Formation, Metabolism, and Action of Hepoxilin A3in the Rat Pineal Gland

Abstract: The present study was undertaken to investigate the possible formation of hepoxilin A₃ in the rat pineal gland and to study the potential physiological role for this compound in this tissue. Incubation of homogenates of rat pineal glands with arachidonic acid (66 μM) led to the appearance of hepoxilin A₃ (HxA₃) analyzed as its stable trihydroxy derivative, trioxilin A₃ by gas chromatography in both the electron impact and negative ion chemical ionization modes. Endogenous formation of HxA₃ is estimated to be 1.43 ± 0.66 ng//μg of protein. This amount is not modified when the tissue is boiled (2.07 ± 0.66 ng/μg of protein). However, the formation of this compound was stimulated to 21.26 ±5.82 ng/μg of protein when exogenous arachidonic acid was added to the homogenate. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (10 /μg) resulted in a partial blockade of hepoxilin formation. Using [1-¹⁴C] H×A₃, we demonstrated that the pineal gland contained hepoxilin epoxide hydrolase, which hydrolyzed HxA₃ into trioxilin A₃. This hydrolysis was inhibited by 1 μmol/L of 3, 3, 3-trichloropropene-1, 2-oxide. In a separate study, HxA₃ in the presence of 3, 3, 3-trichloropropene-1, 2-oxide to block the hydrolysis of HxA₃ decreased the production of cyclic AMP in cultured organ rat pineals after stimulation with 5′-N-ethylcarboxamidoadenosine, an A₁/A₂ adenosine receptor agonist. This effect is stereospecific because the (8S)-enantiomer is more active in decreasing cyclic AMP production (?88.7%) than the (8R)-enantiomer. This is the first demonstration of the presence, metabolism, and action of HxA₃ in the rat pineal gland.