Uptake, stimulated release and metabolism of (1-¹⁴C)-eicosapentaenoic acid in a perfused organ of the rabbit

Summary

• 1. The isolated rabbit ear was prelabeled by perfusion with (1-¹⁴C)-eicosapentaenoic acid (EPA). Uptake and distribution in lipids, basal release and, in particular, stimulated release and metabolism was studied. In a series of experiments a comparison with results obtained by labeling the perfused organ with (1-¹⁴C)-arachidonic acid (AA) was made.

• 2. Approximately 80% of the perfused labeled EPA was incorporated into the tissue. The main part (74% of the incorporated radioactivity) was found in the phospholipids.

• 3. Following incorporation of labeled EPA, basal release of EPA but not of trienoic prostaglandins (PGs) could be observed.

• 4. Bolus injection of bradykinin (3 μg) and the calciuminophore A 23187 (10 μg) led to an immediate increased release of radioactivity in the effluent which declined within 10–20 min.

• 5. Analysis of the extracted effluent by thin layer chromatography (TLC) showed that only the release of EPA and of radioactivity at the R _f-value of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) was increased following stimulation by bradykinin and A 23187. No labeled trienoic PGs could be detected. Upon injection of A 23187 in the presence of indometacin (3 μg/ml) there was no reduction of any peak of radioactivity on the TLC-plate, indicating that no cyclooxygenase product of EPA was generated.

• 6. The extremely high dose of 10 μg bradykinin or 50 μg A 23187 led to a small release of labeled PGI₃ (A 23187) and to a somewhat higher release of labeled PGE₃ (bradykinin, A 23187).

• 7. In some experiments release and metabolism of labeled EPA were compared with those of labeled AA. A 23187 (10 μg) released high amounts of labeled PGI₂ and PGE₂ whereas from the contralateral ear, prelabeled with EPA, neither PGI₃ nor PGE₃ were released. An extremely high dose of A 23187 (50 μg) again released high amounts of PGI₂ and PGE₂, small amounts of PGE₃ and somewhat higher amounts of PGE₃. The amounts of PGI₂ and PGE₃ released were only 7.6% and 15.2% of PGI₂ and PGE₂, respectively. Labeled PGI₂ and PGI₃ were measured in form of their stable degradation products (see methods).

• 8. The results show that EPA release can be stimulated by phospholipase A₂ activators. EPA becomes available since small amounts of radioactivity at the R _f-value of HETE (possibly 12-L-hydroxy-5,8-10,14,17-eicosapentaenoic acid = HEPE) are released. In contrast to AA there is, however, no conversion of released EPA into trienoic PGs. Only following an extremely high stimulus small amounts of PGI₃ and PGE₃ are released.

The present investigation shows that the formation of trienoic PGs from EPA differs greatly from that of bisenoic PGs from AA and does not support the assumption that the antiaggregatory effect seen under EPA feeding (fish oil) may be mediated via release of trienoic PGs.