POSTRECEPTOR SIGNAL-TRANSDUCTION AND REGULATION OF 14(R),15(S)-EPOXYEICOSATRIENOIC ACID (14,15-EET) BINDING IN U-937 CELLS

14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET), a cytochrome P-450 m onooxygenase (epoxygenase) metabolite of arachidonic acid has been rep orted to induce adhesion of a monocyte cell line (U-937) to cultured e ndothelial cells. In this study, we identified a population of specifi c, high affinity binding sites for 14(R),IS(S)-EET in U-937 cell surfa ce with K-d of 13.84 +/- 2.58 nM and B-max of 3.54 +/- 0.28 pmol/10(6) cells. The specific binding of [H-3]-14,15-EET on U-937 cells is more effectively displaced by 14(R),15(S)-EET than the 14(S),15(R)-isomer thus indicating stereospecificity. The binding was sensitive to variou s protease treatments suggesting the binding site is protein in nature . 14,15-EET binding in U937 cells is attenuated by cholera toxin (CT) and dibutyryl cAMP. Mean binding site density (B-max) decreased 31.61% and 34.8% by the pretreatment with cholera toxin (200 mu g/ml) and di butyryl cAMP (300 nM), respectively, without affecting the dissociatio n constant. Under similar conditions, pertussis toxin (20-200 ng/ml) w as less effective as compared to CT and dibutyryl cAMP. The down regul ation of 14,15-EET binding caused by dibutyryl cAMP in U-937 cell was reversed by a specific protein kinase A (PKA) inhibitor, H-89, but not by the PKC inhibitor K252a. Thus, the results suggest that the specif ic binding site of 14,15-EET in U-937 cells is associated with a recep tor that could be down regulated through an increase in intracellular cAMP and activation of a PKA signal transduction mechanism. We propose that the signal transduction mechanism of 14,15-EET begins with the b inding of the receptor, which leads to the increase of intracellular c AMP levels and the activation of PKA, and finally with the down regula tion of 14,15-EET receptor binding. (C) 1997 Elsevier Science B.V.