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

被引:70
作者
Wong, PYK
Lai, PS
Falck, JR
机构
[1] Univ Med & Dent New Jersey, Sch Osteopath Med, Dept Cell Biol, Stratford, NJ 08084 USA
[2] Univ Texas, SW Med Ctr, Dept Biochem, Dallas, TX 75235 USA
关键词
14(R), 15(S)-EET epoxyeicosatrienoic acid(14,15-EET); 14,15-EET binding; GPM;
D O I
10.1016/S0090-6980(00)00079-4
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
14(R), 15(S)-epoxyeicosatrienoic acid (14,15-EET) is a cytochrome P-450 monooxygenase (epoxygenase) metabolite of arachidonic acid (A. 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 mug/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 suggest 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 tsans- 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. (C) 2000 Elsevier Science Inc. All rights reserved.
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页码:321 / 333
页数:13
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