Docosahexaenoic acid causes accumulation of free arachidonic acid in rat pineal gland and hippocampus to form hepoxilins from both substrates

Abstract

Hepoxilins (Hx) are biologically active metabolites of arachidonic acid (AA) formed regioselectively from 12(S)-HPETE by `hepoxilin synthase'. Hx modulate synaptic neurotransmission in hippocampal CA1 neurons, and inhibit norepinephrine release in hippocampal slices. During the course of our studies we investigated whether docosahexaenoic acid (DHA) was a substrate for hepoxilin formation. We used two tissues, the pineal gland and hippocampal slices. Tissues were incubated alone or with AA (20 μg/ml) or DHA (20 μg/ml). After 60 min at 37°C, samples were acid-extracted to convert Hx into their stable trioxilin (TrX) form and analyzed as the Me-TMSi derivatives by EI-GC/MS to determine the structures of the DHA metabolites, and as PFB-TMSi derivatives by GC/MS in the NICI mode using SIM to simultaneously quantify TrX products of the 3-series (derived from AA) monitored at m/z 569, while those of the 5-series (derived from DHA) were monitored at m/z 593. Results show good conversion of both substrate fatty acids by the rat pineal gland and hippocampal slices, into the 3-series (21.3±5.8 and 12.5±2.2 ng/ μg protein, respectively) and 5-series TrX (12.3±2.7 and 2.9±0.4 ng/ μg protein, respectively). Surprisingly though, experiments with DHA, in both tissues, also showed formation of TrX derived from endogenous AA (3-series) (10.4±8.3 and 3.1±2.1 ng/ μg protein, respectively). These experiments demonstrate previously unreported actions of DHA causing the accumulation of AA, which is converted into hepoxilins. In order to prove that AA is accumulated during DHA stimulation of the tissue, we carried out separate experiments with hippocampal slices in which the neutral lipids and phospholipids were labeled with [ 14C]AA. DHA caused a time-dependent appearance of free [ 14C]AA which was released mostly from the TG pool. Measurement of the AA/DHA ratio in the TG pool by GC/MS further indicated that DHA is incorporated into the TG at the expense of AA. These results demonstrate that DHA competes with AA for acylation into the metabolically active TG fraction, and both fatty acids are converted into hepoxilins of the corresponding series.