Post-receptor signal transduction and regulation of 14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET) binding in U-937 cells

Patrick Y K Wong, Pi Shiang Lai, Shu Ying Shen, Yuri Y. Belosludtsev, J R Falck

Research output: Contribution to journalArticlepeer-review

63 Scopus citations

Abstract

14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET), a cytochrome P-450 monooxygenase (epoxygenase) metabolite of arachidonic acid has been reported to induce adhesion of a monocyte cell line (U-937) to cultured endothelial cells. In this study, we identified a population of specific, high affinity binding sites for 14(R),15(S)-EET in U-937 cell surface with K(d) of 13.84 ± 2.58 nM and B(max) of 3.54 ± 0.28 pmol/106 cells. The specific binding of [3H]-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 various 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 μg/ml) and dibutyryl cAMP (300 nM), respectively, without affecting the dissociation constant. Under similar conditions, pertussis toxin (20-200 ng/ml) was less effective as compared to CT and dibutyryl cAMP. The down regulation 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 specific binding site of 14,15-EET in U-937 cells is associated with a receptor that could be down regulated through an increase in intercellular cAMP and activation of a PKA signal transduction mechanism. We propose that the signal transduction mechanism of 14,15-EET begins with the binding of the receptor, which leads to the increase of intracellular cAMP levels and the activation of PKA, and finally with the down regulation of 14,15-EET receptor binding.

Original languageEnglish (US)
Pages (from-to)155-169
Number of pages15
JournalJournal of Lipid Mediators and Cell Signalling
Volume16
Issue number3
DOIs
StatePublished - Jul 1 1997

Keywords

  • 14(R),15(S)-epoxyeicosatrienoic acid binding
  • Regulation
  • U-937 cells

ASJC Scopus subject areas

  • Immunology
  • Pharmacology

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