Mechanism and signal transduction of 14 (R), 15 (S)-epoxyeicosatrienoic acid (14,15-EET) binding in guinea pig monocytes.
14(R), 15(S)-epoxyeicosatrienoic acid (14,15-EET) is a cytochrome P-450 monooxygenase (epoxygenase) 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 Ki 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 (Bmax), decreased 32.0% and 19.1% by the pretreatment with cholera toxin (200 micrograms/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.
Wong
P Y
PY
Department of Cell Biology, University of Medicine and Dentistry of New Jersey-School of Osteopathic Medicine, Stratford 08084, USA.
Lai
P S
PS
Falck
J R
JR
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D003994:Bucladesine / Q000494:pharmacology; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D010450:Endopeptidases / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006168:Guinea Pigs; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism; D015398:Signal Transduction; D014316:Tritium
D004791:Enzyme Inhibitors; D014316:Tritium; D016718:Arachidonic Acid; D003994:Bucladesine; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D002772:Cholera Toxin; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D010105:Oxygenases; D000090762:Cytochrome P-450 CYP2J2; D017868:Cyclic AMP-Dependent Protein Kinases; D010450:Endopeptidases; D015126:8,11,14-Eicosatrienoic Acid