FORMATION, METABOLISM, AND ACTION OF HEPOXILIN A(3) IN THE RAT PINEAL-GLAND

The present study was undertaken to investigate the possible formation of hepoxilin A(3) in the rat pineal gland and to study the potential physiological role for this compound in this tissue. Incubation of hom ogenates of rat pineal glands with arachidonic acid (66 mu M) led to t he appearance of hepoxilin A(3) (HxA(3)) analyzed as its stable trihyd roxy derivative, trioxilin A(3) by gas chromatography in both the elec tron impact and negative ion chemical ionization modes. Endogenous for mation of HxA(3) is estimated to be 1.43 +/- 0.66 ng/mu g of protein. This amount is not modified when the tissue is boiled (2.07 +/- 0.66 n g/mu g of protein). However, the formation of this compound was stimul ated to 21.26 +/- 5.82 ng/mu g of protein when exogenous arachidonic a cid was added to the homogenate. Addition of the dual cyclooxygenase/l ipoxygenase inhibitor BW 755C(10 mu g) resulted in a partial blockade of hepoxilin formation. Using [1-C-14]HxA(3), we demonstrated that the pineal gland contained hepoxilin epoxide hydrolase, which hydrolyzed HxA(3) into trioxilin A(3). This hydrolysis was inhibited by 1 mu mol/ L of 3,3,3-trichloropropene-1,2-oxide. In a separate study, HxA(3) in the presence of 3,3,3-trichloropropene-1,2-oxide to block the hydrolys is of HxA(3) decreased the production of cyclic AMP in cultured organ rat pineals after stimulation with 5'-N-ethylcarboxamidoadenosine, an A(1)/A(2) adenosine receptor agonist. This effect is stereospecific be cause the (8S)-enantiomer is more active in decreasing cyclic AMP prod uction (-88.7%) than the (8R)-enantiomer. This is the first demonstrat ion of the presence, metabolism, and action of HxA(3) in the rat pinea l gland.