Mechanism and signal transduction of 14 ( R), 15 ( S)-epoxyeicosatrienoic acid (14,15-EET) binding in guinea pig monocytes

Abstract

14( R), 15( S)-epoxyeicosatrienoic acid (14,15-EET) is a cytochrome P-450 monooxygenase (epoxy- genase) metabolite of arachidonic acid (AA). In this study, we have identified a population of specific high affinity binding sites for 14,15-EET in the guinea pig mononuclear (GPM) cells. The results of competition studies showed that 14(R), 15(S)-EET was an effective competing ligand with a K i of 226.3 nM followed by 11(R), 12(S)-EET, 14(S), 15(R)-EET, 14,15 thia(S)-ET, and 14,15-aza(N)-ET. The binding was sensitive to various protease treatments suggesting that the binding site is protein in nature. Cholera toxin (CT) and dibutyryl cAMP attenuated 14,15-EET binding in GPM cells. Mean binding site density ( B max), decreased 32.0% and 19.1% by the pretreatment with cholera toxin (200 μg/ml) and dibutyryl cAMP (100 nM), respectively, without changing the dissociation constant. A specific protein kinase A (PKA) inhibitor, H-89, but not the PKC inhibitor K252a reversed the down regulation of 14,15-EET receptor binding caused by dibutyryl cAMP in GPM cells. Thus, the results sug- gest that the specific binding site of 14,15-EET in GPM cells be associated with a receptor that could be down regulated through an increase in intracellular cAMP and activation of a PKA signal trans- duction. We propose that the signal transduction mechanism begins with the binding of 14,15-EET to its receptor that leads to increase intracellular cAMP levels and the activation of PKA, and finally, with the down regulation of 14,15-EET receptor binding.