Arachidonic acid metabolism in gill homogenate and isolated gill cells from rainbow trout, Oncorhynchus mykiss: the effect of osmolality, electrolytes and prolactin.
An assay method based on thin layer chromatography to study the arachidonic acid (AA) metabolism in gill tissues was optimized and the effect of osmotically different incubation mediums on AA metabolism was evaluated. Rainbow trout gill tissues metabolize AA into PGE2 in highest concentration followed by PGD2, PGF2α and 6-keto-PGF1α (the stable metabolite of PGI2) among the prostanoids tested. Approximately 40% of PGE2 is synthesized within the first minute of incubation and is directly dependent on the substrate concentration (AA). As in mammalian tissues, PGE2 synthesis in fish gills is inhibited by the cyclooxygenase inhibitor indomethacin. PGE2 synthesis in gill homogenate and isolated gill cells incubated in trout Ringer was 0.45 and 1.9 ng/mg protein, respectively, and increased to 8.9 and 4.3 ng/mg protein, respectively, when incubated in KPO4 buffer, due to a ten-fold increase in the free AA. The hydroxy acid synthesis of the gill homogenate was higher (13%), and that of the isolated gill cells incubated in KPO4 buffer was lower (44%) compared to gill homogenate and cells incubated in trout Ringer. Gill homogenate incubated in 50 mM phosphate buffer with increasing sodium or potassium concentrations (up to 250 mM) exhibited a concentration-dependent increase in PGE2 synthesis (220% and 72%, respectively). Prolactin stimulated the PGE2 synthesis up to 30% while PGD2, PGF2α and 6-keto-PGF1α synthesis was not affected. This effect of prolactin was maximal when PGE2 synthesis was estimated 30 minutes after prolactin addition and diminished after two hours. These results suggest that rainbow trout gills possess the ability to metabolize AA through the cyclooxygenase and lipoxygenase pathways. PGE2 synthesis may be under the influence of ion balance and prolactin availability, indicating the probable involvement of AA metabolites in the regulation of ion balances across the gill membrane.
Institute of Biology, University of Odense, Campusvej 55, DK-5230, Odense M, Denmark.
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Beckman
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Mustafa