.2) to 2 h (16.3), decreased at 4 h (7.9) then increased again by 8 h (15.5). Indomethacin decreased all PG in follicular tissue but 6-keto-PGF1 alpha rose after 2 h, whereas PGE2 and PGF2 alpha did not. Estradiol-17 beta, progesterone, and androstenedione did not vary with treatment; testosterone was increased (P less than .05) by IN. PGE2 or PGF2 alpha may terminate the first phase of cAMP production, rather than initiate the second phase.     

Effects of dietary butter enrichment on the fatty acid distribution of phospholipid fractions isolated from rat platelets and aortae

Rats were maintained for 2 weeks on a low-fat basal diet (5% energy) and a diet from which 50% of the energy was derived from butter. Lipids were extracted from aortae and platelets and the fatty acid profiles of individual phospholipids were examined. Similar responses to dietary butter enrichment occurred in PI, PS, PE and PC fractions from either tissue: 20:4(n - 6) and all other n - 6 series longer-chain polyunsaturated fatty acids except 20:3(n - 6) decreased in percentage; all n - 3 series polyunsaturated fatty acids increased, including 20:5(n - 3) and 22:6(n - 3); n - 9 series polyunsaturated fatty acids, derived from 18:1(n - 9), increased. Despite the considerable redistribution of polyunsaturated fatty acids, the percentages of total polyunsaturated fatty acids in each phospholipid were, in every case, independent of diet. None of the changes were localized in a particular phospholipid fraction. Quantitation of fatty acids using heptadecanoic acid as an internal standard revealed that the concentrations of 20:4(n - 6) in platelet and aortic PE and PC was higher than in PI fractions. Therefore, in terms of substrate amount, it appears that PC and PE as well as PI have the potential to provide endogenous 20:4(n - 6) for oxygenation to the prostanoids thromboxane A2 and prostacyclin I2.     

Metabolism of polyunsaturated fatty acids by larvae of the waxmoth,Galleria mellonella

The metabolic fates of linoleic (18:2n6) and linolenic (18:3n3) acids injected into the hemocoel of fifth instar larvae of the waxmoth, Galleria mellonella, were examined by radio-high-pressure liquid chromatography and radio-gas-liquid chromatography. In addition to undergoing β-oxidation and incorporation into neutral and phospholipid fractions, a portion of both of these C18 fatty acids was elongated and desaturated to longer chain and more unsaturated polyenoics. Radioactivity from linoleic acid was recovered in components that coeluted with 18:3, 18:4, 20:3, and 20:4. Radioactivity from linolenic acid was recovered in an unidentified component and in components that coeluted with 18:4, 20:3, and 20:5. Labeled arachidonic and eicosapentaenoic acids injected into waxmoth larvae were converted to prostaglandins, suggesting that one aspect of the biological significance of the elongation/desaturation reactions is to generate precursors for prostaglandin biosynthesis.     

Isolation and identification of prostaglandins of the E and F series in testes and semen of lowland-black-white breed of bulls

The prostaglandins of E and F series were obtained from testes and semen of sexually mature bulls of lowland-black-white breed. From 1 g of fresh testes tissue we obtained 7.01 X 10(-9) M prostaglandin of the F series (PGF) and 20.65 X 10(-9) M prostaglandin of the E series (PGE): from 11 of semen 3.28 X 10(-6) M of PGF and 10.58 X 10(-6) M of PGE were obtained as well. The prostaglandins thus obtained displayed biological activity in experiments on the isolated small intestine of the rabbit.     

Prostaglandin synthesis by the cochlea

The exogenous and endogenous syntheses of prostaglandins (PG's) by the cochlea of adult mongolian gerbils were studied in vitro. After incubation of the whole membraneous cochlea with [3H]-arachidonic acid (AA), syntheses of PGF2 alpha, 6-keto PGF1 alpha, PGE2, thromboxane (TX) P2 and PGD2 were evidenced in this order. The synthesis of radioactive PG's was almost completely inhibited by incubation with 10(-5) M indomethacin. No significant amounts of those PG's were detected by radioimmunoassay (RIA) in the cochlea obtained from animals killed by microwave irradiation at 5.0 kw for 0.8 sec. However, when the homogenate of the whole membraneous cochlea obtained from animals without microwave irradiation was incubated at 37 degrees C for 0-15 min, PGD2, PGE2, PGF2 alpha and 6-keto PGF1 alpha were found to be formed from endogenous AA in the cochlea by RIA. PG's were formed already at 0 time to considerable level (PGD2, PGF2 alpha and 6-keto PGF1 alpha, 90-120 pg/cochlea; PGE2, 370 pg/cochlea), reached to the maximum level (PGD2, PGF2 alpha and 6-keto PGF1 alpha, 170-200 pg/cochlea; PGE2, 500 pg/cochlea) at a 5-min incubation, and then gradually decreased. On the other hand, the amount of TXB2 was lower than the detection limit by RIA (less than 50 pg/cochlea) even after the incubation. The cochlea was dissected into three parts: organ of Corti + modiolus (OC + M), lateral wall (LW), and cochlear nerve (CN), and then PG's formed by these tissues were determined after a 5-min incubation of the homogenates. In the CN and OC + M, PGE2 was the major PG (100 and 160 pg/tissue, respectively), and the amounts of PGD2, PGF2 alpha and 6-keto PGF1 alpha were about 1/3 of those of PGE2. In the LW, the amounts of PGD2, PGE2, PGF2 alpha and 6-keto PGF1 alpha were about the same level (70-100 pg/LW).     

Conversion of arachidonic acid to cyclooxygenase and lypoxygenase products,and incorporation into phospholipids in the mouse neuroblastoma clone,Neuro-2A

Arachidonic acid (AA) incorporation into phospholipids and cyclooxygenase and lipoxygenase mediated metabolism of arachidonic acid were studied in homogenized and intact Neuro-2A cells. When 3H8-AA was added to homogenized cells and incubated 20 minutes, 39% of the label was converted to prostaglandins (PGs), 10% to hydroxy-eicosatetraenoic acid (HETE) and 26% was incorporated into phospholipids. PGE2 and PGF2a were the major PGs produced. Synthesis of PGs was blocked by 10 microM indomethacin and synthesis of PGs and HETE was blocked by 10 microM eicosatetraynoic acid (ETYA). The cell homogenate produced the 13,14-dihydro-15-keto metabolites of PGE2 and PGF2a from 3H8-AA and also converted exogenous 3H7-PGE2 and 3H8-PGF2a to metabolites. When intact cells were labeled for 24 hours with 14C1-AA and the cells and media then analyzed, 75% of the radioactivity was incorporated into cellular phospholipids, 0.8% was converted to PGs and metabolites and 0.7% converted to HETE. Cells prelabeled for 24 hours were washed and incubated for 30 minutes in fatty acid free media. There was a 23% release of AA from phospholipids. One-fifth of the released AA was converted to HETE. PG synthesis in the intact resting cells was low. In summary, the Neuro-2A cell provides a good model system for studying arachidonic acid metabolism and incorporation into phospholipids in cells of neuronal origin.