Uptake,incorporation and calcium-ionophore-stimulated mobilization of arachidonic,eicosapentaenoic and docosahexaenoic acids by leucocytes of the rainbow trout,Salmo gairdneri

Rainbow trout leucocytes contain high levels of neutral lipid (about 70% of total lipid on a wt% basis) consisting of mostly triacylglycerol, free sterols and sterol esters (25%, 15% and 52% of neutral lipid, respectively). The phospholipids, separated by thin-layer chromatography, consisted predominantly of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, each present at about 30% of the total phospholipid. Radiolabelling of the leucocytes for 1 h with 1 μCi (approx. 6 μM) 1?¹⁴C]20:4(n?6), 1?¹⁴C]20:5(n?3) or 1?¹⁴C]22:6(n?3) each gave similar uptake values (approx. 1 · 10⁵ cpm/10⁷ leucocytes). The incorporation into total phospholipids was highest for 22:6(n?3) and lowest for 20:4(n?6). A higher percentage of radiolabel from 1?¹⁴C]22:6(n ? 3) was found incorporated into phosphatidylcholine and phosphatidylethanolamine as compared to that from 1?¹⁴C]20:4(n ? 6) and 1?^14C]20:5(n?3), while the reverse situation was found with phosphatidylinositol and phosphatidylserine. The relative rates of incorporation into the different phospholipid classes for all three fatty acids were in the order phosphatidylinositol > sphingomyelin > diphosphatidylglycerol > phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine. Calcium ionophore-challenge did not significantly alter the pattern of phospholipid radiolabel. Ionophore-challenge released large amounts of radiolabel, much of which was recovered after high-performance liquid chromatographic separation as free fatty acid/monohydroxy fatty acids, although only approx. 0.3% was recovered in leukotriene B₄ and leukotriene B₅ for the 1?¹⁴C]20:4(n?6) and 1?¹⁴C]20:5(n?3) labelled leucocytes, respectively. Other lipoxygenase products were also radiolabelled and tentatively identified as 20-carboxy-LTB₄, 20-hydroxy-LTB₄, 6-trans-LTB₄, 6-trans-12-epi-LTB₄, 6-trans-8-cis-12-epi-LTB₄ and the corresponding LTB₅ structures. No ‘6-series’ leukotrienes were produced from 1?¹⁴C]22:6(n?3), nor was there any evidence for the synthesis of ‘5-series’ leukotrienes via retroconversion of 22:6(n?3) to 20:5(n?3). This latter finding shows that, despite the preponderance of 22:6(n?3) in the membranes of trout leucocytes, this fatty acid is not a substrate for leukotriene generation.